Cancers

Invasive lobular carcinoma (ILC) is the most frequently diagnosed special histological subtype of invasive breast cancer and accounts for 10 - 15% of all cases. The pathognomonic hallmark of ILC is the genetic loss of E-cadherin (CDH1) causing the disruption of adherens junctions and resulting in discohesive, linear growth. To better understand the role of E-cadherin in ILC metastasis, we generated three ILC cell lines, MDA-MB-134-VI, SUM44PE, and BCK4, with inducible E-cadherin expression, resulting in successful restoration of functional adherens junctions. E-cadherin expression reduced growth in 2D culture, and that effect was even greater in 3D ultra-low attachment (ULA) conditions where increased cell death was consistent with the previously described role of E-cadherin in anoikis. E-cadherin expression did not rescue the lack of migration and invasion of ILC cell line models; however, it decreased haptotaxis and increased adherence to Collagen I in SUM44 cells. There was no significant effect of E-cadherin expression on primary orthotopic tumor growth, but spontaneous metastasis to the reproductive tract, brain, and GI tract was reduced. Inhibition of metastasis to the reproductive tract and brain was also seen after tail vein injection of MDA-MB-134 E-cadherin-expressing cells. In summary, overexpression of functional E-cadherin in ILC models has some, but limited, effects on 2D growth in vitro and primary tumor growth in vivo, but there are pronounced effects on 3D ULA growth and metastases in vivo, with stronger effects on metastatic sites enriched in patients with ILC, especially the reproductive and GI tracts.

BackgroundRecent evidence suggests that cancer can exhibit splicing alterations that give rise to tumour-specific isoforms. One example is NUMB, which produces four isoforms (p72, p71, p66, and p65) through alternative splicing of exons 3 and 9. Traditionally considered a tumour suppressor, it also has been considered an oncogene. We propose that this duality is due to isoform-specific expression. ResultsUsing public databases, we identified a tumour-associated switch in NUMB isoform expression: p72/p71 are upregulated in tumours, whereas p66/p65 are more expressed in non-tumour tissues. These isoforms correlate differently with cellular processes. NUMBL, a NUMB homolog, behaves similarly to p65. We identified two transcriptional clusters: one characterized by high expression of p72/p71, and another by p66/p65/NUMBL. Each group was associated differently with the Notch, WNT/{beta}-catenin, Hedgehog, and Hippo signalling pathways, suggesting isoform-specific regulatory roles. Analysis of breast cancer cell lines (CCLE) led to a NUMB score based on isoform expression, which classified cell lines into biologically distinct groups. The p72/p71-enriched group showed distinct signatures, pathway activity, and drug sensitivity. Applying this score to TCGA-BRCA samples revealed a significant link between high NUMB-score and poor survival, confirmed by Kaplan-Meier analysis. ConclusionsNUMB emerges as a potential oncogenic contributor and biomarker in splicing-based personalised medicine, highlighting isoform-specific expression as a clinically relevant determinant of tumour behaviour, pathway activity, and therapeutic response.

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

Importance: Non-medullary thyroid cancer (NMTC) and melanoma are associated with inherited long telomeres due to germline pathogenic/likely pathogenic variants (PV/LPV) in POT1, TINF2, and ACD resulting in long-telomere syndrome (LTS) and they commonly have somatic TERT promoter mutations. The genetic relationship between these variants and their clinical associations are defined incompletely and may inform clinical practice. Objective: To test the hypothesis that germline LTS-associated PV/LPV are exclusive from functional somatic TERT variants and assess clinical/genetic associations. Design: Retrospective observational cohort study with/without germline LTS variants, that have somatic sequencing and pathology data. Setting: Participants were enrolled through 18 cancer centers participating in the Oncology Research Information Exchange Network (ORIEN). Participants: 995 adults with NMTC and 993 with melanoma between 2013 and 2025. All adult patients at an ORIEN center were offered enrollment Exposures: All patients with NMTC or melanoma are included. There are no required exposures. Main Outcomes and Measures: The presence/absence of a germline or somatic long-telomere variant; secondary outcomes are associations with tumor stage, telomerase expression, and oncogenes. Results: Germline and somatic variants in POT1/TINF2/ACD, somatic TERT promoter variants, TERT fusions, oncogenes, and telomerase mRNA expression were evaluated in 995 NMTC and 993 melanoma patients. In NMTC, 13 (1.5%) had a germline LTS variant while 0/12 with tumor sequencing had somatic TERT promoter variants/fusions. In melanoma, 7 (0.7%) had a LTS variant; 0/2 with tumor sequencing had a TERT promoter variant/ fusion. Meta-analysis including NMTC and melanoma in the current study, a recent thyroid cancer study, and thyroid TCGA, germline LTS-associated PV/LPV and somatic TERT variants/fusions were mutually exclusive (p=0.036). High telomerase mRNA levels were associated with TERT promoter variants/fusions (p<4e-11) and larger NMTC/distant metastases (p=0.016), but not germline LTS variants. NMTCs with somatic TERT promoter variants/fusions had higher tumor mutation burden (p<0.02) versus tumors from patients with a germline LTS variant. TERT promoter mutant variant allele frequency was lower in smaller and non-metastatic vs larger/metastatic NMTC. Conclusion and Relevance: Germline LTS-associated variants appear to be exclusive from somatic TERT promoter variants/fusions but are not associated with aggressive NMTC, suggesting common roles in tumorigenesis but different biological impacts.

Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide, and the great majority of these deaths are caused by metastatic disease. Whether the immunohistochemical (IHC) phenotype of breast cancer is associated with the anatomical site of metastasis has been characterized mainly in high-income, registry-based populations, while data from ecologically stressed and medically under-served regions such as the Lower Aral Sea basin are lacking. Methods: We retrospectively reviewed 652 women diagnosed with breast cancer at the Khorezm Branch of the Republican Specialized Scientific-Practical Medical Center of Oncology and Radiology (Uzbekistan) between 2020 and 2024, of whom 213 had metastatic disease (306 metastatic foci). Histological type was assessed on hematoxylin-eosin and van Gieson-stained sections; quantitative morphometry was performed in Fiji/ImageJ; and HER2, estrogen receptor (ER), progesterone receptor (PR) and Ki-67 were assessed by IHC. The association between marker expression and metastatic site (liver, lung, lymph node) was tested in 187 foci with adequate tissue using the chi-square test, with significance at p < 0.05. Results: Invasive ductal carcinoma predominated. Metastatic site was significantly associated with the IHC phenotype. Liver metastases showed the highest frequency of HER2 3+ (45.7%), ER-negativity (65.2%), PR-negativity (69.6%) and high proliferation (Ki-67 [&ge;] 60%; 47.8%), whereas lymph-node metastases were more often hormone-receptor-positive (ER+ 58.7%; PR+ 52.4%) with lower HER2 3+ (22.2%); lung metastases were intermediate (all p < 0.05). The combination of HER2 3+ and Ki-67 [&ge;] 60% was associated with multi-organ spread. Morphometry corroborated these patterns: liver lesions had larger atypical cells (up to 132.8 m), a higher nuclear-to-cytoplasmic ratio (0.76 vs 0.51) and more extensive necrosis and microvascularity than lymph-node lesions. A pragmatic 5-criterion morphological score (histological type, Ki-67, HER2, ER/PR status, atypical-cell size) stratified metastatic risk into three tiers. Conclusions: In this regional cohort, the IHC phenotype of breast cancer tracked the anatomical site of metastasis, with an aggressive HER2-driven, hormone-receptor-negative profile concentrated in liver metastases and a hormone-receptor-positive profile in lymph-node metastases. These findings reproduce established organotropism patterns in a previously uncharacterized population and support phenotype-aware, site-specific surveillance together with a low-cost morphological risk score for resource-limited settings.

