copyright by kathlene gravelin

Suspected Germline Variants in SNaPshot NGS Tumor Genotyping and Genetic Counseling Implications

Master’s Thesis Presented to

The Faculty of the Graduate School of Arts and Sciences

Brandeis University Graduate Program in Genetic Counseling Gayun Chan-Smutko, MS, LGC, Advisor

In Partial Fulfillment of the Requirements for the Degree

Master of Science in

Genetic Counseling

by Kathlene Gravelin

May 2016

Copyright by

Kathlene Gravelin

© 2016

iii

ACKNOWLEDGEMENTS

I would like to express my sincerest appreciation to my thesis advisor, Gayun Chan-

Smutko, MS, LGC, and committee members, Elaine Hiller, MS, LGC and Valentina Nardi, MD.

This project would not have been possible without their expertise, insight, and guidance. I am also

deeply grateful for the encouragement I have received from Judith Tsipis, Ph.D., Gretchen

Schneider, MS, CGC, Missy Goldberg, Gayun Chan-Smutko, MS, LGC, and all of the Brandeis

University Genetic Counseling Program faculty throughout the duration of my studies. Most

importantly, I would like to thank my husband Rich for his patience, love and unwavering support

in my pursuit of my dream to become a genetic counselor.

iv

ABSTRACT

Suspected Germline Variants in SNaPshot NGS Tumor Genotyping and Genetic Counseling Implications

A thesis presented to the Graduate Program in Genetic Counseling

Graduate School of Arts and Sciences Brandeis University

Waltham, Massachusetts

By Kathlene Gravelin

SNaPshot next generation sequencing (NGS) tumor genotyping is used to guide clinical

decision-making in the diagnosis, prognosis and treatment of cancer patients at Massachusetts

General Hospital. Analysis focuses on somatic alterations in tumor tissue; however, variants of

suspected germline origin may be incidentally identified. Our study examined how frequently

suspected germline variants (SGVs) were detected for non-lung cancer patients who underwent

SNaPshot NGS between December 1, 2014 and May 31, 2015. We selected cases with at least 1

variant present at an allelic fraction between 42-58% in a gene associated with a hereditary cancer

syndrome in order to identify cases likely to have SGVs warranting genetic counseling referral.

We used ClinVar, COSMIC and ExAC to assess the clinical germline significance for each SGV.

Thirty-eight of 599 patients (6.3%) had at least one deleterious SGV of potential clinical germline

significance; 98 patients had SGVs that were deemed to have low potential for clinical germline

significance. Report annotation indicating suspected germline origin was present for 19.5% of

deleterious SGVs of potential clinical germline significance, and for 32.5% of SGVs of low

potential for clinical germline significance. We determined risk for hereditary cancer syndromes

v

by considering SGV assessment and concordance with personal and/or family history; 11 patients

were considered to be at high or moderate risk for a hereditary cancer syndrome. We did not find

evidence of genetic counseling referrals made based on SGVs in the timeframe of our study. Our

data show that SGVs of clinical significance for hereditary cancer syndromes may be detected

through SNaPshot NGS tumor genotyping, and highlight the challenges in the reporting of and

genetic counseling follow-up for SGVs. A multi-disciplinary effort involving medical oncology,

genetics and molecular pathology practices may be an optimal approach to identifying patients at

high risk for a hereditary cancer susceptibility.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................... iii

ABSTRACT ................................................................................................................................... iv

LIST OF TABLES ........................................................................................................................ vii

LIST OF FIGURES ..................................................................................................................... viii

INTRODUCTION .......................................................................................................................... 1

METHODS ..................................................................................................................................... 5

RESULTS ....................................................................................................................................... 9

DISCUSSION ............................................................................................................................... 19

CONCLUSIONS .......................................................................................................................... 25

REFERENCES ............................................................................................................................. 26

APPENDICES .............................................................................................................................. 28

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LIST OF TABLES

Table 1: NGS SNaPshot genes associated with hereditary cancer syndromes ............................... 7

Table 2: Report annotation for suspected germline variants (SGVs) ........................................... 10

Table 3: Genetic counseling referral outcome by risk for hereditary cancer syndrome ............... 16

Table 4: Patients at high risk for hereditary cancer syndrome based on SNaPshot NGS results . 17

Table 5: Patients at moderate risk for hereditary cancer syndrome based on SNaPshot NGS

results .................................................................................................................................... 18

Supplemental Table 1: SNaPshot NGS genes and targeted exons ............................................... 28

viii

LIST OF FIGURES

Figure 1: Suspected germline variants (SGVs) by gene, potential for clinical germline

significance ........................................................................................................................... 10

Figure 2: Suspected germline variants (SGVs) by tumor type, patients with concordant variants

............................................................................................................................................... 12

1

INTRODUCTION

Genetic profiling of tumor samples is part of an emerging genomics-driven approach to

cancer treatment referred to as personalized or precision cancer medicine in which a patient’s

genetic profile is used to guide clinical decision-making in the diagnosis, prognosis and treatment

of cancer (Garraway, Verweij, & Ballman, 2013; Jackson & Chester, 2015). Its implementation in

clinical practice has grown in response to both an increased body of knowledge of tumor-specific

somatic genetic alterations that drive molecular pathways involved in tumor survival and

progression, and the development of anticancer agents targeting these pathways (Garraway, 2013).

