Search Results (1,048)

Background/Objectives: Cribriform architecture is an adverse histopathologic feature in prostate cancer and has been associated with poor oncologic outcomes. Emerging evidence suggests that cribriform-positive tumors may behave as a biologically non-localized disease, raising the possibility of early occult dissemination. Lymphovascular invasion (LVI) is a key pathological marker of metastatic potential, but its relationship with cribriform architecture has not been evaluated. We examined the association between cribriform morphology and LVI to provide biological context for the aggressive clinical course of cribriform-positive prostate cancer. Methods: We performed a retrospective analysis of patients with prostate adenocarcinoma who underwent radical prostatectomy and had available clinicopathologic data. Cribriform architecture was determined by a centralized pathology review, and LVI status was obtained from original pathology reports. Unadjusted associations were evaluated using contingency tables. Multivariable logistic regression was used to assess whether cribriform architecture was independently associated with LVI after adjustments for Gleason score, tumor stage, and nodal status. Results: Among 338 patients, 28 (8.3%) had LVI and 123 (36.4%) had cribriform architecture. LVI was more common in cribriform-positive than cribriform-negative tumors (17.9% vs. 2.8%; p < 0.001), corresponding to a crude odds ratio (OR) of 7.6 (95% CI, 3.0–19.3). Cribriform architecture remained independently associated with LVI after adjustment (adjusted OR, 5.20; 95% CI, 2.12–1.40; p < 0.001). Conclusions: Cribriform architecture is strongly and independently associated with LVI, supporting a biological link between cribriform morphology and early metastatic dissemination. These findings support the design of prospective, biomarker-driven studies to evaluate treatment intensification strategies in this high-risk subgroup. Full article

The tightly controlled and transient acquisition of a motile phenotype by otherwise static epithelial cells (epithelial–mesenchymal transition, EMT) enables the repair of a damaged epithelium. Conversely, a persistent, dysregulated, and exacerbated EMT characterizes epithelial malignancies such as breast carcinoma (BC) and oral squamous cell carcinoma (OSCC), being key for their metastasis and for their escaping anti-tumor immune responses. Herein, we investigated the relationship between EMT signatures and immune cell infiltration across OSCC and metastatic BC with the aim to identify prognostic markers and/or therapeutic targets common to both these malignancies, or unique to OSCC or BC. To this end, we analyzed publicly available transcriptomic datasets to identify coding genes involved in EMT with strong correlation to immune cell signatures. The methodology consisted of data selection, correlation analysis, signature overlap determination, and validation using independent databases. Results indicated that in both OSCC and BC the expression of EMT-related genes is strongly associated with that of immunosuppressive and pro-tumor macrophages. Notably, the FN1 gene coding for the extracellular matrix glycoprotein fibronectin (FN) emerged as the EMT gene common to either tumor types. In confirmation of this, FN protein levels were higher in OSCC and BC tissues than in their normal counterparts. Given FN capability of favoring tumor invasion and metastasis while hindering antitumor immune responses, these data encourage the development of FN antagonists to be used as an adjunct to conventional therapy in the treatment of both OSCC and BC. Full article

Background and Objectives: The Gustave Roussy Immune (GRIm) score, reflecting systemic inflammation and nutritional status, has emerged as a simple and reproducible prognostic biomarker in various malignancies. However, its prognostic interaction with tumor microenvironmental factors remains unclear in gastric cancer. The primary aim of this study was to evaluate the prognostic value of the GRIm score in patients with resectable gastric adenocarcinoma, while the secondary aim was to determine whether integrating the GRIm score with tumor microenvironment–related pathological markers could improve prognostic stratification. Materials and Methods: This retrospective study analyzed 188 patients with resectable gastric adenocarcinoma treated at the Trakya University Faculty of Medicine between 2007 and 2018. GRIm scores were calculated from preoperative lactate dehydrogenase (LDH), albumin, and neutrophil-to-lymphocyte ratio (NLR) values. Pathologic parameters, including programmed death-ligand 1 (PD-L1) expression (combined positive score [CPS] ≥ 1 vs. <1), tumor–stroma ratio (TSR; stromal component ≥ 50% vs. <50%), and tumor-infiltrating lymphocyte (TIL) density (CD8+ ≥ 10% vs. <10%), were evaluated on surgical specimens. Survival outcomes were assessed using Kaplan–Meier and multivariate Cox analyses. Results: The study population had a mean age of 61.8 years and was predominantly male (72.3%). Patients with low GRIm scores had significantly longer disease-free survival (DFS; 24 vs. 12 months; p = 0.004) and overall survival (OS; 32 vs. 19 months; p = 0.006). In multivariate analysis, the GRIm score remained an independent predictor for both disease-free survival (p = 0.035) and overall survival (p = 0.044). Among combined models, the GRIm–TSR classification provided the most pronounced stratification (median DFS = 35 vs. 12 months; OS = 45 vs. 19 months; p = 0.014 and 0.001, respectively), retaining independent prognostic significance (hazard ratio [HR] = 1.23; p = 0.005). Integrating GRIm with PD-L1 and TIL density also improved prognostic discrimination. Conclusions: The GRIm score is a robust and cost-effective biomarker that independently predicts disease-free survival and overall survival in resectable gastric adenocarcinoma. Its combination with microenvironmental markers—PD-L1, TIL, and TSR—captures complementary biological dimensions of tumor aggressiveness, offering an integrative and clinically feasible framework for individualized risk assessment and postoperative management. Prospective multicenter validation is warranted. Full article

