Babak Litkouhi

Institutional review of primary non-hodgkin lymphoma of the female genital tract: a 33-year experience.

Objective The aim of this is to provide an updated review of the literature and to report our institutional experience with this rare gynecologic malignancy. Methods The medical records of patients with diagnosis of non-Hodgkin lymphoma of the female genital tract from 1980 to 2013 at the Yale–New Haven Hospital were reviewed retrospectively. Histological classification and staging were determined by the World Health Organization and Ann Arbor systems, respectively. Kaplan-Meier was used to calculate the survival. Results There were 36 patients with diagnosis of non-Hodgkin lymphoma of the female genital tract and followed for a median of 61 months (0–361 months). The median age of diagnosis was 44 years (19–87 years), and 76% (n = 28) were classified as stage IV. Of these, 4 patients were asymptomatic on presentation, and 13 were identified incidentally during surgery/radiography (n = 9), on prenatal ultrasound (n = 1), and on Papanicolaou test (n = 3). The location of the disease included the ovary (n = 6), uterine corpus and cervix (n= 9), vagina (n = 1), a pelvic mass (n = 7), isolated pelvic/para-aortic lymph nodes (n = 3), and/or multiple sites (n = 9). There were 6 cases that were concomitant with other gynecologic malignancies. Diffuse large B-cell lymphoma (n= 18) was the most common histologic type. A total of 28 patients underwent surgery. Combination chemotherapy was used in 34 patients, with concomitant radiation therapy in 7 and stem cell transplantation in 3. A total of 5 patients had recurrent disease. The overall median survival from the diagnosis of lymphoma was 70 months (0.3–361 months) with a 91% 1-year survival, 86% 5-year survival, and a 79% 10-year survival. Conclusions Our report is the largest published single-institution experience of this disease. It demonstrates a more favorable prognosis and proposes that with early diagnosis and appropriate therapy, radical gynecologic surgery can be avoided.

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.

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.

Randomized Phase II Trial of Carboplatin-Paclitaxel Versus Carboplatin-Paclitaxel-Trastuzumab in Uterine Serous Carcinomas That Overexpress Human Epidermal Growth Factor Receptor 2/neu

Purpose Uterine serous carcinoma is a rare, aggressive variant of endometrial cancer. Trastuzumab is a humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2)/neu, a receptor overexpressed in 30% of uterine serous carcinoma. This multicenter, randomized phase II trial compared carboplatin-paclitaxel with and without trastuzumab in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu. Methods Eligible patients had primary stage III or IV or recurrent HER2/neu-positive disease. Participants were randomly assigned to receive carboplatin-paclitaxel (control arm) for six cycles with or without intravenous trastuzumab (experimental arm) until progression or unacceptable toxicity. The primary end point was progression-free survival, which was assessed for differences between treatment arms via one-sided log-rank tests. Results From August 2011 to March 2017, 61 patients were randomly assigned. Forty progression-free survival-related events occurred among 58 evaluable participants. Among all patients, median progression-free survival was 8.0 months (control) versus 12.6 months (experimental; P = .005; hazard ratio [HR], 0.44; 90% CI, 0.26 to 0.76). Similarly, median progression-free survival was 9.3 (control) versus 17.9 (experimental) months among 41 patients with stage III or IV disease undergoing primary treatment ( P = .013; HR, 0.40; 90% CI, 0.20 to 0.80) and 6.0 (control) versus 9.2 months (experimental), respectively, among 17 patients with recurrent disease ( P = .003; HR, 0.14; 90% CI, 0.04 to 0.53). Toxicity was not different between treatment arms, and no unexpected safety signals emerged. Conclusion Addition of trastuzumab to carboplatin-paclitaxel was well tolerated and increased progression-free survival. These encouraging results deserve further investigation to determine their impact on overall survival in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu.

