Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

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.

Rearranged T-cell receptor beta genes represent powerful targets for quantification of minimal residual disease in childhood and adult T-cell acute lymphoblastic leukemia.

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 (Vbeta-Dbeta-Jbeta rearrangements in 80%, Dbeta-Jbeta rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the 'end-stage' segment Jbeta2.7 in childhood T-ALL (27%), whereas Jbeta2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vbeta segments (19-1/20-1/21-1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jbeta 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.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR.

PCR of clonally rearranged immunoglobulin heavy chain (IgH) gene sequences is increasingly used for detection of minimal residual disease (MRD) in lymphoid malignancies. Inherent quantitating problems are the main drawbacks of traditional PCR technologies. These limitations have been overcome by the recently developed real-time quantitative PCR (RQ PCR) technology. However, clinical application of the few published RQ PCR assays targeting immune gene rearrangements is hampered by the expensive and time-consuming need for individual hybridization probes for each patient. We have developed a new RQ PCR strategy targeting clonally rearranged IgH sequences that solves this problem. The method uses only two different JH hybridization probes and four downstream JH primers homologous to consensus germline JH gene segments. In combination with an allele-specific upstream (ASO) primer the consensus JH probes and primers allow quantitation of about 90% of possible IgH rearrangements. In a series of 22 B-lineage ALL the new assay allowed the detection of one to 10 blasts in a background of 10(5) normal cells. To prove the clinical utility we quantified MRD in 23 follow-up samples of six ALL patients with the new assay in comparison with a published RQ PCR technique that used individually designed primer/probe sets. We showed that the sensitivity of the new RQ PCR assay was slightly higher for four of the six cases and about 100-fold higher for one case, enabling detection of an increasing MRD level as an indicator of subsequent relapse 44 weeks earlier compared to the ASO probe assay in this particular patient. The results suggest, that the novel RQ PCR assay is a rapid, technically simple, reliable, and sensitive alternative to traditional quantification assays and simplifies current approaches of monitoring MRD in clinical trials.

Segregation of the ability to degrade lupanine in Pseudomonas lupanini.

Two types of mutants differing in the ability to degrade lupanine were obtained from the JD1 strain of Pseudomonas lupanini: lus growing slowly on lupanine, and lun unable to utilize lupanine as a source of carbon and nitrogen. The mutation rate for the spontaneous lus mutants was 0.28%, and for the mitomycin C-induced lus mutants, 3-24%. Rifampicin induced solely lun mutation. The plasmid nature of the lupanine degradation pathway is discussed.