John T. Vetto

Engagement of the OX-40 Receptor In Vivo Enhances Antitumor Immunity

The OX-40 receptor (OX-40R), a member of the TNFR family, is primarily expressed on activated CD4+ T lymphocytes. Engagement of the OX-40R, with either OX-40 ligand (OX-40L) or an Ab agonist, delivers a strong costimulatory signal to effector T cells. OX-40R+ T cells isolated from inflammatory lesions in the CNS of animals with experimental autoimmune encephalomyelitis are the cells that respond to autoantigen (myelin basic protein) in vivo. We identified OX-40R+ T cells within primary tumors and tumor-invaded lymph nodes of patients with cancer and hypothesized that they are the tumor-Ag-specific T cells. Therefore, we investigated whether engagement of the OX-40R in vivo during tumor priming would enhance a tumor-specific T cell response. Injection of OX-40L:Ig or anti-OX-40R in vivo during tumor priming resulted in a significant improvement in the percentage of tumor-free survivors (20–55%) in four different murine tumors derived from four separate tissues. This anti-OX-40R effect was dose dependent and accentuated tumor-specific T cell memory. The data suggest that engagement of the OX-40R in vivo augments tumor-specific priming by stimulating/expanding the natural repertoire of the host’s tumor-specific CD4+ T cells. The identification of OX-40R+ T cells clustered around human tumor cells in vivo suggests that engagement of the OX-40R may be a practical approach for expanding tumor-reactive T cells and thereby a method to improve tumor immunotherapy in patients with cancer.

Adjuvant Immunization of HLA-A2-Positive Melanoma Patients With a Modified gp100 Peptide Induces Peptide-Specific CD8+ T-Cell Responses

PURPOSE To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule. PATIENTS AND METHODS Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freunds adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry. RESULTS Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59). CONCLUSION Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.

Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers.

BACKGROUND The OX-40 antigen is a cell surface glycoprotein in the tumor necrosis factor receptor family that is expressed primarily on activated CD4+ T cells. Selective target organ expression of the OX-40 receptor on autoantigen specific T cells has been found in autoimmune disease. In order to evaluate whether OX-40 is expressed on T cells from patients with nodal-draining carcinomas, OX-40 expression was assessed in tumor infiltrating lymphocytes (TILs), draining lymph node cells (DLNCs), and/or peripheral blood lymphocytes (PBLs) of 13 patients with head and neck squamous cell carcinomas and 9 patients with melanomas. METHODS Cell phenotype was determined by fluorescence cell analysis using a monoclonal antibody to human OX-40, and CD4+ T cell lymphokine production was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS Expression of the OX-40 receptor was found in as many as 31% of the TILs and as many as 28% of the DLNCs tested. Conversely, no OX-40 expression was found in PBLs. In addition, CD4+ T cells isolated from DLNCs (but not from TILs or PBLs) secreted a Th1 pattern of cytokines (IL-2, gamma interferon). Co-culture of autologous CD4+ TILs with an MHC class II+ melanoma cell line transfected with OX-40 ligand cDNA resulted in T cell proliferation and in vitro tumor regression. CONCLUSIONS These findings suggest that OX-40+ CD4+ T cells isolated from tumors and their adjacent draining nodes may represent a tumor-specific population of activated T cells capable of mediating tumor reactivity. These cells may play an exploitable role in future trials of immunotherapy.

Clinicopathologic Predictors of Sentinel Lymph Node Metastasis in Thin Melanoma

PURPOSE Indications for sentinel lymph node biopsy (SLNB) for thin melanoma are continually evolving. We present a large multi-institutional study to determine factors predictive of sentinel lymph node (SLN) metastasis in thin melanoma. PATIENTS AND METHODS Retrospective review of the Sentinel Lymph Node Working Group database from 1994 to 2012 identified 1,250 patients who had an SLNB and thin melanomas (≤ 1 mm). Clinicopathologic characteristics were correlated with SLN status and outcome. RESULTS SLN metastases were detected in 65 (5.2%) of 1,250 patients. On univariable analysis, rates of Breslow thickness ≥ 0.75 mm, Clark level ≥ IV, ulceration, and absence of regression differed significantly between positive and negative SLN groups (all P < .05). These four variables and mitotic rate were used in multivariable analysis, which demonstrated that Breslow thickness ≥ 0.75 mm (P = .03), Clark level ≥ IV (P = .05), and ulceration (P = .01) significantly predicted SLN metastasis with 6.3%, 7.0%, and 11.6% of the patients with these respective characteristics having SLN disease. Melanomas < 0.75 mm had positive SLN rates of < 5% regardless of Clark level and ulceration status. Median follow-up was 2.6 years. Melanoma-specific survival was significantly worse for patients with positive versus negative SLNs (P = .001). CONCLUSION Breslow thickness ≥ 0.75 mm, Clark level ≥ IV, and ulceration significantly predict SLN disease in thin melanoma. Most SLN metastases (86.2%) occur in melanomas ≥ 0.75 mm, with 6.3% of these patients having SLN disease, whereas in melanomas < 0.75 mm, SLN metastasis rates are < 5%. By using a 5% metastasis risk threshold, SLNB is indicated for melanomas ≥ 0.75 mm, but further study is needed to define indications for SLNB in melanomas < 0.75 mm.

