Clinical significance of cytokeratin 19 expression in hepatocellular carcinoma
0 Abstract
Aim: This study aimed to compare the prognostic outcomes of cytokeratin (CK) 19-positive and CK19-negative hepatocellular carcinoma (HCC), focusing on clinicopathological features and the impact of targeted therapy in CK19-positive patients.
Methods: A retrospective analysis was performed on 310 HCC patients who underwent curative resection between 2010 and 2020 at the First Affiliated Hospital of Nanjing Medical University. Among them, 102 were CK19-positive and 168 were CK19-negative. Multivariate Cox regression was used to identify independent predictors of overall survival (OS) and recurrence-free survival (RFS). Kaplan-Meier survival curves were generated from the Cox model.
Results: CK19-positive patients exhibited significantly poorer tumor differentiation (P < 0.001), increased microvascular invasion (P = 0.010), elevated α-fetoprotein (AFP) (P = 0.0001) and were more often asymptomatic at diagnosis (P = 0.014). The median survival time (MST) was 22.1 months in CK19-positive versus 60.3 months in CK19-negative patients. Among those with recurrence, MST was 56.4 months for CK19-positive and 101.6 months for CK19-negative patients. CK19 status significantly impacted OS (P < 0.001) and RFS (P = 0.006) in advanced-stage cases. Independent prognostic factors for RFS included cirrhosis, tumor size, number of tumors, macrovascular invasion, poor differentiation, and CK19 expression. Microvascular invasion and Child-Pugh classification were additional predictors of OS. Targeted therapies did not significantly improve outcomes in CK19-positive patients.
Conclusions: CK19-positive HCC is associated with more aggressive tumor behavior, higher recurrence, and poorer survival. Targeted therapy provided no significant survival benefit in this study.
Keywords
INTRODUCTION
From 2018 to 2022, hepatocellular carcinoma (HCC) has consistently been the 6th most prevalent cancer worldwide. During this period, it has risen from the fourth to the third leading cause of cancer-related deaths, accounting for approximately 758,000 deaths in 2022[1-3]. Asia bears the highest burden, representing 75% of global HCC cases, with China alone contributing to 50% of the total[4]. Despite the treatment techniques currently available, such as surgical resection, ablation, transarterial chemoembolization (TACE), radiotherapy, and systemic therapy, prolonging life expectancy in HCC patients remains a constant challenge. Until now, the 5-year overall survival rate has been 30%-40% after resections, with 70% of recurrences occurring within 5 years[5]. The implementation of semi-annual surveillance programs, particularly among patients with cirrhosis, has markedly improved early tumor detection rates. A
Consequently, researchers have attempted to analyze the various subtypes of HCC in order to improve patient prognosis and treatment outcomes. Prior research has classified HCC into two major molecular subtypes: proliferative and non-proliferative[12-14]. CKs are polygenic biomarkers that can be categorized according to their molecular weight and chemical structures[15]. Among them, CK19 is the least acidic, with a molecular weight of approximately 40 kDa, and is typically expressed in laminated epithelium[16]. The proliferative subtype of HCC that expresses CK19 is also referred to as biphenotypic HCC[5]. CK19 acts as a marker of bipotential progenitor cells capable of differentiating into both hepatocytes and biliary epithelial cells[17]. Approximately 10%-30% of HCC cases express CK19[18-20]. When present, CK19 expression in HCC is associated with pathological features of both HCC and cholangiocarcinoma, resulting in a poor prognosis[21]. Moreover, the combined assessment of pathological features and biomarkers such as CK19 has shown higher specificity and sensitivity in predicting metastatic recurrence in HCC[22].
CK19 promotes epithelial-mesenchymal transition (EMT), thereby enhancing invasiveness and metastatic potential by downregulating E-cadherin and upregulating mesenchymal markers such as vimentin and
Targeted therapies such as sorafenib, lenvatinib, and donafenib have shown clinical efficacy by disrupting key signaling pathways commonly activated in CK19-positive tumors. Sorafenib, a multikinase inhibitor, targets Rapidly Accelerated Fibrosarcoma(RAF), Vascular Endothelial Growth Factor Receptor (VEGFR), and Platelet-Derived Growth Factor Receptor (PDGFR), thereby inhibiting tumor proliferation via the RAF/Mitogen-activated protein kinase (MEK)/Extracellular Signal- Regulated Kinase (ERK) pathway and suppressing angiogenesis[32]. CK19-positive HCCs are often hypervascular, making them potentially responsive to VEGFR inhibition[20]. Lenvatinib exhibits broader activity, targeting VEGFR1-3, FGFR1-4, PDGFRα, RET, and KIT. It also inhibits the Fibroblast Growth Factor (FGF) pathway, which plays a role in the growth of stem-like, CK19-positive cells[33]. Through dual inhibition of VEGF and FGF signaling, lenvatinib may more effectively control tumor angiogenesis and cell proliferation. Its consistent therapeutic efficacy across various liver disease etiologies, including MAFLD, supports its use in CK19-positive HCC, regardless of underlying hepatic pathology[34]. Donafenib, a novel derivative of sorafenib, shares similar molecular targets but provides improved safety and tolerability, particularly in Asian populations, making it a suitable alternative for CK19-positive HCC patients requiring long-term treatment[35]. The introduction of molecular targeted agents such as lenvatinib, alongside immune checkpoint inhibitors like nivolumab and pembrolizumab, has improved survival outcomes. Notably, although the combination of atezolizumab and bevacizumab has demonstrated superior efficacy over sorafenib, establishing a new first-line treatment standard for advanced HCC[36], its effectiveness in CK19-positive HCC remains to be fully confirmed.
CK19-positive HCC is aggressive, poorly differentiated, resistant to therapy, and highly recurrent, yet remains an underrecognized subtype. This makes it a compelling area for further investigation. The aim of this study is to elucidate the prognostic significance of CK19-positive HCC and evaluate the therapeutic efficacy of targeted therapy in these patients.
