Laboratory Investigation最新文献_第5页

Glycolysis Inhibition Restores Immune Sensitivity in GSNOR–Deficient Colorectal Cancer 糖酵解抑制可恢复gsnorn缺陷结直肠癌的免疫敏感性。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-10-11 DOI: 10.1016/j.labinv.2025.104250

Ana Mantrana , María Teresa Sánchez-Montero , Carmen Navarrete-Sirvent , Nerea Herrera-Casanova , Rafael Mena-Osuna , Aurora Rivas-Crespo , Alejandra Díaz-Chacón , Virginia Ávila-Oca , Marta Toledano-Fonseca , María Victoria García-Ortíz , Rafael González-Fernández , María Auxiliadora Gómez-España , Carlos Villar , Francisco Javier Medina-Fernández , Enrique Aranda , Silvia Guil-Luna , Antonio Rodríguez-Ariza

S-nitrosoglutathione reductase (GSNOR) is increasingly recognized as a tumor suppressor, and we have recently reported that its deficiency drives an aggressive and immune-evasive phenotype in colorectal cancer (CRC). However, the mechanisms linking GSNOR loss to immune escape remain incompletely understood. In this study, we uncover a previously unrecognized connection between metabolic reprogramming and immune escape in GSNOR-deficient CRC and identify a therapeutic vulnerability that can be exploited to restore immune responsiveness. A comprehensive analysis of 137 clinical CRC samples revealed that GSNOR-deficient tumors exhibit high-grade tumor budding, an established marker of poor prognosis, and reduced CD4⁺ and CD8⁺ T-cell infiltration, consistent with an immunosuppressive tumor microenvironment. Integrating transcriptomic, immunohistochemical, and single–cell RNA-sequencing data, we demonstrate that GSNOR-deficient tumors undergo a striking metabolic reprogramming toward glycolytic dependence, with elevated lactate production contributing to T-cell exclusion. Based on these findings, we show that pharmacologic glycolysis inhibition with 2-deoxyglucose reverses immune resistance in GSNOR-knockout models, enhancing CD8⁺ T-cell infiltration and sensitizing tumors to anti–PD-1 therapy both in vitro and in vivo. Notably, this is the first demonstration that metabolic intervention can restore immune sensitivity in GSNOR-deficient CRC. Our results identify GSNOR expression as a predictive biomarker for metabolic-immune combinatorial strategies and support the clinical translation of 2-deoxyglucose plus anti–PD-1 as a precision immunotherapy approach for this high-risk CRC phenotype.

s -亚硝基谷胱甘肽还原酶（GSNOR）越来越被认为是一种肿瘤抑制因子，我们最近报道了它的缺乏导致结直肠癌（CRC）的侵袭性和免疫逃避表型。然而，将GSNOR丢失与免疫逃逸联系起来的机制仍然不完全清楚。在这项研究中，我们揭示了gsnorr缺陷CRC中代谢重编程和免疫逃逸之间先前未被认识到的联系，并确定了一种可用于恢复免疫反应性的治疗脆弱性。对137例临床CRC样本的综合分析显示，gsnorr缺陷肿瘤表现为高级别肿瘤出芽，这是一种预后不良的标志，CD4+和CD8+ t细胞浸润减少，与免疫抑制肿瘤微环境一致。综合转录组学、免疫组织化学和单细胞RNA-seq数据，我们证明了gsnorr缺陷肿瘤经历了惊人的代谢重编程，导致糖酵解依赖，乳酸产量升高导致t细胞排斥。基于这些发现，我们发现2-脱氧葡萄糖（2-DG）的药理学糖酵解抑制逆转了gsnoro - ko模型的免疫抵抗，增强了CD8+ t细胞的浸润，并使肿瘤对抗pd -1治疗增敏。值得注意的是，这是首次证明代谢干预可以恢复gsnorr缺陷CRC的免疫敏感性。我们的研究结果确定GSNOR表达作为代谢-免疫组合策略的预测性生物标志物，并支持2-DG加抗pd -1的临床翻译作为这种高风险CRC表型的精确免疫治疗方法。

