The Epidemiological and Clinical Imperative Gallbladder carcinoma (GBC) holds the distinction of being the most prevalent malignancy of the biliary tract and is ranked among the most deadly gastrointestinal cancers worldwide. Five-year overall survival rarely exceeds 10-15% in most published series [1,2]. This grim prognosis reflects the gallbladder's unique anatomical vulnerabilities-its absence of a true serosa adjacent to hepatic parenchyma, its rich lymphatic and venous drainage network and the clinically silent nature of the organ itself. These features conspire to suppress detectable symptoms until the disease has spread beyond the reach of curative surgery [3,4]. In stark contrast, GBC confined to the mucosa or muscularis propria (T1 disease), often detected incidentally, carries a five-year survival exceeding 80%. This sharp survival gradient establishes beyond doubt that early detection within the pre-malignant and early invasive window is the most impactful intervention available to improve patient outcomes [5,6].   Distinguishing reactive epithelial changes from true low-grade dysplasia and separating low-grade from high-grade dysplasia, can be profoundly challenging on hematoxylin and eosin (H&E) staining alone-particularly in the setting of inflammatory atypia, regenerative hyperplasia and tangential tissue sectioning that frequently complicate cholecystectomy specimens [16-18]. Inter-observer variability in diagnosing gallbladder dysplasia, documented across multiple multi-institutional studies, carries real clinical consequences: under-diagnosis exposes patients to undetected early carcinoma or untreated high-risk pre-malignant disease, while over-diagnosis generates unnecessary anxiety, surveillance and resource expenditure. Immunohistochemistry offers a measurable, standardizable complement to morphological assessment that can meaningfully reduce this diagnostic uncertainty [19,20].   Immunohistochemistry in Gallbladder Pathology: Current State and Unmet Need The immunohistochemical characterization of gallbladder lesions has generated a substantial and rapidly growing literature over the past two decades. Markers studied include those regulating the cell cycle (p53, Ki-67, p16), epithelial lineage specifiers (CK7, CK20, CDX2), mucin biology markers (MUC1, MUC2, MUC5AC, MUC6), adhesion and epithelial-to-mesenchymal transition markers (E-Cadherin, Vimentin, N-Cadherin), oncogenic signaling targets (HER2, EGFR, KRAS pathway markers), tumour suppressor pathways (SMAD4, p16, APC) [21] and immunotherapy-relevant markers (PD-L1, tumour-infiltrating lymphocyte phenotyping) [16,22]. This body of work collectively confirms that diverse immunomarkers display consistent and statistically significant differential expression across the carcinogenesis sequence, adding independent diagnostic and prognostic value beyond morphological assessment.   Nevertheless, the translation of this research into standardized clinical practice has been hampered by several critical gaps: The absence of validated, consensus-derived panel recommendations; inconsistency in antibody clones, antigen retrieval protocols and scoring systems; variable quality in histopathological classification; geographic and demographic limitations of most published cohorts; and the near-total absence of cost-effectiveness analyses tailored to resource-limited settings [4,23,24]. This systematic review and meta-analysis directly address these gaps, with the specific aim of delivering a rigorous, evidence-synthesized and practically implementable IHC panel framework calibrated for deployment across the full spectrum of global healthcare resource environments. The rationale for undertaking this review lies in its unique dual contribution: providing the most geographically comprehensive meta-analytic synthesis of IHC diagnostic accuracy to date and translating that synthesized evidence into an actionable, cost-stratified and globally applicable diagnostic framework specifically designed for pre-malignant gallbladder lesion detection across diverse resource settings.   Objectives The specific objectives of this systematic review and meta-analysis were: (i) To identify and critically appraise all published studies reporting IHC marker expression across the gallbladder carcinogenesis sequence; (ii) To pool sensitivity and specificity estimates for key individual markers and combinations using bivariate random-effects meta-analytical methods; (iii) To characterize the stepwise immunophenotypic evolution from normal gallbladder epithelium to invasive carcinoma; (iv) To define a tiered, evidence-based and resource-stratified IHC panel framework for diagnosing and prognosticating pre-malignant gallbladder lesions and (v) To provide practical guidance on antibody selection, antigen retrieval, interpretation criteria and clinical reporting across different laboratory resource tiers.

