Ploidy and large-scale genomic instability consistently identify basal-like breast

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1 Supplementary Data and Figures Ploidy and large-scale genomic instability consistently identify basal-like breast carcinomas with BRCA1/2 inactivation Popova T, Manié E, Rieunier G, Caux-Moncoutier V, Tirapo C, Dubois T, Delattre O, Sigal- Zafrani B, Bollet M, Longy M, Houdayer C, Sastre-Garau X, Vincent-Salomon A, Stoppa- Lyonnet D, Stern MH. METHODS Experimental set of 65 BLCs A series of undifferentiated Basal-Like breast Carcinomas (BLCs) was assembled from patients from the Institut Curie. This series was enriched for tumors arisen in patients carrying deleterious BRCA1 mutations (23 samples thereafter called BRCA1 BLCs). All BRCA1 mutations were different except for the Quebec founder mutation c.4327c>t/p.arg1443stop (5 unrelated patients) and the Ashkenazi founder mutation c.5266dupc/p.gln1756profsx74 (2 unrelated patients). The remaining BLCs arose in patients without evidence of familial predisposition of breast/ovarian cancer, or in patients tested negative for BRCA1/2 mutations (42 samples thereafter called sporadic BLCs). Mean age at diagnosis was 42.8 and 56.3 yearsold for BRCA1 and sporadic BLCs, respectively. Processing SNP-arrays For mining both Illumina and Affymetrix SNP array the GAP method was utilized.(1) Copy number variation and allele imbalance profiles (Log R ratio / B allele frequency and Log2Ratio / Allele Difference in Illumina and Affymetrix platforms, respectively) were segmented; the values were smoothed within the segments by median (copy number variation profile) and mode (allelic imbalance profile); segmental copy numbers, allelic contents (major allele counts) and normal cell contamination were detected; segmentations were optimized

2 with respect to the genomic status detected. R scripts and full details of the application are available at Recognition of absolute copy number ranged from 0 to 8 copies (all segments with the copy number variation exceeding 8-copy level were ascribed 8-copy status). Thus, 22 possible segmental genotypes were discriminated (copy number / major allele count): A (1/1); AA (2/2) and AB (2/1); AAA (3/3) and AAB (3/2); AAAA (4/4), AAAB (4/3) and AABB (4/2); etc. Proportions of segmental genotypes in tumor genomes were calculated. Inferring chromosome number Number of chromosomes was estimated by the sum of the copy numbers detected at the pericentric regions. The status of the pericentric region of each chromosome arm was defined by the corresponding juxta-centromeric segment when the latter contained 500 SNPs or more. When not measurable, missing values were substituted by the modal copy number of the considered chromosome arm (3.4±2.2 out of 41 chromosome arms per genome were substituted in the series). This chromosome counting procedure was validated by comparing estimated chromosome numbers versus available numbers from karyotype or SKY data for 25 breast cancer cell lines ( (Table S1). Error rate was less than 2 chromosomes per sample (1.7±2.3). Detection of tumor ploidy Tumor ploidy was detected based on the inferred chromosome number and further verified by the representative genomic states of the tumor genome. Typical near-diploid and neartetraploid genomic profiles from the considered set of BLCs and their GAP patterns are shown in Figure S1. Near-diploid genomes (defined by the chromosome counts < 50) were mainly distributed between normal state (AB), one copy loss (A), one copy gain (AAB), and

