Jun Koshio

Clinical responses to EGFR-tyrosine kinase inhibitor retreatment in non-small cell lung cancer patients who benefited from prior effective gefitinib therapy: a retrospective analysis

BackgroundGefitinib was the first epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of advanced non-small cell lung cancer (NSCLC). Few treatment options are available for NSCLC patients who have responded to gefitinib treatment and demonstrated tumor progression. The present study was conducted to evaluate the efficacy and toxicity of the 2nd EGFR-TKI administration.MethodsWe retrospectively analyzed 11 patients who had obtained a partial response (PR) or stable disease (SD) with gefitinib treatment and were re-treated with EGFR-TKI after failure of the initial gefitinib treatment.ResultsThree patients (27%) were treated with gefitinib as the 2nd EGFR-TKI, and 8 patients (73%) received erlotinib. Only one patient (9%) showed PR, 7 (64%) achieved SD, and 3 (27%) had progressive disease. The disease control rate was 73% (95% CI, 43% - 91%) and the median progression-free survival was 3.4 months (95% CI, 2 - 5.2). The median overall survival from the beginning of the 2nd EGFR-TKI and from diagnosis were 7.3 months (95% CI, 2.7 - 13) and 36.7 months (95% CI, 23.6 - 43.9), respectively. No statistical differences in PFS or OS were observed between gefitinib and erlotinib as the 2nd EGFR-TKI (PFS, P = 0.23 and OS, P = 0.052). The toxicities associated with the 2nd EGFR-TKI were generally acceptable and comparable to those observed for the initial gefitinib therapy.ConclusionsOur results indicate that a 2nd EGFR-TKI treatment can be an effective treatment option for gefitinib responders.

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Abstract Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133+ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133+ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4+ T cells produced CD133+ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.

Vaccination with CD133+ melanoma induces specific Th17 and Th1 cell–mediated antitumor reactivity against parental tumor

Accumulating evidence suggests that cancer cells possess a small subpopulation that survives during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular-targeting therapy. CD133 is a putative marker that distinguishes a minor subpopulation from normal differentiated tumor cells in many cancers. Although it is necessary to eradicate all cancer cells to obtain a cure, effective treatment to eliminate the CD133+ treatment–tolerant cells has not been elucidated. In this study, we demonstrated that a CD133+ subpopulation in murine melanoma is immunogenic and that effector T cells specific for the CD133+ melanoma cells mediated potent antitumor reactivity, curing the mice of the parental melanoma. CD133+ melanoma antigens preferentially induced type 17 T helper (Th17) cells and Th1 cells but not Th2 cells. CD133+ melanoma cell–specific CD4+ T-cell treatment eradicated not only CD133+ tumor cells but also CD133− tumor cells while inducing long-lasting accumulation of lymphocytes and dendritic cells with upregulated MHC class II in tumor tissues. Further, the treatment prevented regulatory T-cell induction. These results indicate that T-cell immunotherapy is a promising treatment option to eradicate CD133+ drug-tolerant cells to obtain a cure for cancer.

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4+CD25+ Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

Recurrent Interstitial Lung Disease Induced By Various Therapies forNon-Small Cell Lung Cancer

Erlotinib is a human epidermal growth factor receptor type 1 tyrosine kinase inhibitor which is used for non-small cell lung cancer treatment. Interstitial lung disease has been reported as an adverse event of erlotinib. We report the case of a 39-year-old man with erlotinib-induced interstitial lung disease in a non-small cell lung cancer patient. Although interstitial lung disease had improved by steroid therapy, palliative radiotherapy recalled the pneumonitis beyond the radiation fields. After the pneumonitis was well controlled, the patient was started on irinotecan, but the interstitial lung disease recurred shortly thereafter. We may have to abandon further cytotoxic therapies to avoid the recurrence of interstitial lung disease in patients who develop erlotinib-induced interstitial lung disease once.

Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity

MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies.