Adjuvant therapy with agonistic antibodies to CD134 (OX40) increases local control after surgical or radiation therapy of cancer in mice.

The tumor recurrence from residual local or micrometastatic disease remains a problem in cancer therapy. In patients with soft tissue sarcoma and the patients with inoperable nonsmall cell lung cancer, local recurrence is common and significant mortality is caused by the subsequent emergence of metastatic disease. Thus, although the aim of the primary therapy is curative, the outcome may be improved by additional targeting of residual microscopic disease. We display in a murine model that surgical removal of a large primary sarcoma results in local recurrence in approximately 50% of animals. Depletion of CD8 T cells results in local recurrence in 100% of animals, indicating that these cells are involved in the control of residual disease. We further show that systemic adjuvant administration of αOX40 at surgery eliminates local recurrences. In this model, αOX40 acts to directly enhance tumor antigen-specific CD8 T-cell proliferation in the lymph node draining the surgical site, and results in increased tumor antigen-specific cytotoxicity in vivo. These results are also corroborated in a murine model of hypofractionated radiation therapy of lung cancer. Administration of αOX40 in combination with radiation significantly extended the survival compared with either agent alone, and resulted in a significant proportion of long-term tumor-free survivors. We conclude that αOX40 increases tumor antigen-specific CD8 T-cell cytotoxic activity resulting in improved endogenous immune control of residual microscopic disease, and we propose that adjuvant αOX40 administration may be a valuable addition to surgical and radiation therapy for cancer.

A phase 2 trial of complete resection for stage IV melanoma: Results of Southwest Oncology Group Clinical Trial S9430

BACKGROUND On the basis of retrospective experience at individual centers, it appears that patients with stage IV melanoma who undergo complete resection have a favorable outcome compared with patients with disseminated stage IV disease. The Southwest Oncology Group (SWOG) performed a prospective trial in patients with metastatic melanoma who were enrolled before complete resection of their metastatic disease and provided prospective outcomes in the cooperative group setting. METHODS Based on their physical examination and radiologic imaging studies, patients with a stage IV melanoma judged amenable to complete resection underwent surgery within 28 days of enrollment. All eligible patients were followed with scans (computed tomography or positron emission tomography) every 6 months until relapse and death. RESULTS Seventy-seven patients were enrolled from 18 different centers. Of those, 5 patients were ineligible; 2 had stage III disease alone; and 3 had no melanoma in their surgical specimen. In addition, 8 eligible patients had incompletely resected tumor. Therefore, the primary analysis included 64 completely resected patients. Twenty patients (31%) had visceral disease. With a median follow-up of 5 years, the median relapse-free survival was 5 months (95% CI, 3-7 months) whereas median overall survival was 21 months (95% CI, 16-34 months). Overall survivals at 3 and 4 years were 36% and 31%, respectively. CONCLUSIONS In a prospective multicenter setting, appropriately selected patients with stage IV melanoma achieved prolonged overall survival after complete surgical resection. Although median relapse-free survival was only 5 months, patients could still frequently undergo subsequent surgery for isolated recurrences. This patient population is appropriate for aggressive surgical therapy and for trials evaluating adjuvant therapy.

Patient and peri-operative predictors of morbidity and mortality after esophagectomy: American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP), 2005-2008.

PurposeOur aim was to determine what specific patient and peri-operative factors contribute to major complications after esophagectomy.MethodsUsing the American College of Surgeons National Surgical Quality Improvement Program database, data for esophagectomies between the years 2005 and 2008 were extracted and analyzed. Thirty-day post-operative complications were classified into seven major groups: (1) wound infections, (2) respiratory complications (pneumonia, intubation), (3) cardiac complications, (4) deep venous thrombosis, (5) sepsis/septic shock, (6) re-operation, and (7) death. Univariate analysis and logistic regression modeling were performed to determine if a significant association existed between patient factors or peri-operative factors and these post-operative complications.ResultsOne thousand thirty-two patients who underwent esophagectomy were identified. Diabetes was the strongest pre-operative independent predictor of death (odds ratio (OR) 10.98; 95% confidence interval (CI) 1.37–1.15, p < 0.1) or respiratory (OR 1.86; 95% CI 1.03–3.29, p = 0.04) or cardiac (OR 5.14; 95% CI 1.93–13.20, p < 0.01) complications following esophagectomy. Thoracotomy performed during the operation was not associated with an increased risk of respiratory or cardiac complications.ConclusionsThe major predictors of morbidity after an esophagectomy are the patient factors of diabetes, dyspnea, peripheral vascular disease, and cerebrovascular accident while the peri-operative factors are pre-operative international normalized ratio, contaminated wound classification, and American Society of Anesthesiologists class. Similarly, the major predictors of mortality are diabetes, dyspnea, and age for patient factors and contaminated wound classification for peri-operative factors.

