A Novel Multiplexing, Polymerase Chain Reaction-Based Assay for the Analysis of Chromosome 18q Status in Colorectal Cancer

Abstract

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 at the long arm of chromosome 18. Amplicon detection and evaluation was accomplished by capillary electrophoresis using an ABI 310 genetic analyzer. Robustness of the assay when performed on DNA extracted from formalinfixed, paraffin-embedded tissue sections was confirmed by analyzing its repeatability and reproducibility. Allelic loss was assessed in 61 stage II colorectal tumors and was detected in 58% (31 of 53) of tumors not showing instability. As part of the study, results of 207 previous polymerase chain reaction/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 between observers were detected in 16.4% of determinations. In conclusion, we have developed a robust and reliable assay for multiplexed loss of heterozygosity determination that improves assessment of chromosome 18q allelic loss in colorectal tumors 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. 1,2 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 30% of patients with stage II colon cancer will relapse and die of the disease. [3][4][5][6] More significantly, up to 65% of patients with stage III tumors will succumb to colon cancer. The use of adjuvant therapies in patients with stage II lesions is not exempt of controversy. These therapies have sideeffects, and should be given to those patients who would benefit from them. 7 Prognostic markers that complement standard clinical and pathological staging further stratify stage II patients into high-risk and lowrisk groups of relapse after surgery, and better guide adjuvant therapy. Biological 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. 3,5,6,8 -10 Loss of this region, from which several tumor suppressor genes have been c...

A microsatellite based multiplex PCR method for the detection of chromosomal instability in gastric cancer

“…Microsatellite analysis allows the detection of AI by comparing the intensities of the paternal and maternal alleles between normal and tumour tissues of heterozygous patients and then by calculating a quotient of the respective allele ratios between normal and tumour tissue. A standardized cut-off value for the determination of AI is not clearly defined and various studies used cut-off values in the range below 0.5–0.6 or above 1.5–2.0, which correspond to a reduction in the intensity of one of the alleles of at least 40–50% 8 , 9 , 18 – 21 . For our assay, we experimentally determined individual cut-off values for each microsatellite marker.…”

DNA Diagnostics by Capillary Electrophoresis

“…In several publications, the variability in STR identified by CE was demonstrated as being a significant diagnostic marker of cancer. − Mathies' group developed a sensitive two-color labeling method with ET primers for the high-throughput STR-based screening of bladder cancer patients . Detection of a size difference in PCR amplicons from normal and tumor DNA is an indication of expansion or deletion in the microsatellites in the five different loci used as markers.…”

Prognostic Significance of 18q LOH in Sporadic Colorectal Carcinoma

“…The frequency 18q LOH in our series overlaps with the majority of that reported in similar studies. 23 However, looking at single marker level, we found that the loss of D18S61 was paradoxically associated with more favorable prognosis in Stage II and III colon carcinoma. This evidence was especially significant for Stage II and untreated patients (Figs.…”