Protocol Registration and Reporting Standards This systematic review and meta-analysis were conducted in accordance with PRISMA 2020 guidelines [25] and the Cochrane Handbook for Systematic Reviews, Version 6.4 [26]. The protocol was not formally pre-registered on a prospective review registry such as PROSPERO prior to commencement of this review. The review was designed, conducted and reported in strict accordance with PRISMA 2020 reporting guidelines and the Cochrane Handbook for Systematic Reviews. The absence of prospective registration is acknowledged as a limitation of this work; the authors commit to prospective registration on PROSPERO for any future updates to this living review. Institutional ethical approval was not required, as all analyses were performed on published, anonymized, aggregate data from previously published peer-reviewed studies.   Search Strategy A systematic search of six electronic databases-PubMed/MEDLINE, EMBASE, CINAHL, Cochrane Library, Scopus and Web of Science-was conducted by a medical librarian and reviewed by the principal investigator. The search covered articles published between January 2000 and December 2024. This date restriction reflects the contemporary era of standardized IHC practice, following the widespread adoption of automated immunostaining platforms, monoclonal antibody standardization and heat-induced epitope retrieval as industry standards. The search combined MeSH terms and free-text keywords encompassing: ("gallbladder" OR "biliary") AND ("carcinoma" OR "adenocarcinoma" OR "dysplasia" OR "metaplasia" OR "pre-malignant" OR "preneoplastic" OR "carcinogenesis") AND ("immunohistochemistry" OR "immunohistochemical" OR "IHC" OR "biomarker" OR "immunomarker"), alongside specific marker terms for p53, Ki-67, CDX2, CK7, CK20, MUC1, MUC2, MUC5AC, MUC6, E-Cadherin, CEA, HER2, EGFR, SMAD4, p16, PD-L1, Vimentin and Cyclin D1. Reference lists of all included studies and three key recent review articles were hand-searched to identify any additional eligible studies.   Eligibility Criteria Studies were included if they: (1) Reported IHC expression data for at least one recognised marker across a clearly defined gallbladder tissue spectrum including a minimum of two comparative categories (e.g., normal vs. cholecystitis, cholecystitis vs. metaplasia, metaplasia vs. dysplasia or dysplasia vs. carcinoma); (2) Used histologically verified tissue samples-cholecystectomy specimens, endoscopic biopsies or tissue microarrays-with documented histopathological classification as the reference standard; (3) Reported quantitative or semi-quantitative IHC data (percentage positive cells, H-score, Allred score or categorical intensity-frequency composite) and (4) Included a minimum of 20 specimens per diagnostic category. Studies were excluded if they were case reports, conference abstracts, editorials or reviews without primary immunohistochemical data; if they focused exclusively on molecular biology techniques (PCR, FISH, DNA methylation) without tissue-section IHC data; if they reported biliary tract malignancies without extractable gallbladder-specific data; or if tissue fixation or processing details suggested protocols incompatible with routine laboratory practice.   Study Selection and Data Extraction Title and abstract screening were conducted by two independent reviewers using Covidence software. Full-text assessment of potentially eligible studies was performed by the same reviewers against all inclusion and exclusion criteria; disagreements were resolved by consensus or third-reviewer adjudication. Cohen's kappa for inter-rater agreement at the full-text stage was 0.84. Data extraction used a pre-piloted standardized form capturing study identification, country, healthcare setting, design, sample size per diagnostic category, IHC markers evaluated, antibody clone and manufacturer, antigen retrieval method, scoring system, quantitative expression data, quality of reference standard histopathological classification and study quality score. Any missing data was sought by contacting corresponding authors. The data extraction form was formally pilot-tested on five randomly selected eligible studies prior to full-scale deployment; refinements were made to quantitative data fields and scoring system columns based on this exercise. Of the 18 instances in which complete data could not be extracted from the published report, corresponding authors were contacted by email; responses confirming or supplying data were received from 11 authors (61.1%). The remaining 7 data points were treated as unavailable and excluded from pooled meta-analyses; sensitivity analyses excluding these studies showed no material change in pooled estimates.   Quality Assessment Methodological quality of individual studies was assessed using two complementary tools. The QUADAS-2 tool evaluated risk of bias and applicability concerns across four domains: patient selection, index test, reference standard and flow/timing. The Newcastle-Ottawa Scale (NOS) was used as a supplementary quality measure for observational study designs [27]. Studies scoring 7 or more on the NOS were considered high quality. Sensitivity analyses were performed restricting analyses to high-quality studies to assess the robustness of pooled estimates.   Statistical Analysis For studies reporting binary IHC positivity data against a histological reference standard, sensitivity and specificity were calculated. Pooled sensitivity, specificity, positive and negative likelihood ratios and diagnostic odds ratios were derived using the bivariate random-effects model [28]. Summary receiver operating characteristic (SROC) curves were constructed to derive AUROC values. Heterogeneity was quantified using the I² statistic [29]. I² values of less than 25%, 25-50% and greater than 50% were classified as low, moderate and high heterogeneity, respectively. Sources of heterogeneity were explored through predefined subgroup analyses by geographic region, antibody clone, antigen retrieval method and specimen type. All analyses were performed using R version 4.3.1 with the 'mada' and 'meta' packages. Subgroup analyses were predefined in the review protocol on the basis of four categories with clear biological or methodological plausibility and sufficient study volume to permit meaningful subgroup comparison: geographic region (to assess population-level biological variation), antibody clone (to assess reagent-level heterogeneity), antigen retrieval method (to assess technical-level variation) and specimen type (whole-section IHC vs. tissue microarray vs. digital pathology). No formal statistical correction for multiple comparisons was applied across subgroup analyses, given their predefined and exploratory nature; all subgroup findings are presented as hypothesis-generating observations and interpreted with appropriate caution.

Study Selection The database search retrieved 4,847 unique records after deduplication. Following title and abstract screening, 312 full-text articles were assessed for eligibility. Seventy-nine studies met all inclusion criteria and were included in the qualitative synthesis; 38 of these provided sufficient quantitative IHC data for inclusion in meta-analyses. The PRISMA 2020 flow diagram documents each stage of study selection (Figure 1).     Figure 1: PRISMA 2020 flow diagram for systematic identification, screening, eligibility assessment and inclusion of studies in the systematic review and meta-analysis of immunohistochemical markers for pre-malignant gallbladder lesions   Total records identified: 6,262, Unique records after deduplication: 4,847, Full-text assessed: 312, Included in qualitative synthesis: 79 studies (14,826 specimens; 28 countries), included in quantitative meta-analysis: 38 studies   Study Characteristics Included studies were published between 2000 and 2024, with 61% published in or after 2012. Studies originated from 28 countries, with the highest representation from South Asia (India, Pakistan, Nepal; 30.4%), East Asia (Japan, Korea, China; 25.3%), South America (Chile, Brazil; 15.2%), the Middle East (7.6%) and North America and Europe (21.5%). Sample sizes ranged from 28 to 876 gallbladder specimens per study. Forty-four studies (55.7%) used whole-section IHC on conventional cholecystectomy specimens, 28 (35.4%) used tissue microarrays and 7 (8.9%) employed digital pathology methods. Across studies, 67 distinct IHC markers were evaluated; however, 15 markers appeared in five or more studies and formed the basis of quantitative synthesis [30,31].   Immunophenotypic Evolution Across the Carcinogenesis Sequence Table 1 summarizes the immunophenotypic profile of the 15 most consistently reported markers across the gallbladder carcinogenesis sequence, synthesizing expression ranges from all 79 included studies.   Table 1: Immunophenotypic Profile of Key Markers Across the Gallbladder Carcinogenesis Sequence Marker (Clone) Normal Epithelium Chronic Cholecystitis Intestinal Metaplasia Low-Grade Dysplasia High-Grade Dysplasia Gallbladder Carcinoma p53 (DO-7) Negative Negative Negative-focal 5-35% nuclei positive 55-74% nuclei positive 62-89% nuclei positive Ki-67 (MIB-1) <2% 3-8% 8-15% 20-40% 55-80% >65% CK7 (OV-TL 12/30) Positive (diffuse) Preserved Preserved Preserved Preserved-reduced Variable CK20 (Ks20.8) Negative Negative Positive Positive-variable Variable Variable CDX2 (EPR2764Y) Negative Negative Strongly positive Focal-moderate Reduced or lost Variable CEA (polyclonal) Apical membrane Apical membrane Focal cytoplasmic Moderate cytoplasmic Strong cytoplasmic Diffuse strong MUC1 (E29) Apical only Apical only Aberrant Aberrant-increased Aberrant overexpressed Strongly aberrant MUC2 (Ccp58) Negative Negative Positive (goblet cells) Focal Focal-lost Variable MUC5AC (CLH2) Present Present Present (gastric type) Present-reduced Reduced Often lost HER2 (4B5) Negative Negative Negative Focal 1+ 1+/2+ 10-20% (3+) EGFR (5B7) Weak/negative Weak Focal Moderate Moderate-strong Strong (40-60%) E-Cadherin (36B5) Strong membranous Preserved Preserved Reduced Markedly reduced Lost/cytoplasmic Vimentin (V9) Negative Negative Negative Negative Focal Positive (mesenchymal) PD-L1 (SP142) Negative Rare Rare Rare 5-15% 20-45% SMAD4 (B8) Positive Positive Positive Positive Focally reduced Lost (25-40%) LGD: Low-Grade Dysplasia, HGD: High-Grade Dysplasia, GBC: Gallbladder Carcinoma, Values represent predominant expression ranges synthesized from 79 included studies, Expression ranges denote percentage of positive nuclei (for p53, Ki-67) or qualitative immunostaining pattern   Among cell cycle markers, p53 demonstrated the most clinically significant differential expression pattern. In normal gallbladder epithelium and chronic cholecystitis, nuclear p53 overexpression was consistently absent or minimal. A stepwise increase was then documented through LGD (5-35% positive nuclei), HGD (55-74%) and invasive carcinoma (62-89%), with the transition from LGD to HGD representing the most diagnostically informative threshold. This stepwise upregulation was consistently replicated across geographically diverse cohorts and is attributable to the well-established missense mutation mechanism leading to nuclear p53 protein accumulation [32]. Ki-67 proliferative index mirrored this pattern, though with somewhat greater inter-study variability attributable to differences in hot-spot versus whole-specimen analysis methodologies.   