Dual-Phenotype Hepatocellular Carcinoma Exhibits Distinct Clinicopathological Aggressiveness and Poor Prognosis: A Retrospective Cohort Study of 463 Hepatocellular Carcinoma Patients

Dual-Phenotype Hepatocellular Carcinoma Exhibits Distinct Clinicopathological Aggressiveness and Poor Prognosis: A Retrospective Cohort Study of 463 Hepatocellular Carcinoma Patients | 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 Dual-Phenotype Hepatocellular Carcinoma Exhibits Distinct Clinicopathological Aggressiveness and Poor Prognosis: A Retrospective Cohort Study of 463 Hepatocellular Carcinoma Patients Xiaojuan Ouyang, Bohao Hu, Xinjing Gao, Xingfeng Qi, Lijuan Qu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9029042/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Objective: To investigate the clinicopathological characteristics and prognostic differences between the dual-phenotype hepatocellular carcinoma (DPHCC) and classical hepatocellular carcinoma (CHCC), and to identify the key prognostic risk factors for DPHCC. Methods : The Clinicopathological data from 463 hepatocellular carcinoma (HCC) patients were retrospectively analyzed. According to the morphology and immunophenotype (co-expression of HCC and intrahepatic cholangiocarcinoma [ICC] markers in > 15% of tumor cells). The cases were divided into two groups: the DPHCC group (93 cases, 20.09%) and the CHCC group (370 cases, 79.91%). Clinicopathological characteristics (microvascular invasion, tumor size, differentiation grade) and survival outcomes (overall survival [OS]; recurrence-free survival[RFS]) were compared between the two groups. Cox regression models were employed to analyze independent prognostic risk factors for DPHCC. Results: The DPHCC group exhibited significantly higher rates of microvascular invasion (MVI) (44.09% vs. 21.08%), tumor size ≥ 3 cm (80.65% vs. 68.92%), multifocality (33.33% vs. 15.14%), poor differentiation (39.78% vs. 21.08%), and necrosis-positivity (76.34% vs. 49.20%), capsular invasion (31.18% vs. 15.95%) compared to the CHCC group (for all, P < 0.05 ). Survival analysis revealed significantly lower 5-year OS (61.10% vs 80.00%; HR 2.113) and RFS (37.40% vs 55.70%; HR 1.479) rates in the DPHCC group compared to the CHCC group. Multivariate analysis identified tumor size ≥ 3 cm (OS: HR = 3.021), poor differentiation (OS: HR 2.614; RFS: HR 1.927), and cytokeratin 19 (CK19) overexpression (OS: HR 4.903) as independent risk factors for poor prognosis in DPHCC. Additionally, DPHCC patients were significantly younger than CHCC patients (74.19% vs. 57.84% aged < 60 years; P = 0.003), suggesting a potential association with aberrant differentiation of hepatic progenitor cells. Conclusions: DPHCC represents a distinct HCC subtype characterized by high aggressiveness, poor differentiation, and unfavorable prognosis, influenced by tumor size, differentiation grade, and CK19 expression.. It is recommended that patients diagnosed with this distinct HCC subtype receive intensified postoperative surveillance and personalized treatment strategies targeting the CK19-positive subgroup. hepatocellular carcinoma Dual-phenotype hepatocellular carcinoma classical hepatocellular carcinoma microvascular invasion cytokeratin 19 expression Recurrence-free survival Overall survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, continues to pose a significant global health threat due to its persistently high incidence and mortality rates [1] . In recent years, a new histological subtype of HCC with stronger invasivity and poorer prognosis has been reported, namely Dual-phenotype hepatocellular carcinoma(DPHCC) [2, 3] . DPHCC, a distinct clinicopathological subtype defined by the co-expression of hepatocellular (e.g., HepPar-1/ Arg-1) and cholangiocytic (e.g., CK19/CK7/MUC1) immunophenotypic markers, has emerged as a growing focus in hepatobiliary oncology research [4] . According to diagnostic criteria, DPHCC is defined as tumors exhibiting typical HCC histomorphology with co-expression of HCC- and ICC-associated immunophenotypic markers in more than 15% of tumor cells [5] . Although DPHCC accounts for only 10% of HCC cases [5] , clinical observations indicate that DPHCC patients are prone to earlier vascular invasion, higher postoperative recurrence rates, and poorer survival outcomes [6] , underscoring its distinct biological behavior and unmet clinical management needs. Current understanding of DPHCC remains limited, with significant knowledge gaps persisting in both its clinicopathological features and molecular pathogenesis. While the 2024 Chinese Clinical Practice Guidelines for Liver Cancer have established standardized diagnostic criteria for DPHCC, it has not yet been recognized by experts worldwide and there are no international diagnostic criteria. Second, whether the enhanced aggressiveness of DPHCC compared to classical HCC (CHCC) correlates with specific clinicopathological characteristics, such as microvascular invasion (MVI) or tumor differentiation grade, requires validation in large-scale cohorts. Furthermore, the identification of prognostic risk factors for DPHCC and their mechanistic links to cholangiocyte markers (e.g., CK19) remain unclear. Although some studies propose that DPHCC may originate from aberrant differentiation of hepatic progenitor cells (HPCs), emerging experimental evidence indicates that hypoxic microenvironments may drive phenotypic transdifferentiation from conventional HCC to DPHCC through hypoxia-inducible factor (HIF)-mediated pathways [7–9] , this hypothesis lacks robust clinical evidence. Importantly, existing studies are predominantly constrained by small sample sizes or single-center designs, leading to insufficient evidence-based strategies for DPHCC management. Therefore, comprehensive analyses of clinicopathological characteristics and prognostic models based on large cohorts are urgently needed. Building upon this scientific foundation, our investigation conducted a retrospective analysis of 463 HCC cases to perform a comprehensive comparative evaluation of clinicopathological profiles and survival outcomes between DPHCC and CHCC, specifically addressing these key research inquiries: (1) Whether DPHCC has specific pathological characteristics distinct from CHCC; (2) Independent risk factors for poor prognosis of DPHCC and its association with cholangiocyte markers. This investigation seeks to establish a mechanistic framework for advancing diagnostic refinement, prognostic stratification, and personalized therapeutic strategies in DPHCC, and lay the foundation for exploring its molecular mechanism and the research on the classification of special subtypes of HCC. Materials and Methods Patients Consecutive patients who underwent curative hepatic resection for HCC at the 900th Hospital of the PLA Joint Logistic Support Force between January 2011 and December 2021 were retrospectively enrolled. The histological features of all specimens were independently reviewed and confirmed by two pathologists in accordance with the 2019 WHO Classification of Tumours of the Digestive System (5th edition; IARC, Lyon) [10] . The 2024 Chinese Guidelines for the Diagnosis and Treatment of Liver Cancer have established clear diagnostic criteria for DPHCC [11] . The inclusion criteria for this study were as follows:(1) availability of complete clinicopathological documentation with a minimum 24-month postoperative surveillance period; (2) radiologically confirmed absence of macrovascular invasion (portal/hepatic venous or biliary infiltration) and extrahepatic metastases prior to intervention; (3) absence of prior exposure to neoadjuvant anticancer therapies, specifically radiofrequency ablation (RFA), transarterial chemoembolization (TACE) or chemoradiation. Cases meeting DPHCC diagnostic thresholds were further defined by: (a) histopathological confirmation of HCC morphology, and (b) immunohistochemical co-expression of hepatocytic (HepPar-1/ Arg-1) and cholangiocytic (CK19/CK7/MUC1) markers in ≥ 15% of tumor cells [5] . Of 463 patients meeting the inclusion criteria (criteria 1–3), 93 fulfilled the DPHCC enrollment requirements following HCC resection.Exclusion criteria were consistent with those employed in previous studies and included patients with recurrent tumors, other malignant conditions, cases of combined intrahepatic cholangiocarcinoma, presence of extrahepatic metastases, or existence of other malignant tumors. Histopathological Classification Microvascular invasion (MVI) was stratified according to the 2015 Chinese Guidelines for Standardized Pathological Diagnosis of Primary Liver Cancer [12] . The MVI risk stratification was categorized as follows: M0 (no risk: absence of MVI), M1 (low risk: ≤5 MVI sites identified within adjacent liver tissue ≤ 1 cm from the tumor margin), and M2 (high risk: either > 5 MVI sites identified or any MVI presence in adjacent liver tissue > 1 cm from the tumor margin) [11] . Tumor size was determined by measuring the maximum diameter of the lesion. Tumor differentiation was classified according to the Edmondson-Steiner grading system, stratified into two groups: a poorly differentiated group (grades