Abstract
Background
Surgical resection and ablation therapy are both primary treatment options for very early stage hepatocellular carcinoma (HCC). Accurate risk stratification is important, since patients at higher risk of recurrence may derive greater benefit from curative resection than from ablation. We investigated whether the mitotic index is associated with early recurrence in HCC and may serve as a prognostic marker to refine risk assessment in very early stage disease.
Methods
The number of mitoses was counted in representative tumor slides from 942 cases of surgically resected HCC from Samsung Medical Center. A high mitotic index was defined as more than eight mitoses in 10 high-power fields. The relationship between mitotic index, clinicopathological characteristics, and prognosis were analyzed. External validation was performed using 112 HCC cases obtained from Hallym University Sacred Heart Hospital.
Results
High mitotic index was identified in 296 patients and was significantly associated with aggressive clinicopathological features including higher Edmondson grade, advanced American Joint Committee on Cancer T stage, and early tumor recurrence. Patients with a high mitotic index displayed a significantly shorter early recurrence-free survival (e-RFS). In subgroup analysis of patients with very early stage, the high mitotic index group showed unfavorable influences on e-RFS.
Conclusion
High mitotic index is a significant predictor of early recurrence in HCC patients and may provide useful prognostic information in very early stage disease. While its direct role in guiding primary treatment selection is limited, the mitotic index could contribute to risk stratification and postoperative management strategies.
Keywords: hepatocellular carcinoma, prognosis, recurrence, mitosis, mitotic index
Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the fourth leading cause of cancer-related deaths worldwide.1 Surgical resection is the treatment of choice for HCC, but patient prognosis after hepatectomy is unsatisfactory due to the high recurrence rate.2 Thus, accurate prediction of tumor recurrence using appropriate treatment strategies is crucial for improving patient outcomes.3
Several predictive biomarkers for HCC recurrence after surgical resection have been investigated.4,5 However, most of these biomarkers are not routinely analyzed or used in daily pathological examinations. Therefore, time-efficient, easily applicable, and reproducible predictive markers are urgently needed to stratify HCC patients who are likely to experience early tumor recurrence after surgery.
High cell proliferation is a classic hallmark of cancer, and mitosis of tumor cells is a simple and useful method for evaluating cell proliferation under microscopic examination.6,7 The prognostic role of mitotic index has been demonstrated in various types of tumors. Thus, a high mitotic index is now used as a key histologic factor for tumor grading systems in diverse organs, including the breast, ovary, adrenal gland, skin, soft tissue, and gastrointestinal tract.8–13 However, the prognostic role of mitotic index in HCC as a predictive biomarker has not yet been fully elucidated.
There is still a clinically unmet need for the treatment of HCC patients at a very early stage (Barcelona Clinic Liver Cancer [BCLC] stage 0). Both resection and ablation therapy are recommended, but guidelines for the selection between the two treatments have not been clearly established.14 Although many studies reported no significant prognostic difference between the two treatment options in HCC patients at a very early stage,15–23 some studies have demonstrated that the surgical resection may provide superior survival outcomes compared with ablation.24–29 Therefore, identifying reliable biomarkers of recurrence risk could be helpful in refining treatment selection.
We previously demonstrated that a high mitotic index of tumor cells was significantly associated with shorter disease-specific survival in 282 HCC patients.7 In the present study, we sought to further evaluate the prognostic effect of mitotic index in a larger cohort (n = 942) and validate the findings in an independent external cohort (n = 112). Specifically, we investigated whether the mitotic index is associated with early tumor recurrence (within 24 months) and whether it could serve as a potential biomarker to inform clinical decision-making, particularly in very early stage HCC.
Methods
Patient Selection
From January 2008 to December 2012, 1297 patients underwent hepatic resection for HCC at the Samsung Medical Center (SMC), Seoul, Korea. Of the 1297 patients, those who received liver transplantation (n = 166) and other treatments including transarterial chemoembolization, radiofrequency ablation (RFA), and chemotherapy before surgery (n = 89) were excluded. Additionally, 2 patients were excluded because of intraoperative RFA and previous operative history. The remaining 1040 patients underwent curative resection as a first-line treatment for primary HCC, and 98 patients were excluded as follows: 5 patients died within 30 days after surgery, 3 patients displayed tumor involvement of the microscopic resection margin on pathologic examination, 24 patients had lymph node or distant metastasis, 57 patients had portal vein invasion, and 9 patients had unavailable slides. Finally, 942 cases that were appropriate for pathological slide review were considered in the retrospective analysis. Figure 1 shows the flowchart of patient selection.
