Single-cell sequencing defines genetic heterogeneity in pancreatic cancer precursor lesions.

Intraductal papillary mucinous neoplasms (IPMNs) are precursors to pancreatic cancer; however, little is known about genetic heterogeneity in these lesions. The objective of this study was to characterize genetic heterogeneity in IPMNs at the single-cell level. We isolated single cells from fresh tissue from ten IPMNs, followed by whole genome amplification and targeted next-generation sequencing of pancreatic driver genes. We then determined single-cell genotypes using a novel multi-sample mutation calling algorithm. Our analyses revealed that different mutations in the same driver gene frequently occur in the same IPMN. Two IPMNs had multiple mutations in the initiating driver gene KRAS that occurred in unique tumor clones, suggesting the possibility of polyclonal origin or an unidentified initiating event preceding this critical mutation. Multiple mutations in later-occurring driver genes were also common and were frequently localized to unique tumor clones, raising the possibility of convergent evolution of these genetic events in pancreatic tumorigenesis. Single-cell sequencing of IPMNs demonstrated genetic heterogeneity with respect to early and late occurring driver gene mutations, suggesting a more complex pattern of tumor evolution than previously appreciated in these lesions. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aspartate racemase and D-aspartate in starfish; possible involvement in testicular maturation.

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish.

Clinicopathological significance of somatic RNF43 mutation and aberrant expression of ring finger protein 43 in intraductal papillary mucinous neoplasms of the pancreas.

Mutations in RNF43, which encodes the ubiquitin E3 ligase ring finger protein 43, were recently found in intraductal papillary mucinous neoplasms of the pancreas. We evaluated somatic mutations of RNF43 and the expression of ring finger protein 43 as well as their associations with the molecular and clinicopathological features in 176 surgically resected intraductal papillary mucinous neoplasms. Frozen tissues were available for 57 cases and were used for next-generation sequencing analysis of the entire coding exons of RNF43. Formalin-fixed and paraffin-embedded tissues from all 176 cases were used for the immunohistochemical analysis of the expression of ring finger protein 43. Mutations detected with the next-generation sequencing analysis were validated by using Sanger sequencing. Statistical analysis was used to evaluate the associations between RNF43 aberrations and molecular and clinicopathological features including GNAS mutations, KRAS mutations, loss of SMA and MAD4 homologue expression, tumor protein 53 overexpression, tumor grade, histological type, mural nodule detection, macroscopic type, stage, recurrence, and survival. Somatic RNF43 mutations were found in 8 (14%) of the 57 examined cases, and included 5 frameshift mutations (p.F69fs, p.S264fs, p.L311fs, p.R363fs, and p.V490fs), 1 non-sense mutation (p.Q153X), and 2 missense mutations (p.I164N and p.P310A). The expression of ring finger protein 43 was downregulated in 52 (29.5%) of the 176 examined cases. RNF43 mutations were significantly associated with the downregulated expression of ring finger protein 43 (P=0.011), GNAS mutation (P=0.020), and mural nodule detection (P=0.038). The expression of ring finger protein 43 was not associated with any clinicopathological features except RNF43 mutation. These results indicate that RNF43 mutation might cause downregulation of the expression of ring finger protein 43 and play a crucial role and associate synergistically with GNAS mutation during development of intraductal papillary mucinous neoplasm of the pancreas.

The discrete nature and distinguishing molecular features of pancreatic intraductal tubulopapillary neoplasms and intraductal papillary mucinous neoplasms of the gastric type, pyloric gland variant.

Intraductal tubulopapillary neoplasms (ITPNs) are composed of tubulopapillary glands with high-grade dysplasia in the pancreatic duct. Intraductal papillary mucinous neoplasms of the gastric type, pyloric gland variant (IPMN-PGs) are composed of tubular glands mimicking pyloric glands with low-grade dysplasia and were formerly called intraductal tubular adenomas. Because of their apparent common tubular morphology, IPMN-PGs and ITPNs could be associated. While the former might progress to the latter, this has not been fully assessed. In this study, we compared the molecular features of ITPNs and IPMN-PGs to determine their association using formalin-fixed, paraffin-embedded tissues of 14 ITPNs and 15 IPMN-PGs. Somatic mutations in PIK3CA, GNAS, KRAS, and BRAF were determined by Sanger sequencing. Expression of phosphorylated AKT was examined by immunohistochemistry. Somatic PIK3CA mutations were found in 3 of 14 ITPNs (21.4%) but in none of the IPMN-PGs (p = 0.0996). In contrast, GNAS mutations were found in none of the ITPNs but in 9 of 15 IPMN-PGs (60.0%; p < 0.001). KRAS mutations were detected in 1 of 14 ITPNs (7.1%) and 12 of 15 IPMN-PGs (80.0%; p < 0.001). BRAF mutation was found in one ITPN but in none of the IPMN-PGs. Phosphorylated AKT expression in ITPNs was significantly more evident than that in IPMN-PGs (p = 0.0401). These results indicate that ITPNs and IPMN-PGs are molecularly distinct, suggesting that IPMN-PG does not progress to ITPN. Furthermore, the molecular features of IPMN-PGs are confirmed to be identical to those of IPMNs reported elsewhere. These results validate the current World Health Organization system that classifies pancreatic intraductal neoplasms into IPMN and ITPN and confirm that IPMN-PG is not a benign counterpart of ITPN. The term 'intraductal tubular adenoma' should be eliminated and replaced with IPMN-PG.

