Comprehensive phenotypic and genomic characterization of venous malformations.

Venous malformations (VMs) are the most common vascular malformations. TEK and PIK3CA are the causal genes of VMs, and may be involved in the PI3K/AKT pathway. However, the downstream mechanisms underlying the TEK or PIK3CA mutations in VMs are not completely understood. This study aimed to identify a possible association between genetic mutations and clinicopathological features. A retrospective clinical, pathological, and genetic study of 114 patients with VMs was performed. TEK, PIK3CA, and combined TEK/PIK3CA mutations were identified in 49 (43%), 13 (11.4%), and 2 (1.75%) patients, respectively. TEK-mutant VMs more commonly occurred in younger patients than TEK and PIK3CA mutation-negative VMs (other-mutant VMs), and showed more frequent skin involvement and no lymphocytic aggregates. No significant differences were observed in sex, location of occurrence, malformed vessel size, vessel density, or thickness of the vascular smooth muscle among the VM genotypes. Immunohistochemical analysis revealed that the expression levels of phosphorylated AKT (p-AKT) were higher in the TEK-mutant VMs than those in PIK3CA-mutant and other-mutant VMs. The expression levels of p-mTOR and its downstream effectors were higher in all the VM genotypes than those in normal vessels. Spatial transcriptomics revealed that the genes involved in "blood vessel development", "positive regulation of cell migration", and "extracellular matrix organization" were up-regulated in a TEK-mutant VM. Significant genotype-phenotype correlations in clinical and pathological features were observed among the VM genotypes, indicating gene-specific effects. Detailed analysis of gene-specific effects in VMs may offer insights into the underlying molecular pathways and implications for targeted therapies.

Identification of invasive subpopulations using spatial transcriptome analysis in thyroid follicular tumors.

BACKGROUND: Follicular tumors include follicular thyroid adenomas and carcinomas; however, it is difficult to distinguish between the two when the cytology or biopsy material is obtained from a portion of the tumor. The presence or absence of invasion in the resected material is used to differentiate between adenomas and carcinomas, which often results in the unnecessary removal of the adenomas. If nodules that may be follicular thyroid carcinomas are identified preoperatively, active surveillance of other nodules as adenomas is possible, which reduces the risk of surgical complications and the expenses incurred during medical treatment. Therefore, we aimed to identify biomarkers in the invasive subpopulation of follicular tumor cells. METHODS: We performed a spatial transcriptome analysis of a case of follicular thyroid carcinoma and examined the dynamics of CD74 expression in 36 cases. RESULTS: We identified a subpopulation in a region close to the invasive area, and this subpopulation expressed high levels of CD74. Immunohistochemically, CD74 was highly expressed in the invasive and peripheral areas of the tumor. CONCLUSIONS: Although high CD74 expression has been reported in papillary and anaplastic thyroid carcinomas, it has not been analyzed in follicular thyroid carcinomas. Furthermore, the heterogeneity of CD74 expression in thyroid tumors has not yet been reported. The CD74-positive subpopulation identified in this study may be useful in predicting invasion of follicular thyroid carcinomas.

Ovarian high-grade serous carcinoma cells with low SMARCA4 expression and high SMARCA2 expression contribute to platinum resistance.

