Supplementary Information Titles

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1 Supplementary Information Titles Please list each supplementary item and its title or caption, in the order shown below. Note that we do NOT copy edit or otherwise change supplementary information, and minor (nonfactual) errors in these documents cannot be corrected after publication. Please submit document(s) exactly as you want them to appear, with all text, images, legends and references in the desired order, and check carefully for errors. Journal: Nature Medicine Article Title: Corresponding Author: Chromosome 1q21.3 amplification is a trackable biomarker and actionable target for breast cancer recurrence Qiang Yu Supplementary Item & Number (add rows as necessary) Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Figure 9 Supplementary Figure 1 Supplementary Figure 11 Supplementary Table 1 Supplementary Table 2 Title or Caption Characterization of patient-derived tumorspheres. Validation of 1q21.3 amplification in METABRIC dataset and Singapore discovery cohort. Evaluation of ddpcr assay performance with genomic DNA. ddpcr copy number analysis of patient related samples. 1q21.3 amplification is associated with tumor recurrence. ddpcr analysis of cfdna samples. Detection of 1q21.3 amplification in cfdna of breast cancer patients. Flow diagram of patients included in in breast cancer cohort studies. cfdna detection of 1q21.3 amplification in the course of chemotherapy. S1A7/8/9 promotes tumorsphere growth. Pacritinib treatment preferentially inhibits tumor growth in 1q21.3 amplified xenograft tumors. Clinical pathological characteristics of patients used in Singapore TTSH discovery cohort Multivariate Cox regression analysis of 1q21.3 amplification status for overall survival of Singapore TTSH discovery cohort (n = 67).

2 Supplementary Table 3 DNA FISH quantitation of 1q21.3 (Red R) and 1p32.3 (Green G). Supplementary Table 4 Stage Information for Fig. 2c,d. Supplementary Table 5 Clinical information and ddpcr result for Fig. 2c-e and Supplementary Fig. 5b. Supplementary Table 6 Healthy female individuals plasma samples (n = 3) Supplementary Table 7 Clinical information and ddpcr result for Fig. 3a and Supplementary Fig. 7b. Supplementary Table 8 Clinical information and ddpcr result for Fig. 3c. Supplementary Table 9 Clinical pathological characteristics of Denmark OUH earlystage breast cancer cohort. Supplementary Table 1 Clinical information and ddpcr result of Denmark OUH early-stage breast cancer cohort. Supplementary Table 11 Clinical pathological characteristics of Singapore NUHS neoadjuvant breast cancer cohort. Supplementary Table 12 Clinical information and ddpcr result of Singapore NUHS neoadjuvant breast cancer cohort. Supplementary Table 13 Multivariate Cox regression analysis of 1q21.3 amplification status for overall survival of Singapore NUHS neoadjuvant breast cancer cohort. Supplementary Table 14 Clinical information and data for Fig. 4i-k and Supplementary Fig. 11a. Supplementary Table 15 Primers and oligos sequences.

3 a 15 ** ** ** *** rlog Expression 1 5 Primary tumor (n = 12) Patient-derived tumorsphere (n = 12) ALDH1A3 ALDH3A1 ALDH3A2 ALDH3B2 b 25 Patient 3 tumor 25 Patient 5 p tumor 25 Patient 7 tumor 2 3.5% 2 1.4% 2 2.6% SSC 15 1 R5 SSC 15 1 R5 SSC 15 1 R ALDH-FITC ALDH-FITC ALDH-FITC 25 Patient 3 tumorsphere 25 Patient 5 tumorsphere p 25 Patient 7 tumorsphere p % % 2 1% SSC 15 1 SSC 15 1 R5 SSC 15 1 R ALDH-FITC ALDH-FITC ALDH-FITC c Tumorsphere cells / injections Patient 2 tumorsphere (ER positive) 2 / 2 2 / 2 / 2 Patient 3 tumorsphere (ER negative) 2 / 2 2 / 2 1 / 2 Supplementary Figure 1. Characterization of patient-derived tumorspheres. (a) Boxplot of several ALDH family members gene expression in primary tumors versus matching patient-derived tumorpsheres as determined by RNAseq. p-value was calculated with Wilcoxon signed-rank test. ** p <.1, *** p <.1. Box plots with median value (center line), minimum, 25%, 75% and maximum value lines (box lines) are presented. (b) Flow cytometry analysis for the ALDH activity in primary tumors and corresponding tumorsphere cells. (c) In vivo limiting dilution assay showing tumor initiating capability of patient-derived tumorsphere cells in immunodeficient, NOD-SCID gamma (NSG) mice.

4 a c Cumulative Survival (%) 1 mrna expression Expression: No. of patients: 8 6 1q21.3 Neutral Gain Amp All p < HR: 2.68 p < Low (n = 115) High (n = 56) Time (months) Cumulative Survival (%) b Time to death (Days) Days: No. of patients: ER positive HR: p = Low (n = 648) High (n = 13) Time (months) 1q21.3 * n.s. Neutral Gain Cumulative Survival (%) Amp ER negative HR: p = Low (n = 284) High (n = 4) Time (months) d S1A8 copy number amplification Normal Breast Epithelial Breast Tumor Specimen Amp: 11/67 (16.4%) e Cumulative Survival (%) Singapore Discovery Cohort Overall Survival 25 HR: log-rank p = Months Negative (n = 56) Positive (n = 11) (n = 11) (n = 56) Supplementary Figure 2. Validation of 1q21.3 amplification in METABRIC dataset and Singapore discovery cohort. (a) Correlation of 1q21.3 amplification and corresponding gene expressions in METABRIC breast cancer dataset. The number of patients per group is indicated below graph. p-values were calculated with Kruskal-Wallis test. Box plots with median value (center line), minimum, 25%, 75% and maximum value lines (box lines) are presented. (b) Time to death analysis of patients with 1q21.3 amplification in METABRIC breast cancer dataset. The number of patients per group is indicated below graph. p-values were calculated with one-tailed Mann-Whitney test. * p <.5, n.s., not significant. For scatter plots, data points with median value (center line) and error bars are presented. (c) Kaplan-Meier overall survival analysis of the combined gene signature (17 genes on 1q21.3) in METABRIC breast cancer dataset. The number of patients (n) per group is indicated. HR: Hazard ratio (d) Clinical validation of copy number amplification of S1A8 gene in 67 breast primary tumor specimens using real-time PCR of genomic DNA. 1 normal breast epithelial tissues were used as control. A cutoff of copy number ratio for S1A8 gene was used to yield two groups (positive and negative). Data are expressed as means ± s.d. of three technical replicates. (e) Kaplan- Meier overall survival analysis of breast cancer patients with positive or negative S1A8 amplification in 67 primary tumors of Singapore discovery cohort. The number of samples (n) per group is indicated. HR: Hazard ratio.

