Germline Mutation Status, Pathological

Supplementary Online Content

Hahnen E, Lederer B, Hauke J, et al. Germline mutation status, pathological complete response, and disease-free survival in triple-negative breast cancer: secondary analysis of the GeparSixto randomized clinical trial. JAMA Oncol. Published online July 13, 2017. doi:10.1001/jamaoncol.2017.1007

eTable 1. Definition of “Positive Family History” as Defined by the Inclusion Criteria of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC) for BRCA1 and BRCA2 Germline Testing eTable 2. BRCA1/2 Mutations Genotyped by SNP Type Assay eTable 3. Deleterious Heterozygous Germline Mutations in BRCA1, BRCA2, and Other BC Predisposing Genes Identified in 291 TNBC Cases eTable 4. Multivariable Analysis on Prediction of pCR According to Different Definitions eTable 5. Baseline Patient and Tumor Characteristics Associated With Germline Mutations in BRCA1 and BRCA2 eTable 6. Prediction of Response According to Family History: Correlation of Carboplatin Treatment With pCR Endpoints eTable 7. Response Rates (DFS) and Cancer Family History in Patients Carrying Non– BRCA1/2 Gene Mutations eFigure. Kaplan-Meier Analysis of DFS According to BRCA1/2 Mutation Status

This supplementary material has been provided by the authors to give readers additional information about their work.

Downloaded From: https://jamanetwork.com/ on 09/23/2021

eTable 1. Definition of “Positive Family History” as Defined by the Inclusion Criteria of the German Consortium for Hereditary Breast and Ovarian Cancer (GC‐HBOC) for BRCA1 and BRCA2 Germline Testing

 ≥ 3 women with breast cancer  ≥ 2 women with breast cancer, 1 with onset below 51 years of age  ≥ 1 woman with breast cancer and 1 woman with ovarian cancer  ≥ 2 women with ovarian cancer  ≥ 1 woman with breast‐ and ovarian cancer  ≥1 woman with breast cancer below 36 years of age  ≥ 1 woman with bilateral breast cancer with onset below 51 years  ≥ 1 male with breast cancer and 1 woman with breast‐ or ovarian cancer

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 2. BRCA1/2 Mutations Genotyped by SNP Type Assay