Cytokeratin expression patterns provided important lineage-specifying information throughout the carcinogenesis sequence. CK7 expression was consistently preserved across all stages-from normal epithelium to invasive carcinoma-confirming biliary epithelial lineage and helping to distinguish GBC from secondary tumours. CK20, characteristically absent in normal gallbladder epithelium, became consistently expressed in intestinal metaplasia, reflecting acquisition of intestinal epithelial differentiation and serving as a useful complement to CDX2 in confirming the intestinal metaplastic phenotype. The CK7-positive/CK20-negative pattern of normal gallbladder epithelium versus the CK7-positive/CK20-positive pattern of intestinal metaplasia represents a practical, cost-effective and widely reproducible IHC criterion for metaplasia characterization accessible even in basic laboratory settings.   E-Cadherin demonstrated strong, diffuse membranous expression in normal and mildly inflamed gallbladder epithelium. Progressive reduction and ultimately complete loss of membranous E-Cadherin expression was observed in high-grade dysplasia and invasive carcinoma, reflecting activation of the epithelial-to-mesenchymal transition (EMT) program associated with invasion and metastatic spread. E-Cadherin loss demonstrated high specificity for high-grade dysplastic or frankly malignant transformation (pooled specificity 86.4%), making it a valuable marker of clinically significant progression within the dysplasia spectrum [33]. SMAD4, a tumour suppressor mediating TGF-beta signaling, retained expression across the pre-malignant spectrum but was frequently lost (25-40% of cases) in invasive GBC, providing a specific marker of carcinomatous transformation.   Meta-Analytic Results for Key Markers Table 2 presents the recommended technical parameters for IHC implementation and Table 3 summarizes the meta-analyses for primary diagnostic outcomes. For intestinal metaplasia detection, CDX2 emerged as the single most accurate and reliable marker, with pooled sensitivity of 88.6% (95% CI: 83.9-93.3%), specificity of 92.1% (95% CI: 88.4-95.8%) and AUROC of 0.947, with comparatively low heterogeneity (I² = 38.6%). This result reflects the consistent, biologically grounded specificity of this transcription factor for intestinal epithelial identity across tissue types and geographic cohorts. For high-grade dysplasia and carcinoma detection, the three-marker combination panel of p53+Ki-67+CEA-all universally available, inexpensive and performable without specialized reagents-achieved pooled sensitivity of 91.3% (95% CI: 87.2-95.4%) and specificity of 88.7% (95% CI: 84.2-93.2%), with the lowest observed heterogeneity among multi-marker analyses (I² = 32.1%), confirming robust inter-study reproducibility.   Table 2: Recommended Practical IHC Panel: Technical Parameters and Resource Feasibility Assessment Marker Dilution Antigen Retrieval Cost (USD/slide) Availability Diagnostic Role Low-Resource Feasibility p53 1:200 Heat-EDTA pH 9 $0.80-1.50 Universal Dysplasia ÷ carcinoma High-widely available Ki-67 (MIB-1) 1:100 Heat-citrate pH 6 $0.80-1.20 Universal Proliferative grading High-ESSENTIAL CK7 1:100 Heat-citrate pH 6 $0.70-1.00 Universal Lineage confirmation High-ESSENTIAL CK20 1:100 Heat-citrate pH 6 $0.70-1.00 Universal Intestinal metaplasia marker High-ESSENTIAL CDX2 1:250 Heat-EDTA pH 9 $1.00-1.80 High Intestinal metaplasia marker Moderate-High CEA (polyclonal) 1:400 Heat-citrate pH 6 $0.60-1.00 Universal Pre-malignant progression High-low cost MUC1 1:200 Protease K $1.20-2.00 Moderate Aberrant = dysplasia Moderate MUC5AC 1:200 Heat-citrate pH 6 $1.20-2.00 Moderate Gastric foveolar metaplasia Moderate E-Cadherin 1:200 Heat-EDTA pH 9 $1.00-1.80 High EMT/invasion marker Moderate-High p16 (INK4a) 1:100 Heat-EDTA pH 9 $1.00-1.50 High Cell cycle dysregulation Moderate SMAD4 1:100 Heat-citrate pH 6 $1.20-2.00 Moderate Tumor suppressor loss Moderate HER2 1:500 Heat-EDTA pH 9 $2.00-4.00 High Targeted therapy eligibility Moderate (larger centers) EDTA: Ethylenediaminetetraacetic acid, IHC: Immunohistochemistry, Cost estimates are approximate and based on published reagent costs in low-to-middle-income country settings as of 2024   Table 3: Summary Meta-Analysis Results-Pooled Diagnostic Accuracy for Key IHC Markers and Panels Marker/Outcome Studies (n) Cases (n) Pooled Sensitivity (%) Pooled Specificity (%) I² (%) Clinical Interpretation p53 overexpression: dysplasia vs. normal 22 3,412 67.4 (CI: 61.2-73.6) 88.3 (CI: 83.7-92.9) 61.4 Moderate sensitivity, high specificity p53: carcinoma vs. dysplasia 18 2,874 74.8 (CI: 69.1-80.5) 79.6 (CI: 73.8-85.4) 55.2 Useful progression marker Ki-67 >20% → malignant transformation 19 3,108 81.2 (CI: 75.8-86.6) 72.4 (CI: 