A number of cancer research institutions and diagnostic companies offer clinical-level tumor

genetic profiling services that interrogate somatic genetic alterations in tumor biopsy specimens

obtained during the course of a patient’s treatment (Hansen & Bedard, 2013; Shen, Pajaro-Van de

Stadt, Yeat, & Lin, 2015). Due to the non-heritable nature of somatic mutations confined to tumor

tissue, genetic counseling considerations in the genetic profiling of tumor samples have not been

extensively studied. However, recent ACMG guidelines regarding the return of incidental germline

findings in the context of clinical exome and genome sequencing, coupled with preliminary data

indicating the detection of potential incidental germline variants in hereditary cancer susceptibility

genes in the course of tumor somatic mutation analysis, suggests a need for the utilization of

genetic counseling services.

A majority of the currently available tumor genetic profiling assays use a targeted approach

which interrogates single genes or a panel of genes with known clinical implications in cancer

(Shen et al., 2015). Gene panels include known oncogenes and tumor suppressor genes, as well

2

additional genes whose mutational status can be used to predict susceptibility or resistance to

existing drugs or investigational therapies, or inform prognosis (Dias-Santagata et al., 2010;

Frampton et al., 2013; MacConaill et al., 2014). Newer assay platforms utilizing targeted

massively parallel DNA sequencing techniques have expanded both the number of genes that can

be analyzed simultaneously and the types of detectable genetic alterations (Frampton et al., 2013;

Wagle et al., 2012; Zheng et al., 2014).

Although genetic profiling of tumor samples focuses on genes known to have somatic

mutations in cancer, estimates show that 10% of genes involved in cancer can harbor both somatic

and germline mutations (Futreal et al., 2004). Tumor-only assay platforms employ a

bioinformatics-based approach using a pool of unmatched reference normal samples and further

filters to distinguish variants that are somatic in origin. There is, however, evidence to indicate that

the bioinformatics-based approaches in tumor-only analysis are insufficient to fully distinguish

somatic variants from germline variants, and that a matched germline sample is required to identify

true somatic alterations (Jones et al., 2015).

Recent studies have provided a preliminary indication of the frequency with which

germline variants in known hereditary cancer susceptibility genes are detected via NGS-based

tumor profiling platforms. A study by Catenacci et al. revealed the potential for detecting germline

variants in tumor-only analysis platforms. They showed that deleterious mutations in genes

associated with hereditary cancer syndromes were found in 18.9% of gastrointestinal tumors

profiled with FoundationOne®, and were confirmed in germline samples for 42.8% of the patients

for which follow-up was possible. (Catenacci et al., 2015) Additional studies by Schrader et al.

and Meric-Bernstam et al., which looked at data from matched tumor-normal assay platforms,

showed that pathogenic mutations in genes associated with cancer susceptibility were detected in

3

the comparator germline sample at frequencies of 12.6% and 4.6%, respectively; furthermore, the

identified germline variants were retained in the matched tumor sample for a majority of the cases

(Meric-Bernstam et al., 2016; Schrader et al., 2016).

Coupled with emerging data regarding the prevalence of secondary germline findings in

targeted tumor genetic profiling, there are growing concerns about how to proceed with the return

of incidental germline findings to patients. These concerns are in part fueled by the 2013 American

College of Medical Genetics and Genomics (ACMG) recommendations for return of secondary

findings in the context of clinical exome and genome sequencing. The ACMG proposed a minimal

list of genes for which secondary findings should be reported, and it includes 25 genes known to

be associated with hereditary cancer syndromes.(Green et al., 2013). The ACMG included the

matched normal sample of any tumor-normal subtractive analyses in the scope of their

recommendations, but did not provide guidance regarding secondary germline findings

encountered in tumor-only analysis. (Green et al., 2013) There is however a recognized need for

the implementation of a framework for managing the reporting of secondary germline findings as

part of tumor genetic profiling (Bombard, Robson, & Offit, 2013; Parsons, Roy, Plon,

Roychowdhury, & Chinnaiyan, 2014).

The ACMG Working Group on Incidental Findings in Clinical Exome and Genome

sequencing places the responsibility for the management of results interpretation and counseling

issues that may arise in the return of secondary germline findings on the ordering clinician, but

recommends consultation with a clinical geneticist given the complexity of genomic information.

(Green et al., 2013). However, data suggest that ordering oncologists receiving potential germline

results may not be equipped to bear this primary responsibility. A study of US physicians’ attitudes

toward genetic counseling for cancer susceptibility revealed that only 50% of oncologists felt

4

qualified to provide genetic counseling to their patients, and most oncologists felt that clinical

geneticists and genetic counselors were most qualified to provide genetic counseling services

(Freedman et al., 2003). Moreover, in a more recent survey of clinically active adult cancer

physicians at a major cancer center, 22% of responding physicians reported low confidence in their

genomic knowledge. (S. W. Gray et al., 2012)

Although clinical geneticists and genetic counselors are trained to counsel patients about

the implications of genetic tests and their results, and to interpret complex genetic testing data, the

extent of their involvement clinical tumor genetic profiling is not clear. Data from the previously

described research studies suggests a high uptake in the use of genetic counseling services

following receipt of a potential or confirmed germline mutation in clinically actionable gene, with

almost 100% of patients choosing to receive genetic counseling (Catenacci et al., 2015; Meric-

Bernstam et al., 2016). In addition, there is one published case study in which a patient with

incidental germline findings reported as part of tumor only profiling was referred to genetic

counseling prior to confirmatory germline testing as part of their clinical care (Varga, Chao, &

Yeager, 2015).

To gain a better understanding of how frequently incidental suspected germline variants

are being reported in the context of tumor genetic profiling, we performed a retrospective review

of tumor genotyping results for patients with variants identified through the SNaPshot NGS tumor

genotyping platform at the Massachusetts General Hospital (MGH) Center for Integrated

Diagnostics. In addition, we further examined SNaPshot NGS tumor genotyping cases with

suspected germline variants to determine whether genetic counseling services were offered to

individuals considered to be at risk for hereditary cancer syndromes based on their tumor

genotyping results.

5

METHODS Genomic sequencing

The previously described SNaPshot NGS tumor genotyping assay platform, implemented

by the MGH Molecular Pathology Laboratory in April of 2014, utilizes a multiplex polymerase

chain reaction (PCR) technology called Anchored Multiplex PCR (AMP) (Zheng et al., 2014) to

detect single nucleotide variants (SNVs) and insertions/deletions (indels) in genomic DNA using

next generation sequencing (NGS). Briefly, genomic DNA was isolated from formalin-fixed

paraffin embedded tumor specimens (after histological review for tumor enrichment), blood, bone

marrow aspirates, or pancreatic cyst fluid. The genomic DNA was sheared with the Covaris M220

instrument, followed by end-repair, adenylation, and ligation with an adapter. A sequencing

library targeting hotspots and exons in 39 cancer-related genes across 152 exons (Supplemental

Table 1) was generated using two hemi-nested PCR reactions. Illumina MiSeq 2 x 151 base

paired-end sequencing results were aligned to the hg19 human genome reference using BWA-

MEM (Li & Durbin, 2009), MuTect (Cibulskis et al., 2013) and a laboratory-developed

insertion/deletion analysis algorithm were used for SNV and indel variant detection, respectively.

This assay is validated to detect SNV and indel variants at 5% allelic frequency or higher in target

regions with sufficient read coverage. The SNaPshot NGS bioinformatics pipeline employs a

multi-tier filter to exclude common genetic variants including silent variants, or variants found in

a pool of normal blood or FFPE specimens; common confirmed somatic variants found in the

COSMIC database (Futreal et al., 2004) as well as novel variants are however reported.

6

Participants

The participants were MGH patients with a confirmed cancer diagnosis who had a genetic

variant reported after undergoing SNaPshot NGS tumor genotyping in the MGH Molecular

Pathology Laboratory between December 1, 2014 and May 31, 2015; lung cancer patients were

excluded from our analysis due to their over-representation in the pool of SNaPshot NGS cases,

and presumed underrepresentation in hereditary cancer syndromes. We selected this timeframe

because: 1) It encompassed a period during which the SNaPshot NGS tumor genotyping platform

was employed regularly in tumor genotyping analysis. 2) It allowed for a 6-month period after

results reporting in which we could assess genetic counseling follow-up. 3) It limited the record

number to a quantity that could be reviewed in the timeframe of this study. All patients provided

their written informed consent for SNaPshot NGS tumor genotyping. A review of SNaPshot NGS

reports and clinical information was conducted under a protocol approved by the Partners Human

Research Committee and the Brandeis Institutional Review Board.

Variant Curation

In order to focus our review on cases likely to have reported suspected germline variants

(SGVs) warranting genetic counseling referral, we utilized an allelic fraction cut-off of 42-58% to

identify variants that may be of germline origin, and limited our review to cases with SGVs in

SNaPshot NGS genes associated with hereditary cancer syndromes (Table 1); this included cases

with SGVs that may or may not be of clinical significance. We used the ClinVar, COSMIC, and

ExAC databases to assess the clinical germline significance for each unique SGV. SGVs meeting

the following criteria were categorized as having high potential for clinical germline significance:

pathogenic or likely pathogenic germline classification in ClinVar, absent or infrequent somatic

7

annotation in COSMIC, and absent or very low (<1%) minor allelic frequency (MAF) in ExAC.

Variants were categorized as having low potential for clinical germline significance if they met

one or more of the following criteria: absent pathogenic or likely pathogenic classification in

ClinVar, not previously reported in the germline, or reported at high germline frequency in ExAC

(>1%), with or without previous somatic annotations in COSMIC. Variants were categorized as

having unclear potential for clinical germline significance if they had pathogenic or likely

pathogenic annotation in ClinVar, but are also well-known tumor variants as evidenced by multiple

entries in COSMIC.