5 a Total ddpcr copies / 2 µl Total ddpcr copies / 2 µl Normal genomic DNA Normal genomic DNA Normal genomic DNA RPP3 TUFT1 1q21.3 amplified genomic DNA RPP3 TUFT1 RPP3 S1A7 1q21.3 amplified genomic DNA RPP3 S1A7 RPP3 S1A8 1q21.3 amplified genomic DNA RPP3 S1A8 DNA input (ng) DNA input (ng) DNA input (ng) b 2.2 Copy number ratio ng.1ng (25%) (5%) 1ng (5%) ng (75%) 2ng (1%) % amplified tumor DNA in normal diploid genomic DNA Supplementary Figure 3. Evaluation of ddpcr assay performance with genomic DNA. (a) Range of detection for genomic amplification by ddpcr was validated by serial dilutions of normal fibroblast genomic DNA and 1q21.3 amplified genomic DNA (HCC7) starting from 1 ng of input DNA. As low as.156 ng of genomic DNA input with 32-4 copies/reaction was sufficient to detect 1q21.3 amplification. Data are generated from means of two technical replicates. (b) Sensitivity testing of 1q21.3 assay to detect copy number amplification in heterogeneous population. 1q21.3 amplified genomic DNA (HCC7) was serially diluted into normal diploid human fibroblast DNA. 5% of tumor DNA with 1q21.3 amplification can be detected in the presence of 2 ng normal diploid genomic DNA. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Dataare generated from means of two technical replicates.

6 a RPP3 TUFT1 S1A7 S1A8. Normal Tumor Normal Tumor Normal Tumor BT11 BT64 BT95 b ALDH+ vs ALDH- ALDH+ ALDH-.5. RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 Patient 3 Patient 4 Patient 5 Patient 7 ER positive ER negative c S1A8 (1q21.3) PVRL4 (1q23.3) LAMB3 (1q32.2).5. Fibroblast Epithelial Patient 3 Patient 5 Patient 7 Normal Tumorsphere Supplementary Figure 4. ddpcr copy number analysis of patient related samples. (a) ddpcr results showing the relative copy number amplification of TUFT1, S1A8 and S1A7 genes in three positive primary tumors and matched normal adjacent tissues. RPP3 was used as reference gene for normalization. Data are expressed as means ± s.d. of four technical replicates. (b) ddpcr analysis of 1q21.3 genes in FACS-sorted ALDH positive and negative tumorsphere cells. RPP3 was used as reference gene for normalization. Data are expressed as means ± s.d. of two technical replicates. (c) ddpcr analysis of the copy number ratio of S1A8, PVRL4 and LAMB3 in normal tissues and tumorsphere cells. RPP3 was used as reference gene for normalization. Data are expressed as means ± s.d. of four technical replicates.

7 a 2..5 Normal breast tissue Reference gene 3 target genes -3 SD. BT11N BT22N BT31N BT33N BT44N BT48N BT64N BT65N BT74N BT79N BT82N BT9N BT91N BT95N BT96N BT98N BT11N BT13N BT14N BT18N BT118N BT23N BT24N BT27N BT219N BT223N BT224N BT228N BT239N BT26N b 2..5 Primary Tumor OUH Validation Cohort Patients with recurrence within 5 years Reference gene 3 target genes Recurrent Tumor Reference gene 3 target genes. D-2-P D-9-P D-2-P D-25-P D-26-P D-4-P D-47-P D-51-P D-58-P D-59-P D-73-P D-82-P D-86-P D-112-P D-TNBC1-P D-TNBC2-P D-TNBC3-P D-TNBC4-P D-TNBC5-P D-TNBC6-P D-TNBC7-P D-TNBC8-P D-TNBC9-P c Untreated group Paclitaxel treatment group ER+ / HER2- (n = 13) HER2+ TN (n = 9) (n = 1) 2 weeks paclitaxel Untreated tumors n = 4 Residual tumors n = 5 Surgical removal Surgical removal ~8 weeks Recurrent tumors n = 4 Surgical removal d * *** 1q21.3 Supplementary Figure 5. 1q21.3 amplification is associated with tumor recurrence. (a) ddpcr analysis of 1q21.3 amplification in normal adjacent breast tissues from breast cancer patients (n = 3). Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of four technical replicates. Cutoff value for positive 1q21.3 amplification is determined (3 SD above mean). (b) ddpcr analysis of the primary tumors and matched recurrent tumors from patients who had recurrence within 5 years in OUH validation cohort. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates. The number of patients (n) per group is indicated. TN: Triple negative. (c) Breast orthotopic PDX model workflow. NOD-SCID mice bearing a breast PDX tumor were treated with vehicle or 2 mg/kg paclitaxel for 14 days to induce tumor regression. Untreated (n =4)andresidual(n = 5) tumors were harvested at the end of paclitaxel treatment. Recurrent (n =4)PDX tumors were harvested when the tumor relapse and its volume reached ~8 mm 3.(d) ddpcr analysis of 1q21.3 amplification in indicated tumors. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates. p-values were calculated with unpaired two-tailed Student s t-test. * p <.5, *** p <.1.