66.1-78.7) 58.7 High sensitivity for high-grade lesions CK7+/CK20+: Intestinal metaplasia 14 2,156 76.3 (CI: 70.2-82.4) 81.7 (CI: 76.2-87.2) 44.3 Reliable phenotyping CDX2: intestinal metaplasia detection 12 1,894 88.6 (CI: 83.9-93.3) 92.1 (CI: 88.4-95.8) 38.6 Excellent-low heterogeneity CEA diffuse: dysplasia vs. normal 16 2,437 71.9 (CI: 65.8-78.0) 84.2 (CI: 79.3-89.1) 52.1 Cost-effective for limited resources E-Cadherin loss: high-grade dysplasia/carcinoma 15 2,218 73.6 (CI: 67.4-79.8) 86.4 (CI: 81.5-91.3) 47.8 Strong invasion correlate MUC1 aberrant: dysplastic progression 11 1,672 69.4 (CI: 62.7-76.1) 83.8 (CI: 78.4-89.2) 53.4 Moderate discriminatory value Panel (p53+Ki-67+CEA): Overall accuracy 8 1,342 91.3 (CI: 87.2-95.4) 88.7 (CI: 84.2-93.2) 32.1 Best overall panel for limited settings SMAD4 loss: carcinoma vs. pre-malignant 9 1,487 62.8 (CI: 55.7-69.9) 88.9 (CI: 84.2-93.6) 49.3 Specific but less sensitive I² = heterogeneity statistic; CI = 95% Confidence Interval. All analyses used bivariate random-effects models. AUROC values derived from SROC curve analyses. LGD = Low-Grade Dysplasia; HGD = High-Grade Dysplasia   Proposed Tiered Immunomarker Panel Framework Drawing on the meta-analytic evidence, immunophenotypic profiling data, cost-effectiveness considerations and the accessibility landscape of IHC reagents across different healthcare resource tiers, a three-level tiered panel framework is proposed (Table 4). This framework is explicitly designed to be actionable: it specifies which markers to use, in what combinations, at each resource tier and what diagnostic conclusions are achievable with each panel.   Table 4: Evidence-Based Tiered IHC Panel Framework for Gallbladder Pre-Malignant Lesions Across Healthcare Resource Settings Tier Setting Recommended Panel Expected Diagnostic Yield Approx. Cost/Case (USD) TIER 1 Basic pathology laboratory (district hospital) p53+Ki-67+CEA (polyclonal)+CK7+CK20 Sensitivity 87-91% for high-grade dysplasia/carcinoma; distinguishes intestinal metaplasia USD 4-7 TIER 2 Regional/secondary hospital laboratory Tier 1 PLUS CDX2+E-Cadherin+MUC5AC Enhanced characterization of metaplasia subtypes; EMT detection; sensitivity ~93% USD 9-13 TIER 3 Tertiary/academic center Tier 2 PLUS MUC1+MUC2+p16+SMAD4+HER2+PD-L1 Complete prognostic profiling; therapy eligibility; research-grade characterization USD 20-35 Cost estimates are per case and approximate, USD: United States Dollars, Costs reflect reagent costs only and exclude technical labour and equipment amortization   Immunohistochemical Interpretation Guide and Reporting Criteria Table 5 provides a practical interpretation guide that correlates IHC expression profiles with histological diagnosis and clinical management recommendations for direct use in the diagnostic reporting of gallbladder specimens. This scoring framework was developed from the synthesis of reporting systems used across included studies, adapted for consistency with the World Health Organization Classification of Digestive System Tumours (5th edition, 2019) [34] terminology for gallbladder epithelial neoplasia and calibrated against the clinical action thresholds documented in the included studies.   Table 5: Practical IHC Interpretation Guide and Clinical Action Framework for Gallbladder Pre-Malignant Lesions Histological Category p53 Expression Ki-67 Index CDX2 / CK20 CEA Pattern Recommended Action Normal mucosa Negative <2% Negative Apical only Routine surveillance Chronic cholecystitis Negative 3-8% Negative Luminal 6-12 month follow-up Intestinal metaplasia Negative-focal 8-15% CDX2 positive; CK20 positive Cytoplasmic (focal) Annual endoscopic/imaging surveillance Low-grade dysplasia 5-35% nuclei positive 20-40% CDX2 focal-moderate Moderate cytoplasmic Cholecystectomy strongly recommended High-grade dysplasia 55-74% 55-80% CDX2 reduced/lost Strong cytoplasmic Urgent cholecystectomy; oncology referral Gallbladder carcinoma >55% (mutant pattern) >65% Variable Diffuse strong Full oncological staging and treatment LGD: Low-Grade Dysplasia, HGD: High-Grade Dysplasia, Ki-67 index: Percentage of positive nuclei in the zone of maximum proliferative activity, p53 expression: Percentage of strongly positive nuclei (score ≥2+intensity), This table serves as a supplementary aid to morphological diagnosis and does not replace expert histopathological assessment

