Supplementary materials: (Online Resources)

Exome sequencing reveals frequent deleterious germline variants in cancer susceptibility genes in women with invasive breast cancer undergoing neoadjuvant chemotherapy

Marissa S. Ellingson, MSa, Steven N. Hart, PhDb, Krishna R. Kalari, PhDb, Vera Suman, PhDb Kimberly A. Schahl, MSa, Travis J. Dockter, MSb, Sara J. Felten, BAb, Jason P. Sinnwell, MSb, Kevin J. Thompson, PhDb, Xiaojia Tang, PhDb, Peter T. Vedell, PhDb, Poulami Barman, MSb, Hugues Sicotte, PhDb, Jeanette E. Eckel-Passow, PhDb, Donald W. Northfelt, MDc, Richard J. Gray, MDd, Sarah A. McLaughlin, MDe, Alvaro Moreno-Aspitia, MDf, James N Ingle, MDm, Ann M. Moyer, MDg, Daniel W. Visscher, MDg, Katie Jones, MDh, Amy Conners, MDh, Michelle McDonough, MDi, Eric D Wieben, PhDj, Liewei Wang, PhDk, Richard Weinshilboum, MDa,k, Judy C. Boughey, MDl*Matthew P. Goetz, MDk,m*

aCenter for Individualized Medicine;Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

bDepartment of Health Sciences Research; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

cHematology/Oncology; Mayo Clinic, 13400 East Shea Blvd, Scottsdale, AZ, 85259

dDepartment of Surgery; Mayo Clinic, 13400 East Shea Blvd, Scottsdale, AZ, 85259

eDepartment of Surgery; Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224

fHematology/Oncology; Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224

gDepartment of Laboratory Medicine and Pathology; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

hDepartment of Radiology; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

iDepartment of Radiology; Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224

jBiochemistry; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

kDepartment of Molecular Pharmacology & Experimental Therapeutics; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

lDepartment of Surgery; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

mMedical Oncology; Mayo Clinic, 200 First Street SW, Rochester, MN, 55905

*Joint senior author

Corresponding authors:

Dr. Judy C. Boughey and Dr. Matthew P. Goetz

Mayo Clinic

200 First Street SW

Rochester, MN 55906

Phone: 507-284-2511

Fax: 5072841803

ONLINE RESOURCES

Methodology forSample Preparation, Exome Sequencing, and Bioinformatics Analysis

Clinical approximated tumor subtypes were categorized as follows: triple negative (includes ER <10%, PR <10%, HER2 negative, HER2 positive (regardless of ER), and ER positive/HER2 negative. For the latter, Ki-67 (<15% or 15%) was used to differentiate between luminal A and luminal B, respectively. ASCO/CAP guidelines with regard to immunohistochemistry and in-situ-hybridization were used for determination of HER2 status.1

Genomic DNA was extracted from peripheral blood using the Qiagen flexigene chemistry on an Autogen Flex Star.Red cells were lysed to allow white cells to be pelleted by centrifugation.Pellets were lysed and treated with protease to remove proteins from the sample.DNA was precipitated out with isopropanol and washed with 70% ethanol.DNA was then resuspended with TE and quantified using a spectrophotometer.

Paired-end libraries were prepared following the manufacturer’s protocol (Agilent) using the Bravo liquid handler (Agilent). Briefly, 1-3 ug of genomic DNA was fragmented to150-200 bp using the Covaris E210 sonicator. The ends were repaired and an “A” base added to the 3’ ends. Paired end Index DNA adaptors (Agilent) with a single “T” base overhang at the 3’ end were ligated, and the resulting constructs were purified using AMPure SPRI beads (Agencourt). The adapter-modified DNA fragments were enriched by 4 cycles of PCR using SureSelect forward and SureSelect ILM Pre-Capture Indexing reverse primers (Agilent). The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip.