gene HGVS (nucleotide) consequence HGVS (protein) HGMD

BRCA1 c.68_69del frameshift p.Glu23Valfs*17 CD951600

BRCA1 c.181T>G missense p.Cys61Gly CM940172

BRCA1 c.191G>A missense p.Cys64Tyr CM960164

BRCA1 c.676del frameshift p.Cys226Valfs*8 CD972044

BRCA1 c.843_846del frameshift p.Ser282Tyrfs*15 CD982484

BRCA1 c.1504_1508del frameshift p.Leu502Alafs*2 CD972048

BRCA1 c.1621C>T nonsense p.Gln541* CM980219

BRCA1 c.1687C>T nonsense p.Gln563* CM950144

BRCA1 c.1874_1877dup frameshift p.Val627Serfs*4 CI024230

BRCA1 c.1953_1956del frameshift p.Lys653Serfs*47 CD021780

BRCA1 c.2197_2201del frameshift p.Glu733Thrfs*5 CD951613

BRCA1 c.2309C>A nonsense p.Ser770* CM980221

BRCA1 c.2338C>T nonsense p.Gln780* CM950146

BRCA1 c.2411_2412del frameshift p.Gln804Leufs*5 CD021781

BRCA1 c.2475del frameshift p.Asp825Glufs*21 CD951614

BRCA1 c.2515del frameshift p.His839Thrfs*7 CD014638

BRCA1 c.2685_2686del frameshift p.Pro897Lysfs*5 CD961836

BRCA1 c.2722G>T nonsense p.Glu908* CM960175

BRCA1 c.2923C>T nonsense p.Gln975* CM024207

BRCA1 c.2989_2990dup frameshift p.Asn997Lysfs*4 CI972553

BRCA1 c.3018_3021del frameshift p.His1006Glnfs*17 CD982491

BRCA1 c.3108dup nonsense p.Lys1037* CI021885

BRCA1 c.3328_3331del frameshift p.Gln1111Asnfs*5 CD961838

BRCA1 c.3331_3334del frameshift p.Gln1111Asnfs*5 CD961838

BRCA1 c.3485delA frameshift p.Asp1162Valfs*48 CD972058

BRCA1 c.3607C>T nonsense p.Arg1203* CM940176

BRCA1 c.3700_3704del frameshift p.Val1234Glnfs*8 CD951619

BRCA1 c.3756_3759del frameshift p.Ser1253Argfs*10 CD941618

BRCA1 c.4035del frameshift p.Glu1346Lysfs*20 CD961844

BRCA1 c.4065_4068del frameshift p.Asn1355Lysfs*10 CD941619

BRCA1 c.4161_4162del frameshift p.Gln1388Glufs*2 CD020845

BRCA1 c.4183C>T nonsense p.Gln1395* CM960182

BRCA1 c.4327C>T nonsense p.Arg1443* CM940180

BRCA1 c.4675+1G>A splicing p.Ser1496Glyfs*14 CS031769

Downloaded From: https://jamanetwork.com/ on 09/23/2021 BRCA1 c.4689C>G nonsense p.Tyr1563* CM960185

BRCA1 c.4936del frameshift p.Val1646Serfs*12 CD021409

BRCA1 c.4986+3G>C splicing p.Met1663Valfs*14 CS031770

BRCA1 c.4986+6T>G splicing p.Met1663Valfs*14 CS011028

BRCA1 c.5080G>T nonsense p.Glu1694* CM980231

BRCA1 c.5095C>T missense p.Arg1699Trp CM041706

BRCA1 c.5096G>A missense p.Arg1699Gln CM034007

BRCA1 c.5137del nonsense p.Val1713* CD951622

BRCA1 c.5152+1G>C splicing p.Asp1692_Trp1718delinsGly CS982091

BRCA1 c.5153‐2del splicing p.Trp1718Serfs*2 CD003507

BRCA1 c.5177_5180del frameshift p.Arg1726Lysfs*3 CD972067

BRCA1 c.5193+1del splicing p.Glu1731Aspfs*33 CD123578

BRCA1 c.5251C>T nonsense p.Arg1751* CM970175

BRCA1 c.5266dup frameshift p.Gln1756Profs*74 CI941841

BRCA1 c.5345G>A nonsense p.Trp1782* CM032199

BRCA1 c.5503C>T nonsense p.Arg1835* CM960190

BRCA2 c.3G>A start lost p.Met1? CM087365

BRCA2 c.462_463del nonsense p.Asp156* CD021787

BRCA2 c.634_635del frameshift p.Arg212Lysfs*2 CD021788

BRCA2 c.643G>T nonsense p.Glu215* /

BRCA2 c.658_659del frameshift p.Val220Ilefs*4 CD984134

BRCA2 c.1310_1313del frameshift p.Lys437Ilefs*22 CD972071

BRCA2 c.1773_1776del frameshift p.Ile591Metfs*22 CD032734

BRCA2 c.2808_2811del frameshift p.Ala938Profs*21 CD951625

BRCA2 c.3199del frameshift p.Thr1067Leufs*10 CD044116

BRCA2 c.3296C>A nonsense p.Ser1099* /

BRCA2 c.3847_3848del frameshift p.Val1283Lysfs*2 CD961852

BRCA2 c.3865_3868del frameshift p.Cys1290Alafs*3 /

BRCA2 c.4449del frameshift p.Asp1484Thrfs*2 CD021793

BRCA2 c.4478_4481del frameshift p.Glu1493Valfs*10 CD961854

BRCA2 c.4554del frameshift p.Glu1518Aspfs*25 CD021794

BRCA2 c.5213_5216del frameshift p.Thr1738Ilefs*2 CD022535

BRCA2 c.5238dup nonsense p.Asn1747* CI021893

BRCA2 c.5350_5351del frameshift p.Asn1784Hisfs*2 CD972078

BRCA2 c.5621_5624del frameshift p.Ile1874Argfs*34 CD972080

BRCA2 c.5645C>A nonsense p.Ser1882* CM980241

BRCA2 c.5682C>G nonsense p.Tyr1894* CM010781

Downloaded From: https://jamanetwork.com/ on 09/23/2021 BRCA2 c.5722_5723del frameshift p.Leu1908Argfs*2 CD951626

