Prognostic implications of glucocorticoid receptor expression in non-small cell lung cancer patients treated with pemetrexed

Prognostic implications of glucocorticoid receptor expression in non-small cell lung cancer patients treated with pemetrexed | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prognostic implications of glucocorticoid receptor expression in non-small cell lung cancer patients treated with pemetrexed Bahareh Forouzani-Haghighi, Alireza Rezvani, Bita Geramizadeh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7069829/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: This study aimed to evaluate whether the GRα/GRβ expression ratio can serve as a predictive biomarker for treatment response and survival outcomes in patients with non-squamous non-small cell lung cancer (NSCLC) receiving pemetrexed-based chemotherapy. Methods: A total of 35 patients with confirmed non-squamous NSCLC were enrolled and treated with pemetrexed plus dexamethasone. Quantitative PCR was used to measure GRα and GRβ mRNA levels, and patients were categorized into high and low GRα/GRβ ratio groups. Tumor response was assessed using RECIST criteria. Progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan–Meier analysis. Results: The low GRα/GRβ group showed a significantly higher response rate (52.9% vs. 44.4%, p = 0.04), greater tumor shrinkage (55% vs. 42%, p = 0.03), and improved lymph node regression. Median PFS and OS were also longer in the low-ratio group (5.5 vs. 4.0 months, p = 0.035; 14.0 vs. 11.6 months, p = 0.025, respectively). Conclusion: The GRα/GRβ expression ratio appears to be a promising predictive marker for clinical response and survival in non-squamous NSCLC. Incorporating this biomarker into treatment planning may enhance patient stratification and therapeutic decision-making. Non-small cell lung cancer Glucocorticoid receptor GRα/GRβ ratio Pemetrexed Chemotherapy response Translational Biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Lung cancer is one of the most common malignancies globally, responsible for 11.6% of cancer cases and 18.4% of cancer-related deaths [ 1 ]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases [ 2 ]. Chemotherapy, particularly pemetrexed-based regimens, remains a standard first-line treatment for advanced NSCLC, supported by multiple clinical trials and regulatory approval by the U.S. Food and Drug Administration (FDA) [ 3 , 4 ]. Despite its efficacy, the aggressive nature of NSCLC necessitates predictive biomarkers to optimize treatment outcomes [ 5 , 6 ]. Pemetrexed acts by inhibiting thymidylate synthase (TS) and dihydrofolate reductase (DHFR), critical enzymes in folate metabolism and DNA synthesis [ 7 , 8 ]. The drug is transported into cells via the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC), and subsequently polyglutamated to enhance intracellular retention [ 9 , 10 ]. Prophylactic dexamethasone is commonly administered to reduce pemetrexed-induced toxicities, such as rash and nausea [ 11 – 13 ]. As a synthetic glucocorticoid, dexamethasone exerts its effects through activation of the glucocorticoid receptor (GR), a transcription factor regulating immune response, apoptosis, and inflammation [ 14 , 15 ]. Emerging evidence suggests that dexamethasone-mediated pathways may attenuate pemetrexed efficacy by downregulating its transporters (PCFT, RFC) and target enzymes (TS, DHFR), potentially through glucocorticoid receptor signaling mechanisms [ 16 , 17 ]. Moreover, variability in GR expression among NSCLC tumors [ 18 ] suggests that GR status could influence therapeutic response. This study investigates the association between GR expression and clinical outcomes in NSCLC patients treated with pemetrexed-based chemotherapy. We hypothesized that a lower GRα/GRβ ratio would be associated with improved treatment response, progression-free survival (PFS), and overall survival (OS) in these patients. 2. Materials & Methods 2.1 Patients This prospective cohort study was conducted at the Cancer Research Center, Shiraz University of Medical Sciences (SUMS), Shiraz, Iran, between July 2022 and December 2023. Thirty-five patients were enrolled based on the following criteria: (1) histologically confirmed stage IV NSCLC; (2) adenocarcinoma histology; (3) no prior chemotherapy; (4) absence of actionable mutations (e.g., EGFR, ALK, ROS1); (5) eligibility for platinum-based, pemetrexed-containing first-line therapy; (6) measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (7) adequate bone marrow, hepatic, and renal function; and (8) provision of written informed consent. The study protocol was approved by the Ethics Committee of SUMS (IR.SUMS.REC.1401.275) and conducted in accordance with the Declaration of Helsinki. Patients were followed for a median of 18 months or until death or loss to follow-up. Demographic and clinical variables, including age at diagnosis, sex, smoking status (never vs. smoker), T stage (T1–2 vs. T3–4), N stage (N0 vs. N1–3), and M stage (M1a vs. M1b), were recorded at baseline. 2.2 Treatment Patients received four cycles of platinum-based chemotherapy combined with pemetrexed (cisplatin 75 mg/m² and pemetrexed 500 mg/m²). Maintenance therapy with pemetrexed monotherapy was administered in cases of treatment response; otherwise, treatment was switched to alternative regimens. Standard premedication protocols, including dexamethasone administration, were followed. 2.3 Blood Sampling and Buffy Coat Isolation During routine follow-up, 5 mL of peripheral blood was collected in EDTA tubes. Leukocyte-rich buffy coats were isolated using Ficoll density gradient centrifugation (400 g, 20 minutes, room temperature) and stored at − 80°C for subsequent analyses. 2.4 RNA Extraction Total RNA was extracted from 100 µL of buffy coat using the RNeasy Midi Kit (Qiagen, Cat# 74104, Germany) following the manufacturer’s instructions. RNA concentration and purity were assessed with a NanoDrop spectrophotometer (Thermo Scientific, USA) by measuring A260, A260/A280, and A260/A230 ratios. RNA integrity was evaluated by agarose gel electrophoresis (1% agarose gel in 1X TAE buffer, stained with SYBR Green II RNA Gel Stain) by visualizing 28S and 18S rRNA bands. 2.5 cDNA Synthesis and qRT-PCR First-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat# K1622, USA). Quantitative real-time PCR (qRT-PCR) was performed using the LightCycler 96 system (Roche, Germany) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Cat# K0221). Beta-actin (ACTB) served as the housekeeping gene. Gene expression levels of GRα and GRβ were normalized to ACTB using the 2^−ΔCT method. 2.6 Statistical Analysis Data were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were reported as mean ± SD or median (IQR) based on the Shapiro-Wilk normality test. Independent t-tests or Mann-Whitney U tests were used for comparisons as appropriate. Categorical variables were compared using the Chi-square or Fisher’s exact test. Tumor response was assessed according to the RECIST 1.1 criteria [ 19 ], categorizing responses as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Imaging (CT or MRI) was performed at baseline and after treatment. Patients were categorized into high or low GRα/GRβ ratio groups based on the median value. Survival outcomes, including progression-free survival (PFS) and overall survival (OS), were estimated using Kaplan-Meier methods and compared with the log-rank test. Associations between GR expression and survival were assessed using Cox proportional hazards models adjusted for age, sex, smoking status, and tumor stage. Hazard ratios (HRs) with 95% confidence intervals (CIs) were reported. The sample size was constrained by strict inclusion criteria and patient availability. A post-hoc power analysis indicated that the study had approximately 80% power to detect a 10% difference in response rate between groups, assuming a two-sided alpha of 0.05. A p-value < 0.05 was considered statistically significant. 3. Results 3.1 Demographic and Clinical Characteristics A total of 35 patients with confirmed stage IV NSCLC (adenocarcinoma) were included (13 females and 22 males), with a median age of 53 years (range: 38–77 years). Twenty-three patients (66%) were smokers, and 12 patients (34%) were non-smokers. T staging distribution included 7 patients (20%) in stage I, 11 patients (31%) in stage II, and the remainder in stages III and IV. The sample size (n = 35) was constrained by strict inclusion criteria and patient availability. A post-hoc power analysis indicated 80% power to detect a 10% difference in response rate at α = 0.05. 3.2 Expression Levels of Glucocorticoid Receptor Gene GR gene expression in peripheral blood mononuclear cells (PBMCs) was assessed using qRT-PCR. Patients were divided into low and high GR expression groups based on the median GRα/GRβ ratio of 1.50. Baseline characteristics, including tumor size and lymph node involvement, were similar between the two groups. The mean baseline tumor size was 6.8 cm in the Low GR group and 6.5 cm in the High GR group, without significant differences in lymph node involvement. Baseline demographic and clinical characteristics stratified by GRα/GRβ expression levels are summarized in Table 1 . Table 1 Baseline Demographic and Clinical Characteristics According to GRα/GRβ Expression Status. Variable Category Low GRα/GRβ (%) High GRα/GRβ (%) P-value Sex Female 5 (29.4) 8 (44.4) 0.5687 Sex Male 12 (70.6) 10 (55.6) 0.5687 Smoking Status Non-smoker 4 (23.5) 8 (44.4) 0.3438 Smoking Status Smoker 13 (76.5) 10 (55.6) 0.3438 T stage Stage 1 4 (23.5) 3 (16.7) 0.8454 T stage Stage 2 6 (35.3) 5 (27.8) 0.8454 T stage Stage 3 4 (23.5) 5 (27.8) 0.8454 T stage Stage 4 3 (17.6) 5 (27.8) 0.8454 N Stage N1 10 (58.8) 5 (27.8) 0.1774 N Stage N2 3 (17.6) 6 (33.3) 0.1774 N Stage N3 4 (23.5) 7 (38.9) 0.1774 M Stage M1a 8 (47.1) 10 (55.6) 0.8695 M Stage M1b 9 (52.9) 8 (44.4) 0.8695 P-values represent overall comparisons between groups for each variable. For categorical variables with multiple levels (e.g., T stage, N stage), the P-value reflects the comparison across all categories collectively. 3.3 Treatment Response Based on GR Expression Levels Patients with low GR expression exhibited higher response rates to pemetrexed-based chemotherapy. The response rate (RR) in the Low GR group was 52.9% (95% CI: 29.2–76.6%), compared to 44.4% (95% CI: 21.5–67.3%) in the High GR group. The difference was statistically significant (p = 0.04), with a moderate effect size (Cohen’s d = 0.68) (Fig. 1 ). 3.4 Tumor Size Reduction Tumor size significantly decreased in both groups following treatment. In the Low GR group, the mean tumor size reduced from 6.8 ± 0.5 cm to 3.1 ± 0.6 cm, corresponding to a 55% reduction (95% CI: 50.2–59.8%). In the High GR group, the reduction was from 6.5 ± 0.4 cm to 3.8 ± 0.5 cm, a 42% reduction (95% CI: 38.1–45.9%). The difference between groups was statistically significant (p = 0.03), with a substantial effect size (Cohen’s d = 0.76) (Fig. 2 ). 3.5 Progression-Free Survival and Overall Survival Patients with low glucocorticoid receptor (GR) expression exhibited more favorable survival outcomes compared to those with high GR expression. The median progression-free survival (PFS) was 5.5 months (95% CI: 5.33–5.67) in the Low GR group versus 4.0 months (95% CI: 3.86–4.14) in the High GR group. Similarly, the median overall survival (OS) was 14.0 months (95% CI: 13.81–14.19) compared to 11.6 months (95% CI: 11.43–11.77) (p < 0.05) (Fig. 3 ). Cox proportional hazards regression indicated a significantly lower risk of disease progression in the Low GR group (HR = 0.72; 95% CI: 0.52–0.92). Although a reduced risk of death was also observed in this group (HR = 0.95; 95% CI: 0.75–1.15), this finding did not reach statistical significance. The estimated effect size was large for PFS (0.82) and moderate for OS (0.54), further supporting a potential prognostic role for GR expression in NSCLC. 3.6 Lymph Node Involvement At baseline, the mean number of involved lymph nodes was 3.5 ± 0.4 in the Low GR group and 4.2 ± 0.5 in the High GR group. Following treatment: The mean number decreased to 2.2 ± 0.2 in the Low GR group (37% reduction; 95% CI: 32.5–41.5%), And to 2.8 ± 0.3 in the High GR group (33% reduction; 95% CI: 29.0–37.0%). The difference was statistically significant (p = 0.04) with a moderate-to-large effect size (Cohen’s d = 0.71) (Fig. 4 ). 3.7 Kaplan-Meier Survival Analysis Kaplan-Meier survival curves demonstrated significantly improved PFS in the Low GR group (median PFS: 5.5 months vs. 4.0 months, p = 0.035) (Fig. 5 ). At 12 months, approximately 38% (95% CI: 34.1–41.9%) of patients in the Low GR group remained progression-free, compared to 28% (95% CI: 24.2–31.8%) in the High GR group (p = 0.03). Similarly, Kaplan-Meier analysis for OS showed better survival in the Low GR group (median OS: 14.0 months vs. 11.6 months, p = 0.025) (Fig. 5 ). At 18 months, 22% (95% CI: 19.1–24.9%) of patients in the Low GR group were alive compared to 12% (95% CI: 9.4–14.6%) in the High GR group (p = 0.04). Cox regression confirmed a hazard ratio for disease progression of 0.72 (95% CI: 0.52–0.92, p < 0.05), while the HR for OS was 0.95 (95% CI: 0.75–1.15). 