3 uniparental disomy (AA) (44.2±10.6%, 34.6±9.1%, 8.8±2.9%, 7.2±3.1% in average per genome, respectively; 87.6±4.13% in total in the experimental set of BLCs; see Table S2). Near-tetraploid genomes (defined by the chromosome counts 50 chromosome counts) were mainly distributed between AABB, AA and AAB genomic states (13.0±7.8%, 26.7±11.0%, 18.9±8.6%, in average per genome respectively; 58.6±15.7% in total; see Table S2). However, three tumors displayed genomic profiles outlying from this general scheme (Figure S2 A,B,C). The tumor BLC_78 carried 61 chromosomes while displaying the near-diploid pattern of alterations (AB, AAB and AAA accounted for 41%, 14% and 15%, respectively). The tumor BLC_25 displayed the highest range of well represented different genomic states of the series with 108 chromosomes (DNA index = 2.36), probably suggesting higher than near-tetraploid state. The tumor BLC_10 carried more than half of the chromosome set in a triplicated homozygous state (AAA comprised 58% of the genome). The tumor BLC_62 with borderline chromosome counts (52) actually did not globally fit to the near-diploid pattern because of the low proportion of AB state (10% vs 44.2±10.6% in the typical near-diploid genome). The first case was then mined together with the near-diploid BLCs, while the three latter cases together with the near-tetraploid BLCs. Breakpoints estimation Number of breakpoints was estimated depending on the size of associated segments. Based on the distribution of the size of the detected variation we defined a threshold of 3Mb which separates conventionally small-scale alterations and large-scale alterations (Figure S3, A-B). Distribution of the segment size in the experimental set of BLCs clearly follows exponential law starting from approximately 3Mb of the segment size: log2 (f) ~ 0.1 S 5.0, where f is the average proportion of segments of the size more or equal to S. The effective size of the segment (mean of the exponentially distributed random value) is thus ~6.5 Mb. Given that small-scale breakpoints often were clustered within rather small genomic regions (for

4 example, in firestorms ) and thus were not independent and not characteristic of the genomic instability rate, we smoothed and/or filtered small-scale variation and estimated breakpoints number referring only to the large-scale variation. We introduced the Large-scale State Transitions (LSTs) as chromosomal breaks between adjacent regions of at least 10Mb (comprising ~3000 SNPs in Affymetrix SNP6.0) determined after smoothing and filtering of all variation less than 3Mb in size. Validation of LSTs by Next Generation Sequencing and Sanger sequencing Validation of LSTs was performed based on the Next Generation Sequencing (NGS) data of one BRCA1 mutated BLC described in (2) and also included in our cohort (BLC_B1_T06). SNP arrays were available on the two platforms Illumina 300K and Affymetrix SNP6.0. SNP arrays were processed following the same procedure and the number of LSTs was estimated to be 34 and 30 for Illumina and Affymetrix chips respectively (30 LSTs were found to be in common). 4 LSTs detected only by Illumina profiling were missed in Affymetrix profile due to the noise and ambiguous interpretation. The tumor was classified as near-diploid, so both LST numbers exceeded the cut-off of the non-brcaness for the near-diploid tumors. LSTs detected were crossed with the NGS data describing structural rearrangements in the tumor genome (TableS3, Figure S4). All LSTs detected corresponded to the copy number change, also evident from the copy number profile obtained based on the number of reads. 28/34 LSTs were supported by the fine attributed structural rearrangement event, where 19/28 were defined as inter-chromosomal rearrangements, 5/28 as tandem duplications, 3/28 as probable inversions and 1/28 as deletion. 9/28 were validated by the Sanger sequencing and 11/28 were validated by translocation specific PCR (Figure S5). 6/34 LSTs were lacking fine placement of the breaks, while were clearly supported by the copy number change detected for those regions. Those LSTs which failed validation (3 translocations) corresponded to regions with

5 multiple homology across the genome, evidencing possible misplacement of translocation determined by NGS. Features of LSTs 1. LST robustness to the calculation procedure Filtering less than 3Mb variations before calculating the number of LSTs is essential for the robustness of discrimination. Calculation of LSTs without any filtering resulted in reduced number of LSTs detected (78±12% on average per tumor genome of LSTs detected after filtering). The way of filtering and smoothing was not strongly affecting the number of breakpoints detected. We effectuated two ways of filtering/smoothing: 1) simple filtering of all segments less than 3Mb; 2) successive filtering and smoothing starting from the minimal segment. Only 10% cases were affected by the procedure and the difference in the LST number was less than 10% in all cases (Figure S6 A-B). The centromeric breaks were not taken into account when calculating LSTs; the chromosome arms were considered independently. Including centromeric regions enlarges the number of LSTs on 6.7±2.0 on average per genome and did not affect the overall discriminative pattern (Figure S6 C). 2. What type of alteration detects LSTs LSTs comprised 45±12% and 38±9% of all breaks detected after less than 3Mb filtering in the near-diploid and near-tetraploid genomes, respectively. In the near-diploid genomes the following transitions comprised 90.8±8.8% of LSTs: AB B, AB ABB, B BB, ABB BB, ABB B, BB BBB, ABB AABB, ABB ABBB (ordered according to the mean frequency of occurrence). In the near-tetraploid genomes the following transitions comprised 68.8±17.0% of LSTs: AB ABB, ABB AABB, BB AABB, BB BBB, B BB, BB BBBB,

