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Bronchiolar adenoma (BA) is a novel entity in the 2021 WHO classification of lung tumours. The expression profile of mucin core proteins in BAs is not clear. The aim of this study was to clarify the expression profiles of mucins and to validate the clinicopathologic and molecular features of BAs.We examined the clinicopathological, immunohistochemical, and molecular features of 20 BAs. Our cohort comprised 10 proximal and 10 distal BAs. Only seven of 18 patients (39%) were accurately diagnosed with BA at the time of intraoperative consultation. The frequent genetic alterations were BRAF V600E (35%) and KRAS (30%) mutations, which were mutually exclusive. The expression of MUC1, MUC4, and MUC5B was observed in all cases and that of MUC5AC and MUC6 was observed in nine (45%) and five (25%) cases, respectively. MUC4 was diffusely expressed in 18 cases. In contrast, MUC1, MUC5AC, MUC5B, and MUC6 displayed a patchy expression pattern. These expression patterns were similar to that of bronchiolar epithelium in normal lung tissue. In addition, overexpression of MUC1 and MUC4 on the entire cell surface was not observed in any of the BAs, suggesting their benign nature.BA commonly exhibits diffuse MUC4 and patchy MUC1 and MUC5B expression. Its unique expression pattern is probably attributed to mucin expression specific to the bronchiolar epithelium. These results confirm the clinicopathologic and molecular characteristics of BA, including the difficulty in intraoperative frozen section diagnosis and the broad morphologic spectrum of BA derived from the bronchiolar epithelium.

The clinicopathological differences among high-grade fetal lung adenocarcinomas completely comprising tumor cells that resemble fetal lung epithelium (pure type) and those with fetal lung-like components admixed with conventional adenocarcinoma cells (mixed type) remain undetermined. Here, we examined the clinicopathological, immunohistochemical, and molecular features of 11 lung adenocarcinomas with fetal lung-like morphology among 3895 consecutive cases of primary lung cancer based on the expression pattern of transcription factors. According to the current WHO classification, two cases (0.05%) were categorized as low-grade fetal adenocarcinoma, two cases (0.05%) were pure-type high-grade fetal adenocarcinoma, five cases (0.1%) were mixed-type high-grade fetal adenocarcinoma, and the remaining two cases (0.05%) were lung adenocarcinoma with high-grade fetal features (fetal lung-like morphology occupied less than 50%). CTNNB1 mutations were exclusively identified in low-grade fetal adenocarcinomas. In contrast, mixed-type high-grade fetal adenocarcinoma or lung adenocarcinoma with high-grade fetal features frequently harbored mitogenic drivers including EGFR mutations. Furthermore, almost all tumor cells expressed CDX2 and HNF4α in both cases of pure-type high-grade fetal lung adenocarcinoma, but lacked TTF-1 positivity. In contrast, TTF-1 was frequently expressed in mixed-type high-grade fetal lung adenocarcinoma and in lung adenocarcinoma with high-grade fetal features. Our data suggest similar prevalence of low-grade fetal lung adenocarcinoma and pure-type high-grade fetal lung adenocarcinoma, and indicate that pure- and mixed-type high-grade fetal lung adenocarcinomas are distinct, with the former akin to low-grade fetal adenocarcinoma with respect to purely embryonic morphology and absence of common lung adenocarcinoma mitogenic drivers, and the latter being genetically and transcriptionally related to conventional lung adenocarcinoma.

A distinct subset of lung adenocarcinomas (LADs), arising from a series of peripheral lung cells defined as the terminal respiratory unit (TRU), is characterised by thyroid transcription factor 1 (TTF-1) expression. The clinical relevance of transcription factors (TFs) other than TTF-1 remains unknown in LAD and was explored in the present study.Seventy-one LAD samples were subjected to high-throughput transcriptome screening of LAD using cap analysis gene expression (CAGE) sequencing data; CAGE provides genome-wide expression levels of the transcription start sites (TSSs). In total, 1083 invasive LAD samples were subjected to immunohistochemical examination for paired box 9 (PAX9) and TTF-1 expression levels. PAX9 is an endoderm development-associated TF that most strongly and inversely correlates with the expression of TTF-1 TSS subsets. Immunohistochemically, PAX9 expression was restricted to the nuclei of ciliated epithelial and basal cells in the bronchi and bronchioles and the nuclei of epithelial cells of the bronchial glands; moreover, PAX9 expression was observed in 304 LADs (28%). PAX9-positive LADs were significantly associated with heavy smoking, non-lepidic subtype, EGFR wild-type tumours and PD-L1 expression (all P 0.0001). All these characteristics were opposite to those of TRU-type LADs with TTF-1 expression. PAX9 expression was an independent prognostic factor for decreased overall survival (P = 0.022).Our results revealed that PAX9 expression defines an aggressive subset of LADs preferentially occurring in smokers that may arise from bronchial or bronchiolar cells.