MATERIALS AND METHODS
Data collection
Clinical records of 310 patients diagnosed with HCC at the First Affiliated Hospital with Nanjing Medical University between 2010 and 2020 were retrospectively collected. The initial clinical diagnosis of HCC was based on two criteria: the presence of a liver lesion > 10 mm in diameter and an α-fetoprotein (AFP) level ≥ 20 ng/mL[37]. The diagnosis was pathologically confirmed by two independent pathologists based on biopsy samples. The study was conducted in accordance with institutional ethical guidelines and the principles outlined in the Declaration of Helsinki.
Inclusion and exclusion criteria
Patients who underwent curative resection, TACE, or neoadjuvant immunotherapy were considered for inclusion. Forty patients were excluded based on the following criteria: recurrent HCC (n = 20), presence of other malignancies (n = 11), distant metastasis confirmed either preoperatively or within one month postoperatively (n = 3), or palliative resection (n = 6). After applying these criteria, a total of 270 patients were included in the final analysis.
Detection of CK19 expression
Following surgical resection, tumor samples were subjected to immunohistochemical analysis to evaluate CK19 expression. Formalin-fixed, paraffin-embedded tissue sections were stained with anti-CK19 antibodies. CK19 expression was assessed via light microscopy. Tumors were classified as CK19-positive if
Targeted therapy regimen
Targeted therapy was administered to patients with advanced, unresectable tumors. Among the 270 included patients, 56 patients received targeted therapy, 15 of them were CK19-positive. The therapeutic agents used were lenvatinib, sorafenib, and donafenib. Lenvatinib was given at 8 mg once daily for patients weighing less than 60 kg, and at 12 mg once daily for those weighing ≥ 60 kg. Sorafenib was administered at 400 mg twice daily, and donafenib at 200mg twice daily. Treatment continued until the patient no longer derived clinical benefit.
Patient grouping
The 270 patients were categorized into two groups based on CK19 expression: 102 were CK19-positive and 168 were CK19-negative.
Statistical analysis
Patient characteristics were classified as either continuous or categorical variables. Continuous variables were summarized using medians and interquartile ranges (IQRs), and categorical variables as frequencies and percentages. The Student’s t-test or Mann-Whitney U test was used to compare continuous variables, while the Chi-square test or Fisher’s exact test was applied to categorical variables. Differences in recurrence-free survival (RFS) and overall survival (OS) between the two groups were assessed using the log-rank test. RFS was defined as the time to tumor recurrence, and OS as the time to death. Survival curves were plotted using the Kaplan-Meier method. Cox proportional hazards models were employed for univariate and multivariate analyses using a backward stepwise selection approach. Predicted survival plots were constructed based on multivariable Cox regression. A subgroup analysis of RFS was also performed based on factors such as age, gender, cirrhosis status, etiology, Child-Pugh grade, tumor size, tumor number, vascular invasion, and Edmondson-Steiner grade. A two-tailed P-value < 0.05 was considered statistically significant.
All statistical analyses were performed using R software (version 4.2.2, R Project for Statistical Computing; http://www.r-project.org) and Stata (version 17.0).
RESULTS
Patient population and characteristics
A total of 310 patients diagnosed with HCC were initially included in the analysis. After applying the exclusion criteria (outlined in Figure 1), 40 patients were excluded, resulting in a final cohort of 270 patients, comprising 102 CK19-positive and 168 CK19-negative individuals. The median age of the study population was 58 years (interquartile range: 50-66 years), with a predominance of male patients (n = 211, 78.1%).
Figure 1. Flowchart of patient selection following the application of inclusion and exclusion criteria. HCC: Hepatocellular carcinoma.
Among the CK19-positive group, 79.4% were male. CK19-negative expression was identified in 168 patients. A comparative analysis of clinicopathological features between the CK19-positive and CK19-negative groups, as presented in Table 1, revealed statistically significant differences in several parameters.
Baseline characteristics of CK19-negative and -positive patient groups
| Characteristics | All patients (n = 270) | CK19-Negative (n = 168) | CK19- Positive (n = 102) | P-value |
| Median age (years) | 58 (50-66) | 58.5 (51.0-67.5) | 57.0 (49.0-65.0) | 0.325 |
| Gender | 0.695 | |||
| Male | 211 (78.1) | 130 (77.4) | 81 (79.4) | |
| Female | 59 (21.9) | 38 (22.6) | 21 (20.6) | |
| Comorbid illness | 0.133 | |||
| Absent | 181 (67) | 107 (63.7) | 74 (72.5) | |
