T-DM1, a novel antibody-drug conjugate, is highly effective against uterine and ovarian carcinosarcomas overexpressing HER2

Abstract

Ovarian and uterine carcinosarcoma (CS) are characterized by their aggressive clinical behavior and poor prognosis. We evaluated the efficacy of trastuzumab-emtansine (T-DM1), against primary HER2 positive and HER2 negative CS cell lines in vitro and in vivo. Eight primary CS cell lines were evaluated for HER2 amplification and protein expression by fluorescence in situ hybridization, immunohistochemistry, flow cytometry and qRT-PCR. Sensitivity to T-DM1-induced antibody-dependent-cell-mediated-cytotoxicity (ADCC) was evaluated in 4-h-chromium-release-assays. T-DM1 cytostatic and apoptotic activities were evaluated using flow cytometry based proliferation assays. In vivo activity of T-DM1 was also evaluated. HER2 protein overexpression and gene amplification were detected in 25 % (2/8) of the primary CS cell lines. T-DM1 and T were similarly effective in inducing strong ADCC against CS overexpressing HER2 at 3+ levels. In contrast, T-DM1 was dramatically more effective than T in inhibiting cell proliferation (P < 0.0001) and in inducing G2/M phase cell cycle arrest in the HER2 expressing cell lines (shift of G2/M: mean ± SEM from 14.87 ± 1.23 to 66.57 ± 4.56 %, P < 0.0001). Importantly, T-DM1 was highly active at reducing tumor formation in vivo in CS xenografts overexpressing HER2 (P = 0.0001 and P < 0.0001 compared to T and vehicle respectively) with a significantly longer survival when compared to T and vehicle mice (P = 0.008 and P = 0.0001 respectively). T-DM1 may represent a novel treatment option for the subset of HER2 positive CS patients with disease refractory to chemotherapy.

Figure 1. Representative flow cytometry analysis in HER2 positive and negative CS cell lines

High HER2 expressing cell line (SARARK-6, panel A) versus the low HER2 expressing cell line (SARARK-1, panel B). Rituximab control mAb is the solid line and trastuzumab is the dotted line. High HER2 expressing cell line shows significantly higher mean fluorescence intensity with trastuzumab versus low HER2 expressing cell line.

Figure 2. Representative ADCC results of T-DM1 versus T

HER2 overexpressing cell line (SARARK-6) was challenged with PBLs at a ratio of 20:1. No significant difference in ADCC is seen with T versus T-DM1 (P=0.259). Results represent average percent cytotoxicity ± SEM of two independent experiments.

Figure 3. Representative proliferation assay result of T-DM1 versus T

Cells were plated in 6 wells and treated with T-DM1 24 hours later, then counted after 72 hours by flow citometry. T-DM1 significantly inhibits SARARK-6 (high HER2 expressing cell line) proliferation compared to T (P<0.0001). Reported values are representative of 3 independent experiments; bars are mean ± SEM.

Figure 4. Analysis of cell cycle by flow cytometry

Representative experiment showing cell cycle arrest in G2/M phase after treatment with T-DM1 in the high HER2 expressing cell line SARARK-6 (P<0.0001). No change in cell cycle distribution is shown in the HER2 negative cell line SARARK-1. Data are representative of at least 3 independent experiments.

Figure 5. Tumor growth inhibition by T vs T-DM1

Tumors were allowed to establish growth for a week after subcutaneous injection before initiation of treatment. Fifteen mice (5 mice per group) with CS xenografted tumor everexpressing HER2 (SARARK-6) were treated with a series of 5 IV injection of T-DM1 (15 mg/kg/w), T (15 mg/kg/w) and vehicle, respectively. T-DM1 treatment resulted in a dramatic reduction of tumors volumes and complete disappearance of established disease when compared to T and to vehicle group (P=0.0001 and P<0.0001 respectively) (panel A). Points are mean tumor volume (cm³); bars are SEM (standard error of the mean) (n=5 mice per group). T-DM1 treatment significantly prolonged overall survival in treated group when compared to trastuzumab group and to the vehicle group (P=0.008 and P=0.0001 respectively) (panel B).