Chemotherapeutic treatment of breast cancer with Doxorubicin (DOX) can induce tumor and stromal cell senescence leading to therapy-resistance. Senescence-associated secretory phenotype (SASP) promotes secretion of pro-inflammatory and tumorigenic factors causing systemic inflammation. Combined, this can result in immune suppression, tumor growth and secondary spread of cancer. Targeting and removing senescent and cancerous cells using a combination of chemotherapeutic and senolytic drugs may reduce systemic inflammation, improve therapeutic efficacy, and prevent metastasis. Exposure of triple-negative breast cancer (MDA-MB-231), hormone-responsive (MCF-7) and HER2+ (MDA-MB-453) cells, and primary spine osteoblasts to DOX showed significant induction of p21-positive senescent cells. DOX and senolytics (RG-7112, o-Vanillin) treatment of co-culture spheroids showed a significant additive effect in reducing tumor sphere viability and growth, indicating reduced metastatic potential. This was correlated with reduced SASP in triple-negative and hormone responsive lines and decreased levels of senescent cells in all subtypes and primary stromal cells, while proliferation was decreased, and apoptosis increased across all breast cancer subtypes. Future chemotherapeutic treatment in breast cancer models may be optimized by adding senolytic drugs to more effectively clear senescent tumor and stromal cells, reducing risk for relapse and metastatic potential, while allowing for tissue regeneration in the bone metastatic environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724653v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@c4cb8forg.highwire.dtl.DTLVardef@105219org.highwire.dtl.DTLVardef@17e0517org.highwire.dtl.DTLVardef@802bd2_HPS_FORMAT_FIGEXP M_FIG C_FIG Senolytics selectively eliminate senescent cancer and stromal cells and enhance Doxorubicin efficacy in a 3D bone-like tumor microenvironment model.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies. Immune dysregulation is believed to play an important role in ATC. Here, we aimed to characterize the systemic inflammation and the function of circulating immune cells of patients with ATC. First, we retrospectively assessed biochemical parameters of patients with ATC and observed that high systemic inflammation correlated with worse survival. Next, we prospectively investigated the inflammatory proteome, single-cell peripheral blood mononuclear cell transcriptome and epigenetic changes. Circulating concentrations of proinflammatory cytokines were increased in ATC patients. This proinflammatory profile was apparent at the level of gene transcription and chromatin accessibility, especially in monocytes. These findings were substantiated by an increased capacity of peripheral blood mononuclear cells of ATC patients to produce IL-6, IL-8 and lactate. As IL-6 is known to promote tumor cell survival, we assessed its capacity to influence ATC cell proliferation. Blocking IL-6/gp130/Jak/STAT3 pathway inhibited proliferation of ATC cell lines in vitro. In conclusion, these findings show that ATC is characterized by inappropriate systemic inflammation and epigenetic and transcriptional reprogramming of circulating monocytes. Proinflammatory cytokines released by monocytes support survival and proliferation of ATC tumor cells, suggesting a therapeutic potential of targeting this pathway in ATC patients.

Objective To determine if it is possible to assess individual patient risk of the development of colorectal cancer (CRC) in people in high-risk groups due to their family history. Design/Method Retrospective observational study of prospectively collected data from consecutive patients referred for a colonoscopy. 2,478 consecutive patients were referred to a single colorectal surgical practice in Sydney, Australia between 1977 and 2018 for a colonoscopy because of a family history of CRC. Of these, 1,963 have been followed for more than 10 years and are the subject of this paper. Histopathological findings categorised as normal (N), non-advanced adenoma (NAA) or advanced neoplasia (AN) with AN proven to be the precursor to CRC. Intervention Colonoscopic screening on the basis of contemporary practice to 2006 and subsequently according to Australian National Health and Medical Research Council guidelines. Results Participants with normal or low-risk findings in the first decade remain at lower risk of CRC for 30 years from the commencement of screening. Conclusion It is possible to stratify individual patients in a high relative risk cohort into those with high or low personal risk of CRC based on colonoscopic findings in the first 10 years of surveillance. Those with no AN in the first ten years have a lower 30-year risk of developing AN than the general community. This offers the possibility of structuring surveillance programs around individual risk rather than group risk, lessening the need for multiple surveillance colonoscopies in the majority of such patients and improving the cost effectiveness of CRC screening at the population level.

INTRODUCIONTumor cell infiltration in regional lymph nodes is a strong prognostic marker, guiding treatment decisions in breast cancer. While the immune cell composition in primary tumors has been more widely explored in later years, the immune cell composition of the sentinel node (SN) and axillary lymph nodes (ALN) remains understudied. A better understanding of how primary tumor and metastatic tumor cells alter the nodal immune microenvironment can shed light on metastasis and cancer progression to unveil new treatment strategies. MATERIALS AND METHODSFrom a prospective clinical cohort of 458 treatment-naive patients with primary operable breast cancer, we performed comprehensive immunophenotypic analysis using mass cytometry analysis of non-metastatic (SN-) and metastatic (SN+) and ALN (ALN+) lymph nodes. RESULTSAs expected, patients with ALN+ cases had a shorter time to distant metastases than SN+ and SN- cases. We identified an exhausted T-cell phenotype and an increase in Germinal Center B (GC B) cells and plasma cells in ALN+ samples compared to SN- samples, both in the whole cohort as well as when investigating estrogen-receptor positive (ER+) patients only. There were no differences in immune cell composition across breast cancer (BC) subtypes within SN-samples. SN+ samples from triple negative BC (TNBC) showed a trend towards increased abundance of GC B and plasma cells, similar to more advanced ALN+, suggesting that smaller TN metastases may trigger an immune activation at an early stage of dissemination. Further analysis of SN- samples from ER+ patients revealed a subset of patients where the immune response had a more exhausted T-cell phenotype. This group was enriched for lymph nodes that were deemed negative by ordinary pathology examination (microscopy) but had detectable tumor cells by CyTOF analysis. CONCLUSIONThe immune profiles of SN and ALN samples from breast cancer patients are highly diverse, showing limited associations to BC subtype, clinical parameters or patient outcome. Metastatic tumor cells play a significant role in driving T-cell exhaustion and immunosuppression. Notably, in approximately 50% of the ER+ samples, T-cell exhaustion was detectable. This coincides with the presence of tumor cells identified by CyTOF, which were likely missed by conventional pathological examination. These findings suggest that small tumor deposits alter the immune composition, and the immune profile reveals the presence of tumor cells.

