4 research outputs found
NGS better discriminates true MRD positivity for the risk stratification of childhood ALL treated on an MRD-based protocol
We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols
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Epigenetic Changes in CEBPα Gene and Xenotransplantation Model of B Cell Precursor Acute Lymphoblastic Leukemia Switching to Monocytoid Lineage During the Early Phase of the Treatment
Abstract Abstract 876 Immunophenotypic instability during early phase of ALL treatment is a frequent observation during flow cytometric minimal residual disease (FC MRD) monitoring. Antigens typically involved include CD10, CD20, CD34 and CD45 and these changes do not usually revoke initial disease classification and do not hamper FC MRD detection. We previously described a subtype of B-cell precursor (BCP) ALL with striking immunophenotypic instability towards monocytoid lineage within the first month of therapy (switching ALL-swALL). Blasts expressing both B and monocytoid markers emerged at early time points on days 8 and 15 of treatment while later only those blasts with pure monocytoid phenotype were present. Incidence of swALL in childhood was unexpectedly high (3-4% of all pediatric BCP ALL) as confirmed in two national reference labs. This phenomenon was associated with aberrant expression of CD2 (LFA-2) on diagnostic blasts (Mejstrikova et al, ASH 2010). The leukemic origin of monocytoid blasts was proven by the detection of clone-specific immunoreceptor gene rearrangements (Ig-TCR). No common genetics aberration was found in a cohort of swALL (n=17), MLL gene was always in germline configuration. We found an increased rate of alteration in IKZF1 gene compared to control BCP ALL cases (p=0.012). An expression analysis of the key hematopoietic regulators showed a difference in CEBPα expression, which is an important transcription factor in transdifferentiation of B cells into macrophages (Xie et al., Cell 2004). Expression of CEBPα is increased in swALL compared to other BCP ALL cases (p=0.01) already at diagnosis prior switching, however, the expression is lower compared to AML (p=0.002). We analyzed CEBPα the methylation status of the promoter region of this gene. Demethylation of CEBPα promoter region analyzed by bisulfite sequencing (295bp-594bp of promoter region) was found in 10/12 swALL cases, while it was seen in only 6/28 control BCP ALLs (Fisher test, p=0.0004). The only cases having demetylation in CEBPα were 5/5 BCR-ABLpos and 1/4 ETV6-RUNX1pos. Whole genome ERRBS method (Enhanced Reduced Representation Bisulfite Sequencing) confirmed this methylation pattern of CEBPα in 7 patients (4 swALL,3 BCP ALL). In order to establish an in vivo model to study the underlying molecular mechanisms, we transplanted ALL cells from 7 swALL patients intrafemorally into NOD-SCIDIL2Rgammanull (NSG) mice. Successfulstable engraftment was achieved only in 2 out of 7 swALL cases (28%) (Fisher test, p=0.049).Interestingly in these two cases, the 200 bp promoter region of CEBPα was methylated to some extent at diagnosis and completely methylated after engraftment into mice, suggesting the possibility of a selective advantage in this context. We treated engrafted animals with prednisolone and in both cases we observed demethylation of CEBPα promoter. Because the rate of engraftment of ALL in NSG is usually very high, these observations may indicate that the biology of this particular subset of patient is distinct. Conclusion: We described a novel subtype of BCP-ALL with the demethylation of CEBPα promoter region, increased CEBPα expression and immunophenotypic shift towards monocytic lineage during first weeks of the therapy. We identified CEBPα as the potential regulator of this lineage plasticity. Disclosures: No relevant conflicts of interest to declare