No abstract

Objective: Human chorionic gonadotropin (hCG) is a marker of trophoblastic tumors, and the serum concentration of the free β-subunit (hCGβ) is an independent prognostic marker in several nontrophoblastic cancers. hCGβ is encoded by six genes, of which the type II genes (hCGβ 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGβ 6/7) in cancer. Method: We developed a novel quantitative RT-PCR method for the quantification of the relative expression levels of the two groups of hCGβ genes and analyzed 28 bladder tumors and 15 urine samples. Results: We found a higher relative expression level of type II genes in malignant compared with benign urothelia (p = 0.016) and in exfoliated urinary cells from cancer patients compared with those from benign controls (p = 0.026). The expression level was increasing with higher stage (p = 0.014) and grade (p = 0.001) and tended to be higher in relapsing tumors (p = 0.059). Conclusion: The increased hCGβ concentrations in body fluids of patients with aggressive bladder cancer may be due to overexpression of type II genes. Quantification of the relative mRNA expression levels of the hCGβ type I and II genes in urine cells should be further studied as a potential noninvasive tool for the diagnosis and follow-up of bladder cancer.

The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue.

Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.

In general, the best new markers give higher sensitivity than urinary cytology, but specificity is usually lower. By using new markers, the intervals between follow-up cystoscopies can be increased and the detection of relapse can be improved. But to date no non-invasive biomarker has proven to be sensitive and specific enough available to replace cystoscopy, neither in the diagnosis nor in the follow-up of bladder cancer. However, new marker combinations and algorithms for risk assessment hold promise for the future.

Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

Our results indicate that sensitive nested RT-PCR method detects PSM mRNA in the leukocyte fraction of normal blood. This "background" expression is probably caused by a leaky promoter of PSM. We conclude that it is necessary to develop quantitative RT-PCR assays to differentiate PSM mRNA expression derived from circulating cancer cells from background expression in blood cells.