Background: Immune checkpoint inhibitors have transformed cancer treatment, yet a large number of patients fail to respond. Identifying molecular characteristics that predict response before treatment initiation remains an unmet need. Towards that end, this study presents a large-scale integrative analysis of existing single-cell and bulk tissue datasets, aimed at identifying predictive features while providing insights into their cellular origin and potential function within the tumor microenvironment. Methods: A stepwise analysis was performed using single-cell RNA-sequencing data from 60 melanoma patients at baseline, separated into discovery (n=41) and validation (n=19) sets. An integrated bulk transcriptomics dataset (n=128) from melanoma patients and a bladder cancer dataset (n=298) were used for further validation. Results: Integrative analysis of melanoma single-cell datasets revealed that responders exhibit distinct molecular profiles across multiple cell types compared to non-responders. Notably, these included downregulation of the TNFR superfamily and other immunosuppressive genes (TNFRSF18, TNFRSF9, TNFRSF4, LGALS1, BATF, IL12RB2, LINGO1, DUSP4, SDC4, VCAM1) in T-cells. By investigating the findings from the immune cell populations in the bulk tumor context, 13 transcripts were found to be consistently associated with response across all cohorts. These were differentially expressed in T-cells (SELL, EPB41, CD96, UHFR2, LINGO1, LGALS1), B-cells (ALDH5A1), NK cells (PLEC, PDGFRB) and Monocytes (TLR10, ST6GAL1, IKZF1, MPRIP). A predictive model based on these features effectively discriminated responders from non-responders in melanoma (AUC=0.73). The model maintained significant predictive power in an independent bladder cancer dataset (IMvigor210; AUC=0.64). Of high clinical relevance, it demonstrated enhanced performance in identifying responders among patients with low tumor mutational burden (AUC=0.75). Conclusion: Our study reveals pre-treatment molecular features related to immune-cancer crosstalk that are associated with response to immunotherapy. A 13-gene model demonstrates potential added clinical value in stratifying responders, particularly in patients with low tumor mutational burden, meriting further validation.

PurposeBladder cancer (BLCA) is a heterogeneous tumor type. Only one third of muscle-invasive (MIBC) patients respond to immune checkpoint inhibitors (ICIs). Reliable resistance markers are needed to guide clinical decisions. We investigated the nerve growth factor receptor (NGFR) in BLCA and analyzed its correlation with disease progression and response to immunotherapy. Experimental DesignWe analyzed NGFR expression in BLCA cell lines, organoids, mouse models and patient samples. The cohorts used were The Cancer Genome Atlas (TCGA), enriched in muscle-invasive bladder cancer (MIBC) (n=407); IMvigor210, representing MIBC patients treated with ICIs (n=348); and UROMOL2, as a non-muscle-invasive bladder cancer (NMIBC)-specific cohort (n=535). IMvigor010 was also included (n=728). Patients were stratified by NGFR expression quartiles. We analyzed survival and tumor subtypes and performed stromal deconvolution and functional profiling. We assessed stemness- and invasion-related features in SCaBER cells. ResultsNGFR marks a basal tumor cell subcluster and is independently associated with poor prognosis in TCGA and IMvigor210. NGFR-high tumors show stromal content enriched in cancer-associated fibroblasts, lower neoantigen burden, higher CD8+ T effector signature together with an immune-excluded phenotype, and a CAF-specific TGF{beta} signature. In the immunotherapy-treated cohort, high NGFR expression was also associated with poorer outcome. Functionally, NGFR appears to promote a stem-like/pro-invasive program in BLCA cells. ConclusionsNGFR identifies a basal-like BLCA subpopulation linked to poor survival, while its association with immunotherapy response requires further validation. In addition, our in vitro analyses support a role of NGFR in stem-like and invasive traits, highlighting its relevance as a biomarker in BLCA.

