Background: Stage II colorectal cancer(CRC) patients after surgery alone have a five-year survival rate of ~60-80%; the incremental benefit of adjuvant chemotherapy is <5%. Predicting risk of recurrence and selecting effective personalized adjuvant drugs for stage II CRC using formalin-fixed, paraffin-embedded(FFPE) samples is a major challenge. Methods: 1319 stage II CRC patients who enrolled in 2011-2019 at Sun Yat-sen University Cancer Center were screened. RNAseq data of FFPE tumor samples of 222 stage II MSS CRC patients(n.recurrence=47, n.norecurrence=175, median follow-up=41 months) were used to develop a method TFunctionalProg for dissecting heterogeneous subgroups of recurrence, predicting risk of recurrence and proposing adjuvant drugs. Results: TFunctionalProg showed significant predictive values in 222 stage II MSS CRCs. The TFunctionalProg low-risk group had significantly better RFS (validation set (HR=4.78, p-value=1e-4, low-risk group three-year RFS=92.6%, high-risk group three-year RFS=59.7%). TFunctionalProg dissected two subgroups of transition states of stage II MSS CRCs at a high risk of recurrence; each state displays distinct levels of hybrid epithelial-mesenchymal traits, cytotoxic cell inhibition and FOLFOX resistance. Based on mechanisms in two subgroups, TFunctionalProg proposed personalized rational adjuvant drug combinations of immunotherapy, chemotherapy and repurposed CNS drugs. The complementary utility of TFunctionalProg and ctDNA-based prognostic biomarkers were presented. Conclusion: TFunctionalProg was validated using FFPE samples to predict the risk of recurrence and propose rational adjuvant drug combinations for stage II CRCs.

RW and ST owns stocks and shares and/or stock options in Carbon Logic Biotech Ltd. ST is an inventor of a patent application related to this work. All remaining authors have declared no conflicts of interest.

The authors declare no funding.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Medical Ethical Committee of Sun Yat-sen University Cancer Center gave ethical approval for this work(nr. B-0440-1). Patient consent is waived.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.

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RNAseq data of 81 genes used in TFunctioanlProg method will be deposit as online supplementary data.