6 AABB AABBB, AABB AABBBB, AB B, ABB ABBB (ordered according to the mean frequency of occurrence). Transitions corresponding to the change of allelic content without copy number change (for example, transitions AB BB, so called uniparental disomy, or ABB BBB, etc) were observed to be rather rare (at maximum 2 transitions per genome in about 10% of cases). To conclude, LSTs covered such types of alterations as, gains, losses, copy number change within LOH regions and LOH without copy number change. The fact that LSTs were mainly associated with copy number change evidenced possibility of evaluation of LSTs number directly from the copy number variation profile, however, this need to be further verified. 3. Number of LSTs and number of chromosomes in the tumor genome Number of LSTs does not depend on the chromosome number within each ploidy subgroup and in general across samples. (Figure S6 D) 4. LSTs and chromothripsis Based on the only one formal hallmark of chromothripsis, namely, exceptionally high number of alteration events affecting several chromosomes or chromosome arms (3), 7 BLCs from our series could be considered to be affected (20-40 breaks per chromosome arm). However, copy number variation in those chromosomes comprised various states, possibly evidencing subsequent alteration events after chromosome disruption or some other mechanism of appearance. Filtering less than 3 Mb variations resulted in dramatic reduction and homogenization of number of chromosomal breaks (up to 6.2 ± 1.9 maximum breaks number per chromosome arm in our cohort). Maximal number of LSTs per chromosome arm detected in our cohort of tumors was less than 6 in all cases (3.1 ± 1.0), evidencing that LSTs avoid overestimation of the level of genomic instability due to possible catastrophic events affecting individual chromosomes.

7 To conclude, number of LSTs is a good surrogate measure of amount of the large scale rearrangements correlated to BRCA1 inactivation. Number of LSTs reflected well the overall genomic patterns of the tumors, contrary to the total number of breakpoints: tumors with numerous small scale variation displayed low number of LSTs contrary to the high LSTs number observed in BRCA1 tumors (Figure S7). Moreover, the constructive way of definition of LSTs ensures their possible application on various platforms measuring genomic alterations. Distribution of LSTs along the genome Distribution of LSTs along the genome was not uniform, with 11 peaks accumulating more than 6 breaks (at least 7 tumors had LSTs detected in the region) (Figure S8 A). Some of the peaks in LSTs distribution along the genome displayed elevated GC content. One peak with the lowest GC was associated with a common fragile site (4) (Figure S8 B). These observations imply that accumulations of breakpoints in specific genomic region are due to several mechanisms, including, expressed genes, fragile sites, and probably selection in tumor evolution. LSTs in detection of BRCA1 tumors in Experimental and Validation sets The resulting tables for the Experimental and Validation (Tables S4 and S5) contain LSTs number, breakpoints number and some supplementary information. Somatic BRCA1 mutations detected in BLCs with high LST BRCA1 mutations were searched in 27 sporadic BLCs with available material including 12 cases with high LST. Deleterious mutations were found in 7 cases with high LST, including the one case with whole exon deletion. According to French regulations, searching for the presence of a BRCA1 mutation in germline DNA was restricted to the two patients who previously had an oncogenetic council at Institut Curie. In each case, the mutations were

