The tumor microenvironment comprises various cell types, and cancer-associated fibroblasts (CAFs) are crucial contributors to cancer progression and metastasis. CAFs also play an important role in esophageal squamous cell carcinoma and have been extensively studied in this context. However, the association between CAFs and progression across pathological stages has not yet been reported. To identify these specific CAFs, we used a case-oriented approach for single-cell RNA sequencing. Consequently, we identified 3 CAF clusters classified as myofibroblastic CAFs (myCAFs), which increased in number as the cancer progressed. Pathway analysis revealed that the 3 CAF clusters had distinct properties. These CAFs were named MMP13+, COL11A1+, and SFRP4+ myCAFs based on their characteristic gene expression. We also investigated the distribution of various immune cells within the tumor microenvironment associated with the 3 different CAF clusters. The results revealed the presence of different types of immune cells, including M2 macrophages, regulatory T cells, and interferon gamma+ programmed death-1+ T cells and interferon gamma+ programmed death-1− T cells. Next, we evaluated the presence of these 3 CAF subtypes in surgically resected specimens from patients with advanced esophageal squamous cell carcinoma using RNA in situ hybridization. Analysis of the association between these 3 CAF subtypes and prognosis showed that 2 subtypes (MMP13+ and COL11A1+ myCAFs) were associated with poor prognosis. MMP13+ myCAFs were associated with poorly differentiated infiltration patterns, whereas COL11A1+ myCAFs were associated with lymph node metastasis. These results suggest that future treatments targeting these CAFs and patient stratification based on these CAFs are warranted.
{"title":"MMP13-Expressing and COL11A1-Expressing Cancer-Associated Fibroblasts: Key Drivers of Esophageal Squamous Cell Carcinoma Progression and Prognostic Indicators","authors":"Shu Kato , Yuki Kato , Makoto Kodama , Kouhei Yamamoto , Asuka Furukawa , Yoshihiro Nagase , Rinka Miyashiro , Minako Takagi , Masayoshi Sakano , Hisashi Fujiwara , Kenro Kawada , Yusuke Kinugasa , Kenichi Ohashi","doi":"10.1016/j.labinv.2025.104247","DOIUrl":"10.1016/j.labinv.2025.104247","url":null,"abstract":"<div><div>The tumor microenvironment comprises various cell types, and cancer-associated fibroblasts (CAFs) are crucial contributors to cancer progression and metastasis. CAFs also play an important role in esophageal squamous cell carcinoma and have been extensively studied in this context. However, the association between CAFs and progression across pathological stages has not yet been reported. To identify these specific CAFs, we used a case-oriented approach for single-cell RNA sequencing. Consequently, we identified 3 CAF clusters classified as myofibroblastic CAFs (myCAFs), which increased in number as the cancer progressed. Pathway analysis revealed that the 3 CAF clusters had distinct properties. These CAFs were named <em>MMP13</em><sup>+</sup>, <em>COL11A1</em><sup>+</sup>, and <em>SFRP4</em><sup>+</sup> myCAFs based on their characteristic gene expression. We also investigated the distribution of various immune cells within the tumor microenvironment associated with the 3 different CAF clusters. The results revealed the presence of different types of immune cells, including M2 macrophages, regulatory T cells, and interferon gamma<sup>+</sup> programmed death-1<sup>+</sup> T cells and interferon gamma<sup>+</sup> programmed death-1<sup>−</sup> T cells. Next, we evaluated the presence of these 3 CAF subtypes in surgically resected specimens from patients with advanced esophageal squamous cell carcinoma using RNA in situ hybridization. Analysis of the association between these 3 CAF subtypes and prognosis showed that 2 subtypes (<em>MMP13</em><sup>+</sup> and <em>COL11A1</em><sup>+</sup> myCAFs) were associated with poor prognosis. <em>MMP13</em><sup>+</sup> myCAFs were associated with poorly differentiated infiltration patterns, whereas <em>COL11A1</em><sup>+</sup> myCAFs were associated with lymph node metastasis. These results suggest that future treatments targeting these CAFs and patient stratification based on these CAFs are warranted.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104247"},"PeriodicalIF":4.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145176288","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-22DOI: 10.1016/j.labinv.2025.104244
Hongyan Qian , Min Tang , Tianqi Wu , Zhouna Sun , Junjie Mao , Juanjuan Cui , Feng Sun , Yunyan Lu , Hua Jin , Aiguo Shen
Cervical cancer (CC) remains a major global health challenge, with radiotherapy resistance (RR) representing a critical impediment to treatment efficacy. This study investigated the underlying mechanisms of replication stress (RS) in RR and identified potential therapeutic targets for CC. A comprehensive bioinformatics workflow was applied to analyze the expression profiles and prognostic significance of RS-related differentially expressed genes (RSRDs) in patients with RR. The prognostic utility of an RS-based risk score model was subsequently evaluated in the context of the tumor microenvironment, somatic mutation landscape, etc. The clinical relevance of the identified hub RSRDs was validated through immunohistochemistry, univariate and multivariate Cox regression analyses, and a prognostic nomogram using data from a real-world patient cohort. Functional assays conducted both in vitro and in vivo further confirmed the role of the key RSRD. Thus, enrichment analysis of the 124 common differentially expressed genes showed RS-related biological processes were enriched. The RS risk score model, constructed using 2 hub RSRDs (AXIN1 and C-terminal binding protein 1) identified through Least Absolute Shrinkage and Selection Operator (LASSO) regression, showed strong diagnostic and prognostic performance. Enrichment analysis showed the risk score model influenced CC prognosis by tumor microenvironment and mutation, etc. Immunohistochemistry analysis of tissue microarrays explored a significant downregulation of AXIN1 in RR samples. AXIN1 was also an independent prognosis biomarker for CC patients, particularly among patients receiving radiotherapy. Knockdown of AXIN1 significantly inhibited the radiosensitivity in CC cell lines, and in vivo experiments showed AXIN1 knockdown led to increased tumor volume following radiotherapy. Molecular docking analysis illustrated JQ1 may promote AXIN1 expression. This study is the first to identify AXIN1 as a replication stress-associated gene with prognostic value in CC, specifically in the context of radiotherapy. These findings may support personalized treatment strategies and provide a foundation for future investigations into RS-targeted therapies in CC.
