Genomic breakpoint-specific monitoring of measurable residual disease in pediatric non-standard-risk acute myeloid leukemia.

Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application.

Molecular characterization of acute lymphoblastic leukemia with high CRLF2 gene expression in childhood.

BACKGROUND: A high-level expression of the CRLF2 gene is frequent in precursor B-cell acute lymphoblastic leukemia (pB-ALL) and can be caused by different genetic aberrations. The presence of the most frequent alteration, the P2RY8/CRLF2 fusion, was shown to be associated with a high relapse incidence in children treated according to ALL-Berlin-Frankfurt-Münster (BFM) protocols, which is poorly understood. Moreover, the frequency of other alterations has not been systematically analyzed yet. PROCEDURE: CRLF2 mRNA expression and potential genetic aberrations causing a CRLF2 high expression were prospectively assessed in 1,105 patients treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol. Additionally, we determined copy number alterations in selected B-cell differentiation genes for all CRLF2 high-expressing pB-ALL cases, as well as JAK2 and CRLF2 mutations. RESULTS: A CRLF2 high expression was detected in 26/178 (15%) T-cell acute lymphoblastic leukemia (T-ALL) cases, 21 of them (81%) had been stratified as high-risk patients by treatment response. In pB-ALL, a CRLF2 high expression was determined in 91/927 (10%) cases; the P2RY8/CRLF2 rearrangement in 44/91 (48%) of them, supernumerary copies of CRLF2 in 18/91 (20%), and, notably, the IGH/CRLF2 translocation was detected in 16/91 (18%). Remarkably, 7 of 16 (44%) patients with IGH/CRLF2 translocation had already relapsed. P2RY8/CRLF2- and IGH/CRLF2-positive samples (70 and 94%, respectively) were characterized by a high frequency of additional deletions in B-cell differentiation genes such as IKZF1 or PAX5. CONCLUSION: Our data suggest that this high frequency of genetic aberrations in the context of a high CRLF2 expression could contribute to the high risk of relapse in P2RY8/CRLF2- and IGH/CRLF2-positive ALL.

Implications of delayed bone marrow aspirations at the end of treatment induction for risk stratification and outcome in children with acute lymphoblastic leukaemia.

Minimal residual disease (MRD) at the end of induction therapy is important for risk stratification of acute lymphoblastic leukaemia (ALL), but bone marrow (BM) aspiration is often postponed or must be repeated to fulfil qualitative and quantitative criteria for morphological assessment of haematological remission and/or MRD analysis. The impact of BM aspiration delay on measured MRD levels and resulting risk stratification is currently unknown. We analysed paired MRD data of 289 paediatric ALL patients requiring a repeat BM aspiration. MRD levels differed in 108 patients (37%) with a decrease in the majority (85/108). This would have resulted in different risk group allocation in 64 of 289 patients (23%) when applying the ALL-Berlin-Frankfurt-Münster 2000 criteria. MRD change was associated with the duration of delay; 40% of patients with delay ≥7 days had a shift to lower MRD levels compared to only 18% after a shorter delay. Patients MRD-positive at the original but MRD-negative at the repeat BM aspiration (n = 50) had a worse 5-year event-free survival than those already negative at first aspiration (n = 115) (86 ± 5% vs. 94 ± 2%; P = 0·024). We conclude that BM aspirations should be pursued as scheduled in the protocol because delayed MRD sampling at end of induction may result in false-low MRD load and distort MRD-based risk assessment.

Small sizes and indolent evolutionary dynamics challenge the potential role of P2RY8-CRLF2-harboring clones as main relapse-driving force in childhood ALL.

The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non-high-risk group. Of 22 P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2-positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.

Progenitor B-1 B-cell acute lymphoblastic leukemia is associated with collaborative mutations in 3 critical pathways.

B-1 and B-2 lymphocytes are derived from distinct developmental pathways and represent layered arms of the innate and adaptive immune systems, respectively. In contrast to a majority of murine B-cell malignancies, which stain positive with the B220 antibody, we discovered a novel form of B-cell leukemia in NUP98-PHF23 (NP23) transgenic mice. The immunophenotype (Lin- B220- CD19+ AA4.1+) was identical to that of progenitor (pro) B-1 cells, and VH gene usage was skewed toward 3' V regions, similar to murine fetal liver B cells. Moreover, the gene expression profile of these leukemias was most similar to that of fetal liver pro-B fraction BC, a known source of B-1 B cells, further supporting a pro-B-1 origin of these leukemias. The NP23 pro-B-1 acute lymphoblastic leukemias (ALLs) acquired spontaneous mutations in both Bcor and Janus kinase (Jak) pathway (Jak1/2/3 and Stat5a) genes, supporting a hypothesis that mutations in 3 critical pathways (stem-cell self-renewal, B-cell differentiation, and cytokine signaling) collaborate to induce B-cell precursor (BCP) ALL. Finally, the thymic stromal lymphopoietin (Tslp) cytokine is required for murine B-1 development, and chromosomal rearrangements resulting in overexpression of the TSLP receptor (CRLF2) are present in some patients with high-risk BCP-ALL (referred to as CRLF2r ALL). Gene expression profiles of NP23 pro-B-1 ALL were more similar to that of CRLF2r ALL than non-CRLF2r ALL, and analysis of VH gene usage from patients with CRLF2r ALL demonstrated preferential usage of VH regions used by human B-1 B cells, leading to the suggestion that this subset of patients with BCP-ALL has a malignancy of B-1, rather than B-2, B-cell origin.

