A novel multiplexing PCR-based assay of the anaylisis of chromosome 18q status in colorectal cancer

Nadina Erill1, Anna Colomer1, Miquel Calvo2, August Vidal3, Ruth Román1, Montse Verdú3, Carlos Cordón-Cardó4, Xavier Puig1,3.

1BIOPAT, Grup Assistència, Statistics Department, 2Universitat de Barcelona, 3HISTOPAT Laboratoris, Barcelona, and 4Division of Molecular Pathology,  Memorial Sloan-Kettering Cancer Center,  New York.

Chromosome 18q allelic loss has been reported to have prognostic significance in stage II colorectal carcinoma. We have developed a fluorescent multiplex polymerase chain reaction assay to analyze five microsatellite markers (D18S55, D18S58, D18S61, D18S64 and D18S69) for allelic loss determination at the long arm of chromosome 18. Amplicon detection and evaluation is accomplished by capillary electrophoresis using an ABI 310 genetic analyzer. Robustness of the assay when performed on DNA extracted from formalin-fixed, paraffin-embedded tissue sections was confirmed by analyzing its repeatability and reproducibility. Allelic loss was determined in 61 stage II colorectal tumors and detected in 58% of those not showing instability.  As part of the study, results of 207 previous PCR/polyacrylamide based assays were re-evaluated by two independent observers to determine the degree of concordance of visual evaluation.  In the case of stage II colorectal tumors, when electropherogram results were compared with those obtained from visual evaluation of the same markers after polyacrylamide gel electrophoresis, discrepancies (worse observer) were detected in 16.4% of determinations.  We have developed a robust and reliable assay for multiplexed LOH determination that improves assessment of chromosome 18q allelic loss status in colorectal tumor processed as routine formalin fixed, paraffin-embedded specimens.

The prognosis of colorectal cancer is mainly based on tumor stage at time of diagnosis.  However, other clinicopathological features such as tumor location, perineural invasion, lymphatic vessel invasion and differentiation, as well as certain molecular alterations such as microsatellite instability, have also been reported to be predictors of tumor recurrence and patient outcome.  Survival for patients with tumors confined to the muscularis propria (stage I) or with extensive metastatic disease (stage IV) is predictable; however, tumor biological behavior after surgical treatment for stage II and III tumors is not well established.  Approximately thirty percent of patients with stage II colon cancer will relapse and die of the disease. More significantly, up to sixty-five percent of patients with stage III tumors will succumb of colon cancer.  The use of adjuvant therapies in patients with stage II lesions is not exempt of controversy.  These therapies have side-effects, and should be given to those patients who would benefit from them.7  Prognostic markers that complement standard clinical and pathologic staging further stratify stage II patients into high-risk and low-risk groups of relapse after surgery, and better guide adjuvant therapy.  Biologic factors that account for the different outcome among patients presenting with the same clinical stage are still poorly understood, but several studies have revealed the prognostic significance of 18q allelic loss in stage II colorectal carcinomas. Loss of this region, from which several tumor suppressor genes have been cloned and characterized, such as DCC, SMAD2 and SMAD4, can be detected in about 60-70% of colorectal cancer cases.8,11   Within the group of stage II tumors, 18q21-22 loss correlates significantly with appearance of recurrent disease and poor survival. Thus, examination of 18q21-22 LOH in primary stage II colorectal cancers can assist in identifying patients prone to recurrence and candidates for further treatment.

The aim of this study was to analyze chromosome 18q LOH by means of a new multiplex PCR assay and GeneScan analysis after capillary electrophoresis developed in our laboratory.  After the reproducibility of the multiplex assay using DNA obtained from paraffin-embedded tissues had been evaluated, the new multiplex assay was used to assess 18q LOH in 61 stage II colorectal carcinomas and to determine the contribution of each marker to LOH detection.   Finally, also as a part of our experimental design, the degree of discordance between different observers when analyzing gel electrophoresis results was determined by re-evaluating retrospective LOH results from a cohort of 207 colorectal carcinomas.

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J Mol Diagn 2005; 7: 478-485