Cases Of Juvenile Myelomonocytic Leukemia Research Articles

Germline bi-allelic SH2B3/LNK alteration predisposes to a neonatal juvenile myelomonocytic leukemia-like disorder.

Juvenile myelomonocytic leukemia (JMML) is a rare, generally aggressive myeloproliferative neoplasm affecting young children. It is characterized by granulomonocytic expansion, with monocytosis infiltrating peripheral tissues. JMML is initiated by mutations upregulating RAS signaling. Approximately 10% of cases remain without an identified driver event. Exome sequencing of two unrelated cases of familial JMML of unknown genetics and analysis of the French JMML cohort identified 11 patients with variants in SH2B3, encoding LNK, a negative regulator of the JAK-STAT pathway. All variants were absent from healthy population databases, and the mutation spectrum was consistent with a loss of function of the LNK protein. A stoploss variant was shown to affect both protein synthesis and stability. The other variants were either truncating or missense, the latter affecting the SH2 domain that interacts with activated JAK. Of the 11 patients, eight from five families inherited pathogenic bi-allelic SH2B3 germline variants from their unaffected heterozygous parents. These children represent half of the cases with no identified causal mutation in the French cohort. They displayed typical clinical and hematologic features of JMML with neonatal onset and marked thrombocytopenia. They had a hypomethylated DNA profile with fetal characteristics and did not have additional genetic alterations. All patients showed partial or complete spontaneous clinical resolution. However, progression to thrombocythemia and immunity-related pathologies may be of concern later in life. Bi-allelic SH2B3 germline mutations thus define a new condition predisposing to a JMML-like disorder, suggesting that JAK pathway deregulation is capable of initiating JMML, and opening new therapeutic options.

Two Cases of Juvenile Myelomonocytic Leukemia and Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease

BackgroundMyelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an autoimmune demyelinating disorder that often manifests after infections or vaccinations. We report two patients who developed MOGAD out of eight patients with juvenile myelomonocytic leukemia (JMML) that has never been reported. MethodsWe investigated two patients with JMML who developed MOGAD among 127 patients with leukemia from 2012 to 2021. ResultsPatient 1 was treated for JMML and developed fever and impaired consciousness at two years and one month of age. Magnetic resonance imaging revealed high-intensity lesions in the left frontal and left occipital white matter. The serum anti-MOG antibody test was positive, while the test was negative in the stored serum 45 days before the onset of encephalopathy. He had relapse of MOGAD after steroid therapy and plasmapheresis. Patient 2, who was treated for JMML, became apathetic and mute at three years and seven months of age. Magnetic resonance imaging revealed left frontoparietal subcortical high-intensity lesions. Anti-MOG antibody at the onset of encephalopathy was positive, while it was negative in stored serum 57 days before and 47 days after the onset. ConclusionWe treated two patients who developed MOGAD out of eight patients with JMML and none with MOGAD out of 119 patients with acute lymphocytic leukemia, acute myelocytic leukemia, or chronic myelocytic leukemia. The activated autoimmune process via the RAS pathway abnormality may have led to the formation of the anti-MOG antibody and the onset of MOGAD. MOGAD can occur in children with JMML, and abnormalities of the RAS pathway possibly contribute to its onset.

Genetic characteristics and survival analysis of 27 cases of juvenile myelomonocytic leukemia

