Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia

Wattjes, Mike P.; Krauter, Jürgen; Nagel, Stefan; Heidenreich, Olaf; Ganser, Arnold; Heil, Gerhard

doi:10.1038/sj.leu.2401679

Cited by 57 publications

(38 citation statements)

References 36 publications

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“…15 RQ-PCR has proved to be a successful approach when applied to hematological malignancies carrying gene translocations as tumor markers. [27][28][29][30] For B-and T-cell malignancies in which no recurrent well-defined translocations are present, B-and T-cell receptor genes are used as tumor markers for RQ-PCR. 17,20 In the last 4 years, RQ-PCR has become an attractive and useful approach for the detection of MRD in immature B-cell malignancies and the subset of B-CLL without somatic mutations, 17 mainly because of the utilization of consensus probes in the JH region thereby avoiding the need to synthesize allele-specific probes (ASO probes) for each patient, which is very expensive and time-consuming.…”

Section: Discussionmentioning

confidence: 99%

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

González

Alonso

et al. 2003

Leukemia

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The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10 5 polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

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Section: Discussionmentioning

confidence: 99%

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

González

Alonso

et al. 2003

Leukemia

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“…To obtain the standard curve, laboratories used either cell line cDNA 37,39,128,190 or plasmid DNA. 38,42,125,191 So far, few authors used the DDCt method 49,192 and, to our knowledge, a standard curve of diagnostic cDNA has not been used so far. In our EAC network, we discussed four possibilities: (1) cell line RNA dilutions, (2) percentage of positive cell number relative to the diagnostic sample, (3) copy number ratios and (4) the DDCt method.…”

Section: Expression Of Rq-pcr Datamentioning

confidence: 99%

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

et al. 2003

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Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqManbased real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer (EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n ¼ 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic Correspondence: Professor J

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“…24 However, recently, several studies strongly suggested that RQ-RT-PCR might be a more valuable technique to predict relapse in AML with core binding factor (CBF) rearrangement in general or in t(8;21) AML. 13,14,[25][26][27] In those studies, critical thresholds of MRD levels, which could distinguish between patients at risk of relapse and those in continuous CR, were identified. In the present study, no pretreatment characteristics were predictive of the outcome, possibly due to the relatively low number of patients.…”

Section: Prognostic Value Of Rq-pcr In Amlmentioning

confidence: 99%

“…10 Several studies strongly suggest that quantitative PCR (RQ-PCR) methods of minimal residual disease (MRD) detection might improve the predictive results of qualitative PCR techniques in acute leukemia. [11][12][13] Recently, one study reported that the quantification of AML1-ETO fusion transcript at diagnosis with a quantitative real-time PCR method could identify patients at high risk of relapse. 14 We sequentially monitored AML1-ETO rearrangement by RQ-PCR in 21 patients uniformly treated in our center, in order to assess the prognostic value of this technique.…”

Section: Introductionmentioning

confidence: 99%

Prognostic value of real-time quantitative PCR (RQ-PCR) in AML with t(8;21)

et al. 2005

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Despite the favorable prognosis of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22) translocation, relapses still occur in about 30% of the cases but no initial factors can strongly predict the risk of relapse. Several recent studies suggest that monitoring minimal residual disease (MRD) may identify patients at risk of relapse. We prospectively monitored AML1-ETO rearrangement by real-time quantitative PCR (RQ-PCR) in 21 patients uniformly treated in our center. Blood (PB) and bone marrow (BM) samples were collected during and after therapy. At diagnosis, levels of AML1-ETO transcript showed large variations and there was a trend for a higher relapse rate in patients with high pretreatment expression levels (P ¼ 0.065). After induction therapy, absolute transcript levels (below 10 À3 , compared to Kasumi cell line), or a greater than 3 log decrease by comparison to diagnosis levels, were significant predictors of the absence of relapse (P ¼ 0.02 and P ¼ 0.02, respectively). MRD levels after consolidation therapy were also significant indicators of relapse (P ¼ 10 À5 ). Comparison of BM and PB samples showed similar sensitivity for detecting AML1-ETO transcript. In conclusion, RQ-PCR appears to be an early predictive factor of the relapse risk in AML with t(8;21). PB samples can be used adequately to evaluate the level of MRD by this technique.

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Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia

Cited by 57 publications

References 36 publications

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

Prognostic value of real-time quantitative PCR (RQ-PCR) in AML with t(8;21)

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