{"title":"The Role of Replication Stress-Related Genes in Cervical Cancer Radiotherapy Resistance: A Bioinformatic and Experimental Validation","authors":"Hongyan Qian , Min Tang , Tianqi Wu , Zhouna Sun , Junjie Mao , Juanjuan Cui , Feng Sun , Yunyan Lu , Hua Jin , Aiguo Shen","doi":"10.1016/j.labinv.2025.104244","DOIUrl":"10.1016/j.labinv.2025.104244","url":null,"abstract":"<div><div>Cervical cancer (CC) remains a major global health challenge, with radiotherapy resistance (RR) representing a critical impediment to treatment efficacy. This study investigated the underlying mechanisms of replication stress (RS) in RR and identified potential therapeutic targets for CC. A comprehensive bioinformatics workflow was applied to analyze the expression profiles and prognostic significance of RS-related differentially expressed genes (RSRDs) in patients with RR. The prognostic utility of an RS-based risk score model was subsequently evaluated in the context of the tumor microenvironment, somatic mutation landscape, etc. The clinical relevance of the identified hub RSRDs was validated through immunohistochemistry, univariate and multivariate Cox regression analyses, and a prognostic nomogram using data from a real-world patient cohort. Functional assays conducted both in vitro and in vivo further confirmed the role of the key RSRD. Thus, enrichment analysis of the 124 common differentially expressed genes showed RS-related biological processes were enriched. The RS risk score model, constructed using 2 hub RSRDs (AXIN1 and C-terminal binding protein 1) identified through Least Absolute Shrinkage and Selection Operator (LASSO) regression, showed strong diagnostic and prognostic performance. Enrichment analysis showed the risk score model influenced CC prognosis by tumor microenvironment and mutation, etc. Immunohistochemistry analysis of tissue microarrays explored a significant downregulation of AXIN1 in RR samples. AXIN1 was also an independent prognosis biomarker for CC patients, particularly among patients receiving radiotherapy. Knockdown of AXIN1 significantly inhibited the radiosensitivity in CC cell lines, and in vivo experiments showed AXIN1 knockdown led to increased tumor volume following radiotherapy. Molecular docking analysis illustrated JQ1 may promote AXIN1 expression. This study is the first to identify AXIN1 as a replication stress-associated gene with prognostic value in CC, specifically in the context of radiotherapy. These findings may support personalized treatment strategies and provide a foundation for future investigations into RS-targeted therapies in CC.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104244"},"PeriodicalIF":4.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137939","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-22DOI: 10.1016/j.labinv.2025.104243
Ainiah Rushdiana Raquib , Torsten O. Nielsen
Synovial sarcoma is an aggressive cancer generally affecting adolescents and young adults and is characterized by high rates of recurrence and metastasis. It is primarily driven by the fusion oncoprotein SS18::SSX, the product of a pathognomonic chromosomal translocation t(X;18), which facilitates widespread epigenetic dysregulation through interactions with complexes, such as the BRG1/BRM-associated factor complex and polycomb repressive complexes. Previous attempts to perform mass spectrometry (MS) of the SS18::SSX interactome have been limited by the lack of an antibody to detect the endogenous protein, hence relying on single-cell lines with exogenous tags. We previously used a monoclonal antibody, which specifically detects SS18::SSX containing the canonical fusion junction (seen in 95% of cases) and established its utility in several applications. Using that antibody, MS analysis revealed that the SS18::SSX protein undergoes alternative splicing of exon 8 in SS18. We next performed immunoprecipitation MS of SS18::SSX in 6 immortalized human synovial sarcoma cell lines and identified the canonical polycomb repressive complex member chromobox 4 (CBX4), as a novel interactor of the oncoprotein. Immunohistochemical staining of several epigenetic factors on a human synovial sarcoma tissue microarray showed an association of synovial sarcoma samples with higher CBX4 expression. Last, an analysis of CBX4 expression across 337 samples from 12 sarcoma subtypes, carcinomas, and normal tissue demonstrates higher expression in synovial sarcoma samples compared with other tissue types. These results highlight a crucial approach in identifying important partners of SS18::SSX in synovial sarcoma to establish new biological pathways that contribute to the disease.
