Marcus C.B. Tan

Disruption of CCR5-Dependent Homing of Regulatory T Cells Inhibits Tumor Growth in a Murine Model of Pancreatic Cancer

Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of CD4+CD25+Foxp3+ regulatory T cells (Treg). We have previously described increased prevalence of these cells in pancreatic adenocarcinoma, but it remains unclear what mechanisms are involved in recruiting Tregs into the tumor microenvironment. Here, we postulated that chemokines might direct Treg homing to tumor. We show, in both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), that tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, that CD4+ Foxp3+ Tregs, compared with CD4+ Foxp3− effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (N,N-dimethyl-N-{{4-{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl}amino}}benzyl]-N,N-dimethyl-N- {{{4-{{{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocycloheptan-8-yl]carbonyl}amino}}benzyl}}}tetrahydro-2H-pyran-4-aminiumchloride; TAK-779), Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, this study demonstrates the importance of Tregs in immune evasion by tumors, how blockade of Treg migration might inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Tregs. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer.

Tumor-derived TGF-β Mediates Conversion of CD4+Foxp3+ Regulatory T Cells in a Murine Model of Pancreas Cancer

CD4+25+Foxp3+ regulatory T cells (Treg) play a critical role in the induction of tolerance to tumor-associated antigens and suppression of antitumor immunity. How Treg are induced in cancer is poorly understood. We reported previously that Treg are significantly elevated in the peripheral blood of patients with pancreas cancer and that in a murine pancreas cancer model induction of Treg seems to be transforming growth factor (TGF)-β dependent. Here we provide additional evidence that Treg are increased locally within the tumor microenvironment by a mechanism that seems dependent on TGF-β receptor expression and the presence of tumor derived TGF-β. The murine pancreas cancer cell line Pan02 produces high levels of TGF-β both in vitro and in vivo. In contrast, the esophageal murine cancer cell line, Eso2, does not. Immunohistochemical staining of Foxp3 in explanted tumors shows an identifiable population of Treg in the Pan02 (TGF-β positive) tumors but not Eso2 (TGF-β negative). Naive CD4+25−Foxp3− T cells, when adoptively transferred into Rag−/− mice, are converted into Foxp3+ Treg in the presence of Pan02 but not Eso2 tumors. Induction of Treg in Pan02 mice is blocked by systemic injection of an anti–TGF-β antibody. If Rag−/− mice are instead reconstituted with naive CD4+25− T cells expressing a mutated TGF-β receptor, induction of Foxp3+ Treg in Pan02 bearing mice is blocked. Collectively, these observations further support the role of TGF-β in the induction of Treg in pancreas adenocarcinoma.

Chemotherapy-Induced Normalization of FDG Uptake by Colorectal Liver Metastases Does Not Usually Indicate Complete Pathologic Response

Dramatic responses are being observed in colorectal cancer liver metastases treated with newer chemotherapeutic regimens. These have been associated with normalization of [18F]fluoro-2-deoxy-d-glucose (FDG) uptake (complete metabolic response) on follow-up Positron Emission Tomography with [18F]fluoro-2-deoxy-d-glucose (FDG-PET) scans in some patients. It is unclear how often complete metabolic response is indicative of complete tumor destruction. We analyzed a subset of patients who had neoadjuvant chemotherapy for hepatic metastases from colorectal adenocarcinoma. Inclusion criteria were: (1) FDG-avid hepatic lesions before initiation of chemotherapy; (2) complete metabolic response of the same lesions after chemotherapy; and (3) histopathologic examination of hepatic lesions. Complete pathologic response was defined as no histologically identifiable viable tumor. Fourteen patients fit the inclusion criteria. All had synchronous, hepatic-only colorectal metastases. On microscopic examination, complete pathologic response to the neoadjuvant regimen was found in only 5 of 34 lesions (15%) and in only 3 of the 14 patients (21%). Seven lesions had complete metabolic response and disappeared on computed tomography (CT); of these, six still contained viable tumor. We conclude that complete metabolic response on FDG-PET after neoadjuvant chemotherapy is an unreliable indicator of complete pathologic response. Therefore, currently, curative resection of liver metastases in these patients should not be deferred on the basis of FDG-PET findings.