Figure 1.
Flowchart of patient selection process.
Abbreviations: HCC, hepatocellular carcinoma; TACE, Transarterial chemoembolization; RFA, Radiofrequency ablation; CTx, chemotherapy; LN, lymph node.
An external cohort of 112 HCC patients were prepared from Hallym University Sacred Heart Hospital (HUMC), Anyang, Republic of Korea, for validation. All patients were treated with surgical resection as first-line treatment for primary HCC between January 2011 and December 2015, and no patients received previous treatment before surgery, exhibited positive tumor resection margin, lymph node or distant metastasis.
Patients’ medical records were reviewed to obtain clinical information on age, sex, underlying etiology of chronic liver disease, serum α-fetoprotein (AFP) and albumin-bilirubin (ALBI) score. Histopathologic features including tumor differentiation, microvascular invasion, multicentric occurrence, intrahepatic metastasis, tumor necrosis, and background non-tumor liver pathology, were collected by reviewing pathology reports. Histological differentiation of HCC was determined using the Edmondson-Steiner grading system.30 Multicentric occurrence and intrahepatic metastasis were determined according to the criteria of the Liver Cancer Study Group of Japan.31 Two-year cutoffs were used to classify tumor recurrence as early or late.32 Patients were staged according to the American Joint Committee on Cancer (AJCC) staging manual 8th edition and the BCLC staging system.33
After surgery, all patients underwent dynamic contrast-enhanced CT and serum AFP measurement every 3 months for surveillance. Dynamic contrast-enhanced MRI was additionally performed when tumor recurrence was suspected. Tumor recurrence was diagnosed based on radiologic evaluation of CT and/or MRI results.
The primary endpoint was early tumor recurrence within 24 months after surgical resection, which is primarily associated with intrahepatic metastasis, reflecting tumor aggressiveness.32 This study was approved by the Institutional Review Board of the Samsung Medical Center (IRB No. 2019–08-018) and Hallym University Sacred Heart Hospital (IRB No. 2021–09-008).
Evaluation of Mitotic Index
The mitotic index of tumor cells was evaluated as described previously.7 Briefly, two pathologists (SC and SP) examined the hematoxylin and eosin-stained slides and counted the number of mitoses in 10 high-power fields (HPFs). We first identified hotspot areas that contained the most mitotic figures. After counting the mitoses in the hotspot, the count was extended to nine adjacent non-overlapping fields. When mitoses were sparse and randomly distributed throughout the tumor and there was no identifiable hot spot, a representative mitosis was chosen, and the count began with that field. Mitotic figures were assessed based on the criteria of Baak,34 and were included in the count only when a consensus was achieved between the two pathologists using a multi-head microscope. The mitotic index was determined as the highest mitotic count among the assessed fields. For validation in external cohort, one pathologist (YAC) determined the mitotic index in the same way.
Statistical Analysis
We used the X-Tile statistics package (Yale University, New Haven, CT, USA) to determine the optimal cutoff value of the high mitotic index with the best statistical significance related to patient survival,35 and cases were dichotomized into low and high mitotic index groups based on the established cutoff value. The relationships between mitotic index and clinicopathological parameters were analyzed using the chi-squared test or Fisher’s exact test. The Kaplan-Meier method was used to construct the survival curves. Cox regression analysis was performed to assess the factors independently associated with early tumor recurrence. Statistical analyses and visualization were performed using R version 4.2.2 (https://cran.r-project.org), and a p-value <0.05 (two sided) was considered statistically significant.
Results
Patients Characteristics
The clinicopathological characteristics of 942 SMC patients are summarized in Table 1.
Table 1.
