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Vol. III / Nr. 1 / 2016 ISSN 2392 – 7461 ISSN-L 2392 – 7461

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C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY Volume I II/ Issue 1 / March 2016

EDITOR-IN-CHIEF & MANAGING EDITOR Victor Gabriel Clatici, MD, “Carol Davila”University of Medicine and Pharmacy, Bucharest, Romania

ROMANIAN SENIOR EDITORS George Sorin Tiplica, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Virgil Patrascu, Professor, MD, PhD, University of medicine and Pharmacy Craiova, Romania Simona Fica, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Radu Vladareanu, Professor, M.D. PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Oana Andreia Coman, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Alexandru Rafila, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Sabina - Andrada Zurac, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Caius Solovan, Professor, MD, PhD, University of Medicine and Pharmacy ,,Victor Babes,, Timisoara, Romania Luiza Spiru, Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Mihai Nicolescu, Professor, MD, Vicepresident of ASAS - Academy for Agriculture and Forestry Sciences Gheorghe Ionescu-Șișești, Bucharest, Romania Mihai-Lucian Pascu, Professor, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Romania Stefana Jurcoane, Professor, PhD, University of Agronomics and Veterinary Medicine, Bucharest, Romania Carmen Maria Salavastru, MD, PhD, Associate Professor, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Dana Mihaela Jianu, MD, PhD, Associate Professor “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Ruxandra Diana Sinescu, Associate Professor, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Roxana Silvia Bumbacea, MD, PhD, Associate Professor, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Simona Vladareanu, M.D. PhD, Associate Professor, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Horatiu Moldovan, MD, PhD, Associate Professor, University of Medicine and Pharmacy, Tirgu-Mures, Romania Angela Staicu, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Romania

INTERNATIONAL SENIOR EDITORS Cassian Sitaru, Professor, MD, PhD, University of Freiburg, Freiburg, Germany Claude Dalle, Professor, MD, Paris, France Daniel Racoceanu, Professor, PhD, Sorbonne Universités, Université Pierre et Marie Curie, Paris, France Razvan Cristescu, MD, PhD, Merck Research Laboratories, Boston MA, USA Carmen Cantemir-Stone, PhD, Research Scientist, The Ohio State University, Columbus OH, USA Mihaela Balu, Professor, Beckman Laser Institute, University of California, Irvine, CA, USA Leonardo Marini, Professor, Skin Doctors Center, Trieste, Italy Miroslav Blumenberg, MD, PhD, Langone Medical Center, New York, USA Björn Brücher, Professor, MD, PhD, FRCS, FACS, Bon Secours Cancer Institute, Eichmond, Virginia, USA Klaus Fritz, Assoc. Prof. “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Ijaz Jamall, PhD, American Board of Toxicology, Sacramento, USA Marco A. Pelosi III, MD, FACOG, FACS, FICS, FAACS, International Society of Cosmetogynecology Bayonne, New Jersey, USA Mehmet Ufuk Abacioglu, MD, Radiation Oncology Department, Neolife Medical Center, Istanbul, Turkey Uwe Gieler, Professor, MD, PhD, Department of Dermatology and Allergology, University Clinic Giessen (UGKM), Germany Lucia Tomas-Aragones, PhD, Department of Psychology, University of Zaragoza, Spain Jacek C Szepietowski, Professor, MD, PhD, Department of Dermatology, Venereology and Allergology, Medical University, Wroclaw, Poland

ROMANIAN EDITORIAL BOARD Alin Laurentiu Tatu , MD, PhD, University Dunarea de Jos, Faculty of Medicine and Pharmacy, Galati, Romania Cristian Radu Jecan, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Madalina Musat, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Valentin Calu, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Cosmin Balabuc, DDS, Victor Babeș University of Medicine and Pharmacy, Timișoara, Romania Claudia Dima, MD, PhD, National Institute of Public Health, Bucharest, Romania Ileana Turcu, Senior Researcher, “Ana Aslan” International Foundation, Bucharest, Romania Violeta Corina Cristea, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Aurel-Florentin Badiu, PhD, Academy for Agriculture and Forestry Sciences Gheorghe Ionescu-Șișești, Bucharest, Romania Loredana Manolescu, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Catalin Enachescu, MD, PhD, Research, Elias Hospital, Bucharest, Romania Alexandra Maria Hillebrand -Voiculescu, PhD, ,,Emil Racovita,, Institute of Speleology of the Romanian Academy, Bucharest, Romania Loredana Mitran, MD, PhD, Emergency Universitary Hospital Elias , Bucharest, Romania Mihai Mitran, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania Carmen Sorina Martin MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

INTERNATIONAL EDITORIAL BOARD Serban P. Georgescu, MD, Romanian American Biomedical Association, Boston, Massachusetts, USA Ciprian Enachescu, MD, Societé Francaise De Radiothérapie Et Oncologie, LYON, FRANCE Daniel Nichita, Md, Ms, Senior Medical Editor, Borm Bruckmeier Publishing, California, USA Raffaele Rauso, MD, University of Foggia, Foggia, Italy Nikoleta Koini, Md, Greek Medical Association, Athens-Greece Marinela van den Heuvel-Olăroiu, MD, PhD, SOAZ / RACE (Research and Advice in Care of Elderly), Maastricht, The Netherlands Carmen Dolea, MD, MPH, MBA, World Health Organization, Geneva, Switzerland Patrick Treacy, LRCSI, MICGP, MBCAM, DRCOG, DCH, H Dip Dermatology, Dublin, Ireland Tara Nekoroski, Bachelor degree, Halozyme Therapeutics, San Diego, CA, USA Elena Campione, MD, PhD, University Hospital of Rome ,,Tor Vergata,, Italy

EDITORIAL OFFICE TEAM Cristiana Voicu Ana Maria Cristina Medeleanu Ana Maria Veronica Draganita

EDITORIAL

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46 – the magic number?

Victor Gabriel Clătici

ONCOLOGY & DERMATO-ONCOLOGY

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NOTES ON THE 7Th EDITION ON TNM CLASSIFICATION OF SKIN TUMORS Christian Wittekind

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SIGNALLING PATHWAYS IN MALIGNANT SKIN MELANOMA THE PROGNOSTIC AND THERAPEUTIC IMPLICATIONS Ciprian Enachescu, Sena Yossi, Tristan Brahmi, Victor Gabriel Clatici, Alina Tita

REVIEW

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BASAL CELL CARCINOMA: REVIEW OF EPIDEMIOLOGY AND RISK FACTORS

Cristiana Simionescu USEFUL MELANOMA DIAGNOSTIC TOOLS: IMMUNOHISTOCHEMISTRY AND MOLECULAR TECHNIQUES Gencia Ioana, Vincze Dana, Chiticariu Elena, Solovan Caius

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ISSN 2392 – 7461 ISSN-L 2392 – 7461

Andreea-Oana Enache, Virgil Pătrașcu, Raluca Niculina Ciurea, Loredana Elena Stoica, Nicolae Cernea, Stepan Desdemona,

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CLINICAL EVOLUTION OF LYMPHANGIOMA CIRCUMSCRIPTUM: OBSERVATIONS ON THREE CASES Pătraşcu Virgil, Bocîrnea Alexandra Georgiana, Ciurea Raluca

CLINICAL STUDIES

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EMOLLIENTS – A CORNERSTONE IN THE TREATMENT OF EPIDERMOLYSIS BULLOSA Ioana Mitoșeriu-Bonteanu, Alexandra Butacu, Radu-Nicolae Grigore, Cristina Cucu, Mihaela Zaiț, George-Sorin Ţiplica, Carmen-Maria Sălăvăstru

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EPIDERMOLYSIS BULLOSA - PSYCHODERMATOLOGICAL ASPECTS Lucia Tomas-Aragones, Cristiana Voicu.

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Editorial

46 – the magic number?

Dr. Victor Gabriel Clătici Editor-in-Chief & Managing Editor

Skin and genetics represent an exciting topic, because everything we are is based on a number, 46, respectively 44 chromosomes and XX or XY! We are the final results between interaction of our genetic code and the environment, and in this wonderful world we don’t know in this moment all the rules!

We are gathering information, stories, series of cases and we try to help the patients!

We have a lot of dates about implication of genes in our lives but we don’t know why some people develop disease X and other people develop disease Y, but have the same genes!

Maybe, there will be a day when we can treat genetic diseases with a ,,magic scissor,, which will cut the genes with problems!

But, until then, in this issue of Romanian Journal of Clinical and Experimental Dermatology, we present some skin diseases associated with an important genetic factor!

And YES, 46 represents a magic number and we must have the willingness to decode the number!

WHAT ABOUT GERIATRIC DERMATOLOGY? NEW DERMATOSES IN ELDERLY PEOPLE Mihail-Alexandru Badea MA1, Silviu-Horia Morariu1 Dermatology Department, University of Medicine and Pharmacy of Tîrgu Mureș Corresponding author: Mihail-Alexandru Badea Assistant Professor, Dermatology Department University of Medicine and Pharmacy of Tîrgu Mureș, Romania Phone: +40767630246 ; e-mail: badeamihai2011@yahoo.com

To the Editor, The increase in lifespan opens new challenges for healthcare providers worldwide. Skin aging has evolved from a cosmetic problem to a health issue. Dermatologists are faced with a new geriatric disorder, the cutaneous insufficiency syndrome.

This article aims to draw attention on the diversity and the functional impairment of the cutaneous organ in elderly which we will witness in the coming decades. The functional degraded skin, especially due to actinic damage, will be the source of a multi-organ body failure. We will witness real systemic implications due to geriatric skin diseases which far exceed the current approach to aging skin. Dermatoporosis or the chronic cutaneous insufficiency syndrome described in 2007 by Kaya and Saurat is due to a chronic actinic aggression and has 4 stages of evolution, from light manifestations such as skin atrophy to skin dissecting hematomas with significant shedding of the cutaneous tissue ,which can lead to severe systemic manifestations.

Identical events may be encountered in dermatological steroid dependent patients (sufferers of autoimmune bullous diseases, autoimmune connective tissue, etc.). The molecular mechanism is represented by a decrease in the CD44 receptor for hyaluronic acid which causes cutaneous atrophy and the subsequent exposure to trauma of the underlying structures. These minor traumas lead to the development of purpura, pigmentations, ulcerations, stellate pseudoscars and skin dissecting hematomas [1,2] .I have personally seen about 40 cases of dermatoporosis in the last 2 years. So the disease is common but little known.

The location described so far is in the forearms and only rarely in the lower legs where it can mimic very accurately the mani-

festations of chronic venous insufficiency. The injuries are categorized most frequently as adverse reactions to anticoagulant therapy. The treatment includes topical application of either hyaluronic acid or the application of a combination of hyaluronic acid and retinoids which is superior but is not available in Romania. Effective results were also obtained with the application of an epidermal growth factor [3,4]. Sunscreen and the avoidance of even minor local trauma are essential in obtaining effective therapeutic results. We presented during various dermatological manifestations four scientific papers about the chronic cutaneous insufficiency “dermatoporosis” and we also published an article on this subject.

We did not get too many questions...! There are 13 articles on PubMed about dermatoporosis from 2007 to the present and only one in the last year. We wonder why so few... Do we really need to witness serious dermatological events due to dermatoporosis and in the general geriatric dermatologic pathology and only then will we mobilize? Why always in the last moment?

Why not organized? A final aspect is the fact that this dermatosis responds very well to therapy with various topical as well as using preventive measures.

In conclusion it is necessary to recognize the disease and to not just simply catalogue it as a response to anticoagulation therapy, as it is usually categorized due to the Bateman senile purpura lesions that are constantly encountered in the dermatoporosis context. The real issue is the diagnosis. We cannot treat what we don't know. Papers on the matter are scarce and we don’t know the full length of misdiagnosis. It is our opinion that we are faced today with the task of rethinking our approach to dermaporosis for a better patient management.

Figure 1. a) Dermatoporosis on forearms; b) Dermatoporosis on shins

Bibliography 1. Kaya G, Saurat JH.Dermatoporosis: a chronic cutaneous insufficiency/fragility syndrome.

Clinicopathological features, mechanisms, prevention and potential treatments. Dermatology.

2007. 215(4):284-294.

2. Badea MA, Morariu SH, Sin Anca. A Novel Disease Caused by Increase of the Lifespan: Chronic Cutaneous Insufficiency Syndrome or Dermatoporosis. Acta Medica Marisiensis.

2015;61(1):54-56

3. Nikolic DS, Ziori C, Kostaki M, Fontao L, Saurat JH, Kaya G. Hyalurosome gene regulation and dose-dependent restoration of skin atrophy by retinaldehyde and defined-size hyaluronate fragments in dermatoporosis.Dermatology. 2014;229(2):110-115.

4. McKnight B, Seidel R, Moy R. Topical Human Epidermal Growth Factor in the Treatment of Senile Purpura and the Prevention of Dermatoporosis. J Drugs Dermatol. 2015;14(10):11471150.

Letter to the Editor

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Oncology & Dermato-Oncology

Notes on the 7th edition on TNM classification of skin tumors

NOTES ON THE 7TH EDITION ON TNM CLASSIFICATION OF SKIN TUMORS COMENTARII CU PRIVIRE LA CEA DE-A 7-A EDIȚIE A CLASIFICĂRII TNM A TUMORILOR CUTANATE Christian Wittekind Pathology Institute, Clinical University, Leipzig, Germany Corresponding author: Prof. Dr. med. Christian Wittekind, Institut für Pathologie, Universitätsklinikum Leipzig, Liebigstraße 26, D-04103 LEIPZIG,Germany; Tel. ++49 341 9715000, Fax. ++49 341 9715009.

E-Mail: Christian.Wittekind@medizin.uni-leipzig.de

No conflict of interest

Open Access Article

Abstract Keywords: Skin tumours, TNM classification, Skin carcinoma, malignant melanoma of skin, Merkel cell carcinoma of skin.

In the 7th edition of the TNM classification of malignant tumours some alterations and innovations for skin tumours were introduced including an entirely new classification for Merkel Cell Carcinomas. The changes are shown along with notes on some critical issues. The chapter “cutaneous squamous cell carcinoma and other cutaneous carcinomas” has been newly named and supplied with new definitions. The UICC definitions of the T categories in the 7th edition differ from those in the AJCC Cancer staging manual. The classification of carcinomas of the eyelid has been moved from the ophthalmic tumours chapter to the skin tumours chapter and has undergone some changes in the definition of categories T2 – T4 as compared to the 6th edition. In Malignant Melanoma of skin in addition to tumor ulceration, mitotic rate replaces level of invasion as a primary criterion for defining the subcategory of pT1b. The first staging proposal for Merkel Cell Carcinoma of the skin is based on the analysis of over 4,700 patients using the National Cancer Database as well as extensive review of the literature.

Rezumat Cuvinte-cheie: Tumori cutanate, clasificare TNM, carcinom cutanat, melanom cutanat, carcinom cutanat cu celule Merkel.

Cite this article: Christian Wittekind.

Notes on the 7th edition on TNM classification of skin tumors RoJCED 2016; 3(1):8 - 12

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În cadrul celei de a 7-a ediții privind clasificarea TNM a tumorilor maligne, au fost introduse o serie de modificări și informații noi legate de tumorile cutanate, printre care, în premieră, o clasificare a carcinoamelor cutanate cu celule Merkel. Lucrarea de față prezintă modificările aduse precum și câteva comentarii legate de diferite aspecte importante. Capitolul “carcinoame spino-celulare și alte carcinoame cutanate” a fost redenumit și completat cu definiții noi. În cea de-a 7-a ediție, definițiile UICC ale categoriei T sunt diferite de cele din Manualul AJCC de stadializare a cancerului. Comparativ cu cea de 6-a ediție, clasificarea carcinoamelor pleoapei a fost mutată din capitolul de tumori oftalmice în cel de tumori cutanate și a suferit o serie de modificări în ceea ce privește definirea categoriilor T2-T4. Legat de melanomul cutanat, pe lângă ulcerația cutanată, rata mitotică înlocuiește nivelul de invazie ca prim criteriu de definire a subcategoriei pT1b. Prima propunere de stadializare a carcinoamelor cutanate cu celule Merkel are la bază analiza a peste 4 700 de pacienți incluși în Baza de Informații Națională a Cancerului, precum și o analiză extensivă a literaturii de specialitate.

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Oncology & Dermato-Oncology Introduction In the 7 edition of the UICC TNM classification of malignant tumours, published in 2010, several alterations for tumours of the skin have been introduced[1]. The number of entities enrolled in this chapter has been enlarged from two to four.

Carcinoma of the skin of eyelid has been moved from the ophthalmic tumours chapter to the skin tumours chapter and Merkel cell carcinoma TNM classification is entirely new.

The alterations and new definitions will be introduced in this contribution. th

Skin tumours UICC 2002 [2] Carcinoma of skin Malignant melanoma of skin

UICC 2100 [1] Carcinoma of skin Carcinoma of skin of eyelid Malignant melanoma of skin Merkel cell carcinoma of skin

Carcinoma of the skin The chapter “cutaneous squamous cell carcinoma and other cutaneous carcinomas” has been newly named and supplied with new definitions.

A comparison between the definitions of T categories of the 6th and 7th edition is shown in Table 1.

Table 1.

UICC 6th edition 2002 [2] T1 Tumour 2 cm or less in greatest dimension T2 Tumour more than 2 cm,but no more than 5 cm in greatest dimension

UICC 7th edition 2010 [1] T1 Tumour 2 cm or less in greatest dimension T2 Tumour more than 2 cm

T3 Tumour more than 5 cm in greatest dimension

T3 Tumour with invasion of deep structures,e.g.,muscle, bone,cartilage,jaws,and orbit

T4 Tumour invades deep extradermal structures as cartilage,skeletal muscle,or bone

T4 Tumour with direct or perineural invasion of skull base or axial skeleton

The UICC definitions of the T categories in the 7th edition differ from those in the AJCC Cancer staging manual[3].

Table 2.

UICC 7th edition [1] T2 Tumour more than 2 cm in greatest dimension T3 Tumour invades deep extradermal structures T4 Tumour with direct or perineural invasion of skull base or axial skeleton

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AJCC 7th edition [3] T2 Tumour more than 2 cm in greatest dimension or tumour any size with two or more high risk features * T3 Tumour with invasion of maxilla, mandible, orbit or temporal bone T4 Tumour with invasion of skeleton (axial or appendicular ) or perineural invasion of skull base

Notes on the 7th edition on TNM classification of skin tumors

The differences between the TNM classifications of UICC and AJCC are shown in Table 2.

Depth/invasion

m m m

> 2 mm thickness Clark level > IV Perineural invasion

Anatomic location

m m

Primary site ear Primary site non-hair bearing lips

Differentiation

m m

Poorly differentiated Undifferentiated

*High-risk features for the primary tumour /T) staging are: The AJCC TNM classification does focus on tumours of skin of face not really taking into consideration other sites as skin of hands or legs.

Additionally the definitions of the N categories between UICC and AJCC differ in that UICC only has a N2 category and AJCC offers subcategories N2a – N2c (not shown in a Table 2). In the UICC TNM classification the high-risk features are considered in stage grouping, cases with high-risk features being located in a stage II.

Carcinoma of the skin of eyelid According to the rules of classification for these tumours there should be histological confirmation of the disease and division of cases by histological type, eg., basal cell, squamous cell, sebaceous carcinoma. Melanoma of the eyelid is classified with skin tumours. The classification of carcinomas of the eyelid has undergone some changes in the definition of categories T2 – T4 as compared to the 6th edition (are shown in a Table 3).

In these definitions some new elements have been introduced. T3/pT3 is depending on the extent of resection and T4/pT4 is defined by resectability.

Additionally, some changes in the stage grouping of these tumours have been introduced (not shown in a Table 3).

Malignant Melanoma of Skin According to the rules of classification there should be histological confirmation of the disease.

Malignant melanoma of eyelid is classified with skin tumours. The extent of the primary tumor is classified after excision, there is only a pT category.

A T category is not a valid category.

For pT melanomas, in addition to tumor ulceration, mitotic rate replaces level of invasion as a primary criterion for defining the subcategory of pT1b. This has been differently used in the first print of the 7th edition of TNM [1].

Data from the AJCC Melanoma Staging Database demonstrated a highly significant correlation with increasing mitotic rate und poorer survival rates [4] (Table 4).

Since the feature of mitotic rate was not published in the original 7th edition of the UICC TNM classification, it is recommended to use it for the sake of uniformity[5]. The change will appear in subsequent UICC reprinting of the 7th edition. The

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Christian Wittekind

Table 3.

UICC 6th edition 2002 [2]

UICC 7th edition 2010 [1]

T1 Tumour of any size, not invading the tarsal plate; or at eyelid margin 5 mm or less in greatest dimension

T1 Tumour 5 mm or less in greatest dimension not invading the tarsal plate or eyelid margin

T2 Tumour invades tarsal plate; or at eyelid margin, more than 5 mm but not more than 10 mm in greatest dimension

-T2a Tumor more than 5 mm, but not 10 mm or less in greatest dimension or any tumour that invades the tarsal plate or the eyelid margin -T2b Tumour more than 10 mm, but more than 20 mm in greatest dimension, or involves full thickness eyelid

T3 Tumour involves full eyelid thickness; or at eyelid margin, more than 10 mm in greatest dimension

-T3a Tumour more than 20 mm in greatest dimension or any tumour that invades adjacent ocular or orbital structures or any tumour with perineural invasion -T3b Tumour whose complete resection requires enucleation, exenteration, or bone resection

T4 Tumour invades adjacent

T4 Tumour is not resectable due to extensive invasion of ocular, orbital, craniofacial structures or brain

structures

Table 4.

UICC 7th edition [1]

AJCC 7th edition [3]

pT1 - Tumor 1 mm or less in thickness

pT1 - Tumour 1 mm or less in thickness

pT1a - Clark level II or III, without ulceration

pT1a - without ulceration and mitosis < 1/mm2

pT1b - Clark level IV or V, or with ulceration

pT1b - with ulceration or mitoses > 1/mm2

other definitions of the T-, N- and M categories have not changed from the 6th to the 7th edition.

The site of distant metastasis continues to represent the primary component of subcategorizing the M category. In contrast to the AJCC[3] in the UICC classification isolated tumor cells in regional lymph nodes are classified as pN0(i+) whereas in the AJCC TNM classification these findings are classified as pN1. The findings of tumor cells in a lymph node is considered clinically significant. A lower threshold of clinically insignificant metastasis of regional lymph nodes has not been defined based on evidence.

Merkel Cell Carcinoma This is the first staging proposal for Merkel Cell Carcinoma of the skin. Up till this publication several staging proposals have been used, none of which will be mentioned here.

According to the AJCC Cancer staging Manual[3] the new staging system is based on the analysis of over 4,700 patients using the National Cancer Database as well as extensive review of the literature.

According to the AJCC rules for classification, Merkel Cell carcinoma of the eyelid (C44.1) is not included in this chapter but is classified with carcinomas of the eyelid.

Table 5. TNM Clinical Classification T â&#x20AC;&#x201C; Primary Tumour TX Primary tumour cannot be assessed T0 No evidence of primary tumour Tis Carcinoma in situ T1 Tumour 2 cm or less in greatest dimension T2 Tumour more than 2 cm but not more than 5 cm in greatest dimension T3 Tumour more than 5 cm in greatest dimension T4 Tumour invades deep extradermal structures, i.e., cartilage, skeletal muscle, fascia or bone N â&#x20AC;&#x201C; Regional Lymph Nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 N1a N1b N2

Regional lymph node metastasis Microscopic metastasis (clinically occult: cN0 + pN1) Macroscopic metastasis (clinically apparent: cN1 + pN1) In transit metastasis M a rc h 2 0 1 6

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Oncology & Dermato-Oncology

Notes on the 7th edition on TNM classification of skin tumors

Note: In-transit metastasis: a tumor distinct from the primary lesion and located 1) between the primary lesion and the draining regional lymph nodes and/or 2) distal to the primary lesion M â&#x20AC;&#x201C; Distant Metastasis Distant metastasis cannot be assessed No distant metastasis Distant metastasis Skin, subcutaneous tissues or non-regional lymph node(s) Lung Other site(s)

MX M0 M1 M1a M1b M1c

The definitions of the different T-, N-, and M categories are shown below ( Table 5). pTNM Pathological Classification The pT, pN, and pM categories correspond to the T, N, and M categories. pN0 Histological examination of a regional lymphadenectomy specimen will ordinarily include 6 or more lymph nodes. If the examined lymph nodes are negative, but the number ordinarily resected is not met, classify as pN0.

A histopathological Grading is not applicable.

A new element in stage grouping is the use of a cN0 and pN0 in stage grouping. The N1 category is subdivide in N1a with microscopic metastasis (clinically occult: cN0 + pN1) and N1b with macroscopic metastasis (clinically apparent: cN1 + pN1).

Thus, stages I and II are subdivided into IA and IB as well as IIA and IIB, depending on the results of regional lymph node examination cN0 versus pN0, the latter being the more reliable.

Table Anatomical Stage Grouping Stage 0 Stage I Stage IA Stage IB

Tis T1 T1 T1

N0 N0 pN0 cN0

M0 M0 M0 M0

Stage IIA

T2,T3

pN0

M0

Stage IIB

T2,T3

cN0

M0

Stage IIC

T4

N0

M0

Stage IIIA Stage IIIB

Any T Any T

N1a N1b,N2

M0 M0

Stage IV

Any T

Any N

M1

Summarizing commentary The TNM classification of skin tumours has undergone some changes from the 6th to the 7th edition.

These changes have resulted in some differences between the definitions of the UICC and AJCC TNM classification. They become obvious in the definitions of T categories of squamous cell carcinomas of skin, in meanwhile adapted definitions of pT1 and pT1

categories as well as in the definitions of ITCs in regional lymph nodes (UICC pN0 versus AJCC pN1).

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Bibliography [1] Sobin LH, Gospodarowicz MK, Wittekind C (eds.) UICC TNM Classification of Malignant Tumours, Seventh Edition. Oxford: Blackwell Publishing Ltd.; 2010 [2] UICC (International Union Against Cancer) TNM Classification of Malignant Tumours.

6th ed. Sobin LH, Wittekind Ch., eds. New York: Wiley; 2002 [3] American Joint Committee on Cancer (AJCC) Cancer Staging Manual 7th ed. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A. Eds. New York: Springer; 2009

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[4] Balch CM, Soong SJ, Gershenwald JE, et al. Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer Melanoma staging system.

J Clin Oncol 2001;19:22-34 [5] Wittekind Ch, Compton CC, Brierley J, Sobin LH. TNM Supplement. A commentary on uniform use. 4th edition. Wiley-Blackwell, Oxford, 20

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Christian Wittekind

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Signalling pathways in Malignant Skin Melanoma. The prognostic and therapeutic implications

SIGNALLING PATHWAYS IN MALIGNANT SKIN MELANOMA THE PROGNOSTIC AND THERAPEUTIC IMPLICATIONS SEMNALE CELULARE ÎN MELANOMA IMPLICAȚII PROGNOSTICE ȘI TERAPEUTICE 1

Ciprian Enachescu1, Sena Yossi1, Tristan Brahmi1, Victor Gabriel Clatici2 , Alina Tita3 Centre Hospitalier Lyon Sud, 165, Chemin du Grand Revoyet 69495 Pierre-Bénite cedex, France, 2 Dermatology Department, ELIAS Emergency Universitary Hospital, Bucharest, Romania; 3 Medical Student , “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.

Corresponding author: Ciprian Enachescu, Département de Radiothérapie et Oncologie, Centre Hospitalier Lyon Sud, Chemin du Grand Révoyet, 69495 Pierre-Benite Cedex, France, E-mail: ciprian.enachescu@lyon.fr

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: Melanoma, Prognostic, Treatment, BRAF, MEK.