Background Cancer treatment response is highly variable, even among patients with the same tumor type and treatment. Exceptional responders (ERs), who are individuals who experience unusually favorable outcomes, provide critical insights into the biological factors driving treatment success. While prior studies have highlighted the role of somatic changes, the contribution of germline rare variants remains underexplored. This study aimed to uncover the genetic underpinnings of exceptional responses by identifying rare, non-silent and predicted deleterious germline mutations enriched among ERs compared to typical cancer patients. Methods The Network of Enigmatic Exceptional Responders (NEER) project collected clinical and germline whole-genome sequencing (WGS) data from 53 ERs. After quality control procedures and ancestry background checks, 51 ERs were left for final analysis. While non-silent mutations were identified based on allele frequencies and mutation types, multiple pathogenicity predictors were applied for predicted deleterious variants. These were compared to a harmonized and comparable subset from the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort (n=414) using Fisher's exact tests. Kaplan-Meier survival analysis applied to evaluate prognostic associations in PCAWG patients. Additionally, Fisher's exact tests were conducted stratified by cancer type and treatment regimen to identify potential associations between rare germline variants and therapeutic responses. Results Variants in immune-related genes such as CCL26 and GPRC5D were prevalent, suggesting enhanced immune regulation among ERs. Fourteen genes with non-silent and eight with predicted deleterious mutations showed significantly different frequencies between NEER and PCAWG cohorts (FDR < 0.05). IRX3 emerged as a protective gene enriched in ERs, whereas OR6B2 was associated with poor survival in PCAWG lung cancer patients. Moreover, rare non-silent germline variants in drug target genes were enriched among ERs treated with cisplatin and doxorubicin, implicating altered DNA repair and drug-binding mechanisms in their remarkable outcomes. Conclusions This study reveals a distinctive germline mutation landscape in exceptional cancer responders, marked by immune-related and drug-target-associated variants that may enhance therapy response and prolong survival. The findings highlight potential novel prognostic biomarkers, such as IRX3 and OR6B2, providing a foundation for developing personalized cancer treatments informed by rare genetic variation.

Abstract Background Immune-related adverse events (irAEs) involving the breast remain rarely reported. Purpose To characterize clinical and imaging features of camrelizumab-associated breast lesions (CABLs). Materials and Methods This retrospective dual cohort study (October 2019 to February 2026) included 196 female patients. Cohort A comprised 180 non-breast cancer patients; Cohort B comprised 16 breast cancer patients receiving neoadjuvant camrelizumab. Baseline characteristics, treatment response, and CT/MRI features were compared between CABL-positive and CABL-negative groups using Mann-Whitney U and chi-square tests. Results CABLs developed in 34.4% (62/180) of Cohort A and 93.8% (15/16) of Cohort B. CABL-positive patients were younger (median 50.5 vs 54.5 years; P = 0.006) and more often premenopausal (46.8% vs 26.3%; P = 0.009). The objective response rate was relatively high among patients with positive lesions; in Group A, the disease progression rate was lower in the CABL-positive group than in the CABL-negative group (3.2% vs 17.8%), whilst in Group B, the pathological complete response rate was as high as 53.3% (8/15). On CT/MRI, CABLs were predominantly multiple (62.5%), with well-defined margins and unrestricted diffusion. The predominant time-intensity curve (TIC) pattern was washout (46.7%). Median time to onset was 2-3 cycles (the second MRI scan); most lesions disappeared (40.3%) and shrank (46.8%) during follow-up. ADC values of lesions were significantly higher than those of primary tumors (1.847+/-0.284 vs 0.976+/-0.055 x10[-3] mm[2]/s; P < 0.001). Histopathology of four lesions revealed lymphocytic infiltration and fibrosis without malignancy. Conclusion CABLs are benign reactive changes driven by multiple factors. Their recognition prevents misinterpretation as disease progression, thereby avoiding unnecessary treatment discontinuation or biopsy.

