Hiroaki Itamochi

Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells

Expression of the αvβ6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvβ6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αβ) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvβ6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvβ6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvβ6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.

Establishment and characterization of a novel ovarian clear cell carcinoma cell line, TU-OC-2, with loss of ARID1A expression

Abstract A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5xa0h. The IC50 values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7xa0μM, 17.7xa0nM and 301xa0nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.

Whole-genome sequencing revealed novel prognostic biomarkers and promising targets for therapy of ovarian clear cell carcinoma

Background:Ovarian clear cell carcinoma (OCCC) is mostly resistant to standard chemotherapy that results in poor patient survival. To understand the genetic background of these tumours, we performed whole-genome sequencing of OCCC tumours.Methods:Tumour tissue samples and matched blood samples were obtained from 55 Japanese women diagnosed with OCCC. Whole-genome sequencing was performed using the Illumina HiSeq platform according to standard protocols.Results:Alterations to the switch/sucrose non-fermentable (SWI/SNF) subunit, the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway, and the receptor tyrosine kinase (RTK)/Ras signalling pathway were found in 51%, 42%, and 29% of OCCC tumours, respectively. The 3-year overall survival (OS) rate for patients with an activated PI3K/Akt signalling pathway was significantly higher than that for those with inactive pathway (91 vs 40%, hazard ratio 0.24 (95% confidence interval (CI) 0.10–0.56), P=0.0010). Similarly, the OS was significantly higher in patients with the activated RTK/Ras signalling pathway than in those with the inactive pathway (91 vs 53%, hazard ratio 0.35 (95% CI 0.13–0.94), P=0.0373). Multivariable analysis revealed that activation of the PI3K/Akt and RTK/Ras signalling pathways was an independent prognostic factor for patients with OCCC.Conclusions:The PI3K/Akt and RTK/Ras signalling pathways may be potential prognostic biomarkers for OCCC patients. Furthermore, our whole-genome sequencing data highlight important pathways for molecular and biological characterisations and potential therapeutic targeting in OCCC.

ARID1A mutation sensitizes most ovarian clear cell carcinomas to BET inhibitors

Current treatment for advanced stage ovarian clear cell cancer is severely hampered by a lack of effective systemic therapy options, leading to a poor outlook for these patients. Sequencing studies revealed that ARID1A is mutated in over 50% of ovarian clear cell carcinomas. To search for a rational approach to target ovarian clear cell cancers with ARID1A mutations, we performed kinome-centered lethality screens in a large panel of ovarian clear cell carcinoma cell lines. Using the largest OCCC cell line panel established to date, we show here that BRD2 inhibition is predominantly lethal in ARID1A mutated ovarian clear cell cancer cells. Importantly, small molecule inhibitors of the BET (bromodomain and extra terminal domain) family of proteins, to which BRD2 belongs, specifically inhibit proliferation of ARID1A mutated cell lines, both in vitro and in ovarian clear cell cancer xenografts and patient-derived xenograft models. BET inhibitors cause a reduction in the expression of multiple SWI/SNF members including ARID1B, providing a potential explanation for the observed lethal interaction with ARID1A loss. Our data indicate that BET inhibition may represent a novel treatment strategy for a subset of ARID1A mutated ovarian clear cell carcinomas.

Integrative kinome profiling identifies mTORC1/2 inhibition as treatment strategy in ovarian clear cell carcinoma

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models. Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient–derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055. Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient–derived xenograft models. Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928–40. ©2018 AACR.

Establishment and characterization of a novel uterine carcinosarcoma cell line, TU-ECS-1, with mutations of TP53 and KRAS

A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2xa0h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9xa0µM, 154xa0nM, and 219xa0ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.

