Molecular Pathology

Molecular Pathology

George Burghel January 2020

Cancer genomics North West Genomics Laboratory Hub

Overview • • • •

• • • •

Human genome and DNA variation Molecular pathology testing - techniques Cancer Pharmacogenetics Pre-analytical considerations and importance of pathology information Services offered Pathology review – key to success Deceased index services Example Questions

Human genome and variation > 3 billion base pairs

We share most of it (>99.9%)

Human genome and variation > 3 billion base pairs

We share most of it (>99.9%)

Human genome and variation GTGGCGCGAGCTTCTGAAACTAGGCGGCAGAGGCGGAGCCGCTGTGGCACTGC GAGGCGCGAGCTTCTGAAACTAGGCGGCAGAGGCGGAGCCGCTGTGGCACTGC

GTGGCGCGAGCTTCTGAAACTAGGCGGCAGAGGCGGAGCCGCTGTGGCACTGC GTGGCGCGAGCTTCTGAAACTA GCAGAGGCGGAGCCGCTGTGGCACTGC

100,000s of SNVs and small scale variants (<50bp) in any human genome

Human genome and variation • Structural variants (SVs) are large type of DNA variation – CNVs (large deletions and gains >50bp) – Translocations – Inversions

Molecular pathology techniques • • • • • • • •

Southern blot PCR Quantitative PCR Karyotyping FISH Array Comparative Genome Hybridisation DNA Sequencing Tissue microarray

Southern Blot

Principles of PCR • Initial step in many/most downstream mutation detection methods • PCR in itself does not detect the mutations • Create a huge copy number of the region of interest • Primer design is the trick

Principles of PCR • PCR stages – Initial denaturation(~95oC): Complete denaturation of template at start of PCR is vital – 3 major cyclic steps (25-40 cycles) • Denaturation (~95oC) • Annealing (~55oC) • Extension (~72oC)

– Final extension (~72oC): fill in protruding ends of newly synthesised PCR products

Quantitative PCR - The more starting material there is, the quicker the sample will reach threshold cycle Sample A Sample B Sample C

Fluorescence

Fluorescence level too low for detection….

Threshold cycle

Amplification cycles

Karyotyping

Karyotyping

Karyotyping

Translocation between chromosomes 4 and 11

Karyotyping

Karyotyping PML PCR q24

Chromosome

q21

RARA

der(15) 15 der(17) 17

Chromosome translocation t(15;17)(q24;q21)

Acute promyelocytic leukemia

Lancet. 2004 May 15;363(9421):1633-41.Preimplantation genetic diagnosis.Sermon K, Van Steirteghem A, Liebaers I. http://sarcomahelp.org/research_center/chromosomal_translocations.html

all-trans retinoic acid (ATRA)

Fluorescence in situ hybridization (FISH) – Interphase or metaphase chromosomes Chromosome

9

Sample Probe

22

Tissue

aCGH Cancer samples

GACTGAGAATGCTTCGGCAAGACTCAAAAAATAGCTGGACTG A T G C T T C G G C A A G AC T CA A A A A A T A

GACTGAGAATGCTTCGGCAAGACTCAAAAAATAGCTGGACTG AT G G A GA AT G T G A AT G AT G

C C C C C C C AT G C G C A GA AT G C

T T T T T T T T T T

T T T T T T T T T T

C C C C C C C C C C

GG GG GG GG GG GG GG GG GG GG

C C C C C C C C C C

A A A A A A A A A A

A A A A A A A A A A

G AC G AC G AC G AC G AC G AC G AC G AC G AC G AC

T T T T T T T T T T

CA CA CA CA CA CA CA CA CA CA

A A A A A A A A A A

A A A A A A A A A A

A A A A A A A A A A

A A A A A A A A A A

A T A A T AG C

A A A A A A

T T T T T T

A G A AG C T A AG C

Tissue microarray Individual paraffin blocks

Hollow needle used to remove tissue cores from regions of interest

(Recipient block) tissue microarray block

Why test, what can we do?

