Prognostic and therapeutic significance of microscopic tumor focus in combined hepatocellular-cholangiocarcinoma: a multicenter pathological study

Introduction

Primary liver cancer ranks among the most common fatal malignant tumors globally (1). Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare subtype defined by the unequivocal presence of both hepatocytic and cholangiocyte differentiation within the same tumor. cHCC-CCA only accounts for about 2% of the primary liver cancer (2). Owing to its low incidence and complex tumor behaviors, no independent diagnostic and therapeutic paradigm has been well established. For example, cHCC-CCA is staged according to the tumor-node-metastasis (TNM) staging system for intrahepatic cholangiocarcinoma (CCA) (3); however, it lacks a Tis (carcinoma in situ) category. Therefore, it remains necessary to explore personalized clinicopathological parameters with prognostic and therapeutic value for cHCC-CCA.

Two microscopic intrahepatic metastatic patterns are commonly observed in primary liver cancer. The first is microvascular invasion (MVI), which localizes to endothelial-lined lumina, including interlobular vein, interlobular artery, central vein, and vessels within the tumor capsule (4,5); this lesion may eventually progress to portal vein tumor thrombus and hepatic vein tumor thrombus. The second pattern comprises round-shaped tumor nests distributed in hepatic sinusoids, without a vascular endothelial lining. This pathological phenomenon has been referred to as microscopic tumor focus (MTF), satellite nodule, or tumor budding in different studies (6,7), and may ultimately develop into multinodular liver cancer. Given their distinct anatomical localizations, these two patterns are hypothesized to represent different tumor behaviors: MVI, present within vascular lumina, may better reflect the potential for distant metastasis, whereas MTF, located in narrow hepatic sinusoids, may more accurately indicate local invasive capacity (5).

Numerous studies have focused on MVI in primary liver cancer, particularly in hepatocellular carcinoma (HCC); in contrast, research on MTF remains extremely scarce. We postulate that, for cHCC-CCA, in addition to assessing the presence or absence of MTF, their quantity, distribution, and histological components require special attention. Both HCC and CCA components in cHCC-CCA may form various degrees of MTF, exerting differential effects on disease outcomes. Accurate evaluation within MTF may provide a feasible approach to resolve the dilemma of intervention for cHCC-CCA.

Accordingly, we conducted this multicenter pathological study to investigate the significance of MTF in patients with cHCC-CCA, with the aim of providing evidence for personalized pathological evaluation and intervention strategies for this tumor entity. We present this article in accordance with the STROBE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0333/rc).

Methods

Patients

Consecutive patients diagnosed with primary cHCC-CCA who underwent curative-intent hepatectomy between 2013 and 2017 were retrospectively enrolled from 6 medical centers: Eastern Hepatobiliary Surgery Hospital, Changhai Hospital, Changzheng Hospital, Huadong Hospital, Minhang Hospital, and the Affiliated Hospital of Hangzhou Normal University. The diagnosis of cHCC-CCA was based on the 6th edition of the World Health Organization (WHO) Classification of Tumors of the Digestive System. Patients were excluded if they received neoadjuvant therapy or recurrent cHCC-CCA, harbored heterologous differentiation components within the tumor, or lacked essential data (Figure S1). Demographic data were collected at the time of first admission. Serological data were extracted from the latest examinations performed prior to surgery, and perioperative information was obtained from surgical records. The definition of macrovascular invasion and large bile duct invasion in our study referred to radiologically detectable or grossly visible tumor thrombi within intrahepatic vessels and intrahepatic bile ducts that were ascertained during the perioperative period. TNM staging was performed using the 9th edition of American Joint Committee on Cancer Staging Manual. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital (EHBHKY2022-H010-P001). Written informed consent was acquired from each patient. All participating hospitals were informed of and agreed to the study.

After surgery, all patients were recommended to undergo a standardized follow-up protocol: every 2–3 months within the first 2 years postoperatively, and every 4–6 months thereafter. Follow-up assessments included imaging examinations (ultrasound, computed tomography, or magnetic resonance imaging) and serological tests [alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA199)]. Hepatitis B virus (HBV) deoxyribonucleic acid (DNA) load was monitored only in HBV-infected patients, and hepatitis C virus (HCV) ribonucleic acid (RNA) load only in HCV-infected patients. Once tumor recurrence was suspected, individualized treatments were formulated based on tumor burden, performance status, liver function, and other clinical factors.