Abstract 1550: Successful treatment of advanced tumors with chemo-immunotherapy: a combination of cyclophosphamide and inhibition of chemo-resistant immune suppressor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is well known that naive T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have been shown that transfer of naive T cells following sublethal whole body irradiation augmented antitumor immunity and inhibited tumor progression. Further analyses showed that antitumor effector T cells were primed from transferred naive T cells. Although the exact mechanism underlying this enhancement of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be a promising strategy. Although whole body irradiation induces lymphopenia to tumor-bearing animals and enhances antitumor immunity, it has not been routinely used in clinical settings. To test whether cytotoxic agents deplete lymphocytes and enhance antitumor immune responses, we infused cyclophosphamide (CPA), fludarabine, cisplatin and etoposide at the sublethal doses to mice. CPA treatment depleted 95% of lymphocytes in mice, and enhanced antitumor effects of transferred naive T cells. Other cytotoxic drugs failed to augment antitumor immunity in combination with transfer of T cells. Previously, we have demonstrated that CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) inhibited priming of antitumor effector T cells. Depletion of these suppressor cells increased the number of tumor-specific T cells and augmented antitumor immune responses. To examine the effect of sublethal doses of cyclophosphamide on immune suppressor cells, we injected cyclophosphamide into mice and harvested lymph-nodes and spleens for FACS analyses. Unexpectedly, we found a significant increase in the frequency of Treg and MDSC after CPA treatment. Magnetically isolated Treg and MDSC from cyclophosphamide treated mice suppressed tumor-specific responses of effector T cells in vitro. Depletion of Treg with anti-CD25 monoclonal antibodies following CPA administration and transfer of naive T cells increased the number of antitumor effector T cells. Further, the combination of Treg depletion, CPA treatment and transfer of naive T cells succeeded to cure 20-day established skin tumors in mice. We have also been testing whether inhibition of MDSC after CPA treatment augments antitumor immunity. Our results showed that CPA treatment efficiently depletes lymphocytes and enhanced antitumor immune responses despite an increase of immune suppressor cells. Further inhibition of suppressor cells after CPA administration successfully treated advanced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1550. doi:1538-7445.AM2012-1550

Abstract 2685: Irradiation and reconstitution with in vitro-expanded polyclonal T cells from naïve mice augments antitumor response

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Transfer of naive T cells into lymphopenic tumor-bearing hosts induces antitumor effector T cells effectively and inhibits tumor progression. Although the exact mechanism underlying this augmentation of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Thus, the combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be an attractive approach. Because of difficulty to acquire enough number of naive T cells from cancer patients, it is necessary to establish a method for obtaining the large number of naive T cells. The current study seeks to determine whether lymphodepletion and transfer of in vitro expanded T cells enhances antitumor immunity. We stimulated CD4+ and CD8+ T cells from naive C57/BL6 mice with homeostatic cytokines. At day9 of culture, their number increased 4 to 10-fold. FACS analysis demonstrated TCR diversity of stimulated T cells. Resulted cells were injected into mice harboring MCA205 fibrosarcoma after sublethal irradiation (500 rad). As well as transfer of fresh naive T cells, transfer of stimulated T cells strongly inhibited tumor-progression. To confirm whether in vitro stimulated T cells become antitumor effector T cells and retarded tumor growth, GFP+ T cells were expanded in vitro and transferred into irradiated wild mice. On the same day, mice were inoculated s.c. with tumor cells. Twelve days later, tumor-draining LNs were harvested and cells were stimulated with the anti-CD3/IL-2 method. Activated tumor-draining LN cells were analyzed for secretion of IFN-γ after specific tumor stimulation. Intracellular IFN-γ staining revealed that the majority of effector T cells were GFP+ donor cells. We next examined whether dendritic cell vaccination enhances antitumor immune responses of in vitro stimulated T cells. Bone marrow dendritic cells were co-cultured with irradiated tumor cells and were injected into mice reconstituted with in vitro stimulated T cells. Compared with transfer of T cells alone, the combination of vaccination and transfer of T cells significantly delayed tumor progression. We report in this study that even after nonspecific stimulation, naive T cells retain their ability to respond to tumor-antigens when they are transferred into lymphopenic hosts. Irradiation and reconstitution with in vitro stimulated T cells can be useful conditioning for cancer immunotherapy, especially for vaccination with tumor antigens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2685. doi:10.1158/1538-7445.AM2011-2685