Cite this article: Ciprian Enachescu, Sena Yossi, Tristan Brahmi, Victor Gabriel Clatici, Alina Tita.Signalling pathways in Malignant Skin Melanoma.

The prognostic and therapeutic implications.

RoJCED 2016; 3(1):14 - 20

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Malignant melanoma remains one of the most aggressive forms of cancer, being responsible for 1.7 % of all cancer deaths in the USA. To improve the treatment efficacy, molecular targeted therapies have been developed and gradually introduced in clincal practice. Some of these molecules act on the signal transduction pathway of cell proliferation, pathway named Ras/ Raf/MEK/ERK cascade or MAPK /ERK pathway.

The Mitogen - Activated Protein Kinases (MAPKs) are serine-threonine kinases that control fundamental cellular processes such as growth, proliferation, survival or apoptosis and cell transformation, and, in abnormal situations, play a critical role in the development and progression of cancer. Today, the BRAF inhibitors (Vemurafenib and Dabrafenib) and MEKinhibitors (Trametinib and Cobimetinib) demonstrated effectiveness in the treatment of BRAF V600 mutation-positive, unresectable, locally advanced or metastatic melanoma and therefore can be used in these situations. Regarding RAS and ERT directed therapies , there is no clinical evidence to prove any benefit for Farnesyl Transferase Inhibitors (FTIs) and RNA interference (RNAi) neither for the specific and selective, ATP-competitive , inhibitor of ERK1/2, SCH772984.

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Ciprian Enachescu, Sena Yossi, Tristan Brahmi, Victor Gabriel Clatici, Alina Tita

Rezumat Cuvinte-cheie: Melanom, Prognostic, Tratament, BRAF, MEK.

Melanomul malign ramâne una dintre cele mai agresive forme de cancer, responsabil de 1.7% dintre decesele prin cancer în Statele Unite. Pentru a îmbunătăţi eficacitatea terapeutică, tratamente moleculare ţintite au fost dezvoltate şi introduse treptat în practica clinică. Unele dintre aceste molecule acționează la nivelul căii de transmitere a semnalului responsabil de proliferarea celulară, cale numită Ras/Ras/MEK/ERK sau MAPK/ERK. Proteinele efectoare ale căii Mitogen -Activated Protein Kinases (MAPKs) sunt kinaze tip serin-treonina, care controlează procese fundamentale celulare precum creşterea, proliferarea, supravieţuirea sau apoptoza, precum şi transformarea celulară, iar în situaţiile anormale joacă un rol esenţial în apariţia şi progresia cancerului.

Astăzi, inhibitorii de BRAF (Vemurafenib şi Dabrafenib) și inhibitorii de MEK (Trametinib şi Cobimetinib) s-au dovedit eficace în tratamentul melanomului nonrezecabil avansat sau metastatic purtător al mutaţiei BRAF-V600 şi prin urmare au fost validaţi pentru aceste situaţii. În ceea ce priveşte tratamentele anti RAS și anti ERK, nu există la momentul actual evidenţe clinice privind eficacitatea terapeutică pentru Inhibitorii de Farnesil Transferază (FTIi) sau interferenţă RNA (RNA interference, RNAi), nici pentru inhibitorul specific şi selectiv al ERK 1/ERK2, numit SCH772984.

The incidence of malignant skin melanoma, the most aggressive form of skin cancer, continues to rise in recent years, new 73870 cases have been estimated in 2015 in the United State by the Surveillance, Epidemiology and End Results (SEER) database, and represents 4.5% of all new cancer cases. Regarding the mortality rate, the skin melanoma is responsible for 9940 deaths in USA, representing 1.7 % of all cancer deaths. (1) Melanoma originates from melanocyte, a specialized skin cell derived from neural-crest cells (like neurons and glial cells, adrenal medulla, cardiac cells and craniofacial tissue), whose major function is to produce the melanin. (2) Melanin is a complex mixture of tyrosine-derived biopolymers produced in response to UVR in specialised organelles called melanosomes, and is responsible for the pigmentation of the skin, hair and eyes. (3) Although malignant melanoma is mainly a sporadic disease, about 5 to 10% of the cases are hereditary and show familial aggregation (Familial Malignant Melanoma), defined as the occurrence of melanoma in at least two first-degree relatives or families with at least two melanomas irrespective of the degree of relationship. (4) The most common abnormality responsible for tumorogenesis and progression of malignant melanomas concerns the Ras/Raf/MEK/ERK cascade, known as MAPK / ERK pathway (5).

Mitogen -Activated Protein Kinases (MAPKs) These are serine-threonine kinases that couple intracellular signals initiated by extracellular or intracellular stimuli to transcription factors which control fundamental cellular processes such as growth, proliferation, differentiation, migration,

survival or apoptosis and transformation(6) Abnormalities in MAPK signalling impinge on most, if not all these processes, and play a critical role in the development and progression of cancer.(7)

RAS (Rat Sarcoma) genes and proteins Among the 3 RAS known oncogenes (H-RAS, KRAS, and N-RAS), the NRAS (Neuroblastoma RAS viral oncogene homolog), located on the short (p) arm of chromosome 1, is particularly involved in the induction and progression of malignant melanoma. (8) NRAS mutations are found in all melanoma subtypes, but may be slightly more common in melanomas derived from chronic sun-damaged(9).

Normal p21ras protein is a small (21 kDa) G-protein (Guanosine-nucleotide-binding protein), that binds GDP (Guanosine Diphosphate) or GTP (Guanosine Triphosphate) and acts as a binary molecular switch (“on” and “off” states) that controls intracellular signalling networks. (10) In the inactive (“off») state p21ras is bound to the GDP, while in the active growth-promoting signal («on”) state, it is bound to GTP. (11) Therefore, the ratio of p21ras-GDP-bound ("off" state) to p21ras-GTP-bound (“on” state) is crucial for the control of cell growth and proliferation, and this ratio must be controlled precisely. In the activation phase GTP triggers a cascade of mitogen-activated protein kinases which ultimately phosphorylate the target, such as a transcription factor, delivering the message to its final destination. (12) In normal situations (wild-type p21ras) this signal is necessarily self-limiting, the deactivation (GTP to GDP conversion) being achieved by hydrolysis, but mutations of p21ras sends a continuously growth-promoting signal by the active GTPbound form.(13) M a rc h 2 0 1 6

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Incidence of NRAS mutation in malignant melanoma NRAS mutations, present in 20% all melanomas, are mainly represented by 2 oncogenic changes, the position 61 (a point mutation representing more than 80% of all NRAS mutations) and position 12-13 mutations. Position 61 mutation is an activating mutation which locks the p21ras into its activated (GTP-associated) conformation, while mutations at positions 12 and 13 render p21ras insensitive to the physiological mechanisms of inactivation. (14, 15)

Clinical and therapeutically significance In terms of morphological and phenotypic diagnosis, NRAS mutated melanomas do not exhibit any particular characteristics, but the presence of a NRAS mutation has a prognostic significance.

NRAS mutant melanomas are thicker tumours, typically located at the extremities with greater rates of mitosis compared to BRAF mutant melanomas.

Regarding the overall survival, the NRAS mutation is an unfavourable prognostic factor for melanoma patients compared with non mutated patients (16)

Ras-directed therapies There are 2 distinct anti-Ras strategies: actions at the p21ras proteins level and actions at the RAS gene expression level.

At the p21ras level, the main directed therapy is

represented by Farnesyl Transferase Inhibitors (FTIs), a class of drugs designed to inhibit the posttranslational farnesylation of a number of target proteins, including p21ras, preventing its membrane attachment and signalling function.(17) Despite potent target inhibition in tumour tissue (85-98%), FTIs showed no clinical benefit (no clinical responses). (18)

At the RAS gene expression level the idea is to prevent the Ras expression by RNA interference (RNAi), a biological process in which RNA molecules inhibit gene expression, causing the mRNA cleavage, effects dependent on the doubling time of the cells.(19, 20) B-RAF (Rapidly Accelerated Fibrosarcoma) genes and proteins Located on the long (q) arm of chromosome 7, the BRAF gene provides instructions for making a protein that helps transmit chemical signals from outside the cell to the cell’s nucleus. (21) The RAF family proteins (there are three known mammalian RAF isoforms: A, B and C-Raf) are serine/threonine kinases which play a role in various normal physiological processes as cellular metabolism, cell cycle progression, cell death and neurological function. (22) Most RAF kinase protein located in the cytosol where the enzymes lie, are in their dormant state.

Being activated by RAS-GTP complex, the RAF kinases participate in the RAS-RAF-MEK-ERK signal

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transduction cascade. (23) On the contrary, by inhibiting RAF, the MAPK pathway is being blocked and therefore attenuates cell cycle progression by arresting cells at the Go/G1 boundary.(24) Mutations in the BRAF gene leads to a mutant protein whose kinase activity is greatly elevated, which constitutively stimulates ERK activity independent of RAS. As a result, mutated BRAF causes overactive downstream signalling via MEK and ERK , leading to excessive and uncontrolled cell proliferation, independent of growth factors and resistance to apoptosis (programmed cell death). (25)

Incidence of BRAF mutation in malignant melanoma Approximately 40-50% of melanomas harbour activating BRAF mutations without correlation between BRAF mutational status and age, sun exposure, and Clark’s level. (26) Most commonly (90-95% of BRAF mutant melanoma), the valine at amino acid 600 is replaced by glutamate (V600E) through mutation of a single nucleotide (GTG to GAG), which leads to a 5000 fold increase in its kinase activity. (27)

Clinical and prognostic significance For localised skin melanoma BRAF-mutation is correlated with age (younger patient, ≤ 50 years), location (truncal location), histopathology subtype (superficial spreading or nodular melanoma) and lack of cumulative sun-induced damage (CSD) at the primary site (28). In metastatic cases B-RAF mutation, identified in more than 70% of cases is principally correlated with age (younger patients).

The interval from diagnosis of melanoma to first distant metastasis (including first unresectable locoregional recurrence) was not statistically different between BRAF-mutant and BRAF wild-type melanoma. B-RAF mutation is a survival unfavourable prognostic factor, the overall survival time from diagnosis of first distant metastases being 4 time shorter for the BRAF-mutated patients compared with wild-type BRAF patients (11.1 months vs. 46.1 months).(29)

BRAF mutation detection methods A) Tumor biopsy methodes. Several methods can be used to detect BRAF mutations, variations in reported BRAF mutation rates being attributable to differences in the detection methods used.

The Cobas 4800 BRAF V600 Mutation Test is a Real-Time Polymerase Chain Reaction (RT-PCR) on a formalin-fixed, paraffin-embedded (FFPE) tissue in order to amplify and detect the target DNA. It presents cross-reactivity with non-BRAF p.V600E mutations (eg, p.V600K, p.V600D, and p.V600‘E2’) (30) Sanger sequencing is a method of DNA sequencing (determine the precise order of nucleotides within a DNA molecule) used to identify mutations in DNA. However, sequencing has relatively low analytical sensitivity, meaning that a mutation must be present in >15% to 20% of tumor content to be

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detected. (31) Immunohistochemistry (IHC), a rapid and inexpensive test, particularly useful for specimens not suitable for molecular analysis, uses a mouse monoclonal antibody directed against the BRAF V600E mutant epitope, that only recognizes the V600E epitope and not others (eg, BRAF wild type, V600K, V600D, V600R, K601E, etc). (32) A comparative molecular study shows that BRAF immunohistochemistry (IHC) has excellent specificity for detecting BRAF V600 mutations and confers comparable results with cobas test, but concludes that IHC should not be used alone and the cobas test remains the FDA-approved diagnostic standard. (33) B) â&#x20AC;&#x2DC;Liquid biopsiesâ&#x20AC;&#x2122; methods. Liquid biopsies are non-invasive methods to detect Circulating Tumour Cells (CTCs) and Circulating Nucleic Acids (CNA) , especially circulating tumour DNA (Cellfree or circulating tumour DNA , ctDNA) which are fragments shed into the blood from the primary tumour and from metastatic sites. (34) The circulating mutated DNA detection method must be extremely sensitive, because tumor-specific DNA can greatly vary related to individualspecific and tumor-specific factors and, most important, the most cfDNA present in the serum are wild-types and the tumor-derived mutant DNA fraction could be <0.01% .(35) Using a Droplet digital PCR (ddPCR), an analytically sensitive technique for quantifying small concentrations of DNA, BRAF-V600E mutation in circulating DNA (cfDNA ) has being detected in >75% of late-stage melanoma patients with BRAF mutation-positive tumors, with the correspondence between tumor tissue BRAF(V600E) and plasma cfBRAF (V600E) of 84.3%. (36)

B-RAF-V600E mutation and target therapy There are 2 drugs against BRAF protein, Vemurafenib (Zelboraf) and Dabrafenib (Tafinlar) , approved for the treatment of metastatic or unresectable melanoma for patients with BRAF V600E mutation (not indicated in patients with wild-type BRAF melanoma) (37) A) Metastatic melanoma.

BRIM-3, a phase III randomized clinical trial on 675 treatment-naive patients with BRAF V600Emutated stage IIIC/IV metastatic melanoma, compares Vemurafenib (960 mg orally twice daily) with Dacarbazine (1,000 mg/m2 intravenously every 3 weeks). Vemurafenib produced improved rates of overall survival (84% vs. 64% at 6 months) and progression-free survival (5.3 against 1.6 months) than classical chemotherapy with Dacarbazine. (38) BREAK-3 is a phase III trial on 250 patients with stage IV or inoperable stage IIIC BRAFV600E melanoma, with no prior therapy, that compares Dabrafenib (150 mg twice daily) with Dacarbazine (1000 mg/m2 every 3 weeks, IV), with a crossover to Dabrafenib for the patients that progresses in Dacarbazine group. Dabrafenib is superior to classical Dacarbazine regarding objective response rate (52% vs 17%) and progression free survival (5.1 vs 2.7 months). Dabrafenib's toxicity is mainly directed to the skin (hyperkeratosis, papillomas, palmar-plantar erythrodysesthesia), followed by pyrexia, fatigue, headache and arthralgia. (39) B) Nonmetastatic malignant melanoma.

Currently neoadjuvant treatment for resectable/ unresectable stage III melanoma remains an investigational approach, but for inoperable bulky melanoma with positive BRAF V600E mutation, neoadjuvant therapy can be recommended to reduce the tumour burden and facilitate tumor resection.

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A study on 11 patients with advanced locoregional BRAF V600E mutant melanoma treated with 6.0 months ( 1.2-29.4 months) Vemurefenib demonstrate objective responses in 50% of patients without major complications attributable to BRAFtargeted therapy (41)

(40)

MEK (MAP kinase / ERK Kinase ) genes and proteins The MEK family of genes, also known as MAPKK (Mitogen-Activated Protein Kinase Kinase) or MAP2K, located on the long (q) arm of chromosome 15 (15q) for MEK 1 and on the long (q) arm of chromosome 19 (19q) for MEK2 respectively.(42) MEK genes provide instructions for making MEK protein kinases (molecular weight about 45-50 kDa), dual-specificity kinases, kinases that can act as both tyrosine kinase and serine/threonine kinase.

The most potent activator MEK kinase is the BRAF. The RAF kinase causes the phosphorylation and activation of MEK1/2, which in turn phosphorylates and activates Extracellular signalâ&#x20AC;&#x201C;regulated kinase (ERK). Constitutive activation of MEK1 (caMEK) can induce the oncogenic transformation. (43)

MEK mutants actions Constitutively active MEK mutants promote longterm events such as cell differentiation, proliferation and transformation, inducing the formation of aggressive tumors that progress up to the metastatic stage.(44) Conversely, dominant negative MEK mutants prevent cells proliferation and can revert RAF, RAS transformed cells. (45)

Incidence of MEK mutation in malignant melanoma and therapeutic implications The incidence of MEK mutation in malignant melanoma is relativelly low (3 to 8 %) , only 7 MEK1 and one MEK 2 mutation being identified in the analysis of tumor samples of 127 melanoma patients.

(46) The MEK mutation downstream of BRAF causes the reactivation of kinase activity and confers resistance of BRAF- inhibitors ( resistance-associated MEK1 mutations).(47) MEK inhibitors are able to target MAPK-dependent tumors and exhibit distinct efficacies against BRAF- and KRAS-mutant melanomas.

Since 1995 highly selective MEK1/2 inhibitors have been tested clinically or are currently undergoing clinical trial evaluation. (48, 49)

Patients selection.

Pre-clinical studies suggest that patients harboring activating mutations in RAS or BRAF genes are better candidates for treatment with these kinase inhibitors. (50)

Therapeutic scheme selection : Monotherapy versus combination.

Two highly selective allosteric inhibitors of MEK1 and MEK2, Trametinib and Cobimetinib , are

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curently used for the treatment of metastatic melanoma patients carrying the BRAF V600 mutation.

MEK inhibitors as monotherapy Trametinib was studied in a Phase III randomized, open-label, multisite study on 322 patients with metastatic BRAF V600E/K mutation-positive melanoma. Trametinib 2 mg once daily compared to chemotherapy increases median Progressionfree Survival (PFS) from 1.5 months to 4.8 months (P<0.001) and Overall Survival (OS) at 6 months from 67% to 81 % (P=0.01). The objective response rates according to Response Evaluation Criteria In Solid Tumors (RECIST) were 22% for trametinib against 8% for chemotherapy (P=0.001). The results of this study led to approval of trametinib as a single agent for treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations. (51)

MEK inhibitors associated with BRAF inhibitors The BRAF-MEK inhibitor combination tries to realise multistage inhibition of the MAPK pathway to avoid the development of acquired resistance due to intrinsic resistance and MAPK-reactivation. (52) There are 3 Phase III clincal trials that compare the combination of Trametinib or Cobimetinib and Dabrafenib versus Dabrafenib or Vemurafenib alone on patients with unresectable stage IIIC or metastatic stage IV melanoma harboring a BRAF V600E or V600K mutation.

The Phase III trial (COMBI-d) conducted on 423 previously untreated patients, demonstrates that association of Trametinib (2 mg orally once daily) and Dabrafenib (150 mg orally twice daily) is superior to Dabrafenib alone in terms of overall response rate (67% vs 51%) , median progressionfree survival ( 9.3 vs 8.8 months, p=0.03) and interim 6-month overall survival rate (93% vs 85%, P=0.02). Toxicity was similar in the two groups except for pyrexia, which occurred more often in the combination arm than in the monotherapy arm (51% versus 28%). (53) The Phase III open-label trial (COMBI-v), on 704 treatment-naĂŻve patients, shows that combination of Trametinib-Dabrafenib (same dosage as in COMBI-d) are superior to Vemurafenib (960 mg twice daily) alone in terms of overall survival rate at 12 months (72% vs 65%; p=0.005), median progression-free survival (11.4 vs 7.3 months; p<0.001) and objective response rate (64% vs 51 , p<0.001). Rates of severe adverse events and study-drug discontinuations were similar in the two groups. Cutaneous squamous-cell carcinoma and keratoacanthoma occurred in 1% of patients in the combination-therapy group and 18% of those in the vemurafenib group.(54) The results of phase III CoBRIM trial, adding Cobimetinib to Vemurafenib in 495 patients with BRAF V600 mutation-positive, unresectable, locally advanced or metastatic melanoma show an increase in the.. median progression-free survival (from 7.2 to 12.3 months) and overall survival rate at 9 months (from

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73% to 81%) compared to Vemurafenib alone.(55) As a result of these excellent findings The US Food and Drug Administration - FDA has approved the combination of Dabrafenib (150 mg orally twice daily) and Trametinib (2 mg orally once daily) for the treatment of patients with BRAF V600E/K-mutant metastatic melanoma, and their use seem to be currently the best approach. (56)

ERK (Extracellular signal-Regulated Kinases) genes and proteins ERK gene (also known as Mitogen Activated Protein Kinase 3), located on the short (p) arm of chromosome 16 (16p), is responsible for the coding of ERK proteins, a 44 kDa (ERK1) and 42-kDa (ERK2) serine/threonine kinase.(57) ERK1 /2 (MAPK3/1) is regulated by the dual-specificity kinases MEK1/2 through phosphorylation, then activated ERK1/2 translocates to the nucleus, where it phosphorylates many different substrates involved in various cellular responses from cytoskeletal changes to gene transcription. (58) It has been shown that activation of ERK1/2 (MAPK3/1) is crucial for cyclin D1 induction, providing a molecular link between ERK signalling and cell cycle control as cyclin D1 gene is essential for G1 to S-phase progression.(59) The kinetics and duration of ERK activation may play an important role in influencing its effect on cell fate, in the sense of cell survival and proliferation (transient activation) or, to the contrary, in cell apoptosis (prolonged ERK activation). (60) In normal state, melanocytes do not have detectable ERK activity (phosphorylated ERK1/2 levels were low in normal human melanocytes ) and require a number of growth factors, secreted by nearby keratinocytes, to survive. There are at least 2 pathways whereby growth factors can activate ERK in melanoma. The first “classical” pathway involves direct activation of the Ras/Raf pathway, while the other pathway involves the activation of Ras through a novel cAMPdriven exchange factor. (61)

Incidence of ERK activation in malignant melanoma and therapeutic implications In malignant melanoma, activation of ERK (phosphorylation of ERK, p-ERK) is present in 54% of primary and 33% of metastastic tumors respectively, but no correlation was observed between p-ERK expression and melanoma-specific survival. (62, 63) From treatment point of view, a successful therapy needs a significative reduction in ERK1/2 phosphorylation. Studies on Cisplatin, one of the most potent antitumor agents, showed that it not decrease ERK1/2 phosphorylation in vitro; on the contrary, it increased the activation of ERK1/2 and consequently enhanced chemoresistance. (64) ERK activation can also represent one of the principal mechanisms of resistance to BRAF/MEK inhibitors. To block these potentially activating pathways, a novel molecule, SCH772984 a specific and selective, ATP-competitive, inhibitor of ERK1/2, activity has been tested on resistant cell lines.(65) In preclinical studies, SCH772984 inhibits cellular proliferation and causes selective apoptosis in RAS or BRAF mutated tumour cell lines, being effective even in tumor cells that were resistant to either BRAF or MEK inhibitors and in cells that became resistant to the dual combination of these inhibitors.

No clincal studies are available. (66) In conclusion, the development of novel treatments that selectively inhibit the RAS-RAF-MAPK pathway represents a milestone in the malignant melanoma therapeutic strategy that improves the outcome, compared with classical cytotoxic chemotherapy.

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29. Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ, Hughes TM, Thompson JF, Scolyer RA, Kefford RF. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.J Clin Oncol. 2011 Apr 1;29(10):1239-46.

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BASAL CELL CARCINOMA: REVIEW OF EPIDEMIOLOGY AND RISK FACTORS CARCINOMUL BAZOCELULAR - EPIDEMIOLOGIE ȘI FACTORI DE RISC - REVIEW Andreea-Oana Enache1), Virgil Pătrașcu1), Raluca Niculina Ciurea2), Loredana Elena Stoica1), Nicolae Cernea3), Stepan Desdemona4), Cristiana Simionescu2) Dermatology Department, University of Medicine and Pharmacy of Craiova, Romania 2 Pathology Department, University of Medicine and Pharmacy of Craiova, Romania Department of Obstetrics and Gynecology, University of Medicine and Pharmacy of Craiova, Romania 4 Department of Infant Care–Pediatrics–Neonatology, University of Medicine and Pharmacy of Craiova, Romania 1

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Corresponding author: Virgil Pătrașcu, Professor, MD, PhD, University of Medicine and Pharmacy from Craiova, Petru Rareș Street, No2-4, 200345, Craiova, Romania, phone: 004-0724273676, e-mail: vm.patrascu@gmail.com.

No conflict of interest

Open Access Article

Abstract Keywords: basal cell carcinoma, epidemiology, risk factors

Basal cell carcinoma (BCC) is a malignant tumor with slow extension and local malignancy, with an exceptionally rare metastatic potential. BCC incidence is continuously increasing and there are geographical variations, the highest values being reported in Australia. The following are involved in BCC pathogenesis: actinic radiation, ionizing radiation, genetic factor, chemical carcinogens, immunosuppression, smoking etc. Actinic radiation is the main etiologic factor. There are multiple mechanisms of photocarcinogenesis, without being fully elucidated yet. Changing the attitudes towards the sun (tanning fashion), increased life expectancy and the presence of immunosuppression (organ transplant, HIV / AIDS) are factors that will further contribue to the increased incidence of BCC.

Rezumat Cuvinte-cheie:

Cite this article: Andreea-Oana Enache, Virgil Pătrașcu, Raluca Niculina Ciurea, Loredana Elena Stoica, Nicolae Cernea, Stepan Desdemona, Cristiana Simionescu.

Basal cell carcinoma: review of epidemiology and risk factors.

RoJCED 2016; 3(1):22 - 28

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carcinom bazocelular, epidemiologie, factori de risc

Carcinomul bazocelular (CBC) este o tumoră cu extensie lentă şi malignitate locală, având potențial excepțional de rar de metastazare. Incidența CBC este în continuă creștere și există variații geografice, cele mai mari valori raportându-se în Australia. În etiopatogenia CBC sunt implicaţi următorii factori: radiaţiile actinice, radiaţiile ionizante, factori genetici, carcinogeni chimici, imunodepresia, fumatul etc. Radiațiile actinice reprezintă principalul factor etiologic. Mecanismele intime ale fotocarcinogenezei sunt multiple, fără a fi încă elucidate. Modificarea atitudinii față de soare (moda bronzării), creșterea speranței de viață și circumstanțele prezenței imunodepresiei (transplant de organe, infecția HIV/SIDA) sunt factori care vor contribui în continuare la creșterea incidenței CBC.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Andreea-Oana Enache, Virgil Pătrașcu, Raluca Niculina Ciurea, Loredana Elena Stoica, Nicolae Cernea, Stepan Desdemona, Cristiana Simionescu

Figure 1. Pearl-like BCC with central ulceration

Figure 2. Nodular BCC (Prof. dr. Pătrașcu per-

Basal cell carcinoma (BCC) is a slow growing malignant tumor with local extension that develops in the basal cells of the skin or its annexes [1].

BCC was first described in 1824 by Arthur Jacob as “ulcus rodens” and in 1951, Thackrayhad correlated histopathologic data about the specific growth pattern of the tumor with clinical aspect [2,3].

Basal cell carcinoma is characterized by slow extension and invasion (in years), and its frequency of metastasis is exceptionally rare (0.0028) [4-6]. The clinical evaluation of BCC lesions has revealed a clinical polymorphism (Figure 1-4). Despite this relatively benign behavior, some tumors grow aggressively and may cause extensive tissue damage.

The concepts of "low risk" and "high risk" factors for BCC recurrence have been described. Nodular and superficial types fall into the “low risk” category, while the morpheaform type belongs to the “high risk” category [7]. BCC is found in both sexes (sex ratio B / F-1.5-2: 1), with a maximum incidence after 50 years old [8].

Recently, an increased incidence of BCC among young population has been noticed (under 40 years) especially at women. The increased incidence of BCC is a result of the action of multiple factors: increased exposure to UV radiation, the depletion of the ozone layer, increasing life expectancy and taking over by developing countries of some habits from industrialized countries (smoking, changing clothing style, sedentarism)[9]. There are significant geographical variations in the incidence of basal cell carcinoma: the highest values are reported in Australia, where there are over 1000 cases /100 000 inhabitants / year. Minimum

values are found in Africa, where there are less than 1 /100 000 person-years. In Germany, Canada, Italy, Finland, Switzerland, Scotland, Ireland, UK intermediate values have been reported (49-128 / 100,000 inhabitants) [10-13].