8 a Copy number ratio ng.1ng (25%) (5%) 1ng (5%) ng (75%) 2ng (1%) b 2..5 cfdna Healthy Control Reference gene 3 target genes -3 SD. TH-41 TH-117 TH-282 TH-378 TH-386 TH-498 TH-512 TH-564 TH-783 TH-867 TH-943 TH-947 TH-123 TH-1279 TH-1288 TH-1293 TH-1462 TH-147 TH-15 TH-1629 TH-1786 TH-185 TH-1938 TH-23 TH-261 TH-288 TH-2167 TH-2253 TH-2264 TH-2272 % amplified tumor DNA in cfdna Supplementary Figure 6. ddpcr analysis of cfdna samples. (a) Sensitivity testing of 1q21.3 cfdna assay was determined by spiking serial diluted 1q21.3 amplified genomic DNA (HCC7) into healthy control cfdna. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates. (b) ddpcr analysis of cfdna isolated from healthy female blood to establish normal baseline (n = 3). Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates. Cutoff value for positive 1q21.3 amplification is determined (3 SD above mean).

9 a 23 subjects with breast cancer 34 subjects with breast cancer Tumor tissue analysis Plasma analysis Tumor tissue analysis Plasma analysis 2 excluded due to low quantity of DNA in blood 1 excluded due to low quantity of DNA in blood 23 analysed samples 21 analysed samples 33 analysed samples 33 analysed samples Development cohort Concordance study 21 matching pairs Validation cohort Concordance study 33 matching pairs b Tumor DNA Amp: 8/21 (38.1%) Tumor DNA Amp: 7/33 (21.2%) Reference gene 3 target genes. 2. cfdna Amp: 8/21 (38.1%). 2. cfdna Amp: 7/33 (21.2%).5.5. C-BB856 C-BB931 C-BB111 C-BB1112 C-BB1113 C-BB1117 C-BB1122 C-BB1137 C-BB1151 C-BB1156 C-BB1164 C-BB1188 C-BB1328 C-BB133 C-BB1332 C-HOB-44 C-HOB-77 C-HOB-8 C-HOB-85 C-HOB-88 C-HOB-96 Development cohort (n = 21). T-BT49 T-BT55 T-BT65 T-BT82 T-BT95 T-BT11 T-BT24 T-BT216 T-BT224 T-BT225 T-BT235 T-BT239 T-BT443 T-BT46 T-BT462 T-BT476 T-BT529 T-BT539 T-BT559 T-BT562 T-BT564 T-BT566 T-BT621 T-BT649 T-BT65 T-BT878 T-BT9 T-BT92 T-BT924 T-BT932 T-BT943 T-BT986 T-BT99 Validation cohort (n = 33) Supplementary Figure 7. Detection of 1q21.3 amplification in cfdna of breast cancer patients. (a) Studyflow design for primary tumor genomic DNA versus blood cfdna analysis of development cohort (Left) and validation cohort (Right). (b) ddpcr analysis of 1q21.3 amplification in matching primary tumor DNA and plasma cfdna of breast cancer patients from two independent cohorts. Positive 1q21.3 amplification was defined as those respective cutoff values as shown. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates.

10 a 1 early-stage breast cancer patients b 52 locally advanced breast cancer patients with neoadjuvant chemotherapy Eligible samples (n = 1) Eligible samples (n = 52) Serum analysis Serum analysis 2 excluded due to low quantity of DNA in blood Pre-surgery serum samples (n = 8) Baseline serum samples (n = 52) 76 samples were detected non-amplified 4 samples were detected amplified 44 samples were detected non-amplified 8 samples were detected amplified Supplementary Figure 8. Flow diagram of patients included in breast cancer cohort studies. (a) Denmark OUH early-stage breast cancer cohort study and (b) Singapore NUHS neoadjuvant breast cancer cohort study.

11 a Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 G/C G G/C G G/C G G/C G G/C G G/C G b CT scan Legend G/C: Gemcitabine/Carboplatin G: Gemcitabine Patient ID: C-S2 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S19 Post-PD CT scan Collection of blood Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle Baseline Patient ID: C-S21 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Post-PD Baseline Patient ID: C-S26 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S3 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Post-PD CT scan Supplementary Figure 9. cfdna detection of 1q21.3 amplification in the course of chemotherapy. (a) Schematic illustration of the gemcitabine and carboplatin chemotherapy regimen. (b) Serial ddpcr analysis of cfdna of metastatic breast cancer patients treated with gemcitabine and carboplatin chemotherapy regimen in a phase II clinical study. Shown are the averaged ratios of the three target genes TUFT1, S1A8 and S1A7 relative to the reference gene RPP3. Data are generated from means of two technical replicates. Patients were treated with gemcitabine (day 1 and 8) and carboplatin (day 1) every 3-week for a maximum of 6 cycles. Blood samples were taken at the baseline time point before the treatment and indicated time points during the course of treatment. CT scans were performed after every 2 cycles as denoted by the colored arrows. Patients stopped treatment when their CT scan show disease progression. Green arrow indicates partial response, yellow arrow indicates stable disease, and red arrow indicates progressive disease. Overall, 1q21.3 amplification can be detected on average 2.2 months (range.6 to 5.7 months) before the establishment of progressive disease by means of CT scan Patient ID: C-S5 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S2 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S22 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S29 Post-PD Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Patient ID: C-S Baseline Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Post-PD

