The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Minimal residual disease and outcome characteristics in infant KMT2A-germline acute lymphoblastic leukaemia treated on the Interfant-06 protocol.

BACKGROUND: The outcome of infants with KMT2A-germline acute lymphoblastic leukaemia (ALL) is superior to that of infants with KMT2A-rearranged ALL but has been inferior to non-infant ALL patients. Here, we describe the outcome and prognostic factors for 167 infants with KMT2A-germline ALL enrolled in the Interfant-06 study. METHODS: Univariate analysis on prognostic factors (age, white blood cell count at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI; n = 163). Bone marrow minimal residual disease (MRD) was measured in 73 patients by real-time quantitative polymerase chain reaction at various time points (EOI, n = 68; end of consolidation, n = 56; and before OCTADAD, n = 57). MRD results were classified as negative, intermediate (<5∗10-4), and high (≥5∗10-4). RESULTS: The 6-year event-free and overall survival was 73.9% (standard error [SE] = 3.6) and 87.2% (SE = 2.7). Relapses occurred early, within 36 months from diagnosis in 28 of 31 (90%) infants. Treatment-related mortality was 3.6%. Age <6 months was a favourable prognostic factor with a 6-year disease-free survival (DFS) of 91% (SE = 9.0) compared with 71.7% (SE = 4.2) in infants >6 months of age (P = 0.04). Patients with high EOI MRD ≥5 × 10-4 had a worse outcome (6-year DFS 61.4% [SE = 12.4], n = 16), compared with patients with undetectable EOI MRD (6-year DFS 87.9% [SE = 6.6], n = 28) or intermediate EOI MRD <5 × 10-4 (6-year DFS 76.4% [SE = 11.3], n = 24; P = 0.02). CONCLUSION: We conclude that young age at diagnosis and low EOI MRD seem favourable prognostic factors in infants with KMT2A-germline ALL and should be considered for risk stratification in future clinical trials.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Childhood secondary ALL after ALL treatment.

Data on secondary acute lymphoblastic leukaemia (sALL) following ALL treatment are very rare. However, the incidence might be underestimated as sALLs without a significant lineage shift might automatically be diagnosed as relapses. Examination of immunoglobulin and T-cell receptor gene rearrangements brought a new tool that can help in discrimination between relapse and sALL. We focused on the recurrences of childhood ALL to discover the real frequency of the sALL after ALL treatment. We compared clonal markers in matched presentation and recurrence samples of 366 patients treated according to the Berlin-Frankfurt-Munster (BFM)-based protocols. We found two cases of sALL and another three, where the recurrence is suspicious of being sALL rather than relapse. Our proposal for the 'secondary ALL after ALL' diagnostic criteria is as follows: (A) No clonal relationship between diagnosis and recurrence; (B) significant immunophenotypic shift--significant cytogenetic shift--gain/loss of a fusion gene. For the sALL (A) plus at least one (B) criterion should be fulfilled. With these criteria, the estimated frequency of the sALL after ALL is according to our data 0.5-1.5% of ALL recurrences on BFM-based protocols. Finally, we propose a treatment strategy for the patients with secondary disease.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

An activating mutation of GNB1 is associated with resistance to tyrosine kinase inhibitors in ETV6-ABL1-positive leukemia.

Leukemias harboring the ETV6-ABL1 fusion represent a rare subset of hematological malignancies with unfavorable outcomes. The constitutively active chimeric Etv6-Abl1 tyrosine kinase can be specifically inhibited by tyrosine kinase inhibitors (TKIs). Although TKIs represent an important therapeutic tool, so far, the mechanism underlying the potential TKI resistance in ETV6-ABL1-positive malignancies has not been studied in detail. To address this issue, we established a TKI-resistant ETV6-ABL1-positive leukemic cell line through long-term exposure to imatinib. ETV6-ABL1-dependent mechanisms (including fusion gene/protein mutation, amplification, enhanced expression or phosphorylation) and increased TKI efflux were excluded as potential causes of resistance. We showed that TKI effectively inhibited the Etv6-Abl1 kinase activity in resistant cells, and using short hairpin RNA (shRNA)-mediated silencing, we confirmed that the resistant cells became independent from the ETV6-ABL1 oncogene. Through analysis of the genomic and proteomic profiles of resistant cells, we identified an acquired mutation in the GNB1 gene, K89M, as the most likely cause of the resistance. We showed that cells harboring mutated GNB1 were capable of restoring signaling through the phosphoinositide-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, whose activation is inhibited by TKI. This alternative GNB1K89M-mediated pro-survival signaling rendered ETV6-ABL1-positive leukemic cells resistant to TKI therapy. The mechanism of TKI resistance is independent of the targeted chimeric kinase and thus is potentially relevant not only to ETV6-ABL1-positive leukemias but also to a wider spectrum of malignancies treated by kinase inhibitors.

