Implication of cytoplasmic p53 expression in pulmonary neuroendocrine carcinoma using next-generation sequencing analysis.

AIMS: Cytoplasmic p53 expression indicates a high frequency of TP53 abnormalities in gynaecological carcinoma. However, the implication of this expression in pulmonary neuroendocrine carcinoma (NEC) remains unclear. Thus, our study aimed to fill this research gap. METHODS AND RESULTS: Immunohistochemistry (IHC) of p53 was performed on 146 cases of resected small-cell lung carcinoma and large-cell NEC, and next-generation sequencing was conducted on cases showing cytoplasmic and wild-type p53 expression. IHC revealed overexpression in 57% of the cases (n = 83), complete absence in 31% (n = 45), cytoplasmic expression in 8% (n = 12) and wild-type expression in 4% (n = 6) of the cases. TP53 mutations were identified in nine of the 13 cases with available genetic analysis. The TP53 mutation rates in cases with cytoplasmic and wild-type p53 expression were 88% (seven of eight) and 40% (two of five), respectively. All seven cases showing cytoplasmic expression with TP53 mutations harboured loss-of-function type mutations: four had mutations in the DNA-binding domain, two in the nuclear localisation domain and one in the tetramerisation domain. Clinically, cases with cytoplasmic p53 expression had a poor prognosis similar to that in cases with p53 overexpression or complete absence. CONCLUSIONS: Cytoplasmic p53 expression in patients with pulmonary NEC suggests a high TP53 mutation rate, which is associated with a poor prognosis similar to that in patients with p53 overexpression or complete absence. This cytoplasmic expression should not be misidentified as a wild-type expression. This is the first report, to our knowledge, that demonstrates the implication of cytoplasmic p53 expression in pulmonary NEC.

Association of preoperative seizures with reduced expression of soluble CD163, an M2 macrophage marker, in the cerebrospinal fluid in isocitrate dehydrogenase wild-type glioblastoma.

PURPOSE: To investigate the relationship between the tumor microenvironment (TME), tumor-related seizures (TRS), and cerebrospinal fluid (CSF) markers that predict preoperative seizures in patients with glioblastoma. METHODS: In total, 47 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma who underwent preoperative CSF examination, 3-T magnetic resonance spectroscopy (MRS), and neurological surgery between January 2017 and December 2023 were included. We measured the concentrations of soluble CD163 (sCD163), a soluble form of the M2 macrophage marker, in the CSF, the metabolite concentration on MRS, and the number of CD163-positive M2 macrophages in the tumor tissue. Factors associated with preoperative seizures were examined. RESULTS: Twelve patients (25.5%) had preoperative seizures. sCD163 levels in the CSF were positively correlated with the number of CD163-positive M2 macrophages in the tumor tissue, and both were significantly lower in the preoperative seizure group than in the non-preoperative seizure group (p = 0.0124 and p < 0.0001, respectively). MRS indicated that only glutathione (GSH) concentrations were higher in the preoperative seizure group than in the non-preoperative seizure group (2.55 mM and 1.87 mM, respectively; p = 0.0171). CD163-positive M2 macrophages were inversely correlated with GSH levels. sCD163 in the CSF had a high predictive accuracy (sensitivity, 91.7%; specificity, 54.3%; and area under the receiver operator curve, 0.745) for preoperative seizures. CONCLUSIONS: The CSF level of sCD163 is useful for predicting the TME and preoperative seizures in IDH wild-type glioblastoma.

Colostomy-site carcinoma with primitive phenotype in a rectal cancer patient after achieving pathological complete response with neoadjuvant chemoradiotherapy.

