SUPPLEMENTARY MATERIALS Supplementary Methods Clinical

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To assess data quality, a review of a random sample of 1200 (10%) breast and ..... carriers. No. cases. Freq. (%). Breast cancer predisposition genes. ATM. 17.

SUPPLEMENTARY MATERIALS

Supplementary Methods Clinical Cohort Study Population All 8,753 TNBC cases from a cohort of 140,449 individuals subjected to clinical germline cancer panel testing between March 2012 and June 2016 at a clinical testing laboratory (Ambry Genetics-Aliso Viejo, CA) were included in this study. Demographic, clinical history, and family history of cancer information was collected from test requisition forms, clinic notes, and pedigrees provided by ordering clinicians at the time of testing. Information was collected on current age, personal history and age at diagnosis of all cancers, ancestry, family history of cancer with cancer type, and age at diagnosis among relatives. Family history was limited to first and second-degree relatives. Families with breast or ovarian cancer in two or more individuals, on the same parental side, were considered positive for family history for each cancer. TNBC status, defined as ER-negative, PR–negative and HER2–negative breast cancer was provided by ordering clinicians and/or clinical tumor pathology reports. ER, PR, and HER2 status was available on 50,820 of 74,649 (67.3%) breast cancer cases. To assess data quality, a review of a random sample of 1200 (10%) breast and ovarian cancer patient intake forms was conducted. Of these, 43.3% (520 of 1200) with additional clinical history documentation available (clinic notes, pedigrees, detailed letters of medical necessity). Consistent information was observed for 100% (429/429) of breast cancers, 99.5% (409/411) for age at breast cancer diagnosis, 99.7% (331/332) for breast tumor pathology, 97.9% (275/281) for breast tumor hormone receptors status, 100% (90/90) for ovarian cancer, and 100% (88/88) for age at ovarian cancer diagnosis, suggesting that the intake data for the cohort is of reasonably high quality. Triple-Negative Breast Cancer Consortium (TNBCC) study The TNBCC study included 2,148 TNBC cases from 12 clinical centers in the USA (MCBCS, DFCI, OSU, RPCI, KUMC and FCCC), Germany (BBCC and GENICA), Finland (HEBCS), Greece (DEMOKRITOS) and the United Kingdom (UK) (SBCS); and the POSH UK study of women diagnosed under age 40. Selection of TNBC cases was independent of family history of breast and ovarian cancer. All 2,148 TNBC cases were recruited to institutional review board approved studies. Information on age of diagnosis, family history of cancer, and self-reported ethnicity was provided by participating studies. Family history was recorded as all first and second-degree relatives with breast or ovarian cancer. TNBC cases in TNBCC were individuals with ER-negative, PR–negative and HER2–negative (0-1 by immunohistochemistry) breast cancer. Because a subset of patients were recruited from 1995-2011, ER and PR-negative status was defined as 70:30), variants with minor allele frequency (MAF)>0.3%, and low penetrance variants were excluded from both cases and controls (e.g., CHEK2 p.Ile157Thr). Truncating variants in the last 55bp of the penultimate exon or the last exon that potentially avoided nonsense mediated mRNA decay, and did not disrupt a known functional domain, were excluded. Large genomic rearrangements of one or more exons were excluded from case-control comparisons because rearrangements were not validated among reference controls. Associations between combined PVs in each gene and TNBC for the Caucasian population were estimated by odds ratio (OR) and corresponding 95% confidence intervals based on Fisher’s exact test. P-values 60 Unknown Multiple breast cancer Ovarian Colorectal Pancreatic Family History of Cancer* Breast (no ovarian) Breast & Ovarian Ovarian (no breast) Colorectal Pancreatic No Breast, Ovarian, Colorectal or pancreatic Mean age at TNBC diagnosis

