Fig 1.
HER2 gene amplification patterns in different breast cancer cell lines and PDXs.
A, The indicated cell lines were treated with colchicine and metaphase spreads (top panels) or HER2 DISH staining (middle panels) were performed as described under Materials and Methods. As a control, HER2 DISH was also performed on untreated cultures of the same cell lines (lower panels). B, Samples from the indicated PDXs were analyzed by HER2 DISH and the pattern of gene amplification as well as the ratio HER2/CEP17 were determined. Levels of HER2 protein expression were analyzed by immunohistochemistry (IHC). C, Samples from the indicated PDXs were analyzed by HER2 DISH. D, Cell cultures obtained from PDXs with HSR or DM HER2 gene amplifications were treated with colchicine and metaphase spreads (upper panels) or HER2 DISH (lower panels) were performed as in A.
Fig 2.
HER2 amplification patterns and response to trastuzumab.
A, The pattern of amplification of samples from two cohorts of breast cancers patients treated with neoadjuvant or adjuvant trastuzumab was determined as in Fig 1A and samples were classified as HSR, DM or mixed (see text for details). Representative examples are shown. B, Upper graph, percentages of the HER2 gene amplification patterns in tumors from a cohort of breast cancer patients treated with neoadjuvant trastuzumab. Lower graph, pathologic complete response (pCR) rates according to the HER2 gene amplification pattern. P values shown were calculated by the two-sided Fisher’s exact test. C, Paired samples, pre- and post-treatment with trastuzumab, from tumors with DM and HSR amplifications, obtained from patients without pCR, were analyzed by HER2 DISH and the ratio HER2/CEP17 was calculated and represented. D, Upper graph, percentages of the HER2 gene amplification patterns in tumors from a cohort of breast cancer patients treated with adjuvant trastuzumab. Lower graph, disease-free survival (DFS) according to HER2 gene amplification pattern. P values shown were calculated by the Mantel-Cox test comparing the HSR and DM groups.
Fig 3.
HER2 amplification pattern and gene copy number in tumors resistant to trastuzumab or T-DM1.
A, NOD/SCID mice (n = 8 per group) carrying PDX118, which has amplification in DM (see S1B and S1C Fig), were treated with anti-HER2 antibodies as indicated (red and green arrowheads indicate the administrations of trastuzumab (10 mg/kg twice per week, i.p) and T-DM1 (15 mg/kg every three weeks, i.v., respectively). Tumor volumes were calculated using the formula: (length × width2) × (pi/6) and results are expressed as averages. Error bars correspond to 95% confidence intervals. B, Mice carrying PDX118 were chronically treated with trastuzumab (left) or twice with T-DM1(right) and tumor volumes were determined as in A. The growth of representative individual tumors is shown. C, HER2 protein levels and HER2 gene amplification were analyzed by immunohistochemistry and DISH, respectively, in samples from parental PDX118, or samples from the tumors shown in B. D, HER2 protein levels determined by IHC or HER2 gene amplification determined by DISH were quantified in parental PDX118 or in samples from resistant xenografts shown in B and in S2 Fig E, Gene copy number was determined by qPCR as described under Experimental Procedures.
Fig 4.
HER2 amplification pattern and gene copy number in cells resistant to lapatinib or T-DM1.
A, Schematic showing the procedure to obtain cells resistant to anti-HER2 therapies in vitro. Cultures from PDX118 were treated with increasing concentrations of lapatinib or T-DM1 for 45 days. B, Parental PDX118 cell cultures or cells selected in the presence of lapatinib or T-DM1 as shown in A, in two independent experiments, were plated in the presence of the indicated concentrations of the anti-HER2 drugs. At the indicated time points, cell proliferation was determined and normalized. The results were expressed as averages ± standard deviations. C, The same cells as in B were lysed and the cell lysates analyzed by Western blot with antibodies against HER2 in three independent experiments. Representative results are shown. Blots were quantified and the results were expressed as averages ± standard deviations. *, P < 0.05; ***, P < 0.001. D, Gene copy number was determined by qPCR as described under Experimental Procedures.