Background/Objectives: Kidney cancer (RC) is a significant global health burden. Renal cell carcinoma (RCC) is the most common form of kidney cancer. Its predominant histological subtype is clear cell renal cell carcinoma (ccRCC), which is frequently diagnosed at an advanced local stage or with metastases. Detecting cancer at an early stage significantly increases the likelihood of a cure; therefore, research on new markers and a thorough understanding of tumor biology are essential. This study investigated the significance of aromatase (ARO), cathepsin S (CTSS), and matrix metalloproteinase 1 (MMP-1) as potential biomarkers in ccRCC. Methods: ARO, CTSS, and MMP-1 concentrations in plasma were determined using SPRi biosensors. Appropriate antibodies were used as biorecognition molecules in the biosensors. The samples analyzed came from 60 patients with histopathologically confirmed clear cell renal cell carcinoma (ccRCC) and from 26 patients diagnosed with chronic cystitis or benign prostatic hyperplasia (BPH). Results: A statistically significant increase (p < 0.00001) in the concentration of all proteins compared with the control samples was observed at the T3–T4 stage. The ARO concentration was already statistically significantly higher at the T1–T2 stage (p < 0.00001). The ROC curve for aromatase demonstrated high sensitivity and specificity for detecting ccRCC, with a cut-off point of 7.53 ng mL⁻¹. A moderate positive correlation was also found between the concentrations of the three tested substances in renal cancer, which may indicate potential interactions in the tumor’s pathogenesis. Conclusions: SPRI testing has been shown to be an alternative to standard methods for detecting potential ccRCC markers. The biosensors used in the study can simultaneously determine ARO, CTSS, and MMP-1. The results obtained suggest the potential importance of these proteins in the development of ccRCC, and our work proposes a new diagnostic technique that may aid in the diagnosis of ccRCC. Full article

Background: The development of lung cancer is strongly influenced by oxidative stress (OS), which results when the balance between oxidants and antioxidants is disturbed. Evaluation of both specific redox markers such as thiol/disulfide homeostasis (TDH) and overall indicators including total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) may provide a more comprehensive view of oxidative imbalance in lung cancer. We examined OS indices and TDH in patients with lung cancer versus healthy controls. Methods: Eighty participants were enrolled, consisting of 40 patients with newly diagnosed lung cancer and 40 age- and sex-matched healthy controls. Serum levels of native thiol (NT), total thiol (TT), and disulfide were determined using an automated spectrophotometric method. Additionally, TAS, TOS, and the OSI were evaluated to provide an overall assessment of oxidative balance. Routine hematological and biochemical parameters were compared between groups. Results: White blood cell and neutrophil counts were notably higher in lung cancer patients compared with controls (p < 0.05). NT and TT levels were remarkably decreased, whereas disulfide levels, TOS, and OSI were significantly elevated in the lung cancer group (p < 0.05). TAS levels tended to be lower in patients, although not reaching statistical significance. No significant association was observed between oxidative parameters and tumor stage or localization. Conclusions: Patients with lung cancer exhibited a marked oxidative imbalance, characterized by elevated oxidant burden and impaired TDH. Combined assessment of TAS, TOS, OSI, and thiol/disulfide parameters may provide valuable insight into the oxidative pathophysiology of lung cancer and hold potential as complementary biomarkers for disease evaluation. Further large scale studies are needed to confirm these findings. Full article