The Association Between Mitotic Index and Clinicopathologic Parameters in All 942 Hepatocellular Carcinoma Patients
| Mitotic Index | ||||
|---|---|---|---|---|
| Total | Low (<8) | High (≥8) | ||
| n=942 | n=646 (%) | n=296 (%) | p value | |
| Age, year | ||||
| ≤55 | 442 | 279 (43.2) | 163 (55.1) | 0.001 |
| >55 | 500 | 367 (56.8) | 133 (44.9) | |
| Gender | ||||
| Female | 181 | 130 (20.1) | 51 (17.2) | 0.338 |
| Male | 761 | 516 (79.9) | 245 (82.8) | |
| Tumor size, cm | ||||
| ≤5.0 | 747 | 518 (80.2) | 229 (77.4) | 0.365 |
| >5.0 | 195 | 128 (19.8) | 67 (22.6) | |
| Edmondson grade | ||||
| 1 | 61 | 56 (8.7) | 5 (1.7) | <0.001* |
| 2 | 824 | 574 (88.9) | 250 (84.5) | |
| 3 | 54 | 15 (2.3) | 39 (13.2) | |
| 4 | 3 | 1 (0.2) | 2 (0.7) | |
| Microvascular invasion | ||||
| (-) | 516 | 404 (62.5) | 112 (37.8) | <0.001 |
| (+) | 426 | 242 (37.5) | 184 (62.2) | |
| Intrahepatic metastasis | ||||
| (-) | 867 | 597 (92.4) | 270 (91.2) | 0.616 |
| (+) | 75 | 49 (7.6) | 26 (8.8) | |
| Multicentric occurrence | ||||
| (-) | 885 | 601 (93.0) | 284 (95.9) | 0.111 |
| (+) | 57 | 45 (7.0) | 12 (4.1) | |
| Necrosis | ||||
| (-) | 583 | 445 (68.9) | 138 (46.6) | <0.001 |
| (+) | 359 | 201 (31.1) | 158 (53.4) | |
| AJCC T-stage | ||||
| 1a | 199 | 136 (21.1) | 63 (21.3) | <0.001* |
| 1b | 312 | 251 (38.9) | 61 (20.6) | |
| 2 | 399 | 243 (37.6) | 156 (52.7) | |
| 3 | 27 | 16 (2.5) | 11 (3.7) | |
| 4 | 5 | 0 (0.0) | 5 (1.7) | |
| BCLC stage | ||||
| 0 | 199 | 136 (21.1) | 63 (21.3) | 0.754 |
| A | 263 | 176 (27.2) | 87 (29.4) | |
| B | 480 | 334 (51.7) | 146 (49.3) | |
| C | 0 | 0 (0.0) | 0 (0.0) | |
| D | 0 | 0 (0.0) | 0 (0.0) | |
| ALBI Grade | ||||
| 1 | 639 | 431 (66.7) | 208 (70.3) | 0.533 |
| 2 | 281 | 200 (31.0) | 81 (27.4) | |
| 3 | 22 | 15 (2.3) | 7 (2.4) | |
| AFP level, ng/mL | ||||
| ≤200 | 705 | 517 (80.0) | 188 (63.5) | <0.001 |
| >200 | 237 | 129 (20.0) | 108 (36.5) | |
| Etiology | ||||
| Non-viral | 136 | 120 (18.6) | 16 (5.4) | <0.001* |
| HBV | 759 | 494 (76.5) | 265 (89.5) | |
| HCV | 43 | 29 (4.5) | 14 (4.7) | |
| HBV & HCV | 4 | 3 (0.5) | 1 (0.3) | |
| Liver cirrhosis | ||||
| (-) | 531 | 377 (58.4) | 154 (52.0) | 0.080 |
| (+) | 411 | 269 (41.6) | 142 (48.0) | |
| Early recurrence | ||||
| (≤2 years) | ||||
| (-)† | 683 | 494 (76.5) | 189 (63.9) | <0.001 |
| (+) | 259 | 152 (23.5) | 107 (36.1) | |
| Late recurrence | ||||
| (>2 years) | ||||
| (-)† | 566 | 409 (82.8) | 157 (83.1) | 1.000 |
| (+) | 117 | 85 (17.2) | 32 (16.9) | |
Notes: *by Fisher’s exact test, otherwise by chi-square test, † No early or late recurrence.
Abbreviations: AJCC, American Joint Committee on Cancer; BCLC, Barcelona Clinic Liver Cancer; ALBI, albumin-bilirubin; AFP, α-fetoprotein; HBV, hepatitis B virus; HCV, hepatitis C virus.
The median age was 56 years (range, 20–83 years); 761 (80.8%) patients were male and 181 (19.2%) were female. The median tumor size was 3.1 cm, and 195 (20.7%) patients had tumors larger than 5 cm. Seven hundred and fifty-nine patients (80.6%) were infected with hepatitis B virus, and 43 (4.6%) were infected with hepatitis C virus. Combined hepatitis B and C virus infections were identified in 4 (0.4%) patients. Viral markers were not detected in 136 (14.4%) patients. Approximately 54% of patients had AJCC stage 1 disease. The most common BCLC stage was stage B (480/942, 51.0%), followed by stage A (263/942, 27.9%), and stage 0 (199/942, 21.1%). Microvascular invasion, intrahepatic metastasis, and multicentric occurrence were observed in 45.2%, 8.0%, and 6.1% of patients, respectively. Tumor necrosis was observed in 359 patients (38.1%). Approximately 44% of HCCs occur in the background of liver cirrhosis. Tumor recurrence developed in 376 (39.9%) patients, early recurrence in 259 (27.5%), and late recurrence in 117 (12.4%).