Herein, we report a rare case of a carcinoma with primitive phenotype (enteroblastic and/or hepatoid differentiation) occurring at a colostomy site. The patient was an elderly male who underwent neoadjuvant chemoradiotherapy for rectal cancer, followed by abdominoperineal resection. A biopsy specimen for the rectal carcinoma before neoadjuvant chemoradiotherapy was conventional tubular adenocarcinoma. Moreover, a pathological complete response was confirmed in the proctectomy specimen. However, a colostomy-site tumor appeared 6 months after the proctectomy, and it was resected 1 year after the initial proctectomy. The colostomy-site tumor comprised solid to focal glandular growth of atypical polygonal cells with clear to pale eosinophilic cytoplasm and was immunohistochemically positive for cytokeratin, spalt-like transcription factor 4, glypican-3, caudal type homeobox 2, and special AT-rich sequence-binding protein 2. Thus, the tumor was diagnosed as poorly differentiated adenocarcinoma with primitive phenotype, with suggested origin from the colorectal epithelium. Additionally, a multilocular cystic lesion comprising various types of epithelia was found adjacent to the tumor, suggestive of metaplasia or heterotopia. Changes in the histology and immunophenotype, and the findings of an adjacent cystic lesion suggest a metachronous tumor rather than a recurrence of the primary tumor.

Nuclear long diameter as a new criterion to distinguish high-grade urothelial carcinoma cells from benign reactive cells.

OBJECTIVE: Recently, the nuclear area has attracted attention as a morphological parameter to differentiate high-grade urothelial carcinoma (HGUC) cells from benign reactive cells. The nuclear long diameter (NLD) strongly correlates with the nuclear area and is easy to subjectively estimate. Therefore, this study examined the usefulness of the NLD-to-neutrophil diameter ratio for detecting HGUC cells in urine cytology. METHODS: This study included 29, 26 and 18 patients with HGUC, glomerular disease and urolithiasis respectively. An image analysis system was used to measure the NLD of HGUC and benign reactive cells (reactive renal tubular cells and reactive urothelial cells) and the neutrophil diameter that appeared in the voided urine in these cases. The NLD index was calculated using the NLD-to-neutrophil diameter ratio. We subsequently compared HGUC and benign reactive cells with respect to NLD and NLD indices. In addition, the HGUC cell group and benign reactive cell group were compared by selecting the five cells with the largest NLD and NLD index on each slide. RESULTS: The NLD and NLD indices of HGUC cells were significantly higher than those of benign reactive cells in all cells and in the five cells with the largest NLD and NLD indices. The cut-off value of the NLD index for detecting HGUC cells was 1.25 in all cells and 1.80 in the five cells with the largest NLD index. CONCLUSIONS: The NLD index is a useful parameter that can be introduced into routine microscopic examinations to differentiate HGUC cells from benign reactive cells.

Association of Sarcopenia with a Poor Prognosis and Decreased Tumor-Infiltrating CD8-Positive T Cells in Pancreatic Ductal Adenocarcinoma: A Retrospective Analysis.

BACKGROUND: Sarcopenia, defined as a loss of skeletal muscle mass and quality, is found in 30-65% of patients with pancreatic ductal adenocarcinoma (PDAC) at diagnosis, and is a poor prognostic factor. However, it is yet to be evaluated why sarcopenia is associated with poor prognosis. Therefore, this study elucidated the tumor characteristics of PDAC with sarcopenia, including driver gene alterations and tumor microenvironment. PATIENTS AND METHODS: We retrospectively analyzed 162 patients with PDAC who underwent pancreatic surgery between 2008 and 2017. We defined sarcopenia by measuring the skeletal muscle mass at the L3 level using preoperative computed tomography images and evaluated driver gene alteration (KRAS, TP53, CDKN2A/p16, and SMAD4) and tumor immune (CD4+, CD8+, and FOXP3+) and fibrosis status (stromal collagen). RESULTS: In localized-stage PDAC (stage ≤ IIa), overall survival (OS) and recurrence-free survival were significantly shorter in the sarcopenia group than in the non-sarcopenia group (2-year OS 89.7% versus 59.1%, P = 0.03; 2-year RFS 74.9% versus 50.0%, P = 0.02). Multivariate analysis revealed that sarcopenia was an independent poor prognostic factor in localized-stage PDAC. Additionally, tumor-infiltrating CD8+ T cells in the sarcopenia group were significantly less than in the non-sarcopenia group (P = 0.02). However, no difference was observed in driver gene alteration and fib.rotic status. These findings were not observed in advanced-stage PDAC (stage ≥ IIb). CONCLUSIONS: Sarcopenia was associated with a worse prognosis and decreased tumor-infiltrating CD8+ T cells in localized-stage PDAC. Sarcopenia may worsen a patient's prognosis by suppressing local tumor immunity.