Dysregulated magnesium (Mg²⁺) homeostasis contributes to colorectal cancer (CRC), yet its context-dependent function within the tumor microenvironment remains unresolved. This study aimed to determine how sustained low and high extracellular Mg²⁺ environments affect CRC spheroid (SP) growth and Mg²⁺ homeostasis using HT-29 SPs. We analyzed Mg²⁺ flux, the expression of Mg²⁺ transporters (e.g., Transient Receptor Potential Melastatin (TRPM) 6), viability, apoptotic and autophagic markers, and phospho-/oxidoproteomic alterations. Both Mg²⁺ extremes destabilized SP architecture, reduced viability, and induced apoptosis and autophagy, with SPs displaying heightened vulnerability relative to 2D cultures. Mg²⁺ stress impaired Mg²⁺ influx and eliminated adaptive transporter regulation in SPs. Loss of membrane TRPM6/7 heterodimers, driven by altered phosphorylation (e.g., TRPM6 Serine 141, Serine 1252, Threonine 1851) and elevated oxidation (e.g., Methionine 1755), suppressed channel activity. High Mg²⁺ caused profound metabolic failure despite increased total Mg²⁺, reflecting functional Mg²⁺ deficiency. CRC spheroids are acutely susceptible to Mg²⁺ imbalance due to collapsed transporter homeostasis and post-translational inhibition of Mg²⁺ channels. These findings reveal a targetable metabolic vulnerability and support the therapeutic potential of localized Mg²⁺ modulation in CRC. Full article

Background/Objectives: Cancer-associated fibroblasts (CAFs) are key stromal mediators of breast tumor progression and therapy resistance. Carvacrol, a dietary monoterpenic phenol, exhibits antiproliferative activity in cancer cells, but its effects on primary human breast CAFs remain unclear. This study aimed to determine whether carvacrol selectively induces mitochondria-related apoptotic signaling in breast CAFs while sparing normal fibroblasts (NFs). Methods: Primary fibroblast cultures were established from invasive ductal carcinoma tissues (CAFs, n = 9) and nonmalignant breast tissues (NFs, n = 5) and validated by α-SMA and FAP immunofluorescence. Cells were exposed to 400 μM carvacrol. Apoptosis was assessed by TUNEL assay and BAX/BCL-XL Western blotting. Changes in signaling pathways were evaluated by analyzing PPARα/NF-κB, sirtuin (SIRT1, SIRT3), autophagy-related markers (LAMP2A, p62), and matrix metalloproteinases (MMP-2, MMP-3). In silico molecular docking and 100-ns molecular dynamics simulations were performed to examine interactions between carvacrol and caspase-3 and caspase-9. Results: Carvacrol induced a pronounced, time-dependent apoptotic response in CAFs, with TUNEL-based viability declining to approximately 10% of control levels by 12 h and a marked increase in the BAX/BCL-XL ratio. In contrast, NFs exhibited minimal TUNEL positivity and no significant change in BAX/BCL-XL. In CAFs, but not NFs, carvacrol reduced PPARα expression and NF-κB nuclear localization, increased SIRT1 and SIRT3 levels, selectively suppressed MMP-3 while partially normalizing MMP-2, and altered autophagy-related markers (decreased LAMP2A and accumulation of p62), consistent with autophagic stress and possible impairment of autophagic flux. Computational analyses revealed stable carvacrol binding to caspase-3 and caspase-9 with modest stabilization of active-site loops, supporting caspase-dependent, mitochondria-related apoptosis. Conclusions: Carvacrol selectively targets breast cancer-associated fibroblasts by inducing mitochondria-related apoptotic signaling while largely sparing normal fibroblasts. This effect is accompanied by coordinated modulation of PPARα/NF-κB, sirtuin, autophagy, and MMP pathways. These findings support further evaluation of carvacrol as a microenvironment-directed adjunct in breast cancer therapy. Full article