| Present | 89 (33) | 61 (36.3) | 28 (27.5) | |
| Hypertension | 78 (28.9) | 54 (32.1) | 24 (23.5) | 0.130 |
| Diabetes mellitus | 37 (13.7) | 22 (13.1) | 15 (14.7) | 0.709 |
| Asymptomatic HCC | 0.014 | |||
| Yes | 173 (64.1) | 117 (69.6) | 56 (54.9) | |
| No | 97 (35.9) | 51 (30.4) | 46 (45.1) | |
| Smoking | 0.625 | |||
| Yes | 68(25.2) | 44(26.2) | 24()23.5 | |
| No | 202(74.8) | 124(73.8) | 78(76.5) | |
| Alcohol consumption | 0.814 | |||
| Yes | 51(18.9) | 31(18.4) | 20(19.6) | |
| No | 219(81.1) | 137(81.6) | 82(80.4) | |
| AFP | 14.5(3.5-380.2) | 6.6.(3.2-111.8) | 130.6(6.4-1210) | 0.0001 |
| Etiology | 0.926 | |||
| Hepatitis B or C virus | 195 (72.2) | 121 (72) | 74 (72.5) | |
| Others | 75 (27.8) | 47 (28) | 28 (27.5) | |
| Cirrhosis | 0.980 | |||
| Yes | 193 (71.5) | 120 (71.4) | 73 (71.6) | |
| No | 77 (28.5) | 48 (28.6) | 29 (28.4) | |
| Child-Pugh class | 0.133 | |||
| A | 258 (95.6) | 163 (97) | 95 (93.1) | |
| B | 12 (4.4) | 5 (3) | 7 (6.9) | |
| Tumor size (cm) | 4.0 (2.5-7.0) | 4.0 (2.5-6.8) | 4.0 (2.5-7.0) | 0.842 |
| Tumor number | 0.089 | |||
| Solitary | 213 (78.9) | 127 (75.6) | 86 (84.3) | |
| Multiple | 57 (21.1) | 41 (24.4) | 16 (15.7) | |
| Macrovascular invasion | 0.075 | |||
| Absent | 244 (90.4) | 156 (92.9) | 88 (86.3) | |
| Present | 26 (9.6) | 12 (7.1) | 14 (13.7) | |
| Extracapsular invasion | 0.687 | |||
| Absent | 251 (93) | 157 (93.5) | 94 (92.2) | |
| Present | 19 (7) | 11 (6.5) | 8 (7.8) | |
| Lymph node metastasis | 0.780 | |||
| Absent | 261 (96.7) | 162 (96.4) | 99 (97.1) | |
| Present | 9 (3.3) | 6 (3.6) | 3 (2.9) | |
| Edmondson-Steiner grade | < 0.001 | |||
| Well | 121 (44.8) | 88 (52.4) | 33 (32.4) | |
| Moderate | 73 (27) | 47 (28) | 26 (25.5) | |
| Poor | 76 (28.1) | 33 (19.6) | 43 (42.2) | |
| Microscopic vascular invasion | 0.010 | |||
| Absent | 201 (74.4) | 134 (79.8) | 67 (65.7) | |
| Present | 69 (25.6) | 34 (20.2) | 35 (34.3) | |
| Resection margin adjacent to tumor | 0.506 | |||
| No | 235 (87) | 148 (88.1) | 87 (85.3) | |
| Yes | 35 (13) | 20 (11.9) | 15 (14.7) | |
| TNM 8th stage | 0.553 | |||
| IA/IB | 155 (57.4) | 100 (59.5) | 55 (53.9) | |
| II/IIIA | 65 (24.1) | 40 (23.8) | 25 (24.5) | |
| IIIB/IVA | 50 (18.5) | 28 (16.7) | 22 (21.6) | |
| Resection | 0.451 | |||
| Major hepatectomy | 54 (20) | 36 (21.4) | 18 (17.6) | |
| Minor hepatectomy | 216 (80) | 132 (78.6) | 84 (82.4) | |
| HCC-related local treatment | 0.262 | |||
| Absent | 257 (95.2) | 158 (94) | 99 (97.1) | |
| Present | 13 (4.8) | 10 (6) | 3 (2.9) |
Comparison of OS and RFS pattern between CK19-negative and CK19-positive groups
RFS and OS were compared between CK19-positive and CK19-negative HCC patients using the log-rank test, with the results summarized in Table 2. CK19-positive patients exhibited significantly higher recurrence rates across all time points: 64.2% at 1 year, 44.5% at 3 years, 36.2% at 5 years, and 34.0% at 10 years. Moreover, the median survival time following recurrence in CK19-positive patients was approximately three times shorter than that of their CK19-negative counterparts, indicating a more aggressive disease course.
Survival outcomes in CK19-negative vs. CK19-positive patients
| Events (%) | MST (95%CI) | 1 year- | 3 year- | 5 year- | 10 year- | Log-rank χ2 | P-value | |
| RFS | ||||||||
| Total | 156 (57.8%) | 43.1 (34.1-60.8) | 72.3% | 56.0% | 45.4% | 28.5% | ||
| CK19 (-) | 94 (56.0%) | 60.3 (42.5-70.6) | 77.3% | 62.9% | 51.1% | 26.8% | 4.60 | 0.032 |
| CK19 (+) | 62 (60.8%) | 22.1 (14.1-37.8) | 64.2% | 44.5% | 36.2% | 34.0% | ||
| OS | ||||||||
| Total | 113 (41.9%) | 90.2 (69.9-114.1) | 88.1% | 71.0% | 60.9% | 40.9% | ||
| CK19 (-) | 64 (38.1%) | 101.6 (82.2-NA) | 91.6% | 77.6% | 67.9% | 40.5% | 7.69 | 0.006 |
| CK19 (+) | 49 (48.0%) | 56.4 (30.2-NA) | 82.2% | 60.1% | 49.0% | 44.4% |
Similarly, OS was consistently worse in the CK19-positive group, with survival rates of 82.2% at 1 year, 60.1% at 3 years, 49.0% at 5 years, and 44.4% at 10 years. The median OS for CK19-negative patients was nearly twice as long as that of CK19-positive patients, further underscoring the adverse prognostic implications of CK19 expression in HCC.
Predictors of RFS and OS
Multivariate Cox proportional hazards analysis, adjusted for relevant covariates, identified CK19 expression as an independent negative prognostic factor for both RFS and OS. CK19 positivity was associated with a 43% increased risk of recurrence [HR 1.43, 95%CI (1.02-2.01), P = 0.041] and a 60% higher risk of death [HR 1.60, 95%CI (1.07-2.40), P = 0.023], as detailed in Table 3 and illustrated in Figure 2. These findings suggest a significant association between CK19 expression and both increased tumor recurrence and reduced survival outcomes.
Figure 2. Survival curves from multivariate Cox regression for (A) recurrence-free survival and (B) overall survival.