Mitotic Index in HCC and Its Association with Clinicopathological Characteristics
The overall mitotic index of 942 HCC is shown in Figure 2. The mean mitotic index was 7.0 (median, 3.5; range, 0–132). The mitotic index was regarded as high when eight or more mitoses were identified in 10 HPF, which was determined as the best cut-off value related to early recurrence-free survival (e-RFS) via the X-tile software. A high mitotic index was observed in 296 patients (31.4%). The association between high and low mitotic indices and clinicopathological characteristics is summarized in Table 1. A high mitotic index was significantly associated with younger age (p = 0.001), higher Edmondson grade (p < 0.001), microvascular invasion (p < 0.001), tumor necrosis (p < 0.001), advanced AJCC T stage (p < 0.001), increased AFP level (p < 0.001), hepatitis B virus infection (p < 0.001), and early tumor recurrence (p < 0.001).
Figure 2.
Overview of mitotic index in 942 hepatocellular carcinoma patients from Samsung Medical Center.
Very early stage (BCLC stage 0) was identified in 21.1% (199/942) of patients. The correlation between mitotic index and clinicopathological parameters is shown in Table 2. The high mitotic index group was associated with higher Edmondson grade (p = 0.010), microvascular invasion (p = 0.029), tumor necrosis (p = 0.012), and early tumor recurrence (p = 0.006).
Table 2.
The Association Between Mitotic Index and Clinicopathologic Parameters in 199 Very Early Stage Hepatocelluar Carcinoma Patients
| Mitotic Index | ||||
|---|---|---|---|---|
| Total | Low (<8) | High (≥8) | ||
| n=199 | n=136 (%) | n=63 (%) | p value | |
| Age, year | ||||
| ≤55 | 101 | 66 (48.5) | 35 (55.6) | 0.441 |
| >55 | 98 | 70 (51.5) | 28 (44.4) | |
| Gender | ||||
| Female | 43 | 34 (25.0) | 9 (14.3) | 0.128 |
| Male | 156 | 102 (75.0) | 54 (85.7) | |
| Edmondson | ||||
| 1 | 26 | 22 (16.2) | 4 (6.3) | 0.010 |
| 2 | 165 | 112 (82.4) | 53 (84.1) | |
| 3 | 5 | 1 (0.7) | 4 (6.3) | |
| 4 | 3 | 1 (0.7) | 2 (3.2) | |
| Microvascular invasion | ||||
| (-) | 156 | 113 (83.1) | 43 (68.3) | 0.029 |
| (+) | 43 | 23 (16.9) | 20 (31.7) | |
| Intrahepatic metastasis | ||||
| (-) | 197 | 134 (98.5) | 63 (100.0) | 1.000 |
| (+) | 2 | 2 (1.5) | 0 (0.0) | |
| Bile duct invasion | ||||
| (-) | 195 | 134 (98.5) | 61 (96.8) | 0.592 |
| (+) | 4 | 2 (1.5) | 2 (3.2) | |
| Multicentric occurrence* | ||||
| (-) | 194 | 133 (97.8) | 61 (96.8) | 0.653 |
| (+) | 5 | 3 (2.2) | 2 (3.2) | |
| Necrosis | ||||
| (-) | 161 | 117 (86.0) | 44 (69.8) | 0.012 |
| (+) | 38 | 19 (14.0) | 19 (30.2) | |
| ALBI Grade | ||||
| 1 | 133 | 90 (66.2) | 43 (68.3) | 0.936 |
| 2 | 63 | 44 (32.4) | 19 (30.2) | |
| 3 | 3 | 2 (1.5) | 1 (1.6) | |
| AFP level, ng/mL | ||||
| ≤200 | 166 | 115 (84.6) | 51 (81.0) | 0.666 |
| >200 | 33 | 21 (15.4) | 12 (19.0) | |
| Etiology* | ||||
| Non-viral | 13 | 12 (8.8) | 1 (1.6) | 0.069 |
| HBV | 178 | 120 (88.2) | 58 (92.1) | |
| HCV | 7 | 3 (2.2) | 4 (6.3) | |
| HBV & HCV | 1 | 1 (0.7) | 0 (0.0) | |
| Liver cirrhosis | ||||
| (-) | 77 | 54 (39.7) | 23 (36.5) | 0.784 |
| (+) | 122 | 82 (60.3) | 40 (63.5) | |
| Early recurrence | ||||
| (≤2 years) | ||||
| (-)† | 165 | 120 (88.2) | 45 (71.4) | 0.006 |
| (+) | 34 | 16 (11.8) | 18 (28.6) | |
| Late recurrence | ||||
| (>2 years) | ||||
| (-)† | 136 | 101 (84.2) | 35 (77.8) | 0.465 |
| (+) | 29 | 19 (15.8) | 10 (22.2) | |
Notes: *by Fisher’s exact test, otherwise by chi-square test, † No early or late recurrence.