Whole exome capture was performed using the protocol for Agilent’s Sure Select Human All + UTRs 71 MB v4 kit; 750 ng of the prepped library was incubated with whole exon biotinylated RNA capture baits for 24 hours at 65°C. Captured DNA:RNA hybrids were recovered using Dynabeads MyOne Streptavidin T1 (Dynal); after elution, DNA was desalted using Ampure XP beads (Agencourt). Purified capture products were then amplified using SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Concentration and size distribution of the libraries was determined on Qubit (Invitrogen) and Agilent Bioanalyzer DNA 1000 chip. Exome libraries were loaded one sample per lane onto Illumina TruSeq v3 paired end flow cells at concentrations of 9 pM to generate cluster densities of 600,000-800,000/mm2 following Illumina’s standard protocol using the Illumina cBot and TruSeq Rapid Paired end cluster kit version 3.100bp Paired Ends were sequenced. On average, 369M reads were sequenced per sample. A sample passed initial quality control if more than 90% of the capture region was covered at a minimum of 20x and more than 70% of the capture kit covered at 50x. Unless otherwise specified, all bioinformatics tools were run under default configuration. Reads were aligned to the hg19 reference genome using Novoalign ( VN:V2.07.13) with the following options: --hdrhd off -v 120 -c 4 -i PE 425,80 -x 5 -r Random. Realignment and recalibration was performed using GATK (VN: 2.7-4-g6f46d11) Best Practices version 3.2 Germline variations were called with GATK’s UnifiedGenotyper and SNVMix2.3 Variant quality score recalibration was also done with the following command line optimizations: for SNVs, -an QD -an MQRankSum -an ReadPosRankSum -an FS -an DP; and for INDELS, -an DP -an FS -an ReadPosRankSum -an MQRankSum --maxGaussians 4. When comparing these calls to Omni2.5-8 microarray, our genotype concordance was greater than 99.5% (data not shown).

References (Online Resources)

1. Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KJ, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013; 31(31): 3997-4013.

2. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet2011;43(5):491-498.

3.Shah SP, Morin RD, Khattra J, Prentice L, Pugh T, Burleigh A, et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature2009;461(7265):809-813.

Online Resources, Table I. Hereditary Cancer Susceptibility Gene List and Associated Syndromes