Multivariate analysis of predictors for recurrence-free survival and overall survival
| Univariate analysis | Multivariate analysis | |||
| Hazard ratio (95%CI) | P | Hazard ratio (95%CI) | P | |
| RFS analyzes | ||||
| Age (≥ 60 vs. < 60 yr) | 0.79 (0.57-1.08) | 0.142 | ||
| gender (female vs. male) | 0.56 (0.36-0.86) | 0.009 | ||
| Comorbid illness (present vs. absent) | 0.75 (0.53-1.06) | 0.105 | ||
| Asymptomatic HCC (no vs. yes) | 1.34 (0.97-1.85) | 0.071 | ||
| Etiology (hepatitis vs. others) | 1.59 (1.09-2.33) | 0.016 | ||
| Cirrhosis (yes vs. no) | 1.50 (1.03-2.19) | 0.035 | 1.82 (1.23-2.67) | 0.002 |
| Child-Pugh class (B vs. A) | 1.57 (0.77-3.22) | 0.214 | ||
| Tumor size (> 5 vs. ≤ 5 cm) | 1.95 (1.42-2.68) | < 0.001 | 1.91 (1.37-2.68) | < 0.001 |
| Tumor no. (multiple vs. solitary) | 1.71 (1.20-2.46) | 0.003 | 1.63 (1.12-2.37) | 0.011 |
| Macrovascular invasion (present vs. absent) | 3.96 (2.46-6.37) | < 0.001 | 2.60 (1.56-4.34) | < 0.001 |
| Extracapsular invasion (present vs. absent) | 2.34 (1.34-4.08) | 0.003 | ||
| Lymph node metastasis (yes vs. no) | 3.41 (1.67-6.97) | 0.001 | ||
| Edmondson-Steiner grade, well | ||||
| Moderate vs. well | 1.75 (1.19-2.58) | 0.004 | 1.56 (1.06-2.30) | 0.025 |
| Poor vs. well | 2.41 (1.65-3.52) | < 0.001 | 1.97 (1.32-2.94) | 0.001 |
| Microscopic vascular invasion (present vs. absent) | 2.40 (1.69-3.40) | < 0.001 | ||
| Resection margin adjacent to tumor (yes vs. no) | 1.17 (0.73-1.87) | 0.519 | ||
| Resection (major vs. minor hepatectomy) | 0.70 (0.48-1.02) | 0.061 | ||
| HCC-related local treatment (present vs. absent) | 1.45 (0.74-2.85) | 0.285 | ||
| CK19 (positive vs. negative) | 1.42 (1.03-1.96) | 0.033 | 1.43 (1.02-2.01) | 0.041 |
| OS analyzes | ||||
| Age (≥ 60 vs. < 60 yr) | 0.85 (0.58-1.24) | 0.400 | ||
| Gender (female vs. male) | 0.66 (0.40-1.07) | 0.093 | ||
| Comorbid illness (present vs. absent) | 0.81 (0.54-1.24) | 0.338 | ||
| Asymptomatic HCC (no vs. yes) | 1.81 (1.25-2.63) | 0.002 | ||
| Etiology (hepatitis vs. others) | 1.57 (1.00-2.46) | 0.051 | ||
| Cirrhosis (yes vs. no) | 1.22 (0.79-1.87) | 0.366 | ||
| Child-Pugh class (B vs. A) | 2.76 (1.33-5.70) | 0.006 | 2.34 (1.07-5.13) | 0.033 |
| Tumor size (> 5 vs. ≤ 5 cm) | 2.60 (1.79-3.76) | < 0.001 | 2.16 (1.43-3.26) | < 0.001 |
| Tumor no. (multiple vs. solitary) | 1.84 (1.22-2.77) | 0.004 | 1.70 (1.09-2.65) | 0.019 |
| Vascular invasion | ||||
| Microscopic invasion vs. absent | 2.77 (1.70-4.52) | < 0.001 | 1.84 (1.09-3.08) | 0.022 |
| Macrovascular invasion vs. absent | 7.84 (4.68-13.13) | < 0.001 | 3.16 (1.76-5.69) | < 0.001 |
| Extracapsular invasion (present vs. absent) | 3.45 (1.91-6.24) | < 0.001 | ||
| Lymph node metastasis (yes vs. no) | 4.47 (2.25-8.89) | < 0.001 | ||
| Edmondson-Steiner grade | ||||
| Moderate vs. well | 2.89 (1.79-4.65) | < 0.001 | 2.59 (1.58-4.25) | < 0.001 |
| Poor vs. well | 4.02 (2.52-6.43) | < 0.001 | 2.72 (1.64-4.49) | < 0.001 |
| Resection margin adjacent to tumor (yes vs. no) | 1.21 (0.70-2.08) | 0.500 | ||
| Resection (major vs. minor hepatectomy) | 0.60 (0.39-0.91) | 0.015 | ||
| HCC-related local treatment (present vs. absent) | 1.06 (0.43-2.61) | 0.894 | ||
| CK19 (positive vs. negative) | 1.69 (1.16-2.45) | 0.006 | 1.60 (1.07-2.40) | 0.023 |
In the univariate analysis of RFS, several clinicopathologic factors were significantly associated with poorer outcomes, including CK19 positivity, female sex, hepatitis infection, cirrhosis, multiple tumor nodules, tumor size > 5 cm, macrovascular invasion, microvascular invasion, extracapsular invasion, lymph node metastasis, and moderate to poor Edmondson-Steiner grade [Table 3]. In the multivariate analysis, CK19 positivity, cirrhosis, multiple tumors, tumor size > 5 cm, and moderate to poor tumor differentiation remained independent predictors of poor RFS.
Similarly, the univariate analysis of OS identified CK19 positivity, asymptomatic presentation, Child-Pugh class B, multiple tumors, tumor size > 5 cm, macrovascular invasion, microvascular invasion, extracapsular invasion, lymph node metastasis, moderate to poor Edmondson-Steiner grade, and major hepatic resection as significant adverse prognostic factors. Multivariate analysis further confirmed that CK19 positivity, Child-Pugh class B, multiple tumors, larger tumor size (> 5 cm), macrovascular invasion, microvascular invasion, and moderate to poor histological differentiation were independently associated with decreased OS.