The worldwide incidence of BCC is increasing rapidly. It is estimated that approximately 2.8 million new cases of BCC are diagnosed each year in USA, the annual growth rate ranging between 3% and 7% [14,15]. BCC represents 80% of non-melanoma skin cancers and is the most common cancer among Caucasians [16, 17]. Bauer et al. showed an increased frequency of BCC in people with occupations involving excessive sun exposure (farm laborers, fishermen, sailors, builders, pilots of aircraft) [18] . Up to 80% of all the lesions are found on the head and neck (30% nose, cheek 22%, 15% forehead, periorbital 5%, 4% scalp, neck 4%, etc.) [19] The pathogenesis of basal cell carcinomas involves the following factors:

(Prof. dr. Pătrașcu personal photo archive)

sonal photo archive)

Ultraviolet Light

The ground solar spectrum comprises wavelengths between 290 and 3000 nm. The solar energy received at the Earth’s surface is divided as follows: 50% is infrared radiation, visible radiation 40%, 10% is UV (8% UVA and 2% UVB). It still depends on the altitude, latitude, season and is influenced by a number of factors: water vapor content in the troposphere, ozone layer, air masses, molecules and atmospheric particles, the presence of reflective surfaces, etc. As the earth’s protective ozone layer thins continuously, increases in the incidence M a rc h 2 0 1 6

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Figure 3. Cicatricial basal cell carcinoma,

effects of sun damage with solar elastosis and cutis rhomboidalis nuchae (Prof. dr. Pătrașcu personal photo archive)

of skin cancer must be anticipated.

The first experimental evidence on the causal relationship between UV radiation and skin cancer are known from the period 1920-1930 [20].

There are many arguments advocating for the role of solar radiation in the pathogenesis of skin cancer [21]. About 80% of all skin carcinomas are located on the face.

Also in terms of epidemiological aspect, we mention the increased incidence of skin cancer as we approach the Equator.

International Agency for Research on Cancer has classified UV radiation - regardless of its source (solar or artificial) –in Group 1 of risk [22].

Several studies have shown that one exposure to artificial tanning increases the risk of BCC with 29% and 6 sessions of artificial tanning increases the risk by 73% [23-25].

In a meta-analysis of 12 studies on 9328 patients with non-melanoma skin cancers, Wehner et al. have found that artificial tanning is associated with an increased risk of developing basal cell carcinoma and squamous cell carcinoma. The risk was higher among users of tanning beds, under the age of 25 years [26].

A higher incidence of BCC was reported in people who had frequent or severe sunburns in childhood. Thus, it is considered that adolescence and childhood are critical periods for determining an increased risk of BCC and implicitly of other skin disorders in adulthood [27]. Carcinogenesis is considered a multistadial process which comprises

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the following steps: initiation, promotion and neoplastic progression. Ultraviolet radiation can act as initiator, promoter or both of them. Although the mechanisms of photocarcinogenesis are not fully elucidated, it is known that together with the photons action on DNA, UV may intervene through the following: • increased ornithine decarboxylase activity (the enzyme ornithine decarboxylase is involved in the synthesis of the following polyamines - putrescine, spermine and spermidine; • pheomelanin iradiation produces superoxide ions and other free radicals, highly cancerigen; • the action of oxygenated sterols, particularly α 1 - dehydrocholesterol produced by UV irradiation of the cholesterol. This, product is powerful carcinogenic and also an immunosuppressive agent; • depression of cellular immunity.

Studies have shown that the repair of UV-induced damage at DNA level is possible due to the action of the p53 protein (encoded by the p53 gene that belongs to the family of tumor suppressor genes).

This stops cell proliferation, and after repair, the cells resume their cycle. If damages are very important, then p53 leads to programmed death (apoptosis) by inducing the Bax gene and decreasing the Bcl2 gene expression (bax protein has antagonistic action with Bcl2). Mutations in this gene enable the developing of skin carcinomas or others cancers. It has been found that the p53 gene is inactive in 20-50% of cases of skin cancers.

Interestingly, mutations in the p53 gene were found in precancerous lesions and in chronically UV-irradiated skin, data that can be viewed as a warning for the accumulated solar energy which is already dangerous.

Hras mutations were found as well in cutaneous carcinomas, but less frequently [28].

UVB radiation represents the most harmful portion of the solar spectrum for the skin, due to photocarcinogenesis by a continuous and cumulative process [29].

Recent studies, certify the involvement of UVA radiation in skin carcinogenesis [30]. After these studies, in cutaneous carcinogenesis would participate with 65% UVB and UVA with 35%.

We mention that phototherapy (PUVA= psoralen + UVA) can cause in long term, skin cancers. The incidence of basal cell carcinoma among patients with psoriasis has been proved to be dependent on the irradiation dose and the number of PUVA sessions [31, 32].

Regarding the carcinogenesis, the following mechanisms may be involved: - the decrease of immune response, both cutaneous and systemic. The decrease of epidermal Langerhans cells was observed.- the intervention of psoralen on DNA; - the interventions of HPV virus type 16 and 18, capable of inactivate the p53 gene by forming inactive complexes between the E6 protein (encoded by the virus), and the p53 protein. The presence of HPV in skin lesions suggest that immunosuppres-

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Andreea-Oana Enache, Virgil Pătrașcu, Raluca Niculina Ciurea, Loredana Elena Stoica, Nicolae Cernea, Stepan Desdemona, Cristiana Simionescu

Figure 4.

Pearl-like basal cell carcinoma (Prof. dr. Pătrașcu personal photo archive) sion induced by PUVA may have an important role in photocarcinogenesis.

Risk factors for skin cancers in people who underwent PUVA - therapy are: - skin phototypes I and II; - intense and repeated sun exposure; - previous history of skin cancer; - existence of precancerous lesions; - previous treatment with arsenic, ionizing radiation, UVB, methotrexate, cyclosporine.

Ionizing Radiation Ionizing radiation are ionizing particles in different forms: α (helium nucleus consisting of two protons and two neutrons), β (electrons, protons), electromagnetic ionizing radiation (X-rays, gamma rays, cosmic rays).

In 1895 Wilhelm Conrad Röntgen discovered Xrays, and a few years later had established their role in the pathogenesis of skin cancers [33].

The effects of ionizing radiations on tissues and cells are: - determines rapid cell death by apoptosis (may be induced by the overexpression of p53 molecule because of the important alterations in the DNA), or cause delayed cell death (by inhibiting mitosis); - causes mutations and chromosome aberrations: the alteration of tumor suppressor genes or protooncogenes causes cancer. The Study of radiationinduced skin cancer on rats revealed the fact that the activation of C-myc oncogene occurs, gene heavily involved in different stages of cancer; - neoplastic transformation through sublethal alterations and mutations in irradiated cells.

The cutaneous cellular targets of radiation-induced carcinogenesis are mainly, in basal layer and superjacent cells. The other cells, located in the upper epidermis, are more resistant.

Skin carcinogenesis is measured in terms of the years order, the latency depending on irradiation dose. We emphasize that human skin has a medium sensitivity to ionizing radiation and there is no safe minimum dose. The risk of developing skin cancer increases if along with irradiation the patient will be exposed to ultraviolet radiation, chemical carcinogens or oncogenic viruses.

Radiation-induced basal-cell carcinoma is rare.

BCC appears on chronic radiodermatitis 40-50 years after irradiation with low doses, especially in the lumbar region (radiodiagnostic) or on the scalp after roentgen epilation in patients with pilomycosis (method now abandoned).

In a study conducted on a sample of 2224 children treated by roentgen epilation for tinea capitis the risk of developing CBC in the cephalic region was 3.6. We have also noted an increased incidence of CBC in the irradiation field after the treatment of angiomas, Hodgkin’s disease or after accidental exposure [34].

Genetic factors In this context we mention the importance of individual sensitivity to sunlight.

Epidemiological studies have revealed significant differences in the incidence of skin carcinomas according to skin pigmentation [35]. Regarding the skin response to ultraviolet radiation (presence or not of burns and susceptibility to sunburn) there are 6 skin phototypes.

The risk of developing skin carcinomas is maximum in phototype I and minimum in phototype VI [36]. In the USA, it is estimated that the frequency of skin cancers is 7-8 times higher in whites compared with african-americans (phototype VI) [37].

It is known that in some genetic disorders (GorlinGoltz syndrome, Xeroderma pigmentosum) skin carcinomas occur early and are multiple [38, 39].

Chemical carcinogens More frequently cause carcinomas and rarely sarcomas or lymphomas. The target cells for chemical carcinogens have mechanisms of detoxification (Selenium-dependent glutathione peroxidase protein, superoxide dismutase, etc.) which, for a time, succeed to neutralize the effect of these substances. The following substances are considered chemicals carcinogens: - Arsenic: only mineral trivalent arsenic is a human carcinogen [40]. It causes skin cancers but also upper aerodigestive tract, lung, bone and liver cancers. Latency period is 25-30 years. Tseng et al. have found a dose-dependent relation between M a rc h 2 0 1 6

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the arsenic levels in drinking water and the prevalence of skin cancer [41]; - Coal tar: the carcinogen agents found in coal tars are polycyclic aromatic hydrocarbons (PAHs).The main PAHs are benzo (a) pyrene, benzofenantracen, dibenzantracen, actually the same substance involved in smoking-induced malignancies. Regarding the carginogenetic mechanisms the occurence of mutations in the p53 gene and in the proto-ocogene H-ras are currently discussed. Also, the generation of free radicals, the inhibition of Langerhans cell antigen-presenting function and reducing the T cell response are mechanisms brought into discussion; - Local cytostatic agents: caryolysine, BCNU (carmustine); - Other chemical carcinogens: phorbol esters, pesticides, insecticides, fungicides, benzoyl peroxide.

Immunosuppression Immune status appears to be a fundamental parameter in promotion and progression of photoinduced skin cancers. The risk of skin cancer is increased in patients treated with immunosuppressants (transplant patients) or those with AIDS. In these categories of patients, skin cancers appear earlier and often are more aggressive.

The risk of developing BCC in patients with organ transplant is 10 times higher than the general population [42, 43].Risk factors include - skin phototype I-II, cumulative sun exposure, age at transplantation, the degree of immunosuppression.

Smoking The oncogenic effect of smoking is the result of thermal and chemical action. After almost half a century of research for identifying toxic and carcinogenic substances (over 480) contained in cigarette smoke, the list remains open. Recent researche revealed the association of smoking with mutations in the p53 gene (tumor suppresor), which reveals another way of smoking involvement in the process of carcinogenesis. Boyd et al. found that BCC appearance in young women is related to smoking. Smith and Randle have described an increased prevalence of BCC with a diameter greater than 1 cm in smokers [44,45].

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Other factors Repeated micro traumatisms [46], chronic skin infections, ulcers with chronic evolution, chronic alcoholism, a high dietary fat intake diet [47], etc.

As regards the cancers developed on burn scar, it was noted that the latency period is approximately 35 years (7-62 years), although cases in children have been described. Squamous cell carcinoma appears predominantly but basal cell carcinoma and melanoma can develop. Approximately 2% of burn scars, may degenerate.

Although the role of actinic radiation seems preponderantly in the pathogenesis of skin cancers, in many cases, cancer is the result of a complex of factors (external and internal), which makes the assessment of the rate of participation of each of them difficult.

However, identifing the mentioned risk factors and limiting their action on the skin, can promote the prevention of BCC. Thus, the BCC prevention include: knowledge of risk factors, early diagnosis and adoption of preventive measures, especially in susceptible populations (light phototypes, unprotected professional exposure to UV rays)

Conclusions BCC is often the consequence of the action of several etiological factors, whose share of contribution is difficult to assess.

Changing attitudes regarding sun exposure (tanning fashion), increasing life expectancy and the presence of immunosuppression (organ transplant, HIV / AIDS) are factors that will continue to contribue to the increased incidence of BCC.

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Andreea-Oana Enache, Virgil Pătrașcu, Raluca Niculina Ciurea, Loredana Elena Stoica, Nicolae Cernea, Stepan Desdemona, Cristiana Simionescu

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14. Fellner C. Vismodegib (Erivedge) For Advanced Basal Cell Carcinoma. Pharmacy and Therapeutics 2012; 37(12):670-682.

15. Benjamin W. LeSueur, BS; Nancy G. Silvis, MD; Ronald C. Hansen.Basal Cell Carcinoma in Children. Arch Dermatol.2000; 136:370-372 16. Welsch MJ, Troiani BM, Hale L,DelTondo J, Helm KF, Clarke LE. Basal cell carcinoma characteristics as predictors of depth of invasion.J Am Acad Dermatol. 2012 Jul;67(1):47-53.

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28. Reifenberger J, Wolter M, Knobbe SB. Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas. Br J Dermatol 2005; 152:43–51.

29. J. L. Lim and R. S. Stern. High levels of ultraviolet B exposure increase the risk of nonmelanoma skin cancer in psoralen and ultraviolet A-treated patients.J Invest Dermatol. 2005 Mar;124(3):505-13.

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31. Archier E, Devaux S, Castela E, Gallini A, Aubin F, Le Maître M, Aractingi S, Bachelez H, Cribier B, Joly P, Jullien D, Misery L, Paul C, Ortonne JP, Richard MA. Carcinogenic risks of Psoralen UV-A therapy and Narrowband UV-B therapy in chronic plaque psoriasis: a systematic literature review. J EurAcad Dermatol Venereol. 2012 May;26 Suppl 3:22-31.

32. Robert S. Stern. The risk of squamous cell and basal cell cancer associated with psoralen and ultraviolet A therapy: A 30-year prospective study. Journal of the American Academy of Dermatology, 2012, Vol. 66, Issue 4, p553–562.

33. Yoshinaga, S., Hauptmann, M., Sigurdson, A. J., Doody, M. M., Freedman, D. M., Alexander, B. H., Linet, M. S., Ron, E. and Mabuchi, K. (2005), Nonmelanoma skin cancer in relation to ionizing radiation exposure among U.S. radiologic technologists. Int. J. Cancer, 115: 828–834.

34. Armstrong BK, Kricker A: The epidemiology of UV induced skin cancer. J PhotochemPhotobiol B. 2001 Oct;63(1-3):8-18.

35. Gloster HM, Neal K. Skin cancer in skin of color. J Am Acad Dermatol. 2006 Nov;55(5):741-60.

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38. Kim MY, Park HJ, Baek SC, et al. Mutations of the p53 and PTCH gene in basal cell carcinomas: UV mutation signature and strand bias. J DermatolSci 2002; 29:1–9.

39. Vasiliki Nikolaou, Alexander J. Stratigos, HensinTsao.Hereditary Nonmelanoma Skin Cancer.Semin Cutan Med Surg. 2012 Dec; 31(4): 204–210.

40. Cabrera HN, Gómez ML. Skin cancer induced by arsenic in the water. J Cutan Med Surg.

Mar-Apr 2003;7(2):106-11 41. Leiter U, Eigentler T, Garbe C. Epidemiology of skin cancer. AdvExp Med Biol. 2014; 810:120-40.

42. Berg D, Otley CC. Skin cancer in organ transplant recipients: epidemiology, pathogenesis and management. J Am AcadDermatol 2002; 47: 1-17.

43. Wisgerhof HC, Edelbroek JR, de Fijter JW, Haasnoot GW, Claas FH, Willemze R, Bavinck JN.Subsequent squamous- and basal-cell carcinomas in kidney-transplant recipients after the first skin cancer: cumulative incidence and risk factors.Transplantation. 2010 May 27;89(10):1231-8.

44. Boyd AS, Shyr Y, King LE Jr. Basal cell carcinoma in young women: an evaluation of the association of tanning bed use and smoking. J Am AcadDermatol. 2002 May; 46(5):706-9.

45. Smith JB, Randle HW. Giant basal cell carcinoma and cigarette smoking.Cutis. 2001 Jan; 67(1):73-6.

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USEFUL MELANOMA DIAGNOSTIC TOOLS: IMMUNOHISTOCHEMISTRY AND MOLECULAR TECHNIQUES TESTE UTILE ÎN DIAGNOSTICUL MELANOMULUI: TEHNICI IMUNOHISTOCHIMICE ȘI MOLECULARE Gencia Ioana1,2 ,Vincze Dana3, Chiticariu Elena4, Solovan Caius1,2 Dermatology and Venereology University Clinic, Timisoara City Hospital, Timisoara, Timis, Romania 2 Dermatology Department, University of Medicine and Pharmacy “Victor Babes”,Timisoara, Romania 3 PhD student at University of Medicine and Pharmacy “Victor Babes”,Timisoara, Romania 4 University of Lausanne, Lausanne, Switzerland 1

Corresponding author: Vincze Dana E-mail: deak_dana_2@yahoo.ro Adress: Daliei street, no. 17, Timisoara, Romania Telephone number: +40372779220

No conflict of interest

Open Access Article

Abstract Keywords: melanoma, immunohistochemistry, fluorescence in situ hybridization, comparative genomic hybridization, proteomics

The diagnosis of melanoma is currently based on classical histology and immunohistochemistry (IHC). The markers used for immunohistochemical analysis are classified in two main categories: melanocytic differentiating markers and melanoma progression markers.

Nevertheless, challenging cases have led to the development of new molecular techniques. Since as how we are in the molecular biology era, there are molecular techniques that have proven their utility in the diagnosis of ambiguous melanocytic lesions. In this article we overview the main IHC markers and the most used molecular techniques with diagnostic potential in melanoma.

Rezumat Cuvinte-cheie:

Cite this article: Gencia Ioana, Vincze Dana, Chiticariu Elena, Solovan Caius. Useful melanoma diagnostic tools: immunohistochemistry and molecular techniques.

RoJCED 2016; 3(1):30 - 36

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melanom, imunohistochimie, hibridizare fluorescența in situ, hibridizare genomică comparativă, proteomică

În prezent, diagnosticul de melanom se bazează pe histologia clasică și imunohistochimie (IHC). Markerii utilizați pentru analiza imunohistochimică sunt clasificați în două categorii principale: markeri de diferențiere melanocitară și markeri de progresie. Cu toate acestea, cazurile dificile au condus la dezvoltarea unor noi tehnici moleculare.

Având în vedere faptul că suntem în epoca biologiei moleculare există tehnici care și-au dovedit utilitatea în diagnosticul leziunilor melanocitare ambigue. În acest articol vom oferi o privire de ansamblu asupra markerilor principali de IHC și tehnicilor moleculare cele mai utilizate cu potențial de diagnostic în melanom.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Gencia Ioana, Vincze Dana, Chiticariu Elena, Solovan Caius

1. Introduction The gold standard of melanoma diagnosis is currently represented by histology in combination with immunohistochemistry (IHC), but challenging cases are frequently seen. The histopathological diagnosis of melanocytic tumours of uncertain malignant potential seems to be unreliable, even if it is reviewed by dermatopathologists experts in melanocytic tumours.(1) The misdiagnosis between melanoma and benign melanocytic tumours or vice versa has serious implications. This is why additional specific techniques useful in differentiating between these entities are needed and this is the reason why scientists have turned to molecular biology. However, the utility of these new techniques in difficult cases is still controversial. For this purpose, we overview the main diagnostic techniques used in the differentiation of ambiguous melanocytic tumours. In particular, we discuss the potential usefulness of IHC markers in melanoma diagnosis, compared with molecular biology.

Other up and coming diagnostic methods in melanoma are the in vivo imaging diagnostic tools which are showing a lot of promise (reflectance confocal microscopy and multiphoton microscopy).(2) In this article though we will only refer to the IHC, genomics and proteomics.

Figure 1.

IHC, Melanoma, HMB45, 200x

Figure 2.

IHC, Melanoma, S100, 100x

2. Immunohistochemistry (IHC) and melanoma IHC is the most used complementary technique to histology. It has roles in both diagnosis and prognosis, and new markers are continuously introduced in this field. A reliable marker for melanoma (with 100% sensitivity and specificity) has not yet been reported.

The markers used in melanoma diagnosis are classified as melanocytic differentiation markers (specific for the melanocytic cell lineage) and melanoma progression markers.(3) (Table 1) 2.1 Melanocytic differentiating markers The classical differentiating markers are HMB-45 (gp100), MART-1 (Melan-A), tyrosinase, and S100.

The first three markers are specific but lack the sensitivity.(4) Furthermore, 20% of cutaneous melanomas are negative for all these three markers.(5) HMB-45 is a monoclonal antibody that recognizes PMEL17 (known as gp100), a protein which plays roles in maintaining melanosomal structure. (figure 1) HMB-45 stains foetal melanocytes but is negative in adult normal melanocytes. It is also positive in junctional nevi, dysplastic nevi, Spitz nevi, congenital nevi and blue nevi, but is negative in intradermal nevi. In melanomas, the staining has a patchy pattern seen both in superficial and profound tumour areas, whereas in nevi it stains the papillary dermis and the surrounding areas of adnexa.(6) MART-1, also known as Melan A, is exclusively expressed in cutaneous and retinal melanocytes. It is present in all types of nevi and in approximately 90% of melanomas. Tyrosinase is indispensable for melanogenesis. It is expressed in cutaneous melanocytes and ocular pigmented ep-

ithelia. It is expressed in both nevi and melanomas, but most desmoplastic melanomas are tyrosinasenegative.(6) One of the most sensitive markers is S100 (93%-100%) and it is seen in all subtypes of melanoma.(figure 2) It is not specific because it also can be found in non-melanocytic tumours such as carcinomas, sarcomas, peripheral nerve sheath tumours, or Langerhans cell histiocytosis.

It must be used in association with at least one other melanocytic differentiation marker (MART-1/ Melan-A, HMB-45, or tyrosinase). Keratin should be used when a carcinoma is suspected. S100 has a nuclear and cytoplasmic staining pattern which is typically strong and diffuse in melanoma. In other tumours its expression is focal and low.(7) Other differentiating markers that have been suggested as useful in melanoma are as follows: microphthalmia transcription factor (MITF), multiple myeloma 1 (MUM-1), SOX10, melanocortin 1 receptor (MC1R), NKI/beteb, PNL2, KBA.62, and CD146 (Mel-CAM). PNL2 seems very useful, being positive in over 75% of epithelioid melanomas. MUM-1 is expressed in all nevi and in most primary and metastatic melanomas, but not in peripheral nerve sheath tumours. SOX10 seems to be also a useful marker, but the information is limited; its sensitivity is over 95% in both primary and metastatic melanomas. MC1R was reported as a sensitive marker but the information regarding its specificity is also very limited. MITF is present in M a rc h 2 0 1 6

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nearly all melanocytic lesions; although it is a very sensitive marker for desmoplastic melanoma, its specificity is low (5-9).

2.2 Melanoma progression markers These markers derive from both melanoma cells and stromal components and are used for distinguishing between nevi and melanomas. This category includes a few classes of molecules, as follows: proliferation molecules, signalling molecules, transcription factors, growth factors and growth factor receptors, adhesion molecules, proteases and related factors, proteins, immunoregulators, and cancer-testis antigens (3). The proliferation molecules such as Ki67 or PCNA and the cyclins (especially A and D3) are detected at low levels in benign lesions but their level increases in primary and metastatic lesions (10, 11). The expression of CDKN2A and p16 decreases in advanced malignant tumors (12). In contrast, melanoma maintains the expression of p53, other signalling impeding disabling its apoptotic function in melanoma(13).

The signalling molecules with possible application in diagnosis are PTEN, which is downregulated in melanoma (negative immunostaining in melanoma comparing with benign nevi) and Akt, which is expressed in melanoma (14, 15).

The transcription factor AP2 and the transmembrane receptor CD117 (c-Kit) intervene in the transition of melanoma from the radial to the vertical growth phase. They are expressed in nevi and in radial growth phase of the melanoma and are negative in vertical growth phase and metastatic tumors. ATF-1 is negative in normal melanocytes and is frequently positive in metastatic melanoma cells (16, 17). The growth factors such angiogenic factors such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β have diagnostic potential. VEGF is expressed in melanoma, but usually not in benign nevi (18). The epidermal growth factor receptor (EGFR) and osteonectin are strongly expressed in melanomas, moderately in displastic naevi, and weakly in nevi (19). Transferin in strongly expressed in melanoma, whereas in benign nevi is absent (20). The adhesion molecules with potential roles in differentiating malignant melanoma from benign nevi include β-catenin, E- and N-cadherin, αvβ3 and α5β1 integrins, β3integrin, CD44, ICAM-1, VCAM-1, or CD63 (3). The cancer-testis antigens are antigens predominantly expressed in normal testis and malignant tumours.

The most important antigens expressed in melanoma are SPANX ⁄ CTp11, TRAG-3, MAGE-A1, and MAGE-A3 (21, 22).

2.3 Other markers with potential in melanoma diagnosis Abbas et al performed a study on other immunohistochemcal markers which could be useful in the diagnosis of melanoma: cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1 and E-cadherin. In

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their study on 78 melanocitic tumors (including 26 melanomas) showed AMACR to be a potentially useful immunohistochemical marker in the differentiation between melanoma, melanocytic nevi and dysplastic nevi. (23) Mitchell et al, in a study on 107 melanoma samples investigated the correlation between the disregulation of the CXCR4/ CXCL12 axis and known melanoma prognosticators and BRAF status. Their study demonstrated that CXCR4 may be a useful prognosticator seeing how its expression was associated with a lower number of mitoses and lower depth of invasion.

Both CXCR4 and CXCL12 appear not to be influenced by the BRAF status. (24) Matrix metalloproteinasis-12 (MMP-12) has demonstrated its utility as a negative prognosis marker. The results of a study on 298 melanoma samples showed that MMP-12 expression was associated with tumor invasion and metastases. (25) 2.4 Melanoma marker cocktails This term describes a mixture of 2 or more antibodies against one or more melanoma-associated antigens. These cocktails of antibodies increase the sensitivity for detecting melanocytic differentiation. They are useful in the evaluation of sentinel lymph nodes, in metastatic melanoma or for the detection of melanocytic origin in poorly differentiated tumours. They are sensitive but lack the specificity because the reactivity with the nonmelanocytic tumours can be increased by their combination. The diagnosis must be confirmed by individual melanoma markers (6).

2.5 Immunohistochemistry: a two step diagnosis The first step is the identification of the melanocytic origin. From all panel of melanocytic differentiation markers, S100 is the most commonly used, in combination with at least one other marker, more specific for cutaneous melanocytic proliferations (HMB-45, MART1, MUM-1). Desmoplastic and spindle cell melanomas usually do not express melanocytic differentiation markers. However, 29% of these subgroups were positive for MITF. Furthermore, using appropriate antigen-retrieval techniques, gp100 and tyrosinase 2 could also stain desmoplastic melanoma (3, 26, 27). The second step is to differentiate melanoma from benign nevi. For this purpose the tumour cell-related proteins and tumour stromal components can be used, e.g.: Ki67, topoisomerase II, cyclin D1, p16INK4a (3).

The most intensively used remain Ki-67 index and topoisomerase IIa. Benign melanocytic nevi show little or no expression of Ki-67. In borderline melanocytic lesions, a Ki-67 index expressed in less than 5% of cells, without a zonal distribution, is useful for differentiating melanocytic nevi from nevoid melanoma or Spitz nevi (28). Recently, a panel of four markers was proposed for differentiating melanoma from dysplastic nevi: Bim (member of the Bcl-2 family), BRG1 (binding partner of the tumor suppressor p16INK4a), Cul1 (member of Cullin family), and ING4 (a tumour suppressor) (29).