{"title":"Chromobox 4 (CBX4) Is a Novel Interactor of SS18::SSX in Synovial Sarcoma","authors":"Ainiah Rushdiana Raquib , Torsten O. Nielsen","doi":"10.1016/j.labinv.2025.104243","DOIUrl":"10.1016/j.labinv.2025.104243","url":null,"abstract":"<div><div>Synovial sarcoma is an aggressive cancer generally affecting adolescents and young adults and is characterized by high rates of recurrence and metastasis. It is primarily driven by the fusion oncoprotein SS18::SSX, the product of a pathognomonic chromosomal translocation t(X;18), which facilitates widespread epigenetic dysregulation through interactions with complexes, such as the BRG1/BRM-associated factor complex and polycomb repressive complexes. Previous attempts to perform mass spectrometry (MS) of the SS18::SSX interactome have been limited by the lack of an antibody to detect the endogenous protein, hence relying on single-cell lines with exogenous tags. We previously used a monoclonal antibody, which specifically detects SS18::SSX containing the canonical fusion junction (seen in 95% of cases) and established its utility in several applications. Using that antibody, MS analysis revealed that the SS18::SSX protein undergoes alternative splicing of <em>exon 8</em> in SS18. We next performed immunoprecipitation MS of SS18::SSX in 6 immortalized human synovial sarcoma cell lines and identified the canonical polycomb repressive complex member chromobox 4 (CBX4), as a novel interactor of the oncoprotein. Immunohistochemical staining of several epigenetic factors on a human synovial sarcoma tissue microarray showed an association of synovial sarcoma samples with higher CBX4 expression. Last, an analysis of CBX4 expression across 337 samples from 12 sarcoma subtypes, carcinomas, and normal tissue demonstrates higher expression in synovial sarcoma samples compared with other tissue types. These results highlight a crucial approach in identifying important partners of SS18::SSX in synovial sarcoma to establish new biological pathways that contribute to the disease.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104243"},"PeriodicalIF":4.2,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-18DOI: 10.1016/j.labinv.2025.104241
Sandro Bräunig , Carl Dencker , Dang Nghiem Vo , Rong Fan , Alba Lillo Sierras , Jens Enoksson , Anne Hultquist , Hongzhe Li , Stefan Scheding
Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Bone marrow (BM) fibrosis in ALL has been associated with adverse outcomes; however, little is known about the mechanisms that cause fibrosis in ALL. Therefore, we established a novel and advanced analysis method by combining multicolor immunofluorescence (IF) staining with in situ RNA expression analysis (RNAscope) to investigate the spatial expression of putative fibrotic drivers in ALL BMs. We analyzed standard BM biopsies from pediatric patients with ALL. Sequential 5-color IF staining with CD45, CD271, CD31, CD34, and DAPI was used to identify different BM cell types. Combined RNAscope and IF staining was established for spatial messenger RNA expression analysis of transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1), which are known to play major roles in primary myelofibrosis (PMF). PMF and normal BM samples served as controls. As expected, ALL BMs showed high cellularities and prominent populations of blast cells. CD271+ mesenchymal stromal cell density was increased in ALL and was associated with fibrosis in a similar manner as observed for PMF. TGFB1 and PDGFA1 expression was considerably increased in ALL megakaryocytes (MKs) compared with patients with PMF and normal controls. Furthermore, MK TGFB1 and PDGFA1 expression intensities in fibrotic ALL correlated with fibrosis grade. TGFB1 and PDGFA1 were also expressed in leukemic blasts, however, at lower intensities compared with ALL MKs. Taken together, advanced in situ RNA and IF staining not only revealed increased expression of TGFB1 and PDGFA1 in fibrotic pediatric ALL but also identified ALL blasts and MKs as their cellular origin at the single-cell level. These novel data strongly suggest a role of these cytokines as potential fibrosis drivers in ALL. More broadly, our findings demonstrate that combined RNA and surface marker analysis is a powerful tool to provide new and valuable insights into BM pathophysiology.
