MyHVP Newsletter (Vol. 6. Issue 2. Dec 2021)

MyHVP Newsletter Volume 06 | Issue 02 | December 2021

THE IMPETUS AND NEED FOR GENETIC COUNSELLING Written by: Santhiagu Thiagu, Voon Chun Ping, Nurfarahin Saain Universiti Kebangsaan Malaysia Introduction enetic counselling is an official term to define a clinical practice which was introduced by Sheldon Reed in the year 1947 who ambiguously defined it then as a “kind of genetic social work”. The American Society of Human Genetics (ASHG) subsequently proposed a definition for genetic counselling in the early 1970’s which is widely used till now. Genetic counselling is a communication process that deals with human problems associated with the occurrence or risk of occurrence of a genetic disorder in a family.

This process involves an attempt by one or more appropriately trained persons to help the individual or family to comprehend the medical facts including the diagnosis, probable course of the disorder, and the available management. Genetic counselling also appreciates the way heredity contributes to the disorder and the risk of recurrence in specified at-risk relatives, and understand the alternatives for dealing with the risk of recurrence. Genetic counselling helps individuals choose a course of action which seems appropriate to them in view of their risk, their family

G

goals, and their ethical and religious standards and act following the decision, and to make the best possible adjustments to the disorder in affected family members or to the risk of recurrence of that particular disorder. This definition has held up well over the years and articulates the central features of genetic counselling.

Over the past sixty years, the field of genetic counselling has been constantly evolving with the completion of the Human Genome Project and advancement in new technologies to study the genes, to find disease-causing mutations, and certainly to improve genetic testing. According to the definition provided by Secretary’s Advisory Committee on Genetics, Health and Society, a genetic or genomic test involves an analysis of human chromosomes, DNA, RNA, genes, and gene products like enzymes and types of proteins, which is predominately used to detect heritable or somatic mutations, genotypes, or phenotypes related to disease and health. The purpose of genetic tests includes predicting the risk of disease, screening newborns, directing clinical management, identifying carriers, and establishing prenatal or clinical diagnoses or prognosis in individuals, families, or populations. The broad types of genetic tests used are cytogenetic tests or chromosome analysis which will look for changes in the chromosome number, structure, and arrangement; molecular test or DNA analysis which will detect changes in the DNA sequence, methylation, duplications, and deletions; and biochemical test to detect changes in gene products like protein and enzyme level. Genetic tests have different clinical uses such as diagnostic or confirmatory tests, predictive tests, pre-symptomatic tests, carrier risk assessment, newborn screening, prenatal diagnosis, preimplantation diagnosis, pharmacogenetics testing, and prognostic testing which are influenced by the specific selected technology.

Photo is for illustrative purposes only.

Contact us: | Secretariat Office: Human Variome Project Malaysian Node & South-east Asian Node School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kota Bharu, Kelantan, Malaysia | Phone :(60) 097676543 / 6531 | Fax: (60) 097676543 | Email : myhvp@usm.my | Website: hvpmalaysia.kk.usm.my

(See the page 4)

MyHVP Newsletter

Head’s Address

Board of Editors 2021

ISSN: 2550-1747 | Volume 06 | Issue 02| December 2021

Editor in Chief Professor Dr. Zilfalil Alwi

Managing Editor

Dr. Nik Norliza Nik Hassan

Editorial Board Members

Prof. Ida Madieha binti Azmi Assoc. Prof. Dr. Muhammad Farid Johan Assoc. Prof. Dr. Rosnah Bahar Mr. Abdul Halim Fikri Bin Hashim

English Editor Amyzar Alwi

Contents

2 Board of Editors

2021

3 Report

4-12 Article 13 Call for papers

© 2021. All rights reserved. The information in this newsletter is provided by the Malaysian Node of the Human Variome Project (MyHVP) members including South-east Asian Node (HVPSEA Node) for educational / information purpose only. It is not a substitute for professional medical care and medical advice.

The contents express the opinions of the authors who alone are responsible for their view expressed. MyHVP does not accept any legal responsibility for their contents.

Writers, Invited!

MyHVP Newsletter is issued biannually. For the coming issue, the public from various fields and specialties are invited to their lived experience in dealing with the issues of medicine or biomedicine. Writers can contribute their writing based on these following criteria: 1. Length (Max. 1page A4 size, and it may be edited for our use) 2. The committee has the right to share your writing for further issues.

3. To suit the needs of the publication, and your writing won’t be returned 4. Emailed the article to myhvp@usm.my

Published by

Malaysian Node of the Human Variome Project (MyHVP) School of Medical Sciences Universiti Sains Malaysia Health Campus 16150, Kubang Kerian, Kelantan, Malaysia Tel: +6097676531 /6543 | Email: myhvp@usm.my

Thalassaemia is one of the most common genetic disorders which has resulted in increased morbidity and mortality globally especially in low- and middle-income countries (LMIC). While there have been many researches conducted on thalassaemia, studies on genetic modifiers are relatively less common. This can be explained by the diverse clinical manifestations and the varying degrees of disease severity which are less understood.

The International Hemoglobinopathy Research Network (INHERENT) was established by a group of researchers and experts from more than 42 countries worldwide with the aim of providing better understanding of the role of genetic modifiers among the different ethnic groups worldwide. INHERENT brings together existing international and regional consortia in the field of hemoglobinopathies, focussing on standardisation of phenotypic definitions and development of an open research infrastructure that will foster international collaboration for the benefit of patients and their families. This includes Global Globin Network (GGN) as one of the nine participating consortia in INHERENT committed to addressing this challenge.

Prior to 2020, MyHVP actively organised the EduVariome programme at selected secondary schools in the country. However, the Covid-19 pandemic brought this programme to a halt but we plan to continue it when the situation improves. The EduVariome programme is one of our awareness-raising platforms to educate the public on the burden of thalassaemia disease as one of the common diseases in this country. We hope that this initiative will encourage other institutions and organisations conductiong and participating in these activities to improve healthcare and raise awareness on inherited diseases in our country.

We are pleased to inform all the MyHVP members of the successful publication of the second issue of June 2021 of our journal, the Malaysian Journal of Human Genetics (MJHG). For the upcoming first issue for 2022, the Editorial Board seeks submission of more articles from MyHVP members. Your continuous contribution and support is pivotal to the success of our journal publication.

If you have articles of interest to contribute to this newsletter, please contact us with any news, announcements or views in your field of study by emailing it to me at zilfalil@gmail.com or to any members of the editorial board.

Thank you.

Prof. Dr. Zilfalil Alwi Head, Malaysian Node of the Human Variome Project (MyHVP)

MyHVP Newsletter | December 2021 | page 2

Report WORLD THALASSAEMIA DAY 2021

Forum on World Thalassemia Day 2021 • This forum was held virtually on 31 May 2021 in conjunction with the annual celebration of World Thalassemia Day on 8th of May. • Organised by the Malaysian Node of the Human Variome Project (MyHVP) in collaboration with the Institute for Medical Research (IMR), Kuala Lumpur, the forum aimed at providing knowledge and promoting public awareness on thalassaemia.

• This forum was moderated by Dr. Mujahid Bakar from School of Health Sciences, Universiti Sains Malaysia and the panelists of the forum consisted of 3 experts of haematologists; Prof. Zarina Abdul Latiff (UKM), Dr. Ezalia Esa (Institute for Medical Research, Kuala Lumpur), Dr. Norafiza Mohd Yassin (Institute for Medical Research, Kuala Lumpur) and the President of Sabah Thalassaemia Society, Mr. Francis Mujim).

• The forum was attended by approximately 280 participants.

A Forum on thalassaemia awareness in conjunction with National Independence Day at the Sabah state level

• • • •

This was the second virtual forum on thalassaemia organised by MyHVP in collaboration with Sabah Thalassemia Association, Sabah State Education Department, and Malaysian Genetic Association (PGM).

The theme of the forum was “Memerdekakan Pejuang Talasemia” with the aim of raising awareness and basic knowledge on thalassaemia among the patients, educators and public community.

The forum was participated by 9 secondary schools in Sabah with the total number of 800 participants.

This forum was moderated by Ms. Fazilah Suhud, representative from Sabah Thalassaemia Association.

The panelist of this forum consist of 2 thalassaemia experts and 2 patients of thalassaemia.

MyHVP Newsletter | December 2021 | page 3

Article Continued from page 1 (The impetus and need for genetic counselling) Scope of genetic counselling A genetic counsellor is an individual trained in medical genetics and counselling to provide medical care for individuals or families affected by a medical condition.

Genetic counsellors may not be medical officers, but they work along with clinical geneticists to provide care, support, risk evaluation and genetic investigation for their client or patient. A genetic counsellor is also an individual trained to deliver complex and technical information about genetic risks, testing, and diagnosis in a comprehensible manner to an individual or family.

It is also their role to explain the significance of genetic conditions concerning cultural, personal, and familial contexts to their client. When providing further management, a genetic counsellor is someone who provides available options in a non-directive manner and can provide referrals to support groups and other health professionals to better assist their clients.