8 neither found in the germline DNA of the patients nor - in a hypothesis of germinal mosaic - in normal tissue adjacent to the tumor, demonstrating their somatic origin (Figure S10). BRCA2 mutations detected in BLCs with high LST The remaining 5 tumor cases with high LST were tested for BRCA2 mutations. Three tumors were found mutated for the BRCA2: BLC_T33 (c.9026_9030del5/p.tyr3009tyrfsx19 ); BLC_T35 (c.8140c>t/p.gln2714x ); BLC_T62 (c.9106c>t/p.gln3036x). All these tumors displayed LOH on the 13q in the BRCA2 locus. Triple negative cell lines Comparison of LSTs to the deep sequencing data of the TN cell lines {3} showed partial overlap with the inter-chromosomal rearrangements being the most frequent event corresponding to LSTs (Table S6). Stability of LSTs in xenograft passages as compared to primary tumors Copy number alterations and LSTs number were compared between primary tumors and several passages of xenografts from publicly available series GSE from GEO (Table S7, Figure S10) (5). References 1. Popova T, Manie E, Stoppa-Lyonnet D, Rigaill G, Barillot E, Stern MH. Genome Alteration Print (GAP): a tool to visualize and mine complex cancer genomic profiles obtained by SNP arrays. Genome Biol 2009;10:R Natrajan R, Mackay A, Lambros MA, Weigelt B, Wilkerson PM, Manie E, et al. A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptornegative and -positive breast cancers J Pathol 2012; 227: Stephens PJ, Greenman CD, Fu B, Yang F, Bignell GR, Mudie LJ, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 2011; 144:

9 4. Mrasek K, Schoder C, Teichmann AC, Behr K, Franze B, Wilhelm K, et al. Global screening and extended nomenclature for 230 aphidicolininducible fragile sites, including 61 yet unreported ones. Int J Oncol 2010; 36: DeRose YS, Wang G, Lin YC, Bernard PS, Buys SS, Ebbert MT, et al. Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nat Med 2011; 17:

10 Table S1. Number of chromosomes in the cell lines according to ATCC description, SKY images, and GAP estimation Cell line GAP ATCC ATCC min ATCC max SKY Error* HCC HCC Hs578T MDA-MB MDA-MB MDA-MB MDA-MB CAMA BT MCF MDA-MB BT MDA-MB HCC HCC HCC MDA-MB HCC outlier HCC HCC HCC PMC B SKBR ZR *Error was calculated as minimal absolute difference between GAP estimation and ATCC and/or SKY numbers.

11 Table S2. Tumor ploidy and percentage of genomic states in tumor genome SampleID Ploidy Chromosome A AB AA AAB AAA AABB AAAB AAAA AAABB AAAAB AAAAA >5copy BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T40A BLC_B1_T42B BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T78* BLC_B1_T BLC_B1_T07A BLC_B1_T BLC_B1_T BLC_B1_T13A BLC_B1_T BLC_B1_T BLC_B1_T BLC_B1_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T BLC_T MBC_B1_T MBC_T MBC_T BLC_T BLC_T *Despite the chromosome numbers BLC_78 fits to the near-diploid pattern.

12 Table S3. Validation of LSTs by NGS, PCR and Sanger sequencing LST* number Type by NGS or SNP Transition LSTs Comments / PCR or Sanger validation Left Chr Left Position Right Chr Right Position Junction- EventId Id 1 tandem-duplication LST 1 B_AB Copy Number change, but no junction probable-inversion LST 2 ABB_AB Simple transition Inter-chromosomal Validated Inter-chromosomal Validated Transition through 3 LST 3 ABB_B short AB segment Inter-chromosomal Validated LST 4 B_AB Simple transition deletion LST 5 AB_B Copy Number change, but no junction Inter-chromosomal LST 6 B_AB Simple transition Inter-chromosomal Validated LST 7 B_AB Simple transition Inter-chromosomal NotValidated distal-duplication Validated X LST 8 AB_B Simple transition Inter-chromosomal Validated LST 9 BB_ABB Inter-chromosomal Validated probable-inversion Validated LST 10 AB_B Simple transition LST 11 B_BB Simple transition Inter-chromosomal Validated LST 12 BB_BBB Simple transition Inter-chromosomal Validated Inter-chromosomal tandem-duplication Validated Transition with short 13 LST 13 BBB_BB tandem duplication Inter-chromosomal Validated LST 14 BB_B Simple transition

