Effects of Slide Storage on Detection of Molecular Markers by IHC and FISH in Endometrial Cancer Tissues From a Clinical Trial: An NRG Oncology/GOG Pilot Study.

We performed a pilot study in anticipation of using long-aged precut formalin-fixed paraffin-embedded tissue sections stored in real-world conditions for translational biomarker studies of topoisomerase 2A (TOP2A), Ki67, and human epidermal growth factor receptor 2 (HER2) in endometrial cancer. Formalin-fixed paraffin-embedded tissue blocks or unstained slides or both from GOG-0177 were collected centrally (1999-2000) and stored at room temperature. During 2004 to 2011 specimens were stored at 4°C. Matched pairs of stored slides and freshly cut slides from stored blocks were analyzed for TOP2A (KiS1), Ki67 (MIB1), and HER2 (HercepTest) proteins. To assess DNA stability (HER2 PathVision), fluorescence in situ hybridization (FISH) was repeated on stored slides from 21 cases previously shown to be HER2 amplified. Immunohistochemistry (IHC) staining intensity and extent, mean FISH copies/cell, and copy number ratios were compared using the κ statistic for concordance or signed rank test for differences in old cut versus new cut slides. IHC results reflected some protein degradation in stored slides. The proportion of cells with TOP2A staining was lower on average by 12% in older sections (P=0.03). The proportion of Ki67-positive cells was lower in stored slides by an average of 10% (P<0.01). Too few cases in the IHC cohort were FISH positive for any conclusions. HER2 amplification by FISH was unaffected by slide storage. We conclude that use of aged stored slides for proliferation markers TOP2A and Ki67 is feasible but may modestly underestimate true values in endometrial cancer. Pilot studies for particular storage conditions/durations/antigens to be used in translational studies are warranted.

Impact of carboplatin hypersensitivity and desensitization on patients with recurrent ovarian cancer.

PURPOSE: Hypersensitivity reactions (HSRs) to chemotherapy is an ongoing issue in cancer treatments. Strategies to induce tolerance and maximize chemotherapy efficacy include desensitization protocols. The precise impact of these protocols, however, in the long-term treatments remains unclear. We aim to compare overall survival (OS) in hypersensitive patients treated with carboplatin desensitization to patients without hypersensitivity reactions. We also sought to identify new risk factors for HSRs and reconfirm that the DNA repair enzyme, germline BRCA1/2 (gBRCA1/2), is a risk factor for hypersensitivity. EXPERIMENTAL DESIGN: Retrospective study in patients with ovarian cancer tested for gBRCA1/2 mutations who received more than six infusions of carboplatin from August 2005 to November 2016. Two-sided Fisher exact, Student's t test and Gehan-Breslow-Wilcoxon test were used for statistical analysis. Univariate and multivariate analyses were completed to identify independent predictors of survival. Statistical significance was set with a two-sided p value of 0.05. RESULTS: Ninety-one patients with gBRCA1/2 testing met inclusion. Forty patients (44%) were gBRCA1/2-deficient and 51 (56%) were gBRCA1/2-proficient. Patients with gBRCA1/2 deficiencies had a higher likelihood of developing carboplatin hypersensitivity, HR 6.433 (95% CI: 1.868-22.149). None of the patients with carboplatin hypersensitivity were given PARP inhibitors prior to the development of HSRs. The patients with recurrent advanced stage (III-IV) ovarian cancer had a higher likelihood of developing carboplatin hypersensitivity, HR 4.783 (1.008-22.689). Moreover, we found that hypersensitive patients who underwent carboplatin desensitization had a 48-month longer OS than patients without hypersensitivity to carboplatin not undergoing carboplatin desensitization (p = 0.0094). A subgroup analysis indicated that gBRCA1/2-proficient hypersensitive patients undergoing carboplatin desensitization had a 43-month longer OS than gBRCA1/2-proficient patients without HSRs (p = 0.034). CONCLUSIONS: We confirmed that gBRCA1/2 deficiency and advanced stage are independent risk factors for development of carboplatin hypersensitivity in ovarian cancer patients. Our study also shows improved OS in hypersensitive patients receiving CD compared to non-hypersensitive patients, independent of gBRCA1/2.