Subgroup analysis of RFS and OS
A sensitivity analysis of patient characteristics, illustrated in Figure 3, was conducted to validate the robustness of the covariate analyses. The results were consistent with the primary findings. No statistically significant differences in RFS or OS were observed between CK19-positive and CK19-negative patients in subgroups where the confidence intervals crossed the line of no effect. However, a general trend toward poorer outcomes was evident among CK19-positive patients compared to their CK19-negative counterparts. Elevated recurrence rates were observed in nearly all CK19-positive subgroups, with particularly pronounced effects in patients aged < 60 years, males, those with hepatitis B or C infection, Child-Pugh class A liver function, tumors > 5 cm in diameter, solitary tumors, vascular invasion (microscopic or macroscopic), and poor histological differentiation. Similarly, reduced overall survival was also more common among CK19-positive patients, especially within the same clinical subgroups: age < 60 years, male sex, viral hepatitis, preserved liver function (Child-Pugh A), large solitary tumors, vascular invasion (both microscopic and macroscopic), poor differentiation, and underlying cirrhosis.
Figure 3. Subgroup analyses of recurrence-free survival (A) and overall survival (B). Shared clinicopathological characteristics of CK19-positive and CK19 patients were thoroughly examined. CK19: Cytokeratin 19.
RFS and OS according to tumor, node, metastasis tumor stage
Figure 4 presents RFS and OS stratified by tumor, node, metastasis (TNM) stages IA/IB, II/IIIA, and IIIB/IVA in both CK19-positive and CK19-negative patients. A significantly poorer prognosis was observed in CK19-positive patients at the advanced stages, namely TNM stage IIIB/IVA (panels C and F).
Figure 4. Kaplan-Meier analysis of recurrence-free survival (A-C) and overall survival (D-F) by TNM stage and CK19 status. TNM: Tumor, node, metastasis; CK19: cytokeratin 19.
Effect of targeted therapy in CK19-positive HCC
Among the 270 patients included in the study, 56 received postoperative targeted therapy, 15 of whom were CK19-positive. Figure 5 shows that there was no statistically significant relation between CK19 positivity and administration of targeted therapy (P = 0.064). Furthermore, survival outcomes [RFS (P = 0.8888) and OS (P = 0.1721)] were not statistically significant between those who received targeted therapy and those who did not.
Figure 5. The OS (A) and RFS (B) in CK19-positive patients that received targeted therapy postoperatively. OS: Overall survival; RFS: recurrence-free survival; CK19: cytokeratin 19.
DISCUSSION
The present study is consistent with previous reports suggesting that CK19-expressing HCC represents a biologically aggressive subtype[38]. All CK19-positive patients with end-stage disease (TNM stage IIIB/IVA) experienced tumor recurrence or death within two years after curative resection. Similarly, Uenishi et al. reported that 73% of CK19-positive patients developed early postoperative recurrences[39]. Our findings further show that a moderate to poor Edmondson-Steiner grade is significantly associated with reduced OS and RFS in CK19-positive patients, corroborating earlier studies indicating that poorly differentiated HCC more frequently expresses CK19[39]. Subgroup analyses revealed that coexisting hepatitis B or C infection was associated with worse outcomes in both RFS and OS. CK19-positive HCC patients with hepatitis B have been shown to exhibit higher early recurrence rates and poorer prognosis[38,40,41]. The relationship among AFP, CK19, and HCC remains complex. AFP-positive HCC patients tend to present with larger tumors, more advanced stages, elevated HBV markers, and increased liver enzyme levels and blood cell counts compared to AFP-negative patients[42].
Macrovascular invasion, tumor multiplicity, and increased tumor size are well-established predictors of advanced disease and poor prognosis in HCC[43-46]. These features reflect aggressive tumor biology and are frequently associated with higher recurrence rates and reduced OS. CK19 expression has increasingly been recognized as a marker of such biological aggressiveness. Several studies have demonstrated that
Although alcohol consumption and cigarette smoking are well-established etiological factors for HCC, their specific associations with CK19-positive HCC remain poorly defined. Chronic alcohol intake contributes to HCC pathogenesis primarily through mechanisms such as liver cirrhosis, oxidative stress, and DNA damage. Epidemiological data suggest that alcohol-related cirrhosis accounts for approximately 30%-40% of HCC cases in Western populations[48,49]. Similarly, cigarette smoking is an independent risk factor for HCC, with meta-analyses showing a 51% increased risk in current smokers and a 12% increase in former smokers compared to non-smokers[50]. The carcinogenicity of tobacco is partly mediated by the activation of oncogenic pathways, such as p53 mutations, and synergistic interactions with viral hepatitis. However, limited data exist regarding the influence of alcohol and smoking on CK19-positive HCC. While
The benefits of targeted therapy for CK19-positive HCC remain to be confirmed in larger cohorts. In addition to the financial burden, patients may suffer from increased side effects if the treatment proves ineffective. Previous studies have reported that both chemotherapy and targeted therapy yield limited benefit in CK19-positive HCC[51]. Given the complexity of these tumors and their involvement in multiple activated signaling pathways, combination therapies - such as targeted agents with immunotherapies (e.g., anti-PD-1/PD-L1 antibodies) or locoregional approaches (TACE or radiotherapy) - may enhance outcomes by simultaneously addressing both intrinsic tumor resistance and the tumor microenvironment. Our findings support the need for future studies exploring tailored combination strategies based on the molecular profile of CK19-positive HCC. Regorafenib has shown promise as a targeted agent in
Lymph node metastasis in HCC remains relatively rare, with reported incidences ranging from 1.7% to 7.5%[39,54]. Patients with lymph node involvement generally face poor outcomes despite undergoing lymphadenectomy, with a reported 5-year survival rate of only 31%[55,56]. Current European and American guidelines do not recommend routine lymphadenectomy unless there is strong radiological or intraoperative evidence, particularly in aggressive subtypes with suspected extrahepatic spreads. In our study, no significant correlation was observed between CK19 expression and lymph node metastasis. However, Zhuang et al. reported a significantly higher incidence (27.7%) of lymph node metastasis in CK19-positive patients compared to 5.6% in CK19-negative patients[57]. Moreover, CK19-positive patients with lymph node involvement had significantly worse prognoses[57].