13 Table S3. Continue 15 LST 15 AB_ABBB Simple transition, possibly through ABB Inter-chromosomal NotValidated LST 16 ABBB_ABBBB Transition with short tandem duplication Inter-chromosomal Validated LST 17 AB_B Simple transition B 17 Inter-chromosomal Validated tandem-duplication Validated LST 18 ABB_ABBB Big gain probable-inversion Validated Clear deletion, 19 LST 19 AB_B indicated as inversion LST 20 AB_B Simple transition Inter-chromosomal Validated LST 21 B_AB Simple transition Inter-chromosomal NotValidated LST 22 AB_ABB The same as LST tandem-duplication Validated LST 23 ABB_B The same as LST 22; transition through AB tandem-duplication Inter-chromosomal LST 24 B_BB Simple transition probable-inversion Validated Simple transition with 25 LST 25 BB_B small interstitial gain deletion Validated LST 26 B_AB Simple transition LST 27 AB_B Simple transition Inter-chromosomal Validated LST 28 B_AB Copy Number change, but no junction distal-duplication Validated X Copy Number change, 29 LST 29 AB_ABB but no junction X X probable-inversion X X

14 Table S3. Continue 30 tandem-duplication X X LST 30 ABB_AB Copy Number change, but no junction X tandem-duplication X X tandem-duplication Validated LST 31 B_BBB False recognition in both Illum and Affy, breaks but NOT LST LST 32 AB_ABB missing break in Affimetrix tandem-duplication LST 33 AB_B break intermediated by LOH and deletion, detection depends on interpretation interchromosomal distal-duplication Validated tandem-duplication missing break in 34 LST 34 AB_ABB Affymetrix tandem-duplication tandem-duplication tandem-duplication * Color code for the LSTs related to the validated by NGS (orange), not validated by NGS (pink), validated by NGS, Sanger sequencing or translocation specific PCR (green), corresponding genomic positions (yellow).

15 Table S4. Final results obtained on the Experimental set of BLCs SampleID BRCA1 Ploidy LSTs All breaks BRCA1_672_at BLC_B1_T52 mutated BLC_B1_T06 mutated BLC_B1_T22 mutated BLC_B1_T14 mutated NA BLC_T35 BRCA BLC_B1_T42B mutated BLC_B1_T49 mutated BLC_T05 promoter methylated BLC_B1_T51 mutated BLC_T30 promoter methylated BLC_T56 promoter methylated BLC_T32 promoter methylated BLC_T50 somatic mutation BLC_T15 promoter methylated BLC_T74 ND BLC_B1_T46 mutated BLC_B1_T53 mutated BLC_T63 promoter methylated BLC_B1_T45 mutated BLC_B1_T17 mutated BLC_B1_T40A mutated NA BLC_T72 promoter methylated NA BLC_T68 ND BLC_T78 ND BLC_B1_T19 mutated BLC_T40 mutation in tumor BLC_T33 BRCA BLC_T43 mutation in tumor NA BLC_T79 promoter methylated BLC_B1_T03 mutated NA BLC_T60 mutation in tumor BLC_T34 mutated BLC_T52 promoter methylated BLC_T61 promoter methylated BLC_B1_T12 mutated NA BLC_T12 promoter methylated BLC_B1_T103 mutated BLC_B1_T18 mutated NA BLC_T31 somatic mutation BLC_T62 BRCA BLC_B1_T07A mutated BLC_B1_T20 mutated MBC_B1_T26 mutated MBC_T25 mutation in tumor BLC_B1_T13A mutated BLC_B1_T21 mutated MBC_T24 mutation in tumor BLC_T55 ND BLC_T73 WT BLC_T01 ND BLC_T23 WT NA BLC_T25 ND BLC_T69 ND BLC_T70 WT BLC_T71 ND BLC_T37 ND BLC_T64 WT BLC_T66 WT BLC_T07 ND NA BLC_T09 ND BLC_T57 ND BLC_T36 WT BLC_T53 WT BLC_T02 ND NA BLC_T10 ND