Lobularity rather than hyperechoic foci/stranding on endoscopic ultrasonography is associated with more severe histological features in chronic pancreatitis.

BACKGROUND AND AIM: Endoscopic ultrasonography (EUS) findings of the pancreatic parenchyma, such as hyperechoic foci/stranding and lobularity, may be associated with the severity of chronic pancreatitis (CP). However, the correlation between parenchymal EUS findings and histology remains unclear. We designed a large-scale retrospective study analyzing over 200 surgical specimens to elucidate the association between parenchymal EUS findings and histological features. METHODS: Clinical data of 221 patients with pancreatobiliary tumors who underwent preoperative EUS and pancreatic surgery between January 2010 and November 2020 were reviewed to investigate the association between parenchymal EUS findings and histological features at the pancreatic body. None of these patients met the definition of CP. RESULTS: Of the 221 patients, 87 (39.4%), 89 (40.2%), and 45 (20.4%) had normal EUS findings, hyperechoic foci/stranding without lobularity, and hyperechoic foci/stranding with lobularity, respectively. In the multivariate analyses, parenchymal EUS findings significantly correlated with histological CP findings of fibrosis, inflammation, and atrophy (hyperechoic foci/stranding without lobularity vs hyperechoic foci/stranding with lobularity, odds ratio [95% confidence interval]: 4.1 [2.2-7.9] vs 31.3 [9.3-105.6], Ptrend  < 0.001; 3.9 [1.9-8.2] vs 21.8 [8.0-59.4], Ptrend  < 0.001; and 4.0 [2.0-7.8] vs 22.9 [7.0-74.5], Ptrend  < 0.001, respectively). Further, a trend toward higher histological grade was observed in the following order: normal findings, hyperechoic foci/stranding without lobularity, and hyperechoic foci/stranding with lobularity. CONCLUSIONS: Endoscopic ultrasonography findings of the pancreatic parenchyma may be associated with the histological conditions in CP, such as pancreatic fibrosis, inflammation, and atrophy. Lobularity reflects more severe histological conditions than does hyperechoic foci/stranding.

The usefulness of nuclear area in the diagnosis of high-grade urothelial carcinoma cells in voided urine cytology.

OBJECTIVE: The Paris System for Reporting Urinary Cytology considered the nuclear-to-cytoplasmic (N:C) ratio as the most important cytomorphological feature for detecting high-grade urothelial carcinoma (HGUC) cells. Few quantitative studies have been conducted on other features although quantitative studies on the N:C ratio have been reported. Therefore, this study quantitatively analysed important cytomorphological features in distinguishing benign reactive cells from HGUC cells. METHODS: We analysed 2866 cells from the urine of 52 patients. A digital image analyser was used to quantitatively measure the nuclear area, cell area, N:C ratio, and nuclear roundness for HGUC cells and benign reactive cells. Additionally, the diagnostic value of quantitative cytomorphological criteria in HGUC cells was evaluated by the receiver operating characteristic curve. RESULTS: The area under the curve for the prediction of HGUC cells for all cells and the top five cells was in the following order: nuclear area (0.920 and 0.992, respectively), N:C ratio (0.849 and 0.977), cell area (0.781 and 0.920), and nuclear roundness (0.624 and 0.605). The best cutoff value of the N:C ratio to differentiate HGUC cells from benign reactive cells was 0.438, and using the N:C ratio of 0.702, the positive predictive value obtained was 100%. CONCLUSIONS: Our study indicated that nuclear area is a more important cytomorphological criterion than the N:C ratio for HGUC cell detection. Moreover, extracted data of the top five cells were more valuable than the data of all cells, which can be helpful in the routine practice and future criteria definition in urine cytology.

Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B.

Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine.

Comprehensive Analysis of Molecular Biologic Characteristics of Pancreatic Ductal Adenocarcinoma Concomitant with Intraductal Papillary Mucinous Neoplasm.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) concomitant with intraductal papillary mucinous neoplasm (IPMN) is defined as PDAC occurring apart from IPMN. This study comprehensively investigated the molecular biologic characteristics of PDAC concomitant with IPMN in major genetic alterations, tumor microenvironment, and prognosis by contrast with those of conventional PDAC. METHODS: The study retrospectively reviewed the data of 158 surgically resected PDAC patients. The driver gene alteration status (KRAS, TP53, CDKN2A, SMAD4, and GNAS) together with the immune and fibrotic status in tumor was evaluated. The prognosis of PDAC concomitant with IPMN and that of conventional PDAC also were compared. RESULTS: No statistically significant difference was found between PDAC concomitant with IPMN and conventional PDAC in the alteration frequency analysis of the major driver genes and the immune and fibrotic status in the tumor microenvironment. Overall survival and disease-free survival between patients who had PDAC concomitant with IPMN and those who had conventional PDAC did not show statistically significant differences in propensity-matched subjects. Furthermore, the co-existence of IPMN was not a poor prognostic factor in the multivariable-adjusted Cox proportional hazards model (hazard ratio, 0.95; 95 % confidence interval, 0.51-1.78). CONCLUSIONS: In this study, PDAC concomitant with IPMN had tumor characteristics similar to those of conventional PDAC in terms of the major driver gene alterations, tumor microenvironment, and prognosis.

An Animal Model That Mimics Human Herpesvirus 6B Pathogenesis.

Human herpesvirus 6B (HHV-6B), a T-lymphotropic virus, infects almost exclusively humans. An animal model of HHV-6B has not been available. Here, we report the first animal model to mimic HHV-6B pathogenesis; the model is based on humanized mice in which human immune cells were engrafted and maintained. For HHV-6B replication, adequate human T-cell activation (which becomes susceptible to HHV-6B) is necessary in this murine model. Here, we found that an additional transfer of human mononuclear cells to humanized mice resulted in an explosive proliferation of human activated T cells, which could be representative of graft-versus-host disease (GVHD) because the primary transfer of human cells was not sufficient to increase the number and ratio of human T cells. Mice infected with HHV-6B became weak and/or died approximately 7 to 14 days later. Quantitative PCR analysis revealed that the spleen and lungs were the major sites of HHV-6B replication in this model, and this was corroborated by the detection of viral proteins in these organs. Histological analysis also revealed the presence of megakaryocytes, indicating HHV-6B infection. Multiplex analysis of cytokines/chemokines in sera from the infected mice showed secretions of human cytokines/chemokines as reported for both in vitro infection and clinical samples, indicating that the secreted cytokines could affect pathogenesis. This is the first animal model showing HHV-6B pathogenesis, and it will be useful for elucidating the pathogenicity of HHV-6B, which is related to GVHD and idiopathic pneumonia syndrome.IMPORTANCE Human herpesvirus 6B (HHV-6B) is a ubiquitous virus that establishes lifelong latent infection only in humans, and the infection can reactivate, with severe complications that cause major problems. A small-animal model of HHV-6B infection has thus been desired for research regarding the pathogenicity of HHV-6B and the development of antiviral agents. We generated humanized mice by transplantation with human hematopoietic stem cells, and here, we modified the model by providing an additional transfer of human mononuclear cells, providing the proper conditions for efficient HHV-6B infection. This is the first humanized mouse model to mimic HHV-6B pathogenesis, and it has great potential for research into the in vivo pathogenesis of HHV-6B.

Sunitinib decreases the expression of KRT6A and SERPINB1 in 3D human epidermal models.