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Gencia Ioana, Vincze Dana, Chiticariu Elena, Solovan Caius

Table1 Useful IHC markers in melanoma diagnosis Melanocytic differentiation markers S100 HMB-45 (gp100) MART-1 (Melan-A) Tyrosinase MITF MUM-1 SOX10 MC1R NKI/beteb PNL2 KBA.62

Progression markers Ki67 Cycline A and D3 CDKN2A p53 PTEN transcription factor AP2 transmembrane receptor CD117 (c-Kit) ATF-1 angiogenic factors (VEGF, bFGF, TGF-b) adhesion molecules (include β-catenin, E- and N-cadherin, αvβ3 and α5β1 integrins, β3-integrin, CD44, ICAM-1, VCAM-1, CD63) cancer-testis antigens (SPANX ⁄ CTp11, TRAG-3, MAGE-A1, and MAGE-A3)

Other markers with potential in melanoma diagnosis p16Ink4a AMACR CXCR4, CXCL12 MMP-12

CD146 (Mel-CAM)

3. Melanoma in molecular biology era Advances in molecular biology demonstrated the heterogeneity of melanoma, a tumour characterized by chromosomal abnormalities and DNA mutation which leads to oncogene activation and tumour suppressor genes inactivation.

Melanoma is the place of a complex pathogenesis involving several signalling pathways. The most studied are the mitogen activated protein kinase (MAPK) pathway, also known as RAS-BRAFMEK-ERK pathway and the phosphotidylinositol3-kinase-PTEN pathway or PI3K/AKT/PTEN/mTOR (figure 3).

The majority of cutaneous melanomas contain activating mutations of BRAF (which interacts with MAPK pathway) and loss of CDKN2A locus (which encodes p16 and p14) (27).

CDKN2A or CDK4 mutations are found in 25-40% of familial melanomas (31-33). BRAF and NRAS activating mutations are identified in 70%, respectively 20% of lentigo maligna, but are negative in mucosal and acral melanoma, which are associated with C-kit upregulation in 40% of cases (34-36).

Gross chromosomal aneuploidy is a characteristic of most malignant tumours, including melanoma.

Different chromosomal break points, gains, losses, specific allelic imbalances, or specific aneusomies are involved in melanoma pathogenesis. (37) These changes can be identified through the use molecular biology techniques.

Among them, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), next generation sequencing and mass spectrometry are the most intensively used (38-43).

Other molecular techniques which can be used to increase the accuracy of diagnosis are as follows: in situ hybridization (ISH), chromosomal G banding (CGB), gene microarray, polymerase chain reaction (PCR and RT-PCR), multiplex ligationdependent probe amplification (MLPA), high-resolution melting analysis (HRMA) or imaging mass spectrometry (42, 44).

3.1 Fluorescence in situ hybridization (FISH) FISH detects the presence or absence of specific gene sequences and cytogenetic abnormalities such as chromosomal deletion, gain, or translocation. For this purpose, the selection of an individual FISH probe is an important step, more exactly “one must know what one is FISHing for” (45).

In melanoma, FISH detects subtle chromosome aberrations, including targeted genes such as: CCND1, MYB, CEN6, and RREB1 (46-49). Gerami et al. (49) studied the reliability of FISH in melanocytic tumour diagnosis on a cohort of ambiguous melanocytic tumour compared with three cohorts of unequivocal nevi and melanomas of varying levels of atypia. They demonstrated that four probes offer the best sensitivity and specificity (86.7 and 95.4% respectively) in differentiating melanoma from benign nevi. These are represented by one locus on chromosome 11 (11q13, which encodes cyclin D1 gene) and three locuses on the chromosome 6 (6q23 encoding MYB gene, 6p25 encoding RREB1, and CEN6). This method is applicable to formalin-fixed paraffin-embedded tissues and is claimed to have the most accuracy in discriminating between melanoma and benign nevi (48, 49). This panel of four FISH probes has been patented by Abbott Molecular and is now commercially available (50). These results support the fact that FISH is a useful complementary technique in distinguishing nevoid melanoma from benign nevi with mitotic activity (51). A gap of this technique is the risk of technical omission of chromosome segments by partial entrapping of the nuclei in the tissue sections (52). Other limitations include imperfect hybridization, nonspecific binding, interobserver variability, or false results (45).

3.2 Comparative genomic hybridization (CGH) CGH analyzes copy number aberrations of the entire genome in a single experiment, by comparing DNA from a lesion of interest to DNA from a normal reference tissue of the same patient. The results are M a rc h 2 0 1 6

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Figure 3.

There are two major signalling pathways involved in melanoma pathogenesis represented by MAPK pathway (green) and PI3K/AKT pathway (yellow). Other pathogenetic pathways are represented by (N)RAS, c-KIT, CDK (4/6), GNAQ/11, MITF.

presented as gain or loss of copy number (53). CGH has shown some genome aberrations and imbalances in melanoma comparing with benign melanocytic tumours. CGH demonstrated that 96% of melanomas had multiple genetic aberrations. The most common alterations found by CGH or karyotyping are the chromosomal losses 6q, 8p, 9p and 10q, and the chromosomal gains 1q, 6p, 7, 8q, 17q and 20q (35). CGH is currently used in research settings (54, 55) for the analysis of genomic signatures in melanoma subtypes (54, 55, 56). CGH analysis stood at the basis of the FISH probes selection in melanoma. Bastian et al identified a combination of 13 regions on chromosomes 1, 6, 7, 9, 10, 11, 17, and 20, which is a powerful discriminatory tool between nevi and melanomas. A probe targeting C-kit (chromosome 4) was subsequently added because of its potential as a therapeutic target (49, 50) . The identification of chromosomal aberrations is justified by the fact that, excepting a few Spitz nevi, melanocytic nevi do not present these changes. Although some Spitz nevi have been found to have genomic aberrations, FISH and CGH can also be used for differentiation of spitzoid melanocytic neoplasms; Spitz nevi show an increased copy number of chromosome 11p, aberration not seen in melanoma (58). Other differences are the presence of BRAF or NRAS mutations in 86% of Spitzoid melanomas, mutations absent in common and atypical Spitz nevi (59). Progressive loss of p16 was associated with the transformation of benign nevi to melanoma and with the metastasizing process (60) . FISH and CGH can also identify mutated cells, including cells at distance from the tumour, in the apparently normal skin. Histological normal skin

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adjacent to acral melanoma (up to 6.1 mm from visible melanoma in situ, and 4.5 mm from invasive malignant melanoma) was found to have genetic amplifications in 84% of cases. The number of cells with molecular abnormalities was not correlated with the depth or diameter. This phenomenon explains the reoccurrence of melanoma excised with clear margins. The clinical applicability of these techniques is the possibility of genetic profiling of margins, which can aid in planning for reexcision (61, 62) . Another clinical applicability of molecular biology in melanoma is the identification of mutations and genetic aberrations which nowadays can be therapeutically targeted using inhibitors of BRAF, MEK, mTOR, MAPK, PIK3, CDK, or c-KIT (63). FISH can be performed on fresh or formalin-fixed tissue, on nuclei spreads, or on DNA microarrays (40).

The technique requires no more than 20â&#x20AC;&#x201C;30 cells to provide an accurate signal. Even if FISH detects only specific chromosomal abnormalities targeted by the probes used, it can identify balanced translocations not detectable by CGH resolution (50).

CGH is more expensive and laborious. It requires microdissection from paraffin-embedded tissues.

The changes should be present in at least 30â&#x20AC;&#x201C;50% of the cells in order to be evident on CGH analysis (44) . Looking back, the most suitable FISH probes are the cytogenetic abnormalities identified by CGH. Thus, FISH and CGH could be complementary used for an accurate molecular diagnosis.

3.3 Next generation sequencing (NGS) NGS is a second generation sequencing method, which can be used for whole genome sequencing (seq). This type of technique is used to sequence

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Gencia Ioana, Vincze Dana, Chiticariu Elena, Solovan Caius

both DNA and ARN. NGS is a cheaper and faster (sequence a hole genome in a day) alternative to Sanger sequencing. The DNA seq facilitates the discovery of gene mutations regulatory elements associated with disease or the identification of disease causing mutations for diagnosis. The ARN seq provides information regarding the entire transcriptome of a sample.(42) Furney SJ et. al used whole genome and exome sequencing to identify the mutational burden of different types of melanoma and demonstrated that there were some differences between cutaneous melanoma and the mucosal one. The primary cutaneous melanoma presented an average of 84495 somatic single nucleotide variants whilst the mucosal one only 8193 somatic single nucleotide variants. The mutational burden in cutaneous melanoma is 5-10 times higher than that in mucosal melanoma. On the other hand they demonstrated a much lower rate of copy number and structural variants in cutaneous melanoma as compared to the mucosal ones. (64)

Imaging mass spectrometry is an important technique that analyses metabolites, peptides, proteins DNA segments and lipids directly from the tissue sample. It reveals the molecular signature of different diseases. Lazova et al used matrix assisted imaging mass spectrometry to analyze 30 samples of Spitz nevus and 33 samples of Spitzoid Melanoma.

They have proven this method potential by identifying 5 peptides that were expressed differently in these melanocytic tumors. The method demonstrated a 97% sensitivity and 90% specificity. (43)

5. Conclusions Although recent studies have demonstrated that molecular techniques can highlight the atypical features and support the diagnosis of malignancy, they must not replace the traditional histopathologic analysis.

Acknowledgements: None

4. Proteomics While genomics analyzes the changes in DNA or ARN, proteomics investigates the post translational changes that appear due to genetic mutations.

One of the techniques used besides imunohistochemistry is the imaging mass spectrometry.

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45. Chen AY, Chen A, Fluorescence In Situ Hybridization, J Invest Dermatol, 2013, 133(5):e8 46. Newman MD, Mirzabeigi M, Gerami P, Chromosomal copy number changes supporting the classification of lentiginous junctional melanoma of the elderly as a subtype of melanoma, Modern Pathology, 2009, 22(9):1258–1262.

47. Newman MD, Lertsburapa T, Mirzabeigi M, Mafee M, Guitart J, Gerami P, Fluorescence in situ hybridization as a tool for microstaging in malignant melanoma, Mod Pathol, 2009, 22(8):989–995 48. Morey AL, Murali R, Mccarthy SW, Mann GJ, Scolyer RA, Diagnosis of cutaneous melanocytic tumours by four-colour fluorescence in situ hybridisation, Pathology, 2009, 41(4):383–387.

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Neurofibromatosis – one disease for a multidisciplinary team

NEUROFIBROMATOSIS – ONE DISEASE FOR A MULTIDISCIPLINARY TEAM NEUROFIBROMATOZA – O BOALĂ PENTRU O ECHIPĂ MULTIDISCIPLINARĂ Smaranda Cristina Nica(1), Gabriela Mihăilescu (2,3), Sanda Maria Nica (2,3), Cristina Băetu (2,3), Victor Gabriel Clătici (4), Ioan Buraga (2,3) (1) Dermatology, Dermestet Private Practice, Bucharest, Romania (2).

Neurology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania (3).

Neurology Department 1, Colentina Clinical Hospital, Bucharest, Romania (4) Dermatology Department, ELIAS Emergency Universitary Hospital, Bucharest, Romania Corresponding author: Smaranda Cristina Nica, MD – dermatology-venerology specialist Dermestet Private Practice, Address: 257A Sos. Olteniței, Bucharest, Romania Phone: 0040213180661, Fax: 0040213180661, email: drsmarandanica@yahoo.ro

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: neurofibromatosis type 1, neurofibromatosis type 2, schwannoma, genetic disease, dermatologic, neurologic, multidisciplinary team

Cite this article: Smaranda Cristina Nica, Gabriela Mihăilescu, Sanda Maria Nica, Cristina Băetu, Victor Gabriel Clătici, Ioan Buraga.Neurofibromatosis – one disease for a multidisciplinary team RoJCED 2016; 3(1):38 - 49

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Neurofibromatosis is a genetic disease with an autosomal dominant inheritance pattern, mainly characterised by neurologic and cutaneous findings. Several types of neurofibromatosis have been identified and there have been several attempts to classify them. Among them, two are more frequent: type 1 (von Recklinghausen) and type 2 (central/acoustic). Both sexes and all races are affected, type 1 being more frequent than type 2.

The mutations are situated for every type on different chromosomes, affecting different genes and different proteins. These mutations have high penetrance, but variable expressivity. There are also sporadic cases which are caused by new gene mutations. The pathogenesis is also different as the pathways are not the same. The clinical findings vary between the two types, as well as between patients within a certain type. Different organs and systems can be affected: nervous, eyes, skin, mucosae, bones, endocrine glands, a.s.o. However, for an accurate diagnosis, certain diagnostic criteria, specific for each type should be considered by a multidisciplinary team. Usually additional workup tests are not needed for the diagnosis, but sometimes can be useful, especially if differential diagnoses are taken into consideration. There is no ethiological treatment available for neurofibromatosis; therefore the only options are symptomatic treatments associated to the information and education of the patients regarding the disease and the complications as well as a genetic counselling.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Smaranda Cristina Nica, Gabriela Mihăilescu, Sanda Maria Nica, Cristina Băetu, Victor Gabriel Clătici, Ioan Buraga.

Rezumat Cuvinte-cheie: neurofibromatoza tip 1, neurofibromatoza tip 2, schwanom, boala genetică, dermatologic, neurologic, echipă multidisciplinară

Neurofibromatoza este o afecțiune genetică transmisă autozomal dominant, caracterizată în principal prin afectare neurologică și cutanată. Au fost identificate mai multe tipuri de neurofibromatoză, existând mai multe clasificări ale acestora. Dintre acestea, două tipuri sunt mai frecvente: tipul 1 (von Recklinghausen) și tipul 2 (centrală/acustică). Boala afectează toate rasele și ambele sexe, neurofibromatoza de tip 1 fiind mai frecvent întâlnită decât cea de tip 2. Mutațiile genetice sunt localizate pe cromozomi diferiți, fiind afectate gene și proteine diferite. Aceste mutații au penetranță crescută, dar expresivitate variabilă. Există însă și cazuri sporadice, apărute în urma unor mutații noi. Patogeneza variază și ea, mecanismele fiind diferite. Clinica diferă de la un tip la altul, precum și de la un pacient la altul în cadrul aceleași forme. Manifestările sunt variate, afectarea fiind multisistemică: neurologică, oculară, cutaneo-mucoasă, osoasă, endocrinologică etc. Pentru a pune un diagnostic corect criterii de diagnostic specifice fiecărui tip, trebuiesc luate în considerație de o echipă multidisciplinară. Teste diagnostice speciale nu sunt necesare, dar uneori pot fi folosite pentru stabilirea unor diagnostice diferențiale. Nu este disponibil un tratament etiologic pentru neurofibromatoză, ci doar tratamente simptomatice la care se adaugă informarea și educarea pacienților cu privire la boală și la complicațiile ei, cât și sfatul genetic.

Introduction The neurofibromatosis are genetic diseases with an autosomal dominant inheritance pattern, which is mainly characterised by cutaneous and neurological findings. It is part of the big family of phakomatoses (neurocutaneous syndromes).

Phakomatoses (“phakos” = lentil, spot, lens; “oma” = tumour) represent a heterogeneous group of diseases that associate different malformations of the nervous system and benign tumours of the skin, mucosae, eye and nervous system, with ectodermic origin.

Neurofibromatosis, is part of the phakomatoses, together with the tuberous sclerosis of Bourneville, Sturge–Weber syndrome, Von Hippel-Lindau syndrome, ataxia-telangiectasia, and some others with lower incidence.

In neurofibromatosis, neuroectodermal abnormalities are frequently found in the skin and nervous system, but also in other tissues (bones, eyes, endocrine glands and others) and the signs and symptoms can vary from one patient to another, needing a multidisciplinary approach in order to establish the type of each neurofibromatosis.

Classification Several types of neurofibromatosis (NF) have been identified. In 1982, Riccardi has proposed one classification that included 8 forms, based on the clinical findings and on the inheritance pattern: NF1 (von Recklinghausen), NF2 (acoustic), NF3 (mixed NF1 and NF2 or schwannomatosis), NF4 (variant), NF5 (segmental), NF6 (familial café-au-lait spots), NF7 (late onset) and NF8 (unspecified) (1).

Once the genetic diagnosis was possible, based on it and the clinical particularities of the disease, Carey et al. proposed in 1986 a new classification including only 5 types of neurofibromatosis: NF1 (classical/von Recklinghausen), NF2 (acoustic), NF3 (segmental/mosaic), NF4 (familial café-aulait spots) and NF5 (NF-Noonan phenotype)( 2,3).

The consensus conference of the National Institute of Health in Bethesda (MD, USA) defined in 1988, 7 main criteria for the diagnosis of NF1.

Despite all this, due to the rarity of the other forms than type 1 and 2, there isn’t a consensus regarding a final and universal classification.

Epidemiology It is estimated that globally this disorder affects at least one million individuals (4). Among the neurofibromatosis types that have been described, the most frequent forms are type 1 (NF1, von Recklinghausen) and type 2 (NF2, central/acoustic). The incidence of NF1 is 1:3,000 births (5) (one of the most common genetic diseases), while NF2 is much rare (1:33,000 births)(6).

This disease affects all races, men and women equally and almost a half of the cases have a family history of neurofibromatosis, the rest developing the disease due to a new genetic mutation (7).

Etiopathogenesis The two most frequent types of neurofibromatosis (NF1 and NF2) are both genetic diseases, with an autosomal dominant inheritance pattern, but the mutations are situated at different levels and on different chromosomes.

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Figure 1.

Personal archives of Victor Gabriel Clatici

Neurofibromatosis type 1 (Von Recklinghausen’s Disease) NF1 is a multisystem neurocutaneous disorder, and the most common phakomatosis. It is caused by a genetic mutation localised on the long arm of the chromosome 17, at 17q11.2. The affected gene is NF1, encoding neurofibromin, a 327kDa protein, composed of 2818 amino acids (8, 9). This protein is found in many types of cells (neurons, oligodendrocytes, Schwann cells) and has an important role in regulating cellular proliferation and differentiation, being required especially during the embryonic development, in the differentiation of the cells derived from the neural crest (cells for the peripheral and enteric nervous system, glia but also nonneural cell types including smooth muscle cells of the cardiovascular system, melanocytes – pigment cells in the skin, and craniofacial bone cells, cartilage and connective tissue) (10). After birth, the main role of this protein is to regulate by negative feedback the RAS-mitogen-activated-protein kinase (MAPK) pathway which allows cellular proliferation and supports the cells survival (11). If neurofibromin is deficient, the RAS-MAPK pathway is no longer blocked, it becomes hyperactive, the cells proliferate uncontrolled and the final result is the appearance of tumours.

Over 1400 different pathogenic mutations of the NF1 gene have been discovered.(12). The penetrance is high (almost 100% - autosomal dominant transmission), but the expressivity is variable (13).

30-50% of the cases are sporadic, being caused by new gene mutations, neither of their parents having NF1 (14-16). A patient afflicted with NF1 has a 50% chance of transmitting the disease to each of his/her children, regardless of sex.

Neurofibromatosis type 2 Neurofibromatosis type 2 (NF2) is caused by a genetic mutation situated on the short arm of the chromosome 22, at 22q12.2. The affected gene is NF2, encoding merlin (neurofibromin 2 or schwannomin), a 69.7 kDa protein, composed of 595

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Figure 2.

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amino acids. This protein is produced in the nervous structures and is part of the cytoskeleton. It is a tumour suppressor protein and regulates different processes in Schwann cells (17, 18). The sequence analysis of this protein resembles with that of the protein family Ezrin-Radixin-Moesin so that the name merlin protein is an acronym for Moesin-Ezrin-Radixin-Like Protein. All it’s action mechanisms are not very well understood, but most of the data show that it is involved in cell signalling and migration pathways. It seems that merlin regulates the surface receptors’ activity and turnover by modulating their interactions with the actin cytoskeleton, acts at the adherence junctions and suppresses tumour development, probably by inhibiting cell growth(18). Due to it’s dysfunction, tumours in the nervous system develop and more frequently bilaterally vestibular schwannoma (acoustic neurinoma) can be diagnosed. Like NF1, NF2 is also a genetic disease with high penetrance (almost 100% - autosomal dominant transmission), but with variable expressivity (19). Still, over 50% of the patients are developing the disorder due to a novel mutation and almost a third has mosaic mutations, responsible for the appearance of the disease (20).

Clinical findings The diagnosis will start with a family history/medical history and sometimes photographs of family members can be useful. Genetic prenatally tests are necessary in selected cases. Due to the fact that the clinical manifestations and diagnostic criteria are different, the two main forms of the disease (NF1 and NF2) will be described separately and due to the multifaceted appearance and it’s complexity a team work, (a multidisciplinary team) is very useful in diagnosing, monitoring and managing patients with NF. A. Neurofibromatosis type 1 (Von Recklinghausen’s Disease) The onset of NF1 is much earlier than that of NF2 and NF3 and the clinical findings of NF1 vary, as

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Smaranda Cristina Nica, Gabriela Mihăilescu, Sanda Maria Nica, Cristina Băetu, Victor Gabriel Clătici, Ioan Buraga.

Figure 3.

Personal archives of Victor Gabriel Clatici

different organs or systems can be affected such as nervous system, eye, skin, mucosae, bones, endocrine glands and other. The diagnosis of NF1 is more easy to be done in adults, as the symptoms and signs develop with age, but it is more difficult to be done in children under 5 years of age, where café-au-lait spots (macules) can be the only pathological sign, increasing in number and size with the age and associating other diagnosis criteria.

The diagnosis criteria take into consideration the cutaneous, ocular, neurologic and skeletal manifestations to which the genetic component is added. These criteria have been established in 1988 during the Neurofibromatosis - National Institutes of Health Consensus Development Conference (21).

Clinical diagnosis of NF 1 requires the presence of two or more of the following criteria: 1. 6 or more café-au-lait macules over 5 mm in greatest diameter in prepubertal individuals and over 15 mm in greatest diameter in post pubertal individuals, 2. 2 or more neurofibromas of any type or at least one plexiform neurofibroma, 3. freckling in the axillary or inguinal region (Crowe sign), 4. optic nerve glioma, 5. 2 or more Lisch nodules (iris hamartoma), 6. a characteristic bone lesion such as sphenoid wing dysplasia or thinning of long bone cortex with or without pseudarthrosis, 7. a first-degree relative (parent, sibling or offspring) with NF-1 diagnosed using the above criteria.

Clinical manifestations As shown, NF is affecting, is affecting a variety of organs and systems, this is why, the clinical manifestations will be described separately taking into account the affected organ or system.

I. The skin is frequently affected in NF1.

From the diagnostic criteria mentioned above, 3 out of 7 are assessed by the dermatologist, but

Figure 4.

Personal archives of Victor Gabriel Clatici

some others can be added, not so characteristic but also important. The café-au-lait spots (macules) are well-demarcated, uniformly hyperpigmented macules which may vary in colour from light brown (the colour of coffee with milk - „caféau-lait”) to dark brown, depending on the patients’ phototype. They can be localised anywhere on the skin, but they usually spare the scalp, genital region, palms and soles. Their shape is usually oval, but the size differs in diameter from 5 mm to 500 mm, commonly being under 100 mm (10cm) (22).

Café-au-lait spots are usually the first sign of the disease and can be present at birth or can appear later in life. Generally they develop during the first year of life, but they may also appear at older ages, regardless of the moment, having normally a tendency to grow though some authors have described lesions that diminished or disappeared spontaneously in older individuals (23). A correlation between the number of café-au-lait spots and the severity of the disease hasn’t been proven, but the number and appearance of café-au-lait spots has to be monitored yearly by the dermatologist.

Larger café-au-lait spots, especially if associated with hypertrichosis, should always be palpated in order to exclude an underlying plexiform neurofibroma (24). Axillary or inguinal freckles are smaller café-au-lait spots, having the same characteristics (colour, borders), but smaller sizes. They resemble solar lentigines, but are localised on non-photoexposed areas, such as axillary and inguinal regions, as well as in other intertriginous areas (e.g. inframammary fold). The most common location is the axillary fossa, axillary freckling, also referred to as Crowe’s sign, being present in almost 80% of the cases (25, 26). They appear after development of the café-au-lait spots, at 4-6 years of age, but before the development of neurofibromas (27).

Neurofibromas are benign peripheral nerve sheaths tumours (formed of Schwann cells, fibroblasts, perineural cells and mast cells). They can be situated more superficially (cutaneous neurofibromas) or deeper (subcutaneous, deep nodular M a rc h 2 0 1 6

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dermal or plexiform neurofibromas). The deeper tumours are not usually present from childhood, as they appear later, at teenagers or mostly after puberty. With aging they may grow in size and number.

Cutaneous neurofibromas are pink, brown or skin coloured papules, nodules or exophitic domeshaped tumours, pedunculated or sessile, with well-defined borders. Their surface may be smooth or ridged, but the overlying skin is normal. When palpated they can be soft or slightly renitent. The size may vary from a couple of millimetres to several centimetres. Very characteristic for the cutaneous neurofibromas is that they invaginate into the skin when lateral pressure is applied (the “button hole” sign). Usually, neurofibromas are asymptomatic, but sometimes they can itch. Cutaneous neurofibromas can be situated anywhere on the body surface, but they are mostly found on the trunk and limbs. Women can have larger lesions if situated on the breast. The total number of lesions may vary from one case to another, and sometimes it can reach hundreds.

Subcutaneous neurofibromas are localised more profoundly than the cutaneous ones, in the dermis and subcutaneous tissues. Clinically they are more firm and less well defined. Subcutaneous neurofibromas can be found anywhere on the body surface, and if present in the cervical region they can be easily misdiagnosed as lymph nodes(28). Almost 20% of the patients diagnosed with NF 1 have at least one subcutaneous neurofibroma(25).

Plexiform neurofibromas are fibromas that are localised along the length of cranial, peripheral and enteric nerves, histologically similar to the other neurofibromas but involving one or more nerve bundles. There are two types of plexiform neurofibromas, regarding their location: superficial and deep. The superficial type of plexiform neurofibromas present as nodules or firm masses situated in the subcutis, with firm cords which can be palpated inside, giving the feeling there is a “bag of worms”.

The overlying skin can be pigmented (including a café-au-lait macule) and it can be covered with excess hair (hypertrichosis). Superficial plexiform neurofibromas can be found almost anywhere, but more frequently on the trigeminal nerves and the nerves originating in the cervical spinal cord (22) . The size of the tumours may vary, the larger ones generating overhanging folds of loose skin that produce deformities of the face, neck or limbs, sometimes with functional impairment (eyesight, movement). Sometimes, in its most extreme form, thickening of the skin and underlying tissues may occur, and the affected region can become very large and deformed, condition known as “elephantiasis neuromatosa”.

The deep type of plexiform neurofibroma (or visceral, enteric, plexiform neurofibroma) can reach deeper, to the muscles fascia, muscles and even internal thoracic or abdominal structures (blood vessels, organs). The consequences can be very serious if these tumours produce an invasion of

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the vital structures such as the spinal cord, gut or ureters. Almost 25% of the patients have visible and superficial plexiform neurofibromas, most of them being observed from the birth or developing in the first years of life (on an average at 4-5 years of age) (24, 28, 29). However, the deeper ones can remain unnoticed / undetected or can be found accidentally during imaging tests (e.g. ultrasound, magnetic resonance imaging MRI) (30). The plexiform neurofibromas’ growth is variable, there can be times of rapid growth alternating with stationary periods, but the probability of developing a new plexiform neurofibroma after childhood is low (24). In about 3-15% of the young adult cases there is the risk for a plexiform neurofibroma to undergo malignant transformation, turning into a neurofibrosarcoma(25,27). The tumour is growing fast, becomes firmer and may associate persistent local pain or some neurological deficits. However, sometimes, these signs are absent and the malignant transformation is discovered only after the tumour has metastasized (24). The evolution of neurofibrosarcomas is very aggressive, often fatal (31).

