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Adult patients with acute lymphoblastic leukemia (ALL) who are stratified into the standard-risk (SR) group due to the absence of adverse prognostic factors relapse in 40% to 55% of the cases. To identify complementary markers suitable for further treatment stratification in SR ALL, we evaluated the predictive value of minimal residual disease (MRD) and prospectively monitored MRD in 196 strictly defined SR ALL patients at up to 9 time points in the first year of treatment by quantitative polymerase chain reaction (PCR). Frequency of MRD positivity decreased from 88% during early induction to 13% at week 52. MRD was predictive for relapse at various follow-up time points. Combined MRD information from different time points allowed definition of 3 risk groups (P < .001): 10% of patients with a rapid MRD decline to lower than 10 ؊4 or below detection limit at day 11 and day 24 were classified as low risk and had a 3-year relapse rate (RR) of 0%. A subset of 23% with an MRD of 10 ؊4 or higher until week 16 formed the high-risk group, with a 3-year RR of 94% (95% confidence interval [CI] 83%-100%). The remaining patients whose RR was 47% (31%- 63% IntroductionInvestigation of minimal residual disease (MRD) has been proven to be a valuable tool for predicting outcome in childhood acute lymphoblastic leukemia (ALL). [1][2][3][4][5] In contrast, only a few studies have focused on adult ALL, and they were based mostly on patients with heterogeneous risk profiles and different kinds and intensities of treatment. [6][7][8] However, monitoring homogeneous patient cohorts at different time points during therapy might provide additional insight into the nature and clinical relevance of MRD kinetics in adult ALL, which is particularly relevant for the large population of standard-risk (SR) patients without conventional risk factors. Relapses in this patient group occur in about 40% to 55% of cases and cannot be predicted by any known conventional risk factor. [9][10][11] In a number of clinical studies this led to a policy of stem cell transplantation (SCT) in first remission, 12-14 causing overtreatment and additional expenses for those patients who are cured by conventional chemotherapy alone. Therefore, definition of prognostic factors allowing discrimination of SR patients with poor outcome after standard chemotherapy from those with a favorable prognosis is highly warranted. Currently, the most widely used techniques to detect and quantify residual disease in patients with ALL are multiparameter immunophenotypic evaluation of aberrant protein expression 2,3,8 and clone-specific polymerase chain reaction (PCR) amplification of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements. 1,4,5 Such molecular targets can be identified in more than 90% of patients with ALL by the use of various PCR primer sets. Besides its large applicability and high sensitivity, a main advantage of PCR-based assays is the use of DNA as a stable and easy conveyable specimen, which is particularly relevant in large multicenter studies....

Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vb-Db-Jb rearrangements in 80%, Db-Jb rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jb2.7 in childhood T-ALL (27%), whereas Jb2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vb segments (19-1/20-1/21-1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jb primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 10 4 polyclonal cells in 93% of cases and will be of high value for future MRD studies.

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The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.