Hand-foot skin reaction (HFSR) is a common side effect caused by several tyrosine kinase inhibitors, including sunitinib. However, the nature of the cornifying factors related to the molecular biological mechanisms underlying HFSR remains poorly understood. We used human keratinocyte models to investigate the key cornifying factors for dermatological and biological abnormalities induced by sunitinib. On the basis of the results of microarray analysis using the three-dimensional (3D) human epidermal model, keratin (KRT)6A, serine protease inhibitor (SERPIN)B1, KRT5, and SERPIN Kazal-type 6 were selected as candidate genes related to HFSR. Sunitinib treatment significantly decreased the expression of SERPINB1 and KRT6A in the immunohistochemical staining of the 3D epidermal model. In PSVK1 cells, but not in normal human epidermal keratinocyte cells, both of which are human normal keratinocyte cell lines, sunitinib decreased the expression of KRT6A with a concomitant decrease in levels of phosphorylated extracellular signal-regulated kinases (ERK)1/2 and phosphorylated p38 mitogen-activated protein kinase (MAPK). Inhibitors of the ERK and p38 MAPK signal pathways also significantly decreased KRT6A expression. Sunitinib-induced decrease in KRT6A expression was suppressed by the inhibition of glycogen synthase kinase-3ß by enhancing ERK1/2 and p38 MAPK phosphorylation. Thus, sunitinib reduces the expression of KRT6A and SERPINB1 by inhibiting the ERK1/2 and p38 MAPK signalling pathways in the skin model. These changes in expression contribute to the pathology of HFSR.

Acute pancreatitis in intraductal papillary mucinous neoplasms correlates with pancreatic volume and epithelial subtypes.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is associated with acute pancreatitis (AP) in some cases, however its causes have not been fully elucidated. We investigated the association of the incidence of AP with epithelial subtypes and pancreatic volume in IPMN. METHODS: This retrospective study included 182 consecutive surgically resected IPMN patients between January 2000 and December 2018. The relationship between the incidence of AP and epithelial subtypes of IPMN and pancreatic volume was investigated. Epithelial subtypes of IPMN were classified into gastric (G type: N = 116), intestinal (I type: N = 49), pancreatobiliary (PB type: N = 14), and oncocytic types (O type: N = 3). Pancreatic volume of the contrast-enhanced computed tomography scan was measured using Ziostation2 software. Histological pancreatic parenchymal atrophy was also evaluated. RESULTS: AP occurred more frequently in I-types (I-type vs. G-type, 22.4% [11/49] vs 3.4% [4/116], P = 0.003) and PB-types (PB type vs. G-type, 35.7% [5/14] vs. 3.4% [4/116], P = 0.007) in comparison with G-types, which constituted the majority of the resected IPMNs. AP occurred more frequently in I-type patients with high pancreatic volumes (I-type with high pancreatic volume vs. I-type with low pancreatic volume, 37.0% [10/27] vs. 4.7% [1/21], P = 0.02). However, histological atrophy did not show an additional influence on the association between the incidence of AP and epithelial subtypes. The elevation of serum pancreatic enzymes was not significantly related to epithelial subtypes. CONCLUSION: Epithelial subtypes and the degree of pancreatic volume may be closely associated with the incidence of AP in IPMN.

Hyperintense signal on diffusion-weighted imaging for monitoring the acute response and local recurrence after photodynamic therapy in malignant gliomas.

