Comparison of a Quantitative PCR Method with FISH for the Assessment of the Four Aneuploïdies Commonly Evaluated in CLL Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4950-4950
Author(s):  
Christian Bastard ◽  
Christophe Fruchart ◽  
Gregory Raux ◽  
Francoise Parmentier ◽  
Dominique Vaur ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Target Genes ◽  
In Situ Hybridisation ◽  
Low Frequency ◽  
Proliferation Index ◽  
Trisomy 12 ◽  
Binet Stage ◽  
Pcr Method ◽  
Conventional Cytogenetics ◽  
13Q Deletion

Abstract Four chromosomal defects associated with prognosis have been identified in CLL patients, namely deletions of the 13q13-q14, 11q22 and 17p13 regions and trisomy 12. Due to the low proliferation index of these tumors and to the fact that some defects can be cryptic, the detection of these abnormalities by conventional cytogenetics is difficult. Therefore, the resulting aneuploidies are usually evaluated by fluorescent in situ hybridisation (FISH). As probes are expensive and FISH time consuming, we aimed to compare a quantitative PCR method - quantitative PCR of short fluorescent fragments (QMPSF) - with FISH for the detection of these acquired aneuploidies in a series of 110 patients with Binet stage A CLL. Genes located in the deleted or gained regions were selected as target genes - DLEU2 at 13q14, ATM at 11q22, p53 at 17p13, POU6F1 and MDM2 at 12q13 and 12q15 respectively - and amplified using a method based on the simultaneous amplification of short fluorescent genomic fragments under quantitative conditions. A chromosomal imbalance involving one or several of the four loci was detected in 76 patients (69%) either by FISH or QMPSF or both. A deletion of chromosome 13 was found in 61 patients (55%). A 11q22 deletion was present in 9 patients (8%), a trisomy 12 in 9 (8%) and a 17p deletion in one. The rather low frequency of the three latter defects reflects the fact that only patients with stage A CLL at diagnosis were studied, and neither stage B or C CLL. The 13q deletion was isolated in 53 patients and associated with a second defect in 8: these were six of the 11q22 deletions, one trisomy 12 and the 17p deletion. When the 13q deletion was associated with either a 11q deletion or a trisomy 12 both abnormalities were present in the same proportion of cells. This was not the case for the 17p deletion which appeared to be a secondary event, since, as evaluated by FISH, it was present in 24% of cells whereas the 13q deletion was present in 85% of interphase nuclei. FISH and QMPSF results were identical for 103 of 110 patients. The secondary 17p deletion was not detected whereas all other discrepancies could be explained. This study demonstrates that a single multiplex PCR can replace FISH in CLL patients. However, whereas QMPSF is perfectly adapted to the detection of primary defects, care should be taken when searching for minor clonal evolutions present in a small proportion of tumor cells.

Detection of 13q14 Deletion by FISH Is Not Sufficient to Define a Group of Good Prognosis Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2332-2332
Author(s):  
Virginie Eclache ◽  
Vincent Levy ◽  
Fanny Baran-Marszak ◽  
Remi Letestu ◽  
Florence Cymbalista
Keyword(s):  
Prognostic Markers ◽  
Prognostic Significance ◽  
Genetic Alterations ◽  
Large Cohort ◽  
Fish Analysis ◽  
Prognostic Impact ◽  
Trisomy 12 ◽  
Conventional Cytogenetics ◽  
The Impact ◽  
13Q Deletion

Abstract Abstract 2332 Poster Board II-309 Deletion of a 13q14.3 region is by far the most common genomic alteration in CLL. In a large cohort of CLL patients, the presence of deletion 13q as sole anomaly detected by FISH was predominantly found in Binet stage A CLL and associated with a favorable outcome (Dohner et al., N Engl J Med 2000). Further studies have evidenced some heterogeneity among CLL cases with 13q deletion, such as the size of the clone carrying the deletion, the existence of mono versus biallelic deletions, and the presence of other concomitant genetic aberrations. Therefore, we aimed at analysing the impact of this heterogeneity on the prognostic value of 13q14 deletion (del13q) in CLL. Patients and methods In a cohort of 329 previously untreated newly diagnosed stage A CLL, we detected del13q by FISH in 172 patients (52%) using the D13S319 probe. Conventional cytogenetics was performed in the 105 cases with del13q followed in our institution. The other important prognostic markers ( ZAP70, IgVH, CD38, proliferation markers) and clinical progression were also available for all patients. Results We first studied the large cohort of stage A patients and found that deletion 13q had no prognostic impact on PFS. When considering more specifically the presence of deletion 13q as sole anomaly (n=143), PFS was not significantly different from that of patients with no aberration detected by FISH analysis (del 13q, del11q, del17p and trisomy 12) (n=98). Moreover, the distribution of prognostic factors (ZAP70, sTK, mutational status, CD38 expression, lymphocytosis) was not statistically different among these two groups. We aimed at deciphering further these del13q cases through analysis of the percentage of deleted cells, the presence of mono versus biallelic deletions, and the presence of additional aberrations as detected by FISH and conventional cytogenetics in the 105 del13q cases followed in our institution. The size of the del13q clone, as reflected by the percentage of del13q cells by FISH, was highly variable, ranged from 7 to 90 %, and had no prognostic significance on PFS. Monoallelic deletions were present in 77 cases, fully biallelic deletions in 9 cases, and concomitant bi and monoallelic deletions in 19 cases. The 9 cases with biallelic deletions had a significantly shorter PFS and were associated with other unfavorable prognostic markers. As biallelic deletions are most likely to represent progression from monoallelic cases, it is understandable that no clear prognostic impact was evidenced between cases with monoallelic deletions and with concomitant variable amount of bi and monoallelic deleted cells. Twenty cases (20 monoallelic and 6 biallelic) were further studied by array-CGH. Minimal deleted region (MDR) was included in all deletions but the size of the deletion was variable and in most cases much larger than MDR. Among the 6 bi allelic cases, one of the deletions was restricted to the MDR in all cases, pointing out to the importance of the level of miRNA expression. Additional aberrations were found in 44/105 del13q cases. In 17 patients, one or more alterations were detected by FISH techniques : del11q (n=8), trisomy 12 (n=8) or del17p (n=5). By conventional cytogenetic all these aberrations were also detected, as well as other rare ones in 16 additional cases, either isolated or associated in a complex karyotype in 5 cases. Presence of additional aberrations had a significant unfavourable impact on PFS, even when excluding del11q, del17p and tri12, and considering the non recurrent aberrations that were detected by conventional cytogenetics only. Conclusion Presence of 13q deletion should not be considered as a good prognostic marker by itself among stage A patients. Moreover, del13q cases are highly heterogeneous, and the presence of deletion 13q should not be interpreted without considering both alleles or the presence of concomitant genetic alterations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Pricila da Silva Cunha ◽  
Heloisa B. Pena ◽  
Carla Sustek D’Angelo ◽  
Celia P. Koiffmann ◽  
Jill A. Rosenfeld ◽  
...  
Keyword(s):  
Real Time ◽  
Diagnostic Test ◽  
Quantitative Pcr ◽  
Target Genes ◽  
Cost Effective ◽  
Qpcr Assay ◽  
Deletion Syndrome ◽  
Comparative Genomic ◽  
Real Time Quantitative Pcr ◽  
Subtelomeric Deletion

Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36:PRKCZandSKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescentin situhybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR ofPRKCZandSKIis a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

Multiplex Quantitative PCR Assays for the Detection and Quantification of the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces†

2016 ◽  
Vol 79 (1) ◽  
pp. 66-74 ◽  
Author(s):  
P. B. SHRIDHAR ◽  
L. W. NOLL ◽  
X. SHI ◽  
B. AN ◽  
N. CERNICCHIARO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Virulence Genes ◽  
Target Genes ◽  
Culture Methods ◽  
Fecal Samples ◽  
E Coli ◽  
Cattle Feces ◽  
Before And After

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, called non-O157 STEC, are important foodborne pathogens. Cattle, a major reservoir, harbor the organisms in the hindgut and shed them in the feces. Although limited data exist on fecal shedding, concentrations of non-O157 STEC in feces have not been reported. The objectives of our study were (i) to develop and validate two multiplex quantitative PCR (mqPCR) assays, targeting O-antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2); (ii) to utilize the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae), to quantify seven serogroups and three virulence genes in cattle feces; and (iii) to compare the three mqPCR assays to a 10-plex conventional PCR (cPCR) targeting seven serogroups and three virulence genes and culture methods to detect seven E. coli serogroups in cattle feces. The two mqPCR assays (1 and 2) were shown to be specific to the target genes, and the detection limits were 4 and 2 log CFU/g of pure culture–spiked fecal samples, before and after enrichment, respectively. A total of 576 fecal samples collected from a feedlot were enriched in E. coli broth and were subjected to quantification (before enrichment) and detection (after enrichment). Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥4 log CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. The three mqPCR assays detected higher proportions of postenriched fecal samples (P < 0.01) as positive for one or more serogroups compared with cPCR and culture methods. This is the first study to assess the applicability of qPCR assays to detect and quantify six non-O157 serogroups in cattle feces and to generate data on fecal concentration of the six serogroups.

CD79b Expression in Chronic Lymphocytic Leukemia

2003 ◽  
Vol 127 (5) ◽  
pp. 561-566 ◽  
Author(s):  
Ellen Schlette ◽  
L. Jeffrey Medeiros ◽  
Michael Keating ◽  
Raymond Lai
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Initial Study ◽  
Lymphocytic Leukemia ◽  
Study Group ◽  
Cell Marker ◽  
Trisomy 12 ◽  
Conventional Cytogenetics

Abstract Context.—CD79b is a relatively newly characterized B-cell marker that is expressed in a minority of chronic lymphocytic leukemia (CLL) cases. Objective.—To systematically correlate CD79b expression with specific morphologic and immunophenotypic findings and trisomy 12. Design.—We assessed CD79b expression in 100 consecutively accrued CLL cases that were also analyzed by conventional cytogenetics. Based on the association between trisomy 12 and CD79b expression, we then assessed 43 additional CLL cases with trisomy 12. CD79b expression was correlated with morphology and expression of other immunophenotypic markers. Results.—Eighteen (18%) of 100 consecutively accrued cases were CD79b positive. No significant association was found between CD79b expression and atypical morphology. CD79b expression correlated with CD22 and FMC7 positivity. Eight (8%) cases had trisomy 12; 4 (50%) of these were CD79b positive, suggesting an association with trisomy 12. Examination of a second group of 51 CLL cases with trisomy 12 (including 8 cases from the initial study group) showed that CD79b was positive in 26 cases (49%), a frequency significantly higher than that of the consecutively accrued CLL cases without trisomy 12 (P < .05). Conclusions.—We conclude that CD79b immunoreactivity is positive in approximately 20% of CLL cases and that expression correlates with trisomy 12 and atypical immunophenotypic findings.

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