Caucasian n % 5498 100

Clinical TNBC African American % PV n % % PV 14.0 1271 100 14.6

5492 5 1

99.9 0.1 0.0

14.0 ND ND

1270 1 0

99.9 0.1 0.0

14.6 ND ND

0 292 0 5206 0 0

0.0 5.3 0.0 94.7 0.0 0.0

NA 14.3 NA 14.0 NA NA

1271 0 0 0 0 0

100.0 0.0 0.0 0.0 0.0 0.0

14.6 NA NA NA NA NA

671 1772 2648 4454 1022 22 881 79 63 21

18.9 17.0 16.1 14.7 11.4 ND 17.6 ND ND ND

180 487 709 1105 162 4 196 9 11 1

14.2 38.3 55.8 86.9 12.7 0.3 15.4 0.7 0.9 0.1

26.3 20.7 18.8 15.0 11.4 ND 24.5 ND ND ND

2584 396 273 1340 517

12.2 32.2 48.2 81.0 18.6 0.4 16.0 1.4 1.1 0.4 0.0 47.0 7.2 5.0 24.4 9.4

14.1 25.7 23.5 13.1 16.0

610 63 47 200 95

48.0 5.0 3.7 15.7 7.5

17.1 ND ND 15.3 ND

1292

23.5

10.6

338

26.6

10.1

Mean ± SD

Range

Mean ± SD

50 ±11.4 18-90 48.6 ±10.8 ND = not determined, SD = Standard deviation; PV = pathogenic variant *1st and 2nd degree relatives with the relevant cancer

Range 19-83

Supplementary Table 2. TNBC gene-based variant frequency by race and ethnicity in the clinical and TNBCC studies Clinical Cohort TNBCC Cohort All race/ethnicity Caucasian African American All race/ethnicity Caucasian Mutated alleles BARD1 48 BRCA1 513 BRCA2 201 BRIP1 27 PALB2 111 RAD51C 31 RAD51D 16 TP53 14 Total frequency (%)

Cases 6464 8537 8537 6464 6980 6464 6095 8741

Freq (%)

Mutated alleles

Cases

0.74 6.01 2.35 0.42 1.59 0.48 0.26 0.16 12.01

27 297 119 18 75 20 10 10

4144 5332 5332 4144 4441 4144 3868 5490

Freq (%) 0.65 5.57 2.23 0.43 1.69 0.48 0.26 0.18 11.49

Mutated Freq Mutated alleles Cases (%) alleles TNBC predisposition genes 11 886 1.24 10 74 1257 5.89 166 39 1257 3.10 58 4 886 0.45 10 8 964 0.83 22 5 886 0.56 8 3 859 0.35 8 3 1270 0.24 2 12.66

Freq (%)

Mutated alleles

Cases

2148 2148 2148 2148 2148 2148 2148 2148

0.47 7.73 2.70 0.47 1.02 0.37 0.37 0.09 13.22

10 164 54 10 22 8 8 2

2095 2095 2095 2095 2095 2095 2095 2095

0.48 7.83 2.58 0.48 1.05 0.38 0.38 0.10 13.28

2148 ND ND 2148 ND 2148 372 372 ND 2148 ND 2148 2148 2148

0.19 ND ND 0.09 ND 0.19

4 ND ND 2 ND 4 0 1 ND 2 ND 1 5 2

2095 ND ND 2095 ND 2095 372 372 ND 2095 ND 2095 2095 2095

0.19 ND ND 0.10 ND 0.19

Cases

Freq (%)

Other cancer predisposition genes ATM 17 6652 0.26 13 4265 0.30 1 CDH1 5 8505 0.06 1 5329 0.02 3 CDKN2A 5 1790 0.28 3 1213 0.25 CHEK2* 22 6639 0.33 19 4252 0.45 MLH1 5 3497 0.14 4 2301 0.17 MRE11A 6 6464 0.09 2 4144 0.05 3 MSH2 3 3497 0.09 2 2301 0.09 MSH6 9 3497 0.26 7 2301 0.30 NF1 9 6097 0.15 9 3870 0.23 NBN 12 6464 0.19 10 4144 0.24 1 PMS2 9 3497 0.26 4 2301 0.17 3 PTEN 4 8719 0.05 2 5471 0.04 1 RAD50 14 6464 0.22 9 4144 0.22 3 XRCC2 ND ND ND ND ND ND ND Total frequency (%) 2.38 2.53 (-) no mutated alleles; Freq = variant frequency in percent; ND = not determined * Excluding CHEK2 p.Ile157Thr and p.Ser428Phe

