Introduction: The new European Kidney Function Consortium (EKFC) eGFR equation uses rescaled serum creatinine (SCr) to an age-and sex-specific normal median value, making it applicable across the entire age spectrum. We evaluated the performance of the recalibrated EKFCcrea equation using Indian-specific Q (for median SCr) and K (for normal glomerular filtration rate [GFR]) in a large Indian cohort and the ability to correctly classify chronic kidney disease (CKD). Methods: Indian adults (healthy and patients with CKD) were recruited. Plasma iohexol clearance was used to assess measured GFR (mGFR). Indian-specific Q values were derived from healthy individuals, and K from those under the age of 40 years. We evaluated EKFCcrea, CKD-Epidemiology Collaboration (EPI)2021, and recalibrated the EKFCcrea equations, incorporating the Indian Q (ISQ), Indian constant (ISK), and both (ISQ + ISK). We evaluated bias and accuracy (P 30 ) for all equations. Results: A total of 1174 participants (615 healthy, 559 CKD), mean age 48 years, with equal sex distribution were recruited. The EKFCcrea equation showed a mean bias of 9.9 (95% CI: 8.7-11.1) ml/min per 1.73 m 2 with P 30 ; 58.1%. EKFCISQ reduced bias to 4.9 (95% CI: 3.7 6.0) ml/min per 1.73 m 2 and improved P 30 to 64%. EKFCISK achieved a bias of-1.2 (95% CI:-0.1 to-2.2) ml/min per 1.73 m 2 and P 30 of 69%. 30%, 48%, and 67% of CKD patients were categorized into lower categories using the EKFCISQ, EKFCISK, and EKFCISQ+ISK equations, respectively, against the CKD-EPI 2021 . Conclusion: Recalibrating EKFCcrea with Indian-specific Q and K reduced bias and improved accuracy, with EKFCISK emerging as the best model. Adopting this recalibrated equation in clinical practice and public health initiatives after validation in diverse cohorts could lead to more accurate CKD diagnosis and staging in India.

Abstract Background: HNSCC has significant burden with high morbidity and mortality. Majority of the HNSCC patients receive Radiotherapy (RT), which is associated with development of radiation resistance and disease-recurrence, which needs in-depth evaluation. Lipocalin2(LCN2), found to be dysregulated in the saliva of HNSCC patients in our previous study using LC/MS shotgun proteomics, is also found associated with radio resistance (RR). To establish the potential role as a predictor of RR, we evaluated LCN2 in HNSCC patients. Methodology: 356 biopsy-proven treatment-naïve HNSCC, 26 Oral Pre-Malignant Diseases (OPMDs) and 118 healthy subjects were recruited for the study. The patients were treated with curative intent with 66Gy of RT. 5ml of unstimulated saliva and 3ml of whole blood were collected at baseline and during follow-ups in No evidence of disease (NED) and Residual Disease (RD)patients post therapy completion. To evaluate the role LCN2 as a marker of RR, radioresistant cells were generated by repeated sub-lethal exposure of Cal27 cells to γ-rays from radioactive Co60. The RR phenotype was confirmed by phosphorylated H2AX(γH2AX) expression using Western Blot (WB) and Immunofluorescence (IF). Various RR assays such as clonogenic survival assay, apoptosis assay and cell cycle analysis, were performed on the successfully generated RR cells, which were later analysed for the expression of LCN2. Results: Significantly higher(p≤0.0001) levels of LCN2 in saliva and serum (median- 609.15ng/ml & 186.18ng/ml) of patients compared to OPMD (median-358.47ng/ml & 124.05ng/ml) and healthy controls (median-95.70ng/ml & 85.53ng/ml) respectively. The increased level of these proteins also correlated with patients’ response post therapy completion with significantly higher(p≤0.0001) LCN2 in RD (median-1919.61ng/ml & 133.48ng/ml) group compared to NED (median-537.27ng/ml & 90.68ng/ml), indicating that the increased LCN2 level estimated after therapy completion is associated with therapy resistance. Poorly differentiated patients had significantly higher(p≤0.0001) LCN2(H-score148.12) as compared to moderately(H-score34.04) and well differentiated(H-score17.12) on IHC analysis. In the generated RR cell line, on IF analysis γH2AX foci was significantly higher validated by western blot. Further using clonogenic survival assay, apoptosis and cell cycle analysis assay it was henceforth proven that the cell lines generated had RR characteristics. The proven RR cell lines were further checked for LCN2 expression, which had significantly higher (3.6 fold) expression as compared to wild type cell lines. The results were validated with western blot. Conclusion: LCN2 is proposed as a predictive biomarker to prognosticate resistance to radiation in patients with HNSCC. Citation Format: Smriti Suri, Sushmita Ghoshal, Jaimanti Bakshi, Arnab Pal. Potential role of Lipocalin 2 as a biomarker of radio resistance in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7365.