Multi-gene expression assays have been developed with the aim of predicting late recurrence in patients with estrogen receptor (ER)-positive breast cancer. However, establishment of alternative markers based on immunohistochemistry is also important for achieving practical use. Based on our previous study, forkhead box A1 (FOXA1) protein was tested as a potentially useful predictive marker for late recurrence. 117 patients with ER-positive HER2-negative invasive breast cancer who developed distant metastasis following curative surgery were retrospectively investigated. We also evaluated responsiveness to endocrine therapy according to FOXA1 expression. Furthermore, publicly available mRNA microarray data were analyzed to examine patterns of metastasis according to FOXA1 mRNA expression, employing the Kaplan–Meier plotter. High expression of FOXA1 was an independent factor predicting long disease-free survival (DFS), along with small tumor size (p = 0.010 and 0.016, respectively). Discrimination of DFS was improved by combining these two factors, i.e., patients with FOXA1-high small tumors had the longest DFS while those with FOXA1-low large tumors had the shortest DFS. Moreover, we revealed that risk of distant metastasis started to increase after the completion of adjuvant endocrine therapy in patients with FOXA1-high tumors. Among patients who developed distant metastasis, those with FOXA1-high tumors had significantly longer DFS. We believe our data to raise the possibility of FOXA1 being a useful predictive marker for late recurrence and to provide new insights into the biology of FOXA1-high breast cancers.

Intraductal papillary neoplasms of the bile duct (IPNB) are histologically and clinically classified as type 1 and 2. This study aimed to identify the differences between these two types.Based on multiple gene expression analysis (MGEA) using type 1, type 2, and pancreatic intraductal papillary mucinous neoplasms (n=4, 6, and 5, respectively), immunohistochemistry of DNMT1 and methylation-specific PCR for p16, APC, BRCA1, hMLH1, TIMP3, and SOX17 were performed on type 1 and 2 IPNBs (n=14, each).The DNMT1 protein was highly expressed (p0.001) in 28.6% of type 1 cases and all type 2 cases. The DNA methylation ratio for the six genes in total as well as for SOX17 was lower in type 1 than in type 2 (p0.05 each).Type 2 IPNB showed increased DNMT1 protein expression and increased DNA methylation frequency of the examined tumor suppressor genes compared to type 1. DNMT1 IHC may be helpful in discriminating between these two types.

Invasive mucinous adenocarcinoma (IMA) of the lung is a unique variant of lung adenocarcinoma. Aberrant mucin expression is associated with cancer development and metastasis. However, the clinicopathological significance of mucin expression in IMA is not fully understood. Herein, we evaluated the clinicopathological, immunohistochemical, and molecular characteristics of 70 IMA tumors. EGFR, KRAS, GNAS, and TP53 mutations were assessed by PCR-based sequencing. Next-generation sequencing was used to assess cases without EGFR/KRAS mutations. A NanoString-based screening for fusions was performed in all IMAs without mitogenic driver mutations. Expression of mucins (MUC1, MUC2, MUC4, MUC5AC, and MUC6) was evaluated by immunohistochemistry and categorized as follows: negative (

Background/Aims: Gastric adenocarcinoma of fundic gland type (GAFG), which is a rare variant of gastric cancer, is reportedly associated with both Wnt/β-catenin signaling activation and guanine nucleotide binding protein, alpha stimulating complex (GNAS) mutations. This study aimed to elucidate potential roles of the Sonic hedgehog (Shh) signaling pathway in GAFG. Methods: We performed immunostaining for β-catenin and Shh signal-associated proteins, including Patched (Ptch), Smoothened (Smo), and Glioma-associated oncogene-1 (Gli1), and the direct sequencing of GNAS/BRAF/KRAS in 27 GAFGs, and compared them with 30 conventional gastric adenocarcinomas (CGAs). Results: GAFGs exhibited significantly lower immunoreactivity scores for Ptch, Smo, and Gli1 than CGAs. Moreover, while the Ptch score was significantly lower in the GAFG tumor areas than in the non-neoplastic areas adjacent to GAFG, the score was significantly higher in the CGA tumor areas than in the non-neoplastic areas. Similar trends were observed in the scores for Smo and Gli1. β-Catenin expression and GNAS mutations were found in 22 (81%) and 8 (30%) of the 27 GAFGs respectively. Gli1 expression was significantly associated with mutations in GNAS. Conclusion: GAFG and CGA exhibited distinct Ptch, Smo, and Gli1 expression patterns. Downregulation of the Shh signaling pathway, as well as activation of the Wnt/β-catenin signaling pathway, may therefore be associated with tumorigenesis in GAFG.

Background Colorectal sessile serrated adenoma/polyps (SSA/Ps) are considered early precursor lesions in the serrated neoplasia pathway. Recent studies have shown associations of SSA/Ps with lost MLH1 expression, a CpG island methylator phenotype, and BRAF mutations. However, the molecular biological features of SSA/Ps with early neoplastic progression have not yet been fully elucidated, owing to the rarity of cases of SSA/P with advanced histology such as cytologic dysplasia or invasive carcinoma. In this study, we aimed to elucidate the molecular biological features of SSA/Ps with dysplasia/carcinoma, representing relatively early stages of the serrated neoplasia pathway. Methods We performed immunostaining for β-catenin, MLH1, and mucins (e.g., MUC2, MUC5AC, MUC6, and CD10); targeted next-generation sequencing; and microsatellite instability (MSI) testing in 8 SSA/P lesions comprised of 4 SSA/Ps with high-grade dysplasia and 4 SSA/Ps with submucosal carcinoma. Results Lost MLH1 expression was found in 5 cases. All lesions studied were positive for nuclear β-catenin expression. Regarding phenotypic mucin expression, all lesions were positive for MUC2, but negative for CD10. MUC5AC and MUC6 positivity was observed in 7 cases. Genetically, the most frequently mutated gene was BRAF (7 cases), and other mutations were detected in FBXW7 (3 cases); TP53 (2 cases), and KIT, PTEN, SMAD4, and SMARCB1 (1 case each). Furthermore, 4 of 8 lesions were MSI-high and the remaining 4 lesions were microsatellite-stable (MSS). Interestingly, all 4 MSI-high lesions displayed MLH1 loss, 3 of which harbored a FBXW7 mutation, but not a TP53 mutation. However, 2 MSS lesions harbored a TP53 mutation, although none harbored a FBXW7 mutation. Conclusions SSA/Ps with dysplasia/carcinoma frequently harbored BRAF mutations. Activation of the WNT/β-catenin signaling pathway may facilitate the development of dysplasia in SSA/Ps and progression to carcinoma. Furthermore, our results suggested that these lesions might be associated with both MSI-high and MSS colorectal cancer, which might be distinguished by distinct molecular biological features such as lost MLH1 expression, FBXW7 mutations, and TP53 mutations.

Eosinophilic myenteric ganglionitis (EMG) is characterised by eosinophilic infiltration of the myenteric plexus. EMG has been rarely reported as a cause of chronic intestinal pseudo-obstruction (CIPO), and its histopathological features are not fully elucidated. We analysed seven patients with CIPO. Three of them were diagnosed with EMG and four patients were categorised as non-EMG. Clinicopathological features were similar in both groups. These features included subtle to mild lymphocytic infiltration at the myenteric ganglia/muscularis propria, loss of myenteric ganglions and interstitial cells of Cajal (ICC), and no significant findings in the mucosa. The exceptions were moderate to severe degree of eosinophilic infiltration at the myenteric ganglia/muscularis propria in EMG. Functional gastrointestinal obstruction may be associated with inflammatory cell infiltration at the myenteric ganglia/muscularis propria, leading to subsequent hypoganglionosis and deficiency of ICC in EMG. Pathologists and clinicians should be aware of this distinction during differential diagnosis of patients with CIPO.

Twenty-four surgically resected, gallbladder pyloric gland adenomas (GB-PGAs) were examined and their features were compared with the reported features of stomach, duodenum, and pancreatic PGAs to better understand GB-PGAs. Clinical information on background gallbladder lesions and histologic data, including tumor grade, existence of squamoid morules, intratumoral cholesterosis, and intracytoplasmic mucins were collected. Immunohistochemical staining for MUC2, MUC5AC, MUC6, CDX2, pepsinogen I, p53, and MIB-1/nuclear β-catenin were evaluated. Targeted mutational analyses of KRAS exon2, GNAS exon 7, and CTNNB1 exon 3 were conducted. We found that 29.2% of the GB-PGAs were histologically high-grade dysplasias/carcinomas; 70.8% were low grade; and 20.8% and 33.3% contained squamoid morules and intratumoral cholesterosis, respectively. In addition, 45.8% and 54.2% of GB-PGAs were mucin-rich and mucin-poor types, respectively. Immunohistochemically, MUC6 was diffusely positive in all GB-PGAs; MUC2, MUC5AC, and CDX2 were only focally positive, and no pepsinogen-I positive cells were observed. Nuclear β-catenin accumulation was observed in all cases; however, the ratio varied among cases. Mucin-poor types were significantly associated with high histologic grade dysplasias/carcinomas and high nuclear β-catenin labeling indices. Mutational analyses identified CTNNB1 mutations in 100% of GB-PGAs (21/21), KRAS in 4.2% (1/23), and GNAS in 0% (0/22). The present study clarified the unique histologic features, phenotypic differentiation, and molecular statuses frequently associated with GB-PGAs. Altogether, our data suggest that tumorigenesis of GB-PGA is distinct from that of stomach, duodenum, and pancreatic PGAs.

