Tsuyoshi Saito

Introduction: Intravascular fasciitis (IVF) is a rare variant of nodular fasciitis characterized by intraluminal, intramural, and extramural involvement of blood vessels, accounting for less than 3% of all nodular fasciitis cases. Case presentation: A 59-year-old man with a history of treated laryngeal squamous cell carcinoma was found to have a 22-mm nodule in the fifth intercostal space on follow-up computed tomography, which raised clinical suspicion of chest wall metastasis. The patient underwent a left thoracotomy with combined resection of the fifth and sixth ribs. Histologically, spindle cells proliferated in a tissue culture-like growth pattern within a fibromyxoid stroma. Immunohistochemically, the spindle cells were positive for muscle-specific actin (M-actin) and smooth muscle actin and negative for cytokeratins. Break-apart fluorescence in situ hybridization (FISH) demonstrated USP6 rearrangement. Based on these findings and the intravascular involvement of small blood vessels, a final diagnosis of IVF was made. The patient has been followed for 15 months after surgery without any recurrence or metastasis. Discussion: IVF is frequently misdiagnosed as intravascular invasion of a malignant spindle cell neoplasm. In the present case, metastatic carcinoma with sarcomatoid change was initially suspected because of the patient’s history of laryngeal carcinoma. Accurate diagnosis of IVF requires careful evaluation of the histological and immunohistochemical findings, together with confirmation of USP6 rearrangement by FISH. Conclusions: We report a case of IVF arising in the chest wall of a man with a history of laryngeal cancer. When an intravascular spindle cell lesion is encountered, IVF should be considered in the differential diagnosis.

High-grade sarcomas often lack typical morphological features and exhibit no clear differentiation, often leading to a diagnosis of undifferentiated sarcoma (US). Pleomorphic leiomyosarcoma (PLMS) is a high-grade sarcoma consisting of a typical leiomyosarcoma (LMS) component alongside dedifferentiated high-grade areas. A few decades ago, PLMS was regarded as a subtype of high-grade sarcoma previously referred to as malignant fibrous histiocytoma; it is now classified as a variant of LMS. The mechanisms underlying myogenic differentiation and their relevance to the pathological diagnosis of high-grade sarcomas remain poorly understood. To investigate the gene expression networks associated with myogenic differentiation, we employed Cap Analysis of Gene Expression (CAGE) to distinguish PLMS from other high-grade sarcoma subtypes. We analyzed 27 frozen high-grade sarcoma samples, comprising 10 PLMSs, 11 high-grade myxofibrosarcomas, 3 dedifferentiated liposarcomas, 2 USs, and 1 high-grade sarcoma not otherwise specified, using CAGE profiling. Hierarchical clustering based on differentially expressed genes identified by CAGE separated 7 of the 10 PLMSs from other high-grade sarcomas, while the remaining 3 PLMSs clustered with a single US case. CAGE analysis also revealed that the myostatin (MSTN) promoter (false discovery rate [FDR] < 0.05) was more strongly activated in the high-grade sarcoma group lacking morphological and immunohistochemical smooth muscle differentiation than in the PLMS group, whereas the alpha smooth muscle actin (ACTA2) promoter (FDR < 0.05) was more prominently activated in the PLMS group. Immunohistochemical analysis showed reduced or absent myostatin expression in PLMSs, in contrast to diffuse myostatin expression in other high-grade sarcomas. Smooth muscle actin, encoded by ACTA2, was expressed in all 10 PLMS cases but only in 11 of 17 other high-grade sarcomas. Furthermore, both conventional immunohistochemistry and double immunostaining revealed that myostatin and myogenic markers exhibited largely mutually exclusive expression patterns within these tumors. A validation study was performed using 59 soft tissue sarcoma cases, including 27 PLMSs and 16 LMSs. Loss or reduction in myostatin expression was confirmed in both LMS and PLMS, and the ratio of myostatin loss was comparable (62.5% in LMS vs. 63% in PLMS). Collectively, these findings suggest that myostatin contributes to smooth muscle differentiation in high-grade sarcomas and has potential utility as a diagnostic marker.