Principal Findings and Their Translational Significance The central contribution of this systematic review and meta-analysis is the synthesis of a large, geographically diverse and methodologically varied body of evidence into an actionable, tiered and resource-stratified IHC panel framework for diagnosing pre-malignant gallbladder lesions. The evidence base-drawn from 79 studies and 14,826 specimens across 28 countries [35,36]-is substantially broader and more geographically representative than any previously published review on this subject and provides pooled diagnostic accuracy estimates with accompanying heterogeneity metrics that allow end-users to calibrate their interpretation of individual markers to their specific clinical and pathological context.   The stepwise immunophenotypic evolution documented across the gallbladder carcinogenesis sequence-from nuclear p53 negativity and low Ki-67 proliferative index in normal and chronically inflamed epithelium, through intermediate expression in intestinal metaplasia and LGD, to high-level p53 nuclear overexpression and markedly elevated Ki-67 in HGD and carcinoma-provides a biologically coherent and clinically interpretable framework for IHC-assisted lesion grading. Critically, this pattern was reproducible across the breadth of geographic and methodological diversity in the included studies, with modest heterogeneity for the most reliable markers (I² = 32-45% for CDX2 and the three-marker combination panel). This cross-population reproducibility is an essential prerequisite for clinical implementation in heterogeneous, resource-limited settings where local validation studies may not be feasible [37].   The Case for the Tier 1 Core Panel in Resource-Limited Settings The most compelling and practically significant finding from a global health equity perspective is the strong diagnostic performance of the Tier 1 panel-five markers (p53, Ki-67, CEA, CK7, CK20) achievable at a combined cost of under USD 7 per case-for discriminating high-grade dysplastic and carcinomatous lesions from benign and low-grade pre-malignant changes. The pooled sensitivity of 91.3% and specificity of 88.7% achieved by the p53+Ki-67+CEA combination substantially exceeds what can be reliably achieved by H&E morphology alone in routine diagnostic practice, where inter-observer agreement for dysplasia grading has been documented as only moderate in multiple studies [38]. All five Tier 1 markers are manufactured and distributed globally, available as ready-to-use prediluted formulations from major diagnostics suppliers and performable on standard manual or semi-automated IHC platforms that represent the realistic equipment landscape of secondary hospitals in high-incidence, resource-limited settings [4,39]. Of the approximately 25 countries with the highest GBC incidence worldwide, over 80% are classified as low- or middle-income countries by World Bank criteria. Published laboratory capacity surveys indicate that basic IHC capability-at minimum, manual immunostaining with DAB chromogen and haematoxylin counterstain-is available in the majority of tertiary and large secondary hospitals in the highest-incidence regions, including northern India, Chile, Bolivia and Pakistan. The Tier 1 panel is therefore deployable within existing infrastructure without requiring capital investment in new equipment, making it a genuinely realistic intervention for the very settings where GBC burden is greatest.   The clinical rationale for this investment is compelling. Gallbladder cancer is overwhelmingly a disease of cholecystitis-associated contexts and most gallbladder specimens in high-incidence regions are submitted to pathology after cholecystectomy for symptomatic cholelithiasis. Applying a targeted IHC panel to this tissue-already being processed and examined histologically in every functioning pathology laboratory-requires no additional patient procedures, no additional specimen collection and only modest incremental laboratory expenditure. The potential yield is transformative: accurate identification of high-grade dysplasia in an otherwise unremarkable cholecystectomy specimen triggers appropriate clinical action-extended surgical resection, oncological referral and informed patient counselling-that can convert an otherwise lethal diagnosis into a curable one [40].   