Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1-45 years with Philadelphia ...chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1-9 years (A), 266 were 10-17 years (B) and 221 were 18-45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1-3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS
) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.
Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) ...is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.
Abstract 955
Hematopoietic stem cell transplantation (HSCT) has improved survival of children with JMML considerably. Since it became apparent that JMML is a heterogeneous disease it has been ...questioned whether HSCT is the therapy of choice for all subtypes of JMML. Here we report the outcome of children with JMML registered to the retrospective or prospective studies of the European Working Group of MDS and JMML in Childhood (EWOG-MDS) with respect to clinical and mutational subtypes and presence or absence of HSCT. Among the 488 children with JMML studied by EWOG-MDS 43 patients (8.8%) were known to have Noonan syndrome, while 48 (9.7%) carried the clinical diagnosis of neurofibromatosis type 1 (NF1). Mutational analysis was carried out in 254 of the remaining 397 children. Somatic mutations in PTPN11, KRAS and NRAS were seen in 116, 46 and 47 children, respectively; 8 patients were noted to have germline CBL mutations in the absence of Noonan phenotype and 37 children with complete typing (N=203) had none of these abnormalities (all negative group). For the 51 patients with Noonan syndrome and/or germline CBL mutations overall survival at 5 years was 0.72 (0.58-0.86) irrespective whether HSCT was performed or not. A normal karyotype was observed in all but 3 of these 51 children. Normal chromosomal studies were also observed in 74%, 66% and 60% of the NF1, PTPN11 mutated and the all negative group, respectively. Interestingly, monosomy 7, and other aberrations were noted in 52% and 10% of KRAS mutated patients, respectively, but only in 7% and 5% of the NRAS mutated group (P<0.05). Five of the patients with NRAS mutation and normal karyotype are long term survivors without HSCT (5.5 to 27 years after diagnosis) compared to none of the children from the NF1, PTPN11 mutated, KRAS mutated or all negative group. Event-free survival (EFS) following HSCT differed significantly among the mutational groups with 0.38 (0.29-0.49), 0.43 (0.25-0.61), 0.45 (0.24-0.66), 0.69 (0.54-0.84) and 0.72 (0.56-0.88) for children with PTPN11 mutation, NF1, NRAS mutation, KRAS mutation and the all negative group (P<0.01). Likewise, relapse rate varied between 0.41 (PTPN11 mutation), 0.39 (NF1), 0.31 (NRAS mutation), 0.13 (all negative group) and 0.10 (KRAS mutation) (P< 0.01). Multivariate analysis identified mutational type and age at diagnosis as independent prognostic factors for EFS following HSCT. Interestingly, of the 7 children with JMML and NRAS mutation who relapsed post HSCT 5 had a normal karyotype. Considering that other children with JMML sharing the same oncogenic exon 1 mutations in NRAS experienced long-term survival without HSCT these data indicate that a NRAS mutation by itself may not be sufficient to cause lethal disease. Our results also demonstrate that clinical characteristics and outcome following HSCT differ between patients with JMML and oncogenic NRAS and KRAS mutations. Most importantly, we showed that in JMML subtypes with a low relapse incidence following HSCT (KRAS mutated and all negative group) transplant related mortality exceeds the relapse incidence, while in other subtypes (PTPN11 mutations, NF1) leukemic relapse is the most common failure. Current transplant strategies for children with JMML need to be revised to accommodate these differences among mutational subgroups.
No relevant conflicts of interest to declare.