IntroductionCellular differentiation and lineage commitment are known to be associated with differences in DNA methylation. Leiomyosarcoma (LMS) is a tumor thought to originate from smooth muscle cells in the walls of vessels in the soft tissue (STLMS) or from the uterine myometrium (ULMS). Here, we identify the methylation signatures of normal smooth muscle cells from blood vessels and the uterine wall and compare these with those found in STLMS and ULMS. We hypothesized that these methylation signatures could be used to assign a smooth muscle subtype of origin to individual leiomyosarcomas, and that tumors of different origin would show biological differences with potential therapeutic relevance. MethodsTo define methylation profiles for smooth muscle from vessel walls versus those found in myometrium, EPIC methylation profiling was performed on DNA from 49 formalin-fixed paraffin-embedded (FFPE) normal smooth muscle samples. A supervised machine learning algorithm (Random Forest) was used to distinguish the methylation patterns of normal smooth muscle cells in vessel walls from those in the myometrium. The resulting classifier was applied to methylation data on 67 cases of LMS with corresponding bulk RNAseq data to identify which tumors showed a methylation signature most consistent with either blood vessel wall (LMSvessel) or myometrial smooth muscle (LMSwall). A custom signature matrix derived from scRNAseq data from 6 samples of LMS was used in CIBERSORTx analysis to compare the cellular composition of LMS cases with a vessel or uterine wall methylation signature. ResultsA high degree of correlation was found between the known site of origin for LMS (STLMS vs ULMS) and the methylation signature derived from different types of normal smooth muscle. LMSwall tumors compared to LMSvessel tumors had significantly higher activation of the PD-1 checkpoint pathway in RNAseq analysis. Digital flow cytometry by CIBERSORTx analysis showed an increased expression of transcriptomic signatures of several immune cell subtypes in LMSvessel tumors. ConclusionUsing a supervised machine learning approach we classified LMS samples as either showing a high similarity in methylation patterns to normal smooth muscle cells of either the vessel wall or the myometrium. We found a correlation between LMS showing either a "vessel" or "muscle wall" methylation signature and their site of origin, but notably we also identified some exceptions. When classified based on their methylation signature LMSwall and LMSvessel differed in their PD-1 pathway activation and in their predicted immune cell populations, suggesting potential implications for immunotherapeutic approaches.

Introduction: With recent approvals of multiple targeted therapies for triple-negative breast cancer (TNBC), including antibody-drug conjugates and immunotherapy in biomarker-selected populations, it is critical to define the temporal evolution of cell-surface target expression from early-stage to metastatic disease, the co-expression patterns across these markers, and optimal quantification methodologies. Here we report biomarker expression profiles measured by multi-omics and pathology-based platforms in patients with TNBC using a large cohort of matched longitudinal tumor samples. Methods: Patients who underwent neoadjuvant chemotherapy (NAC) for stage I-III TNBC or were diagnosed with any stage TNBC and developed metastatic recurrence were retrospectively identified from an institutional database and prospective research metastatic biopsy protocol. Tumor samples from diagnosis (DX), residual disease (RD) post-NAC (if applicable), and metastasis/recurrence (MR) were collected. Quantification of HER2, TROP2, and PD-L1 expression was performed by immunohistochemistry (IHC), whole-exome sequencing, transcriptome sequencing, and targeted mass spectrometry (MS). For HER2, TROP2, and stromal tumor-infiltrating lymphocytes (sTILs), both manual pathologist assessment and computational pathology quantification were obtained. HER2 status was categorized as HER2-0 or HER2-low by local (L-IHC) and central (C-IHC) review, TROP2 status was defined as low (H-score <100), medium (H-score 100-200) or high (H-score >200), and PD-L1 as low (tumor area positivity, TAP <5%) or high (TAP [&ge;]5%). Pathologist-assessed sTILs were classified as low (<10%), medium ([&ge;]10% and <40%) or high ([&ge;]40%). Biomarkers were compared between primary (DX/RD) and MR, and between pre- vs post-NAC (DX-RD) samples. Correlations between markers, quantification methods, inferred PAM50 subtype, and clinical variables of interest were evaluated. Results: A total of 359 samples from 110 patients with TNBC with data available from at least one platform were included in the analysis. HER2-low prevalence at DX, RD, and MR was: 51% (50/98), 40% (21/53), and 27% (16/60); TROP2 high/medium was 90% (47/52), 91% (42/46), and 88% (28/32); PD-L1-high was 51%, 50%, and 38% (9/24); and sTILs-high/medium was 88% (59/67), 80% (40/50), and 49% (17/35), respectively. While TROP2-high/medium vs low remained stable over time, HER2 IHC and sTILs significantly decreased from DX/RD to MR samples, both at the cohort-level (HER2, p=0.0081; sTILs, p=4.6x10e-5) and longitudinal patient-level (HER2, p=0.030; sTILs, p=0.0077), with a similar decreasing trend for PD-L1 that did not reach statistical significance. HER2 concordance (0 vs low) between L-IHC and C-IHC was 78% (91/116). ERBB2, TACSTD2 and CD274 mRNA expression were significantly correlated with IHC protein levels, though only TACSTD2 had limited overlap in distribution of gene expression between high/medium vs low groups. Strong correlation between protein membrane staining intensity from computational pathology, protein expression measured by MS, and pathologist-assed IHC was observed across all biomarkers tested by each method. In comparisons between biomarkers, pathologist-assessed PD-L1 IHC and sTILs were significantly correlated (p=0.0001); 94% (51/54) of PD-L1-high tumors were classified as sTILs high/medium. PAM50 subtype was not significantly correlated with time point or biomarker status, although there was a trend toward more HER2-enriched tumors in HER2-low (20%, 5/25) vs HER2-0 (6%, 3/52) (p=0.086). Across biomarkers and clinical variables, an association between age and sTILs was observed (p=0.038, FDR=0.42) due to a decrease in sTILs high/medium tumors with age, primarily driven by post-treatment (RD/MR) but not DX samples. Conclusions: Multi-platform and multi-omics profiling in this large unique cohort of longitudinal TNBC samples revealed distinct patterns of expression and dynamic changes of key biomarkers of interest for targeted therapies. Given variability with manual IHC scoring, improved methods for quantification of expression may help optimize treatment selection in an individualized manner.

