Supplemental data

1. Supplemental data

2. 3. Ex vivo irradiation (5Gy) 4. Ex vivo culture 2 hours 1. Fresh, viable tumor 2. Tissue slicing 5. Formalin fixation overnight 6. Paraffin embedding overnight 7. Sectioning and drying 8. RAD51/GEMININ immunostaining 9. Quantification of RAD51IRIF+/GEMININ+ Day 3 Day 2 Day 4 Day 1 24hr 2hr 8 1 2 3 5 6 7 9 Supplemental figure S1: Schematic workflow RAD51IRIF assay Tumor samples were obtained from breast resection tissue and were sliced into smaller tissue slices. One such a slice was irradiated ex vivo (5Gy) and subsequently cultured for 2 hours at 37°C before being fixed with formalin solution overnight. The next day samples were embedded in paraffin and the day after microscopy slides were generated on which the RAD51/Geminin staining was performed. Finally, the ratio of RAD51 IRIF positive cells among the Geminin positive cells was quantified. Lower figure panel shows the timeline of corresponding actions.

3. B. A. Supplemental figure S2: Validation of RAD51 IRIF immunostaining Tissue slices were irradiated (5Gy) directly after slicing. (A) Two hours after IR is the timepoint displaying the most RAD51 IRIF positive cells compared to 4 and 6 hours post irradiation. (B) Unirradiated slices display minimal RAD51 IRIF and this accounts also for cells negative for Geminin. Results of 3 independent tumor samples displayed. n ≥ 30, Error bars indicate standard error assuming binomial distribution.

4. 0 Gy 5 Gy – 2hr A. Sample # ᵞ-H2AX / DAPI 53BP1 / DAPI ᵞ-H2AX / DAPI 53BP1 / DAPI # 1 # 2 # 20 # 32 # 54 B. 0 µM Olaparib – 24hr 10 µM Olaparib – 24hr ᵞ-H2AX / DAPI 53BP1 / DAPI ᵞ-H2AX / DAPI 53BP1 / DAPI # 20 # 17 Supplemental figure S3: Induction of DSBs in tissue slices ᵞ-H2AX and 53BP1 nuclear foci indicate the induction of DSBs in tissue slices. A. RAD51 IRIF negative tumors all show DSB induction after 5Gy IR. B. Representative images of Olaparib treated tumors display induction of DSBs after 24 hour of incubation with 10µM Olaparib. Blue = DAPI, Green = 53BP1, Red = ᵞ-H2AX.

5. A. B. 5µm 5µm 5µm 5µm C. 5µm 5µm Supplemental figure S4: Non-tumor cells display normal RAD51 IRIF Normal RAD51 IRIF formation in non-tumor cells from RAD51 IRIF negative samples exclude technical issues as reasons for impaired RAD51 IRIF formation. Cells having normal RAD51 IRIF were observed in A. fat tissue, B. stromal tissue and C. normal mammary ducts from the same fluorescently stained sections. Black and white pictures give an overview of the tissue structure. Color panels represent single cells expressing Geminin and RAD51 foci. blue= dapi, green= Geminin, red= RAD51. Scale bars represent 5µm.

6. A. Normal “reference” DNA Patient tumor DNA Patient normal breast DNA B. Sample #20 Sample #1 CEPH control DNA normal tumor normal tumor Without restriction enzyme (copy number aberrations) With restriction enzyme (promoter methylation) Supplemental figure S5: Mutation and methylation analysis of impaired RAD51 IRIF tumors A. Validation by Sanger sequencing of BRCA2 mutation in sample #2 versus normal DNA of the same patient. Results show a germ line G>T mutation (c.7617+1G>T) which is predicted to cause aberrant splicing. B. Copy number variation (upper panels) and promoter methylation (lower panels) analysis of BRCA1 gene using MS-MLPA kit. Methylation specific peaks were observed in tumor DNA (red arrows indicate BRCA1 specific promoter sequence) and not in normal tissue DNA from the same patient. The low methylation specific peak in #1 DNA corresponds with the low fraction of tumor cells in this sample (approximately 30%). No copy number aberrations for BRCA1 were observed between tumor and normal DNA from these patients.

7. Probe POLR2A DapB BRCA1 Sample #32 Sample #20 Supplemental figure S6: In situ detection of BRCA1 mRNA In situ detection of BRCA1 mRNA in two TNBC samples. No hypermethylation of the BRCA1 promoter (table 1) and normal expression of BRCA1 mRNA was seen in tumor sample #32. Breast tumor sample #20 showed hypermethylation of the BRCA1 promoter (table 1) and no expression of BRCA1 mRNA indicating functional silencing of BRCA1 by promoter methylation. Human RNA Polymerase 2 (POLR2A) probes served as a positive control and Bacterial DapB probes as a negative control in this assay. Red = amplified signal of respective hybridized probe.

8. Supplemental table S1: Clinico-pathological characteristics of tumor samples Complete overview of each tumor with corresponding clinicopathological data, RAD51 IRIF, size and age of patient at time of breast surgery. ND = Not determined RAD51 IRIF because of low Geminin expression.

9. Supplemental table S2: Clinico-pathological characteristics of Geminin positive vs Geminin negative tumor samples Geminin expression in tumor cells was not correlated with clinicopathological tumor characteristics. ER= Estrogen receptor, PR= Progesterone receptor, Her2= Human Epidermal Growth factor receptor 2, TN= Triple Negative. Variables tumor size and age are represented as median and range, other data represent proportions. For categorical data the p-values were calculated using Fisher’s Exact test and for continuous data (age and tumor size) p-values were calculated using Mann-Whitney test. Asterisks indicate statistically significant differences (p<0.05).