Dual-Targeting Nanoparticles for In Vivo Delivery of Suicide Genes to Chemotherapy-Resistant Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecologic cancer. Claudin-3 and -4, the receptors for Clostridium perfringens enterotoxin (CPE), are overexpressed in more than 70% of these tumors. Here, we synthesized and characterized poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) modified with the carboxy-terminal-binding domain of CPE (c-CPE-NP) for the delivery of suicide gene therapy to chemotherapy-resistant ovarian cancer cells. As a therapeutic payload, we generated a plasmid encoding for the diphtheria toxin subunit-A (DT-A) under the transcriptional control of the p16 promoter, a gene highly differentially expressed in ovarian cancer cells. Flow cytometry and immunofluorescence demonstrated that c-CPE-NPs encapsulating the cytomegalovirus (CMV) GFP plasmid (CMV GFP c-CPE-NP) were significantly more efficient than control NPs modified with a scrambled peptide (CMV GFP scr-NP) in transfecting primary chemotherapy-resistant ovarian tumor cell lines in vitro (P = 0.03). Importantly, c-CPE-NPs encapsulating the p16 DT-A vector (p16 DT-A c-CPE-NP) were significantly more effective than control p16 DT-A scr-NP in inducing ovarian cancer cell death in vitro (% cytotoxicity: mean ± SD = 32.9 ± 0.15 and 7.45 ± 7.93, respectively, P = 0.03). In vivo biodistribution studies demonstrated efficient transfection of tumor cells within 12 hours after intraperitoneal injection of CMV GFP c-CPE-NP in mice harboring chemotherapy-resistant ovarian cancer xenografts. Finally, multiple intraperitoneal injections of p16 DT-A c-CPE-NP resulted in a significant inhibition of tumor growth compared with control NP in chemotherapy-resistant tumor-bearing mice (P = 0.041). p16 DT-A c-CPE-NP may represent a novel dual-targeting therapeutic approach for the selective delivery of gene therapy to chemotherapy-resistant ovarian cancer cells. Mol Cancer Ther; 16(2); 323-33. ©2016 AACR.

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

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.

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.

BACKGROUND: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. METHODS: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. RESULTS: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. CONCLUSIONS: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.

SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine Serous Carcinoma with HER2/Neu Expression.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer. Up to 35% of USC may overexpress the HER2/neu oncogene at strong (i.e., 3+) levels by IHC while an additional 40% to 50% express HER2/neu at moderate (2+) or low (1+) levels. We investigated the efficacy of SYD985, (Synthon Biopharmaceuticals), a novel HER2-targeting antibody-drug conjugate (ADC) composed of the mAb trastuzumab linked to a highly potent DNA-alkylating agent (i.e., duocarmycin) in USC. We also compared the antitumor activity of SYD985 in head-to-head experiments to trastuzumab emtansine (T-DM1), a FDA-approved ADC, against multiple primary USC cell lines expressing different levels of HER2/neu in in vitro and in vivo experiments. Using antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing assays as well as propidium iodide-based flow cytometry assays and multiple in vivo USC mouse xenograft models, we demonstrate for the first time that SYD985 is a novel ADC with activity against USC with strong (3+) as well as low to moderate (i.e., 1+/2+) HER2/neu expression. SYD985 is 10- to 70-fold more potent than T-DM1 in comparative experiments and, unlike T-DM1, it is active against USC demonstrating moderate/low or heterogeneous HER2/neu expression. Clinical studies with SYD985 in patients harboring chemotherapy-resistant USC with low, moderate, and high HER2 expression are warranted. Mol Cancer Ther; 15(8); 1900-9. ©2016 AACR.

Molecular diagnosis and molecular profiling to detect treatment-resistant ovarian cancer.