913 1233 192 913 407 886 407 407 859 886 407 1269 886 ND

0.11 0.24 ND ND ND 0.34 ND ND ND 0.11 0.74 0.08 0.34 ND 1.96

4 ND ND 2 ND 4 0 1 ND 2 ND 1 6 2

0.27 ND 0.09 ND 0.05 0.28 0.09 1.25

0.27 ND 0.10 ND 0.05 0.24 0.10 1.24

Supplementary Table 3. Gene-based variant frequency by personal and family history of cancer in the TNBC clinical cohort 1st or 2nd degree family history of cancer Personal history of Overall TNBC cases bilateral cancer Breast Ovarian Mutation carriers ATM 17 BARD1† 48 BRCA1† 513 BRCA2† 201 CHEK2 22 PALB2† 111 PTEN 4 RAD51D† 16 TP53† 14 Total frequency (%)

No. cases

Freq. (%)

6652 6464 8537 8537 6639 6980 8719 6095 8741

0.26 0.74 6.01 2.35 0.33 1.59 0.05 0.26 0.16 11.75

Mutation carriers

No. cases

Freq. (%)

Mutation carriers

Breast cancer predisposition genes 5 1037 0.48 8 9 1010 0.89 27 113 1264 8.94 250 41 1264 3.24 108 4 1034 0.39 9 20 1076 1.86 64 1 1305 0.08 3 2 928 0.22 6 6 1307 0.46 6 16.55

Other cancer predisposition genes BRIP1* 27 6464 0.42 5 1010 0.50 10 CDH1 5 8505 0.06 2 1267 0.16 4 CDKN2A 5 1790 0.28 2 280 0.71 1 MLH1 5 3497 0.14 2 544 0.37 3 MRE11A 6 6464 0.09 2 1010 0.20 2 MSH2 3 3497 0.09 544 ND 1 MSH6 9 3497 0.26 2 544 0.37 4 NBN 12 6464 0.19 1 1010 0.10 7 NF1 9 6097 0.15 2 928 0.22 4 PMS2 9 3497 0.26 544 ND 1 RAD50 14 6464 0.22 1 1010 0.10 6 RAD51C* 31 6464 0.48 3 1010 0.30 13 Total frequency (%) 2.62 3.01 (-) no mutated alleles; Freq = variant frequency in percent; ND = not determined * Established TNBC predisposition genes

No. cases

Freq. (%)

Mutation carriers

No. cases

Freq. (%)

3091 3016 3858 3858 3087 3249 3971 2804 3978

0.26 0.90 6.48 2.80 0.29 1.97 0.08 0.21 0.15 13.14

3 1 35 20 1 4 2 -

295 288 394 394 295 306 402 275 402

1.02 0.35 8.88 5.08 0.34 1.31 ND 0.73 ND 17.70

3016 3891 827 1492 3016 1492 1492 3016 2805 1492 3016 3016

0.33 0.10 0.12 0.20 0.07 0.07 0.27 0.23 0.14 0.07 0.20 0.43 2.23

2 3 1 1 3 3 2

288 379 82 237 288 237 237 288 275 237 288 288

0.69 ND 3.66 0.42 ND ND ND 0.35 ND 1.27 1.04 0.69 8.12

Supplementary Table 4. Estimated risks of TNBC in Caucasian patients associated with mutations in non-TNBC hereditary cancer panel genes Non-TNBC predispositio n gene ATM CDH1† CDKN2A† CHEK2 MLH1 MRE11A MSH2† MSH6 NBN NF1 PMS2† PTEN† RAD50 XRCC2†

Clinical Cohort

TNBCC Cohort

ExAC controls

Clinical TNBC Risk

TNBCC TNBC Risk

Mutated Alleles

No. Cases

Mutated Alleles

No. Cases

Mutated Alleles

No. Controls

OR

95%CI lower

95%CI upper

p-value*

OR

95%CI lower

95%CI upper

p-value*

13 1 3 17 3 2 1 7 10 9 3 2 7 ND

4210 5263 1195 4197 2266 4090 2266 2266 4090 3816 2266 5404 4090 ND

4 ND ND 2 ND 4 0 1 2 NA ND 1 4 2

2003 ND ND 2003 ND 2003 372 372 2003 NA ND 2003 2003 2003

102 3 9 232 10 25 6 34 41 29 32 1 58 14

26644 25961 24312 25215 26639 26767 25329 26151 26265 26131 26230 24166 26474 27085