III + IV) and a moderately/well-differentiated group (grades I + II).Immunoreactivity scores (IRS) were employed to categorize marker expression levels. Case stratification additionally incorporated three critical histoprognostic parameters: Tumor lesions (single vs. multiple), tumor necrosis (positive vs negative), and capsular invasion (positive vs negative) . Follow-up Postoperative surveillance was implemented according to a phased schedule: monthly evaluations during the initial 30-day postoperative period, bimonthly to quarterly assessments through the second year, and semiannual monitoring thereafter. The surveillance intervals were dynamically tailored according to individual survival status and disease progression risk. Follow-up data were collected through outpatient clinical evaluations and routine telephone follow-ups that focused on symptom status, treatment adherence, and adverse events. Comprehensive post-treatment surveillance incorporated multimodal assessments, including: (1) cross-sectional imaging of the thoracoabdominal regions using triphasic dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) or contrast-enhanced computed tomography (CECT) for detecting anatomical abnormalities.; (2) serum biomarker profiling with quantitative alpha-fetoprotein (AFP) quantification using chemiluminescent immunoassay. Survival outcomes were analyzed using two primary endpoints: recurrence-free survival (RFS) was defined as the time interval from curative resection to radiologically or histologically confirmed tumor recurrence (assessed according to RECIST 1.1 criteria), while overall survival (OS) was calculated from the date of surgical intervention until all-cause mortality or the last documented follow-up. All survival data were right-censored at the study cutoff (December 2023) and analyzed via the Kaplan-Meier method to account for incomplete follow-up intervals. This integrated approach ensured systematic tracking of disease progression and objective quantification of oncologic outcomes. Tissue Processing and Immunohistochemical Evaluation All HCC specimens were fixed in 10% neutral buffered formalin (NBF, pH 7.4)) for 24–48 hours, processed using standard histological procedures, and paraffin-embedded. Consecutive 3-µm sections were cut with a rotary microtome (Leica RM2235) for hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis. IHC analysis was performed using the EnVision™ FLEX/HRP two-step system (Dako; Agilent Technologies) with 3,3′-diaminobenzidine (DAB) chromogen development. Primary antibodies were commercially obtained from Fuzhou Maixin Biotech Co., Ltd., with appropriate positive controls included in each staining batch. Samples were analyzed by light microscopy and IHC. The IHC panel included the following markers: Arg-1, HepPar-1, CK7, CK19, CD10, MUC-1, CD34, Ki67 and p53. Marker classifications were as follows: HepPar-1 and Arg-1 (hepatocyte differentiation markers); CK7, CK19, and MUC-1 (biliary lineage markers); CD34 (MVI indicator). Two pathologists independently evaluated the immunohistochemical slides using a validated semiquantitative scoring protocol in a blinded manner,and when consensus was not reached, average scores were used. IHC evaluation employed two semiquantitative parameters: (1) staining intensity graded on a 0–3 ordinal scale (0 = negative; 1 = weak; 2 = moderate; 3 = strong), and (2) positive cell proportion categorized into four tiers (0: ≤1%; 1: 2–25%; 2: 26–75%; 3: 76–100%). The immunoreactivity score (IRS) was derived by multiplying the intensity and proportion scores, yielding a 9-point composite scale (range 0–9). Specimens were dichotomized per consensus criteria into low expression (IRS 0–3) and high expression (IRS 4–9) groups. This dual-parameter scoring system ensures standardized quantification while accounting for both qualitative staining characteristics and quantitative cellular distribution patterns. Statistical Analysis Continuous variables were presented as mean±standard deviation or median (interquartile range). Categorical variables were analyzed using the Chi-square test or Fisher's exact test. Survival curves were generated with the Kaplan-Meier method, with between-group differences assessed by log-rank testing. Univariate and multivariate Cox proportional hazards regression models were employed to identify prognostic factors. All statistical tests utilized a two-tailed α level of 0.05 for significance determination. Analyses were performed using IBM SPSS Statistics (Version 26.0; IBM Corp., Armonk, NY, USA). Results Clinicopathological Characteristics A cohort of 463 HCC patients meeting the inclusion criteria was retrospectively analyzed. The study population comprised 370 cases (79.91%) with CHCC and 93 cases (20.09%) with DPHCC. The cohort's demographic and clinicopathological characteristics are detailed in Table 1. Patient ages ranged from 21 to 85 years (mean age ± SD: 55.68 ± 11.57 years; median: 56.0 years). Tumor sizes varied between 0.4cm and 25.0 cm (mean ± SD: 5.56 ± 3.93 cm; median:5.20cm), with 133 patients (28.73%) harboring tumors < 3 cm and 330 patients (71.27%) presenting tumors ≥ 3 cm. With regard to tumor multiplicity, 376 patients (81.20%) exhibited single lesions, while 87 patients (18.80%) had multiple. MVI stratification revealed 344 patients (74.30%) classified as M0 (no MVI), and 119 patients (25.70%) categorized into MVI risk subgroups (M1: low-risk; M2: high-risk). Histopathological grading demonstrated moderately to well-differentiated tumors (grades I-II) in 348 cases (75.16%) and poorly differentiated carcinomas (grades III-IV) in 115 cases (24.84%). Capsular invasion was identified in 375 patients (80.99%), contrasting with 88 cases (19.01%) lacking capsular involvement. Tumor necrosis was observed in 210 patients (45.36%), whereas 253 patients (54.64%) showed no necrotic features. Histopathologic Features and IHC In this study of 463 HCC cases, 370 (79.91%) demonstrated immunohistochemical expression of HCC-specific markers (e.g., Hepar-1 and/or Arg1) without cholangiocyte markers, defined as CHCC. The remaining 93 cases (20.09%) exhibited co-expression of HCC markers (e.g., Hepar-1 and/or Arg1) and marker (e.g., CK19/CK7/MUC1), classified as DPHCC (Fig. 1). Among DPHCC cases, CK19, CK7, and MUC-1 expression was observed in 62, 39 and 25 cases, respectively. According to the immunoreactivity score (IRS), CK19 expression was low in 49 cases (52.69%) and high in 44 cases (47.31%). Histomorphologically, DPHCC resembled CHCC, predominantly displaying solid, thick trabecular, or steatotic growth patterns, with variable nuclear atypia, eosinophilic cytoplasm, frequent mitotic figures (Fig. 2), and occasional necrosis. Both DPHCC and CHCC were associated with chronic hepatitis changes in the surrounding liver parenchyma. Comparative Analysis of Clinicopathological Characteristics in DPHCC and CHCC Patients The study cohort included 93 histologically confirmed DPHCC patients (male: female ratio 76:17; mean age 55.69 ± 11.60years) and 370 CHCC patients (male: female ratio 321:49; mean age55.65 ± 11.56years). Comparative analysis revealed significant intergroup disparities in clinicopathological parameters (all P < 0.05), as detailed in Table 2. Specifically, the DPHCC cohort exhibited higher prevalence of: MVI (44.09% vs. 21.08%), younger age (≤ 60 years) (74.19% vs. 57.84%), larger tumor size (≥ 3 cm) (80.65% vs. 68.92%), multiple tumor lesions (33.33% vs. 15.14%), poor histological differentiation (39.78% vs. 21.08%), necrosis- positivity (76.34% vs. 49.20%), capsular invasion (31.18% vs. 15.95%). Comparative Survival Outcomes in DPHCC versus CHCC Cohorts Kaplan-Meier analysis was performed to assess long-term prognosis in the HCC cohort, revealing significant survival disparities between the DPHCC and CHCC groups. Patients with DPHCC exhibited significantly poorer 5-year outcomes compared to CHCC cases: OS (61.10% vs 80.00%; P < 0.001) and RFS: (37.40% vs 55.70%; P = 0.015), as detailed (Fig. 3). To further delineate the prognostic impact of histological parameters in DPHCC, survival outcomes were stratified by pathological characteristics. Kaplan-Meier survival analysis identified six predictors of reduced survival: MVI-positive, tumor size ≥ 3 cm, multiple tumor lesions, poor differentiation (grades III-IV), necrosis positivity, and cytokeratin 19 (CK19) overexpression (all P < 0.05, Fig. 4). Prognostic factors for OS and RFS Variables attaining statistical significance (P < 0.05) in univariate analysis were retained for multivariate Cox proportional hazards regression modeling. Univariate analysis identified DPHCC as a significant prognostic factor for both survival endpoints: OS (HR 2.11, 95% CI 1.41–3.18; P < 0.001) and RFS (HR 1.48, 95% CI 1.08–2.03; P = 0.016 ) (Tables3-4). In the DPHCC subgroup analysis, three independent predictors of OS emerged: tumor diameter ≥ 3 cm (HR 5.85, 95% CI 1.25–27.38; P = 0.025), poorly differentiated histology (HR 2.37, 95% CI 1.10–5.08; P = 0.027), and CK19 over expression (HR 4.90, 95% CI 1.90-12.62; P = 0.001)(Table 5). Additionally, tumor size ≥3cm (HR 3.554, 95% CI 1.150-10.986, P = 0.028) and poor