International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia.

PURPOSE: Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation. EXPERIMENTAL DESIGN: Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients). RESULTS: Analysis of pooled, published data established three DNA methylation subgroups as a de facto standard. Unfavorable prognostic parameters (PTPN11 mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS. CONCLUSIONS: This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.

Human leukocyte antigen disparities reduce relapse after hematopoietic stem cell transplantation in children with juvenile myelomonocytic leukemia: A single-center retrospective study from China.

BACKGROUND: HSCT is the only proven curative therapy for JMML. Matching donor and recipient HLA alleles is considered optimal to reduce the risk of GVHD after HSCT but is not always possible. Only a limited number of studies have compared the influence of HLA disparities on HSCT outcomes for patients with JMML. METHODS: We conducted a retrospective study among 47 children with JMML who received related or unrelated unmanipulated HSCT (March 2010-October 2018). Among our participants, 27 (57.4%) donor-recipient pairs had 0-1 HLA disparities (Group 1: HLA-matched or ≤1 allele/antigen mismatch donor) and 20 (42.6%) had ≥2 HLA disparities (Group 2: 2-3 mismatched/haploidentical donors). RESULTS: The median follow-up period was 26.0 months (range: 1-105 months), and the 5-year probabilities of DFS and RI for the whole cohort were 54.6 ± 7.7% and 34.8 ± 15.0%, respectively. Compared to Group 1, Group 2 patients had a significantly lower RI (5.3 ± 10.5% vs 55.5 ± 20.9%, P Ë‚ .001), though similar rates of grade II-IV acute GVHD (60.0 ± 22.4% vs 33.3 ± 18.2%, P = .08), grade III-IV acute GVHD (25.0 ± 19.5% vs 7.4 ± 10.1%, P = .08), chronic GVHD (30.0 ± 20.9% vs 34.9 ± 18.8%, P = .85), NRM (20.0 ± 18.0% vs 3.9 ± 7.7%, P = .07), and DFS (74.4 ± 9.9% vs 41.3 ± 10.0%, P = .08). CONCLUSIONS: Disease relapse remains the major cause of treatment failure in JMML patients, especially in patients receiving HLA-matched and limited HLA-mismatched HSCT. Our findings suggest that donor-recipient HLA disparities may improve the outcome of HSCT in children with JMML.

Vitamin D status in children with leukemia, its predictors, and association with outcome.

BACKGROUND: Children and adolescents with leukemia are potentially at high risk of vitamin D inadequacy, which may have clinical relevance for skeletal morbidity, infections, and cancer outcome. This study aimed to evaluate vitamin D status at the time of diagnosis to investigate its predictors and association with overall survival in children with leukemia. PROCEDURE: We included all 295 children and adolescents diagnosed with leukemia at our institution between 1990 and 2016 who had available serum sample from the time of diagnosis. We analyzed serum 25-hydroxyvitamin D and parathyroid hormone levels and correlated them with clinical data. RESULTS: The 25-hydroxyvitamin D level was deficient (< 25 nmol/L), insufficient (25-50 nmol/L), sufficient (50-75 nmol/L), and optimal (> 75 nmol/L) in 6.4%, 26.8%, 39.7%, and 27.1% of the children, respectively. Older age and a more recent time of sampling (calendar year) predicted lower 25-hydroxyvitamin D level. In preschool children (age ≤6 years), lower 25-hydroxyvitamin D level was also associated with acute myeloid leukemia, and a 25-hydroxyvitamin D level < 50 nmol/L was associated with inferior overall survival. In school-aged children (age > 6 years), the 25-hydroxyvitamin D level showed significant seasonal variation. CONCLUSION: It remains unclear whether vitamin D supplementation in pediatric leukemia patients will improve outcome.

PTPN11 mutation with additional somatic alteration indicates unfavorable outcome in juvenile myelomonocytic leukemia: a retrospective clinical study from a single center.

Juvenile myelomonocytic leukemia (JMML) is a heterogeneous childhood leukemia. The management of patients with JMML requires accurate assessment of genetic and clinical features to help in patient risk stratification. This study aimed to investigate the association between genomic alterations and prognosis in children with JMML. Genomic DNA was extracted from a total of 93 patients with JMML for targeted sequencing. Univariable and multivariable analysis were used to evaluate the correlation between gene mutations and prognosis of the patients. Patients with PTPN11 mutation exhibited significantly lower event-free survival (EFS) compared with non-PTPN11 mutations (P = 0.005). Patients without or with one somatic alteration at diagnosis showed significantly better prognosis in comparison with those with more than two alterations (P = 0.009). PTPN11 mutation with additional alterations showed significantly the poorest outcome in comparison with those with only one non-PTPN11 mutation, only one PTPN11 mutation, and combined mutations without PTPN11, respectively (P < 0.0001).Conclusion: Both PTPN11 mutation and the number of somatic alterations detected at diagnosis are likely to be the major determinant of outcome in JMML. The subgroup of patients with PTPN11 mutation showed the shortest survival which was even worsened when a secondary mutation was present.

Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.

RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML.

