Abstract
Background/Aim: Tumor budding (TB) has recently been recognized worldwide as a prognostic predictor in several solid cancers. The objective of this study was to explore the relationship between TB and clinicopathological characteristics, postoperative relapse, and survival in patients with stage II colon cancer. Patients and Methods: A total of 213 patients with stage II colon cancer were retrospectively enrolled at Saitama Medical Center, Dokkyo Medical University from 2010 to 2016. TB was evaluated in hotspot areas on hematoxylin and eosin-stained slides at the invasive front of the tumor to define a low-grade group (BD1) and a high-grade group (BD2 or BD3). Results: High-grade TB was found in 38.3% of cases, and was associated with pT4, presence of lymphovascular invasion, and tumor relapse (p=0.02, p=0.03, p=0.002, respectively). Patients with highgrade TB showed worse relapse-free survival (RFS) and overall survival (OS) rates than patients with low-grade TB (5-year RFS: High 75.6% vs. Low 92.1%, p=0.001; 5-year OS: High 93.7% vs. Low 93.7%, p=0.001). On multivariate analysis for predictors of RFS and OS, high-grade TB was significant for both RFS and OS (RFS, p=0.003; OS, p=0.005). Patients with high-grade TB experienced lung and liver relapses significantly more frequently than patients with low-grade TB (p=0.03 each). Among patients who received adjuvant chemotherapy (AC), no patients showed lung or liver relapse even in the presence of high-grade TB. Conclusion: TB may offer a useful predictor of relapse in patients with stage II colon cancer after surgery, and AC should be considered for patients with high-grade TB.
Curative surgical resection is the fundamental treatment for patients with stage I and II colon cancer, whereas surgical resection and adjuvant chemotherapy (AC) using the combination of fluoropyrimidine and oxaliplatin is the standard treatment for patients with stage III colon cancer. Three landmark trials have shown that AC for patients with curatively resected stage III colon cancer reduced deaths by about 15% (1-3). However, the adaption of AC to patients with stage II colon cancer has been debated (4-6). The QUASAR trial showed only a small (3%) benefit to overall survival (OS) from AC with fluorouracil and folinic acid (7). Other large meta-analyses and randomized trials have shown survival rates in high-risk patients with stage II colon cancer comparable to those of patients with metastatic disease (8-10).
Current international guidelines have identified T4 lesions, lymphovascular or perineural invasions, poorly differentiated histology, examination of less than 12 lymph nodes, elevated levels of carcinoembryonic antigen (CEA), bowel perforation, and obstruction as poor prognostic factors for patients with stage II colon cancer (11, 12). Although sufficient supporting evidence is still lacking, these guidelines presently recommend AC for high-risk patients with stage II colon cancer.
Tumor budding (TB) is presently recognized as a morphological and adverse prognostic factor in many solid cancers, particularly colorectal cancer (CRC) (13). TB is generally defined as a single cancer cell or clusters of up to four cancer cells at the invasive front of the tumor, and standardized scoring systems for TB in CRC have been assessed at the International Tumor Budding Consensus Conference (ITBCC) (14). Regardless of clinical scenarios, the ITBCC scoring system has shown the potential to influence patients with pT1 (stage I) and stage II CRC.
The ITBCC scoring system addresses the lack of standardized and methodological assessments for TB but requires further refinement for the precise selection of high-risk patients. The present study aimed to evaluate the relationships between TB and clinicopathological findings, patterns of postoperative relapse, and survival in patients with stage II colon cancer, and whether TB is useful for deciding the adoption of AC to patients with stage II colon cancer.
Patients and Methods
Patients. This retrospective cohort study was conducted at Saitama Medical Center, Dokkyo Medical University using records from 2010 to 2016 regarding patients with stage II colon cancer who underwent curative resection. The study included 213 consecutive patients (112 men, 101 women) who had undergone surgery either alone or with fluorouracil-based AC. The primary and secondary end points of the present study were to explore the relationships between TB and RFS and OS.