New disease entities have been emerging based on molecular pathological findings, such as pseudoendocrine sarcoma and mesenchymal neoplasm with GLI1 gene alterations, which resemble well-differentiated neuroendocrine tumors. We report a unique case of a gastric submucosal neoplasm of approximately 1.5 cm in size with CTNNB1 mutation showing GLI1 overexpression and epithelial differentiation in a 66-year-old man. It was incidentally identified by routine health screening, and was a slowly growing tumor. Macroscopically, it was a slightly protruded tumor into the mucosa, and was primarily located from the submucosa to the muscularis propria. It was a well-defined lesion measured approximately 20 mm, and was almost stable during almost 5 years after initial identification of the tumor. Uniform round-to-epithelioid cells arranged in solid trabeculae with a microtubular/acinar appearance were seen microscopically. Occasional mitotic figures were noted, but no necrosis was observed. Immunohistochemistry (IHC) demonstrated diffuse expression of pan-cytokeratin, CD10, and CD56 without neuroendocrine markers (chromogranin A, synaptophysin, and INSM1). Molecular analysis confirmed the presence of a hot spot CTNNB1 mutation (S33C), supported by diffuse β-catenin nuclear expression by IHC. Further molecular investigations revealed the absence of GLI1 gene rearrangements, GLI1 amplification, and other fusions. Several differential diagnoses were considered; however, none adequately fit the criteria. The patient remained disease-free for 24 months postoperatively without further adjuvant therapy.

Aims: In non‐small cell lung cancer, alterations in mesenchymal‐epithelial transition (MET) have been recognized as novel therapeutic targets. In particular, the MET exon 14 skipping mutation (METex14s) is a rare oncogenic driver. Targeted therapy with MET tyrosine kinase inhibitors has recently been approved for this mutation. However, c‐MET expression and METex14s frequency and their clinicopathological effects in Japanese patients with breast cancer (BC) remain unknown.Methods: Tissue microarray‐based immunohistochemistry (IHC) was performed to measure c‐MET expression in 930 patients with BC (808 with invasive and 122 with noninvasive BC). Reverse transcription polymerase chain reaction was performed to analyse METex14s in patients exhibiting c‐MET expression. Clinicopathological characteristics, including patient prognosis based on c‐MET expression and METex14s, were elucidated.Results: IHC staining revealed c‐MET expression in 91/930 (9.7%) patients. Notably, IHC expression was frequently observed in apocrine carcinomas (11/26 cases). Among the c‐MET IHC‐positive cases, METex14s frequency was 25.9% (14/54 cases) in invasive BC and 54.1% (20/37 cases) in noninvasive BC. Furthermore, 4/11 informative noninvasive and invasive BC cases with apocrine differentiation carried METex14s. The nuclear grade was significantly higher in the METex14s‐positive group among invasive BC with c‐met IHC expression. Furthermore, patients' age and negative rate for PgR IHC was significantly lower in the METex14s‐positive group among noninvasive BC. Regarding the factors associated with patient outcomes, both c‐MET IHC staining and METex14s expression did not affect survival, regardless of the hormone receptor status.Conclusion: High c‐MET expression and METex14s are common in apocrine carcinoma.

