Carlton L. Schwab

Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial–mesenchymal transition

Significance Some cancers, termed carcinosarcomas (CSs), have mixed cell types, with either epithelial or mesenchymal features. Sequencing the genomes of uterine and ovarian CSs demonstrated that these different cell types derive from a common precursor cell that has many mutations typical of epithelial cancers. In addition, we find that these tumors have a significant burden of point mutations and amplification of histone genes, suggesting a potential role of these mutations in sarcomatous transformation. Consistent with this finding, expression of specific histone gene mutations in uterine carcinoma cells changed gene expression toward a mesenchymal state. These findings have potential implications for the treatment of these cancers. Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC. Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial–mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

Polymerase ε (POLE) ultra-mutated tumors induce robust tumor-specific CD4 + T cell responses in endometrial cancer patients

OBJECTIVE Around 7-10% of endometrial carcinomas are characterized by polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. METHODS We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (-) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or ([3H])thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-h-(51)Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level was also performed. RESULTS DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (-) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P<0.05). CONCLUSIONS POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (-) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their enhanced immunogenicity.

Taselisib, a selective inhibitor of PIK3CA, is highly effective on PIK3CA-mutated and HER2/neu amplified uterine serous carcinoma in vitro and in vivo

OBJECTIVE To evaluate the efficacy of taselisib, a selective inhibitor of PIK3CA, against primary uterine serous carcinomas (USC) harboring PIK3CA mutations and HER2/neu gene amplification. METHODS Sensitivity to taselisib was evaluated by flow-cytometry viability assays in vitro against nine primary USC cell lines. Cell cycle distribution and downstream signaling were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. Preclinical efficacy of taselisib was also evaluated in vivo in a mouse model. RESULTS Four USC cell lines harbored HER2/neu gene amplification by FISH and two of them harbored oncogenic PIK3CA mutations. Taselisib caused a strong differential growth inhibition in both HER2/neu FISH positive and HER2/neu FISH positive/PIK3CA mutated USC cell lines when compared to lines that were FISH negative and PIK3CA wild type (taselisib IC50 mean±SEM=0.042±0.006μM in FISH+ versus 0.38±0.06μM in FISH-tumors, P<0.0001). Taselisib growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and dose-dependent decline in the phosphorylation of S6. Taselisib was highly active at reducing tumor growth in vivo in USC mouse xenografts harboring PIK3CA mutation and overexpressing HER2/neu (P=0.007). Mice treated with taselisib had significantly longer survival when compared to control mice (P<0.0001). CONCLUSIONS Taselisib represents a novel therapeutic option in patients harboring PIK3CA mutations and/or HER2/neu gene amplification.

Clostridium perfringens enterotoxin C‐terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy‐resistant ovarian cancer

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin‐3‐ and ‐4‐overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy‐naïve and chemotherapy‐resistant human ovarian cancer xenografts or patient‐derived xenografts (PDXs) were treated with the carboxyl‐terminal binding domain of the Clostridium perfringens enterotoxin (c‐CPE) conjugated to FITC (FITC‐c‐CPE) or the near‐infrared (NIR) fluorescent tag IRDye CW800 (CW800‐c‐CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC‐c‐CPE and the CW800‐c‐CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800‐c‐CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor‐to‐background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c‐CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c‐CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real‐time detection of micrometastatic ovarian disease overexpressing the Claudin‐3 and ‐4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

Neratinib shows efficacy in the treatment of HER2/neu amplified uterine serous carcinoma in vitro and in vivo