This study highlights the clinical relevance of CK19 expression in four key domains: (1) Prognostic stratification - CK19 status enables early risk assessment and identification of aggressive tumor subtypes prior to overt clinical progression; (2) Therapeutic decision making - CK19 positivity indicates potential resistance to tyrosine kinase inhibitors (TKIs), suggesting the need for alternative or combination regimens; (3) Biomarker-driven therapies - CK19 serves as a foundation for targeted therapies focused on stemness, EMT, and cholangiocytic differentiation. It may also be useful in identifying candidates for precision oncology trials; (4) Postoperative management - Due to the high recurrence risk, CK19-positive patients may benefit from intensified surveillance, earlier adjuvant or neoadjuvant therapy, and more accurate preoperative diagnostics. Semi-annual surveillance in cirrhotic patients has been shown to significantly improve early detection and access to curative treatments. A meta-analysis involving 15,158 patients demonstrated a 2.08-fold increase in early-stage HCC diagnosis and a 2.24-fold rise in curative therapy, leading to improved overall survival (OR 1.90).
The aggressive nature of CK19-positive HCC suggests that effective management may require multifaceted strategies. Combination approaches - including immunotherapy, targeted therapy, ablation, and resections - may improve outcomes. For instance, combining TACE with a Programmed Cell Death 1 (PD1) -inhibitor and Lenvatinib was shown to significantly prolong survival compared to TACE plus PD1 inhibition alone[58]. Despite resistance to several chemotherapies, regorafenib, which targets the STAT3/mitochondria axis, exhibits preferential efficacy against CK19-positive HCC[52]. In patient-derived xenograft models, regorafenib achieved an 80% tumor control rate in CK19-positive tumors, compared to 0% in CK19-negative cases[59]. However, mechanisms such as inflammation, hypoxia, and EMT may limit its effectiveness. Research is ongoing into enhancing regorafenib’s efficacy through combination with immunotherapy techniques[60].
The limitations of this study include population selection bias, retrospective design, endpoint definition inconsistencies, and non-uniform follow-up schedules. Although around 2,500 patients were diagnosed with HCC between 2010 and 2020, only 310 underwent immunohistochemical evaluation, and further exclusions reduced the cohort size. Multivariate Cox regression was employed to adjust for confounding variables, but the variability in follow-up duration remains a concern. Routine lymphadenectomy was not performed in CK19-positive patients, limiting conclusions regarding the association between CK19 expression and lymph node metastasis. Additionally, liver disease etiology was broadly classified as viral or non-viral, preventing more nuanced subgroup analysis [e.g., MAFLD, alcohol-related liver disease(ALD)]. Future studies with more granular etiological data are necessary to fully understand how CK19 expression interacts with underlying liver conditions and affects treatment response.
Research in this area has evolved from focusing on prognostic evaluation to exploring mechanisms of CK19 action, therapeutic strategies, and non-invasive diagnostic tools. Given the aggressive behavior of CK19-positive HCC, early and accurate diagnosis using imaging modalities such as contrast-enhanced ultrasound (US)[61], triphasic Computed Tomography (CT)[62], Gadolinium ethoxylbenzylenetriamine pentaaceetic axid (Gd-EOB-DTPA)-enhanced Magnetic Reasonance Imaging (MRI)[63], Fluorodeoxyglucose-Posittron Emission Tomography (FDG-PET)[64], Magnetic Resonance Elastography (MRE)[65], and deep learning radiomics based on Gadoxetic Acid-Enhanced MRI[66] may significantly improve outcomes. Personalized treatment strategies and clinical trials are essential to developing evidence-based approaches for managing this subtype. Furthermore, elucidating the underlying causes of CK19 expression could inform preventive strategies.
Conclusion
In summary, this study confirms that CK19-positive HCC is associated with a poorer prognosis than
DECLARATIONS
Authors’ contributions
Study conceptualization: Khodabocus Z, Wang X, Mu X, Chen Z
Data curation and analysis: Khodabocus Z, Zhang H, Mu X
Manuscript drafting and editing: Khodabocus Z, Zhang H, Li D, Chen Z
Manuscript draft revision: all authors revised the manuscript for important intellectual content and approved the final version of the article.
Availability of data and materials
The datasets used and analyzed in this study are available from the corresponding author upon reasonable request.
Financial support and sponsorship
This work was supported by the National Natural Science Foundation of China (8202556, 82272791, 31930020), the Natural Science Foundation of Jiangsu Province (BK20201083), the Jiangsu Medical Innovation Center (CXZX202203), and the Jiangsu Provincial Medical Key Laboratory (ZDXYS202201).
Conflicts of interest
All authors declared that there are no conflicts of interest
Ethics approval and consent to participate
Ethics approval was provided by the institutional review board of the First Affiliated Hospital of Nanjing Medical University (Approval number: 2023-SRFA-096). Informed consent to participate was obtained from all participants.
Consent for publication
Not applicable
Copyright
© The Author(s) 2025.
REFERENCES
1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394-424.
2. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209-49.
3. 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. CA Cancer J Clin. 2024;74:229-63.
5. Zhuo JY, Lu D, Tan WY, Zheng SS, Shen YQ, Xu X. CK19-positive hepatocellular carcinoma is a characteristic subtype. J Cancer. 2020;11:5069-77.
6. Singal AG, Pillai A, Tiro J. Early detection, curative treatment, and survival rates for hepatocellular carcinoma surveillance in patients with cirrhosis: a meta-analysis. PLoS Med. 2014;11:e1001624.