Platinum resistance is a major obstacle to the treatment of ovarian cancer and is correlated with poor clinical outcomes. Intratumor heterogeneity plays a key role in chemoresistance. Recent studies have emphasized the contributions of genetic and epigenetic factors to the development of intratumor heterogeneity. Although the clinical significance of multi-subunit chromatin remodeler, switch/sucrose nonfermenting (SWI/SNF) complexes in cancers has been reported, the impacts of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4/subfamily A, member 2 (SMARCA4/A2) expression patterns in human cancer tissues have not been fully elucidated. Here, we show that low expression of SMARCA4 and high expression of SMARCA2 are associated with platinum resistance in ovarian high-grade serous carcinoma (HGSC) cells. We used fluorescence multiplex immunohistochemistry (fmIHC) to study resected specimens; we examined heterogeneity in human HGSC tissues at the single-cell level, which revealed that the proportion of cells with the SMARCA4low /SMARCA2high phenotype was positively correlated with clinical platinum-resistant recurrence. We used stable transfection of SMARCA2 and siRNA knockdown of SMARCA4 to generate HGSC cells with the SMARCA4low /SMARCA2high phenotype; these cells had the greatest resistance to carboplatin. Bioinformatics analyses revealed that the underlying mechanism involved in substantial alterations to chromatin accessibility and resultant fibroblast growth factor (FGF) signaling activation, MAPK pathway activation, BCL2 overexpression, and reduced carboplatin-induced apoptosis; these were confirmed by in vitro functional experiments. Furthermore, in vivo experiments in an animal model demonstrated that combination therapy with carboplatin and a fibroblast growth factor receptor (FGFR) inhibitor promoted cell death in HGSC xenografts. Taken together, these observations reveal a specific subpopulation of HGSC cells that is associated with clinical chemoresistance, which may lead to the establishment of a histopathological prediction system for carboplatin response. Our findings may facilitate the development of novel therapeutic strategies for platinum-resistant HGSC cells. © 2023 The Pathological Society of Great Britain and Ireland.

Tumor cell plasticity in endometrioid carcinoma is regulated by neuronal membrane glycoprotein M6-b.

Tumor cell plasticity and tumor heterogeneity are involved in therapy resistance. Cancer stem cells (CSCs) refer to tumor cells that have the ability to self-renew, and generate the diverse cells that comprise the tumor and complicate tumor heterogeneity. In recent years, CSCs have been reported to emerge from non-CSCs, which is known as tumor cell plasticity; however, the mechanism has not been fully elucidated. The present study investigated tumor cell plasticity from the viewpoint of aldehyde dehydrogenase 1 family member A1 (ALDH1A1) activity, which is one of the markers of CSCs. In the endometrioid carcinoma cell line HEC-1B, the ALDH1A1-low population spontaneously yielded an ALDH1A1-high population, mimicking tumor cell plasticity, and it was revealed that the mixture of the ALDH1A1-high population with the ALDH1A1-low population sometimes accelerated the transition from an ALDH1A1-low to ALDH1A1-high population. Two distinct HEC-1B sublines were established. One of the two sublines accelerated such a transition and the other did not show such acceleration. In the former subline, the effect of the ALDH1A1-high population was abolished when the direct cell-cell contact between ALDH1A1-high and ALDH1A1-low populations was inhibited. By comparing the two sublines, the neuronal membrane glycoprotein M6-b (GPM6B) was identified as the candidate mediating tumor cell plasticity. GPM6B was expressed in the border of ALDH1A1-expressing tumor cells and non-expressing tumor cells in clinical samples of EC. Notably, knockout of GPM6B decreased ALDH1A1 expression, whereas its overexpression increased the expression of ALDH1A1, suggesting that GPM6B mediated the induction of ALDH1A1 and the plasticity of CSCs.

Colorectal liver metastasis with bile duct tumor thrombus discovered 15 years post primary tumor resection: a case report and literature review.

Colorectal liver metastasis (CRLM) appears in 25-50% of patients with colorectal cancer (CRC). However, CRLM with bile duct tumor thrombus (BDTT) is rare and often diagnosed after surgical resection. We report a case of CRLM associated with BDTT in a 79 year-old woman. She underwent sigmoid colectomy for sigmoid colon carcinoma at the age of 64. Fifteen years later, abdominal computed tomography revealed a liver tumor with a biliary tumor thrombus in segment IV/V and localized dilation of the intrahepatic bile duct. Additionally, magnetic resonance imaging confirmed a tumor in liver segment IV/V and mass in the bile duct (B4). Extended left hepatic lobectomy was performed under the diagnosis of intrahepatic cholangiocarcinoma or metastatic liver tumor with tumor development in the intrahepatic bile duct. The resected specimen showed significant cancer infiltration into the intrahepatic bile duct (B4), forming a tumor thrombus. The tumor was a moderately differentiated adenocarcinoma, histologically similar to sigmoid colon cancer. CRLM with BDTT may have a relatively low invasive potential of malignancy with a long interval after primary resection. When a patient with a history of CRC presents with BDTT, the possibility of CRLM with BDTT and surgical treatment should be considered, because resection could lead to a good prognosis. It is important to ensure a secure surgical margin in the bile ducts during surgery and anatomical hepatic resection should be considered. Supplementary Information: The online version contains supplementary material available at 10.1007/s13691-022-00583-6.

