Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

Ribozyme-mediated inhibition of PKCalpha sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis.

BACKGROUND: Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Calpha (PKCalpha) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCalpha by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS: An active ribozyme (PKCalphaRZ) targeting codon 4 in human PKCalpha mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCalphamutRZ) was also made by deleting G(12) from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCalpha expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS: Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50%) lower PKCalpha protein level than parental DU145 cells, whereas no reduction in PKCalpha expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS: Results of the study highlight the importance of PKCalpha in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.