Long-term serial xenotransplantation of juvenile myelomonocytic leukemia recapitulates human disease in Rag2-/-γc-/- mice.

Juvenile myelomonocytic leukemia is a clonal malignant disease affecting young children. Current cure rates, even with allogeneic hematopoietic stem cell transplantation, are no better than 50%-60%. Pre-clinical research on juvenile myelomonocytic leukemia is urgently needed for the identification of novel therapies but is hampered by the unavailability of culture systems. Here we report a xenotransplantation model that allows long-term in vivo propagation of primary juvenile myelomonocytic leukemia cells. Persistent engraftment of leukemic cells was achieved by intrahepatic injection of 1×10(6) cells into newborn Rag2(-/-)γc(-/-) mice or intravenous injection of 5×10(6) cells into 5-week old mice. Key characteristics of juvenile myelomonocytic leukemia were reproduced, including cachexia and clonal expansion of myelomonocytic progenitor cells that infiltrated bone marrow, spleen, liver and, notably, lung. Xenografted leukemia cells led to reduced survival of recipient mice. The stem cell character of juvenile myelomonocytic leukemia was confirmed by successful serial transplantation that resulted in leukemia cell propagation for more than one year. Independence of exogenous cytokines, low donor cell number and slowly progressing leukemia are advantages of the model, which will serve as an important tool to research the pathophysiology of juvenile myelomonocytic leukemia and test novel pharmaceutical strategies such as DNA methyltransferase inhibition.

Phase II/III trial of a pre-transplant farnesyl transferase inhibitor in juvenile myelomonocytic leukemia: a report from the Children's Oncology Group.

BACKGROUND: Juvenile myelomonocytic leukemia (JMML) is not durably responsive to chemotherapy, and approximately 50% of patients relapse after hematopoietic stem cell transplant (HSCT). Here we report the activity and acute toxicity of the farnesyl transferase inhibitor tipifarnib, the response rate to 13-cis retinoic acid (CRA) in combination with cytoreductive chemotherapy, and survival following HSCT in children with JMML. PROCEDURE: Eighty-five patients with newly diagnosed JMML were enrolled on AAML0122 between 2001 and 2006. Forty-seven consented to receive tipifarnib in a phase II window before proceeding to a phase III trial of CRA in combination with fludarabine and cytarabine followed by HSCT and maintenance CRA. Thirty-eight patients enrolled only in the phase III trial. RESULTS: Overall response rate was 51% after tipifarnib and 68% after fludarabine/cytarabine/CRA. Tipifarnib did not increase pre-transplant toxicities. Forty-six percent of the 44 patients who received protocol compliant HSCT relapsed. Five-year overall survival was 55 ± 11% and event-free survival was 41 ± 11%, with no significant difference between patients who did or did not receive tipifarnib. CONCLUSIONS: Administration of tipifarnib in the window setting followed by HSCT in patients with newly diagnosed JMML was safe and yielded a 51% initial response rate as a single agent, but failed to reduce relapse rates or improve long-term overall survival.

Criteria for evaluating response and outcome in clinical trials for children with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia is a rare myeloproliferative disease in young children. While hematopoietic stem cell transplantation remains the only curative therapeutic option for most patients, children with juvenile myelomonocytic leukemia increasingly receive novel agents in phase I-II clinical trials as pre-transplant therapy or therapy for relapse after transplantation. However, response criteria or definitions of outcome for standardized evaluation of treatment effect in patients with juvenile myelomonocytic leukemia are currently lacking. Here we propose criteria to evaluate the response to the non-transplant therapy and definitions of remission status after hematopoietic stem cell transplantation. For the evaluation of non-transplant therapy, we defined 6 clinical variables (white blood cell count, platelet count, hematopoietic precursors and blasts in peripheral blood, bone marrow blast percentage, spleen size and extramedullary disease) and 3 genetic variables (cytogenetic, molecular and chimerism response) which serve to describe the heterogeneous picture of response to therapy in each individual case. It is hoped that these criteria will facilitate the comparison of results between clinical trials in juvenile myelomonocytic leukemia.

Transplantation for juvenile myelomonocytic leukemia: a retrospective study of 30 children treated with a regimen of busulfan, fludarabine, and melphalan.

We report the outcomes of 30 patients with juvenile myelomonocytic leukemia (JMML) who received unmanipulated hematopoietic stem cell transplantation (HSCT) with oral or intravenous busulfan, fludarabine, and melphalan between 2001 and 2011. Mutations in PTPN11 were detected in 15 patients. Six patients received human leukocyte antigen (HLA)-matched HSCT from related donors, and 24 patients received HSCT from alternative donors, including 13 HLA-mismatched donors. Primary engraftment failed in five patients, all of whom had received allografts from HLA-mismatched donors. HLA-mismatched HSCT resulted in poorer event-free survival than HLA-matched HSCT (28.8 vs. 70.6 %). Three patients died of transplantation-related causes, and eight patients experienced hematological relapse (including five patients who died due to disease progression). Eight patients received a second HSCT, and four of these patients have survived. The 5-year estimated overall survival for all patients was 72.4: 88.9 % for the patients without a mutation in PTPN11 (n = 10) and 58.3 % for the patients with a mutation in PTPN11 (n = 15) (P = 0.092). The conditioning regimen reported in the present study achieved hematological and clinical remission in >50 % of patients with JMML who received HSCT from alternative donors, and may also be effective for JMML patients with PTPN11 mutation.