The clinical endpoints evaluated in this study were as follows: recurrence-free survival (RFS), defined as the interval from surgery to radiologically or histologically confirmed tumor recurrence or censoring; overall survival (OS), defined as the interval from hepatectomy to death or censoring; early RFS (≤2 years); late RFS (>2 years). Recurrence patterns were classified as intrahepatic (involvement of hepatic parenchyma, intrahepatic vessels or bile ducts), extrahepatic (distant metastasis or lymph node dissemination), or combined intrahepatic and extrahepatic recurrence. Follow-up was conducted via telephone interviews and medical record review, and was completed by February 2025.

Pathological assessment

Pathological information was retrieved from pathology reports and histological slides. The maximum tumor diameter, tumor number, and distance of surgical margin were extracted from macroscopic descriptions. Two pathologists independently re-evaluated all histological slides for tumor differentiation, predominant tumor component, perineural invasion (PNI), intratumoral mature tertiary lymphoid structure (mTLS), tumor necrosis, MVI, MTF, tumor capsule, liver cirrhosis, and hepatocellular steatosis. To avoid potential bias caused by the evaluation of multinodular tumors, all pathological features were recorded based on the largest cHCC-CCA lesion. In general, 6 to 8 histological slides were archived for this lesion, of which 4 to 6 slides were obtained from the tumor-liver interface or tumor center. One or two slides were sampled from the non-tumorous liver tissue. For detailed information of gross specimen sampling, please see in Appendix 1.

Tumor differentiation was graded according to the WHO criteria for HCC (2) and intrahepatic CCA (8), with the poorer differentiation component documented. The predominant tumor component was classified as HCC-dominant type (>70% HCC component), balanced type, or CCA-dominant type (>70% CCA component), consistent with our previous research (9). PNI was defined as the tumor cells within any of the 3 layers of the nerve sheath or tumor cells outside of the nerve with involvement of ≥33% of the nerve’s circumference (10). Intratumoral mTLS was defined as ectopic lymphoid aggregates with distinct germinal center formation within the tumor bed (11). Tumor necrosis was defined as spontaneous necrotic areas within the tumor without prior intervention, presenting as eosinophilic amorphous regions with or without predominant neutrophilic infiltration (12). MVI was defined as tumor cells observed within endothelium-lined vascular lumina outside the tumor mass under microscopy. Tumor capsule was defined as the presence of mature fibrous tissue surrounding the tumor edge. Liver cirrhosis was defined by the presence of regenerative pseudolobules in the hepatic parenchyma. Hepatic steatosis was defined as intracellular lipid accumulation in more than 5% of hepatocytes, showing macrovesicular or microvesicular vacuolar changes histologically (13).

MTF was defined as microscopically visible, isolated, round-like tumor nests located in hepatic sinusoids without endothelial lining. In this study, MTF was required to be limited to a maximum dimension of <0.2 cm, for the following reasons: during gross specimen measurement, the smallest scale used is 0.1 cm; therefore, the “desirable maximum diameter” of MTF is defined as <0.1 cm. However, we consider the <0.1 cm cut-off value to be overly stringent. According to the rounding-down method, all numbers of 0.1× are regarded as approximately equal to 0.1, hence the “essential maximum diameter” of MTF is defined as <0.2 cm. Unlike MVI, MTF is not confined to the intrahepatic vasculature (Figure 1A). Of note, some studies define grossly visible peritumoral lesions as tumor foci, satellite foci, or satellite lesions; however, these were regarded as multinodular tumors rather than MTF in this study (14). Since MTF was only microscopically detectable, their identification was influenced by sampling sites of gross specimens. According to our sampling protocol, the vast majority of MTFs were observed in slides from the tumor-liver interface and hepatic parenchyma adjacent to the tumor (≤1 cm from the main tumor), and only a few were incidentally found in slides from hepatic parenchyma distant from the tumor (>1 cm from the main tumor). To better characterize the prognostic impact of the number and distribution of MTF, a three-tiered grading system was adopted in reference to the Chinese MVI grading system: MTF 0/MTF-negative: no MTF identified; MTF 1: 1–5 MTFs, all located in the peritumoral region (≤1 cm); MTF 2: >5 MTFs (all peritumoral) or ≥1 MTF detected in distant hepatic parenchyma (>1 cm). MTF was further subclassified into HCC-type MTF (Figure 1B) and CCA-type MTF (Figure 1C) based on morphological features. Additionally, cHCC-CCA-type MTF (Figure 1D) were observed in four patients, with each case recorded as one HCC-type MTF and one CCA-type MTF. In most cases, the subtype of MTF could be determined by morphological similarity to the adjacent main tumor. For morphologically ambiguous cases, a panel of immunohistochemical markers was used, including Hepatocyte Paraffin-1 (Hep Par-1), Arginase-1, AFP, Glypican-3 (GPC3), Cytokeratin (CK) 7, CK19, and Mucin (MUC) 1. MTF was classified as HCC-type if it or the corresponding main tumor components expressed Hep Par-1, Arginase-1, or AFP; as CCA-type if they were negative for Hep Par-1, Arginase-1, and AFP but positive for CK7, CK19, or MUC1; and as HCC-type if negative for Hep Par-1, Arginase-1, AFP, CK7, CK19, and MUC1 but positive for GPC3. Intermediate cell carcinoma components, which show morphological features between HCC and CCA as well as co-express both lineages of markers, were occasionally present in the main tumor but were not observed in MTF and thus not analyzed. In addition, rare cases required the differentiation of HCC-type MTF from the cirrhotic nodule, as well as CCA-type MTF from the bile duct adenoma. For distinguishing the former, markers including GPC3, glutamine synthetase (GS), heat shock protein 70 (HSP70), cluster of differentiation 34 (CD34), and reticulin staining were utilized; for the latter, markers enhancer of zeste homolog 2 (EZH2) and p16 were employed. HCC-type MTF typically demonstrated positive expression of GPC3, GS, and HSP70, along with sinusoidal staining of CD34 and disruption of the reticulin framework. CCA-type MTF typically showed positive expression of EZH2 and negative expression of p16.

