Mikiya Ishihara

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer

Purpose: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene–engineered T cells. Experimental Design: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4–expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. Results: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. Conclusions: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

HER2 peptide-specific CD8[+] T cells are proportionally detectable long after multiple DNA vaccinations

We prepared a plasmid encoding 147 amino acid residues from the N terminus of c-erbB-2/HER2/neu (HER2), which included both a cytotoxic T lymphocyte (CTL) epitope (HER2p63) and a helper epitope (HER2p1), using the mammalian expression vector pCAGGS-New (pCAGGS147HER2). In a parallel analysis with a Tetramer assay and CTL assay, good specificity and sensitivity of a quantitative enzyme-linked immunospot (ELISPOT) assay to detect functional HER2p63-specific CD8+ T cells were demonstrated after intramuscular immunization of pCAGGS147HER2. In an ELISPOT assay for HER2p63, spots of IFNγ-producing cells were first detected 10 days after the first immunization, and additional immunizations increased the number of spots. HER2p63-specific CD8+ T cells were detected over a period of more than 10 months after the last immunization. In hosts receiving more than three immunizations, surprisingly high numbers of specific CD8+ T cells were persistently detectable. HER2 protein-specific antibodies of IgG class with dominance of IgG2a remain detectable 6 months after single or multiple immunizations. The antibodies however, were not reactive with cell surface HER2 antigens. Total suppression of tumor growth was observed when syngeneic HER2+ tumor cells (2 × 106) were injected subcutaneously 14 days after a single immunization with pCAGGS147HER2. Furthermore, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection (1 × 106 cells).

Retrospective analysis of risk factors for central nervous system metastases in operable breast cancer: effects of biologic subtype and Ki67 overexpression on survival.

Objective: Identifying factors that predispose patients to central nervous system (CNS) metastases may hasten disease detection and improve treatment outcomes. Methods: We reviewed the records of patients who were diagnosed with clinical stage I–III primary breast cancer at the National Cancer Center Hospital East from 2003 to 2005. Cox proportional hazard models were fitted to reveal risk factors for CNS metastases. Results: The median follow-up period after the operation was 53.5 months. Among the 591 identified patients with breast cancer, 76 experienced a relapse. Seventeen patients developed CNS metastases. Multivariate analysis indicated that the triple negative (TN) subtype (hazard ratio = 5.5) and a high Ki67 labeling index (LI; hazard ratio = 3.9) were associated with a higher risk for CNS metastases. At 4 years, the TN subtype was associated with significantly worse overall and disease-free survival rates and a higher cumulative incidence of CNS metastases compared with hormone receptor-positive/ human epidermal growth factor receptor-2-negative tumors. Breast cancers with a Ki67 LI ≥30% were also associated with lower overall and disease-free survival rates and a higher cumulative incidence of CNS metastases compared with cancers with a Ki67 LI <30%. Conclusion: TN or Ki67-overexpressing breast cancer produced earlier CNS metastases and lower disease-free and overall survival rates.

Systemic CD8+ T cell-mediated tumoricidal effects by intratumoral treatment of oncolytic herpes simplex virus with the agonistic monoclonal antibody for murine glucocorticoid-induced tumor necrosis factor receptor.

Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8+ T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4+ Foxp3+ T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites.

A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity

Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment.

Pertuzumab, trastuzumab and eribulin mesylate therapy for previously treated advanced HER2-positive breast cancer: a feasibility study with analysis of biomarkers

The standard treatment for advanced human epidermal growth factor receptor 2 (HER2)-positive breast cancer is the triple combination of pertuzumab, trastuzumab and docetaxel, but some patients cannot tolerate taxane. To explore a non-taxane triple therapy, we conducted a feasibility study of pertuzumab, trastuzumab and eribulin mesylate (PTE) therapy for previously treated advanced HER2-positive breast cancer with analyses of quality of life and biomarkers. Ten patients were enrolled, two of whom had a history of docetaxel allergy. The median number of prior regimens was 3. The most common Grade 3 toxicities were leukopenia (70%) and neutropenia (70%). Grade 4 or 5 adverse events were not observed. An improving trend for the Functional Assessment of Cancer Therapy-Breast (FACT-B) score at 3 months was observed. Eight cases were included in the biomarker analysis. The peripheral CD8+ T cell/ CD4+Foxp3+ regulatory T cells (Tregs) ratio was significantly increased (p = 0.039). The frequency of peripheral Tregs was associated with the trastuzumab trough concentration (p = 0.019). In a non-clinical analysis, Eribulin mesylate significantly inhibited Ser473 Akt phosphorylation in PIK3CA wild-type cells and mutated cells. These results suggest that PTE therapy is a feasible and promising option for advanced HER2-positive breast cancer. Further investigation is warranted.