12 a Fold Change.5 S1A7 S1A8 S1A9 MB436 Fold Change.5 S1A7 S1A8 S1A9 MB468 Fold Change 2..5 MB231 S1A7 S1A8 S1A9... b Cell viability (Relative to Day ) MB436 shluc shs1a7 shs1a8 shs1a9 Cell viability (Relative to Day ) MB468 shluc shs1a7 shs1a8 shs1a9 Cell viability (Relative to Day ) MB231 shluc shs1a7 shs1a8 shs1a9 c f S1A7 - + S1A8 - - S1A9 - - p-irak1 (T29) * Relative TS growth (%) IRAK1 Actin Days MCF1A MCF1A *** *** *** * MW (kda) g Fold Change d Relative TS growth (%) S1A7 S1A8 S1A MCF1A * S1A7 6. S1A Days MCF1A tumorsphere * S1A9 e Days Cell viability (Relative to Day ) MCF1A Control S1A7 S1A8 S1A Days Supplementary Figure 1. S1A7/8/9 promotes tumorsphere growth. (a) Real-timeRT-PCRanalysisofshRNA knockdown efficiency in MB231, MB436 and MB468 cell lines. Data are expressed as means ± s.d. of three technical replicates. (b) Cell proliferation of MB231, MB436 and MB468 after individual shrna knockdown of S1A7, S1A8 and S1A9. Data are expressed as means ± s.d. of three technical replicates. (c) Representative western blot analysis showing induction of phospho-irak1 in MCF1A cells after treatment of S1A7/8/9 recombinant protein for 1 days. (d) Quantification of the number of tumorspheres after long term treatment of S1A7/8/9 recombinant protein separately in MCF1A. Data are expressed as means ± s.d. of three independent experiments. (e) Cell proliferation of MCF1A after long term treatment of S1A7/8/9 recombinant protein separately. Data are expressed as means ± s.d. of four technical replicates. (f) Quantification of the number of tumorspheres after IRAK1 wild-type (WT) and K239S mutant overexpression in MCF1A. Data are expressed as means ± s.d. of three independent experiments. (g) Real-time RT-PCR analysis of S1A7/8/9 gene expression in MCF1A tumorspheres overexpressing IRAK1 WT and K239S mutant. Data are expressed as means ± s.d. of three technical replicates. All p-values were calculated with two tailed Student s t-test. * p <.5, *** p <.1.

13 a Relative S1A8 expression (Normalized to GAPDH) Non-amplified Amplified Relative S1A9 expression (Normalized to GAPDH) b HCC7 xenograft Legend S1A7.5. Legend S1A8.5. S1A9.5. Fold Change Fold Change Fold Change c Tumor volume (mm 3 ) q21.3 amplified MB468 xenograft *** Days Tumor volume (mm 3 ) q21.3 amplified MCF7 xenograft Days Vehicle (n = 11) Vehicle (n = 8) * Tumor volume (mm 3 ) q21.3 non-amplified BT474 xenograft n.s Days Vehicle (n = 7) 15 mg/kg pacritinib (n = 11) 15 mg/kg pacritinib (n = 8) 15 mg/kg pacritinib (n = 6) d % change in body weight HCC7 xenograft Days n.s MB231 xenograft Days Vehicle (n = 1) 1 mg/kg pacritinib (n = 1) 5 mg/kg pacritinib (n = 1) 15 mg/kg pacritinib (n = 1) n.s e % change in body weight PDX3 xenograft 4 1st Break 2nd line After 2 line surgery Days n.s 1st line vehicle (n = 4) 1st line paclitaxel (n = 27) 2nd line vehicle (n = 4) 2nd line paclitaxel (n = 4) 2nd line pacritinib (n = 4) 2nd line combination (n = 4)

14 Supplementary Figure 11. Pacritinib treatment preferentially inhibits tumor growth in 1q21.3 amplified xenograft tumors. (a) Correlation analysis of S1A8 and S1A9 gene expression in 1q21.3 amplified and nonamplified breast cancer cell lines. Red: Amplified, Blue: Non-amplified. (b) Real-time RT-PCR analysis of S1A7/8/9 expression in HCC7 xenograft (n = 4 per group) treated with pacritinib (Pac). Data are expressed as means ± s.e.m. of four xenograft tumor per group. (c) NOD-SCID mice bearing MB468, MCF7 and BT474 tumors were treated with 15 mg/kg pacritinib by oral gavage. Shown are the changes in tumor volume over time. The sample size (n) per group is indicated. p-value was calculated with two-tailed unpaired t-tests. * p <.5, *** p <.1, n.s., not significant. (d) Percentage body weight change of NOD-SCID mice bearing HCC7 and MB231 xenograft tumors during drug treatment. The sample size (n) per group is indicated. p-value was calculated with twotailed unpaired t-tests. n.s., not significant. (e) Percentage body weight change of NOD-SCID mice bearing PDX3 tumors during drug treatment. The sample size (n) per group is indicated. p-value was calculated with two-tailed unpaired t-tests. n.s., not significant.

