Background

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms, ranking among the top five leading causes of cancer-related deaths worldwide [1, 2]. Surgical resection remains the primary potentially curative treatment, yet long-term survival remains unsatisfactory, mainly due to a high incidence of recurrence and mortality. Currently, there is no reliable method for predicting the fate of individual patients with HCC [3]. Serum alpha-fetoprotein (AFP), although it is considered the most reliable biomarker of HCC diagnosis, performs poorly as a prognostic indicator. For example, 40–60% of HCC patients exhibit normal AFP levels [4], making it unreliable as a marker for monitoring recurrence after resection. Other prognostic factors for HCC have been proposed, including the tumor / node / metastases (TNM) staging system and the Barcelona Clinic Liver Cancer (BCLC) staging system; however, the accuracy of these systems can vary substantially [5]. Thus, a sensitive and effective biomarker is urgently required to help predict patients’ outcomes, which may improve patient management and therefore survival.

One such biomarker may be circulating tumor cells (CTCs), which have potent proliferative and metastatic abilities. CTCs may shed from the primary tumor into the bloodstream before or even during surgical resection, then travel throughout the body via the systemic circulation. Detecting CTCs by liquid biopsy may substantially improve prognosis prediction and detection of recurrence [6, 7]. CTCs can be classified into three subpopulations: epithelial (E-CTCs), mesenchymal (M-CTCs), or an intermediate subtype undergoing the epithelial-to-mesenchymal transition (E/M-CTCs) [8]. These subtypes can be distinguished based on expression of surface markers. The epithelial-to-mesenchymal transition (EMT), a phenotypic change marked by the loss of epithelial characteristics and the acquisition of invasive mesenchymal properties, is considered essential for metastasis [9]. CTCs gain mesenchymal features via EMT and drive HCC metastasis [10, 11]. Levels of M-CTC not only represent the progression and state of the disease, but also serve as a prognostic marker. In fact, recent studies have demonstrated that M-CTCs are better at predicting prognosis than the total number of CTCs in patients with lung [12], gastric [13], or breast cancer [14].

Accurately predicting the prognosis of HCC patients will likely require taking into account other factors in addition to M-CTCs. One such factor may be cell proliferation, as assessed based on expression of the DNA-binding nuclear protein Ki67. This protein is expressed in all phases of mitosis except the G0 phase, and it consistently shows high sensitivity and specificity as a marker of proliferative cells [15]. Ki67 upregulation may help drive the proliferation of malignant tumor cells, making it a potential biomarker of tumor aggressiveness and poor prognosis [16].

Combining the characteristics of tumor proliferation and CTCs may lead to more accurate prediction of prognosis of patients with various solid neoplasms, including non-small cell lung cancer [17] and renal cell carcinoma [18]. However, we are unaware of reports assessing the relationship between M-CTC and Ki67 in patients with HCC. Therefore, the present study aimed to investigate whether the M-CTC count in peripheral blood and Ki67 expression in tumor tissues correlate with each other and whether they can, alone or together, predict survival of patients with HCC.

Methods

Study population

A total of 105 patients were enrolled between March 2014 and May 2017 at the Guangxi Medical University Cancer Hospital (Nanning, China). The inclusion criteria were as follows: (1) definitive HCC diagnosis based on World Health Organization criteria [19]; (2) liver function in Child–Pugh A stage and Performance Status Test score of 0–1; (3) treatment by R0 resection, defined as complete macroscopic removal of the tumor, negative resection margin and no detectable residual intra- or extrahepatic metastatic lesions; (4) no history of anticancer treatment, such as trans-arterial chemoembolization or targeted therapy; and (5) availability of complete medical records. Patients with other systemic tumors and autoimmune diseases were excluded. Peripheral venous punctures within one week before surgery were used to analyze preoperative blood markers, including AFP and hepatitis B virus DNA (HBV-DNA). Cut-off values were 400 ng/mL for AFP and 5.0 × 102 IU/mL for HBV-DNA, as recommended by the assay manufacturer.

