NGS for Cancer Predisposition

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1 NGS for Cancer Predisposition Colin Pritchard MD, PhD University of Washington Dept. of Lab Medicine AMP Companion Society Meeting USCAP Boston March 22, 2015

2 Disclosures I am an employee of the University of Washington I have no financial stake in any of the tests I will discuss

3 Next Generation Sequencing (NGS) in Clinical Cancer Genetics

4 Outline of The Talk Types of NGS Cancer Predisposition Panels Not all NGS is the Same: Methods Matter Variant Interpretation and Reporting Clinical Vignettes

5 Rationale For NGS Predisposition Panels Multiple genes often considered in suspected hereditary cancers Traditional testing is stepwise, at high cost to the patient Cost of testing + cost of multiple visits

6 NGS Cancer Predisposition Panels Cancer Specific Risk 7 20 genes typically, colon cancer risk, other risk Pan Cancer Risk Panels genes typically, comprehensive cancer risk

7 Example of Cancer Specific Panel: ColoSeq Gene Primary Syndrome MLH1 Lynch syndrome MSH2 Lynch syndrome MSH6 Lynch syndrome PMS2 Lynch syndrome EPCAM Lynch syndrome APC Familial adenomatous polyposis MUTYH MUTYH associated polyposis CDH1 Hereditary diffuse gastric cancer PTEN Cowden syndrome STK11 Peutz Jeghers syndrome TP53 Li Frameni syndrome BMPR1A Juvenile polyposis SMAD4 Juvenile polyposis POLE Hereditary colon cancer POLD1 Hereditary colon cancer GALNT12 Hereditary colon cancer GREM1 Mixed polyposis AKT1 Cowden like syndrome PIK3CA Cowden like syndrome

8 How Are The Genes Selected?

9 Clinical Need Common hereditary cancer syndromes have overlapping clinical features Lynch Syndrome Familial Adenomatous Polyposis (FAP) Especially attenuated FAP (AFAP) MUTYH associated polyposis (MAP) Other rare colon cancer syndromes

10 Lynch/Polyposis Overlap Feature Lynch FAP (APC) AFAP (APC) MAP (MUTYH) # Polyps 0 50 usually > to >700 Inheritance dominant dominant dominant recessive Cancer(s) colon, endometrial, gastric, ovarian colon, small bowel, other colon, small bowel colon Median onset age cancer 39 cancer (16 polyps) cancer

11 Case 35 year old woman multiple colonic adenomas, but less than 100 Father died of colon cancer at age 55; Paternal uncle had colon cancer at 52 Differential diagnosis includes Lynch syndrome, attenuated FAP

12 Optimal Testing Strategy?

13 Case: Approach Taken 1 st APC and MUTYH common mutations negative 2 nd MUTYH full gene sequencing negative 3 rd MLH1, MSH2, MSH6 sequencing, EPCAM del/dup analysis negative 4 th PMS2 sequencing and del/dup analysis COST: ~$10,000

14 Comprehensive Panels: Clinical Need Hereditary cancer syndrome suspected but gene(s) to test first not clear Common cancer syndromes frequently have overlapping clinical features Breast/ovarian cancer Screen for multiple genes simultaneously Particularly useful in high risk BRCA1/2 negative

15 Many Genes Beyond BRCA1/2 Ovarian Cancer Series Walsh et al PNAS (PMID: )

16 Example of Comprehensive Panel BROCA panel developed by Tom Walsh and Mary Claire King

17 Outline of The Talk Types of NGS Cancer Predisposition Panels Not all NGS is the Same: Methods Matter Variant Interpretation and Reporting Clinical Vignettes

18 Myth #1: NGS is inferior to Sanger sequencing in terms of analytic sensitivity and specificity This myth is based on older NGS technology and exome or genome sequencing at low depth of coverage. Targeted clinically validated deep sequencing panels generally outperform Sanger.

19 Myth #2: NGS cannot reliably detect copy number changes and therefore alternate methods are required for deletion/duplication analysis NGS that preserves gene dosage coupled with the appropriate bioinformatics generally outperforms traditional copy number detection methods.