Abstract Metastatic dissemination is driven by rare Circulating Tumour Cells (CTCs) that exhibit pronounced phenotypic plasticity and dynamic interactions with different components in the bloodstream. In Oral Squamous Cell Carcinoma (OSCC), limited resolution of epithelial, mesenchymal, and platelet-associated CTC-states has constrained biological understanding of early tumour spread and hindered translational interpretation. In this study, we identify, count and characterize distinct epithelial, mesenchymal, and platelet-associated CTC-states in a cohort of 33 patients using a strategic workflow involving hematopoietic cell depletion with multiparametric flow-cytometric analysis. The FACS (Fluorescence-assisted cell sorting)-strategy incorporated epithelial tumour-associated markers (EpCAM, EGFR, and Cytokeratin), mesenchymal markers (VIM and N-Cadherin), and platelet/leukocyte markers (CD41 and CD45) to resolve biologically distinct CTC-subpopulations. Analytical robustness was established through spike-in experiments using OSCC cell line i.e., Cal27, demonstrating linear detection across clinically relevant ranges(1-100cells/mL of blood, Spearman’s r = 0.8738, p < 0.0001) and sensitive recovery of rare tumour cells with a Limit of Detection of 1 cell/mL, Limit of Quantification of 10 cells/mL, and Limit of Blank of 1.779 cells/mL. Molecular validation of sorted populations using whole transcriptome amplification-quantitative PCR confirmed tumour-associated marker expression and absence of hematopoietic contamination. Application of this approach in OSCC patients revealed detectable CTCs in 70% of the cases (ranging from 14.20 to 340.5 CTCs-related events/mL). Importantly, CTCs were identified across discrete phenotypic states, including platelet-associated CTC-clusters that were not captured by epithelial marker-restricted strategies. Quantitative comparison of CTC subtype proportions revealed majority (nearly 83%) of CTCs present as platelet clusters, followed by Epithelial-Single CTCs (21.21%). In comparison, CTCs with only mesenchymal phenotype constitute only 3.22% of the total CTC-population. Receiver operating characteristic analysis supported the discriminatory capacity of CTC enumeration and enabled the definition of a biologically relevant threshold of > 19.40 events mL for CTC positivity. Taken together, our findings outline a practical workflow for identifying and quantifying different CTC-states in OSCC, with platelet-associated CTCs emerging as the predominant population detected. These findings support the importance of incorporating phenotypic heterogeneity and platelet-associated tumour cell interactions into future liquid biopsy-based translational pathology research and clinical biomarker development in OSCC.