BackgroundCDH1 (E-cadherin) is a key epithelial adhesion molecule traditionally associated with tumor suppression and epithelial-mesenchymal transition (EMT). However, its roles across cancers remain incompletely understood, particularly within multilayer regulatory contexts involving genomic, epigenetic, transcriptional, and immune mechanisms. MethodsCDH1 expression, survival associations, EMT-correlated gene profiles (VIM, SNAI1, ZEB1), immune infiltration patterns, immune checkpoint correlations (PDCD1, CD274, CTLA4), promoter methylation, and genomic alterations were assessed across five epithelial cancers, breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), ovarian cancer (OV), and stomach adenocarcinoma (STAD). Cross-platform validation was performed using TCGA/GDC datasets, GEPIA2, UALCAN, TIMER, KM Plotter, cBioPortal, and g:Profiler. ResultsCDH1 was overexpressed but showed variable prognostic significance; higher expression predicted better survival in COAD, LUAD and STAD, worse survival in BRCA and had no impact in OV. Classic inverse relationships between CDH1 and VIM or ZEB1 were evident only in STAD, and SNAI1 showed no consistent association. Immune infiltration patterns were tumor-specific, ranging from cytotoxic T-cell dominance in LUAD to macrophage-rich profiles in OV; immune checkpoint correlations were similarly context-dependent. Co-expressed genes were enriched for endomembrane transport rather than adhesion pathways. Promoter methylation patterns varied by cancer, whereas genomic alterations of CDH1 were rare. ConclusionsCDH1 does not function as a universal epithelial or EMT marker across epithelial cancers. Instead, its associations with EMT, immune contexture, methylation, and prognosis are context-dependent, supporting a model of CDH1 as a heterogeneous regulator of epithelial plasticity. These findings challenge single-function interpretations and support cancer-specific CDH1 evaluation in translational research.

Purpose: Chemotherapy-induced peripheral neuropathy (CIPN) affects many people receiving taxane treatment for breast cancer. Symptom trajectories vary, with some recovering, and others experiencing persistent, or delayed worsening (coasting) symptoms. The prevalence and predictors of these trajectories remain unclear. This study identified the prevalence and biopsychosocial predictors of CIPN persistence, improvement, and coasting within three months post-treatment. Methods: This secondary analysis included participants treated with taxanes for stage I-III breast cancer who completed the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity-4 (FACT/GOG-NTX-4) at baseline, post-chemotherapy, and three months later. A minimally important difference (MID) from baseline on the FACT/GOG-NTX-4 defined persistence, improvement, coasting, and no MID-CIPN (below the MID threshold at each assessment) trajectories. Baseline assessments included self-reported pain/well-being, sensory, balance, and lower limb physical functioning measures, and sociodemographic and treatment data were collected. Results: Among 102 participants (51.57{+/-}11.24 years), persistence occurred in 34.3%, improvement in 25.5%, coasting in 6.9%, and no MID-CIPN in 33.3%. Compared to no MID-CIPN, older age (OR=1.120; 95%CI: 1.026-1.222), higher expected pain (OR=1.630; 95%CI: 1.082-2.456), and cold hyperalgesia at the foot (OR=1.130; 95%CI: 1.018-1.254) predicted persistence. Lower fatigue predicted improvement (OR=0.904; 95%CI: 0.845-0.968). No predictors were identified for coasting. Conclusion: CIPN trajectories are heterogeneous. Age and pre-treatment pain expectations, cold hyperalgesia, and fatigue differentiate patients with persistent CIPN and those likely to improve from those with no CIPN. Implications for Cancer Survivors: Early identification of individuals at risk for persistent neurotoxicity may support risk stratification and guide targeted supportive care strategies.