{"title":"Combined Multicolor Immunofluorescence Staining and Spatial In Situ messenger RNA Expression Analysis Identifies Potential Fibrosis Drivers in Acute Lymphoblastic Leukemia","authors":"Sandro Bräunig , Carl Dencker , Dang Nghiem Vo , Rong Fan , Alba Lillo Sierras , Jens Enoksson , Anne Hultquist , Hongzhe Li , Stefan Scheding","doi":"10.1016/j.labinv.2025.104241","DOIUrl":"10.1016/j.labinv.2025.104241","url":null,"abstract":"<div><div>Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Bone marrow (BM) fibrosis in ALL has been associated with adverse outcomes; however, little is known about the mechanisms that cause fibrosis in ALL. Therefore, we established a novel and advanced analysis method by combining multicolor immunofluorescence (IF) staining with in situ RNA expression analysis (RNAscope) to investigate the spatial expression of putative fibrotic drivers in ALL BMs. We analyzed standard BM biopsies from pediatric patients with ALL. Sequential 5-color IF staining with CD45, CD271, CD31, CD34, and DAPI was used to identify different BM cell types. Combined RNAscope and IF staining was established for spatial messenger RNA expression analysis of transforming growth factor beta 1 (<em>TGFB1</em>) and platelet-derived growth factor alpha 1 (<em>PDGFA1</em>), which are known to play major roles in primary myelofibrosis (PMF). PMF and normal BM samples served as controls. As expected, ALL BMs showed high cellularities and prominent populations of blast cells. CD271<sup>+</sup> mesenchymal stromal cell density was increased in ALL and was associated with fibrosis in a similar manner as observed for PMF. <em>TGFB1</em> and <em>PDGFA1</em> expression was considerably increased in ALL megakaryocytes (MKs) compared with patients with PMF and normal controls. Furthermore, MK <em>TGFB1</em> and <em>PDGFA1</em> expression intensities in fibrotic ALL correlated with fibrosis grade. <em>TGFB1</em> and <em>PDGFA1</em> were also expressed in leukemic blasts, however, at lower intensities compared with ALL MKs. Taken together, advanced in situ RNA and IF staining not only revealed increased expression of <em>TGFB1</em> and <em>PDGFA1</em> in fibrotic pediatric ALL but also identified ALL blasts and MKs as their cellular origin at the single-cell level. These novel data strongly suggest a role of these cytokines as potential fibrosis drivers in ALL. More broadly, our findings demonstrate that combined RNA and surface marker analysis is a powerful tool to provide new and valuable insights into BM pathophysiology.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104241"},"PeriodicalIF":4.2,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145103029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-16DOI: 10.1016/j.labinv.2025.104242
Margaret Tulessin , Ludwig Beer , Stephanie Roessler , Anika Beckers , Darko Castven , Diego Francesco Calvisi , Xin Chen , Sebastian Lange , Nicole Pfarr , Jens Marquardt , Theresa Hildegard Wirtz , Marie-Luise Berres , Katja Steiger , Tanja Groll , Carolin Mogler
Cholangiocarcinoma (CCA) is an aggressive malignancy that originates in the bile ducts and is characterized by late-stage diagnosis and limited treatment options. CCA accounts for approximately 10% to 15% of primary liver tumors. Recent genetic studies have shed new light on this disease, exploring CCA’s complexity and finding more effective treatment strategies, particularly based on identifying actionable mutations. Various mouse models for CCA have been established; however, the extent to which these models reflect the complexity of human is not well investigated. Therefore, this study aimed to characterize the available mouse models for CCA studies and compare their characteristics, advantages, and challenges in resemblance to human CCA. We applied tissue-based techniques using classical hematoxylin and eosin, Sirius red, and immunohistochemistry of 16 markers for in-depth characterization of tumor cells, and the tumor microenvironment of 11 different mouse models. Our findings demonstrate that CCAs present with various tumor subtypes, tumor growth patterns, morphologic subtypes, and tumor microenvironment activity. Furthermore, we report here that neoplastic lesions other than CCA, such as hepatocellular carcinoma and nonneoplastic changes in the liver parenchyma (eg, steatosis), occur with significant differences among the investigated models. Nine out of 11 investigated models were suitable for CCA studies as they resemble human CCA features. Overall, our data show that mouse models of CCA represent a valid tool to investigate this deadly disease, but they should be carefully selected, depending on the study’s aims and targets in advance.