PURPOSE: Photodynamic therapy (PDT) subsequent to surgical tumor removal is a novel localized treatment for malignant glioma that provides effective local control. The acute response of malignant glioma to PDT can be detected as linear transient hyperintense signal on diffusion-weighted imaging (DWI) and a decline in apparent diffusion coefficient values without symptoms. However, their long-term clinical significance has not yet been examined. The aim of this study was to clarify the link between hyperintense signal on DWI as an acute response and recurrence after PDT in malignant glioma. METHODS: Thirty patients (16 men; median age, 60.5 years) underwent PDT for malignant glioma at our institution between 2017 and 2020. We analyzed the signal changes on DWI after PDT and the relationship between these findings and the recurrence pattern. RESULTS: All patients showed linear hyperintense signal on DWI at the surface of the resected cavity from day 1 after PDT. These changes disappeared in about 30 days without any neurological deterioration. During a mean post-PDT follow-up of 14.3 months, 19 patients (63%) exhibited recurrence: 10 local, 1 distant, and 8 disseminated. All of the local recurrences arose from areas that did not show hyperintense signal on DWI obtained on day 1 after PDT. CONCLUSIONS: The local recurrence in malignant glioma after PDT occurs in an area without hyperintense signal on DWI as an acute response to PDT. This characteristic finding could aid in the monitoring of local recurrence after PDT.

Pineal gland differentiation in mature teratoma: An under-recognized condition and potential pitfalls for overdiagnosis.

We herein report three cases of mature teratomas with pineal gland differentiation, which is a less recognized phenomenon. Case 1 was a 6-year-old male with a neck mass, Case 2 was a 23-year-old female with a retroperitoneal mass, and Case 3 was a 45-year-old female with a retroperitoneal mass. Each case showed the typical macroscopic and histological findings of mature teratoma, such as solid and cystic lesions mainly lined with a mature squamous epithelium. All cases also showed glial differentiation. Small foci of lobulated cell nests were detected in the center of or adjacent to mature glial tissue. Cells had a clear to pale eosinophilic cytoplasm with small round nuclei. Immunohistochemically, cells were positive for synaptophysin, neurofilament protein with a perivascular "club-shaped swelling" pattern, and cone-rod homeobox protein. To the best of our knowledge, this is the first report of pineal gland differentiation arising in mature teratoma, which may be easily overlooked or misdiagnosed as somatic-type tumors, particularly neuroendocrine tumors. To avoid overtreatment, pathologists need to be aware that pineal gland differentiation may occur in mature teratomas.

Quantitative cytomorphological comparison of SurePath and ThinPrep liquid-based cytology using high-grade urothelial carcinoma cells.

OBJECTIVE: In The Paris System for Reporting Urinary Cytology (TPS), the important cytomorphological features for diagnosing high-grade urothelial carcinoma (HGUC) are a nuclear-to-cytoplasmic (N:C) ratio exceeding 0.7, hyperchromasia, coarse chromatin, and irregular nuclear borders. However, quantitative cytomorphological assessments of HGUC cells using SurePath slides are rare. Therefore, we evaluated HGUC cells on SurePath slides quantitatively using a digital image analysis system and compared these data with ThinPrep data. METHODS: The same urine samples were divided into two aliquots and used to prepare SurePath and ThinPrep slides. We used ImageJ to measure the N:C ratio, hyperchromasia, and irregular nuclear borders for HGUC cells on SurePath and ThinPrep slides. RESULTS: The total number of analysed HGUC cells on SurePath slides was 981, versus 889 on ThinPrep slides. Hyperchromasia and irregular nuclear borders were significantly more severe on SurePath than on ThinPrep slides. Conversely, the N:C ratio did not differ between the methods. Additionally, HGUC cells with N:C ratios exceeding 0.7 were present on almost all slides for both methods. CONCLUSIONS: Our data indicated the reasonableness of using the N:C ratio as the major criterion for TPS on both SurePath and ThinPrep slides, and an N:C ratio cut-off of 0.7 as suitable for identifying HGUC cells. However, the severity of hyperchromasia and irregular nuclear borders differed between the processing methods.

A novel EWSR1-VGLL1 gene fusion in a soft tissue malignant myoepithelial tumor.