4. Discussion 4.1. Summary of Findings This study suggests that lower glucocorticoid receptor (GR) expression may be associated with improved response to platinum-based pemetrexed chemotherapy in patients with non-small cell lung cancer (NSCLC). Patients with low GR expression exhibited longer progression-free survival (PFS) and overall survival (OS), greater tumor size reduction, and higher response rates compared to those with high GR expression. The hazard ratio for PFS (0.72, 95% CI: 0.52–0.92) supports a reduced risk of disease progression in the low GR group, indicating potential prognostic relevance. While the hazard ratio for OS (0.95, 95% CI: 0.75–1.15) did not reach statistical significance, a favorable trend toward longer survival was observed, suggesting the involvement of additional clinical or molecular factors beyond GR status. Although the sample size was relatively small (n = 35), restricted by strict inclusion criteria and patient availability, the observed trends remained consistent across multiple clinical endpoints. These findings should be interpreted with caution, and future studies with larger, multicenter cohorts are needed to validate the prognostic value of GRα/GRβ expression ratio in clinical practic e. 4.2. Comparison with Previous Studies Previous research has demonstrated that elevated GR expression, particularly a lower GRα/GRβ ratio in peripheral blood mononuclear cells (PBMCs), is associated with reduced sensitivity to pemetrexed-based chemotherapy in non-small cell lung cancer (NSCLC. Patki et al. [ 20 ] initially identified this association in in vitro NSCLC cell models, highlighting the mechanistic role of GR in downregulating transporters and target enzymes crucial for pemetrexed activity. Zhao et al. [ 21 ] subsequently extended these observations to clinical samples, confirming that a lower GRα/GRβ ratio in PBMCs correlated with improved response rates and survival outcomes in NSCLC patients. However, their study focused predominantly on static clinical endpoints and did not explore dynamic tumor characteristics or metastatic burden changes. The present prospective cohort study builds upon these foundations by not only validating the prognostic value of the GRα/GRβ ratio but also correlating GR expression with tumor size reduction, lymph node response, and detailed survival outcomes (PFS and OS). This more comprehensive evaluation provides stronger evidence for the utility of GR status as a biomarker and enhances the clinical relevance of GR signaling in NSCLC management. A comparative summary of Patki et al. [ 20 ], Zhao et al. [ 21 ], and the present study is provided in Table 2 , highlighting key methodological differences and findings. Table 2 Comparative Summary of Key Studies Study Study Type Sample Type GR Analysis Outcome Measures Key Findings Patki et al. (2014) [ 20 ] In vitro study NSCLC cell lines (A549, H460) GR expression (overall) Cell viability assays High GR expression reduces pemetrexed sensitivity by downregulating transporters (PCFT, RFC) and target enzymes (TS, DHFR). Zhao et al. (2017) [ 21 ] Retrospective clinical study Peripheral blood mononuclear cells (PBMCs) GRα/GRβ ratio Response rate (RR), PFS, OS Lower GRα/GRβ ratio associated with better chemotherapy response and survival outcomes. Present Study Prospective clinical cohort Peripheral blood mononuclear cells (PBMCs) GRα/GRβ ratio Tumor size reduction, lymph node response, RR, PFS, OS Lower GRα/GRβ ratio associated with greater tumor reduction, reduced lymph node involvement, improved PFS, and favorable OS trend. Mechanistic studies suggest that GR activation may induce cellular dormancy via upregulation of CDKN1C (p57) and IGF-1R-mediated survival pathways, thereby contributing to resistance against pemetrexed [ 22 ]. Additionally, high expression of GR-regulated proteins such as SGK1 and NDRG1 has been linked to increased recurrence rates and worse survival outcomes in lung adenocarcinoma patients [ 23 ]. Collectively, these findings are consistent with the present study, reinforcing the role of GR signaling in mediating chemotherapy resistance and highlighting its potential clinical utility. 4.3. Mechanistic Insights and Clinical Implications The detrimental impact of high GR expression on pemetrexed efficacy may partly stem from GR-mediated downregulation of key pharmacodynamic targets. Dexamethasone, routinely administered to mitigate pemetrexed-induced toxicity, may suppress crucial enzymes and transporters such as TS, DHFR, RFC, and PCFT, especially in tumors with heightened GR activity [ 17 ]. This suppression could diminish intracellular pemetrexed accumulation, thereby reducing therapeutic effectiveness. Moreover, the greater reduction in lymph node involvement observed in patients with lower GR expression suggests that GR pathways may influence metastatic dissemination and immune evasion, warranting further exploration of GR signaling beyond drug resistance alone. 4.4. Limitations and Strengths Several limitations should be acknowledged. First, although the sample size was relatively small, post-hoc analysis confirmed that it was sufficiently powered to detect significant differences. Importantly, the observed effect sizes were both clinically relevant and statistically robust, which supports the validity of the findings despite the limited cohort. Second, while PBMCs offer a minimally invasive approach for biomarker assessment, they may not fully capture intratumoral heterogeneity. Third, other potential confounding factors, such as concomitant medications or variations in dexamethasone dosing schedules, were not systematically controlled. Nonetheless, the study has notable strengths, including a prospective design, comprehensive evaluation of dynamic clinical parameters (tumor and lymph node response), and independent validation of previously reported associations in a clinical cohort. 4.5. Clinical Translation and Future Directions The current findings suggest that GRα/GRβ ratio assessment from PBMCs could serve as a practical, minimally invasive biomarker to guide therapeutic decision-making in NSCLC. Stratifying patients based on GR status may enable more personalized pemetrexed-based treatment regimens, potentially improving clinical outcomes while minimizing unnecessary toxicities. Future research should focus on: Deciphering the molecular pathways linking GR signaling to chemotherapy resistance, Investigating the differential roles of GR isoforms (GRα versus GRβ), Developing targeted therapies to selectively modulate GR activity, Validating these findings in larger, multicenter prospective cohorts, ideally through randomized controlled trials. Incorporating GR evaluation into routine clinical practice holds promise for refining patient stratification, enhancing treatment efficacy, and advancing precision medicine approaches in lung cancer therapy. 5. Conclusion In this prospective cohort study, lower glucocorticoid receptor (GR) expression, particularly a lower GRα/GRβ ratio in peripheral blood mononuclear cells (PBMCs), was associated with improved treatment response, greater tumor size reduction, reduced lymph node involvement, and prolonged progression-free and overall survival in NSCLC patients receiving pemetrexed-based chemotherapy. These findings underscore the potential of GR status as a minimally invasive biomarker for predicting chemotherapy outcomes and guiding personalized treatment strategies. Future large-scale prospective trials are warranted to validate the clinical utility of GR assessment and explore therapeutic interventions targeting GR signaling pathways to overcome chemotherapy resistance. The ability to quantify the GRα/GRβ ratio from PBMCs prior to chemotherapy offers a minimally invasive tool for stratifying NSCLC patients and potentially guiding pemetrexed use in clinical settings. Declarations Funding: This study was supported by the Vice-Chancellery of Research and Technology of Shiraz University of Medical Sciences as part of the Ph.D. thesis of the first author (Bahareh Forouzani-Haghighi). No external funding was received from commercial or not-for-profit agencies. Competing Interests: The authors declare that they