Jarrod D. Predina

Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T cells Expressing a Mesothelin-Specific Chimeric Antibody Receptor

Purpose: Adoptive T-cell immunotherapy with tumor infiltrating lymphocytes or genetically-modified T cells has yielded dramatic results in some cancers. However, T cells need to traffic properly into tumors to adequately exert therapeutic effects. Experimental Design: The chemokine CCL2 was highly secreted by malignant pleural mesotheliomas (MPM; a planned tumor target), but the corresponding chemokine receptor (CCR2) was minimally expressed on activated human T cells transduced with a chimeric antibody receptor (CAR) directed to the MPM tumor antigen mesothelin (mesoCAR T cells). The chemokine receptor CCR2b was thus transduced into mesoCAR T cells using a lentiviral vector, and the modified T cells were used to treat established mesothelin-expressing tumors. Results: CCR2b transduction led to CCL2-induced calcium flux and increased transmigration, as well as augmentation of in vitro T-cell killing ability. A single intravenous injection of 20 million mesoCAR + CCR2b T cells into immunodeficient mice bearing large, established tumors (without any adjunct therapy) resulted in a 12.5-fold increase in T-cell tumor infiltration by day 5 compared with mesoCAR T cells. This was associated with significantly increased antitumor activity. Conclusions: CAR T cells bearing a functional chemokine receptor can overcome the inadequate tumor localization that limits conventional CAR targeting strategies and can significantly improve antitumor efficacy in vivo. Clin Cancer Res; 17(14); 4719–30. ©2011 AACR.

Inhibition of p300 impairs Foxp3 + T regulatory cell function and promotes antitumor immunity

Forkhead box P3 (Foxp3)+ T regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3+ Treg cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3+ Treg cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3+ Treg cells increased T cell receptor–induced apoptosis in Treg cells, impaired Treg cell suppressive function and peripheral Treg cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3+ Treg cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish Treg cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery

Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b+F4/80hiCD206hi and CD11b+F4/80hiCD124hi) macrophages and CD4+Foxp3+ regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.

Local and Systemic Recurrence is the Achilles Heel of Cancer Surgery

This year approximately 569,490 Americans, more than 1,500 people per day, are expected to die of cancer. Cancer has supplanted heart disease as the leading cause of death in the United States in men and women younger than age 85 years (American Cancer Society, Facts and Figures 2010). Cancer also has a major economic impact on the U.S. economy, with 2010 estimates of cancer associated costs of

Improved Survival after Pulmonary Metastasectomy for Soft Tissue Sarcoma

263.8 billion (American Cancer Society, Facts and Figures 2010). The four most common cancers (lung, colorectal, prostate, and breast cancer) account for more than half of all cancer incidences in the United States. Surgery, chemotherapy, and radiotherapy are the most established therapies for patients with these malignancies (American Cancer Society, Facts and Figures 2010). Surgery continues to be the most effective therapy for cancer in the United States. It is several-fold more effective than chemotherapy and radiation in curing patients with cancer. Unfortunately, many people are not surgical candidates due to advanced stage disease or comorbid conditions. During the past two decades, both mortality and observed cancer survival statistics have improved dramatically both in patients who do and do not undergo surgery. This likely reflects an improvement in cancer care in the United States. Since 1973, the Survival Epidemiology and End Results (SEER) Program sponsored by the National Cancer Institute has kept a publicly accessible database that has catalogued the outcome of cancer patients (Surveillance, Epidemiology, and End Results Program. Bethesda, SEER Stat Database). These data include information regarding incidence and mortality, as well as the initial surgical and adjuvant therapy provided for the treatment of cancer. This improvement in survival and mortality in this database during the past 20 years is likely due to better screening and detection, biologically targeted systemic therapies, improved surveillance, improved surgical methods, improved patient selection, and more effective adjuvant therapies. Cancer surveillance also has improved dramatically with the improvement in preoperative imaging and minimally invasive and noninvasive staging techniques, such as mammography, colonoscopy, endobronchial ultrasound, and prostate-specific antigen testing. The goal of this short discussion is to highlight some of the trends that have been observed in cancer and surgery during the past 20 years and the role that surgery continues to play in the oncologic community. We found that surgery remains an effective therapy for solid tumors in the United States and dramatically improves survival rates for patients with solid tumors. The proportion of cancer patients who undergo surgery has declined during the past 20 years for all major cancer types. Additionally, we found that local and systemic recurrences continue to be the Achilles heel of Surgical Oncology; although surgery does improve survival, almost one-third of surgical patients will ultimately recur locally and/or systemically. This is an important reminder to surgeons that we must work diligently to improve cancer care for our patients preoperatively, intraoperatively, and postoperatively to prevent these recurrences. Research and clinical trials should be focused on this major objective in the coming decades. Louis A. Aliperti and Jarrod D. Predina contributed equally to this work.

Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness.