15 Supplementary Table 1: Clinical pathological characteristics of patients used in Singapore TTSH discovery cohort (Refer to Supplementary Fig. 2d,e). Singapore TTSH Discovery Cohort Characteristics Patients (n = 67) Age < > 6 28 Tumor size pt1 (< 2. cm) 12 pt2 (2. to 5. cm) 39 pt3 (> 5. cm) 6 pt4 1 Stage I 11 II 29 III 18 IV 9 Subtype ER+ 4 HER2+ 13 TNBC 14 Lymph node status pn 27 pn1 19 pn2 11 pn3 1 Race Chinese 51 Malay 7 Indian 5 Others 4

16 Supplementary Table 2: Multivariate Cox regression analysis of 1q21.3 amplification status for overall survival of Singapore TTSH discovery cohort (n = 67). Tumor DNA Clinical variable HR 95% CI p-value CNA status (Negative vs Positive) to ER status (Positive vs Negative) to HER2 status (Negative vs Positive) to Tumor size (T1-2 vs T3-T4) to Lymph nodes (N vs N1-3) to Stage (Stage 1-2 vs Stage 3-4) to

17 Supplementary Table 3: DNA FISH quantitation of 1q21.3 (Red R) and 1p32.3 (Green G). Cells were quantitated based on number of both 1q21.3 and 1p32.3 FISH positive cells. Total of 1 cells were enumerated for primary mammary epithelial, ER positive tumorsphere and ER negative tumorsphere 2. 2 cells were enumerated for ER negative tumorsphere 1 specimen due to presence of minor subclones within population. Samples 2R2G 3R3G 4R3G 5R3G 5R4G 6R3G 6R4G 6R5G 6R6G 7R3G 7R4G 7R5G 8R4G 8R6G 12R6G Total Primary mammary epithelial 1 1 ER positive tumorsphere ER negative tumorsphere ER negative tumorsphere

18 Supplementary Table 4: Stage Information for Fig. 2c,d. TumorDNAsamplesfrompatientsof various stages were collected to validate 1q21.3 amplification association with tumor recurrence. Patients without Recurrence Patients with Recurrence Characteristics Patients (n = 28) Patients (n = 34) Stage I 2 (7.1%) 5 (14.7%) II 1 (35.7%) 8 (23.5%) III 14 (5.%) 14 (41.2%) IV 2 (7.1%) 2 (5.9%) Unknown NA 5 (14.7%)

19 Supplementary Table 5: Clinical information and ddpcr result for Fig. 2c-e and Supplementary Fig. 5b. Patients without recurrence within 5 years Primary Tumor SAMPLE Age Ethnicity ER status PR status HER2 Disease Subtype T N M Treatment RPP3 TUFT1 S1A7 S1A8 Combined status stage Ratio T-BT82T 53 Chinese Positive Negative Negative ER+ HER2- IIA T2 N M TCx4; Tamoxifen, Femara; Radiotherapy T-BT83T 42 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M Radiotherapy T-BT96T 59 Indian Positive Negative Negative ER+ HER2- I T1c N M Femara T-BT14T 56 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M Tamoxifen; Radiotherapy T-BT18T 76 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M Tamoxifen, Femara T-BT27T 37 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M ACx4; Tx4; Tamoxifen; Radiotherapy T-BT211T 87 Chinese Positive Positive Negative ER+ HER2- IIIB T4 N2 M Tamoxifen T-BT219T 64 Chinese Positive Not done Negative ER+ HER2- IV T2 N M1 Femara T-BT222T 53 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M ACx4; Tx12; Arimidex; Radiotherapy T-BT223T 6 Chinese Positive Positive Negative ER+ HER2- IIIC T2 N3 M ACx4; Tx12; Femara; Radiotherapy T-BT224T 64 Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M ACx4; Tx12; Tamoxifen, Femara; Radiotherapy T-BT248T 63 Chinese Positive Positive Negative ER+ HER2- IIIA T2 N2 M ACx4; Tx12; Femara; Radiotherapy T-BT26T 45 Chinese Positive Positive Negative ER+ HER2- IIIC T4 N3 M ACx4; Tx4; Tamoxifen, Femara; Radiotherapy T-BT89T 47 Chinese Positive Positive Positive ER+ HER2+ IIB T2 N1 M ACx4; Tx4,Tamoxifen, Arimidex, Aromasin; Radiotherapy T-BT92T 46 Malay Positive Positive Positive ER+ HER2+ IIIA T3 N2 M HRT (unknown); Radiotherapy T-BT12T 46 Malay Positive Positive Positive ER+ HER2+ I T1c N M ACx4; Tx12; Tamoxifen T-BT118T 88 Indian Positive Positive Equivocal ER+ HER2+ IV T4 N3 M1 Arimidex T-BT225T 69 Chinese Positive Positive Positive ER+ HER2+ IIIC T2 N3 M ACx4; Tx12; Femara; Radiotherapy T-BT233T 63 Chinese Positive Negative Positive ER+ HER2+ IIIA T2 N2 M ACx4; Tx12; Tamoxifen, Arimidex; Radiotherapy T-BT258T 44 Chinese Positive Positive Positive ER+ HER2+ IIIC T2 N3 M Chemotherapy (Unknown); Tamoxifen, Arimidex; Radiotherapy T-TP Chinese Negative Negative Positive HER2 IIIB T4 N1 M ACx4; Tx4; Radiotherapy T-TP Chinese Negative Negative Negative TNBC IIIA T3 N2 M FACx5; Tx2; Gem/T x4; Radiotherapy T-TP Chinese Negative Negative Negative TNBC IIIC T4 N3 M ACx4; Tx4; Radiotherapy T-TP Chinese Negative Negative Negative TNBC IIIA T3 N2 M ACx4; Tx4; Radiotherapy T-TP Indian Negative Negative Negative TNBC IIB T3 N M ACx4; Tx12; Radiotherapy T-TP Others Negative Negative Negative TNBC IIB T3 N M ACx4; Tx4; Radiotherapy T-TP Others Negative Negative Negative TNBC IIIA T3 N2 M ACx4; Tx T-TP Chinese Negative Negative Negative TNBC IIIB T4 N1 M ACx4; Tx12; Radiotherapy Patients with recurrence within 5 years Primary Tumor Recurrent Tumor SAMPLE Age Ethnicity ER status PR status HER2 status Subtype Disease stage T N M Treatment RPP3 TUFT1 S1A7 S1A8 Combined Ratio RPP3 TUFT1 S1A7 S1A8 Combined Ratio J-S A 55 Caucasian Positive Unknown Negative ER+ HER2- n/a n/a n/a n/a ACT (neoadjuvant) J-S G 45 Caucasian Positive Unknown Negative ER+ HER2- n/a n/a n/a n/a ACT J-S D 36 Caucasian Positive Unknown Negative ER+ HER2- n/a n/a n/a n/a ACT, Avastin, Tamoxifen J-S E 51 Asian Positive Unknown Negative ER+ HER2- n/a n/a n/a n/a ACT, Cisplatin, Etoposide, Cyclophosphamide, Tamoxifen T-TP Chinese Positive Negative Negative ER+ HER2- IIA T2 N M Arimidex T-TP Indian Positive Positive Negative ER+ HER2- IIA T2 N M Tamoxifen, Radiotherapy T-TP Malay Positive Negative Negative ER+ HER2- IIB T2 N1 M Tamoxifen T-TP Chinese Positive Positive Negative ER+ HER2- IIA T2 N M None T-TP Chinese Positive Positive Negative ER+ HER2- I T1mic N M Tamoxifen T-TP Indian Positive Negative Negative ER+ HER2- IIIC T2 N3 M None T-TP Indian Positive Negative Negative ER+ HER2- I T1b N M Arimidex, Radiotherapy T-TP Malay Positive Negative Negative ER+ HER2- IIIA T2 N2 M Arimidex, Radiotherapy T-TP Chinese Positive Positive Negative ER+ HER2- I T1b N M None T-TP Chinese Positive Positive Negative ER+ HER2- IIB T2 N1 M Arimidex T-TP Malay Positive Negative Negative ER+ HER2- IIIA T3 N2 M ACx T-TP Chinese Positive Positive Negative ER+ HER2- I T1c N M TCx T-TP Chinese Positive Positive Negative ER+ HER2- I T1c N M None T-TP Malay Positive Positive Positive ER+ HER2+ IIIA T3 N2 M CAFx4, Tx1, Herceptin T-TP Chinese Positive Positive Positive ER+ HER2+ IIB T2 N1 M ACx4, Tx4, Herceptin, Tamoxifen, Radiotherapy T-TP Chinese Positive Negative Positive ER+ HER2+ IIIA T1c N2 M ACx4, Tx4, Herceptin, Tamoxifen, Radiotherapy T-TP Chinese Positive Positive Positive ER+ HER2+ IIIB T4 N2 M Arimidex J-S E 45 Caucasian Negative Unknown Positive HER2 IIA T1c N1 M ACT (neoadjuvant) J-S E 56 Asian Negative Unknown Positive HER2 IIIA pt2 pn2a pmx Adriamycin based chemotherapy T-TP Chinese Negative Negative Positive HER2 IIIC T3 N3 M ACx T-TP Chinese Negative Negative Positive HER2 IIIB T4 N1 M T/Gemcitabine x T-TP Chinese Negative Negative Positive HER2 IIIA T3 N1 M ACx T-TP Chinese Negative Negative Positive HER2 IV T3 N1 M Tx T-TP Malay Negative Negative Positive HER2 IV T4 N1 M1 Tx12; ACx J-S A 53 Asian Negative Negative Negative TNBC n/a n/a n/a n/a Chemotherapy J-S I 5 Caucasian Negative Negative Negative TNBC IIB T2 N1a Mx TAC, Avastin, Tamoxifen (core biopsy (ER +)), Femara J-S D 32 Caucasian Negative Negative Negative TNBC IIIA pt1c N2a Mx AC, Taxol T-TP Malay Negative Negative Negative TNBC IIIC T4 N3 M ACx4; Tx T-TP Chinese Negative Negative Negative TNBC IIIC T4 N3 M ACx4; Tx T-TP Chinese Negative Negative Negative TNBC IIIB T4 N M ACx4; Tx