Targeting of MAPK-associated molecules identifies SON as a prime target to attenuate the proliferation and tumorigenicity of pancreatic cancer cells.

BACKGROUND: Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinase (MAPK). Activation of MAPK is associated with the upregulation of genes implicated in the proliferation and survival of pancreatic cancer cells. We hypothesized that knockdown of these MAPK-associated molecules could produce notable anticancer phenotypes. METHODS: A RNA interference-mediated knockdown screening of 78 MAPK-associated molecules previously identified was performed to find molecules specifically associated with proliferation of pancreatic cancer cells in vitro. Expression of an identified molecule in pancreatic cancer tissues was examined by immunohistochemistry. In vivo tumorigenicity of cancer cells with stable knockdown of the molecule was assayed by using xenograft models. Flow cytometry and live cell imaging were employed to assess an association of the molecule with cell cycle. RESULTS: The knockdown screening revealed that knockdown of SON, the gene encoding SON, which is a large serine/arginine-rich protein involved in RNA processing, substantially suppressed pancreatic cancer cell proliferation and survival in vitro and tumorigenicity in vivo. SON expression was higher in ductal adenocarcinomas than in cells of normal ducts and precursor lesions in pancreatic cancer tissues. Knockdown of SON induced G2/M arrest and apoptosis in cultured cancer cells. The suppressive effect of SON knockdown on proliferation was less pronounced in cultured normal duct epithelial cells. SON formed nuclear speckles in the interphase of the cell cycle and dispersed in the cytoplasm during mitosis. Live cell imaging showed that SON diffusely dispersed in the early mitotic phase, accumulated in some foci in the cytoplasm in the late mitotic phase, and gradually reassembled into speckles after mitosis. CONCLUSION: These results indicate that SON plays a critical role in the proliferation, survival, and tumorigenicity of pancreatic cancer cells, suggesting that SON is a novel therapeutic molecular target for pancreatic cancer.

Molecular characteristics and biological behaviours of the oncocytic and pancreatobiliary subtypes of intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasm (IPMN) consists of four epithelial subtypes. Of those, pancreatobiliary and oncocytic types are recently recognized and relatively uncommon, and usually exhibit high-grade dysplasia. The biological properties and molecular characteristics of these two types have not been well documented. The few molecular studies of the oncocytic type showed absence of KRAS mutations commonly seen in the other subtypes, raising the possibility that the oncocytic type is distinct from the other subtypes. Thus, we examined clinicopathological features and molecular alterations of the two subtypes. The study cohort consisted of 12 pancreatobiliary and 18 oncocytic IPMN cases. KRAS, BRAF, and PIK3CA mutations and TP53, SMAD4, and ß-catenin expression were analysed, and the results of molecular and clinicopathological profiles were compared between the two subtypes. KRAS mutations were identified in the oncocytic type, but less frequently than the pancreatobiliary type (17% versus 58%, p = 0.048). BRAF mutation was found in a single oncocytic tumour, and no PIK3CA mutations were seen in any of the study cohort. TP53 overexpression was less frequent in the oncocytic type than in the pancreatobiliary type (11% versus 58%, p = 0.013). Invasive components were present in 50% of the oncocytic and 92% of the pancreatobiliary types, with lymph node metastasis more frequently seen in the latter, corresponding to better outcomes in the former (5-year survival rates: 93% versus 32%, p = 0.014). Our demonstration of KRAS and BRAF mutations in the oncocytic-type IPMN supports a role for the activation of the RAS-MAPK pathway in this tumour type. However, the less frequent TP53 overexpression associated with the significantly lower rates of invasion and nodal disease in the oncocytic type correlates with better outcomes compared to the pancreatobiliary type.