Abstract P4-13-21: A pilot study of pertuzumab, trastuzumab and eribulin for patients with advanced HER2 positive breast cancer

[Introduction] The triple therapy of pertuzumab, trastuzumab and taxanes (docetaxel or paclitaxel) is coming into widespread use, because of the beneficial effects on HER2 positive breast cancer. However, we don9t have enough information about the efficacy and safety of other agents with trastuzumab and pertuzumab (TP). We studied triple therapy of pertuzumab, trastuzumab and eribulin (PTE) for advanced HER2 positive breast cancer to assess the efficacy, safety and QOL prospectively (UMIN000012018). [Patients and methods] Responses were assessed by RECIST criteria v1.1. Adverse events (AEs) were graded according to CTCAE v4.0. Patients with advanced HER2 positive breast cancer were treated with pertuzumab (840 mg loading then 420 mg, day 1), trastuzumab (8 mg/kg loading then 6 mg/kg, day 1), and eribulin (1.4 mg/m2, day 1 and 8) every 3 weeks. Dose reduction was allowed when patients developed febrile neutropenia, grade 3-5 non-hematologic toxicity or skipped day 8 eribulin administration because of neutrophil count [Results] Ten patients were enrolled. Median age of patients was 60 years-old (35-75). Median number of prior chemoregimen for metastatic disease was 3 (0-5). Two patients had a history of docetaxel allergy. Median number of PTE cycle was 6 (3-12). Eight patients reduced eribulin doses 1.4 mg/m2 to 1.1 mg/m2 because of AEs (2 patients), skipped day 8 eribulin (4 patients), or physician9s choice (2 patients). One complete response, 1 partial response and 5 stable disease were achieved at 3 months. Two patients (1 CR and 1 SD) stopped eribulin and received TP as maintenance therapy. At 3 months, all 3 patients with progressive disease developed brain metastasis. Two patients had extracranial progressive lesions, but 1 patient had partial response for extracranial disease. The common treatment-related AEs were leukopenia, neutropenia, lymphopenia diarrhea, hypokalemia and stomatitis. Grade 3 AEs were leukopenia (7 patients), neutropenia (8 patients), lymphopenia (2 patients), febrile neutropenia (1 patient), hypokalemia (1 patient) and peripheral neuropathy (1 patient). Grade 4/5 AEs were not observed. Nine patients could be assessed QOL. FACT-B TOI, FACT-G and FACT-B total score had a tendency to be improved at 3 months. [Conclusion] The PTE therapy showed appropriate clinical effect for extracranial lesions and maintained QOL of patients with advanced HER2 positive breast cancer. It may be a choice for patients who have taxane-resistant diseases or a history of taxane allergy. Many patients needed to reduce eribulin dosage. When the PTE therapy is referred to advanced HER2 positive breast cancer patients as a palliative chemotherapy, eribulin (1.1mg/m2) might be a reasonable dosage. Citation Format: Ishihara M, Tamaru S, Oda H, Yamashita Y, Tono Y, Mizuno T, Katayama N. A pilot study of pertuzumab, trastuzumab and eribulin for patients with advanced HER2 positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-21.

1012PTHE PRESENCE OF SECOND PRIMARY ESOPHAGEAL TUMOR (SPET) IS STILL AN INDEPENDENT NEGATIVE PROGNOSTIC FACTOR FOR HEAD AND NECK CANCER (HNC) PATIENTS: A RETROSPECTIVE STUDY