Background:Genotype-3 HCV cirrhosis is a major risk for hepatocellular carcinoma (HCC). We assessed DAA treatment-related SVR rates, and HCC characteristics and outcomes in a prospective, predominantly genotype-3 compensated cirrhosis cohort participating in the National Viral Hepatitis Control Programme (NVHCP) for HCV elimination in India. Methods:Viremic HCV-related HCC diagnosed at presentation and within 6 months (pre-DAAs), and those with post-SVR-12 HCC (post-DAAs) between 15 October 2018 and 31 December 2024, were evaluated. Results:Among 51,865 HCV patients, 4646 had compensated cirrhosis, 652 had HCC, of whom 399 were treated at the hub. 368/399 (92.2%) at presentation had viremic HCV-cirrhosis related HCC (aged 61.5 ± 10.5 years, 65.2% men, 77.8% genotype-3, median ALBI -2.0, with presentation in BCLC stages A (26.1%), B (25.3%), C (12.8%), and D (35.9%), 16% had portal vein thrombosis while 31 (7.8%) did not have cirrhosis. Ultimate SVR-12 after one or more DAAs courses was in 360/368 (97.8%) in HCV-cirrhosis with HCC(at presentation) and in 3874/3994 (96.9%; P = 0.366) with HCV-cirrhosis without HCC. De novo HCC post SVR-12 was in 128/3994 (3.2%), median follow-up of 4 years, an incidence of 985 per 100,000 person-years; aged 57.6 ± 8.6 years, 77.3% men, 71.6% genotype-3, BCLC stages were A (60.2%), B (28.9%), and C (10.9%). Liver stiffness at SVR-48 (aHR 1.01, 95% CI: 1.001-1.030, P = 0.021) and ALBI score at SVR-12 (aHR 1.31, 95% CI: 1.1-1.4, P = 0.034) independently predicted de novo HCC. The recurrence rate was lower in patients with de novo HCC than in those with HCC at presentation (2.2 vs 17.6 per 100 person-years). 27 (21.1%) died, 4 (3.1%) underwent transplantation. Conclusion:DAAs achieved an SVR-12 rate exceeding 95% in genotype-3 predominant HCV-compensated cirrhosis, with and without HCC. Incidence of de novo HCC post SVR-12 was 985 per 100,000 person-years, and 60% had BCLC Stage A. Post-SVR LSM and ALBI score predicted risk of HCC. Our data argue for mandatory HCC surveillance under the NVHCP for all patients with HCV-cirrhosis in India.

Abstract Background Oral squamous cell carcinoma (OSCC) exhibits high incidences of relapse and treatment failure. To understand the molecular mechanisms driving these poor outcomes, we explored the spatial and temporal heterogeneity of primary tumors and their circulating tumor cells (CTCs) using whole transcriptome analysis. Methods Bulk RNAseq was performed on three tumor regions (T1-invasive front, T2, T3) and the tumor-free margin (TFM) from early (EarlyR) and no recurrence till 2 years (NoR) patients (N=5 per group). Ultra-low cell RNAseq of CTCs was performed at diagnosis and at post-therapy/recurrence. Differential gene expression analysis and pathway enrichment using fgsea and clusterProfiler, with the Hallmark and Reactome databases, were performed to identify biologically relevant genes/pathways. Results Spatially distant tumor regions of EarlyR patients were highly diversified from their adjacent free margin in comparison to the NoR group, as indicated by a significantly high number of DEGs - T1 vs TFM (EarlyR-2969, NoR-23), T2 vs TFM (EarlyR-4220 vs NoR-50), and T3 vs TFM (EarlyR-3883 vs NoR-1239). We ruled out that these increases in DEGs were not due to differences in TFMs of the EarlyR and NoR groups (DEGs-17). The pathway analysis revealed enrichment of metastasis-related pathways, including EMT, TNFA signalling via NFκB, and ECM remodelling, in the T1-invasive front of the EarlyR tumor, while the other regions(T2/T3) showed enrichment of proliferation-related pathways, indicating that the invasive front -T1 might be the source of release of aggressive CTCs. We identified SERPINE1, BMP2, CXCL10, CXCL11, ICAM1, IFIH1, IFIT2, IL15, IL15RA, IRF1, JUNB, TGFB1, and TNC as hub genes across multiple pathways enriched in T1, which may contribute to the aggressiveness of this tumor region. Among these genes, IFIT2, IRF1, and JUNB were also upregulated in baseline CTCs of the EarlyR group, indicating the importance of tracking these genes as markers of aggressive CTCs. The PCA analysis of CTC data revealed that, although baseline CTCs are transcriptomically similar between the EarlyR and NoR groups, the post-therapy CTCs are more evolved in the EarlyR group than in the NoR group. We also identified a key molecular marker, FNBP1L, overexpressed in the tumor invasive front (T1) of EarlyR and in their CTCs at pretherapy and posttherapy. However, FNBP1L was downregulated in other regions of the primary tumor (T2/T3), suggesting that aggressive CTCs originate primarily from the invasive front, and FNBP1L (implicated in cytoskeletal reorganisation) might be a key player driving early recurrence and metastasis. Conclusion OSCC tumors from EarlyR patients undergo extensive spatial and temporal transcriptomic evolution, and the invasive front is plausibly involved in the release of aggressive CTCs marked by expression of IFIT2, IRF1, JUNB, and FNBP1L. Citation Format: Geeta S. Boora, Anshika Chauhan, Suvradeep Mitra, Arindam Maitra, Sushmita Ghoshal, Arnab Pal. Spatial gene expression profile of primary tumor is associated with the release of aggressive CTCs from the invasive front, leading to early recurrence in OSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2119.