Association of epidermal growth factor receptor and mitogen-activated protein kinase with cystic neoplasms of the pancreas.

The molecular pathobiology of pancreatic cystic neoplasms is poorly understood. The aim of this study was to know the involvement of epidermal growth factor receptor (EGFR) and its downstream targets in the serous cystic neoplasms and the mucinous cystic neoplasms of the pancreas. In a total of 72 pancreatic cystic neoplasms, including 39 serous cystic neoplasms and 33 mucinous cystic neoplasms, we examined the expression of native and phosphorylated EGFR, mitogen-activated protein kinase (MAPK), and AKT by immunohistochemistry and somatic mutations in EGFR, KRAS, BRAF, and PIK3CA, by direct sequencing. We also assessed the copy numbers of EGFR transcripts and the amplification of the EGFR gene in some of the samples. We found that EGFR, phosphorylated EGFR, MAPK, and phosphorylated MAPK were evidently expressed in 100, 54, 100, and 69% of the serous cystic neoplasms, and in 12%, none, 33, and 27% of the mucinous cystic neoplasms, respectively; the expression was significantly higher and more prevalent in the serous cystic neoplasms than in the mucinous cystic neoplasms. The expression of AKT and phosphorylated AKT was low in both the types of neoplasms. On average, EGFR transcripts in the serous cystic neoplasms and the mucinous cystic neoplasms increased 53.5- and 2.5-fold, respectively, as compared with that in normal tissues, with the increase in the former being significantly greater than that in the latter. Amplification of the EGFR gene was not detected in any of the examined serous cystic neoplasms. None of the tumors had mutations in any of the examined portions of the genes, except two mucinous cystic neoplasms with mutations in codon-12 of KRAS. These results indicate that EGFR and MAPK are actively involved in the pathobiology of serous cystic neoplasms and may therefore be potential diagnostic markers and therapeutic targets in patients with the above mentioned types of neoplasms.

Molecular biomarkers for progression of intraductal papillary mucinous neoplasm of the pancreas.

OBJECTIVES: We aimed to identify molecular biomarkers for assessing the progression of intraductal papillary mucinous neoplasm of the pancreas (IPMN). METHODS: We retrospectively investigated molecular aberrations and their associations with clinicopathological features in 172 IPMNs. RESULTS: GNAS and KRAS mutations were detected in 48% and 56% of IPMNs, respectively. No mutations of EGFR, PIK3CA GNAO1, GNAQ, or GNAI2 were observed. Significant associations were observed between IPMN morphological types and GNAS mutations, KRAS mutations, the expression of phosphorylated MAPK (pMAPK), AKT, and phosphorylated AKT (pAKT), nuclear accumulation of ß-catenin, SMAD4 loss, and TP53 overexpression; histological grades and the expression of EGFR, pMAPK, AKT, and pAKT, the nuclear ß-catenin, SMAD4 loss, and TP53 overexpression; invasive phenotypes and KRAS mutations, the nuclear ß-catenin, and SMAD4 loss; and prognosis and SMAD4 loss and TP53 overexpression. Multivariate analysis to compare prognostic impacts of multiple molecular features revealed that TP53 overexpression was an independent prognostic factor (P = 0.030; hazard ratio, 5.533). CONCLUSIONS: These results indicate that mutations in GNAS and KRAS, the expression of EGFR and pMAPK, the nuclear ß-catenin, SMAD4 loss, and TP53 overexpression may be relevant for assessing the clinical course of IPMN, including its progression into different morphological types, invasion, and prognosis.

Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas.

Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we identified recurrent mutations of BRCA2 and FAT genes. BRCA2 showed somatic or germline premature termination mutations, with loss of the wild-type allele in 3 of 7 tumors. FAT1, FAT3, and FAT4 showed somatic or germline missense mutations in 4 of 7 tumors. The germline FAT mutations were with loss of the wild-type allele. Loss of BRCA2 expression was observed in 5 of 11 tumors. One patient with a BRCA2-mutated tumor experienced complete remission of liver metastasis following cisplatinum chemotherapy. In conclusion, acinar cell carcinomas show a distinct mutation pattern and often harbor somatic or germline mutations of BRCA2 and FAT genes. This result may warrant assessment of BRCA2 abrogation in patients with the carcinoma to determine their sensitivity to chemotherapy.

Intraductal tubulopapillary neoplasm of the pancreas with somatic BRAF mutation.

