Influence of 17q gain and promoter polymorphisms on mRNA expression of somatostatin receptor type 2 in neuroblastoma.

BACKGROUND: Neuroblastoma, the most frequent solid extracranial tumor in children, is characterized by a wide spectrum of clinical behaviours. We previously reported that high expression of somatostatin receptor type-2 (sst2) mRNA is associated to increased overall and event free survival. Several genetic abnormalities are detected in neuroblastomas, frequently involving balanced and/or unbalanced gain on the long arm on chromosome 17, the same region containing sst2 gene. METHODS: In this study we detected balanced and/or unbalanced 17q gain in 50 neuroblastomas. Since two polymorphisms in sst2 promoter (-57 C>G and -83 A>G) were previously described as responsible for an in vitro reduction of sst2 mRNA expression, promoter sequencing was also performed in the same samples. The results were compared to sst2 mRNA expression, measured by real-time RT-PCR. RESULTS: The frequency of 17q gain (14/50 neuroblastomas) was significantly associated to sst2 mRNA over-expression (Fischer's exact test: p=0.0012). The sst2 expression was significant higher both in balance and unbalance 17q amplifications (ANOVA: p=0.04). Conversely, we found a reduction of sst2 mRNA in neuroblastomas with -57 C>G promoter polymorphism (ANOVA: p=0.03). CONCLUSION: We highlighted that 17q gain and promoter polymorphisms can play a role into the regulation of sst2 expression in neuroblastomas.

Prognostic value of somatostatin receptor subtype 2 expression in colorectal cancer.

The clinical relevance of the somatostatin receptor subtype 2 (sst2) is well defined in neuroendocrine tumors but it is still a matter of debate whether its expression may have a role also in other tumors not arising from the neuroectoderm. We investigated the prognostic value of the expression levels of sst2 mRNA in a consistent group of patients affected by colorectal cancer. Survival analysis of cancer-related death showed that patients with a high sst2 mRNA expression had an unfavourable outcome (p=0.037) and a significantly shorter disease-free survival (p=0.008). Surprisingly, our findings suggest that sst2 gene overexpression is a feature of colorectal tumors that have a negative outlook; in addition, it may allow additional insight into conventional therapeutic approaches for more aggressive tumors, whose prognosis needs to be improved.

Tankyrase, a positive regulator of telomere elongation, is over expressed in human breast cancer.

Tankyrase promotes telomere elongation by interaction with the telomeric protein binding factor TRF1, a negative regulator of telomere extension. We measured tankyrase mRNA by real-time RT-PCR in 66 breast cancers and in paired normal tissues. Results were compared with hTERT mRNA expression. The levels of tankyrase in breast cancers were significantly higher in comparison to normal tissues (P<0.0001) and significantly related to the status of progesterone receptors. No relationship was found between tankyrase and hTERT mRNA expression in breast cancers. According to our results, tankyrase expression appeared up regulated in breast cancers.

An Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Man probes.

Quantitative real-time PCR techniques are increasingly being used for the measurement of nucleic acids in research applications as well as in the clinical laboratory. It is therefore important that external quality control programs (EQA) are implemented for the evaluation of the analytical aspects common to molecular tests based on quantitative PCR. The aim of this study was the development of an Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Mantrade mark probes to compare the analytical performance of 42 laboratories. Participants were provided with a set of reagents (cDNA for reference curve preparation, primers-probe mix and three unknown samples) and requested to perform a conventional assay using the master mix employed in their laboratories. The quantitative results in unknown samples were analyzed. The results of our study showed clear heterogeneity in performance. Two of the 42 laboratories provided results indicating contamination during the experiment, whereas six did not provide values for at least one of the six standard points. Only 12 laboratories gave results that were both precise and accurate for all the samples tested. Regarding imprecision, 17 laboratories appeared to deviate in at least one result, whereas inaccuracy showed an inverse dose-dependent trend. Finally, 12 laboratories were not able to measure the sample with the lowest concentration. Ten of these laboratories were equipped with the same instruments. The results of this first round of analytical EQA of real-time PCR-based methods seem to indicate high variability among laboratories carrying out the same experimental protocol. These findings could have implications for any assay based on this type of technique. This survey demonstrates the importance of experimental EQAs of methodological proficiency testing. Our approach has proved useful for comparing the analytical aspects shared by all diagnostic laboratories applying quantitative assays for the measurement of nucleic acids based on the use of Taq-Mantrade mark probes and real-time platforms.

External quality assurance program for PCR amplification of genomic DNA: an Italian experience.

BACKGROUND: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable. METHODS: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation. RESULTS: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53-63 points; n = 13), sufficient (42-52 points; n = 15), poor (31-41 points; n = 3), and not acceptable (<31 points; n = 4). CONCLUSIONS: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.