BRCA2 c.5857G>T nonsense p.Glu1953* CM970181

BRCA2 c.5864C>A nonsense p.Ser1955* CM021511

BRCA2 c.5946del frameshift p.Ser1982Argfs*22 CD961857

BRCA2 c.6275_6276del frameshift p.Leu2092Profs*7 CD951627

BRCA2 c.6405_6409del frameshift p.Asn2135Lysfs*3 CD972084

BRCA2 c.6591_6592del frameshift p.Glu2198Asnfs*4 CD951628

BRCA2 c.6998dup frameshift p.Pro2334Thrfs*6 /

BRCA2 c.7558C>T nonsense p.Arg2520* CM970183

BRCA2 c.7913_7917del nonsense p.Phe2638* CD972091

BRCA2 c.8165C>G missense p.Thr2722Arg CM021956

BRCA2 c.8167G>C missense p.Asp2723His CM042310

BRCA2 c.8243G>A missense p.Gly2748Asp CM074074

BRCA2 c.8434_8435insC frameshift p.Gly2812Alafs*8 /

BRCA2 c.8961_8964del frameshift p.Ser2988Phefs*12 CD032103

BRCA2 c.9317G>A nonsense p.Trp3106* CM119498

BRCA2 c.9501+3A>T splicing p.Gly3086GlufsX2 CS083876

BRCA2 c.9672dup frameshift p.Tyr3225Ilefs*30 CI022172

BRCA2 c.9699_9702del frameshift p.Cys3233Trpfs*15 CD063469

Legend to etable 2: By SNP genotyping, all 291 DNA samples were pre‐screened for recurrent BRCA1 and BRCA2 mutations identified in the German population. For each variant, the consequences on nucleotide and protein level according to the HGVS nomenclature and the HGMD accession number are given.

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 3. Deleterious Heterozygous Germline Mutations in BRCA1, BRCA2, and Other BC Predisposing Genes Identified in 291 TNBC Cases

gene HGVS (nucleotide)a HGVS (protein)b HGMDc detection methodd referencese Nr. of patients; patient IDsf

BRCA1 (Del Ex1+2) / CG136869 MLPA [1, 2] 2; 121C10, 121H11

BRCA1 (Del Ex13‐15) / CG015438 MLPA [3‐5] 2; 121C05, 122A06

BRCA1 (Dup Ex13) / CN035372 MLPA [6, 7] 1; 121F05 4; 120C12, 121H01, 122A01, BRCA1 c.181T>G p.Cys61Gly CM940172 SNP‐genotyping [8‐10] 122D06 BRCA1 c.1621C>T p.Gln541* CM980219 SNP‐genotyping [11] 1; 122B01

BRCA1 c.1961del p.Lys654Serfs*47 CD951610 SNP‐genotyping [12, 13] 1; 121A07

BRCA1 c.2197_2201del p.Glu733Thrfs*5 CD951613 SNP‐genotyping [14] 1; 121D12

BRCA1 c.2338C>T p.Gln780* CM950146 SNP‐genotyping [14, 15] 2; 122C10, 123A05

BRCA1 c.2411_2412del p.Gln804Leufs*5 CD021781 SNP‐genotyping [16] 1; 120H12

BRCA1 c.2515del p.His839Thrfs*7 CD014638 NGS [17] 1; 121D04

BRCA1 c.3485del p.Asp1162Valfs*48 CD972058 SNP‐genotyping [18] 2; 120C04, 122D01

BRCA1 c.3700_3704del p.Val1234Glnfs*8 CD951619 SNP‐genotyping [19, 20] 2; 122E04, 122F02

