Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

Changes in cytogenetics and molecular genetics in acute myeloid leukemia from childhood to adult age groups.

BACKGROUND: To obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime. METHODS: This study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin-Frankfurt-Münster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory). RESULTS: The frequencies of cytogenetic subgroups were age-dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to < 18 years; 33%) to the oldest groups (<5% for > 70 years; P < .0001). Unfavorable cytogenetics (-7/del(7), -5/del(5q) or 5p, inv(3)/t(3;3), t(6;9), complex karyotype, 12p, 17p, and 11q23/mixed-lineage leukemia aberrations, excluding t(9;11)) were frequent (42%) in infants (<2 years), had a low frequency in children and young adults (<22%), and increased in frequency up to 36% in patients older than 85 years (P = .01). This was even more significant for complex karyotypes (P ≤ .0001), which also showed a strong increase in the absolute age-specific incidence with age. Interestingly, the frequency of 11q23 abnormalities decreased from infants to older patients. The proportion of clinically relevant molecular aberrations of CCAAT/enhancer binding protein α, nucleophosmin (NPM1), and NPM1/fms-related tyrosine kinase 3-internal tandem duplication increased with age. CONCLUSIONS: Altogether, with the exclusion of infants, a significant decrease in the proportion of favorable cytogenetic subtypes and an increase in unfavorable cytogenetics were observed with increasing age. These findings indicate different mechanisms for the pathogenesis of AML; these different mechanisms also suggest directions for etiological research and contribute to the more unfavorable prognosis with increasing age. Cancer 2016;122:3821-3830. © 2016 American Cancer Society.

Sequential karyotyping in Burkitt lymphoma reveals a linear clonal evolution with increase in karyotype complexity and a high frequency of recurrent secondary aberrations.

Typical Burkitt lymphoma is characterized by an IG-MYC translocation and overall low genomic complexity. Clinically, Burkitt lymphoma has a favourable prognosis with very few relapses. However, the few patients experiencing disease progression and/or relapse have a dismal outcome. Here we report cytogenetic findings of seven cases of Burkitt lymphoma in which sequential karyotyping was performed at time of diagnosis and/or disease progression/relapse(s). After case selection, karyotype re-review and additional molecular analyses were performed in six paediatric cases, treated in Berlin-Frankfurt-Münster-Non-Hodgkin lymphoma study group trials, and one additional adult patient. Moreover, we analysed 18 cases of Burkitt lymphoma from the Mitelman database in which sequential karyotyping was performed. Our findings show secondary karyotypes to have a significant increase in load of cytogenetic aberrations with a mean number of 2, 5 and 8 aberrations for primary, secondary and third investigations. Importantly, this increase in karyotype complexity seemed to result from recurrent secondary chromosomal changes involving mainly trisomy 21, gains of 1q and 7q, losses of 6q, 11q, 13q, and 17p. In addition, our findings indicate a linear clonal evolution to be the predominant manner of cytogenetic evolution. Our data may provide a biological framework for the dismal outcome of progressive and relapsing Burkitt lymphoma.

DNA copy number alterations mark disease progression in paediatric chronic myeloid leukaemia.

Early recognition of children with chronic phase chronic myeloid leukaemia (CML-CP) at risk for developing a lymphoid blast crisis (LyBC) is desirable, because therapy options in CML-LyBC are limited. We used Multiplex Ligation-dependent Probe Amplification to determine whether B-cell lymphoid leukaemia-specific copy number alterations (CNAs) (e.g. IKZF1, PAX5, CDKN2A deletions) could be detected in CML-CP and may be used to predict disease progression to LyBC. CNAs were detected in all patients with CML-LyBC, but in none of the 77 patients with CML-CP. Based on this study we conclude that CNAs remain a hallmark of disease progression.

Next-generation-sequencing-based risk stratification and identification of new genes involved in structural and sequence variations in near haploid lymphoblastic leukemia.

