An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

Müller, Martin C.; Saglio, Giuseppe; Feng, Lin; Pfeifer, Heike; Press, Richard D.; Tubbs, Raymond R.; Paschka, Peter; Gottardi, Enrico; O'Brien, Steven G.; Ottmann, Oliver G.; Stockinger, Hubertus; Wieczorek, Lothar; Merx, Kirsten; König, Heiko; Schwindel, Uwe; Hehlmann, Rüdiger; Hochhaus, Andreas

doi:10.3324/haematol.11172

Cited by 48 publications

(30 citation statements)

References 13 publications

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“…In particular, Cp-value was used, which represents the timepoint at which the fluorescence of the sample rises above the background fluorescence during the real-time PCR process and the results were converted to ratios based on the reference of Müller and his co-workers [27] using G6PDH as gene reporter in order to have comparable results. LHR expression was categorized into three groups based on the quartiles of the overall distribution.…”

Section: Real-time Pcrmentioning

confidence: 99%

LH receptor gene expression in cumulus cells in women entering an ART program

Papamentzelopoulou

Mavrogianni

Partsinevelos

et al. 2012

J Assist Reprod Genet

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Purpose Luteinizing hormone (LH) exerts its actions through its receptor (LHR), which is mainly expressed in theca cells and to a lesser extent in oocytes, granulosa and cumulus cells. The aim of the present study was the investigation of a possible correlation between LHR gene and LHR splice variants expression in cumulus cells and ovarian response as well as ART outcome. Methods Forty patients undergoing ICSI treatment for male factor infertility underwent a long luteal GnRH-agonist downregulation protocol with a fixed 5-day rLH pretreatment prior to rFSH stimulation and samples of cumulus cells were collected on the day of egg collection. RNA extraction and cDNA preparation was followed by LHR gene expression investigation through real-time PCR. Furthermore, cumulus cells were investigated for the detection of LHR splice variants using reverse transcription PCR. Results Concerning LHR expression in cumulus cells, a statistically significant negative association was observed with the duration of ovarian stimulation (odds ratio00.23, p00.012). Interestingly, 6 over 7 women who fell pregnant expressed at least two specific types of LHR splice variants (735 bp, 621 bp), while only 1 out of 19 women that did not express any splice variant achieved a pregnancy. Conclusions Consequently, the present study provide a step towards a new role of LHR gene expression profiling as a biomarker in the prediction of ovarian response at least in terms of duration of stimulation and also a tentative role of LHR splice variants expression in the prediction of pregnancy success.

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Section: Real-time Pcrmentioning

confidence: 99%

LH receptor gene expression in cumulus cells in women entering an ART program

Papamentzelopoulou

Mavrogianni

Partsinevelos

et al. 2012

J Assist Reprod Genet

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“…This agrees with systematic differences between LC and TM platforms, which have been recognized previously and will be overcome by the introduction of external calibrators and regular control rounds. 10,15 Control rounds like this turn out to be efficient tools for detection of PCR assay deficits in individual laboratories, providing an opportunity for correction. Since the SNP in the GUS gene has not been considered in the EAC protocol, we suggest changing the TM protocol by using a chimeric primer taking into account both SNP and wild-type sequences or by avoiding the polymorphic sequence.…”

Section: Discussionmentioning

confidence: 99%

“…[7][8][9] Thus, there is an unmet need for harmonization of both procedures and expression of results. 10 In a series of consensus meetings on the initiative of the European LeukemiaNet, a catalogue of prerequisites to achieve optimal sensitivity and standardization has been elaborated: (a) use of at least 10 ml peripheral blood (PB); (b) processing within 36 h; 8 (c) bedside RNA stabilization for multicenter trials; 11 (d) standardized PCR protocols; 12,13 (e) use of a plasmid containing target and housekeeping genes to avoid dilution errors; (f) use of total ABL, BCR and/or glucuronidase-b (GUS) as internal controls. 14 To substantiate this catalogue, an international multicenter trial involving 39 laboratories in 14 countries was initiated.…”

Section: Introductionmentioning

confidence: 99%

Harmonization of BCR-ABL mRNA quantification using a uniform multifunctional control plasmid in 37 international laboratories

et al. 2007

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Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-b (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.

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“…With respect to conventional cytogenetic analysis, RQ PCR can not only allow to monitor the first steps of reduction of the leukemic burden occurring within the first months of TKI therapy, but it may also allow to estimate the amount of the residual disease once CCyR is achieved, as the sensitivity that can be reached with the present RQ PCR procedures in a sample of good quality is in most cases between 1×10 −4 /10 −5 that corresponds to an amount between 2 and 3 logs below the threshold of the achievement of CCyR [14]. According to the established international scale (IS), the relevant BCR-ABL% to be achieved are 1 % (2-log reduction with respect to the median BCR-ABL amount present at diagnosis and that roughly corresponds to the threshold of CCyR), 0.10 % BCR-ABL (major molecular response (MMR)), and 0.01 % and to 0.0032 % BCR-ABL corresponding, respectively, to MR 4 (4-log reduction) and MR 4.5 (4.5-log reduction) [14][15][16].…”

Section: Introductionmentioning

confidence: 99%

“…Based on these principles, a panel of CML experts on behalf of the European Leukemia Net (ELN) as well as members of the National Comprehensive Cancer Network (NCCN) have previously established and more recently revised treatment milestones to be achieved during CML treatment with TKIs [12,13]. This obviously implies that, to optimize CML treatment with TKIs, an appropriate and timely follow-up with cytogenetic and standardized molecular methods of adequate reliability is needed [14][15][16]. In particular, molecular monitoring of BCR-ABL transcript levels by real-time quantitative PCR (RQ PCR) is progressively becoming the most useful and precise way to monitor CML patients.…”

Section: Introductionmentioning

confidence: 99%

The Choice of First-Line Chronic Myelogenous Leukemia Treatment

Fava¹,

Rege‐Cambrin²,

Dogliotti³

et al. 2016

Hematologic Malignancies

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Imatinib has represented a revolution in the treatment of chronic myeloid leukemia (CML), inducing an overall survival never seen with previous therapies. However, with the commonly used dosage of 400 mg, one third of the treated patients does not reach the criteria associated with an optimal outcome and could potentially benefit from a different treatment strategy. Several trials exploring modified imatinibbased treatments or second-generation tyrosine-kinase as front-line therapy have been performed. In some studies, high-dose (800 mg per day) or dose-adapted imatinib or imatinib plus interferon was reported to be able to induce better cytogenetic and molecular responses compared with standarddose imatinib, although no improvements in progression-free survival (PFS) or overall survival (OS) have been so far reported. At the moment, these approaches are still considered investigational. On the other side, on the basis of their capacity to induce very fast and deep molecular responses, including major molecular responses (MMRs) and the newly defined very deep molecular responses MR 4 and MR 4.5 , and to prevent at least part of the early progressions to AP/BC that still occur during the first 2-3 years from diagnosis, dasatinib and nilotinib have been approved and registered by FDA and EMA as the first-line therapy for CML patients, opening the possibility to use different therapeutic strategies for newly diagnosed CML patients and a consequent intense debate among hematologists.

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An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

Cited by 48 publications

References 13 publications

LH receptor gene expression in cumulus cells in women entering an ART program

LH receptor gene expression in cumulus cells in women entering an ART program

Harmonization of BCR-ABL mRNA quantification using a uniform multifunctional control plasmid in 37 international laboratories

The Choice of First-Line Chronic Myelogenous Leukemia Treatment

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