The essence of genetic counselling is to provide information to a client or patient about their health concerns that run in their family that may include a review of family history, health history, and pregnancy history. Although not all genetic counselling sessions will end up with genetic testing, but risk evaluation and surveillance can be offered to those at-risk families or individuals. Couples that have a concern with family planning may benefit from a genetic counselling session, to address their concern about factors that may affect the health of the baby or ability to conceive.

These concerns may include known genetic condition inherited in the couple’s family, history of infertility, multiple miscarriage or stillbirth, or previous pregnancy or child affected by birth defect or genetic condition.

With genetic counselling, the couple can get accurate information to assist their decision in family planning or referral to an assisted reproductive technology clinic. A pregnant mother can seek genetic counselling when the screening of the fetus is abnormal, to be assisted in the investigation of the fetus’ health condition, which may help with future management or evaluate recurrence risk.

Every individual may have a few unusual or abnormal features, but when multiple abnormal features are found in an individual, they may be affected by a genetic condition. Parents are especially concerned about their child when they receive abnormal newborn screening reports, the child appears to be dysmorphic, the child has some degree of intellectual disability or developmental delay, features of autism spectrum disorders and vision or hearing problems.

Those concerns can be addressed by seeking genetic counselling to investigate the nature of the condition, whether it is hereditary or acquired. Genetic counselling

services can benefit the adult individual as well because some genetic conditions may affect the individual later in life. Adults with a family history of medical condition that is of hereditary origin can seek genetic counselling to get support and information about the condition. Some of these conditions may include adult-onset muscular dystrophy or similar conditions, inherited neurodegenerative conditions, inherited blood disorders, familial hypercholesterolemia amongst others. Although most cancers are sporadic, some cancer cases can be clustered in a family. Adults with a strong family history of cancer can benefit from genetic counselling for risk evaluation.

Processes in genetic counselling When a case is referred to a genetic counsellor or clinical geneticist, the investigation should start with a family pedigree and medical history intake. The role of the genetic counsellor here is to construct a pedigree of the proband via a questionnaire, by phone or in-person regarding both family and medical history. When the pedigree is constructed, the genetic counsellor will request and review pertinent medical records of the proband. For genetic conditions with dysmorphic features, photos of family members and proband at different ages will be requested if necessary. Before the proband is counselled, a genetic counsellor will gather the latest information on the investigated genetic condition by searching relevant medical and scientific literature on reference books or genetics databases such as GeneReview and OMIM. Genetic counselling is not complete without risk calculation. To perform a risk assessment, a genetic counsellor needs to know the genetic aetiology of the condition through their reading and evaluate the proband’s pedigree. If it is a hereditary condition, a genetic counsellor may need to calculate risk or provide empirical risk for the proband and other relevant family members.

The genetic counsellor needs to determine if genetic testing for the condition is available and indicated.

If molecular genetic testing is available, genetic counsellors may need to explore the genetic testing options to strategise the genetic testing plan according to cost, turnaround time and reliability of the laboratory before laying the information to the proband. Other than genetic tests and counselling, a genetic counsellor may need to identify information about support and advocacy groups or resources for the condition if available. The proband’s investigation journey may end with the genetic counsellor but coping and living with the condition can last for a lifetime. National or support groups may improve the proband’s experience living with the condition. Brochures and fact sheets may be given by genetic counsellors to assist proband and their family members to understand and explain their condition better. Lastly, a genetic counsellor needs to formulate a clinic visit plan for the proband MyHVP Newsletter | December 2021 | page 4

Article with the frequency of follow-up with the proband and the amount of information to deliver to the proband.

During the visit, genetic counselling may develop or use existing counselling aids to improve the proband’s comprehension of the information delivered. A genetic counsellor works in a team, therefore, if necessary, they may need to discuss the proband’s case with their colleagues or superior for better management of the case.

Evolution of genetic counselling in Malaysia In Malaysia, genetic counselling was first introduced in University of Malaya Medical Centre (UMMC) when medical genetic services was first established in 1994. This was followed by medical genetic services in Hospital Kuala Lumpur (in 1999) followed by University Kebangsaan Malaysia Medical Centre (UKMMC) and Universiti Sains Malaysia (Hospital Universiti Sains Malaysia, Kubang Kerian). Medical or clinical genetics services were essentially run by clinical geneticists based on accreditation by the Malaysian National Specialist Register. Genetic counselling service are also offered at Hospital Pulau Pinang under the Ministry of Health, in addition to several private institutions/sector such as Cancer Research Malaysia, Loh Guan Lye Specialist Centre and Genetic Counselling Asia. The field of medical genetic in Malaysia has been developing in a relatively slow but steady pace for the past ten years. The Malaysian Ministry of Health has recently acknowledged the importance of genetic counselling which includes establishing a National Framework for Rare Diseases in 2019 that facilitates in providing funding for medical genetics services in Malaysia. Recognising the needs for a local training programme for genetic counselling, the National University of Malaysia also established the MSc in Genetic Counselling in 2015.

