1 SUPPLEMENTARY FILES – DESCRIPTION AND LEGENDS Supplementary Data: Validation of amplicons, analysis of grade III IDC-NST of indeterminate phenotype and quantification of PPM1D protein levels by densitometric analysis. Supplementary Figure 1: Validation of CCND1 and EGFR amplifications in a series of 91 grade III-IDC-NST and CCNE1 amplifications on selected cases. (A) i) H&E ii) CISH iii) IHC for a) luminal tumour for CCND1 showing amplification and protein over-expression, b) basal-like tumour for CCND1 with normal copy number and no protein expression, c) EGFR non-amplified tumour with no protein expression d) EGFR amplified tumour showing strong protein expression. Amplification of CCNE1 in basal-like breast cancers. (B) Genome plots of two cases exhibiting amplification with the smallest region of overlap highlighted (i). FISH confirmation of CCNE1 amplification with RP11-327I05 (CCNE1) (red), showing amplification > 5 copies per nucleus (Bii). Supplementary Figure 2: Microarray-based comparative genomic hybridisation analysis of five grade III invasive ductal carcinomas of no special type of indeterminate phenotype. A) Representative genome plots. Log2 ratios are plotted on the Y axis against each clone according to genomic location on the X axis. The centromere is represented by a vertical dotted line. BACs categorised as displaying genomic gains as defined by aws ratios > 0.08 are highlighted in green and those categorised as genomic losses as defined by aws ratios < -0.08 are highlighted in red. Bi) The proportion of tumours in which each clone is gained (green bars) or lost (red bars) is plotted (Y axis) for each BAC clone according to genomic location (X axis). Vertical dotted lines represent chromosome centromeres. Bii) The proportion of tumours in which each clone is amplified (green bars) is plotted (Y axis) for each 2 BAC clone according to genomic location (X axis). Vertical dotted lines represent chromosome centromeres. Supplementary Figure 3: Densitometric analysis of western blots comparing the protein expression levels of PPM1D and β-tubulin. Cell lines harbouring PPM1D gene amplification: MCF-7, KPL-1, MCF-3B, BT474 and ZR-75.3; cell lines with normal PPM1D copy numbers: HeLa, MDA-MB-361, ZR-75.1, CAMA1, T47D and MDA-MB-231. Supplementary Table S1: Summary of the results of the immunohistochemical expression of oestrogen receptor, HER2, progesterone receptor, cytokeratins 5/6 and 14, EGFR and p53; chromogenic in situ hybridisation with probes for HER2, EGFR and CCND1; amplification status of PPM1D and mRNA expression levels of PPM1D as defined by qRT-PCR of all grade III invasive ductal carcinomas of no special type subjected to microarray-based comparative genomic hybridisation analysis and used for the construction of tissue microarrays 1 and 2. Supplementary Table S2: Summary of antibodies, clones, dilutions and scoring systems used for immunohistochemical analysis. Supplementary Table S3: Results of multi-Fisher’s exact test of gains and losses between basal-like, luminal and HER2 tumours. Supplementary Table S4: Recurrent amplifications in basal-like breast cancers. Supplementary Table S5: Results of multi-Fisher’s exact test of gains and losses between basal-like, luminal and HER2 tumours compared in a pair-wise fashion. 3 Supplementary Table S6: Results of multi-Fisher’s exact test of amplifications between basal-like, luminal and HER2 tumours. Supplementary Table S7: Results of multi-Fisher’s exact test of amplifications between basal-like, luminal and HER2 tumours compared in a pair-wise fashion. Supplementary Table S8: Recurrent amplifications in luminal breast cancers. Supplementary Table S9: Recurrent amplifications in HER2 breast cancers. Supplementary Table S1 aCGH series sample ER HER 2 PgR ck5/6 ck14 EGFR TP53 basal marker Nielsen groups Genomic pattern HER2 aCGH HER2 CISH CCND1 CCND1 CISH EGFR aCGH EGFR CISH PPM1D aCGH PPM1D expression number aCGH (Taqman) 1004 Positive Negative Positive Negative Negative Negative Negative Negative Luminal amplifier not amplified not amplified not amplified not amplified not amplified not amplified amplified 3.1256 1005 Positive Negative Positive Negative Negative Negative Positive Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified not amplified 1.1132 1008 Positive Negative Positive Negative Negative Negative Positive Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified not amplified 0.5775 1010 Negative Negative Negative Positive Positive Negative Negative Positive Basal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified not amplified 0.4085 1011 Positive Negative Positive Negative Negative Negative Negative Negative Luminal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified not amplified 0.1381 1012 Negative Positive Negative Negative Negative Negative Negative Negative HER2 amplifier amplified amplified not amplified not amplified not amplified not amplified 1.2885 1013 Negative Positive Negative Negative Negative Negative Positive Negative HER2 amplifier amplified amplified not amplified not amplified not amplified not amplified not amplified 0.6439 1014 Negative Positive Negative Negative Negative Negative Positive Negative HER2 amplifier amplified amplified not amplified not amplified not amplified not amplified amplified 3.3754 1015 Positive Positive Positive Negative Negative Negative Negative Negative HER2 amplifier amplified amplified not amplified not amplified not amplified not amplified not amplified 0.7452 1016 Negative Negative Negative Positive Positive Negative Positive Positive Basal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified not amplified 0.8112 1019 Positive Negative Positive Negative Negative Negative Positive Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified not amplified 1020 Positive Negative Positive Negative Negative Negative Negative Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified 0.8683 1021 Positive Negative Positive Negative Negative Negative Negative Negative Luminal amplifier not amplified not amplified not amplified not amplified not amplified not amplified not amplified 1.5526 1022 Positive Negative Negative Negative Negative Negative Negative Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified not amplified 0.5418 1024 Positive Negative Positive Negative Negative Negative Negative Negative Luminal simplex not amplified not amplified not amplified not amplified not amplified not amplified not amplified 1.2073 1025 Positive Positive Negative Negative Negative Negative Negative Negative HER2 amplifier amplified amplified amplified amplified not amplified not amplified amplified 2.0300 1026 Positive Negative Negative Negative Positive Luminal amplifier not amplified not amplified amplified amplified not amplified not amplified not amplified 0.9993 1027 Positive Negative Negative Negative Negative Negative Negative Negative Luminal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified not amplified 1028 Negative Negative Negative Positive Negative Negative Negative Positive Basal sawtooth not amplified not amplified not amplified 0.5787 1029 Positive Negative Negative Positive Negative Negative Negative Positive Luminal simplex not amplified not amplified not amplified 0.5330 1030 Positive Positive Negative Negative Negative Negative Negative Negative HER2 sawtooth amplified amplified amplified not amplified not amplified not amplified not amplified 1.0201 1031 Negative Positive Negative Positive Negative Negative Positive Positive HER2 amplifier amplified amplified not amplified not amplified not amplified not amplified 0.4661 1032 Positive Negative Negative Negative Negative Negative Positive Negative Luminal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified 0.6805 1033 Positive Negative Positive Negative Negative Negative Negative Negative Luminal amplifier not amplified not amplified not amplified not amplified not amplified not amplified 0.4493 1035 Positive Negative Positive Negative Negative Negative Negative Negative Luminal amplifier not amplified not amplified amplified amplified not amplified not amplified not amplified 0.4849 1037 Positive Negative Negative Negative Positive Negative Negative Positive Luminal sawtooth not amplified not amplified amplified amplified not amplified not amplified not amplified 2.4002 1038 Positive Positive Negative Negative Negative Negative Negative Negative HER2 sawtooth amplified amplified not amplified not amplified not amplified not amplified amplified 2.3594 1040 Negative Negative Negative Positive Positive Positive Negative Positive Basal sawtooth not amplified not amplified not amplified not amplified not amplified not amplified 0.2890 1041 Positive Negative Positive Negative Negative Negative Negative Negative Luminal sawtooth not amplified not amplified amplified amplified not amplified not amplified not amplified 0.5934 1042 Positive Negative Positive Negative Negative Negative Positive Negative Luminal amplifier not amplified not amplified not amplified not amplified not amplified not amplified amplified 2.7133 1043 Positive Positive Negative Positive Negative Positive