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Aims: Traditional serrated adenoma (TSA) is an uncommon type of colorectal serrated polyp. RSPO fusions, which potentiate WNT signalling, are common and characteristic genetic alterations in TSA. The aim of this study was to further characterise the prevalence and variation of RSPO fusions in TSA., Methods and Results: Quantitative polymerase chain reaction (PCR) analysis of 99 TSAs revealed overexpression of RSPO2 and RSPO3 in six and 29 lesions, respectively. Reverse transcription PCR identified previously reported PTPRK-RSPO3 fusion transcripts in all 29 TSAs with RSPO3 overexpression, confirming that PTPRK-RSPO3 is the predominant RSPO fusion in TSAs. Among the six lesions with RSPO2 overexpression, two overexpressed full-length RSPO2. An EIF3E-RSPO2 fusion, which is a known recurrent RSPO fusion in colorectal cancer, was detected in three lesions. In addition, rapid amplification of cDNA ends identified a novel PIEZO1-RSPO2 fusion in one TSA. All of the four TSAs with RSPO2 fusions concurrently had KRAS mutations and showed the classic histological features., Conclusions: The present study identified EIF3E-RSPO2 and PIEZO1-RSPO2 in TSAs. Our observations expand the spectrum of RSPO fusions in TSAs, and suggest that TSAs are precursors of colorectal cancers with these RSPO2 fusions., (© 2019 John Wiley & Sons Ltd.)

Aims: Sessile serrated adenoma/polyp (SSA/P) is regarded as a genetically homogeneous entity, with most lesions harbouring the BRAF V600E mutation. The present study aimed to reappraise the genetic heterogeneity of SSA/Ps and its clinicopathological significance., Methods and Results: We performed next-generation sequencing of 272 SSA/Ps without dysplasia and evaluated morphological and molecular features associated with the respective genotypes. BRAF V600E, BRAF non-V600E, KRAS and NRAS mutations were found in 223 (82.0%), three (1.2%), 28 (10.3%) and one lesion (0.4%), respectively. Notably, all lesions with BRAF non-V600E mutations had either KRAS or NRAS mutations concurrently. Twenty SSA/Ps (7.4%) were negative for these mutations. KRAS-mutated SSA/Ps were located more often in the distal colon (42%) compared to those with the BRAF V600E mutation (14%). Histologically, minimally serrated crypts and goblet cell-rich crypts were more common in KRAS-mutated and mutation-negative SSA/Ps. However, in most instances, SSA/Ps lacking the BRAF V600E mutation were indistinguishable morphologically from those with the BRAF V600E mutation. MUC5AC and MUC6 expression was common regardless of the mutation status, but more extensive in SSA/Ps with the BRAF V600E mutation. CpG island methylator phenotype-high was more frequent in SSA/Ps with the BRAF V600E mutation (60%), followed by mutation-negative SSA/Ps (40%) and KRAS-mutated SSA/Ps (16%)., Conclusions: The present study confirmed the common presence of the BRAF V600E mutation in SSA/Ps, but also demonstrated a degree of molecular heterogeneity of SSA/Ps. SSA/Ps with and without the BRAF V600E mutation showed slightly different but overlapping histological and molecular features., (© 2018 John Wiley & Sons Ltd.)

Aims: Traditional serrated adenoma (TSA) is a rare but distinct type of colorectal polyp. Our previous study showed that PTPRK-RSPO3 fusions are frequent and characteristic genetic alterations in TSAs. This study aimed to characterize comprehensively the prevalence and variability of RSPO fusions in colorectal TSAs., Methods and Results: We examined RSPO expression and explored novel RSPO fusions in 129 TSAs, including 66 lesions analysed previously for WNT pathway gene mutations. Quantitative polymerase chain reaction (qPCR) analyses identified three and 43 TSAs overexpressing RSPO2 and RSPO3, respectively, whereas the expression of RSPO1 and RSPO4 was marginal or undetectable in all cases. RSPO overexpression was always mutually exclusive with other WNT pathway gene mutations. Known PTPRK-RSPO3 fusions were detected in 37 TSAs, all but one of which overexpressed RSPO3. In addition, rapid amplification of cDNA ends revealed three novel RSPO fusion transcripts, an NRIP1-RSPO2 fusion and two PTPRK-RSPO3 fusion isoforms, in six TSAs. Overall, 43 TSAs had RSPO fusions (33%), whereas four TSAs (3%) overexpressed RSPO in the absence of RSPO fusions. TSAs with RSPO fusions showed several clinicopathological features, including distal localization (P = 0.0063), larger size (P = 0.0055), prominent ectopic crypt foci (P = 8.4 × 10 -4 ), association of a high-grade component (P = 1.1 × 10 -4 ), and the presence of KRAS mutations (P = 4.5 × 10 -5 )., Conclusions: The present study identified RSPO fusion transcripts, including three novel transcripts, in one-third of colorectal TSAs and showed that PTPRK-RSPO3 fusions were the predominant cause of RSPO overexpression in colorectal TSA., (© 2017 John Wiley & Sons Ltd.)

Aims: The aim of this study was to examine the expression of mismatch repair (MMR) proteins in Lynch syndrome (LS)-associated colorectal adenomas and to evaluate their relationship with clinicopathological variables and potential utility in LS screening., Methods and Results: We performed immunohistochemistry for MLH1, PMS2, MSH2 and MSH6 in 134 adenomas obtained from 26 genetically confirmed LS patients. MMR deficiency, as determined by loss of any MMR protein, was observed in 113 adenomas (84%). All the MMR-deficient adenomas exhibited homogeneous loss of MMR proteins, which reflected underlying germline mutations. MMR deficiency was more frequent in adenomas obtained from older patients (aged ≥60 years; 81 of 86, 94%), with larger tumour size (>5 mm; 71 of 73, 97%) and with high-grade dysplasia (50 of 51, 98%). Multivariate analyses indicated that increased age and larger tumour size were associated independently with MMR deficiency., Conclusions: This study shows that MMR deficiency is associated significantly with increased age, in addition to two previously reported factors-larger size and high-grade dysplasia. When adenomas are analysed during LS screening, high sensitivity is expected if the adenomas are associated with any of these three factors., (© 2016 John Wiley & Sons Ltd.)

Aims: Pyloric gland adenoma (PGA) is a unique gastric neoplasm expressing mucin 6 (MUC6), and is often associated with high-grade dysplasia and/or adenocarcinoma. MUC6 secreted from the gastric gland mucous cells, such as pyloric gland cells, carries unique O-glycans with terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues on its molecule. As we recently demonstrated that αGlcNAc serves as a tumour suppressor for gastric adenocarcinoma, this study aimed to investigate the significance of αGlcNAc expression in PGA., Methods and Results: Eighteen patients with PGA were examined with immunohistochemistry for αGlcNAc and MUC6. αGlcNAc and MUC6 were coexpressed in 12 of 18 PGAs. However, reduced αGlcNAc expression relative to MUC6 expression was observed in six cases. When the MIB-1 labelling index (LI) of tumour cells was examined with respect to reduced αGlcNAc expression, the MIB-1 LI was significantly higher in PGAs showing decreased αGlcNAc expression relative to MUC6 expression than in PGAs with unchanged αGlcNAc expression (P = 0.023)., Conclusions: The present study indicates that coexpression of αGlcNAc and MUC6 in PGA suggests the presence of fully glycosylated MUC6 on tumour cells, consistent with pyloric gland differentiation. However, the decreased glycosylation of αGlcNAc on MUC6 is associated with high mitotic activity of tumour cells, indicative of malignant potential of PGA., (© 2015 John Wiley & Sons Ltd.)

Aims: Familial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome caused by a germline APC mutation. A recent study showed the enrichment of pyloric gland adenomas (PGAs) of the stomach, in addition to fundic gland polyps (FGPs) and foveolar-type adenomas (FAs), in patients with FAP. In the present study, we analysed the genetic alterations in these FAP-associated gastric lesions., Methods and Results: Mutational statuses of GNAS and KRAS, which are frequently mutated in sporadic PGAs, as well as those of APC, were examined in PGAs, FAs and FGPs in patients with FAP using Sanger sequencing. Our analysis identified GNAS mutations in five of six PGAs (83%), but in none of the three FAs or the 40 FGPs examined. KRAS mutations were identified in four PGAs (67%), one FA (33%) and one FGP (3%). Somatic truncating APC mutations were found in all PGAs (100%), two FAs (67%) and 14 FGPs (47%). We additionally analysed sporadic PGAs of the stomach and duodenum and identified truncating APC mutations in 11 of 25 lesions (44%)., Conclusions: FAP-associated and sporadic PGAs not only show similar morphologies, but also share common genetic aberrations, including mutations of GNAS, KRAS and APC., (© 2015 John Wiley & Sons Ltd.)

Aims: The purpose of this study was to compare the immunohistochemical staining profiles of PAX8-polyclonal, PAX8-monoclonal, PAX5-monoclonal and PAX6-monoclonal antibodies in several histological types of primary thoracic and thyroid tumours. In addition, we analysed PAX8 mRNA expression by using in-situ hybridization., Methods and Results: We compared polyclonal PAX8 and monoclonal PAX8, PAX5 and PAX6 antibodies in 962 samples (687 lung carcinomas, 40 malignant pleural mesotheliomas, 138 thymic tumours and 97 thyroid tumours) using the tissue microarray technique. Among thyroid tumours, the monoclonal and polyclonal PAX8 antibodies showed a high positive rate (98.0%). Of 167 polyclonal PAX8 antibody-positive tumours, except for thyroid tumours, 54 cases tested positive for PAX5 and/or PAX6 (31 lung carcinomas and 23 thymic tumours). No PAX8 mRNA expression was detected using RNAscope (in-situ hybridization technique) other than in thyroid tumours. A portion of polyclonal PAX8 antibody-positive tumours showed cross-reactivity for PAX5 or PAX6 protein., Conclusions: Monoclonal PAX8 antibody showed high specificity to thyroid tumours and was superior to the polyclonal antibody., (© 2014 John Wiley & Sons Ltd.)

Aims: To determine HER2 amplification status and HER2 overexpression status in neuroendocrine carcinomas (NECs) of the stomach., Methods and Results: We analysed 51 gastric NECs, including 15 pure NECs and 36 NECs associated with adenocarcinoma and/or dysplasia, for HER2 amplification by dual-colour chromogenic in-situ hybridization, and for HER2 expression by immunohistochemistry. HER2 amplification was observed in three NECs (6%) and in seven (19%) cases of adenocarcinoma/dysplasia associated with NEC. Immunohistochemically, all of the NECs, including those showing HER2 amplification, lacked HER2 expression. On the other hand, positive and equivocal HER2 overexpression was observed in three (8%) and six (17%) cases of adenocarcinoma/dysplasia associated with NEC, respectively., Conclusions: HER2 expression is consistently absent in gastric NECs, regardless of HER2 amplification status or association with HER2-positive adenocarcinoma/dysplasia components. Accordingly, HER2 is unlikely to be a valid therapeutic target in gastric NECs. Also, the absence of HER2 expression in NECs could be one of the causes of intratumoral heterogeneity of HER2 expression in gastric cancers., (© 2013 John Wiley & Sons Ltd.)

Aims: To determine the prevalence of MED12 mutations in smooth muscle tumours of different organs., Methods and Results: A total of 142 smooth muscle tumours of the uterus, gastrointestinal tract, retroperitoneum and soft tissue were analysed for MED12 mutations using Sanger sequencing. Among the uterine tumours that were examined, MED12 mutations were identified in 36 of 45 conventional leiomyomas (80%), two of six cellular leiomyomas (33%), one of four bizarre leiomyomas (25%), none of four lipoleiomyomas (0%), and two of 12 leiomyosarcomas (17%). The two MED12-mutated leiomyosarcomas were associated with benign leiomyomatous components that also harboured MED12 mutations identical to those in the respective leiomyosarcomatous components. None of the extrauterine smooth muscle tumours, including the leiomyomas, leiomyosarcomas, and angioleiomyomas, had MED12 mutations., Conclusions: Among uterine smooth muscle tumours, MED12 mutations are frequently present in conventional leiomyomas, but are significantly less common in histological variants of leiomyoma and leiomyosarcoma. In contrast to uterine lesions, none of the extrauterine smooth muscle tumours had MED12 mutations., (© 2012 Blackwell Publishing Ltd.)

Aims: Mucinous adenocarcinoma (MUC) is a histological variant of colorectal adenocarcinoma. The aim of the present study was to characterize clinicopathological features and identify prognostic factors of MUCs., Methods and Results: A total of 181 patients with MUC who underwent surgery between 1975 and 2003 were reviewed. The clinicopathological features of these patients were compared with those of 4125 non-MUC patients. Univariate and multivariate analyses were conducted to identify significant prognostic factors in 102 patients with pT3 or pT4 tumour who underwent curative surgery. Patients with MUCs tended to present with more advanced clinical stages. The overall 5-year survival rate of MUC patients was lower than that of non-MUC patients; however, no prognostic difference was found when patients with the same clinical stages were compared. Multivariate analysis revealed male sex, bowel obstruction and infiltrating growth type as independent prognostic factors. Five-year cancer-specific survival rates for MUC patients with ≤1, 2 and 3 risk factors identified by multivariate analysis were 95.5%, 52.1% and 0.0%, respectively (P < 0.001)., Conclusions: Mucinous adenocarcinoma represents a distinct clinicopathological entity. Sex, bowel obstruction and growth patterns might be useful prognostic factors to identify patients with a high risk of recurrence after curative resection of advanced MUCs., (© 2012 Blackwell Publishing Ltd.)

Aims: α-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs., Methods and Results: The expression of AMACR and GLUL, an established downstream target of β-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production., Conclusions: The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of β-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of β-catenin in bile acid metabolism., (© 2011 Blackwell Publishing Limited.)