BRCA1 c.3915del p.Leu1306* / NGS / 1; 122C07

BRCA1 c.4183C>T p.Gln1395* CM960182 SNP‐genotyping [15, 21, 22] 1; 122D02

BRCA1 c.4251_4252del p.Leu1418Argfs*9 CD972063 NGS [23] 1; 121G06

BRCA1 c.4675+1G>A p.Glu1556Glyfs*14 CS031769 SNP‐genotyping [9, 24, 25] 1; 120B03

BRCA1 c.4689C>G p.Tyr1563* CM960185 SNP‐genotyping [26, 27] 2; 120A11, 121C04

BRCA1 c.4986+3G>C p.Met1663Valfs*14 CS031770 SNP‐genotyping [28, 29] 1; 121G05

BRCA1 c.5096G>A p.Arg1699Gln CM034007 SNP‐genotyping [9, 30‐33] 1; 120G02

BRCA1 c.5109T>G p.Tyr1703* CM107813 NGS [34] 1; 122C03

BRCA1 c.5177_5180del p.Arg1726Lysfs*3 CD972067 SNP‐genotyping [22, 35] 1; 122G09

BRCA1 c.5179A>T p.Lys1727* CM950156 NGS [12] 1; 122E08 11; 120E06, 120G04, 121B11, [15, 20, 22, 36, 121C06, 123A10, 122A12, BRCA1 c.5266dup p.Gln1756Profs*74 CI941841 SNP‐genotyping 37] 122E01, 122H07, 121G01, 122A04, 122D12 BRCA1 c.5309G>T p.Gly1770Val CM133533 NGS [31, 38] 1; 122E11

BRCA2 c.1608_1609del p.Glu537Lysfs*22 / NGS / 1; 120G03

BRCA2 c.1796_1800del p.Ser599* CD972072 SNP‐genotyping [39, 40] 1; 122A10

BRCA2 c.4449del p.Asp1484Thrfs*2 CD021793 SNP‐genotyping [41] 1; 122C08

BRCA2 c.5645C>A p.Ser1882* CM980241 SNP‐genotyping [20] 1; 122D08

BRCA2 c.5857G>T p.Glu1953* CM970181 SNP‐genotyping [42] 1; 120B02

BRCA2 c.6937+2T>G p.? / NGS / 1; 120F11

BRCA2 c.9371A>T p.Asn3124Ile CM993726 NGS [9, 43‐45] 1; 120H03

BARD1 c.2002‐2A>G p.? / NGS / 1; 122F03

Downloaded From: https://jamanetwork.com/ on 09/23/2021 FANCM c.3898G>T p.Glu1300* / NGS 1; 122D05