INTRODUCTION: Epithelial ovarian cancer remains the gynecologic tumor with the highest rate of recurrence after initial optimal cytoreductive surgery followed by adjuvant chemotherapy. Unfortunately, with the development of recurrent ovarian cancer often comes the discovery of chemo-resistant disease. The absence of improvement in long term survival, notwithstanding the use of newer agents as is seen in other cancers, emphasizes the need for improved understanding of the processes that lead to chemo-resistant disease. AREAS COVERED: This review will cover the following topics: 1. Molecular and cellular mechanisms in platinum and paclitaxel resistance 2. Other molecular mediators of chemo-resistance 3. Expression of stem cell markers in ovarian cancer and relationship to chemo-resistance 4. MicroRNA and long non-coding RNA expression in chemo-resistant ovarian cancer 5. Determination of chromosomal aberrations as markers of chemo-resistance 6. Molecular profiling in chemo-resistant disease. A standard MEDLINE search was performed using the key words; ovarian cancer, chemo-resistant disease, molecular profiling, cancer stem cells and chemotherapy. Expert Commentary: Over the next few years the challenge remains to precisely determine the mechanisms responsible for the onset and maintenance of chemo-resistance and to effectively target these mechanisms.

Incidental injury and repair of obturator nerve during laparoscopic pelvic lymphadenectomy.

OBJECTIVE: To demonstrate a surgical video wherein left obturator nerve was iatrogenically injured during pelvic lymphadenectomy and repaired immediately with laparoscopic epineural end-to-end tension free anastomosis. METHODS: This is a step-by-step demonstration of an incidental injury and laparoscopic repair of left obturator nerve during pelvic lymphadenectomy. The patient was a 59year-old Hispanic female who was found to have endometrial adenocarcinoma. She was referred to our division for laparoscopic staging during which left obturator nerve was iatrogenically injured. After completion of left pelvic lymphadenectomy, proximal and distal cut ends of the obturator nerve were identified. Careful inspection revealed that the nerve was transected cleanly without any fraying of the edges. Tension-free reattachment of the edges seemed possible without further mobilization of the nerve since the resected part was approximately 5mm. The obturator nerve edges were oriented and stay sutures were placed in order to perform tension-free anastomosis. Epineural end-to-end coaptation was completed with 5-0 polypropylene sutures [1,2]. RESULTS: Postoperatively, the patient did not exhibit any clinically apparent loss of adductor function or any other neurologic deficiency and was discharged home on postoperative day one. Over 6months of follow-up, the patient experienced no residual neuropathy or deficit in the left thigh. CONCLUSION: Laparoscopic repair of a transected obturator nerve during gynecologic surgery is feasible. In this case, immediate repair of the damaged nerve by an experienced laparoscopic gynecologic surgeon did not result in any neurologic deficit postoperatively.

Immune checkpoint inhibitors in gynecologic cancers with lessons learned from non-gynecologic cancers.

INTRODUCTION: The immune system plays a critical role in controlling cancer through a dynamic relationship with cancer cells. Immunotherapy can establish a sustained immune response against recurring cancer cells leading to long-term remissions for cancer patients. The use of immune checkpoint inhibitors, which work by targeting molecules that regulate immune responses, might be a promising avenue of immunotherapeutic research in gynecologic cancers. AREAS COVERED: This review focuses on the use of immune checkpoint inhibitors targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) and the programmed death receptors such as PD-1 and PD1-ligand in gynecologic cancers. Combinatorial approaches utilizing immunotherapeutic agents with conventional treatments as well as immune-related response criteria and the adverse effects arising due to checkpoint inhibitor immunotherapy have been also discussed in the review. EXPERT OPINION: After years of very little success, a better understanding of the pivotal role of the tumor microenvironment in suppressing anticancer immunity and the exploration of treatment regimens using immune checkpoint inhibitors alone or in combination have finally led to the development of effective cancer immunotherapies and to improve survival of patients with certain types of advanced cancers. While the clinical experience with immune checkpoint inhibitors in gynecologic cancer patients remains limited, early signal of clinical activity strongly suggest that immunotherapy will play a significant role in the year to come in at least a subset of gynecologic cancer patients.

The Role of the Immune System in Ovarian Cancer and Implications on Therapy.

Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. While the treatment options have improved with conventional cytotoxic chemotherapy and advanced surgical techniques, 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. 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 role of immune system in disease pathogenesis and different immunotherapies available for the treatment of ovarian cancer as well as current ongoing studies and potential future directions.

Laparoscopic Resection of the Diaphragmatic Tumor Nodule for Management of Recurrent Endometrial Cancer.

STUDY OBJECTIVE: To show a surgical video in which an isolated hemidiaphragmatic tumor nodule was resected laparoscopically in a patient with isolated recurrence of endometrial cancer. DESIGN: Case report (Canadian Task Force Classification study design III). SETTING: Tertiary referral center in New Haven, CT. INTERVENTIONS: This is a step-by-step illustration of tumor nodule resection from the right hemidiaphragm. The patient was a 55-year-old white woman who was diagnosed with stage IIIA endometrioid endometrial adenocarcinoma in June 2011 after surgical debulking. She received adjuvant carboplatin and paclitaxel and vaginal brachytherapy. She was disease free until March 2015 when she presented with right upper abdominal pain. A computed tomographic scan showed a 1-cm implant on the right hepatic dome. The implant was noted to be enlarged to 1.8 cm on a subsequent computed tomographic scan in August 2015. The patient was taken to the operating room for exploratory laparoscopy and resection of the hepatic dome/hemidiaphragmatic tumor nodule. The tumor nodule was noted to involve the full thickness of the right hemidiaphragm. The resection of the entire nodule required perforation of the diaphragm, which was reapproximated after the excision. MAIN RESULTS: The procedure was performed without any complications. The patient had an uneventful postoperative course and was discharged home on postoperative day 1. Pathology revealed metastatic endometrioid endometrial adenocarcinoma with negative resection margins. CONCLUSION: Laparoscopic resection of the diaphragmatic tumor nodule and the reapproximation of the diaphragm were successfully performed in this patient with isolated disease recurrence. The laparoscopic approach should be considered for management of isolated recurrences in gynecologic cancers by experienced laparoscopic surgeons because it might otherwise be associated with significant morbidity and mortality [1-3].

Overexpression of HE4 (human epididymis protein 4) enhances proliferation, invasion and metastasis of ovarian cancer.

Overexpression of Human epididymis protein 4 (HE4) related with a role in ovarian cancer tumorigenesis while little is known about the molecular mechanism alteration by HE4 up regulation. Here we reported that overexpressed HE4 promoted ovarian cancer cells proliferation, invasion and metastasis. Furthermore, human whole genome gene expression profile microarrays revealed that 231 differentially expressed genes (DEGs) were altered in response to HE4, in which MAPK signaling, ECM receptor, cell cycle, steroid biosynthesis pathways were involved. The findings suggested that overexpressed HE4 played an important role in ovarian cancer progression and metastasis and that HE4 has the potential to serve as a novel therapeutic target for ovarian cancer.

Immunotherapy and targeted therapy for cervical cancer: an update.

The prognosis of patients with metastatic cervical cancer is poor with a median survival of 8-13 months. Despite the potency of chemotherapeutic drugs, this treatment is rarely curative and should be considered palliative only. In the last few years, a better understanding of Human papillomavirus tumor-host immune system interactions and the development of new therapeutics targeting immune check points have renewed interest in the use of immunotherapy in cervical cancer patients. Moreover, next generation sequencing has emerged as an attractive option for the identification of actionable driver mutations and other markers. In this review, we provide background information on the molecular biology of cervical cancer and summarize immunotherapy studies, targeted therapies, including those with angiogenesis inhibitors and tyrosine kinase inhibitors recently completed or currently on-going in cervical cancer patients.

An update on the current pharmacotherapy for endometrial cancer.