16 Table S5. Final results obtained on the Validation set of BLCs SampleID BRCA1 GAP quality Ploidy Chromosomes content LSTs breaks Origin Tumor All X50467 promoter methylated ok GSE19177 BC_TN_YW_T13 promoter methylated ok Institut Curie X80553 mutated ok GSE19177 GSM805884_HCI_010 ND ok GSE32350 BC_TN_YW_T5 promoter methylated ok Institut Curie X70441 mutated ok GSE19177 BC_TN_YW_T4 promoter methylated ok Institut Curie X70672 mutated ok GSE19177 BC_TN_YW_T3 ND ok Institut Curie BC_TN_YW_T9 ND ok Institut Curie KFAM_142 mutated ok Institut Bergonie BC_TN_YW_T12 promoter methylated ok Institut Curie X20091 mutated ok GSE19177 X60034 mutated ok GSE19177 medull05 promoter methylated ok Institut Curie GSM805876_HCI_004 ND ok GSE32350 BC_TN_YW_T7 promoter methylated ok Institut Curie medull04 promoter methylated ok Institut Curie medull08 promoter methylated ok Institut Curie BLC_B2_T37B BRCA2 ok Institut Curie medull44 promoter methylated ok Institut Curie BC_TN_YW_T10 ND ok Institut Curie medull38 ND ok Institut Curie GSM805871_HCI_002 ND ok GSE32350 X30130 mutated ok GSE19177 BC_TN_YW_T1 ND ok Institut Curie BC_TN_YW_T6 promoter methylated ok Institut Curie KFAM_135 mutated ok Institut Bergonie X20034 mutated ok GSE19177 BC_TN_YW_T11 promoter methylated ok Institut Curie GSM805868_HCI_001 ND ok GSE32350 KFAM_136 mutated questionable Institut Bergonie KFAM_139 mutated ok Institut Bergonie BC_TN_YW_T14 promoter methylated ok Institut Curie KFAM_134 mutated ok Institut Bergonie BLC_T41V promoter methylated ok Institut Curie X11396 mutated ok GSE19177 BC_TN_YW_T8 promoter methylated ok Institut Curie X70020 mutated ok GSE19177 X21349 ND ok GSE19177 KFAM_137 mutated questionable Institut Bergonie medull43 ND ok Institut Curie BC_TN_YW_T15 ND questionable Institut Curie X60681 ND questionable GSE19177 medull06 ND questionable Institut Curie BLC_T20V ND contamination70% Institut Curie medull36 ND ok Institut Curie BC_TN_YW_T2 ND questionable Institut Curie medull01 ND ok Institut Curie BC_TN_YW_T17 ND ok Institut Curie BC_TN_YW_T19 ND ok Institut Curie BC_TN_YW_T18 ND ok Institut Curie BLC_T65V ND questionable Institut Curie BC_TN_YW_T16 ND ok Institut Curie medull12 ND questionable Institut Curie GSM805908_HCI_008 ND excluded contamination100% GSE32350 KFAM_145 mutated excluded bad quality Institut Bergonie KFAM_146 BRCA2 Excluded contamination 85% Institut Bergonie KFAM_147 mutated Excluded contamination 100% Institut Bergonie X20021 promoter methylated excluded bad quality GSE19177