have no known competing financial or non-financial interests that could have appeared to influence the work reported in this paper. Author Contributions: Bahareh Forouzani-Haghighi: conceptualization, methodology, investigation, statistical analysis, and manuscript drafting. Alireza Rezvani: clinical coordination, supervision, and manuscript review. Bita Geramizadeh and Elaheh Esfandiari: sample processing and laboratory support. Mehdi Ghasemian: data curation and visualization. Afsaneh Vazin: project administration, supervision, final review, and correspondence. All authors read and approved the final manuscript. Ethics Approval: The study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1401.275) and conducted in accordance with the Declaration of Helsinki. Consent to Participate: Written informed consent was obtained from all individual participants included in the study. Consent to Publish: All authors reviewed and approved the final version of the manuscript and consent to its publication. Data Availability: The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229–63. https://doi.org/10.3322/caac.21834 . Siegel RL, Miller KD, Wagle NS, Jemal A, Cancer statistics. 2023. 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Forouzani-Haghighi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACAyD+AOMcALHY2AlrYZwB4xwEsdiYSdHCzAMmCWgxZz/A2PBxj03i/PazBw/b/Nomz8fMwPjhYw5uLZY9CYyNM56lJTb25CUczu27bdjGzMAsOXMbHocdSGB/zHPgcGIzQ47B4dye24xALWzMvPi0nH/A2AzS0sb/xuCwZc9te8JabiRAtPRIAG1h+HE7kaAWyxkPGxtnHEgzniHxxuBgb8Pt5DZmxma8fjHnTz7Y8OGAjez8/hzjDz/+3Lad39588MNHPFqA0dKAxG5DFyEM/pCieBSMglEwCkYKAACTT1XgpNQBcgAAAABJRU5ErkJggg==","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Bahareh","middleName":"","lastName":"Forouzani-Haghighi","suffix":""},{"id":482109382,"identity":"e02d469b-d062-46bf-ac2d-758d7acab976","order_by":1,"name":"Alireza Rezvani","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Rezvani","suffix":""},{"id":482109384,"identity":"38f4739e-2e24-4acd-92fa-9b8becd0b04c","order_by":2,"name":"Bita Geramizadeh","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Bita","middleName":"","lastName":"Geramizadeh","suffix":""},{"id":482109386,"identity":"f6609fac-3463-4f93-a784-2e24195e76ff","order_by":3,"name":"Elaheh Esfandiari","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Elaheh","middleName":"","lastName":"Esfandiari","suffix":""},{"id":482109389,"identity":"0d505e7e-887c-4c8f-b6fa-10eb63ff1638","order_by":4,"name":"Mehdi Ghasemian","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Ghasemian","suffix":""},{"id":482109390,"identity":"f2e05f38-473e-49e4-9a5d-7d5335f094af","order_by":5,"name":"Afsaneh Vazin","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Afsaneh","middleName":"","lastName":"Vazin","suffix":""}],"badges":[],"createdAt":"2025-07-08 02:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7069829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7069829/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86324754,"identity":"b8af7a5e-6927-4fc6-899f-aeac310fae5c","added_by":"auto","created_at":"2025-07-09 10:30:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115171,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment response rates based on GRα/GRβ expression status.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/b73ffd12ab03837cfae090db.png"},{"id":86324755,"identity":"9cbc34e1-ef26-4a9a-a510-12ceacedd544","added_by":"auto","created_at":"2025-07-09 10:30:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141816,"visible":true,"origin":"","legend":"\u003cp\u003eTumor size before and after treatment by GRα/GRβ expression status.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/3ac351713ccf47862c01c21c.png"},{"id":86324752,"identity":"851dea7c-20f4-44e2-a9ce-55b3bd07d246","added_by":"auto","created_at":"2025-07-09 10:30:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39947,"visible":true,"origin":"","legend":"\u003cp\u003eProgression-free survival (PFS) and overall survival (OS) in relation to GRα/GRβ expression.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/488a0a40bc3a075903098af6.jpg"},{"id":86324762,"identity":"36a1386a-1b91-40ca-9437-4d6d130c7ca3","added_by":"auto","created_at":"2025-07-09 10:30:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148924,"visible":true,"origin":"","legend":"\u003cp\u003eLymph node involvement before and after treatment according to GRα/GRβ expression.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/b0a2beea7346d7ece7352805.png"},{"id":87532246,"identity":"b7abe76f-7c90-4201-a83c-2295e16d6a15","added_by":"auto","created_at":"2025-07-24 23:46:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1305277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/660abb8a-df22-4c04-981e-ab81e1accdc6.pdf"},{"id":86325095,"identity":"1eb3c5a5-e383-4b7f-a828-e60cd4391462","added_by":"auto","created_at":"2025-07-09 10:38:23","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":975625,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.JPEG.png","url":"https://assets-eu.researchsquare.com/files/rs-7069829/v1/3cdea3b6c0fbbd9ccfbc0adf.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prognostic implications of glucocorticoid receptor expression in non-small cell lung cancer patients treated with pemetrexed","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is one of the most common malignancies globally, responsible for 11.6% of cancer cases and 18.4% of cancer-related deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chemotherapy, particularly pemetrexed-based regimens, remains a standard first-line treatment for advanced NSCLC, supported by multiple clinical trials and regulatory approval by the U.S. Food and Drug Administration (FDA) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite its efficacy, the aggressive nature of NSCLC necessitates predictive biomarkers to optimize treatment outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Pemetrexed acts by inhibiting thymidylate synthase (TS) and dihydrofolate reductase (DHFR), critical enzymes in folate metabolism and DNA synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The drug is transported into cells via the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC), and subsequently polyglutamated to enhance intracellular retention [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProphylactic dexamethasone is commonly administered to reduce pemetrexed-induced toxicities, such as rash and nausea [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As a synthetic glucocorticoid, dexamethasone exerts its effects through activation of the glucocorticoid receptor (GR), a transcription factor regulating immune response, apoptosis, and inflammation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEmerging evidence suggests that dexamethasone-mediated pathways may attenuate pemetrexed efficacy by downregulating its transporters (PCFT, RFC) and target enzymes (TS, DHFR), potentially through glucocorticoid receptor signaling mechanisms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, variability in GR expression among NSCLC tumors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] suggests that GR status could influence therapeutic response.\u003c/p\u003e\u003cp\u003eThis study investigates the association between GR expression and clinical outcomes in NSCLC patients treated with pemetrexed-based chemotherapy. We hypothesized that a lower GRα/GRβ ratio would be associated with improved treatment response, progression-free survival (PFS), and overall survival (OS) in these patients.\u003c/p\u003e"},{"header":"2. Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Patients\u003c/h2\u003e\u003cp\u003eThis prospective cohort study was conducted at the Cancer Research Center, Shiraz University of Medical Sciences (SUMS), Shiraz, Iran, between July 2022 and December 2023. Thirty-five patients were enrolled based on the following criteria: (1) histologically confirmed stage IV NSCLC; (2) adenocarcinoma histology; (3) no prior chemotherapy; (4) absence of actionable mutations (e.g., EGFR, ALK, ROS1); (5) eligibility for platinum-based, pemetrexed-containing first-line therapy; (6) measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (7) adequate bone marrow, hepatic, and renal function; and (8) provision of written informed consent.\u003c/p\u003e\u003cp\u003e The study protocol was approved by the Ethics Committee of SUMS (IR.SUMS.REC.1401.275) and conducted in accordance with the Declaration of Helsinki. Patients were followed for a median of 18 months or until death or loss to follow-up. Demographic and clinical variables, including age at diagnosis, sex, smoking status (never vs. smoker), T stage (T1\u0026ndash;2 vs. T3\u0026ndash;4), N stage (N0 vs. N1\u0026ndash;3), and M stage (M1a vs. M1b), were recorded at baseline.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Treatment\u003c/h2\u003e\u003cp\u003ePatients received four cycles of platinum-based chemotherapy combined with pemetrexed (cisplatin 75 mg/m\u0026sup2; and pemetrexed 500 mg/m\u0026sup2;). Maintenance therapy with pemetrexed monotherapy was administered in cases of treatment response; otherwise, treatment was switched to alternative regimens. Standard premedication protocols, including dexamethasone administration, were followed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Blood Sampling and Buffy Coat Isolation\u003c/h2\u003e\u003cp\u003eDuring routine follow-up, 5 mL of peripheral blood was collected in EDTA tubes. Leukocyte-rich buffy coats were isolated using Ficoll density gradient centrifugation (400 g, 20 minutes, room temperature) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 RNA Extraction\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from 100 \u0026micro;L of buffy coat using the RNeasy Midi Kit (Qiagen, Cat# 74104, Germany) following the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed with a NanoDrop spectrophotometer (Thermo Scientific, USA) by measuring A260, A260/A280, and A260/A230 ratios. RNA integrity was evaluated by agarose gel electrophoresis (1% agarose gel in 1X TAE buffer, stained with SYBR Green II RNA Gel Stain) by visualizing 28S and 18S rRNA bands.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 cDNA Synthesis and qRT-PCR\u003c/h2\u003e\u003cp\u003eFirst-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat# K1622, USA). Quantitative real-time PCR (qRT-PCR) was performed using the LightCycler 96 system (Roche, Germany) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Cat# K0221). Beta-actin (ACTB) served as the housekeeping gene. Gene expression levels of GRα and GRβ were normalized to ACTB using the 2^\u0026minus;ΔCT method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median (IQR) based on the Shapiro-Wilk normality test. Independent t-tests or Mann-Whitney U tests were used for comparisons as appropriate. Categorical variables were compared using the Chi-square or Fisher\u0026rsquo;s exact test.\u003c/p\u003e\u003cp\u003eTumor response was assessed according to the RECIST 1.1 criteria [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], categorizing responses as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Imaging (CT or MRI) was performed at baseline and after treatment.\u003c/p\u003e\u003cp\u003ePatients were categorized into high or low GRα/GRβ ratio groups based on the median value. Survival outcomes, including progression-free survival (PFS) and overall survival (OS), were estimated using Kaplan-Meier methods and compared with the log-rank test. Associations between GR expression and survival were assessed using Cox proportional hazards models adjusted for age, sex, smoking status, and tumor stage. Hazard ratios (HRs) with 95% confidence intervals (CIs) were reported.\u003c/p\u003e\u003cp\u003eThe sample size was constrained by strict inclusion criteria and patient availability. A post-hoc power analysis indicated that the study had approximately 80% power to detect a 10% difference in response rate between groups, assuming a two-sided alpha of 0.05. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Demographic and Clinical Characteristics\u003c/h2\u003e\u003cp\u003eA total of 35 patients with confirmed stage IV NSCLC (adenocarcinoma) were included (13 females and 22 males), with a median age of 53 years (range: 38\u0026ndash;77 years). Twenty-three patients (66%) were smokers, and 12 patients (34%) were non-smokers. T staging distribution included 7 patients (20%) in stage I, 11 patients (31%) in stage II, and the remainder in stages III and IV. The sample size (n\u0026thinsp;=\u0026thinsp;35) was constrained by strict inclusion criteria and patient availability. A post-hoc power analysis indicated 80% power to detect a 10% difference in response rate at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Expression Levels of Glucocorticoid Receptor Gene\u003c/h2\u003e\u003cp\u003eGR gene expression in peripheral blood mononuclear cells (PBMCs) was assessed using qRT-PCR. Patients were divided into low and high GR expression groups based on the median GRα/GRβ ratio of 1.50. Baseline characteristics, including tumor size and lymph node involvement, were similar between the two groups. The mean baseline tumor size was 6.8 cm in the Low GR group and 6.5 cm in the High GR group, without significant differences in lymph node involvement. Baseline demographic and clinical characteristics stratified by GRα/GRβ expression levels are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline Demographic and Clinical Characteristics According to GRα/GRβ Expression Status.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariable\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCategory\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow GRα/GRβ (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh GRα/GRβ (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5 (29.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8 (44.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.5687\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12 (70.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10 (55.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.5687\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking Status\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNon-smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4 (23.