A ten markers panel provides a more accurate and complete microsatellite instability analysis in mismatch repair-deficient colorectal tumors.
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.


CONCLUSIONS
a complete panel of ten markers including four dinucleotide and six mononucleotide microsatellites allows accurate evaluation of tumor MSI status.
Agostini
Marco
M
II Section of Surgery Clinic, Department Oncologic and Surgical Sciences, University of Padova, Padova, Italy. m.agostini@unipd.it
Enzo
Maria Vittoria
MV
Morandi
Luca
L
Bedin
Chiara
C
Pizzini
Silvia
S
Mason
Silvia
S
Bertorelle
Roberta
R
Urso
Emanuele
E
Mescoli
Claudia
C
Lise
Mario
M
Pucciarelli
Salvatore
S
Nitti
Donato
D
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D014408:Biomarkers, Tumor / Q000235:genetics*; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000145:classification* / Q000235:genetics*; D019175:DNA Methylation; D053843:DNA Mismatch Repair / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D053842:Microsatellite Instability; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein