Jean-Luc Gala

Clin Chem Lab Med 2007;45:1283–7.

Specific gene rearrangements are used for minimal residual disease (MRD) assessment, but are frequently lacking in leukaemias. In these cases, the quantification of PRAME (preferentially expressed antigen of melanoma) transcripts could be useful. PRAME transcripts were quantified by real-time RT-PCR in normal and leukaemic samples, and the results were compared with those of conventional RT-PCR. Basal expression of PRAME was determined in 25 blood samples and 25 bone marrow samples from healthy donors, as well as in 12 haematological cell lines (Jurkat, K562, HL60, DOHH2, IM9, Daudi, CEM, KG1, DG75, 8226, U937, Raji). In paediatric acute myeloid leukaemia (AML) (n=22) and acute lymphoblastic leukaemia (ALL) (n=17), and in adult AML (n=20), abnormal PRAME expression was found in 41%, 35% and 40% of cases, respectively. To assess the sensitivity of PRAME for monitoring MRD, PRAME-positive t(8;21) AML samples with detectable AML1/ETO expression by conventional RT-PCR (n=17) were assessed for quantitative expression of AML1/ETO and PRAME. The expression of these genes was closely correlated. To confirm that PRAME expression was correlated with clinical data, the expression of PRAME was also sequentially followed in patients (n=13) from onset to cytological remission or relapse. The cytological and molecular data were highly correlated in all patients. Our data confirm that PRAME quantification by real-time RT-PCR appears suitable for monitoring MRD in PRAME-positive leukaemia.

Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic ofMycobacterium bovisinfection due to gross and microscopic similarities with lesions caused byMycobacterium aviumsubsp.paratuberculosisorM. aviumsubsp.avium. The aim of this study was to improve molecular methods for the species-specific identification ofM. bovis,M. aviumsubsp.avium, andM. aviumsubsp.paratuberculosisin mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiatedM. bovisfromM. aviumsubsp.paratuberculosisandM. aviumsubsp.aviuminfections, whereas the restriction step differentiated singleM. aviumsubsp.paratuberculosisorM. aviumsubsp.aviuminfections from mixedM. aviumsubsp.paratuberculosis/M. aviumsubsp.aviuminfections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in allM. aviumsubsp.aviumstrains tested, regardless of their origin and the results of IS901PCR. In contrast, the restriction site was abrogated in allM. aviumsubsp.paratuberculosisstrains tested, independent of their origin, Mycobactin J dependency, and IS900PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to excludeM. bovisinfection and to identify single (M. aviumsubsp.paratuberculosisorM. aviumsubsp.avium) or mixed (M. aviumsubsp.paratuberculosis/M. aviumsubsp.avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition ofM. aviumsubsp.paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.

The ABCB1 (MDR1) multidrug transporter plays a key role in determining drug bioavailability. Differences in drug response exist among different ethnic groups. However, until now, no haplotype data are available in a Black African population.

Objective: Blood cultures and cultures of disc material are required to identify and treat bacterial agents responsible for septic spondylodiscitis, but these methods have limited sensitivities. We undertook this study to compare nonculture amplification‐based DNA analysis with conventional culture of disc aspirate.Methods: Nineteen patients with spondylodiscitis, including 11 with a history of spinal surgery, presented with negative blood cultures and underwent percutaneous disc or epidural abscess puncture for bacterial diagnosis. Amplification by polymerase chain reaction was performed on 16S ribosomal DNA universal target genes and femA staphylococci‐specific target genes in all patients, and on the upstream p34 mycobacterial gene in 1 patient. Species identification relied on amplicon sequencing and comparison with templates from GenBank. Amplification of the femA gene led to subsequent testing for methicillin resistance by amplification of the mecA gene. Further assessment using a staphylococci‐ and methicillin resistance–specific DNA array was performed on 3 samples.Results: Microbiologic and molecular assays identified the causative organism in 14 of 19 patients (74%) and 19 of 19 patients (100%), respectively. In culture‐positive patients, DNA‐based and microbiologic results were highly correlated. Five agents (Staphylococcus simulans, Staphylococcus sciuri, Brucella species, Actinomyces israelii, and Mycobacterium tuberculosis complex) were identified only by DNA‐based methods. In 1 sample, Corynebacterium jeikeium and coagulase‐negative Staphylococcus were both cultured, whereas DNA analysis identified only Staphylococcus hominis.Conclusion: DNA‐based methods are highly sensitive and specific. They can usefully complement standard microbiologic methods for identifying the cause of infectious spondylodiscitis and contribute to species‐specific therapeutic orientation in patients with negative blood and disc aspirate cultures.

Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (

Aims  To investigate the distribution of cytochrome P450 2C9 (CYP2C9) and 2C19 (CYP2C19) genotype frequencies in the Beninese and Belgian Caucasian populations.Methods  Beninese (n = 111) and Belgian (n = 121) were genotyped for CYP2C9*2, *3, *4, *5, and *11 as well as for CYP2C19*2 and*3.Results  The distribution of alleles was: CYP2C9*1: 95.5 vs. 82.2% (P < 0.001); CYP2C9*2: 0 vs. 10% (P < 0.001); CYP2C9*3: 0 vs. 7.4% (P < 0.01); CYP2C9*4: both 0%; CYP2C9*5: 1.8 vs. 0% (P = 0.05); and CYP2C9*11: 2.7 vs. 0.4% (P < 0.05). The frequencies of the CYP2C19*2 allele were 13 vs. 9.1%, respectively. CYP2C19*3 was not detected in either population. The 95% confidence intervals for the differences of frequencies of CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, CYP2C9*11, CYP2C19*1, CYP2C19*2 and CYP2C19*3 between Belgian and Beninese were 7%, 19%; − 14%, − 6%; − 11%, − 4%; − 1%, 1%; 0%, 4%; 0%, 5%; − 10%, 2%; − 2%, 10%; − 1%; respectively. The distributions of CYP2C9 genotypes in the Beninese and Belgian individuals were: CYP2C9*1/*1: 91 vs. 67% (P < 0.00001); CYP2C9*1/*2: 0 vs. 18.2% (P < 0.0001); CYP2C9*1/*3: 0 vs. 11.6% (P < 0.001); CYP2C9*1/*5: 3.6 vs. 0% (P = 0.05); CYP2C9*1/*11: 5.4 vs. 0.8% (P = 0.05); CYP2C9*2/*3: 0 vs. 1.6% (NS); CYP2C9*3/*3: 0 vs. 0.8% (NS). The distributions of CYP2C19 genotypes between these ethnic groups were: CYP2C19*1/*1: 73.9 vs. 83.5% (NS); CYP2C19*1/*2: 26.1 vs. 14.9% (P < 0.05); CYP2C9*2/*2: 0 vs. 1.6% (NS).Conclusions  Differences of allele frequencies between Beninese and Belgian populations were statistically significant for CYP2C9*2, *3, *5 and *11, but not for CYP2C9*4 or for CYP2C19*2 and *3.

BACKGROUND To predict poor outcome in patients with a biochemical recurrence (rising PSA) after radical prostatectomy (RP), urologists rely primarily on Gleason score, PSA doubling time, and time from surgery to biochemical (i.e., PSA) recurrence. In the present study, we assess the value of RT‐PCR detection circulating prostate cells in blood of patients with a rising PSA. METHODS RNA from blood samples was obtained from 55 patients with a rising PSA and from 45 patients without evidence of biochemical failure (PSA < 0.1 ng/ml). Both groups were matched for age, Gleason score, pT stage, and interval between radical prostatectomy and PCR testing. RESULTS PSA positive cells were detected in 1/45 (2%) patients without a PSA recurrence and 19/55 (34%) patients with a PSA recurrence. In the rising PSA group, mean PSA doubling time was significantly shorter in patients with positive RT‐PCR (5 months) than in patients with negative RT‐PCR (16 months; P = 0.001). An earlier onset of recurrence was also detected in patients with a positive RT‐PCR (31 months for positive RT‐PCR vs. 50 months for negative RT‐PCR) but this result did not achieve statistical significance (P = 0.102). Salvage radiation therapy was administered in 15 patients. Three of the five patients with a positive RT‐PCR progressed during radiotherapy whereas 7 of the 10 patients with a negative RT‐PCR obtained a complete response and none have progressed. CONCLUSIONS These preliminary results suggest that RT‐PCR detection of prostate cells in blood of patients after RP correlates with rapidly progressing biochemical failure after RP. Prostate 56: 163–170, 2003.