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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.

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.

Detailed Southern blot and PCR analysis of Ig heavy (IGH), Ig kappa (IGK), T-cell receptor delta (TCRD), and TCR gamma (TCRG) genes were performed in 289 children with precursor-B-ALL in order to determine age-related Ig/TCR patterns and their implications for detection of minimal residual disease (MRD). Overall, IGH, IGK, TCRD, and TCRG gene rearrangements were detected in 98, 62, 90, and 58% of patients, respectively. The frequency of IGH and TCRD rearrangements was independent of rearrangements in one of the other three loci, whereas Ig kappa deleting element and TCRG rearrangements preferentially coincided. Southern blot analysis showed that oligoclonality of IGH, IGK, and TCRD was interrelated, that is, oligoclonality in one locus was related with a higher chance of oligoclonality in another locus. Combined Southern blot and PCR analysis revealed that Ig/TCR patterns were age related: children younger than 3 years or older than 10 years showed a higher prevalence of incomplete IGH rearrangements and a lower prevalence of IGK deletions, TCRG rearrangements, and TCRD rearrangements than children between 3 and 10 years. In addition, IGH oligoclonality was more frequent in the younger and older children. These age-related differences probably reflect ALL subsets with different cellular origin and differences in the duration of the preleukemic phase between the initial and final leukemogenetic hit. The more immature Ig/TCR gene rearrangement pattern in children younger than 3 years or older than 10 years resulted in relatively low numbers of potential MRD-PCR targets per patient, particularly if only monoclonal rearrangements were taken into account. These data provide insight into the immunobiological characteristics of Ig/TCR gene rearrangements in childhood precursor-B-ALL and form a useful basis for designing improved strategies for the identification and selection of MRD-PCR targets.

We analyzed the safety and efficacy of Mylotarg (gemtuzumab ozogamicin, an antibody-targeted chemotherapy consisting of a humanized anti-CD33 antibody linked to calicheamicin, a potent antitumor antibiotic) in the treatment of 101 patients > or =60 years of age with acute myeloid leukemia (AML) in untreated first relapse in three open-label trials. Mylotarg is administered as a 2-h intravenous infusion at 9 mg/m(2) for two doses with 14 days between doses. The overall remission rate was 28%, with complete remission (CR) in 13% of patients and complete remission with incomplete platelet recovery (CRp) in 15%. Median survival was 5.4 months for all patients and 14.5 months and 11.8 months for patients achieving CR and CRp, respectively. CD33 antigen is present on normal hematopoietic progenitor cells; thus, an expected high incidence of grade 3 or 4 neutropenia (99%) and thrombocytopenia (99%) was observed. The incidences of grade 3 or 4 elevations of bilirubin and hepatic transaminases were 24% and 15%, respectively. There was a low incidence of grade 3 or 4 mucositis (4%) and infections (27%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Mylotarg is an effective treatment for older patients with CD33-positive AML in first relapse and has acceptable toxicity.

The general order of the immunoglobulin (Ig) gene rearrangement process in human precursor-B cells is largely known. However, the exact Ig rearrangement patterns reflecting this process, especially those of the Ig light chain genes, are not well established. This requires detailed analysis of the gene configuration of all six IGH, IGK and IGL alleles at the single cell level. As such extensive analyses are difficult to perform in a reliable way within a single normal precursor-B cell, we used 169 precursor-B-ALL (ie six pro-B-ALL, 112 common ALL, and 51 pre-B-ALL) as clonal 'single cell' model system. The Ig gene recombinations show hierarchy starting with IGH gene rearrangements in all cases, followed by IGK rearrangements, IGK deletions and/or IGL rearrangements in 71% of cases. IGK deletions were found in the absence of IGL rearrangements in 34% of cases, which might be explained by the continuous recombinase activity in precursor-B-ALL, resulting in 'end-stage' IGK rearrangements, together with an apparently limited accessibility of the IGL locus. Remarkably, in 5% of cases IGL rearrangements took place in the absence of IGK rearrangements. In addition we found that in-frame IGH rearrangements are not necessarily required for the induction of Ig light chain gene rearrangements and that IGL rearrangements can be induced irrespective of the frame of the accompanying IGK rearrangements. In conclusion, precursor-B-ALL constitute a model system for studying Ig gene rearrangement processes without selection for functionality of the rearrangements or the influence of somatic hypermutations. Nevertheless, the hierarchy of IGH, IGK and IGL rearrrangements is apparent in precursor-B-ALL.

Sensitive and quantitative detection of minimal residual disease (MRD) in bone marrow (BM) samples of children with acute lymphoblastic leukemia (ALL) is essential for evaluation of early treatment response. In this study, we evaluated whether the traumatic BM samplings can be replaced by peripheral blood (PB) samplings. MRD levels were analyzed in follow-up samples of 62 children with precursor-B-ALL (532 paired BM-PB samples) and 22 children with T-ALL (149 paired BM-PB samples) using real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T cell receptor gene rearrangements with sensitivities of 10(-3) to 10(-5) (one ALL cell in 10(3) to 10(5) normal cells). In 14 of the 22 T-ALL patients, detectable MRD levels were found in 67 paired BM-PB samples: in 47 pairs MRD was detected both in BM and PB, whereas in the remaining pairs very low MRD levels were detected in BM (n = 11) or PB (n = 9) only. The MRD levels in the paired BM-PB samples were very comparable and strongly correlated (r(s) = 0.849). Comparable results were obtained earlier by immunophenotyping in 26 T-ALL patients (321 paired BM-PB samples), which also showed a strong correlation between MRD levels in paired BM and PB samples (r(s) = 0.822). In 39 of the 62 precursor-B-ALL patients, MRD was detected in 107 BM-PB pairs: in 48 pairs MRD was detected in both BM and PB, in 47 pairs MRD was solely detected in BM (at variable levels), and in 12 pairs only the PB sample was MRD-positive at very low levels (

