**5. Timing of chemotherapy**

*Colorectal Cancer*

**4.2 Other molecular markers**

**4.3 Circulating tumor DNA (ctDNA)**

Gene sequencing of colorectal cancer have identified number of common somatic mutations and these tumor-specific mutations can be utilized to detect the tumor DNA (ctDNA) in the cell free component of peripheral blood. Detectable ctDNA after surgical resection or after completion of adjuvant chemotherapy seem to be associated with high risk of recurrence. In a study of 230 patients with resected stage II colon cancer, 14 patients out of 178 who did not receive adjuvant chemotherapy had detectable ctDNA. Eleven of the 14 (79%) developed recurrence at a median follow up of 27 months. Among those who received chemotherapy 3/44 had detectable ctDNA and all of them have relapsed within 11 months [47]. In metastatic setting, changes in ctDNA correlate with radiological responses [48]. Consistently in the early stage colon cancer, patients who clear ctDNA after adjuvant therapy have favorable prognosis [49]. Currently available data suggest that ctDNA is robust marker of minimal residual disease after surgery or after adjuvant chemotherapy with good prognostic and predictive value. Although current assays used to detect ctDNA have high specificity and positive predictive value, the sensitivity of these assays need optimization. In addition, a consensus on the methodology and larger number of prospective trials are needed before their routine use in clinical practice.

Lack of CDX2 was associated with lower 5-year survival rate compared to CDX2 positive tumors, especially in stage II tumors (49% versus 87%, p = 0.003). CDX2 was also predictive of treatment benefit with higher disease-free survival in CDX2 negative tumors in both stage II and III tumors. This need to be further validated in prospective studies. The presence of BRAF V600E mutation confers a poor prognosis in colon cancer; however, concomitant loss of one or more MMR enzymes (dMMR) seems to improve the survival. In an analysis of three adjuvant chemotherapy trials of stage II and III colon cancer, BRAF mutation was not prognostic, however overall survival was poor among those with pMMR [32]. While another study of 2299 patients from two NSABP trials showed similar results, where BRAF mutation was not predictive of oxaliplatin benefit [37]. The presence of RAS (KRAS and NRAS) mutation is associated with resistance to EGFR targeted therapy in metastatic colon cancer. Although the presence of KRAS mutation seems to confer poor prognosis, not all studies are consistent [32, 37–39]. Number of other molecular markers such as DCC, TP53, thymidylate synthase and POL-E are also found to have prognostic significance [40–43]. Despite emerging evidence of these molecular markers, their predictive value is still not validated in clinical practice and they are not routinely considered in decision making regarding adjuvant therapy, except for MMR status. Gene expression profiling has been utilized to characterize colon cancers and to identify gene signatures that could be predictive and prognostic. A number of commercial assays are developed in the recent past (OncoDefender-CRC, ColonPRS, ColoPrint colon cancer recurrence assay, GeneFx colon) but none have been approved for routine use in clinical practice. The Oncotype-DX colon cancer assay is perhaps the most validated tool which is a 12-gene assay developed to predict the recurrence score in stage II colon cancer. It was validated using prospective data from large studies including QUASAR, CALGB9581 and SUNRISE [44–46]. Despite the ability in predicting the risk of recurrence with confidence, it is unclear whether patients in higher risk category will benefit from adjuvant chemotherapy. A treatment score was developed using the data from QUASAR, but it was not predictive of the treatment effect. At this stage the data are insufficient to recommend routine use of multi-gene assays when deciding adjuvant therapy for stage II colon cancer.

**170**

Adjuvant therapy should be initiated as soon as patient has recovered from surgery with complete healing of surgical wounds which usually takes about 2–4 weeks. A meta-analysis in 2019 which included 34 comparative studies of resected colon cancer reported that delay in treatment beyond 6–8 weeks was associated with inferior survival (HR 1.27,95% CI 1.21–1.33; p < 0.001) [50]. Another review which included more than 15,000 patients concluded that a 4-week increase in time to initiate adjuvant chemotherapy was associated with a 14% relative decrease in disease free survival and overall survival [51]. A number of other studies have consistent findings suggesting inferior outcomes when chemotherapy was initiated more than 6–8 weeks. However, most of these studies are retrospective in nature and potentially biased by confounding factors such as comorbidities, post-operative complications, and emergency resections which are all likely to delay the recovery.