20 OUH validation cohort Primary Tumor Recurrent Tumor SAMPLE ER status PR status HER2 Disease Neoadjuvant Combined T N M Adjuvant Treatment RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 Combined status stage treatment Ratio Ratio D-2 Positive Unknown Negative 2 T2 N M None None D-9 Positive Unknown Negative 2 T2 N1 M None Tamoxifen:1yr+Arimedex D-2 Positive Positive Negative 3 T3 N3 M CEF x4 CEF x 3 + Tamoxifen (5 yrs) D-25 Positive Positive Negative 3 T2 N3 M CEF x4 C x2 + CEF x 4 + Tamoxifen 5 yrs D-26 Positive Negative Positive 2 T1 N1 M None Tamoxifen 2½ yrs + Arimidex 2 yrs D-4 Positive Positive Negative 2 T1 N1 M None CEF x 7 + Tamoxifen D-47 Positive Unknown Negative 3 T2 N3 M Unknown Tamoxifen (4 mo), Arimidex, back to Tamoxifen (side-effects) D-51 Positive Unknown Negative 3 T3 N3 M Unknown tamoxifen, Arimidex (3 yrs) D-58 Positive Positive Negative 3 T3 N2 M ECx3 + Tax x4 Taxol x2 + tamoxifen D-59 Positive Negative Negative 1 T1 N M None None D-73 Positive Positive Negative 3 T2 N2 M None Letrozol D-82 Positive Unknown Negative 2 T2 N M None None D-86 Positive Positive Negative 2 T2 N1 M None Tamoxifen/Arimidex (5+1 yrs) D-112 Positive Positive Negative 3 T2 N2 M CEF x4 Taxotere x3 + Tamoxifen (5 yrs) -> Letrozol D-TNBC1 Negative Negative Negative 2 T2 N1mi M None CE x 3, Tax x D-TNBC2 Negative Negative Negative 3 T1 N3 M None EC x 3, Tax x D-TNBC3 Negative Negative Negative 2 T1 N1 M None CEF x D-TNBC4 Negative Negative Negative 1 T1 N1mi M None None D-TNBC5 Negative Negative Negative 2 T2 N1 M None Cyclophosphamide/Taxotere (READ) D-TNBC6 Negative Negative Negative 2 T1 N1 M None EC x 3, Tax x D-TNBC7 Negative Negative Negative 1 T1 N M None CEF x D-TNBC8 Negative Negative Negative 2 T2 N1mi M None EC x 3, Tax x 3. + Tamoxifen D-TNBC9 Negative Negative Negative 3 T2 N2 M None EC x 3, Tax x