Gene / Tier / Syndrome Name or Associated Cancer Risk / Eligible for return of results?
ALK / 2 / Hereditary neuroblastoma / No - lack of medical management guidelines for cancer risk
APC / 2 / Familial adenomatous polyposis / Yes
ATM / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
ATR / 2 / Seckel / No - lack of medical management guidelines for cancer risk
AXIN2 / 2 / Oligodontia-colorectal cancer / No - lack of medical management guidelines for cancer risk
BAP1 / 2 / Tumor predisposition syndrome / No - lack of medical management guidelines for cancer risk
BARD1 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
BMPR1A / 2 / Juvenile polyposis / Yes
BRCA1 / 1 / Hereditary breast and ovarian cancer / Yes
BRCA2 / 1 / Hereditary breast and ovarian cancer / Yes
BRIP1 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
BUB1B / 2 / Mosaic variegated aneuploidy / No - lack of medical management guidelines for cancer risk
CDC73 / 2 / Familial isolated hyperparathyroidism; Hyperparathyroidism-jaw tumor syndrome; Parathyroid carcinoma / Yes
CDH1 / 1 / Hereditary diffuse gastric cancer / Yes
CDK4 / 2 / Familial atypical multiple mole melanoma / Yes
CDKN1B / 2 / Multiple endocrine neoplasia type 4 / No - lack of medical management guidelines for cancer risk
CDKN1C / 2 / Beckwith-Wiedemann / No - childhood onset
CDKN2A / 2 / Familial atypical multiple mole melanoma / Yes
CEBPA / 2 / Acute myeloid leukemia / No - lack of medical management guidelines for cancer risk
CEP57 / 2 / Mosaic variegated aneuploidy / No - lack of medical management guidelines for cancer risk/autosomal recessive
CHEK2 / 1 / Li-Fraumeni-like / No - lack of medical management guidelines for cancer risk
CYLD / 2 / Familial cylindromatosis / No - lack of medical management guidelines for cancer risk
CYP17A1 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
DDB2 / 2 / Xeroderma pigmentosum / No - autosomal recessive
DICER1 / 2 / Familial pleuropulmonary blastoma / No - lack of medical management guidelines for cancer risk
DIS3L2 / 2 / Perlman syndrome / No - autosomal recessive
DKC1 / 2 / Dyskeratosis congenita / Yes
EPCAM / 1 / Lynch syndrome / Yes
ERCC2 / 2 / Xeroderma pigmentosum / No - autosomal recessive
ERCC3 / 2 / Xeroderma pigmentosum / No - autosomal recessive
ERCC4 / 2 / Xeroderma pigmentosum / No - autosomal recessive
ERCC5 / 2 / Xeroderma pigmentosum / No - autosomal recessive
EXT1 / 2 / Hereditary multiple exostoses/hereditary multiple osteochondromas / No - childhood onset
EXT2 / 2 / Hereditary multiple exostoses/hereditary multiple osteochondromas / No - childhood onset
EZH2 / 2 / Weaver / No - lack of medical management guidelines for cancer risk
FAM175A / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
FANCA / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCB / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCC / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCD2 / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCE / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCF / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCG / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCI / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCL / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FANCM / 2 / Fanconi anemia / No - autosomal recessive/childhood onset
FGFR2 / 2 / Apert / No - lack of medical management guidelines for cancer
FH / 2 / Hereditary leiomyomatosis and renal cell cancer / Yes
FLCN / 2 / Birt-Hogg-Dube / Yes
GALNT12 / 2 / Colorectal cancer risk / No - lack of medical management guidelines for cancer risk
GATA1 / 2 / Diamond-Blackfan anemia / No - childhood onset
GATA2 / 2 / AML and myelodysplastic syndrome / No - lack of medical management guidelines for cancer risk
GEN1 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
GPC3 / 2 / Simpson-Golabi-Behmel / No - childhood onset
GPC4 / 2 / Simpson-Golabi-Behmel / No - childhood onset
HNF1A / 2 / Renal cell carcinoma and hepatic adenoma / No - lack of medical management guidelines for cancer risk
HNF1B / 2 / Prostate cancer and renal cell carcinoma / No - lack of medical management guidelines for cancer risk
HOXB13 / 1 / Breast/prostate cancer risk / No - lack of medical management guidelines for cancer risk
HRAS / 2 / Costello / No - childhood onset
KIT / 2 / Familial GIST / Yes
LMO1 / 2 / T-cell acute lymphoblastic leukemia / No - lack of medical management guidelines for cancer risk
MAX / 2 / Hereditary paraganglioma / Yes
MEN1 / 2 / Multiple endocrine neoplasia type 1 / Yes
MET / 2 / Papillary renal cancer / Yes
MITF / 2 / Melanoma / No - lack of medical management guidelines for cancer risk
MLH1 / 1 / Lynch / Yes
MLH3 / 2 / Lynch? / No - lack of medical management guidelines for cancer risk
MPL / 2 / Myelofibrosis; congenital amegakaryocytic thrombocytopenia / No - lack of medical management guidelines for cancer risk; autosomal recessive
MRE11A / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
MSH2 / 1 / Lynch / Yes
MSH6 / 1 / Lynch / Yes
MUTYH / 1 / MYH-associated polyposis / No - autosomal recessive
NBN / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
NF1 / 2 / Neurofibromatosis type I / No - childhood onset
NF2 / 2 / Neurofibromatosis type II / Yes
NHP2 / 2 / Dyskeratosis congenita / No - autosomal recessive