differentiation (HR 1.911, 95% CI 1.044–3.496, P = 0.036) independently predicted diminished RFS in this cohort (Table 6). Discussion This study conducted a retrospective analysis of clinicopathological characteristics and survival data from 463 patients with HCC,. It systematically compared the biological behavior differences between DPHCC and HCC, clarified the clinicopathological profile of DPHCC as a distinct subtype in a large cohort, and for the first time identified key prognostic risk factors—including MVI and CK19—within the DPHCC subgroup. In HCC, DPHCC, as a subtype of HCC with unique biological behaviors, has significantly different clinicopathological characteristics from CHCC. DPHCC maintains histopathological features consistent with HCC and is diagnosed when tumors exhibit singular HCC morphological characteristics with > 15% of neoplastic cells co-expressing both HCC and ICC protein markers [5] . Despite exhibiting a significantly lower incidence rate compared to CHCC, DPHCC manifests distinct biphenotypic characteristics that encompass both hepatocytic and cholangiocytic differentiation patterns. This unique molecular profile correlates with enhanced biological aggressiveness, as evidenced by accelerated metastatic progression, heightened vascular invasion potential, and significantly reduced overall survival rates when compared to other HCC subtypes [13, 14] .In the present cohort of 463 histologically confirmed HCC cases, DPHCC constituted 20.09% (93/463) of the total HCC population, consistent with the documented 10%-20% proportion of DPHCC among HCC cases reported in previous literature [13, 14] . Our findings demonstrated that DPHCC patients exhibited significantly higher proportions of multiple aggressive histopathological features compared to CHCC, including MVI, tumor size ≥ 3 cm, multiple tumor lesions, poor differentiation, tumor necrosis, and capsular invasion. These characteristics collectively suggest that DPHCC may possess enhanced local invasiveness and metastatic potential, a conclusion aligned with prior investigations [6, 15, 16] . The observed discrepancies reflect multifaceted differences in biological behavior and malignant progression mechanisms between DPHCC and CHCC. Primary analysis revealed that DPHCC demonstrates significantly elevated MVI prevalence .This study revealed that DPHCC demonstrated a significantly higher MVI incidence rate of 44.09% compared to 21.08% in CHCC controls (p < 0.001), indicating its enhanced invasive capacity. As a critical pathological process involving tumor cell penetration through basement membranes into vascular systems, In HCC MVI has been consistently recognized as an independent prognostic factor for both intrahepatic metastasis and postoperative recurrence [17–19] .However, existing MVI research predominantly focuses on CHCC, with scarce investigations addressing DPHCC-specific mechanisms. Our preliminary investigations similarly identified MVI as an independent prognostic determinant in DPHCC [20] . The elevated MVI prevalence in DPHCC may stem from its unique cellular biology. The biphenotypic differentiation pattern - simultaneous hepatocellular and cholangiocytic characteristics - potentially drives extracellular matrix remodeling and upregulates pro-angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF specifically promotes endothelial cell proliferation, migration, and tubulogenesis, thereby facilitating vascular infiltration and creating a permissive microenvironment for intrahepatic dissemination and recurrence in DPHCC [21] . Nevertheless, the molecular mechanisms underlying MVI development in DPHCC remain poorly characterized, warranting systematic exploration through integrated multi-omics approaches. Furthermore, DPHCC exhibits more aggressive clinicopathological characteristics, characterized by larger tumor dimensions, higher rates of multifocality, increased prevalence of poor differentiation, and greater susceptibility to necrotic degeneration. Regarding tumor size and multiplicity, the DPHCC cohort demonstrated a significantly higher proportion of lesions ≥ 3 cm (80.65% vs. 68.92%) and multifocal presentations (33.33% vs. 15.14%) compared to CHCC controls. These findings strongly suggest accelerated tumor growth kinetics in DPHCC. When combined with its elevated rates of poorly differentiated histology (39.78% vs. 21.08%) and necrotic foci (76.34% vs. 49.20%), the collective data imply that DPHCC may harbor heightened genomic instability or epigenetic dysregulation. Such molecular aberrations likely drive uncontrolled cellular proliferation and tumor microenvironment remodeling, potentially through disruption of differentiation programs and aberrant activation of pro-survival signaling pathways. Survival analysis in this study revealed significantly inferior 5-year OS (61.10% vs. 80.00%) and RFS(: 37.40% vs. 55.70%) rates in DPHCC patients compared to CHCC counterparts, further substantiating its clinical significance as a distinct poor-prognosis subtype (OS: HR = 2.113; RFS: HR = 1.479). Six clinicopathological parameters–MVI positivity, tumor diameter ≥ 3 cm, multifocal lesions, poor differentiation (grade III-IV), necrotic foci, and CK19 overexpression–demonstrated significant associations with reduced OS and RFS. Multivariate Cox regression identified tumor diameter ≥ 3 cm, poor differentiation, and CK19 overexpression as independent prognostic determinants for OS, while tumor size ≥ 3 cm and poor differentiation concurrently impacted RFS. DPHCC patients exhibiting these three risk factors warrant stratification into a high-risk surveillance cohort with intensified imaging follow-up intervals and monitoring of serum AFP levels. Notably, CK19 overexpression demonstrated the most significant prognostic value for OS (HR = 4.903), which may be associated with its mediation of cholangiocyte-like differentiation phenotypes or its role in promoting MV development [22, 23] . CK19 is a well-established marker for both biliary/progenitor cells and cancer stem cells. [24–26] . critically potentiates HCC malignancy. Cumulative evidence correlates CK19 positivity with adverse outcomes, including elevated recurrence rates and diminished long-term survival [27–29] . Mechanistically, CK19 positive HCC cells acquire migratory and invasive capacities via EMT activation (e.g., E-cadherin downregulation, N-cadherin/Vimentin upregulation), with KRT19 knockdown significantly attenuating invasiveness and EMT marker expression [30] . Further investigations reveal KRT19-mediated tumor vasculature remodeling through angiogenic gene regulation (FGFR1/VASH1 upregulation, VASH2 suppression), facilitating MVI development [22] . Of critical relevance, KRT19 orchestrates epigenetic dedifferentiation by forming complexes with HDAC1 and COREST to suppress histone acetylation at hepatocyte differentiation gene promoters, thereby maintaining stem-like plasticity [31] . Our findings underscore CK19's dual role as both prognostic biomarker and therapeutic target in DPHCC management strategies. Finally, compared with the CHCC controls, the DPHCC cohort in this study had a significantly higher proportion of patients under 60 years old (74.19% vs. 57.84%, P < 0.05), aligning with previous reports on the age-specific epidemiology of DPHCC [29, 32] . This demographic divergence suggests distinct pathogenic mechanisms underlying DPHCC development. A prevailing hypothesis posits that DPHCC originates from aberrant differentiation of hepatic progenitor cells (HPCs), which remain quiescent under physiological conditions but activate proliferative and bipotential differentiation programs (hepatocytic/cholangiocytic lineages) during hepatic injury repair. In younger individuals, HPCs retain heightened proliferative plasticity and differentiation competence. Chronic hepatic insults, persistent inflammatory stimuli, or carcinogen exposure may drive these hyperactive HPCs toward malignant transdifferentiation, ultimately culminating in DPHCC pathogenesis.Moreover, the robust immune activity characteristic of younger hosts paradoxically creates a selective pressure favoring DPHCC progression. DPHCC cells likely employ advanced immune evasion strategies – potentially through PD-L1 upregulation, MHC class I downregulation, or immunosuppressive cytokine secretion – to circumvent immune surveillance within this immunologically dynamic microenvironment. Such adaptations confer a survival advantage, predisposing younger populations to DPHCC incidence. However, the precise mechanisms underlying this age-related predisposition require systematic investigation through lineage-tracing models, immune profiling, and comparative multi-omics analyses of age-stratified DPHCC cohorts. A critical methodological consideration in this study is the definition of DPHCC, which relies on a > 15% co-expression threshold for biliary markers, a criterion initially proposed by Lu et al. and adopted here for operational consistency with certain regional studies. It must be acknowledged, however, that this cut-off value lacks broad international validation and is not universally accepted. Variations in diagnostic thresholds—for