Juvenile myelomonocytic leukaemia and Noonan syndrome.

BACKGROUND: Infants with Noonan syndrome (NS) are predisposed to developing juvenile myelomonocytic leukaemia (JMML) or JMML-like myeloproliferative disorders (MPD). Whereas sporadic JMML is known to be aggressive, JMML occurring in patients with NS is often considered as benign and transitory. However, little information is available regarding the occurrence and characteristics of JMML in NS. METHODS AND RESULTS: Within a large prospective cohort of 641 patients with a germline PTPN11 mutation, we identified MPD features in 36 (5.6%) patients, including 20 patients (3%) who fully met the consensus diagnostic criteria for JMML. Sixty percent of the latter (12/20) had severe neonatal manifestations, and 10/20 died in the first month of life. Almost all (11/12) patients with severe neonatal JMML were males. Two females who survived MPD/JMML subsequently developed another malignancy during childhood. Although the risk of developing MPD/JMML could not be fully predicted by the underlying PTPN11 mutation, some germline PTPN11 mutations were preferentially associated with myeloproliferation: 10/48 patients with NS (20.8%) with a mutation in codon Asp61 developed MPD/JMML in infancy. Patients with a p.Thr73Ile mutation also had more chances of developing MPD/JMML but with a milder clinical course. SNP array and whole exome sequencing in paired tumoral and constitutional samples identified no second acquired somatic mutation to explain the occurrence of myeloproliferation. CONCLUSIONS: JMML represents the first cause of death in PTPN11-associated NS. Few patients have been reported so far, suggesting that JMML may sometimes be overlooked due to early death, comorbidities or lack of confirmatory tests.

Analysis of risk factors influencing outcomes after cord blood transplantation in children with juvenile myelomonocytic leukemia: a EUROCORD, EBMT, EWOG-MDS, CIBMTR study.

We retrospectively analyzed 110 patients with juvenile myelomonocytic leukemia, given single-unit, unrelated donor umbilical cord blood transplantation. Median age at diagnosis and at transplantation was 1.4 years (age range, 0.1-6.4 years) and 2.2 years (age range, 0.5-7.4 years), respectively. Before transplantation, 88 patients received chemotherapy; splenectomy was performed in 24 patients. Monosomy of chromosome 7 was the most frequent cytogenetic abnormality, found in 24% of patients. All but 8 patients received myeloablative conditioning; cyclosporine plus steroids was the most common graft-versus-host disease prophylaxis. Sixteen percent of units were HLA-matched with the recipient, whereas 43% and 35% had either 1 or 2 to 3 HLA disparities, respectively. The median number of nucleated cells infused was 7.1 × 10(7)/kg (range, 1.7-27.6 × 10(7)/kg). With a median follow-up of 64 months (range, 14-174 months), the 5-year cumulative incidences of transplantation-related mortality and relapse were 22% and 33%, respectively. The 5-year disease-free survival rate was 44%. In multivariate analysis, factors predicting better disease-free survival were age younger than 1.4 years at diagnosis (hazard ratio [HR], 0.42; P = .005), 0 to 1 HLA disparities in the donor/recipient pair (HR, 0.4; P = .009), and karyotype other than monosomy 7 (HR, 0.5; P = .02). Umbilical cord blood transplantation may cure a relevant proportion of children with juvenile myelomonocytic leukemia. Because disease recurrence remains the major cause of treatment failure, strategies to reduce incidence of relapse are warranted.

Exome sequencing identifies secondary mutations of SETBP1 and JAK3 in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

[Prognosis of 75 patients with juvenile myelomonocytic leukemia: prospective study by MDS committee in the Japanese Society of Pediatric Hematology].

Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative disorder of young children. Because the disease is rare and the diagnosis is difficult, a prospective registration of patients suspected of having JMML with a pathological central review have been conducted by the MDS Committee of the Japanese Society of Pediatric Hematology. Between 1999 and 2006, 75 children with JMML were enrolled and diagnosed through this system. Median age at diagnosis was 20 months (1∼85 months). Cytogenetic abnormalities were detected in 21 patients, including 11 with monosomy 7. The 5-year overall survival (OS) was 60%. Regarding the treatment, 61 of the 75 patients received stem cell transplantation (SCT). Conditioning regimen varied widely, and the source of grafts was bone marrow for 43 patients, peripheral blood for 5, and cord blood for 13. The 5-year OS after SCT was 61%. Notably, patients who received cord blood transplantation had inferior survival than those who received grafts from other sources (38 vs. 68%; P=0.03). Given better recognition of the disease, a multi-center protocol study on SCT, JMML11, is now being planned by the Japanese Pediatric Leukemia/Lymphoma Study Group.