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  • 1
    Keywords: CELLS ; BLOOD ; Germany ; DISEASE ; POPULATION ; HYBRIDIZATION ; PATIENT ; DONOR ; SEQUENCE ; FREQUENCY ; TRIAL ; LYMPHOMA ; HUMANS ; AGE ; PCR ; LYMPHOCYTES ; INDIVIDUALS ; sensitivity ; specificity ; VALIDITY ; PREVALENCE ; real-time PCR ; SEQUENCE-ANALYSIS ; FOLLICULAR LYMPHOMA ; MINIMAL RESIDUAL DISEASE ; REARRANGEMENT ; BREAKPOINTS ; GENDER ; healthy individuals ; CONTAMINATION ; BCL-2/J(H) TRANSLOCATION ; nested PCR ; T(14/18) ; t(14 ; 18)
    Abstract: This Study assessed prevalence, frequency, age and gender distribution and breakpoint locations. and detection method validity for the bcl-2/IgH rearrangement in 204 healthy individuals. For this purpose, both classic two-step, nested. semi-quantitative PCR as well as a newly established sequence-specific, hybridization probe-based real-time quantitative PCR (RQ-PCR) were employed and tested for their sensitivity and specificity for detecting t(14; 18) positive cells in healthy blood donors. Interestingly, almost a quarter (24%; 39/204) of all healthy individuals carried the translocation, confirming data of one large prior report [Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon J. Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 2001; 19(2):420-4]. Regarding presence as well as frequency of the translocation, no correlation to age (mean frequency 2.0: 10(4), with a median of 〈 1: 10(4), for 〈 40 years, and mean frequency 1.9:10(4), with a median of 〈 1:10(4) for individuals 〉= 40 years) nor gender was detected. Comparing the two PCR approaches, a 95.1% concordance (194/204) regarding t(I 4; 18) detection was determined for nested and RQ-PCR, with nested PCR being slightly more sensitive (reproducible detection limit 1: 105 cells versus 1:10(4); maximum detection limit 1:10(6) versus 1:10(5)). Sequence analysis confirmed individual breakpoints for all samples analyzed (29/29), indicating detection validity for both PCR approaches and ruling out contamination. The break-point location distribution pattern appeared to be comparable to the pattern seen with follicular lymphoma (FL) patient collectives. In conclusion, clonal bcl-2/IgH rearrangements are indeed a very frequent observation in healthy individuals, and appear to be independent of age and gender in regard to presence and frequency. This represents a conflicting finding in context of potential biological significance, and presents a potential disruptive factor for minimal residual disease (MRD) monitoring in FL patients. Prospective future trials will have to clarify the biological significance of this important observation. (c) 2005 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16297448
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  • 2
    Keywords: CELLS ; BLOOD ; CELL ; COMBINATION ; Germany ; THERAPY ; DISEASE ; RISK ; DRUG ; TIME ; PATIENT ; CYCLE ; treatment ; STAGE ; TRIAL ; TRIALS ; LYMPHOMA ; NUMBER ; REDUCED RISK ; RATES ; chemotherapy ; PCR ; PROBES ; CLEARANCE ; PERIPHERAL-BLOOD ; FOLLICULAR LYMPHOMA ; MINIMAL RESIDUAL DISEASE ; NON-HODGKINS-LYMPHOMA ; CAPACITY ; RESIDUAL DISEASE ; ANTI-CD20 MONOCLONAL-ANTIBODY ; rituximab ; REMISSION ; prospective ; COMBINATION THERAPY ; E ; B-CELL ; peripheral blood ; nested PCR ; t(14 ; 18) ; molecular monitoring ; RQ-PCR ; t(14 : 18)
    Abstract: Accurate monitoring of the t(14; 18) translocation is regarded as highly desirable in patients with follicular lymphoma (FL) as absence of bcl-2/IgH positive cells has been correlated with a reduced risk of recurrence and, more recently, pre-treatment t(14;18) load with response to rituximab (R: Blood 2004; 103:4416-23). With the arrival of R clinical and molecular remission rates for various lymphoma entities improved significantly, creating the need to carefully review and reassess the role of PCR negativity for clinical outcome, specifically when considering the prolonged presence of the drug as compared to chemotherapy. To determine the rate of molecular clearance achieved by the addition of different doses of R 16 newly