instance, the use of > 5% or > 10% in other research—likely contribute to the heterogeneity in reported incidence rates and prognostic associations of DPHCC across the literature. This inconsistency underscores a significant challenge in comparing findings between studies and highlights the urgent need for an international consensus on the standardized pathological criteria for diagnosing DPHCC. Future collaborative efforts should focus on establishing evidence-based, reproducible definitions, possibly incorporating molecular correlates, to ensure improved classification consistency and prognostic accuracy across diverse cohorts. Conclusion In summary, DPHCC represents a distinct subtype of HCC marked by an aggressive nature, poor differentiation, and poor prognosis. Its prognosis is influenced by tumor size, differentiation status, and CK19 expression; clinicians should consider establishing intensified surveillance protocols and implementing enhanced follow-up strategies for patients diagnosed with this aggressive HCC subtype to address its propensity for rapid progression and high recurrence rates.This study addresses dual-phenotype hepatocellular carcinoma (DPHCC), a clinically significant yet historically underrecognized subtype of liver cancer. Despite its distinctive immunophenotypic profile—concurrent expression of both hepatocellular and cholangiocytic markers—which suggests unique tumor biology, and multiple reports associating it with more aggressive behavior and poorer clinical outcomes, DPHCC has not received sufficient attention in international diagnostic guidelines, clinical management consensus, or translational research. This neglect largely stems from the lack of standardized diagnostic criteria and an incomplete understanding of its clinicopathological and molecular basis. Through a systematic analysis of a large single-center cohort, our study further confirms that DPHCC patients exhibit more aggressive tumor features (such as higher rates of microvascular invasion and lymph node metastasis) and shortened survival. These findings strongly underscore the clinical necessity of distinguishing DPHCC from other types of HCC. Our work not only deepens the understanding of the clinicopathological characteristics of this distinct entity but, more importantly, calls for heightened awareness among researchers and pathologists to recognize and report DPHCC in routine diagnostic practice. Promoting in-depth molecular mechanistic studies and ultimately establishing internationally unified diagnostic guidelines are of paramount importance for developing more targeted therapeutic strategies and improving outcomes for this high-risk patient population. Abbreviations DPHCC Dual-phenotype hepatocellular carcinoma HCC hepatocellular carcinoma CHCC classical hepatocellular carcinoma CK19 cytokeratin 19 MVI microvascular invasion CK7 cytokeratin 7 MUC1 mucin1 OS Overall survival RFS Recurrence-free survival HR hazard ratio CI confidence interval. Declarations Ethics approval and consent to participate Ethical approval for this study was granted by the Institutional Review Board of the 900th Hospital of PLA Joint Logistic Support Force (Approval No. 2019-18) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all participants or their legally authorized representatives. Competing interests The authors declare no competing interests. Funding Author Contribution Xiaojuan Ouyang conducted the research, collected experimental data, performed statistical analyses, and drafted the manuscript; Yongqin Yan conceptualized the study design, interpreted analytical results, obtained research funding, and critically revised the manuscript for substantive intellectual content; Bohao Hu and Xinjing Gao assisted in data collection; Xingfeng Qi assisted in performing the Immunohistochemical experiments.Lijuan Qu contributed administrative coordination, technical assistance, and material resources;Xianzong Ye and Haicong Wu contributed to critical appraisal of the scholarly content, and all authors reviewed and approved the final manuscript prior to submission. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Bray F, Laversanne M, Sung H, et al. 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Targeting PELP1 Attenuates Angiogenesis and Enhances Chemotherapy Efficiency in Colorectal Cancer[J]. Cancers (Basel), 2022,14(2). Fatourou E, Koskinas J, Karandrea D, et al. Keratin 19 protein expression is an independent predictor of survival in human hepatocellular carcinoma[J]. Eur J Gastroenterol Hepatol, 2015,27(9):1094–1102. Zhuo J, Lu D, Tan W, et al. CK19-positive Hepatocellular Carcinoma is a Characteristic Subtype[J]. J Cancer, 2020,11(17):5069–5077. Yang C, Song R, Hu J, et al. Integrating single-cell and bulk RNA sequencing reveals CK19 + cancer stem cells and their specific SPP1 + tumor-associated macrophage niche in HBV-related hepatocellular carcinoma[J]. Hepatol Int, 2024,18(1):73–90. Mishra L, Banker T, Murray J, et al. Liver stem cells and hepatocellular carcinoma[J]. Hepatology, 2009,49(1):318–329. Chiba T, Kamiya A, Yokosuka O, et al. Cancer stem cells in hepatocellular carcinoma: Recent progress and perspective[J]. Cancer Lett, 2009,286(2):145–153. Qin S, Zhang J, Qi Y, et al. Individual and joint influence of cytokeratin 19 and microvascular invasion on the prognosis of patients with hepatocellular carcinoma after hepatectomy[J]. World J Surg Oncol, 2022,20(1):209. Sun D, Zhang Y, Sun X, et al. Prognostic value of cytokeratin 19 in hepatocellular carcinoma: A meta-analysis[J]. Clin Chim Acta, 2015,448:161–169. Yang C, Xiang W, Wu Z, et al. CK19 protein expression: the best cutoff value on the prognosis and the prognosis model of hepatocellular carcinoma[J]. BMC Cancer, 2025,25(1):55. Kim H, Choi G H, Na D C, et al. Human hepatocellular carcinomas with "Stemness"-related marker expression: keratin 19 expression and a poor prognosis[J]. Hepatology, 2011,54(5):1707–1717. Han S, Fan H, Zhong G, et al. Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis[J]. Hepatology, 2025,81(3):808–822. Zhang Q, Xing W, Zhang J, et al. Circulating Tumor Cells Undergoing the Epithelial-Mesenchymal Transition: Influence on Prognosis in Cytokeratin 19-Positive Hepatocellular Carcinoma[J]. Onco Targets Ther, 2021,14:1543–1552. Tables Tables 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files table1.xlsx table2.xlsx table3.xlsx table4.xlsx table5.xlsx table6.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 26 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9029042","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619227923,"identity":"4e77a88d-758a-4f8f-9a13-d8dd6de8e676","order_by":0,"name":"Xiaojuan 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07:42:07","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12497,"visible":true,"origin":"","legend":"","description":"","filename":"table6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9029042/v1/e7d90382652973465cab0a74.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual-Phenotype Hepatocellular Carcinoma Exhibits Distinct Clinicopathological Aggressiveness and Poor Prognosis: A Retrospective Cohort Study of 463 Hepatocellular Carcinoma Patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC), one of the most common cancers worldwide, continues to pose a significant global health threat due to its persistently high incidence and mortality rates\u003csup\u003e[1]\u003c/sup\u003e. In recent years, a new histological subtype of HCC with stronger invasivity and poorer prognosis has been reported, namely Dual-phenotype hepatocellular carcinoma(DPHCC)\u003csup\u003e[2, 3]\u003c/sup\u003e. DPHCC, a distinct clinicopathological subtype defined by the co-expression of hepatocellular (e.g., HepPar-1/ Arg-1) and cholangiocytic (e.g., CK19/CK7/MUC1) immunophenotypic markers, has emerged as a growing focus in hepatobiliary oncology research\u003csup\u003e[4]\u003c/sup\u003e. According to diagnostic criteria, DPHCC is defined as tumors exhibiting typical HCC histomorphology with co-expression of HCC- and ICC-associated immunophenotypic markers in more than 15% of tumor cells\u003csup\u003e[5]\u003c/sup\u003e. Although DPHCC accounts for only 10% of HCC cases\u003csup\u003e[5]\u003c/sup\u003e, clinical observations indicate that DPHCC patients are prone to earlier vascular invasion, higher postoperative recurrence rates, and poorer survival outcomes\u003csup\u003e[6]\u003c/sup\u003e, underscoring its distinct biological behavior and unmet clinical management needs.