Near haploidy (23-29 chromosomes) is a numerical cytogenetic aberration in childhood acute lymphoblastic leukemia (ALL) associated with particularly poor outcome. In contrast, high hyperdiploidy (51-67 chromosomes) has a favorable prognosis. Correct classification and appropriate risk stratification of near haploidy is frequently hampered by the presence of apparently high hyperdiploid clones that arise by endoreduplication of the original near haploid clone. We evaluated next-generation-sequencing (NGS) to distinguish between "high hyperdiploid" leukemic clones of near haploid and true high hyperdiploid origin. Five high hyperdiploid ALL cases and the "high hyperdiploid" cell line MHH-CALL-2, derived from a near haploid clone, were tested for uniparental isodisomy. NGS showed that all disomic chromosomes of MHH-CALL-2, but none of the patients, were of uniparental origin, thus reliably discriminating these subtypes. Whole-exome- and whole-genome-sequencing of MHH-CALL-2 revealed homozygous non-synonymous coding mutations predicted to be deleterious for the protein function of 63 genes, among them known cancer-associated genes, such as FANCA, NF1, TCF7L2, CARD11, EP400, histone demethylases, and transferases (KDM6B, KDM1A, PRDM11). Only eight of these were also, but heterozygously, mutated in the high hyperdiploid patients. Structural variations in MHH-CALL-2 include a homozygous deletion (MTAP/CDKN2A/CDKN2B/ANRIL), a homozygous inversion (NCKAP5), and an unbalanced translocation (FAM189A1). Together, the sequence variations provide MHH-CALL-2 with capabilities typically acquired during cancer development, e.g., loss of cell cycle control, enhanced proliferation, lack of DNA repair, cell death evasion, and disturbance of epigenetic gene regulation. Poorer prognosis of near haploid ALL most likely results from full penetrance of a large array of detrimental homozygous mutations.

Treatment outcome of CRLF2-rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups.

The prognostic relevance of CRLF2 -rearrangements in childhood acute B-cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non-Down-syndrome patients (99 P2RY8-CRLF2+ , 15 IGH@-CRLF2+ ), 76 from the AIEOP-BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6-year cumulative relapse incidence of P2RY8-CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@-CRLF2+ AIEOP-BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8-CRLF2+ patients had an intermediate protocol-independent outcome while the different prognosis of IGH@-CRLF2+ patients could be related to the different structures of the applied treatment protocols.

Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study.

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10(-4)); MRD high risk (MRD-HR) if 10(-3) or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP.

Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations.

Acute leukaemias of ambiguous lineage (ALAL) represent a rare type of leukaemia, expressing both myeloid and lymphoid markers. This study retrospectively analyzed data from 92 children (biphenotypic n = 78, bilineal n = 6, lineage switch n = 8) with ALAL registered in the Berlin-Frankfürt-Münster (BFM) acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) studies between 1998 and 2006 (2.4% of all cases with acute leukaemia). Our cohort of ALAL patients was characterized by comparatively high median age (8.9 years), high median white blood cell count (14.9 x 10(9)/l), as well as frequent hyperleucocytosis (18.5%) and central nervous system involvement (24.1%). The most frequent cytogenetic abnormalities were ETV6/RUNX1 fusion (16%) and trisomy 8 (14.6%). Complete remission rate was significantly lower than in ALL-BFM patients (91.8% vs. 99.1%, P < 0.001), but comparable to AML-BFM patients (87.9%). Event-free survival (EFS) and overall survival (OS) of ALAL patients were low, at 62 +/- 5%. 5-year probability of EFS was significantly worse than in ALL patients (80 +/- 1%, P < 0.001), but better than for AML patients (49 +/- 2%, P = 0.027). Our data suggest that an intensive therapy regimen including stem cell transplantation may be favourable for bilineal or lineage switch cases, whereas patients with ETV6/RUNX1 fusion, lymphoid morphology and patients with expression of cyCD22 and cyCD79a should be treated with an ALL-directed therapy.

Distribution of NPM1-ALK and X-ALK fusion transcripts in paediatric anaplastic large cell lymphoma: a molecular-histological correlation.

Anaplastic large cell lymphomas (ALCL) in children express anaplastic lymphoma kinase (ALK) fusion genes, most commonly NPM1-ALK. The distribution of X-ALK among 66 childhood ALCLs was analysed. One ALCL was ALK-negative. Reverse transcription polymerase chain reaction detected NPM1-ALK in 58 tumours, all showing nuclear and cytoplasmic ALK staining. The remaining seven ALCL stained for ALK in the cytoplasm only: two expressed TPM3-ALK, one ATIC-ALK, one MYH9-ALK; three no TPM3-, TFG-, ATIC-, CLTC- or MYH9-ALK. Almost 90% of paediatric ALK-positive ALCLs express NPM1-ALK. There was complete concordance between ALK staining pattern and the presence of a typical/variant ALK fusion partner.

Imatinib treatment of paediatric Philadelphia chromosome-positive acute lymphoblastic leukaemia (EsPhALL2010): a prospective, intergroup, open-label, single-arm clinical trial.

