Adjuvant therapy in patients with stage II colon cancer is a subject of controversy because of the small gains in survival for this group of patients
. Apparently, that was confirmed by our results, showing that the addition of adjuvant treatment to patients with node-negative cancers did not improve DFS and OS (Figure
1). Uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colon cancers is due to the fact that we don’t know which of the stage II patients are at high risk of recurrence and which may benefit from chemotherapy
. To deepen investigation on these aspects, in the current study a combination of pathological factors and molecular markers was assessed in 120 cases of stage II colon cancer independently of 5-FU treatment.
Considering the entire case study, we found that patients showing c.1799 T > A mutation in the BRAF gene showed a worse prognosis, independently of 5-FU treatment, as already reported
[42, 43]. In addition, even though non statistically significant, patients having tumours with a pushing border, or showing a tumour inflammatory response, were associated with better survival. The first finding could be explained by the fact that an infiltrating tumour margin is strongly associated with the presence of tumour budding
, which has been shown to have independent negative prognostic value in colorectal cancer
. The latter is related to the reported good prognostic value of generalized inflammatory cell infiltrate in colorectal cancers of stage II patients
[45, 46] and may be explained by a tumour-related immune response.
Interestingly the adverse effect of BRAF mutation on patients’ outcome appeared to be independent from CIMP phenotype, since in our case study we could not find an association between these two parameters, contrarily to other reports
After splitting the group of patients according to treatment, we found a differential effect of 5-FU-based adjuvant therapy according to thymidylate synthetase (TYMS) mRNA levels, the presence of a defect in the MMR system (dMMR) and the presence of the CpG island methylator phenotype, suggesting that these are important parameters for therapy decision. In patients treated with surgery alone, a high TYMS expression was inversely correlated with survival, as previously reported
[24, 47]. On the contrary, an improved clinical outcome was seen for patients submitted to 5-FU therapy and having a high TYMS expression. Controversial results have been reported on the relationship between TS and 5-FU treatment
[24–27, 31]. Such contradictory results may be related to differences in patient cohorts and methodological inconsistencies. In particular, our results showed that TYMS levels measured by real time qRT-PCR did not correlate with the corresponding protein levels measured by IHC and the effect on the outcome was evident for TYMS only when evaluated at the mRNA level.
Consistent with other studies
[19, 48], our data suggest a favourable prognosis in patients with dMMR tumours if untreated with adjuvant therapy (HR, 0.15; p = 0.2) and agree with other reports, indicating that 5-FU treatment should be avoided in patients with dMMR tumours
[18, 19]. Of note, in our case study, 75% of dMMR patients who developed a recurrence were treated with 5-FU adjuvant therapy. A possible antitumor immune response by the lymphocytic infiltrate characteristic of dMMR tumours
[3, 19], which may be abrogated by the immunosuppressive effects of chemotherapy, could explain these findings. The favourable prognosis of patients with dMMR cancers and their lack of benefit from 5-FU therapy supports a non-adjuvant treatment approach in these patients, whereas pMMR could be considered as a risk factor to recommend adjuvant chemotherapy
. Cancers with a proficient MMR system, however, comprise the majority (80% to 85%) of colorectal cancers and adjuvant therapy decision in such patients should be addressed to other factors
, for instance TYMS expression, which we found associated to therapy benefit in patients with pMMR tumours (Figure
Different studies reported that CIMP tumours have a distinct clinical, pathological, and molecular profile
. Our results confirm the associations between CIMP and proximal location, poor tumour differentiation and MMR deficiency, also showing the recently reported association between CIMP-low and male sex
. On the other hand, studies on CIMP phenotype and survival in colon cancer have yielded somewhat contrasting results
[21, 22, 49]. Our findings of good prognosis and therapy benefit in CIMP-High tumours are in agreement with other reports
[22, 49], even though other studies suggested a lack of benefit from 5-FU adjuvant therapy in these patients
. Evidence was also supplied about an interplay between CIMP and MMR status in affecting prognosis
[50, 51]. Unfortunately the absolute number of dMMR and CIMP-High occurrences are modest in our case study, therefore conclusive results cannot be drawn for the latter consideration. The comparison between our results and the other studies is also complicated by the fact that some studies considered only stage III patients
, while in others the impact of adjuvant 5-FU chemotherapy relative to the CIMP status was not addressed in specific subgroups (stage II versus stage III patients)
. A possible explanation for the apparent chemosensitivity of CIMP-High tumours could be related to the link between CIMP-High and intracellular folate methabolism. It has been reported that CIMP-High tumours show elevated concentrations of intracellular folates, which have a critical importance in determining the response to 5-FU
. Moreover, results from microarray analysis have revealed that several genes involved in the pathways of nucleotide metabolism, as well as folate and glutamine metabolism are differentially expressed between CIMP-positive and CIMP-negative tumours
. Of note, the reported study also shows that TYMS levels are significantly higher in CIMP-positive tumours with respect to CIMP-negative ones, confirming our results
. We also found higher TYMS levels in patients with a defect in the MMR system, in agreement with Ricciardiello et al.
. We recognise that the relationship between TYMS, MMR, and CIMP in colon cancer could be complex. In our cohort, 50% of CIMP-High tumours showed a defect in the MMR system and 72% of dMMR tumours exhibited a high TYMS level. High TYMS levels were more common also in patients with the CpG island methylator phenotype, but while TYMS expression and CIMP-High status have been associated to a positive outcome in patients treated with 5-FU, dMMR has been linked to worse outcome, highlighting the complexity of these molecular relationships. Stratification of patients according to combinations of TYMS expression, MMR and CIMP statuses to assess their joint effect on patient outcome was not performed in this study because of the small number of patients. Larger case-studies will be necessary to deepen investigation on the prognostic and predictive role of such markers in patients with stage II colon cancers, also adopting a multisegmental approach to bowel subsites
 in order to improve tailored preventive and therapeutic strategies.