• Better cancer diagnosis • Cancer Prevention • Stratified Therapy Identification of actionable mutations help in informing clinical management and personalising therapy (diagnosis, prognosis and treatment) – Cancer Pharmacogenetics

Services offered at MCGM

FFPE BRCA1/2 for treatment purposes

Pre-analytical considerations

DNA extraction – FFPE tissue

• • • •

Formalin fixation preserves tissue structure well BUT DNA is cross-linked to other DNA or proteins Breaking cross links - DNA becomes damaged Damaged DNA may contain errors or not be analysable • • •

• •

KEY FACTORS FOR SUCCESSFUL GENETIC ANALYSIS Rapid fixation, use fresh 4% neutral buffered formalin Controlled fixation time 24 – 36hrs Freshly cut sections (unstained slide mounted/scrolls) Avoid cross contamination, clean microtome blade Sterile/clean plasticware for transport

DNA Extraction

DNA damage • DNA in FFPE tissue becomes damaged during fixation and storage • Damage can take several different forms: single strand nicks, double strand breaks, oxidised bases, depurination, deamination, DNA-protein cross-links • Most DNA damage results in a DNA molecule/strand resistant to analysis BUT deaminated DNA leads to artefactual C>T or G>A base change

Deaminated base > in vitro mutation

Depurinated base > not analysable

Pathology review – key issues • Genetic tests are DNA based – Neoplastic cell content is number of nucleated cells, not cross sectional area – Necrotic or calcified tissue contributes little/no DNA e.g neoadjuvant radiotherapy/chemotherapy Necrotic cells, few nuclei, reduced DNA contribution Large cells, few nuclei, reduced DNA contribution Small cells, many nuclei, increased DNA contribution

• Remember to include tumour infiltrating lymphocytes as normal cells

Macrodissection – increase NCC Macrodissection can increase sensitivity for somatic mutation detection

Whole tissue section

Macrodissected Depends on distribution of neoplastic tissue being suitable H&E guide slide should be from a neighbouring section Best practice to always macrodissect when possible

7.6% mutant

30.7% mutant

Heterogeneity within pathology blocks • KRAS mutation in tumours • Heterogeneity present in 8% of tumours

200uM 10uM 200uM

KRAS c.38G>A p.(Gly13Asp)

Normal

10uM 200uM

Normal

10uM 200uM

Mutant – 13.5%

10uM 200uM

Mutant – 20.0%

• Serial sections from an FFPE block • Known KRAS c.38G>A p.(Gly13Asp) • EQA validation • 6/12 (50%) blocks showed heterogeneity across 1000uM • Estimate neoplastic cell content from neighbouring section

Multiple testing options

NGS Real Time PCR

Pyro-sequencing

??

MALDI-TOF

Sanger sequencing

FISH

Which test method – Sensitivity vs LOD • Two key aspects affecting genetic test effectiveness are relevant

– Ability to detect wide spectrum of clinically relevant mutations - sensitivity – Ability to detect clinically relevant mutations at low admixture levels in normal DNA – limit of detection (LOD) More mutations - sensitivity Sanger

Low levels - LOD

15% MAF All KRAS & NRAS muts

10% MAF KRAS & NRAS mut hotspots <5% MAF KRAS codon 12,13 & 61

Pyroseq Therascreen Fragment Cobas

NGS

2% MAF KRAS/NRAS/BRAF/PIK3CA

Colorectal Cancer • NICE approved the use of antiEGFR monoclonal antibodies as 1st line treatment for metastatic CRC with resectable liver mets • Recombinant monoclonal antibody that blocks EGFR • Likelihood of response high if tumour is KRAS (and NRAS) wild type

KRAS and NRAS (BRAF and PIK3CA) • Tested by NGS – 2% sensitivity • Oncogenes • RAS family of genes • Involved in the MAPK pathway • This pathway is a cascade pathway downstream of EGFR

EGFR blocking with Monoclonal antibodies

Anti-EGFR monoclonal antibodies (cetuximab & panitumumab) prevent ligand binding~10% of CRC tumours respond to monotherapy Ciardello & Tortora (2008) N Engl J Med;358:1160-1174

Response to EGFR Mab Inhibition • •

0% KRAS/NRAS mutant tumours respond to antibody therapy 17% KRAS wild type responded

