(word count: 239):Murine syngeneic tumor models are critical to novel immuno-based therapy development but the molecular and immunological features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly-used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We employed array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor 'inflamed' and 'non-inflamed' tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo.4

Deregulated expression of SPARC/osteonectin, a secreted glycoprotein with multiple biological functions, has been associated with the progression of various cancers. Using microarrays, we previously identified SPARC as one of the genes induced by treatment with a DNA methylation inhibitor in pancreatic cancer cells. We therefore analysed the expression pattern and methylation status of the SPARC gene in pancreatic cancer. Gene expression profiling by oligonucleotide microarray and reverse transcription-PCR analyses demonstrated that SPARC mRNA was expressed in non-neoplastic pancreatic ductal epithelial cells, but was not expressed in a majority of pancreatic cancer cell lines. The loss of SPARC expression was associated with aberrant hypermethylation of its CpG island. Immunohistochemical labeling revealed that the SPARC protein was overexpressed in the stromal fibroblasts immediately adjacent to the neoplastic epithelium in primary pancreatic cancers, but rarely expressed in the cancers themselves. Primary fibroblasts derived from pancreatic cancer strongly expressed SPARC mRNA and secreted SPARC protein into the conditioned media, and treatment of pancreatic cancer cells with exogenous SPARC resulted in growth suppression. SPARC expression in fibroblasts from noncancerous pancreatic tissue was augmented by coculture with pancreatic cancer cells. These findings suggest that SPARC is a frequent target for aberrant methylation in pancreatic cancer and that SPARC expression in fibroblasts adjacent to pancreatic cancer cells is regulated through tumor-stromal interactions.

Purpose: Patients with pancreatic ductal adenocarcinoma usually present with advanced-stage disease and a dismal prognosis. One effective strategy likely to improve the morbidity and mortality from pancreatic cancer would be the identification of accurate, noninvasive diagnostic markers that would enable earlier diagnosis of symptomatic patients and earlier detection of cancer in asymptomatic individuals at high risk for developing pancreatic cancer. In this study, we evaluated serum macrophage inhibitory cytokine-1 (MIC-1) as a marker of pancreatic cancer.Experimental Design: MIC-1 expression in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines was determined using the National Center for Biotechnology Information serial analysis of gene expression database, oligonucleotide microarrays analysis, in situ hybridization, and immunohistochemistry. Serum MIC-1 levels were determined by ELISA in 80 patients with pancreatic adenocarcinomas, in 30 patients with ampullary and cholangiocellular carcinomas, in 42 patients with benign pancreatic tumors, in 76 patients with chronic pancreatitis, and in 97 healthy control subjects. The diagnostic performance of serum MIC-1 as a marker of pancreatic cancer was compared with that of serum CA19 -9.Results: Oligonucleotide microarray and serial analysis of gene expression data demonstrated that MIC-1 RNA levels were higher in primary pancreatic cancers, intraductal papillary mucinous neoplasms, and pancreatic cancer cell lines than in nonneoplastic pancreatic ductal epithelium. MIC-1 expression was localized to the malignant epithelium in pancreatic adenocarcinomas by in situ hybridization. MIC-1 protein was expressed in 14 of 16 primary pancreatic adenocarcinomas (88%) by immunohistochemistry and was also expressed in some pancreata affected by pancreatitis but not in normal pancreas. Serum MIC-1 levels were significantly higher in patients with pancreatic ductal adenocarcinoma (mean ؎ SD, 2428 ؎ 2324 pg/ml) and in patients with ampullary and cholangiocellular carcinomas (2123 ؎ 2387 pg/ml) than in those with benign pancreatic neoplasms (940 ؎ 469 pg/ml), chronic pancreatitis (1364 ؎ 1236 pg/ml), or in healthy controls (546 ؎ 262 pg/ml). An elevated serum MIC-1 (defined as 2 SD above the mean for healthy controls) performed as well as CA19 -9 (area under the receiver operating characteristic curve, 0.81 and 0.77, respectively), and the combination of MIC-1 and CA19 -9 significantly improved diagnostic accuracy (P < 0.05; area under the receiver operating characteristic curve, 0.87; sensitivity, 70%; specificity, 85%).Conclusion: Serum MIC-1 measurement can aid in the diagnosis of pancreatic adenocarcinoma.

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Human cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, beta2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face.

Purpose: Each year in the United States, ϳ 30,000 people die from pancreatic cancer. Fewer than 5% of patients survive >5 years after diagnosis, because most patients present with advanced disease. Early diagnosis may improve the prognosis of patients with pancreatic cancer.Experimental Design: In an attempt to improve on current approaches to the serological diagnosis of pancreatic cancer, we analyzed serum samples from patients with and without pancreatic cancer using surface-enhanced laser desorption and ionization (SELDI) protein chip mass spectrometry. Using a case-control study design, serum samples from 60 patients with resectable pancreatic adenocarcinoma were compared with samples from 60 age-and sex-matched patients with nonmalignant pancreatic diseases, as well as 60 age-and sex-matched healthy controls. To increase the number of proteins potentially identifiable, serum was fractionated using anion exchange and profiled on two ProteinChip surfaces (metal affinity capture and weak cation exchange).Results: We determined a minimum set of protein peaks able to discriminate between patient groups and used the unified maximum separability algorithm to compare the performance of the individual marker panels alone or in conjunction with CA19 -9. Among the peaks identified by SELDI profiling that had the ability to distinguish between patient groups, the 2 most discriminating protein peaks could differentiate patients with pancreatic cancer from healthy controls with a sensitivity of 78% and specificity of 97%. These 2 markers performed significantly better than the current standard serum marker, CA19 -9 (P < 0.05).The diagnostic accuracy of the 2 markers was improved by using them in combination with CA 19 -9. Similarly, a combination of 3 SELDI markers and CA19 -9 was superior to CA19 -9 alone in distinguishing individuals with pancreatic cancer from the combined pancreatic disease controls and healthy subject groups (P ‫؍‬ 0.078). SELDI markers were also better than CA19 -9 in distinguishing patients with pancreatic cancer from those with pancreatitis.Conclusion: SELDI profiling of serum can be used to accurately differentiate patients with pancreatic cancer from those with other pancreatic diseases and from healthy controls.

Purpose: More accurate serum markers of pancreatic cancer could improve the early detection and prognosis of this deadly disease. We compared the diagnostic utility of a panel of candidate serum markers of pancreatic cancer. Experimental Design: We collected preoperative serum from 50 patients with resectable pancreatic adenocarcinoma, as well as sera from 50 patients with chronic pancreatitis and 50 age/ sex-matched healthy controls from our institution. Sera were analyzed for the following candidate markers of pancreatic cancer: CA19-9, macrophage inhibitory cytokine 1 (MIC-1), osteopontin, tissue inhibitor of metalloproteinase 1, and hepatocarcinoma-intestine-pancreas protein levels. Results: By logistic regression analysis, MIC-1and CA19-9 were significant independent predictors of diagnosis. Receiver operating characteristic curve analysis showed that MIC-1was significantly better than CA19-9 in differentiating patients with pancreatic cancer from healthy controls (area under the curve is 0.99 and 0.78, respectively; P = 0.003), but not in distinguishing pancreatic cancer from chronic pancreatitis (area under the curve of 0.81 and 0.74, respectively; P = 0.63). Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein, osteopontin, and tissue inhibitor of metalloproteinase 1serum levels did not provide additional diagnostic power. Conclusion: In the differentiation of patients with resectable pancreatic cancer from controls, serum MIC-1outperforms other markers including CA19-9.

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