Liquid Biopsy for Pancreatic Cancer by Serum Extracellular Vesicle-Encapsulated Long Noncoding RNA HEVEPA.

OBJECTIVES: The role of long noncoding RNAs (lncRNAs) in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Extracellular vesicle (EV)-encapsulated RNAs could be effective targets for liquid biopsy. We aimed to identify previously unknown EV-encapsulated lncRNAs in PDAC and establish highly accurate methods for isolating EVs. MATERIALS AND METHODS: Extracellular vesicles were isolated using existing and newly developed methods, namely, PEViA-UC and PEViA-IP, from serum samples of 20 patients with PDAC, 22 patients with intraductal papillary mucinous neoplasms, and 21 healthy individuals. Extracellular vesicle lncRNA expression was analyzed using digital PCR. RESULTS: Gene expression analysis using cDNA microarray revealed a highly expressed lncRNA, HEVEPA , in serum EVs from patients with PDAC. We established PEViA-UC and PEViA-IP using PEViA reagent, ultracentrifugation, and immunoprecipitation. Although detection of EV-encapsulated HEVEPA using existing methods is challenging, PEViA-UC and PEViA-IP detected EV HEVEPA , which was highly expressed in patients with PDAC compared with non-PDAC patients. The detection sensitivity for discriminating PDAC from non-PDAC using the combination of HEVEPA and HULC , which are highly expressed lncRNAs in PDAC, and carbohydrate antigen 19-9 (CA19-9), was higher than that of HEVEPA , HULC , or CA19-9 alone. CONCLUSIONS: Extracellular vesicle lncRNAs isolated using PEViA-IP and CA19-9 together could be effective targets in liquid biopsy for PDAC diagnosis.

Pancreatic hamartoma: detection of harbouring NAB2::STAT6 fusion gene.

Hamartomas in the pancreas are rare and are often histologically and morphologically similar to solitary fibrous tumours (SFTs). We examined the differences between hamartomas and SFTs at the molecular level. METHODS AND RESULTS: Thirteen patients histopathologically diagnosed with pancreatic hamartoma were included in the study. We also performed STAT6 immunohistochemistry (IHC), which is used in the diagnosis of SFT. Furthermore, for the three cases in which RNA was extracted, reverse transcription polymerase chain reaction to search for NAB2::STAT6 fusions was used. Macroscopically, 13 patients had well-demarcated tumour lesions. Histologically, no islets of Langerhans were observed in the lesions, acinar tissue and ducts were unevenly distributed and elastic fibres were not observed around the ducts by Elastica van Gieson staining. One case contained a lipomatous hamartoma composed mainly of adipose tissue. Seven of the 13 cases demonstrated expression of STAT6 in the nuclei of intervening spindle cells. NAB2::STAT6 fusions were observed in two of the three cases in which RNA was extracted. These two cases also demonstrated STAT6 expression in spindle cells using STAT6 IHC. In one case of lipomatous hamartoma, we did not confirm NAB2::STAT6 fusion or STAT6 expression in STAT6 IHC. CONCLUSION: Of the 13 patients histopathologically diagnosed with hamartoma, two demonstrated NAB2::STAT6 fusions, suggesting the existence of pancreatic hamartomas with molecular-level components identical to those of SFT.

HOPS-R01 phase II trial evaluating neoadjuvant S-1 therapy for resectable pancreatic adenocarcinoma.

Although neoadjuvant therapy (Nac) is recommended for high-risk resectable pancreatic cancer (R-PDAC), evidence regarding specific regimes is scarce. This report aimed to investigate the efficacy of S-1 Nac for R-PDAC. In a multicenter phase II trial, we investigated the efficacy of Nac S-1 (an oral fluoropyrimidine agent containing tegafur, gimeracil, and oteracil potassium) in R-PDAC patients. The protocol involved two cycles of preoperative S-1 chemotherapy, followed by surgery, and four cycles of postoperative S-1 chemotherapy. Two-year progression-free survival (PFS) rates were the primary endpoint. Overall survival (OS) rates and median survival time (MST) were secondary endpoints. Forty-nine patients were eligible, and 31 patients underwent resection following Nac, as per protocol (31/49; 63.3%). Per-protocol analysis included data from 31 patients, yielding the 2-year PFS rate of 58.1%, and 2-, 3-, and 5-year OS rates of 96.8%, 54.8%, and 44.0%, respectively. MST was 49.2 months. Intention-to-treat analysis involved 49 patients, yielding the 2-year PFS rate of 40.8%, and the 2-, 3-, and 5-year OS rates of 87.8%, 46.9%, and 33.9%, respectively. MST was 35.5 months. S-1 single regimen might be an option for Nac in R-PDAC; however, the high drop-out rate (36.7%) was a limitation of this study.