Synchronous colon primaries have the same prognosis as solitary colon cancers.

PURPOSE: This study was designed to determine the prognosis of patients with synchronous colon primary tumors. METHODS: An 18-year, multi-institutional database of 4,878 colon cancer patients was reviewed, and patients with synchronous tumors were identified. Survival for each group was calculated by the Kaplan-Meier method and compared using log-rank analysis. RESULTS: There were 160 patients (3.3 percent) with 339 synchronous tumors. Eight percent of these patients had more than two tumors at the time of diagnosis. TNM staging of all synchronous tumors was 12 percent Stage 0, 41 percent Stage I, 21 percent Stage II, 16 percent Stage III, and 7 percent Stage IV. Based on highest stage lesion, 1 percent of patients were at Stage 0, 28 percent Stage I, 33 percent Stage II, 25 percent Stage III, and 11 percent Stage IV. Disease-specific five-year survival by highest stage was 87 percent for Stage O or I, 69 percent for Stage II, 50 percent for Stage III, and 14 percent for Stage IV (all differences significant by log-rank test). These “highest stage” survivals for patients with synchronous tumors were not significantly different from survival of patients with same stage solitary tumors in our database or from survival of patients with solitary colon cancer in national tumor databases. CONCLUSION: For patients with synchronous colon cancers, survival is the same as for patients with solitary colon tumors on a stage-for-stage basis, when highest stage synchronous tumor is considered.

Stages at presentation, prognostic factors, and outcome of breast cancer in males

BACKGROUND In order to support or refute conventional notions of breast cancer in males as a late-presenting disease associated with a worse prognosis than the same disease in females, we reviewed a recent, multi-institutional experience. METHODS A case series from three area hospital system cancer data bases was reviewed. Demographics, pathology, stages at presentation, and treatment were determined from the data set and correlated with outcomes (recurrence/survival). RESULTS Fifty-four patients (mean age 64.5, SD = 12.8) were identified; half of the tumors were stage T0 or T1, 62% were node negative (N0), and 57% had an American Joint Committee on Cancer (AJCC) stage grouping of IIA or less. Eighty-five percent of tumors examined expressed hormone receptors. There were no local-only recurrences in the 50 cases resected for cure, including 5 cases of minimal breast cancer treated by lumpectomy only. Five- and 10-year overall disease-free survival was AJCC stage related: 100% and 71%, respectively, for early stage (0-IIA) disease, and 71% and 20%, respectively, for advanced (IIB-IV) stage (P = 0.0051 by log-rank). Only AJCC stage and its components (tumor size, nodal status, presence of metastases) correlated with survival by multivariate analysis; other factors such as age, family history, and presenting symptoms/signs did not. CONCLUSIONS The majority of breast cancers in males present at early stages and are hormone receptor positive. In contrast to older notions of this disease as uniformly aggressive, we conclude that prognostic factors and stage-for-stage outcomes for breast cancer in males are similar to those published for the disease in females.

Induction of anti-mammary cancer immunity by engaging the OX-40 receptor in vivo.

The OX-40 receptor (OX-40R) is a member of the tumor necrosis factor receptor (TNF-R) superfamily that is expressed on activated CD4+ T cells. The OX-40R is a costimulatory molecule that induces CD4+ T cell activation when engaged by its ligand (OX-40L; found on antigen presenting cells). In human and murine tumors, we have shown upregulation of the OX-40R on CD4+ T cells from tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (TDLNC) but not on systemic CD4+ T cells, such as peripheral blood lymphocytes (PBL) or splenocytes. In order to examine potentially heightened anti-tumor immunity through enhanced costimulation when engaging OX-40R in vivo, we inoculated mice with a murine mammary cancer cell line (SM1) and then treated with a soluble form of the OX-40L. Mice injected with a lethal inoculum of SM1 cells were given two intraperitoneal injections (days 3 and 7 post-inoculation) of 100μg soluble OX-40L. Seven of 28 treated mice survived the lethal tumor inoculum, as compared to one of 28 control mice, demonstrating a significant survival benefit with treatment (p=0.0136, log rank analysis). Mice that did not develop tumor by day 90 were rechallenged; all remained tumor-free. Mice were also injected with a second mammary tumor line (4T1) and treated with OX-40L:Ig with similar therapeutic results. Activation of OX-40R+ CD4+ T cells during mammary cancer priming stimulated an anti-tumor immune response resulting in enhanced survival and protective anti-tumor immunity. These results should have practical applications for treatment modalities for patients with breast cancer.