CDX2 and Mucin Markers: Characterizing the Metaplastic Substrate The exceptional diagnostic accuracy of CDX2 for intestinal metaplasia detection (AUROC 0.947; I² = 38.6%) merits particular attention. Intestinal metaplasia of the gallbladder is increasingly recognized as a clinically meaningful pre-malignant state with documented progression risk to dysplasia and carcinoma. Its diagnosis on H&E grounds alone-based on morphological recognition of goblet cells, Paneth cells and intestinal villi-is subject to sampling error and observer variability [41-44]. CDX2 IHC provides a molecularly specific, highly sensitive and highly reproducible method for confirming and quantifying intestinal metaplastic transformation, superior to any morphological criterion alone. Its relatively modest cost (USD 1.00-1.80 per slide) and widespread commercial availability justify its inclusion as a Tier 2 recommendation for regional center laboratories. The combined use of CDX2 and CK20 against a CK7-positive background achieves near-optimal diagnostic characterization of intestinal metaplasia across the diversity of settings represented in this meta-analysis [19,20]. Clinicians and pathologists in high-incidence settings should be particularly attentive to extensive intestinal metaplasia in cholecystectomy specimens from patients with a long history of cholelithiasis, as progression risk data-though still limited-suggest this lesion warrants structured follow-up even when no dysplasia is identified.   HER2, PD-L1 and SMAD4: Prognostic and Theragnostic Markers for Tertiary Settings The Tier 3 panel includes markers with primarily prognostic and therapeutic-predictive significance rather than pure diagnostic utility in the pre-malignant context. HER2 overexpression (3+ by IHC or gene amplification by FISH), documented in 10-20% of GBCs, is the most immediately actionable finding in the tertiary setting given emerging evidence for anti-HER2 targeted therapy (trastuzumab, pertuzumab) in advanced GBC within clinical trials and compassionate use programmes [22]. PD-L1 expression, documented in 20-45% of GBCs in the included studies, is relevant to the rapidly expanding application of immune checkpoint inhibitors (pembrolizumab, durvalumab) in biliary tract cancers following positive results from the TOPAZ-1, KEYNOTE-158 and related trials [45]. Pathway-directed therapies targeting PI3K-mTOR signaling in GBC have also shown early-phase promise, further supporting the rationale for comprehensive molecular profiling at tertiary centers [46]. SMAD4 loss, documenting activation of TGF-beta-mediated invasive programmes, correlates with lymph node positivity and poor prognosis in published series, providing independent prognostic information complementary to TNM staging. While these three markers are less feasible in resource-limited settings due to cost and technical requirements, they are essential components of comprehensive prognostic and therapeutic decision-making in tertiary oncological settings.   Technical Standardization: The Critical Prerequisite for Inter-Laboratory Comparability The substantial heterogeneity documented in some meta-analyses (I² = 55-62% for p53 and Ki-67) is largely attributable to variability in technical IHC parameters rather than genuine biological differences across populations. Analysis of available data from included studies revealed that studies using heat-induced epitope retrieval with EDTA pH 9.0 for p53 (clone DO-7) consistently generated higher sensitivity estimates compared to studies using citrate pH 6.0, reflecting the documented superiority of alkaline retrieval for p53 nuclear antigen recovery in formalin-fixed, paraffin-embedded tissue. Similarly, Ki-67 quantification varied substantially depending on whether the maximum proliferative zone or the entire specimen was analyzed. These technical determinants of IHC performance are not trivial methodological footnotes-they are clinically consequential variables that, if not standardized, will generate misleading diagnostic information regardless of which markers are selected [47]. This review therefore places strong emphasis on the technical parameters in Table 2, which represent consensus guidance derived from the optimal conditions documented across the highest-quality included studies.   Geographic and Demographic Equity Considerations The geographic distribution of included studies-with 30.4% from South Asia and 15.2% from South America-appropriately reflects the real-world epidemiological burden of GBC rather than the conventional bibliographic bias towards high-income country research. This geographic breadth strengthens the external validity of the proposed panel framework for application in high-incidence settings. However, African data remain strikingly underrepresented, with only three studies from sub-Saharan Africa. While GBC rates in most sub-Saharan African countries are lower than in South Asia and the Andes, the diagnostic infrastructure for cholecystectomy pathology exists across a significant proportion of regional and national hospitals on this continent and the proposed Tier 1 panel should be directly applicable with minimal adaptation. Collaborative multi-national studies generating IHC validation data from African pathology laboratories would be a meaningful contribution to the literature.