Figure 1 Different subtypes of MTF for cHCC-CCA patients. (A) MTF and MVI; (B) HCC-type MTF; (C) CCA-type MTF; (D) HCC + CCA-type MTF. Red arrows: MTF; Black arrow: MVI. Hematoxylin and eosin staining; magnification, 10×. cHCC-CCA, combined hepatocellular-cholangiocarcinoma; MTF, microscopic tumor focus; MVI, microvascular invasion.

Statistical analysis

Continuous variables were presented as mean ± standard deviation or median [interquartile range], and categorical variables as frequency (percentage). Comparisons between groups were performed using parametric tests (Student’s t-test or one-way analysis of variance) or non-parametric tests (Mann-Whitney U test or Kruskal-Wallis test) for continuous variables. Categorical variables were analyzed using the Chi-squared test or Fisher’s exact test. The interobserver concordance in MTF assessment between two pathologists was evaluated using Cohen’s Kappa coefficient. According to conventional interpretation, the Kappa value was categorized as follows: weak agreement ≤0, slight agreement 0–0.2, moderate agreement 0.21–0.40, average agreement 0.41–0.60, basic agreement 0.61–0.80, and 0.81–1.00 almost perfect. Survival analyses were conducted using the Kaplan-Meier method and the log-rank test. Univariate and multivariate Cox regression models were applied to identify prognostic factors for survival. The discriminative ability of the models was evaluated using Harrell’s concordance index (C-index). Univariate and multivariate logistic regression analyses were performed to screen predictive factors associated with MTF. The predictive performance was further validated using the area under the receiver operating characteristic curve (AUC). Variables with a marginal association in univariate analysis (P<0.2) were entered into the multivariate models. Multivariate models were visualized using forest plots. A two-sided P value <0.05 was considered statistically significant. All statistical analyses were performed using R software (Version 4.4.2; R Foundation for Statistical Computing, Vienna, Austria).

Results

Baseline characteristics

A total of 310 patients diagnosed with cHCC-CCA were included in this study. Of these patients, 271 were male (87.4%) and 39 were female (12.6%). The mean age ± standard deviation was 52.7±10.3 years. The median tumor diameter [interquartile range] was 4.4 cm [2.8–6.9 cm]. Solitary and multiple tumors were detected in 208 and 102 patients, respectively. Regarding TNM staging, 236 patients (76.1%) were classified as stage I–II, while 74 patients (23.9%) were classified as stage III–IV. MTF was identified in 149 patients (48.1%), comprising 134 (43.2%) with MTF 1 and 15 (4.8%) with MTF 2 disease. Among these, 55 patients (17.7%) had only HCC-type MTF, 64 (20.6%) had only CCA-type MTF, and 30 (9.7%) had both types (Table 1 and Table S1). The interobserver agreement, as measured by Kappa values, was 0.858 for assessing the presence or absence of MTF, 0.844 for MTF grading, and 0.778 for MTF histology.

MTF, prognosis, and recurrence pattern

Among 310 patients, the median follow-up duration, median RFS, and median OS were 8.52 years [7.61–9.81 years], 0.43 years [0.14–2.02 years], and 2.20 years [0.78–10.04 years], respectively.

