Claudia Rengucci¹, Giulia De Maio¹, Andrea Casadei Gardini², Mattia Zucca¹, Emanuela Scarpi³, Chiara Zingaretti⁴, Giovanni Foschi¹, Maria Maddalena Tumedei¹, Chiara Molinari¹, Luca Saragoni⁵, Maurizio Puccetti⁵, Dino Amadori², Wainer Zoli¹ and Daniele Calistri¹

¹Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS
²Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS
³Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS
⁴National Institute of Molecular Genetics
⁵Pathology Unit, Morgagni-Pierantoni Hospital

Abstract

Background
Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence.

Methods
A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry.

Results
Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P?=?0.001) and 17.68 (95% CI 1.91-163.54, P?=?0.011), respectively.

Conclusions
Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

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