BackgroundTriple-negative breast cancer (TNBC) is a clinically aggressive breast cancer subtype. It is a heterogeneous disease that remains difficult to stratify and that still lacks durable and biomarker-guided therapeutic options. Low expression of the tumour suppressor MTUS1 is associated with aggressive breast cancer features, but the biological properties of MTUS1-low TNBC remain insufficiently defined. Our goal was to determine whether low MTUS1 expression defines shared proliferative and stress-adaptation mechanisms that could guide candidate therapeutic strategies and corresponding target/drug pairs in MTUS1-low TNBC. MethodsWe labelled tumours from seven public TNBC RNA-seq cohorts based on the lowest and highest MTUS1 expression tertiles. Differential gene expression was analysed using gene set enrichment analysis (GSEA) on the Hallmark pathway database to identify deregulated biological pathways between MTUS1-low TNBC tumours and their MTUS1-high counterparts. Reproducibility was examined across independent TNBC cohorts and secondarily in broader breast cancer and selected TCGA tumour cohorts. Gene essentiality scores from CRISPR-Cas9 experiments in TNBC cell-line models were correlated to MTUS1 expression in these cell lines, to propose therapeutic strategies and their corresponding candidate target/drug pairs. ResultsMTUS1-low tumours showed a reproducible pathway-level proliferation mechanism driven by the MYC oncogene and sustained by up-regulated oxidative phosphorylation, combined with stress adaptation mechanisms involving unfolded protein response (UPR), and DNA repair Hallmark gene sets. Based on CRISPR data, we propose 3 therapeutic strategies: (1) targeting MYC to reduce its transcriptional activity, (2) targeting proteins from UPR, (3) targeting DNA-repair. We also propose corresponding candidate target/drug pairs to allow experimental validation of these strategies. ConclusionsProliferation in low MTUS1 TNBC is driven by MYC and stress-adaptation mechanisms. By linking this tumour profile to CRISPR-derived dependency signals, our analysis prioritises experimentally testable target-pathway hypotheses centred on MYC, UPR/proteostasis, and DNA-repair or checkpoint control. Although the proposed therapeutic strategies and candidate targets remain to be experimentally tested, the latter finding is consistent with published work showing that ATIP3-deficient TNBC cell line models are sensitive to inhibition of the WEE1 PKMYT1 G2/M checkpoint kinases.

Background: The use of immune checkpoint inhibitors (ICIs) in the treatment of cancer has rapidly expanded over the last decade. However, there are several knowledge gaps in understanding how tumor cells evade the immune system. There is paucity of data in HPV negative oral cancer, particularly of the gingivobuccal region. Understanding the mechanism of immune system evasion in this cancer is vital for improving patient outcomes. Methods: We characterized the baseline immune milieu of oral cancer using immunohistochemistry (IHC) on whole tumor sections from 124 cases. Tumors were classified as hot or cold and further stratified into high-risk and low-risk groups. High-risk patients included those with lymph node metastasis at diagnosis/recurrence or distant metastasis within 2 years of treatment completion. Patients without these features were categorized as low risk. Validation by RNA-Seq and Joint Enrichment Analysis of Oncogenic and Immunologic Pathways was carried out in a subset of 46 cases. Results: Hot high-risk tumors (by IHC) were distinguished by elevated PD-L1 expression and reduced NK-cell, PD1, and CTLA-4 expression. There was no difference in the expression levels of CD3+, CD8+, granzyme, or perforin compared to hot low-risk tumors, findings that align with the definition of hot tumors. RNA-Seq revealed a gene signature associated with exhausted T-cells in hot high-risk tumors. Gene and pathway analyses identified differential upregulation of isoform-specific TOX, TCF, CXCR, RUNX, IRF, BRD and BCL6 genes, implicating immune cell exhaustion and tumor aggressiveness. Significantly downregulated genes included PDCD1, HAVCR2, ZAP70, and STAT, indicative of a disabled immune microenvironment. These findings support that a state of immune exhaustion in HHR tumors is driven by progenitor exhausted T-cells and terminally exhausted T-cells; independent of PD1-TIM3. Conclusion: These findings suggest that combining TOX/TCF/BCL6 inhibitors with immune checkpoint inhibitors in the adjuvant setting might benefit patients with hot high-risk tumors. Given the results, testing for a targeted exhaustion-related gene panel at diagnosis is recommended for oral cancers to stratify tumors as high-risk or low-risk. Larger validation studies and clinical trials are now warranted.

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.