Acute lymphoblastic leukemia incidence during socioeconomic transition: selective increase in children from 1 to 4 years.

Pre-school acute lymphoblastic leukemia (ALL) peak is consistent in developed but not in developing countries and its magnitude apparently correlates with the socioeconomic status. A population-based study describing ALL incidence during socioeconomic transition has been lacking. Central European post-communist countries (with very low foreign migration and centralized statistics) offer reliable data for the period before and during major socioeconomic changes. Population-based data on Czech ALL patients younger than 18 years were taken from two independent Czech national registries partially overlapping in time (1980-1998, n = 1236 and 1991-1999, n = 570). During the 1980s and 1990s, ALL incidence among children 1-4 years increased 1.5 times (P = 0.01). This increase was more prominent in females than in males (slopes 0.13 and 0.09, P values 0.03 and >0.05, respectively). No significant change was observed in other age groups (0, 5-9, 10-14, 15-17 years or all others combined). We discuss possible underlying socioeconomic factors including infant care and breast-feeding, hygiene, birth order, industry and pollution. Moreover, we try to pinpoint the immunophenotypic/molecular-genetic subsets of ALL that might be socioeconomically affected. Selective increase of ALL in children 1-4 years old provides epidemiological evidence that etiology and/or trigger mechanisms are different for a considerable proportion of these children and that these mechanisms are exogenous.

TEL/AML1 positivity in childhood ALL: average or better prognosis? Czech Paediatric Haematology Working Group.

The presence of TEL/AML1 fusion gene in childhood acute lymphoblastic leukaemia (ALL) defines a subgroup of patients with better than average outcome. However, the prognostic significance of this aberration has recently been disputed by the Berlin-Frankfurt-Münster (BFM) study group due to its relatively high incidence found in relapsed patients (19.6% and 21.9%, in two cohorts). In contrast, only four out of 45 (8.9%) unselected relapsed patients (all of whom had been treated according to BFM protocols) in the Czech Republic carry this fusion. From March 1995 to June 1998, 41 out of 190 (21.6%) newly diagnosed children with ALL were TEL/AML1-positive. There is a statistically significant difference between the incidence of TEL/AML1 fusion at diagnosis and at relapse within our group (P = 0.035). Interim analysis of the minimal residual disease (MRD) detection shows heterogeneity within the group of newly diagnosed TEL/AML1-positive leukaemias--10 out of 24 patients tested at the end of induction therapy had detectable levels of MRD. However, only one of these patients reached relapse-predictive level (10(-3)) of MRD. In conclusion, we corroborate low frequency of TEL/AML1 positivity among relapsed patients with ALL among Czech children who are treated by the BFM protocols. Moreover, we demonstrate different patterns of bone marrow clean-up in TEL/AML1-positive patients.

Aberrant expression of KOR-SA3544 antigen in childhood acute lymphoblastic leukemia predicts TEL-AML1 negativity. The Pediatric Hematology Working Group in the Czech Republic.

The antigen KOR-SA3544 is physiologically expressed exclusively on granulocytes. Aberrant expression of KOR-SA3544 has been invariably found in BCR/ABL-positive acute lymphoblastic leukemia (ALL) and in some BCR/ABL-negative ALL. In an interim analysis of a prospective clinical and cytometric study data of 73 children with newly diagnosed or relapsed ALL with and without TEL/AML1 fusion are presented. KOR-SA3544 expression over 3% was detected in the majority of TEL/AML1-negative patients with newly diagnosed common or preB ALL (19 of 31) and not in TEL/AML1-positive patients (0 of 18, P < 0.0001). The level of expression of KOR-SA3544 was 0.02-90% (median 6.0%) and 0.03-2.4% (median 0.23%) in TEL/AML1-negative and TEL/AML1-positive patients, respectively. All five newly diagnosed patients with DNA index > or =1.16 and <1.6 exhibited high levels of KOR-SA3544 expression. Membrane expression of CD79a was found to correlate with TEL/AML1 negativity, although less significantly than KOR-SA3544 (P = 0.03). Furthermore, our data confirm that TEL/AML1 positivity correlates with non-hyperdiploidy and low presenting age. In conclusion, KOR-SA3544 correlated strongly with TEL/AML1 negativity, it was a better predictor of TEL/AML1 status than other factors tested and was found at high levels in hyperdiploidy. In combination with age, KOR-SA3544 predicted TEL/AML1 status in 86% newly diagnosed preB/cALL patients.

Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry.

