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Pancreatic intraductal papillary mucinous neoplasms (IPMN) are cystic mucin-secreting tumors that arise within the pancreatic ductal system and are considered precursor lesions of pancreatic ductal adenocarcinoma. Profiling the immune infiltrate of IPMNs is essential to understand the immune mechanisms associated with the progression of IPMN. The aim of this study was to characterize the immune profiling of IPMN. We examined 31 formalin-fixed and paraffin-embedded tumor samples from surgically resected IPMN. The diagnostic slides were histologically reviewed by 2 expert pathologists in pancreas and classified as low-grade IPMN (low to intermediate grade dysplasia, N=11), high-grade IPMN (high grade dysplasia, N=18) and IPMN with invasive adenocarcinoma (N=2). Multiplex immunofluorescence (mIF) was used to characterize the immune profiling using two panels: Panel 1: PD-L1, PD1, CD3 (pan T cells), CD8 (T-cytotoxic), CD68 (macrophages), CK (AE1/AE3), DAPI. Panel 2: CD20 (B cells), CD45RO (T-memory), CD57 (NK), Granzyme B (NK and cytotoxic), FOXP3 (T reg), CK (AE1/AE3), DAPI. The slides were scanned using the Vectra multispectral microscope and analyzed by the InForm software (PerkinElmer). We quantified and co-localized immune phenotypes (epithelial cells PD-L1+; Total T-cell lymphocytes; T-cells antigen-experienced; Cytotoxic T-cells; Total macrophages; Macrophages PD-L1+; Total B-cell lymphocytes; Memory T cells; Granzyme B+ cells; Regulatory T cells; NK cells) in low grade dysplasia (LGD), high grade dysplasia (HGD), and invasive carcinoma areas in epithelial and stromal compartments. The Wilcoxon rank sum test or the Wilcoxon singed-rank test was used to test the equality of medians between two independent groups or paired samples, respectively. In all patients, HGD areas showed significantly higher density of macrophages (P= 0.041) and lower density of B-cell lymphocytes (P= 0.028) compared with LGD areas in the epithelial/stromal and epithelial compartments, respectively. Similar differences were observed when patients with high-grade IPMN (n=18) were analyzed for immune infiltrates in the HGD areas compared with LGD areas in the same tumor samples: HGD areas showed significantly higher density of macrophages (P= 0.035) and lower density of B-cell lymphocytes (P= 0.031). Patients with high-grade IPMN had significantly lower densities of B-cell lymphocytes (P= 0.011), T-cell lymphocytes (P= 0.034), and memory T-cells (P= 0.031) in the HGD areas when compared with LGD areas from patients with low-grade IPMN, in the epithelial compartment. No significant differences were found between LGD areas from patients who had high-grade IPMN and LGD areas from patients with low-grade IPMN. In conclusion, we characterized the immune landscape of IPMN and identified immune cell biomarkers associated with progression of IPMN. Citation Format: Luisa M. Solis, Naohiro Uraoka, Edwin Roger Parra, Yu Shen, Wei Wei, Mei Jiang, Barbara Mino, Kajsa Affolter, Courtney L. Scaife, Michele T. Yip-Schneider, C. Max Schmidt, Matthew Firpo, Sean Mulvihill, Eugene J. Koay,, Huamin Wang, Ignacio I. Wistuba, Anirban Maitra, Subrata Sen. Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3121.

Purpose: CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19.9. We have carried out sequential validations starting with 32 protein biomarker candidates to develop a marker panel to improve detection of early stage disease compared with CA19.9 alone. Patients and Methods: Multiple independent sample sets consisting of PDAC cases, benign pancreatic disease and healthy controls were utilized for immunoassays of candidate markers which after sequential validations yielded a panel of three markers that was further tested in a blinded setting. Results: The 32 biomarker candidates were initially tested in a sample set consisting of 75 PDAC cases, 29 healthy controls, and 19 chronic pancreatitis cases which narrowed the list to seven biomarker candidates. A second independent set of 73 early-stage PDAC cases, 60 healthy controls, and 74 benign pancreatic disease cases (60 chronic pancreatitis cases and 14 benign pancreatic cyst cases) yielded a panel of three markers, including CA19-9, with an AUC (95%CI) of 0.949 (0.917-0.981) and sensitivity of 0.849 at 95% specificity. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy controls, yielding an AUC of 0.887. The performance of the biomarker panel was significantly improved compared to CA19-9 alone (P < 0.001) in both plasma sample sets. Conclusion: We validated a protein-based three marker panel with significantly improved performance compared to CA19-9 with the potential to contribute to strategies for detecting early-stage PDAC. Citation Format: Michela Capello, Leonidas E. Bantis, Ghislaine Scelo, Yang Zhao, Dilsher S. Dhillon, Hong Wang, James L. Abbruzzese, Anirban Maitra, Margaret A. Tempero, Randall Brand, Matthew A. Firpo, Sean Mulvihill, Matthew H. Katz, Paul Brennan, Ziding Feng, Samir M. Hanash, Ayumu Taguchi. Sequential validation of blood-based protein biomarker candidates for early-stage pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-134.