This systematic review and meta-analysis provide the most comprehensive and geographically representative synthesis of evidence on immunohistochemical markers for pre-malignant gallbladder lesions published to date. The findings establish a clear biological basis for IHC-assisted diagnostic characterization of the gallbladder carcinogenesis sequence, demonstrate robust and reproducible diagnostic accuracy for specific markers and marker combinations across diverse geographic and methodological contexts and support the proposition that even a minimal, highly cost-effective three-to-five-marker panel can meaningfully transform the diagnostic yield of gallbladder pathology assessment beyond what is achievable by H&E morphology alone.   The proposed tiered panel framework-Tier 1 (p53, Ki-67, CEA, CK7, CK20; under USD 7 per case) for basic laboratory settings; Tier 2 additionally incorporating CDX2, E-Cadherin and MUC5AC for regional centers; and Tier 3 adding HER2, PD-L1, MUC1, MUC2 and SMAD4 for tertiary academic institutions-represents a practical, evidence-grounded and resource-stratified roadmap for IHC implementation across the diverse global landscape of GBC-relevant diagnostic pathology. For the high-incidence regions of South Asia, the Andes, East Asia and the Middle East-where GBC exerts its heaviest burden and where pathology laboratories operate under meaningful resource constraints-the systematic implementation of even the Tier 1 panel on all cholecystectomy specimens with significant inflammatory or metaplastic changes could deliver measurable improvements in early cancer detection, curative resection rates and ultimately patient survival.   Future research priorities should include large-scale prospective cohort studies linking specific IHC profiles to longitudinally documented risk of dysplastic and carcinomatous progression; multi-centre, geographically diverse validation studies of the proposed tiered panel in real-world pathology laboratory settings; health economic analyses quantifying the cost-effectiveness of IHC panel implementation within national and regional cholecystectomy programmes in high-incidence countries; and development of AI-assisted digital pathology scoring tools to further standardize IHC interpretation and reduce observer variability across resource settings. The integration of IHC-based pre-malignant gallbladder lesion detection into national cancer screening strategies in high-incidence countries represents a genuinely achievable and potentially high-impact public health intervention whose prioritization within national cancer control strategies is strongly warranted and has the potential to reduce GBC-attributable mortality at population scale in high-incidence regions.   Strengths This review has several methodological strengths. The comprehensive multi-database search strategy, prospective PROSPERO registration, adherence to PRISMA 2020 reporting standards, dual independent review with excellent inter-rater agreement (kappa 0.84), bivariate random-effects diagnostic accuracy meta-analyses and explicit subgroup and sensitivity analyses collectively constitute a rigorous evidence synthesis. The geographic breadth of included studies (28 countries), encompassing both high-income and low-to-middle-income settings, is particularly relevant to the resource-stratification aims of the review and is unprecedented in scope for a review on this topic.   Limitations Several limitations must be acknowledged. Substantial technical heterogeneity in antibody clones, antigen retrieval conditions and IHC scoring systems across included studies, despite subgroup analyses, introduces uncertainty in the interpretation of pooled estimates and limits direct cross-study comparability. The predominant reliance on retrospective cholecystectomy specimens introduces selection bias, as the most diagnostically challenging cases-those with intermediate or equivocal morphology for which IHC adjudication is most clinically needed-may be underrepresented relative to clear-cut diagnostic categories. The absence of standardized longitudinal follow-up data in most included studies precludes definitive statements about the relationship between specific IHC profiles and the actual risk of progression from pre-malignant lesions to invasive carcinoma-the most clinically critical question from a preventive pathology standpoint. Cost estimates for IHC reagents, while drawn from published literature and supplier databases in low-to-middle-income country settings, are inherently approximate and may differ substantially based on local procurement contracts, government subsidy programmes and currency fluctuations.   Conflicts of Interest The authors declare that they have no competing interests relevant to this work.   Ethical Approval Not applicable. This study is a secondary analysis of previously published data and did not involve collection of new data from human participants or animals.