BACKGROUND: The EsPhALL2004 randomised trial showed a 10% advantage in disease-free survival for short, discontinuous use of imatinib after induction compared with no use of imatinib in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia receiving Berlin-Frankfurt-Münster chemotherapy and haemopoietic stem-cell transplantation (HSCT). Other contemporary studies showed an advantage from continuous protracted exposure to imatinib, challenging the indications to transplant. The EsPhALL2010 study was designed to assess whether imatinib given from day 15 of induction and continuously throughout chemotherapy led to a different outcome to that obtained in EsPhALL2004, despite decreasing the number of patients having HSCT. METHODS: This prospective, intergroup, open-label, single-arm clinical trial (EsPhALL2010) was done at 11 study groups across Europe, Chile, and Hong Kong. Patients aged 1-17 years with the translocation t(9;22)(q34;q11) who were recruited into national front-line trials for acute lymphoblastic leukaemia were eligible for this trial. Patients with abnormal renal or hepatic function or an active systemic infection were ineligible. Patients received imatinib 300 mg/m2 continuously from day 15 of induction during chemotherapy. Eligibility to HSCT depended on early morphological response and minimal residual disease. Imatinib was recommended throughout the first year after transplant. The co-primary endpoints were event-free survival and overall survival. All analyses were done in the intention-to-treat population. The trial is registered with the European Clinical Trials Database (EudraCT 2004-001647-30) and with ClinicalTrials.gov (NCT00287105) and is completed. FINDINGS: 158 patients were screened for eligibility, of whom 155 were enrolled between Jan 1, 2010, and Dec 31, 2014. 151 (97%) patients achieved first complete remission after induction and four after the consolidation phase, with 102 (66%) patients categorised as good risk and 53 (34%) as poor risk according to EsPhALL risk stratification criteria. 59 (38%) patients had HSCT during their first complete remission. 40 (26%) patients relapsed and 41 (26%) patients died during the study (25 [61%] during complete continuous remission, and 16 [39%] after relapse). The 5-year event-free survival was 57·0% (95% CI 48·5-64·6) and 5-year overall survival was 71·8% (63·5-78·5). 154 serious adverse events were reported in 80 (52%) of 155 patients. The most common toxicity was infection (61 [39%] patients, mostly bacterial); gastrointestinal disorders occurred in ten (6%) patients and osteonecrosis in eight (5%). Serious adverse events occurred mainly during high-risk blocks and delayed intensifications, including 14 fatal events (one in the consolidation phase, six in high-risk blocks, six in first delayed intensification, and one in second delayed intensification). INTERPRETATION: Although HSCT was done in a smaller proportion of patients in EsPhALL2010 than in EsPhALL2004, event-free and overall survival were similar between the two studies. Our data suggest that imatinib given early and continuously with intensive chemotherapy might increase toxicity. FUNDING: Projet Hospitalier de Recherche Clinique-Cancer and Novartis France; Bloodwise and Cancer Research UK; Ministry of Health, Czech Republic.

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients.

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.

Computer aided analysis of additional chromosome aberrations in Philadelphia chromosome positive acute lymphoblastic leukaemia using a simplified computer readable cytogenetic notation.

BACKGROUND: The analysis of complex cytogenetic databases of distinct leukaemia entities may help to detect rare recurring chromosome aberrations, minimal common regions of gains and losses, and also hot spots of genomic rearrangements. The patterns of the karyotype alterations may provide insights into the genetic pathways of disease progression. RESULTS: We developed a simplified computer readable cytogenetic notation (SCCN) by which chromosome findings are normalised at a resolution of 400 bands. Lost or gained chromosomes or chromosome segments are specified in detail, and ranges of chromosome breakpoint assignments are recorded. Software modules were written to summarise the recorded chromosome changes with regard to the respective chromosome involvement. To assess the degree of karyotype alterations the ploidy levels and numbers of numerical and structural changes were recorded separately, and summarised in a complex karyotype aberration score (CKAS). The SCCN and CKAS were used to analyse the extend and the spectrum of additional chromosome aberrations in 94 patients with Philadelphia chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) and secondary chromosome anomalies. Dosage changes of chromosomal material represented 92.1% of all additional events. Recurring regions of chromosome losses were identified. Structural rearrangements affecting (peri)centromeric chromosome regions were recorded in 24.6% of the cases. CONCLUSIONS: SCCN and CKAS provide unifying elements between karyotypes and computer processable data formats. They proved to be useful in the investigation of additional chromosome aberrations in Ph-positive ALL, and may represent a step towards full automation of the analysis of large and complex karyotype databases.