Nevertheless, the country still faces a shortage of qualified genetic counsellors to maximise genetic counselling services.

In summary, genetic counselling is essential in providing medical care as the study of genetics and genomics are becoming part of routine healthcare and it is found to be a useful approach for more accurate diagnosis. Genetic counselling may not be an exclusive skill to genetic counsellors as other medical professionals can perform it as well, but lack of awareness and knowledge in genetics are hindering the process of integrating genetic counselling in most healthcare systems in Malaysia. Therefore, efforts and dedication towards promoting the awareness of genetic counsellors and counselling should be adopted by genetic professionals for the better development of genetic counselling in Malaysia.

A Guide to Genetic Counselling, 2nd Edition. WileyBlackwell.

2. Genetic Alliance. 2008. Understanding Genetics: A New York, Mid- Atlantic Guide for Patients and Health Professionals. Genetic Alliance, The New York - Mid-Atlantic Consortium for Genetic and Newborn Screening Services (Appendix E Inheritance Patterns): 105.

3. Lee, J. M., & Thong, M. (2013). Genetic Counseling Services and Development of Training Programs in Malaysia. 911–916. https://doi.org/10.1007/ s10897-013-9589-z 4. Resta, R., Rosseau, J., Kant, I., Watt, J., Hutton, J., & Franklin, B. (2019). Testing , and Humanism. 1–12. https://doi.org/10.1101/cshperspect.a036673

REFERENCES 1. Uhlmann, W.R., Schuette, J.L. & Yashar, B.M. 2009.

MyHVP Newsletter | December 2021 | page 5

Article FROM PATIENT DERIVED XENOGRAFT (PDX) TO 3D SPHEROIDS: MAXIMIZING THE MOUSE MODELS OF CANCER Marini Marzuki, Susan Ling Ling Hoe, Norazlin Abdul Aziz Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research (IMR), National Institutes of Health (NIH), Ministry of Health Malaysia 40170 Shah Alam, Selangor, Malaysia

CANCER: WHAT WE SHOULD KNOW? Human body is made up of trillions of cells. Our blood and skin contain stem cells that multiply constantly (a process called “cell division”) to regenerate, whereas other cells such as the skeletal muscle and nerve cells become specialised (“terminally differentiated”) and stop growing [1, 2]. During the process of cell division, mistakes in copying the genes and/or malfunction in division may occur [3]. This leads to uncontrolled cell growth or cell division, which in turn results in formation of a lump (“tumor”) that may be cancerous (“malignant”), or non-cancerous (“benign”) [4].

Examples of solid malignant tumors are cancers of the breast, colorectum, lung and nasopharynx, while “liquid tumors” include cancers of the blood (leukemias and lymphomas). Cancer cells can extent and penetrate into nearby tissues (“invasion”), travel to distant places in the body to form new tumors (“metastasis”) and/or grow back (“recurrence”). Metastasis and recurrence are the major causes of death of cancer patients. On the other hand, benign tumors do not spread to the nearby tissues.

Benign tumors usually do not grow back after surgical removal, However, benign tumors can sometimes be quite large and can cause serious symptoms or be life threatening, such as benign tumors in the brain [4].

How Does Cancer Happen?

Cancer is caused by changes to the genes or malfunctions in cell growth and division that can arise from: • errors that occur in the cell division process, • DNA damage caused by harmful substances such as ultraviolet rays, chemicals in tobacco smoke and carcinogens in food/drink, and pathogens including H. pylori, human papillomavirus (HPV) and EpsteinBarr virus (EBV), • genetic defects inherited from parents [4].

Illustration 1. Cells are made up from the tightly packed DNA molecules, called chromosomes which contains a few thousands of genes, which were arranged in long strands of DNA bases [4].

Cancer Incidence in Malaysia The Malaysia National Cancer Registry report (20122016) presented data through 13 states and two federal territories [5]. From such reports, the epidemiological profile of cancer in Malaysia can be used to assist medical and health professionals, researchers, policymakers, and agencies in the planning of the National Cancer Control Programme.

The trends of cancer incidence in Malaysia are likely a result of interaction among multiple factors such as lifestyle and exposure (e.g. obesity, physical inactivity and smoking), population ageing, and a declining fertility rate. It is important to encourage an individual to live and practise a healthful behaviour to minimise the risk of cancers [5].

The ten most common cancers among Malaysians (2012-2016) were breast, colorectal, lung, lymphoma, nasopharynx, leukaemia, prostate, liver, cervix uteri and ovary (Figure 1) [5].

