Anne-France Dekairelle

Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction

Abstract Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3′-untranslated region of factor II) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman® assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; −/−), heterozygous (+/−) or homozygous (+/+) variants (n=362) were selected for factor V (n=115, with −/−, 40; +/−, 40; +/+, 35), factor II (n=122, with −/−, 60; +/−, 60; +/+, 2), and MTHFR (n=120, with −/−, 40; +/−, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting.

Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater solid tissue storage methods.

Abstract Background: The aim of the present study was to compare RNALater® with the usual method of liquid nitrogen snap freezing as a surrogate mRNA preservation method for functional analysis of separated alleles in yeast (FASAY). Methods: A total of 81 patients with transitional cell carcinoma of the bladder underwent fresh tissue biopsies directly transferred into RNALater® and stored at room temperature or at 4°C for increasing time intervals until RNA processing. From this cohort of patients, 53 paired snap-frozen and RNALater® preservative-suspended tissues were obtained. Samples immediately frozen in liquid nitrogen were further stored at −80°C. Results: Of the 81 RNALater® samples, 14 were not processed for FASAY because of RNA degradation. Of the remaining 67 samples, 15 (22%) were FASAY-positive. Identical FASAY results were found for 50 of 53 (94.4%) paired samples and the percentage of red yeast colonies was highly correlated (Cohens κ<0.82; p<0.00001). A single p53 missense mutation was found in each of the three discordant positive FASAY and was identical in each concordant positive sample (10/53). Storing samples in RNALater® at room temperature for 3 days and at 4°C for less than 1 month provided high-quality mRNA suitable for FASAY. Conclusions: Our results demonstrate that RNALater® is a suitable and flexible alternative to snap freezing for FASAY analysis. Clin Chem Lab Med 2007;45:1283–7.

Assessment of the transcriptional activity of p53 improves the prediction of recurrence in superficial transitional cell carcinoma of the bladder.

Purpose: To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas. Experimental Design: p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters. TP53 and p21 gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis. Results: A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (∼40%) or underestimated (∼10%) the true incidence of p53 transcriptional abnormalities, especially in Ta-T1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) nor TP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of Ta-T1 grade 3 tumors. Conclusions: FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry.

Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5–8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Assessment of p53 functional activity in tumor cells and histologically normal mucosa from patients with head and neck squamous cell carcinoma.

The purpose of this study was to investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration in head and neck squamous cell carcinoma (HNSCC).

Does genotyping of risk-associated single nucleotide polymorphisms improve patient selection for prostate biopsy when combined with a prostate cancer risk calculator?

Genome‐wide association studies have identified single nucleotide polymorphisms (SNPs) associated with higher risk of prostate cancer (PCa). This study aimed to evaluate whether published SNPs improve the performance of a clinical risk‐calculator in predicting prostate biopsy result.

Regorafenib induced severe toxic hepatitis: characterization and discussion.

Regorafenib is the first small‐molecule multikinase inhibitor which showed survival benefits in pretreated metastatic colorectal cancer (mCRC) patients. Besides classical adverse events of this drug class, hepatotoxicity has been described as a frequent side effect.

Comparative pharmacogenetic analysis of risk polymorphisms in Caucasian and Vietnamese children with acute lymphoblastic leukemia: prediction of therapeutic outcome?

AIMS Acute lymphoblastic leukemia (ALL) is the most common of all paediatric cancers. Aside from predisposing to ALL, polymorphisms could also be associated with poor outcome. Indeed, genetic variations involved in drug metabolism could, at least partially, be responsible for heterogeneous responses to standardized leukemia treatments, hence requiring more personalized therapy. The aims of this study were to (a) to determine the prevalence of seven common genetic polymorphisms including those that affect the folate and/or thiopurine metabolic pathways, i.e. cyclin D1 (CCND1-G870A), γ-glutamyl hydrolase (GGH-C452T), methylenetetrahydrofolate reductase (MTHFR-C677T and MTHFR-A1298C), thymidylate synthase promoter (TYMS-TSER), thiopurine methyltransferase (TPMT*3A and TPMT*3C) and inosine triphosphate pyrophosphatase (ITPA-C94A), in Caucasian (n = 94, age < 20) and Vietnamese (n = 141, age < 16 years) childhood ALL and (b) to assess the impact of a multilocus genetic risk score (MGRS) on relapse-free survival (RFS) using a Cox proportional-hazards regression model. RESULTS The prevalence of MTHFR-677TT genotype was significantly higher in Caucasians (P = 0.008), in contrast to the prevalence of TYMS-TSER*3R/3R and ITPA-94AA/AC genotypes which were significantly higher in Vietnamese (P < 0.001 and P = 0.02, respectively). Compared with children with a low MGRS (≤ 3), those with a high MGRS (≥ 4) were 2.06 (95% CI = 1.01, 4.22; P = 0.04) times more likely to relapse. Adding MGRS into a multivariate Cox regression model with race/ethnicity and four clinical variables improved the predictive accuracy of the model (AUC from 0.682 to 0.709 at 24 months). CONCLUSION Including MGRS into a clinical model improved the predictive accuracy of short and medium term prognosis, hence confirming the association between well determined pharmacogenotypes and outcome of paediatric ALL. Whether variants on other genes associated with folate metabolism can substantially improve the predictive value of current MGRS is not known but deserves further evaluation.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.

Abstract Background: This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies. Methods: A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast). Results: Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples. Conclusions: Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization. Clin Chem Lab Med 2009;47:294–301.

A bias in quantitative RT-PCR limit of detection is induced by the use of cancer cell lines in the molecular detection of circulating tumor cells.

9 species of caddisflies (Trichoptera) are reported for the first time in Saxony, (Germany): Rhyacophila philopotamoides, Synagapetus iridipennis, Hydroptila vectis, Orthotrichia tragetti, Ithytrichia lamellaris, Plectrocnemia brevis, Hydropsyche tennis, Phacopteryx brevipennis, Ylodes simulans. Additional 12 species were rediscovered in reference to Robert (2001): Hydroptila forcipata, Hydropsyche fulvipes, Oligostomis reticulata, Brachycentrus maculatus, Ironoquia dubia, Drusus chrysotus, Grammotaulius nitidus, Limnephilus subcentralis, Halesus tesselatus, Ceraclea annulicornis, Ceraclea nigronervosa, Oecetis testacea.