FLASH Irradiation Results in Reduced Severe Skin Toxicity Compared to Conventional-Dose-Rate Irradiation

Radiation therapy, along with surgery and chemotherapy, is one of the main treatments for cancer. While radiotherapy is highly effective in the treatment of localized tumors, its main limitation is its toxicity to normal tissue. Previous preclinical studies have reported that ultra-high dose-rate (FLASH) irradiation results in reduced toxicity to normal tissues while controlling tumor growth to a similar extent relative to conventional-dose-rate (CONV) irradiation. To our knowledge this is the first report of a dose-response study in mice comparing the effect of FLASH irradiation vs. CONV irradiation on skin toxicity. We found that FLASH irradiation results in both a lower incidence and lower severity of skin ulceration than CONV irradiation 8 weeks after single-fraction hemithoracic irradiation at high doses (30 and 40 Gy). Survival was also higher after FLASH hemithoracic irradiation (median survival >180 days at doses of 30 and 40 Gy) compared to CONV irradiation (median survival 100 and 52 days at 30 and 40 Gy, respectively). No ulceration was observed at doses 20 Gy or below in either FLASH or CONV. These results suggest a shifting of the dose-response curve for radiation-induced skin ulceration to the right for FLASH, compared to CONV irradiation, suggesting the potential for an enhanced therapeutic index for radiation therapy of cancer

Interaction between Host Natural Killer T Cells and Donor CD4+CD25+ Treg Cells Protects Against GVHD after TLI/ATS Host Conditioning and Bone Marrow Transplantation

Abstract The murine non-myeloablative regimen of total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) was recently translated into a transplantation strategy for human hematolymphoid malignancies, preventing recipients from acute graft-versus-host disease (GVHD) without tumor relapses (Lowsky et al, NEJM, 2005). To investigate GVHD protection, we transplanted 50 x 106 bone marrow cells and 60 x 106 splenocytes (BMT) from wild-type, CD4−/−, IL-4−/, or IL-10−/− C57BL/6 (H-2b) donor mice into wild-type or natural killer (NK) T cell deficient Jα18−/− BALB/c (H-2d) hosts following non-myeloablative host conditioning with TLI/ATS. Control wild-type BALB/c hosts received a conventional regimen, either 800cGy (lethal) or 450 cGy (sublethal) total body irradiation (TBI) and ATS with BMT from wild-type C57BL/6 donors. TLI/ATS-conditioned wild-type hosts given BMT from wild-type donors survived 100 days without evidence of GVHD. TBI/ATS-conditioned wild-type hosts given BMT from wild-type donors and TLI/ATS conditioned wild-type hosts given BMT from CD4−/−, IL-4−/−, or IL-10−/− donors succumbed to GVHD, with marked donor CD8+ T cell accumulation demonstrated in liver, mesenteric lymph nodes (MLN), and colon at day 6. Given the dependence of GVHD protection on donor CD4+ cells and the donor cytokines IL-4 and IL-10, we investigated the role of donor CD4+CD25+Foxp3+regulatory T cells (CD4+Tregs) in GVHD protection after TLI/ATS and BMT. Wild-type TLI/ATS conditioned hosts given BMT from wild-type donors demonstrated a significant (p< 0.01) ten-fold increase in both the percentage and absolute number of donor CD4+CD25+Foxp3+Tregs present in the host spleen at day 6. These donor CD4+CD25+Foxp3+cells demonstrated typical IL-4 and IL-10 dominant cytokine secretion profiles on PMA/ionomycin stimulation and were functional both in vitro in donor-host MLR suppression and in vivo in secondary GVHD suppression assays. Using congenic markers (CD45.1/CD45.2), we demonstrate that these donor CD4+ Tregs are derived from splenic (peripheral) T cells within the donor cell inoculum. When wild-type donor bone marrow and spleen cells were rigorously CD25-depleted using magnetic bead selection prior to BMT into wild-type TLI/ATS treated hosts, donor CD4+CD25+Foxp3+ Tregs were not present in the host spleen at day 6, and all hosts demonstrated markedly increased donor CD8+ T cell accumulation in the liver, MLN, and colon at day 6 accompanied by GVHD after BMT. TLI/ATS-conditioned Jα18−/− hosts given BMT from wild-type donors demonstrated low numbers of donor CD4+CD25+Foxp3+ Tregs in host spleen and GVHD similar to TBI/ATS controls. When wild-type host NK T cells were adoptively transferred before BMT into TLI/ATS-conditioned Jα18−/− hosts, the use of non-CD25-depleted donor cells resulted in increased numbers of donor CD4+ Tregs and markedly reduced donor CD8+ T cell accumulation in the host liver, MLN, and colon at day 6, whereas CD25 depletion of donor cells resulted in loss of donor CD4+CD25+Foxp3+ Tregs in the host spleen and significantly increased donor CD8+ T cell accumulation. Our previous studies demonstrated that host invariant NK T cells are critical for GVHD protection after TLI/ATS host conditioning. The present studies show that host regulatory NK T cells can facilitate the expansion of donor CD4+CD25+Foxp3+ Tregs, and that this expansion plays a pivotal role in protecting TLI/ATS treated hosts from lethal GVHD after allogeneic BMT

Host NKT Cells Can Prevent Graft-versus-Host Disease and Permit Graft Antitumor Activity after Bone Marrow Transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL(1) B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d(−/−) and Jα-18(−/−) host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8(−/−) or perforin(−/−) donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jα-18(−/−) host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells

L-selectin and β7 integrin on donor CD4 T cells are required for the early migration to host mesenteric lymph nodes and acute colitis of graft-versus-host disease

The homing receptors L-selectin and α4β7 integrin facilitate entry of T cells into the gut-associated organized lymphoid tissues such as the mesenteric lymph nodes and Peyer patches. We studied the impact of inactivation of genes encoding these receptors on the ability of purified donor CD4+ T cells to induce acute lethal graft-versus-host disease (GVHD) associated with severe colitis in irradiated major histocompatibility complex (MHC)–mismatched mice. Whereas lack of expression of a single receptor had no significant impact on the severity of colitis and GVHD, the lack of expression of both receptors markedly ameliorated colitis and early deaths observed with wild-type (WT) T cells. The changes in colitis and GVHD were reflected in a marked reduction in the early accumulation of donor T cells in the mesenteric lymph nodes and subsequently in the colon. The purified WT donor CD4+ T cells did not accumulate early in the Peyer patches and failed to induce acute injury to the small intestine. In conclusion, the combination of CD62L and β7 integrin is required to induce acute colitis and facilitate entry of CD4+ donor T cells in the mesenteric nodes associated with lethal GVHD in allogeneic hosts

Density of CD3+ and CD8+ cells in gingivo-buccal oral squamous cell carcinoma is associated with lymph node metastases and survival.

The tumor immune microenvironment is emerging as a critical player in predicting cancer prognosis and response to therapies. However, the prognostic value of tumor-infiltrating immune cells in Gingivo-Buccal Oral Squamous Cell Carcinoma (GBOSCC) and their association with tumor size or lymph node metastases status require further elucidation. To study the relationship of tumor-infiltrating immune cells with tumor size (T stage) and lymph node metastases (N stages), we analyzed the density of tumor-infiltrating immune cells in archived, whole tumor resections from 94 patients. We characterized these sections by immune-histochemistry using 12 markers and enumerated tumor-infiltrating immune cells at the invasive margins (IM) and centers of tumors (CT). We observed that a higher density of CD3+ cells in the IM and CT was associated with smaller tumor size (T1-T2 stage). Fewer CD3+ cells was associated with larger tumor size (T3-T4 stage). High infiltration of CD3+and CD8+ cells in IM and CT as well as high CD4+ cell infiltrates in the IM was significantly associated with the absence of lymph node metastases. High infiltrates of CD3+ and CD8+ cells in CT was associated with significantly improved survival. Our results illustrate that the densities and spatial distribution of CD3+ and CD8+ cell infiltrates in primary GBOSCC tumors is predictive of disease progression and survival. Based on our findings, we recommend incorporating immune cell quantification in the TNM classification and routine histopathology reporting of GBOSCC. Immune cell quantification in CT and IM may help predict the efficacy of future therapies

Abstract 2466: Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy

International audienceThe mainstay of HER2+ breast cancer treatment has been monoclonal antibody therapy with Trastuzumab, a humanized antibody that targets HER-2. However, the efficacy of Trastuzumab monotherapy is only 10-15%. Therefore, strategies are being developed to enhance its therapeutic efficacy. Our previous study demonstrated that stimulation of NK cells with an anti-CD137 agonistic mAb enhanced Trastuzumab-mediated Antibody Dependent Cellular Cytotoxicity (ADCC). Anti-CD137 agonistic mAb enhanced anti-breast cancer activity of Trastuzumab in vivo in a xenotransplanted human breast cancer model (Kohrt et al. J Clin. Invest, 2012, 22:3). We have developed a treatment regimen consisting of sequential administration of Trastuzumab followed by CD137 antibody as three weekly injections. This regimen demonstrated more potent antitumor activity than administration of anti-CD137 mAb followed by Trastuzumab. Our combination therapy was superior to Traztuzumab alone in a primary HER2-overexpressing-breast tumor xenotransplant model but was ineffective in low HER-2 expressing breast cancer model. Next, we evaluated the therapeutic efficacy of FDA approved antibody drug conjugate Trastuzumab Emtansine (T-DM1) in combination with CD137 antibody. We used HER2 over-expressing HER18 xenograft model and an SU-258 primary breast cancer model in our studies. Following tumor inoculation, mice received either T-DM1 on day 3 (for HER18 xenograft), or day 30 (for SU-258) and anti-CD137 antibody on day 4(or HER18) or day 31(for SU-258) with each treatment repeated weekly for a total of three weeks. Sequential antibody strategy with T-DM1 and CD137 significantly improved survival compared to T-DM1 alone or with Trastuzumab and CD137 antibody. Our results demonstrate that T-DM1 retains its potency and demonstrates synergistic activity with anti-CD137 mAb therapy against HER2-overexpressing breast cancer models. Our results support a novel, sequential antibody approach against HER-2+breast cancer, by targeting first the tumor with an antibody drug conjugate and then the host immune system. Clinical investigations are now planned to determine the clinical outcome of our immunotherapy strategy. Citation Format: Suparna Dutt, Narendiran Rajasekaran, Aurelien Marabelle, Roch Houot, Mohith Sadaram, Jonathan Hebb, Idit Sagiv-Barfi, Sid Ambulkar, Amanda Rajapaksa, Cariad Chester, Erin Waller, Holbrook Kohrt. Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2015-246