Molecular Pathology by IHC001

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

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

Sample Probe

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

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

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 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

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

Inversion

<- EML4

ALK -> 20

EML4-ALK ->

EML4-ALK Genomic Re-arrangement

EML4 & ALK - Chrom 2p Opposite orientations

Inversion

<- EML4

ALK -> 6

Chrom 2 inversion/translocati on creates a fusion oncogene

EML4-ALK ->

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

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 N

30% tumour

N N

<20% tumour

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