Cross Validation of the Monoclonal Antibody Das-1 in Identification of High-Risk Mucinous Pancreatic Cystic Lesions.

BACKGROUND & AIMS: Although pancreatic cystic lesions (PCLs) are frequently and incidentally detected, it is a challenge to determine their risk of malignancy. In immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analyses of tissue and cyst fluid from pancreatic intraductal papillary mucinous neoplasms, the monoclonal antibody Das-1 identifies those at risk for malignancy with high levels of specificity and sensitivity. We aimed to validate the ability of Das-1 to identify high-risk PCLs in comparison to clinical guidelines and clinical features, using samples from a multicenter cohort. METHODS: We obtained cyst fluid samples of 169 PCLs (90 intraductal papillary mucinous neoplasms, 43 mucinous cystic neoplasms, and 36 non-mucinous cysts) from patients undergoing surgery at 4 tertiary referral centers (January 2010 through June 2017). Histology findings from surgical samples, analyzed independently and centrally re-reviewed in a blinded manner, were used as the reference standard. High-risk PCLs were those with invasive carcinomas, high-grade dysplasia, or intestinal-type intraductal papillary mucinous neoplasms with intermediate-grade dysplasia. An ELISA with Das-1 was performed in parallel using banked cyst fluid samples. We evaluated the biomarker's performance, generated area under the curve values, and conducted multivariate logistic regression using clinical and pathology features. RESULTS: The ELISA for Das-1 identified high-risk PCLs with 88% sensitivity, 99% specificity, and 95% accuracy, at a cutoff optical density value of 0.104. In 10-fold cross-validation analysis with 100 replications, Das-1 identified high-risk PCLs with 88% sensitivity and 98% specificity. The Sendai, Fukuoka, and American Gastroenterological Association guideline criteria identified high-risk PCLs with 46%, 52%, and 74% accuracy (P for comparison to Das-1 ELISA <.001). When we controlled for Das-1 in multivariate regression, main pancreatic duct dilation >5 mm (odds ratio, 14.98; 95% confidence interval, 2.63-108; P < .0012), main pancreatic duct dilation ≥1 cm (odds ratio, 47.9; 95% confidence interval, 6.39-490; P < .0001), and jaundice (odds ratio, 6.16; 95% confidence interval, 1.08-36.7; P = .0397) were significantly associated with high-risk PCLs. CONCLUSIONS: We validated the ability of an ELISA with the monoclonal antibody Das-1 to detect PCLs at risk for malignancy with high levels of sensitivity and specificity. This biomarker might be used in conjunction with clinical guidelines to identify patients at risk for malignancy.

Neutrophil-to-lymphocyte Ratio as a Predictor of Malignancy of Intraductal Papillary Mucinous Neoplasms.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) can lead, via the adenoma-carcinoma sequence, to invasive adenocarcinoma, which has a poor prognosis. Most IPMNs do not meet the indications for surgery and instead are monitored regularly, but no biomarkers of malignant transformation during surveillance have been established. PATIENTS AND METHODS: A total of 50 patients with IPMN who underwent pancreatectomy were evaluated retrospectively. Clinicopathological parameters during the surveillance period before surgery were assessed to explore biomarkers for predicting malignancy. RESULTS: The serum level of carbohydrate antigen 19-9 was significantly higher in IPMN-derived invasive adenocarcinoma. The neutrophil-to-lymphocyte ratio was significantly lower in low-grade IPMN than high-grade and Inv-IPMN cases by univariate and multivariate analysis. Disease-free survival was significantly shorter in patients with high-grade and invasive IPMN compared with those with low-grade IPMN. In patients whose preoperative surveillance was performed for more than 12 months, the neutrophil-to-lymphocyte ratio was significantly higher in those with high-grade and invasive IPMNs compared with patients with low-grade IPMN in the year before surgery. CONCLUSION: The NLR is a useful biomarker for distinguishing between low-grade and high-grade IPMN and thus should be monitored during surveillance.

Cytology adds value to monoclonal antibody Das-1 testing for detection of high-risk pancreatic cysts.

INTRODUCTION: Determining the risk of malignancy in a pancreatic cyst (PC) is a clinical and diagnostic challenge. Monoclonal antibody (mAb) Das-1 test was shown to have high sensitivity, specificity, and accuracy in detecting high-risk (HR) cysts. Das-1 mAb test detects HR mucinous cysts with high-grade dysplasia (HGD), invasive carcinoma, and/or intestinal-type epithelium. Correlation of mAb Das-1 testing of PC fluids with cytomorphologic findings has not been evaluated. MATERIALS AND METHODS: We correlated cytology with mAb Das-1 test results and resection histology in 26 PCs. There were 18 intraductal papillary mucinous neoplasms (IPMN), 1 intraductal oncocytic papillary neoplasm (IOPN), 4 mucinous cystic neoplasms (MCN), 2 serous cystadenomas, and 1 cystic pancreatic neuroendocrine tumor (PanNET). HR cysts included cysts with high-grade atypia on cytology or HGD on histology, invasive carcinoma, IOPNs, and cystic PanNETs. Intestinal type IPMNs were also HR cysts on histology. RESULTS: In 17 cases (65.38%), cytology and mAb Das-1 test correlated with histology. There were 2 (7.69%) mAb Das-1 test negative HR PCs diagnosed by cytology. Five (19.23%) mAb Das-1 test positive HR PCs had mucin only or cells with low-grade dysplasia on cytology. Two mAb Das-1 test positive HR PCs had nondiagnostic cytology. HR IOPN and cystic PanNET were not detected by mAb Das-1 test. CONCLUSION: The mAb Das-1 is a sensitive and specific biomarker for detecting HR mucinous PCs. Adding cytology to mAb Das-1 testing improves the sensitivity for the detection of nonmucinous HR PC. Together, cytology with mAb Das-1 testing is more accurate than either one alone.