{"title":"Glycolysis Inhibition Restores Immune Sensitivity in GSNOR–Deficient Colorectal Cancer","authors":"Ana Mantrana , María Teresa Sánchez-Montero , Carmen Navarrete-Sirvent , Nerea Herrera-Casanova , Rafael Mena-Osuna , Aurora Rivas-Crespo , Alejandra Díaz-Chacón , Virginia Ávila-Oca , Marta Toledano-Fonseca , María Victoria García-Ortíz , Rafael González-Fernández , María Auxiliadora Gómez-España , Carlos Villar , Francisco Javier Medina-Fernández , Enrique Aranda , Silvia Guil-Luna , Antonio Rodríguez-Ariza","doi":"10.1016/j.labinv.2025.104250","DOIUrl":"10.1016/j.labinv.2025.104250","url":null,"abstract":"<div><div>S-nitrosoglutathione reductase (GSNOR) is increasingly recognized as a tumor suppressor, and we have recently reported that its deficiency drives an aggressive and immune-evasive phenotype in colorectal cancer (CRC). However, the mechanisms linking GSNOR loss to immune escape remain incompletely understood. In this study, we uncover a previously unrecognized connection between metabolic reprogramming and immune escape in GSNOR-deficient CRC and identify a therapeutic vulnerability that can be exploited to restore immune responsiveness. A comprehensive analysis of 137 clinical CRC samples revealed that GSNOR-deficient tumors exhibit high-grade tumor budding, an established marker of poor prognosis, and reduced CD4<sup>+</sup> and CD8<sup>+</sup> T-cell infiltration, consistent with an immunosuppressive tumor microenvironment. Integrating transcriptomic, immunohistochemical, and single–cell RNA-sequencing data, we demonstrate that GSNOR-deficient tumors undergo a striking metabolic reprogramming toward glycolytic dependence, with elevated lactate production contributing to T-cell exclusion. Based on these findings, we show that pharmacologic glycolysis inhibition with 2-deoxyglucose reverses immune resistance in GSNOR-knockout models, enhancing CD8<sup>+</sup> T-cell infiltration and sensitizing tumors to anti–PD-1 therapy both in vitro and in vivo. Notably, this is the first demonstration that metabolic intervention can restore immune sensitivity in GSNOR-deficient CRC. Our results identify GSNOR expression as a predictive biomarker for metabolic-immune combinatorial strategies and support the clinical translation of 2-deoxyglucose plus anti–PD-1 as a precision immunotherapy approach for this high-risk CRC phenotype.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104250"},"PeriodicalIF":4.2,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145286480","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}

引用次数: 0

Assessing the Status of Cyclin E1 (CCNE1) From Gene to Protein Level in Ovarian and Endometrial Carcinomas: A Systematic Review 评估周期蛋白E1 （CCNE1）在卵巢癌和子宫内膜癌中从基因到蛋白水平的状态：一项系统综述。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-10-10 DOI: 10.1016/j.labinv.2025.104249

Alexis Trecourt , Catherine Genestie , Alexander Valent , Mojgan Devouassoux-Shisheboran , Etienne Rouleau , Elisa Yaniz-Galende , Audrey Leformal , Valeria Naim , Alexandra Leary

Twenty percent and 45.4% of high-grade ovarian carcinomas (OC) and endometrial carcinomas (EC) exhibit CCNE1 amplification (CCNE1-amp), respectively, which is related to poor prognosis, but could serve as predictive biomarker for response to innovative targeted therapies. However, there is no consensus regarding how to evaluate the CCNE1 status (at the DNA, RNA, and/or protein level). Therefore, we conducted a systematic review of CCNE1 status testing in tubo-ovarian neoplasms and EC, comparing their performance for clinical purposes and highlighting the test’s interpretation criteria (CRD420250651291). Among the 734 records initially found on PubMed and Google Scholar, 48 reports were finally included. Molecular analyses and immunohistochemistry (IHC) were reported on 9774 tubo-ovarian neoplasms and 750 EC, and 6966 tubo-ovarian neoplasms and 856 EC, respectively. The most frequently morphological used method to detect CCNE1-amp was fluorescent in situ hybridization (13/16 studies, 81.3%), with quite consensual criteria to defined amplification (ie, CCNE1/chromosome 19 ratio ≥2, and/or >8/≥8 copies of CCNE1 per nucleus, and/or ≥4 CCNE1 copies in ≥40% of cells). The proportion of tubo-ovarian neoplasms with CCNE1 immunohistochemical overexpression varied from 13.5% to 96%, and 14.6% to 86.1% in EC. The sensitivity and specificity of CCNE1 IHC to detect/exclude CCNE1-amp varied from 54.5% to 100% and 59.3% to 90.1%, respectively. Given the reported data, CCNE1 overexpression should be considered either when an H-score is ≥100 or when the staining is >60% with >5% of cells strongly stained. Both CCNE1-amp and CCNE1 overexpressions were associated with poor prognosis and with response to Wee1 and CDK2 inhibitors in high-grade serous OC (overall response rate up to 53%, objective response rate of 32%-40%). In contrast, CCNE1 messenger RNA overexpression had no prognostic value. Thus, both CCNE1-amp detection by fluorescent in situ hybridization and CCNE1 protein levels quantification using IHC represent today the most validated tools to determine the CCNE1 status in OC/EC.

