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Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Real-time quantitative RT-PCR (RQ-PCR) is a sensitive tool to monitor minimal residual disease (MRD) in leukemic patients through the amplification of a fusion gene (FG) transcript. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript. To identify suitable CGs, a study group within the Europe Against Cancer (EAC) program initially focused on 14 potential CGs using a standardized RQ-PCR protocol. Based on the absence of pseudogenes and the level and stability of the CG expression, three genes were finally selected: Abelson (ABL), beta-2-microglobulin (B2M), and beta-glucuronidase (GUS). A multicenter prospective study on normal (n=126) and diagnostic leukemic (n=184) samples processed the same day has established reference values for the CG expression. A multicenter retrospective study on over 250 acute and chronic leukemia samples obtained at diagnosis and with an identified FG transcript confirmed that the three CGs had a stable expression in the different types of samples. However, only ABL gene transcript expression did not differ significantly between normal and leukemic samples at diagnosis. We therefore propose to use the ABL gene as CG for RQ-PCR-based diagnosis and MRD detection in leukemic patients. Overall, these data are not only eligible for quantification of fusion gene transcripts, but also for the quantification of aberrantly expressed genes.

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 TaqMan-based 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 = 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 samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

Mutations in the kinase domain of BCR-ABL result in impaired drug binding and are thought to be the leading cause of acquired resistance to the tyrosine kinase inhibitor imatinib (Gleevec®). While imatinib is a highly effective therapy in all stages of chronic myelogenous leukemia (CML), relapse after an initial response is common in patients with advanced disease. The T315I point mutation is one of the most common imatinib-resistant mutations and patients with this mutation are also resistant to two second generation tyrosine kinase inhibitors, dasatinib and nilotinib. Recently, the U.S. FDA approved dasatinib for treatment of imatinib-resistant, Philadelphia chromosome-positive acute and chronic leukemias. Thus, appropriate detection of this mutation is essential to optimal management of patients with imatinib resistance and may be useful for clinical trials of agents that target patients with the T315I mutation. Current methods for mutation detection, such as direct DNA sequencing, are not sensitive enough for detection of point mutations at low levels of BCR-ABL transcript. Conversely, ultrasensitive detection methods such as allele specific PCR (AS-PCR) may be too sensitive and can be plagued by false positive test results. In addition the clinical relevance/significance of mutation detection at ultra sensitive levels ( < 1% mutant) is questionable and not yet known. We developed a novel T315I mutation detection assay, using Fluorescent Resonance Energy Transfer (FRET)-based hybridization probes and melting curve analysis. BCR-ABL amplicons generated from a first round of PCR are amplified using primers flanking the ABL kinase region encoding for codon 315. The resultant amplicon is hybridized with fluorescein-labelled anchor probe and a LC Red 640-labelled T315I mutation specific probe. Wild-type and T315I mutant amplicons are distinguished by melting curve analysis (Roche Light Cycler 480™). Using a series of plasmid and cell line dilutions we determined that the sensitivity of this assay for detection of T315I mutations was 5–10%. Using patient-derived samples we were able to successfully genotype samples containing as few as 20–50 BCR-ABL transcripts. To date, the assay sensitivity and specificity are 100%. The assay is performed in a plate based format (96 or 384 wells) and commercially available software allows automated genotype assignment. This approach is suitable for high-throughput detection of T315I mutations for clinical management of CML patients and/or screening of patients to determine eligibility for clinical studies.

Introduction: Major Molecular Response (MMR) is defined as a three-log reduction from a standardized baseline of BCR-ABL/control gene transcript ratio in CML patients at diagnosis. MMR has prognostic significance for progression-free survival for patients on Imatinib® therapy. Day-to-day monitoring of the MMR value in clinical laboratories is challenging due to the absence of a commercially available standardized MMR control RNA. To improve the reliability of BCR-ABL quantitation, MolecularMD has evaluated the feasibility of a single MMR control RNA valid for blood samples drawn in EDTA or PAXgene™ tubes. Material and Methods: Patient sample RNAs were interchanged between our laboratory and an International Randomized Interferon versus STI571 study (IRIS) laboratory, which had established an MMR value and international scale reporting. This exchange enabled our laboratory to establish an MMR value and reporting on an international scale using a validated conversion factor. A serial dilution of a BCR-ABL positive cell line into a human BCR-ABL negative cell line was prepared. These dilutions were tested in IRIS laboratories with established MMR value and international scale reporting and at our laboratory by QRT-PCR to determine the BCR-ABL/control gene ratio using respectively BCR and ABL control genes. We compared the BCR-ABL/ABL ratio in 104 paired PB CML patient samples drawn either in EDTA and PAXgene tubes and the BCR-ABL/BCR ratio in 32 patient samples. Stability studies were performed to evaluate the degradation of liquid and dried forms of the MMR RNA. Results: We established a conversion factor (CF) of 0.81 with an MMR value of 0.123%. Using this CF and MMR value, we created appropriate RNA dilutions that matched the MMR value using ABL as a control gene. Repeated analyzes of this MMR control RNA confirmed the accuracy of the sample with a median value of 0.124%, very close to the MMR value defined previously (0.123%). Stability studies demonstrated that the dried RNA samples could be stored several days at 37°C and freeze-thawed up-to 10 times without significant degradation. These RNA samples once reconstituted with water could also be used several times for BCR-ABL monitoring without any significant degradation. Comparison of BCR-ABL/ABL ratio between EDTA and PAXgene tubes revealed differences unlikely to have clinical impact on disease management suggesting that the MMR RNA created would be suitable under both EDTA and PAXgene extraction methodologies. Conclusions: We produced a stable MMR control RNA in large quantity for accurate monitoring of the MMR value. This MMR control RNA is now be tested in several laboratories to confirm the stability and reliability of this reagent. The MMR control RNA will be an important tool for standardizing MMR value in laboratories, and an integral part of a BCR-ABL QRT-PCR diagnostic kit.