Background and Significance The cure rate of pediatric acute myeloid leukemia (AML) has improved significantly; however, relapses still occur in up to 30% of the total patient population in ...high-income countries (HICs), and in an even higher percentage of patients in low- and middle-income countries (LMICs). Early treatment response, cytogenetics and minimal residual disease (MRD) are well-known prognostic factors for treatment outcome; however, sophisticated techniques for identifying prognostic factors are often lacking in LMICs, and are sometimes unsuccessful in HICs. Early treatment response assessed by a bone marrow (BM) aspirate on day 15 or day 22 (D22) (depending on the applicable protocol) after initiation of chemotherapy is a known prognostic factor for outcome in patients with pediatric AML, but does not allow modifications of the first induction chemotherapy course (Creutzig 2014, Abrahamsson 2011, Kern 2003). In both pediatric acute lymphoid leukemia (ALL) and adult AML, early peripheral blood blast clearance (PBBC) is a known early indicator of treatment response (Manabe 2008, Arellano 2012), whereas to the best of our knowledge, this has not yet been reported in pediatric AML. In this study, we evaluated the significance of time to complete PBBC, and rate of PBBC after initiation of chemotherapy, in relation to disease status in BM D22, as measure of early treatment response that has prognostic significance. Study Design and Methods Pediatric patients with AML, aged 0-18 years, that were treated according to the NOPHO-DBH AML-2012 protocol were included. The first 5 days of induction therapy consisted of etoposide, followed by 7 days of low-dose cytarabine and mitoxantrone or liposomal daunorubicin. Peripheral blood (PB) was assessed by morphology at the time of diagnosis, daily in the first week, and thrice weekly until D22 or until occurrence of complete clearance of blasts. BM D22 was assessed by morphology and flow cytometry. A receiver operating characteristic (ROC) analysis was performed to evaluate the predictive power of days from starting chemotherapy to complete PBBC. The correlation between time to complete PBBC and BM D22 results was analyzed using a logistic regression model. Multivariable logistic regression models were applied for the association after adjusting for effects of other covariates. Rate of PBBC was defined as the percentage of the absolute PB blast count on the day of diagnosis that cleared with each day of therapy, on average, until D22 or the day of complete PBBC. Results In total, 319 patients were included, out of whom 241 were eligible for analysis, and 78 not eligible due to missing or incomplete data. Day 9 after initiation of chemotherapy was identified as the most discriminating cut-off point for predicting BM D22 response. Patients were categorized into early PBBC (≤9 days; n=159) and delayed PBBC (>9 days; n=82) groups. Early PBBC was associated with having <5% AML cells in BM D22 as assessed by both morphology (OR 3.11; P=0.005) and flow cytometry (OR 2.73; P=0.041). Early PBBC also showed a much lower likelihood of having >15% AML cells in BM D22 by morphology (OR 0.08; P=0.002). None of the early PBBC patients had >15% AML cells in BM D22 by flow cytometry. For 217 patients (selection due to missing data n=24), the rate of PBBC could be calculated, and a discriminative cut-off value of 11% was identified. Patients were categorized into high rate of PBBC (>11%; n=132) and low rate of PBBC (≤11%; n=85). High rate of PBBC was associated with having <5% AML cells in BM D22 by flow cytometry (OR 4.30; P=0.007) and negative MRD status (OR 2.02; P=0.004), while also showing a strong association with a lower likelihood of >15% AML cells in BM D22 (OR 0.11; P=0.007) by morphology. None of the high rate PBBC patients had >15% AML cells in BM D22 by flow cytometry. Conclusion Both early complete PBBC and rate of PBBC after initiation of chemotherapy holds promise as prognostic marker for clinical outcome in pediatric AML, as it correlates significantly with BM D22 response. These findings may offer an early and easily accessible prognostic factor, particularly beneficial for LMICs, as well as for patients with unsuccessful MRD analyses.
Germline GATA2 mutations cause cellular deficiencies with high propensity for myeloid disease. We investigated 426 children and adolescents with primary myelodysplastic syndrome (MDS) and 82 cases ...with secondary MDS enrolled in 2 consecutive prospective studies of the European Working Group of MDS in Childhood (EWOG-MDS) conducted in Germany over a period of 15 years. Germline GATA2 mutations accounted for 15% of advanced and 7% of all primary MDS cases, but were absent in children with MDS secondary to therapy or acquired aplastic anemia. Mutation carriers were older at diagnosis and more likely to present with monosomy 7 and advanced disease compared with wild-type cases. For stratified analysis according to karyotype, 108 additional primary MDS patients registered with EWOG-MDS were studied. Overall, we identified 57 MDS patients with germline GATA2 mutations. GATA2 mutations were highly prevalent among patients with monosomy 7 (37%, all ages) reaching its peak in adolescence (72% of adolescents with monosomy 7). Unexpectedly, monocytosis was more frequent in GATA2-mutated patients. However, when adjusted for the selection bias from monosomy 7, mutational status had no effect on the hematologic phenotype. Finally, overall survival and outcome of hematopoietic stem cell transplantation (HSCT) were not influenced by mutational status. This study identifies GATA2 mutations as the most common germline defect predisposing to pediatric MDS with a very high prevalence in adolescents with monosomy 7. GATA2 mutations do not confer poor prognosis in childhood MDS. However, the high risk for progression to advanced disease must guide decision-making toward timely HSCT.
