INTRODUCTION
Colon cancer is an important part of colorectal cancer, which remains one of the most common malignancies worldwide and accounts for more than one million new cases occurring every year.[1,2] Although substantial advances in credible diagnosis and comprehensive therapy strategy have been made in recent years, the long-term prognosis of colon cancer patients remains little changed with relatively high mortality and morbidity.[3-5] Moreover, the American Joint Committee on Cancer (AJCC) TNM staging system, the strongest and most widely used prognostic parameter, is less satisfactory in predicting prognosis in early-stage colon cancer patients, and patients with the same pathological stage may experience significantly different clinical outcomes. These issues call for continued studies on reliable and significant prognostic factors.
In the clinical practice, many clinicopathological factors have been proposed to predict prognosis and identify high-risk group in colorectal cancer patients,[6-11] but substantial evidence is still lacking in early-stage colon cancer patients. An emerging pathological criterion that indicates a more aggressive cancer characteristic and worse prognosis in colorectal cancer patients is the presence of tumor necrosis in routine pathological slide evaluation.[7,12-14] Tumor necrosis is associated with local recurrence, lymph node metastasis, distant metastasis, and reduced long-term survival in types of cancers. The proposed underlying mechanisms of necrosis-associated aggressiveness include rapid outgrowth, extensive hypoxia, and increased invasiveness.[15,16]
Tumor necrosis seems a promising prognostic biomarker for colon cancer patients, but related reports are rare and it deserves further exploration. Therefore, the aim of our study is to identify the independent role of tumor necrosis in prognosis stratification in early-stage colon cancer patients by applying multivariate adjusted statistical analysis. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for observational studies.
MATERIALS AND METHODS
Study design
Colon cancer patients, who received radical resection and lymphadenectomy in Tianjin Hospital between January 1, 2010, through December 31, 2018, were enrolled in our study. Then, they received adjuvant chemotherapy (capecitabine plus 5-FU/leucovorin, the regimens recommended by NCCN guidelines) for six months. These patients were regularly followed up to record their overall survival and progression-free survival status. The inclusion criteria were: (1) the diagnosis of colon cancer was confirmed by positive histology examination other than by autopsy or a death certificate; (2) colon cancer was restricted as the first and only cancer at diagnosis; (3) the absence of lymph node metastasis and distant metastasis was confirmed by histology examination and intensive imaging examination; (4) patient age was between 18 and 80 years old. The exclusion criteria were: (1) patients received neoadjuvant anti-cancer treatments before radical resection; (2) the follow-up was incomplete; (3) patients had perforation or severe obstructive colitis before surgery; (4) the tumor sections were missing or inadequate (<1 section/cm of tumor).
Matched clinicopathological characteristics and clinical outcomes data were collected from medical records and internal database in Tianjin Hospital. The obtained clinicopathological characteristics included demographic characteristics (patient age at diagnosis, gender), laboratory examination (serum CEA level with a cutoff of 5 ng/mL), tumor features (tumor location, tumor size, histological grade, vascular invasion, perineural invasion, T stage), and therapy information (surgical margin status, adjuvant chemotherapy). The clinical outcomes were overall survival (OS), defined as the total survival time from the diagnosis to death or last follow-up, and progression-free survival (PFS), defined as the interval from the diagnosis to first tumor recurrence or metastasis, which was evaluated by follow-up CT or MRI examination every six months after surgery.
This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Ethical approval was approved by the local institutional review board, and the informed consent of enrolled patients was waived due to the retrospective nature. The sample size for the analysis was based on pathological evaluation availability.
Definition of tumor necrosis
Histopathological slides of primary colon cancer were independently reviewed by two experienced pathologists, who were blinded to clinical data. A mean number of 3.7 (median, 3; range, 1–12) routine hematoxylin–eosin staining tissue slides were available for evaluation. Tumor necrosis was characterized by the presence of agglomerated cells with nuclear and cytoplasmic fragmentation, or condensates containing extensive erythrocytes (bleeding) or neutrophils [Figure 1] according to previous reports.[7,12] Before formal evaluation, a random subset of 30 cases were selected from the study population to test the interpathologist compliance, and the final compliance reached 96.7% (29/30) that guaranteed the accuracy of pathological reviewing in the formal evaluation.