Exclusion criteria were as follows: patients with all rectal cancers including rectosigmoid cancer; patients with hereditary CRC syndromes, inflammatory bowel disease, or familial adenomatous polyposis; patients who had received neoadjuvant chemotherapy; patients who had received non-curative surgery; patients with suspected stage IV disease; patients with an insufficient observation period (less than 6 months); patients who had died within 90 days; patients with other advanced malignant diseases; and patients with poorly differentiated adenocarcinoma or signet-ring cell carcinoma. Patients with poorly differentiated or signet ring cell carcinoma were excluded from the study, and those with mucinous carcinoma were included based on our previous study (15).
Tumor location was divided into right-side colon (including cecum, ascending colon, or transverse colon) and left-side colon (including descending colon or sigmoid colon). Tumor stage was diagnosed according to the TNM classification, and this study included patients with T3N0, T4aN0, and T4bN0. Lymph node dissection was considered adequate if at least 12 lymph nodes were investigated, in compliance with the recommendation by the American Joint Committee on Cancer (16). The application of AC after surgery was performed according to the ESMO guideline for patients with high-risk stage II colon cancer, physical state and patient preference, after obtaining sufficient informed consent (11). Regarding AC, oral (UFT/LV: uracil-tegafur/leucovorin or capecitabine) or intravenous [FOLFOX: oxaliplatin with folinic acid and 5-fluorouracil (5-FU) or XELOX: capecitabine plus oxaliplatin] treatments were administrated for 6 months.
This study was reviewed and approved by the Saitama Medical Center, Dokkyo Medical University (approval no. 2081).
Assessment of TB. TB was defined as a single cancer cell or a cell cluster consisting of ≤4 cancer cells at the invasive front of the primary tumor, as scored by two observers (K.S. and Y.O.). The process of counting TBs was performed in 10 high-power fields at medium power (10× objective) along the invasive front of the primary tumor using hematoxylin and eosin (HE)-stained sections. Among those “hotspots”, the number of TBs was counted under a 20× objective lens for the hotspot showing the most frequent TBs (hotspot method). These processes were evaluated for TB according to ITBCC criteria (14). The grade of TB was classified as low-grade (BD1, 0-4 TBs) or high-grade (BD2, 5-9 TBs or BD3, ≥10 TBs) (Figure 1). Inter-observer reliability as assessed using K-statistics was 0.68.
Hematoxylin and eosin-stained images for tumor budding (TB) in colon cancer. Tumor budding is evaluated at the hotspot at the invasive front of the tumor. A) Low-grade TB (BD1; 0 TBs per hotspot, magnification 200×); B) High-grade TB (BD3, 19 TBs per hotspot, magnification 200×).
Postoperative surveillances. Postoperative surveillance was based on the Japanese guideline for CRC (17). Physical examinations and determination of serum concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 were conducted every 3 months for 3 years, then every 6 months for 2 years. Computed tomography of the chest, abdomen, and pelvis was performed every 6 months for 5 years. Colonoscopy was performed within 1 year after surgery, if no abnormality was detected, the subsequent colonoscopy was performed within 3 years. If tumor relapse was suspected, abdominal ultrasound, magnetic resonance imaging, and positron imaging tomography-computed tomography were immediately conducted for confirmation.
Statistical analysis. Continuous variables are shown as median and interquartile range (IQR) and were analyzed using the Mann-Whitney U-test. Categorical variables are presented as number and percentage and were analyzed using the chi-squared test or Fisher’s exact test. Relapse-free survival (RFS) rate was calculated from the date of operation to the date of relapse diagnosis, whereas OS rate was calculated from the date of operation to the date of death by any cause or last follow-up. RFS and OS rates were estimated using the Kaplan-Meier method and compared using the log-rank test. Values of p<0.05 were considered statistically significant, and all analyses were performed using SPSS statistical software (version 27; IBM Japan, Tokyo, Japan).
Results
Demographics. Median tumor diameter was 47.0 cm (IQR=35-60 cm). Median RFS and OS were 62 months (IQR=42-81 months) and 66 months (IQR=47-84 months), respectively. Thirty-four patients underwent surgery with 5-FU-based AC. As the AC, UFT/LV, capecitabine, FOLFOX, and XELOX therapies were provided to 13, 7, 6, and 1 patient, respectively. High-grade TB was found in 38.3% of patients. Among patients who experienced relapse (27/213, 12.7%), 15 patients showed lung or liver relapse, with hematogenous metastasis suspected as the main route. Twenty-three patients (23/213, 14.1%) died of any causes within the observation period, of which 13 patients (56.5%, 13/23) died of causes other than colon cancer. The baseline characteristics are shown in Table I.