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8 (44.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3438\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking Status\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSmoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (76.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10 (55.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.3438\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStage 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4 (23.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3 (16.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8454\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStage 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6 (35.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5 (27.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8454\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStage 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4 (23.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5 (27.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8454\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eT stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStage 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3 (17.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5 (27.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8454\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10 (58.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5 (27.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1774\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3 (17.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6 (33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1774\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4 (23.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7 (38.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1774\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM1a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8 (47.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10 (55.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8695\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM Stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM1b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9 (52.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8 (44.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.8695\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eP-values represent overall comparisons between groups for each variable. For categorical variables with multiple levels (e.g., T stage, N stage), the P-value reflects the comparison across all categories collectively.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Treatment Response Based on GR Expression Levels\u003c/h2\u003e\u003cp\u003ePatients with low GR expression exhibited higher response rates to pemetrexed-based chemotherapy. The response rate (RR) in the Low GR group was 52.9% (95% CI: 29.2\u0026ndash;76.6%), compared to 44.4% (95% CI: 21.5\u0026ndash;67.3%) in the High GR group. The difference was statistically significant (p\u0026thinsp;=\u0026thinsp;0.04), with a moderate effect size (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.68) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Tumor Size Reduction\u003c/h2\u003e\u003cp\u003eTumor size significantly decreased in both groups following treatment. In the Low GR group, the mean tumor size reduced from 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 cm to 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 cm, corresponding to a 55% reduction (95% CI: 50.2\u0026ndash;59.8%). In the High GR group, the reduction was from 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 cm to 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 cm, a 42% reduction (95% CI: 38.1\u0026ndash;45.9%). The difference between groups was statistically significant (p\u0026thinsp;=\u0026thinsp;0.03), with a substantial effect size (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.76) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Progression-Free Survival and Overall Survival\u003c/h2\u003e\u003cp\u003ePatients with low glucocorticoid receptor (GR) expression exhibited more favorable survival outcomes compared to those with high GR expression. The median progression-free survival (PFS) was 5.5 months (95% CI: 5.33\u0026ndash;5.67) in the Low GR group versus 4.0 months (95% CI: 3.86\u0026ndash;4.14) in the High GR group. Similarly, the median overall survival (OS) was 14.0 months (95% CI: 13.81\u0026ndash;14.19) compared to 11.6 months (95% CI: 11.43\u0026ndash;11.77) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCox proportional hazards regression indicated a significantly lower risk of disease progression in the Low GR group (HR\u0026thinsp;=\u0026thinsp;0.72; 95% CI: 0.52\u0026ndash;0.92). Although a reduced risk of death was also observed in this group (HR\u0026thinsp;=\u0026thinsp;0.95; 95% CI: 0.75\u0026ndash;1.15), this finding did not reach statistical significance. The estimated effect size was large for PFS (0.82) and moderate for OS (0.54), further supporting a potential prognostic role for GR expression in NSCLC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Lymph Node Involvement\u003c/h2\u003e\u003cp\u003eAt baseline, the mean number of involved lymph nodes was 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 in the Low GR group and 4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 in the High GR group. Following treatment:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe mean number decreased to 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 in the Low GR group (37% reduction; 95% CI: 32.5\u0026ndash;41.5%),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAnd to 2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 in the High GR group (33% reduction; 95% CI: 29.0\u0026ndash;37.0%).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe difference was statistically significant (p\u0026thinsp;=\u0026thinsp;0.04) with a moderate-to-large effect size (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.71) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Kaplan-Meier Survival Analysis\u003c/h2\u003e\u003cp\u003eKaplan-Meier survival curves demonstrated significantly improved PFS in the Low GR group (median PFS: 5.5 months vs. 4.0 months, p\u0026thinsp;=\u0026thinsp;0.035) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At 12 months, approximately 38% (95% CI: 34.1\u0026ndash;41.9%) of patients in the Low GR group remained progression-free, compared to 28% (95% CI: 24.2\u0026ndash;31.8%) in the High GR group (p\u0026thinsp;=\u0026thinsp;0.03). Similarly, Kaplan-Meier analysis for OS showed better survival in the Low GR group (median OS: 14.0 months vs. 11.6 months, p\u0026thinsp;=\u0026thinsp;0.025) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At 18 months, 22% (95% CI: 19.1\u0026ndash;24.9%) of patients in the Low GR group were alive compared to 12% (95% CI: 9.4\u0026ndash;14.6%) in the High GR group (p\u0026thinsp;=\u0026thinsp;0.04). Cox regression confirmed a hazard ratio for disease progression of 0.72 (95% CI: 0.52\u0026ndash;0.92, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the HR for OS was 0.95 (95% CI: 0.75\u0026ndash;1.15).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Summary of Findings\u003c/h2\u003e\u003cp\u003e\u003cem\u003eThis study suggests that lower glucocorticoid receptor (GR) expression may be associated with improved response to platinum-based pemetrexed chemotherapy in patients with non-small cell lung cancer (NSCLC). Patients with low GR expression exhibited longer progression-free survival (PFS) and overall survival (OS), greater tumor size reduction, and higher response rates compared to those with high GR expression. The hazard ratio for PFS (0.72, 95% CI: 0.52\u0026ndash;0.92) supports a reduced risk of disease progression in the low GR group, indicating potential prognostic relevance. While the hazard ratio for OS (0.95, 95% CI: 0.75\u0026ndash;1.15) did not reach statistical significance, a favorable trend toward longer survival was observed, suggesting the involvement of additional clinical or molecular factors beyond GR status.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAlthough the sample size was relatively small (n\u0026thinsp;=\u0026thinsp;35), restricted by strict inclusion criteria and patient availability, the observed trends remained consistent across multiple clinical endpoints. These findings should be interpreted with caution, and future studies with larger, multicenter cohorts are needed to validate the prognostic value of GRα/GRβ expression ratio in clinical practic\u003c/em\u003ee.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Comparison with Previous Studies\u003c/h2\u003e\u003cp\u003ePrevious research has demonstrated that elevated GR expression, particularly a lower GRα/GRβ ratio in peripheral blood mononuclear cells (PBMCs), is associated with reduced sensitivity to pemetrexed-based chemotherapy in non-small cell lung cancer (NSCLC. Patki et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] initially identified this association in in vitro NSCLC cell models, highlighting the mechanistic role of GR in downregulating transporters and target enzymes crucial for pemetrexed activity. Zhao et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] subsequently extended these observations to clinical samples, confirming that a lower GRα/GRβ ratio in PBMCs correlated with improved response rates and survival outcomes in NSCLC patients. However, their study focused predominantly on static clinical endpoints and did not explore dynamic tumor characteristics or metastatic burden changes.\u003c/p\u003e\u003cp\u003eThe present prospective cohort study builds upon these foundations by not only validating the prognostic value of the GRα/GRβ ratio but also correlating GR expression with tumor size reduction, lymph node response, and detailed survival outcomes (PFS and OS). This more comprehensive evaluation provides stronger evidence for the utility of GR status as a biomarker and enhances the clinical relevance of GR signaling in NSCLC management. A comparative summary of Patki et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Zhao et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and the present study is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, highlighting key methodological differences and findings.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative Summary of Key Studies\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStudy\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStudy Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSample Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGR Analysis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutcome Measures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eKey Findings\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatki et al. (2014) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIn vitro study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNSCLC cell lines (A549, H460)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGR expression (overall)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCell viability assays\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eHigh GR expression reduces pemetrexed sensitivity by downregulating transporters (PCFT, RFC) and target enzymes (TS, DHFR).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZhao et al. (2017) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetrospective clinical study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeripheral blood mononuclear cells (PBMCs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGRα/GRβ ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResponse rate (RR), PFS, OS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLower GRα/GRβ ratio associated with better chemotherapy response and survival outcomes.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent Study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProspective clinical cohort\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeripheral blood mononuclear cells (PBMCs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGRα/GRβ ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTumor size reduction, lymph node response, RR, PFS, OS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLower GRα/GRβ ratio associated with greater tumor reduction, reduced lymph node involvement, improved PFS, and favorable OS trend.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMechanistic studies suggest that GR activation may induce cellular dormancy via upregulation of CDKN1C (p57) and IGF-1R-mediated survival pathways, thereby contributing to resistance against pemetrexed [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, high expression of GR-regulated proteins such as SGK1 and NDRG1 has been linked to increased recurrence rates and worse survival outcomes in lung adenocarcinoma patients [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Collectively, these findings are consistent with the present study, reinforcing the role of GR signaling in mediating chemotherapy resistance and highlighting its potential clinical utility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Mechanistic Insights and Clinical Implications\u003c/h2\u003e\u003cp\u003eThe detrimental impact of high GR expression on pemetrexed efficacy may partly stem from GR-mediated downregulation of key pharmacodynamic targets. Dexamethasone, routinely administered to mitigate pemetrexed-induced toxicity, may suppress crucial enzymes and transporters such as TS, DHFR, RFC, and PCFT, especially in tumors with heightened GR activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This suppression could diminish intracellular pemetrexed accumulation, thereby reducing therapeutic effectiveness.