There are some other rare forms of neurofibromas with different manifestations, they either look like blue-red macules (due to the thickening of the vessel walls and presence of fibromatous tissue in the papillary dermis) or like pseudoatrophic macules (due to a replacement of the collagen in the reticular dermis with fibromatous tissue) (32). Other cutaneous findings, which may also occur, are represented by pruritus (itch), juvenile xanthogranulomatosis (almost 18% of the cases, especially in the first three years of life) and benign glomus tumours (modified smooth muscle cells in blood vessels under the hand of foot finger nails) (33, 34). In 5-10% of the cases patients may develop lesions on the oral mucosa, usually symmetric, like papillomatous tumours on the palate, membranes on the buccal mucosa, tongue or lips, or macroglossia (35) frequently diagnosed by stomatologists.

II. The nervous system (peripheral, enteric or cranial nerves) may also be affected, as partially described, and this is the reason why patients need a neurological examination yearly, to establish new clinical findings and to monitor the evolution of the older diagnosed problems.

Nerurofibromas can be associated with other nervous system tumors such as single or multiple gliomas of the optic nerve and hemispheric visual pathways or hamartoma of the iris (Lisch nodules), glioblastoma, meningioma. Brain computed tomography (CT) or Magnetic resonance imaging (MRI) can be useful for the diagnosis in detecting the tumors.

Other neurological findings can be macrocephaly without hydrocephalus, stenosis of the Sylvius aqueduct which causes non-communicating hydrocephalus, Chiary type 1 malformations, mild mental retardation and learning disabilities. 60% of the patients have impaired learning and thinking skills, speech impairment and visual-spatial skills, or may

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develop attention deficit hyperactivity disorder (ADHD), attributed to dysplasia of the cortex. Cognitive tests, head circumference measurements, neurological and psychiatric evaluation for ADHD can be useful in these children.

Uncommon, 4-7 % of patients may present with epilepsy (seizures) diagnosed using the electroencephalogram, hydrocephalus or stroke (imagistic diagnosis by brain CT scan or MRI).

Nerve tumors in the facial area, large neurofibromas of the trigeminal or facial nerve or extensive café-au-lait spots may generate concern with the patients’ appearance, resulting in anxiety and emotional distress.

Gliomas in other parts of the brain and spinal cord (usually low grade astrocytomas) also occur. Peripheral nerve neurofibromas (e.g on the spinal nerve roots) are diagnostic.

Of note, high T2W1 MRI lesions are seen in 80% of children, commonly in basal ganglia, internal capsule, optic radiation and brainstem or cerebellum.

These lesions are of unknown clinical significance and usually regress after 10 years of age.

More than 1% of patients with NF1 develop a symmetric sensory axonal neuropathy, and some cases may associate polyneuropathy. Nerve conduction studies may be helpful for the diagnosis.

III. Bones – or bone tissue abnormalities can be present. Signs of abnormal development in children can suggest orthopedic evaluation and further investigations (X-rays, CT, MRI) or sometimes a specialized clinical examination may be sufficient.

Long bones may present deformities due to abnormalities in bones growth, formation, and deficiency in bone mineral density (thin cortical layer, bowed legs, decreased bone mineral density, angulations of the long bones).

Sometimes fractures don’t heal, and there is a high risk of osteoporosis, rising a diagnosis problem.

The spine can be abnormal curved and scoliosis may be diagnosed, or bones’ growth is abnormal leading to a short stature (height below average) associated or not with sphenoid wing dysplasia.

Enlarged mandibular canal or mandibular foramen and/or mental foramen can be seen, especially by stomatologists. Pseudoarthrosis is frequently associated with bone development problems.

IV. Endocrine glands – the cells with ectodermal origin can be also affected. Pheochromocytomas (tumors of the adrenal glands) can be diagnosed

by CT or MRI, or using the plasma or urine catecholamine level, and 10% of them can be cancerous. Puberty, menopause and pregnancy increase the risk of appearance of neurofibromas, or the dimensions of the existing ones. Precocious puberty with growth acceleration can cause disruption of the hypothalamic-pituitary axis.

V. The eyes – may also be involved, due to the presence of optic nerve or optic pathway gliomas causing visual difficulties, or hamartomas of the iris

(tiny bumps of the iris, called Lisch nodules) which are to be monitored yearly after diagnosis by the ophthalmologist. This pathologic situations may associate cataracts.

VI. Other organs affected are represented by the blood vessels, due to the smooth muscles involved, present in their walls, causing increased risk of high blood pressure, blood vessel abnormalities (rarely) and hepatomegalia. Gastrointestinal stromal tumors, often multiple, localized more frequent in the proximal part of the small bowel can be found after an episode of gastrointestinal bleeding or intestinal obstruction.

B. Neurofibromatosis type 2 (acoustic neurinoma /vestibular schwannoma) Much less common than NF1, NF2 (central NF) is characterized by the presence of schwannomas (benign tumors) bilaterally.

The diagnosis criteria for NF2 are established by a consensus of experts: Definite (confirmed) NF2 - bilateral vestibular schwannoma (acoustic neurinoma) Probable NF2 : - family history of NF2 and - unilateral vestibular schwannoma or - any 2 of the following tumor types: meningioma, glioma, schwannoma, neurofibroma, juvenile posterior subcapsular lenticular opacity, juvenile cortical cataract Possible NF2 : - unilateral vestibular schwannoma and at least 2 of any of the following: meningioma, glioma, schwannoma, neurofibroma, juvenile posterior subcapsular lenticular opacity, juvenile cortical cataract - multiple (2 or more) meningiomas and unilateral vestibular schwannoma or any 2 of the following : glioma, schwannoma, neurofibroma, cataract.

Clinical manifestations and diagnosis The main clinical findings in NF 2 are the neurologic ones, while the cutaneous manifestations are less specific. However, the skin may help the diagnosis.

I. The skin - cutaneous lesions in NF 2 are usually tumours (most frequently schwannomas) and are present in 70% of cases, but in small numbers/ patient (90% of the patients have less than 10 tumours) (20).

There are at least 3 types of tumours: - cutaneous plaques, slightly elevated, hyperpigmented and with their surface covered with hair (the most frequent type), - nodular fusiform tumours situated in the subcutis which cannot be noticed at the skin surface unless the skin is palpated - cutaneous tumours, similar to those found in NF 1 (relatively rare).

There have also been described café-au-lait spots (fewer and smaller than those found in NF1) and hypopigmented patches (36, 37).

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II. Cranial and peripheral nerves are usually affected by the presence of tumours. Signs and symptoms may appear in the late teens and early adult years. The signs and symptoms may be different, and will be described separately, regarding the affected cranial nerve. Schwannomas (neurinomas) on the VIII-th cranial nerve (acoustic-vestibular) are characteristic and can induce gradual hearing loss (partial/total deafness) and tinnitus if the acoustic nerve is affected and balance impairment, vertigo or dizziness and uncoordinated walking, if the vestibular nerve is affected. If the schwannoma grow, they can compress the brainstem, inducing focal neurological problems (weakness, foot drop, pathologic reflexes, cranial nerve palsies, headache). The clinical examination may be completed with paraclinical investigations including: hearing and balance tests (audiometry, acoustic reflex testing, electronystagmography, brainstem auditory evoked potentials, balance tests – computerized dynamic posturography, walking tests, CT scan and MRI – 3D of the brain can be used for diagnosing and monitoring the dimensions and risk of complications of the schwannoma).

The II-nd cranial nerve (optic nerve) can be affected by the presence of tumors, the retina by specific retinal hamartomas or epiretinal membranes that cause visual difficulties. The visual problems can be increased by the presence of lens opacities and juvenile cataracts that may develop, even before a child shows clinical signs or symptoms of vestibular schwannoma. That’s why ophtalmologic examination yearly is necessary in order to monitor the patients if diagnosed before, or diagnose if they are still undiagnosed with ophtalmologic problems. If the patients are young children who do not cooperate for an ophtalmologic examination (being too young or with cognitive disorders), MRI of the optic nerve pathways and brain must be performed in order to detect a potentially aggressive glioma. If the V-th cranial nerve (trigeminal nerve) is affected, the patient may complain about numbness of the face, maybe on the side and in the distribution territory of the only trigeminal branch which is affected or about weakness of the muscles used for chewing (masticator muscles).

If the VII-th cranial nerve (facial nerve) is affected bilaterally, we will find facial drop, due to weakness of the muscles of the face (mimic muscles).

Spinal nerve roots and peripheral nerves may also be affected by the presence of schwannomas, producing numbness or weakness in the upper or lower limbs and sometimes pain. If symptomatic, MRI of the spinal cord and spinal nerves is indicated in order to establish the diagnosis and appreciate the treatment options. Schwannomas are slightly hyperintense on T2W1 MRI. There is no cognitive impairment in patients with NF2.

III. Cerebral and spinal tumors – can be present as meningioma, glioma, astrocytoma, ependymoma.

Meningiomas are more frequent and may develop in 50-75% of the patients, being localized inside

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the skull and/or spine. MRI can be used in order to establish the diagnosis. Schwannomas are slightly hyperintense on T2W1 MRI, and meningioma hypointense, often multiple and in atypical locations.

Both enhance with contrast substance administration.

Ependymoma are more common than meningioma in the spinal cord.

The genetic diagnosis can be used, the detection rates for molecular-based testing approaches 72% in simplex cases and 93% in familial cases. When a parent has NF2, prenatal testing can be done on amniocytes or chorionic villi, either through direct gene mutation analysis, when such a change has been identified or through linkage analysis. Prenatal testing may not be possible if the affected parent is the first affected person in the family and a mutation cannot be found.

Paraclinical Diagnosis Usually the skin lesions of NF1 don’t need additional paraclinical tests for diagnosis, as this can be easily established by the dermatologist just by clinical examination.

A biopsy is useful in the case of plexiform neurofibromas, when a malignant transformation is suspected.

If a biopsy is performed from a characteristic lesion, the histological findings can show one of the following: - The café-au-lait spots show histopathologically a high quantity of melanin in the epidermis and sometimes a greater number of melanocytes (compared to the rest of the skin) and giant melanosomes (38).

- Cutaneous neurofibromas are characterised histopathologically by the presence in the dermis of groups of well-defined, but nonencapsulated small nervous fibres with interlacing bundles of elongated cells. These cells have a pale cytoplasm and elongated nuclei and are fibroblasts, Schwann cells and perineural cells. All of them are found within a stroma composed of collagen, mucin and scattered mast cells (24).

- Plexiform neurofibromas are quite similar histopathologically to cutaneous neurofibromas, except that the number and size of the nervous fibres is larger, as they show hypertrophy. These nervous bundles are encircled by fibroblasts and Schwann cells, all in a myxoid matrix.

Malignant transformation of a plexiform neurofibroma into a neurofibrosarcoma shows histopathologically cellular pleomorphism, nuclear atypia with hyperchromasia and increased mitotic activity.

The cells are located around the vessels, in a wavy pattern and immunohistochemistry is positive for S-100 (24). NF 2 tumours histopathologically don’t differ from the sporadic lesions (e.g. cutaneous schwannoma). Molecular testing may be helpful in patients with a single clinical finding and no positive family history of neurofibromatosis. Sequencing of the neurofibromin gene offers the highest detection rate. Prenatal diagnosis of NF1 using

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amniocentesis or chorionic villus sample if one parent is affected or preimplantation genetic diagnosis for couples using in vitro fertilization when a specific mutation is known. Urinary free catecholamines (norepinephrine and epinephrine) and their metabolites in the 24 hour urine collection or plasma catecholamines can be used to investigate a suspected pheochromocytoma.

Differential diagnosis (NF1 and NF2) Using the clinical criteria, the positive diagnosis of neurofibromatosis is easy, but at the beginning, especially in young children, when not all the criteria are met, establishing the diagnosis can be quite difficult.

From dermatological point of view the differential diagnosis can be made with some other diseases presenting with café-au-lait spots, axillary or intertriginous freckling or neurofibroma.

Café-au-lait spots are the first and, at the beginning, the only signs of disease that appear. Initially they can be less than 6 and a differential diagnosis can be made with isolated café-au-lait macules (found in 10-20% of the normal population)(14, 39), spilus and Becker naevi. Café-au-lait spots can also appear in other syndromes such as McCuneAlbright, Westerhof, Legius, tuberous sclerosis and Fanconi anaemia. Until the whole clinical picture is revealed the only tests that can be helpful for diagnosis are the genetic ones.

McCune-Albright syndrome is a genetic sporadic disease that has café-au-lait spots present from birth or which appear from infancy, but usually the macules are more asymmetrical and larger in comparison with those in NF1 and also the borders are more imprecisely defined and they never cross the median line. The syndrome also associates bone problems (polyostotic fibrous dysplasia) and endocrine problems (more often early puberty).

Westerhof syndrome is a rare disorder characterised by the presence of hypo- and hyperpigmented macules on the trunk and limbs, without other systemic manifestations.

Legius syndrome is a genetic disorder, with autosomal dominant inheritance, which resembles to NF1, due to the presence of the café-au-lait spots and axillary freckling, but without the other clinical findings (40).

Tuberous sclerosis can also have café-au-lait spots, but without the presence of intertriginous freckling or the neurofibromas and the other associated clinical findings, dermatologic as well as systemic.

Fanconi anaemia is a rare form of aplastic anaemia that associates abnormalities of the bones, microcephaly, hypogonadism, ocular, renal and cutaneous abnormalities, among which café-au-lait spots are present.

Intertriginous freckling may also appear within other syndromes: LEOPARD, Watson or Carney syndromes. LEOPARD syndrome, whose name is a mnemonic and stands for the characteristic signs and symptoms of this disease (Lentigines, Electrocardiographic (ECG) conduction defects, Ocular

hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth and Deafness) presents with multiple lentigines on the face, neck and trunk.

Watson syndrome can have café-au-lait spots as well as freckling in the axillary or inguinal region and neurofibromas, but is associated with pulmonary stenosis, short stature and mental retardation.

Carney complex is an autosomal dominant transmitted syndrome that associates cutaneous findings, endocrinopathy and tumours (myxomas of the heart, breast, psammomatous melanotic schwannomas, testicular tumours, etc).

Dermatologically hyperpigmented macules can be found on the skin and mucosae, lentigines and blue naevi, as well as cutaneous myxomas, the latter presenting as translucent or skin-coloured papules.

Neurofibromas have very characteristic features, clinically as well as histopathologically, but sometimes, especially the plexiform hyperpigmented ones, covered excessively with hair, in children, can clinically resemble to congenital melanocytic naevi.

Cutaneous tumours in NF 2 can be mistaken for the ones found in neurofibromatosis type 1 (NF1) or type 3 (NF3). In NF3 (Riccardi classification), also known as schwannomatosis, the patients have multiple cutaneous schwannomas and different tumours of the central nervous system, but they spare the VIII-th cranial nerves, so the vestibular tumours of NF 2 are not present (41).

Among the differential diagnoses of NF1 and NF2, one should never forget the other forms of neurofibromatosis: NF3 Riccardi (discussed above), type 5 Riccardi or type 3 Carey (known as segmental neurofibromatosis as the lesions are situated on one or more dermatomes or in a mosaic fashion on one segment of the body that is limited by the Blaschko’s lines), type 6 Riccardi or 4 Carey (familial café-au-lait spots, without other stigmata or systemic involvement), type 5 Carey (NF-Noonan phenotype which presents, besides the NF 1 features the characteristic clinical elements of the Noonan syndrome, such as short stature, peculiar facial features, skeletal malformations and cardiac abnormalities) (24, 42, 43).

The neurological differential diagnosis can be made with brainstem glioma, low grade astrocytoma, meningioma, cauda equina and conus medullaris syndromes, vascular lesions of the spinal cord.

Complications Though apparently a benign disease, complications may appear in the evolution of the disease so monitoring the patients for complications is mandatory. The complications may have very different etiologic mechanisms like distorting nerve tissue or compression on internal organs (in the thoracicabdominal cavity) due to tumor growth, or malignant transformation of the tumors.

Tumors (neurofibromas under the skin or plexiform neurofibromas involving multiple nerves) can M a rc h 2 0 1 6

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develop to malignant ones (less than 10% in NF1).

In NF1 there is a higher risk of also developing other forms of cancer such as breast cancer, leukemia, malignant brain tumors, soft tissue cancer, rhabdomyosarcoma or Wilms renal tumor. This high rate of malignancy or association of other malignant tumors have a strong indication of monitoring the tumor appearance, size, number of neurofibromas, or signs of cancerous changes. If malignancy is diagnosed early treatment should be prescribed.

Treatment Neurofibromatosis can’t be cured, there is no specific treatment available! As a predominantly genetic disease, there is no etiological treatment available for this disorder but it requires life-long management adapted to the form and age; therefore the only option is a symptomatic treatment associated with the information and education of patients regarding the disease and it’s complications, the necessity to be monitored for evolution and complications yearly by a multidisciplinary team as well as a genetic counselling.

Dermatologic treatment The dermatologist can excise bigger tumours which produce functional discomfort or deformities. Simple surgical excision is the most frequent method, but ablative lasers (such as C02 Laser) or electrodessication can also be used, with a higher risk of recurrences and hypertrophic or keloid scars (44 – 46). Deeper tumours are treated by surgeons due to the fact that the tumour may extend to the internal structures.

However the results are often unsatisfactory because of the deeper infiltration and the higher risk of recurrences. Chemotherapy, antifibrotic agents, antiangiogenic therapy and genetic and cytokine modulators are studied (47). For plexiform neurofibromas that arise suspicions for malignancy (see above) biopsy and histopathological analysis are mandatory and if malignancy is confirmed, early specific treatment must be prescribed. Treatment of the café-au-lait spots can be tried due to cosmetic reasons, but they respond badly to non-invasive treatments, such as lasers (they do not disappear completely or they reappear) (24). If intense itching is present, antihistaminic treatment can bring relief, some authors considering that ketotifen is the best option (48).

Symptomatic treatment Pain relief can be often needed as pain may be one of the most bothering symptoms in patients with affected peripheral or spinal nerves. Monitoring of blood pressure and specific treatment in order to lower high blood pressure may be useful, if smooth muscles of the blood vessels are involved.

Surgery (laparoscopic/classical methods), in order to remove compressing or invasive tumors,

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or tumors affecting nerves, recidivating tumors in order to reduce complications or cutaneous large tumors in order to help in recovering self-esteem.

Microsurgery and stereotactic radiosurgery are used to remove vestibular schwannomas or schwannomas with other localization. Meningiomas are growing slower than schwannomas, and surgical treatment can be considered only if they are causing serious disabling symptoms.

Radiation therapy can be used for localized tumors or malignant ones, but there is a risk, as it can cause radiation induced cancer if applied repetitively and especially in young patients.

Orthopedic treatments for scoliosis and other treatable bone deformities can be used, as well as medical treatment for osteoporosis in order to reduce complications. Implants (cochlear, auditory brainstem implants) are used for augmentation and amplification of hearing, and in young children with NF1 and speech problems and learning disabilities speech therapy can be a choice.

Treatment for cancer, disabling ependymoma, unresectable schwannoma may include surgery, chemotherapy (lomustine, vincristine, prednisone, or carboplatin and vincristine, or erlotinib) and radiation therapy (49).

Evolution and prognosis NF1 and NF2 are progressive and generalized diseases. For NF1 the overall life expectancy may be reduced by on average 8 years due to complications (hypertension, malignancy, spinal cord or brain lesions) (49). Prenatal diagnosis is difficult, not only because of the high costs, but also as the disease has a wide variability of clinical expressions within the same family, so it is difficult to predict the severity of the disease in a given patient.

Conclusions A multidisciplinary team represented by dermatologist, neurologist, ophthalmologist, orthopedist, pediatrician, geneticist will be very efficient in diagnosing and monitoring on an annual basis for changes in the evolution of the disease.

Together they are preventing, treating symptoms and complications.

The team can be enlarged with neurosurgeon, plastic surgeon, oncologist, otolaryngologist, endocrinologist or some other specialists if necessary as neurofibromatosis is a rare but multifacetated disease.

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Smaranda Cristina Nica, Gabriela Mihăilescu, Sanda Maria Nica, Cristina Băetu, Victor Gabriel Clătici, Ioan Buraga.

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24. Bolognia JL, Jorizzo JL, Schaffer JV, Callen JP, Cerroni L, Heymann WR, HruzaGJ, Mancini AJ, Patterson JW, Röcken M, Schwarz T. Bolognia Textbook of Dermatology, Third Edition. Elsevier Limited, 2012.

25. Pivnick EK, Riccardi VM. The neurofibromatoses. In: Freedberg IM, Eisen AZ, Wolff K,

et al. Fitzpatrick’s Dermatology in General Medicine, 6th Edition. New York: McGraw-Hill, 2003:1825–33.

26. McGaughran JM, Harris DI, Donnai D, et al. A clinical study of type 1 neurofibromatosis in north west England. J Med Genet. 1999;36:197–203.

27. Huson SM, Compston DAS, Harper PS. A genetic study of Von Recklinghausen neurofibromatosis in South East Wales. II. Guidelines for genetic counselling. J Med Genet 1989; 26: 712–21.

28. Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ. Fitzpatrick’s Dermatology in General Medicine, 7th Edition. New York: McGraw-Hill, 2008 29. Gutmann DH, Collins FS. Neurofibromatosis type 1. In: Vogelstein B, Kinzler KW (eds). The Genetic Basis of Human Cancer. New York: McGraw-Hill, 1998:423–42.

30. Mautner VF, Asuagbor FA, Dombi E, et al. Assessment of benign tumor burden by wholebody MRI in patients with neurofibromatosis 1. Neuro Oncol. 2008;10:593–8.

31. Storm FK, Eilber FR, Mirra J, Morton DL. Neurofibrosarcoma. Cancer. 1980;45:126–9.

32. Westerhof W, Konrad K. Blue-red macules and pseudoatrophic macules: additional cutaneous signs in neurofibromatosis. Arch Dermatol. 1982;118:577–81.

33. Cambiaghi S, Restano L, Caputo R. Juvenile xanthogranuloma associated with neurofibromatosis 1:14 patients without evidence of hematologic malignancies. Pediatr Dermatol. 2004;21:97–101.

34. Brems H, Park C, Maertens O, et al. Glomus tumors in neurofibromatosis type 1: genetic, functional, and clinical evidence of a novel association. Cancer Res. 2009;69:7393–401.

35. Canale DJ, Bebin J. In: Vinken PJ, Bruyn GW, eds. Handbook of Clinical Neurology, Vol. 14.

Amsterdam: Elsevier, 1972: 132–62.

36. Asthagiri AR, Parry DM, Butman JA et al. Neurofibromatosis type 2. Lancet 2009; 373: 1974–86.

37. Casado-Verrier B, Feito-Rodriguez M, Noval S, Martinez-Glez V, Lapunzina P, De LucasLaguna R. Cutaneous Cutaneous and ophthalmic signs as a clue to early diagnosis of severe neurofibromatosis type 2: report of a novel mutation that predicts this poor prognosis. Clinical and Experimental Dermatology [serial online]. June 2014;39(4):557-559. Available from MEDLINE, Ipswich, MA 38. Johnson BL, Charneco DR. Café au lait spot in neurofibromatosis and in normal individuals.

Arch Dermatol. 1970;102:442–6.

39. Benedict PH, Szabo G, Fitzpatrick TB et al. Melanotic macules in Albright’s syndrome and in neurofi bromatosis. JAMA 1968; 205: 618–626.

40. Messiaen L, Yao S, Brems H, et al. Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome. JAMA. 2009;302:2111–18.

41. Min H, Kim K, Jun S, Lee Y. Schwannomatosis on a single foot: a case report. Foot & Ankle Specialist [serial online]. June 2015; 8(3):226-229. Available from MEDLINE, Ipswich, MA 42. Madson JG. Multiple or familial café-au-lait spots is neurofibromatosis type 6: clarification of a diagnosis. Dermatol Online J. 2012 May 15;18(5):4.

43. Romano AA, Allanson JE, Dahlgren J, et al. Noonan syndrome: clinical features, diagnosis, and management guidelines. Pediatrics. 2010 Oct. 126(4):746-59.

44.Becker DWJr.Use of the carbon dioxide laser in treating multiple cutaneous neurofibromas.

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2008;61:958–62.

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47. Packer R, Rosser T.Therapy for Plexiform Neurofibromas in Children with Neurofibromatosis 1: An Overview. Journal of Child Neurology [serial online]. Aug2002, 17(8):638. Available from Biomedical Reference Collection: Comprehensive, Ipswich, MA 48. Braun-Falco O, Burgdorf WHC, Plewig G, Wolff HH, Landthaler M. Braun-Falco’s Dermatology, Third Edition. Heidelberg: Springer Medizin Verlag. 2009.

49. Hsieh DT, Rohena LO, Kao A. Neurofibromatosis Type 2. Medscape 2014.

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P AZITROMICINA, O ALEGERE TERAPEUTICĂ DE SUCCES ÎN TRATAMENTUL ACNEEI Introducere

Acneea este o afecțiune inflama­ torie comună a pielii care afec­ tează un procent important din populația generală. Etiologia este multifactorială printr-un complex de factori genetici, hormonali, de mediu și locali.

Este privită ca o disfuncție a unității pilosebacee la care se adaugă un proces inflamator cu exacerbări periodice.

Chiar dacă acneea nu este o boală infecțioasă, flora microbiană joacă un rol important în formarea come­ doanelor și apariția leziunilor inflamatorii.

Principala bacterie incriminată în acnee este Propionibacterium acnes care contribuie in mod semnificativ în patogeneza bolii fără să existe corelație între severitatea clinică și concentrația bacteriilor în zona afectată.

Diferite tulpini de P. acnes s-au dovedit a induce diferite grade de răspuns a sebocitelor și citokinelor proinflamatorii/chemokine.

Antibioterapia Ca parte a arsenalului terapeutic, tratamentul cu antibiotice este considerat necesar în abordarea acneei vulgaris moderat-severe.

Mecanismul de acțiune include pe lângă supresia dezvoltării P. Acnes și efectele imuno-modulatoare și antiinflamatorii. În mod frecvent sunt recomandate antibiotice din clasa ciclinelor sau macrolidelor.

pe P. Acnes și are un puternic efect antiinflamator și imuno-modulator, fără să producă imunosupresie.

Administrată timp de 8 săptămâni, de trei ori pe săptămână, azitromicina oferă rate înalte de răspuns terapeutic situate între 80% și 90%.

Procentul pacienților care au avut un răspuns bun și foarte bun la tratament este de peste 70%.

Răspunsul terapeutic se situează la același nivel și pentru pacienții care au o recădere după tratamentul cu alte antibiotice.

Timpul de înjumătățire mare și concentrarea în țesuturile inflamate permit administrarea azitromicinei de 3 ori pe săptămână, ceea ce crește semnificativ complianța. În studiile care au avut și o componentă subiectivă legată de calitatea vieții, pacienții au afirmat că tratamentul a fost unul ușor de respectat.

Este dovedit că azitromicina are o incidență mai mică a reacțiilor adverse față de alte macrolide și un profil de siguranță și tolerabilitate superior ciclinelor. În cazul azitromicinei nu au fost raportate cazuri de fotosensibilitate.

De aceea ghidurile terapeutice în vigoare recomandă utilizarea antibioticelor macrolide (clasă de recomandare I A pentru azitromicină si eritromicină) în tratamentul pacienților cu acnee vulgaris moderat severă.

