Affiliations: [a] II Section of Surgery Clinic, Department Oncologic and Surgical Sciences, University of Padova, Italy | [b] Department of Ematology and Oncological Science "L. & A. Seragnoli" Section of Pathology, Bellaria Hospital, University of Bologna, Italy | [c] AB ANALITICA s.r.l. Padova, Italy | [d] Istituto Oncologico Veneto, IRCCS, Padova, Italy | [e] Section of Pathology, University of Padova Italy | [f] Surgical Oncology, Regional Oncologic Center (CRO), Aviano, Italy
Correspondence:
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Corresponding author: Marco Agostini PhD, Clinica Chirurgica II, Dipartimento di Scienze Oncologiche e Chirurgiche, Università di Padova, Via Giustiniani 2, 35128 Padova, Italy. Tel.: +39 049 8214374; Fax: +39 049 651891; E-mail: [email protected].
Note: [1] The first three authors contributed equally to this article.
Abstract: Tumour microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC) with defective DNA mismatch repair (MMR} genes. A reference Bethesda panel has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumours with MMR deficiencies. We developed a multiplex PCR assay with additional four mononucleotide markers and one dinucleotide marker (NR-21, NR-24, BAT-40, TGF-BetaR and D18S58) for a rapid and proper classification of MSI-H, MSI-L and MSS colorectal cancers. Two tetranucleotide markers were added to identify sample mix-ups and/or contamination. Results: all the 44 cases test cases were in agreement with previous classification except for three cases: one case MSI-H-Bethesda unstable only for dinucleotides markers shifted to MSI-L category and two cases MSI-L-Bethesda unstable for mononucleotide markers shifted to MSI-H category. Immunohistochemistry analysis revealed that these two MSI-H cases did not expressed hMLH1 and they were found to be methylated at the MLH1 promoter, while the first one that shifted to MSI-L showed MMR protein expression. Conclusion: a complete panel of ten markers including four dinucleotide and six mononucleotide microsatellites allows accurate evaluation of tumor MSI status.