AIMS Recently, several morphological variants of traditional serrated adenoma (TSA) of the colorectum have been recognised, and mucin-rich TSA (MR-TSA) and serrated tubulovillous adenoma (S-TVA) were introduced as distinct morphological variants separate from conventional TSA (C-TSA). This aim of this study was to elucidate the immunohistochemical and genetic characteristics of MR-TSAs. METHODS AND RESULTS We performed immunostaining for cytokeratins (CKs) (e.g. CK7 and CK20), mucins (e.g. MUC2, MUC5AC, MUC6, and CD10), β-catenin, and MLH1, and direct sequencing of BRAF/KRAS, in 32 MR-TSAs, 35 C-TSAs, and 23 S-TVAs. Immunohistochemically, all studied cases were positive for CK20, whereas few cases were positive for CK7, with no significant differences between the three groups. Regarding mucin-phenotypic expression, all cases were positive for MUC2 but negative for MUC6 and CD10. MUC5AC positivity was found significantly more frequently in MR-TSAs (53%) than in C-TSAs (26%; P = 0.026). Nuclear β-catenin expression in MR-TSAs was significantly less frequent than in S-TVAs (P = 0.002). MLH1 nuclear staining was retained in all cases. Genetically, MR-TSAs (75%) more frequently harboured BRAF mutation than C-TSAs (49%; P = 0.044) or S-TVAs (4%; P < 0.001), whereas only two cases (6%) of MR-TSA harboured a KRAS mutation, a frequency that was significantly lower than that in C-TSAs (26%; P = 0.047) or S-TVAs (57%; P < 0.001). CONCLUSIONS MR-TSAs more frequently harboured BRAF mutations than C-TSAs, and had distinct immunohistochemical characteristics. Our findings indicated that MR-TSAs could be important precursors of BRAF-mutated, microsatellite-stable subtypes of colorectal carcinoma.

The combination of CD44 and CD24, or aldehyde dehydrogenase 1 (ALDH1) alone, is a widely used cancer stem cell marker in breast cancer. However, no conclusion has yet been reached as to which marker is the best for characterizing cancer stemness. Immunohistochemical evaluation using cancer stem cell markers is clearly less common clinically than in basic experiments and how the expressions of these markers relate to patient outcomes remains controversial. To investigate whether combining these markers might improve the prediction of patient outcomes, we immunohistochemically examined clinical samples. Primary invasive breast cancer samples from 61 patients who eventually developed distant metastases after curative surgery were immunohistochemically examined. All patients were free of metastatic disease at the time of surgery and received standard adjuvant systemic treatments. CD44+/24- and ALDH1-positive rates in primary tumors differed according to intrinsic subtype. ER-positive patients with CD44+/24- tumors had significantly longer disease-free-survival than all other ER-positive patients (p = 0.0047). On the other hand, CD44+/24- tumors were associated with poor outcomes of ER-negative patients (p = 0.038). Finally, expression patterns of CD44 and ALDH1 in single tumors were strikingly different and there were virtually no individual double-stained cells. Thus, this combination does not allow evaluation of relationships with patient outcomes. Our results raise the possibility of CD44+/24- being a good prognostic marker, one which would allow treatment effects and outcomes to be predicted in patients with recurrent breast cancer.

To investigate expression of estrogen receptors (ER) and progesterone receptors (PR) in smooth muscle metaplasia (SMM) outside the endometriotic foci of rectovaginal endometriosis (RVE). One hundred and ninety-five specimens were obtained from the rectovaginal areas of the 63 patients who were underwent laparoscopic surgery for RVE. The patients were divided into 3 groups: a gonadotropin-releasing hormone (GnRH) agonist group, a non-GnRH group, including a proliferative phase group, and a secretory phase group. Expression of ER and PR in the rectovaginal tissues of RVE were determined using immunohistochemical methods. Smooth muscle metaplasia occurred in 172 specimens (88.2%), and ER and PR expression were found in the smooth muscle cells in the SMM areas outside the endometriotic foci of RVE. The expression of ER and PR in the GnRH agonist group were significantly lower than those in the non-GnRH agonist group. This is the first report demonstrating ER and PR in the smooth muscle cells in SMM outside the endometriotic foci of RVE. The ER and PR were expressed in the SMM areas, but these receptors were not recognized in fibrotic areas. We could identify the expression ratio of these receptors during each menstrual phase, with or without administered GnRH agonist.

Mucinous cystic neoplasms (MCN) of the pancreas are mucin-producing cystic tumors with an ovarian-like stroma (OLS). In the present study MCN were obtained from 27 patients. These MCN were derived from 22 pancreas, three livers, spleen, and mesentery. MCN in various organs have common clinicopathological profiles, being unilocular or multilocular cystic tumors, with a fibrous capsule and lined by mucin-secreting epithelium associated with an underlying subepithelial OLS. The OLS showed strong positivity for alpha-smooth muscle actin (alpha-SMA) and vimentin and weak, focal positivity for desmin. Both estrogen receptors and progesterone receptors were expressed in the nuclei of OLS cells. In addition, 20 ovarian MCN and 13 normal ovaries were studied with particular attention to the stroma. The stroma of ovarian MCN was strongly immunopositive for alpha-SMA and vimentin and focally positive for desmin, whereas normal ovarian stroma was immunonegative for both alpha-SMA and desmin. The OLS of MCN mentioned here was similar to the septa of ovarian MCN but not to ovarian stroma. In conclusion, MCN in various organs should be lumped together as 'extra ovarian' MCN. The OLS was identified on the basis of myofibroblastic proliferation both in response to neoplastic development and dependent on hormones.

Rectovaginal (RV) endometriosis presents with a nodular lesion composed of fibromuscular and endometriotic tissue, and the fibromuscular tissue is the major component in the severe stage. The purpose of our study was to examine the extending process of fibromuscular tissue in RV endometriosis. Histological examinations using immunostains, were performed in 90 RV tissue specimens from 37 women. Fibrosis was present in 89 specimens. In each specimen, the intensity of the fibrosis differed from area to area: in mildly fibrotic areas, the collagen fibers were present around the endometriotic tissue, and in severely fibrotic areas, the fibrosis widely extended into fat and connective tissus as well as within the endometriotic tissue. In the 60 specimens containing endometriotic tissue, the increase in the amount of endometriotic tissue significantly correlated to the increase in degree of fibrosis in the entire tissue. The presence of aggregated smooth muscles, unassociated with blood vessels, was defined as smooth muscle metaplasia (SMM), which was always present within the fibrotic areas, and was observed in 80 specimens. The degree of SMM in the entire tissue was significantly correlated with the degree of fibrosis. From these findings, the following was hypothesized. Initially, endometriotic tissue was present sporadically and fibrosis was present around the endometriotic tissue. Thereafter, proliferation of endometriotic tissue and an increase in fibrosis occur consecutively. The SMM was present within the fibrotic areas, and it became more severe, correlating with the increase in fibrosis. In conclusion, this is the first report describing the extending process of the fibromuscular tissue of RV endometriosis from a histological viewpoint, and we think that recognization of this process is useful for histological diagnosis and clinical management of RV endometriosis.

Squamous cell carcinoma (SCC) of the skin shows an indolent prognosis in general. However, the prognosis of SCC arising in a scar (scar carcinoma) is considered to be worse than that of SCC without any clinical history of injury (nonscar SCC). The aim of this study was to compare several indices, p53, Ki-67, E-cadherin, and beta-catenin, which are related to tumor behavior, between scar carcinoma and nonscar SCC clinicopathologically and immunohistochemically. The materials were from 10 cases of scar carcinoma and 10 cases of nonscar SCC. Clinicopathologically, the mean ages at diagnosis of scar carcinoma and nonscar SCC were 59.2 and 71.2, respectively. The most frequent anatomic site of scar carcinoma was the limbs. The most common cause of scars in our study was burns. The mean duration from the initial injury to the diagnosis of carcinoma was 30.5 years. Immunohistochemically, the mean labeling index (calculated as the percentage of positive cells) of p53 was 16.5 and 58.6 in scar carcinoma and nonscar SCC, respectively (P0.01, Welch test). The LI of Ki-67 was 19.1 in scar carcinoma and 52.1 in nonscar SCC (P0.01, Welch test). The rates of positivity of the other proteins, such as E-cadherin and beta-catenin, were similar between scar carcinoma and nonscar SCC. In this study, the follow-up time was short and the number of patients was small, and for these reasons it might not have been possible to obtain evidence that scar carcinoma is aggressive.