Abbreviations: AJCC, American Joint Committee on Cancer; BCLC, Barcelona Clinic Liver Cancer; ALBI, albumin-bilirubin; AFP, α-fetoprotein; HBV, hepatitis B virus; HCV, hepatitis C virus.
Effect of Mitotic Index on Early Recurrence of HCC
Patients in the high mitotic index group showed significantly greater e-RFS (2-year recurrence rate 37.5% vs 24.3%; p < 0.001) than those in the low mitotic index group (Figure 3A). Differences in e-RFS were statistically significant in specific subgroups of very early (Figure 3B) and early stage (BCLC stage A) tumors (Figure 3C and D).
Figure 3.
Kaplan-Meier survival curves for early recurrence-free survival in 942 hepatocellular carcinoma patients from Samsung Medical Center. (A) All patients. (B) very early stage patients. (C) very early and early stage (single or multiple tumors) patients. (D) very early and early stage (single tumor) patients.
We performed univariate and multivariate analyses twice, including all patients and patients with very early stage HCC, respectively. On univariate analysis of all patients, early recurrence of HCC was significantly correlated with male sex (p = 0.038), larger tumor size (p < 0.001), higher Edmondson grade (p < 0.001), microvascular invasion (p < 0.001), intrahepatic metastasis (p < 0.001), multicentric occurrence (p = 0.005), advanced AJCC T stage (p < 0.001), higher BCLC stage (p < 0.001), and a high mitotic index (p < 0.001). Multivariable analysis showed that larger tumor size (p = 0.016), microvascular invasion (p < 0.001), intrahepatic metastasis (p < 0.001), multicentric occurrence (p = 0.002), and a high mitotic index (p = 0.009) were independent predictors of early recurrence (Table 3). The discrimination of this multivariable model exhibited C-index of 0.714 (Standard error[SE], 0.016).
Table 3.
Univariable and Multivariable Analysis for Early Recurrence-Free Survival in All 942 Hepatocellular Carcinoma Patients
| Univariable Analysis | Multivariable Analysis | |||
|---|---|---|---|---|
| HR (95% CI) | p value | HR (95% CI) | p value | |
| Age, year | ||||
| >55 vs ≤55 | 0.92 (0.72–1.17) | 0.478 | 1.01 (0.78–1.31) | 0.938 |
| Gender | ||||
| Male vs Female | 1.44 (1.02–2.03) | 0.038 | 1.41 (0.99–2.01) | 0.054 |
| Tumor size, cm | ||||
| >5.0 vs ≤5.0 | 2.52 (1.95–3.25) | < 0.001 | 1.49 (1.08–2.05) | 0.016 |
| Edmondson grade | ||||
| 3,4 vs 1,2 | 2.26 (1.51–3.36) | < 0.001 | 1.39 (0.91–2.13) | 0.124 |
| Microvascular invasion | ||||
| (+) vs (-) | 3.09 (2.38–4) | < 0.001 | 1.87 (1.37–2.55) | < 0.001 |
| Necrosis | ||||
| (+) vs (-) | 1.93 (1.51–2.47) | < 0.001 | 1.25 (0.95–1.64) | 0.112 |
| Intrahepatic metastasis | ||||
| (+) vs (-) | 4.07 (2.98–5.57) | < 0.001 | 2.64 (1.87–3.72) | < 0.001 |
| Multicentric occurrence | ||||
| (+) vs (-) | 1.81 (1.2–2.74) | 0.005 | 1.96 (1.28–2.99) | 0.002 |
| AJCC T-stage | ||||
| 2,3,4 vs 1 | 1.94 (1.35–2.78) | < 0.001 | 0.90 (0.58–1.4) | 0.649 |
| BCLC stage | ||||
| B vs 0,A | 2.01 (1.55–2.59) | < 0.001 | 1.38 (0.98–1.96) | 0.066 |
| ALBI grade | ||||
| 2 vs 0.1 | 0.93 (0.38–2.25) | 0.873 | 1.08 (0.44–2.65) | 0.867 |
| AFP level, ng/mL | ||||
| >200 vs ≤200 | 1.29 (0.98–1.69) | 0.069 | 1.00 (0.75–1.34) | 0.998 |
| Etiology | ||||
| Viral vs Non-viral | 1.01 (0.71–1.42) | 0.978 | 1.17 (0.81–1.71) | 0.402 |
| Liver cirrhosis | ||||
| (+) vs (-) | 0.99 (0.78–1.27) | 0.953 | 1.11 (0.85–1.43) | 0.445 |
| Mitotic index | ||||
| High vs Low | 1.79 (1.39–2.29) | < 0.001 | 1.44 (1.1–1.89) | 0.009 |
| Model performance: C-index (SE) | 0.714 (0.016) | |||
Abbreviations: AJCC, American Joint Committee on Cancer; BCLC, Barcelona Clinic Liver Cancer; ALBI, albumin-bilirubin; AFP, α-fetoprotein; SE, standard error.
