Edmond Marzbani

The Invisible Arm of Immunity in Common Cancer Chemoprevention Agents

Immunoprevention refers to a strategy of preventing pathogen-associated and spontaneous cancers through the use of vaccines, antibodies, and immune modulators. Immune modulators function by enhancing the endogenous ability of the immune system to monitor for malignancy, so-called “immunosurveillance.” There is growing evidence that many of the most promising cancer chemoprevention agents including aspirin, COX-2 inhibitors, aromatase inhibitors, and bisphosphonates mediate their effects, in part, by enhancing immunosurveillance and reversing the immune evasive mechanisms that premalignant lesions use. In the following review, we introduce critical components of the human immune surveillance system—dendritic cells, T cells, and immune suppressive cells—and discuss the emerging data suggesting that common chemoprevention agents may modulate the function of these immunologic cells. Cancer Prev Res; 6(8); 764–73. ©2013 AACR.

Abstract P5-04-06: Oral immunomodulatory agents prevent tumor growth and increase tumor CD8 T cell infiltrate; bexarotene further improves tumor response to conventional chemotherapy in breast tumors

The tumor immune environment is important in breast cancer with greater than 50% immune infiltrate (LPBC) prior to neoadjuvant chemotherapy predicting improved pathologic complete response, and LPBC and CD8 infiltrate prior to adjuvant therapy predicting improved survival. Unfortunately, the majority of breast cancers do not have LPBC or robust CD8+ infiltrate. Evidence has emerged that conventional chemotherapy can increase CD8+ T cells therefore discovering ways to boost this response should further enhance the anti-tumor function. Three oral agents have modest anti-tumor function: metformin (oral biguanide), bexarotene (retinoic receptor agonist), and celecoxib (COX2 inhibitor). In vitro data suggest a role for these agents in increasing Th1 immunity: metformin was shown to increase MHC class I expression on tumor cells, bexarotene was shown to decrease CD8+ T cell apoptosis, and celecoxib has been shown to decrease MDSC cells. The goal of this study was to demonstrate whether addition of these oral agents to conventional chemotherapy enhanced the anti-tumor function of chemotherapy, possibly by modifying the immune environment in the transgenic mouse mammary tumor model TgMMTV-neu (genetically similar to luminal breast cancer). Two active chemotherapies in human breast cancer, doxorubicin and paclitaxel, inhibited tumor growth and increased CD8+ T cell tumor infiltrate in transgenic mice. Treatment of mice with 100 mm3 tumors with doxorubicin (5 mg/kg weekly for four weeks) showed a 32% increase in CD8+ T cells and 85% decrease in tumor growth as compared to control mice (p=0.0001) and treatment with paclitaxel (10 mg/kg weekly for four weeks) showed a 40% increase in CD8+ tumor infiltrate (p=0.0068) and 60% decrease in tumor growth as compared to control treated mice (p=0.0026). A third chemotherapy cyclophosphamide (100 mg/kg weekly for four weeks) increased CD8+ tumor infiltrate by 45% (p=0.011) but did not show a significant decrease in tumor volume (p=0.57). Metformin and bexarotene also demonstrated increased CD8+ tumor infiltration and decreased breast tumor growth but celecoxib did not. Metformin treated mice had a 46% increase in CD8+ tumor infiltrate (p=0.001) and a 52% decrease in mean tumor volume (p=0.011) as compared to controls (75 mg/m2 of metformin for four weeks). Bexarotene treated mice had 44% increase in CD8+ tumor infiltrate (p=0.05) and 60% decrease in mean tumor growth (p=0.03) compared to control (treated with 50 mg/m2 bexarotene for four weeks). Of all three oral therapies, bexarotene was most effective inhibiting spontaneous tumor growth in the mice. Furthermore, addition of bexarotene to chemotherapy was superior to chemotherapy alone. Adding bexarotene to weekly doxorubicin decreased tumor growth by 98% (p=0.008 compared to doxorubicin alone), adding bexarotene to weekly paclitaxel decreased tumor growth by 86% (p=0.02 compared to paclitaxel alone), and adding bexarotene to weekly cyclophosphamide inhibited tumor growth by 82% (p=0.04 compared to cyclophosphamide alone). These results suggest that addition of bexarotene, a well-tolerated oral agent, to chemotherapy may improve tumor response in breast cancer. Citation Format: Sasha E Stanton, Ekram Gad, Edmond Marzbani, Lauren Rastetter, Mary L Disis. Oral immunomodulatory agents prevent tumor growth and increase tumor CD8 T cell infiltrate; bexarotene further improves tumor response to conventional chemotherapy in breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-06.