Soft tissue myoepithelial tumors are very rare mesenchymal tumors that are currently categorized as miscellaneous neoplasms with uncertain differentiation. Although the molecular pathogenesis of soft tissue myoepithelial tumors remains unclear, EWSR1 gene fusions with a variety of partner genes are regarded as one of the major pathogenic driver events in these tumors. We herein present a case of a deep soft tissue malignant myoepithelial tumor arising in the thigh with multiple pulmonary metastases. This tumor displayed diverse and unique histological features, namely, an epithelioid glandular growth pattern, pseudorosette-like formation, and a diffuse nest and cord-like pattern within an abundant myxoid matrix. Next-generation RNA sequencing identified a novel fusion transcript, in which the in-frame junctional reads contained exon 9 of EWSR1 and exon 2 of VGLL1, resulting in the formation of a putative chimeric protein with the N-terminal transcriptional activation domain of EWSR1 and C-terminal full length of the VGLL1 protein. EWSR1-VGLL1 fusion has not been described in neoplasm before. Further molecular and functional experiments on the present EWSR1-VGLL1 fusion gene are required to elucidate its tumorigenic effect.

Biliary intraductal tubule-forming neoplasm: a whole exome sequencing study of MUC5AC-positive and -negative cases.

AIMS: Biliary intraductal tubular neoplasms that are non-mucinous and negative for mucin 5AC (MUC5AC) are called intraductal tubulopapillary neoplasms (ITPNs). Intraductal tubular neoplasms with mucinous cytoplasm and MUC5AC positivity also occur and their nature remains unclear, although some pathologists may classify these as 'intraductal papillary neoplasms of the bile duct (IPNBs) of gastric type'. This study aimed to elucidate genetic features of biliary intraductal tubular neoplasms. METHODS AND RESULTS: Six resected cases of biliary intraductal neoplasm with >70% tubular configuration were characterised by clinicopathological examination and whole exome sequencing, and the findings obtained were compared between MUC5AC-negative (n = 2) and -positive cases (n = 4). The intraductal tumours consisted of the pancreatobiliary-type epithelium with high-grade dysplasia arranged in back-to-back tubules. Both of the two MUC5AC-negative cases were non-invasive neoplasms and developed in the liver, whereas all MUC5AC-positive cases had invasive carcinoma and were present in the intrahepatic (n = 2), perihilar (n = 1) and distal bile ducts (n = 1). In an exome-sequencing study, MUC5AC-negative cases harboured mutations in CTNNB1, SF3B1, BAP1 and BRCA1 (one case each). KRAS mutations were observed in three of four MUC5AC-positive cases (75%) but none of the MUC5AC-negative neoplasms. Compared to published data, known driver genes of other intraductal neoplasms of the pancreatobiliary system (e.g. APC, CTNNB1, STK11, GNAS and PIK3CA) were wild-type in all but one MUC5AC-negative case with CTNNB1 mutation. Chromatin modifiers (ARID1A, BAP1 and KMT2C) were also altered in MUC5AC-positive cases, similar to usual cholangiocarcinomas. CONCLUSIONS: This exome-sequencing study suggested that MUC5AC-negative biliary ITPNs are genetically distinct from pancreatic ITPNs and IPNBs. They may also biologically differ from MUC5AC-positive tubular neoplasms despite morphological resemblance.

Insulinoma-associated protein 1 is a prognostic biomarker in pulmonary high-grade neuroendocrine carcinoma.

BACKGROUNDS AND OBJECTIVES: Recent studies have suggested that insulinoma-associated protein 1 (INSM1) is a useful marker for pathological diagnosis of pulmonary neuroendocrine tumors. In the present study, we investigated the association between INSM1 expression and prognosis in patients with pulmonary high-grade neuroendocrine carcinomas (HGNEC) and assessed the usefulness of INSM1 as a prognostic biomarker in these patients. METHODS: Seventy-five consecutive patients with HGNEC who underwent complete surgical resections from January 2000 to December 2018 were enrolled in this study. We classified these patients into two groups: the INSM1-positive group (n = 59) and INSM1-negative group (n = 16). We compared the clinicopathological characteristics, overall survival (OS), and recurrence-free survival (RFS) between the groups. In addition, we performed univariate and multivariate analyses to identify the prognostic factors associated with postoperative survival. RESULTS: Significant differences in tumor diameter and vascular invasion between the groups were found. OS and RFS were significantly poorer in the INSM1-positive group than in the INSM1-negative group. Univariate and multivariate analyses revealed that INSM1 expression was the strongest predictor of poor prognosis for OS and RFS. CONCLUSIONS: INSM1 expression had the greatest influence on the prognosis in patients with HGNEC and may be a prognostic biomarker in these patients.