20 Target Enrichment Approach Matters Capture Multiplex PCR Scalability Higher Lower Cost Higher Lower Turnaround Time Longer Shorter Single nucleotide variants Yes Yes Copy number variants Yes, robust Usually no Structural variants Yes Usually no Large indel variants Yes Challenging

21 Example Capture-Based Target Enrichment Prepare DNA in Libraries Capture Genes of Interest Molecular Bar Coding (up to 96 per lane) Sonicate DNA Millions of 101 bp reads Sequencing Slide courtesy of Tom Walsh, modified

22 Copy Number Variant Detection By NGS Read Depth Deletion of MSH2 Exons 1-6 Adapted from Pritchard et al JMD (PMID: )

23 NGS One Stop Genetics Shopping Genome Variation Large Del/Dup Structural Rearrangements Indels SNVs Technology Used Clinically MLPA, Microarray, FISH G banding, FISH Various, Sanger Sequencing Various, Sanger Sequencing NGS? Yes Yes Yes Yes Indel= small insertion/deletion, SNV= single nucleotide variant

24 Outline of The Talk Types of NGS Cancer Predisposition Panels Not all NGS is the Same: Methods Matter Variant Interpretation and Reporting Clinical Vignettes

25 Cancer Predisposition Panel Reporting Population frequency is key Consider ethnicity Not all genes are equal, consider pre test probability in interpretation 0 or 1 finding reported is typical Expect follow up

26 Number of Genes Matters Probability a Rare Missense Variant is Pathogenic (PPV) Number of Genes Probability of Detecting a Rare Missense Variant

27 Probability a Rare Missense Variant is Pathogenic (PPV) Reporting Risk/Benefit net benefits favor reporting potential harms favor not reporting Number of Genes

28 Example Filters for Short List Internal database ExAC, EVS, DGV, ClinVar 1000 genomes, mutation databases Filter common variants Pathogenic mutations Variants of uncertain significance Done Evolutionary conservation, splice prediction in silico tools

29 Consider Multi Director Data Review Short list of variants selected by each member of an interpretation team Combined lists reviewed together by interpretation group Decisions made on interpretation

30 Outline of The Talk Types of NGS Cancer Predisposition Panels Not all NGS is the Same: Methods Matter Variant Interpretation and Reporting Clinical Vignettes

31 Case 1 Woman with clinical Cowden Syndrome Dysplastic gangliocytoma of the cerebellum (Lhermitte Duclos disease) PTEN full gene sequencing and del/dup analysis negative by Sanger sequencing NGS colon predisposition panel ordered

32 Mosaic Mutations Detected NGS del AG Sanger PTEN 2bp deletion, frameshift Present in 21 out of 1184 reads (1.7%) Pritchard et al Genet Med (PMID: ) Frameshift visible but below limit of reliable detection

33 Mosaic Mutations Detected del AG Tissue % Mutant Allele Peripheral Blood (PB) <5% Skin Fibroblasts ~50% Cerebellar Tumor ~50% Colonic Mucosa ~25% Endocervical Mucosa ~25% Pritchard et al Genet Med (PMID: )

34 Case 2 Woman in 40s with numerous colorectal polyps (too many to count) Family history of colon and breast cancers NGS colon cancer predisposition panel ordered

35 APC Single Exon Inversion Detected APC exon 10 RNA Studies: Skipping of APC exon 10, premature stop exon 9 exon 11 Shirts et al Genet Med (PMID: )

36 Summary/Conclusions Next generation sequencing is replacing older methods for cancer predisposition testing Targeted gene panels allow efficient testing with high sensitivity and accuracy Variant interpretation is aided by databases, multi director review, and clinical history

Thank You UW Lab Medicine and Pathology John Tait

Angie Jacobson Lauren Thomas Tina Lockwood Larry True

of Chicago Jane Churpek Funmi Olopade UW Medical

Bennett SCCA and FHCRC Pete Nelson Heather Cheng Liz

37 Thank You UW Lab Medicine and Pathology John Tait Brian Shirts Steve Salipante David Wu Noah Hoffman Angie Jacobson Lauren Thomas Tina Lockwood Larry True Yajuan Liu Univ. of Chicago Jane Churpek Funmi Olopade UW Medical Genetics Tom Walsh Mary-Claire King Ming Lee Robin Bennett SCCA and FHCRC Pete Nelson Heather Cheng Liz Swisher Bill Grady Ed Lin The Ohio State University Heather Hampel Albert de la Chapelle Paul Goodfellow

COLON CANCER & GENETICS VERMONT COLORECTAL CANCER SUMMIT NOVEMBER 15, 2014

COLON CANCER & GENETICS VERMONT COLORECTAL CANCER SUMMIT NOVEMBER 15, 2014 WENDY MCKINNON, MS, CGC CERTIFIED GENETIC COUNSELOR FAMILIAL CANCER PROGRAM UNIVERSIT Y OF VERMONT MEDICAL CENTER 1 CHARACTERISTICS