Baseline clinicopathological characteristics.
Relationship between TB and clinicopathological findings. Lymphatic and venous invasion and tumor relapse were significantly more frequent in patients with high-grade TB than in those with low-grade TB (p=0.025, p=0.044 and p=0.001, respectively). A marginal relationship between high-grade TB and pT4 was found (p=0.07). Relationships between TB and the investigated clinicopathological findings are presented in Table II.
The relationship between tumor budding and clinicopathological characteristics.
Influence of TB grade on RFS and OS rates. Five-year RFS and OS rates were 86.7% and 90.3% for the entire cohort. Patients with high-grade TB showed significantly lower 5 year RFS and OS rates than patients with low-grade TB (RFS: 75.2% vs. 93.7%, p<0.001; OS: 81.3% vs. 94.8%, p=0.001, respectively). Figure 2 shows the Kaplan-Meier survival curves for RFS and OS according to TB grade.
Relapse-free (A) and overall survival (B) curves in patients with low- and high-grade TB.
Uni- and multi-variate analyses of risk factors correlated with RFS and OS. Uni- and multivariate analyses for risk factors correlated with RFS and OS were performed using Cox proportional hazard modeling (Table III). Both pT category and TB were significant risk factors for postoperative relapse in univariate analysis (p=0.043 and p=0.001, respectively). In multivariate analysis, TB was an independent risk factor for postoperative relapse [hazard ratio (HR)=4.01, 95% confidence interval (CI)=1.744-9.201; p=0.001]. Regarding OS, univariate analysis identified TB as a significant risk factor, and preoperative serum CA19-9 level and pT category were marginally associated with OS (p=0.002, p=0.06 and p=0.06, respectively). In the multivariate analysis including these factors, TB remained a significant risk factor for OS (HR=4.02, 95%CI=1.643-9.841; p=0.002).
Uni- and multi-variate analyses of risk factors correlated with RFS and OS.
Influence of TB grade and AC on relapse site. The relationship between TB grade and site of first relapse is summarized in Table IV. Lung and liver relapses were significantly more frequent among patients with high-grade TB than among patients with low-grade TB (p=0.03 each). None of the 27 patients who received AC developed lung or liver relapses, even in the presence of high-grade TB (Table V).
Comparison between the TB grade and first relapse site.
Comparison between the TB grade and relapse sites in patients who received postoperative adjuvant chemotherapy (n=27).
Discussion
The present findings suggest TB as a useful predictive marker for postoperative tumor relapse and survival in patients with stage II colon cancer who undergo curative surgery. In this study, patients with high-grade TB experienced tumor relapse more frequently than patients with low-grade TB. Patients with high-grade TB had significantly worse RFS and OS rates than patients with low-grade TB. In addition, TB was an independent risk factor for low RFS and OS rates in multivariate analyses. Lung or liver relapses were more frequently seen in patients with high-grade TB than in those with low-grade TB. Interestingly, no patients who received AC developed lung or liver relapses, even among patients with high-grade TB. Such results suggest that TB may represent an important pathological feature for selecting high-risk patients after surgery for stage II colon cancer.
Morodomi et al. first proposed the concept of TB for tumor cells detaching from the primary tumor along the invasive edge more than 30 years ago (18). TB is still recognized as a prognostic biomarker in CRC, despite many advances in surgical treatment and molecular biology. The usefulness of TB as a histological marker has been confirmed in a number of solid cancers, including esophageal, pancreatic, breast, nasopharyngeal, lung, gastric, and endometrioid cancers (19-24). Collective evidence supports the usefulness of TB as a histological feature relevant to prognosis in patients with CRC. The ITBCC grading recommendation published in 2017 provided the standard method for TB assessment in CRC (14).
