Highly specific detection of prostate-specific antigen-positive cells in the blood of patients with prostate cancer or benign prostatic hyperplasia, using a real-time reverse-transcription-polymerase chain reaction method with improved sensitivity.

OBJECTIVE: To assess the presence of circulating prostate-specific antigen (PSA)-expressing cells in patients with prostate cancer or benign prostatic hyperplasia (BPH), and to determine their diagnostic usefulness using a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) method. PATIENTS, SUBJECTS AND METHODS: Venous blood samples were obtained from 175 patients with prostate cancer (12 metastatic and 163 not metastatic), 49 with BPH, and 50 healthy volunteers. To improve the specificity and sensitivity of the qRT-PCR three innovative features were combined; a primer overlapping two adjacent exons to inhibit nonspecific amplification; a no-end-point first round amplification to increase the sensitivity; and a target-specific primer for the RT phase to increase the specificity. RESULTS: The sensitivity of the method was 1 cell/mL of blood and the interassay coefficient of variation was 10.5%. None of the healthy subjects tested positively, while 9% of those with prostatic cancer and 14% with BPH had PSA-positive cells in the blood. There was a positive association between a positive test and the National Comprehensive Cancer Network classification in the patients with newly diagnosed prostate cancer (P = 0.022). There were no additional statistically significant associations. CONCLUSION: Our results strongly indicate that although there were no false-positive results and the sensitivity of the method was increased to maximal levels, a low frequency of positive results in patients with prostatic cancer and a high frequency of positive results in those with BPH seems to discourage the use of PSA-positive circulating cells in the search for a clinical diagnosis of prostate cancer.

Specific detection of cytokeratin 20-positive cells in blood of colorectal and breast cancer patients by a high sensitivity real-time reverse transcriptase-polymerase chain reaction method.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.

Identification of Krit1B: a novel alternative splicing isoform of cerebral cavernous malformation gene-1.

Cerebral cavernous malformations (CCM) are vascular malformations, mostly located in the central nervous system, which occur in 0.1-0.5% of the population. They are characterized by abnormally enlarged and often leaking capillary cavities without intervening neural parenchyma. Some are clinically silent, whereas others cause seizures, intracerebral haemorrhage or focal neurological deficits. These vascular malformations can arise sporadically or may be inherited as an autosomal dominant condition with incomplete penetrance. At least 45% of families affected with cerebral cavernous malformations harbour a mutation in Krev interaction trapped-1 (Krit1) gene (cerebral cavernous malformation gene-1, CCM1). This gene contains 16 coding exons which encode a 736-amino acid protein containing three ankyrin repeats and a FERM domain. Neither the CCM1 pathogenetic mechanisms nor the function of the Krit1 protein are understood so far, although several hypotheses have been inferred from the predicted consequences of Krit1 mutations as well as from the identification of Krit1 as a binding partner of Rap1A, ICAP1A and microtubules. Here, we report the identification of Krit1B, a novel Krit1 isoform characterized by the alternative splicing of the 15th coding exon. We show that the Krit1B splice isoform is widely expressed in mouse cell lines and tissues, whereas its expression is highly restricted in human. In addition, we developed a real-time PCR strategy to accurately quantify the relative ratio of the two Krit1 alternative transcripts in different tissues, demonstrating a Krit1B/Krit1A ratio up to 20% in mouse thymus, but significantly lower ratios in other tissues. Bioinformatic analysis using exon/gene-prediction, comparative alignment and structure analysis programs supported the existence of Krit1 alternative transcripts lacking the 15th coding exon and showed that the splicing out of this exon occurs outside of potentially important Krit1 structural domains but in a region required for association with Rap1A, suggesting a subtle, yet important effect on the protein function. Our results indicate that maintenance of a proper ratio between Krit1A and Krit1B could be functionally relevant and suggest that the novel Krit1B isoform might expand our understanding of the role of Krit1 in CCM1 pathogenesis.

Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway.

The injury of podocytes is associated with alterations of the glomerular size-selective barrier to proteins. In this study, oxidized LDL (oxLDL) but not native LDL induced apoptosis in human cultured podocytes and reduced Akt activity and P-Akt/Akt ratio. Moreover, oxLDL-induced redistribution and loss of nephrin, an adhesion molecule specific for the glomerular slit diaphragm. Nephrin reduction was preceded by inhibition of nephrin tyrosine phosphorylation and of its association with p85 phosphatidylinositol 3-kinase (PI3K). Moreover, three different statins, mevastatin, pravastatin, and simvastatin, inhibited in a dose-dependent manner apoptosis and loss of nephrin induced by oxLDL by stimulating Akt activity. In addition, simvastatin significantly increased the expression of nephrin protein and mRNA by podocytes. The protective effects of statins were blocked by treatment of podocytes with two unrelated pharmacologic inhibitors of PI3K, LY294002 and wortmannin, suggesting a role for PI3K, and by mevalonate, indicating dependency on HMG-CoA reductase activity. Statins directly stimulated Akt phosphorylation ad activity. Finally, oxLDL induced a retraction of cultured podocytes and an increase in the albumin diffusion across their monolayer that was inhibited by treatment with statins. In conclusion, statins reduced the oxLDL-induced apoptosis and loss of nephrin in glomerular podocytes. The statin-induced Akt activation may protect from the loss of nephrin by an inhibition of its redistribution and shedding and by a stimulation of its synthesis. These data provide a rationale for the anti-proteinuric effect of statins.