{"title":"Detailed Characterization and Comparison of Mouse Models for Cholangiocarcinoma","authors":"Margaret Tulessin , Ludwig Beer , Stephanie Roessler , Anika Beckers , Darko Castven , Diego Francesco Calvisi , Xin Chen , Sebastian Lange , Nicole Pfarr , Jens Marquardt , Theresa Hildegard Wirtz , Marie-Luise Berres , Katja Steiger , Tanja Groll , Carolin Mogler","doi":"10.1016/j.labinv.2025.104242","DOIUrl":"10.1016/j.labinv.2025.104242","url":null,"abstract":"<div><div>Cholangiocarcinoma (CCA) is an aggressive malignancy that originates in the bile ducts and is characterized by late-stage diagnosis and limited treatment options. CCA accounts for approximately 10% to 15% of primary liver tumors. Recent genetic studies have shed new light on this disease, exploring CCA’s complexity and finding more effective treatment strategies, particularly based on identifying actionable mutations. Various mouse models for CCA have been established; however, the extent to which these models reflect the complexity of human is not well investigated. Therefore, this study aimed to characterize the available mouse models for CCA studies and compare their characteristics, advantages, and challenges in resemblance to human CCA. We applied tissue-based techniques using classical hematoxylin and eosin, Sirius red, and immunohistochemistry of 16 markers for in-depth characterization of tumor cells, and the tumor microenvironment of 11 different mouse models. Our findings demonstrate that CCAs present with various tumor subtypes, tumor growth patterns, morphologic subtypes, and tumor microenvironment activity. Furthermore, we report here that neoplastic lesions other than CCA, such as hepatocellular carcinoma and nonneoplastic changes in the liver parenchyma (eg, steatosis), occur with significant differences among the investigated models. Nine out of 11 investigated models were suitable for CCA studies as they resemble human CCA features. Overall, our data show that mouse models of CCA represent a valid tool to investigate this deadly disease, but they should be carefully selected, depending on the study’s aims and targets in advance.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104242"},"PeriodicalIF":4.2,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-15DOI: 10.1016/j.labinv.2025.104240
Ekaterina Menshikova , Kristin Deeb , Elizabeth M. Genega , Krisztina Hanley , Gulisa Turashvili
Targeted therapy directed against human epidermal growth factor receptor 2 (HER2) has shown promising results in HER2-positive endometrial cancer. Recent limited data suggest significant intratumoral heterogeneity in HER2 expression in serous carcinomas. We aimed to evaluate HER2 heterogeneity at the protein and gene levels and the impact of variable definitions in endometrial carcinomas including nonserous subtypes. We retrospectively identified biopsies and surgical specimens with available HER2 immunohistochemical (IHC) stains and fluorescence in situ hybridization (FISH) results. IHC stains and FISH data were reevaluated to assess variability in the HER2 protein expression and gene amplification. The overall HER2-positivity rate was 31% using the endometrial criteria, and 42.6% by the gastric criteria. Heterogeneous IHC staining was observed in 45.7% of tumors, predominating in 2+/3+ scores (P < .001). Re-evaluation of FISH revealed a 31% rate of heterogeneous gene amplification. Our study demonstrates a high frequency of HER2 heterogeneity at both the protein and the gene levels. Further studies on HER2 heterogeneity and treatment response are warranted for refining standardized testing and reporting algorithms.
{"title":"Analysis of Human Epidermal Growth Factor Receptor 2 (HER2) Heterogeneity at the Protein and Gene Levels in Endometrial Cancer: Refining HER2 Reporting and HER2-Directed Therapies","authors":"Ekaterina Menshikova , Kristin Deeb , Elizabeth M. Genega , Krisztina Hanley , Gulisa Turashvili","doi":"10.1016/j.labinv.2025.104240","DOIUrl":"10.1016/j.labinv.2025.104240","url":null,"abstract":"<div><div>Targeted therapy directed against human epidermal growth factor receptor 2 (HER2) has shown promising results in HER2-positive endometrial cancer. Recent limited data suggest significant intratumoral heterogeneity in HER2 expression in serous carcinomas. We aimed to evaluate HER2 heterogeneity at the protein and gene levels and the impact of variable definitions in endometrial carcinomas including nonserous subtypes. We retrospectively identified biopsies and surgical specimens with available HER2 immunohistochemical (IHC) stains and fluorescence in situ hybridization (FISH) results. IHC stains and FISH data were reevaluated to assess variability in the HER2 protein expression and gene amplification. The overall HER2-positivity rate was 31% using the endometrial criteria, and 42.6% by the gastric criteria. Heterogeneous IHC staining was observed in 45.7% of tumors, predominating in 2+/3+ scores (<em>P</em> < .001). Re-evaluation of FISH revealed a 31% rate of heterogeneous gene amplification. Our study demonstrates a high frequency of HER2 heterogeneity at both the protein and the gene levels. Further studies on HER2 heterogeneity and treatment response are warranted for refining standardized testing and reporting algorithms.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104240"},"PeriodicalIF":4.2,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145081173","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-12DOI: 10.1016/j.labinv.2025.104239
Hetian Bai , Jinyong Li , Yeting Tu , Chongyun Bao
Cystic lesions are more prevalent in jawbones than in other bones. Odontogenic cysts are typically painless and asymptomatic and often reach significant sizes before detection. Unlike odontogenic cysts of inflammatory origin, understanding of the mechanisms of pathogenesis and progression of developmental odontogenic cysts remains incomplete. This study aimed to elucidate the cause and mechanisms of bone resorption in developmental odontogenic cysts. First, single-cell RNA sequencing was conducted for one of the most common developmental odontogenic cysts, dentigerous cysts (DCs), and the obtained data were compared with those of dental follicles of embedded teeth. Most DEGs in DCs and dental follicles were associated with immunoglobulin secretion, and an activated immunoglobulin-secreting plasma cell subtype was confirmed. Cell-to-cell interaction analysis revealed strong interactions between the activated plasma cells and leptin receptor–expressing (LepR+) fibroblasts via a “bone morphogenetic protein 6 and its receptor type 2" interaction. These LepR+ fibroblasts constituted the majority of fibroblasts in DCs. And these fibroblasts highly expressed genes were related to osteoclastogenesis, such as CSF1, IL6, and IL34. Mouse bone marrow–derived monocytes and bone marrow mesenchymal stem cells were treated with culture supernatants of the LepR+ fibroblasts. The treatment led to osteoclast formation and bone resorption, and inhibition of bone morphogenetic protein signaling suppressed the osteoclastogenesis effect. Thus, the LepR+ fibroblasts distinguished developmental odontogenic cysts from benign follicles; such cells interacted with activated plasma cell through the bone morphogenetic protein signaling pathway. And the LepR+ fibroblasts were crucial in osteoclast induction and bone resorption in DCs. This study confirmed a novel LepR+ fibroblast–induced bone erosion mechanism in developmental odontogenic cysts, which may inspire future pharmacological or surgical therapies.