Poor survival rates for pancreatic adenocarcinoma (PDAC) are in part due to late presentation. Earlier diagnosis is recognized as a means to improve outcomes by increasing the possibility for potentially curative treatment. Identification of early detection biomarkers is a research priority. The stromal compartment is a potential source for biomarkers as a strong desmoplastic response is a hallmark of PDAC. The interaction of mesenchymal, cancer, and inflammatory cells in the tumor induce secretion of autocrine and paracrine effectors and extracellular matrix remodeling enzymes that contribute to tumor growth, invasion, angiogenesis, and metastasis. Basigin (BSG), a cell surface glycoprotein, plays a role in the interaction of cancer and mesenchymal cells through the stimulation of extracellular matrix remodeling enzymes. Circulating BSG has also been demonstrated as a diagnostic/prognostic biomarker for other cancers. We evaluated the potential utility of plasma BSG as a PDAC diagnostic and prognostic biomarker. BSG levels were measured by ELISA in plasma from 106 healthy control subjects (mean = 3.29 ng/ml, 95% CI =3.0 – 3.6), 77 patients with chronic pancreatitis (3.72 ng/ml, 3.3 – 4.1 CI), and 112 pre-treatment samples from patients with PDAC (4.19 ng/ml, 3.9 – 4.5 CI). Nonparametric analysis revealed a significant difference for the model (P = 0.02, Wilcoxon). By ANOVA and Tukey-Kramer post-hoc tests, the distributions of BSG levels in PDAC and healthy control subjects were significantly different (P = 0.0003), but the comparison of PDAC to chronic pancreatitis cases was not significant (P = 0.147). A striking difference was noted after separating the PDAC cases into early stage (I-II) and late stage (III-IV) groups. ANOVA indicated that early stage disease (N = 70, 4.61 ng/ml, 4.2 – 5.0 CI) had significantly elevated BSG levels compared to healthy controls (P < 0.0001), chronic pancreatitis cases (P = 0.006), and late stage PDAC cases (P = 0.003, N = 42, 3.48 ng/ml, 2.9 – 3.9 CI). Increased plasma BSG levels were also significantly related to decreased survival in univariate Cox proportional hazards models (P = 0.01) indicating a 14% increase in risk per 1 ng/ml. This relationship remained significant in multivariate models adjusting for stage and post-diagnosis treatments. The results suggest that circulating BSG is an early stage phenomenon during PDAC development and would apparently be unique in its ability to specifically identify early stage PDAC cases. If validated, this biomarker should substantially contribute to the accuracy of an early detection biomarker panel. Further investigations are warranted into the usefulness of circulating BSG for early detection, selection of patients for surgical resection, identification of recurrence, screening high-risks groups and/or inclusion in panel screens. Citation Format: Samaa Kamal, Matthew A. Firpo, Courtney L. Scaife, Adler G. Douglas, Kenneth M. Boucher, Sean J. Mulvihill. Plasma basigin as an early detection biomarker for pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B23.

Background: Although resectable pancreatic cancers are associated with better survival, only about 10% of pancreatic patients present with localized disease. Imaging modalities, notably endoscopic ultrasound and Magnetic resonance cholangiopancreatography, allow detection of early stage pancreatic cancer or small pancreatic cysts. However, these imaging modalities are not suitable for screening in terms of throughput, cost effectiveness, or invasiveness. Blood-based biomarkers could be ideal for screening of early stage pancreatic cancer. The performance of CA19-9 as a pancreatic cancer biomarker is limited and moreover CA 19-9 is not detectable in 5-10% of subjects with fucosyltransferase deficiency. As a result there is a need for additional markers that complement CA 19-9 for reliable detection of early stage pancreatic cancer. We have previously identified and obtained initial validation data for a set of blood based biomarkers which distinguished cases from control in the pre-diagnostic setting (Faca et al PLoS Medicine, 2008). We have undertaken further validation of this marker panel augmented with novel candidates including autoantibodies to tumor antigens identified by recombinant protein array. Methods: The selected biomarker candidates as well as CA19-9 were further assayed in a training set, consisting of plasmas from 138 pancreatic cancer patients and 81 controls (52 healthy subjects and 29 subjects with chronic pancreatitis) resulting in a combination rule which was tested in an independent cohort consisting of plasmas from 42 early stage pancreatic cancer patients, 50 healthy controls, 29 subjects with chronic pancreatitis, and 14 subjects with benign pancreatic cysts. Results: A logistic regression model was built in the training set using 5 markers. The AUCs of the model and CA19-9 alone were 0.80 (95% CI = 0.71-0.89) and 0.76 (95% CI = 0.67-0.86) (P value for difference in AUC = 0.014) in the training set. In the test set, the AUCs of the model in a comparison of early stage pancreatic cancer vs. (1) healthy controls, (2) subjects with chronic pancreatitis, and (3) subjects with benign pancreatic cysts were 0.91, 0.85, and 0.94 respectively. The marker panel substantially improved the negative predictive values (NPV) at 98% sensitivity in comparison of early stage pancreatic cancer vs. (1) healthy controls and (2) subjects with chronic pancreatitis (NPVs of the panel = (1) 0.96, (2) 0.95, and (3) 0.88; NPVs of CA19-9 alone = (1) 0.83, (2) 0.83, and (3) 0.88). Stratification based on CA19-9 positivity (cut off = 37 units/ml) yielded AUCs of (1) 0.79, (2) 0.72, and (3) 0.90, and NPVs were (1) 0.99, (2) 0.98, and (3) 0.95 in CA19-9 negative subjects. Conclusions: We have validated a marker combination with a potential to differentiate early stage pancreatic cancer from healthy controls, subjects with chronic pancreatitis and subjects with pancreatic cysts. Citation Format: Ayumu Taguchi, Michela Capello, Yang Zhao, Ingrid Babel, Gary Goodman, Margaret A. Tempero, Matthew A. Firpo, Matthew H. Katz, Ziding Feng, Samir Hanash. Development and validation of diagnostic biomarker model for detection of early stage pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B20.