17 Table S6. LSTs fit validated rearrangements in triple-negative cell lines Sample Simplified Nomenclature CN transition Chr Position 1 LST Position LST Position 1_NGS Strand 1Chr 2 Position 2_NGS HCC1143 Amplified 3/3_4/ HCC1143 Tandem Duplication 4/3_3/ HCC1143 Inter-chromosomal 6/6_4/ HCC1143 Amplified 6/4_8/ HCC1143 Amplified 8/6_7/ E+08-1 HCC1143 Inter-chromosomal 5/4_3/ HCC1143 Deletion 2/2_5/ HCC1187 Inter-chromosomal 2/2_4/ HCC1187 Inter-chromosomal 4/2_2/ E HCC1187 Inter-chromosomal 2/2_1/ HCC1187 Deletion 3/3_2/ HCC1187 Tandem Duplication 4/2_2/ HCC1187 Inter-chromosomal 5/3_3/ HCC1187 Deletion 3/2_2/ HCC1187 Deletion 2/2_3/ HCC1187 Inter-chromosomal 3/3_1/ HCC1187 Inter-chromosomal 2/2_3/ HCC1187 Inverted orientation 4/2_2/ HCC1395 Inter-chromosomal 3/3_2/ HCC1395 Inter-chromosomal 3/3_1/ HCC1395 Tandem Duplication 1/1_2/ HCC1395 Inter-chromosomal 2/2_3/ HCC1395 Inter-chromosomal 2/2_3/ E+08-3 HCC1395 Inter-chromosomal 3/2_2/ HCC1395 Inter-chromosomal 3/3_2/ HCC1395 Tandem Duplication 4/2_2/ HCC1395 Inter-chromosomal 4/2_2/ HCC1395 Inter-chromosomal 3/2_4/ HCC1395 Inter-chromosomal 2/1_3/ HCC1395 Inter-chromosomal 3/2_2/ HCC1599 Inter-chromosomal 4/3_5/ E HCC1599 Inverted orientation 3/3_4/ E+08-2 HCC1599 Inter-chromosomal 2/1_4/ E HCC1599 Inter-chromosomal 4/3_3/ E+08-3 HCC1599 Inter-chromosomal 3/2_2/ E+08-4 HCC1599 Inter-chromosomal 1/1_2/ E HCC1599 Amplified 1/1_2/2 X E Strand 2 No. Reads

18 Table S6. Cont. HCC1937 Inter-chromosomal 3/3_6/ E+08-7 HCC1937 Inter-chromosomal 4/4_2/ E+08-5 HCC1937 Inter-chromosomal 5/5_3/ E+08-2 HCC1937 Inter-chromosomal 5/5_8/ HCC1937 Deletion 2/2_4/ HCC1937 Tandem Duplication 5/3_4/ E+08-6 HCC1937 Inter-chromosomal 4/2_2/ HCC1937 Inverted orientation 5/5_3/ HCC1937 Inverted orientation 5/3_8/ HCC1937 Inter-chromosomal 3/2_5/ HCC1937 Inter-chromosomal 2/2_3/ HCC1937 Inter-chromosomal 7/4_6/ HCC1937 Inter-chromosomal 3/2_2/ HCC1937 Inter-chromosomal 6/4_4/ HCC38 Tandem Duplication 3/2_6/ E HCC38 Tandem Duplication 6/4_6/ E+08-2 HCC38 Inverted orientation 6/4_3/ E HCC38 Deletion 3/2_4/ HCC38 Inter-chromosomal 4/2_5/ HCC38 Inter-chromosomal 6/6_3/ HCC38 Inter-chromosomal 3/3_2/ E HCC38 Inverted orientation 4/4_5/ E+08-3 HCC38 Tandem Duplication 2/2_4/ E HCC38 Deletion 4/4_2/ E HCC38 Deletion 2/2_4/ E+08-4 HCC38 Tandem Duplication 4/3_2/ HCC38 Tandem Duplication 2/1_4/ E HCC38 Inter-chromosomal 4/3_2/ HCC38 Tandem Duplication 6/3_8/ HCC38 Inter-chromosomal 3/3_2/ E HCC38 Inter-chromosomal 4/2_2/ HCC38 Deletion 4/3_1/ HCC38 Deletion 1/1_4/ HCC38 Inter-chromosomal 4/3_3/ HCC38 Inter-chromosomal 4/2_3/ E+08-8

