Mj Willemse

Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present in 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde TaqMan-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of ⩽10−4 was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis (95%) are concerned. Furthermore, most IGK-Kde rearrangements (90%) can be used for sensitive detection of MRD (⩽10−4) by RQ-PCR analysis.

Molecular detection of minimal residual disease is a strong predictive factor of relapse in childhood B-lineage acute lymphoblastic leukemia with medium risk features. A case control study of the International BFM study group

The medium-risk B cell precursor acute lymphoblastic leukemia (ALL) accounts for 50–60% of total childhood ALL and comprises the largest number of relapses still unpredictable with diagnostic criteria. To evaluate the prognostic impact of minimal residual disease (MRD) in this specific group, a case control study was performed in patients classified and treated as medium (or intermediate)-risk according to the criteria of national studies (ALL-BFM 90, DCLSG protocol ALL-8, AIEOP-ALL 91), which includes a good day 7 treatment response. Standardized polymerase chain reaction (PCR) analysis of patient-specific immunoglobulin and T cell receptor gene (TCR) rearrangements were used as targets for semi-quantitative estimation of MRD levels: ⩾10−2, 10−3, ⩽10−4. Twenty-nine relapsing ALL patients were matched with the same number of controls by using white blood cell count (WBC), age, sex, and time in first complete remission, as matching factors. MRD was evaluated at time-point 1 (end of protocol Ia of induction treatment, ie 6 weeks from diagnosis) and time-point 2 (before consolidation treatment, ie 3 months from diagnosis). MRD-based high risk patients (⩾10−3 at both time-points) were more frequently present in the relapsed cases than in controls (14 vs 2), while MRD-based low risk patients (MRD negative at both time-points) (1 vs 18) showed the opposite distribution. MRD-based high risk cases experienced a significantly higher relapse rate than all other patients, according to the estimated seven-fold increase in the odds of failure, and a much higher rate than MRD-based low risk patients (OR = 35.7; P = 0.003). Using the Cox model, the prediction of the relapse-free interval at 4 years was 44.7%, 76.4% and 97.7% according to the different MRD categories. MRD-based risk group classification demonstrate their clinical relevance within the medium-risk B cell precursor ALL which account for the largest number of unpredictable relapses, despite the current knowledge about clinical and biological characteristics at diagnosis. Therefore, MRD detection during the first 3 months of follow-up can provide the tools to target more intensive therapy to those patients at true risk of relapse.

Regenerating normal B-cell precursors during and after treatment of acute lymphoblastic leukaemia: Implications for monitoring of minimal residual disease

We studied 57 childhood acute lymphoblastic leukaemia (ALL) patients who remained in continuous complete remission after treatment according to the Dutch Childhood Leukaemia Study Group ALL‐8 protocols. The patients were monitored at 18 time points during and after treatment [640 bone marrow (BM) and 600 blood samples] by use of cytomorphology and immunophenotyping for the expression of TdT, CD34, CD10 and CD19. Additionally, 60 BM follow‐up samples from six patients were subjected to clonality assessment via heteroduplex polymerase chain reaction (PCR) analysis of immunoglobulin V h‐J h gene rearrangements. We observed substantial expansions of normal precursor B cells in regenerating BM not only after maintenance therapy but also during treatment. At the end of the 2‐week intervals after consolidation and reinduction treatment, B‐cell‐lineage regeneration was observed in BM with a large fraction of immature CD34+/TdT+ B cells. In contrast, in regenerating BM after cessation of maintenance treatment, the more mature CD19+/CD10+ B cells were significantly increased, but the fraction of immature CD34+/TdT+ B cells was essentially smaller. Blood samples showed a profound B‐cell lymphopenia during treatment followed by a rapid normalization of blood B cells after treatment, with a substantial CD10+ fraction (10–30%). Heteroduplex PCR analysis confirmed the polyclonal origin of the expanded precursor B cells in regenerating BM. This information regarding the regeneration of BM is essential for the correct interpretation of minimal residual disease studies.

Precursor-B-ALL with D(H)-J(H) gene rearrangements have an immature immunogenotype with a high frequency of oligoclonality and hyperdiploidy of chromosome 14.

The IGH gene configuration was investigated in 97 childhood precursor-B-ALL patients at initial diagnosis. Rearrangements were found by Southern blotting in all but three patients (97%) and in 30 cases (31%) we observed oligoclonal IGH gene rearrangements. Heteroduplex PCR analysis revealed at least one clonal PCR product in all Southern blot-positive cases. In 89 patients (92%) complete V(D)J rearrangements were found, while incomplete DH–JH rearrangements occurred in only 21 patients (22%). In 5% of cases the DH–JH rearrangements were the sole IGH gene rearrangements. Sequence analysis of the 31 identified incomplete rearrangements revealed preferential usage of segments from the DH2, DH3 and DH7 families (78%). While DH2 and DH3 gene rearrangements occur frequently in normal B cells and B cell precursors, the relatively frequent usage of DH7–27 (19%) in precursor-B-ALL patients is suggestive of leukemic transformation during prenatal lymphopoiesis. Among JH gene segments in the incomplete DH–JH rearrangements, the JH6 segment was significantly overrepresented (61%). This observation together with the predominant usage of the most upstream DH genes indicates that many of the identified clonal DH–JH gene rearrangements in precursor-B-ALL probably represent secondary recombinations, having deleted pre-existing DH–JH joinings. The patients with incomplete DH–JH gene rearrangements were frequently characterized by hyperdiploid karyotype with additional copies of chromosome 14 and/or by IGH oligoclonality. The presence of incomplete DH–JH joinings was also significantly associated with a less mature immunogenotype: overrepresentation of VH6–1 gene segment usage, absence of biallelic TCRD deletions, and low frequency of TCRG gene rearrangements. This immature immunogenotype of precursor-B-ALL with incomplete IGH gene rearrangements was not associated with more aggressive disease.

Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not 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 103 to 105 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 (rs = 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 (rs = 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 (≤10−4). Furthermore, in the 48 double-positive pairs, MRD levels in BM and PB varied enormously with MRD levels in BM being up to 1000 times higher than in the corresponding PB samples. Consequently, BM samples cannot easily be replaced by PB sampling for MRD analysis in childhood precursor-B-ALL, in line with their BM origin. In T-ALL, which are of thymic origin, BM sampling might be replaced by PB sampling, because the dissemination of T-ALL cells to BM and PB appears to be comparable.

T cell receptor gamma ( TCRG ) gene rearrangements in T cell acute lymphoblastic leukemia reflect ‘end-stage’ recombinations: implications for minimal residual disease monitoring

The T cell receptor gamma (TCRG) gene configuration was established in a large series of 126 T cell acute lymphoblastic leukemia (T-ALL) patients using combined Southern blotting (SB) and heteroduplex PCR analyses. The vast majority of T-ALL (96%) displayed clonal TCRG gene rearrangements, with biallelic recombination in 91% of patients. A small immature subgroup of CD3− T-ALL (n = 5) had both TCRG genes in germline configuration, three of them having also germline TCRD genes. In five patients (4%) combined SB and PCR results indicated oligoclonality. In five rearrangements detected by SB, the Vγ gene segment could not be identified suggesting illegitimate recombination. Altogether, 83% of TCRG gene rearrangements involved either the most upstream Vγ2 gene (including four cases with interstitial deletion of 170 bp in Vγ2) and/or the most downstream Jγ2.3 segment, which can be perceived as ‘end-stage’ recombinations. Comparative analysis of the TCRG gene configuration in the major immunophenotypic subgroups indicated that TCRγδ+ T-ALL display a less mature immunogenotype as compared to TCRαβ+ and most CD3− cases. This was reflected by a significantly increased usage of the more downstream Vγ genes and the upstream Jγ1 segments. Comparison between adult and pediatric T-ALL patients did not show any obvious differences in TCRG gene configuration. The high frequency, easy detectability, rare oligoclonality, and frequent ‘end-stage’ recombinations make TCRG gene rearrangements principal targets for PCR-based detection of minimal residual disease (MRD) in T-ALL. We propose a simple heteroduplex PCR strategy, applying five primer combinations, which results in the detection of approximately 95% of all clonal TCRG gene rearrangements in T-ALL. This approach enables identification of at least one TCRG target for MRD monitoring in 95% of patients, and even two targets in 84% of T-ALL.

Fusion gene transcripts and Ig/TCR gene rearrangements are complementary but infrequent targets for PCR-based detection of minimal residual disease in acute myeloid leukemia

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.

Clearance of maternal leukaemic cells in a neonate

A 36‐week pregnant woman was diagnosed with acute lymphoblastic leukaemia. Delivery was initiated prematurely, and a healthy child was born. Cord blood and peripheral blood samples from the neonate (obtained at 6 weeks, 3 months and 6 months) were analysed for the presence of minimal residual disease by polymerase chain reaction analysis of a leukaemia‐specific IGH gene rearrangement and the E2A–PBX1 fusion gene transcript. In the cord blood sample, a tumour load of ≈ 4 × 10−4 was found, whereas all later blood samples were negative. Our data indicate that the maternal leukaemic cells did not engraft in the neonate.

Two consecutive immunophenotypic switches in a child with immunogenotypically stable acute leukaemia

A 12‐year‐old girl presented with a CD33+ precursor B‐acute lymphoblastic leukaemia (ALL) and seemed to respond well to ALL treatment. However, 2 weeks after diagnosis her leucocyte count rose rapidly with a predominance of myeloid blasts with M5b morphology and CD19+ myeloid immunophenotype. Acute myeloid leukaemia (AML) treatment was started and remission was achieved after one course of chemotherapy; the AML treatment was continued for 6 months. Two months after cessation of chemotherapy, the patient developed a bone marrow relapse, this time with an undifferentiated blast morphology and a precursor B immunophenotype. Molecular analysis of the immunoglobulin and T‐cell receptor genes showed several clonal gene rearrangements at diagnosis: two IGH, two IGK and two TCRD gene rearrangements. All rearrangements were also detected during the AML phase of the disease, suggesting a phenotypic shift of the same leukaemia. At relapse, 8 months later, all rearrangements were preserved except for one TCRD (Vδ2–Dδ3) rearrangement. The first phenotypic shift in the genotypically stable leukaemia was remarkably fast. The most probable explanation for our observations is an oncogenic event in an undifferentiated haematopoetic progenitor clone, with a highly versatile phenotype.