Figure 1. The ten most common cancers in Malaysia from 2012 to 2016 [5].

MyHVP Newsletter | December 2021 | page 6

Article PATIENT DERIVED XENOGRAFT (PDX) Why We Need PDX as Cancer Model?

Traditionally, cancer research was and still is performed with cancer cell lines that are established from fresh patient tumors (“2D cell cultures”). Cell lines are grown (“cultured”) in plastic vessels in artificially controlled environment (CO2 incubator). Recently, increasing number of publications had revealed the limited relevance of cell lines in preclinical assessments of chemotherapeutics. This is largely due to the nonphysiologic nature of cell cultures whereby only cells that can survive in man-made conditions will be retained [6], the acquired mutations due to extended period of continuous culture of the cell lines (usually many years) as well as selection for phenotypes that are developed in laboratories and not previously present in the original tumour.

Table 1 shows the advantages, challenges and limitations of the PDX as a cancer model.

Patient Derived Xenograft (PDX) is a study model of cancer which is generated by transplanting fresh patient tumors directly into immune-compromised murine hosts [7]. The tumor may comprise mixed cancer cell populations that contain different genetic profiles due to unique somatic mutation events. Continuous passages of these tumors through subsequent generations of murine hosts permit propagation of PDX tumor lines (Illustration 2).

Nasopharyngeal Carcinoma (NPC) is among the top three most common cancers affecting Malaysian males of age 35 to 55 years old [5]; however, it is very rare among Caucasians and hence often being neglected in drug discovery. The Molecular Pathology Unit, Cancer Research Centre, IMR have established numerous PDX models from local NPC patients (Malay, Chinese and Bidayuh). We have optimised in vitro and in vivo techniques for drug discovery pipeline as well as tumor growth and metastasis study using these PDX models.

Illustration 2. Development of Patient Derived Xenograft (PDX) line from fresh human cancer tissues. Credit: Mohd. Firdaus b. Che Mat.

Examples of Applications of PDX in Cancer Research a) Cancer stem cells (CSCs) NOD scid gamma (NSG) mice, being the most immunocompromised strain of mice to date, are excellent models for studies on cancer stem cells (CSCs). CSCs are a sub-group of cancer cells with an almost indefinite lifespan and are able to develop into cells with differing growth rates and resistance to therapy, amongst others.

With these properties, they are believed to be the seeds of cancers that could initiate tumor growth when other contributing factors are present.

In one of our studies, we evaluated potential CSC biomarkers by analyzing the surface receptors of MyHVP Newsletter | December 2021 | page 7

Article NPC cell lines and PDXs established in our laboratory.

The PDX cells expressing high levels of EpCAM marker (EpCAMbr) seemed to have higher tumor formation ability (“tumorigenicity”), a behavior that is consistent among all CSCs [8]. As compared to controls, these EpCAMbr cells grew faster, resulted in tumors of larger sizes, and could stably form new tumors in other NSG mice for at least 3 consecutive generations (Figure 2).

Figure 2. Growth properties of EpCAM cells from NPC PDXs [8] b) Metastatic Model For the study of cancer metastasis, we transduced the NPC PDX cells with lentiviral vector expressing Green Fluorescent Protein and luciferase (GFP-Luc2), inoculated the cells into NSG mice and track the invasion and/or spreading of cancer cells to other organs via Xenoluc assay [9,10]. Real-time monitoring of the inoculated GFP-Luc2 expressing cells were carried out using a noninvasive bio-luminescence imaging technique (Figure 3).

Figure 3. Metastatic model of NPC. (A) Orthotropic injection, (B) Coronal section of mouse head showing nasopharynx area with surrounding organs, adjacent tissue and invading tumor, (C) Intracardiac injection leading to metastatic deposits in (D) bone and (E) liver [9]. c) Therapeutic Model Vascular endothelial growth factor (VEGF) is overexpressed in the majority of solid tumors and is critical in mediating blood vessel formation in tumors (“tumor angiogenesis”) [11, 12]. Bevacizumab blocks the binding of VEGF ligand to its receptor. Clinical trials of bevacizumab with chemotherapy showed increased overall survival and/or

progression-free survival in cancers such as colorectal, cervical, ovarian and renal cell [13, 14, 15, and 16].