In a subgroup analysis among patients with very early stage, microvascular invasion (p < 0.001), and high mitotic index (p = 0.002) were significantly associated with early recurrence on univariate analysis (Table 4). In multivariable analysis of very early stage patients, microvascular invasion (p = 0.007) and high mitotic index (p = 0.013) were independent predictors of early recurrence. The corresponding model yielded a C-index of 0.707 (SE, 0.044), indicating modest discriminatory performance.
Table 4.
Univariable and Multivariable Analyses for Early Recurrence-Free Survival in 199 Very Early Stage Hepatocellular Patients
| Univariable Analysis | Multivariable Analysis | |||
|---|---|---|---|---|
| HR (95% CI) | p value | HR (95% CI) | p value | |
| Age, year | ||||
| >55 vs ≤55 | 1.23 (0.63–2.43) | 0.544 | 1.38 (0.64–2.95) | 0.413 |
| Gender | ||||
| Male vs Female | 1.12 (0.49–2.57) | 0.791 | 1.13 (0.46–2.80) | 0.790 |
| Edmondson grade | ||||
| 3,4 vs 1,2 | 1.76 (0.42–7.36) | 0.436 | 0.96 (0.21–4.32) | 0.955 |
| Microvascular invasion | ||||
| (+) vs (-) | 3.08 (1.56–6.11) | 0.001 | 2.73 (1.32–5.64) | 0.007 |
| Necrosis | ||||
| (+) vs (-) | 1.74 (0.81–3.72) | 0.156 | 0.98 (0.42–2.27) | 0.959 |
| ALBI grade | ||||
| 2 vs 0.1 | 0 (0-Inf) | 0.997 | 0 (0-Inf) | 0.997 |
| AFP level, ng/mL | ||||
| >200 vs ≤200 | 0.98 (0.38–2.53) | 0.968 | 0.93 (0.34–2.56) | 0.893 |
| Etiology | ||||
| Viral vs Non-viral | 2.26 (0.31–16.52) | 0.422 | 2.00 (0.26–15.4) | 0.506 |
| Liver cirrhosis | ||||
| (+) vs (-) | 1.24 (0.61–2.55) | 0.550 | 1.06 (0.50–2.27) | 0.880 |
| Mitotic index | ||||
| High vs Low | 2.95 (1.5–5.79) | 0.002 | 2.48 (1.21–5.08) | 0.013 |
| Model performance: C-index (SE) | 0.707 (0.044) | |||
Abbreviations: AJCC, American Joint Committee on Cancer; BCLC, Barcelona Clinic Liver Cancer; ALBI, albumin-bilirubin; AFP, α-fetoprotein; SE, standard error.
In the main SMC cohort, time-dependent receiver operating characteristic analysis at 24 months revealed that mitotic index ≥8 showed modest predictive ability for early recurrence. The area under the curve was 0.571 for all HCC patients and 0.636 for those with very early HCC (Supplementary Figure 1).
External Validation of Mitotic Index in Independent HUMC Cohort
For 112 HUMC patients, the median age was 59 years (range, 30–95 years); 96 (85.7%) patients were male and 16 (14.3%) were female. The median tumor size was 3.0 cm, and 26 (23.2%) patients had tumors larger than 5 cm. Seventy-three patients (65.2%) were infected with hepatitis B virus, and 7 (6.3%) were infected with hepatitis C virus. Combined hepatitis B and C virus infection were observed in 1 patient (8.9%). Viral markers were not identified in 31 patients. Sixty patients (53.6%) had AJCC stage 1 disease. BCLC stage A (97/112, 86.6%) was most common, followed by stage B (10/112, 8.9%), 0 (4/112, 3.6%), and D (1/112, 0.9%). Microvascular invasion, intrahepatic metastasis, and multicentric occurrence were observed in 39 (34.8%), 6 (5.4%), and 11 (9.8%) patients, respectively. Sixty-four patients (57.1%) had background liver cirrhosis. Early and late tumor reccurences was developed in 29 (25.9%) and 22 (19.6%) patients, respectively. (Supplementary Table 1).