BACKGROUND AND OBJECTIVE:Serum periostin and carcinoembryonic antigen (CEA) are markers of type 2 inflammation. However, their role in diagnosing and monitoring treatment responses in allergic bronchopulmonary aspergillosis (ABPA) remains uncertain. The objective of the study was to assess the diagnostic performance of serum CEA and periostin in distinguishing ABPA from asthma. We also evaluate their usefulness in monitoring treatment responses. METHODS:We enrolled consecutive subjects with ABPA (cases) and asthmatic patients without ABPA (controls). Serum periostin and CEA levels were measured at baseline and again 2 months after oral prednisolone. We constructed receiver operating characteristic (ROC) curves and determined sensitivity and specificity using the optimal cut-off derived from Youden's index. RESULTS:We enrolled 112 and 108 subjects with ABPA and asthma (median age: 34 years) respectively. At baseline, the median serum periostin (22.03 vs 16.36 ng/mL; P < 0.001) and CEA levels (4.80 vs 2.35 ng/mL; P < 0.001) were significantly higher in the ABPA group than in the controls. CEA (AUROC = 0.77) showed better diagnostic accuracy than serum periostin (AUROC = 0.64) in differentiating asthma from ABPA. After 2 months of treatment, median serum CEA (4.8 ng/mL vs 3.7 ng/mL) and periostin levels (22.03 vs 17.74 ng/mL) declined significantly. However, 34% and 30% of subjects exhibited increased periostin and CEA levels following treatment. CONCLUSION:While serum CEA and periostin exhibit modest diagnostic performance in differentiating ABPA from asthma, they are suboptimal for monitoring treatment responses. Further studies are required to validate our findings.

Oral Squamous Cell Carcinoma (OSCC), one of the most prevalent cancers in developing countries. It is associated with poor prognosis due to relapse in a significant number of patients. Circulating tumour cells (CTCs) are precursors for metastasis and thought to be key players in early relapse in various cancers including OSCC. Though CTC enumeration has been associated with disease outcome, in-depth molecular analysis of CTCs remained minimal as the techniques for CTC isolation and analysis are challenging. While exploring gene expression in CTCs, we performed whole transcriptome analysis of paired primary tumour and CTCs isolated from Oral Squamous Cell Carcinoma (OSCC) patients. Various genes were found to be differentially expressed in CTCs. We found PADI4 gene was significantly upregulated in CTCs. PADI4 gene encodes for an enzyme that converts arginine to citrulline.PADI4 expression in primary tumours was previously observed to be associated with metastasis. Here, we are reporting PADI4 expression for the first time in CTCs and its association with relapse. Interestingly in our data, PADI4 expression was more seen in CTCs with EMT (epithelial to mesenchymal transition)-phenotype than with CTCs only epithelial-phenotype. In conclusion, this is the first study presenting the potential prognostic utility of PADI4 expression in CTCs isolated from OSCC patients.