Concordance of human equilibrative nucleoside transporter-1 expressions between murine (10D7G2) and rabbit (SP120) antibodies and association with clinical outcomes of adjuvant chemotherapy for pancreatic cancer: A collaborative study from the JASPAC 01 trial.

BACKGROUND: Expression of human equilibrative nucleoside transporter-1 (hENT1) is reported to predict survival of gemcitabine (GEM)-treated patients. However, predictive values of immunohistochemical hENT1 expression may differ according to the antibodies, 10D7G2 and SP120. AIM: We aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis. METHODS: The subjects of this study were totally 332 whose formalin-fixed paraffin-embedded specimens and/or unstained sections were obtained. The individual H-scores and four classifications according to the staining intensity were applied for the evaluation of hENT1 expression by 10D7G2 and SP120, respectively. RESULTS: The highest concordance rate (79.8%) was obtained when the cut-off between high and low hENT1 expression using SP120 was set between moderate and strong. There were no correlations of hENT1 mRNA level with H-score (p = .258). Although the hENT1 mRNA level was significantly different among four classifications using SP120 (p = .011), there was no linear relationship among them. Multivariate analyses showed that adjuvant GEM was a significant predictor of the patients with low hENT1 expression using either 10D7G2 (Hazard ratio [HR] 2.39, p = .001) or SP120 (HR 1.84, p < .001). In contrast, agent for adjuvant chemotherapy was not significant predictor for the patients with high hENT1 expression regardless of the kind of antibody. CONCLUSION: The present study suggests that the two antibodies for evaluating hENT1 expression are equivalent depending on the cut-off point and suggests that S-1 is the first choice of adjuvant chemotherapy for pancreatic cancer with low hENT1 expression, whereas either S-1 or GEM can be introduced for the pancreatic cancer with high hENT1 expression, no matter which antibody is used.

The Interaction Between Long Non-coding RNA HULC and MicroRNA-622 via Transfer by Extracellular Vesicles Regulates Cell Invasion and Migration in Human Pancreatic Cancer.

Although non-coding RNAs (ncRNAs) are involved in disease pathogenesis, their contributions to pancreatic ductal adenocarcinoma (PDAC) remain unclear. Recently, the interrelationship between two classes of ncRNA, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs), has been reported to contribute to the epigenetic regulation of gene expression in several diseases including cancers. Moreover, some ncRNAs can be transferred by extracellular vesicles (EVs) from their donor cells to recipient cells. We previously verified that lncRNA HULC is up-regulated in PDAC cells and the intercellular transfer of HULC by EVs can promote PDAC cell invasion and migration through the induction of epithelial-mesenchymal transition (EMT). Therefore, we identified the miRNA that could target HULC and investigated the functional contributions of the miRNA-HULC interaction and EV transfer of miRNA to the EMT pathway in PDAC. Microarray analysis revealed 187 miRNAs that were decreased to <0.87-fold in Panc-1 cells treated with TGF-ß compared with the control. Of these, miR-622 was predicted to target HULC directly by bioinformatics analysis. Expression of miR-622 was significantly down-regulated by TGF-ß in a panel of PDAC cells. miR-622 overexpression by a miRNA mimic significantly decreased HULC expression, increased E-cadherin expression, and decreased expression of Snail, N-cadherin, and vimentin. Moreover, overexpression of miR-622 significantly reduced cell invasion and migration whereas inhibition of miR-622 increased HULC expression and promoted EMT signaling, invasion, and migration of PDAC cells. Furthermore, incubation with miR-622-overexpressing EVs could transfer miR-622, which significantly elevated miR-622 expression and decreased cell invasion and migration via inhibition of the EMT pathway in recipient PDAC cells. These results provide mechanistic insights into the development of PDAC by demonstrating that miR-622, as a miRNA downregulated by TGF-ß, could target HULC and suppress invasion and migration by inhibiting EMT signaling via EV transfer. These observations may identify EV-encapsulated miRNA as a novel therapeutic target for human PDAC.