Myeloid-Derived Suppressor Cells: General Characteristics and Relevance to Clinical Management of Pancreatic Cancer

Recent studies describe a heterogeneous population of cells of the myeloid lineage, termed myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread. Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice. Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to reverse immune tolerance to tumor. We discuss here what is currently known about MDSC as well as some potential strategies targeting MDSC in the context of our work on pancreatic cancer and recent literature. Due to the number of new reports on MDSC, the most pertinent ones have been selected.

Predictors of complete pathological response after neoadjuvant systemic therapy for breast cancer

BACKGROUND The aim of the current study was to identify predictors of pathologic complete response (pCR) following neoadjuvant therapy. METHODS From 2000 to 2007, 518 breast cancer patients received neoadjuvant therapy. Data were compared using chi(2) and Fishers exact tests and multivariate analysis of variance, as appropriate. RESULTS Of 518 breast cancer patients receiving neoadjuvant therapy, 81 (16%) had pCR (77 of 456 [17%] with chemotherapy, 4 of 62 [6%] with endocrine therapy; P < .05). Four factors were associated with pCR: higher tumor grade (P = .015), lack of estrogen receptor (ER) and progesterone receptor (PR) expression (P < .0001), HER2/neu amplification (P = .025), and negative lymph node status (P < .0001). On multivariate analysis, ER and PR negativity, HER2/neu amplification, and negative lymph node status were found to significantly correlate with pCR. CONCLUSIONS Patients with ER-negative and PR-negative and HER2/neu-amplified breast cancer phenotypes are more likely to experience pCR to neoadjuvant therapy. Although pCR is more frequently observed following neoadjuvant chemotherapy, it is rare following neoadjuvant endocrine therapy.

Adjuvant interferon-based chemoradiation followed by gemcitabine for resected pancreatic adenocarcinoma: a single-institution phase II study.

Purpose:This is a phase II, single-center, single-arm study of patients with resectable adenocarcinoma of the pancreas who were treated with adjuvant interferon-based chemoradiation followed by gemcitabine. The primary end point was 2-year overall survival, with secondary endpoints being 2-year disease-free survival, and the frequency of grade 3 or 4 toxicity. Patients and Methods:From April 2002 to September 2005, 53 patients with adenocarcinoma of the pancreas underwent curative resection at a single institution, and subsequently received interferon- and gemcitabine-based adjuvant therapy consisting of external-beam irradiation at a dose of 5040 cGy (25 fractions per 5 weeks) and simultaneous 3-drug chemotherapy consisting of (1) continuous infusion 5-fluorouracil (175 mg/m2); (2) weekly intravenous bolus cisplatin (25 mg/m2); and (3) interferon-α (3 million units subcutaneously 3 times per week) during the 6 weeks of radiation. This was followed by two 4-week courses of weekly intravenous infusion of gemcitabine (1000 mg/m2, 3 of 4 weeks). Results:Median follow-up is 38 months. Seventy-seven percent of patients had node-positive disease. Sixteen patients (30%) failed to complete adjuvant therapy, due to disease progression (7 patients), toxicity (7 patients), and consent withdrawal (2 patients). No patients completed planned therapy without dose modification. Median overall survival was 25 months (confidence interval [CI] = 21.5–48.5 months). Actuarial overall survival for the 1-, 2- and 3-year periods were 75% (CI = 61–85%), 56% (CI = 41–69%), and 41% (26–55%), respectively. Conclusions:This phase II trial demonstrated increased patient survival compared with historical controls, and equivalent survival compared with the regimen combining interferon-α with 5-fluorouracil-based chemoradiation. Despite these encouraging results, significant concerns regarding dose- and treatment-limiting toxicities remain.

Histology-based Classification Predicts Pattern of Recurrence and Improves Risk Stratification in Primary Retroperitoneal Sarcoma.