In the recently concluded American Association for Cancer Research Congress 2021 (AACR 2021), Wong et al. presented the work of comparing the effect of a new bispecific nanobody (VEGF/Ang-2) with bevacizumab in NPC PDXs. There were no signs of animal stress and loss of weight throughout the drug administration period. At the end of the experiment, the anti-VEGF/Ang-2 nanobody remarkably lower the numbers of microvessels and also increased the tumor internal dead cell areas as compared to bevacizumab and vehicle control [17]. d) Cancer Immunotherapy The PDX model is also used in immuno-oncology, a field of study that aims to enhance body’s own immune system to fight cancers. For example, the immune system of NSG mice can be reconstituted by introducing purified human hematopoietic stem cells (HSCs) into the NSG mice [18]. These “humanized” mice with PDX inoculation would serve as better tools for testing immunotherapeutic agents against cancers. Zhao et al. [19] reported that the liver cancer PDX tumours from their humanized mice experiment had similar types of immune cells surrounding the tumour cells, as observed in patients. Another study on humanized melanoma PDX mice found that antigenspecific T cells produced by these mice could be expanded in vitro and these cells demonstrated anti-tumor activity when being inoculated into other melanoma PDX mice [20].

IN VITRO CULTURE USING PDX CELLS 3D in vitro culture models Murine hosts require housing, care and facilities to support their living, of which not all laboratories are well equipped with as well as the costs for their continuous maintenance and propagation. With some optimizations, most PDX cells can be cultured in vitro to grow from single cell suspensions to form multicellular aggregates, known as 3D spheroids.

The structural and dimensional arrangement of PDX cells within the 3D spheroid model, perhaps together with the presence of other cell types and extracellular matrix components, could mimic their natural microenvironment and hence may reinstate the important interactions and signalling that are lost or compromised in monolayer 2D cell line culture systems [21]. For these reasons, 3D spheroid models offer greater clinical and biological relevance than monolayer 2D models and therefore, could bridge the gap between the monolayer 2D culture and animal works [10].

Example of Application of 3D Spheroid Models in Cancer Research In our laboratory, PDX cells were transduced with lentiviral vector expressing GFP-Luc2 to facilitate the monitoring of in vitro growth during drug treatment experiments (Figure 5). PDX cell viability and death was then quantified in MyHVP Newsletter | December 2021 | page 8

Article real-time using the XenoLuc assay [10].

Figure 5. Immunofluorescent staining of NPC PDX cells transduced with lentiviral vector expressing GFPluc2 and grown as 3D spheroids. In sequence, bright field, green fluorescent protein (GFP), Hoechst and epithelial cell marker (EpCAM) images of 3D spheroids at 20x magnification. Cells were stained with Hoechst to locate the nucleus of the cells. EpCAM indicates the characteristic of epithelial cells in the spheroids.

CONCLUSION Patient’s tumor cells can be inoculated into immunocompromised mice to generate PDXs and be continuously maintained as PDX lines. The cells from these PDXs can be cultured in vitro as 2D and 3D cultures.

These cell culture and PDX models are important tools for researchers to study cancer stem cells and metastasis, tumor biology, tumor invasion as well as to screen new drugs for treatment of cancer.

ACKNOWLEDGEMENT We thank the Director General of Health Malaysia, Ministry of Health Malaysia for the approval of this article. The work of our unit described in this article is supported by a series of MOH grants, MREC and ACUC approvals from 2011 to 2021. We thank Dr Tan Lu Ping for her critical review. Our gratitude also goes to past and present members of the Molecular Pathology Unit, Cancer Research Centre, IMR as well as the patients and staff from participating hospitals for their support.

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CD24, CD44 and EpCAM enrich for tumour-initiating cells in a newly established patient-derived xenograft of nasopharyngeal carcinoma. Sci. Rep. 7(1): 12372.

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Mohd Firdaus Che Mat, Nor Linda Abdullah, Hoe Susan Ling Ling, Munirah Ahmad, Khoo Alan Soo Beng (2019). Laboratory Mice as preclinical animal models in biomedical research. IMR Research Highlight issue 02/20196. Rosalyn W. Sayaman, Mohamad Saad, Vésteinn Thorsson, Jérôme Galon, Elad Ziv, Davide Bedognetti et. al. (2021). Germline genetic contribution to the immune landscape of cancer. Immunity. Vol. 54:2.

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Chi-Hang Wong, Gigi Lam, Connie Hui, Rachel C.T. Lam, K.W. Lo, Edwin P. Hui, Anthony T.C. Chan, Brigette B.Y. Ma (2021). In vivo evaluation of VEGF/ Ang-2 bispecific nanobody BI836880 in nasopharyngeal carcinoma (NPC). https://www.inoncology.com/events/ congresses/aacr-2021. Poster.

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Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice. Exp. Mol. Med; 50:99 19.

Yue Zhao,Timothy Wai Ho Shuen, Tan Boon Toh, Xue Ying Chan, Min Liu, Sue Yee Tan, Yong Fan et al.

(2018). Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy. Gut; 67:1845–1854.