\u003c/p\u003e \u003cp\u003eCurrent understanding of DPHCC remains limited, with significant knowledge gaps persisting in both its clinicopathological features and molecular pathogenesis. While the 2024 Chinese Clinical Practice Guidelines for Liver Cancer have established standardized diagnostic criteria for DPHCC, it has not yet been recognized by experts worldwide and there are no international diagnostic criteria. Second, whether the enhanced aggressiveness of DPHCC compared to classical HCC (CHCC) correlates with specific clinicopathological characteristics, such as microvascular invasion (MVI) or tumor differentiation grade, requires validation in large-scale cohorts. Furthermore, the identification of prognostic risk factors for DPHCC and their mechanistic links to cholangiocyte markers (e.g., CK19) remain unclear. Although some studies propose that DPHCC may originate from aberrant differentiation of hepatic progenitor cells (HPCs), emerging experimental evidence indicates that hypoxic microenvironments may drive phenotypic transdifferentiation from conventional HCC to DPHCC through hypoxia-inducible factor (HIF)-mediated pathways\u003csup\u003e[7\u0026ndash;9]\u003c/sup\u003e, this hypothesis lacks robust clinical evidence. Importantly, existing studies are predominantly constrained by small sample sizes or single-center designs, leading to insufficient evidence-based strategies for DPHCC management. Therefore, comprehensive analyses of clinicopathological characteristics and prognostic models based on large cohorts are urgently needed.\u003c/p\u003e \u003cp\u003eBuilding upon this scientific foundation, our investigation conducted a retrospective analysis of 463 HCC cases to perform a comprehensive comparative evaluation of clinicopathological profiles and survival outcomes between DPHCC and CHCC, specifically addressing these key research inquiries: (1) Whether DPHCC has specific pathological characteristics distinct from CHCC; (2) Independent risk factors for poor prognosis of DPHCC and its association with cholangiocyte markers. This investigation seeks to establish a mechanistic framework for advancing diagnostic refinement, prognostic stratification, and personalized therapeutic strategies in DPHCC, and lay the foundation for exploring its molecular mechanism and the research on the classification of special subtypes of HCC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eConsecutive patients who underwent curative hepatic resection for HCC at the 900th Hospital of the PLA Joint Logistic Support Force between January 2011 and December 2021 were retrospectively enrolled. The histological features of all specimens were independently reviewed and confirmed by two pathologists in accordance with the 2019 WHO Classification of Tumours of the Digestive System (5th edition; IARC, Lyon)\u003csup\u003e[10]\u003c/sup\u003e. The 2024 Chinese Guidelines for the Diagnosis and Treatment of Liver Cancer have established clear diagnostic criteria for DPHCC\u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe inclusion criteria for this study were as follows:(1) availability of complete clinicopathological documentation with a minimum 24-month postoperative surveillance period; (2) radiologically confirmed absence of macrovascular invasion (portal/hepatic venous or biliary infiltration) and extrahepatic metastases prior to intervention; (3) absence of prior exposure to neoadjuvant anticancer therapies, specifically radiofrequency ablation (RFA), transarterial chemoembolization (TACE) or chemoradiation. Cases meeting DPHCC diagnostic thresholds were further defined by: (a) histopathological confirmation of HCC morphology, and (b) immunohistochemical co-expression of hepatocytic (HepPar-1/ Arg-1) and cholangiocytic (CK19/CK7/MUC1) markers in \u0026ge;\u0026thinsp;15% of tumor cells\u003csup\u003e[5]\u003c/sup\u003e. Of 463 patients meeting the inclusion criteria (criteria 1\u0026ndash;3), 93 fulfilled the DPHCC enrollment requirements following HCC resection.Exclusion criteria were consistent with those employed in previous studies and included patients with recurrent tumors, other malignant conditions, cases of combined intrahepatic cholangiocarcinoma, presence of extrahepatic metastases, or existence of other malignant tumors.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological Classification\u003c/h3\u003e\n\u003cp\u003eMicrovascular invasion (MVI) was stratified according to the 2015 Chinese Guidelines for Standardized Pathological Diagnosis of Primary Liver Cancer\u003csup\u003e[12]\u003c/sup\u003e. The MVI risk stratification was categorized as follows: M0 (no risk: absence of MVI), M1 (low risk: \u0026le;5 MVI sites identified within adjacent liver tissue\u0026thinsp;\u0026le;\u0026thinsp;1 cm from the tumor margin), and M2 (high risk: either \u0026gt;\u0026thinsp;5 MVI sites identified or any MVI presence in adjacent liver tissue\u0026thinsp;\u0026gt;\u0026thinsp;1 cm from the tumor margin)\u003csup\u003e[11]\u003c/sup\u003e. Tumor size was determined by measuring the maximum diameter of the lesion. Tumor differentiation was classified according to the Edmondson-Steiner grading system, stratified into two groups: a poorly differentiated group (grades III\u0026thinsp;+\u0026thinsp;IV) and a moderately/well-differentiated group (grades I\u0026thinsp;+\u0026thinsp;II).Immunoreactivity scores (IRS) were employed to categorize marker expression levels. Case stratification additionally incorporated three critical histoprognostic parameters: Tumor lesions (single vs. multiple), tumor necrosis (positive vs negative), and capsular invasion (positive vs negative) .\u003c/p\u003e\n\u003ch3\u003eFollow-up\u003c/h3\u003e\n\u003cp\u003ePostoperative surveillance was implemented according to a phased schedule: monthly evaluations during the initial 30-day postoperative period, bimonthly to quarterly assessments through the second year, and semiannual monitoring thereafter. The surveillance intervals were dynamically tailored according to individual survival status and disease progression risk. Follow-up data were collected through outpatient clinical evaluations and routine telephone follow-ups that focused on symptom status, treatment adherence, and adverse events.\u003c/p\u003e \u003cp\u003eComprehensive post-treatment surveillance incorporated multimodal assessments, including: (1) cross-sectional imaging of the thoracoabdominal regions using triphasic dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) or contrast-enhanced computed tomography (CECT) for detecting anatomical abnormalities.; (2) serum biomarker profiling with quantitative alpha-fetoprotein (AFP) quantification using chemiluminescent immunoassay. Survival outcomes were analyzed using two primary endpoints: recurrence-free survival (RFS) was defined as the time interval from curative resection to radiologically or histologically confirmed tumor recurrence (assessed according to RECIST 1.1 criteria), while overall survival (OS) was calculated from the date of surgical intervention until all-cause mortality or the last documented follow-up. All survival data were right-censored at the study cutoff (December 2023) and analyzed via the Kaplan-Meier method to account for incomplete follow-up intervals. This integrated approach ensured systematic tracking of disease progression and objective quantification of oncologic outcomes.\u003c/p\u003e\n\u003ch3\u003eTissue Processing and Immunohistochemical Evaluation\u003c/h3\u003e\n\u003cp\u003eAll HCC specimens were fixed in 10% neutral buffered formalin (NBF, pH 7.4)) for 24\u0026ndash;48 hours, processed using standard histological procedures, and paraffin-embedded. Consecutive 3-\u0026micro;m sections were cut with a rotary microtome (Leica RM2235) for hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemical (IHC) analysis. IHC analysis was performed using the EnVision\u0026trade; FLEX/HRP two-step system (Dako; Agilent Technologies) with 3,3\u0026prime;-diaminobenzidine (DAB) chromogen development. Primary antibodies were commercially obtained from Fuzhou Maixin Biotech Co., Ltd., with appropriate positive controls included in each staining batch. Samples were analyzed by light microscopy and IHC. The IHC panel included the following markers: Arg-1, HepPar-1, CK7, CK19, CD10, MUC-1, CD34, Ki67 and p53. Marker classifications were as follows: HepPar-1 and Arg-1 (hepatocyte differentiation markers); CK7, CK19, and MUC-1 (biliary lineage markers); CD34 (MVI indicator).\u003c/p\u003e \u003cp\u003eTwo pathologists independently evaluated the immunohistochemical slides using a validated semiquantitative scoring protocol in a blinded manner,and when consensus was not reached, average scores were used. IHC evaluation employed two semiquantitative parameters: (1) staining intensity graded on a 0\u0026ndash;3 ordinal scale (0\u0026thinsp;=\u0026thinsp;negative; 1\u0026thinsp;=\u0026thinsp;weak; 2\u0026thinsp;=\u0026thinsp;moderate; 3\u0026thinsp;=\u0026thinsp;strong), and (2) positive cell proportion categorized into four tiers (0: \u0026le;1%; 1: 2\u0026ndash;25%; 2: 26\u0026ndash;75%; 3: 76\u0026ndash;100%). The immunoreactivity score (IRS) was derived by multiplying the intensity and proportion scores, yielding a 9-point composite scale (range 0\u0026ndash;9). Specimens were dichotomized per consensus criteria into low expression (IRS 0\u0026ndash;3) and high expression (IRS 4\u0026ndash;9) groups. This dual-parameter scoring system ensures standardized quantification while accounting for both qualitative staining characteristics and quantitative cellular distribution patterns.