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Figure 1: The representative pathological images of tumor necrosis (a), tumor grade (b), vascular invasion (c), and perineural invasion (d). Scale bars, 200 μm
Statistical analysis
The primary analysis was to evaluate the prognostic impact of tumor necrosis on early-stage colon cancer patients. The secondary analysis was to assess the association between clinicopathological characteristics and tumor necrosis.
Clinicopathological characteristics of enrolled patients were compared using χ2 tests or Fisher’s exact test for categorical variables, or unpaired t test for continuous variables. Kaplan–Meier plots and the log-rank test were conducted to investigate the association between tumor necrosis and survival. Univariate and multivariate Cox proportional hazard models were applied to identify prognostic factors and calculate the survival hazard ratio (HR) with a 95% confidence interval (95% CI) of OS and PFS. Most statistical analysis was performed using R software (version 4.1.2). All P values were two-tailed, and a P value < 0.05 was considered statistically significant.
RESULTS
Patient characteristics
A total of 173 eligible early-stage (AJCC TNM stage I–II) colon cancer patients were identified from 2010 to 2018 at Tianjin Hospital and enrolled in our cohort. The summary statistics for the demographics and clinicopathological characteristics of colon cancer patients are shown in Table 1. The median (interquartile range) age of patients at diagnosis was 62 (52–71) years and 53.2% of patients were older than 60 years. The proportion of female patients surpassed that of male patients, accounting for 60.7% and 39.3%, respectively. Nearly one-third of these patients had abnormal serum CEA level (>5 ng/mL). As for the tumor location, 61.8% of primary colon cancer was located on the right side. Among these early-stage colon cancer patients, mild tumor characteristics were observed, including a relatively low proportion of poor tumor grade (22.5%), large tumor size (>5 cm, 32.4%), vascular invasion (15.6%), perineural invasion (8.1%), and high T stage (T3 or T4, 34.1%). Due to the nature of the lower aggressiveness of early-stage colon cancer, radical resection achieved a high R0 tumor surgical margin (89.0%). Among the 173 patients, 32 (18.5%) received adjuvant chemotherapy after surgery.
Table 1: Patient and tumor characteristics of stage I–II colon cancer patients grouped by the presence (n=78) or absence (n=95) of tumor necrosis
After an intensive review of pathological slides, enrolled early-stage colon cancer patients were dichotomized according to the presence (78, 45.1%) or absence (95, 54.9%) of tumor necrosis (histological examples were demonstrated in Figure 1). The demographic comparison showed no significant differences between the two groups in clinicopathological characteristics, including age, gender, serum CEA level, tumor location, grade, perineural invasion, T stage, tumor surgical margin, or receipt of adjuvant chemotherapy [Table 1]. However, we observed larger tumor size (>5cm) in colon cancer with tumor necrosis (41.0% vs. 25.3%, P = 0.028), and a marginal association with vascular invasion (20.5% vs. 11.6%, P = 0.107) was also found in our cohort. These findings suggested that even in early-stage colon cancer without lymph node metastasis or distant metastasis, tumor necrosis remained associated with higher aggressiveness.
Association of tumor necrosis with prognosis
The median follow-up (interquartile range) of these early-stage colon cancer patients was 58.3 months (27.6–79.2 months). The 2-year and 5-year OS rates were 88.3% and 68.2%, respectively, and the 2-year and 5-year PFS rates were 85.6% and 62.7%, respectively. During the follow-up, 94 deaths and 89 tumor progressions were observed in the 173 patients, and 46 disease progressions were observed in 78 patients with tumor necrosis (59.0%), while 43 disease progressions were observed in 95 patients without tumor necrosis (45.3%). Kaplan–Meier survival curves demonstrated that tumor necrosis was associated with worse OS (Figure 2, log-rank P = 0.003) and PFS (Figure 3, log-rank P = 0.002). In addition to the presence of tumor necrosis, the extent of tumor necrosis was also found associated with unfavorable prognosis in advanced tumor patients. Thus, we analyzed the association between the extent of tumor necrosis and patient prognosis [Figure 4], revealing no significant prognostic difference. The results suggest that the presence of tumor necrosis is an independent prognostic factor for patients with early colon cancer, while the extent of tumor necrosis holds no prognostic significance.