Evolution of the immune landscape during progression of pancreatic intraductal papillary mucinous neoplasms to invasive cancer.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions of pancreatic cancer, which is characterized by an immunosuppressive microenvironment. Yet, the spatial distribution of the immune infiltrate and how it changes during IPMN progression is just beginning to be understood. METHODS: We obtained tissue samples from patients who underwent pancreatic surgery for IPMN, and performed comprehensive immunohistochemical analyses to investigate the clinical significance, composition and spatial organization of the immune microenvironment during progression of IPMNs. Survival analysis of pancreatic cancer patients was stratified by tumour infiltrating immune cell subtypes. FINDINGS: The immune microenvironment evolves from a diverse T cell mixture, comprising CD8+ T cells, Th/c1 and Th/c2 as major players combined with Th9, Th/c17, Th22, and Treg cells in low-grade IPMN, to a Treg dominated immunosuppressive state in invasive pancreatic cancer. Organized lymphoid clusters formed in IPMN surrounding stroma and accumulated immunosuppressive cell types during tumour progression. Survival of pancreatic cancer patients correlated with Th2 signatures in the tumour microenvironment. INTERPRETATION: The major change with regards to T cell composition during IPMN progression occurs at the step of tissue invasion, indicating that malignant transformation only occurs when tumour immune surveillance is overcome. This suggests that novel immunotherapies that would boost spontaneous antitumor immunity at premalignant states could prevent pancreatic cancer development. FUNDING: The present work was supported by German Cancer Aid grants (70,112,720 and 70,113,167) to S. R., and the Olympia Morata Programme of the Medical Faculty of Heidelberg University to S. R.

Circulating and Salivary Antibodies to Fusobacterium nucleatum Are Associated With Cystic Pancreatic Neoplasm Malignancy.

Objectives: Intraductal papillary mucinous neoplasms (IPMNs) are cystic precursor lesions to pancreatic cancer. The presence of oral microbes in pancreatic tissue or cyst fluid has been associated with high-grade dysplasia (HGD) and cancer. The present study aims at investigating if humoral immunity to pancreas-associated oral microbes reflects IPMN severity. Design: Paired plasma (n = 109) and saliva (n = 65) samples were obtained from IPMN pancreatic cystic tumor cases and controls, for anti-bacterial antibody analysis and DNA quantification by enzyme-linked immunosorbent assay (ELISA) and qPCR, respectively. Tumor severity was graded by histopathology, laboratory, and clinical data. Circulating plasma and salivary antibody reactivity to a pancreas-associated oral microbe panel were measured by ELISA and correlated to tumor severity. Results: The patient group with high-risk cystic tumors (HGD and/or associated invasive cancer) shows ample circulating IgG reactivity to Fusobacterium nucleatum (F. nucleatum) but not to Granulicatella adiacens (G. adiacens), which is independent of the salivary bacteria DNA levels. This group also shows higher salivary IgA reactivity to F. nucleatum, Fap2 of F. nucleatum, and Streptococcus gordonii (S. gordonii) compared to low-risk IPMN and controls. The salivary antibody reactivity to F. nucleatum and Fap2 are found to be highly correlated, and cross-competition assays further confirm that these antibodies appear cross-reactive. Conclusion: Our findings indicate that humoral reactivity against pancreas-associated oral microbes may reflect IPMN severity. These findings are beneficial for biomarker development.

HHLA2 is a novel immune checkpoint protein in pancreatic ductal adenocarcinoma and predicts post-surgical survival.

HHLA2 is a newly identified member of the B7 immune checkpoint family, but its function and crosstalk with immune cells is not fully understood. To gain insights into the HHLA2 expression profile and to determine the clinical significance and function of HHLA2 in pancreatic cancer, we performed immunohistochemistry (IHC) analyses on tissue microarrays (TMAs) of pancreatic ductal adenocarcinoma (PDAC, n = 92) with matched peritumoral tissues as well as in cohorts of precancerous lesions: pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN). We found that HHLA2 was rarely detected in normal acinar, islet, and ductal cells but widely expressed from early pancreatic precancerous lesions to invasive PDAC. The overall HHLA2 positivity was 95% (19/20) in low grade PanIN and 70.73% (29/41) in IPMN. HHLA2 expression was detected in 77.17% (71/92) of the PDAC cases and was significantly associated with better prognosis in this cohort. Our findings suggest that HHLA2 may behave as a costimulatory ligand in pancreatic cancer, which differs from other B7 family members that are largely characterized as checkpoint inhibitors. Further investigation of the HHLA2 signaling pathway and its receptors is warranted by our data and may lead to novel therapeutic interventions.