Objective: To investigate the genetic and genomic profiling of juvenile myelomonocytic leukemia (JMML) and factors affecting its survival rate. Methods: Clinical characteristics, cytogenetics, molecular biology results and survival status of children with 27 JMML cases admitted to the Hematology Department of Children's Hospital, Capital Institute of Pediatrics from December 2012 to December 2021 were analyzed retrospectively, and the outcomes of the children were followed up. Kaplan-Meier method was used for survival analysis. Univariate analysis was used for analyzing factors affecting the overall survival (OS) rates of patients who received hematopoietic stem cell transplantation (HSCT). Log-Rank test was used for comparison of survival curves. Results: Among 27 JMML cases, there were 11 males and 16 females. The age of disease onset was 28 (11,52) months. There are 20 cases of normal karyotype, 4 cases of monosomy 7, 1 case of trisomy 8,1 case of 11q23 rearrangement and 1 case of complex karyotype. A total of 39 somatic mutations were detected.Those involved in RAS signal pathway were the highest (64%(25/39)), among which PTPN11 mutation was the most frequent (44% (11/25)). A total of 17 cases (63%) received HSCT, 8 cases (30%) did not receive HSCT, and 2 cases (7%) lost follow-up. For children receiving transplantation, the follow-up time after transplantation was 47 (11,57) months. The 1-year OS rate of high-risk transplantation group (17 cases) and high-risk non transplantation group (6 cases) was (88±8)% and (50±20)% respectively, with a statistically significant difference (χ2=5.01, P=0.025). The 5-year OS rate of the high-risk transplantation group was (75±11)%. The survival time of those who relapsed or progressed to acute myeloid leukemia after transplantation was significantly shorter than that of those who did not relapse (χ2=6.80, P=0.009). The OS rate of patients with or without PTPN11 mutation was (81±12) % and (67±19)% respectively (χ2=0.85, P=0.356). Conclusions: The main pathogenesis involved in JMML is gene mutation related to RAS signaling pathway, and the most common driver gene of mutation is PTPN11. Allogeneic HSCT can significantly improve the survival rate of high-risk JMML patients. The recurrence or progression after transplantation was related to poor prognosis.

High serum cystatin C levels in juvenile myelomonocytic leukemia patients without abnormal kidney function.

In pediatric cancer patients, the estimated glomerular filtration rate based on serum cystatin C (CysC) was reported to be suitable for estimating kidney function because of low serum creatinine (Cr) and high serum β2-microglobulin. Recently, however, serum CysC levels have been reported to be elevated in some cancer patients other than those with juvenile myelomonocytic leukemia (JMML), regardless of normal kidney function. We describe two pediatric cases of JMML with an elevated serum CysC level. Urinalysis tests showed no abnormalities and no evidence of nephritis or nephropathy, and there were no findings indicating abnormal kidney function, such as Cr clearance in one case or the estimated glomerular filtration rate based on serum Cr in both cases, except for the serum CysC levels. There were no other causes of a high serum CysC level, including hyperthyroidism and steroid administration. The patients were treated with a conventional dosage of drugs, and their serum CysC levels decreased to the normal range when they were in complete remission after treatment. An elevated serum CysC level may reflect tumor burden independent of kidney function in JMML patients. Therefore, creatinine or inulin clearance should be determined to more accurately estimate kidney function for administering an optimal dose of anticancer drugs. In addition, a high serum CysC level may be a potential biomarker of cancer progression.

Juvenile Myelomonocytic Leukemia (JMML) presenting as bilateral periorbital swelling in a ten year old - A case report

Juvenile Myelomonocytic Leukemia (JMML) is an infrequent pediatric myelodysplastic – myeloproliferative neoplasm characterized by the proliferation of myelomonocytic cells. The incidence of JMML is estimated to be 0.13 cases annually per 100000 children aged from 0-14 years. JMML is difficult to diagnose due to its similar clinical and bone marrow findings with that of other myeloproliferative neoplasms such as Chronic Myeloid Leukemia. Some characteristic features that establish the diagnosis of JMML are the presence of monocytosis (>/=1x 10/L), blast percentage in blood and bone marrow of <20%, splenomegaly, absence of Philadelphia (Ph) chromosome or BCR-ABL1 fusion and raised fetal hemoglobin. Leukemic infiltration to any hematopoietic organ is also seen in JMML but orbital tissue infiltration is unusual. Here in, we report an unusual case of JMML in a ten year old child with emphasis on the distinguishing features for appropriate diagnosis and management plan.