Figure 2: Kaplan–Meier curve showing overall survival of stage I–II colon cancer patients by the presence or absence of tumor necrosis
Figure 3: Kaplan–Meier curve showing progression-free survival of stage I–II colon cancer patients by the presence or absence of tumor necrosis
Figure 4: (a) The representative pathological images of different extents of tumor necrosis. (b) Kaplan–Meier curve showing overall survival of stage I–II colon cancer patients by the extent of tumor necrosis. (c) Kaplan–Meier curve showing progression-free survival of stage I–II colon cancer patients by the extent of tumor necrosis. Scale bars, 200 μm
Then, univariate and multivariate Cox regression analyses were conducted to identify the clinicopathological factors of OS [Table 2]. In univariate analysis, patient age (>60 years old, hazard ratio [HR] =2.33 (1.72–3.16), P = 0.003), tumor grade (moderate, HR = 1.33 (1.09–1.62), P = 0.032; poor, HR = 1.89 (1.42–2.52), P = 0.008), vascular invasion (HR = 2.16 (1.58–2.95), P = 0.006), T stage (T3, HR = 1.47 (1.23–1.76), P = 0.028; T4, HR = 2.08 (1.59–2.72), P = 0.009), tumor surgical margin (R1/R2, HR = 2.27 (1.74–2.96), P = 0.002), tumor necrosis (HR = 1.97 (1.59–2.44), P = 0.003), and receipt of adjuvant chemotherapy (HR = 0.72 (0.57–0.91), P = 0.036) were found significantly associated with OS. These characteristics were then included in the multivariate Cox regression analysis, identifying their independent prognostic roles in early-stage colon cancer patients: patient age (>60 years old, HR = 2.09 (1.51–2.89), P = 0.007), tumor grade (poor, HR = 1.63 (1.25–2.13), P = 0.026), vascular invasion (HR = 2.08 (1.46–2.96), P = 0.013), T stage (T3, HR = 1.44 (1.19–1.74), P = 0.037; T4, HR = 1.86 (1.33–2.60), P = 0.012), tumor surgical margin (R1/R2, HR = 1.96 (1.55–2.48), P = 0.004), tumor necrosis (HR = 1.91 (1.52–2.40), P = 0.004), and receipt of adjuvant chemotherapy (HR = 0.82 (0.72–0.94), P = 0.042). These results revealed a significant association between the presence of tumor necrosis and worse prognosis in early-stage colon cancer patients, which has not been demonstrated in previous reports.
Table 2: Prognostic factors for overall survival in stage I–II colon cancer patients
DISCUSSION
The current study confirmed the independent prognostic role of tumor necrosis from pathological review in early-stage colon cancer patients after tumor radical resection. This pathological criterion promises to help in identifying high-risk subgroup from early-stage colon cancer patients, who may benefit from strict follow-up and adjuvant therapy.