高级别卵巢癌（OC）和子宫内膜癌（EC）分别有20%和45.4%表现出CCNE1扩增（CCNE1-amp），这与预后不良有关，但可以作为对创新靶向治疗反应的预测性生物标志物。然而，关于如何评估CCNE1状态（在DNA、RNA和/或蛋白质水平上）尚无共识。因此，我们对输卵管卵巢肿瘤和EC的CCNE1状态检测进行了系统综述，比较了它们在临床中的表现，并强调了检测的解释标准（CRD420250651291）。在PubMed和b谷歌Scholar上最初发现的734条记录中，最终收录了48篇报告。分别对9774例输卵管卵巢肿瘤和750例EC、6966例输卵管卵巢肿瘤和856例EC进行了分子分析和免疫组化（IHC）。检测CCNE1-amp最常用的形态学方法是荧光原位杂交（FISH； 13/16项研究，81.3%），对扩增的定义标准是相当一致的（即CCNE1/ 19号染色体的比率≥2，和/或每个细胞核有8/≥8个CCNE1拷贝，和/或≥4个CCNE1拷贝在≥40%的细胞中）。CCNE1免疫组化过表达的输卵管卵巢肿瘤比例为13.5% ~ 96%，EC为14.6% ~ 86.1%。CCNE1 IHC检测/排除CCNE1-amp的敏感性和特异性分别为54.5% ~ 100%和59.3% ~ 90.1%。根据已报道的数据，当h评分至少为>00时，或者当>染色为60%，>染色为5%的细胞强烈染色时，应考虑CCNE1过表达。CCNE1-amp和CCNE1过表达均与HGSOC患者预后不良以及对Wee1和CDK2抑制剂的反应相关（总缓解率高达53%，客观缓解率为32-40%）。相比之下，CCNE1 mRNA过表达没有预后价值。因此，FISH检测CCNE1-amp和IHC定量CCNE1蛋白水平是目前确定OC/EC中CCNE1状态最有效的工具。

{"title":"Assessing the Status of Cyclin E1 (CCNE1) From Gene to Protein Level in Ovarian and Endometrial Carcinomas: A Systematic Review","authors":"Alexis Trecourt , Catherine Genestie , Alexander Valent , Mojgan Devouassoux-Shisheboran , Etienne Rouleau , Elisa Yaniz-Galende , Audrey Leformal , Valeria Naim , Alexandra Leary","doi":"10.1016/j.labinv.2025.104249","DOIUrl":"10.1016/j.labinv.2025.104249","url":null,"abstract":"<div><div>Twenty percent and 45.4% of high-grade ovarian carcinomas (OC) and endometrial carcinomas (EC) exhibit <em>CCNE1</em> amplification (<em>CCNE1</em>-amp), respectively, which is related to poor prognosis, but could serve as predictive biomarker for response to innovative targeted therapies. However, there is no consensus regarding how to evaluate the CCNE1 status (at the DNA, RNA, and/or protein level). Therefore, we conducted a systematic review of CCNE1 status testing in tubo-ovarian neoplasms and EC, comparing their performance for clinical purposes and highlighting the test’s interpretation criteria (CRD420250651291). Among the 734 records initially found on PubMed and Google Scholar, 48 reports were finally included. Molecular analyses and immunohistochemistry (IHC) were reported on 9774 tubo-ovarian neoplasms and 750 EC, and 6966 tubo-ovarian neoplasms and 856 EC, respectively. The most frequently morphological used method to detect <em>CCNE1</em>-amp was fluorescent in situ hybridization (13/16 studies, 81.3%), with quite consensual criteria to defined amplification (ie, <em>CCNE1</em>/chromosome 19 ratio ≥2, and/or >8/≥8 copies of <em>CCNE1</em> per nucleus, and/or ≥4 <em>CCNE1</em> copies in ≥40% of cells). The proportion of tubo-ovarian neoplasms with CCNE1 immunohistochemical overexpression varied from 13.5% to 96%, and 14.6% to 86.1% in EC. The sensitivity and specificity of CCNE1 IHC to detect/exclude <em>CCNE1</em>-amp varied from 54.5% to 100% and 59.3% to 90.1%, respectively. Given the reported data, CCNE1 overexpression should be considered either when an H-score is ≥100 or when the staining is >60% with >5% of cells strongly stained. Both <em>CCNE1</em>-amp and CCNE1 overexpressions were associated with poor prognosis and with response to Wee1 and CDK2 inhibitors in high-grade serous OC (overall response rate up to 53%, objective response rate of 32%-40%). In contrast, <em>CCNE1</em> messenger RNA overexpression had no prognostic value. Thus, both <em>CCNE1</em>-amp detection by fluorescent in situ hybridization and CCNE1 protein levels quantification using IHC represent today the most validated tools to determine the CCNE1 status in OC/EC.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104249"},"PeriodicalIF":4.2,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145280579","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}

引用次数: 0

Prevalence, Immune Checkpoint Expression, and Spatial Interplay of Immune Cells Are Linked to Favorable Tumor Phenotype in 4915 Human Carcinomas 在4915例人类癌症中，患病率、免疫检查点表达和免疫细胞的空间相互作用与有利的肿瘤表型有关。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-10-09 DOI: 10.1016/j.labinv.2025.104248

Zhihao Huang , Tim Mandelkow , Jonas B. Raedler , Elena Bady , Jan H. Müller , Ronald Simon , Eik Vettorazzi , Guido Sauter , Julia Ebner , Niclas C. Blessin

