CNV analysis in a diagnostic setting using target panel.

OBJECTIVE: Copy-number variation (CNV) is an important source of genetic diversity in humans. It can cause Mendelian or sporadic traits or be associated with complex diseases by various molecular mechanisms, including gene dosage, gene disruption, gene fusion and position effects. In clinical diagnostics, it is therefore fundamental to be able to identify such variations. The preferred techniques for CNV detection are MLPA, aCGH and qPCR, which have proven to be valuable, and they are complex, costly and require prior knowledge of the region to analyze. CNV calling from NGS data still suffers from data variability. Coverage can vary greatly from one region of the genome to another, depending on many factors like complexity, GC content, repeated regions and many others. In this paper, we describe how we developed a method for CNV detection. MATERIALS AND METHODS: Our method exploits CoNVaDING to detect single- and multiple-exon CNVs in targeted NGS data. RESULTS: We demonstrated that our CNV analysis has 100% specificity and 99.998% sensitivity. We also show how we evaluated the performance of this method based on internal analysis. CONCLUSIONS: The results indicate that the method can be used to screen prior to standard labs technologies, thus reducing the number of analyses, as well as costs, and increasing test conclusiveness.

Characterization of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in prostate cancer cell lines.

Stromal expression of some matrix metalloproteinases (MMPs) has been associated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaP-derived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer.

Beta-human chorionic gonadotropin in semen: a marker for early detection of prostate cancer?

BACKGROUND AND PURPOSE: The beta subunit of human chorionic gonadotropin (beta-hCG) has been detected in prostate cancer by immunologic and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Recently, prostate cells have been detected in human ejaculate. This study aimed to determine if beta-hCG could be detected by RT-PCR from prostatic mRNA isolated from semen, thus providing a noninvasive procedure for prostate cancer detection. RESULTS: Expression of beta-hCG in prostate cancer was confirmed by immunohistochemistry methods. The protein was associated with low-grade disease: Gleason Score 4 through 7 (N = 26; 69%) but not high-grade (Gleason 8 or 9) or metastatic (lymph node) disease (N = 12; 8%). Normal prostate tissue was negative for beta-hCG (N = 14). The beta-hCG RT-PCR was performed on RNA extracted from seven human prostate cancer cell lines, which showed variable expression of beta-hCG mRNA. Semen was collected from patients suspected of having carcinoma of the prostate (N = 94) and from volunteers who were under the age of 30 years and had no family history of prostate cancer (N = 9). mRNA for beta-hCG was detected in the ejaculates of 12% of the patients with confirmed prostate cancer (N = 42) but not in any patients found to be negative for cancer (N = 52). Expression of beta-hCG mRNA was found in 22% of the control samples. CONCLUSIONS: The beta-hCG protein is expressed in low-grade prostate cancer and can be detected by RT-PCR in both prostate cancer cell lines and human ejaculate. However, the low percentage of detection in ejaculate suggests that beta-hCG in semen does not provide a useful marker for early prostate cancer detection.

In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors.

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.

The detection and isolation of protease activity associated with purified preparations of human chorionic gonadotropin.

The highly purified human CG (hCG) CR series is widely used as a reference material in immunological and biological assays. However these hormone preparations, specifically hCG (CR127), exhibit Arg-specific peptidase activity with synthetic peptide substrates. The putative serine protease-like activity associated with hCG (CR127) was almost completely inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone and to a lesser extent by N-alpha-p-tosyl-L-lysine chloromethyl ketone and was isolated after hydrophobic interaction and affinity chromatography with soybean trypsin inhibitor, which indicated the presence of exogenous protease contaminants rather than intrinsic peptidase activity. 3H-DFP labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated contaminants revealed two possible serine proteases of apparent mol wts 60,000 and 20,000. The presence of these contaminants had no apparent effect on the receptor binding capability of hCG, however the in vitro biological activity of hCG determined by maximal cAMP production, was decreased after hydrophobic interaction chromatographic purification of the hormone. These observations suggest that the serine protease-like contaminants enhance cAMP production, thereby introducing a significant source of error in biological assays that use hCG (CR127). Further purification of hCG by hydrophobic interaction and affinity chromatography is recommended before its use in bioassays or research.