The Most Probable Limit of Detection (MPL) for rapid microbiological methods.

Classical microbiological methods have nowadays unacceptably long cycle times. Rapid methods, available on the market for decades, are already applied within the clinical and food industry, but the implementation in pharmaceutical industry is hampered by for instance stringent regulations on validation and comparability with classical methods. Equivalence studies become less relevant when rapid methods are able to detect only one single microorganism. Directly testing this capability is currently impossible due to problems associated with preparing a spiked sample with low microbial counts. To be able to precisely estimate the limit of detection of rapid absence/presence tests, the method of the most probable limit is presented. It is based on three important elements; a relatively precise quantity of microorganisms, a non-serial dilution experiment and a statistical approach. For a set of microorganisms, a limit of detection of one was demonstrated using two different rapid methods.

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 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 (

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 < or =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 (< or =10(-4)) by RQ-PCR analysis.

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.

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 D(H)-J(H) rearrangements occurred in only 21 patients (22%). In 5% of cases the D(H)-J(H) rearrangements were the sole IGH gene rearrangements. Sequence analysis of the 31 identified incomplete rearrangements revealed preferential usage of segments from the D(H)2, D(H)3 and D(H)7 families (78%). While D(H)2 and D(H)3 gene rearrangements occur frequently in normal B cells and B cell precursors, the relatively frequent usage of D(H)7-27 (19%) in precursor-B-ALL patients is suggestive of leukemic transformation during prenatal lymphopoiesis. Among J(H) gene segments in the incomplete D(H)-J(H) rearrangements, the J(H)6 segment was significantly overrepresented (61%). This observation together with the predominant usage of the most upstream D(H) genes indicates that many of the identified clonal D(H)-J(H) gene rearrangements in precursor-B-ALL probably represent secondary recombinations, having deleted pre-existing D(H)-J(H) joinings. The patients with incomplete D(H)-J(H) gene rearrangements were frequently characterized by hyperdiploid karyotype with additional copies of chromosome 14 and/or by IGH oligoclonality. The presence of incomplete D(H)-J(H) joinings was also significantly associated with a less mature immunogenotype: overrepresentation of V(H)6-1 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.

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 approximately 4 x 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 (Vdelta2-Ddelta3) 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 haematopoietic progenitor clone, with a highly versatile phenotype.

Molecular discrimination between relapsed and secondary acute lymphoblastic leukemia: proposal for an easy strategy.

BACKGROUND: Discrimination between late relapse of acute lymphoblastic leukemia (ALL) and secondary ALL might be clinically important, because the former might still respond favorably to chemotherapy and/or bone marrow transplantation, whereas secondary ALL is associated with poor prognosis. PROCEDURE: We present a pre-B-ALL patient in whom disease recurred 2 years after completion of treatment. Differences in cytomorphology and immunophenotyping raised a suspicion of secondary ALL. We performed detailed molecular studies of immunoglobulin and T-cell receptor genes for discrimination between relapsed and secondary ALL. RESULTS: Southern blot analysis showed an oligoclonal immunoglobulin heavy chain (IGH) gene configuration at diagnosis and a monoclonal configuration at relapse. The size of one of the rearranged bands at relapse was identical to one of the faint rearranged bands at diagnosis. However, heteroduplex PCR analysis demonstrated that none of the clonal IGH gene rearrangements at diagnosis and at relapse was fully identical. Sequencing of several clonal PCR products revealed an identical DH6-13<-->JH6b junction shared by two different rearrangements at diagnosis and one rearrangement at relapse, thereby proving the clonal relationship between diagnosis and late relapse in this patient. CONCLUSIONS: We propose a stepwise molecular approach for discrimination between relapsed and secondary ALL based on the rapid and cheap heteroduplex PCR technique, including mixing of clonal (homoduplex) PCR products identified at diagnosis and at relapse. Direct sequencing and comparative sequence analysis of IGH gene rearrangements at diagnosis and at relapse should be regarded as an ultimate standard, but can be limited to the rare cases, in which no identical clonal PCR products at diagnosis and at relapse were detected with the mixed heteroduplex PCR analyses.

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: > or =10(-2), 10(-3), < or =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 (> or =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 VH-JH 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.

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

T cell receptor gamma (TCRG) gene rearrangements in T cell acute lymphoblastic leukemia refelct '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 TALL (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 Vgamma gene segment could not be identified suggesting illegitimate recombination. Altogether, 83% of TCRG gene rearrangements involved either the most upstream Vgamma2 gene (including four cases with interstitial deletion of 170 bp in Vgamma2) and/or the most downstream Jgamma2.3 segment, which can be perceived as 'end-stage' recombinations. Comparative analysis of the TCRG gene configuration in the major immunophenotypic subgroups indicated that TCRgammadelta+ T-ALL display a less mature immunogenotype as compared to TCRalphabeta+ and most CD3- cases. This was reflected by a significantly increased usage of the more downstream Vgamma genes and the upstream Jgamma1 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.

