Christos Colovos

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity

Regional CAR T cell delivery provides superior protection compared to systemic administration. An Amazon Model for CAR T Cell Delivery The success of chimeric antigen receptor (CAR) T cells in treating otherwise untreatable hematological malignancies has been a flag-bearer for cancer immunotherapy. Yet, moving these therapies to solid tumors has been restricted by T cell infiltration and persistence. Now, Adusumilli et al. take a page from online sellers—setting up regional distribution centers to improve delivery. They found that mesothelin-targeted CAR T cells administered directly to the lung outpaced those administered systemically by both efficacy and persistence in an orthotopic model of lung cancer. What’s more, these effects weren’t restricted to lung, as tumors in extrathoracic sites were also eliminated. If these results hold in patients, regional delivery could improve both the efficacy and efficiency of solid tumor immunotherapy. Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intrapleurally administered CAR T cells vastly outperformed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. After intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced antitumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4+ T cell activation associated with a higher intratumoral CD4/CD8 cell ratios and CD28-dependent CD4+ T cell–mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication, or persistence. The ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through “regional distribution centers.” On the basis of these results, we are opening a phase 1 clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.

Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients

Purpose: Mesothelin (MSLN) is a tumor-associated antigen, being investigated as a biomarker and therapeutic target in malignant pleural mesothelioma (MPM). The biologic function of MSLN overexpression in MPM is unknown. We hypothesized that MSLN may promote tumor invasion in MPM, a tumor characterized primarily by regional aggressiveness and rare distant metastases. Experimental Design: Human and murine MPM cells with MSLN forced expression and short hairpin RNA knockdown were examined for proliferation, invasion, and matrix metalloproteinase (MMP) secretion. The influence of MSLN overexpression on MPM cell invasion was assessed in an orthotopic mouse model and in patient samples. Results: MSLN expression promotes MPM cell invasion and MMP secretion in both human and murine MPM cells. In an orthotopic MPM mouse model characterized by our laboratory, MPM cells with MSLN overexpression preferentially localized to the tumor invading edge, colocalized with MMP-9 expression, and promoted decreased survival without an increase in tumor burden progression. In a tissue microarray from epithelioid MPM patients (n = 139, 729 cores), MSLN overexpression correlated with higher MMP-9 expression at individual core level. Among stage III MPM patients (n = 72), high MSLN expression was observed in 26% of T2 tumors and 51% of T3 tumors. Conclusions: Our data provide evidence elucidating a biologic role for MSLN as a factor promoting tumor invasion and MMP-9 expression in MSLN expressing MPM. As regional invasion is the characteristic feature in MSLN expressing solid cancers (MPM, pancreas, and ovarian), our observations add rationale to studies investigating MSLN as a therapeutic target. Clin Cancer Res; 18(9); 2478–89. ©2012 AACR.

A nuclear grading system is a strong predictor of survival in epitheloid diffuse malignant pleural mesothelioma

Epithelioid mesothelioma is the most prevalent subtype of diffuse malignant pleural mesothelioma in which only staging is prognostic for survival. In this study of epithelioid diffuse malignant pleural mesothelioma, we investigate the prognostic utility of nuclear features. The slides of 232 epithelioid diffuse malignant pleural mesothelioma patients (14 stage I, 54 stage II, 130 stage III, and 34 stage IV) from a single institution were reviewed for the following seven nuclear features: nuclear atypia, nuclear/cytoplasmic ratio, chromatin pattern, intranuclear inclusions, prominence of nucleoli, mitotic count, and atypical mitoses. MIB-1 immunohistochemistry was performed using tissue microarray, and MIB-1 labeling index was recorded as the percentage of positive tumor cells. Median overall survival of all patients was 16 months and correlated with nuclear atypia (P<0.001), chromatin pattern (P=0.031), prominence of nucleoli (P<0.001), mitotic count (P<0.001), and atypical mitoses (P<0.001) by univariate analysis. Multivariate analysis revealed nuclear atypia (P=0.012) and mitotic count (P<0.001) as independent prognostic factors, and these two factors were utilized to create a three-tier nuclear grade score. The resulting nuclear grade stratified patients into three distinct prognostic groups: grade I (n=107, median overall survival=28 months), grade II (n=91, 14 months), and grade III (n=34, 5 months). Not only was nuclear grade an independent predictor of overall survival (P<0.001), but it was also a stronger discriminator of survival than all currently available factors. Furthermore, nuclear grade was associated with time to recurrence (P=0.004) in patients who underwent complete surgical resection (n=159). MIB-1 labeling index correlated with mitotic count (P<0.001) and nuclear atypia (P=0.037) and stratified overall survival (P<0.001) and time to recurrence (P=0.048), confirming the prognostic value of the nuclear grade. Nuclear grading in epithelioid mesothelioma provides a simple, practical, and cost-effective prognostic tool that better stratifies clinical outcome and time to recurrence than currently available clinicopathologic factors.

Immune responses and immunotherapeutic interventions in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive, primary pleural malignancy with poor prognosis, hypothesized to originate from a chronic inflammatory state within the pleura. Similar to what has been observed in other solid tumors (melanoma, ovarian and colorectal cancer), clinical and pre-clinical MPM investigations have correlated anti-tumor immune responses with improved survival. As such, a better understanding of the complex MPM tumor microenvironment is imperative in strategizing successful immunotherapies. Herein, we review the immune responses vital to the development and progression of MPM, as well as assess the role of immunomodulatory therapies, highlighting recent pre-clinical and clinical immunotherapy investigations.