Abstract 2694: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked is a CD133+ tumor-specific protein and induces antitumor immunity

Cancer cells that exclusively maintain the ability of self-renewal and differentiation are termed cancer stem cells (CSCs). It is still controversial if the classical CSC hierarchy exists in all of solid tumors, however, accumulating evidence suggests that heterogeneity within cancer cells exists and that cancer survives as the cells with CSC features during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular targeting therapy. Although it is necessary to eradicate CSCs to obtain cure of cancer, effective treatment has not been elucidated. Interestingly, most of the identified immunogenic tumor antigens are cancer/testis (CT) antigens. One of the reasons why CT antigens are immunogenic is that they are unlikely to maintain peripheral tolerance, owing to restricted expression in the testis and in immortal malignant cells, because Tregs are maintained with antigen stimulation by dendritic cells (DCs) that acquire dying cells in the steady state. Since CSCs are highly immortal, it is possible that they possess immunogenic antigens that are not expressed in differentiated cancer cells or normal epithelial cells, and that these antigens may be ideal therapeutic targets for cancer treatment. Recently it was reported that CT antigens are mainly expressed in CSCs. We isolated CD133+ tumor cells, which possessed CSC properties, from B16 melanoma cells. We found that CSC-specific LN T cells primed by the CD133+ tumor vaccine mediated potent antitumor therapeutic efficacy by eradicating CSCs in tumor, thereby curing parental melanomas that comprised Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2011-2694

Abstract 1920: Chemo-resistant regulatory T cells inhibit the augmentation of antitumor immunity during homeostatic T cell proliferation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It has been demonstrated that lymphodepletion by cytotoxic regimens, such as chemotherapy and radiotherapy, augments antitumor immune response. Although the mechanism underlying this stimulation of antitumor immunity remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. We previously reported that CD4+CD25+Foxp3+ regulatory T cells were increasing in sublethally (500 rad) irradiated mice. Those radio-resistant regulatory T cells inhibited the development of antitumor immunity during recovery from lymphopenia. Depletion of regulatory T cells after irradiation enhanced generation of antitumor effector T cells and significantly suppressed tumor progression. We hypothesized that chemotherapy-resistant, as well as radio-resistant, regulatory T cells inhibited antitumor immune response. In this study, C57/BL6 mice were administered 200 or 400 mg/kg of cyclophosphamide to deplete lymphocytes. On the same day, naive spleen T cells were transferred intravenously followed by subcutaneous injection of MCA205 tumor cells. We found that the percentage of regulatory T cells increased from 4-5% in normal mice to 16% in lymphopenic mice injected with cyclophosphamide. Transfer of naive T cells into cyclophosphamide injected lymphopenic mice showed some antitumor effects, and further elimination of regulatory T cells by anti-CD25 antibodies (PC61) following administration of cyclophosphamide significantly inhibited skin-tumor growth. It is well known that transforming growth factor-beta (TGF-beta) is critical differentiation cytokine for regulatory T cells. Thus, we examined the effects of anti-TGF-beta antibodies (1D11) on regulatory T cells during recovery from lymphopenia. Injection of anti-TGF-beta antibodies after lymphodepletion stimulated antitumor immune responses and delayed tumor progression. The results indicate that regulatory T cells are resistant to cytotoxic regimens. The combination of cytotoxic therapy and inhibition of regulatory T cells efficiently increases antitumor immune response. 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 1920.