A Novel Three-Dimensional Imaging System Based on Polysaccharide Staining for Accurate Histopathological Diagnosis of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Tissue-clearing and three-dimensional (3D) imaging techniques aid clinical histopathological evaluation; however, further methodological developments are required before use in clinical practice. METHODS: We sought to develop a novel fluorescence staining method based on the classical periodic acid-Schiff stain. We further attempted to develop a 3D imaging system based on this staining method and evaluated whether the system can be used for quantitative 3D pathological evaluation and deep learning-based automatic diagnosis of inflammatory bowel diseases. RESULTS: We successfully developed a novel periodic acid-FAM hydrazide (PAFhy) staining method for 3D imaging when combined with a tissue-clearing technique (PAFhy-3D). This strategy enabled clear and detailed imaging of the 3D architectures of crypts in human colorectal mucosa. PAFhy-3D imaging also revealed abnormal architectural changes in crypts in ulcerative colitis tissues and identified the distributions of neutrophils in cryptitis and crypt abscesses. PAFhy-3D revealed novel pathological findings including spiral staircase-like crypts specific to inflammatory bowel diseases. Quantitative analysis of crypts based on 3D morphologic changes enabled differential diagnosis of ulcerative colitis, Crohn's disease, and non-inflammatory bowel disease; such discrimination could not be achieved by pathologists. Furthermore, a deep learning-based system using PAFhy-3D images was used to distinguish these diseases The accuracies were excellent (macro-average area under the curve = 0.94; F1 scores = 0.875 for ulcerative colitis, 0.717 for Crohn's disease, and 0.819 for non-inflammatory bowel disease). CONCLUSIONS: PAFhy staining and PAFhy-3D imaging are promising approaches for next-generation experimental and clinical histopathology.

Deep Learning Analysis of Histologic Images from Intestinal Specimen Reveals Adipocyte Shrinkage and Mast Cell Infiltration to Predict Postoperative Crohn Disease.

Most patients with Crohn disease (CD), a chronic inflammatory gastrointestinal disease, experience recurrence despite treatment, including surgical resection. However, methods for predicting recurrence remain unclear. This study aimed to predict postoperative recurrence of CD by computational analysis of histopathologic images and to extract histologic characteristics associated with recurrence. A total of 68 patients who underwent surgical resection of the intestine were included in this study and were categorized into two groups according to the presence or absence of postoperative disease recurrence within 2 years after surgery. Recurrence was defined using the CD Activity Index and the Rutgeerts score. Whole-slide images of surgical specimens were analyzed using deep learning model EfficientNet-b5, which achieved a highly accurate prediction of recurrence (area under the curve, 0.995). Moreover, subserosal tissue images with adipose cells enabled highly accurate prediction. Adipose cell morphology showed significant between-group differences in adipose cell size, cell-to-cell distance, and cell flattening values. These findings suggest that adipocyte shrinkage is an important histologic characteristic associated with recurrence. Moreover, there was a significant between-group difference in the degree of mast cell infiltration in the subserosa. These findings show the importance of mesenteric adipose tissue in patient prognosis and CD pathophysiology. These findings also suggest that deep learning-based artificial intelligence enables the extraction of meaningful histologic features.

PIK3CA mutation correlates with mTOR pathway expression but not clinical and pathological features in Fibfibroipose vascular anomaly (FAVA).