{"title":"Leptin Receptor–Expressing (LepR+) Fibroblasts Activated by BMP Signaling Promote Bone Resorption in Developmental Odontogenic Cysts","authors":"Hetian Bai , Jinyong Li , Yeting Tu , Chongyun Bao","doi":"10.1016/j.labinv.2025.104239","DOIUrl":"10.1016/j.labinv.2025.104239","url":null,"abstract":"<div><div>Cystic lesions are more prevalent in jawbones than in other bones. Odontogenic cysts are typically painless and asymptomatic and often reach significant sizes before detection. Unlike odontogenic cysts of inflammatory origin, understanding of the mechanisms of pathogenesis and progression of developmental odontogenic cysts remains incomplete. This study aimed to elucidate the cause and mechanisms of bone resorption in developmental odontogenic cysts. First, single-cell RNA sequencing was conducted for one of the most common developmental odontogenic cysts, dentigerous cysts (DCs), and the obtained data were compared with those of dental follicles of embedded teeth. Most DEGs in DCs and dental follicles were associated with immunoglobulin secretion, and an activated immunoglobulin-secreting plasma cell subtype was confirmed. Cell-to-cell interaction analysis revealed strong interactions between the activated plasma cells and leptin receptor–expressing (LepR<sup>+</sup>) fibroblasts via a “bone morphogenetic protein 6 and its receptor type 2\" interaction. These LepR<sup>+</sup> fibroblasts constituted the majority of fibroblasts in DCs. And these fibroblasts highly expressed genes were related to osteoclastogenesis, such as <em>CSF1</em>, <em>IL6</em>, and <em>IL34</em>. Mouse bone marrow–derived monocytes and bone marrow mesenchymal stem cells were treated with culture supernatants of the LepR<sup>+</sup> fibroblasts. The treatment led to osteoclast formation and bone resorption, and inhibition of bone morphogenetic protein signaling suppressed the osteoclastogenesis effect. Thus, the LepR<sup>+</sup> fibroblasts distinguished developmental odontogenic cysts from benign follicles; such cells interacted with activated plasma cell through the bone morphogenetic protein signaling pathway. And the LepR<sup>+</sup> fibroblasts were crucial in osteoclast induction and bone resorption in DCs. This study confirmed a novel LepR<sup>+</sup> fibroblast–induced bone erosion mechanism in developmental odontogenic cysts, which may inspire future pharmacological or surgical therapies.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 11","pages":"Article 104239"},"PeriodicalIF":4.2,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145065219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-04DOI: 10.1016/j.labinv.2025.104238
Dong Ren , Chenchen Niu , Nyein Htun , Xin Zhang , Jiadi He , Ronggang Li , Qiongru Liu , Vincent Lee , Robert A. Edwards , Grace G. Zhou , Brandon M. Lehrich , Derek H. Liu , Angie Nguyen , Jeff Chan , Nicholas R. Pannunzio , Edward C. Kuan , Beverly Y. Wang
Sinonasal mucosal melanoma (SNMM) is a rare aggressive malignancy of the sinonasal tract. Due to its advanced clinical presentation and frequent late-stage diagnosis, the 5-year survival rate is <30%, with an even worse prognosis in patients with distant metastasis (SNMM-M). Therefore, characterizing the molecular landscape of SNMM may provide novel therapeutic targets for SNMM-M. This study aimed to decipher the histopathological and molecular landscape of SNMM-M and yields novel insights into potential therapeutic approaches using targeted DNA and RNA next-generation sequencing, immunohistochemistry, and fluorescence in situ hybridization. SNMM-M cases were characterized by epithelioid-predominant morphology, frequent tumor necrosis, minimal pleomorphism, and sparse tumor-infiltrating lymphocytes (TILs). A significant association between the absence of brisk TILs and metastasis in SNMM was noted. Moreover, the presence of lymphovascular invasion and absence of brisk TILs were both significantly associated with poorer overall survival in patients with SNMM. Both DNA and RNA sequencing identified no gene fusions, whereas DNA sequencing revealed 304 genomic alterations across 186 genes, including 9 multihit genes. Notably, missense mutations in structure-specific endonuclease subunit (SLX4), a key homologous recombination repair scaffold protein, were exclusively detected in SNMM-M, and patients with SLX4 mutations exhibited significantly worse survival (median, 9.9 vs 41.2 months without SLX4 mutations; P < .0001). A comparative analysis of genomic alterations and copy number variations of clinically actionable genes between SNMM-M and SNMM without distant metastasis (SNMM-nM) revealed that CDK4 gains/amplifications were commonly seen in SNMM-M cases, whereas receptor tyrosine kinase and homologous recombination repair gene alterations were highly enriched in SNMM-nM cases. Additionally, PD-L1 was more commonly expressed in SNMM-nM. These exploratory findings suggest that SLX4 mutation may serve as a potential prognostic biomarker and that CDK4 inhibitors could represent promising therapeutic options for SNMM-M. However, given the limited sample size, further validation in larger studies is essential to confirm these findings.