PAX5 fusion genes in t(7;9)(q11.2;p13) leukemia: a case report and review of the literature.

BACKGROUND: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent genetic alterations including chromosomal translocations. The transcription factor PAX5, which is pivotal for B-cell commitment and maintenance, is affected by rearrangements, which lead to the expression of in-frame fusion genes in about 2.5% of the cases. RESULTS: Using conventional cytogenetics, fluorescence in situ hybridization (FISH), and molecular methods, an additional case with a der(9)t(7;9)(q11.23;p13) resulting in the expression of a PAX5-ELN fusion gene was identified. Furthermore, a general review of leukemia harboring a t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which occurs more often in children than in adults and shows a remarkably high male preponderance, is given. These cytogenetically highly similar translocations lead to the expression of one of three different in frame PAX5-fusions, namely with AUTS2 (7q11.22), ELN (7q11.23), or POM121 (7q11.23), which constitute the only currently known cluster of PAX5 partner genes. CONCLUSION: Our report underlines the recurrent involvement of PAX5 in different fusion genes resulting either from t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which cannot be distinguished cytogenetically and whose discrimination requires molecular analysis.

MicroRNAs distinguish cytogenetic subgroups in pediatric AML and contribute to complex regulatory networks in AML-relevant pathways.

BACKGROUND: The role of microRNAs (miRNAs), important post-transcriptional regulators, in the pathogenesis of acute myeloid leukemia (AML) is just emerging and has been mainly studied in adults. First studies in children investigate single selected miRNAs, however, a comprehensive overview of miRNA expression and function in children and young adults is missing so far. METHODOLOGY/PRINCIPAL FINDINGS: We here globally identified differentially expressed miRNAs between AML subtypes in a survey of 102 children and adolescent. Pediatric samples with core-binding factor AML and promyelocytic leukemia could be distinguished from each other and from MLL-rearranged AML subtypes by differentially expressed miRNAs including miR-126, -146a, -181a/b, -100, and miR-125b. Subsequently, we established a newly devised immunoprecipitation assay followed by rapid microarray detection for the isolation of Argonaute proteins, the hallmark of miRNA targeting complexes, from cell line models resembling core-binding factor and promyelocytic leukemia. Applying this method, we were able to identify Ago-associated miRNAs and their targeted mRNAs. CONCLUSIONS/SIGNIFICANCE: miRNAs as well as their mRNA-targets showed binding preferences for the different Argonaute proteins in a cell context-dependent manner. Bioinformatically-derived pathway analysis suggested a concerted action of all four Argonaute complexes in the regulation of AML-relevant pathways. For the first time, to our knowledge, a complete AML data set resulting from carefully devised biochemical isolation experiments and analysis of Ago-associated miRNAs and their target-mRNAs is now available.

PAX5-AUTS2: a recurrent fusion gene in childhood B-cell precursor acute lymphoblastic leukemia.

PAX5 rearrangements resulting in the expression of fusion transcripts account for 2-3% of childhood B-cell precursor acute lymphoblastic leukemia. Most PAX5 fusions are rare and many of them have only been described in a couple of, or even only in single, cases. We have identified the third case with a PAX5-AUTS2 fusion, which results from unbalanced t(7;9)(q11.2;p13.2) rearrangements. Our findings substantiate that PAX5-AUTS2 is a recurrent fusion gene in pediatric B-cell precursor acute lymphoblastic leukemia, and we summarize the clinical characteristics of such patients.

Quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) using amplicon-fusion-site polymerase chain reaction (AFS-PCR).

The amplification of putative oncogenes is a common finding within the genome of various cancer types. Identification and further targeting of specific junction sites within the sequence of genomic amplicons (amplicon fusion sites, AFS) by PCR (AFS-PCR) is suitable for quantification of minimal residual disease (MRD). This approach has recently been developed and described for MYCN amplified neuroblastomas. To compare AFS-PCR directly to routinely used MRD diagnostic strategies, we mapped the amplified genomic regions (ampGR) of an iAMP21-amplicon in high resolution of a patient with acute lymphoblastic leukemia (ALL). Successfully, we established AFS-PCR covering junction sites between ampGR within the iAMP21-amplicon. Quantification of MRD by AFS-PCR was directly comparable to IgH/TCR based real time quantitative PCR and fluorescence activated cell sorting (FACS) analysis in consecutive bone marrow (BM) specimens. Our data give an additional proof of concept of AFS-PCR for quantification of MRD. The method could be taken into account for ALL patients with genomic amplifications as alternative MRD diagnostic, if no or qualitatively poor Ig/TCR-PCRs are available.