BACKGROUND: Fibro-adipose vascular anomaly (FAVA) is a rare and new entity of vascular anomaly. Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene were identified at a frequency of 62.5% in FAVA cases. The PIK3CA mutations excessively activate mammalian target of rapamycin (mTOR) pathway, which promotes angiogenesis and lymphangiogenesis, implying that PIK3CA mutations may act as drivers of FAVAs. This study investigated the correlations between PIK3CA mutational status, clinicopathological features and immunohistochemical expression of the mTOR pathway in a series of FAVA. METHODS: We retrospectively evaluated the clinical and pathological findings of four FAVA cases. We performed next-generation sequencing (NGS) with a custom panel of genes associated with the mTOR pathway and genes responsible for other vascular anomalies; followed by direct sequencing and immunohistochemical analysis of the mTOR pathway. RESULTS: Two PIK3CA-mutation cases and two PIK3CA-wild-type (wt) cases exhibited similar typical clinical features of FAVA. Histological analysis revealed venous malformation, lymphatic malformation, nerves containing enlarged abnormal vessels and fibrofatty tissue were observed regardless of PIK3CA mutational status. In contrast to clinical and histological findings, the immunohistochemical expression of activated AKT and mTOR that are upstream of the mTOR pathway was detected in abnormal vessels of PIK3CA-mutation cases but not in those of PIK3CA-wt cases. However, activated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase 1 (S6K1), both of which are downstream effectors of the mTOR pathway, were expressed in abnormal vessels of both PIK3CA-mutation and PIK3CA-wt cases. Furthermore, targeting NGS did not find any common genetic mutations involved in the mTOR pathway among PIK3CA-wt cases. CONCLUSIONS: There was no significant association between the presence of PIK3CA mutations and the clinicopathological features of FAVA, suggesting that the PIK3CA gene is not necessarily involved in the onset of FAVA. FAVAs lacking PIK3CA mutations may be caused by other gene mutations that activate 4EBP1 and S6K1.

Strong expression of PD-L1 in invasive front of MELF pattern in endometrioid carcinoma.

Endometrioid carcinoma (EC) is classified into 3 histological subtypes; Grade 1 (G1), Grade 2 (G2), and Grade 3 (G3). Although the prognosis is relatively good in G1, some G1 cases are more aggressive, which are called G1 with MELF (microcystic, elongated, and fragmented) pattern. Current therapy, such as radiotherapy and chemotherapy, is not effective in MELF, and more effective treatment is needed. The Cancer Genome Atlas (TCGA) performed an integrated genomic, transcriptomic, and proteomic analysis and classified EC into 4 groups: DNA polymerase epsilon (POLE) ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high, in which MELF was associated with microsatellite instability hypermutated. Microsatellite instability is detected in a wide variety of cancer, and PD-1 (programmed cell death 1) and PD-L1 (programmed cell death-ligand 1) are received a lot of attention as a therapeutic target. To date, no studies have been focused on PD-L1 expression in EC with MELF pattern. Then we performed immunohistochemical analysis of the distribution of PD-L1 expressing cells in G1 with MELF pattern. In cases of G1 with MELF pattern, tumor cells expressed PD-L1 significantly higher in invasive front area than in surface area. We often found lymphovascular invasion of PD-L1 expressing tumor cells. PD-L1 expressing tumor cells in MELF would be the cause of recurrence or lymph node metastasis. Moreover, in most G1 cases with MELF pattern, PD-L1 was expressed in inflammatory cells as well as tumor cells in invasive front area. PD-L1 expression in both tumor and immune cells contribute to immune suppression and both cells could be sensitive to therapeutic agents targeting the PD-L1/PD-1 axis. Therefore, significant therapeutic effect can be expected by applying PD-1/PD-L1 immunotherapy to the treatment of G1 with MELF.

Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma.