{"title":"Exploring Molecular Characteristics and Therapeutic Strategies for Primary Sinonasal Mucosal Melanoma With Distant Metastasis","authors":"Dong Ren , Chenchen Niu , Nyein Htun , Xin Zhang , Jiadi He , Ronggang Li , Qiongru Liu , Vincent Lee , Robert A. Edwards , Grace G. Zhou , Brandon M. Lehrich , Derek H. Liu , Angie Nguyen , Jeff Chan , Nicholas R. Pannunzio , Edward C. Kuan , Beverly Y. Wang","doi":"10.1016/j.labinv.2025.104238","DOIUrl":"10.1016/j.labinv.2025.104238","url":null,"abstract":"<div><div>Sinonasal mucosal melanoma (SNMM) is a rare aggressive malignancy of the sinonasal tract. Due to its advanced clinical presentation and frequent late-stage diagnosis, the 5-year survival rate is <30%, with an even worse prognosis in patients with distant metastasis (SNMM-M). Therefore, characterizing the molecular landscape of SNMM may provide novel therapeutic targets for SNMM-M. This study aimed to decipher the histopathological and molecular landscape of SNMM-M and yields novel insights into potential therapeutic approaches using targeted DNA and RNA next-generation sequencing, immunohistochemistry, and fluorescence in situ hybridization. SNMM-M cases were characterized by epithelioid-predominant morphology, frequent tumor necrosis, minimal pleomorphism, and sparse tumor-infiltrating lymphocytes (TILs). A significant association between the absence of brisk TILs and metastasis in SNMM was noted. Moreover, the presence of lymphovascular invasion and absence of brisk TILs were both significantly associated with poorer overall survival in patients with SNMM. Both DNA and RNA sequencing identified no gene fusions, whereas DNA sequencing revealed 304 genomic alterations across 186 genes, including 9 multihit genes. Notably, missense mutations in structure-specific endonuclease subunit (<em>SLX4</em>), a key homologous recombination repair scaffold protein, were exclusively detected in SNMM-M, and patients with <em>SLX4</em> mutations exhibited significantly worse survival (median, 9.9 vs 41.2 months without <em>SLX4</em> mutations; <em>P</em> < .0001). A comparative analysis of genomic alterations and copy number variations of clinically actionable genes between SNMM-M and SNMM without distant metastasis (SNMM-nM) revealed that <em>CDK4</em> gains/amplifications were commonly seen in SNMM-M cases, whereas receptor tyrosine kinase and homologous recombination repair gene alterations were highly enriched in SNMM-nM cases. Additionally, PD-L1 was more commonly expressed in SNMM-nM. These exploratory findings suggest that <em>SLX4</em> mutation may serve as a potential prognostic biomarker and that CDK4 inhibitors could represent promising therapeutic options for SNMM-M. However, given the limited sample size, further validation in larger studies is essential to confirm these findings.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104238"},"PeriodicalIF":4.2,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145008311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ameloblastoma (AM) is a benign epithelial odontogenic tumor that occurs in the jawbone. Although benign, AM can exhibit aggressive features, including locally invasive growth. In addition, local recurrence or distant metastasis may occur. Therefore, understanding the mechanisms underlying AM-cell migration is essential for improving clinical therapy. The role of the tumor microenvironment in disease progression has been extensively studied in various tumors. In the microenvironment, it has been reported that the extracellular matrix plays many roles. Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is highly expressed during tissue development and remodeling. In this study, we investigated the involvement of TN-C in AM progression. Immunohistochemical analysis of AM specimens revealed high TN-C protein expression in the stroma, particularly at the invasive front. In contrast, RNA in situ hybridization demonstrated that TNC was localized within tumor cells, suggesting that the TN-C protein in the stroma is secreted by tumor cells rather than produced by stromal cells. In in vitro analyses, TN-C expression was significantly upregulated in cocultures of the AM cell line, AM-1, and primary human periodontal ligament fibroblasts, indicating that tumor-stroma interactions enhance tumor-derived TN-C expression. Functionally, TN-C stimulation promoted AM-cell migration, whereas TNC knockdown suppressed it. Spatial transcriptomics revealed elevated TNC expression in regions undergoing malignant transformation. Our results demonstrate that tumor-derived TN-C promotes AM progression. The expression of TN-C at the invasive front and in malignant regions suggests its potential as both a prognostic marker and a therapeutic target in tumor progression.