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eContinuous variables were presented as mean\u0026plusmn;standard deviation or median (interquartile range). Categorical variables were analyzed using the Chi-square test or Fisher's exact test. Survival curves were generated with the Kaplan-Meier method, with between-group differences assessed by log-rank testing. Univariate and multivariate Cox proportional hazards regression models were employed to identify prognostic factors. All statistical tests utilized a two-tailed α level of 0.05 for significance determination. Analyses were performed using IBM SPSS Statistics (Version 26.0; IBM Corp., Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinicopathological Characteristics\u003c/h2\u003e \u003cp\u003eA cohort of 463 HCC patients meeting the inclusion criteria was retrospectively analyzed. The study population comprised 370 cases (79.91%) with CHCC and 93 cases (20.09%) with DPHCC. The cohort's demographic and clinicopathological characteristics are detailed in Table\u0026nbsp;1. Patient ages ranged from 21 to 85 years (mean age\u0026thinsp;\u0026plusmn;\u0026thinsp;SD: 55.68\u0026thinsp;\u0026plusmn;\u0026thinsp;11.57 years; median: 56.0 years). Tumor sizes varied between 0.4cm and 25.0 cm (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD: 5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93 cm; median:5.20cm), with 133 patients (28.73%) harboring tumors\u0026thinsp;\u0026lt;\u0026thinsp;3 cm and 330 patients (71.27%) presenting tumors\u0026thinsp;\u0026ge;\u0026thinsp;3 cm. With regard to tumor multiplicity, 376 patients (81.20%) exhibited single lesions, while 87 patients (18.80%) had multiple. MVI stratification revealed 344 patients (74.30%) classified as M0 (no MVI), and 119 patients (25.70%) categorized into MVI risk subgroups (M1: low-risk; M2: high-risk). Histopathological grading demonstrated moderately to well-differentiated tumors (grades I-II) in 348 cases (75.16%) and poorly differentiated carcinomas (grades III-IV) in 115 cases (24.84%). Capsular invasion was identified in 375 patients (80.99%), contrasting with 88 cases (19.01%) lacking capsular involvement. Tumor necrosis was observed in 210 patients (45.36%), whereas 253 patients (54.64%) showed no necrotic features.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathologic Features and IHC\u003c/h3\u003e\n\u003cp\u003eIn this study of 463 HCC cases, 370 (79.91%) demonstrated immunohistochemical expression of HCC-specific markers (e.g., Hepar-1 and/or Arg1) without cholangiocyte markers, defined as CHCC. The remaining 93 cases (20.09%) exhibited co-expression of HCC markers (e.g., Hepar-1 and/or Arg1) and marker (e.g., CK19/CK7/MUC1), classified as DPHCC (Fig.\u0026nbsp;1). Among DPHCC cases, CK19, CK7, and MUC-1 expression was observed in 62, 39 and 25 cases, respectively. According to the immunoreactivity score (IRS), CK19 expression was low in 49 cases (52.69%) and high in 44 cases (47.31%).\u003c/p\u003e \u003cp\u003eHistomorphologically, DPHCC resembled CHCC, predominantly displaying solid, thick trabecular, or steatotic growth patterns, with variable nuclear atypia, eosinophilic cytoplasm, frequent mitotic figures (Fig.\u0026nbsp;2), and occasional necrosis. Both DPHCC and CHCC were associated with chronic hepatitis changes in the surrounding liver parenchyma.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparative Analysis of Clinicopathological Characteristics in DPHCC and CHCC Patients\u003c/h2\u003e \u003cp\u003eThe study cohort included 93 histologically confirmed DPHCC patients (male: female ratio 76:17; mean age 55.69\u0026thinsp;\u0026plusmn;\u0026thinsp;11.60years) and 370 CHCC patients (male: female ratio 321:49; mean age55.65\u0026thinsp;\u0026plusmn;\u0026thinsp;11.56years). Comparative analysis revealed significant intergroup disparities in clinicopathological parameters (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as detailed in Table\u0026nbsp;2. Specifically, the DPHCC cohort exhibited higher prevalence of: MVI (44.09% vs. 21.08%), younger age (\u0026le;\u0026thinsp;60 years) (74.19% vs. 57.84%), larger tumor size (\u0026ge;\u0026thinsp;3 cm) (80.65% vs. 68.92%), multiple tumor lesions (33.33% vs. 15.14%), poor histological differentiation (39.78% vs. 21.08%), necrosis- positivity (76.34% vs. 49.20%), capsular invasion (31.18% vs. 15.95%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparative Survival Outcomes in DPHCC versus CHCC Cohorts\u003c/h2\u003e \u003cp\u003eKaplan-Meier analysis was performed to assess long-term prognosis in the HCC cohort, revealing significant survival disparities between the DPHCC and CHCC groups. Patients with DPHCC exhibited significantly poorer 5-year outcomes compared to CHCC cases: OS (61.10% vs 80.00%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and RFS: (37.40% vs 55.70%; P\u0026thinsp;=\u0026thinsp;0.015), as detailed (Fig.\u0026nbsp;3). To further delineate the prognostic impact of histological parameters in DPHCC, survival outcomes were stratified by pathological characteristics. Kaplan-Meier survival analysis identified six predictors of reduced survival: MVI-positive, tumor size\u0026thinsp;\u0026ge;\u0026thinsp;3 cm, multiple tumor lesions, poor differentiation (grades III-IV), necrosis positivity, and cytokeratin 19 (CK19) overexpression (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrognostic factors for OS and RFS\u003c/h2\u003e \u003cp\u003eVariables attaining statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in univariate analysis were retained for multivariate Cox proportional hazards regression modeling. Univariate analysis identified DPHCC as a significant prognostic factor for both survival endpoints: OS (HR 2.11, 95% CI 1.41\u0026ndash;3.18; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and RFS (HR 1.48, 95% CI 1.08\u0026ndash;2.03; P\u0026thinsp;=\u0026thinsp;0.016\u003cb\u003e)\u003c/b\u003e(Tables3-4). In the DPHCC subgroup analysis, three independent predictors of OS emerged: tumor diameter\u0026thinsp;\u0026ge;\u0026thinsp;3 cm (HR 5.85, 95% CI 1.25\u0026ndash;27.38; P\u0026thinsp;=\u0026thinsp;0.025), poorly differentiated histology (HR 2.37, 95% CI 1.10\u0026ndash;5.08; P\u0026thinsp;=\u0026thinsp;0.027), and CK19 over expression (HR 4.90, 95% CI 1.90-12.62; P\u0026thinsp;=\u0026thinsp;0.001)(Table\u0026nbsp;5). Additionally, tumor size \u0026ge;3cm (HR 3.554, 95% CI 1.150-10.986, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) and poor differentiation (HR 1.911, 95% CI 1.044\u0026ndash;3.496, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036) independently predicted diminished RFS in this cohort (Table\u0026nbsp;6).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study conducted a retrospective analysis of clinicopathological characteristics and survival data from 463 patients with HCC,. It systematically compared the biological behavior differences between DPHCC and HCC, clarified the clinicopathological profile of DPHCC as a distinct subtype in a large cohort, and for the first time identified key prognostic risk factors\u0026mdash;including MVI and CK19\u0026mdash;within the DPHCC subgroup.\u003c/p\u003e\n\u003cp\u003eIn HCC, DPHCC, as a subtype of HCC with unique biological behaviors, has significantly different clinicopathological characteristics from CHCC. DPHCC maintains histopathological features consistent with HCC and is diagnosed when tumors exhibit singular HCC morphological characteristics with \u0026gt;\u0026thinsp;15% of neoplastic cells co-expressing both HCC and ICC protein markers\u003csup\u003e[5]\u003c/sup\u003e. Despite exhibiting a significantly lower incidence rate compared to CHCC, DPHCC manifests distinct biphenotypic characteristics that encompass both hepatocytic and cholangiocytic differentiation patterns. This unique molecular profile correlates with enhanced biological aggressiveness, as evidenced by accelerated metastatic progression, heightened vascular invasion potential, and significantly reduced overall survival rates when compared to other HCC subtypes\u003csup\u003e[13, 14]\u003c/sup\u003e.In the present cohort of 463 histologically confirmed HCC cases, DPHCC constituted 20.09% (93/463) of the total HCC population, consistent with the documented 10%-20% proportion of DPHCC among HCC cases reported in previous literature\u003csup\u003e[13, 14]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur findings demonstrated that DPHCC patients exhibited significantly higher proportions of multiple aggressive histopathological features compared to CHCC, including MVI, tumor size\u0026thinsp;\u0026ge;\u0026thinsp;3 cm, multiple tumor lesions, poor differentiation, tumor necrosis, and capsular invasion. These characteristics collectively suggest that DPHCC may possess enhanced local invasiveness and metastatic potential, a conclusion aligned with prior investigations\u003csup\u003e[6, 15, 16]\u003c/sup\u003e. The observed discrepancies reflect multifaceted differences in biological behavior and malignant progression mechanisms between DPHCC and CHCC.