Laboratory Issues

Although over the last decade numerous methods to monitor MRD in acute leukemias have been developed and new technologies are being used, standardization and quality control still are needed to apply molecular diagnostic procedures in hematopathology. This is particularly true in efforts to assure reproducible results within multicenter international studies.

The molecular approach for detection of antigen receptor gene rearrangements requires standardization and quality control, which has been addressed within the same BIOMED-1 framework. One of the major problems resides in the fact that each patient's leukemia has a unique rearrangement, requiring the use of patient-specific assays and individual optimization. Use of patient-specific assays represents a major difficulty in transferring this method to clinical practice. In an effort to maximize detection of virtually all ALL patients and to prevent false-negative results, study PCR primers have been designed for multiple targets: TCRD, TCRG, and IGLK rearrangements, as well as the SIL/TAL rearrangement.18 The method uses a total of 54 primers with ASO probes in single or nested PCR for target identification at diagnosis, sequence analysis of junctional regions, and MRD detection in follow-up samples. A total of 25 PCR reactions are performed at diagnosis to identify the PCR targets. This standardized approach allows rapid detection of clonal IG and TCR rearrangements in ALL with high sensitivity and high specificity, and enables discrimination between mono- and oligoclonal gene rearrangements. The combination of the four PCR target types allowed PCR monitoring in more than 90% of B-cell precursor ALL and 95% of T-ALL cases. In the vast majority of childhood and adult ALL cases, two or more PCR targets were available for MRD

monitoring. The sensitivity of detecting PCR targets depended at least partially on the size of the junctional region, with a level of 1 x 10-4 reached in most cases. Recently, to increase the percentage of cases successfully stratified by MDR, newly identified molecular targets have been incorporated. The monoclonal V82-]a rearrangements in precursor B-ALL were used as patient-specific targets for MDR detection, because they show high sensitivity and good stability.27 A sensitive TCRB RQ-PCR assay was developed99 with utility for MDR studies in T-ALL, in which the repertoire of IG/TCR rearrangements is limited and less sensitive.

Careful standardization and quality control of MRD techniques were also the aims of the European BIOMED-1 Concerted Action ("Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease"), with participants from several laboratories in European countries.43 In particular, the five most-frequent, well-defined ALL chromosomal aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(9;22) with BCR-ABL (p190 and p210), t(12;21) with TEL-AML1, and microdeletion 1p32 with SIL-TAL1. PCR primers, positioned to cover several different transcript versions, were designed according to predefined criteria for single and nested PCR (Figure 31-3).

More recently, a Europe Against Cancer program was established to achieve standardization and quality control on the new RQ-PCR technique for detection and quantification of fusion gene transcripts. Twenty-five European laboratories in ten countries have collaborated and established consensus standards for RQ-PCR (Taqman technology) for the main translocations seen in a spectrum of hematologic malignancies, including ALL. A set of 12 primers and 9 probes has been selected to cover the most frequent chimeric transcripts, with a threshold of detection of 100 molecules and/or a 1 x 10-4 dilution.47 A representative experiment of RQ-PCR detection of the fusion transcript generated by the translocation t(9;22)is shown in Figure 31-5.

Figure 31-5. MRD monitoring by quantitative PCR for a patient with ALL carrying the t(9;22) translocation during the course of treatment.

This work is the first coordinated international effort on standardization and quality control methods for a molecular diagnosis procedure in hematopathology across therapeutic protocols. It should allow accurate quantitative measurement of fusion transcripts with an international consensus protocol for diagnosis and MRD assessment in follow-up samples from leukemia patients.

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