Table 1: NGS SNaPshot genes associated with hereditary cancer syndromes

Gene Cancer syndrome OMIM® phenotype number(s)a

AKT1 Cowden-like syndrome 615109 ALK Familial neuroblastoma 613014 APC Familial adenomatous polyposis 175100 CDH1 Hereditary diffuse gastric cancer 137215 CDKN2A Melanoma-pancreatic cancer syndrome 606719 EGFR Familial lung cancer 211980 HRAS Costello syndrome 218040 KIT Familial GIST 606764 MET Familial papillary renal cancer 605074 PDGFRA GIST (rarely familial) 606764 PIK3CA Cowden-like 615108 PTEN Cowden syndrome, and related PTEN

hamartosis syndromes 158350

RET Multiple Endocrine Neoplasia 2A/2B 171400, 162300 SMAD4 Juvenile polyposis/HHT 174900, 175050 STK11 Peutz-Jeghers syndrome 175200 TP53 Li-Fraumeni syndrome 151623 VHL Von Hippel-Lindau syndrome 193300

aCancer syndrome information taken from Online Mendelian Inheritance in Man (OMIM®)

8

Criteria for clinical concordance and genetic counseling referral

We used the National Comprehensive Cancer Network (NCCN) guidelines ("NCCN

Clinical Practice Guidelines in Oncology," 2016), relevant literature (OMIM®, GeneReviews), and

clinical judgment to assess personal and family histories for the hereditary cancer syndromes listed

in Table 1. A personal and/or family cancer history was considered to be concordant with the

reported SGV if it displayed features consistent with the hereditary cancer syndrome associated

with the relevant gene. Similarly we used NCCN guidelines ("NCCN Clinical Practice Guidelines

in Oncology," 2016) for genetic risk evaluation, age of onset, relevant literature (OMIM®,

GeneReviews), and clinical judgment to identify cases with the strongest personal and family

histories for which genetic counseling referral would be indicated independent of clinical

concordance with identified SGV(s).

9

RESULTS Patients

One-hundred-thirty-six 136 of 599 (22.7%) non-lung cancer patients who had tumors analyzed

via SNaPshot NGS genotyping in the MGH Molecular Pathology Laboratory between December

1, 2014 and May 31, 2015 had at least one reported SGV. Patients with SGVs were 45.6% male

and 54.4% female, with a mean age of cancer diagnosis of 59.6 years. The patients had a diverse

range of tumor types, with colon and hematological cancers being the most prevalent (Figure 2).

Suspected germline variants

A total of 168 SGVs were reported for the 136 patients; 107 patients (78.7%) had 1 SGV,

26 patients (19.1%) had 2 SGVs, and 3 patients (2.2%) had 3 SGVs. The 168 SGVs consisted of

116 unique SGVs in 14 genes. The most commonly reported SGVs were in TP53 (n=46) and APC

(n=24); other genes with 5 or more unique SGVs included PTEN (n=10), CDH1 (n=9), and EGFR

(n=5) (Figure 1). The majority of SGVs (n = 89, 76.7%) were of low potential for clinical germline

significance. Of the remaining SGVs, 7 (6.0%) were determined to be of high potential for clinical

germline significance, and 20 (17.2%) were considered to be of unclear potential for clinical

germline significance.

10

Figure 1: Suspected germline variants (SGVs) by gene, potential for clinical germline significance

Report annotation

Further review of SNaPshot NGS reports showed that 42 of 136 patients (30.9%) had a report

with annotation indicating that one or more of their variants may be of germline origin. Suspected

germline annotation was more frequently present for variants of high (33.3%) and low (31.5%)

potential for clinical germline significance than for variants of unclear (17.1%) potential for

clinical germline significance (Table 2). Samples with suspected germline annotation had a lower

tumor content (43.5%) than samples without annotation (58.2%).

Table 2: Report annotation for suspected germline variants (SGVs)

SGV categorya No. SGVs (% of total)

No. SGVs with annotation (% of category)

High 6 (3.6) 2 (33.3) Unclear 35 (23.2) 6 (17.1)

Low 127 (73.2) 40 (31.5) Total 168 48 (28.6)

a Potential for clinical germline significance

0

5

10

15

20

25

30

35

40

45

Uni

que

SG

Vs,

No.

HighUnclearLow

Potential for clinical germline significance

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Clinical correlation

Thirty-five patients (25.8%) had a personal cancer history that was concordant with at least

one of their SGVs (Figure 2). Eleven colorectal cancer patients had SGVs in genes associated with

hereditary colon cancer (APC, STK11, and SMAD4). This included four patients with 2 unique

SGVs in APC; 3 patients with single APC SGVs, one of which had a second SGV in SMAD4; one

patient with a single SGV in SMAD4; and three patients with single SGVs in STK11. Eight

patients with TP53 SGVs had cancer diagnoses that fell within the Li-Fraumeni syndrome (LFS)

cancer spectrum. These included 4 patients with brain tumors; 3 patients with early-onset HER2+

breast cancer, and 1 patient with a soft tissue sarcoma. For one breast cancer patient with a TP53

SGV, concern for LFS was lower due to diagnosis at age 64. Four breast cancer patients had SGVs

in genes associated with other hereditary breast cancers (PIK3CA, CDH1, STK11). Four patients

with PTEN SGVs had uterine cancer diagnoses, which has been reported in Cowden syndrome.

Four pancreatic cancer patients had SGVs in the APC gene which is associated with Familial

Adenomatous Polyposis, one of whom had a second SGV in CDKN2A. Two additional patients

had tumors consistent with CDKN2A-associated cancers including one patient with melanoma and

one patient with a metastatic lesion suggestive of pancreaticobiliary origin. Eight patients had

cancer diagnoses that were not suggestive of the hereditary cancer syndrome associated with the

gene in which a SGV was reported, but had a family cancer history that was potentially consistent.

