[HER-2 oncogene amplification assessment in invasive breast cancer by dual-color in situ hybridization (dc-CISH): a comparative study with fluorescent in situ hybridization (FISH)]. / Détermination de l'amplification de l'oncogène HER-2 dans le cancer du sein invasif par hybridation chromogénique in situ double couleur (dc-CISH) : étude comparative avec l'hybridation fluorescente in situ (FISH).

OBJECTIVES: The amplification of the gene encoding for the human epidermal growth factor receptor 2 (HER-2 oncogene), located on chromosome 17 (17q21-q22), or the overexpression of this receptor have prognostic and therapeutic implications in invasive breast cancer. An evaluation of the HER-2 status by immunohistochemistry (IHC) is performed on all invasive breast cancer cases. Fluorescent in situ hybridization (FISH) is considered as the gold standard for the detection of HER-2 gene amplification for IHC equivocal cases (score 2+). A more recent in situ hybridization technique, the dual-color chromogenic in situ hybridization (dc-CISH), has been proposed as an alternative to FISH. The aim of this study was to measure the correlation between dc-CISH and FISH for HER-2 oncogene amplification assessment in invasive breast cancer. METHODS AND RESULTS: We built four tissue micro-array (TMA) blocs with 100 breast invasive cancer cases that had been previously tested by IHC for HER-2 detection: 10 score 0 cases, 10 score 3+cases, 39 score 1+and 41 score 2+cases. Both FISH and dc-CISH techniques were applied on all TMA cases as well as on two additional slides serving as controls. Interpretation of dc-CISH was carried out by a pathologist using an optical microscope. For FISH, the interpretation was done by a professional from the medical genetics department using a fluorescent microscope linked to a computer system for image capturing and analysis. The interpretation of the HER-2/CEN-17 ratio for both tests was in accordance with the values of the updated recommendations from the Canadian National Consensus Meeting on HER-2/neu testing in breast cancer and from the ASCO/CAP. Among the 100 cases initially included in the study, eight were excluded from the analysis due to sampling or technical flaws. From the 92 remaining cases, we obtained a concordance of 97.8% (90/92 cases) between the two techniques (Kappa coefficient 0.97, 95% confidence interval). The correlation coefficient (rho) between ratios was estimated at 0.57. CONCLUSION: This study shows a strong concordance between FISH and dc-CISH techniques and indicates that dc-CISH is a good alternative method for HER-2 gene amplification assessment in breast cancer.

DNAc: A clustering method for identifying kinship relations between DNA profiles using a novel similarity measure.

After decades of refinement, DNA testing methods have become essential tools in forensic sciences. They are essentially based on likelihood ratio test principle, which is utilized specifically, by using as prior knowledge the allele frequencies in the population, to confirm or refute a given kinship hypothesis made on two genotypes. This makes these methods ill suited when allele frequencies or kinship hypotheses are unavailable. In this paper, we introduce DNAc, a new clustering methodology for DNA testing based on a new similarity measure that allows an accurate retrieval of the degree of relatedness among two or more genotypes, without relying on kinship hypotheses or allele frequencies in the population. We used DNAc in analyzing microsatellite DNA sequences distributed among 12 genotypes from normal individuals from two distinct families. The results show that DNAc accurately determines kinship among genotypes and further gathers them in the appropriate kinship groups.

[Dominant negative activity of mutated p53 proteins]. / Activité dominante négative des protéines p53 mutées.

Tumor suppressor gene inactivation as proposed by the Knudson model implies a sequential inactivation of two alleles of a gene. For example, the first allele is inactivated by a missense mutation, and the second one is inactivated by a deletion or insertion. The alteration of the p53 tumor suppressor gene is far to correspond only to this model. In the great majority of cancers, the mutated allele of p53 coexists with the normal allele. It is well known that the transcriptional activity is one of the most important functions of p53. The p53 protein is active as a tetramer (this complex activates the expression of targeted genes by binding to its consensus DNA sequence called the p53 response element). Experimental evidence shows that wild-type p53 interacts with mutant proteins to form heterotetramers. In association with wild-type proteins, mutant proteins drive the wild-type subunits into a mutant conformation. This association leads to a loss of trans-activating function. The capacity of mutant subunits to form heterotetramers with wild-type subunits and to commit them into a mutant conformation is called << dominant negative effect >>. Many p53 mutant proteins possess this dominant negative activity. Recently, several factors, which are implicated in the control of the dominant negative activity of p53 mutants, have been identified. The elucidation of these complex molecular functions, which are implicated in the dominant negative activity of the p53 mutated protein represents an important aspect in the comprehension of the biological mechanisms involved in carcinogenesis.

[From the conception of the PRINS to its coronation]. / De la conception du PRINS à son couronnement.

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).

Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique.

Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.

Genetic cooperation between the Werner syndrome protein and poly(ADP-ribose) polymerase-1 in preventing chromatid breaks, complex chromosomal rearrangements, and cancer in mice.

Werner syndrome is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for Werner syndrome encodes a DNA helicase/exonuclease protein. Participation in a replication complex is among the several functions postulated for the WRN protein. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme, which is known to bind to DNA strand breaks, is also associated with the DNA replication complex. To determine whether Wrn and PARP-1 enzymes act in concert during cell growth, mice with a mutation in the helicase domain of the Wrn gene (Wrn(Deltahel/Deltahel) mice) were crossed to PARP-1-null mice. Both Wrn(Deltahel/Deltahel) and PARP-1-null/Wrn(Deltahel/Deltahel) cohorts developed more neoplasms than wild-type animals. The tumor spectrum was the same between PARP-1-null/Wrn(Deltahel/Deltahel) mice and Wrn mutants. However, PARP-1-null/Wrn(Deltahel/Deltahel) mice developed neoplasms at a younger age. Mouse embryonic fibroblasts derived from such PARP-1-null/Wrn(Deltahel/Deltahel) mice stop dividing abruptly unlike Wrn(Deltahel/Deltahel) or PARP-1-null cells. PARP-1-null/Wrn(Deltahel/Deltahel) fibroblasts were distinguished by an increased frequency of chromatid breaks, complex chromosomal rearrangements, and fragmentation. Finally, experiments have indicated that the PARP-1 enzyme co-immunoprecipitates with the WRN protein in human 293 embryonic kidney cells. These results suggest that Wrn and PARP-1 enzymes may be part of a complex involved in the processing of DNA breaks.