High mitotic index was observed in 27 patients (24.1%) of HUMC cohort, and showed significant association with higher Edmondson grade (p = 0.010), tumor necrosis (p = 0.021), advanced AJCC T stage (p = 0.010), increased AFP level (p = 0.014), and early tumor recurrence (p = 0.005) (Supplementary Table 1). Very early and early stage (BCLC stage 0 and A) was identified in 3.6% (4/112) and 86.6% (97/112) of patients, respectively (Supplementary Table 2). The high mitotic index group showed significant association with higher Edmondson grade (p = 0.007), tumor necrosis (p = 0.042) increased AFP level (p = 0.017) in a subgroup analysis among patients with very early or early stage HCC.
In similar to SMC cohort, high mitotic index group exhibited significantly greater e-RFS (2-year recurrence rate 50.6% vs 20.3%; p < 0.001) than those in the low mitotic index group (Figure 4A). Statistical significance was also observed when very early and early stage patients were analyzed (Figure 4B and C).
Figure 4.
Kaplan-Meier survival curves for early recurrence-free survival in 112 hepatocellular carcinoma patients from Hallym University Medical Center. (A) All patients. (B) very early and early stage (single or multiple tumors) patients. (C) very early and early stage (single tumor) patients.
Univariate analysis revealed that early tumor recurrence was significantly associated with larger tumor size (p = 0.013), advanced AJCC T stage (p = 0.036), and a high mitotic index (p = 0.001). In multivariate analysis, only a high mitotic index (p = 0.011) was independent predictor of early recurrence (Supplementary Table 3). In univariate and multivariate analysis for HUMC patients with very early or early stage, only a high mitotic index showed significant association (p = 0.017) and was an independent predictor (p = 0.029) of early tumor recurrence. (Supplementary Table 4) The discrimination of these multivariable models showed C-indices of 0.744 (SE, 0.050) for all HCC patients and 0.730 (SE, 0.049) for the BCLC 0/A subgroup, comparable to the values observed in the main cohort.
In the external validation HUMC cohort, the time-dependent receiver operating characteristic analysis at 24 months demonstrated that mitotic index ≥8 had modest discriminatory power for predicting early recurrence. The area under the curve was 0.655 for all HCC patients and 0.625 for the subgroup with BCLC stage 0/A (Supplementary Figure 2).
Discussion
In the present study, we evaluated the prognostic significance of the mitotic index in a large cohort of HCC patients who underwent surgical resection as a primary treatment from two independent institutes. We established an optimal cut-off value for a high mitotic index showing the most significant RFS. In particular, we assessed whether a high mitotic index could accurately predict early tumor recurrence in very early stage HCC patients after surgery.
Several studies have investigated the relationship between the mitotic index and clinicopathological factors in HCC. Ouchi et al reported that a high mitotic index was associated with multicentric occurrence and advanced tumor stage and was also an independent prognostic factor for OS.36 Nanashima et al demonstrated that a high mitotic index was significantly correlated with vascular invasion, poorly differentiated histology, and tumor recurrence.37 They also noted that a high mitotic index was associated with a shorter OS in univariate analysis. In our previous study, we also found that a high mitotic index was an independent predictor of shorter disease-specific survival.7 However, none of the studies have described an association between the mitotic index and early tumor recurrence of HCC. To the best of our knowledge, this is the first report to demonstrate that a high mitotic index is an independent predictor of early recurrence, especially in very early stage HCC.
According to recent treatment strategies used in the management of HCC, two options are recommended as the primary treatment for very early stage HCC: resection or ablation therapy.14 However, due to conflicting data across studies, there are no standardized criteria for the selection of these two options because it is still controversial which treatment provides better survival outcomes in very early stage HCC patients. Many studies have reported that there was no significant difference in DFS and OS between patients who underwent surgical resection and ablation therapy.15–23 In contrast, several other studies have reported that patients with very early stage HCC who underwent surgical resection showed better OS and DFS than those who received ablation therapy.24,26–29,38 Combining the previous results, it can be estimated that the surgical resection group had a better, or at least similar, prognosis to RFA in terms of RFS. Thus, surgical resection should be considered as the first treatment option to achieve better RFS for HCC with a high risk of recurrence.