Visceral Pleural Invasion Does Not Affect Recurrence or Overall Survival Among Patients With Lung Adenocarcinoma ≤ 2 cm: A Proposal to Reclassify T1 Lung Adenocarcinoma

BACKGROUND T1 (≤ 3 cm) tumors with visceral pleural invasion (VPI) are upstaged to T2a (stage IB) in the TNM classification. We investigated the effect of VPI on the cumulative incidence of recurrence (CIR) and overall survival (OS) of lung adenocarcinoma (ADC) ≤ 2 cm (T1a) and 2 to 3 cm (T1b). METHODS OS and CIR among patients with or without VPI were examined by tumor size (≤ 2 and 2-3 cm) in 777 patients with node-negative lung ADC ≤ 3 cm who underwent resection. RESULTS Among patients with tumors ≤ 2 cm, VPI was not associated with either increased CIR (P = .90) or decreased OS (P = .11). Among patients with tumors 2 to 3 cm in size, the presence of VPI was associated with increased CIR (P = .015) and decreased OS (P < .001), even after adjusting for histologic subtype. When stage I lung ADCs ≤ 3 cm were regrouped as either new stage IA (≤ 2 cm with or without VPI, 2-3 cm without VPI) or new stage IB (2-3 cm with VPI), there was a statistically significant difference in 5-year CIR and OS between new stage IA and new stage IB tumors (CIR, 18% vs 40% [P = .004]; OS, 76% vs 51% [P < .001]). CONCLUSIONS VPI stratifies prognosis in patients with lung ADC 2 to 3 cm but not in those with tumors ≤ 2 cm. Our proposed regrouping of a new stage IB better stratifies patients with poor prognosis, similar to published outcomes in patients with stage II disease, who may benefit from adjuvant chemotherapy.

An In Vivo Platform for Tumor Biomarker Assessment

Tumor biomarkers provide a quantitative tool for following tumor progression and response to therapy. However, investigations of clinically useful tumor biomarkers are time-consuming, costly, and limited by patient and tumor heterogeneity. In addition, assessment of biomarkers as indicators of therapy response is confounded by the concomitant use of multiple therapeutic interventions. Herein we report our use of a clinically relevant orthotopic animal model of malignant pleural mesothelioma for investigating tumor biomarkers. Utilizing multi-modality imaging with correlative histopathology, we demonstrate the utility and accuracy of the mouse model in investigating tumor biomarkers – serum soluble mesothelin-related peptide (SMRP) and osteopontin (OPN). This model revealed percentage change in SMRP level to be an accurate biomarker of tumor progression and therapeutic response – a finding consistent with recent clinical studies. This in vivo platform demonstrates the advantages of a validated mouse model for the timely and cost-effective acceleration of human biomarker translational research.

Animal models and molecular imaging tools to investigate lymph node metastases

Lymph node metastasis is a strong predictor of poor outcome in cancer patients. Animal studies of lymph node metastasis are constrained by difficulties in the establishment of appropriate animal models, limitations in the noninvasive monitoring of lymph node metastasis progression, and challenges in the pathologic confirmation of lymph node metastases. In this comprehensive review, we summarize available preclinical animal cancer models for noninvasive imaging and identification of lymph node metastases of non-hematogenous cancers. Furthermore, we discuss the strengths and weaknesses of common noninvasive imaging modalities used to identify tumor-bearing lymph nodes and provide guidelines for their pathological confirmation.

Pre‐Clinical Mouse Models of Primary and Metastatic Pleural Cancers of the Lung and Breast and the Use of Bioluminescent Imaging to Monitor Pleural Tumor Burden

Malignant pleural disease (MPD) results in an estimated 150,000 cases of malignant pleural effusions (MPE) annually. The most common malignancies associated with MPD are primary malignant pleural mesothelioma (MPM) and metastatic lung cancer, breast cancer, and lymphoma. MPM is a rare, regionally aggressive malignancy whose incidence is increasing secondarily to the latency of disease progression. MPD is characteristic of advanced‐stage pleural disease and portends a grave clinical prognosis with a median survival between 3 and 12 months. Preclinical investigations conducted in flank and intraperitoneal tumor models do not fully recapitulate the pleural tumor microenvironment, and the results are not directly translatable to the clinical setting. The protocol described herein provides a mouse model of MPM and MPD from nonhematogenous tumors, resulting in reproducible tumor location, tumor progression, animal survival, and histopathology. Pleural tumor growth in this model resembles the regionally aggressive clinical course and tumor microenvironment of human pleural cancers and provides an optimal animal model to investigate MPD biology and therapies. Curr. Protoc. Pharmacol. 54:14.21.1‐14.21.18.

Safety and stability of retrovirally transduced chimeric antigen receptor T cells

Evaluation of: Scholler J, Brady TL, Binder-Scholl G et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl Med. 4(132), 132-153 (2012). Adoptive cellular therapy with genetically engineered T cells is predicated on generating effective and persisting T-cell-mediated immunity. Gammaretroviral vector-mediated gene transfer in T cells is the technological basis for promising therapy in both HIV and cancer. Because of concerns over delayed adverse events caused by persisting retroviral vector-engineered cells, the US FDA mandates long-term follow-up of clinical trials. Scholler et al. report FDA-mandated safety data and demonstrate that retrovirally transduced T cells persist in the blood of patients for more than a decade after treatment. These persisting T cells proliferated ex vivo in the presence of antigens and showed no evidence of either insertional oncogenesis or clonal expansion in 11 subjects followed for up to 11 years, supporting the long-term safety and persistence of retrovirally transduced T cells.