Introduction:Survival after pulmonary metastasectomy for soft tissue sarcoma (STS) has been lower than in osteosarcoma (14–40% versus 40–50%). With improved patient selection criteria and advanced chemotherapy agents, we hypothesized that survival after metastasectomy for STS has improved in recent years. Methods:Retrospective study of 48 patients undergoing pulmonary metastasectomy for STS between 1995 and 2007. Potential predictors of overall survival and disease-free survival (DFS) were examined using the log-rank test or Cox regression. Multivariate analysis was conducted using Cox regression. Results:Overall survival after initial metastasectomy was 67% and 52% at 3 and 5 years, respectively; DFS was 17% and 10% at 3 and 5 years. Univariate analysis indicated that ≤2 pulmonary metastases (p = 0.03), diameter of largest metastasis ≤2 cm (p = 0.09), and the absence of extrapulmonary metastases (p = 0.10) were associated with longer overall survival. Absence of extrapulmonary metastases (p = 0.07) and smaller size of the largest pulmonary metastasis (p = 0.06) were associated with longer DFS. Before 2001, 46.7% of patients received adjuvant chemotherapy versus 72.7% after (p = 0.10). Neither use of chemotherapy nor chemotherapy type was related to overall survival or DFS. Conclusion:Five-year overall survival is substantially higher after pulmonary metastasectomy for STS in our study relative to previously published results (52% versus 14–40%). This improvement does not seem to be the result of greater use of, or newer, chemotherapeutic regimens. Among potential explanations, improved patient selection is the most likely factor.

Comparison of Folate Receptor Targeted Optical Contrast Agents for Intraoperative Molecular Imaging

BackgroundMultiple immunotherapy approaches have improved adaptive anti-tumor immune responses in patients with early stage disease; however, results have been less dramatic when treating patients with late stage disease. These blunted responses are likely due to a host of factors, including changes in the tumor microenvironment and systemic immunosuppressive features, which accompany advanced tumor states. We hypothesized that cytoreductive surgery could control these immunosuppressive networks and restore the potency of immunotherapy in advanced disease scenarios.MethodsTo test these hypotheses, two representative intratumoral immunotherapies (an adenoviral vector encoding a suicide gene, AdV-tk, or a type-I interferon, Ad.IFNα) were tested in murine models of lung cancer. Cytoreductive surgery was performed following treatment of advanced tumors. Mechanistic underpinnings were investigated using flow cytometry, in vivo leukocyte depletion methods and in vivo tumor neutralization assays.ResultsAdV-tk and Ad.IFNα were effective in treating early lung cancers, but had little anti-tumor effects in late stage cancers. Interestingly, in late stage scenarios, surgical cytoreduction unmasked the anti-tumor potency of both immunotherapeutic approaches. Immune mechanisms that explained restoration in anti-tumor immune responses included increased CD8 T-cell trafficking and reduced myeloid derived suppressor cell populations.ConclusionThis study demonstrates that surgical resection combined with immunotherapy may be a rational therapeutic option for patients with advanced stage cancer.

Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models

Background. Intraoperative imaging can identify cancer cells in order to improve resection; thus fluorescent contrast agents have emerged. Our objective was to do a preclinical comparison of two fluorescent dyes, EC17 and OTL38, which both target folate receptor but have different fluorochromes. Materials. HeLa and KB cells lines were used for in vitro and in vivo comparisons of EC17 and OTL38 brightness, sensitivity, pharmacokinetics, and biodistribution. In vivo experiments were then performed in mice. Results. The peak excitation and emission wavelengths of EC17 and OTL38 were 470/520 nm and 774/794 nm, respectively. In vitro, OTL38 required increased incubation time compared to EC17 for maximum fluorescence; however, peak signal-to-background ratio (SBR) was 1.4-fold higher compared to EC17 within 60 minutes (p < 0.001). Additionally, the SBR for detecting smaller quantity of cells was improved with OTL38. In vivo, the mean improvement in SBR of tumors visualized using OTL38 compared to EC17 was 3.3 fold (range 1.48–5.43). Neither dye caused noticeable toxicity in animal studies. Conclusions. In preclinical testing, OTL38 appears to have superior sensitivity and brightness compared to EC17. This coincides with the accepted belief that near infrared (NIR) dyes tend to have less autofluorescence and scattering issues than visible wavelength fluorochromes.

A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRASG12V and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma.

The virtual-patient pilot: testing a new tool for undergraduate surgical education and assessment.

Up to 30% of cancer patients undergoing curative surgery develop local recurrences due to positive margins. Patients typically receive adjuvant chemotherapy, immunotherapy and/or radiation to prevent such relapses. Interestingly, evidence supporting these therapies is traditionally derived in animal models of primary tumors, thus failing to consider surgically induced tumor microenvironment changes that may influence adjuvant therapy efficacy. To address this consideration, we characterized a murine model of local cancer recurrence. This model was reproducible and generated a postoperative inflammatory tumor microenvironment that resembles those observed following human cancer surgery. To further validate this model, antagonists of two pro-inflammatory mediators, TGFβ and COX-2, were tested and found to be effective in decreasing the growth of recurrent tumors. We appreciated that preoperative TGFβ inhibition led to wound dehiscence, while postoperative initiation of COX-2 inhibition resulted in a loss of efficacy. In summary, although not an exact replica of all human cancer surgeries, our proposed local recurrence approach provides a biologically relevant and reliable model useful for preclinical evaluation of novel adjuvant therapies. The use of this model yields results that may be overlooked using traditional preclinical cancer models that fail to incorporate a surgical component.