Median RFS was 0.89 years [0.24–2.95 years] in MTF-negative patients and 0.23 years [0.11–1.00 years] in MTF-positive patients (Figure 2A, P<0.001). The 1-, 2-, and 5-year RFS rates were 45.0%, 31.2%, and 20.5% in the MTF-negative group versus 25.1%, 18.8%, and 13.0% in the MTF-positive group. Early RFS differed significantly between the two groups (Figure 2B, P<0.001), whereas late RFS did not (Figure 2C, P=0.47). Median OS was 2.95 years [1.32 years–not reached] and 1.53 years [0.54–6.88 years], respectively (Figure 2D, P<0.001), with corresponding OS rates of 79.7%, 62.1%, and 39.8% versus 60.1%, 40.1%, and 26.2%.

Figure 2 Impact of MTF on the prognosis of cHCC-CCA patients. (A) RFS; (B) early RFS; (C) late RFS; (D) OS. cHCC-CCA, combined hepatocellular-cholangiocarcinoma; MTF, microscopic tumor focus; OS, overall survival; RFS, recurrence-free survival.

Median RFS was 0.23 years [0.11–1.03 years] in the MTF 1 group and 0.21 years [0.10–0.36 years] in the MTF 2 group (Figure 3A, P<0.001). The 1-, 2-, and 5-year RFS rates were 26.5%, 21.0%, and 14.6% versus 13.3%, 0.0%, and 0.0%, respectively. Early RFS differed significantly among the three groups (Figure 3B, P<0.001). Late RFS was not evaluated for the three groups because all patients in the MTF 2 group experienced recurrence within 2 years. Median OS was 1.56 years [0.55–7.29 years] and 1.18 years [0.48–1.70 years] in the MTF 1 and MTF 2 groups, respectively (Figure 3C, P<0.001), with corresponding OS rates of 60.2%, 41.5%, and 28.6% versus 58.3%, 25.0%, and 0.0%.

Figure 3 Impact of MTF grading system on the prognosis of cHCC-CCA patients. (A) RFS; (B) early RFS; (C) OS. MTF 0: absence of MTF; MTF 1: 1–5 proximal MTFs; MTF 2: >5 proximal MTFs/≥1 distal MTF. cHCC-CCA, combined hepatocellular-cholangiocarcinoma; MTF, microscopic tumor focus; OS, overall survival; RFS, recurrence-free survival.

Median RFS was 0.21 years [0.11–1.93 years] for the HCC-type MTF group, 0.33 years [0.13–1.00 years] for the CCA-type MTF group, and 0.15 years [0.09–0.27 years] for the HCC + CCA-type MTF group (Figure 4A, P<0.001). The 1-, 2-, and 5-year RFS rates were 27.8%, 24.1%, and 16.7% versus 25.8%, 19.4%, and 12.9% versus 18.3%, 7.3%, and 7.3%, respectively. Early RFS differed significantly among the four groups (Figure 4B, P<0.001), whereas late RFS did not (Figure 4C, P=0.67). Median OS was 1.87 years [0.59 years–not reached], 1.74 years [0.58–6.88 years], and 0.61 years [0.39–1.61 years] for the three subgroups, respectively (Figure 4D, P<0.001), with corresponding OS rates of 63.4%, 48.1%, and 34.2% versus 67.2%, 43.1%, and 27.1% versus 40.0%, 20.0%, and 10.0%.

Figure 4 Impact of MTF histology on the prognosis of cHCC-CCA patients. (A) RFS; (B) early RFS; (C) late RFS; (D) OS. CCA, cholangiocarcinoma; cHCC-CCA, combined hepatocellular-cholangiocarcinoma; HCC, hepatocellular carcinoma; MTF, microscopic tumor focus; OS, overall survival; RFS, recurrence-free survival.

The recurrence patterns of 256 patients who experienced recurrence with and without TS are shown in Figure S2. The patients with TS had a higher incidence of intrahepatic recurrence (62.8% versus 58.3%).

MTF and surgical margin

In patients with MTF-negative cHCC-CCA, no significant statistical difference was observed in RFS between patients who underwent hepatectomy with a surgical margin ≤0.1 cm and those with a surgical margin >0.1 cm (Figure S3A, median RFS: 0.89 versus 0.87 years, P=0.54). The corresponding 1-, 2-, and 5-year RFS rates were 43.4%, 33.2%, and 20.0% versus 46.9%, 29.0%, and 21.0%, respectively. No significant differences were found in either early RFS (Figure S3B, P>0.99) or late RFS (Figure S3C, P=0.14) between the two groups. Similarly, OS did not differ significantly between groups (Figure S3D, median OS: 2.23 versus 3.26 years, P=0.11). The corresponding 1-, 2-, and 5-year OS rates were 76.2%, 50.8%, and 35.1% versus 83.7%, 75.2%, and 45.2%, respectively.