\u003c/p\u003e\n\u003cp\u003ePrimary analysis revealed that DPHCC demonstrates significantly elevated MVI prevalence .This study revealed that DPHCC demonstrated a significantly higher MVI incidence rate of 44.09% compared to 21.08% in CHCC controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating its enhanced invasive capacity. As a critical pathological process involving tumor cell penetration through basement membranes into vascular systems, In HCC MVI has been consistently recognized as an independent prognostic factor for both intrahepatic metastasis and postoperative recurrence\u003csup\u003e[17\u0026ndash;19]\u003c/sup\u003e.However, existing MVI research predominantly focuses on CHCC, with scarce investigations addressing DPHCC-specific mechanisms. Our preliminary investigations similarly identified MVI as an independent prognostic determinant in DPHCC\u003csup\u003e[20]\u003c/sup\u003e. The elevated MVI prevalence in DPHCC may stem from its unique cellular biology. The biphenotypic differentiation pattern - simultaneous hepatocellular and cholangiocytic characteristics - potentially drives extracellular matrix remodeling and upregulates pro-angiogenic factors such as vascular endothelial growth factor (VEGF). VEGF specifically promotes endothelial cell proliferation, migration, and tubulogenesis, thereby facilitating vascular infiltration and creating a permissive microenvironment for intrahepatic dissemination and recurrence in DPHCC\u003csup\u003e[21]\u003c/sup\u003e. Nevertheless, the molecular mechanisms underlying MVI development in DPHCC remain poorly characterized, warranting systematic exploration through integrated multi-omics approaches.\u003c/p\u003e\n\u003cp\u003eFurthermore, DPHCC exhibits more aggressive clinicopathological characteristics, characterized by larger tumor dimensions, higher rates of multifocality, increased prevalence of poor differentiation, and greater susceptibility to necrotic degeneration. Regarding tumor size and multiplicity, the DPHCC cohort demonstrated a significantly higher proportion of lesions\u0026thinsp;\u0026ge;\u0026thinsp;3 cm (80.65% vs. 68.92%) and multifocal presentations (33.33% vs. 15.14%) compared to CHCC controls. These findings strongly suggest accelerated tumor growth kinetics in DPHCC. When combined with its elevated rates of poorly differentiated histology (39.78% vs. 21.08%) and necrotic foci (76.34% vs. 49.20%), the collective data imply that DPHCC may harbor heightened genomic instability or epigenetic dysregulation. Such molecular aberrations likely drive uncontrolled cellular proliferation and tumor microenvironment remodeling, potentially through disruption of differentiation programs and aberrant activation of pro-survival signaling pathways.\u003c/p\u003e\n\u003cp\u003eSurvival analysis in this study revealed significantly inferior 5-year OS (61.10% vs. 80.00%) and RFS(: 37.40% vs. 55.70%) rates in DPHCC patients compared to CHCC counterparts, further substantiating its clinical significance as a distinct poor-prognosis subtype (OS: HR\u0026thinsp;=\u0026thinsp;2.113; RFS: HR\u0026thinsp;=\u0026thinsp;1.479). Six clinicopathological parameters\u0026ndash;MVI positivity, tumor diameter\u0026thinsp;\u0026ge;\u0026thinsp;3 cm, multifocal lesions, poor differentiation (grade III-IV), necrotic foci, and CK19 overexpression\u0026ndash;demonstrated significant associations with reduced OS and RFS. Multivariate Cox regression identified tumor diameter\u0026thinsp;\u0026ge;\u0026thinsp;3 cm, poor differentiation, and CK19 overexpression as independent prognostic determinants for OS, while tumor size\u0026thinsp;\u0026ge;\u0026thinsp;3 cm and poor differentiation concurrently impacted RFS. DPHCC patients exhibiting these three risk factors warrant stratification into a high-risk surveillance cohort with intensified imaging follow-up intervals and monitoring of serum AFP levels. Notably, CK19 overexpression demonstrated the most significant prognostic value for OS (HR\u0026thinsp;=\u0026thinsp;4.903), which may be associated with its mediation of cholangiocyte-like differentiation phenotypes or its role in promoting MV development\u003csup\u003e[22, 23]\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv\u003e\n\u003cp\u003eCK19 is a well-established marker for both biliary/progenitor cells and cancer stem cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003e[24\u0026ndash;26]\u003c/sup\u003e. critically potentiates HCC malignancy. Cumulative evidence correlates CK19 positivity with adverse outcomes, including elevated recurrence rates and diminished long-term survival\u003csup\u003e[27\u0026ndash;29]\u003c/sup\u003e. Mechanistically, CK19 positive HCC cells acquire migratory and invasive capacities via EMT activation (e.g., E-cadherin downregulation, N-cadherin/Vimentin upregulation), with KRT19 knockdown significantly attenuating invasiveness and EMT marker expression\u003csup\u003e[30]\u003c/sup\u003e. Further investigations reveal KRT19-mediated tumor vasculature remodeling through angiogenic gene regulation (FGFR1/VASH1 upregulation, VASH2 suppression), facilitating MVI development\u003csup\u003e[22]\u003c/sup\u003e. Of critical relevance, KRT19 orchestrates epigenetic dedifferentiation by forming complexes with HDAC1 and COREST to suppress histone acetylation at hepatocyte differentiation gene promoters, thereby maintaining stem-like plasticity\u003csup\u003e[31]\u003c/sup\u003e. Our findings underscore CK19's dual role as both prognostic biomarker and therapeutic target in DPHCC management strategies.\u003c/p\u003e\n\u003cp\u003eFinally, compared with the CHCC controls, the DPHCC cohort in this study had a significantly higher proportion of patients under 60 years old (74.19% vs. 57.84%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), aligning with previous reports on the age-specific epidemiology of DPHCC\u003csup\u003e[29, 32]\u003c/sup\u003e. This demographic divergence suggests distinct pathogenic mechanisms underlying DPHCC development. A prevailing hypothesis posits that DPHCC originates from aberrant differentiation of hepatic progenitor cells (HPCs), which remain quiescent under physiological conditions but activate proliferative and bipotential differentiation programs (hepatocytic/cholangiocytic lineages) during hepatic injury repair. In younger individuals, HPCs retain heightened proliferative plasticity and differentiation competence. Chronic hepatic insults, persistent inflammatory stimuli, or carcinogen exposure may drive these hyperactive HPCs toward malignant transdifferentiation, ultimately culminating in DPHCC pathogenesis.Moreover, the robust immune activity characteristic of younger hosts paradoxically creates a selective pressure favoring DPHCC progression. DPHCC cells likely employ advanced immune evasion strategies \u0026ndash; potentially through PD-L1 upregulation, MHC class I downregulation, or immunosuppressive cytokine secretion \u0026ndash; to circumvent immune surveillance within this immunologically dynamic microenvironment. Such adaptations confer a survival advantage, predisposing younger populations to DPHCC incidence. However, the precise mechanisms underlying this age-related predisposition require systematic investigation through lineage-tracing models, immune profiling, and comparative multi-omics analyses of age-stratified DPHCC cohorts.\u003c/p\u003e\n\u003cp\u003eA critical methodological consideration in this study is the definition of DPHCC, which relies on a\u0026thinsp;\u0026gt;\u0026thinsp;15% co-expression threshold for biliary markers, a criterion initially proposed by Lu et al. and adopted here for operational consistency with certain regional studies. It must be acknowledged, however, that this cut-off value lacks broad international validation and is not universally accepted. Variations in diagnostic thresholds\u0026mdash;for instance, the use of \u0026gt;\u0026thinsp;5% or \u0026gt;\u0026thinsp;10% in other research\u0026mdash;likely contribute to the heterogeneity in reported incidence rates and prognostic associations of DPHCC across the literature. This inconsistency underscores a significant challenge in comparing findings between studies and highlights the urgent need for an international consensus on the standardized pathological criteria for diagnosing DPHCC. Future collaborative efforts should focus on establishing evidence-based, reproducible definitions, possibly incorporating molecular correlates, to ensure improved classification consistency and prognostic accuracy across diverse cohorts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, DPHCC represents a distinct subtype of HCC marked by an aggressive nature, poor differentiation, and poor prognosis. Its prognosis is influenced by tumor size, differentiation status, and CK19 expression; clinicians should consider establishing intensified surveillance protocols and implementing enhanced follow-up strategies for patients diagnosed with this aggressive HCC subtype to address its propensity for rapid progression and high recurrence rates.This study addresses dual-phenotype hepatocellular carcinoma (DPHCC), a clinically significant yet historically underrecognized subtype of liver cancer. Despite its distinctive immunophenotypic profile\u0026mdash;concurrent expression of both hepatocellular and cholangiocytic markers\u0026mdash;which suggests unique tumor biology, and multiple reports associating it with more aggressive behavior and poorer clinical outcomes, DPHCC has not received sufficient attention in international diagnostic guidelines, clinical management consensus, or translational research. This neglect largely stems from the lack of standardized diagnostic criteria and an incomplete understanding of its clinicopathological and molecular basis. Through a systematic analysis of a large single-center cohort, our study further confirms that DPHCC patients exhibit more aggressive tumor features (such as higher rates of microvascular invasion and lymph node metastasis) and shortened survival. These findings strongly underscore the clinical necessity of distinguishing DPHCC from other types of HCC. Our work not only deepens the understanding of the clinicopathological characteristics of this distinct entity but, more importantly, calls for heightened awareness among researchers and pathologists to recognize and report DPHCC in routine diagnostic practice. Promoting in-depth molecular mechanistic studies and ultimately establishing internationally unified diagnostic guidelines are of paramount importance for developing more targeted therapeutic strategies and improving outcomes for this high-risk patient population.