KRAS codon 12/13 Mutant

KRAS codon 12/13 Wild type

Siena et al (2009) J Nat Cancer Inst; 101: 1168-1176

Reporting

Melanoma • NICE approved the use of Vemurafenib (Zelboraf) for treatment of BRAF V600 mutation positive unresectable or metastatic melanoma • Positive test for BRAF V600 mutation is required before starting treatment

BRAF (NRAS and KIT) • Family of serine-threonine protein kinases (RAF) • Central mediators in MAP kinase pathway ‒ therefore involved in many cellular processes including proliferation, differentiation and transcriptional regulation

• Mutant BRAF implicated in pathogenesis of several cancers, including melanoma, colorectal cancer, glioma and GIST

Reporting

NSCLC • NICE approved the use of Erlotinib as 1st line treatment for locally advanced or metastatic NSCLC if tumour is positive for an EGFR mutation • EGFR activating mutations seen in 10-15% of NSCLC patients • Erlotinib (Tarceva): active inhibitor of EGFR tyrosine kinase (EGFR-TKI)

Genetics of NSCLC Driver mutations identified in >50% of NSCLC cases

Potential for drug therapy or clinical trials

Sharma et al (2010) Nature Reviews Cancer 10, 241-253 Sharma et al (2010) Nature Reviews Cancer 10, 241-253

Mok, T.S., et al. (2009).NEJM, 361(10), pp.947-957.

Spectrum of EGFR Mutations

Reporting

Reporting

ALK testing • NICE approved the use of Crizotinib as an option for previously treated ALK-positive advanced NSCLC • Crizotinib (Xalkori): active inhibitor of ALK tyrosine kinase and its variants

EML4-ALK Genomic Re-arrangement

EML4 & ALK - Chrom 2p Opposite orientations

EML4-ALK Genomic Re-arrangement

EML4 & ALK - Chrom 2p Opposite orientations

7

6

Inversion

5

19

<- EML4

20

ALK -> 20

6

EML4-ALK ->

21

21

5

EML4-ALK Genomic Re-arrangement

EML4 & ALK - Chrom 2p Opposite orientations

7

6

Inversion

5

19

<- EML4

21

ALK -> 6

5

Chrom 2 inversion/translocati on creates a fusion oncogene

20

20

EML4-ALK ->

21

ALK Sensitive to inhibition by crizotinib

Sample processing • FISH testing to detect ALK fusion genes • Pathology review is key to guiding which cells to target • Thickness of sections also important • Positive result: at least 15% cells show a signal pattern consistent with a rearrangement

Example of results

Analysing Fusion Genes with NGS Technology

Ovarian cancer • NICE approved the use of Olaparib as monotherapy for maintenance treatment of patients with platinum-sensitive relapsed BRCA-mutated tumours • Olaparib (Lynparza): poly-ADP-ribose polymerase (PARP) enzyme inhibitor that selectively kills tumour cells with an impaired homologous recombination DNA repair pathway while sparing normal cells

Synthetic Lethality

• Poly (ADP-ribose) polymerase (PARP) • Inhibiting PARP mediated DNA repair combined with DNA damage from Platinum chemotherapy promotes apoptosis • Olaparib (Lynparza) is a PARP inhibitor • All coding exons in BRCA1 and BRCA2

Reporting

Pathology Review – key to success •

Pathology Review is key to success

N N N N N

SANGER SEQUENCING T T

x

T T T

Only HALF signal is mutant if ALL cells are tumour

Neoplastic cell content Neoplastic cell content (TCC) of sample is important N

N

T

N

N N

T

30% tumour

N

N N

T

N N

N

<20% tumour

• Lower neoplastic cell content > below LOD of assay > higher risk of false negative result • Neoplastic cell content 2x test LOD

Macrodissection increases DNA from neoplastic cells

Negative

BRAF V600E

Macrodissection increases DNA from neoplastic cells

Negative

BRAF V600E

Macrodissection increases DNA from neoplastic cells

Negative

BRAF V600E

Patient X sample

Macrodissection increases DNA from neoplastic cells

Negative

BRAF V600E

Patient X sample

Macrodissection increases DNA from neoplastic cells

Negative

BRAF V600E

Patient X sample

Patient X sample - macrodissected

Negative reports and NCC

Negative reports and NCC

Negative reports and NCC

Deceased index cases • Identification of germline cancer predisposition mutations in deceased patients • Hereditary colorectal (Lynch and FAP) and HBOC

Deceased Colorectal Cancer case 44 year old woman referred to discuss her family history of polyposis. Father underwent ileostomy in his early 30’s due to multiple bowel polyps and died age 70 in 2016.