The present study assessed TB using HE-stained slides in accordance with the ITBCC recommendation (14). Assessment using immunohistochemistry (IHC) has been reported to be as superior as that using HE staining, in terms of interobserver agreement and reproducibility (25-27). However, the ITBCC recommends evaluating TB using HE staining because of the possibility of miscounting stained apoptotic bodies and cellular debris, and the costeffectiveness and amount of data available from HE staining (14). In the terms of interobserver agreement, the present study showed a ĸ value of 0.68, representing adequate reliability. Interestingly, several meta-analyses have found no significant difference in prognostic power between HE staining and IHC (28, 29).
In the current study, lung and liver relapses were significantly more frequent among patients with high-grade TB than among patients with low-grade TB. The SACURA trial showed a significant correction between three-tier TB grade and organ of first relapse including the lung, liver, lymph nodes, and peritoneum (30). The difference in results between this study and the SACURA trial may be attributable to the sample size or differences in study populations. Further studies using molecular techniques may be required to clarify the relationship between TB and site of relapse in CRC patients. Interestingly, no patients in this study who received AC showed lung or liver relapses even if high-grade TB was present. The administration of AC to stage II colon cancer patients with high-grade TB may reduce postoperative relapse among high-risk patients.
A clinical advantage of TB is that TB in an intensitydependent prognostic factor (31). The number of TBs affects the risk of lymph node and distant metastases. The ITBCC has recommended the three-tier system and described only BD-3 as associated with an increased risk of relapse in patients with stage II CRC (14). The present study showed no difference in the relapse rate between patients with BD-2 and BD-3 (BD2 grade: 21% vs. BD-3 grade: 27%). This discrepancy might be due to the different patient characteristics or populations between the present study and those examined by the ITBCC. The ITBCC has indicated that the difference between 9 TBs (BD-2) and 10 TBs (BD-3) may be questionable in terms of differences in biological attitude of the primary cancer (14, 32). Further studies are necessary to clarify the utility of the grading system.
The molecular attributes of cancer cells influence the morphologic appearance on pathologic slides. Several studies have suggested that TB may represent the morphological feature of epithelial-mesenchymal transition (EMT) (33). EMT is commonly known as cellular plasticity in epithelial cells and represents the process of difference in wound healing and stem cell behaviors, contributing pathologically to fibrosis and cancer progression (34). EMT is known to allow migration of cancer cells through complex molecular mechanisms regulated by several genes. Molecular features such as loss of E-cadherin expression, β-catenin translocation, and acquisition of vimentin expression have been correlated with TB by several researchers (35, 36).
EMT-cancer cells need anoikis resistance to survive in the environment after detachment from the primary tumor. Neurotropic tyrosine receptor kinase B has been reported as a potent anoikis suppressor and is significantly overexpressed in TBs compared to the primary tumor (37). Although these findings suggest an association between TB and EMT, other studies have failed to verify the expression of EMT-related genes in TB (38). A better understanding of the molecular and pathogenetic mechanisms involved in TB and its interactions with the cellular microenvironment might provide key insights into the mechanisms governing cancer metastasis.
The present study suffered several limitations that deserve consideration when interpreting the results. This study had a retrospective design, analyzed only a small sample size, and encountered difficultly exploring the exact influence of TB and AC. Another limitation was that some biomarkers that could affect prognosis in patients with stage II colon cancer, including microsatellite instability, mismatch repair, and perineural invasion were not examined in the present study, which would have contributed some bias to the results.
Conclusion
The present study showed that 2-tier TB classification offers a predictive pathological marker of tumor relapse and survival in patients with stage II colon cancer. Although further large studies are warranted, tumors with ≥5 TBs at the invasive front may show high risk of relapse and poor prognosis compared with those with <5 TBs. Evaluation of TBs may be useful for identifying patients suitable for neoadjuvant therapy.
Footnotes
Authors’ Contributions
KS, TO and MO designed the study. KS, TO, SM, HO, ET, HY and MO reviewed the clinical records. KS and YO reviewed the pathological records. All Authors participated in the study design, data interpretation, and critical discussion. KS, TO, and MO wrote the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received March 5, 2022.
- Revision received April 16, 2022.
- Accepted April 18, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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