In patients with MTF-positive cHCC-CCA, RFS was significantly different between the ≤0.1 and >0.1 cm surgical margin groups (Figure 5A, median RFS: 0.18 versus 0.33 years, P=0.02). The corresponding 1-, 2-, and 5-year RFS rates were 19.7%, 14.8%, and 9.5% versus 32.3%, 24.2%, and 17.7%, respectively. A significant difference was detected in early RFS (Figure 5B, P=0.03), whereas no significant difference was observed in late RFS (Figure 5C, P=0.20). OS differed significantly between the two groups (Figure 5D, median OS: 0.99 versus 1.86 years, P=0.004). The corresponding 1-, 2-, and 5-year OS rates were 49.6%, 35.6%, and 20.1% versus 74.2%, 46.2%, and 34.3%, respectively.

Figure 5 Impact of surgical margin on the MTF-positive patients. (A) RFS; (B) early RFS; (C) late RFS; (D) OS. MTF, microscopic tumor focus; OS, overall survival; RFS, recurrence-free survival.

Predictive factors of MTF

Table S2 shows the results of univariate logistic regression analyses of MTF. For the multivariate logistic model, age, tumor diameter, MVI, and PNI were the independent factors of MTF (Table 2, AUC value: 0.704).

Prognostic factors

Tables S3-S6 show the results of univariate Cox regression analyses of RFS, early RFS, late RFS, and OS. For the multivariate Cox regression models, age, lymphocyte (LYM), macrovascular invasion, tumor diameter, tumor number, tumor component, MVI, and MTF histology were the independent factors of RFS [Figure 6A, C-index =0.722, 95% confidence interval (CI): 0.691–0.753]; age, LYM, macrovascular invasion, tumor diameter, tumor number, tumor differentiation, MVI, and MTF histology were the independent factors of early RFS (Figure 6B, C-index =0.721; 95% CI: 0.688–0.754); platelet, tumor number, and tumor component were the independent factors of late RFS; (Figure 6C, C-index =0.658, 95% CI: 0.560–0.756); LYM, macrovascular invasion, tumor diameter, tumor number, MVI, PNI, tumor necrosis, and MTF histology were the independent factors of OS (Figure 6D, C-index =0.728, 95% CI: 0.695–0.761).

Figure 6 Multivariate Cox regression analyses of prognosis. (A) RFS; (B) early RFS; (C) late RFS; (D) OS. CCA, cholangiocarcinoma; CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; LYM, lymphocyte; MTF, microscopic tumor focus; MVI, microvascular invasion; OS, overall survival; PLT, platelet; PNI, perineural invasion; RFS, recurrence-free survival.

Discussion

Although anti-recurrence therapy has long been a major focus in the field of primary liver cancer, MTF, a highly common form of intrahepatic metastasis, have received surprisingly little attention. To date, there is no universally accepted definition for MTF, including its standardized terminology, as well as criteria for its size, distance from the main tumor, and clonal origin with the primary lesion. We therefore aimed to propose our recommendations regarding this pathological phenomenon.

In terms of terminology, several terms have been used, including satellites (15), satellite tumors (16), satellite nodules (17), satellite foci (18), satellite lesions (14), satellite neoplastic nodules (19), and tumor budding (20,21). We strongly recommend “microscopic tumor focus” as the standard term. First, the word “satellite” inherently implies derivation from the main tumor. However, even when tumors are closely located and morphologically similar, a multicentric origin cannot be entirely excluded (22). For primary liver cancer, a highly heterogeneous tumor entity, “tumor” rather than “satellite” avoids arbitrary assumptions about hierarchical relationships between tumors without molecular evidence. Of note, “satellite” may be appropriate if the clonal relationship between MTF and the main tumor is firmly confirmed by rigorous analyses. Furthermore, among terms such as nodule, focus, lesion, and budding, we discourage the use of “tumor budding”. This concept is most widely applied in colorectal cancer, with a strict definition (≤4 tumor cells per focus) (23), which is not applicable to the vast majority of MTF in primary liver cancer. We propose adopting the nomenclature used for dysplastic nodules and dysplastic foci of liver, in which “nodules” and “foci” distinguish macroscopic visible lesions from microscopic-only lesions. Since MTF was a microscopic finding, the term “microscopic tumor focus” is justified. In contrast, macroscopically visible lesions are more reasonably classified as multinodular neoplasm, which can minimize controversies in TNM staging. Detection of MTF relies on sampling. The seven-point baseline sampling protocol has been widely adopted in China, which defines liver tissue adjacent to the tumor and distant to the tumor using a 1 cm cutoff (24). We therefore suggest maintaining this threshold to distinguish proximal MTF and distal MTF, as it is well aligned with this sampling approach.