Clinical Outcomes of Biliary Drainage during a Neoadjuvant Therapy for Pancreatic Cancer: Metal versus Plastic Stents.

Neoadjuvant chemotherapy/neoadjuvant chemoradiotherapy (NAC/NACRT) can be performed in patients with pancreatic cancer to improve survival. We aimed to clarify the clinical outcomes of biliary drainage with a metal stent (MS) or a plastic stent (PS) during NAC/NACRT. Between October 2013 and April 2016, 96 patients with pancreatic cancer were registered for NAC/NACRT. Of these, 29 patients who underwent biliary drainage with MS or PS before NAC/NACRT and a subsequent pancreatoduodenectomy were retrospectively analyzed with regard to patient characteristics, preoperative recurrent biliary obstruction rate, NAC/NACRT delay or discontinuation rate, and operative characteristics. The median age of the patients was 67 years. NAC and NACRT were performed in 14 and 15 patients, respectively, and MS and PS were used in 17 and 12 patients, respectively. Recurrent biliary obstruction occurred in 6% and 83% of the patients in the MS and PS groups, respectively (p<0.001). NAC/NACRT delay was observed in 35% and 50% of the patients in the MS and PS groups, respectively (p=0.680). NAC/NACRT discontinuation was observed in 12% and 17% of the patients in the MS and PS groups, respectively (p=1.000). The operative time in the MS group tended to be longer than that in the PS group (625 minutes vs 497 minutes, p=0.051), and the operative blood loss volumes and postoperative adverse event rates were not different between the two groups. MS was better than PS from the viewpoint of preventing recurrent biliary obstruction, although MS was similar to PS with regards to perioperative outcomes.

Human equilibrative nucleoside transporter-1 expression is a predictor in patients with resected pancreatic cancer treated with adjuvant S-1 chemotherapy.

The high expression of human equilibrative nucleoside transporter-1 (hENT1) and the low expression of dihydropyrimidine dehydrogenase (DPD) are reported to predict a favorable prognosis in patients treated with gemcitabine (GEM) and 5-fluorouracil (5FU) as the adjuvant setting, respectively. The expression of hENT1 and DPD were analyzed in patients registered in the JASPAC 01 trial, which showed a better survival of S-1 over GEM as adjuvant chemotherapy after resection for pancreatic cancer, and their possible roles for predicting treatment outcomes and selecting a chemotherapeutic agent were investigated. Intensity of hENT1 and DPD expression was categorized into no, weak, moderate or strong by immunohistochemistry staining, and the patients were classified into high (strong/moderate) and low (no/weak) groups. Specimens were available for 326 of 377 (86.5%) patients. High expression of hENT1 and DPD was detected in 100 (30.7%) and 63 (19.3%) of 326 patients, respectively. In the S-1 arm, the median overall survival (OS) with low hENT1, 58.0 months, was significantly better than that with high hENT1, 30.9 months (hazard ratio 1.75, P = 0.007). In contrast, there were no significant differences in OS between DPD low and high groups in the S-1 arm and neither the expression levels of hENT1 nor DPD revealed a relationship with treatment outcomes in the GEM arm. The present study did not show that the DPD and hENT1 are useful biomarkers for choosing S-1 or GEM as adjuvant chemotherapy. However, hENT1 expression is a significant prognostic factor for survival in the S-1 arm.

Circulating extracellular vesicle-encapsulated HULC is a potential biomarker for human pancreatic cancer.

The role of long noncoding RNAs (lncRNAs) in the epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) is unclear. Some lncRNAs can be transferred by extracellular vesicles (EVs) and have potential as biomarkers. Here, we identify an lncRNA that could serve as a biomarker for PDAC and show the functional roles of the lncRNA. Expression profiling of lncRNAs revealed that highly upregulated in liver cancer (HULC) was highly expressed, and induced, by transforming growth factor-ß in PDAC cells and their EVs. Knockdown of HULC decreased PDAC cell invasion and migration by inhibiting the EMT. Thus, HULC could be transferred by EVs, and promote EMT, invasion, and migration in recipient PDAC cells. To assess the roles of HULC, PDAC cell xenografts in nude mice were established. Knockdown of HULC in PDAC cells implanted in mice inhibited tumor growth. Moreover, microRNA-133b suppressed PDAC cell invasion and migration by inhibiting the EMT through targeting HULC. Furthermore, serum samples were obtained from 20 PDAC and 22 intraductal papillary mucinous neoplasm (IPMN) patients, as well as 21 healthy individuals. Analysis of serum EV HULC expression by digital PCR showed that HULC expression was significantly increased in PDAC patients compared to healthy individuals or IPMN patients. Additionally, HULC showed good predictive performance for discriminating PDAC, suggesting that the analysis of EV-encapsulated HULC would contribute to the diagnosis for human PDAC. Extracellular vesicle-transported HULC promotes cell invasion and migration by inducing the EMT, and microRNA-133b suppresses the EMT by targeting HULC. Extracellular vesicle-encapsulated HULC could be a potential circulating biomarker for human PDAC.