Although there is rising evidence that immune cell subpopulations that are in direct contact with the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patients’ outcome, a comprehensive assessment of intraepithelial immune cells and their spatial interplay is lacking. To assess intraepithelial leukocyte densities, immune checkpoint expression, and spatial interactions in 43 carcinoma entities, 4915 tumor samples in a tissue microarray format were analyzed using a deep learning framework and BLEACH&STAIN multiplex fluorescence immunohistochemistry. This approach enabled single-cell resolution quantification of 21 biomarkers through 7 sequential staining and imaging rounds. Immune and tumor cells were classified into 54 subpopulations. The mean intraepithelial immune cell density of CD8⁺ cytotoxic T cells, CD4⁺ T-helper cells, FOXP3⁺ regulatory T cells, CD20⁺ B cells, M1/M2 macrophages, and CD11c⁺ dendritic cells varied markedly between tumor entities and individual tumors. For instance, 88(±90) cells/mm² were found in tubular breast cancer, 661(±729) cells/mm² in colorectal cancer, and up to 2325(±2131) cells/mm² in squamous cell cancers from various origins. Unsupervised cluster analysis revealed a “cluster a” of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells that was characterized by a unique interaction profile along with the highest immune checkpoint expression. Across all analyzed tumor entities, the intraepithelial highly inflamed cluster a was significantly linked to low pathologic tumor stage (P < .001). The data from this study provide a comprehensive characterization of intraepithelial immune cells across 43 different human carcinomas and identified an inflamed pan-cancer phenotype characterized by strong interactions of intraepithelial CD8⁺ cytotoxic T cells, CD4⁺ T cells, dendritic cells, and M2 macrophages, along with highest levels of TIM3, PD-1, and CTLA-4 expression that is linked to a favorable tumor phenotype.

尽管越来越多的证据表明，与肿瘤细胞（上皮内）直接接触的免疫细胞亚群可以预测对免疫检查点治疗的反应和患者的预后，但缺乏对上皮内免疫细胞及其空间相互作用的全面评估。为了评估43种癌症实体的上皮内白细胞密度、免疫检查点表达和空间相互作用，使用深度学习框架和BLEACH&STAIN多重荧光免疫组织化学（mfIHC）分析了组织微阵列格式的4915例肿瘤样本。该方法通过7轮连续染色和成像，实现了21种生物标志物的单细胞分辨率定量。免疫细胞和肿瘤细胞可分为54个亚群。上皮内CD8+细胞毒性t细胞、CD4+ t辅助细胞、FOXP3+Tregs细胞、CD20+ b细胞、M1/ m2巨噬细胞和CD11c+树突状细胞的平均免疫细胞密度在肿瘤实体和个体之间存在显著差异。例如，在管状乳腺癌中发现88（±90）个细胞/mm2，在结直肠癌中发现661（±729）个细胞/mm2，在各种来源的鳞状细胞癌中发现高达2325（±2131）个细胞/mm2。无监督聚类分析显示，来自几乎所有不同肿瘤实体的634例患者的“聚类a”具有异常高密度的上皮内免疫细胞，其特征是独特的相互作用谱以及最高的免疫检查点表达。在所有分析的肿瘤实体中，上皮内高度炎症的簇a与低pT (p) +细胞毒性t细胞、CD4+ t细胞、树突状细胞和M2巨噬细胞以及最高水平的TIM3、PD-1和CTLA-4表达显著相关，这与有利的肿瘤表型有关。

{"title":"Prevalence, Immune Checkpoint Expression, and Spatial Interplay of Immune Cells Are Linked to Favorable Tumor Phenotype in 4915 Human Carcinomas","authors":"Zhihao Huang , Tim Mandelkow , Jonas B. Raedler , Elena Bady , Jan H. Müller , Ronald Simon , Eik Vettorazzi , Guido Sauter , Julia Ebner , Niclas C. Blessin","doi":"10.1016/j.labinv.2025.104248","DOIUrl":"10.1016/j.labinv.2025.104248","url":null,"abstract":"<div><div>Although there is rising evidence that immune cell subpopulations that are in direct contact with the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patients’ outcome, a comprehensive assessment of intraepithelial immune cells and their spatial interplay is lacking. To assess intraepithelial leukocyte densities, immune checkpoint expression, and spatial interactions in 43 carcinoma entities, 4915 tumor samples in a tissue microarray format were analyzed using a deep learning framework and BLEACH&STAIN multiplex fluorescence immunohistochemistry. This approach enabled single-cell resolution quantification of 21 biomarkers through 7 sequential staining and imaging rounds. Immune and tumor cells were classified into 54 subpopulations. The mean intraepithelial immune cell density of CD8<sup>+</sup> cytotoxic T cells, CD4<sup>+</sup> T-helper cells, FOXP3<sup>+</sup> regulatory T cells, CD20<sup>+</sup> B cells, M1/M2 macrophages, and CD11c<sup>+</sup> dendritic cells varied markedly between tumor entities and individual tumors. For instance, 88(±90) cells/mm<sup>2</sup> were found in tubular breast cancer, 661(±729) cells/mm<sup>2</sup> in colorectal cancer, and up to 2325(±2131) cells/mm<sup>2</sup> in squamous cell cancers from various origins. Unsupervised cluster analysis revealed a “cluster a” of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells that was characterized by a unique interaction profile along with the highest immune checkpoint expression. Across all analyzed tumor entities, the intraepithelial highly inflamed cluster a was significantly linked to low pathologic tumor stage (<em>P</em> < .001). The data from this study provide a comprehensive characterization of intraepithelial immune cells across 43 different human carcinomas and identified an inflamed pan-cancer phenotype characterized by strong interactions of intraepithelial CD8<sup>+</sup> cytotoxic T cells, CD4<sup>+</sup> T cells, dendritic cells, and M2 macrophages, along with highest levels of TIM3, PD-1, and CTLA-4 expression that is linked to a favorable tumor phenotype.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104248"},"PeriodicalIF":4.2,"publicationDate":"2025-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145258496","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}

