Background:HER-2/neu gene amplification has predictive value in breast cancer patients responding to trastuzumab. We wanted to investigate the frequency and clinical significance of HER-2/neu amplification in gastric carcinoma. Patients and methods: The frequency of HER-2/neu and Topoisomerase IIα gene amplification was studied in adenocarcinomas of the stomach (n=131) and the gastroesophageal junction (n=100) by chromogenic in situ hybridization (CISH). Sensitivity of a gastric cancer cell line N87 with HER-2/neu amplification to trastuzumab was studied by a cell viability assay and compared with that of a HER-2 amplified breast cancer cell line SKBR-3. Growth inhibition of N87 cells was also verified in vivo in N87 xenograft tumors. Results:HER-2/neu amplification was present in 16 (12.2%) of the 131 gastric and in 24 (24.0%) of the 100 gastroesophageal adenocarcinomas. Co-amplification of Topoisomerase IIα was present in the majority of gastric (63%) and esophagogastric junction cancers (68%) with HER-2/neu amplification. HER-2/neu amplification was more common in the intestinal histologic type of gastric cancer (21.5%) than in the diffuse (2%) or the mixed/anaplastic type (5%, P=0.0051), but it was not associated with gender, age at diagnosis or clinical stage. Presence of HER-2/neu amplification was associated with poor carcinoma-specific survival (P=0.0089). HER-2/neu targeting antibody trastuzumab inhibited the growth of a p185^HER-2/neu overexpressing gastric and breast carcinoma cell lines (N87 and SKBR-3) with equal efficacy. Conclusions:HER-2/neu amplification is common in the intestinal type of gastric carcinoma, and it is associated with a poor outcome. HER-2 might be a useful target in this disease, and this hypothesis deserves to be investigated in clinical trials.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is effective and generally well tolerated when administered as a single agent to treat advanced breast cancer. Efficacy has now been demonstrated in randomized trials as first line, second line, and later than the second line treatment of advanced breast cancer. T-DM1 is currently being evaluated as adjuvant treatment for early breast cancer. It has several mechanisms of action consisting of the anti-tumor effects of trastuzumab and those of DM1, a cytotoxic anti-microtubule agent released within the target cells upon degradation of the human epidermal growth factor receptor-2 (HER2)-T-DM1 complex in lysosomes. The cytotoxic effect of T-DM1 likely varies depending on the intracellular concentration of DM1 accumulated in cancer cells, high intracellular levels resulting in rapid apoptosis, somewhat lower levels in impaired cellular trafficking and mitotic catastrophe, while the lowest levels lead to poor response to T-DM1. Primary resistance of HER2-positive metastatic breast cancer to T-DM1 appears to be relatively infrequent, but most patients treated with T-DM1 develop acquired drug resistance. The mechanisms of resistance are incompletely understood, but mechanisms limiting the binding of trastuzumab to cancer cells may be involved. The cytotoxic effect of T-DM1 may be impaired by inefficient internalization or enhanced recycling of the HER2-T-DM1 complex in cancer cells, or impaired lysosomal degradation of trastuzumab or intracellular trafficking of HER2. The effect of T-DM1 may also be compromised by multidrug resistance proteins that pump DM1 out of cancer cells. In this review we discuss the mechanism of action of T-DM1 and the key clinical results obtained with it, the combinations of T-DM1 with other cytotoxic agents and anti-HER drugs, and the potential resistance mechanisms and the strategies to overcome resistance to T-DM1.