The increasing prevalence of obesity-related primary arterial hypertension (PAH) in the pediatric population emphasizes the need to develop new biomarkers that can aid in clinical practice for prevention or early diagnosis of the cardiovascular disease. The objective of the present study was to evaluate the relationship between selected adipokines, cytokines, and blood pressure (BP) values in children with obesity. A total of 78 children participated in the study: 60 children with obesity (study group) and 18 children with normal weight (control group). Blood pressure was measured according to guidelines. Serum levels of metabolic and inflammatory markers, including leptin, adiponectin, resistin, ghrelin, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α), vascular endothelial growth factor (VEGF), and insulin were determined using multiplex immunoassays. Statistical analysis included correlation and ROC tests to identify potential predictors of PAH. The study group had significantly higher systolic and diastolic BP compared to the control group (p < 0.0001). Serum levels of leptin, IL-6, VEGF, insulin, and resistin were increased in the study group. Leptin, IL-6 and resistin correlated positively with BP values (p < 0.05), while ghrelin and adiponectin correlated negatively. ROC analysis identified leptin, IL-6, and VEGF as the most promising biomarkers for predicting PAH. The results confirm the role of adipokines and cytokines in the pathogenesis of PAH. The assessment of adipokine and cytokine profiles complements traditional anthropometric parameters such as BMI in assessing cardiovascular risk. Leptin, IL-6, and VEGF presented the strongest correlation with hypertension, suggesting their potential in future diagnostic and preventive strategies. Full article

Introduction: Prompt diagnosis and differentiation between non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) are essential in the treatment of bladder cancer. Inflammation-based biomarkers have recently emerged as potential tools for improving cancer diagnostics and prognostication. This study aims to evaluate the potential value of the Systemic Inflammation Index (SII), Systemic Inflammation Response Index (SIRI), Pan-immune Inflammation Value (PIV), and Platelet-to-Lymphocyte Ratio (PLR) as predictors of muscle-invasive disease. Materials and methods: Analyzed data included 310 bladder tumors. The SII, SIRI, PIV, and PLR were calculated from pre-TURBT complete blood-count results. Differences in inflammatory markers between pathological stages (pTa–pT4) were examined using ANOVA with Tukey’s post hoc testing. Optimal cutoff values for distinguishing NMIBC from MIBC were identified using ROC curve analysis and Youden’s J statistic. Logistic regression models incorporating age, sex, the number of recurrences, and each inflammatory index were developed to evaluate their predictive performance in patients treated with curative intent. Results: All investigated inflammation indices significantly differed across tumor stages (p < 0.001), with lower values observed in pTa tumors compared with muscle-invasive disease. Determined cutoff values for muscle-invasive disease were 865.63 for SII, 2.02 for SIRI, 579.28 for PIV, and 166.35 for PLR. Logistic regression models demonstrated promising diagnostic performance, achieving AUC values of 0.812 (SII), 0.816 (SIRI), 0.821 (PIV), and 0.795 (PLR); sensitivity and specificity were 76% and 75% for SII, 79% and 77% for SIRI, 80% and 72% for PIV, and 88% and 59% for PLR. Discussion: The presented results indicate that inflammation-based indices vary meaningfully between bladder cancer stages and may be utilized in early identification of muscle-invasive disease. As inexpensive and widely available biomarkers, they offer potential value in the evaluation of suspected MIBC before the final pathology report and could enhance the diagnostic process. Full article

Background/Objectives: Acetaminophen (paracetamol or APAP) overdose is a major cause of acute liver injury mediated by oxidative stress, inflammation, and hepatocellular necrosis. The present study investigates the in vivo hepatoprotective potential of morin (M), lignin nanoparticles (LN), and morin-encapsulated lignin nanoparticles (LMN) against APAP-induced hepatotoxicity in mice. The specific goal was to determine whether LMN could strengthen hepatic antioxidant and anti-inflammatory defenses prior to toxic insult, which aligns with a prophylactic model rather than a post-injury clinical rescue approach. This study was guided by the primary hypothesis that LMN pretreatment would markedly reduce APAP-induced hepatic injury. Methods: Experimental groups included control, APAP, M, LN, LMN, M+APAP, LN+APAP, and LMN+APAP treatments. Serum hepatic biomarkers, oxidative stress parameters, and inflammatory cytokines were analyzed to assess protective responses. Results: APAP exposure markedly elevated aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels, indicating severe hepatic dysfunction, accompanied by increased lipid peroxidation and pro-inflammatory cytokine production. LMN+APAP treatment significantly restored hepatic enzyme levels to approximately normal values and suppressed malondialdehyde (MDA) formation, while enhancing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities. LMN also downregulated interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β), while upregulating interleukin 10 (IL-10), suggesting effective attenuation of inflammatory signaling. Correlation analyses demonstrated positive interactions between MDA, cytokines, and hepatic enzymes, whereas antioxidant enzyme levels were inversely correlated with liver injury markers. Histopathological analysis revealed that treatment with LMN enhanced hepatoprotection, demonstrating predominantly mild, reversible lesions and suggesting a synergistic antioxidant and immunomodulatory effect. Conclusions: It could be concluded that LMN provided superior hepatoprotection compared to M or LN. These findings establish LMN as a promising bio-based nanotherapeutic agent for mitigating drug-induced hepatotoxicity through coordinated antioxidant and anti-inflammatory mechanisms. Full article