Grade 1 (G1) endometrioid carcinoma (EC) is relatively a good prognosis. However, in a minority of cases, G1 shows an aggressive histological pattern known as the microcystic, elongated, and fragmented (MELF) pattern. We previously reported that EC with high expression levels of S100A4 and serum deprivation-response protein (SDPR) was related to MELF pattern invasion. However, the molecular features of the invasive front area of the MELF pattern have not been investigated. In this study, we searched for genes preferentially expressed in the invasive front area of EC with the MELF pattern using laser microdissection and RNA sequencing, and showed that nicotinamide N-methyltransferase (NNMT) is related to MELF pattern invasiveness. Immunohistochemical analyses confirmed high NNMT expression in the invasive front area of the MELF pattern. Moreover, NNMT promoted migration, invasion, colony formation, epithelial-mesenchymal transition (EMT), and chemoresistance using EC cell lines. We speculate that depletion of NNMT promotes histone methylation and leads to tumor suppression because NNMT consumes S-adenosyl methionine (SAM), which is an essential methylation cofactor. NNMT knockout cells showed enhanced expression of H3K9me2. RNA sequencing using NNMT knockout cell lines suggested that methylation of H3K9 leads to repression of the transcription of various oncogenic genes. Our findings demonstrate the possibility that NNMT inhibitors, which are expected to be used for the treatment of metabolic disorders, would be effective for the treatment of aggressive EC. This is the first report of gene analyses focusing on the morphological changes associated with MELF pattern invasion of EC.

An immature inhibin-α-expressing subpopulation of ovarian clear cell carcinoma cells is related to an unfavorable prognosis.

Inhibin-α, a member of transforming growth factor-ß, is elevated in multiple tumors, but its specific roles are poorly understood. Here, we examined the feature of inhibin-α-expressing cells in ovarian tumors. Immunohistochemically, inhibin-α-expressing tumor cells were detected only in ovarian clear cell carcinoma (OCCC) among various types of ovarian tumors. By comparing the expression of inhibin-α and Ki-67, inhibin-α-expressing tumor cells were revealed to be less proliferative. When spheroids and chemoresistant cells were derived from OCCC cell lines, the expression level of inhibin-α was elevated, and that of an immature marker, aldehyde dehydrogenase, was also elevated. In consistent with this, inhibin-α expression was correlated with other immature markers, such as OCT3/4 and SOX2, and inversely correlated with proliferative marker MKI67 in public database on OCCC. Knockdown of inhibin-α in OCCC cell decreased chemoresistance. Moreover, prognostic analysis with 69 surgically resected OCCC cases revealed that the increased inhibin-α expression was an independent unfavorable prognostic factor. These findings suggested that inhibin-α-expressing subpopulation of OCCC tumor cells appeared to be less proliferative, immature, and angiogenic and to be related to acceleration of malignant progression.

The expression of trefoil factor 3 is related to histologic subtypes and invasiveness in lung adenocarcinoma.

Adenocarcinoma is the most common histological type of lung cancer and has various histologic subtypes, including lepidic, papillary, acinar and invasive mucinous adenocarcinoma. Histologic subtypes are associated with tumor invasiveness. For example, the lepidic subtype is less invasive than the papillary/acinar subtype. Trefoil factor 3 (TFF3) is a small secreting protein that is a member of the trefoil factor family, which is involved in mucosal stabilization and repair through its mitogenic and antiapoptotic activities. TFF3 overexpression is associated with various types of cancer. In lung cancer, TFF3 is expressed significantly in adenocarcinoma. However, the relationship between TFF3 expression and histologic subtypes in lung adenocarcinoma is unclear. The current study immunohistochemically revealed that, beside invasive mucinous carcinoma, the expression of TFF3 in papillary and acinar adenocarcinoma was significantly higher than that in lepidic adenocarcinoma. To further confirm these results, the expression of TFF3 in cases with both lepidic and papillary/acinar areas were examined. The expression of TFF3 in papillary/acinar areas was significantly higher when compared with lepidic areas in a single sample. Furthermore, using the lung adenocarcinoma cell line A549, TFF3-knockdown cells were generated. The results revealed that knockdown of TFF3 attenuated invasion. In vitro and immunohistochemical assays using clinical samples demonstrated that TFF3 expression was associated with lung adenocarcinoma invasiveness. To the best of our knowledge, the current study is the first to report that TFF3 expression was associated with the histologic subtypes of lung adenocarcinoma.

HTR3A is correlated with unfavorable histology and promotes proliferation through ERK phosphorylation in lung adenocarcinoma.

Lung cancer is the leading cause of cancer death around the world. Adenocarcinoma is the most common histological type and has various histologic subtypes: lepidic, acinar, papillary, solid, and invasive mucinous adenocarcinoma. Histologic subtypes are related to invasiveness of tumors; eg, lepidic subtype is less invasive than acinar/papillary subtype. HTR3A is the main subunit of 5-hydroxytryptamine 3 (5-HT3) receptors, which are the only ligand-gated ion channels in seven families of 5-HT receptors. Although 5-HT3 receptor is expressed mainly throughout the central and peripheral nervous systems, some papers report the effect of 5-HT3 receptors on tumor cells, including lung cancer. However, whether HTR3A correlates with histopathological findings such as the histologic subtypes or the distribution in an individual sample remains unclear. Immunohistochemically, we revealed that the higher expression level of HTR3A was detected in acinar, papillary, and solid adenocarcinoma than in adenocarcinoma in situ and lepidic adenocarcinoma; the former was a more aggressive subtype than the latter. We also showed the relationship between HTR3A expression and Ki-67 positivity, widely used as a proliferation marker. Moreover, we generated HTR3A-knockdown lung adenocarcinoma cells and showed that the HTR3A knockdown attenuated proliferation by ERK phosphorylation. Our results indicated that HTR3A expression was related to proliferation in lung adenocarcinoma, by means of both in vitro and immunohistochemical assays on clinical samples. We showed the therapeutic potential of a 5-HT3 receptor antagonist, tropisetron, for the treatment of lung adenocarcinoma.

Serine racemase enhances growth of colorectal cancer by producing pyruvate from serine.

Serine racemase (SRR) catalyses not only the racemization but also the dehydration of L-serine and D-serine, resulting in the formation of pyruvate and ammonia. Although SRR activity is important in the central nervous system, SRR has not been linked to cancer metabolism before. Here we show that SRR supports proliferation of colorectal-cancer cells. We find that SRR expression is upregulated in colorectal adenoma and adenocarcinoma lesions compared with non-neoplastic mucosa in human colorectal-cancer specimens. SRR-mediated dehydration of serine contributes to the pyruvate pool in colon-cancer cells, enhances proliferation, maintains mitochondrial mass and increases basal reactive oxygen species production, which has anti-apoptotic effects. Moreover, SRR promotes acetylation of histone H3 by maintaining intracellular acetyl-CoA levels. Inhibition of SRR suppresses growth of colorectal tumours in mice and augments the efficacy of 5-fluorouracil treatment. Our findings highlight a previously unknown mechanism through which a racemase supports cancer-cell growth and suggest that SRR might be a molecular target for colorectal-cancer therapy.

FAM111B enhances proliferation of KRAS-driven lung adenocarcinoma by degrading p16.

Lung cancer is a common type of cancer that represents a health problem worldwide; lung adenocarcinoma (LUAD) is a major subtype of lung cancer. Although several treatments for LUAD have been developed, the mortality rate remains high because of uncontrollable progression. Further biological and clinicopathological studies are therefore needed. Here, we investigated the role of family with sequence similarity 111 member B (FAM111B), which is highly expressed in papillary-predominant LUAD; however, its role in cancer is unclear. An immunohistochemical analysis confirmed that papillary-predominant adenocarcinomas exhibited higher expression of FAM111B, compared with lepidic-predominant adenocarcinomas. Additionally, FAM111B expression was significantly correlated with clinical progression. In vitro functional analyses using FAM111B-knockout cells demonstrated that FAM111B plays an important role in proliferation and cell cycle progression of KRAS-driven LUAD under serum-starvation conditions. Furthermore, FAM111B regulated cyclin D1-CDK4-dependent cell cycle progression by degradation of p16. In summary, we revealed the clinical importance of FAM111B in human tumor tissues, as well as its function as a degradative enzyme. Therefore, FAM111B has potential as a clinicopathological prognostic marker for LUAD.