引用次数: 0

Corrigendum to “Tumor Necrosis Factor-α–Dependent Inflammation Upregulates High Mobility Group Box 1 To Induce Tumor Promotion and Anti–Programmed Cell Death Protein-1 Immunotherapy Resistance in Lung Adenocarcinoma” [Laboratory Investigation 105 (2025) 102164] “肿瘤坏死因子-α -依赖性炎症上调高迁移率组1诱导肺腺癌肿瘤促进和抗程序性细胞死亡蛋白-1免疫治疗耐药性”的勘误表[实验室研究105 (2025)102164]

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-10-02 DOI: 10.1016/j.labinv.2025.104228

Lifei Kang , Jingjing Cao , Wenli Guo , Xiaohui Cui , Yangxuan Wei , Jiayu Zhang , Feiran Liu , Chenyang Duan , Qiang Lin , Ping Lvx , Zhiyu Ni , Jing Zuo , Haitao Shen

引用次数: 0

Ultrarapid EGFR Testing in Non–Small Cell Lung Carcinoma Patients: Findings From a Canadian Clinical Testing Workflow 非小细胞肺癌患者的超快速EGFR检测：来自加拿大临床检测工作流程的发现

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-24 DOI: 10.1016/j.labinv.2025.104245

Kate Fitzsimmons , Curtis Hughesman , Reka Pataky , Deirdre Weymann , Marie-Frédérique D’Amours , Deepu Alex , Diana N. Ionescu , Barb Melosky , Hannah Carolan , Kelly McNeil , Cheryl Ho , Anna McGuire , Stephen Yip , Julia R. Naso

Single vs multigene molecular testing modalities for lung cancer offer distinct advantages and risks. We examined lung cancer cases with clinically requested ultrarapid EGFR testing to (1) identify clinical features of rapid tested cases and their association with EGFR mutations, (2) evaluate performance of single-gene and multigene panel testing for ultrarapid tested patients, and (3) estimate laboratory costs and clinical outcomes. We include all retrospectively identified lung cancer patients who had ultrarapid Idylla EGFR testing during the study period. Demographic data were retrieved from clinical charts, and cost estimates were obtained from the BC Cancer Genetics and Genomics Laboratory. Of the 109 ultrarapid tests, 94 (86%) were technically successful, yielding a positive or negative result. Of these, 62 tests (66%) identified an EGFR mutation. Patients with negative or failed testing were offered panel sequencing (n = 47, 43%). Ultrarapid testing had a median 1-day turnaround time and 95% sensitivity for EGFR mutation detection relative to panel sequencing. East/Southeast Asian ethnicity and female sex were significantly associated with EGFR mutation positivity in a multivariate logistic regression model (P = .0001 and .029, respectively). The mean molecular testing cost per ultrarapid tested patient, including panel sequencing for cases with negative/failed rapid tests, was $550.53 (SD: $284), slightly less than the $571 cost for panel sequencing. Single-gene testing of patients with urgent clinical need or high probability of mutation may allow a rapid time to treatment at similar testing costs.

肺癌单基因与多基因分子检测方式具有明显的优势和风险。我们对肺癌病例进行了临床要求的超快速EGFR检测，以(i)确定快速检测病例的临床特征及其与EGFR突变的关系，（ii）评估超快速检测患者的单基因和多基因面板检测的性能；（iii）估计实验室费用和临床结果。我们纳入了所有在研究期间进行超快速Idylla EGFR检测的回顾性肺癌患者。人口统计数据来自临床图表，成本估算来自BC省癌症遗传学和基因组学实验室。在109项超快速检测中，94项（86%）在技术上是成功的，产生阳性或阴性结果。其中，62项（66%）检测发现EGFR突变。阴性或检测失败的患者接受小组测序（n=47, 43%）。与面板测序相比，超快速检测的平均周转时间为1天，EGFR突变检测的灵敏度为95%。多因素logistic回归模型显示，东亚/东南亚族裔和女性与EGFR突变阳性显著相关（P分别为0.0001和0.029）。每位超快速检测患者的平均分子检测成本，包括快速检测阴性/失败病例的小组测序，为550.53美元（标准偏差284美元），略低于小组测序的571美元。对有迫切临床需要或突变可能性高的患者进行单基因检测，可以在相同的检测费用下快速获得治疗。