OBJECTIVES Uterine serous carcinoma (USC) represents an aggressive variant of endometrial cancer and accounts for a large proportion of deaths annually. HER2/neu amplification is associated with USC in approximately 30-35% of cases. The objective of this study was to determine the sensitivity of a panel of primary USC cell lines to the small tyrosine kinase inhibitor neratinib, an ErbB1 and HER2 inhibitor, both in vitro and in vivo. METHODS HER2/neu amplification was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 24 USC cell lines. Flow cytometry was used to determine the effects of neratinib on cell viability, cell cycle distribution and signaling in vitro. Mice harboring HER2/neu amplified xenografts were treated with neratinib to assess the efficacy of the drug in vivo. RESULTS HER2/neu amplification was noted in 8/24 primary cell lines. Data regarding the efficacy of neratinib was determined using 4 HER2 amplified cell lines and 4 non-amplified cell lines with similar growth rates. Data revealed that cell lines with HER2/neu amplification were exquisitely more sensitive to neratinib compared to non-amplified cell lines (mean ± SEM IC50: 0.011μM ± 0.0008 vs. 0.312μM ± 0.0456 p<0.0001). Neratinib caused arrest in the G0/G1 phase of the cell cycle and resulted in decreased autophosphorylation of HER2 and activation of S6. Neratinib treated mice harboring xenografts of HER2/neu amplified USC showed delayed tumor growth and improved overall survival compared to vehicle (p=0.0019). CONCLUSIONS Neratinib may be a potential treatment option for patients harboring HER2/neu amplified USC. Clinical trials for this subset of endometrial cancer patients are warranted.

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy‐resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo

Solitomab is a novel, bispecific, single‐chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T‐cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy‐resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites.

Past, present and future targets for immunotherapy in ovarian cancer

Ovarian cancer is the leading cause of death from gynecologic malignancy in the US. Treatments have improved with conventional cytotoxic chemotherapy and advanced surgical techniques but disease recurrence is common and fatal in nearly all cases. Current evidence suggests that the immune system and its ability to recognize and eliminate microscopic disease is paramount in preventing recurrence. Ovarian cancer immunotherapy is targeting tumors through active, passive and adoptive approaches. The goal of immunotherapy is to balance the activation of the immune system against cancer while preventing the potential for tremendous toxicity elicited by immune modulation. In this paper we will review the different immunotherapies available for ovarian cancer as well as current ongoing studies and potential future directions.

T‐DM1, a novel antibody–drug conjugate, is highly effective against primary HER2 overexpressing uterine serous carcinoma in vitro and in vivo

Amplification of c‐erbB2 has been reported in over 30% of uterine serous carcinoma (USC) and found to confer poor survival because of high proliferation and increased resistance to therapy. In this study, we evaluated for the first time Trastuzumab emtansine (T‐DM1), a novel antibody–drug conjugate, against multiple epidermal growth factor receptor‐2 (HER2)‐positive USC cells in vitro followed by developing a supportive in vivo model. Fifteen primary USC cell lines were assessed by immunohistochemistry (IHC) and flow cytometry for HER2 protein expression. C‐erbB2 gene amplification was evaluated using fluorescent in situ hybridization. Sensitivity to T‐DM1 and trastuzumab (T)‐induced antibody‐dependent cell‐mediated cytotoxicity was evaluated in 5‐h chromium release assays. T‐DM1 and T cytostatic and apoptotic activities were evaluated using flow‐cytometry‐based proliferation assays. In vivo activity of T‐DM1 versus T in USC xenografts in SCID mice was also evaluated. High levels of HER2 protein overexpression and HER2 gene amplification were detected in 33% of USC cell lines. T‐DM1 was considerably more effective than trastuzumab in inhibiting cell proliferation and in causing apoptosis (P = 0.004) of USC showing HER2 overexpression. Importantly, T‐DM1 was highly active at reducing tumor formation in vivo in USC xenografts overexpressing HER2 (P = 0.04) and mice treated with TDM‐1 had significantly longer survival when compared to T‐treated mice and control mice (P ≤ 0.0001). T‐DM1 shows promising antitumor effect in HER2‐positive USC cell lines and USC xenografts and its activity is significantly higher when compared to T. T‐DM1 may represent a novel treatment option for HER2‐positive USC patients with disease refractory to trastuzumab and traditional chemotherapy.

Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines In Vitro and In Vivo

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC) and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib, and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA-mutated and PIK3CA wild-type HER2/neu–amplified USC cell lines. Cell viability and cell-cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC xenografts. We found both single-agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long-lasting growth inhibition in both USC xenografts when compared with single-agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0–G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single-agent–treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA and pan-ErbB inhibitor–resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild-type PIK3CA resistant to chemotherapy. Mol Cancer Ther; 14(11); 2519–26. ©2015 AACR.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo

Background:Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts.Methods:Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival.Results:Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean±s.e.m. IC50=0.0056±0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean±s.e.m. IC50=0.563±0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017).Conclusions:Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.