Background The pathogenesis of pemphigus is multifactorial, and pesticides have been implicated as one of the triggering factors due to their interaction with acetylcholine receptors. Aim The aim of this study was to observe the effect of exposure to pesticides (by blood pesticide levels) on serological markers of pemphigus (anti-Dsg1, anti-Dsg3, and anti-M3 ACh receptor antibodies) amongst healthy individuals involved in pesticide use. Methods This was an observational, cross-sectional pilot study performed in Punjab. The study subjects were 45 couples (husband and wife) wherein the husband was involved in activities like spraying and handling pesticides, and the wife was considered a non-genetically related control (not occupationally exposed to pesticides). Clinical data were collected by a structured questionnaire, and venous blood was collected to test blood pesticide levels. Gas chromatography - mass spectroscopy (GC-MS) and enzyme-linked immunosorbent assay (ELISA) were used for anti-Dsg-1, anti-Dsg-3, and anti-M3Ach receptor antibody level detection. Results Pesticides were detected in both husband and wife. There was no correlation between the duration of pesticide exposure and their blood levels. Chlorpyrifos was most frequently detected in husbands and β- endosulfan in wives. The levels of anti-Dsg-1 and anti-Dsg-3 titres were below the diagnostic cut-off values (20 RU/mL) for pemphigus in all participants. Husbands had significantly higher anti-Dsg3 values (p=0.029). There was no statistically significant association between the presence of pesticides and anti-Dsg-1, anti-Dsg-3, and anti-M3 ACh receptor antibodies in the sera. Anti-Dsg-1 antibodies had a significant positive correlation with chlorpyrifos serum levels (r= 0.3, p=0.005). Limitation Determining aspects like expression of adhesion proteins on keratinocyte cell lines, long term follow up of healthy subjects and correlation with pesticide levels with cases of pemphigus was not done as it is cost intensive. Conclusion The exposure to pesticides did not significantly increase any of the biomarkers above clinical cut-off levels in any of the healthy participants. Considering the rarity of clinically manifest pemphigus, many healthy subjects need to be studied with the current study design. Alternatively, the expression of adhesion proteins or their adhesion function can be assessed in keratinocyte cell lines upon incubation with varying concentrations of pesticides.

In search of salivary biomarkers for Head Neck Squamous Cell Carcinoma (HNSCC), we found Cornulin to be the most downregulated(~10-fold) protein during our previous study, which identified 135 dysregulated proteins in the saliva of the patients. The current study aimed to explore the role(s) of Cornulin in pathophysiology of HNSCC and its translational application. The sandwich ELISA and immunohistochemistry assessed the levels of Cornulin in the saliva and primary tumour tissues, respectively, in a cohort of 128 HNSCC patients. The effects of Cornulin modulation were evaluated in-vitro and in-vivo HNSCC models and associated mechanisms were explored by analysis of transcriptomic and proteomic signatures. Cornulin was significantly downregulated in saliva and tumour tissue, which was also associated with tumour differentiation and poor survival. Cell proliferation, migration, viability, and invasion were decreased in Cornulin overexpressed HNSCC cell lines, which was substantiated using in-vivo model, suggesting the anti-tumour role of Cornulin. We identified the probable mechanism through cell cycle arrest in the G1 phase by upregulating p18 and reducing the free intracellular calcium. In conclusion, we report the multifaceted roles of Cornulin in HNSCC, as a potential prognostic biomarker with anti-tumour properties.

Interrelationship of non-alcoholic fatty liver disease (NAFLD) with Gilbert syndrome (GS, homozygous UGT1A1*28/*28 or TA7/TA7 state) is intriguing as GS’ inherited unconjugated hyperbilirubinemia may protect against oxidative damage to hepatocytes and prevent the development of metabolic syndrome. Only a few prior papers report the frequencies of GS in NAFLD patients. We studied 152 adult NAFLD patients (69.7