{"title":"Ultrarapid EGFR Testing in Non–Small Cell Lung Carcinoma Patients: Findings From a Canadian Clinical Testing Workflow","authors":"Kate Fitzsimmons , Curtis Hughesman , Reka Pataky , Deirdre Weymann , Marie-Frédérique D’Amours , Deepu Alex , Diana N. Ionescu , Barb Melosky , Hannah Carolan , Kelly McNeil , Cheryl Ho , Anna McGuire , Stephen Yip , Julia R. Naso","doi":"10.1016/j.labinv.2025.104245","DOIUrl":"10.1016/j.labinv.2025.104245","url":null,"abstract":"<div><div>Single vs multigene molecular testing modalities for lung cancer offer distinct advantages and risks. We examined lung cancer cases with clinically requested ultrarapid <em>EGFR</em> testing to (1) identify clinical features of rapid tested cases and their association with <em>EGFR</em> mutations, (2) evaluate performance of single-gene and multigene panel testing for ultrarapid tested patients, and (3) estimate laboratory costs and clinical outcomes. We include all retrospectively identified lung cancer patients who had ultrarapid Idylla <em>EGFR</em> testing during the study period. Demographic data were retrieved from clinical charts, and cost estimates were obtained from the BC Cancer Genetics and Genomics Laboratory. Of the 109 ultrarapid tests, 94 (86%) were technically successful, yielding a positive or negative result. Of these, 62 tests (66%) identified an <em>EGFR</em> mutation. Patients with negative or failed testing were offered panel sequencing (n = 47, 43%). Ultrarapid testing had a median 1-day turnaround time and 95% sensitivity for <em>EGFR</em> mutation detection relative to panel sequencing. East/Southeast Asian ethnicity and female sex were significantly associated with <em>EGFR</em> mutation positivity in a multivariate logistic regression model (<em>P</em> = .0001 and .029, respectively). The mean molecular testing cost per ultrarapid tested patient, including panel sequencing for cases with negative/failed rapid tests, was $550.53 (SD: $284), slightly less than the $571 cost for panel sequencing. Single-gene testing of patients with urgent clinical need or high probability of mutation may allow a rapid time to treatment at similar testing costs.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104245"},"PeriodicalIF":4.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145176379","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}

引用次数: 0

Comparison of QuPath and HALO Platforms for Analysis of the Tumor Microenvironment in Prostate Cancer QuPath与HALO平台在前列腺癌肿瘤微环境分析中的比较

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-24 DOI: 10.1016/j.labinv.2025.104246

Wei Zhang , Qin Zhou , Jonathan V. Nguyen , Erika Egal , Qian Yang , Michael R. Freeman , Siwen Hu-Lieskovan , Gita Suneja , Anna Coghill , Beatrice S. Knudsen

QuPath, an open-source digital pathology platform, has gained widespread use for image analysis in biomedical research since its release in 2016. However, its reproducibility and reliability compared with commercial software, such as HALO, require further validation, particularly for multiplex immunofluorescence analysis. In this study, we performed a direct comparison of QuPath and HALO using a multiplex immunofluorescence–stained prostate cancer tissue microarray inclusive of 192 unique cores. We evaluated performance across 3 key analytical modules: immune cell phenotyping, tumor infiltration with immune cells, and nearest neighbor analysis. Furthermore, we integrated QuPath with CytoMap, an open-source spatial analysis tool, to perform unsupervised clustering of immune cell infiltration—a feature not available in HALO. Our results demonstrated high concordance between 2 platforms, with correlation coefficients >0.89 for immune cell density, distance, and pattern of cell organization in tumor microenvironment. A neighborhood analysis using CytoMap was further performed and provided a more detailed spatial analysis of immune cell distribution across different prostate cancer grades. A significant increase of CD103+ T-cell infiltration into tumor microenvironment was observed in prostate cancer. In conclusion, our findings validate QuPath as a robust and reproducible alternative to commercial platforms for fluorescence-based digital pathology. By demonstrating QuPath’s capability to perform high-quality quantitative analysis with additional flexibility for integration with external tools, our study underscores its potential for advancing tumor microenvironment research in translational oncology.

QuPath是一个开源的数字病理平台，自2016年发布以来，在生物医学研究中获得了广泛的图像分析应用。然而，与商业软件（如HALO）相比，其再现性和可靠性需要进一步验证，特别是对于多重免疫荧光（mIF）分析。在这项研究中，我们使用包含192个独特核心的mif染色前列腺癌组织微阵列（TMA）对QuPath和HALO进行了直接比较。我们评估了三个关键分析模块的性能：免疫细胞表型、免疫细胞浸润肿瘤和最近邻分析。此外，我们将QuPath与开放源代码的空间分析工具CytoMap结合起来，对免疫细胞浸润进行无监督聚类，这是HALO中不具备的功能。我们的结果表明，两个平台之间的一致性很高，免疫细胞密度、距离和肿瘤微环境（TME）中细胞组织模式的相关系数超过0.89。进一步使用细胞图谱进行邻域分析，并对不同级别前列腺癌的免疫细胞分布提供了更详细的空间分析。前列腺癌中CD103+ T细胞对TME的浸润明显增加。总之，我们的研究结果验证了QuPath作为基于荧光的数字病理商业平台的强大且可重复的替代方案。通过展示QuPath进行高质量定量分析的能力，以及与外部工具集成的额外灵活性，我们的研究强调了其在转化肿瘤学中推进肿瘤微环境研究的潜力。