diagnosed, t(14;18) positive patients with FL (Ann Arbor stages III/IV) were investigated before, during and after primary chemotherapy with six cycles of CHOP combined with varying (1, 3 or 6) cycle numbers of R (varR1-, varR3- or varR6-CHOP, respectively) regarding molecular remission status. For this purpose classic nested PCR as well as a newly established RQ-PCR employing juxtaposed hybridisation probes were employed to assess molecular remission prior, during and post therapy. Interestingly, administering just a single cycle of R (varR1-CHOP) in combination with a standard regimen of CHOP resulted in rapid and effective clearance of t(14;18) carrying cells from the peripheral blood of 4/5 patients in this treatment group. 6/8 (6/8) varR1-/varR3-CHOP patients were fully cleared and 8/8 (7/8) var6-CHOP patients as assessed by RQ- (nested) PCR. This indicates the high clearance capacity of this combination therapy approach even when adding a very low cycle number of R (1 and 3, respectively) to CHOP in primary therapy of FL. In summary, the relationship established between molecular clearance and minimal residual disease/risk of recurrence may have to be redefined in the times of R. Upcoming large prospective trials will have to elucidate to what degree the clearance of peripheral blood from t(14;18) positive cells can still be regarded as informative regarding absence of minimal residual disease, remission status and/or risk of recurrence. (c) 2006 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16530831
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  • 3
    ISSN: 1437-1596
    Keywords: Urine ; DNA-typing ; Storage ; PCR-inhibition ; Coextraction of non-human DNA ; Urin ; DNA-Typisierung ; Lagerung ; PCR ; Hemmung ; Koextraktion nicht humaner DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Law
    Description / Table of Contents: Zusammenfassung Die Typisierbarkeit von DNA-Polymorphismen an gelagerten Urinproben wurde in einem systematischen Lagerungsexperiment und stichprobenartig au 1-7 Jahre alten Proben untersucht. In beiden Untersuchungen zeigte sich bei Frauen eine erhöhte Zahl von Epithelzellen im Urin und damit eine größere Menge an extrahierbarer DNA. Mit dem Verfahren der Genamplifikation konnten für den untersuchten Zeitraum von sechs Monaten alle Proben typisiert werden. Die mikroskopische Begutachtung der mehrere Jahre alten Proben zeigte verschiedene Grade von Kontamination mit Bakterien, Hefen und Pilzen, aber auch das Vorhandensein von intakten Epithelzellen. Nur 20% der Urinproben von Männern und 32% der Proben von Frauen ergaben spezifische Amplifikationsergebnisse. Durch die Trennung von humanen Zellen und kontaminierenden Organismen vor der DNA-Extraktion konnte diese Quote auf 35% der männlichen und 77% der weiblichen Proben erhöht werden. Dieses Ergebnis zeigt, daß ein hoher Anteil koextrahierter nicht menschlicher DNA einen hemmenden Effekt auf die spezifische Amplifikation humaner Zielsequenzen hat.
    Notes: Summary The possibility of typing DNA polymorphisms on urine samples was investigated in a controlled storage experiment and for samples that were 1-7 years old. Female urine samples showed a higher amount of epithelial cells and therefore a higher DNA yield. Employing the polymerase chain reaction, specific amplification results were achieved for all samples over a 6 month storage period. The microscopical examination of the samples revealed not only differing degrees of contamination with bacteria, yeasts and fungi, but also the presence of still intact epithelial cells. Only 20% of the male samples and 32% of the female samples yielded specific amplification results. By separating the human cells from the contaminating organisms prior to DNA extraction, the number of successfully typed samples could be improved to 35% of the male and 77% of the female samples. This result confirms that excess amounts of coextracted non-human DNA can inhibit the specific amplification of human target sequences.
    Type of Medium: Electronic Resource
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