Abstract 3678: DNA methylation profiling of colorectal cancer identifies molecular subtypes and subtype-specific biomarkers for improved prediction of patient prognosis
Authors: Mattesen Trine Block , Rasmussen Mads Heilskov , Bramsen Jesper , Andersen Claus Lindbjerg 
Published: August 2018

By analysis of RNA profiles from &gt;1000 fresh-frozen (FF) colorectal cancer (CRC) samples we recently identified distinct molecular subtypes of CRC with different etiology, prognosis and prognostic biomarkers. The clinical validation and routine utilization of this exiting knowledge is, however, complicated by the unavailability of high quality RNA from clinical collected formalin-fixed paraffin-embedded (FFPE) samples and the high cost/complexity of RNA sequencing. As part of a solution we have established a novel CRC subtyping approach “methCORR”, which only require DNA from either FF or FFPE tissue to classify CRC into subtypes based on their genome-wide DNA methylation profile. Using methCORR we were able to stratify CRC in independent datasets into two major cancer epithelial subtypes, “CRC1” and “CRC2", which are distinguished by their chromosomal/microsatellite stability status, the activity of the BMP-, WNT- and NOTCH signaling pathways and cellular composition of their tumor microenvironment (TME). Very notably, we also found evidence of subtype-specific prognostic biomarkers reflecting inter-tumor TME heterogeneity: aggressive CRC1 tumors were associated with an absence of infiltrating immune cells, whereas aggressive CRC2 tumors were instead associated with immune cell infiltration and stromal fibroblast activation. To derive cost-efficient prognostic biomarkers from our genome-wide methCORR analysis we next exploited that CRC1- and CRC2 cancer epithelial cells as well as TME cell types, such as T-lymphocytes and fibroblasts, have unique and robust DNA methylation patterns that can be interrogated by quantitative methylation-specific PCR (QMSP). Indeed, QMSP biomarker assays specific for CRC1 and CRC2 cancer epithelial cells successfully classified our discovery cohort (n=174) according to methCORR CRC1 and CRC2 epithelial cancer subtypes. Furthermore, additional quantification using T-lymphocytes and fibroblast-specific QMSP biomarker assays confirmed our methCORR estimations: CRC1 aggressive tumors were associated with a high content of cancer epithelial cells and absence of T-lymphocytes whereas aggressive CRC2 tumors were, in contrast, associated with a high content of T-lymphocytes and fibroblasts. Finally, we applied our top 5 QMSP biomarker panel to an independent validation cohort of 192 FFPE samples, which confirmed our observations from the discovery cohort. In conclusion: we have developed and validated novel QMSP-based biomarkers for CRC molecular subtyping and subtype-specific prediction of patient prognosis. Our assays are cost-efficient and compatible with FFPE samples hereby making them potentially relevant for clinical applications.

Citation Format: Trine Block Mattesen, Mads Heilskov Rasmussen, Jesper Bramsen, Claus Lindbjerg Andersen. DNA methylation profiling of colorectal cancer identifies molecular subtypes and subtype-specific biomarkers for improved prediction of patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3678.