Objective: Impairment of the Fas/FasL apoptotic pathway is a mechanism contributing to the malignant transformation of multiple cell types. This study aimed to investigate the clinical and prognostic relevance of serum soluble Fas (sFas) and soluble Fas ligand (sFasL) levels in patients with pancreatic and papilla of Vater adenocarcinomas. Methods: An ELISA was used to determine sFas and sFasL levels. Serum samples were obtained from 53 healthy controls, 82 pancreatic and 14 papilla of Vater carcinoma patients. Sera from carcinoma patients were obtained before surgery and 30 days after surgery. The relationships of preoperative levels with clinicopathological features and patient survival were evaluated. Changes in serum sFas and sFasL levels after surgery were also evaluated. Results: Higher sFas and lower sFasL levels were found in the serum of carcinoma patients in comparison to healthy controls. Serum sFas and sFasL levels correlated significantly with both lymph node and distant metastases and an advanced stage of disease. Elevated sFas and decreased sFasL levels correlated significantly with poor overall survival when the entire study population was considered with sFas being an independent prognostic factor. After patient stratification, their prognostic value was evident in pancreatic carcinoma patients only. Preoperative sFas levels decreased and sFasL levels increased after radical resection of the tumor but remained unchanged in cases of unresectable disease. Conclusions: These findings suggest that serum levels of sFas and sFasL could be useful tumor markers with prognostic value in pancreatic adenocarcinomas. Increased sFas secretion may reflect a mechanism for apoptotic escape of cancer cells. Full article

Although excessive manganese (Mn) exposure is known to cause neuromotor function in cases of poisoning, its effect on grip strength (a neuromotor marker) in older adults at environmental levels remains unclear. To investigate this issue, we conducted an integrated investigation combining epidemiology and animal experimentation to examine the association between urinary manganese and grip strength. A cross-sectional study of 375 elderly men (60–74 years) was conducted in Guangxi, China, from 2016 to 2017. Urinary Mn concentrations were determined by ICP-MS, and their associations with grip strength were evaluated using generalized linear models and restricted cubic splines. In parallel, 32 six-week-old male C57BL/6J mice were exposed to 0, 5, 10, or 15 mg/kg MnCl₂·4H₂O via intraperitoneal injection for 6 weeks. Forelimb grip strength of the mice was measured after the final exposure, and mRNA expression of inflammatory markers and cytokines (C reactive protein (CRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α in triceps) in triceps tissue was quantified. The median urinary Mn concentration in the study population was 0.22 μg/g creatinine. After adjusting for confounders, urinary Mn was inversely associated with hand grip strength (highest vs. lowest tertile: β = −3.57 kg; 95% CI: −5.68 to −1.47; p-trend = 0.007). Similarly, in male C57BL/6J mice, grip strengths declined significantly with increasing Mn exposure (p-trend < 0.0001), accompanied by upregulation of the mRNA levels of CRP, IL-6 and TNF-α in muscle tissue. Together, our findings suggest that environmental manganese exposure is inversely associated with grip strength in elderly men. While the manganese doses used in the animal study exceeded typical human environmental exposure, the experimental results further indicate that such grip strength reduction may be linked to muscle inflammation. Full article