Introduction: Mesenchymal chondrosarcoma (MCS) is a rare subtype of chondrosarcoma that occurs at widespread anatomical locations, such as bone, soft tissue, and intracranial sites. The central nervous system (CNS) is one of the most common origins of extraosseous MCS. However, alternative HEY1::NCOA2 fusions have not been reported in this tumor.Case report: We report a case of intracranial MCS with HEY1::NCOA2 rearrangement. A 52-year-old woman presented with a 15-mm calcified mass around the sella turcica. She initially underwent transsphenoidal surgery for tumor resection and then additional resections for five local recurrences over 5 years. Histologically, the tumor was composed of small round to spindle-shaped cells admixed with well-differentiated hyaline cartilaginous islands. A hemangiopericytoma-like vascular pattern and small sinusoid-like vessels were also observed. RNA sequencing using RNA extracted from formalin-fixed paraffin-embedded samples from the last operation revealed two alternative variants of the HEY1::NCOA2 fusion: HEY1(ex4)::NCOA2 (ex13) and HEY1(ex4)::NCOA2(ex14). Both variants were confirmed as in-frame fusions using reverse transcription-polymerase chain reaction.Discussion: Cartilaginous components were often not apparent during the recurrences. In addition to the non-typical pathological finding, the correct diagnosis was hampered by the poor RNA quality of the surgical specimens and non-specific STAT6 nuclear staining.Conclusion: This is the first reported case of intracranial MCS with an alternative HEY1::NCOA2 fusion.

Aims: Ulcerative colitis‐associated neoplasia (UCAN) is characterised by multifocal tumourigenesis. A wide range of metachronous lesions have been reported to occur after endoscopic treatment of UCAN, which suggests the development of sporadic tumours in lesions treated as UCAN. Therefore, we aimed to evaluate differences of immunohistochemistry (IHC) in features and clinicopathological characteristics of intramucosal lesions in patients with ulcerative colitis (UC).Methods and results: We examined 35 intramucosal lesions resected for carcinoma or dysplasia by total colectomy from patients with UC and 71 sporadic adenomas (SAs) endoscopically resected from patients without UC. UC lesions were divided into the conventional UCAN group, defined as p53 mutant pattern and normal expression of β‐catenin, and the non‐conventional UCAN group, defined as the rest. Ki‐67 distribution, α‐methylacyl‐CoA racemase (AMACR) expression and mucin phenotypes were compared using IHC, and clinicopathological characteristics were investigated. Conventional and non‐conventional UCAN lesions were located in the left colon and rectum. Relative to the SA lesions, UCAN lesions occurred in much younger patients and exhibited more frequent basal distribution of Ki‐67 in tumour crypts. Conventional UCAN lesions tended to be non‐polyploid and exhibited a higher frequency of normal AMACR expression than SA lesions. UC lesions were heterogeneous—only two of the eight patients with multiple lesions had lesions (both non‐conventional UCAN lesions) exhibiting concordant IHC staining features.Conclusions: The basal pattern of Ki‐67 distribution, normal expression of AMACR and a non‐intestinal mucin phenotype were determined as characteristic features suggestive of UCAN. Non‐polypoid growth was another a key feature of UCAN.

Gastrointestinal stromal tumors (GISTs) are typically characterized by activating mutations of the KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Recently, the neurotrophic tyrosine receptor kinase (NTRK) fusion was reported in a small subset of wild-type GIST. We examined trk IHC and NTRK gene expressions in GIST. Pan-trk immunohistochemistry (IHC) was positive in 25 (all 16 duodenal and 9 out of 16 small intestinal GISTs) of 139 cases, and all pan-trk positive cases showed diffuse and strong expression of c-kit. Interestingly, all of these cases showed only trkB but not trkA/trkC expression. Cap analysis of gene expression (CAGE) analysis identified increased number of genes whose promoters were activated in pan-trk/trkB positive GISTs. Imbalanced expression of NTRK2, which suggests the presence of NTRK2 fusion, was not observed in any of trkB positive GISTs, despite higher mRNA expression. TrkB expression was found in duodenal GISTs and more than half of small intestinal GISTs, and this subset of cases showed poor prognosis. However, there was not clear difference in clinical outcomes according to the trkB expression status in small intestinal GISTs. These findings may provide a possible hypothesis for trkB overexpression contributing to the tumorigenesis and aggressive clinical outcome in GISTs of duodenal origin.