21 Supplementary Table 6: Healthy female individuals plasma samples (n = 3) (Refer to Supplementary Fig. 6b). S/N Sample ID Sex Age Ethnicity RPP3 TUFT1 S1A7 S1A8 Combined Ratio 1 TH-41 Female 6 Chinese TH-117 Female 41 Chinese TH-282 Female 34 Chinese TH-378 Female 24 Chinese TH-386 Female 52 Malay TH-498 Female 28 Chinese TH-512 Female 37 Chinese TH-564 Female 3 Chinese TH-783 Female 29 Chinese TH-867 Female 35 Chinese TH-943 Female 33 Indian TH-947 Female 4 Chinese TH-123 Female 23 Chinese TH-1279 Female 51 Chinese TH-1288 Female 36 Chinese TH-1293 Female 43 Indian TH-1462 Female 26 Chinese TH-147 Female 24 Chinese TH-15 Female 6 Chinese TH-1629 Female 56 Chinese TH-1786 Female 26 Malay TH-185 Female 22 Chinese TH-1938 Female 46 Malay TH-23 Female 29 Malay TH-261 Female 23 Chinese TH-288 Female 29 Chinese TH-2167 Female 25 Chinese TH-2253 Female 41 Indian TH-2264 Female 29 Chinese TH-2272 Female 44 Chinese

22 Supplementary Table 7: Clinical information and ddpcr result for Fig. 3a and Supplementary Fig. 7b. Development cohort Tumor genomic DNA Blood cfdna SAMPLE Age Ethnicity ER status PR status HER2 Disease Combined Combined T N M RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 status stage Ratio Ratio C-BB Chinese Positive Positive Negative 2A T1 N1 M C-BB Chinese Negative Negative Negative 1 T1 N M C-BB Caucasian Positive Positive Negative Unknown NA NA NA C-BB Eurasian Negative Negative Borderline 2B T3 N M C-BB Filipino Positive Negative Negative 2B T3 N M C-BB Chinese Positive Positive Borderline Unknown NA NA NA C-BB Chinese Positive Positive Negative 4 T3 N1 M C-BB Malay Positive Positive Negative 1 T1 N M C-BB Chinese Positive Positive Positive 2B T2 N1 M C-BB Chinese Negative Negative Positive 3C T2 N3 M C-BB Indian Negative Negative Positive 2A T2 N M C-BB Chinese Positive Positive Negative 1A T1 N M C-BB Chinese Negative Negative Positive 3A T1 N2 M C-BB Malay Positive Positive Positive 3A T3 N1 M C-BB Chinese Positive Positive Negative 3A T2 N2 M C-HOB Malay Negative Negative Unknown 3A T3 N2 M C-HOB Chinese Positive Negative Unknown 4 T4 N1 M C-HOB-8 58 Malay Positive Negative Unknown 3A T3 N1 M C-HOB Chinese Positive Positive Unknown 3B T4 Nx M C-HOB Chinese Negative Negative Unknown 4 T4 N3 M C-HOB Chinese Negative Negative Unknown 3A T3 N2 M Validation cohort Tumor genomic DNA Blood cfdna SAMPLE Age Ethnicity ER status PR status HER2 Disease Combined Combined T N M RPP3 TUFT1 S1A7 S1A8 RPP3 TUFT1 S1A7 S1A8 status stage Ratio Ratio T-BT49 54 Chinese Positive Positive Negative IIB T2 N1 M T-BT55 36 Chinese Positive Negative Negative IIB T2 N1 M T-BT65 76 Chinese Positive Negative Positive IIA T2 N M T-BT82 53 Chinese Positive Negative Negative IIA T2 N M T-BT95 45 Others Positive Positive Negative IIA T1 N1 M T-BT11 57 Malay Positive Positive Negative IIIC T2 N3 M T-BT24 36 Chinese Negative Negative Positive IIIA T2 N2 M T-BT Chinese Negative Negative Negative IIIA T2 N2 M T-BT Chinese Positive Positive Negative IIB T2 N1 M T-BT Chinese Positive Positive Positive IIIC T2 N3 M T-BT Chinese Positive Positive Negative I T1 N M T-BT Chinese Negative Negative Positive I T1 N M T-BT Chinese Negative Positive Positive IV T4 N1 M T-BT46 49 Chinese Positive Negative Positive IIIB T4 N1 M T-BT Chinese Positive Positive Negative IV T4 N1 M T-BT Chinese Positive Positive Negative IIIB T4 N1 M T-BT Chinese Positive Positive Negative IIB T2 N1 M T-BT Chinese Positive Negative Positive IIIB T4 N2 M T-BT Chinese Positive Positive Negative IIIC T2 N3 M T-BT Indian Negative Negative Negative IIIC T2 N3 M T-BT Indian Positive Positive Positive IIA T1c N1 M T-BT Chinese Positive Positive Negative IIIA T3 N2 M T-BT621 5 Malay Positive Positive Negative IIIA T2 N2 M T-BT Chinese Positive Positive Negative IIIA T3 N2 M T-BT65 73 Chinese Positive Positive Negative IIIA T1c N2 M T-BT Others Negative Negative Positive IIIA T2 N2 M T-BT9 45 Chinese Negative Negative Negative IIIA T2 N2 M T-BT92 79 Chinese Positive Positive Negative IIIC T4 N3 M T-BT Malay Positive Positive Negative IIIC T2 N1 M T-BT932 7 Chinese Positive Negative Negative IIIA T3 N2 M T-BT Chinese Positive Positive Negative IV T4 N3 M T-BT Indian Negative Negative Negative IIIA T2 N2 M T-BT99 63 Chinese Positive Positive Negative IIIC T2 N3 M

