As recommended by current international guidelines [5], adjuvant chemotherapy was limited to patients with at least one of the high risk factors aforementioned in localized colon cancer. However, there is significant variation in clinical practice among countries, especially in stage II CRC, despite the fact that adjuvant treatment of clinical low risk patients was not associated with improvements in outcomes [28]. Considering the toxic and side effects, high expenses, and many inconveniences including transportation and difficulties in admission to hospital of treatment, as well as the insufficient survival benefit of only an absolute improvement of 3.6% [2], it’s necessary to explore novel biomarkers that can stratify stage II CRC more precisely and can hopefully be accepted by international criteria.

In the present study, we have proven that nucleotyping was the dominant prognostic factor using whether univariate or multivariable Cox analyses in our whole cohorts as well as in high-risk group, as was demonstrated in a previous study [22]. Tumor progression is accompanied by genomic and epigenetic changes, which alter the nuclei in multiple ways including the size of the nucleus, the density of DNA, and the structure of chromatin. Nucleotyping, as a quantitative analysis of the degree of nuclear disorder of tumors, provides descriptions of cell nuclei and in particular the chromatin structure in cancer cell nuclei [29] based on the theory of statistical texture analysis. It can help to distinguish between normal tissue and precancerous tissue [19] and its prognostic significance has been shown in several cancers [30,31,32,33]. As for CRC, a pan-cancer study involving stage I and II CRC [20] showed a predictive function towards cancer-specific survival. Our results were similar by finding that patients with CHE had a shorter OS (a median of 69.5 months) than those with CHO (a median of 94.4 months) in all patients. The above evidence confirmed nucleotyping as an independent prognostic factor and could add value to traditional tumor-node-metastasis staging system.

It’s believed that more than 70% solid tumors are aneuploid [7], resulting from chromosome missegregation and correlating with the aggressiveness of the tumor. Though aneuploidy, describing the state and CIN, referring to the elevated rate of chromosome gain or loss, are not synonymous, they are interrelated and aneuploidy can be used representing CIN. Unlike point mutations that affect only a small number of genes, the number alterations of chromosomes alter the transcription of hundreds of genes and can disturb a large array of cellular processes [6]. We detected cellular DNA ploidy as a proxy for the degree of aneuploidy and assumed it to be prognostic in stage II colon cancer as in previous studies of breast, endometrioid endometrial, ovarian and prostate cancers [34]. There was a significant survival advantage in diploidy patients, whose median OS was 87.5 months compared to non-diploidy patients, whose median OS was 93.4 months. But in multivariable analysis, significance was not observed. Considering the result that there was a negative correlation between DNA ploidy and MSI and the view that CIN was strongly negatively associated with MSI in a previous analysis of VICTOR trial [10], the difference might be offset in multivariable models. We also observed a superior survival in diploidy patients of low-risk group with dMMR, and the consequence was consistent with the study of VICTOR trail [10] too. The unobserved significance in multivariable analysis was possibly attributed to our small sample of low-risk patients.

It was identified that CHE patients were more likely to have non-diploid phenotypes, and diploid patients were also more likely to be CHO by previous researchers [23], suggesting that both biomarkers correlated on a cellular level and the correlation was demonstrated in our data (with a coefficient of 0.404). It was reasonable to observe significance when we carried out analysis of the combination of DNA ploidy and nucleotyping based on the above results. Patients with CHE and non-ploidy had the worst prognosis in the whole cohorts and in the low-risk group as well. The large-scale genomic instability owing to aneuploidy must correlate with large-scale rearrangement of interphase nuclear chromatin, which can be reflected by examining nucleotyping. Thus the correlation could be explained properly. After verification by multivariable models, the combination of DNA ploidy and nucleotyping was proven to be an independent predictor of survival in stage II colon cancer and a candidate marker to select those with higher risk even in low-risk clinical group with MSI.

Stroma surrounding tumors was associated with tumor initiation, progression, and metastasis and held prognostic value [15]. Researchers suggested that stroma supplied the tumor with growth factors, cytokines, and metabolites, and stimulated blood vessel formation, which could cause tumorigenesis and induction of epithelial-mesenchymal transition (EMT) [35]. And an expanded tumor stroma might influence disease progression by promoting tumor growth and enhancing invasive capabilities. Vascular and lymphatic metastases were more likely with the increasing of stroma ratio [36]. Contradictory to the results earlier that high stroma was associated with poorer survival whether in domestic populations [17] or in European populations [21, 37], we found no significance between low stroma and high stroma whether using OS or DFS as an endpoint despite a tendency in DFS. The possible causes might include the variations in methods when calculating the stroma-tumor fraction, as we evaluated all tumor areas on the whole scanned images rather than studied the areas with the deepest tumor infiltrating margin, and the small sample size, as well as the correlated low power calculation. Given the characteristics of peritumoral stroma in angiogenesis, the stroma-tumor fraction, or tumor stroma percentage according to Park JH and colleagues [36], was a potential biomarker to select patients who could be considered for treatment targeted at the stroma itself and beneficial from anti-angiogenic therapies.