19 Table S7. LSTs are conserved in xenografts Sample Ploidy LST BRCAness Comments GSM805868_HCI_ Yes GSM805869_HCI.001_x Yes GSM805870_HCI.001_x Yes GSM805871_HCI_ No GSM805872_HCI.002_x1 2 7 No GSM805873_HCI.002_x5 2 7 No GSM805876_HCI_ Yes GSM805877_HCI.004_x Yes GSM805884_HCI_ No GSM805885_HCI.009_x No Second passage acquired a number of additional aberrations: +9LSTs Perfect correspondence of three profiles and LSTs Xenograft had +5 LSTs 9 LSTs in common GSM805886_HCI_ Yes Pattern of recent duplication, 29 LSTs in common, 4 LSTs were modified after GSM805887_HCI.010_x Yes duplication + 3 new LSTs in xenograft

20 Figure S1. Near-diploid (A) and near-tetraploid (B) profiles and patterns of alteration detected with the GAP method in the series of BLCs (Affymetrix SNP6.0).

21 Figure S2. Four outlying patterns of alteration found in the series of BLCs. A. Near-tetraploid tumor, where 41% of AB genomic status evidenced ploidy of 2. B. Over-tetraploid tumor with DNA index of C. Tumor with 58% of genome with AAA status (triplicated homozygous genomic state). D. Tumor with 52 chromosomes, where low AB content (10%) contradicted to the ploidy of 2.

22 Log2(f) ~ 0.1 S 5.0 A. B. Figure S3. Deciphering the cutoff for the small-scale variation. A. Distribution of the log2(s) (S=size of segments) found in tumor genomic profiles in the set of 65 BLCs and two fitted normal distributions. The shape of distribution suggests two populations of segments, one (red curve) accounts for small-scale variation and another (blue curve) accounts for largescale variation. B. Average frequency of number of breakpoints depending on the size of a segment calculated for 65 BLC. Prominent change of the decreasing rate around 3 Mb is pointed by the arrow. Linear model was fitted starting from 4 Mb segment size and excluding outliers.

23 Figure S4. LSTs detected in SNP array profile and validated by the NGS, Sanger sequencing and translocation specific PCR. A. All validated LSTs; B. Mistakes in LST recognition due to the noise or misinterpretation. Red arrows: validated in NGS; Green arrows: validated in NGS and Sanger sequencing/pcr; Black arrows: fine resolution of the break not found; Dashed arrow: misinterpretation of SNP array profile.

24 Figure S5. Translocation specific PCR for validation of inter-chromosomal translocations detected by NGS and found within LSTs boundaries.

25 A. B. number of breaks length of the segment number of breaks number of breaks length of the segment C. D length of the segment Figure S6. Persistence and robustness of the discriminating window independently of the method of LSTs evalutation (supplementary to Figure 2B). A. Simple filtering of all variation less than 3 Mb. B. Consequential filtering (starting from the smallest size segment) and smoothing of all variation up to 3 Mb in size. C. The same as B, but including centromeric breaks; D. Number of LSTs does not depend on the chromosome counts estimated from the tumor genome profile.

26 Figure S7. Two tumors with similar total numbers of breakpoints. A. BLC_53, a non-brca1 BLC; B. BRCA1_07A, a BRCA1 BLC

27 A. B. Figure S8. Distribution of LSTs along the genome, GC content and common fragile site for the chromosome bands with LSTs detected in more than 6 tumors. A. Number of tumors having LST detected in a corresponding chromosome band, 11 LST hotspots are indicated by the orange triangles; (B). Average GC content in the whole genome (green box) and in the 14 chromosome bands covering the 11 LST hotspots (one hotspot corresponding to the common fragile site is a red box).

28 Figure S9. Somatic mutation in BRCA1 detected in two BLCs.

29 Figure S10. LSTs in xenograft passages from GSE Alteration profiles are shown after filtering all variation less than 3 Mb. Violet fragments of CN profile corresponded to segments determining LSTs. Horizontal lines corresponded to copy number counts: dashed grey 1 copy; two gray lines 2 and 4 copies and read dashed 6 copies. Pr: primary tumor; X1, X2, X5: 1, 2, 5 xenograft passages, respectively.