NOP10 / 2 / Dyskeratosis congenita / No - autosomal recessive
NTRK1 / 2 / Familial medullary thyroid cancer / No - lack of medical management guidelines for cancer risk
PALB2 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
PALLD / 2 / Pancreatic cancer risk / No - lack of medical management guidelines for cancer risk
PDGFRA / 2 / Familial GIST / Yes
PHOX2B / 2 / Hereditary neuroblastoma / No - lack of medical management guidelines for cancer risk
PMS1 / 2 / Lynch? / No - lack of medical management guidelines for cancer risk
PMS2 / 1 / Lynch / Yes
POLD1 / 2 / Colorectal cancer risk / No - lack of medical management guidelines for cancer risk
POLE / 2 / Colorectal cancer risk / No - lack of medical management guidelines for cancer risk
POLH / 2 / Xeroderma pigmentosum / No - autosomal recessive
PRF1 / 2 / Non-Hodgkin lymphoma / No - lack of medical management guidelines for cancer risk; autosomal recessive
PRKAR1A / 2 / Carney complex / Yes
PTCH1 / 2 / Nevoid basal cell carcinoma syndrome (Gorlin) / Yes
PTCH2 / 2 / Nevoid basal cell carcinoma syndrome (Gorlin) / No - lack of medical management guidelines for cancer risk
PTEN / 1 / Cowden / Yes
RAD50 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
RAD51C / 1 / Breast/ovarian cancer risk / No - lack of medical management guidelines for cancer risk
RAD51D / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
RB1 / 2 / Retinoblastoma / No - childhood onset
RBBP8 / 2 / Pancreatic cancer risk / No - lack of medical management guidelines for cancer risk
RECQL3 / 2 / Bloom / No - autosomal recessive
RECQL4 / 2 / Rothmund-Thomson / No - autosomal recessive
RET / 2 / MEN2 / Yes
RHBDF2 / 2 / Tylosis with esophageal cancer / No - lack of medical management guidelines for cancer risk
RPL11 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPL26 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPL35A / 2 / Diamond-Blackfan anemia / No - childhood onset
RPL5 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS10 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS17 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS19 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS24 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS26 / 2 / Diamond-Blackfan anemia / No - childhood onset
RPS7 / 2 / Diamond-Blackfan anemia / No - childhood onset
RUNX1 / 2 / Autosomal dominant familial platelet disorder / No - lack of medical management guidelines for cancer risk
SBDS / 2 / Shwachman-Diamond / No - autosomal recessive
SCG5 / 2 / Hereditary mixed polyposis syndrome / Yes
SDHA / 2 / Paraganglioma / No - lack of medical management guidelines for cancer risk
SDHAF2 / 2 / Hereditary paraganglioma / Yes
SDHB / 2 / Hereditary paraganglioma / Yes
SDHC / 2 / Hereditary paraganglioma / Yes
SDHD / 2 / Hereditary paraganglioma / Yes
SLX4 / 2 / Fanconi anemia / No - autosomal recessive
SMAD4 / 2 / Juvenile polyposis / Yes
SMARCA4 / 2 / Rhabdoid tumor predisposition / No - lack of medical management guidelines for cancer risk
SMARCB1 / 2 / Rhabdoid tumor predisposition / No - childhood onset
SMARCE1 / 2 / Spinal meningiomas / No - lack of medical management guidelines for cancer risk
STK11 / 1 / Peutz-Jeghers / Yes
SUFU / 2 / Medulloblastoma predisposition / No - lack of medical management guidelines for cancer risk
TERC / 2 / Dyskeratosis congenita / Yes
TERT / 2 / Dyskeratosis congenita / No - the effect of heterozygosity for one mutant TERT allele is unknown
TINF2 / 2 / Dyskeratosis congenita / Yes
TMEM127 / 2 / Hereditary paraganglioma / No - limited research; no medical management guidelines specific to TMEM127
TP53 / 1 / Li-Fraumeni / Yes
TSC1 / 2 / Tuberous sclerosis / Yes
TSC2 / 2 / Tuberous sclerosis / Yes
TSHR / 2 / Thyroid carcinoma / No - lack of medical management guidelines for cancer risk
VHL / 2 / Von Hippel-Lindau / Yes
WAS / 2 / Wiskott-Aldrich / No - childhood onset
WRN / 2 / Werner / No - autosomal recessive
WT1 / 2 / Familial Wilms tumor / No - childhood onset
XPA / 2 / Xeroderma pigmentosum / No - autosomal recessive
XPC / 2 / Xeroderma pigmentosum / No - autosomal recessive
XRCC2 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk
XRCC3 / 1 / Breast cancer risk / No - lack of medical management guidelines for cancer risk

Online Resources, Table II. Clinical genetic testing performed in in 124 breast cancer patients

Clinical Genetic Testing Discussed with Provider / N
Yes / 81
No / 43
Total / 124
Clinical Genetic Testing Pursued
Single site BRCA1 / 2
BRCA1/BRCA2 sequencing / 7
BRCA1/BRCA2 sequencing and deletion/duplication analysis / 45
BRCA1/BRCA2 and TP53 sequencing and deletion/duplication analysis / 2
Hereditary cancer panel* / 9
IHC screening for Lynch syndrome / 1
Total / 66
Clinical Genetic Test Results
BRCA1 deleterious mutation / 4
BRCA2 deleterious mutation / 5
Likely benign MSH6 variant / 1
Likely benign BRCA2 variant / 1
NBN VUS / 1
BRCA2 VUS / 1
Negative / 54
Total / 67+

VUS = Variant of Uncertain Significance

*BRCAPlus Panel at Ambry Genetics, Breast/Ovarian Cancer Panel at GeneDx, or MyRisk Panel at Myriad Genetics

+Total does not equal 66 as one patient tested positive for a BRCA2 deleterious mutation and a BRCA2 VUS

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