Azitromicina este de preferat eritromicinei datorită tolerabilității Numeroase studii clinice au do­ vedit că azitromicina este activă superioare.

Atuurile azitromicinei

Bibliography 1. Zaenglein et al.:Guidelines of care for the management of acne vulgaris.JAMACAD DERMATOL doi:10.1016/j.jaad.2015.12.037 2. Federico B et al: Azithromycin: A new therapeutical strategy for acne in adolescents. Dermatology Online Journal 13(4):4

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3.Carl Llor et al: The higher the number of daily doses of antibiotic treatment in lower respiratory tract infection the worse the compliance.Journal of Antimicrobial Chemotherapy (2009) 63, 396–399 4. Abdullateef A. et al: Macrolides in Chronic Inflammatory Skin Disorders. Mediators of Inflammation, Volume 2012, Article ID 159354, doi:10.1155/2012/15935

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Case Presentation

Spitzoid skin lesions diagnosis engages hight expertise – clinical and pathologic interpretation of two cases

SPITZOID SKIN LESIONS DIAGNOSIS ENGAGES HIGHT EXPERTISE – CLINICAL AND PATHOLOGIC INTERPRETATION OF TWO CASES DIAGNOSTICUL LEZIUNILOR CUTANATE SPITZOIDE NECESITĂ UN ÎNALT GRAD DE EXPERTIZĂ – ANALIZĂ CLINICO-PATOLOGICĂ A DOUĂ CAZURI Virginia Chiţu1,3, Sabina Zurac 2,3, Alina E. Cipi 4 1

First Department of Dermatology, Colentina Clinical Hospital, Bucharest, Romania 2 Department of Pathology , Colentina Clinical Hospital, Bucharest, Romania 3 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania 4 Private Healthcare Network Regina Maria, Bucharest, Romania Corresponding author: Virginia Chiţu, “Colentina” University Hospital, First Department of Dermatology, 19-21 Stefan cel Mare str., sector 2, 020125, Bucharest, Romania, Tel. +40213173245, Fax +40213165512 E-mail: virginiachitu@gmail.com

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: spitzoid lesions, nevi, melanoma, dermoscopy, aberration, genetic, molecular, tests

Cite this article: Virginia Chiţu, Sabina Zurac, Alina E. Cipi.Spitzoid skin lesions diagnosis engages hight expertise – clinical and pathologic interpretation of two cases. RoJCED 2016; 3(1):50 - 56

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Spitzoid skin lesions cause diagnosis difficulties due to their clinical, dermoscopic and histopathological similarities to melanomas. A correct diagnosis implies a high expertise and close collaboration between the dermatologist and the pathologist as there are no algorithms or methods of diagnosis with a 100% accuracy. This type of lesions continues to be of interest for scientists considering the fact that 30% of the malpraxis cases of misdiagnosed melanomas are caused by Spitzoid lesions. In most cases, Spitz nevi do not show any chromosomal aberrations and when present they are totally different from those seen in melanomas. In this paper we present two cases of Spitzoid lesions: desmoplastic Spitz nevus and atypical Spitz nevus. The paper underlines the clinical, dermoscopic, histopathological and immunohistochemical features and their impact on final diagnosis. Management of Spitzoid lesions is a challange even for experts. Cytogenetic evaluation of atypical Spitzoid lesions, using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) respectively, can be very useful when differentiating them from melanomas and establishing their most probable biological behaviour.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Virginia Chiţu,Sabina Zurac, Alina E. Cipi

Rezumat Cuvinte-cheie: leziuni spitzoide, nevi, melanom, dermatoscopie, aberaţii, citogenetică, comportament.

Leziunile cutanate spitzoide ridică dificultăţi de diagnosticare, din cauza asemănărilor cu melanomul la nivel clinic, dermatoscopic şi histopatologic. Stabilirea unui diagnostic corect implică un nivel înalt de expertiză şi o colaborare strânsă între clinician şi anatomopatolog, neexistând încă un algoritm sau o metodă de diagnosticare cu sensibilitate de 100% . Această categorie de leziuni continuă să suscite interesul comunităţii ştiinţifice, în contextul în care 30% din cazurile de malpraxis prin subdiagnosticarea melanomului sunt cauzate de leziunile spitzoide. Nevii Spitz în marea majoritate a cazurilor, nu prezintă aberaţii cromozomiale iar atunci când există sunt complet diferite de cele găsite în melanom. În această lucrare sunt prezentate două leziuni cutanate spitzoide : un nev Spitz desmoplazic şi un nev Spitz atipic. În articol sunt discutate aspectele clinice, dermatoscopice, histopatologice şi de imunohistochimie întâlnite şi răsunetul lor asupra diagnosticului final. Managementul leziunilor spitzoide reprezintă o provocare chiar şi pentru experţi.

Evaluarea citogenetică a leziunilor spitzoide atipice poate fi foarte utilă în diferenţierea lor de melanom şi în stabilirea comportamentului biologic cel mai probabil.

INTRODUCTION Spitzoid skin lesions are a heterogeneous group of melanocytic proliferations with different biological potential varying from the benign nature of the classical Spitz nevus to the obvious malignancy of Spitzoid melanomas, between the two being the atypical Spitz nevus with unpredictable potential(1,2,3). Diagnosis of Spitzoid lesions is usually a real challenge because of clinical, dermoscopic and histopathological similarities with melanomas(4,5). Unlike melanomas, genetic aberrations are rarely found in classical Spitz nevi, in just about 25% of the cases being observed an increase in the number copies of the p-arm of chromosome 11(4,69) . This genetic aberration has not been described in melanomas, which present multiple, frequent cromosomal aberrations which affect totally or just fragments of chromosomes(8,9). Although Spitz nevi represent a low percentage of the amount of excised nevi, medical reports reveal the fact that about one third of misdiagnosed melanoma cases are initially interpreted as Spitz nevi(4,10-12).

Over the past decades, current use of dermoscopy on evaluation of melanocytic lesions has increased the accuracy of diagnosis of Spitz nevus, these lesions being most often interpreted as suspicious for malignity(1,4,13,14,16,17).

Unfortunately, the accuracy of histopathological diagnosis for Spitzoid lessions is less than 100%, as there is a gray zone of uncertainty of atypical Spitz nevus also called Spitzoid tumour or atypical Spitzoid tumour, whose risk of metastasis is well known(2,4).

The diagnosis difficulties are even higher for the variants of Spitz nevus (SN) such as halo SN, pigmented SN, desmoplastic SN, recurrent SN, combined SN, angiomatoid SN, acral SN and the mucous membranes SN(1,4,6,18,19). Cytogenetic evaluation of Spitzoid lesions characterised by conflicting histopathological and immunohistochemical

Figure 1A. Desmoplastic Spitz nevus. Clinical appearance.

Figure 1B. Desmoplastic Spitz nevus. Dermo-

scopic image showed an unspecific pattern

features hard to interpret in the diagnosis process, can provide valuable information which makes it M a rc h 2 0 1 6

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Case Presentation

Spitzoid skin lesions diagnosis engages hight expertise â&#x20AC;&#x201C; clinical and pathologic interpretation of two cases

Figure 1C. Desmoplastic Spitz nevus. HE x 100.

Figure 1D. Desmoplastic Spitz nevus. HE x 200.

Figure 1E. Desmoplastic Spitz nevus. HE x 400.

Figure 1F.

Figure 1G. Desmoplastic Spitz nevus. HE x 200.

Figure 1H. Desmoplastic Spitz nevus. HE x 400.

Dermal melanocytic proliferation with overall symmetric architecture; moderate cellular pleomorfism; epidermal hyperplasia.

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Desmoplastic Spitz nevus. HE x 200

Transition to a sclerotic dermis with sparse pigmentation with plump spindle melanocytes interspersed between collagen bundles.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Virginia ChiĹŁu,Sabina Zurac, Alina E. Cipi

Figure 1I.

Desmoplastic Spitz nevus.

HMB45 x 100. HMB45 positive in superficial part of the tumor and negative in deepest part; nontumor active epidermal melanocytes also positive.

Figure 1K. Desmoplastic Spitz nevus.

i67 x 200. Occasional nuclei positive for ki67 (ki67 index less than 1%). Basal and parabasal epidermal cells positive for ki67 (internal control).

gically excised with a 5 mm safe margin and the histopathological exam followed by immunohistochemical tests, confirmed the diagnosis of intradermic Spitz nevus with desmoplastic component (Fig. 1C-H).

CASE PRESENTATION 2

Desmoplastic Spitz nevus. T311 x

Figure 1L. 400. Plump spindle tumor melanocytes positive for T311. possible to obtain a clear-cut differentiation from melanomas in some cases(4,8). Below we report two cases of Spitz nevus resembling melanoma in varying degrees.

CASE PRESENTATION 1 Case 1. A 35 year old male came to our clinic for a 3/5 mm diameter light-brown nodule with a dark brown halo around (Fig. 1A), that had appeared 2 years before on the right side of the abdomen.

Dermoscopic exam established the melanocytic nature of the lesion and revealed a fine typical pigmented network in the halo area and an unspecific global pattern with astructural gray-brownish areas on the nodular part of the lesion (Fig.1B).

The history along with the clinical and dermoscopic features raised the suspicion for melanoma, the diagnosis of Spitz nevus being considered only because of the patient`s age. The lesion was sur-

Case 2. A 53 year old female came to our clinic for a round pale-pink plaque, 8/8mm in diameter, slightly raised, surrounded by an incomplete pale-brown halo, that had appeared on the right shoulder three years before (Fig.1A). Considering the clinical appearance of the lesion, we took into acount a dermatofibroma, amelanotic melanoma and less likely Spitz nevus because of the patient`s age. Dermoscopic examination showed an unspecific global pattern with homogenous pale-brown colour at the periphery, associated with a palepink hue in the central part (Fig.2B). The lesion was surgically excised and the histopathological exam confirmed the diagnosis of atypical Spitzoid tumour with 0.5 mm thickness having a desmoplastic dermal component and a pagetoid epidermal component (Fig. 2C-F). The immunohistochemical tests revealed the Ki67 index <1% in the superficial tumour component and absent in the dermis. Considering the patient`s age and the metastatic potential of the lesion, routine blood tests and imagistical exams (chest X-ray, abdominal ecography) were performed with negative results. The patient will be followed up every 6 months for 2 years and then annually.

DISCUSSION Diagnosis difficulty of Spitzoid lesions is well known and requires a close collaboration between the dermatologist and the pathologist(6).

The final diagnosis can be established based on the close correlation between the clinical and hisM a rc h 2 0 1 6

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Case Presentation

Spitzoid skin lesions diagnosis engages hight expertise â&#x20AC;&#x201C; clinical and pathologic interpretation of two cases

Figure 2A. Atypical Spitz Tumor. Clinical appearance.

Figure 2B. Atypical Spitz Tumor. Dermoscopic image showed an unspecific pattern.

Figure 2C-D. Atypical Spitz Tumor. HE x 100 Irregular pattern of growth with asymmetry and focal

epidermal effacement associated with architectural disordered of the intraepidermal component consisting in prominent lentiginous melanocytic proliferation.

topathological criteria(2,13). Molecular tests CGH and FISH have been recently added to the diagnosis methods for Spitzoid lesions, being applied in difficult cases since their high costs limit a wide scale use(4,6-8,13).

Typical Spitz nevus is usually a symmetrical, monochromatic nodule, with a diameter under 6 mm and affects children and young people and has a worrisome rapid growth(4,20,21).

Histopathological Spitz nevus is in most cases a compound nevus, but it can also be junctional or intradermal, with a symmetrical architecture, an absent or low mitotic activity in the superficial nevus cells with cell maturation in depth and central pagetoid spreading(4,6,13,20,21).The genetic aberrations are infrequent, the increase in the number of

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chromosomal copies 11p present in 25 % of the cases and they are commonly associated with H-RAS oncogenic mutation(4,6,7,9,20). Neither of the above mentioned genetic aberrations have been decribed in melanomas(4,22). Recently, Spitz nevus has been considered to be similar to other melanocytic nevi, showing a growth period, faster than that of other melanocytic nevi, followed by a stagnation period and with a spontaneous involution occuring in the final(13,19,21). Because of its similarity to melanomas and the possibility of acquiring atypical features over its evolution, difficult to interpret, the surgical excision with a 3-5mm safe margin is recommended in the patients older than 11 years(4,13). In younger children, the decision to follow up or to excise will be taken according to

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Virginia ChiĹŁu,Sabina Zurac, Alina E. Cipi

Figure 2E.

Atypical Spitz Tumor. HE x 200.

Figure 2F.

Atypical Spitz Tumor.HE x 200.

clinical and dermoscopic aspects. In the case of such children this decision will be taken together with their parents, considering the fact that melanoma cases in childhood are extremely rare yet possible(3,13,23).

The lesion described in case 1, despite its worrying clinical appearance, had typical histopathological features showing intradermal proliferation of epithelioid cells, mitotic activity under 2/mm2 within the upper part of the lesion, the immunohistochemical analysis supporting this diagnosis (Ki67<1%, p16 positive in frequent nuclei, cyclin D1 positive in few nuclei, HMB 45 positive in the upper part of the tumour, tyrosinase T311 positive in the entire tumour) (Fig. 1I-L).

Unlike dysplastic melanocytic nevus, the diagnosis of atypical Spitz nevus is worrying and does not mean a certain benign biological potential since sentinel lymph node involvement occurs in 30-50% of the cases(2,6). The patients with atypical Spitz nevus should be carefully followed up and if the tumour thickness is larger than 1 mm the therapeutical approach should be as for melanomas making sentinel node biopsy, although its value is still debated(2,6,13).

The immunohistochemical and molecular tests can contribute to establishing the diagnosis; however, they are not always able to make a sharp differentiation of Spitz nevi from melanomas(8,6,19,24). A subset of atypical Spizoid nevi may show chromosomal aberrations that may indicate an aggresive biological behaviour(22,25). Unfortunately, such clinical and histopathological borderline Spitzoid lesions can often reveal borderline cytogenetic features(6).

The lesion presented in case 2 showed on histopathological exam a symmetrical proliferation of epithelioid and fusiform cells, rare mitosis, with pagetoid spreading in epidermis and desmoplastic aspect in papillary and reticular dermis (Fig. 2C-F). The immunohistochemical tests revealed the following: Ki 67 proliferation index under 1%, p21 negative, HMB 45 positive in the upper part of the tumour, S100 highly positive in the entire tumour. However, the diagnosis of atypical Spitz nevus on the upper thorax in a 53 year old patient, a common site also for desmoplastic melanomas, requires a careful follow-up of the patient. The low tumour thickness and rare mitotic activity are good prognosis factors for our patient.

The histopathological diagnosis of Spitzoid melanomas is not yet a standard procedure, the importance of each parameter found is established according to all existing parameters which causes considerable subjectivism and disagreement among experts(4,6,26). The conflicting clinical, dermoscopic and histopathological data should compel physicians to make a cautious categorising of such lesions considering the medico-legal and therapeutic impact(7,11,12). The molecular tests CGH and FISH are valuable tools to acquire a higher accuracy of the diagnosis. Melanoma chromosomal aberrations - present in 95% of the cases - are multiple and result from telomeric crisis; they are structural and numerical(7,9). The most frequent chromosomal aberrations found in melanomas are by losses of 9p, 10q, 6q, 8p and by gains of 7, 8, 6p, (1q)(7,8,22,25). Most melanomas have BRAF or NRAS mutations. The type and number of mutations depend on the evolution span of lesions, on the patientâ&#x20AC;&#x2122;s age and melanoma type(9,25).

CONCLUSIONS The Spitzoid term for pigmented melanocytic lesions needs a very careful evaluation of clinical, dermoscopic, histopathological, immunohistochemical and molecular features by expert teams(24). Both dermatologist and pathologist are responsible for the medico-legal impact.

All participants in the diagnosis process should be aware of discrepancies between clinical, dermoscopic and histopatological features of such lesions and must evaluate the promalignant potential, avoiding misdiagnosis of melanomas.

For atypical Spitz nevus the therapeutical approach should be similar to that for melanomas, according to tumor thickness(4). Molecular tests CGH and FISH represent valuable tools for categorizing Spitzoid lesions with confidence(25). Atypical Spitz M a rc h 2 0 1 6

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Case Presentation

Spitzoid skin lesions diagnosis engages hight expertise – clinical and pathologic interpretation of two cases

nevi compel a cautious post-surgical attitude, the management of such lesions depending on the thickness of the tumor, on the genetic aberration pattern and on the patient’s age(13,24). Where the molecular tests do not reveal any chromosomal aberrations or show only an increased of the copy number of 11p, the lesions should be approached like a Spitz nevus(2,4,9). The future directions of Spitz nevus management remain its proper identification and the avoidance of needless therapies(19).

Acknowledgements This paper was supported by the Sectoral Operational Programme Human Resources Develop-

ment (SOP HRD), financed from the European Social Fund and by the Romanian Government under the contract POSDRU/159/1.5/S/137390.

This work is licensed under a Creative Commons Attribution 4 .0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc/4.0/

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18. Fabrizi G, Massi G. Angiomatoid Spitz naevus: a close simulator of regressing malignant melanoma. Br J Dermatol. 2001 Nov;145(5):845-6.

19. Philip E. Leboit. Spitz Nevus. In: Darrell S Rigel, Clay J Cockerell, June K. Robinson,HenryW. Lim,Merrick Ross,Eggert Stockfleth,Robert J. Friedman, John M Kirkwood.Cancer of the Skin.2th Edition. China.Elsevier Saunders; 2011 20. William D James, Timothy G Berger, Dirk M Elston. Melanocytic Nevi and Neoplasms.

In: William D James, Timothy G Berger, Dirk M Elston. Andrews` Diseases of the Skin Clinical Dermatology. 11th Edition.China. Saunders Elsevier;2011 21. Grichnik JM, Rhodes AR, Sober AJ. Benign Neoplasias and Hyperplasias of Melanocytes.In: Lowell a. Goldsmith, Stephen I. Katz, Barbara A. Gilchrest, Amy S. Paller, David J. Leffell, Klaus Wolff.

Fitzpatrick`s Dermatology In General Medicine.8th Edition,NewYork.The McGraw-Hill Medical,2012 22. Cesinaro AM, Schirosi L, Bettelli S, Migaldi M, Maiorana A. Alterations of 9p21 analysed by FISH and MLPA distinguish atypical spitzoid melanocytic tumours from conventional Spitz’s nevi but do not predict their biological behaviour. Histopathology. 2010 Oct;57(4):515-27.

23. Primoz Strojana, Janez Lamovecb. Prepubertal Malignant Melanoma: Report of Three Cases.

Pediatric Hematology and Oncology. 2000Vol 17( 2):163-169 24. Diaconeasa A, Boda D, Solovan C, Enescu DM, Vîlcea AM, Zurac S. Histopathologic features of Spitzoid lesions in different age groups. Rom J Morphol Embryol. 2013;54(1):51-62.

25. Ali L, Helm T, Cheney R, Conroy J, Sait S, Guitart J, Gerami P. Correlating array comparative genomic hybridization findings with histology and outcome in spitzoid melanocytic neoplasms.

Int J Clin Exp Pathol. 2010 Jun 28;3(6):593-9.

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Clinical evolution of lymphangioma circumscriptum:observations on three cases.

CLINICAL EVOLUTION OF LYMPHANGIOMA CIRCUMSCRIPTUM:OBSERVATIONS ON THREE CASES LIMFANGIOMUL CIRCUMSCRIS, OBSERVAŢII CLINICO-EVOLUTIVE PRIVIND TREI CAZURI Pătraşcu Virgil1, Bocîrnea Alexandra Georgiana2, Ciurea Raluca3 Department of Dermatology, University of Medicine and Pharmacy, Craiova, Romania 2 Clinic of Dermatology, Emergency County Clinical Hospital, Craiova, Romania 3 Department of Pathology, University of Medicine and Pharmacy, Craiova, Romania Corresponding author: Virgil Pătraşcu, Professor, MD, PhD, Department of Dermatology, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, 2-4 Petru Rares Street, 200349 Craiova, Romania; Phone +40724273676, e-mail: vm.patrascu@gmail.com 1

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: lymphangioma circumscriptum, clinical, treatment.

Introduction: Lymphangiomas are rare congenital dysplasia of lymphatic vessels, which may affect the tegument, mucous membranes and viscera.

They are superficial cavernous or cystic. In 80-90% of cases they are diagnosed prior to the age of 2 years.

Patients and Methods: We present 3 patients with lymphangioma circumscriptum, aimed to highlight aspects of this tumor clinical course, based on clinical examination, laboratory investigations, histopathological examination and Doppler ultrasound. Patients were investigated for diagnosis and comorbidities. The treatment was surgical (excision and electrocautery of tumors) for the first two cases and by medication in the third case.

Results: Our patients had no associated comorbidities. Correct diagnosis was established based on histopathological examination and the treatment has conducted good results.

We obtained healing by surgically treating cases I and II and correction by medical treatment bleeding complications in case III. However regular follow up is necessary in these patients.

Conclusion: Lymphangioma circumscriptum is a benign condition, but difficult to treat and recurrent due to the deep hypodermic component.

Cite this article: Pătraşcu Virgil, Bocîrnea Alexandra Georgiana, Ciurea Raluca.

Clinical evolution of lymphangioma circumscriptum: observations on three cases.

RoJCED 2016; 3(1):58 - 64

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R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Pătraşcu Virgil, Bocîrnea Alexandra Georgiana, Ciurea Raluca

Rezumat Cuvinte-cheie: Limfangiom circumscris, aspecte clinice, tratament.

Introducere: Limfangioamele sunt displazii congenitale rare ale vaselor limfatice, care pot interesa tegumentul, mucoasele şi viscerele. Ele sunt superficiale, cavernoase sau chistice. În 80-90% din cazuri sunt diagnosticate înainte de vârsta de 2 ani.

Pacienţi şi metodă: Prezentăm 3 bolnavi cu limfangiom circumscris având ca obiectiv evidenţierea aspectelor clinico-evolutive ale acestei tumori, pe baza examenului clinic, investigaţiilor paraclinice, a examenului histopatologic şi ecografiei Doppler. Bolnavii au fost exploraţi şi pentru diagnosticarea comorbidităţilor. Tratamentul a fost chirurgical (excizia şi electrocauterizarea formaţiunilor) la cazurile I şi II şi medicamentos la cazul III. Rezultate: Pacienţii noştri nu au avut comorbidităţi asociate. Diagnosticul de certitudine a fost stabilit pe baza examenului histopatologic, iar tratamentul efectuat a condus la rezultate bune.

Am obţinut vindecarea cazurilor I şi II tratate chirurgical şi corectarea prin tratament medicamentos a complicaţiei hemoragice la cazul III. Cu toate acestea urmărirea periodică este necesară în cazul acestor pacienţi.

Concluzie: Limfangiomul circumscris este o afecţiune benignă, însă dificil de tratat şi recidivantă datorită componentei profunde hipodermice.

INTRODUCTION

Lymphangiomas are rare congenital dysplasia of lymphatic vessels, which may affect the skin, mucous membranes and viscera. About 80-90% of cases are diagnosed before the age of 2 years.

They are classified based on microscopic features in the following subtypes: capillary, cavernous and cystic hygroma. A fourth subtype is hemolymphangioma. The classification of lypmhangiomas has no clearly standard defined and universal application due to the clinicopathological nature of those lesions. The classification most commonly used divided lymphangiomas into two major groups based on depth and the size of these lymphatic vessels tumors: superficial group are called lymphangioma circumscriptum, the deeper group including cavernous lymphangioma and cystic hygroma. Some of the literature data classifies cystic hygroma as a variant of lymphangioma cavernosum.(1) Capillary lymphangiomas are composed of small size vessels and are located in the epidermis.

Cavernous lymphangiomas usually arise at birth or within the first 2 years of life, with an equal incidence by gender. Most common sites are the head and neck area, especially the oral cavity and rarely extremities. It appears as subcutaneous masses fluctuent diffuse delineated and consist in lymphatic dilated and irregularly shaped spaces and they can invade surrounding tissues. Frequently recur after a limited excision. Cystic hygroma are large, making the overall appearance of a “honeycomb” and they are developing within 75% of cases in the head or neck, most commonly on the left side of the body, but can also occur in the axilla, groins or other areas, equally affecting both genders. They tend to be better circumscribed

Figure 1.

The first case: two vegetable tumors (1.5cm and 2 cm), on the left thigh and internal side.

than cavernous lymphangiomas and also have the tendency for local recurrence if the excision is not wide enough.

Hemolymphangiomas have both types of components in their structure: lymphatic vessels and blood vessels also.

Another classification of lymphangiomas divides them into the following subtypes: microcystic, macrocystic and mixed type. Microcystic lymphangiomas measure less than 2-3 cm and the macrocystic lymphangiomas have more than 2-3 cm in diameter. Mixed lymphangiomas contain both components. (2, 3) M a rc h 2 0 1 6

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Case Presentation

Figure 2.

Clinical evolution of lymphangioma circumscriptum:observations on three cases.

Lymphangioma: network of spaces identifiable as lymphatic vessel, HE stain, x 40.

Figure 3.

Lymphangioma: lymphatic spaces containing a protein-rich liquid and a small number of lymphocyte, HE stain, x 100.

PATIENTS AND METHOD

We present 3 patients with lymphangioma circumscriptum, with the objective of highlighting aspects of this tumor clinical course, based on clinical examination, laboratory investigations, histopathological examination and Doppler ultrasound.

Patients were also investigated for possible comorbidities. In all three cases lesion biopsy was obtainned and surgical specimens were sent in order to achieve histopathological result.

The pieces were processed by classical histological technique and histopathological interpretation was performed usingHemaoxiline -Eosin standard staining. Two of the cases were treated by surgical excision and electrosurgery of the tumor, while the third case was treated with drugs.

RESULTS

The first case is a 22-years-old male, from urban area, requesting a dermatological consult for two vegetable tumors, having dimensions of 1.5 cm and 2 cm, partially keratotic and also with ulcerated areas, located left thigh and internal side.

The tumors are located on the edge of a surgical procedure scar at the age of 4 years for similar lesions and several translucent vesicles were located in the neighborhood (Fig.1).

From history we retain electrocautery at 7 years old of other injuries with the same location. The usual investigations were normal and Doppler ultrasound excluded other vascular malformations.

Histopathology revealed microscopic structure of lymphangioma, containing a network of spaces coated by endothelial cells, identifiable as lymphatic vessels only because they do not contain red blood cells (Fig.2).

These spaces contain a protein-rich liquid identifiable in HE staining as a material eosinophilic homogeneous. A small number of lymphocytes is present in the stroma (Fig 3). We performed exci-

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Figure 4.

The second case: tumor with irregular surface, with crusts and purulent deposit (2 5 / 3.5 cm), front of the right leg and a similar lesion (1 cm) in proximity.

sion of tumor lesions and electrocautery for the translucent vesicles.

The second case that we present is a 10-year-old boy, from rural area, underweight (BMI = 20), who was hospitalized in Dermatology Clinic for a tumor with irregular surface, covered by haematic crusts and purulent deposit, dimensions 2 5 / 3.5 cm, mobile on deep tissues, located on the front of the right leg, upper third and a similar lesion having 1 cm size located in proximity of the first (Fig 4). From history we retain the appearance of this tumor when he was 7 years old, after a local me-

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Pătraşcu Virgil, Bocîrnea Alexandra Georgiana, Ciurea Raluca

Figure 5.

Hemolymphangioma: lymphatic vessels and blood vessels, HE stain x100.

chanical trauma. Over time the tumor has grown relatively slowly in size, and later a new injury occurred near the first one. X-ray of right leg did not show bone alterations near the tumor.