A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine‐specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia‐telangiectasia and Rad3‐related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome‐wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with ‘cell cycle’, whereas downregulated genes in tumors with KDM5D copy number loss were associated with ‘immune response’. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor‐infiltrating CD8⁺/T‐bet⁺ T cells was lower in SCCs with KDM5D copy number loss than in wild‐type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be ‘cold tumors’, which are characterized by the paucity of tumor T‐cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.

Gastric adenocarcinoma (GA) with enteroblastic differentiation (GAED) is an aggressive carcinoma histologically characterized by a glycogen-rich clear cytoplasm and fetal gut-like structures. GAED shows the expression of at least one of the following enteroblastic markers (EMs): glypican-3 (GPC3), spalt-like transcription factor 4 (SALL4), and α-fetoprotein (AFP). Despite the absence of clear cytoplasm, we often encounter GA with EMs expression (GA with EM); however, the clinicopathological characteristics of GA with EM remain unclear. Immunohistochemical (IHC) expression of three EMs (AFP, GPC3, and SALL4) was examined on tissue microarray. According to the status of the clear cytoplasm of tumor cells, GAs showing IHC expression of EMs were classified as either GAED or GA with EM, and this analysis categorized 688 GAs into 94 GAEDs (13.7%), 58 GAs with EM (8.4%), and 536 conventional GAs (CGAs). Both GAED and GA with EM showed frequent lymphovascular invasion, lymph node metastasis, and liver metastasis compared to CGA. However, a higher frequency of venous invasion, but not of lymphatic invasion, was noted for GAED in comparison to CGA. GAED and GA with EM showed similar overall survival. GAED had significantly poorer prognosis than CGA; however, not for GA with EM. Furthermore, GA showing EM expression had a worse prognosis than CGA. Interestingly, GA showing EM-positive group was more aggressive than CGA group as they had frequent venous invasion and liver metastasis despite its smaller tumor size. GAED and GA with EM can be clinically classified as aggressive tumors but pathologically they seem to be slightly different.

Aims: Tyrosine kinase (TK) alterations, such as anaplastic lymphoma kinase (ALK) fusion, neurotrophic tyrosine receptor kinase (NTRK) fusion, c‐ros oncogene 1 (ROS1) fusion and mesenchymal–epithelial transition factor (MET) exon 14 skipping, have been reported in colorectal cancers (CRC). We have previously reported CRCs with NTRK fusion among our cohort. However, their clinicopathological features have not been fully elucidated.Methods and results: Tissue microarray (TMA)‐based immunohistochemistry (IHC) was performed on 951 CRC lesions from 944 patients. IHC was evaluated as positive or negative for ALK and ROS1 and 0 to 3+ for c‐MET. For ALK and ROS1 IHC‐positive cases, RNA‐based imbalanced gene expression assays, Archer FusionPlex assays and reverse transcription–polymerase chain reaction (RT‐PCR) followed by Sanger sequencing were performed. For c‐MET IHC 3+ cases, RT‐PCR followed by Sanger sequencing were performed. ALK IHC was positive in three cases (0.2%) and all showed imbalanced ALK gene expression. The following ALK fusions were confirmed: EML4 (exon 21)::ALK (exon 20), EML4 (exon 6)::ALK (exon 19) and HMBOX1 (exon 6)::ALK (exon 20). Two showed microsatellite instability‐high/mismatch repair (MMR)‐deficient, and all were located in the right colon. ROS1 IHC was positive in one case; however, imbalanced expression and ROS1 fusion was negative. Forty‐two cases (4.4%) showed c‐MET IHC3+. MET exon 14 skipping was confirmed in nine cases. All cases were microsatellite stable/MMR‐proficient, and eight were located in the left colon and rectum.Conclusions: CRCs with these TK alterations had distinct clinicopathological features. Together with our previous study, 15 cases (1.6%) harboured targetable TK alterations (three NTRK fusion, three ALK fusion, nine MET exon 14 skipping).