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Zheng Hu, Jinxing Xia, Wei Fan, Jennifer Wargo and Yong-Guang Yang (2016). Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice. Oncotarget; 7:No. 64486459.

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Breslin S, O’Driscoll L (2013). Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today. 18(5–6):240–9.

Acute Myeloid Leukemia Updates

Written by Dr. Rabiatul Basria S. M. N. Mydin1* (Ph.D) and Dr. Eman S. Algariri1,2 (MD) 1 Oncological and Radiological Sciences Cluster, Advanced Medical and Dental Institute Universiti Sains Malaysia 13200, Pulau Pinang, Malaysia 2 Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Hadhramout University, Mukalla, Hadhramout, Yemen. *Corresponding author email: rabiatulbasria@usm.my In the last decade, the incidence of acute myeloid leukemia (AML) has increased worldwide. In Malaysia, AML represented the highest incidence and mortality rate among other leukemia type, as shown in Figure 1 (Global Health Data Exchange, 2019). AML is more common in adults, with a median age of 67 and around 30% of AML patients over 75 years. However, Pediatric AML is the fifth most common cancer in children and its prognosis is still poor compared to ALL, the most common type of leukemia in children (Chen et al., 2019). AML is characterized as a highly heterogeneous hematologic malignancy and is associated with the clonal expansion of immature (blast) myeloid precursors and disturbance of hematopoiesis in the bone marrow.

Figure 1. Incidence and mortality rate of leukemia subtypes in Malaysia in 2019. Comparison of incidence and mortality rate between leukemia subtypes in Malaysia in 2019.

AML: Acute myeloid leukemia, ALL: Acute lymphocytic leukemia, CML: Chronic myelogenous leukemia, CLL: Chronic lymphocytic leukemia. Data was obtained from Global health data exchange (2019).

Recently, the AML survival rate revealed certain improvements that were prominent across all age groups, except for the age group older than 70 years.

From 2010 to 2017, the estimated 5-year survival rate was 65 %-70 % in childhood AML, 50-63 % in patients less than 40 years old, and 40% for patients aged 40-60 years old. The survival rate did not show any change among elderly AML patients, where the survival rate MyHVP Newsletter | December 2021 | page 10

Article remained just around 5% (Sasaki et al., 2021). Despite the higher AML survival rate among childhood AML, it is still less than the childhood ALL survival rate that reached 90% (Chen et al., 2019). Relapse is another big challenge in AML, which occurs in 40-50% of younger and the great majority of elderly patients (Thol & Ganser, 2020).

In summary, AML treatments is still a major medical challenge, especially for adult and elderly patients.

Since AML has a complex etiology, multiple targeted therapies could be a promising therapeutic strategy to overcome the AML challenges. Further comprehensive studies on relapse, precision medicine and targeted therapy are critically needed better AML managements.

Survival rates for AML have improved in tandem with advances in molecular characterization and therapeutic strategy. AML mutations such as the FMS-like tyrosine kinase 3 (FLT3) that is found in around 30% of AML patients and the IDH mutations that happen in 15-20% of newly diagnosed AML patients are currently targeted with approved targeted therapy such as midostaurin and Ivosidenib, which when added to standard therapy results in improving the complete remission and survival rate of AML patients, especially for those with very poor prognosis (relapsed and refractory AML) and the elderly (Puccini et al., 2021; Wu et al., 2018). The therapeutic targets that have recently been approved for subgroups of AML are presented in Table 1.

Acknowledgements The authors would like to thank The Ministry of Higher Education Malaysia for sponsoring this work under the Fundamental Research Grant Scheme (FRGS) with Project Code: FRGS/1/2019/SKK15/USM/02/2.

Drug

Class

Target

References 1.

Global health data exchange. (2019). GBD results tool. http://ghdx.healthdata.org/gbd-resultstool.

2.

Chen, X., Pan, J., Wang, S., Hong, S., Hong, S., & He, S. (2019). The epidemiological trend of acute myeloid leukemia in childhood: a population-based analysis. Journal of Cancer, 10(20), 4824.

Inclusion criteria

Out come

Reference

Median OS

Median EFS

CR

Midostaurin

Tyrosine kinase inhibitor

FLT3, KIT, PDGFRα/β, SYK, VEGFR1/2

Adult, newly diagnosed, untreated AML with FLT3 mutation

74.7 months in the midostaurin group vs.

25.6 months in the control group

8.2 months in the midostaurin group and 3.0 months in the control group

58.9% in the midostaurin group and 53.5% in the control group

(7)

Gilteritinib

Tyrosine kinase inhibitor

Highly specific FLT3 inhibitor

Adult R/R AML with FLT-3 mutation

Longer among the gilteritinib group than the chemotherapy group (9.3 months vs. 5.6 months

2.8 months in the gilteritinib group and 0.7 months in the chemotherapy group.