In addition, accurate distinction between MTF and MVI is warranted, although it is challenging in some cases. MTF and MVI may represent distinct modes of tumor dissemination: the former contributes to some multinodular tumors, whereas the latter precedes the macrovascular invasion. When MVI do not completely fill the vessel, they can be distinguished by the presence of a distinct luminal structure and flattened endothelial cells. However, when the lumen is entirely occupied by tumor cells, the endothelial cells become obscured, leading to difficulties in morphological identification. Under such circumstances, the immunohistochemistry of CD34 or elastic fiber staining may also be limited, as tumor cell themselves can give rise to specialized vascular structures (25). In our experience, attention should be paid to the accompanying structures adjacent to tumor nests. Generally, MVI are most frequently observed in vessels within the portal tract. Therefore, the presence of bile ducts adjacent to tumor nests strongly favors a diagnosis of MVI. Conversely, tumor nests isolated within the hepatic parenchyma are more likely to represent MTF. Furthermore, the diagnosis of MTF should be made cautiously only when it is located outside the fibrous background of the tumor bed and within the hepatic parenchyma, to avoid overestimating its positivity rate. Tumor nests still embedded within the desmoplastic stroma of the tumor bed should be more appropriately regarded as the invasive region of the main tumor.

Against the above background, we investigated the prognostic value and intervention strategies of MTF in cHCC-CCA. Consistent with findings from other studies (26,27), we also confirmed that MTF was indicative of poor prognosis in patients with cHCC-CCA. Building on this, we established a grading system based on the number and location of MTF, which enabled further prognostic stratification of cHCC-CCA. This suggests that providing quantitative information regarding MTF in the pathological diagnosis of cHCC-CCA may offer better stratification for tailoring the intensity of clinical intervention strategies. In addition, we classified the histological types of MTF in cHCC-CCA. We found that the prognosis of patients with only HCC-type or CCA-type MTF was intermediate between that of patients without MTF and those with both histological types of MTF. This indicates that incorporating qualitative information of MTF into the pathological diagnosis of cHCC-CCA can provide more granular information for tailoring the direction of clinical intervention strategies. The prognostic impacts of both the quantity and distribution of MTF, as well as their histological types, were predominantly manifested in early recurrence. Meanwhile, correlation analysis between recurrence patterns and MTF revealed that MTF was more closely associated with intrahepatic recurrence. Both early recurrence and intrahepatic recurrence are closely related to the potential minimal residual tumor adjacent to the surgical margin. Accordingly, we further explored the influence of MTF based on surgical margin distance.

We observed that MTF was mainly distributed adjacent to the main tumor, so we used 0.1 cm, the smallest macroscopically measurable scale, to evaluate the relationship between surgical margin and MTF (28). The results showed no significant correlation between surgical margin distance and survival in cHCC-CCA patients without MTF. In contrast, maintaining an adequate surgical margin distance was associated with significantly better prognosis in patients with MTF. These findings further support the intervention implications of MTF. Consequently, we constructed a predictive model for MTF, several parameters of which are preoperatively available. This model allows hepatic surgeons to perform wide-margin resection whenever feasible in patients at high risk of MTF. Furthermore, postoperative interventions such as adjuvant radiotherapy may represent a viable therapeutic option for cHCC-CCA patients with MTF who underwent narrow-margin resection (29).

This study firstly standardized the definition of MTF, and systematically delineated its prognostic and therapeutic implications in cHCC-CCA. Nevertheless, several limitations warrant due acknowledgment. First, the retrospective design of this study inevitably introduces inherent selection and observational biases, thereby mandating prospective cohort studies to rigorously validate the robustness and generalizability of our current conclusions. Second, the vast majority of patients enrolled in this study were HBV-associated patients. Accordingly, whether MTF exerts comparable prognostic value in cHCC-CCA patients triggered by other etiological factors remains to be further elucidated. Third, although the vast majority of resected specimens in this study were sampled using the seven-point baseline sampling protocol, a small number of patients did not undergo this approach due to subjective or objective reasons, which may cause some bias in the evaluation of MTF. Therefore, the validation of the prognostic and therapeutic relevance of MTF based on a standardized sampling protocol remains valuable. Fourth, despite our meticulous efforts to differentiate MVI from MTF via comprehensive pathological assessment, low-probability misdiagnosis or misclassification might still arise, attributable to the limitations of histological sampling and indistinct histomorphological features. This dilemma urgently necessitates the development and application of more advanced auxiliary diagnostic modalities to achieve accurate identification. The China Liver Cancer Guidelines for the Diagnosis and Treatment of Hepatocellular Carcinoma propose the integration of both MVI and MTF into the MVI grading system, aiming to address this prevailing pathological challenge (30).