0.81 1.64 6.79 0.44 3.53 0.52 1.86 2.38 1.57 2.13 1.09 8.95 0.78 ND

0.43 0.06 1.58 0.26 0.83 0.09 0.08 1.03 0.75 0.99 0.28 0.70 0.35 ND

1.45 14.91 24.22 0.73 12.72 2.03 13.90 5.33 3.19 4.46 3.38 259.36 1.68 ND

0.59 0.52 0.02 3.63 x10-4 0.08 0.57 0.45 0.04 0.22 0.05 0.76 0.09 0.72 ND

0.52 ND ND 0.11 ND 2.14 ND 2.07 0.64 NA ND 12.07 0.91 1.93

0.18 ND ND 0.02 ND 0.68 ND 0.10 0.11 NA ND 0.31 0.30 0.31

1.36 ND ND 0.40 ND 5.96 ND 12.67 2.48 NA ND 464.53 2.50 8.01

0.25 ND ND 6.75 x10-6 ND 0.14 ND 0.39 0.77 NA ND 0.15 1.00 0.30

ND: not determined; OR = Odds ratio; CI = confidence interval; TNBCC = Triple Negative Breast Cancer Consortium * Statistical significance of associations were estimated using Fisher’s exact test. All tests were two-sided. †Insufficient events for estimation of risk

Supplementary Table 5. Estimated risks of TNBC in Caucasian patients using gnomAD controls gnomAD Clinical Cohort TNBCC Cohort Clinical TNBC Risk controls Gene Mutated No. Alleles Cases TNBC associated genes BARD1 25 4090 BRCA1 255 5265 BRCA2 115 5265 BRIP1 17 4090 PALB2 70 4383 RAD51C 15 4090 RAD51D 8 3814 TP53 10 5423 TP53† 6 1055

TNBCC TNBC Risk

Mutated Alleles

No. Cases

Mutated Alleles

No. Controls

OR

95%CI lower

95%CI upper

p-value*

9 158 51 9 17 8 7 2 2

2003 2003 2003 2003 2003 2003 2003 2003 504

56 194 231 119 98 51 28 20 20

59601 60628 60033 60616 60675 60657 60537 60671 60671

6.52 15.49 5.73 2.12 9.96 4.37 4.54 5.60 17.30

3.99 12.80 4.56 1.23 7.23 2.42 1.89 2.41 6.80

10.46 19.11 7.21 3.55 13.65 7.74 10.51 11.99 42.26

2.27x10 -16 1.05x10 -41 8.52x10 -3 6.98x10 -37 2.13x10 -5 1.23x10 -3 1.03x10 -5 8.96x10 -6 4.25x10

-11

Other predisposition genes ATM 13 4210 4 2003 236 60572 0.79 0.43 1.39 0.52 CDH1‡ 1 5263 ND ND 8 59483 1.41 0.06 9.01 0.53 -3 CDKN2A‡ 3 1195 ND ND 13 56936 11.01 2.68 36.95 3.98 x10 -3 CHEK2 17 4197 2 2003 488 59768 0.50 0.29 0.80 2.12 x10 MLH1 3 2266 ND ND 12 60372 6.66 1.61 23.26 0.02 MRE11A 2 4090 4 2003 54 60581 0.55 0.09 2.07 0.58 MSH2‡ 1 2266 0 372 14 60246 1.90 0.09 11.50 0.43 MSH6 7 2266 1 372 67 59898 2.76 1.25 6.20 0.02 -5 NF1 9 3816 ND ND 23 60462 6.21 2.85 13.60 7.31 x10 NBN 10 4090 2 2003 96 60497 1.54 0.78 2.91 0.23 PMS2‡ 3 2266 ND ND 58 59272 1.35 0.35 4.15 0.49 PTEN‡ 2 5404 1 2003 4 60242 5.57 0.75 29.94 0.08 RAD50 7 4090 4 2003 143 60466 0.72 0.33 1.51 0.50 ND = not determined or no PVs; OR = Odds ratio; CI = confidence interval; TNBCC = Triple Negative Breast Cancer Consortium * Statistical significance of associations were estimated using Fisher’s exact test. All tests were two-sided. †Age at diagnosis of 40 years or younger ‡ Insufficient events for estimation of risk

OR

95%CI lower

95%CI upper

p-value*

4.79 25.62 6.69 2.29 5.27 4.76 7.57 3.03 12.06

2.31 20.67 4.90 1.12 3.07 2.06 3.20 0.51 2.01

9.67 31.79 9.09 4.46 8.86 9.83 17.78 12.44 49.65

2.49 x10 -16