•Germline GATA2 mutations account for 15% of advanced and 7% of all primary pediatric MDS and do not influence overall survival.•The majority (72%) of adolescents with MDS and monosomy 7 carry an underlying GATA2 deficiency.
Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation ...t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia‐associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC‐derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down‐regulation of genes associated with megakaryocyte differentiation and up‐regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.
What's new?
Acute myeloid leukemia results from aberrant hematopoietic processes that are often initiated by chromosomal translocations. The mechanisms underlying leukemogenesis in pediatric patients with the t(7;12) translocation remain poorly understood. Here, the authors have created induced pluripotent stem cells (iPSC) with the t(7;12)(q36;p13) translocation using CRISPR/Cas9 technology. Characterization of these iPSC shows cell differentiation and growth properties as well as a gene expression signature similar to those seen in patients with t(7;12) acute myeloid leukemia. This model represents a valuable tool for further studies of the mechanisms behind the development of pediatric leukemia caused by this translocation.
We evaluated the outcome of children with acute lymphoblastic leukemia (ALL) in second remission (2CR), comparing bone marrow transplantation (BMT) using either matched sibling donors or unrelated ...donors (URDs).
A total of 65 patients, aged 2 months to 20 years at BMT, with ALL in 2CR underwent allogeneic BMT at seven Nordic centers during 1990 to 1997. Of the first relapses, 85% were in bone marrow; 46% occurred on therapy, and 54%, off therapy. The preparative regimens were cyclophosphamide plus total-body irradiation +/- antithymocyte/antilymphocyte globulin, busulfan plus cyclophosphamide +/- antithymocyte/antilymphocyte globulin, or cytarabine plus total-body irradiation. Of the allografts, 37 were from HLA-matched siblings and 28 were from URDs.
In the sibling versus URD graft recipient groups, the posttransplantation 5-year event-free survival was 39% versus 54% (P =.4), the estimated posttransplantation relapse rate was 76% versus 40% (P = not significant NS), and the toxic death rate was 19% versus 11% (P = NS). The incidence of significant (grade 2 to 4) acute graft-versus-host disease (GVHD) was 38% versus 64% (P <.05) and was 14% versus 32% (P <.10) for severe (grade 3 to 4) acute GVHD; the incidence of chronic GVHD was 26% versus 57% (P <.05) and was 13% versus 22% (P = NS) for extensive chronic GVHD in the sibling and URD groups.
BMT with matched URD allografts offers at least equal survival for children with ALL in 2CR, as compared with allografts from matched sibling donors. URD allografts were not associated with a higher toxic mortality rate, although both acute and chronic GVHD were more frequent with URD. Indications for using matched URD allografts in ALL 2CR can be considered the same as for using matched sibling donors.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves event-free survival in acute myeloid leukemia (AML); however, the burden of late effects may be increased. We compared ...health-related outcomes in childhood AML survivors treated according to the NOPHO-AML protocols either with or without allo-HSCT at age < 21 years. Out of the 147 eligible AML survivors treated with allo-HSCT, 95 (65%) and 53 (75%) of their eligible siblings completed a questionnaire. Their data were compared to corresponding data collected previously from NOPHO-AML survivors treated with chemotherapy only (CT) (n = 101). The median follow-up was 12 (range 2-28) years after allo-HSCT and 47% had received total body irradiation (TBI)-based conditioning. Allo-HSCT survivors reported significantly more physical health limitations (39% vs 7%, p < 0.005), medications for cardiovascular disease (10% vs 1%, p < 0.05) and use of analgesics (32% vs 11%, p < 0.01) than CT survivors. Health problems prevented 16% of the allo-HSCT survivors from attending school or managing a job vs. 3% among CT survivors (p < 0.05). Among 73 allo-HSCT survivors (age ≥ 15 years), seven females reported natural pregnancies and three males reported unassisted conceptions in partners. Survivors of childhood AML treated with allo-HSCT experienced more physical health limitations and used more medications than the survivors treated with chemotherapy only.