Unusual immunoglobulin and T-cell receptor gene rearrangement patterns in acute lymphoblastic leukemias.

Immunoglobulin (Ig) and T-cell receptor (TCR) genes are rearranged in virtually all acute lymphoblastic leukemia (ALL) cases. However, the recombination patterns display several unusual features as compared to normal lymphoid counterparts. Cross-lineage gene rearrangements occur in more than 90% of precursor-B-ALL and in approximately 20% of T-ALL, whereas they are rare in normal lymphocytes. Approximately 25-30% of the Ig and TCR gene rearrangements at diagnosis are oligoclonal, and can undergo continuing or secondary recombination events during the disease course. Based on our extensive molecular studies we hypothesize that the unusual Ig and TCR gene rearrangements in ALL occur as an early postoncogenic event resulting from the continuing V(D)J recombinase activity on accessible gene loci. This hypothesis is on the one hand supported by the virtual absence of cross-lineage gene rearrangements in normal lymphocytes and mature lymphoid malignancies and on the other hand by the presence of oligoclonality and secondary Ig and TCR gene rearrangements in ALL.

Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor alpha beta lineage.

Rearranged IGH genes were detected by Southern blotting in 22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and 31% (8/26) of these cases, respectively. IGH gene rearrangements were found in 19% (13/69) of CD3(-) T-ALL and in 50% of TCRgammadelta+ T-ALL (12/24), whereas only a single TCRalpha beta+ T-ALL (1/25) displayed a monoallelic IGH gene rearrangement. The association with the T-cell receptor (TCR) phenotype was further supported by the striking relationship between IGH and TCR delta (TCRD) gene rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelic TCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex polymerase chain reaction (PCR) analysis with VH and DH family-specific primers in combination with a JH consensus primer showed a total of 39 clonal products, representing 7 (18%) VH-(DH-)JH joinings and 32 (82%) DH-JH rearrangements. Whereas the usage of VH gene segments was seemingly random, preferential usage of DH6-19 (45%) and DH7-27 (21%) gene segments was observed. Although the JH4 and JH6 gene segments were used most frequently (33% and 21%, respectively), a significant proportion of joinings (28%) used the most upstream JH1 and JH2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of incomplete DH-JH rearrangements, the frequent usage of the more downstream DH6-19 and DH7-27 gene segments, and the most upstream JH1 and JH2 gene segments suggests a predominance of immature IGH rearrangements in immature (non-TCRalpha beta+) T-ALL as a result of continuing V(D)J recombinase activity. More mature alpha beta-lineage T-ALL with biallelic TCRD gene deletions apparently have switched off their recombination machinery and are less prone to cross-lineage IGH gene rearrangements. The combined results indicate that IGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes and completed TCR alpha (TCRA) gene rearrangements.

Cross-lineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease.

A large series of 202 childhood precursor-B cell acute lymphoblastic leukemia (ALL) patients was analyzed by Southern blotting (SB) for cross-lineage rearrangements and/or deletions in the T cell receptor TCRB, TCRG and TCRD loci. In 93% (187/201) of the precursor-B-ALL patients one or more genes were rearranged and/or deleted. TCRB gene rearrangements were found in 35% (69/196), TCRG gene rearrangements in 59% (113/192), TCRD gene rearrangements in 55% (112/202), and isolated monoallelic or biallelic deletions of TCRD loci in 34% (68/202) of the cases. TCRB gene rearrangements involved exclusively the Jbeta2 locus with complete V(D)Jbeta2 joinings in 53% of gene rearrangements and incomplete Dbeta-Jbeta2 gene rearrangements in 33%. TCRG gene rearrangements frequently occurred on both alleles (65% of cases) and in approximately 70% concerned rearrangements to Jgamma1 gene segments. Most rearranged TCRD alleles (80%) represented incomplete Vdelta2-Ddelta3 or Ddelta2-Ddelta3 gene rearrangements, while the remaining TCRD gene rearrangements remained unidentified. Subsequently, we evaluated, whether heteroduplex PCR analysis of rearranged TCRG and TCRD genes can be used for reliable identification of PCR targets for detection of minimal residual disease (MRD). The concordance between SB and heteroduplex PCR analysis for detection of the various types of clonal TCRG and TCRD gene rearrangements ranged between 78% and 87%. The discrepancies could be assigned to the presence of 'atypical' TCRD gene rearrangements or translocations only detectable by SB, but also to efficient PCR-based detection of rearrangements derived from small subclones, which are difficult to detect with SB. Indications for oligoclonality were observed in 38% and 30% of patients with TCRG and TCRD gene rearrangements, respectively, which is comparable to the frequency of oligoclonality in IGH locus. Based on the combined data it was possible to reduce the broad panel of six TCRD and 12 TCRG primer combinations for MRD studies to two TCRD combinations (Vdelta2-Ddelta3 and Ddelta2-Ddelta3) and six TCRG combinations (VgammaI, VgammaII, VgammaIV family-specific primers with Jgamma1.1/2.1 and Jgamma1.3/2.3 primers) resulting in the detection of 80% and 97% of all TCRD and TCRG gene rearrangements, respectively. Finally, the heteroduplex PCR data indicate that MRD monitoring with TCRG and/or TCRD targets is possible in approximately 80% of childhood precursor-B-ALL patients; approximately 55% of patients even have two TCRG and/or TCRD targets.

Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.

It is now widely accepted that the detection of minimal residual disease (MRD) has prognostic value in acute leukemia. However clinical MRD studies need standardized techniques. Therefore, several European laboratories have aligned their goals and performed comparative studies to achieve optimization and standardization of MRD techniques. This was achieved via the BIOMED-1 Concerted Action "Investigation of minimal residual disease in acute leukemia: International standardization and clinical evaluation." This report describes the development of PCR primers and protocols for the detection of MRD in acute lymphoblastic leukemia (ALL) using clone-specific junctional regions of immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. A total of 54 primers was developed (1) to amplify rearrangements of the TCRD, TCRG, and IGK (Kde) genes as well as TAL1 deletions; (2) to sequence the junctional regions and breakpoint fusion regions; and (3) to perform MRD detection in bone marrow or peripheral blood samples during follow-up of ALL patients. Protocols were established to identify PCR targets at diagnosis by performing 25 PCR reactions per patient using appropriate positive and negative controls. Standardized protocols were developed for MRD monitoring via single amplification of the PCR target followed by dot blot hybridization with the corresponding patient-specific junctional region probe. In addition, alternative approaches were designed for cases where the target sensitivity of at least 10(-4) was not obtained. The standardization described here of MRD-PCR techniques is essential for the process of translating MRD research into clinical practice.

Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes.

Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-based risk group assignment was based on the kinetics of tumor reduction. Consequently, the level of MRD has to be defined precisely in follow-up samples. However, current PCR methods do not allow easy and accurate quantification. We have tested 'real-time' quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle ('real-time') by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the design of patient-specific TaqMan probes at the position of the junctional regions. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-blot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.

Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood.

BACKGROUND: Sensitive techniques for detection of minimal residual disease (MRD) at degrees of one leukaemic cell per 10(3)-10(6) cells (10(-3)-10(-6)) during follow-up of children with acute lymphoblastic leukaemia (ALL) can provide insight into the effectiveness of cytotoxic treatment. However, it is not yet clear how information on MRD can be applied to treatment protocols. METHODS: We monitored 240 patients with childhood ALL who were treated according to national protocols of the International BFM Study Group. 60 patients relapsed and the patients in continuous complete remission (CCR) had a median event-free follow-up of 48 months. Bone-marrow samples were collected at up to nine time points during and after treatment. Standardised PCR analysis of patient-specific immunoglobulin and T-cell receptor gene rearrangements and TAL1 deletions were used as targets for semiquantitative estimation of MRD. Amount of MRD was classed as 10(-2) or more, 10(-3), and 10(-4) or less. FINDINGS: MRD negativity at the various follow-up times was associated with low relapse rates (3-15% at 3 years), but five-fold to ten-fold higher relapse rates (39-86% at 3 years) were found in MRD-positive patients. The distinct degrees of MRD appeared to have independent prognostic value (p [trend]<0.001) at all separate time points, especially at the first two time points (at the end of induction treatment and before consolidation treatment). At these two time points a high degree of MRD (> or = 10(-2)) was associated with a three-fold higher relapse rate when compared with patients with a low degree of MRD (< or = 10(-4)). At later time points (including the end of treatment) even a low degree of MRD was associated with a poor outcome. Positivity in patients in CCR after treatment was rare (< 1%). With the combined MRD information from the first two follow-up time points, it was possible to recognise three different risk groups--55 (43%) were in a low-risk group and had a 3-year relapse rate of only 2% (95% CI 0.05-12%); 19 (15%) were in a high-risk group and had a relapse rate of 75% (55-95%); and 55 (43%) were in an intermediate-risk group and had a 3-year relapse rate of 23% (13-36%). INTERPRETATION: Our collaborative MRD study shows that monitoring patients with childhood ALL at consecutive time points gives clinically relevant insight into the effectiveness of treatment. Combined information on MRD from the first 3 months of treatment distinguishes patients with good prognoses from those with poor prognoses, and this helps in decisions whether and how to modify treatment.