{"title":"Comparison of QuPath and HALO Platforms for Analysis of the Tumor Microenvironment in Prostate Cancer","authors":"Wei Zhang , Qin Zhou , Jonathan V. Nguyen , Erika Egal , Qian Yang , Michael R. Freeman , Siwen Hu-Lieskovan , Gita Suneja , Anna Coghill , Beatrice S. Knudsen","doi":"10.1016/j.labinv.2025.104246","DOIUrl":"10.1016/j.labinv.2025.104246","url":null,"abstract":"<div><div>QuPath, an open-source digital pathology platform, has gained widespread use for image analysis in biomedical research since its release in 2016. However, its reproducibility and reliability compared with commercial software, such as HALO, require further validation, particularly for multiplex immunofluorescence analysis. In this study, we performed a direct comparison of QuPath and HALO using a multiplex immunofluorescence–stained prostate cancer tissue microarray inclusive of 192 unique cores. We evaluated performance across 3 key analytical modules: immune cell phenotyping, tumor infiltration with immune cells, and nearest neighbor analysis. Furthermore, we integrated QuPath with CytoMap, an open-source spatial analysis tool, to perform unsupervised clustering of immune cell infiltration—a feature not available in HALO. Our results demonstrated high concordance between 2 platforms, with correlation coefficients >0.89 for immune cell density, distance, and pattern of cell organization in tumor microenvironment. A neighborhood analysis using CytoMap was further performed and provided a more detailed spatial analysis of immune cell distribution across different prostate cancer grades. A significant increase of CD103+ T-cell infiltration into tumor microenvironment was observed in prostate cancer. In conclusion, our findings validate QuPath as a robust and reproducible alternative to commercial platforms for fluorescence-based digital pathology. By demonstrating QuPath’s capability to perform high-quality quantitative analysis with additional flexibility for integration with external tools, our study underscores its potential for advancing tumor microenvironment research in translational oncology.</div></div>","PeriodicalId":17930,"journal":{"name":"Laboratory Investigation","volume":"105 12","pages":"Article 104246"},"PeriodicalIF":4.2,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145176351","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}

引用次数: 0

MMP13-Expressing and COL11A1-Expressing Cancer-Associated Fibroblasts: Key Drivers of Esophageal Squamous Cell Carcinoma Progression and Prognostic Indicators 表达MMP13-和col11a1的癌症相关成纤维细胞：食管鳞状细胞癌进展和预后指标的关键驱动因素

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-24 DOI: 10.1016/j.labinv.2025.104247

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

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.

肿瘤微环境包括多种细胞类型，癌症相关成纤维细胞是癌症进展和转移的关键因素。癌症相关成纤维细胞在食管鳞状细胞癌中也起重要作用，并在此背景下被广泛研究。然而，癌症相关成纤维细胞与病理阶段进展之间的关系尚未报道。为了鉴定这些特定的癌症相关成纤维细胞，我们使用了一种以病例为导向的单细胞RNA测序方法。因此，我们确定了三种癌症相关成纤维细胞簇，归类为肌成纤维细胞癌症相关成纤维细胞，其数量随着癌症的进展而增加。通路分析显示，三种与癌症相关的成纤维细胞簇具有不同的特性。这些癌症相关成纤维细胞根据其特征基因表达被命名为MMP13+、COL11A1+和SFRP4+肌成纤维细胞癌症相关成纤维细胞。我们还研究了与三种不同的癌症相关成纤维细胞簇相关的肿瘤微环境中各种免疫细胞的分布。结果显示存在不同类型的免疫细胞，包括M2巨噬细胞、调节性T细胞、干扰素-γ+程序性死亡-1+ T细胞和干扰素-γ+程序性死亡-1- T细胞。接下来，我们使用RNA原位杂交技术评估了晚期食管鳞状细胞癌患者手术切除标本中这三种癌症相关成纤维细胞亚型的存在。对这三种癌症相关成纤维细胞亚型与预后的相关性分析显示，两种亚型（MMP13+和COL11A1+肌成纤维细胞癌症相关成纤维细胞）与预后不良相关。MMP13+肌成纤维细胞癌相关成纤维细胞与低分化浸润模式相关，而COL11A1+肌成纤维细胞癌相关成纤维细胞与淋巴结转移相关。这些结果表明，未来针对这些癌症相关成纤维细胞的治疗和基于这些癌症相关成纤维细胞的患者分层是有必要的。

{"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}

引用次数: 0

The Role of Replication Stress-Related Genes in Cervical Cancer Radiotherapy Resistance: A Bioinformatic and Experimental Validation 复制应激相关基因在宫颈癌放疗抵抗中的作用：生物信息学和实验验证。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-22 DOI: 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.