The study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethics Review Committee of the Affiliated Cancer Hospital of Guangxi Medical University (approval number: LW2022121). The requirement for informed consent to participate was waived by the Ethics Review Committee of the Affiliated Cancer Hospital of Guangxi Medical University. The reason for the waiver of informed consent is because all patients, on admission, consented for their anonymized medical data to be analyzed and published for research purposes.

CTC isolation and in situ hybridization

At 1–2 days before resection, peripheral blood (5 mL) was collected into anticoagulant-coated tubes for assays of CTCs. CTCs were isolated using the CanPatrol enrichment method [31, 32]. This implies a link between M-CTCs and cancer progression. Indeed, M-CTC count is significantly higher in advanced HCC than in early disease [33], and we found the M-CTC count to predict OS better than the total CTC count. Our study also found that M-CTC positivity was associated with several features of HCC malignancy, including high AFP, larger and multiple tumors, poor tumor differentiation, incomplete tumor capsule, and the presence of MVI and PVTT. This may help explain why patients in our study who were positive for M-CTCs had significantly worse outcomes than those negative for the cells, and why M-CTC was an independent predictor of shorter OS. Previous work has shown that patients positive for M-CTCs are more likely to experience early recurrence [34]. Our previous work revealed that anatomical resection may not be more beneficial than non-anatomical resection for patients positive for M-CTCs [24]. Therefore, M-CTCs should be considered an aggressive subtype, and CTC analysis before surgery can help guide the choice of resection method. We further recommend that HCC patients positive for M-CTCs should be treated with more effective therapies against recurrence and should be monitored closely after treatment.

Our study suggests that in addition to M-CTCs, Ki67 can help individualize treatment and surveillance. Higher Ki67 levels reflect the enhanced proliferative activity of tumor cells. A meta-analysis of 54 studies involving 4,996 HCC patients revealed that Ki67 expression was associated with more advanced tumor stage [35]. However, the tumors in the meta-analyzed studies were dichotomized arbitrarily into those expressing low or high Ki67 values [36,37,38]. In the present study, we obtained the optimal cut-off value of Ki67 using X-tile software [25], a widely recognized and applied method in many studies. Based on this cut-off, high Ki67 expression correlated significantly with unfavorable clinicopathological features and with significantly worse OS after hepatectomy. In addition, multivariate analysis identified Ki67 as an independent predictor for OS after hepatectomy.

The observed weak link between M-CTC and Ki67 may reflect that CTCs are derived from the primary tumor and therefore share similar characteristics, yet the tumor microenvironment undergoes substantial changes when tumor cells detach from the primary tumor and enter the bloodstream [39]. Our patients classified as high-risk based on positive M-CTC and high Ki67 expression showed significantly shorter OS than those classified as medium- or low-risk, even if their HCC was in early stages. Similarly, prognosis was significantly worse for patients with non-small cell lung cancer who were positive for M-CTCs and had high Ki67 expression than for patients with only one of these two risk factors [17]. We hypothesize that the proliferative effects of Ki67 synergize with the EMT of CTCs to promote HCC progression, ultimately leading to poor prognosis. These results may help improve the risk stratification of patients with HCC. In particular, high-risk patients may benefit from a higher intensity of adjuvant therapy and follow-up.

This study has several limitations. First, it was a single-center retrospective study with a relatively small sample, which increases the risk of selection bias and lack of generalizability to other patient populations. Our findings should be validated and extended in larger, multi-center, prospective studies. Second, the current study was stratified the Ki67 index according to the cut-off point determined by the X-Title software, but other studies have shown different stratification parameters [40]; the precise cut-off value needs to be established with more research for further verification. Furthermore, we did not examine possible mechanistic explanations for the observed association of M-CTC count or Ki67 expression with poor OS. Future studies should elucidate how elevated Ki67 expression and M-CTCs worsen the prognosis of HCC patients.

Conclusion

Our results suggest that an increase in the number of M-CTCs in preoperative peripheral blood closely correlates with high Ki67 expression in HCC tissues, and that both events promote tumor progression, ultimately leading to poor OS. Our study provides strong evidence that M-CTCs and Ki67 together may serve as a prognostic biomarker for stratifying HCC patients by risk of poor prognosis. These findings may help clinical decision-making and management of HCC patients.