Abstract 279: Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu

In breast cancer, increased immune infiltrate prior to neoadjuvant chemotherapy predicts improved pathologic complete response (pCR) and improved survival, however almost half of breast tumors have no CD8+ T cell infiltrate. Evidence has emerged that conventional breast cancer chemotherapy can increase CD8+ T cells (doxorubicin) and decrease the immunosuppressive regulatory CD4+ T cells (paclitaxel and cyclophosphamide); therefore discovering well tolerated agents that further enhance the anti-tumor immune function of these chemotherapies may further improve response in breast cancer patients. The oral agent bexarotene (a retinoic receptor agonist) showed a 20% disease response as a single agent in metastatic breast cancer and has been shown to increase CD8+ T cell tumor infiltration and decrease CD8+ T cell apoptosis in vitro. The goal of this study is to demonstrate whether this well tolerated oral agent can enhance the anti-tumor response of conventional chemotherapy through increasing intratumoral CD8+ T cells in TgMMTV-neu transgenic mice that are immunologically competent and genetically similar to human luminal B breast cancer. Two of the most active chemotherapies in human breast cancer, doxorubicin and paclitaxel, each could inhibit tumor growth by 70-80% and increase CD8+ T cell tumor infiltrate by approximately 40% in tumor bearing TgMMTV-neu mice, a third breast cancer chemotherapy cyclophosphamide increased CD8+ tumor infiltrate by 45% (p = 0.011) but did not show a statistically significant decrease in tumor volume. When TgMMTV-neu mice with ∼100 mm3 tumors were treated with 50 mg/m2 bexarotene for four weeks they demonstrated a 44% increase in CD8+ tumor infiltrate (p = 0.05) and 60% decrease in mean tumor growth (p = 0.03) compared to control. However, the addition of bexarotene to chemotherapy was superior to chemotherapy alone, and significantly more effective than bexarotene alone. This enhanced anti-tumor function was even seen with cyclophosphamide that by itself had not inhibited tumor growth. Adding bexarotene to weekly doxorubicin decreased tumor growth by 98% (p = 0.008 compared to doxorubicin alone), adding bexarotene to weekly paclitaxel decreased tumor growth by 86% (p = 0.02 compared to paclitaxel alone), and adding bexarotene to weekly cyclophosphamide inhibited tumor growth by 82% (p = 0.04 compared to cyclophosphamide alone). Further studies are now ongoing to identify the anti-tumor role of bexarotene particularly its immune modulatory role. These results suggest that the addition of bexarotene, a relatively well-tolerated oral agent, may modify the immune environment and improve tumor response to chemotherapy in breast cancer possibly improving response to neoadjuvant chemotherapy. Citation Format: Sasha E. Stanton, Ekram Gadd, Edmond Marzbani, Lauren Rastetter, Mary L. Disis. Bexarotene increases tumor CD8+ T cells and improves response to conventional breast chemotherapy in the transgenic mouse mammary tumor model TgMMTV-neu. [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 279. doi:10.1158/1538-7445.AM2015-279

Abstract 3135: HER2 specific adoptive T cells shown to localize and infiltrate all sites of disease using combined SPECT and PET imaging