Conclusions

Our results establish MTF as a novel pathological parameter for cHCC-CCA, of which its stratification by number/distribution and histology enables precise prognostic assessment. Moreover, MTF demonstrates its value in guiding personalized therapeutic decision-making, particularly in the selection of the surgical margin. This study addresses the lack of tailored pathological markers for cHCC-CCA and provides a translational framework for integrating MTF into routine pathological reporting and clinical management of this rare tumor entity.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0333/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0333/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0333/prf

Funding: This work was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (No. 2023ZD0500100), Shanghai Leading Talent Program of Eastern Talent Plan (No. BJWS2024084), Shanghai Municipal Health Commission Seed Program for Research and Translation of Novel Medical Technologies (No. 2024ZZ1026), and Take-off Project Talent Program of EHBH (No. TF2024XSJJ02).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0333/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital (EHBHKY2022-H010-P001). Written informed consent was acquired from each patient. All participating hospitals were informed of and agreed to the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. 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. [Crossref] [PubMed]
2. Arends MJ, Esposito I, Gill AJ, et al. Changes in the 6th edition of the World Health Organization classification of tumours of the digestive system. Histopathology 2026;88:1295-314.
3. James B, Meredith G, Brian OS, et al. TNM Classification of Malignant Tumours 9th Edition. Bognor Regis: John Wiley & Sons Ltd.; 2025.
4. Isik B, Gonultas F, Sahin T, et al. Microvascular Venous Invasion in Hepatocellular Carcinoma: Why Do Recurrences Occur? J Gastrointest Cancer 2020;51:1133-6. [Crossref] [PubMed]
5. Erstad DJ, Tanabe KK. Prognostic and Therapeutic Implications of Microvascular Invasion in Hepatocellular Carcinoma. Ann Surg Oncol 2019;26:1474-93. [Crossref] [PubMed]
6. Li C, Ouyang W, Yang T. The association of microvascular invasion with satellite nodule, tumor multiplicity, tumor encapsulation and resection margin of hepatocellular carcinoma. J Hepatol 2022;77:890-1. [Crossref] [PubMed]
7. Wu G, Luo R, Xu Q, et al. Tumor budding in pre-neoadjuvant biopsy and post-neoadjuvant resection specimens is associated with poor prognosis in intrahepatic cholangiocarcinoma-a cohort study of 147 cases by modified ITBCC criteria. Virchows Arch 2024;485:913-23. [Crossref] [PubMed]
8. Fred TB, Fátima C, Ralph HH, et al. WHO Classification of Tumours. Digestive System Tumours. 4th Edition. Lyon: IARC Press; 2010.
9. Wang H, Qian YW, Sheng X, et al. Tumor component proportion in combined hepatocellular-cholangiocarcinoma shows a significant correlation with its biological behavior and prognosis. Hepatobiliary Pancreat Dis Int 2026; [Crossref]
10. Wang H, Zhou YY, Chen HZ, et al. Perineural invasion as a prognostic determinant in combined hepatocellular-cholangiocarcinoma: a multicenter pathological study. J Gastrointest Surg 2025;29:102155. [Crossref] [PubMed]
11. Wang H, Qian YW, Zhao Y, et al. A Grading System of Intratumoral Tertiary Lymphoid Structure for Combined Hepatocellular-Cholangiocarcinoma: A Multicenter Pathological Study. J Hepatocell Carcinoma 2025;12:3011-27. [Crossref] [PubMed]
12. Wang H, Qian YW, Sheng X, et al. Deciphering the significance of tumor necrosis and developing a grading system in combined hepatocellular-cholangiocarcinoma: A multicenter pathological study. Pathol Res Pract 2026;278:156335. [Crossref] [PubMed]
13. Wang H, Cao Z-Y, Xia C-Y, et al. Severe hepatic steatosis as a protective prognostic factor in combined hepatocellular-cholangiocarcinoma: A multicenter pathological study. World J Gastrointest Oncol 2026;18:116764.