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDPHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDual-phenotype hepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eclassical hepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK19\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytokeratin 19\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMVI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrovascular invasion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK7\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytokeratin 7\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMUC1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emucin1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOverall survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRecurrence-free survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehazard ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econfidence interval.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e Ethical approval for this study was granted by the Institutional Review Board of the 900th Hospital of PLA Joint Logistic Support Force (Approval No. 2019-18) and adhered to the Declaration of Helsinki. Written informed consent was obtained from all participants or their legally authorized representatives.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiaojuan Ouyang conducted the research, collected experimental data, performed statistical analyses, and drafted the manuscript; Yongqin Yan conceptualized the study design, interpreted analytical results, obtained research funding, and critically revised the manuscript for substantive intellectual content; Bohao Hu and Xinjing Gao assisted in data collection; Xingfeng Qi assisted in performing the Immunohistochemical experiments.Lijuan Qu contributed administrative coordination, technical assistance, and material resources;Xianzong Ye and Haicong Wu contributed to critical appraisal of the scholarly content, and all authors reviewed and approved the final manuscript prior to submission.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024,74(3):229\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Huang K, He Y, Liang T, et al. Analysis of clinicopathologic and imaging features of dual-phenotype hepatocellular carcinoma[J]. Sci Rep, 2024,14(1):3314.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chung G E, Lee J, Yoon J, et al. Prognostic implications of tumor vascularity and its relationship to cytokeratin 19 expression in patients with hepatocellular carcinoma[J]. Abdom Imaging, 2012,37(3):439\u0026ndash;446.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Cong W, Bu H, Chen J, et al. Practice guidelines for the pathological diagnosis of primary liver cancer: 2015 update[J]. World J Gastroenterol, 2016,22(42):9279\u0026ndash;9287.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lu X, Xi T, Lau W, et al. Hepatocellular carcinoma expressing cholangiocyte phenotype is a novel subtype with highly aggressive behavior[J]. Ann Surg Oncol, 2011,18(8):2210\u0026ndash;2217.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yu Z, Fang Z, Yu Y, et al. The Clinical Characteristics, Patterns of Recurrence, and Long-Term Survival Outcomes of Dual-Phenotype Hepatocellular Carcinoma After Curative Liver Resection[J]. J Hepatocell Carcinoma, 2025,12:183\u0026ndash;192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin[J]. 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Onco Targets Ther, 2021,14:1543\u0026ndash;1552.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hepatocellular carcinoma, Dual-phenotype hepatocellular carcinoma, classical hepatocellular carcinoma, microvascular invasion, cytokeratin 19 expression, Recurrence-free survival, Overall survival","lastPublishedDoi":"10.21203/rs.3.rs-9029042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9029042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate the clinicopathological characteristics and prognostic differences between the dual-phenotype hepatocellular carcinoma (DPHCC) and classical hepatocellular carcinoma (CHCC), and to identify the key prognostic risk factors for DPHCC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: The Clinicopathological data from 463 hepatocellular carcinoma (HCC) patients were retrospectively analyzed. According to the morphology and immunophenotype (co-expression of HCC and intrahepatic cholangiocarcinoma [ICC] markers in \u0026gt; 15% of tumor cells). The cases were divided into two groups: the DPHCC group (93 cases, 20.09%) and the CHCC group (370 cases, 79.91%). Clinicopathological characteristics (microvascular invasion, tumor size, differentiation grade) and survival outcomes (overall survival [OS]; recurrence-free survival[RFS]) were compared between the two groups. Cox regression models were employed to analyze independent prognostic risk factors for DPHCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe DPHCC group exhibited significantly higher rates of microvascular invasion (MVI) (44.09% vs. 21.08%), tumor size ≥ 3 cm (80.65% vs. 68.92%), multifocality (33.33% vs. 15.14%), poor differentiation (39.78% vs. 21.08%), and necrosis-positivity (76.34% vs. 49.20%), capsular invasion (31.18% vs. 15.95%) compared to the CHCC group (for all, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 ). Survival analysis revealed significantly lower 5-year OS (61.10% vs 80.00%; HR 2.113) and RFS (37.40% vs 55.70%; HR 1.479) rates in the DPHCC group compared to the CHCC group. Multivariate analysis identified tumor size ≥ 3 cm (OS: HR = 3.021), poor differentiation (OS: HR 2.614; RFS: HR 1.927), and cytokeratin 19 (CK19) overexpression (OS: HR 4.903) as independent risk factors for poor prognosis in DPHCC. Additionally, DPHCC patients were significantly younger than CHCC patients (74.19% vs. 57.84% aged \u0026lt; 60 years; \u003cem\u003eP\u003c/em\u003e = 0.003), suggesting a potential association with aberrant differentiation of hepatic progenitor cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eDPHCC represents a distinct HCC subtype characterized by high aggressiveness, poor differentiation, and unfavorable prognosis, influenced by tumor size, differentiation grade, and CK19 expression.. It is recommended that patients diagnosed with this distinct HCC subtype receive intensified postoperative surveillance and personalized treatment strategies targeting the CK19-positive subgroup.\u003c/p\u003e","manuscriptTitle":"Dual-Phenotype Hepatocellular Carcinoma Exhibits Distinct Clinicopathological Aggressiveness and Poor Prognosis: A Retrospective Cohort Study of 463 Hepatocellular Carcinoma Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-12 07:41:58","doi":"10.21203/rs.3.rs-9029042/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-19T00:53:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141035800658843333118906316614814378457","date":"2026-04-07T23:00:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T12:29:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T11:32:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T14:59:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Diagnostic Pathology","date":"2026-03-26T09:08:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"diagnostic-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dpat","sideBox":"Learn more about [Diagnostic Pathology](http://diagnosticpathology.biomedcentral.com)","snPcode":"13000","submissionUrl":"https://submission.nature.com/new-submission/13000/3","title":"Diagnostic Pathology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f4ba232-3253-41cb-a5bb-22e1f58ff831","owner":[],"postedDate":"April 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-12T07:41:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-12 07:41:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9029042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9029042","identity":"rs-9029042","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}