BackgroundMutation in the tumor suppressor gene TP53 is an early event in the development of high-grade serous (HGS) ovarian cancer and is identified in more than 96 % of HGS cancer patients. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 protein by refolding it to wild type conformation, thus inducing apoptosis. APR-246 has been tested as monotherapy in a Phase I/IIa clinical study in hematological malignancies and prostate cancer with promising results, and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing. In the present study, we investigated the anticancer effects of APR-246 in combination with conventional chemotherapy in primary cancer cells isolated from ascitic fluid from 10 ovarian, fallopian tube, or peritoneal cancer patients, 8 of which had HGS cancer.MethodsCell viability was assessed with fluorometric microculture cytotoxicity assay (FMCA) and Combination Index was calculated using the Additive model. p53 status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by western blotting.ResultsWe observed strong synergy with APR-246 and cisplatin in all tumor samples carrying a TP53 missense mutation, while synergistic or additive effects were found in cells with wild type or TP53 nonsense mutations. Strong synergy was also observed with carboplatin or doxorubicin. Moreover, APR-246 sensitized TP53 mutant primary ovarian cancer cells, isolated from a clinically platinum-resistant patient, to cisplatin; the IC50 value of cisplatin decreased 3.6 fold from 6.5 to 1.8 μM in the presence of clinically relevant concentration of APR-246.ConclusionThese results suggest that combination treatment with APR-246 and DNA-damaging drugs could significantly improve the treatment of patients with TP53 mutant HGS cancer, and thus provide strong support for the ongoing clinical study with APR-246 in combination with carboplatin and pegylated liposomal doxorubicin in patients with recurrent HGS cancer.
Introduction: Mesothelin (MSLN) is overexpressed in several tumors including ovarian cancer and is the target of current trials. There is limited and conflicting data on MSLN prognostic impact in ovarian cancer. Methods: We performed a retrospective study on patients with high-grade serous ovarian cancer, analyzing MSLN expression by immunohistochemistry and examining the correlation of its expression to overall and progression-free survival. Correlations of expression of MSLN, CD8, and macrophage markers in different tumor compartments were also investigated. Results: Positive MSLN expression was detected in 55.1% of primary tumors and 51.5% of the metastases. MSLN expression was not correlated with survival. We observed a significant positive correlation (r = 0.34, p = 0.01) between MSLN expression in the metastatic site and CD11c expression in total tumor area and perivascular area in the primary tumor. Isabelle Magalhaes and Josefin Fernebro contributed equally to this work as joint first authors. Jonas Mattsson and Hanna Dahlstrand also contributed equally to this work.
Background: Mutations in the TP53 gene occur in at least 60% of ovarian tumors and are associated with chemoresistance and poor prognosis. APR-246 (PRIMA-1MET) is the first mutant p53-reactivating compound in clinical development and has been tested as monotherapy in hematological malignancies and prostate cancer with promising results (Lehmann et al. J Clin Oncol 30, 2012). The aim of this study was to investigate the anticancer effects of APR-246 in combination with conventional chemotherapy in cancer cells isolated from ascites fluid from ovarian cancer patients. Methods: Ascites cells were purified by Ficoll and viably frozen, and the quality and purity were confirmed by May Grünwald/Giemsa staining. For some samples, immunocytochemical stainings with anti-Ber-EP4 and anti-calretinin antibodies were used to distinguish between mesothelial and cancer cells. Cell viability was assessed with FMCA assay and Combination Index (CI) calculated using Additive model. CI < 0.8 indicates synergy and CI < 0.5 strong synergy. TP53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Results: Eight of ten samples tested were from patients with recurrent ovarian cancer previously treated with platinum drugs. Cancer cells from seven patients possessed TP53 core domain missense mutations L111Q, C135Y, P151H, Y163H, C238F, P278R and R280K, respectively; two had nonsense mutations E346* and E204*, and one was wild type. All the missense mutations have been predicted to severely affect p53 tumor suppressor function. Missense mutant p53 proteins were expressed at high levels while no p53 expression was detected in cells with wild type or nonsense mutant p53. Synergistic or strong synergistic effects with APR-246 and cisplatin were observed in all ten samples tested. Synergy was also observed with the platinum analogue carboplatin and the anthracycline doxorubicin. The IC50 values for cisplatin ranged from 3 to 40 μM and for APR-246 from 5 to 37 μM. We also tested the ability of APR-246 to sensitize the primary ovarian cancer cells carrying Y163H mutant p53, to cisplatin; the IC50-value of cisplatin decreased from 10 to 2.6 μM in the presence of 6 μM APR-246. Conclusions: We observed striking synergy with APR-246 and platinum drugs or doxorubicin. These results are consistent with our previous results in ovarian cancer cell lines showing synergy not only in p53 mutant but also in p53 null cancer cell lines. In these cells, the synergy may be related to the fact that APR-246 decreases intracellular glutathione level. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with carboplatin and doxorubicin in patients with recurrent ovarian cancer and suggest that combination treatment with APR-246 and DNA-damaging drugs could allow significantly improved treatment for ovarian cancer carrying mutant p53. Citation Format: Åsa Fransson, Daria Glaessgen, Jessica Alfredsson, Klas G. Wiman, Svetlana Bajalica Lagercrantz, Nina Mohell. Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2015-1639
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