FANCM c.5101C>T p.Gln1701* CM147953 NGS [46] 1; 121F07

MRE11A c.350A>G p.Asn117Ser CM993183 NGS [47] 1; 120G09

NBN c.643C>T p.Arg215Trp CM044022 NGS [48, 49] 1; 122H08

NBN c.657_661del p.Lys219Asnfs*16 CD982819 NGS [50‐52] 1; 121G07

RAD50 c.1253_1254del p.Phe418Cysfs*13 / NGS / 1; 120A09

RAD50 c.2165dup p.Glu723Glyfs*5 / NGS / 1; 122G06

RAD51C c.409C>T p.Gln137* / NGS / 1; 120A08

XRCC2 c.96_99del p.Phe32Leufs*29 / NGS / 1; 121E09

Legend to etable 3: All 291 DNA samples have been pre‐screened for deleterious BRCA1 and BRCA2 mutations by MLPA and SNP genotyping. MLPA revealed large heterozygous rearrangements in BRCA1 in a total of 5 patients, another 32 BRCA1 and 4 BRCA2 mutation carriers were identified by SNP genotyping. The remaining samples were subjected to next generation sequencing (NGS). NGS identified another 6 BRCA1 and 3 BRCA2 mutation carriers, while a total of 10 patients carry deleterious alterations in BARD1, FANCM, MRE11A, NBN, RAD50, RAD51C or XRCC2. For each variant, the consequences on nucleotide and protein level according to the HGVS nomenclature (a, b), the HGMD accession number (c), detection method (d) and number of mutation carriers and patient IDs are given (f). The relevant literature is indicated (e). No patient had deleterious mutations on more than one gene. For the bioinformatic analysis of NGS data, the VARBANK pipeline version 2.1 and interface were used. Primary data were filtered according to signal purity using the Illumina Realtime Analysis (RTA) software version 1.8. Subsequently, the reads were mapped to the human genome reference build GRCh37 using the BWA‐aln version alignment algorithm[53]. GATK version 1.6 [54] was used to mark duplicated reads, perform local realignment around short insertions and deletions, recalibrate the base quality scores; and call SNPs and short Indels together with SAMtools version 0.1.7 [53]. Scripts developed at the Cologne Center for Genomics were applied to detect protein changes, affected donor and acceptor splice sites, and overlaps with known variants. Acceptor and donor splice site mutations were analyzed using a Maximum Entropy model [55]. The filter parameters to select relevant variants used in this study were 1) variant fraction 25%; 2) minor allele frequency (MAF) <1%; 3) minimal read depth of 10x; and 4) variants in the exons and flanking intronic regions (+/‐25bp). The remaining variants were classified in accordance with the regulations of the international ENIGMA consortium (Evidence‐based Network for the Interpretation of Germline Mutant Alleles; https://enigmaconsortium.org). Deleterious or likely deleterious variants were validated by Sanger sequencing.

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eReferences:

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Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 4. Multivariable Analysis on Prediction of pCR According to Different Definitions

pCR (ypT0/is ypN0) OR 95% CI p‐value Mutation wildtype 1.00 mutant 2.56 0.93‐7.07 0.070 Carboplatin no 1.00 yes 2.08 1.19‐3.63 0.010 Age <40 1.00 >=40 1.01 0.49‐2.05 0.986 Tumor stage cT1 1.00 cT2‐4 0.59 0.34‐1.04 0.069 Nodal status cN0 1.00 cN+ 0.36 0.21‐0.61 <0.001 Grading G1/2 1.00 G3 2.37 1.27‐4.41 0.007 Ki67 <60% 1.00 ≥60% 1.38 0.80‐2.41 0.251 BRCA risk according to family history no 1.00 yes 1.24 0.68‐2.25 0.482 Interaction test mutation:carbo 0.68 0.17‐2.68 0.579

pCR (ypT0 ypN0) OR 95% CI p‐value Mutation wildtype 1.00 mutant 1.44 0.54‐3.82 0.462 Carboplatin no 1.00 yes 1.97 1.13‐3.43 0.016 Age <40 1.00 >=40 0.89 0.44‐1.79 0.744 Tumor stage cT1 1.00 cT2‐4 0.79 0.46‐1.38 0.415 Nodal status cN0 1.00 cN+ 0.36 0.21‐0.63 <0.001 Grading G1/2 1.00 G3 1.96 1.06‐3.64 0.033 Ki67 <60% 1.00 ≥60% 1.19 0.69‐2.04 0.542 BRCA risk according to family history no 1.00 yes 1.04 0.58‐1.87 0.905 Interaction test mutation:carbo 1.15 0.31‐4.34 0.833

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 5. Baseline Patient and Tumor Characteristics Associated With Germline Mutations in BRCA1 and BRCA2

Overall mutant wildtype p‐value* N % N % N % Overall 291 100.0 50 17.2 241 82.8

Carboplatin treated 0.776 no 145 49.8 24 16.6 121 83.4 yes 146 50.2 26 17.8 120 82.2 Age <0.001 <40years 65 22.3 23 35.4 42 64.6 ≥40years 226 77.7 27 11.9 199 88.1 Family history <0.001 no 181 62.2 19 10.5 162 89.5 yes 110 37.8 31 28.2 79 71.8 Tumor stage at baseline 0.721 cT1 87 29.9 16 18.4 71 81.6 cT2‐4 204 70.1 34 16.7 170 83.3 Nodal status at baseline 0.267 cN0 182 64.5 35 19.2 147 80.8 cN+ 100 35.5 14 14.0 86 86.0 Grading 0.894 G1/2 72 24.7 12 16.7 60 83.3 G3 219 75.3 38 17.4 181 82.6 Ki67 0.966 <60% 104 35.7 18 17.3 86 82.7 ≥60% 187 64.3 32 17.1 155 82.9 *Pearson Chi2 test

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 6. Prediction of Response According to Family History: Correlation of Carboplatin Treatment With pCR Endpoints

Overall no Carboplatin Logistic regression (N=181) Carboplatin (N=89) (univariate) (N=92) no BRCA risk N % N % N % p‐value* OR (95% CI) p‐ according to risk vs no risk value family history Primary endpoint pCR (ypT0/is ypN0) 0.022 yes 82 45.3 34 37.0 48 53.9 2.0 (1.10‐3.62) 0.022 no 99 54.7 58 63.0 41 46.1 Secondary endpoint pCR (ypT0 ypN0) 0.022 yes 76 42.0 31 33.7 45 50.6 2.0 (1.11‐3.67) 0.022 no 105 58.0 61 66.3 44 49.4

Overall no Carboplatin Logistic regression (N=110) Carboplatin (N=57) (univariate) (N=53) BRCA risk N % N % N % p‐value* OR (95% CI) p‐ according to risk vs no risk value family history Primary endpoint pCR (ypT0/is ypN0) 0.193 yes 61 55.5 26 49.1 35 61.4 1.65 (0.77‐3.53) 0.194 no 49 44.5 27 50.9 22 38.6 Secondary endpoint pCR (ypT0 ypN0) 0.083 yes 53 48.2 21 39.6 32 56.1 1.95 (0.91‐4.17) 0.085 no 57 51.8 32 60.4 25 43.9 *Pearson Chi2 test

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eTable 7. Response Rates (DFS) and Cancer Family History in Patients Carrying Non–BRCA1/2 Gene Mutations

gene patient HGVS (nucleotide) Age at onset Cancer family history pCR (ypT0/is ypN0) pCR (ypT0 ypN0) ID BARD1 122F03 c.2002‐2A>G 34 yes yes yes

FANCM 122D05 c.3898G>T 54 no yes yes

FANCM 121F07 c.5101C>T 42 no yes yes

MRE11A 120G09 c.350A>G 61 no no no

NBN 122H08 c.643C>T 62 no no no

NBN 121G07 c.657_661del 63 no no no

RAD50 120A09 c.1253_1254del 47 no no no

RAD50 122G06 c.2165dup 61 no no no

RAD51C 120A08 c.409C>T 46 no yes yes

XRCC2 121E09 c.96_99del 42 no no no

Downloaded From: https://jamanetwork.com/ on 09/23/2021 eFigure. Kaplan‐Meier Analysis of DFS According to BRCA1/2 Mutation Status (A) DFS, by BRCA1/2 mutation status and pCR (ypT0/is, ypN0 definition). More than 95% of patients who achieved pCR remained disease‐free after a three years cut‐off, irrespective of the BRCA1/2 mutation status (w/o BRCA1/2 mutations: 96.1%, 95%CI:89.9‐98.5%; with BRCA1/2 mutations: 95.5%, 95%CI:71.9‐99.3%). In contrast, approximately 63‐69% of the patients without pCR remained disease‐free after 3 years, again with no differences observed between mutation carriers and non‐carriers (BRCA1/2: w/o mutations 65.4%, 95%CI:55.9‐73.3%; with mutations 62.7%, 95%CI:34.8‐81.4%; figure 1 A). A significant correlation of pCR with DFS rates was also observed for the ypT0 ypN0 definition (B).

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