Type-2 somatostatin receptor mRNA levels in breast and colon cancer determined by a quantitative RT-PCR assay based on dual label fluorogenic probe and the TaqMan™ technology

Abstract

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neuroblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measurement and its applicability to large groups of patients, we present here an original ‘real-time’ quantitative RT-PCR method, based on a dual-labeled fluorogenic probe and the TaqMan™ technology. By this method, we have measured sst2 mRNA expression in 24 breast cancer samples and 26 colon carcinomas as well as on the corresponding non-adjacent non-neoplastic tissue of the same patients. The proposed method has a dynamic range of 4×10 4 to 4×10 8 molecules of sst2 mRNA. The intra-assay precision of the test, evaluated as signal detection variability, was 2.4%. Accuracy, evaluated by the addition of standard RNA to unknown samples, provided a mean recovery of 98±2%. A significant correlation has been observed in a study performed in 24 neuroblastoma samples measured both with the proposed method and with a competitive RT-PCR assay ( r=0.913, p<0.001). In our preliminary clinical study, no significant differences were observed in sst2 mRNA levels between normal and tumor specimens in both colorectal (normal tissue 5.1×10 7±2.0×10 7 molecules/μg total RNA, cancer tissue 9.7×10 7±4.2×10 7) and breast tumors (normal tissue 5.5×10 8±2.0×10 8, cancer tissue 4.4×10 8±3,7×10 8). However, in colorectal cancer, sst2 mRNA values of subjects with high circulating carcinoembryonic antigen (CEA) levels (>5 ng/ml) were statistically lower (2.3×10 7±6.2×10 6 molecules/μg total RNA; p<0.05) than those of subjects with low CEA concentration (1.4×10 8±6.7×10 7). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T ratio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T ratio of negative (below 10 fmol/mg protein) and positive estrogen receptor tumors ( p<0.05). Analogous results were found selecting breast tumors on the basis of the progesterone receptor status ( p<0.05). The proposed method is accurate, precise, sensitive and less labor-intensive than the competitive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it seems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens.