The molecular analysis on archival materials was conducted in the context of Institutional Protocol approved by the Internal Review Boards of the Institutions listed in this work. the mutation and amplification is indeed more prevalent in post-treatment samples than in treatment-na?ve tumors (MUT-AMP: 11/13 [85%] vs AMP: 74/171[43%]. However, this analysis was insufficiently powered to identify robust differences between the two groups possibly due to the small numbers of patients with concomitant mutated and amplified tumors (Exact p = 0.076; fig. S1). Coincident HER2 mutation and amplification was also associated with poor response to a trastuzumab/pertuzumab/paclitaxel combination therapy compared to HER2 amplification alone [on the basis of progression-free survival, which was 7.3 months in the coincident group (95% AM 103 confidence interval: 4.8, not reached) and 22.6 months in the amplification-alone group (95% confidence interval: 15.8, 53.7), log-rank = 0.0136; Fig. 1D]. Notably, further SF3a60 adjustment for sample type (metastatic vs primary) or line of therapy in the AM 103 metastatic setting did not change these results. Exogenous expression of a HER2 mutation limits the sensitivity to trastuzumab and lapatinib in HER2 amplified breast cancer cells Our results, together with both preclinical (14) and clinical (15) observations recently reported, suggest that the acquisition of HER2 mutation in the metastatic setting may affect the sensitivity to anti-HER2 therapy in HER2-positive breast cancer patients. To confirm that the presence of HER2 mutations is sufficient to limit the sensitivity to trastuzumab or lapatinib in a HER2-amplified background, we stably transduced Sk-Br-3 and BT474 cells with lentiviral vectors encoding for wild-type or mutant (L755S) HER2 (or an empty vector control). We chose the L755S mutation because is the most frequently found in breast cancer and was detected in one of the 4 patients treated with anti-HER2 therapy described above. Treatment with lapatinib markedly suppressed the phosphorylation of HER2 downstream signaling proteins, namely the kinases AKT and extracellular signal-regulated kinase (ERK) and ribosomal protein S6, in both wild-typeC and empty-vectorCtransduced cells. The same concentrations of lapatinib were found to be less effective in inhibiting AKT, ERK and S6 signaling in the HER2 L755S-transduced cells (Fig. 2, A and ?andC).C). Consequently, lapatinib had significantly less anti-proliferative activity in Sk-Br-3 and BT474 cells expressing the L755S HER2 mutation compared to cells expressing wild-type HER2 (IC50= 1424 nM vs 171.3 nM and 583.8 nM vs 44.5 nM for the Sk-Br-3 and the BT474 clones, respectively; value obtained by two-tailed Students t-test. Open in a separate window Fig. 3. HER2 L755S mutation induces resistance to HER2-targeted therapy.Clonogenic growth assays performed by crystal violet staining on Sk-Br-3 (A) and BT-474 (B) cells expressing empty-vector, wild-type or mutant (L755S) HER2 constructs and cultured with trastuzumab (20 g/ml), lapatinib (500 nM) or the combination, as indicated, for ten days. Results from treated cultures were quantified relative to non-treated cells. Data are means SEM from three independent experiments. *value obtained by two-tailed Students t-test. In vivo efficacy of neratinib To test the efficacy of neratinib in vivo against breast tumors with coincident amplification and mutation of HER2, we established sub-cutaneous patient-derived xenografts (PDXs) from a HER2-positive breast cancer patient whose metastatic lung lesions carried both a D769Y mutation and amplification of HER2. Mice were treated with vehicle, trastuzumab, lapatinib, or neratinib for just under 5 weeks. As expected, xenografts were refractory to both trastuzumab and lapatinib (Fig. 5), suggesting that the presence of this HER2 mutation may lead to cross-resistance to multiple anti-HER2 agents. Conversely, neratinib significantly inhibited tumor growth and, in enough cases to have a cohort-level effect, neratinib induced durable tumor shrinkage (Fig. 5). Open in a separate window Fig. 5. In vivo efficacy of neratinib against HER2-amplified and AM 103 mutant PDXs.Antitumor effects of neratinib in xenografts bearing in ERBB2. Growth of patient-derived xenografts containing coincident amplification and HER2 D769Y mutation, in mice treated with vehicle (control), trastuzumab (10 mg/kg; ip; 2xwkly), lapatinib (100mg/kg daily) or neratinib (40mg/kg daily). Data are k means SEM from 8 mice each condition. by two-tailed Students t-test Neratinib has clinical activity in breast cancer patients with tumors bearing ERBB2 amplification and mutations Our findings indicate that the acquisition of HER2 mutations confer resistance to current standard anti-HER2 therapies AM 103 but not to neratinib. Moreover, we have recently shown that neratinib has clinical activity in breast cancer patients with HER2-negative tumors bearing HER2 mutations (11). Thus, we posited that neratinib would be effective in tumors with co-existing HER2 amplification and mutation. We tested this hypothesis in six breast cancer.
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