Introduction: Accurate assessment of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 is essential in the histopathologic diagnostics of breast cancer. Commercially available image analysis systems are usually bundled with dedicated analysis hardware and, to our knowledge, no easily installable, free software for immunostained slide scoring has been described. In this study, we describe a free, Internet-based web application for quantitative image analysis of ER, PR, and Ki-67 immunohistochemistry in breast cancer tissue sections. Methods: The application, named ImmunoRatio, calculates the percentage of positively stained nuclear area (labeling index) by using a color deconvolution algorithm for separating the staining components (diaminobenzidine and hematoxylin) and adaptive thresholding for nuclear area segmentation. ImmunoRatio was calibrated using cell counts defined visually as the gold standard (training set, n = 50). Validation was done using a separate set of 50 ER, PR, and Ki-67 stained slides (test set, n = 50). In addition, Ki-67 labeling indexes determined by ImmunoRatio were studied for their prognostic value in a retrospective cohort of 123 breast cancer patients. Results: The labeling indexes by calibrated ImmunoRatio analyses correlated well with those defined visually in the test set (correlation coefficient r = 0.98). Using the median Ki-67 labeling index (20%) as a cutoff, a hazard ratio of 2.2 was obtained in the survival analysis (n = 123, P = 0.01). ImmunoRatio was shown to adapt to various staining protocols, microscope setups, digital camera models, and image acquisition settings. The application can be used directly with web browsers running on modern operating systems (e.g., Microsoft Windows, Linux distributions, and Mac OS). No software downloads or installations are required. ImmunoRatio is open source software, and the web application is publicly accessible on our website. Conclusions: We anticipate that free web applications, such as ImmunoRatio, will make the quantitative image analysis of ER, PR, and Ki-67 easy and straightforward in the diagnostic assessment of breast cancer specimens.

Clinical resistance to the HER-2 oncogene–targeting drug trastuzumab (Herceptin) exists, but studies of the resistance mechanisms are hampered by the lack of suitable experimental model systems. We established a carcinoma cell line (designated JIMT-1) from a pleural metastasis of a 62-year old patient with breast cancer who was clinically resistant to trastuzumab. JIMT-1 cells grow as an adherent monolayer and form xenograft tumors in nude mice. JIMT-1 cells have an amplified HER-2 oncogene, which showed no identifiable mutations in its coding sequence. JIMT-1 cells overexpress HER-2 mRNA and protein, and the levels of HER-1, HER-3, and HER-4 mRNA and protein were similar to the trastuzumab-sensitive cell line SKBR-3. The cell line lacks expression of hormone receptors (estrogen receptors and progesterone receptors) and is phenotypically of epithelial progenitor cell origin, as evidenced by immunohistochemical positivity for both cytokeratins 5/14 and 8/18. JIMT-1 cells were insensitive to trastuzumab and another HER-2-inhibiting drug, pertuzumab (2C4), in vitro and in xenograft tumors. Small molecule tyrosine kinase inhibitors Ci1033 and ZD1839 inhibited the JIMT-1 cell growth but to a lesser degree than in trastuzumab-sensitive BT-474 cells. The lack of growth inhibition was rationalized by the unaltered Akt phosphorylation in JIMT-1 cells. Erk1/2 phosphorylation was slightly reduced but still evident in JIMT-1 cells. We conclude that the JIMT-1 cell line provides a valuable experimental model for studies of new trastuzumab-resistance mechanisms.

Introduction: Some molecular subtypes of breast cancer have preferential sites of distant relapse. The protein expression pattern of the primary tumor may influence the first distant metastasis site. Methods: We identified from the files of the Finnish Cancer Registry patients diagnosed with breast cancer in five geographical regions Finland in 1991-1992, reviewed the hospital case records, and collected primary tumor tissue. Out of the 2,032 cases identified, 234 developed distant metastases after a median follow-up time of 2.7 years and had the first metastatic site documented (a total of 321 sites). Primary tumor microarray (TMA) cores were analyzed for 17 proteins using immunohistochemistry and for erbB2 using chromogenic in situ hybridization, and their associations with the first metastasis site were examined. The cancers were classified into luminal A, luminal B, HER2+ enriched, basal-like or non-expressor subtypes. Results: A total of 3,886 TMA cores were analyzed. Luminal A cancers had a propensity to give rise first to bone metastases, HER2-enriched cancers to liver and lung metastases, and basal type cancers to liver and brain metastases. Primary tumors that gave first rise to bone metastases expressed frequently estrogen receptor (ER) and SNAI1 (SNAIL) and rarely COX2 and HER2, tumors with first metastases in the liver expressed infrequently SNAI1, those with lung metastases expressed frequently the epidermal growth factor receptor (EGFR), cytokeratin-5 (CK5) and HER2, and infrequently progesterone receptor (PgR), tumors with early skin metastases expressed infrequently E-cadherin, and breast tumors with first metastases in the brain expressed nestin, prominin-1 and CK5 and infrequently ER and PgR. Conclusions: Breast tumor biological subtypes have a tendency to give rise to first distant metastases at certain body sites. Several primary tumor proteins were associated with homing of breast cancer cells.