14. Wei T, Ma ZJ, Guo S, et al. Satellite lesions versus intrahepatic metastasis in multifocal intrahepatic cholangiocarcinoma: Prognostic impact and genomic profiling. Hepatology 2025; Epub ahead of print. [Crossref]
15. Gonvers S, Martins-Filho SN, Hirayama A, et al. Macroscopic Characterization of Hepatocellular Carcinoma: An Underexploited Source of Prognostic Factors. J Hepatocell Carcinoma 2024;11:707-19. [Crossref] [PubMed]
16. D’Silva M, Varty G, Patkar S, et al. Navigating early recurrences in post-hepatectomy hepatocellular carcinoma: Preventive strategies and prognostic indicators. Surg Oncol 2026;66:102438.
17. Wu YH, Tseng YF, Yang YJ, et al. Synchronous two-nodule hepatocellular carcinoma without satellite nodules is associated with better survival outcomes compared with satellite-nodule hepatocellular carcinoma. J Clin Pathol 2026;79:332-9. [Crossref] [PubMed]
18. Xia F, Yan J, Liu X, et al. A novel pathologic scoring system for predicting postoperative recurrence in BCLC stage 0-A hepatocellular carcinoma patients: a nationwide multicenter study. Sci China Life Sci 2026;69:611-21. [Crossref] [PubMed]
19. Vernuccio F, Porrello G, Cannella R, et al. Benign and malignant mimickers of infiltrative hepatocellular carcinoma: tips and tricks for differential diagnosis on CT and MRI. Clin Imaging 2021;70:33-45. [Crossref] [PubMed]
20. Sarelin N, Kairaluoma V, Saarnio J, et al. Tumour budding and tumour-stroma ratio in hepatocellular carcinoma. Hum Pathol 2025;165:105937. [Crossref] [PubMed]
21. Nguyen Thi Q, Nguyen Canh H, Li Z, et al. New insights into the adverse prognostic role of squamous differentiation in intrahepatic cholangiocarcinoma: A comprehensive analysis. Hum Pathol 2025;160:105824. [Crossref] [PubMed]
22. Boot GF, Haak F, Coto-Llerena M, et al. Multi-omics profiling reveals divergent biology and liver microenvironment in HCC of metastatic and de novo origin. Mol Cancer 2026;25:101. [Crossref] [PubMed]
23. Li P, Liu N, Wang Y, et al. Preoperative prediction of tumor budding in colorectal cancer based on quantitative parameters of dual-layer detector spectral computed tomography: a preliminary study. J Gastrointest Oncol 2025;16:1971-84. [Crossref] [PubMed]
24. Sheng X, Ji Y, Ren GP, et al. A standardized pathological proposal for evaluating microvascular invasion of hepatocellular carcinoma: a multicenter study by LCPGC. Hepatol Int 2020;14:1034-47. [Crossref] [PubMed]
25. Taniai T, Haruki K, Väyrynen JP, et al. Immunological and prognostic significance of vessels encapsulating tumor clusters (VETC) in hepatocellular carcinoma. Eur J Surg Oncol 2025;51:110381. [Crossref] [PubMed]
26. Na BG, Hwang S, Ahn CS, et al. Limited survival benefit of preoperative transarterial chemoembolization in huge hepatocellular carcinoma due to the prognostic impact of satellite nodules: a retrospective cohort study. Ann Surg Treat Res 2025;109:194-206. [Crossref] [PubMed]
27. Xu XF, Wu H, Gu LH, et al. Development and Validation of an Individualized Prediction Model for Postoperative Late Recurrence After Hepatectomy for Hepatocellular Carcinoma (POLAR-HCC): A Multicenter Study. Ann Surg Oncol 2025;32:9573-83. [Crossref] [PubMed]
28. Long L, Chen B, Zheng X, et al. Postoperative radiotherapy following null-margin hepatectomy in patients with hepatocellular carcinoma adhering to the major vessels: A propensity score-matched survival analysis cohort study. Clin Transl Radiat Oncol 2024;45:100727. [Crossref] [PubMed]
29. Choi SH, Koom WS, Lee IJ. The role of radiotherapy in the management of combined hepatocellular-cholangiocarcinoma: current evidence and future perspectives. J Liver Cancer 2026;26:55-64. [Crossref] [PubMed]
30. Zhou J, Sun H, Wang Z, et al. China Liver Cancer Guidelines for the Diagnosis and Treatment of Hepatocellular Carcinoma (2024 Edition). Liver Cancer 2025;14:779-835. [Crossref] [PubMed]