As pathological examination was not routinely performed before HCC treatment, none of the previous studies evaluated histopathological findings as independent variables, which might have affected the conflicting results among previous studies. Tumor differentiation, Edmondson grade, microvascular invasion, necrosis, and mitotic index can be included as variables for predicting RFS and may offer individualized risk stratification for recurrence after RFA or surgical resection.
In the present study, we demonstrated that a high mitotic index was an independent predictor of early recurrence in a large cohort of HCC patients and independent external cohort from other institute. Moreover, subgroup analysis among patients with very early stage HCC showed that a high mitotic index was associated with unfavorable early recurrence. Importantly, the discriminatory performance of mitotic index was reproducible across both cohorts, with time-dependent AUC values ranging from 0.57 to 0.66 and C-indices around 0.71–0.74. Although these values indicate only modest predictive ability, they strengthen the robustness of our findings and suggest that mitotic index may provide clinically meaningful information when used in conjunction with other pathological or imaging factors. These findings support the role of mitotic index in early recurrence risk estimation and suggest its potential value in providing additional guidance for treatment decision-making or postoperative surveillance in very early stage HCC.
We acknowledge, however, that the immediate clinical applicability of mitotic index is limited, since it is most commonly evaluated in resection specimens and not routinely available prior to treatment. Nevertheless, mitotic figures can also be assessed in liver biopsy samples when performed, which indicates that mitotic index is not entirely restricted to postoperative assessment. Although liver biopsy is not performed in all very early HCC patients, preoperative assessment in selected cases may provide additional prognostic information. Furthermore, as counting mitoses is a simple, inexpensive, and widely available histologic method, mitotic index could contribute to histological staging systems and postoperative surveillance strategies.
While direct histologic assessment of mitotic index is not routinely available before surgery, non-invasive imaging approaches may provide complementary information. In particular,18 F-fluorodeoxyglucose (FDG) uptake on positron emission transmission (PET)/CT has been widely studied as a prognostic marker in HCC, and several reports have shown that higher FDG uptake correlates with unfavorable tumor biology and poorer postoperative outcomes.39–41 Moreover, FDG uptake has been positively associated with Ki-67 expression in HCC,42 and mitotic index and Ki-67 show a strong correlation.36 Taken together, these findings raise the possibility that FDG uptake may indirectly reflect mitotic activity and thus serve as a surrogate marker for tumor proliferative potential. Although FDG PET/CT is not universally applied in all very early HCC patients, its integration with histologic factors such as mitotic index could enhance risk stratification and provide additional guidance for surveillance or adjuvant strategies in selected cases. While further validation is required, the convergence of histologic and metabolic imaging markers represents a promising direction for improving individualized prognostic assessment in HCC.
This study has several limitations. First, its retrospective design may be subject to inherent selection bias, and both cohorts were derived from institutions within a single country, which may limit the generalizability of our findings to other populations. Second, although mitotic figures were assessed by two pathologists with consensus, some degree of interobserver variability cannot be excluded, and the cut-off value used in this study has not yet been universally validated. Third, while we attempted external validation, the relatively small sample size of the external cohort reduced statistical power, particularly for subgroup analyses. Finally, the clinical applicability of mitotic index remains limited, since it is usually assessed in surgical specimens and not routinely available preoperatively; future studies incorporating biopsy assessment or integration with non-invasive imaging markers will be necessary to establish its role in guiding treatment decisions.
Conclusion
In conclusion, we demonstrated that a high mitotic index of HCC is associated with aggressive clinicopathological parameters, including a higher risk of early recurrence, particularly in very early stage disease. Mitotic index may serve as a useful predictive and prognostic biomarker, contributing to risk stratification in clinical practice. While its direct role in primary treatment decision-making is limited, it could provide additional information to support individualized management strategies and postoperative surveillance in patients with very early HCC.
Funding Statement
This research was supported by the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00253353, RS-2023-00222838 and RS-2024-00405650).
Ethics Statement
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Review Board of the Samsung Medical Center (IRB No. 2019-08-018) and Hallym University Sacred Heart Hospital (IRB No. 2021-09-008). The requirement for informed consent was waived because this was a retrospective study using de-identified medical records, which posed minimal risk to participants. All data were anonymized prior to analysis to ensure patient confidentiality.
Disclosure
The authors declare that they have no conflict of interest.
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