Intraductal tubulopapillary neoplasm (ITPN) is a rare pancreatic tumor belonging to a newly recognized entity that is coined in the 2010 WHO classification. We present a case of ITPN-associated microinvasive adenocarcinoma that developed in an asymptomatic 78-year-old patient. The tumor demonstrated all the clinical, radiological, and pathological characteristics of ITPN, but it differs from other reported cases of ITPN in molecular analysis, which revealed a somatic mutation in BRAF (c.1799T>A; p.V600E) but no mutation in PIK3CA. Post-operative recurrence was discovered 34 months after tumor resection with negative margins and a 6-month course of adjuvant chemotherapy. To the best of our knowledge, this is the first case of ITPN with BRAF mutation. This case suggests that an activation of RAS-mitogen-activated protein kinase signaling pathway may play a role in development of some of ITPNs. A possible mechanism of tumor recurrence in ITPN is also discussed. Further case series with molecular study are awaited to delineate the clinicopathological and molecular characteristics of ITPN.

Whole-exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas.

Intraductal papillary mucinous neoplasm (IPMN) is a common pancreatic cystic neoplasm that is often invasive and metastatic, resulting in a poor prognosis. Few molecular alterations unique to IPMN are known. We performed whole-exome sequencing for a primary IPMN tissue, which uncovered somatic mutations in KCNF1, DYNC1H1, PGCP, STAB1, PTPRM, PRPF8, RNASE3, SPHKAP, MLXIPL, VPS13C, PRCC, GNAS, KRAS, RBM10, RNF43, DOCK2, and CENPF. We further analyzed GNAS mutations in archival cases of 118 IPMNs and 32 pancreatic ductal adenocarcinomas (PDAs), which revealed that 48 (40.7%) of the 118 IPMNs but none of the 32 PDAs harbored GNAS mutations. G-protein alpha-subunit encoded by GNAS and its downstream targets, phosphorylated substrates of protein kinase A, were evidently expressed in IPMN; the latter was associated with neoplastic grade. These results indicate that GNAS mutations are common and specific for IPMN, and activation of G-protein signaling appears to play a pivotal role in IPMN.

Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas.

Intraductal tubulopapillary neoplasm (ITPN) is a recently recognized rare variant of intraductal neoplasms of the pancreas. Molecular aberrations underlying the neoplasm remain unknown. We investigated somatic mutations in PIK3CA, PTEN, AKT1, KRAS, and BRAF. We also investigated aberrant expressions of phosphorylated AKT, phosphatase and tensin homolog (PTEN), tumor protein 53 (TP53), SMAD4, and CTNNB1 in 11 cases of ITPNs and compared these data with those of 50 cases of intraductal papillary mucinous neoplasm (IPMN), another distinct variant of pancreatic intraductal neoplasms. Mutations in PIK3CA were found in 3 of 11 ITPNs but not in IPMNs (P = 0.005; Fisher exact test). In contrast, mutations in KRAS were found in none of the ITPNs but were found in 26 of the 50 IPMNs (P = 0.001; Fisher exact test). PIK3CA mutations were associated with strong expression of phosphorylated AKT (P < 0.001; the Mann-Whitney U test). Moreover, the expression of phosphorylated AKT was apparent in most ITPNs but only in a few IPMNs (P < 0.001; the Mann-Whitney U test). Aberrant expressions of TP53, SMAD4, and CTNNB1 were not statistically different between these neoplasms. Mutations in PIK3CA and the expression of phosphorylated AKT were not associated with age, sex, tissue invasion, and patients' prognosis in ITPNs. These results indicate that activation of the phosphatidylinositol 3-kinase pathway may play a crucial role in ITPNs but not in IPMNs. In contrast, the mutation in KRAS seems to play a major role in IPMNs but not in ITPNs. The activated phosphatidylinositol 3-kinase pathway may be a potential target for molecular diagnosis and therapy of ITPNs.

A case of solid pseudopapillary neoplasm of the pancreas presenting with left-sided extrahepatic portal hypertension.

A 53-year-old woman was referred to our hospital for management of gastric varices that ran transversely across the greater curvature of the gastric body, detected during routine upper gastrointestinal endoscopy. CT identified a low-density calcified mass near the tail of the pancreas and the splenic hilum. Based on the results of radiographic and pathological investigations, the tumor was diagnosed as solid pseudopapillary neoplasm (SPN), and the gastric varices were considered to have developed secondary to occlusion of the splenic vein by the tumor mass. This is a rare case of SPN associated with splenic vein occlusion and left-sided extrahepatic portal hypertension.