Background:Rituximab-mediated B-cell depletion in pemphigus patients is majorly caused by antibody-dependent cellular cytotoxicity. This is mediated by Fcγ receptors on immune cells, particularly FcγRIIIa (CD16) on natural killer (NK) cells. This study investigates the impact of FcγRIII expression on response to rituximab in pemphigus patients. Patients and Methods:A cross-sectional study was carried out on 85 pemphigus patients who had been treated with rituximab using a rheumatoid arthritis protocol. Patients who achieved complete remission within 4.5 months were arbitrarily defined as responders. CD16 expression on NK cells, NK-T cells, and monocytes, as well as FcγRIIIa-158V/F polymorphism, were assessed using flow cytometry and genetic assays, respectively. Results:CD16 expression on NK cells and NK-T cells was higher in responders than in resistant patients but did not reach statistical significance. A significant negative correlation was observed between NK-T cell percentage and time to remission (P < 0.001), suggesting NK-T cell percentage as a possible marker for prediction of time to remission. Distribution of FcγRIIIa genotypes (V/V, V/F, F/F) did not significantly differ between responders and resistant patients (P = 0.574). A statistically non-significant trend suggested that FF homozygotes may have a slightly worse survival outcome in achieving remission compared to FV/VV genotypes, with a hazard ratio of 1.255 (95% CI: 0.6112-2.577). Limitations:Small, single-center sample, potential selection bias, and lack of long-term follow-up or functional assays. Conclusion:This study underscores the potential influence of CD16 expression and FcγRIIIa polymorphism in response to rituximab in pemphigus patients, emphasizing the need for further research to establish definitive relationships for personalized treatment strategies.

The lack of effective stem cell protocols for generating personalized neurovascular niches poses a critical challenge in precision medicine. While iPSC-based methods are explored, their clinical use is hindered by high costs, long timelines, and cancer risks. Recent advancements in plasma-driven differentiation, using circulating monocytes, offer a promising solution as they can be reprogrammed into neuron-like, endothelial-like, and hematopoietic cells without genetic manipulation , by inducing growth factors mediated transdifferentiation. Vasculature is integral to neurodevelopment, with early blood supply transitioning from the perineural to intrinsic vascular plexus, driven by neuro-hematovascular signaling. The choroid plexus selectively transports proteins and growth factors from blood to CSF, supporting neural proliferation and differentiation. Building on these insights, we leveraged the innate reprograming potential of blood-derived cells to generate neuro-hematovascular niches using a novel PITTRep methodology, devoid of transgene and growth factor mediated transdifferentiation opening new avenues for regenerative and investigative neurovascular studies. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES:The study evaluated neonatal cerebro-spinal fluid (CSF) leucocyte esterase (LE) estimation by reagent strips to diagnose CSF leucocytosis in situations where the CSF leucocyte count is delayed or unavailable. METHODS:This prospective diagnostic study had a cohort-selection design. CSF samples of neonates with clinical sepsis were analysed for LE by reagent strips, cytochemistry, gram stain and culture. CSF LE was measured on an ordinal scale from nil to large. Correlations of CSF LE with CSF leucocyte and neutrophil count were determined. CSF LE was compared versus CSF leucocytosis (defined a priori ≥ 25 leucocytes/μL) and versus CSF neutrophilia (defined a priori ≥ 15 neutrophils/μL) as the reference standard. RESULTS:A total of 341 neonates were enrolled. CSF LE values correlated with leucocyte count (rho = 0.49, p < 0.001) and neutrophil count (rho = 0.44, p < 0.001). LE had a sensitivity (95% CI) of 63.5% (51.2, 74.1), specificity 90.7% (88.4, 92.6), LR+ (likelihood ratio) 6.79 (4.43, 9.98) and LR- 0.40 (0.28, 0.55) to diagnose CSF leucocytosis. When expressed as an ordinal variable, LE had an area under the receiver operator characteristic curve of 0.77 (95% CI: 0.69, 0.85; p < 0.001) to detect leucocytosis. With respect to all parameters, the diagnostic performance of CSF LE to detect neutrophilia was inferior to the detection of leucocytosis. CONCLUSIONS:CSF leucocyte esterase estimation by reagent strips has a moderately high specificity and a good LR+ but, overall, does not have sufficiently robust diagnostic accuracy to substitute CSF leucocyte counts.