34% in the gilteritinib group and 15.3% in the chemotherapy group.

(8,9)

Ivosidenib

Small-molecule inhibitor

Mutated IDH1

Adult R/R AML with IDH1 mutation

9.0 months after follow-up 15.3 months

-

24% (with 10.1 months median CRD)

(10)

Venetoclax

Small-molecule inhibitor

Bcl-2

> 65 years, newly diagnosed AML patients, ineligible for intensive chemotherapy

10.1-month in the venetoclax plus LDAC group vs. 5.5 months in the LDAC group

-

54% in the venetoclax plus LDAC group vs. 11% to 19% in the LDAC group

(11)

Gemtuzumab ozogamicin (GO)

Monoclonal antibody

CD33 transmembrane protein

Newly diagnosed de novo adult AML patients

27.5 months in the GO group and 21.8 months in the control group

longer for the GO group (median 17.3 months) than in the control group (median 9.5 months)

No significant difference in the rate of CR in the GO group compared with the control group

(12)

MyHVP Newsletter | December 2021 | page 11

Article 3.

Sasaki, K., Ravandi, F., Kadia, T. M., DiNardo, C. D., Short, N. J., Borthakur, G., ... & Kantarjian, H. M. (2021). De novo acute myeloid leukemia: A population‐ based study of outcome in the United States based on the Surveillance, Epidemiology, and End Results (SEER) database, 1980 to 2017. Cancer, 127(12), 2049-2061.

4.

Thol, F., & Ganser, A. (2020). Treatment of relapsed acute myeloid leukemia. Current Treatment Options in Oncology, 21(8), 1-11.

5.

Puccini, M., Pilerci, S., Merlini, M., Grieco, P., Scappini, B., Bencini, S., ... & Gianfaldoni, G. (2021).

Venetoclax-Based Regimens for Relapsed/Refractory Acute Myeloid Leukemia in a Real-Life Setting: A Retrospective Single-Center Experience. Journal of Clinical Medicine, 10(8), 1684.

6.

Wu, M., Li, C., & Zhu, X. (2018). FLT3 inhibitors in acute myeloid leukemia. Journal of hematology & oncology, 11(1), 1-11.

7.

Stone, R. M., Mandrekar, S. J., Sanford, B. L., Laumann, K., Geyer, S., Bloomfield, C. D., ... & Döhner, H. (2017). Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. New England Journal of Medicine, 377(5), 454-464.

8.

Perl, A. E., Martinelli, G., Cortes, J. E., Neubauer, A., Berman, E., Paolini, S., ... & Levis, M. J. (2019).

Gilteritinib or chemotherapy for relapsed or refractory FLT3-mutated AML. New England Journal of Medicine, 381(18), 1728-1740.

9.

McMahon, C. M., & Perl, A. E. (2019). Gilteritinib for the treatment of relapsed and/or refractory FLT3-mutated acute myeloid leukemia. Expert review of clinical pharmacology, 12(9), 841-849.

10.

Megías-Vericat, J. E., Ballesta-López, O., Barragán, E., & Montesinos, P. (2019). IDH1-mutated relapsed or refractory AML: current challenges and future prospects. Blood and lymphatic cancer: targets and therapy, 9, 19.

11.

Wei, A. H., Strickland Jr, S. A., Hou, J. Z., Fiedler, W., Lin, T. L., Walter, R. B., ... & Roboz, G. J. (2019).

Venetoclax combined with low-dose cytarabine for previously untreated patients with acute myeloid leukemia: results from a phase Ib/II study. Journal of Clinical Oncology, 37(15), 1277.

12.

Lambert, J., Pautas, C., Terré, C., Raffoux, E., Turlure, P., Caillot, D., ... & Castaigne, S. (2019).

Gemtuzumab ozogamicin for de novo acute myeloid leukemia: final efficacy and safety updates from the open-label, phase III ALFA-0701 trial. Haematologica, 104(1), 113.

MyHVP Newsletter | December 2021 | page 12

Journal | Call for papers Malaysian Journal of Human Genetics The Malaysian Journal of Human Genetics (MJHG) (eISSN: 2716-649X) is the official journal of Malaysian Node of the Human Variome Project and Malaysian Society of Human Genetics. The MJHG publishes high quality peer reviewed original research, case report, short report and review articles that covers all aspect of human genetics including molecular, clinical, pharmacogenetics, population genetics and functional genomics. MJHG is now indexed in MyJurnal and Google Scholar.

MyHVP Newsletter | December 2021 | page 13