Feasibility study on combination chemotherapy using nogitecan hydrochloride (topotecan) and cisplatin for patients with metastatic, persistent, or recurrent uterine cervical cancer

BackgroundThe standard chemotherapeutic regimen for stage IVB, persistent, or recurrent uterine cervical cancer is platinum-based combination chemotherapy such as cisplatin (CDDP)/paclitaxel and CDDP/nogitecan hydrochloride (NGT, topotecan). Because it is unclear whether the CDDP/NGT combination chemotherapy is tolerable for Japanese patients, we conducted the present study to assess the feasibility of CDDP/NGT combination chemotherapy.MethodsBetween June 2012 and April 2014, 15 patients with stage IVB, persistent, or recurrent uterine cervical cancer were enrolled in this study. Patients underwent six cycles of NGT at a dose of 0.75xa0mg/m2, followed immediately by CDDP at a dose of 50xa0mg/m2 on day 1 by intravenous infusion, and then NGT at a dose of 0.75xa0mg/m2 on days 2 and 3.ResultsOf 15 patients, 9 patients underwent at least 6 cycles of NGT/CDDP combination chemotherapy. Of a total of 83 cycles, 70 cycles (84.3xa0%) of NGT/CDDP combination chemotherapy could be continued at the starting dose of NGT (0.75xa0mg/m2). Grade 3/4 hematological toxicities included leukopenia in 10 patients (66.7xa0%), neutropenia in 15 (100xa0%), anemia in 6 (40.0xa0%), thrombocytopenia in 4 (26.7xa0%), and febrile neutropenia in 4 (26.7xa0%). The response rate according to RECIST was 27xa0% (3/11), with partial response in 3 patients.ConclusionsNGT/CDDP combination chemotherapy may be a tolerable and effective regimen for Japanese patients with stage IVB, persistent, or recurrent uterine cervical cancer. Based on the results of this study, NGT/CDDP combination chemotherapy was approved in Japan in November 2015.

Measurement of endometrial thickness in premenopausal women in office gynecology

To define the median endometrial thickness (ET) in office gynecology is thought to be important for clinical practice. However, there are few reports about ET that have included the general female population on a large scale. The median ET was determined prospectively in premenopausal women who attended office gynecology for cervical cancer screening.

Gemcitabine and docetaxel in a patient with primary ovarian leiomyosarcoma: a case report and review of literature

Primary ovarian leiomyosarcoma (POLMS) is extremely rare, and optimal therapy for this disease is unknown. A 40-year-old woman presented at a local hospital with abdominal pain. Tumor resection of the left ovary was performed. The pathological diagnosis was leiomyoma of the left ovary. Nine months after surgery, she developed of severe back pain and a subcutaneous tumor on her left shoulder. Magnetic resonance imaging and computed tomography revealed left ovarian tumor recurrence, pelvic bone metastasis, and multiple liver masses. Biopsy of the subcutaneous tumor on her left shoulder demonstrated metastatic leiomyosarcoma. The previously resected left ovarian tumor was re-examined, and the tumor was found to be a leiomyosarcoma. The patient received gemcitabine 800xa0mg/m2 and docetaxel 60xa0mg/m2 (GD therapy), administered at 3-week intervals. After three cycles of GD therapy, the patient experienced dyspnea and was diagnosed with mild interstitial pneumonia. Oral corticosteroid therapy resulted in complete symptom improvement. Thereafter, the dosage of GD was decreased, and after 13 cycles of GD therapy, radiofrequency ablation was performed twice for liver metastases. The tumors have shrunk by 65.5% after 23 cycles of GD. She remains alive after undergoing 24 cycles of GD. GD therapy may be effective for POLMS.

Signal Transduction and Targeted Therapy for Gynecologic Cancer

Numerous agents that target specific gynecologic cancer-related molecules have been developed and are now entering clinical trials. These agents target aberrant molecules/processes in tumor tissues, including angiogenesis, poly(ADP-ribose) polymerase (PARP), human epidermal growth factor receptor family, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, and a-folate receptor (a-FR). The anti-angiogenic compound bevacizumab is reportedly the most effective targeted agent for ovarian cancer. Bevacizumab plus chemotherapy prolonged progression-free survival (PFS) both for first-line treatment and recurrent ovarian cancer and may increase overall survival (OS) among high-risk patients. Bevacizumab with nonplatinum chemotherapy also prolonged OS in recurrent cervical cancer. Maintenance treatment with a PARP inhibitor, olaparib, improved PFS in platinum-sensitive relapsed ovarian cancer. Furthermore, mTOR inhibitor therapy, alone or with chemotherapy, is an attractive treatment strategy for endometrial cancer. An understanding of tumor molecular biology and identification of predictive biomarkers are essential steps in optimal treatment selection. This article reviews available clinical data of the most promising targeted agents for gynecologic cancer.