We have established the diagnosis of hemolymphangioma based on clinical examination, laboratory and histopathological exams, which revealed lymphatic vessels and blood vessels (Fig 5).

We performed excision of the two tumors with the scalpel blade from the skin plane, then curettage and electrocautery of the bases. In Fig. 6 we present the clinical aspect one month after this treatment.

The third case is a 43-years old woman who requested dermatological consult, being concerned about some old skin lesions changing their appearance, after a recently mechanical trauma at this level. The patient reports this lesion since she was 5 years old.

Clinical examination revealed multiple vesicles, some with yellowish others with hemorrhagic content, located on the left iliac region, the proximal part of the left thigh and the buttock on the same side.

She also presents a painful induration about 4/3 cm below the left inguinal fold and one bruising about 5 / 2.5 cm wide on the left buttock (Fig.7).

Following clinical examination, histopathological exam, skin ultrasound, CT scanner and biological investigations we have established the diagnosis of lymphangioma circumscriptum.

Under general treatment with antibiotics, pain relievers, hepatoprotectors and local care of the injury the outcome of complications was favorable.

DISCUTION Lymphangiomas consist in isolated lymph cisterns of varying sizes located in the dermis and hypodermis, without connections with the normal lymphatic system. Depending on the size of those cisterns the lymphangiomas can have different

aspects: microcystic (lymphangioma simplex and lymphangioma circumscriptum) and macrocystic (cavernous lymphangioma and cystic hygroma). (3) Etiopathogeny A racial predominance in lymphangiomas was not reported. It is an approximately equal gender incidence.(4) In 1976, Whimster (5) studied the pathogenesis of lymphangioma circumscriptum. According to him, the basic pathological process is the formation of lymphatic cisterns deep into the subcutaneous tissue. These reservoirs are separated from the normal network of lymphatic vessels. However they communicate with superficial lymphatic vesicles and with vertical channels through the dilated lymphatics. Whimster found that those cisterns could arise from a primitive lymphatic vessel who fails to connect with the rest of the lymphatic system during its embryonic development. The rhythmic contractions of muscle fibers can cause a sequester of a primitive lymphatic vessel.

These contractions increase intramural pressure, forming channels that cross the walls of the dilated cisterns to skin. Whimster’s observations are supported by lymphangiographic and radiographic studies.

These studies have shown that large cisterns, multilobulated, are extended deep into the dermis and laterally beyond the clinically apparent lesions.(6) Cases of lymphangiomas secondary to trauma have been reported, likewise our second case or following an infection.

Clinical aspects Lymphangioma circumscriptum is the most common type of lymphangioma and it is characterized by translucent vesicles having dimensions around 2-4 mm, which can be present at birth or appear shortly after, having a tendency to expansion.

The lesions are asymptomatic, but occasionally patients may experience spontaneous episodes of minor bleeding or leakage of fluid from broken vesicles.

These vesicles represent dilated superficial lymphatic vessels involving the upper dermis and containing clear or pinkish - purple fluid (serosanguinolent or haemorrhagic content).

Lymphangioma circumscriptum has a high rate of recurrence after excision, because of its deep component. (3) Cavernous lymphangioma is less common and usually occurs in childhood. It is located at the cephalic extremity frequently and rarely in other areas. The lesions develop as nodules with elastic consistency, painless, in the dermis and subcutaneous tissue.

Unlike lymphangioma circumscriptum, the covering tegument is usually undamaged. These lesions often have a rapid growth phase similar to hemangiomas. The tumor can measure 1 cm or involve an entire anatomical area. There is no family history of the lymphangioma. Some patients experience pain when tumor makes compression on adjacent strucures.

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Figure 6.

Clinical evolution of lymphangioma circumscriptum:observations on three cases.

Clinical aspect one month after treatment.

Lymphangioma circumscriptum may be associated in some cases with cavernous lymphangioma and cystic hygroma.(3) Cystic hygroma occurs in childhood, it is large and located in the soft tissues, usually in the head and neck but can occur in axillaries or inguinal areas. It has elastic consistency, varies in size and shape, and tends to increase if it is not excised. Typical lesions are multilocular cysts filled with clear or serocitrin lymph fluid.

These congenital lesions are located deep into the connective tissue. When drained, they tend to fill up quickly with lymphatic fluid. Usually cystic hygroma is diagnosed clinically based on the large size, location and translucency (1). Cystic hygroma may be associated with congenital disorders as follows: Turner syndrome, Chromosome Aneuploidy, fetal hydrops, fetal alcohol syndrome, Noonan syndrome, Down syndrome and other disorders with trisomy(7). Patients with cystic hygroma should be cytogenetic investigated for chromosome aneuploidy. Parents will receive genetic counseling because chromosome changes may recur in future pregnancies. (8) Diffuse lymphangiomas are characterized by asymptomatic, erythematous, indurate or atrophic plaques without changes in their surface and sometimes can go unnoticed. If not completely excised the lesions may grow to large sizes.(2) Hemolymphangiomas develop from vascular and lymphatic congenital malformation. They occur mostly in younger patients, especially newborns and infants, and 6.5% of them are located on the extremities.

Diagnosis Lymphangioma diagnosis is mainly based on lesion history, clinical examination and histopathological exam. Microscopically the vesicles in lymphangio-

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Figure 7.

The third case: indurations (4/3 cm) below the left inguinal fold and one bruising (5/2.5 cm) on the left buttock.

ma circumscriptum are dilated lymphatic channels that develop into the papillary dermis. This may be associated with hyperkeratosis and acanthosis of the epidermis.

Channels are numerous in the superficial dermis and often extend into the deep dermis.

These deep vessels seem to have a wide caliber and thick wall containing smooth muscle fibers.

The lumen is filled with lymphatic fluid, but often contains red cells, lymphocytes, macrophages and neutrophils. These channels are lined by flattened endothelial cells. The gap has often numerous lymphatic cells and presents evidence of fibrosis.

The nodules in cavernous lymphangioma are characterized by large, irregular channels comprising a single layer of endothelial cells, being located in the reticular dermis and subcutaneous tissue.

The surrounding stroma consist in loose connective tissue or fibrous tissue and contains a big amount of inflammatory cells. Cystic hygroma is histologically indistinguishable from cavernous lymphangioma.(6) Imunohistochemical study is effective to differentiate lymphangiomas from hemangiomas in difficult cases.

Test results for VIII factor antigen reveal the fact that this is positive for hemangiomas endothelium and negative or weakly positive for lymphangiomaâ&#x20AC;&#x2122;s endothelium.

In case of normal blood vessels and hemlymphangiomas, imunohistochemistry for laminine indicates a typical multilayered basal membrane, and a discontinued basal membrane in lymphangiomas. Dermoscopy can be helpful in lymphangioma circumscriptum diagnostic. (9) Clear fluid lesions present brown lightning gaps, surrounded by palisading septae.

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Pătraşcu Virgil, Bocîrnea Alexandra Georgiana, Ciurea Raluca

Those with haemorrhagic areas can show focal reddish shades inside the gaps while the difused pink and/or red color is present in the purple gap structures.

Thus, they are characterised by a gap model with a blood content that may not be differentiated from a hemangioma. Monoclonal D2-40 antibody expression or Podoplanin and VEGFR-3 markers may establish the differential diagnostic with a hemangioma.(10,11) Invasion of the surrounding tissues and tumor relation with them is evidenced by MRI. This can help to prevent unnecessary extensive resection or incomplete resection.(12) Differential Diagnostic Usually, lymphangioma circumscriptum must be differentiated from skin metastases, lymphangiectasias, simplex herpes, zoster herpes(13), molluscum contagiosum.

Other cystic lesions and elastic subcutaneous masses must be considered. Other differential diagnostics regarding the clinical aspects are: melanoma, Dabska tumor, lipomas, type 1 neurofibromatosis, Stewart-Treves syndrome, angiokeratomas.

Treatment First choice treatment for lymphangiomas is complete surgical excision. (14) Based on Whimster hypothesis, the large subcutaneous cisterns must be removed to prevent tumor recurrence.

The complete excision of lymphangiomas may be difficult and sometimes unachievable and this is the main reason for the high rate of recurrence.

The tumors limited to the superficial dermis are surgically removed easily, being associated with a higher success rate.

Lymphangiomas are not responding to radiotherapy and corticosteroids. Although, Propanolol is a new treatment option, which can be helpful even for the diffuse lymphangiomas.(15) The antibiotic therapy is indicated only in secondary cellulitis.

Cryotherapy, sclerotherapy and electrocautery represent other possibilities of therapy.(16) Lymphangioma circumscriptum can also be treated by electroextraction.(17) It does not always respond to the pulsed laser, and the CO2 laser vaporization has been successfully tested(18), but also other types of lasers (argon, pulsed-dye) can be used.(19) Another therapeutical option for lymphangioma circumscriptum is

sclerotherapy with bleomycin solution or NaCl 23,4% hypertonic saline(20).

Intralesional OK432 (Picibanil) is a new and efficient treatment in macrocystic lesions (21), but in the cases of microcystic or cavernous lesions, the response to OK-432 was disappointing, surgery remaining the main option.(22) Postoperative vacuum assisted closure devices may decrease the recidive and infection risk. (23) Evolution Rarely, lymphangiomas interferes with the wellbeing of the patients. They usually present to the medical doctor for aesthetic issues. Lymphangioma circumscriptum is associated with minor bleedings, recurent cellulitis and lymphorrhea.

Two cases of lymphangiosarcoma complicating a lymphangioma circumscriptum were reported.

Lesions from both patients were treated for a long time with X-ray therapy.

Therefore, radiotherapy must be avoided in lymphangiomas therapy. Dabska tumor can complicate a lymphangioma circumscriptum.(24) In case of cervical cystic hygroma, a complete surgical excision is necessary to prevent complications like: breath alteration, aspiration and superinfections.(25) Lymphangiomas tends to recur, except the ones with complete surgical excision. In the first and the second cases the evolution was favorable after the surgical treatment obtaining healing, and in the third case we obtained the correction of bleeding complication by oral medication. Patients will be surveilled to capture possible recidives.

CONCLUSIONS Lymphangioma circumscriptum is a benign lesion, but difficult to treat and recurrent due to the deep hypodermic component.

During lifetime, lymphangiomas can lead to complications like spontaneous or posttraumatic hemorrhages. Regular examination of skin is necessary to evaluate the treatment response and to capture possible recurrence or complications.

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Clinical evolution of lymphangioma circumscriptum:observations on three cases.

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3. Rook’s Textbook of Dermatology Edited by Tony Burns, Stephen Breathnach, Neil Cox, Christopher Griffiths In four volumes, Volume 1 :4826-4828 4. Amir J, Metzker A, Krikler R, Reisner SH. Strawberry hemangioma in preterm infants. Pediatr Dermatol. 1986 Sep. 3(4):331-2.

5. Whimster IW. The pathology of lymphangioma circumscriptum. Br J Dermatol. 1976 May. 94(5):473-86.

6. Itakura E, Yamamoto H, Oda Y, Furue M, Tsuneyoshi M. VEGF-C and VEGFR-3 in a series of lymphangiomas: is superficial lymphangioma a true lymphangioma?. Virchows Arch. 2009 Mar. 454(3):317-25.

7. Pearce JM, Griffin D, Campbell S. Cystic hygromata in trisomy 18 and 21. Prenat Diagn. 1984 Sep-Oct. 4(5):371-5 8. Marchese C, Savin E, Dragone E, et al. Cystic hygroma: prenatal diagnosis and genetic counselling. Prenat Diagn. 1985 May-Jun. 5(3):221-7.

9. Amini S, Kim NH, Zell DS, Oliviero MC, Rabinovitz HS. Dermoscopic-histopathologic correlation of cutaneous lymphangioma circumscriptum. Arch Dermatol. 2008 Dec. 144(12):1671-2.

10. Arpaia N, Cassano N, Vena GA. Dermoscopic features of cutaneous lymphangioma circumscriptum. Dermatol Surg. 2006 Jun. 32(6):852-4.

11. Massa AF, Menezes N, Baptista A, Moreira AI, Ferreira EO. Cutaneous Lymphangioma circumscriptum - dermoscopic features. An Bras Dermatol. 2015 Mar-Apr. 90(2):262-4.

12. Park SH, Moon SK, Sung JY. Sonographic findings in a case of scrotal lymphangioma. J Clin Ultrasound. 2014 May. 42(4):234-6.

13. Patel GA, Siperstein RD, Ragi G, Schwartz RA. Zosteriform lymphangioma circumscriptum.

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15. Ozeki M, Fukao T, Kondo N. Propranolol for intractable diffuse lymphangiomatosis. N Engl J Med. 2011 Apr 7. 364(14):1380-2.

16. Mirza B, Ijaz L, Saleem M, Sheikh A. Different Modalities Used to Treat Concurrent Lymphangioma of Chest wall and Scrotum. J Cutan Aesthet Surg. 2010 Sep. 3(3):189-90.

Emer J, Gropper J, Gallitano S, Levitt J. A case of lymphangioma circumscriptum successfully treated with electrodessication following failure of pulsed dye laser. Dermatol Online J. 2013 Mar 15. 19(3):2 17. Haas AF, Narurkar VA. Recalcitrant breast lymphangioma circumscriptum treated by UltraPulse carbon dioxide laser. Dermatol Surg. 1998 Aug. 24(8):893-5.

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Clinical studies

Emollients – a cornerstone in the treatment of Epidermolysis bullosa

EMOLLIENTS – A CORNERSTONE IN THE TREATMENT OF EPIDERMOLYSIS BULLOSA EMOLIENTELE – ELEMENTE DE BAZĂ ÎN TRATAMENTUL EPIDERMOLIZEI BULOASE Ioana Mitoșeriu-Bonteanu[1], Alexandra Butacu[1], Radu-Nicolae Grigore[1], Cristina Cucu[1], Mihaela Zaiț[1], George-Sorin Ţiplica[1,2], Carmen-Maria Sălăvăstru[2,3] 1 Clinica Dermatologie 2, Spitalul Clinic Colentina, București, România 2 Universitatea de Medicina si Farmacie”Carol Davila”, București, România 3 Clinica Dermatologie Copii, Spitalul Clinic Colentina, București, România Corresponding author: George-Sorin Ţiplica Clinica Dermatologie 2, Spitalul Clinic Colentina Şos. Ştefan cel Mare nr. 19-21, sector 2, Bucureşti cercetare1@spitalulcolentina.ro

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: epidermolysis bullosa, emollients, xerosis.

Cite this article: Ioana Mitoșeriu-Bonteanu, Alexandra Butacu, RaduNicolae Grigore, Cristina Cucu, Mihaela Zaiț, GeorgeSorin Ţiplica, Carmen-Maria Sălăvăstru.Emollients – a cornerstone in the treatment of Epidermolysis bullosa.

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Introduction: Xerosis is a common finding in Epidermolysis bullosa (EB) patients, and it can lead to the formation of new blisters and erosions. The objective of our study was to asses the importance of emollient use in patients with EB. Methods: From a total of 23 patients with EB registered in our Clinic, 18 agreed to participate in a survey regarding the treatments used for their disease, the use of emollients and how these improved their quality of life.

Results: Emollients were used by all the patients. Most of the subjects applied emollients two or more times daily with the following results: 77.8% noticed an improvement in erythema; 88.9% an improvement of xerosis and 44.4% an improvement in pruritus after use of emollients. An accelerated healing process after treatment with emollients was noticed by 64.7% of subjects.

Discussions: Emollients play an important role in alleviating the symptoms of EB patients, preventing the formation of new skin lesions. Emollients should be used on a daily basis. Their cost is often prohibitive for the EB patient and such a patient must use large amounts of them.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Ioana Mitoșeriu-Bonteanu, Alexandra Butacu, Radu-Nicolae Grigore, Cristina Cucu, Mihaela Zaiț, George-Sorin Ţiplica, Carmen-Maria Sălăvăstru

Rezumat Cuvinte-cheie: epidermoliză buloasă, emoliente, xeroză

Introducere: Xeroza cutanată este prezentă în mod obișnuit la pacienții cu epidermoliză buloasă (EB), iar aceasta poate duce la formarea de noi bule sau eroziuni. Obiectivul studiului nostru a fost să evaluăm importanța folosirii emolientelor la pacienții cu EB. Metode: Din totalul de 23 de pacienți cu EB aflați în evidența Clinicii noaastre, 18 au fost de acord să participe la un chestionar privind tratamentul folosit pentru afectiunea lor, utilizarea de emoliente şi modul cum acestea le îmbunătațesc calitatea vieții.

Rezultate: Emolientele au fost folosite de toți pacienții din studiu.

Majoritatea subiecților le aplică de 2 sau mai multe ori pe zi. 77,8% din pacienți au observat o ameliorare a eritemului după aplicarea de emolient, 88,9% au avut o imbunătățire a xerozei și 44,4% o ameliorare a pruritului. 64,7% au observat o accelerare a procesului de vindecare a leziunilor după utilizarea de emoliente.

Discutii: Emolientele joacă un rol important în ameliorarea simptomelor la pacienții cu EB, previn formarea de leziuni cutanate noi si ar trebui folosite zilnic. Costul lor este din păcate de multe ori prohibitiv, iar pacienții cu EB trebuie să le folosească în cantități mari.

Introduction The term epidermolysis bullosa encompasses a group of clinically distinctive genodermatoses that have in common three major features: genetic transmission, mechanical fragility of the skin, and blister formation following seemingly minor or insignificant trauma or traction to the skin.[1] Mutations within genes that encode structural proteins at different levels in the skin determine the major clinical forms of the disease, depending on the location where the blisters arise.

There are four major forms of inherited EB: EB simplex (EBS – with blisters within the epidermis), junctional EB (JEB – with blisters at the dermo-epidermal junction), dystrophic EB (DEB – with blisters formed in the uppermost papillary dermis) and Kindler syndrome (photosensibility is present).

[2] There are at least 30 distinctive clinical phenotypes.[3] Every aspect of the EB patient’s life is affected. Patients with EB may experience moderate to severe pain, caused simply by basic activities like standing, walking or even sitting.

The disease is preventing patients from participating in many activities or limiting them at school or work. Even when it comes to everyday clothing and shoes, a special attention must be paid because they have to be very soft, have no pressure points and should not be too tightly fitted. [3] Only a day-to-day special management of EB which consists in the prevention of mechanical trauma and infection is actual possible.[2] This daily routine takes a long time, being a constant stress factor for the patient and the family. A patient with EB needs special care throughout all his life. [3] Of tremendous importance is the access to home care and specialized caring teams. [4] Currently there are no curative therapies for any form of inherited EB, but in the future, an effective gene therapy may be possible for at least some forms of EB.[5] In terms of incidence, data shows the occurrence of 50 new

epidermolysis bullosa cases per one million live births. There are 72 families with different types of epidermolysis bullosa in Romania. Since 2012 a National Program for the treatment of these patients is run by the National House of Health Insurance[4] covering bandages and drugs (antibiotic and corticosteroids). Xerosis is an important factor in EB and can lead to the formation of new blisters and erosions but emollients are not included in the National Program being registered and marketed as cosmetics. It is worth mentioning that emollients are used on an everyday basis, while the antibiotic and corticosteroid ointments usage is less frequent. Despite the fact that the application of moisturizers increases hydration of the skin, as measured subjectively by patients and objectively by assessment of conductance and microscopy [68] , a survey of the literature indicates that there is little primary evidence as to how emollients should be effectively used. [9] Only a few randomized controlled trials have assessed the efficacy of emollients and none of these trials have examined xerosis as the main endpoint. [5]

Methods and subjects In order to see if the daily use of emollients in patients with EB is beneficial we started a pilot-study in the 2nd Department of Dermatology, Colentina Clinical Hospital – Bucharest.

In January 2016 we monitored the use and results of emollient applications in improving clinical appearance and increasing the quality of life in EB patients. A telephone-administered questionnaire was conducted to discuss some of these aspects with the patients. There were 20 questions regarding the use of emollients in EB scheme of treatment, most frequently used creams in this pathology, ways of applying it, quantities used, results after treatment and financial aspects. In our clinic there are registered 23 patients with EB in the NaM a rc h 2 0 1 6

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Clinical studies

Emollients – a cornerstone in the treatment of Epidermolysis bullosa

Table1 Group structure Sex

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Age

Females

Males

Children (<18 years)

Adults (>18 years)

Number

8

10

12

6

Percentage

44.44%

55,56%

66,67%

33,33%

tional Program for Rare Diseases PN 6.2. Only 18 patients (both adults and children) accepted to participate in this survey. The group structure is presented in Table 1. All data were entered into a relational database (Microsoft Excel 2010). Quality checks were made for all data.

Discussions

Results The first set of questions was designed to study the use of emollients as part of treatment in patients with EB. All 18 patients (100%) answered that they use emollients in their treatment plan.

The next set of questions was about the way of applying the emollients. Out of 18 patients, 7 use emollients more than 2 applications per day, 6 patients use 2 applications per day and only 5 use one application per day. Most patients (83,8%) apply these products in a thin layer, only 16,7% use a thick layer application. Only 1 patient uses index phalanx measurement unit, most of them (94,4%) do not use a measurement unit. 38,9% of patients apply emollient on a body surface equivalent of 1-5 palms. An equal percent applies it on a greater surface of 5-10 palms. Regarding the quantity used per week, 38.9% of the patients use less than 250g/week, 33.3% patients use more 250g/week and 27.8% patients use approximately 250g/week.

Out of 18 patients, 8 use emollients in combination with both antibiotics and topical corticosteroids, 4 patients use with topical antibiotics and 3 patients only with topical corticosteroids (Fig. 1).

When it comes to the effects of emollient use, 77.8% of the patients noticed an improvement in erythema, 44.4% an improvement in pruritus, 88.9% an improvement in xerosis and 77.8% stated an improvement in scaling after the treatment (Fig. 2). Almost a quarter of the patients (22,2%) observed a delay in bullae and vesicles apparition after treatment with emollients. Also, 64.7% stated noticing an accelerated healing process after treatment with emollients (Fig. 3). Discussing about the products they have tried in their daily routine, the majority of patients (83.3%) found the emollients’ texture as appealing and 77.8% said they had no problem with the smell, because the products are usually fragrance-free. Another important percent of the patients (83,3%) stated that emollients are easily absorbed into the skin, leaving a smooth texture of the tegument. Most of the patients, 15 out of 18, read the ingredient list of products before applying it.

An important problem for the patients when it comes to emollients’ use is the financial aspect.

Only 1 patient found the emollients’ prices accessible. Most patients (61.1%) spend more than

A complete emollient therapy is a very important step in the treatment of all patients suffering from diseases that involve a dry skin, which consists of: emollient creams or ointments, emollient wash products and emollient bath and shower products.

[10-11] The patients suffering from EB have xerosis in various degrees and they are using emollients in their daily treatment routine. The emollients have different modes of action, depending on the constituents of the product. Emollients draw moisture into the stratum corneum. They contain humectants (such as propylene glycol, lactic acid, urea and glycerol - molecules with a low molecular weight and water-attracting properties) which penetrate the epidermis and draw water in from the dermis. [9] Some of the ointment and lotion emollients contain a mixture of occlusive and humectant substances, so they work in the both ways: the humectant draws water into the epidermis while the occlusive element ensures that it is trapped there. [9, 12] Also, while penetrating the stratum corneum, humectants found in emollients, such as urea and glycerine imitate the natural moisturizers.[20, 21] It has been shown recently that emollients accelerate regeneration of skin barrier function following disruption[6], especially the lipid-rich emollients.[22] This was also stated by our patients, that have noticed an accelerated healing process of the erosions following bullae by applying emollients. Also, almost a third of them have also observed a smaller incidence of bullae and erosions when the skin is well hydrated. It has been also proved in various studies that the emollients have anti-inflammatory, antimitotic, antipruritic (by increasing the amount of water in the stratum corneum and reducing the xerosis), exfoliative (especially when combined with products such as salicylic acid) and steroid-sparing effects.[9, 13, 14] Also, the additional excipients used in the emollients provide excellent effects: preservatives (such as chlorocresol and parabens) that reduce the microbial contamination and improve product stability, antiseptics (such as benzalkonium chloride and triclosan) that prevent infection, antipruritics (such as lauromacrogols that inhibits the transmission of itch sensations through unmyelinated C fibres).[15] Anti-pruritic formulas are available in both leaveon emollients and bath emollients.[9] Our patients confirmed that by using emollients, the erythema,

100 RON/month for this kind of treatment, 22.2% spend between 50 and 100 RON / month and only 3 patients spend less than 100 RON / month (Fig. 4). (1EUR – 4,5 RON).

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Ioana Mitoșeriu-Bonteanu, Alexandra Butacu, Radu-Nicolae Grigore, Cristina Cucu, Mihaela Zaiț, George-Sorin Ţiplica, Carmen-Maria Sălăvăstru

Figure 1.

Figure 2.

Figure 3.

Figure 4.

pruritus and scales were significantly diminished. It is often difficult for the dermatologist to prescribe an emollient in generic terms, because there are so many products that broadly contain the same ingredients, but to which patients will respond in very different ways.[9] Also the patients remember the brand names of the emollients to which they responded the best and to which they partially responded or not at all, so it is very important to discuss with the patient before choosing an emollient for a long-term use. The complex ways in which emollients affect the stratum corneum structure and barrier function are only now just beginning to be understood.[16] This underlines the need for an individualized treatment with emollients. Due to the various ways of applying the emollients, a large number of formulations from different brands are available on the market e.g.: wash products (bath additives, soap substitutes, skin cleansers etc.) or skin-care preparations (creams, ointments, gels, mousses, lotions etc.). This only shows that there is no particular product for all individuals, even though we received from our patients a positive feedback regarding a certain product or a brand.

Fragrances added as aditives are known sensitisers, almost 1% of the general population being allergic to them, and this percent grows up to 14% in those with a sensitive skin.[23] Patients with EB should look for emollients with a special, true fragrance-free formula, because many of them con-

tain masking fragrances.[9] Our patients claimed that all the products they use have a pleasant smell, mentioning that it is almost fragrance-free, well tolerated. The majority also said that they read the label before trying a new product. The recommendation would be to apply a small amount of product on a “test area” where it would be left for 24 or 48 hours, especially for a patient who cannot find an emollient that suits him/her.[9] It is recommended for people with a xerotic condition of the skin to use both wash products and skin-care formulations.[13, 18] The bath additives (or bath oils) contain liquid paraffin or soya oil and are added in the water in quantities indicated by the manufacturer.[9] They are all non-foaming and many of them fragrance-free. Even though bathing with water can hydrate the skin and remove the scales, crusts and irritants[29], sometimes the water itself can have an accentuated drying effect and so, the bath oils protect the skin by leaving a layer of oil on the skin after bathing. Also, some of them have even anti-pruritic properties (these contain lauromacrogols) or antiseptic properties (those who contain benzalkonium chloride, chlorhexidine hydrochloride or triclosan). It is well known that soaps, more specifically the surfactants found in soaps, produce a dry skin and irritation.[32,33] Also, most soaps have an alkaline pH, whereas the skin’s normal pH is between 4 - 5.5. For a better tolerance, non-soap-based surfactants and synthetic M a rc h 2 0 1 6

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Clinical studies detergents (syndets) are recommended. This indication is based on only a few supportive clinical studies [34,35] but the patients’ experience confirms the positive effect of syndets or hypoallergenic and fragrance free non-soap cleansers that have a pH neutral to low. Beside the use of special bath products, it is recommended for people who have a xerotic condition of the skin to avoid the following: excessively hot water (which increases the water loss through evaporation), vigorous rubbing with a towel (which disrupt barrier function and may produce irritation and trauma), baths longer than 15 minutes (which can also disrupt the barrier function).[9] Immediately after bath, the application of moisturizers is necessary to maintain a good hydration status.[30, 31] In every chronic skin condition that develops xerosis, emollients should be taken into consideration as an adjuvant treatment, alongside other topical or systemic therapies regardless of what those treatments are.[14, 24, 25] Our patients stated that they frequently associate them with antibiotics or topical steroids or even both. Studies have shown that emollients have significantly reduced the amount of high-potency topical steroids in chronic dermatoses [9, 13, 14, 26], leading to fewer side-effects connected to this therapy. There is a lack of systematic studies to define an optimal amount or frequency of application of emollients in various skin diseases [28].