ABSTRACT Aim: Head and neck cancer (HNC) patients have a high incidence of primary esophageal tumor. Moreover, second primary esophageal tumor (SPET) also had a negative impact on survival of HNC patients and previous studies reported that 3-year survival rate of HNC patients with SPET was 0-15%. Therefore, the establishment of optimal management for HNC patients with SPET is crucial. Recent advances in endoscopy such as magnifying endoscopy enabling early detection of esophageal tumor and the novel treatment may lead to improve survival for HNC patients with SPET. Methods: A total of 226 HNC patients who underwent magnifying esophagogastroduodenoscopy at our institute from October 2005 to September 2012 were retrospectively examined in this study. We investigated the incidence of SPET, the difference in the therapeutic outcome between HNC patients with and without SPET and the prognostic value of the presence of SPET in HNC patients. Results: Out of 226 HNC patients, 34 patients (15%) had SPET during their clinical course. The median age was 71 years (range 51 to 88) and 33 patients were male. All patients had squamous cell esophageal carcinoma. The majority of patients (79%) had SPET with stage 0 (26%) or 1(53%) disease. Out of 34 patients with SPET, 10 patients (29%) underwent endoscopic resection for SPET and 10 patients (29%) underwent simultaneous chemoradiation therapy with cisplatin plus continuous infusional 5-fluorouracil for HNC and SPET. The median over survival was 23.9 months in HNC patients with SPET, as compared with 27.0 months in HNC patients without SPET. The 3-years survival rates in HNC patients with SPET and in HNC patients without SPET were 50.9% and 72.0 %, respectively. Multivariate analysis identified the clinical stage of HNC (p = 0.01, hazard ratio 2.36) and the presence of SPET (p = 0.03, hazard ratio 1.9) as significant and independent determinants of overall survival. Conclusions: Our study suggested that although survival of HNC patients with SPET might be considerably improved due to early detection and treatment of SPET, the presence of SPET still remained an independent negative prognostic factor for HNC patients. Disclosure: All authors have declared no conflicts of interest.

In vivo persistence of adoptively transferred TCR gene-transduced lymphocytes with anti-tumor reactivity in patients with MAGE-A4 expressing esophageal cancer

The application of adoptive immunotherapy with tumor-specific T cells has been limited because of the short life span of the transferred T cells unless the host has been manipulated. Engineering the antigen receptor gene in patients’ lymphocytes is one promising strategy to create antigen-specific lymphocytes without senescent phenotypes. The strategy provides an opportunity to broaden the types of cancer to be treated. However, this concept has not been tested in the epithelial cancer patients. We completed a phase I clinical trial of TCR gene therapy targeting MAGE-A4 to treat esophageal cancer patients without lympho-depleting pre-conditioning. The trial was designed as a cell-dose escalation consisting of three cohorts, 2x10E8, 1x10E9 and 5x10E9 cells/patient. The treatment was tolerable with no adverse events associated with transferred cells. In all ten patients of the 3 cell-doses, the transferred lymphocytes were detected in their peripheral blood in a dose-dependent manner during the first 14 days. In 4 patients, the infused cells have been persisting more than 5 months after the transfer. The T cell clones were established from the transferred lymphocytes that were harvested more than 100 days after the transfer. These clones sustained the reactivity to the antigen-expressing tumor cells. Three patients showed SD or long tumor free status. These results suggest that this approach may extend the availability of adoptive T cell therapy for epithelial cancer patients by providing tumor-reactive and long surviving lymphocytes reducing the risk of intensive pre-treatments.

Abstract 2600: Effect of green tea intake on pharmacokinetics of oral etoposide in cancer patients

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Habitual consumption of green tea has been epidemiologically correlated with low risk of cancer. Among various constituents of green tea, catechins are experimentally suggested to have benefitial activity. Especially, (-)-epigallocatechin gallate (EGCG) is the most bioactive constituent. Decreased activation of carcinogenic compounds by inhibition CYP3A4 is proposed as one of mechanisms of anticarcinogenic effect. Green tea is one of the most popular beverages in Japan and Asian countries, and patients often take drugs with green tea beverages. Co-administration of oral cancer medicine which is metabolized by CYP3A4 with green tea may cause harmful interactions. PURPOSE: We investigated the effect of green tea beverage intake on pharmacokinetics of etoposide in an open label, single-dose, randomized, two-way crossover study. METHODS: Patients with various cancers were randomly assigned to receive an etoposide 50-mg capsule either with 600mL green tea beverage or 600mL of water, separated by at least 72-hours washout period. Blood samples were collected for 24 hours. Etoposide concentrations in plasma were determined by high performance liquid chromatography. Plasma pharmacokinetic parameters, including area under the curve (AUC), maximum concentration (Cmax) and elimination half-life (t1/2) were calculated by non-compartment analysis using WinNonlin software. RESULTS: Twenty-five cancer patients (11 males, 14 females; median (range) age, 63 years (46 to 81 years)) participated in this study. Co-administration with green tea resulted in an unexpected decrease by 24.8% in the AUC (p= 0.0003) of etoposide (Table). Carry-over effect was denied. CONCLUSION: Green tea reduces rather than increases exposure to orally administered etoposide. View this table: Mean pharmacokinetic parameters of etoposide Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2600.