7. Ghazanfar H, Javed N, Qasim A, et al. Metabolic dysfunction-associated steatohepatitis and progression to hepatocellular carcinoma: a literature review. Cancers. 2024;16:1214.
8. Yoon JK, Choi JY, Rhee H, Park YN. MRI features of histologic subtypes of hepatocellular carcinoma: correlation with histologic, genetic, and molecular biologic classification. Eur Radiol. 2022;32:5119-33.
9. Fulgenzi CAM, Talbot T, Murray SM, et al. Immunotherapy in hepatocellular carcinoma. Curr Treat Options Oncol. 2021;22:87.
10. Komatsu S, Ueshima K, Kido M, et al. Hepatectomy versus sorafenib for advanced hepatocellular carcinoma with macroscopic portal vein tumor thrombus: a bi-institutional propensity-matched cohort study. J Hepatobiliary Pancreat Sci. 2023;30:303-14.
11. Sun P, Li Z, Yan Z, et al. Lenvatinib targets STAT-1 to enhance the M1 polarization of TAMs during hepatocellular carcinoma progression. BMC Cancer. 2024;24:922.
12. Boyault S, Rickman DS, de Reyniès A, et al. Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology. 2007;45:42-52.
13. Lee JS, Heo J, Libbrecht L, et al. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med. 2006;12:410-6.
14. Chiang DY, Villanueva A, Hoshida Y, et al. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer Res. 2008;68:6779-88.
15. Mehrpouya M, Pourhashem Z, Yardehnavi N, Oladnabi M. Evaluation of cytokeratin 19 as a prognostic tumoral and metastatic marker with focus on improved detection methods. J Cell Physiol. 2019;234:21425-35.
16. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11-24.
17. Haruna Y, Saito K, Spaulding S, Nalesnik MA, Gerber MA. Identification of bipotential progenitor cells in human liver development. Hepatology. 1996;23:476-81.
18. 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. Histopathology. 2006;49:138-51.
19. Lee JI, Lee JW, Kim JM, Kim JK, Chung HJ, Kim YS. Prognosis of hepatocellular carcinoma expressing cytokeratin 19: comparison with other liver cancers. World J Gastroenterol. 2012;18:4751-7.
20. Govaere O, Komuta M, Berkers J, et al. Keratin 19: a key role player in the invasion of human hepatocellular carcinomas. Gut. 2014;63:674-85.
21. Llovet JM, Villanueva A, Lachenmayer A, Finn RS. Advances in targeted therapies for hepatocellular carcinoma in the genomic era. Nat Rev Clin Oncol. 2015;12:408-24.
22. Qin LX, Tang ZY. Recent progress in predictive biomarkers for metastatic recurrence of human hepatocellular carcinoma: a review of the literature. J Cancer Res Clin Oncol. 2004;130:497-513.
23. Yamashita T, Forgues M, Wang W, et al. EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res. 2008;68:1451-61.
24. Yamashita T, Wang XW. Cancer stem cells in the development of liver cancer. J Clin Invest. 2013;123:1911-8.
25. Scheau C, Badarau IA, Costache R, et al. The Role of matrix metalloproteinases in the epithelial-mesenchymal transition of hepatocellular carcinoma. Anal Cell Pathol. 2019;2019:9423907.
26. Zhu Y, He Y, Gan R. Wnt signaling in hepatocellular carcinoma: biological mechanisms and therapeutic opportunities. Cells. 2024;13:1990.
27. Zhu Z, Hao X, Yan M, et al. Cancer stem/progenitor cells are highly enriched in CD133+CD44+ population in hepatocellular carcinoma. Int J Cancer. 2010;126:2067-78.
28. Kim H, Park YN. Hepatocellular carcinomas expressing ‘stemness’-related markers: clinicopathological characteristics. Dig Dis. 2014;32:778-85.
29. Idowu SA, Olaniyi OO, Oluwole KA. Cytokeratin 19 (CK19) expression by thyroid neoplasms in a Nigerian tertiary health centre. Pan Afr Med J. 2023;44:176.
30. Verma V, Rao RN. Cytokeratin 19 expression in intrathoracic neoplasms: first study utilizing cellblocks, evaluating the role of a rarely used cytokeratin for lung cancers. Diagn Cytopathol. 2022;50:105-11.
31. Jia JJ, Cheng YF, Feng MB, et al. Diagnosis and treatment of biliary mucinous cystic neoplasms: a single-center experience. Hepatobiliary Pancreat Dis Int. 2024;23:495-501.
32. Llovet JM, Ricci S, Mazzaferro V, et al; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359:378-90.
33. Kudo M, Finn RS, Qin S, et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet. 2018;391:1163-73.
34. Sacco R, Ramai D, Tortora R, et al; A. I.G.O. (Italian Association of Hospital Gastroenterologists). Role of etiology in hepatocellular carcinoma patients treated with lenvatinib: a counterfactual event-based mediation analysis. Cancers. 2023;15:381.
35. Qin S, Bi F, Gu S, et al. Donafenib versus sorafenib in first-line treatment of unresectable or metastatic hepatocellular carcinoma: a randomized, open-label, parallel-controlled phase II-III trial. J Clin Oncol. 2021;39:3002-11.
36. Finn RS, Qin S, Ikeda M, et al; IMbrave150 Investigators. Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. N Engl J Med. 2020;382:1894-905.
37. Lee YT, Wang JJ, Zhu Y, Agopian VG, Tseng HR, Yang JD. Diagnostic criteria and LI-RADS for hepatocellular carcinoma. Clin Liver Dis. 2021;17:409-13.
38. Shuyao W, Mingyang B, Feifei M, Xiaoqin H. CK19 Predicts recurrence and prognosis of HBV positive HCC. J Gastrointest Surg. 2022;26:341-51.
39. Uenishi T, Kubo S, Yamamoto T, et al. Cytokeratin 19 expression in hepatocellular carcinoma predicts early postoperative recurrence. Cancer Sci. 2003;94:851-7.