Conclusions Even though the emollients are a very effective adjuvant therapy in the daily routine of patients

Emollients – a cornerstone in the treatment of Epidermolysis bullosa

suffering from EB, with results recognized by both patients and doctors, they are still not included in the list of medications and medical devices that the patients with inherited EB receive monthly, through the National Program for Rare Diseases PN 6.2. In Romania, the emollients still remain an expensive therapy. Patients with EB usually have a large surface of skin affected and they require large amounts of emollients, which they can’t afford, thus limiting their beneficial effects. Even if, unlike many other topical preparations, emollients have a few insignificant side-effects, are usually easy to use and often significantly improve symptoms, as our patients confirmed, they don’t use them regularly because they are too expensive.

That the lack of systematic information about the mechanism of action and the proper use of emollients leads to an inadequate usage by the patients. In the future, developing a National Program for Teledermatology would improve the access to precise information for both doctors and patients, educating the last about the proper treatment plan. Because many of the activities of a patient suffering from EB are limited and the daily routine takes a lot of time and attention, a nursing program for home-care would greatly improve not only the life of the patient, but also of its family.

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3. Diem A, Fine JD., Hintner H. Living with EB - Impact on Daily Life in Life with Epidermolysis Bullosa (EB) Etiology, Diagnosis, Multidisciplinary Care and Therapy. Springer Wien NewYork; 2009:313334 4. Salavastru CM, Sprecher E, Panduru M, Bauer J, Solovan CS, Patrascu V, Morariu HS, Tudorache A, Lotti T, Tagliente I, Ciasulli A, Marchili MR, Sabatino G, Burciu E, Cosgarea R, Fritz K, Tiplica GS. Recommended Strategies for Epidermolysis Bullosa Management in Romania. MAEDICA – a Journal of Clinical Medicine. 2013; 8(2): 200-205.

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6. Breternitz M, Kowatzki D, Langenauer M, Elsner P, Fluhr JW. Placebo-controlled, double-blind, randomized, prospective study of a glycerol-based emollient on eczematous skin in atopic dermatitis: biophysical and clinical evaluation. Skin Pharmacol Physiol 2008;21:39-45.

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11. Voegeli D (2007). The role of emollients in the care of patients with dry skin. Nursing Standard.

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15.LodenM(2012).Effectof moisturizersonepidermalbarrierfunction.ClinDermatol30(3):286-296.

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18. Downing D, Stewart M (2000). Epidermal composition. In: Lodén M and Maibach HI (Eds). Dry Skin and Moisturizers – Chemistry and Function. CRC Press, Boca Raton.

19. Grieve K (2012) Glycerine: the naturally effective humectant. Dermatol Nurs 11(1): 30-34.

20. Locke J, Baird S, Hendry G (2012). The use of urea-based creams in the prevention of diabetic ulceration. Dermatol Nurs 11(2): 26-32.

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22. de Groot A (2000). Sensitizing substances. In: Lodén M and Maibach HI (Eds). Dry Skin and Moisturizers – Chemistry and Function. CRC Press, Boca Raton.

23. Van Onselen J (2001) Psoriasis. Dermatology Nursing — A practical guide. Churchill Livingstone, Edinburgh.

24. Boguniewicz M, Nicol N (2002). Conventional Therapy for Atopic Dermatitis. Immunology and Allergy Clinics of North America,Atopic Dermatitis.WB Saunders, Philadelphia.

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26.McAuley WJ,Kravitz L (2012).Pharmacokinetics of topical products.Dermatol Nurs 11(2): 40-44.

27. Hon KL, Ching GK, Leung TF, Choi CY, Lee KK, Ng PC. Estimating emollient usage in patients with eczema. Clin Exp Dermatol 2010;35:22-6.

28. Gutman AB, Kligman AM, Sciacca J,James WD. Soak and smear: a standard technique revisited.

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30.Simpson E,Trookman NS,Rizer RL,Preston N,Colon LE,Johnson LA,et al.Safety and tolerability of a body wash and moisturizer when applied to infants and toddlers with a history of atopic dermatitis: results from an open-label study. Pediatr Dermatol 2012;29:590-7.

31. Ananthapadmanabhan KP, Moore DJ, Subramanyan K, Misra M, Meyer F. Cleansing without compromise: the impact of cleansers on the skin barrier and the technology of mild cleansing.

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32.White MI, Jenkinson DM, Lloyd DH. The effect of washing on the thickness of the stratum corneum in normal and atopic individuals. Br J Dermatol 1987;116:525-30.

33. Solodkin G, Chaudhari U, Subramanyan K, Johnson AW, Yan X, Gottlieb A. Benefits of mild cleansing: synthetic surfactant based (syndet) bars for patients with atopic dermatitis. Cutis 2006;77:317-24.

34. Cheong WK. Gentle cleansing and moisturizing for patients with atopic dermatitis and sensitive skin.Am J Clin Dermatol 2009; 10(Suppl):13-7.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Ioana Mitoșeriu-Bonteanu, Alexandra Butacu, Radu-Nicolae Grigore, Cristina Cucu, Mihaela Zaiț, George-Sorin Ţiplica, Carmen-Maria Sălăvăstru

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Psychodermatology

Epidermolysis bullosa - psychodermatological aspects

EPIDERMOLYSIS BULLOSA PSYCHODERMATOLOGICAL ASPECTS EPIDERMOLIZA BULOASĂ - ASPECTE PSIHODERMATOLOGICE Lucia Tomas-Aragones¹, Cristiana Voicu² Department of Psychology, University of Zaragoza, Spain Dermatology Department, POLISANO Clinic, Bucharest, Romania Corresponding author: Lucia Tomas-Aragones Paseo Independencia 28, 4-3 50004 Zaragoza, Spain Phone: +34 606 973 090 Fax: +34 978 871 146 luciatomas@cop.es 1

2

Conflict of interest: The authors do not have any conflict of interest to declare.

Open Access Article

Abstract Keywords: Epidermolysis Bullosa, QoL measurements, psychological interventions, patient QoL, caregiver burden.

Epidermolysis bullosa is an inherited disease characterized by severe skin fragility and blister formation in response to minor trauma. Apart from the main features mentioned above, patients suffer from chronic pain, pruritus, skin infections, but also from more severe systemic symptoms and even mutilations. All these aspects can potentially influence the psychological well being of both patients and family members.

This review article summarizes the information published in the literature regarding quality of life (QoL), generic and specific questionnaires and also psychological interventions available for epidermolysis bullosa patients and caregivers.

Rezumat Cuvinte-cheie:

Cite this article: Lucia Tomas-Aragones, Cristiana Voicu.

Epidermolysis bullosa - psychodermatological aspects. RoJCED 2016; 3(1):74 - 78

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Calitatea vieții în epidermoliza buloasă, evaluarea calității vieții, intervenții psihologice, calitatea vieții pacienților, impactul asupra familiei.

Epidermoliza buloasă este o afecțiune genetică caracterizată prin fragilitate cutanată marcată și formarea consecutivă de leziuni buloase ca răspuns la traumatisme minore. În afara trăsăturilor principale menționate anterior, pacienții sunt afectați de durere, prurit, infecții cutanate dar și de manifestări sistemice și chiar mutilări. Toate aceste aspecte au abilitatea de a influența starea psihică a pacienților dar și a familiilor acestora.

Articolul de față rezumă informațiile existente în literatura de specialitate cu privire la calitatea vieții (QoL), chestionare generice sau specifice dar și intervențiile psihologice disponibile în cazul pacienților suferind de epidermoliză buloasă și a familiilor acestora.

R O M A N I A N J O U R N A L o f C L I N I CA L a n d E X P E R I M E N TA L D E R M ATO LO GY

Lucia Tomas-Aragones, Cristiana Voicu

Introduction: Inherited epidermolysis bullosa is a genodermatosis characterized by skin and mucosa fragility with subsequent formation of blisters in response to minor trauma. Because of the skin fragility, children affected by the disease are also referred to as “butterfly children”. Epidermolysis bullosa subtypes vary depending on the distribution of the lesions, the level of blister formation, the underlying molecular defect, the internal organ involvement and the wound healing response (1).

Therefore, the spectrum of epidermolysis bullosa varies from minimal - or no internal organ involvement and a normal lifespan - to multiorgan disorders, severe mutilation, profound impact on the quality of life of both patients and caregivers, as well as a shorter expectancy of life (1).

Quality of life (QoL) is a complex concept designed to reflect the negative impact of an individual’s disease on a physical, psychological and social level. However, the burden of the disease on the individual is often extended to family members and caregivers, therefore a holistic approach in QoL assessment should also include the persons in close contact or who take care of the patient (2).

Quality of life measurements: The assessment of QoL in epidermolysis bullosa has major benefits because the information provided by these measurements can be used in clinical setting, treatment management and also research (3).

A research conducted in Scotland in 2002 assessed QoL using the Dermatology Life Quality Index (DLQI) and the Children’s Dermatology Life Quality Index (CDLQI). The conclusion was that the effect of epidermolysis bullosa on the QoL was similar to that of atopic dermatitis and psoriasis (4).

An observational, cross-sectional postal survey study conducted by Tabolli et al. using different questionnaires (Short Form-36 SH-36, Skindex-29, General Health Questionnaire-12 GHQ-12, EuroQo15, Patient Global Assessment five-point scale and the Family Strain Questionnaire) aimed to asses the burden of different types of epidermolysis bullosa on a group of Italian patients and their families. The results highlighted the fact that the mental components were not significantly impaired, but the physical components of the SF-36 showed lower scores. Regarding the impact of different epidermolysis bullosa types, the researchers concluded that patients with severe generalized dystrophic and junctional forms reported lower values in the GHQ-12 scores than patients with epidermolysis bullosa simplex. No significant differences were observed regarding EB subtypes for Skindex-29, but women proved to be more affected than males in all Skindex-29 and SF-36 scales, suggesting a higher risk for anxiety and depression. Also, the QoL of both patients and caregivers decreased with increasing patient body surface involvement. The analysis of the differences in mo-

bility and self-care in children versus adult patients revealed that anxiety and depression regarding the aspects mentioned above were higher among adults(5).

The first epidermolysis bullosa specific QoL questionnaire, QOLEB (Quality of Life Evaluation in Epidermolysis Bullosa), an English 17 item measurement tool, was developed in 2009. The QOLEB gives different QoL scores for different epidermolysis bullosa subtypes and was statistically proven to be valid and reliable in assessing the impact of epidermolysis bullosa on patients’ lives (6). It allows the stratification of QoL impairment into very mild, mild, moderate, severe and very severe, as well as monitoring the changes in QoL during the administration of new treatments in clinical trials (7).

Recently, the QOLEB was translated into Brazilian Portuguese (8) and Dutch. The latter translation was used together with other measurement tools to assess the impact of Epidermolysis bullosa on QoL in the Dutch population. The authors concluded that the results obtained in their study are similar to those of Tabolli et al. described above (9).

Many quality of life measurement tools, both quantitative and qualitative, have been used in the attempt to assess the impact of epidermolysis bullosa on the patients, as well as on their families.

The quantitative tools prove to be the most statistically accurate and valid, but generic dermatology tools have been used with success in some circumstances (3).

Patients’ quality of life: Patients’ lives are affected mainly by the clinical manifestations and the disease severity (10). Studies have shown that these patients also suffer from different psychological and psychiatric problems (11).

Adolescents have a need to be accepted and liked. According to Eric Erikson, the quest for identity is the principal developmental task confronting every individual throughout the lifespan. Body image affects our emotions, thoughts, and behaviours in everyday life. But, above all, body image influences our relationships. Furthermore, body image has the potential to dramatically influence our quality of life.

Being visibly different, as is the case in many children and adults with EB, can be the cause of dissatisfaction with appearance, psychological difficulties and social problems. The difficulties with physical appearance experienced by youngsters are also a cause of concern for the parents. Williams et al. (12) relate the experiences of young people with EB simplex. Even with a mild form of EB, the children interviewed felt visibly different to others and sometimes excluded by their peers. The subjects in this study also experienced others’ fear of contagion.

In another qualitative study, this time with adults, Dures et al. (13) look into the impact of the disease on the beholder. A continuum can be drawn with patients who found their skin condition to affect all

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Psychodermatology areas of their lives on one end, with those who had learned to live with it on the other end. Some of the participants interviewed expressed their desire to have more personalized therapy, a one-to-one support to help them cope with their emotional distress.

Recently, a group of researchers aimed to determine the relationship between hand function in epidermolysis bullosa (EB) and quality of life, conducting a cross-sectional study that included children suffering from EB. After measuring parent and patient-reported hand function with the ABILHAND-Kids questionnaire, they concluded that hand impairment has a negative effect on the quality of life, probably because it interferes with daily activities (14). The influence of pain due to skin, joint and bone lesions on the quality of life in young patients suffering from epidermolysis bullosa has been assessed during a study conducted between 2011-2014 in Southampton. The research highlighted the prevalence of pain in patients with EB and its negative effect on their own and their families’ lives. Patients’ QoL is impaired because of frequent pain but also due to fright of pain. The authors concluded that on-line materials about psychological interventions for pain management are necessary in order to help patients and caregivers with everyday challenges, which will contribute to improving QoL (15). A 3-year project, 2015-2018, is currently being conducted in Dublin. The epidemiological and pathophysiological mechanisms of pruritus in patients with epidermolysis Bullosa are being studied. Similar to pain, itch has a profound impact on the quality of life of both patients and their families, for which, up until now there is no effective therapy. The study aims to assess the impact of itch on the quality of life, as well as the pathophysiological mechanisms that lead to therapy-resistance itch in EB, in order to identify new treatment options for this troublesome symptom (16) . Itch can be a source of social embarrassment due to the presence excoriated skin and social scratching (17).

Families’ quality of life: Taking care of a chronically ill person poses a great deal of stress on family members, phenomenon referred to as the caregiver burden.

The family burden in EB children is a heavy one.

Caregiver burden can develop in many ways and affect parents both as individuals and as a couple.

It can affect areas such as a couple’s marital relationship, their sense of ‘private life’, conversation, the ability to stay close (physically and emotionally), their sex life and it can influence the decision to have more children. Their children’s disease can be a major cause for divorce in some cases (18). According to a Polish Study (19), parents of children with EB seem to have a greater need of social support and a lower level of life satisfaction. Tabolli et al. used the Family Strain Questionnaire (FSQ), the Family Dermatology Life Quality Index (FDLQI) and the General Health Questionnaire (GHQ-12)

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to assess the burden of epidermolysis bullosa on patient’s caregivers. The study concludes that the family burden is associated with the clinical severity of the disease and with the body surface involved, similar to that observed in neurological or oncological diseases. Furthermore, the impact of the burden may vary depending on the EB subtype. Parents of children with junctional EB and recessive dystrophic EB reported a more profound impact on their lives than those of children with EB simplex and dominant dystrophic EB (18). In order to better asses the parental burden of epidermolysis bullosa regarding medical and socio-economic aspects, a specific 20 item questionnaire has been recently developed: the Epidermolysis Bullosa Burden of Disease (EB-BoD) (20).

Psychological interventions: It is important for health professionals to help patients define their problem and not to make assumptions about the nature of their problem and the way that it affects them. In doing this, there is a danger of over-emphasizing certain issues while ignoring others that may be more pertinent to the patient. In order to avoid making assumptions, the healthcare giver should allow the patient to explain what the issues are that are worrying him/her, and not assume that patients will be able to “open up” and discuss their feelings immediately; be patient and let them “set the pace”. The patient and his/her problem needs to be approached in such a way that takes into account the complete procedure of their “illness experience” (21).

Programs providing patients with detailed information about their skin disease, including etiology, therapeutic options, and prognosis, can be helpful in enhancing compliance with treatment regimen.

Additionally, psychoeducation directed at educating the patient with regard to common emotional reactions to their skin disease can be helpful in reducing the patient’s sense of isolation (22).

Stress can cause exacerbation of disease and this in turn can cause more stress, leading to a vicious cycle. Relaxation can be effective in reducing arousal levels and hence feelings of anxiety and stress. Relaxation training can be accomplished by numerous techniques, all mainly directed at minimizing sympathetic reactivity and enhancing parasympathetic function.

Included in this group of procedures are progressive muscle relaxation, autogenic training, guided imagery, as well as transcendental and other meditation techniques, such as mindfulness.

Breathing exercises and self-talk have also shown to be helpful (22).

Psychological intervention should initially be targeted towards patients’ symptoms and areas of burden and distress, as for example: pain, itch, feelings of isolation, body image issues, emotional problems, and social difficulties.

Some authors (17) suggest that cognitive behavioural therapy (CBT) and hypnosis may prove helpful in controlling pain symptoms. Both are evi-

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Psychodermatology dence-based interventions and have proven to be effective in other pain related conditions. For the control of itch: hypnosis, biofeedback, meditation, eye movement desensitization and processing (EMDR), and CBT. Based on their findings, Williams et al. (12) suggest that psychological interventions focused on the patient may place the causes of their distress on them, while wider communityfocused educational programs could be offered in order to address misinformation and negative attitudes and behaviors in their communities, for example, in their schools. Support groups may also prove helpful as subjects with EB can share experiences and feelings about their condition and thereby reduce their sense of loneliness. Parents can also benefit from support groups as many feel they lack information about their children’s condition. In a group setting, patients are encouraged to understand their own and one another’s difficulties. There are many different modalities of group therapy, including family therapy that can help families develop more functional patterns of organizing and interacting with each other (23).

Epidermolysis bullosa - psychodermatological aspects

on their own with their disease, others are profoundly affected on a psychological level, leading to anxiety and depression. In addition, their families and caregivers are also subject of distress (1).

Quality of life evaluation is an important topic in epidermolysis bullosa since it can improve treatment management, as well as to assess new therapeutic modalities in order to help patients and their families to cope better with the disease (3).

A multidisciplinary approach is therefore mandatory, involving dermatologists, psychologists and psychiatrists, working together to support and provide both patients and caregivers with a holistic attention, identifying any sign of emotional distress or mental disorder and addressing it in the best possible manner. In addition, support groups, psychotherapy sessions and organizations such as Dystrophic Epidermolysis Bullosa Research Association (DEBRA) and Epidermolysis Bullosa Medical Research Foundation can help to provide patients and their families with information, psychoeducation and support (1).

Conclusions: Patients with severe forms of epidermolysis bullosa suffer from chronic pain, mutilations, residual scarring and, sometimes, underlying systemic involvement. While some of them, despite severe manifestations, seem to find the resources to cope

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2. S. Tabolli, P. Giannantoni, D. Abeni. Family burden of Epidermolysis Bullosa assessed using a generic and a specialty-specific questionnaire. Clinical dermatology ,2014 April-June; 2(2): 67–72.

3. John W. Frew, Dédée F. Murrell. Quality of Life Measurements in Epidermolysis Bullosa: Tools for Clinical Research and Patient Care. DERMATOLOGIC CLINICS, 2010, Vol 28, Issue 1, 185-190.

4. H. M.Horn and M.J.Tidman.Quality of life in epidermolysis bullosa.Clin Exp Dermatol. 2002 Nov;27(8):707-10.

5. S Tabolli, F Sampogna, C Di Pietro, A Paradisi, C Uras et all. Quality of life in patients with epidermolysis bullosa. Br J Dermatol 2009 Oct 11;161(4):869-77 6. Frew JW, Martin LK, Nijsten T, Murrell DF. Quality of life evaluation in epidermolysis bullosa (EB) through the development of the QOLEB questionnaire: an EB-specific quality of life instrument. Br J Dermatol. 2009 Dec; 161(6):1323-30 7. JW Frew, SS Venugopal, WF Yan ,LK Martin, T Nijsten et all. Quality of Life Evaluation in Epidermolysis Bullosa. www.debracongress2011.com (accesed online 16.01.2016) 8. Tania Cestari, Clarissa Prati, Dóris Baratz Menegon, Dédée F Murrell. Translation, crosscultural adaptation and validation of the Quality of Life Evaluation in Epidermolysis Bullosa instrument in Brazilian Portuguese. www.researchgate.net (accesed online 17.01.2016).

9. W. Y. Yuen , J. W. Frew, K. Veerman, E. R. van den Heuvel , D. F. Murrell , Marcel F. Jonkman.

Health-related Quality of Life in Epidermolysis Bullosa: Validation of the Dutch QOLEB Questionnaire and Assessment in the Dutch Population. Acta Derm Venereol 2014; 94: 442–447.

10. Calogero Pagliarello, Stefano Tabolli. Factors affecting quality of life in epidermolysis bullosa. Expert Review of Pharmacoeconomics & Outcomes Research 10 (3):329-38, jun 2010.

11. Francesco Margari, Paola Alessandra Lecce, Wanda Santamato, Ernesto Bonifazi. Psychiatric Symptoms and Quality of Life in Patients Affected by Epidermolysis Bullosa. JOURNAL OF CLINICAL PSYCHOLOGY IN MEDICAL SETTINGS 17(4):333-9 DECEMBER 2010.

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12. Williams EF, Gannon K, Soon K. The experiences of young people with Epidermolysis Bullosa Simplex: A qualitative study. Journal of Health Psychology. 2011 Jul 1;16(5):701–10.

13. Dures E, Morris M, Gleeson K, Rumsey N. The Psychosocial Impact of Epidermolysis Bullosa. Qualitative Health Research. 2011 Jun 1;21(6):771–82.

14. Eismann EA, Lucky AW, Cornwall R. Hand function and quality of life in children with epidermolysis bullosa. Pediatr Dermatol. 2014 Mar-Apr;31(2):176-82.

15. Pain and quality of life in young people with Epidermolysis Bullosa. http://www.debra.org. uk/uk-funded-projects/liossi-qol-in-young-people (accesed online 26.01.2016) 16. Epidemiological and Pathophysiological Studies about the Problem of Itch (Pruritus) in Patients with Epidermolysis bullosa (EB). http://www.debra.org.uk/uk-funded-projects/ steinhoff-itch-project (accesed online 26.01.2016) 17. Goldschneider KR, Good J, Harrop E, Liossi C, Lynch-Jordan A, Martinez AE, et al. Pain care for patients with epidermolysis bullosa: best care practice guidelines. BMC medicine.

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18. Fine J-D, Johnson LB, Weiner M, Suchindran C. Impact of inherited epidermolysis bullosa on parental interpersonal relationships, marital status and family size. British Journal of Dermatology. 2005 May;152(5):1009–14.

19. Dorota Mącik, Monika Kowalska-Dąbrowska.The need of social support, life attitudes and life satisfaction among parents of children suffering from epidermolysis bullosa. Przegl Dermatol 2015, 102, 211-20.

20. H Dufresne, S Hadj-Rabia, Charles Taieb, Christine Bodemer. Development and Validation of an Epidermolysis Bullosa family/parental Burden Score. Br J Dermatol. 2015 dec; 173(6):1405-10.

21. Papadopoulos L, Bor R. Ed. Psychological approaches to dermatology. Leicester: BPS Books; 1999.

22 Fried RD. Nonpharmacological treatments in psychodermatology. In: Koo JY, Lee CS, Eds. Psychocutaneous medicine. New York: Marcel Decker Inc 2003; pp. 411-26.

23. Tomas-Aragones L, Marron SE. Body dysmorphic disorder in adolescents. In: Tareen RS, Greydanus DE, Jafferany M, Patel DR, Merrick J. Pediatric Psychodermatology: A Clinical Manual of Child and Adolescent Psychocutaneous Disorders. Berlin; Boston: De Gruyter, 2013: 201-215.

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INTERNATIONAL EVENTS CALENDAR

2016

INTERNATIONAL EVENTS CALENDAR CALENDAR EVENIMENTE INTERNAŢIONALE mMARTIE 2016 16-20: 1st International Dermatology and Cosmetology Congress (INDERCOS). Istanbul, Turkey. www.idercos.org 30 mar- 2 apr: 14th Aesthetic & Anti-aging Medicine World Congress AMWC 2016, Monte Carlo, Monaco. www.euromedicom.com mAPRILIE 2016 12-14: 5th Continental Congress of Dermatology Dubai Derma. Dubai, United Arab Emirates. http://dubaiderma.com/ 15-17: International Congress of Dermatology, Cometic & Laser. Teheran, Iran. http://crtsdl.tums.ac.ir/ mMAI 2016 11-14: 75th Annual Meeting of the Society for Investigative Dermatology (SID), Scottsdale, AZ – USA www.sidnet.org 19-22: 13th EADV Spring Symposium. Athens, Greece www.eadv.org 26: 13th ESPD (European Society for Pediatric Dermatology) Congress. Paris, France. http://www.espd2016.com/ mIUNIE 2016 16-19: Facial Aesthetic Conference & Exhibition FACE 2016. London, UK. www.euromedicom.com 11-15: EAACI Congress. Vienna, Austria. www.eaaci.org 16-18: 3rd International Congress of Aesthetic Dermatology and Healthy Aging Medicine ICAD Brazil. Sao Paolo, Brazil euromedicom.com mIULIE 2016 5-7: 96th Annual Meeting of the British Association of Dermatologists.Birmingham – UK www.bad.org.uk

5th Congress of the Psoriasis International Network-Psoriasis 2016.Paris,France.www.pso2016.com 27-31: Summer meeting of the American Academy of Dermatology (AAD). Boston, MA – USA www.aad.org 7-9:

mAUGUST 2016 11-14: International Congress of Tropical Dermatology.

Colombo, Srilanka. http://www.ictd2016.org/ 31 aug-3 sept: 16th World Congress on Cancers of the Skin; 12th Congress of the EADO. Vienna, Austria. www.wccs2016.com mSEPTEMBRIE 2016 8-10: 23rd Biennal Conference on Diseases of the Vulva & Vagina. Chicago, USA. http://issvd.org/event/23rd-biennial-confeence-on-diseases-of-the-vulva-vagina/ 14-17: 13th ESCD ( European Society of Contact Dermatitis) Congress, Manchester, United Kingdom.www.escd2016.com 16-17: 4th Aesthetic & Anti-aging Medicine World Congress Eastern Europe AMWC Eastern Europe. Moscow, Russia. www.euromedicom.com 28-sept-2 oct: 25th CONGRESS OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VEN REOLOGY (EADV) , Vienna – Austria. www.eadv.org mOCTOMBRIE 2016 20-22: 2nd International Conference of dermatology.

Kathmandu, Nepal. www.icderm2016.com mNOIEMBRIE 2016 24-26: International Congress of Aesthetic Dermatology ICAD 2016. Bankok, Thailand. www.euromedicom.com

NATIONAL EVENTS CALENDAR CALENDAR EVENIMENTE NAŢIONALE mAPRILIE 2016 Campania „Adolescenţa fără acnee” 15-17: International Congress of Dermatology, Cometic & Laser. Teheran, Iran. http://crtsdl.tums.ac.ir/

mOCTOMBRIE 2016 19 - 22: Congresul Naţional de Dermatologie, Sinaia 29: Ziua Mondială a Psoriazisului

mMAI - AUGUST 2016 Campania Euromelanoma Day

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