{"title":"Tenascin-C Expression in Relation to Tumor-Stroma Interaction in Ameloblastoma","authors":"Satoko Sumi , Shohei Yoshimoto , Kanako Suyama , Masahide Taguchi , Hiromitsu Morita , Akimitsu Hiraki , Kyoko Oka","doi":"10.1016/j.labinv.2025.104237","DOIUrl":"10.1016/j.labinv.2025.104237","url":null,"abstract":"<div><div>Ameloblastoma (AM) is a benign epithelial odontogenic tumor that occurs in the jawbone. Although benign, AM can exhibit aggressive features, including locally invasive growth. In addition, local recurrence or distant metastasis may occur. Therefore, understanding the mechanisms underlying AM-cell migration is essential for improving clinical therapy. The role of the tumor microenvironment in disease progression has been extensively studied in various tumors. In the microenvironment, it has been reported that the extracellular matrix plays many roles. Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is highly expressed during tissue development and remodeling. In this study, we investigated the involvement of TN-C in AM progression. Immunohistochemical analysis of AM specimens revealed high TN-C protein expression in the stroma, particularly at the invasive front. In contrast, RNA in situ hybridization demonstrated that <em>TNC</em> was localized within tumor cells, suggesting that the TN-C protein in the stroma is secreted by tumor cells rather than produced by stromal cells. In in vitro analyses, TN-C expression was significantly upregulated in cocultures of the AM cell line, AM-1, and primary human periodontal ligament fibroblasts, indicating that tumor-stroma interactions enhance tumor-derived TN-C expression. Functionally, TN-C stimulation promoted AM-cell migration, whereas <em>TNC</em> knockdown suppressed it. Spatial transcriptomics revealed elevated <em>TNC</em> expression in regions undergoing malignant transformation. Our results demonstrate that tumor-derived TN-C promotes AM progression. The expression of TN-C at the invasive front and in malignant regions suggests its potential as both a prognostic marker and a therapeutic target in tumor progression.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 11","pages":"Article 104237"},"PeriodicalIF":4.2,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145006405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-02DOI: 10.1016/j.labinv.2025.104236
Victoria Mahar, Zachary Colburn, Joshua Sakai
Challenging pathology case consultations require the shipment of irreplaceable patient materials to the consultants’ location for evaluation. In the military, consultants and generalists span geographically diverse locations. Shipped cases risk diagnostic delays, loss, and irreparable damage in transit over extensive distances. Using digital pathology for consultation eliminates these risks. Digital pathology implementation efforts in the Military Health System have been unsuccessful; however, triservice pathologists’ attitudes toward this innovation have never been investigated. Our explanatory mixed-methods study used a web-based needs assessment and interviews to understand pathologists’ facilitators and barriers to using digital pathology for consultation. We believe that understanding their perceptions is critical if further implementation efforts are to be successful. Analyses showed that pathologists were receptive to enterprise-wide implementation, especially if it improved turnaround time and allowed immediate subspecialist feedback. Future implementation efforts may benefit from comprehensive technical support combined with a consolidated digital pathology program office for implementation and sustainment guidance.
{"title":"Telepathology for Consultation in the Military Health System: An Evaluation of Pathologists’ Impressions of Facilitators and Barriers Prior to Implementation","authors":"Victoria Mahar, Zachary Colburn, Joshua Sakai","doi":"10.1016/j.labinv.2025.104236","DOIUrl":"10.1016/j.labinv.2025.104236","url":null,"abstract":"<div><div>Challenging pathology case consultations require the shipment of irreplaceable patient materials to the consultants’ location for evaluation. In the military, consultants and generalists span geographically diverse locations. Shipped cases risk diagnostic delays, loss, and irreparable damage in transit over extensive distances. Using digital pathology for consultation eliminates these risks. Digital pathology implementation efforts in the Military Health System have been unsuccessful; however, triservice pathologists’ attitudes toward this innovation have never been investigated. Our explanatory mixed-methods study used a web-based needs assessment and interviews to understand pathologists’ facilitators and barriers to using digital pathology for consultation. We believe that understanding their perceptions is critical if further implementation efforts are to be successful. Analyses showed that pathologists were receptive to enterprise-wide implementation, especially if it improved turnaround time and allowed immediate subspecialist feedback. Future implementation efforts may benefit from comprehensive technical support combined with a consolidated digital pathology program office for implementation and sustainment guidance.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 11","pages":"Article 104236"},"PeriodicalIF":4.2,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145000987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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