Adoptive T cell therapy has been shown to stimulate anti-tumor response in multiple cancers, however these responses have not been robust or durable. In previous studies, indium-111 labeled T cells have functioned normally but were either not present at all sites of metastatic disease or not able to infiltrate the sites of disease. In breast cancer, adoptive HER2 targeted T cells were unable to penetrate visceral metastases. Previous work in our laboratory has demonstrated that HER2 vaccine-primed autologous adoptive T cells were safe and well tolerated. In this phase I study, HER2 positive breast cancer patients received three HER2 peptide vaccines before plasmapheresis and ex-vivo expansion of HER2 specific autologous T cells to evaluate the immune and clinical response to adoptive T cell therapy in breast cancer. In one patient, the trafficking of indium-111 labeled T cells was also evaluated using SPECT and PET imaging. An aliquot of 1×107 expanded T cells were labeled with 300 uCi of indium-111 and given with the third T cell infusion. Prior in vitro studies had demonstrated that labeled HER2 expanded T had similar viability (at 24 hours 97±1% viability with unlabeled cells and 90.9±1.1% viability with 480 uCI labeled cells) and interferon gamma release (263±8 pg/mL released in unlabeled cells and 208.5±11 released in 480 uCi labeled cells) as unlabeled HER2 expanded T cells when stimulated by IL2. SPECT imaging demonstrated that the T cells trafficked to all the metastatic sites of disease by 24 hours and completely infiltrated the tumor. The patient studied had metastases to her skull, left axilla, sternum, bilateral proximal humeri, and sacrum. The corrected indium-111 uptake at 24 hours varied from 2.27 counts per pixel in the R proximal humerus to 6.28 counts per pixel in the R sacrum and remained elevated at 48 hours (for example a continued 6.9 counts/pixel signal at the R sacrum). Concurrent PET CT imaging demonstrated FDG flare at 48 hours at all sites of metastatic disease including a 1.3 fold increase in the R proximal humerus and a 1.3 fold increase in the L proximal humerus signal over baseline scans. This increased FDG uptake had resolved 1 month after therapy. After this study, the patient had stable disease for 18 months. She had a robust response to each T cell infusion and the booster vaccines, including fevers, headaches, and increased pain at the sites of metastatic disease. These symptoms have been associated with a disease specific T cell response. This study demonstrates by a novel method of concurrent SPECT and PET imaging that the ex-vivo expanded HER2 specific T cells were able to traffic to and fully infiltrate all sites of metastatic disease causing an acute FDG-PET flare and prolonged stable disease in a HER2 positive breast cancer patient with bone-only metastatic disease. Further studies are now needed to confirm if this imaging method can be used universally in adoptive T cell studies. Citation Format: Sasha E. Stanton, Janet Eary, Edmond Marzbani, David Mankoff, Lupe Salazar, Doreen Higgins, Jessica Reichow, Yushe Dang, Mary L. Disis. HER2 specific adoptive T cells shown to localize and infiltrate all sites of disease using combined SPECT and PET imaging. [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 3135. doi:10.1158/1538-7445.AM2015-3135

Abstract 2742: Identification of tumor antigens in neu transgenic mice may provide biomarkers useful for the early detection and diagnosis of human breast cancer

Early detection of breast cancer is crucial for better prognosis and successful treatment of the patients. Development of novel methods for screening for breast cancer is needed for early detection and diagnosis. The human immune system responds to tumor-specific antigens in the pre-malignant stage of breast cancer and produces specific antibodies, which can be detected as potential breast cancer biomarkers. Due to the limited availability of well-annotated pre-cancer and post-cancer sera from human subjects, we performed serial serum collection in a neu transgenic mouse model from the age of 10 weeks to the terminal stage of disease. Using a technique called serological screening of cDNA expression library (SEREX), a high-throughput method to rapidly screen recombinant proteins expressed in a bacteriophage-based cDNA library, we have previously identified tumor antigens in the late stage of neu breast cancers. The mouse tumors have similar immunogenicity as their human counterparts due to the fact that some of these antigens are also immunogenic in human. Indeed, we have found that one mouse antigen called gelsolin, shares 93% homology with human gelsolin and has the highest response to human serum antibody among other antigens. In an ELISA analysis using the serum samples from 50 breast cancer cases and 50 normal donors, the area-under-curve (AUC) value of the receiver-operating-characteristic (ROC) curve of gelsolin ELISA detects more than 70% of cancer patients. Using the SEREX technique, we sought to further identify the early-stage serum antibody biomarkers by comparing the pre-cancer, early stage, and late stage sera from the same mouse. We have identified three early-stage biomarkers, A20/TNFaip3, Rpl5, and Pdhx, in neu mice. In summary, we conclude that identification of tumor antigens in the neu transgenic animal model is useful for the discovery of serum antibody biomarkers for early detection and diagnosis. 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 2742.