Colorectal cancer remains one of the most common malignancies all over the world and results in more than one million new cases every year.[1,2] Colorectal cancer is composed of colon cancer with a superior prognosis and rectal cancer with an inferior prognosis.[3,4] Thus, early-stage colon cancer (without lymph node metastasis or distant metastasis) represents the subtype of colorectal cancer with the best prognosis and patients are usually waived from routine adjuvant chemotherapy. However, substantial progress in diagnosis and therapeutic strategies in recent decades have not significantly improved the long-term prognosis of early-stage colon cancer patients. On the other hand, the most widely used tumor staging system, i.e., AJCC TNM staging system, is not very satisfactory in prognosis stratification in colon cancer patients. For example, IIC (T4bN0) colorectal cancer patients held worse survival than IIIA (T1/T2N1) cases, suggesting that there are underlying factors, beyond TNM staging, that account for the worse prognosis for cancer patients with lower pathological stage.[17]
As reported in previous studies, numerous pathological features have been proposed to predict prognosis and identify cancer patients with high risk, who may benefit from strict follow-up and adjuvant treatments.[6,7,10] Vascular invasion and perineural invasion are two commonly recognized pathological features that are significantly associated with tumor relapse, chemoresistance, and worse survival of types of cancers.[18,19] In addition to these acknowledged histopathological parameters, there are other pathological findings that have been proposed to be associated with tumor aggressiveness. A tumor bud is defined as several malignant cells at the leading edge of an epithelial tumor, which can be evaluated from pathological reviewing. The presence of tumor buds is found associated with local recurrence, distant metastasis, and poor prognosis of cancer patients, probably because tumor buds represent an EMT phenotype characterized by loss of adhesion molecules and increased invasiveness.[9,20-22] Crohn’s-like lymphoid reaction refers to the aggregating nodular lymphoid lining the tumor periphery, and it significantly reduced the overall survival of colorectal cancer.[7,8]
Tumor necrosis is another emerging pathological criterion that can be evaluated from routine pathological slide reviewing, which suggests stronger invasiveness and tumor progression in colorectal cancer and other epithelial cancers.[14,19,23] Tumor necrosis is associated with tumor relapse, lymph node metastasis, distant metastasis, and worse prognosis in types of cancers. The underlying mechanisms are distinct, such as hypoxia-regulated aggressiveness caused by tumor rapid outgrowth,[24] vascular insufficiency resulted from aberrant IL-6-related inflammation stimulation,[25] refractory hypoxia microenvironment brought about by extensive incomplete neovascularization,[26,27] etc. Anyway, tumor necrosis reflects a more aggressive phenotype of cancer, but substantial studies on its role in early-stage colon cancer patients are still lacking, which is also of great importance.
Therefore, we conducted the current study to explore the prognostic role of tumor necrosis in early-stage colon cancer patients. The results demonstrated that tumor necrosis was a stable and independent risk factor of early-stage colon cancer, independent of T stage, vascular invasion, and perineural invasion [Table 2]. On the other hand, no significant association between tumor buds and tumor necrosis was found (χ2 testing, P = 0.304). A number of previous reports focused on the impact of tumor necrosis in late-stage colorectal cancer, and few researches investigated its role in early-stage patients since their prognosis was favorable and mainly influenced by tumor residual. To our knowledge, our study is the first report that confirmed the unfavorable impact of tumor necrosis in early-stage colon cancer and proved that early-stage cancer patients could be further stratified into subgroups with different progression risks according to valuable pathological criteria. These findings had the potential ability in identifying colon cancer patients with high risk and making personalized follow-up plan and therapeutic strategies.
Although our findings deepened the understanding and role of tumor necrosis in the progression and prognosis of early-stage colon cancer, there are still several limitations that should be acknowledged. First, the main drawback of our study is that it is retrospective and patient selection bias is inevitable. Thus, prospective cohort studies are needed to verify the prognostic role of tumor necrosis in routine clinical practice. Secondly, the evaluation of tumor necrosis from pathological slides relies on manual reviewing by pathologists, which will cause interpathologist heterogeneity. However, in our study, a random subset of 30 cases were selected from the enrolled population to test the interpathologist compliance in evaluating tumor necrosis. A clear definition of tumor necrosis and uniform training can reduce the heterogeneity to a great extent, and future automatic evaluation by artificial intelligence may bring about more benefits.
In conclusion, the current study demonstrates that tumor necrosis reviewed from pathological slides is significantly associated with tumor aggressiveness and can serve as an independent prognostic factor in early-stage colon cancer patients, which should be validated in further prospective clinical trials. Regardless of the limitations, these findings promise to facilitate the identification of high-risk subgroup from early-stage colon cancer patients, aiding in making personalized and interactive follow-up and therapy decisions.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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