Interlaboratory comparison of K-ras testing by real-time PCR and RFLP in colorectal cancer samples.

Activating mutations of K-ras have been described in approximately 40% of patients with colorectal cancer, and are associated with resistance to epidermal growth factor receptor-targeted antibodies, such as cetuximab and panitumumab. Cost-effective and easy methods to determine K-ras mutations are urgently needed. Samples from 31 patients were tested. In laboratory 1, a real-time polymerase chain reaction (PCR)-based technique was used. All samples (n=31) were additionally tested using a restriction fragment length polymorphism (RFLP) analysis in laboratory 2. All results were confirmed by direct sequencing. In the first run, a concordance of real-time PCR and RFLP was observed in 77.4% (24 of 31) of samples. After resampling and reevaluation, a concordance of 93.5% (30 of 31) could be achieved. One of 7 (6.5%) initial discordant cases showed a mutation using real-time PCR and no mutation using RFLP, but the mutation was confirmed by direct sequencing. Real-time PCR and RFLP can be considered as valid K-ras mutation detection techniques. However, in patient probes with lower amounts of tumor cells and wild-type K-ras, reanalysis of further tumor tissue is recommended.

T-cell receptor excision circles: a novel prognostic parameter for the outcome of transplantation in multiple myeloma patients.

This study investigated whether T-cell receptor excision circles (TRECs) are a prognostic marker for the outcome of myeloma patients undergoing a tandem autologous peripheral blood stem cell transplantation (PBSCT). Twenty-five patients were enrolled. Samples were obtained at study enrollment, after conventional therapy, between first and second transplantation and 3, 6, 12 and 24 months after the second PBSCT. TRECs were quantified using real-time polymerase chain reaction. A high variation in TREC levels was found at diagnosis (median TREC level 136/10(5) peripheral blood mononuclear cells (PBMCs); range 1-1729), suggesting individual differences in thymic output of naive T cells. Patients with more than 136 TRECs/10(5) P BMCs at diagnosis had a statistically significant better overall survival (P=0.05) and event-free survival (P=0.045), whereas low TREC levels correlated with a higher incidence of infectious complications. Median TREC values were lowest after the first PBSCT (52/10(5) PBMCs) and reached the baseline 12 months after the second transplantation. Patients with high TREC levels after the second PBSCT had a significantly higher probability of being in complete or partial remission 30 months after the second PBSCT. TREC levels were not correlated with beta2-microglobulin and C-reactive protein levels at diagnosis. These data suggest that TRECs could be a relevant prognostic factor for patients who receive high-dose chemotherapy and autologous PBSCT.

Human parvovirus Bb19 detection in asymptomatic blood donors: association with increased neopterin concentrations.

Serum neopterin concentrations were determined in 20,000 blood donations. For the 400 donations with neopterin concentrations above the 98 th percentile and another 1200 donations with neopterin concentrations in the lower range, results of human parvovirus (HPV) B19 tests were compared. Infectious specimens were identified by dot blot hybridization assay and polymerase chain reaction (PCR) that used the outer primers and detected 1 pg of HPV B19 DNA, corresponding to approximately 10(5) copies of the genome, in the specimens and by a nested PCR that detected 1-10 fg of DNA, corresponding to 10(2)-10(3) copies of the genome. Of 400 specimens with neopterin concentrations > or =12 nmol/L (98th percentile, current cutoff), 10 tested positive by dot blot hybridization assay (9 of these were confirmed by nested PCR). Among 1200 specimens with low neopterin concentrations (<12 nmol/L), no specimen containing HPV B19 DNA was detected. These findings suggest an association between elevated neopterin concentrations and HPV B19 infectivity.