(a) Values on the ordinate are the PAM score. Genes with significant PAM scores are displayed according to their annotated location on chromosome 8 (b) metaphase FISH in normal human lymphoid cells using 8q22 probe RP11-347C18 (orange) (c) interphase FISH with 8q22 probe RP11-347C18 (orange), chromosome 8 centromere probe (green), and DAPI nuclear stain (blue) in a human tumor sample (d) Correlation between 8q22 copy number detected by FISH and the 8q22 twelve gene expression-based index (8qEI) (regression R = 0.65). (e) Kaplan-Meier analysis for distant recurrence-free survival (DRFS) of 85 Boston cases with or without 8q22 amplification identified by FISH (Cox Hazard Ratio (HR) = 7.77). (f) DRFS for 52 Boston cases treated with doxorubicin and cyclophosphamide, according to 8q22 amplification identified by FISH (HR = 7.66). (g) Disease free survival (DFS) for 361 patients from six independent cohorts who received adjuvant chemotherapy, according to 8qEI high and low based on 8qEI levels above or below median level in each of the six cohorts (HR = 1.9).

(a) mRNA levels of LAPTM4B and YWHAZ relative to a reference sample (log2 scale, left axis) and doxorubicin IC₅₀ concentration (µM, right axis) in three breast cancer cell lines, as indicated below the bars. (b, c, and g) Merged fluorescence analysis for doxorubicin (drug autofluorescence, red) and nuclear staining (DAPI, blue). Doxorubicin localized in the nucleus appears purple. Scale bars indicate 10 µm. (b) Intracellular doxorubicin distribution after 24 h of drug exposure in three breast tumor cell lines. (c) Intracellular doxorubicin distribution in MDA-MB-231 cells transfected with control siRNA and LAPTM4B-specific siRNA as indicated to the right. Time points during drug exposure and after removal of drug from culture medium are shown above the panels. (d) Western blot with anti-phospho-H2AX antibody and β-actin control antibody in lysates of MDA-MB-231 cells transfected with control and gene-specific siRNA oligonucleotides, as indicated along the left side, at the indicated time points of drug exposure and after removal of drug from culture medium. (e) Plot of inhibition of cell growth by doxorubicin, paclitaxel and cisplatin in HMEC cells transfected with vectors containing GFP control, LAPTM4B-HA, or YWHAZ-HA. The percent viable cells, compared to transfected cells without drug treatment, are indicated on the Y-axis (mean of triplicates ± S.D.). ** indicates >250% increase in IC₅₀. (f) Western blot of HMEC cells transfected with vectors containing LacZ control, LAPTM4B-HA and YWHAZ-HA, as indicated above, using HA tag antibody and β-actin antibody. (g) Intracellular doxorubicin distribution in HMEC cells transfected with LacZ control vector or with LAPTM4B-HA vector after 16 hours of doxorubicin exposure.

(a) siRNA knockdown of the twelve 8q22 genes in BT549 breast tumor cells. Bars indicate the ratio of IC₅₀ for the indicated drugs in gene-specific siRNA treated cells, relative to control siRNA treated cells. Percent reduction in IC₅₀ is indicated as follows: ** 63% reduction, *** 82%, **** >85%. (b) Relative mRNA levels for LAPTM4B and YWHAZ in the three breast cancer cell lines, after treatment with control or specific siRNAs as indicated. Levels are log 2 scale and relative to HMEC reference sample. (c) Drug concentration-dependent cell survival curves for doxorubicin (upper panels), cisplatin (middle panels), and paclitaxel (lower panels) in cells transfected with control siRNA or gene-specific siRNAs, as indicated. The cell lines are shown at top. Approximate percent reduction in IC50 is indicated as follows: *** 85%, ** 75%, and * 50%. (d) Western blot for pro-caspase 3 (pr-cas3), active caspase 3 (ac-cas) and cleaved lower molecular weight PARP (cl-PARP), in MB231 and BT549 cells transfected with siRNA for LAPTM4B (L), YWHAZ (Y), or scramble control (C) as indicated across the top. Cells were treated with carrier (no marker) or with doxorubicin or cisplatin (+) for 48 hours, as indicated above each lane.

(a, b, c) Cases ranked according to mean sum expression of LAPTM4B and YWHAZ and (d, e, f) Receiver Operating Characteristic plots for the performance of the LAPTM4B and YWHAZ genes to predict pCR for (a, d) epirubicin in the whole breast cancer cohort (pCR, n = 17; no pCR, n = 101), (b, e) epirubicin in the ER⁻ and HER2⁻ sub-cohort (pCR, n = 9; no pCR, n = 78), and (c, f) cisplatin in ER⁻ PR⁻ HER2⁻ breast cancer patients (pCR, n = 4; no pCR, n = 20). In the ROC plots (e, f, g), the solid diagonal lines indicate the performance of a random predictor and the dashed line indicates the performance of the mean LAPTM4B and YWHAZ levels to predict pCR.