SYD985, a novel duocarmycin-based HER2-targeting antibody-drug conjugate, shows promising antitumor activity in epithelial ovarian carcinoma with HER2/Neu expression

BACKGROUND Epithelial ovarian cancer (EOC) is an aggressive and heterogeneous disease. <10% of EOC demonstrate HER2/neu 3+ receptor over-expression. However, moderate to low (i.e., 2+ and 1+) HER2/neu expression is reported in up to 50% of EOC. The objective of this study was to compare the anti-tumor activity of SYD985, a novel HER2-targeting antibody-drug conjugate (ADC), to trastuzumab emtansine (T-DM1) in EOC models with differential HER2/neu expression. METHODS The cytotoxicity of SYD985 and T-DM1 was evaluated using ten primary EOC cell lines with 0/1+, 2+, and 3+ HER2/neu expression in antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments. Finally, the in vivo activity of SYD985 and T-DM1 was also studied in ovarian cancer xenografts. RESULTS SYD985 and T-DM1 induced similar ADCC in the presence of peripheral blood lymphocytes (PBL) against EOC cell lines with differential HER2/neu expression. In contrast, SYD985 was 3 to 42 fold more cytotoxic in the absence of PBL when compared to T-DM1 (p<0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ tumor cells when admixed with HER2/neu 3+ EOC cells. In vivo studies confirmed that SYD985 is significantly more active than T-DM1 against HER2/neu 3+ EOC xenografts. CONCLUSIONS SYD985 is a novel ADC with remarkable activity against EOC with strong (3+) as well as moderate to low (i.e., 2+ and 1+) HER2/neu expression. SYD985 is more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against EOC demonstrating moderate/low or heterogeneous HER2/neu expression.

Polymerase ε (POLE) ultra-mutation in uterine tumors correlates with T lymphocyte infiltration and increased resistance to platinum-based chemotherapy in vitro

OBJECTIVE Up to 12% of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. METHODS Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9-14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. RESULTS Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P=0.001) and CD8+ (32.8 vs 13.5; P<0.001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P<0.001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P<0.004). CONCLUSIONS POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity.

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.

In Vitro and In Vivo Activity of IMGN853, an Antibody–Drug Conjugate Targeting Folate Receptor Alpha Linked to DM4, in Biologically Aggressive Endometrial Cancers

Grade 3 endometrioid and uterine serous carcinomas (USC) account for the vast majority of endometrial cancer deaths. The purpose of this study was to determine folic acid receptor alpha (FRα) expression in these biologically aggressive (type II) endometrial cancers and evaluate FRα as a targetable receptor for IMGN853 (mirvetuximab soravtansine). The expression of FRα was evaluated by immunohistochemistry (IHC) and flow cytometry in 90 endometrioid and USC samples. The in vitro cytotoxic activity and bystander effect were studied in primary uterine cancer cell lines expressing differential levels of FRα. In vivo antitumor efficacy of IMGN853 was evaluated in xenograft/patient-derived xenograft (PDX) models. Semiquantitative IHC analysis indicated that 41% of the USC patients overexpress FRα. Further, overexpression of FRα (i.e., 2+) was detected via flow cytometry in 22% (2/9) of primary endometrioid and in 27% (3/11) of primary USC cell lines. Increased cytotoxicity was seen with IMGN853 treatment compared with control in 2+ expressing uterine tumor cell lines. In contrast, tumor cell lines with low FRα showed no difference when exposed to IMGN853 versus control. IMGN853 induced bystander killing of FRα = 0 tumor cells. In an endometrioid xenograft model (END(K)265), harboring 2+ FRα, IMGN853 treatment showed complete resolution of tumors (P < 0.001). Treatment with IMGN853 in the USC PDX model (BIO(K)1), expressing 2+ FRα, induced twofold increase in median survival (P < 0.001). IMGN853 shows impressive antitumor activity in biologically aggressive FRα 2+ uterine cancers. These preclinical data suggest that patients with chemotherapy resistant/recurrent endometrial cancer overexpressing FRα may benefit from this treatment. Mol Cancer Ther; 17(5); 1003–11. ©2018 AACR.

