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 (TM) technology

We reported previously that the expression of type 2 somatostatin receptor (sst2) was positively related to patient outcome in the childhood tumor neu roblastoma. To quantitate the expression of mRNA sst2 expression, we used a competitive RT-PCR assay. To improve the practicability of this measuremen t and its applicability to large groups of patients, we present here an ori ginal 'real-time' quantitative RT-PCR method, based on a dual-labeled fluor ogenic probe and the TaqMan(TM) technology. By this method, we have measure d 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 s ame patients. The proposed method has a dynamic range of 4 x 10(4) to 4 x 1 0(8) molecules of sst2 mRNA. The intra-assay precision of the test, evaluat ed as signal detection variability, was 2.4%. Accuracy, evaluated by the ad dition 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 clinica l study, no significant differences were observed in sst2 mRNA levels betwe en normal and tumor specimens in both colorectal (normal tissue 5.1 x 10(7) <plus/minus> 2.0 X 10(7) molecules/mug total RNA, cancer tissue 9.7 x 10(7 ) +/- 4.2 X 10(7)) and breast tumors (normal tissue 5.5 x 10(8) +/- 2.0 x 1 0(8), cancer tissue 4.4 x 10(8 +/-) 3,7 x 10(8)). However, in colorectal cancer, sst2 mRNA values of subjects with high circu lating carcinoembryonic antigen (CEA) levels (> 5 ng/ml) were statistically lower (2.3 x 10(7) +/- 6.2 x 10(6) molecules/mug total RNA; p < 0.05) than those of subjects with low CEA concentration (1.4 x 10(8) <plus/minus> 6.7 x 10(7)). Also, the sst2 mRNA ratio between normal and tumor tissue (N/T r atio) resulted significantly inversely related to CEA levels. In breast cancer, a significant difference was found between the mean N/T r atio 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 meth od is accurate, precise, sensitive and less labor-intensive than the compet itive RT-PCR assay. For a correct evaluation of sst2 mRNA expression, it se ems very important to measure the sst2 expression both in tumor and in the non-tumoral non-adjacent tumor specimens. (C) 2001 Elsevier Science B.V. Al l rights reserved.