Objective:To determine the prognostic significance of histologic type/subtype in a large series of patients with primary resected retroperitoneal sarcoma. Background:The histologic diversity and rarity of retroperitoneal sarcoma has hampered the ability to predict patient outcome. Methods:From a single-institution, prospective database, 675 patients treated surgically for primary, nonmetastatic retroperitoneal sarcoma during 1982 to 2010 were identified and histologic type/subtype was reviewed. Clinicopathologic variables were analyzed for association with disease-specific death (DSD), local recurrence (LR), and distant recurrence (DR). Results:Median follow-up for survivors was 7.5 years. The predominant histologies were well-differentiated liposarcoma, dedifferentiated liposarcoma, and leiomyosarcoma. Five-year cumulative incidence of DSD was 31%, and factors independently associated with DSD were R2 resection, resection of 3 or more contiguous organs, and histologic type. Five-year cumulative incidence for LR was 39% and for DR was 24%. R1 resection, age, tumor size, and histologic type were independently associated with LR; size, resection of 3 or more organs, and histologic type were independently associated with DR. Liposarcoma and leiomyosarcoma were associated with late recurrence and DSD (as long as 15 years from diagnosis). For solitary fibrous tumor, LR was uncommon (<10%), but early distant recurrence was common (36% at 5 years). Nomograms were developed to predict DSD, LR, and DR. Conclusions:Histologic type/subtype is the most important independent predictor of DSD, LR, and DR in primary retroperitoneal sarcoma. Histology predicts the pattern and incidence of LR and DR and will aid in more accurate patient counseling and selection of patients for adjuvant therapy trials.

Circulating Mesothelin Protein and Cellular Antimesothelin Immunity in Patients with Pancreatic Cancer

Purpose: Mesothelin is a glycoprotein expressed on normal mesothelial cells and is overexpressed in several histologic types of tumors including pancreatic adenocarcinomas. A soluble form of mesothelin has been detected in patients with ovarian cancer and malignant mesothelioma, and has prognostic value. Mesothelin has also been considered as a target for immune-based therapies. We conducted a study on the potential clinical utility of mesothelin as a biomarker for pancreatic disease and therapeutic target pancreatic cancer. Experimental Design: Tumor cell–bound and soluble mesothelin in patients was evaluated by immunohistochemistry and ELISA, respectively. The in vitro cellular immune response to mesothelin was evaluated by INFγ ELISA and intracellular cytokine staining for IFNγ in CD4+ and CD8+ T cells. The level of circulating antibodies to mesothelin was measured by ELISA. Results: All tumor tissue from patients with pancreatic adenocarcinoma expressed mesothelin (n = 10). Circulating mesothelin protein was detected in patients with pancreatic adenocarcinoma (73 of 74 patients) and benign pancreatic disease (5 of 5) but not in healthy individuals. Mesothelin-specific CD4+ and CD8+ T cells were generated from peripheral blood lymphocytes of patients with pancreatic cancer in 50% of patients compared with only 20% of healthy individuals. Antibodies reactive to mesothelin were detected in <3% of either patients or healthy individuals. Conclusions: Circulating mesothelin is a useful biomarker for pancreatic disease. Furthermore, mesothelin-specific T cells can be induced in patients with pancreatic cancer. This suggests that mesothelin is a potential target for immune-based intervention strategies in pancreatic cancer. (Clin Cancer Res 2009;15(21):6511–8)

Anti-Mesothelin Antibodies and Circulating Mesothelin Relate to the Clinical State in Ovarian Cancer Patients

Most human ovarian carcinomas express mesothelin, which is shed as a diagnostically useful biomarker. We applied an ELISA to measure antibodies to native mesothelin in serum from a series of patients with divergent clinical outcomes. The level of anti-mesothelin antibodies determined as OD450 nm and referred to as absorption units (AU) for 1:20 diluted serum was higher in patients who remained disease-free after therapy [no evidence of disease (NED); n = 14] than in patients whose disease recurred [clinical evidence of disease (CED); n = 21; P < 0.01]. Applying AU ≥ 0.5 at a serum dilution of 1:20 as cutoff, 10 of 14 (71%) ovarian carcinoma patients with NED and 9 of 21 (43%) patients with CED had antibodies to mesothelin compared with 6 of 23 (26%) healthy women (P < 0.008) and 5 of 24 (21%) women with other benign gynecologic diseases (P < 0.003), whereas 7 of 9 (78%) of women with pelvic inflammatory disease were positive. Three of the 14 (21%) NED patients had circulating mesothelin detected as an AU ≥ 0.2 at a serum dilution of 1:40 (P < 0.005) compared with 15 of 21 (71%) CED patients, and 9 of 14 (64%) NED patients (P < 0.0002) were positive for antibodies and negative for antigen compared with 1 of 21 (5%) CED patients. Although our data indicate that an antibody response to mesothelin is an important correlate of ovarian carcinoma, prospective studies are needed to show whether the measurement of such antibodies (alone or together with antigen) aids the diagnosis and monitoring of patients. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1520–6)

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

OBJECTIVES:There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery.METHODS:WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity.RESULTS:We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion.CONCLUSIONS:WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.