A novel multiple biomarker panel for the early detection of high-grade serous ovarian carcinoma

INTRODUCTION Since the majority of patients are diagnosed at an advanced stage, ovarian cancer remains the most lethal gynecologic malignancy. There is no single biomarker with the sensitivity and specificity required for effective cancer screening; therefore, we investigated a panel of novel biomarkers for the early detection of high-grade serous ovarian carcinoma. METHODS Twelve serum biomarkers with high differential gene expression and validated antibodies were selected: IL-1Ra, IL-6, Dkk-1, uPA, E-CAD, ErbB2, SLPI, HE4, CA125, LCN2, MSLN, and OPN. They were tested using Simple Plex™, a multi-analyte immunoassay platform, in samples collected from 172 patients who were either healthy, had benign gynecologic pathologies, or had high-grade serous ovarian adenocarcinomas. The receiver operating characteristic (ROC) curve, ROC area under the curve (AUC), and standard error (SE) of the AUC were obtained. Univariate ROC analyses and multivariate ROC analyses with the combination of multiple biomarkers were performed. RESULTS The 4-marker panel consisting of CA125, HE4, E-CAD, and IL-6 had the highest ROC AUC. When evaluated for the ability to distinguish early stage ovarian cancer from a non-cancer control, not only did this 4-marker panel (AUC=0.961) performed better than CA 125 alone (AUC=0.851; P=0.0150) and HE4 alone (AUC=0.870; P=0.0220), but also performed significantly better than the 2- marker combination of CA125+HE4 (AUC=0.922; P=0.0278). The 4-marker panel had the highest average sensitivity under the region of its ROC curve corresponding to specificity ranging from 100% down to ~95%. CONCLUSION The four-marker panel, CA125, HE4, E-CAD, and IL-6, shows potential in detecting serous ovarian cancer at earlier stages. Additional validation studies using the biomarker combination in ovarian cancer patients are warranted.

Superior in vitro and in vivo activity of trastuzumab-emtansine (T-DM1) in comparison to trastuzumab, pertuzumab and their combination in epithelial ovarian carcinoma with high HER2/neu expression

BACKGROUND Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. The objective of this study was to compare the anti-tumor activity of HER2/neu-targeting monoclonal antibodies, trastuzumab (T), pertuzumab (P), combination of trastuzumab and pertuzumab (T+P) and trastuzumab-emtansine (T-DM1) in EOC with high HER2/neu expression. METHODS Primary EOC cell lines were established and cell blocks were analyzed for HER2/neu expression. Cytostatic, apoptotic and antibody-dependent cell-mediated cytotoxicity (ADCC) activities of T, P, T+P and T-DM1 were evaluated in vitro. The in vivo antitumor activity was tested in xenograft models with 3+ HER2/neu expression. RESULTS High (3+) HER2/neu expression was detected in 40% of the primary EOC cell lines. T, P, T+P, and T-DM1 were similarly effective in inducing strong ADCC against primary EOC cell lines expressing 3+ HER2/neu. The combination of T and P was more cytostatic when compared with that of T or P used alone (p<0.0001 and p<0.0001, respectively). T-DM1 induced significantly more apoptosis when compared with T+P (p<0.0001). Finally, T-DM1 was significantly more effective in tumor growth inhibition in vivo in EOC xenografts overexpressing HER2/neu when compared to T alone, P alone and T+P (p=0.04). CONCLUSION In vitro and in vivo experiments with 3+ HER2/neu expressing EOC revealed limited anti-tumor activity of T or P. T-DM1 showed superior anti-tumor activity to T and P as single agents and as a combination. Our preclinical data support the design of clinical studies with T-DM1 for the treatment of chemotherapy-resistant EOC overexpressing HER2/neu.