Background: Breast cancer is the most prevalent cancer in women, and metastatic breast cancer remains a major cause of cancer-related deaths. Resveratrol (RSV) is a natural compound found in various plants and is known to exhibit various anti-cancer effects. The present study aims to investigate the therapeutic effects and mechanisms of RSV in inhibiting breast cancer metastasis in a murine model of 4T1 breast tumor that shares close molecular features with human triple negative breast cancer. Methods: Murine breast cancer 4T1 cells were used to examine the effects of RSV on breast cancer metastasis and epithelial–mesenchymal transition (EMT). In vitro cell proliferation and Transwell migration assays and in vivo 4T1 tumor transplantation models were established in female Balb/c mice to determine the anti-metastatic effects of RSV and its mechanism of action. Results: RSV significantly inhibited 4T1 tumor cell migration and significantly decreased expression levels of EMT markers Snail and Vimentin, as well as the nuclear translocation of β-catenin both in vitro and in vivo. Knockdown of β-catenin similarly reduced the expression levels of EMT markers. RSV significantly decreased the number of lung metastases in 4T1-implanted mice by inhibiting Wnt/β-catenin signaling pathway activation. RSV (150 mg/kg/day) reduced the number of visible tumor metastatic nodules and the histological count of metastatic lung carcinomas by 51.82% and 62.58%, respectively, compared to vehicle administration. Conclusions: Our study provides important new mechanistic insight into the strong anti-cancer effects of RSV in inhibiting 4T1 breast cancer metastasis by preventing Wnt/β-catenin signaling pathway-mediated epithelial–mesenchymal transition. These findings suggest the therapeutic potential of RSV as a promising drug in the treatment of metastatic breast cancer. Full article

Background/Objectives: Metabolic reprogramming is an essential feature of tumors. Mitochondrial sirtuins SIRT3 and SIRT5 differently regulate glutamine metabolism with SIRT5 inhibiting glutaminase (GLS) and SIRT3 increasing glutamate dehydrogenase (GDH). Considering the important and interconnected role of glutamine, SIRT3 and SIRT5 for cancer growth and progression, our hypothesis is that a simultaneous modulation of SIRT3 and SIRT5 could represent a valid anti-tumoral strategy. Methods: wt and GLS1-silenced triple negative breast cancer spheroids were treated with 3-TYP, a selective SIRT3 inhibitor, and with MC3138, a new selective SIRT5 activator, both alone and in combination. The effects of such treatments on hypoxia, autophagy and mitophagy markers were determined by immunofluorescence and Western blot. Mitochondria morphology was studied by transmission electron microscopy (TEM) and mitochondrial ROS production by confocal analysis. Results: We observed that 3-TYP+MC3138 treatment decreased the size of spheroids by affecting HIF-1α, c-Myc, glutamine transporter SLC1A5 and autophagy (LC3II) and mitophagy (BNIP3) markers. Moreover, such treatments altered the morphology and conformation of the mitochondria. Finally, we also documented an increase in mitochondria reactive oxygen species (mtROS). Conclusions: The combined inhibition of SIRT3 and activation of SIRT5 greatly reduces the size of spheroids through the inhibition of hypoxic response, which is then followed by the alteration of the autophagic and mitophagic process and the toxic accumulation of mitochondrial ROS, representing a new anti-tumoral strategy. Full article

Background/Objectives: Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis, creating an urgent need for novel biomarkers to improve risk stratification. The prognostic significance of the transmembrane glycoprotein CD44 and its isoforms (CD44s, v5, v6) in CCA remains controversial. This preliminary study aimed to investigate whether the combined loss of these isoforms could serve as a distinct prognostic indicator. Methods: We evaluated the expression of CD44s, CD44v5, and CD44v6 via immunohistochemistry on a retrospective cohort of 61 paraffin-embedded CCA patient tissue blocks from Ramathibodi Hospital, Bangkok, Thailand. Expression levels were correlated with clinicopathological parameters. Survival analyses, including Kaplan–Meier and Cox proportional hazards models, were used to determine the prognostic value of individual isoforms and the complete absence of all three. Results: Expression of CD44s, CD44v5, and CD44v6 was found in 52.5%, 47.5%, and 82.0% of tumors, respectively. In univariate and multivariate analyses, the expression of any single isoform was not a significant predictor of overall survival. However, a subgroup of 8 patients (13.1%) was identified whose tumors were negative for all three isoforms, a phenotype we termed “CD44s-v5-v6 Null”. This status was significantly associated with advanced TNM stages (p = 0.022). Patients with these Null tumors also showed a clinically relevant, though not statistically significant, trend towards poorer survival (median 7.0 vs. 12.0 months, p = 0.336). Conclusions: Individual CD44 isoforms did not serve as reliable independent prognostic markers in this cohort. Instead, the complete loss of the CD44 expression program characterizes a potential “CD44s-v5-v6 Null” phenotype associated with advanced-stage disease. Full article