40. Wang ZS, Wu LQ, Yi X, et al. CK19 can be used to predict the early recurrence and prognosis of HBV-related hepatocellular carcinoma patients with low AFP serum concentration after R0 radical hepatectomy. Chin J Oncol. 2012;34:753-8.
42. Chi X, Jiang L, Yuan Y, et al. A comparison of clinical pathologic characteristics between alpha-fetoprotein negative and positive hepatocellular carcinoma patients from Eastern and Southern China. BMC Gastroenterol. 2022;22:202.
43. Bruix J, Sherman M; American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma: an update. Hepatology. 2011;53:1020-2.
45. Ariizumi S, Kotera Y, Katagiri S, Nakano M, Yamamoto M. Combined hepatocellular-cholangiocarcinoma had poor outcomes after hepatectomy regardless of Allen and Lisa class or the predominance of intrahepatic cholangiocarcinoma cells within the tumor. Ann Surg Oncol. 2012;19:1628-36.
46. Connell LC, Harding JJ, Shia J, Abou-Alfa GK. Combined intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Chin Clin Oncol. 2016;5:66.
47. Gong SC, Cho MY, Lee SW, Kim SH, Kim MY, Baik SK. The meaning of gross tumor type in the aspects of cytokeratin 19 expression and resection margin in patients with hepatocellular carcinoma. J Gastroenterol Hepatol. 2016;31:206-12.
48. Morgan TR, Mandayam S, Jamal MM. Alcohol and hepatocellular carcinoma. Gastroenterology. 2004;127:S87-96.
50. Chuang SC, Lee YC, Hashibe M, Dai M, Zheng T, Boffetta P. Interaction between cigarette smoking and hepatitis B and C virus infection on the risk of liver cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev. 2010;19:1261-8.
51. Huang A, Yang XR, Chung WY, Dennison AR, Zhou J. Targeted therapy for hepatocellular carcinoma. Signal Transduct Target Ther. 2020;5:146.
52. Zhuo J, Lu D, Lin Z, et al. The distinct responsiveness of cytokeratin 19-positive hepatocellular carcinoma to regorafenib. Cell Death Dis. 2021;12:1084.
53. Wu MS, Zhong JH, Chen K, et al. Association of CK19 expression with the efficacy of adjuvant transarterial chemoembolization after hepatic resection in hepatocellular carcinoma patients at high risk of recurrence. J Clin Transl Res. 2022;8:71-9.
54. Ercolani G, Grazi GL, Ravaioli M, et al. The role of lymphadenectomy for liver tumors: further considerations on the appropriateness of treatment strategy. Ann Surg. 2004;239:202-9.
55. Sun HC, Zhuang PY, Qin LX, et al. Incidence and prognostic values of lymph node metastasis in operable hepatocellular carcinoma and evaluation of routine complete lymphadenectomy. J Surg Oncol. 2007;96:37-45.
56. Uenishi T, Hirohashi K, Shuto T, et al. The clinical significance of lymph node metastases in patients undergoing surgery for hepatocellular carcinoma. Surg Today. 2000;30:892-5.
57. Zhuang PY, Zhang JB, Zhu XD, et al. Two pathologic types of hepatocellular carcinoma with lymph node metastasis with distinct prognosis on the basis of CK19 expression in tumor. Cancer. 2008;112:2740-8.
58. Xiang YJ, Wang K, Yu HM, et al. Transarterial chemoembolization plus a PD-1 inhibitor with or without lenvatinib for intermediate-stage hepatocellular carcinoma. Hepatol Res. 2022;52:721-9.
59. Juengpanich S, Topatana W, Lu C, et al. Role of cellular, molecular and tumor microenvironment in hepatocellular carcinoma: Possible targets and future directions in the regorafenib era. Int J Cancer. 2020;147:1778-92.
60. Granito A, Forgione A, Marinelli S, et al. Experience with regorafenib in the treatment of hepatocellular carcinoma. Therap Adv Gastroenterol. 2021;14:17562848211016959.
61. Liang L, Pang JS, Gao RZ, et al. Development and validation of a combined radiomic and clinical model based on contrast-enhanced ultrasound for preoperative prediction of CK19-positive hepatocellular carcinoma. Abdom Radiol. 2025.
62. Huang K, He Y, Liang T, et al. Analysis of clinicopathologic and imaging features of dual-phenotype hepatocellular carcinoma. Sci Rep. 2024;14:3314.
63. Hu X, Wang Q, Huang G, et al. Gadoxetic Acid-Enhanced MRI-Based Radiomics Signature: A Potential Imaging Biomarker for Identifying Cytokeratin 19-Positive Hepatocellular Carcinoma. Comput Math Methods Med. 2023;2023:5424204.
64. Lv J, Yin H, Yu H, Shi H. The added value of 18F-FDG PET/MRI multimodal imaging in hepatocellular carcinoma for identifying cytokeratin 19 status. Abdom Radiol. 2023;48:2331-9.
65. Gu Y, Jin K, Gao S, et al. A preoperative nomogram with MR elastography in identifying cytokeratin 19 status of hepatocellular carcinoma. Br J Radiol. 2025;98:210-9.
Cite This Article
Open AccessHow to Cite
Download Citation
Export Citation File:
Type of Import
Tips on Downloading Citation
Citation Manager File Format
Type of Import
Direct Import: When the Direct Import option is selected (the default state), a dialogue box will give you the option to Save or Open the downloaded citation data. Choosing Open will either launch your citation manager or give you a choice of applications with which to use the metadata. The Save option saves the file locally for later use.
Indirect Import: When the Indirect Import option is selected, the metadata is displayed and may be copied and pasted as needed.
About This Article
Copyright
Data & Comments
Data
0
Comments
Comments must be written in English. Spam, offensive content, impersonation, and private information will not be permitted. If any comment is reported and identified as inappropriate content by OAE staff, the comment will be removed without notice. If you have any queries or need any help, please contact us at [email protected].