23 Supplementary Table 8: Clinical information and ddpcr result for Fig. 3c. Treatment naïve patients without metastasis SAMPLE Age Ethnicity ER status PR status HER2 status RPP3 TUFT1 S1A7 S1A8 Combined Ratio C-BB Chinese Positive Positive Negative C-BB Chinese Negative Negative Negative C-BB Caucasian Positive Positive Negative C-BB Eurasian Negative Negative Borderline C-BB Filipino Positive Negative Negative C-BB Chinese Positive Positive Borderline C-BB Malay Positive Positive Negative C-BB Chinese Positive Positive Positive C-BB Chinese Negative Negative Positive C-BB Indian Negative Negative Positive C-BB Chinese Positive Positive Negative C-BB Chinese Negative Negative Positive C-BB Malay Positive Positive Positive C-BB Chinese Positive Positive Negative C-HOB Malay Negative Negative Unknown C-HOB-8 58 Malay Positive Negative Unknown C-HOB Chinese Positive Positive Unknown C-HOB Chinese Negative Negative Unknown T-BT49 54 Chinese Positive Positive Negative T-BT55 36 Chinese Positive Negative Negative T-BT65 76 Chinese Positive Negative Positive T-BT82 53 Chinese Positive Negative Negative T-BT95 45 Others Positive Positive Negative T-BT11 57 Malay Positive Positive Negative T-BT24 36 Chinese Negative Negative Positive T-BT Chinese Negative Negative Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Positive T-BT Chinese Positive Positive Negative T-BT Chinese Negative Negative Positive T-BT46 49 Chinese Positive Negative Positive T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Negative Positive T-BT Chinese Positive Positive Negative T-BT Indian Negative Negative Negative T-BT Indian Positive Positive Positive T-BT Chinese Positive Positive Negative T-BT621 5 Malay Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT65 73 Chinese Positive Positive Negative T-BT Others Negative Negative Positive T-BT9 45 Chinese Negative Negative Negative T-BT92 79 Chinese Positive Positive Negative T-BT Malay Positive Positive Negative T-BT932 7 Chinese Positive Negative Negative T-BT Indian Negative Negative Negative T-BT99 63 Chinese Positive Positive Negative Treatment naïve patients with metastasis SAMPLE Age Ethnicity ER status PR status HER2 status RPP3 TUFT1 S1A7 S1A8 Combined Ratio C-BB Chinese Positive Positive Negative C-HOB Chinese Positive Negative Unknown C-HOB Chinese Negative Negative Unknown C-SCN/P2A2-BL 48 Chinese Positive Positive Positive C-CLC/P2A7-BL 53 Chinese Positive Positive Negative C-TSE/P2A1-BL 6 Chinese Positive Positive Negative C-LMS/P2A29-BL 55 Malay Positive Positive Negative C-ZLL/P2B12-BL 54 Chinese Negative Negative Positive C-ULM/P2B18-BL 63 Chinese Negative Positive Negative C-FFL/P2B2-BL 52 Chinese Positive Positive Negative T-BT Chinese Negative Positive Positive T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT72 6 Malay Negative Negative Negative T-BT Others Positive Positive Negative T-BT Malay Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT Malay Positive Positive Negative

24 Recurrent patients SAMPLE Age Ethnicity ER status PR status HER2 status RPP3 TUFT1 S1A7 S1A8 Combined Ratio C-S Malay Positive Positive Negative C-S Malay Negative Negative Negative C-S Chinese Negative Negative Negative C-S Malay Positive Negative Negative C-S9-1 4 Chinese Positive Positive Positive C-S Malay Negative Positive Negative C-S Malay Negative Positive Negative C-S Chinese Negative Positive Negative C-S Malay Negative Positive Negative C-S Chinese Negative Negative Negative C-S Malay Negative Positive Negative C-S Chinese Negative Negative Positive C-S Malay Positive Positive Positive C-S Indian Positive Unknown Negative C-S Malay Positive Positive Negative C-S Malay Negative Positive Positive C-S Chinese Positive Negative Negative C-S Indian Positive Positive Negative C-S Malay Positive Positive Negative C-S Malay Negative Negative Negative C-S Chinese Positive Positive Negative C-S Indian Negative Negative Negative C-S Indian Positive Positive Positive C-S Malay Negative Negative Negative C-S Chinese Positive Positive Negative C-S Malay Positive Negative Positive C-S Chinese Positive Positive Negative C-S Malay Positive Positive Negative C-S Others Negative Positive Negative N-BC94 Unknown Unknown Positive Positive Negative N-BC1 Unknown Unknown Positive Negative Negative N-BC114 Unknown Unknown Positive Positive Positive N-BC116 Unknown Unknown Negative Negative Positive N-BC117 Unknown Unknown Positive Negative Positive T-BT114 6 Chinese Negative Negative Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT51 49 Chinese Negative Negative Positive T-BT Chinese Positive Positive Negative T-BT63 64 Chinese Positive Positive Equivocal T-BT Chinese Positive Negative Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Equivocal T-BT Chinese Negative Positive Positive T-BT Chinese Positive Positive Negative T-BT96 55 Chinese Negative Negative Negative T-BT Malay Positive Positive Negative T-BT Chinese Positive Positive Negative T-BT Chinese Positive Positive Positive T-BT Chinese Positive Positive Negative T-BT Indian Positive Positive Negative