宫颈癌（CC）仍然是一个主要的全球健康挑战，放射治疗耐药性（RR）是治疗效果的一个严重障碍。本研究探讨了复制应激（RS）在RR中的潜在机制，确定了CC的潜在治疗靶点，并应用综合生物信息学工作流程分析了RR患者中RS相关差异表达基因（RSRDs）的表达谱及其预后意义。基于rs的风险评分模型的预后效用随后在肿瘤微环境，体细胞突变景观等背景下进行了评估。通过免疫组织化学（IHC）、单变量和多变量Cox回归分析以及使用现实世界患者队列数据的预后nomogram来验证所确定的枢纽RSRDs的临床相关性。体外和体内的功能分析进一步证实了关键RSRD的作用。因此，对124个共同差异表达基因的富集分析表明，RS相关的生物过程得到了富集。RS风险评分模型采用LASSO回归确定的两个枢纽RSRDs （AXIN1和CTBP1）构建，显示出较强的诊断和预后性能。富集分析显示，风险评分模型通过肿瘤微环境、突变等因素影响CC预后。组织微阵列的免疫组化分析发现了RR样本中AXIN1的显著下调。AXIN1也是CC患者的独立预后生物标志物，特别是在接受放疗的患者中。敲低AXIN1可显著抑制CC细胞系的放射敏感性，体内实验显示，敲低AXIN1可导致放疗后肿瘤体积增大。分子对接分析表明JQ1可能促进AXIN1的表达。这项研究首次确定了AXIN1是一种复制应激相关基因，在CC中具有预后价值，特别是在放疗的背景下。这些发现可能支持个性化治疗策略，并为未来研究CC的rs靶向治疗提供基础。

{"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}

引用次数: 0

Chromobox 4 (CBX4) Is a Novel Interactor of SS18::SSX in Synovial Sarcoma CBX4是滑膜肉瘤中SS18::SSX的一种新型相互作用因子。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-22 DOI: 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.

滑膜肉瘤是一种侵袭性癌症，通常影响青少年和年轻人，其特点是高复发和转移率。它主要由融合癌蛋白SS18::SSX驱动，这是一种致病染色体易位的产物（X;18），通过与BAF复合物和多梳抑制复合物等复合物的相互作用，促进了广泛的表观遗传失调。以前对SS18::SSX相互作用组进行质谱分析的尝试由于缺乏检测内源性蛋白的抗体而受到限制，因此依赖于带有外源性标签的单株细胞系。我们之前使用了一种单克隆抗体，它可以特异性检测含有典型融合连接的SS18::SSX（95%的病例中见过），并在几种应用中建立了它的实用性。使用该抗体，质谱分析显示SS18::SSX蛋白在SS18中经历了外显子8的选择性剪接。接下来，我们在6个永生化的人滑膜肉瘤细胞系中对SS18::SSX进行了免疫沉淀质谱分析，并鉴定出典型的多梳抑制复合体成员4号染色体盒CBX4是一种新的肿瘤蛋白相互作用物。在人滑膜肉瘤组织芯片上的几个表观遗传因子的免疫组织化学染色显示滑膜肉瘤样品与较高的CBX4表达相关。最后，对来自12种肉瘤亚型、癌和正常组织的337个样本的CBX4表达分析表明，与其他组织类型相比，滑膜肉瘤样本中的CBX4表达更高。这些结果强调了识别滑膜肉瘤中SS18::SSX的重要伴侣以建立促进该疾病的新生物学途径的关键方法。

{"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}

引用次数: 0

Combined Multicolor Immunofluorescence Staining and Spatial In Situ messenger RNA Expression Analysis Identifies Potential Fibrosis Drivers in Acute Lymphoblastic Leukemia 联合多色免疫荧光染色和空间原位mRNA表达分析确定急性淋巴细胞白血病潜在的纤维化驱动因素。

IF 4.2 2区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Laboratory Investigation

Pub Date : 2025-09-18 DOI: 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.

急性淋巴细胞白血病（ALL）是最常见的儿童癌症。ALL患者骨髓纤维化与不良预后相关，然而，对ALL患者骨髓纤维化的机制知之甚少。因此，我们建立了一种新的先进的分析方法，将多色免疫荧光染色与原位RNA表达分析（RNAscope®）相结合，研究ALL骨髓中可能的纤维化驱动因子的空间表达。我们分析了儿科ALL患者的标准脑脊髓瘤活检。采用CD45、CD271、CD31、CD34和DAPI的顺序5色免疫荧光（IF）染色对不同类型的BM细胞进行鉴定。建立RNAscope®和IF联合染色，分析转化生长因子β 1 （TGFB1）和血小板源性生长因子α 1 （PDGFA1）的空间mRNA表达，这两个已知在原发性骨髓纤维化（PMF）中起主要作用。PMF和正常BM作为对照。正如预期的那样，ALL骨髓显示出高细胞性和突出的胚细胞群。CD271+ MSC密度在ALL中升高，与PMF中观察到的纤维化相似。与PMF患者和正常对照相比，ALL巨核细胞（mk）中TGFB1和PDGFA1的表达显著增加。此外，MK TGFB1和PDGFA1在纤维化性ALL中的表达强度与纤维化级别相关。TGFB1和PDGFA1也在白血病原细胞中表达，但与ALL mk相比表达强度较低。总之，先进的原位RNA和IF染色不仅揭示了TGFB1和PDGFA1在纤维化儿童ALL中的表达增加，而且在单细胞水平上确定了ALL母细胞和mk是它们的细胞来源。这些新的数据强烈提示这些细胞因子在ALL中作为潜在的纤维化驱动因素的作用。更广泛地说，我们的研究结果表明，结合RNA和表面标记分析是一个强大的工具，为骨髓病理生理学提供新的和有价值的见解。

{"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}

引用次数: 0