Background: In the last few years, there has been increasing evidence in the literature for promoter hypermethylation of various host genes following infection with various pathogens. Therefore, in the present study, the role of epigenetic reprogramming of host cells through DNA methylation was evaluated which indicates that the disease progression takes place through changes in host immune response related genes. Objective: To elucidate the differential genome-wide DNA methylation profile of peripheral blood mononuclear cells in pulmonary tuberculosis (PTB) patients. Materials and methods: To decipher the methylation profile of PBMCs in PTB patients whole genome bisulphite sequencing was performed using 4 DNA samples from each study group i.e. PTB, healthy controls and diseased controls. Briefly, the samples were subjected to bisulphite conversion followed by library preparation and whole genome bisulphite sequencing. The data then were analysed for differential methylation analysis followed by gene set enrichment analysis to select DMRs for validation by sanger sequencing. Further the GEOR2 online server was used to investigate the mRNA expression of selected genes associated with hypermethylated DMRs in promoter region. Results: Differential methylation region analysis was performed for the following comparisons. TB v/s Diseased (Hypermethylated=1756; Hypomethylated=1886), TB v/s Healthy( Hypermethylated=2203; Hypomethylated=2466), Diseased v/s Healthy (Hypermethylated=1656; Hypomethylated=3936). Further gene set enrichment analysis showed that the hypermetylated regions belonged to genes that are involved in immune responses mainly T cell functioning and T cell mediated immune processes. There were 8 DMR belonging to TAF8,FZD5, HLA-DRB, MIR483, PVRIG, SH2B2, ZAP70, TNFRSF13C simultaneously (n=10 from each study group). mRNA expression analysis with GEOR2 (Datasets used n= 12) revealed significant downregulation of TNFRSF13C, ZAP70, PVRIG Among the selected genes associated with hypermethylated DMRs in promoter region. Conclusion: The altered differential methylation profile of PBMCs from TB patients shows that on onset the active disease there occurs aberrant methylation of expression regulating regions that in turn results in decreased T cell functions and hence suboptimal immune responses in TB. ### Competing Interest Statement The authors have declared no competing interest.

Oral Squamous Cell Carcinoma (OSCC) often presents with bad prognosis due to high frequency of loco-regional metastasis and relapse. Circulating Tumour Cells (CTCs) are precursors for metastasis, can be identified through liquid biopsy, but their molecular analysis has been challenging due to limitations in current isolation and downstream analysis techniques. To address this, we developed an innovative FACS-based method for CTC isolation and explored the prognostic potential of CTC gene expression in OSCC. We developed an innovative technique that isolates CTCs by depleting CD45+ hematogenous cells and positively selecting CTCs using four markers— EpCAM, EGFR, CK, and Vimentin (Chauhan et al., 2024) using FACS. Next, we performed whole transcriptome analysis of isolated CTCs and paired primary tumours from 24 OSCC patients. Gene expression signatures in CTCs were validated in separate cohort of 60 patients. Data analysis revealed distinct gene expression profiles between CTCs and primary tumours, with 521 differentially expressed genes. Interestingly, PADI4 gene, an enzyme that is involved in the citrullination of various proteins was significantly upregulated in CTCs (p<0.0001). Validation of this finding in the separate patient cohort showed that patients with detectable PADI4 levels in CTCs had significantly poorer disease-free survival (p=0.038, HR=2.571). Interestingly, PADI4 expression was more in CTCs which having EMT signatures. Upon unsupervised hierarchical clustering of CTC transcriptome data, two subclusters of CTCs (namely C1A and C1B) with a total of 372 differentially- expressed between genes between them were found. Patients in CTC cluster C1B had significantly poorer overall survival (p=0.0028) and disease-free survival (p=0.0185), with a higher incidence of loco-regional metastasis (p=0.0082). We also validated topmost upregulated genes in separate cohort of patients and identified that TNFAIP2, NFKBIZ, CTSS and PTGS2 are significantly associated with increased probability of cancer relapse. Multiple logistic regression modelling using these genes identified that cumulative expression of baseline TNFAIP2, NFKBIZ, CTSS and PTGS2 expression can successfully predict recurrence in our cases. The developed model was validated using calibration plot analysis. Our study demonstrates that the gene expression profiles of CTCs can serve as prognostic indicators in OSCC and we successfully developed a four-gene panel model for predicting relapse in OSCC patients. Anshika Chauhan, Geeta S. Boora, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Gene expression profiling of circulating tumor cells provides prognostic insights in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1934.