Supplemental data

1. Fresh, viable tumor

2. Tissue slicing 3. Ex vivo irradiation (5Gy)4. Ex vivo culture 2 hours

5. Formalin fixation overnight6. Paraffin embedding overnight7. Sectioning and drying

8. RAD51/GEMININ immunostaining9. Quantification of RAD51IRIF+/GEMININ+

Day 1

1 2 3

2hr

5

Day 2

6

Day 3 Day 4

7 8 9

24hr

Supplemental figure S1: Schematic workflow RAD51IRIF assayTumor 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.

IR -Geminin +

IR+Geminin +

IR+Geminin -

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10

20

30

40

50

60

70

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90

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Cel

ls w

ith R

AD

51 IR

IF (%

)

B.

2 4 60

102030405060708090

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Time post IR (hours)

RA

D51

IRIF

+/ G

emin

in+

(%)

A.

Supplemental figure S2: Validation of RAD51 IRIF immunostainingTissue 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.

# 1

# 2

# 20

# 32

# 54

# 20

# 17

ᵞ-H2AX / DAPI ᵞ-H2AX / DAPI53BP1 / DAPI 53BP1 / DAPI

0 Gy 5 Gy – 2hr

ᵞ-H2AX / DAPI ᵞ-H2AX / DAPI53BP1 / DAPI 53BP1 / DAPI

0 µM Olaparib – 24hr 10 µM Olaparib – 24hr

A.

B.

Supplemental figure S3: Induction of DSBs in tissue slicesᵞ-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.

Sample #

A. B.

C.

5µm

5µm

5µm

5µm

5µm

5µm

Supplemental figure S4: Non-tumor cells display normal RAD51 IRIFNormal 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.

Normal “reference” DNA

Patient normal breast DNA

Patient tumor DNA

A.

B.CEPH

control DNA tumor normaltumor normal

Sample #1 Sample #20

Without restriction enzyme(copy number aberrations)

With restriction enzyme(promoter methylation)

Supplemental figure S5: Mutation and methylation analysis of impaired RAD51 IRIF tumorsA. 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.

Sample #32

Sample #20

Probe

BRCA1DapBPOLR2A

Supplemental figure S6: In situ detection of BRCA1 mRNAIn 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.

Sample #Lab code

Histologicalsubtype Grade ER PR HER2 TN GMN

RAD51 IRIF (%)

Sizeø cm

Age(years)

1 P1 metaplastic 3 - - - + + 1 5.5 482 P2 ductolobular 3 + + - - + 1 4.8 523 P3 ductal 3 + + - - + 88 3.0 514 P5 ductal 2 + - - - - ND 2.5 635 P8 ductal 3 + - - - + 76 2.2 706 P9 ductal 1 + + - - + 75 1.5 667 P12 ductal 3 - - + - - ND 5.1 478 M002 ductal 2 + - + - - ND 3.2 839 M003 lobular 2 + - - - - ND 2.8 61

10 M005 ductal 1 + - - - - ND 5.5 4911 M007 ductal 3 + + - - - ND 2.6 6512 M009 lobular 2 + + - - + 71 2.8 8513 M018 ductal 3 + + - - + 71 12.5 6014 M019 lobular 3 + + - - + 77 8.2 4715 M021 ductal 3 - - + - + 67 2.4 6216 M022 ductal 1 + + - - + 81 1.6 5317 M023 ductal 3 + + - - + 89 3.4 6718 M025 ductal 2 + + - - + 67 1.7 5719 M026 ductal 2 + + - - + 86 4.5 7920 M028 ductal 3 - - - + + 3 4.8 7521 M029 ductal 3 + + + - + 62 3.5 3922 M030 ductal 2 + + - - + 67 10.0 6523 M031 ductal 2 + + - - + 70 2.1 6824 M043 ductal 3 + - - - + 75 4.3 6925 M046 ductal 3 - - + - + 70 2.3 5626 M047 ductal 3 + + - - - ND 2.5 8027 M048 lobular 3 + - - - + 86 5.8 6628 M051 ductal 3 + + - - + 75 2.7 4929 M052 ductal 3 + + - - + 71 7.5 6930 M055 ductal 3 + + - - + 38 5.4 10231 M056 ductal 3 + - - - + 74 2.9 5632 M057 ductal 3 - - - + + 13 2.2 7433 M058 ductal 3 + + - - + 79 2.6 5934 M059 ductal 2 + - - - + 84 3.4 6435 M060 ductal 2 + + - - + 84 2.2 6736 M061 ductal 2 + + - - + 71 1.4 8037 M062 ductal 3 - - - + + 83 3.4 6538 M063 ductal 2 + + - - + 71 1.4 6939 M064 ductal 2 + + - - - ND 3.4 3240 M065 ductal 3 + + + - + 57 2.1 2941 M066 ductal 1 + - - - + 76 1.8 7442 M067 lobular 2 + + - - + 74 1.3 6343 M068 ductal 1 + + - - + 86 1.5 5344 M069 ductal 3 + + - - - ND 1.9 6445 M070 lobular 2 + + - - + 71 4.0 4946 M071 lobular 2 + + - - + 75 5.5 4847 M072 ductal 2 + + - - + 74 11.0 7748 M073 lobular 2 + + - - + 69 1.8 4649 M074 ductal 2 + + - - + 77 10.5 6650 M075 ductal 3 + + - - + 67 10.0 5751 BC288 ductal 1 + + - - + 83 2.4 7252 BC289 ductal 1 + + - - + 90 1.8 5653 BC290 ductal 2 - - + - + 94 0.8 6254 BC291 ductal 2 + + - - + 6 1.1 84

Supplemental table S1: Clinico-pathological characteristics of tumor samplesComplete 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.

n=54Geminin -positive

n=45Geminin-negative

n=9 P-value

Histological subtype Ductal carcinoma Lobular carcinoma Other

36 (80%) 7 (16%) 2 ( 4%)

8 (89%) 1 (11%) 0 ( 0%)

1.000

Histological grade 1 2 3

6 (13%) 17 (38%) 22 (49%)

1 (11%) 4 (44%) 4 (44%) 1.000

Receptor status ER/PR-positive ER/PR-negative

HER2-positive HER2-negative

TN ER/PR/HER2-positive

38 (84%) 7 (16%)

5 (11%) 40 (89%)

4 (3%) 41 (91%)

8 (89%) 1 (11%)

2 (22%) 7 (78%)

0 (0%) 9 (100%)

1.000

0.330

1.000

Tumor size (ø cm)(median – range) 2.8 – 11.7 2.8 – 3.6 0.745

Age (years) at surgery(median ̶ range) 64 – 73 63 – 51 0.634

Supplemental table S2: Clinico-pathological characteristics of Geminin positive vs Geminin negative tumor samplesGeminin 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).