Our results were consistent with what Danielsen HE and colleagues [21] had suggested that the combination of DNA ploidy and stroma-tumor fraction provided a prognostic stratifier for stage II CRC even superior to RNA signature. Similarly, we found that this combined indicator was statistically significant based on whether OS or DFS, displaying predictive functions in survival as well as in recurrence and metastasis in colon patients. An assumption was that the effect of aneuploidy may not be driven by a particular combination of chromosomes per se, but rather by the specific interaction of the karyotype with the various genetic contexts and microenvironments found in different tissues [6]. Although mechanism not elucidated, tumor stroma as the most important microenvironment could have an effect on ploidy phenotypes and evolving directions [38]. Thus a deeper understanding of the relationship between ploidy and stroma might reveal new avenues for anti-cancer therapies. Though we did not found significant statistical difference between low-stroma and high-stroma patients except in high-risk group, the combination of DNA ploidy and stroma as well as the combination of nucleotyping and stroma turned out to have prognostic value. This might be due to the significance of DNA ploidy and nucleotyping take dominant position when we combine the two factors with stroma.

Our current study emphasized the important prognostic roles of conventional risk factors including age and pathological T stage, and added new biomarkers that were valuable to decision-making in postoperative adjuvant treatment. MSI was the most validated prognostic marker next to clinical prognostic factors, which has been demonstrated by a meta-analysis [39], and showed statistical significance only in DFS in the whole cohort (P = 0.043), suggesting a decreased rate of events including relapse and metastasis. Our bias when selecting cases in different risk groups may affect the accuracy. While previous studies have shown that in multivariable analysis, the absence of adjuvant chemotherapy was risk factor for stage II colon cancer [37], our study showed that adjuvant chemotherapy had no significant effect on prognosis whether in all patients or in each risk group. The inferred reasons were as following: variations between countries and ethnic groups; the limitations of diagnostic and surgical level twenty years ago; the younger median age in our cohort than that of study mentioned above (with a median age of 73 years old); the refusal to chemotherapy in our cohorts due to financial reason or utilization of Chinese medicine as a substitute.

According to Chinese Protocol of Diagnosis and Treatment of Colorectal Cancer (2023 Edition), adjuvant therapy is needed only in high-risk group with a recommended chemotherapy regimen of CapeOx or FOLFOX based on Oxaliplatin, or monotherapy with 5-FU/LV and Capecitabine. However, using the two independent prognostic factors produced in our study, we tried adjusting treatment strategies, considering if patients with diploidy and CHO could avoid adjuvant treatment even in high-risk group. Postoperative adjuvant chemotherapy was not recommended in CRC with dMMR or MSI-H. A possible treatment change may be that patients with non-diploidy and CHE should accept at least monotherapy, though they belonged to low-risk group. On the basis of these results, we can infer that stratification of patients according to nucleotyping and the combination of ploidy and nucleotyping will help select stage II colon patients who may benefit from postoperative adjuvant therapy, avoid overtreatment and insufficient clinical intervention. By this way, the novel biomarkers may help guide clinical decisions, optimize prognosis assessments, and tailor individual treatments.

In patients who were older than 61 years old, CHE and the combination of non-ploidy and CHE represented shorter OS time (P = 0.009 for nucleotyping and P = 0.016 for the combination of ploidy and nucleotyping). While in patients who were at pT4 stage, CHE and the combination of non-ploidy and CHE were associated with inferior DFS (P = 0.013 for nucleotyping and P = 0.034 for the combination of ploidy and nucleotyping). It was possible to take the novel parameters and existing conventional indicators such as age and pathological T stage into comprehensive consideration in clinical practice to develop the best diagnosis and treatment plan.

One of the obvious restrictions is the single-centre design with a relatively small sample size. Though we tried to collect patients who met our inclusion criteria regardless of gender or age, limited cases were included. The reasons were that electronic information might lost due to time factor and that there were a lower early cancer diagnosis rate and a lower proportion of stage II colon cancer patients in China due to the limited coverage of cancer screening. Other restrictions included the retrospective character, inconsistence of follow-up time, variations in adjuvant chemotherapy regiments, different compliance of regular examinations, and an inherent disadvantage of OS susceptible to non-cancer-related deaths. However, we still successfully identified two independent prognostic indicators—nucleotyping and the combination of nucleotyping and DNA ploidy, and might stratify stage II colon cancers more accurately, therefore reducing adjuvant overtreatment and intensifying adjuvant treatment on those at a higher risk of death [20]. We expanded the application of nucleotyping as a predictor from high risk stage II colon cancer [22] to whole risks. Given the speed, simplicity, accuracy, batch processing, and low cost of nucleotyping measurements [19], it will become more competitive in the clinical environment and contribute to personalized medication in oncology.