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analysed WT1 expression at presentation in an unselected group of 47 childhood AML patients using real-time quantitative reverse-transcription PCR. We also showed that within the first 30 h following aspiration RQ-RT-PCR results were not influenced by transportation time. We observed lower levels of WT1 transcript in AML M5 (P = 0.0015); no association was found between expression levels and sex, initial leukocyte count and karyotype-based prognostic groups. There was significant correlation between very low WT1 expression at presentation and excellent outcome (EFS P = 0.0014). Combined analysis of WT1 levels, three-colour flow cytometry residual disease detection and the course of the disease in 222 samples from 28 children with AML showed remarkable correlation. Fourteen patients expressed high WT1 levels at presentation. In eight of them, who suffered relapse or did not reach complete remission, dynamics of WT1 levels clearly correlated with the disease status and residual disease by flow cytometry. We conclude that very low WT1 levels at presentation represent a good prognostic factor and that RQ-RT-PCR-based analysis of WT1 expression is a promising and rapid approach for monitoring of MRD in approximately half of paediatric AML patients.

Importance of using comparative genomic hybridization to improve detection of chromosomal changes in childhood acute lymphoblastic leukemia.

We used comparative genomic hybridization (CGH) and conventional cytogenetics (CC) to define chromosomal changes and to evaluate the usefulness of CGH in 65 patients having childhood acute lymphoblastic leukemia (ALL). Subsequently, fluorescence in situ hybridization (FISH) was used to evaluate the CGH and cytogenetic results. Comparative genomic hybridization revealed DNA copy number changes in 49 (75%) patients (including 7 patients with unsuccessful cytogenetics and 2 patients with normal karyotype). A total of 85 losses and 195 gains were detected. The most commonly gained chromosomes were 21 (35%), X (31%), 18 (27%), 10 (26%), 6 (25%), 17 (25%), 4 (23%), and 14 (22%). Losses were most frequently observed on chromosomes 9p (18%) and 12p (11%). Other losses were detected on chromosomes 13q (9%), 6q (9%), 7p (8%), and chromosome X (6%). Conventional cytogenetics revealed chromosomal changes in 53 (82%) patients. The employment of CGH and FISH together with CC analysis revealed chromosomal changes in 62 (95%) of the childhood ALL patients investigated. The CGH completed CC results in 36 patients; in 9 patients, the changes escaped detection without using CGH. The results of our study were compared to 6 other CGH studies previously reported. Our observations underline the benefits of supplementing routine cytogenetic investigation in childhood ALL by FISH and CGH, because small unbalanced changes may escape detection when conventional cytogenetics is the only diagnostic method used.

[The role of TEL and AML1 genes in the pathogenesis of hematologic malignancies]. / Uloha genu TEL a AML1 v patogenezi hematologickych malignit.

TEL and AML1 genes occur in a markedly high number of different aberrations in haematological malignancies. Besides the AML1, TEL is often fused to genes, which encod thyrosin-kinases. AML1 gene is a part of CBF transcription factor. AML1 can be altered in childhood acute lymphoblastic leukaemia (ALL) and also in a substantial number of acute myeloid leukaemias (most frequently as an AML1/ETO fusion). TEL/AML1 fusion gene (derived from t(12;21)(p13;q22) translocation) became recently one of the most important genetic aberrations in children with ALL. TEL/AML1 act presumably as dominant inhibitors of the second AML1 allele and thus they block transcription of genes dependent on CBF factor. Childhood ALL with TEL/AML1 hybrid gene is very frequent (approximately 22% of overall childhood ALL in the Czech Republic) and patients with this fusion form relatively homogenous group. These children are diagnosed mostly in pre-school age as a B cell precursor leukaemias and they have very good treatment results.

[Detection of BCR/ABL, MLL/AF4 and TEL/AML1 hybrid genes and monitoring of minimal residual disease in pediatric patients with acute lymphoblastic leukemia]. / Detekce hybridních genu BCR/ABL, MLL/AF4 a TEL/AML1 a sledování minimální reziduální nemoci u detských pacientu s akutní lymfoblastickou leukémií.

BACKGROUND: The BCR/ABL and MLL/AF4 fusion genes--resulting from t(9;22)(q34;q11) and t(4;11)(q21;q23) translocations, respectively--are considered as a high risk prognostic factors in children with acute lymphoblastic leukaemia (ALL). Their presence in malignant cells indicates patient for the most intensive antileukaemic therapy regardless of the other criteria. In contrast, the most common non-random chromosomal aberration in paediatric ALL--translocation t(12;21)(q12;q22)--is associated with a favourable prognosis. The examination of these rearrangements is important for the stratification of patients to the risk groups and also provides the most sensitive and specific tool for minimal residual disease (MRD) follow-up. METHODS AND RESULTS: This study comprises 241 patients with ALL from Czech and Slovak Republics younger than 18 years at diagnosis. They were examined for presence of m-RNA of fusion genes BCR/ABL, MLL/AF4 and TEL/AML1 by reverse transcriptase-polymerase chain reaction (RT-PCR) method. Seven out of 197 (3.6%) carried MLL/AF4 fusion gene, but among infants it was 56% (5 out of 9). BCR/ABL positivity was found in 2.5% (7 out of 240) and TEL/AML1 in 21.7% (41 out of 189) cases. Event free survival (EFS) curves demonstrate the clinical impact of these hybrid genes on patients' prognosis. Moreover, we present the possibility of the monitoring of MRD levels in follow-up samples of these patients. CONCLUSIONS: All particular rearrangements were found only in a cohort of patients with B-precursor ALL (or hybrid leukaemia), which constitutes 85% of our group. Presence of BCR/ABL or MLL/AF4 fusion gene is associated with poor prognosis and is indispensable condition for correct stratification of patients to the risk groups according to treatment protocols. Hybrid gene TEL/AML1 defines subgroup of children with better prognosis and due to its high frequency provides us with a very useful tool for MRD detection.