Keratin 19-expressing hepatocellular carcinoma and small-duct type intrahepatic cholangiocarcinoma show a similar postoperative clinical course but have distinct genetic features.

AIMS: The present study aimed to systematically compare clinicopathological and genetic features between keratin 19 (K19)-expressing hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). METHODS AND RESULTS: Consecutive cases of HCC (n = 430) were classified into K19+ and K19- using immunohistochemistry. ICCA cases were also separated into small-(S-iCCA; n = 36) and large-duct types (n = 22) based on recently proposed criteria, with the former being used in the present study. Mutational hot-spots in TERT, CTNNB1, KRAS and IDH1 were sequenced. Twenty-six cases (6%) of HCC expressed K19. K19+ HCC was more strongly associated with chronic hepatitis B than K19- HCC and S-iCCA (46% versus 17% and 6%; both P < 0.001). Lymph node metastasis was observed in K19+ HCC (8%) and S-iCCA (22%), but was exceptional in K19- HCC (1%). K19+ HCC had TERT promoter mutations less frequently than K19- HCC (31% versus 59%; P = 0.022), and lacked alterations in KRAS and IDH1. CTNNB1 mutations were similarly observed in K19+ and K19- HCC (23% and 19%, respectively), but rare in S-iCCA (3%). The postoperative survival curve of K19+ HCC was almost identical to that of S-iCCA in the first 5 years (approximately 50% at 5 years), and significantly worse than that of K19- HCC (P = 0.040). Extrahepatic recurrence was more common in K19+ HCC (50%) and S-iCCA (35%) than in K19- HCC (15%) (P = 0.001). CONCLUSIONS: Although K19+ HCC and S-iCCA showed similar biological behaviours, they did not share any driver gene mutations, suggesting the possible involvement of epigenetic alterations in the iCCA-like features of K19+ HCC.

IL-33 overexpression in gallbladder cancers associated with pancreatobiliary maljunction.

AIMS: To investigate whether genetic or inflammatory pro-oncogenic factors are relevant to the increased risk of gallbladder cancers in patients with pancreaticobiliary maljunction (PBM). METHODS AND RESULTS: Mutations in KRAS exon 2 were examined by a highly sensitive, droplet digital PCR platform using surgically resected specimens of PBM-associated (n = 31) and non-associated gallbladder cancers (n = 49). The tissue expression of IL-6 and IL-33, which are suspected to promote biliary carcinogenesis, was analysed by quantitative real-time PCR and in-situ hybridisation. The incidence of KRAS mutations was similarly low in PBM-associated (five of 32 cases; 16%) and non-associated cancers (four of 49 cases; 8%) (P = 0.272). The tissue expression of IL-33 mRNA, but not IL-6 mRNA, was significantly higher in PBM-associated gallbladder cancers than in gallbladder cancers without PBM (P = 0.004). A similar degree of IL-33 overexpression was also observed in the background non-cancerous mucosa in cases of PBM-associated gallbladder cancers, and was significantly greater than that in PBM cases with cholecystitis alone (P < 0.001). The results of in-situ hybridisation indicated that the source of IL-33 production in PBM-associated carcinomas was the endothelium, cancer cells and non-neoplastic biliary epithelium. In a combined PBM-associated and non-associated cohort, IL-33 overexpression in gallbladder cancers correlated with less aggressive features (e.g. a lower pT stage and longer overall survival), similar to recently reported findings on large-duct cholangiocarcinomas. CONCLUSIONS: KRAS mutations do not appear to be associated with a high risk of malignancy in PBM, while IL-33 overexpression may provide a pro-oncogenic microenvironment in the gallbladder mucosa of patients with PBM.