Figure 2: Suspected germline variants (SGVs) by tumor type, patients with concordant variants

a Patients with personal and/or family cancer history concordant with at least one reported SGV b SGV concordant with personal cancer history; SGV may also be concordant with family history c SGV not concordant with personal cancer history, but concordant with family cancer history

SGVs(SGVsconcordantwithcancerhistory) Patients

TP53

APC

CDH1

MET

PTEN

STK1

1

PIK3

CA

CDKN

2A

EGFR

VHL

ALK

SMAD

4

KIT

PDGF

RA

Total

Total

Concorda

nta

CRC 14(1) 11(11) 2 1 3(3) 1 1 2(2) 23 24 12Bonemarrow 6 5 3 3 2 1 1 21 19 0Esophagus 11(1) 1 1 1 1 15 12 1Pancreas 3(1) 4(4) 2(2) 1 1 1 12 11 7Breast 5(4) 2 1(1) 2 1(1) 4(4) 15 10 7Brain 4(4) 2 4(1) 1 11 10 5Melanoma 7 1 3(1) 1 1(1) 13 8 2Other 3 1 2(1) 1 7 7 1Uterus 3 5(5) 8 5 4Liver 1 1(1) 1 1 1 5 5 1HeadandNeck 1 1 1 2 5 4 0Stomach 2 2 4 4 0Biliarytract 2 1 3 3 0Bladder 2 1 3 3 0Thyroidgland 1 1 1 3 3 0Ovary 3 3 3 0Unknown 2 2 2 0Sarcoma 1(1) 1 1 1Prostate 1 1 1 0SquamousCellCarcinoma 1 1 1 1

Total 62 33 14 11 10 10 8 5 5 3 3 3 1 1 169 136 42 Concordantwithpersonalcancerhistoryb 35 Concordantwithfamilycancerhistoryc 8

12

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Identification of patients at risk for hereditary cancer syndromes We determined risk for hereditary cancer syndromes based on SNaPshot NGS tumor

genotyping results by considering SGV category and concordance with personal and/or family

history. If patients had greater than 1 reported SGV, the variant with the highest potential for

clinical germline significance was used for risk assessment (Table 3). Patients were considered to

be at a high risk for a hereditary cancer syndrome if they had a SGV of high potential for clinical

germline significance that was concordant with their personal and/or family cancer history.

Patients were considered to be at moderate risk for a hereditary cancer syndrome if they had a

SGV with unclear potential for germline significance that was concordant with personal and/or

family cancer history. In addition, patients with SGVs of high potential for clinical germline

significance that were discordant with personal and/or family cancer history were also considered

to be at moderate risk due to the potential for de novo germline mutations. Patients with SGVs of

unclear potential for clinical germline significance that were discordant with personal and/or

family cancer history, and patients with SGVs with low potential for clinical germline significance

were considered to be at low risk for a hereditary cancer syndrome.

We also examined personal and/or family cancer history for patients not meeting high risk

criteria based on SGV category and concordance. Thirty-seven patients had a personal and/or

family cancer history that was suggestive of a hereditary cancer syndrome either related or

unrelated to their reported SGV. These patients were also considered to be at high risk for a

hereditary cancer syndrome.

Review of genetic counseling follow-up We reviewed genetic counseling follow-up for all patients. Two patients considered to be

at high risk for a hereditary cancer syndrome based on SNaPshot NGS results had genetic risk

14

assessment performed by a genetic counselor (Table 4). In both cases, the assessment was made

based on clinical history, and neither patient had SNaPshot NGS report annotation indicating that

his/her reported variant may be of germline origin. One patient with endometrial cancer, a

component feature of Cowden syndrome, had a reported variant in PTEN. A genetic counselor

reviewed the patient’s immunohistochemistry (IHC), BRAF V600E mutation, and MLH1

promoter methylation analysis that was run concurrently with SNaPshot NGS tumor genotyping

per standard institutional Lynch syndrome screening practices. Further evaluation for Lynch

Syndrome was not suggested due to MLH1 promoter hypermethylation, but a genetics assessment

for additional hereditary cancer syndromes was recommended. There was no record of a follow-

up consult with a genetic counselor for this patient. A second patient, with early-onset colorectal

cancer and two reported variants in APC, was seen by a genetic counselor prior to SNaPshot NGS

and underwent ColoNext (Ambry) germline testing. No germline variants in APC were reported.

Of the 38 patients determined to be at high risk based on a personal and/or family cancer history

suggestive of a hereditary cancer syndrome, 17 (45%) met with a genetic counselor. The majority

of these patients were referred before SNaPshot NGS tumor genotyping

A referral to genetics was not evident for the 9 patients considered to be at moderate risk

for a hereditary cancer syndrome based on SNaPshot NGS results (Table 5). One patient had a

SNaPshot NGS report with annotation noting that the reported variant may be of germline origin.

The reported APC variant (c.3920T>A), which is a known risk factor for colorectal cancer in the

Ashkenazi Jewish population (Gryfe, Di Nicola, Lal, Gallinger, & Redston, 1999) , was discordant

with the patient’s smoldering myeloma diagnosis, and her medical record did not indicate

additional family cancer history; however, the patient’s medical record noted Eastern European

Ashkenazi Jewish descent.