Multiple bowel polyps Ileostomy 1976

Normal tissue sample 2015 from Whipple procedure Pathology review confirms normal tissue 43yo

44yo

13yo

18yo

10yo

Adult family members undergoing regular gastroscopies and colonoscopies for several years

Deceased Colorectal Cancer case 44 year old woman referred to discuss her family history of polyposis. Father underwent ileostomy in his early 30’s due to multiple bowel polyps and died age 70 in 2016.

Multiple bowel polyps Ileostomy 1976

>99.9% APC gene coverage at 100x APC c.1863_1866delTTAC p.(Tyr622GlyfsTer7) 48.5% reads

43yo

44yo

Testing offered to daughter 1 Normal for APC c.1863_1866delTTAC p.(Tyr622GlyfsTer7)

13yo

18yo

10yo

APC c.1863_1866delTTAC p.(Tyr622GlyfsTer7) confirmed heterozygous on Sanger analysis

Testing offered to daughter 2 Normal for APC c.1863_1866delTTAC p.(Tyr622GlyfsTer7)

MCQ

Question Answer

Using this technique, cores from hundreds of paraffin blocks are transferred into a recipient block used for high throughput immunohistochemical staining and analysis: A. Tissue microarrays B. DNA microarrays C. Western blot D. q-PCR E. CGH

MCQ

Question Answer

To screen a particular genetic alterations in DNA extracted from a tumour, an appropriate test to do is: A. Northern blot B. Western Blot C. Gene expression microarray D.qPCR E. Comparative genomic hybridization (CGH) There may be multiple correct answers

MCQ

Question Answer Which of the chromosome on the right indicate a chromosomal translocation?

B C

A. B. C. D.

A D

EMQ Option A

Option B

Option C

Option D

Option E

Quantitative PCR

PCR

Microarray

Western blot

Northern blot

Option F

Option G

Option H

Option I

Option J

FISH

Comparative genomic hybridization

Laser capture microdissection

Proteomics

Southern blot

Question Answer Which refers to the large-scale study of proteins, particularly their structures and functions

EMQ Option A

Option B

Option C

Option D

Option E

Quantitative PCR

PCR

Microarray

Western blot

Northern blot

Option F

Option G

Option H

Option I

Option J

FISH

Comparative genomic hybridization

Laser capture microdissection

Proteomics

Southern blot

Question Answer Allows quantitative measurement of the amount of DNA or RNA in a sample

EMQ Option A

Option B

Option C

Option D

Option E

Quantitative PCR

PCR

Microarray

Western blot

Northern blot

Option F

Option G

Option H

Option I

Option J

FISH

Comparative genomic hybridization

Laser capture microdissection

Proteomics

Southern blot

Question Answer Based on the hybridization of fluorescently labelled tumour DNA and normal DNA to normal human metaphase preparations.

EMQ Option A

Option B

Option C

Option D

Option E

Quantitative PCR

PCR

Microarray

Western blot

Northern blot

Option F

Option G

Option H

Option I

Option J

FISH

Comparative genomic hybridization

Laser capture microdissection

Proteomics

Southern blot

Question Answer It analyses copy number changes in the DNA content of a given subject's DNA/tumour cells.

EMQ Option A

Option B

Option C

Option D

Option E

Quantitative PCR

PCR

Microarray

Western blot

Northern blot

Option F

Option G

Option H

Option I

Option J

FISH

Comparative genomic hybridization

Laser capture microdissection

Proteomics

Southern blot

Question Answer A method for probing for the presence of a specific DNA sequence within a DNA sample