Clinical usefulness of conversion surgery for unresectable pancreatic cancer diagnosed on multidetector computed tomography imaging: Results from a multicenter observational cohort study by the Hokkaido Pancreatic Cancer Study Group (HOPS UR-01).

BACKGROUND AND AIM: Effective multidisciplinary approaches for unresectable pancreatic cancer (UR-PC) that include modern chemotherapeutic regimens and subsequent conversion surgery (CS) are being developed. The aim of this study was to evaluate outcomes of patients clinically diagnosed with UR-PC, focusing on the efficacy of CS. METHODS: Patients ineligible for two multicenter phase II studies conducted by the Hokkaido Pancreatic Cancer Study Group (HOPS) were recruited. Sequential treatment regimens, conversion to radical surgery, and overall survival (OS) were analyzed by multidetector computed tomography (MDCT)-based UR factors. Univariate and multivariate analyses were performed to identify predictors of OS. RESULTS: Sixty-six of 247 intended recruits for HOPS studies from October 2013 to April 2016 were included. Unresectability was due to locally advanced (LA) disease and metastasis (M) in 42 and 24 patients, respectively. Induction therapy began with chemotherapy (CT) and chemoradiotherapy (CRT) in 44 and 17 patients, respectively, of whom 23 received modern CT regimens. Radical surgery was completed in 12 (LA, 10; M, two) with a median treatment interval of 10.3 months (range, 2-32). Eleven patients (91.6%) achieved pathological R0 resection. Median OS was significantly longer in patients who underwent CS than those who did not (44.1 vs 14.5 months, P < 0.0001). CS was an independent predictor of OS (hazard ratio, 0.078; 95% confident interval, 0.017-0.348; P = 0.001). CONCLUSION: Conversion surgery after a favorable response to sequential treatment might prolong survival in patients with UR-PC. Precise diagnosis on MDCT followed by sequential multimodal anticancer treatment is essential.

Distinctive histopathologic findings of pancreatic hamartomas suggesting their "hamartomatous" nature: a study of 9 cases.

Pancreatic hamartoma is a rare tumor, and its characteristic histopathologic features have not yet been fully evaluated. In this study, we collected 9 cases of pancreatic hamartoma to elucidate distinctive histopathologic features that can serve to establish this tumor as a clear disease entity and thus formulate useful histopathologic criteria for this tumor. The cases comprised 4 men and 5 women with a mean age of 62.7 years. The average tumor diameter was 3.3 cm. All patients underwent surgical treatment, and none showed any recurrence postoperatively. Macroscopically, pancreatic hamartomas were well-demarcated tumors with a solid or solid and cystic appearance. Microscopically, these tumors comprised mature acini and small-sized to medium-sized ducts showing a distorted architecture with various amounts of fibrous stroma. Strikingly, the tumors consistently lacked concentric elastic fibers in their duct walls, peripheral nerves, and well-formed islets of Langerhans, all of which exist in both the normal and atrophic pancreas. Immunohistochemically, scattered chromogranin A-positive neuroendocrine cells were observed in the acinar and ductal components. Ductal components were positive for S-100 protein. Spindle-shaped stromal cells expressed CD34 and/or c-kit. These histopathologic features were distinct from those of 5 cases of pancreatic ductal adenocarcinoma, 3 cases of type 1 autoimmune pancreatitis (lymphoplasmacytic sclerosing pancreatitis), 3 cases of alcoholic chronic pancreatitis, and 5 cases of normal pancreas. In conclusion, pancreatic hamartomas share some distinctive histopathologic features and clinical outcomes (neither recurrence nor metastasis) that allow them to be interpreted as malformative lesions. The term "hamartoma" is appropriate for these unique lesions.