In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution.
We analyzed ALK mutations in ...54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%.
All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%).
In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.
Background: Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein, predominantly located in the nucleolus that regulates a multiplicity of different biological processes. NPM1 localization ...in the cell is finely tuned by the nuclear export signal domain, where two tryptophan residues (Trp) are essential for the nucleolar localization. In acute myeloid leukemia (AML), several genomic subsets of NPM1 frameshift mutations of 4 base pair insertions have been identified, with the type A variant (insertion of TCTG) being the most common, followed by type B (CATG) and D (CCTG). More recently, a series of different and rarer frameshifts have been found in adult and pediatric AML. We collected and studied the NPM1 insertion genotypes in a large cohort of children with de novo AML enrolled in the AIEOP, BFM, ELAM02, NOPHO, DCOG, COG trials. Methods: NPM1 mutations were sequenced in a group of 345 pediatric patients with de novo AML. 166 patients were enrolled in recent AIEOP, BFM, ELAM02, NOPHO, DCOG trials, and 179 in the CCG/COG AML trials (CCG-2961, AAML03P1, AAML0531 and AAML1031). A cohort of 75 patients of SWOG young adult (age 18-60) AML trial (S0106) were also included as validation cohort. The Kaplan-Meier method was used to estimate the probabilities of overall survival (OS) and event-free survival (EFS). Results: In this cohort of 345 pediatric patients sequencing of exon 12 demonstrated multiple 4 bp insertion subtypes, with type A variant detected in 188 patients (55%), type B in 64 cases (19%) and type D mutation in 26 patients (7.5%). Other variants with the loss of the 2 Trp residues (namely A-like) were found in 42 patients (12%), whereas in 25 patients (7.5%) we identified mutations with the loss of 1 Trp residue (namely non-A-like). Comparison of disease characteristics demonstrated that NPM1 variants were prevalent in older patients (median age=13.3 years). WBC count was not significantly different among subgroups (Type A=30.3; Alike=25.2; B=16.8; D=41; non-A-like= 14.8x10 3/ul, p=0.14). FLT3-ITD prevalence was higher in Type A, B and D compared to the other Type A-like variants (42%, 44%, 46% vs. 26%); interestingly, FLT3-ITD was absent in non-A-like variants. Evaluation of clinical outcome based on NPM1 genotypes showed that although patients with type A, A-Like, non-A-like and type B had favorable outcomes, those with Type D had a significantly worse outcome with OS of 63% versus 86% for other genotypes (p=0.0053). Type D variant adverse outcome was confirmed in most of the trials and will be further examined considering different treatments. Each NPM1 variant mediated a different EFS, with most of early events occurring within the D group (EFS 51.4%), and type A (EFS 68.8%) compared to the non-A-like NPM1, A like and B mutations (79.8%, 73%, and 75.5%, respectively) (p=ns). Validation data from adult SWOG trial (SWOG0106) demonstrated similar adverse outcome with EFS of 40% for type D vs. 61.4% for other NPM1 variants, indicating D as a peculiar NPM1 genotype /Figure A). Evaluation of overlapping mutations demonstrated that except for lack of FLT3-ITD in non-A-like genotype, there was similar distribution of high-risk mutations (FLT3-ITD, WT1, etc.) in those with different genotypes, thus not explaining the underlying biology responsible for the Type D adverse outcome. Even if the aminoacidic sequence of D and A variants is the same, the different prognosis suggests a further layer of regulation. We inquired whether Type D genotype might have a different NPM1 localization pattern compared to that in Type A variant. Immunofluorescence staining of primary AML cells from patients with Type D vs. Type A variant demonstrated that, in contrast to the canonical Type A variant with aberrant cytoplasmic localization, Type D variant maintains nuclear localization (Figure B). Further studies will provide the underlying biology of Type D being a silent mutation mediating a clinical outcome like NPM1 wild type AML. Conclusion: This Eu/COG/SWOG collaborative study of over 400 pediatric and adult patients suggest a different functional biology behind NPM1 genotype where those with Type D variants have a significantly inferior outcome resembling feature of non-NPM1 mutated AML. Our data suggest that the specific genotype of NPM1 mutations can contribute to a more accurate risk-stratification, with D cases eventually shifting from standard to higher risk groups.
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