Normal IgM(-)IgD(+) CD38(+) B cells and IgM(-)IgD(+) multiple myelomas (MM) are characterized by Cmu deletion, biased Iglambda expression and hypermutated IgV regions. The predominant Iglambda usage has been proposed as resulting from secondary Ig gene rearrangements during extensive clonal expansion in the germinal center environment. Here, four cases of IgDlambda MM were studied to address the question of light chain receptor revision in a 'single cell' model. Detailed analyses of both IGK and IGL alleles of each case were performed by Southern blotting, (RT-) PCR, and sequencing. The expressed IgV genes were extensively mutated and Cmu deletion was confirmed in two cases. In addition, in the four MM a total of six non-functional deletional IGK rearrangements were identified, which proved to be unmutated. We conclude that IgD myelomas indeed originate from (post) germinal center B cells in which, in spite of the fact that they are hypermutated, there is no evidence of receptor revision.

Several studies have shown that quantitative detection of minimal residual disease (MRD) predicts clinical outcome in childhood acute lymphoblastic leukemia (ALL). In this report we investigated the applicablility of T cell receptor gamma (TCRG) gene rearrangements as targets for MRD detection by real-time quantitative PCR analysis. Seventeen children with precursor-B-ALL and 15 children with T-ALL were included in this study. Using an allele-specific (ASO) forward primer in combination with germline Jgamma reverse primers and Jgamma TaqMan probes, a reproducible sensitivity of < or =10(-4) (defined by strict criteria) was obtained in only four out of 19 (21%) TCRG gene rearrangements in precursor-B-ALL patients and in 10 out of 15 (67%) TCRG gene rearrangements in T-ALL patients. The main reason for not obtaining a reproducible sensitivity of < or =10(-4) in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of < or =10(-4) (defined by less strict criteria) was obtained in 42% of TCRG gene rearrangements in precursor-B-ALL patients. The number of inserted nucleotides was significantly higher in T-ALL (mean: 8.5) as compared to precursor-B-ALL (mean: 6.8) and appeared to be the most important predictor for reaching a reproducible sensitivity < or =10(-4). The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vgamma member forward primers and TaqMan probes did not clearly improve the overall results. Nevertheless, RQ-PCR analysis of TCRG gene rearrangements in follow-up samples obtained from 12 ALL patients showed the applicability of this method for MRD detection. We conclude that RQ-PCR analysis of TCRG gene rearrangements can be used for the detection of MRD, but that sensitivities might be limited due to non-specific amplification. This method is applicable in the majority of T-ALL patients and in almost half of precursor-B-ALL patients, particularly when used as second-choice target for confirmation of the MRD results obtained via the first-choice target.

About 30% of T cell acute lymphoblastic leukemias (T-ALL) carry TAL1 gene aberrations. In the majority of cases (approximately 25%), this concerns a submicroscopic deletion of approximately 90 kb in chromosome region 1p32, which deletes the coding regions of the SIL gene and the untranslated region of the TAL1 gene, thereby placing the TAL1 gene under control of the SIL promoter region. Translocation (1;14)(p32;q11) involving the TAL1 gene occurs at a much lower frequency (3%), whereas some other rare variant translocations have been described as well. In this study we developed a set of TAL1 FISH probes based on the split-signal FISH principle that enables detection of both types of TAL1 gene aberrations in single test. For this purpose, one probe was designed downstream of the TAL1 gene (TAL1-D) and the second probe in the region upstream of the TAL1 gene, partly covering the SIL gene (SIL-U). We show that this split-signal FISH probe set allows reliable detection of the unaffected SIL-TAL1 gene region with a fusion signal, SIL-TAL1 fusion genes with loss of the SIL-U signal, and TAL1 gene translocations with a split-signal, independent of the involved partner gene.

PCR-based monitoring of minimal residual disease (MRD) in acute leukemias can be achieved via detection of fusion gene transcripts of chromosome aberrations or detection of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements. We wished to assess whether both PCR targets are complementary in acute myeloid leukemia (AML). We investigated 105 consecutive AML cases for the presence of fusion gene transcripts by reverse transcriptase polymerase chain reaction (RT-PCR): AML1-ETO associated with t(8;21), CBFB-MYH11 with inv(16), PML-RARA with t(15;17), BCR-ABL with t(9;22), and MLL-AF4 with t(4;11). In 17 out of 105 AML cases (16%), fusion gene transcripts were found. Ninety-five of these AML patients (13 with fusion gene transcripts) were also investigated for the presence of IGH, IGK, TCRG and TCRD rearrangements by Southern blot and/or PCR heteroduplex analysis and sequencing. In nine out of 95 patients (9.5%), such rearrangements were found. Combined data revealed that only one patient with a fusion gene transcript had a coexistent Ig/TCR rearrangement. The nine AML patients with Ig/TCR rearrangements, as well as five additional AML patients from a previous study were investigated in more detail, revealing that Ig/TCR rearrangements in AML are immature and unusual. The presence of Ig/TCR rearrangements in AML did not correlate with RAG gene expression levels as determined by real-time quantitative PCR. In conclusion, fusion gene transcripts and Ig/TCR rearrangements are infrequent, but complementary MRD-PCR targets in AML.