[Fluorescent in situ hybridization (FISH) in the diagnosis of acute childhood lymphatic leukemia (ALL)]. / Fluorescencní in situ hybridizace (FISH) v diagnostice detských akutních lymfatických leukémií (ALL).

Classical cytogenetic analysis plays an important role in the diagnosis, classification, therapy monitoring and prognosis of patients with leukemia. Many recurrent cytogenetic abnormalities with major prognostic values have been described in childhood ALL. Hyperdiploidy and/or t(12;21) are associated with good prognosis, whereas t(9;22) and/or rearrangements of MLL gene correlate with poor outcome and therefore early detection of these abnormalities is very important. FISH can overcome some limitations of conventional cytogenetic and molecular-genetic analyses and due to high sensitivity specific chromosomal aberrations in mitoses and/or interphase nuclei can be detected. In the Center of Oncocytogenetics of the 3rd Medical Department for assessment of hyperdiploidy and structural rearrangements we use double-color FISH with centromeric and/or locus-specific probes and complex aberrations are ascertained by whole chromosome painting probes and multicolor FISH. Among 275 children with ALL examined during the last 8 years by different FISH methods we found seven patients with translocation t(9;22) and 14 patients with MLL rearrangements in bone marrow cells. Since 1988 we focus on detection of hyperdiploidy and/or t(12;21). High hyperdiploidy was found in 35 children, 10 of them had further complex rearrangements. Translocation t(12;21) was proved in 37 patients and complex rearrangements were found in 22 of them. FISH, cytogenetic and molecular-genetic analyses become obligatory for the first diagnostic examination as well as for monitoring of treatment effect in children with ALL.

[Detection of monoclonality in childhood lymphoma with the polymerase chain reaction]. / Detekce monoklonality u detských lymfomu polymerázovou retezovou reakcí.

We studied monoclonality of tumour cells by polymerase chain reaction (PCR) in paediatric lymphomas. In B-lymphomas we detected monoclonal rearrangement of the immunoglobulin of the T-cell receptor delta and gamma chain (TCR, TCR-delta, TCR-gamma) by an analogous way. We examined a group of 37 paediatric patients with lymphomas (26 with B-lymphomas and 11 with T-lymphomas). Monoclonal gene rearrangement was found in 34 cases, i.e. the method used in this study proved to be successful in 92% of cases. We confirmed our results by correlation with histologic and immunohistologic diagnoses. The method may be used as a complementary diagnostic tool in differential diagnosis of lymphoma versus non-neoplastic proliferation of lymphatic tissue, in distinction between B- and T-lymphomas, and to separate lymphomas and other malignancies. Beside the pure diagnostic usage we perceive the main importance of the method in monitoring minimal residual disease (MRD), because amplified products of PCR may be sequenced and used for preparing tumour specific primers and probes.

[Monitoring minimal residual disease in pediatric patients with acute lymphoblastic leukemia]. / Sledování minimální reziduální nemoci u detských pacientu s akutní lymfoblastickou leukémií.

The level of minimal residual disease is an important prognostic factor in childhood acute lymphoblastic leukaemia. The end of induction therapy is the most significant time-point for prediction of treatment outcome. Within a pilot study covered by the Paediatric Haematology Working Group in the Czech Republic 51 childhood patients were analysed at diagnosis of acute lymphoblastic leukaemia and at the end of induction using method based on detection of clonal rearrangements of immuno-receptor genes. The majority of tested patients (32/51, 63%) had a low or non-detectable levels of residual disease, a group of patients with the highest levels and thus the highest risk of relapse included 10% of patients (5/51). Within each of three risk groups one patient has relapsed so far. Therefore, the relapse rate in particular subgroups is 3% (1/32), 7% (1/14) and 20% (1/5) to date, respectively. The results are compared with these published by the BFM group (van Dongen et al., Lancet 1998). The pilot phase of a new BFM treatment protocols includes examination of residual disease for stratification of patients into the different risk groups.

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage.

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.