\u003c/p\u003e\u003cp\u003eMoreover, the greater reduction in lymph node involvement observed in patients with lower GR expression suggests that GR pathways may influence metastatic dissemination and immune evasion, warranting further exploration of GR signaling beyond drug resistance alone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Limitations and Strengths\u003c/h2\u003e\u003cp\u003eSeveral limitations should be acknowledged. First, although the sample size was relatively small, post-hoc analysis confirmed that it was sufficiently powered to detect significant differences. Importantly, the observed effect sizes were both clinically relevant and statistically robust, which supports the validity of the findings despite the limited cohort. Second, while PBMCs offer a minimally invasive approach for biomarker assessment, they may not fully capture intratumoral heterogeneity. Third, other potential confounding factors, such as concomitant medications or variations in dexamethasone dosing schedules, were not systematically controlled. Nonetheless, the study has notable strengths, including a prospective design, comprehensive evaluation of dynamic clinical parameters (tumor and lymph node response), and independent validation of previously reported associations in a clinical cohort.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Clinical Translation and Future Directions\u003c/h2\u003e\u003cp\u003eThe current findings suggest that GRα/GRβ ratio assessment from PBMCs could serve as a practical, minimally invasive biomarker to guide therapeutic decision-making in NSCLC. Stratifying patients based on GR status may enable more personalized pemetrexed-based treatment regimens, potentially improving clinical outcomes while minimizing unnecessary toxicities.\u003c/p\u003e\u003cp\u003eFuture research should focus on:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eDeciphering the molecular pathways linking GR signaling to chemotherapy resistance,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eInvestigating the differential roles of GR isoforms (GRα versus GRβ),\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eDeveloping targeted therapies to selectively modulate GR activity,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eValidating these findings in larger, multicenter prospective cohorts, ideally through randomized controlled trials.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eIncorporating GR evaluation into routine clinical practice holds promise for refining patient stratification, enhancing treatment efficacy, and advancing precision medicine approaches in lung cancer therapy.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this prospective cohort study, lower glucocorticoid receptor (GR) expression, particularly a lower GRα/GRβ ratio in peripheral blood mononuclear cells (PBMCs), was associated with improved treatment response, greater tumor size reduction, reduced lymph node involvement, and prolonged progression-free and overall survival in NSCLC patients receiving pemetrexed-based chemotherapy. These findings underscore the potential of GR status as a minimally invasive biomarker for predicting chemotherapy outcomes and guiding personalized treatment strategies. Future large-scale prospective trials are warranted to validate the clinical utility of GR assessment and explore therapeutic interventions targeting GR signaling pathways to overcome chemotherapy resistance. The ability to quantify the GRα/GRβ ratio from PBMCs prior to chemotherapy offers a minimally invasive tool for stratifying NSCLC patients and potentially guiding pemetrexed use in clinical settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Vice-Chancellery of Research and Technology of Shiraz University of Medical Sciences as part of the Ph.D. thesis of the first author (Bahareh Forouzani-Haghighi). No external funding was received from commercial or not-for-profit agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial or non-financial interests that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBahareh Forouzani-Haghighi: conceptualization, methodology, investigation, statistical analysis, and manuscript drafting.\u003cbr\u003eAlireza Rezvani: clinical coordination, supervision, and manuscript review.\u003cbr\u003eBita Geramizadeh and Elaheh Esfandiari: sample processing and laboratory support.\u003cbr\u003eMehdi Ghasemian: data curation and visualization.\u003cbr\u003eAfsaneh Vazin: project administration, supervision, final review, and correspondence.\u003cbr\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1401.275) and conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final version of the manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, Jemal A. 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Anticancer Res. 2023;43(7):2965\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21873/anticanres.16467\u003c/span\u003e\u003cspan address=\"10.21873/anticanres.16467\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, Glucocorticoid receptor, GRα/GRβ ratio, Pemetrexed, Chemotherapy response, Translational Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-7069829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7069829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e This study aimed to evaluate whether the GRα/GRβ expression ratio can serve as a predictive biomarker for treatment response and survival outcomes in patients with non-squamous non-small cell lung cancer (NSCLC) receiving pemetrexed-based chemotherapy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A total of 35 patients with confirmed non-squamous NSCLC were enrolled and treated with pemetrexed plus dexamethasone. Quantitative PCR was used to measure GRα and GRβ mRNA levels, and patients were categorized into high and low GRα/GRβ ratio groups. Tumor response was assessed using RECIST criteria. Progression-free survival (PFS) and overall survival (OS) were evaluated using Kaplan–Meier analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eThe low GRα/GRβ group showed a significantly higher response rate (52.9% vs. 44.4%, p = 0.04), greater tumor shrinkage (55% vs. 42%, p = 0.03), and improved lymph node regression. Median PFS and OS were also longer in the low-ratio group (5.5 vs. 4.0 months, p = 0.035; 14.0 vs. 11.6 months, p = 0.025, respectively).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The GRα/GRβ expression ratio appears to be a promising predictive marker for clinical response and survival in non-squamous NSCLC. Incorporating this biomarker into treatment planning may enhance patient stratification and therapeutic decision-making.\u003c/p\u003e","manuscriptTitle":"Prognostic implications of glucocorticoid receptor expression in non-small cell lung cancer patients treated with pemetrexed","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 10:30:18","doi":"10.21203/rs.3.rs-7069829/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c29edab-d26c-4e44-8bbb-1b044f6bb472","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-24T23:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-09 10:30:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7069829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7069829","identity":"rs-7069829","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}