Pathway Level Subtyping Identifies a Slow-cycling Biological Phenotype Associated with Poor Clinical Outcomes in Colorectal Cancer

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5 stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1 stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.

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References

Durinck S, Spellman P, Birney E, Huber W . Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc. 2009; 4(8):1184-91. PMC: 3159387. DOI: 10.1038/nprot.2009.97. View

Karras P, Bordeu I, Pozniak J, Nowosad A, Pazzi C, Van Raemdonck N . A cellular hierarchy in melanoma uncouples growth and metastasis. Nature. 2022; 610(7930):190-198. PMC: 10439739. DOI: 10.1038/s41586-022-05242-7. View

Gao S, Yan L, Wang R, Li J, Yong J, Zhou X . Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing. Nat Cell Biol. 2018; 20(6):721-734. DOI: 10.1038/s41556-018-0105-4. View

Joanito I, Wirapati P, Zhao N, Nawaz Z, Yeo G, Lee F . Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer. Nat Genet. 2022; 54(7):963-975. PMC: 9279158. DOI: 10.1038/s41588-022-01100-4. View

Lawler M, Kaplan R, Wilson R, Maughan T . Changing the Paradigm—Multistage Multiarm Randomized Trials and Stratified Cancer Medicine. Oncologist. 2015; 20(8):849-51. PMC: 4524758. DOI: 10.1634/theoncologist.2015-0014. View

Jones S, Chen W, Parmigiani G, Diehl F, Beerenwinkel N, Antal T . Comparative lesion sequencing provides insights into tumor evolution. Proc Natl Acad Sci U S A. 2008; 105(11):4283-8. PMC: 2393770. DOI: 10.1073/pnas.0712345105. View

Satija R, Farrell J, Gennert D, Schier A, Regev A . Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015; 33(5):495-502. PMC: 4430369. DOI: 10.1038/nbt.3192. View

Sirinukunwattana K, Domingo E, Richman S, Redmond K, Blake A, Verrill C . Image-based consensus molecular subtype (imCMS) classification of colorectal cancer using deep learning. Gut. 2020; 70(3):544-554. PMC: 7873419. DOI: 10.1136/gutjnl-2019-319866. View

Malla S, Fisher D, Domingo E, Blake A, Hassanieh S, Redmond K . In-depth Clinical and Biological Exploration of DNA Damage Immune Response as a Biomarker for Oxaliplatin Use in Colorectal Cancer. Clin Cancer Res. 2020; 27(1):288-300. PMC: 7614625. DOI: 10.1158/1078-0432.CCR-20-3237. View

10.

Marisa L, De Reynies A, Duval A, Selves J, Gaub M, Vescovo L . Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value. PLoS Med. 2013; 10(5):e1001453. PMC: 3660251. DOI: 10.1371/journal.pmed.1001453. View

11.

Burki T . UK and US governments to fund personalised medicine. Lancet Oncol. 2015; 16(3):e108. DOI: 10.1016/S1470-2045(14)71204-5. View

12.

Guinney J, Dienstmann R, Wang X, De Reynies A, Schlicker A, Soneson C . The consensus molecular subtypes of colorectal cancer. Nat Med. 2015; 21(11):1350-6. PMC: 4636487. DOI: 10.1038/nm.3967. View

13.

Papatheodorou I, Oellrich A, Smedley D . Linking gene expression to phenotypes via pathway information. J Biomed Semantics. 2015; 6:17. PMC: 4404592. DOI: 10.1186/s13326-015-0013-5. View

14.

Seymour M, Maughan T, Ledermann J, Topham C, James R, Gwyther S . Different strategies of sequential and combination chemotherapy for patients with poor prognosis advanced colorectal cancer (MRC FOCUS): a randomised controlled trial. Lancet. 2007; 370(9582):143-152. DOI: 10.1016/S0140-6736(07)61087-3. View

15.

Sato T, Vries R, Snippert H, van de Wetering M, Barker N, Stange D . Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009; 459(7244):262-5. DOI: 10.1038/nature07935. View

16.

Gil Vazquez E, Nasreddin N, Valbuena G, Mulholland E, Belnoue-Davis H, Eggington H . Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia. Cell Stem Cell. 2022; 29(11):1612. PMC: 9807457. DOI: 10.1016/j.stem.2022.09.005. View

17.

Mayakonda A, Lin D, Assenov Y, Plass C, Koeffler H . Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res. 2018; 28(11):1747-1756. PMC: 6211645. DOI: 10.1101/gr.239244.118. View

18.

Jackstadt R, van Hooff S, Leach J, Cortes-Lavaud X, Lohuis J, Ridgway R . Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis. Cancer Cell. 2019; 36(3):319-336.e7. PMC: 6853173. DOI: 10.1016/j.ccell.2019.08.003. View

19.

Van Cutsem E, Labianca R, Bodoky G, Barone C, Aranda E, Nordlinger B . Randomized phase III trial comparing biweekly infusional fluorouracil/leucovorin alone or with irinotecan in the adjuvant treatment of stage III colon cancer: PETACC-3. J Clin Oncol. 2009; 27(19):3117-25. DOI: 10.1200/JCO.2008.21.6663. View

20.

Franzen O, Gan L, Bjorkegren J . PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data. Database (Oxford). 2019; 2019. PMC: 6450036. DOI: 10.1093/database/baz046. View