15

Eighty-seven patients were considered to be at low risk for a hereditary cancer syndrome

based on SNaPshot NGS results; however, 29 patients had a SNaPshot NGS report noting that one

or more of their reported variants may be of germline origin. One colorectal cancer patient

considered to be at low risk for a hereditary cancer syndrome based on SNaPshot NGS results and

personal and/or family cancer history received a genetic counseling assessment as part of standard

Lynch syndrome screening.

Table 3: Genetic counseling referral outcome by risk for hereditary cancer syndrome

SUSPPECTEDGERMLINEVARIANT

PERSONALAND/ORFAMILYCANCERHISTORY

SNAPSHOTNGSREPORTANNOTATION

GENETICCOUNSELINGREFERRALS

RISKFORHEREDITARYCANCERSYNDROME

Potentialforclinicalgermlinesignificance

ConcordantwithSGV

Suggestiveofhereditarycancersyndrome

TotalPatients

Patientswithpotentialgermlineannotation

BeforeSNaPshotNGS

AfterSNaPshotNGS

Totalreferred

%referred

BASEDONSNAPSHOTNGSRESULTS

HIGH High + + 1 0 1 0 1 100 High + - 1 0 0 1 1 100MODERATE Unclear + - 6 0 0 0 0 0 High - - 3 1 0 0 0 0 11 1 1 1 2 2LOW Unclear - - 17 3 0 0 0 0 Low + - 12 4 0 1 0 9 Low - - 58 22 0 0 0 0 87 29 0 1 1 1

BASEDONPERSONALAND/ORFAMILYHISTORYHIGH High - + 1 1 1 0 1 100 Unclear + + 5 1 1 0 1 20 Unclear - + 4 2 1 0 1 25 Low + + 15 4 5 2 7 47 Low - + 13 4 5 2 7 54 38 12 13 4 17 45

136 42 14 6 20 15

16

Table 4: Patients at high risk for hereditary cancer syndrome based on SNaPshot NGS results

Variant(s) Report annotation

Age at diagnosis

Personal cancer history

Family cancer history GC referral outcome

HIGH POTENTIAL FOR GERMLINE SIGNIFICANCE; PERSONAL AND/OR FAMILY HISTORY CONCORDANT WITH VARIANT APC c.4473dupT ClinVar: VP germline; COSMIC: 5x somatic

No 45 Metastatic colon adenocarcinoma; intact MMR proteins.

No family history of malignancy

GC prior to SNaPshot; ColoNext multi-gene panel (Ambry); no reported germline variants in APC

PTEN c.493-1G>A ClinVar: VP germline; COSMIC: 5x somatic

No 88 Endometriod carcinoma; loss of MLH1/ PMS2, BRAF V600E-, MLH1 meth+

No history of GYN, colon cancers

GC reviewed IHC per Lynch screening; recommended genetics F/U for additional hereditary cancer syndromes. No record of F/U

17

Table 5: Patients at moderate risk for hereditary cancer syndrome based on SNaPshot NGS results

Variant(s) Report annotation

Age at diagnosis

Personal cancer history

Family cancer history GC referral outcome

HIGH POTENTIAL FOR GERMLINE SIGNIFICANCE; PERSONAL AND/OR FAMILY CANCER HISTORY DISCORDANT APC c.3920T>A ClinVar: 5x risk factor, 2x VP germline; COSMIC 4x somatic; ExAC: 17% MAF

Yes 63 Smoldering myeloma Eastern European Ashkenazi Jewish descent

No evidence of referral

SMAD4 c.1081C>T ClinVar: VP germline; COSMIC: 42x somatic

No 49 Metastatic esophageal adenocarcinoma

Mother: Colon, 65; Sibling: Lip Ca., 47


TP53 c.920-2A>G ClinVar: VLP germline; COSMIC: 20x somatic

No 67 Esophageal cancer Possible cancers in 1st/2nd degree relatives >65y


MODERATE POTENIAL FOR GERMLINE SIGNIFICANCE; PERSONAL AND/OR FAMILY CANCER HISTORY CONCORDANT APC c.4348C>T ClinVar: 2x VP germline; COSMIC:246x somatic

Yes 71 Invasive adenocarcinoma of sigmoid colon

Mother died of leukemia at “very young” age


APC c.2626C>T ClinVar: 1x VP germline; COSMIC: 113x somatic

No 65 Rectal adenocarcinoma

No CRC or GI cancer in the family


TP53 c.524G>A ClinVar: 5x VLP germline; COSMIC: >1100x somatic

No 42 Astrocytoma; malignant glioma

None available No evidence of referral

TP53 c.524G>A ClinVar: 5x VP germline; COSMIC: >1100x somatic; ExAC .082%

No 47 Paravertebral sarcoma Mother: lung Father: Prostate


TP53 c.817C>T ClinVar: 4x VP, 1X VLP, 1X VUS germline; COSMIC: 710x somatic

No 26 Glioblastoma Negative for brain tumors


TP53 c.1024C>T ClinVar: 2x VP germline; COSMIC: 146x somatic

No 62 Esophageal adenocarcinoma

Mother: colorectal polyps at 40; Son d. 33 glioblastoma


18

19

DISCUSSION

Tumor genetic profiling assays available at major cancer research institutions and through

commercial diagnostic companies are being utilized in the course of clinical care for cancer

patients. (Dias-Santagata et al., 2010; Frampton et al., 2013; Hansen & Bedard, 2013; MacConaill

et al., 2014; Shen et al., 2015) Although the intent is generally limited to characterizing genetic

information specific to patients’ tumor samples, it is becoming increasingly clear that the potential

exists to encounter information about a patient’s risk for heritable cancers in the course of tumor

genetic profiling. (Catenacci et al., 2015; Jones et al., 2015; Madlensky et al., 2014; Varga et al.,

2015) This can occur not only in the analysis of the normal germline sample in subtractive analysis

of matched tumor-normal pairs, but also in tumor-only analysis. (Jones et al., 2015) The present

study examined how frequently SGVs in hereditary cancer-associated genes were reported for

patients undergoing SNaPshot NGS tumor genotyping at the MGH Center for Integrated

Diagnostics in the course of their clinical care, and genetic counseling follow-up for patients with

suspected germline variant(s).