Quantitation of the sterol regulatory element-binding protein mRNA in mononuclear blood cells by competitive RT-PCR.

BACKGROUND: The genes for the sterol regulatory element-binding protein-1a (SREBP-1a), -1c, and -2, the low-density lipoprotein (LDL) receptor, and the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play a key role in the intracellular cholesterol and lipid metabolism. METHODS: To enable the absolute and relative quantitation of the mRNA levels of these genes we developed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The inclusion of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for reference and normalization enabled us to accurately discriminate between a twofold variance in the expression levels of these genes. We used this assay to study their expression in mononuclear peripheral blood cells (PBMNC). RESULTS: We found that the relative expression of SREBP-1a is tenfold higher than that of SREBP-1c, but only half of that of SREBP-2. The level of SREBP-1a transcripts correlated with that of the SREBP-1c, LDL receptor, HMG-CoA reductase, and SREBP-2 genes, whereas the amount of SREBP-1c mRNA did not show a relationship with that of the latter three genes. The most abundant transcript in PBMNC is that of SREBP-2, followed by that of SREBP-1a, whereas SREBP-1c mRNA is only found in smaller amounts. CONCLUSIONS: This competitive RT-PCR method is very well suited for the accurate quantitation of the respective mRNAs.

Screening for leukemia- and clone-specific markers at birth in children with T-cell precursor ALL suggests a predominantly postnatal origin.

Childhood T-cell precursor acute lymphoblastic leukemia (TCP ALL) is an aggressive disease with a presumably short latency that differs in many biologic respects from B-cell precursor (BCP) ALL. We therefore addressed the issue of in utero origin of this particular type of leukemia by tracing oncogenic mutations and clone-specific molecular markers back to birth. These markers included various first- and second-hit genetic alterations (TCRD-LMO2 breakpoint regions, n = 2; TAL1 deletions, n = 3; Notch1 mutations, n = 1) and nononcogenic T-cell receptor rearrangements (n = 13) that were derived from leukemias of 16 children who were 1.5 to 11.2 years old at diagnosis of leukemia. Despite highly sensitive polymerase chain reaction (PCR) approaches (1 cell with a specific marker among 100,000 normal cells), we identified the leukemic clone in the neonatal blood spots in only 1 young child. These data suggest that in contrast to BCP ALL most TCP ALL cases are initiated after birth.

Rapid determination of Epstein-Barr virus latent or lytic infection in single human cells using in situ hybridization.

Epstein-Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein-Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein-Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.

Low frequency of clonotypic Ig and T-cell receptor gene rearrangements in t(4;11) infant acute lymphoblastic leukaemia and its implication for the detection of minimal residual disease.

Infant t(4;11) acute lymphoblastic leukaemia (ALL) is a rare but cytogenetically well defined subgroup of immature B-cell precursor (BCP) ALL. To date, the configuration of their antigen receptor genes has not been studied in a large group of patients so far. In this study on 27 t(4;11) infant ALL, we have used standardized primer sets for the detection of all incomplete and complete immunoglobulin (Ig) heavy chain (IGH) rearrangements, as well as for the Ig light chain kappa (IGK), T-cell receptor delta (TCRD) and gamma (TCRG) rearrangements that are most common in childhood BCP ALL. Only 52% of cases displayed clonotypic antigen receptor gene rearrangements (IGH in 48%, IGK, TCRD and TCRG in 12%, 41% and 6% respectively). This low frequency suggests, together with the findings of predominantly incomplete DJh joins and monoallelic IGH rearrangements, that they are derived from an immature progenitor cell. As 48% of the t(4;11) infant ALL cases had no detectable antigen receptor gene rearrangements that could be used for minimal residual disease (MRD) analysis, we established an expression-independent, leukaemia-specific polymerase chain reaction (PCR) using the genomic sequence of the MLL-AF4 fusion genes. This method had high sensitivity and specificity and resulted in identical estimations of tumour loads when compared with IGH targets. Thus, genomic MLL-AF4 fusion genes are a good alternative target for the analysis of MRD in patients with t(4;11) leukaemias.