INTRODUCTION: Endometrial cancer (EC) is the most common gynecologic malignancy in the developed world and is increasing in incidence. While the mainstay of treatment for EC is surgery followed by chemotherapy and/or radiation therapy, the available pharmacotherapies are rapidly and constantly evolving. Understanding these new therapies is an important part of the research and clinical care of women with EC. A review of available literature from MEDLINE (1879-2015) was conducted for the historic treatments and current therapies available for endometrial tumors. AREAS COVERED: This article reviews the current conventional therapies and discusses novel therapeutic agents, some of which are available to clinicians while others are currently being investigated in the preclinical setting. EXPERT OPINION: Genomic and immunohistochemical characterization of endometrial cancer may soon be the best approach for the identification of aggressive forms of tumor. Targeted therapies will soon be standard in the management of endometrial cancer.

Solitomab, an EpCAM/CD3 bispecific antibody construct (BiTE), is highly active against primary uterine serous papillary carcinoma cell lines in vitro.

BACKGROUND: Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the in vitro activity of solitomab against primary uterine serous carcinoma cell lines in vitro and ex-vivo in the ascites of patients with uterine serous carcinoma. OBJECTIVE: The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule-positive cell lines or epithelial cell adhesion molecule-positive ascitic fluid in vitro. STUDY DESIGN: Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired t test. T-cell activation marker increase and cytokine release were analyzed by a paired t test. RESULTS: Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule-positive cell lines were found resistant to natural killer cells or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule-positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule-positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites (P < .001). CONCLUSION: Solitomab induces robust immunologic responses in vitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma-overexpressing epithelial cell adhesion molecule.

Solitomab, an EpCAM/CD3 bispecific antibody construct (BiTE®), is highly active against primary uterine and ovarian carcinosarcoma cell lines in vitro.

BACKGROUND: Uterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which carry an extremely poor prognosis. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab, a bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region, against primary uterine and ovarian CS cell lines. METHODS: EpCAM expression was evaluated by flow cytometry in a total of 5 primary CS cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against the panel of primary CS cell lines expressing different levels of EpCAM in standard 4 h (51)Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the pleural fluid of a CS patient were also evaluated by CFSE and flow-cytometry assays. RESULTS: Surface expression of EpCAM was found in 80.0 % (4 out of 5) of the CS cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-h chromium-release assays (mean killing ± SEM = 1.1 ± 1.6 %, range 0-5.3 % after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive CS cells became highly sensitive to T-cell-cytotoxicity (mean killing ± SEM of 19.7 ± 6.3 %; range 10.0-32.0 %; P < 0.0001). Ex vivo incubation of autologous TAL with EpCAM expressing malignant cells in pleural effusion with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable CS cells in the exudate (P < 0.001). CONCLUSIONS: Solitomab may represent an effective treatment for patients with recurrent/metastatic and/or chemo-resistant CS overexpressing EpCAM.

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.

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas.

OBJECTIVES: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. METHODS: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. RESULTS: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. CONCLUSIONS: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.

Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo.

OBJECTIVE: Carcinosarcoma is a deadly gynecologic malignancy with few effective treatment options. The study of new therapies is difficult because of its rarity. The objective of this study was to determine the efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma. METHODS: The efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma was determined in vitro using seven primary carcinosarcoma cell lines with differential expression of HER2/neu. Data regarding IC50, cell cycle distribution, and cell signaling changes were assessed by flow cytometry. The efficacy of neratinib was determined in treating mice harboring HER2 amplified carcinosarcoma xenografts. RESULTS: Two of seven (28.5%) carcinosarcoma cell lines were HER2/neu amplified. HER2/neu amplified cell lines SARARK6 and SARARK9 were significantly more sensitive to neratinib than the five non-HER2/neu amplified carcinosarcoma cell lines (mean±SEM IC50:0.014µM±0.004vs.0.164µM±0.019 p=0.0003). Neratinib treatment caused a significant build up in G0/G1 phase of the cell cycle, arrest auto phosphorylation of HER2/neu and activation of S6. Neratinib inhibited tumor growth (p=0.012) and prolonged survival in mice harboring HER2 amplified carcinosarcoma xenografts (p=0.0039). CONCLUSIONS: Neratinib inhibits HER2 amplified carcinosarcoma proliferation, signaling, cell cycle progression and tumor growth in vitro. Neratinib inhibits HER2/neu amplified xenograft growth and improves overall survival. Clinical trials are warranted.