Overall, we found that 136 of 599 (22.7%) of non-lung cancer patients who had SNaPshot

NGS tumor genotyping in the timeframe of our study had at least one SGV in a gene associated

with a hereditary cancer syndrome. Ninety-eight patients (16.4%) had SGVs that were

subsequently deemed to have low potential for clinical germline significance, and 38 patients

(6.3%) had at least one deleterious SGV of potential clinical germline significance. Our observed

frequency of deleterious SGVs was lower than the 18.9% reported in study of gastrointestinal

20

patients who underwent FoundationOne® tumor genotyping (Catenacci et al., 2015), and was

more similar to the 3% - 12.6% confirmed germline deleterious mutations in cancer susceptibility

genes reported for cancer patients with unselected tumor types analyzed via matched tumor-normal

genotyping platforms (Jones et al., 2015; Meric-Bernstam et al., 2016; Schrader et al., 2016).

Although the frequencies observed in our study and previous studies cannot be directly compared

due to differences in assay platforms and patient populations, our results support the potential for

encountering SGVs associated with hereditary cancer syndromes in the course of tumor

genotyping.

We reviewed SNaPshot NGS reports to get gain a better understanding of current practices

for reporting SGVs at the MGH Center for Integrated Diagnostics. We found that 42 patients

(30.9%) with SGVs had a SNaPshot NGS report with annotation indicating that one or more of

the variants may be of germline origin. A low proportion (8 of 41; 19.5%) of SGVs of potential

clinical germline significance had annotation to this effect; conversely, a higher proportion (40 of

123; 32.5%) of SGVs of low potential for germline significance had annotation. This underscores

the inherent challenges of variant annotation in the tumor-only genotyping setting. While the 2013

ACMG recommendations for return of secondary findings in the context of clinical exome and

genome sequencing include reporting of findings in the normal sample in matched tumor-normal

genotyping, there are currently no guidelines for reporting incidental suspected germline variants

in the context of tumor-only genotyping (Green et al., 2013). Interpretation of the potential

germline significance for reported variants is secondary to the intent of tumor genotyping, which

is to identify tumor variants that may help inform treatment decisions or prognosis (Dias-Santagata

et al., 2010; Frampton et al., 2013; MacConaill et al., 2014). Variant interpretation is a time

21

consuming process, and it is often infeasible for busy pathology laboratories to perform a detailed

curation to investigate the potential clinical germline significance for each detected variant.

Annotation indicating suspected germline origin for variants detected in SNaPshot NGS

samples analyzed at the MGH Center for Integrated Diagnostics is currently added at the discretion

of the reviewing pathologist. It is unclear what criteria were used in selecting variants for which

to include suspected germline annotation for the cases in our study; however, the use of criteria

such as high frequency in ExAC or absence in COSMIC may have introduced annotation bias for

variants of low potential for clinical germline significance. We hypothesized that samples with

low tumor content might have more frequent annotation indicating suspected germline origin;

however, mean tumor content was similar for variants with or without annotation. Our data suggest

that if SGVs are to be reported in the context of tumor-only genotyping, standardized criteria that

select for SGVs with clinical significance may be warranted.

The primary aim of our study was to assess whether patients considered to be at risk for a

hereditary cancer syndrome based on SNaPshot NGS tumor genotyping results were routinely

referred for a genetic counseling assessment. We could not find evidence indicating that patients

considered to be at high or moderate risk for a hereditary cancer syndrome based on their SNaPshot

NGS tumor genotyping results received a genetic counseling referral; however, only one patient

in in this category had a variant of suspected germline origin indicated in his/her SNaPshot NGS

report. The absence of variant interpretation and annotation regarding suspected germline

significance in a majority of the SNaPshot NGS reports for patients in this category may be a

contributory factor in the low referral rate. Previous studies have shown that oncologists report

low confidence in their genomic knowledge (Stacy W. Gray, Hicks-Courant, Cronin, Rollins, &

Weeks, 2014). Variants in TP53, the most frequently somatically mutated gene in human cancers

22

(Kandoth et al., 2013), were reported for a number of the patients determined to be at high or

moderate risk for a hereditary cancer syndrome based on their SNaPshot NGS tumor genotyping

results. Without supporting annotation, the treating oncologist may not have recognized potential

clinical germline significance of the TP53 variants.

Conversely, our medical record review for all patients with SNaPshot NGS tumor

genotyping reports including annotation indicating that one or more of the variants may be of

germline origin revealed only genetic counseling referrals made based on personal and/or family

cancer history. This suggests that report annotation alone may not trigger a genetic counseling

referral. It is unclear why we did not find evidence for genetic counseling referrals for patients in

our study with SGVs. Although tumor genotyping is intended to provide information about a

patient’s tumor to inform treatment decisions, data indicate that patients are receptive to genetic

counseling follow-up if suspected germline findings are reported (Catenacci et al., 2015; Meric-

Bernstam et al., 2016).

Secondary to our assessment of genetic counseling referral for patients with SGVs, we

found that 38 of 136 (27.9%) of patients who underwent SNaPshot NGS tumor genotyping in the

timeframe of our study met genetic risk assessment referral criteria based on personal and/or family

cancer history alone independent of their SNaPshot NGS tumor genotyping results. We found

evidence of a genetic counseling referral for 17 of 38 (44.7%) patients. This finding suggests that

personal and family cancer history review of patients undergoing SNaPshot NGS tumor

genotyping may capture additional patients who could benefit from genetic risk assessment.

Overall, we found a high degree of variability in SNaPshot NGS reporting practices within

the timeframe of this study. Furthermore, the clinical application of SNaPshot NGS and other

tumor profiling platforms are in patients at advanced stages of disease progression. In this setting,

23

genetic risk assessment for hereditary susceptibility may not be of primary concern for ordering

physicians and for molecular pathologists characterizing suspected germline variants. Given the

additional finding of low genetic counseling referral rates within this patient population we suggest

a cooperative, multi-disciplinary effort to efficiently identify and refer high-risk patients to genetic

counseling.

Study limitations The aim of our study was to gain a better understanding of how frequently SGVs are

reported in the context of SNaPshot NGS tumor genotyping at the MGH Center for Integrated

Diagnostics. Our study examined SNaPshot NGS tumor genotyping results generated for non-lung

cancer patients between December 1, 2014 and May 1, 2015. Although this timeframe was chosen

carefully to encompass a period during which SNaPshot NGS tumor genotyping was regularly

employed in clinical management of cancer cancer patients and to allow for adequate time to assess

genetic counseling follow-up, the data observed in this timeframe may not reflect the overall

frequency with which SGVs are reported in the context of SNaPshot NGS tumor genotyping and

current practices for genetic counseling referrals based on SGVs.

Furthermore, we utilized an allelic fraction cut-off of 42-58% to select patients with SGVs.

Although germline variants are expected to be present in 50% of reads in absence of copy number

gains or losses, a recent study of incidental germline variants in somatic tumor genotyping showed

that the MAF for confirmed germline variants ranged from .6 - 83% in the corresponding tumor

sample (Meric-Bernstam et al., 2016). Our study may therefore underestimate the number of

patients with SGVs.

Lastly, we relied on COSMIC reporting in determining whether reported variants were of

high potential for germline significance. COSMIC annotations are largely based on what has been

24

studied and submitted, and infrequent annotation does not necessarily mean that a variant has a

higher likelihood of being germline in origin. Nonetheless, variants classified as having high

potential for clinical germline significance also had pathogenic or likely pathogenic germline

classification in ClinVar, and confirmatory germline testing is warranted even if COSMIC reports

underestimate the frequency with which the variant has been detected in tumor tissue.

25

CONCLUSIONS Our study shows that SGVs in hereditary cancer susceptibility-associated genes many be

detected in the context of SNaPshot NGS tumor genotyping at the MGH Center for Integrated

Diagnostics. A subset of patients had deleterious SGVs, that when evaluated in conjunction with

personal and/or family cancer history, raised concern for a hereditary cancer syndrome; however,

we did not find evidence of genetic counseling referrals based on SGVs in the timeframe of our

study. We identified inconsistencies in laboratory annotation for SGVs, but it is unclear whether

this contributed to the absence of genetic counseling referrals. Our data highlight the challenges

in reporting of and genetic counseling follow-up for SGVs encountered in SNaPshot NGS tumor

genotyping. A multi-disciplinary approach to addressing these challenges may aid in timely

referral of patients at risk of an inherited cancer susceptibility.

26

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APPENDICES

Supplemental Table 1: SNaPshot NGS genes and targeted exons

Gene ID Targeted exons AKT1 3 ALK 22, 23, 25 APC 16 BRAF 11, 15 CDH1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 CDKN2A 1, 2, 3 CTNNB1 3 DDR2 12, 13, 14, 15, 16, 17, 18 EGFR 7, 15, 18, 19, 20, 21 ERBB2 10, 20 ESR1 8 FBXW7 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 FGFR1 4, 8, 15, 17 FGFR2 7, 9, 12,14 FGFR3 7, 8, 9, 14, 16 FOXL2 1 GNA11 5 GNAQ 4, 5 GNAS 6, 7, 8, 9 HRAS 2, 3 IDH1 3, 4 IDH2 4 KIT 8, 9, 11, 17 KRAS 2, 3, 4, 5 MAP2K1 2, 3 MET 14, 16, 19, 21 NOTCH 25, 26, 34 NRAS 2, 3, 4, 5 PDGFRA 12, 14, 18, 23 PIK3CA 2, 5, 8, 10, 21 PIK3R1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 PTEN 1, 2, 3, 4, 5, 6, 7, 8, 9 RET 11, 16 ROS1 38 SMAD4 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 SMO 9 STK11 1, 2, 3, 4, 5, 6, 7, 8, 9 TP53 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 VHL 1, 2, 3

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