Analysis of the Short-Term Efficacy of Decitabine Combined with Low-Dose Cytarabine in the Treatment of JMML

Objective: Juvenile myelomonocytic leukemia (JMML) has both the disease characteristics of myelodysplastic syndrome and myeloproliferative neoplasm. Currently, hematopoietic stem cell transplantation (HSCT) is the only possible cure. Most of the newly diagnosed pediatric patients with JMML have high tumor burden, rapid disease progression, and may not tolerate HSCT. This study explored the short-term efficacy of decitabine combined with low-dose chemotherapy in the treatment of JMML before transplantation. Methods: A retrospective analysis of the patient files of 9 cases of JMML was performed from January 2019 to May 2020. All patients were given decitabine 20mg/m2× 5 days, supplemented with a small dose of cytarabine (50-100mg/m2×3~5 days), and/or etoposide (50mg/m2×3~5 days) chemotherapy. Each treatment interval is 3~4 weeks, bridging with HSCT after 3~4 treatment courses. Results: The median age of onset of 9 cases of JMML was 2 years old (0.5~4 y), male to female ratio was 8:1, the median size of spleen was 6.4cm (2.9~9.8cm) under the costal arch, and WBC was 28.03×109/L (7.3~127.69×109/L), monocytes were 9.25×109/L (1.66~15.79×109/L) at diagnosis. There were 8 cases in the high-risk group and 1 case in the low-risk group. Second-generation sequencing results show that 7 cases carried PTPN11 somatic mutations. Five in 9 cases had two kinds of classic JMML mutations, and 1 case had only NRAS mutation. Seven patients had normal chromosomal karyotypes, and 3 patients had abnormal 8, 11, and 18 chromosomes, respectively. Median treatment courses with decitabine are 3 courses (1~5 courses), the response rate of one course is 77.8% (7/9), the response rate of three courses is 80% (4/5), one case from high-risk group achieved complete remission after treated with 4 courses of decitabine and low-dose chemotherapy. The five-month progression-free survival rate was 77.8% (7/9). Conclusion: Treating JMML with decitabine combined with low-dose chemotherapy, can reduce patients' tumor burden, improve the general condition, and obtain approximately 80% clinical response rate. Decitabine combined with low-dose chemotherapy can be used as a treatment option for JMML before HSCT. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Safety and effecacy of decitabine applicated in JMML patients have not been confirmed.

Role of lncRNA Morrbid in PTPN11(Shp2)E76K-driven juvenile myelomonocytic leukemia

AbstractMutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, contribute to ∼35% of cases of juvenile myelomonocytic leukemia (JMML). A common clinical picture in children with JMML is that it presents as a constitutive hyperinflammatory syndrome, partially reminiscent of chronic myelomonocytic leukemia in adults. Thus, a component of JMML is associated with a hyperinflammatory state and abundant innate immune cells such as neutrophils and monocytes. Recently, we showed that the evolutionarily conserved mouse lncRNA Morrbid is specifically expressed in myeloid cells and uniquely represses the expression of the proapoptotic gene Bim to regulate the lifespan of myeloid cells. However, its role in JMML has not been investigated. In this study, we characterized the role of Morrbid and its target Bim, which are significantly dysregulated in Shp2E76K/+-bearing myeloid cells, in driving JMML. Loss of Morrbid in a mouse model of JMML driven by the Shp2E76K/+ mutation resulted in a significant correction of myeloid and erythroid cell abnormalities associated with JMML, including overall survival. Consistently, patients with JMML who had PTPN11, KRAS, and NRAS mutations and high expression of MORRBID manifested poor overall survival. Our results suggest that Morrbid contributes to JMML pathogenesis.

Clinical Study on Treatment of Juvenile Myelomonocytic Leukemia with Haploidentical-Hematopoietic Stem Cell Transplantation

To analyze the therapeutic efficacy of haploidentical-hematopoietic stem cell transplantation (hi-HSCT) for patients with juvenile myelomonocytic leukemia (JMML). The engraftment of hematopoietic stem cells, incidence of graft versus host disease (GVHD), infection, relapse, and survival of 6 JMML patients received hi-HSCT were retrospectively analyzed. Six (6 males) JMML patients received hi-HSCT from haplo-HLA-matched related donors. The results showed that the hematopoictic stem cells in all 6 patients were grafted successfully. Two cases of JMML died of pulmenary infections, other 4 cases survive without disease. Acute GVHD occurred in 3 patients and chronic GVHD occurred in 1 patients. The overall survival, disease free survival and relapse rates were 66.7%, 66.7%, 0%, respectively. The hi-HSCT is an effective method for treatment of patients with JMML, but there also is a serial problems to be resolved.

Leucémie myélomonocytaire juvénile : à propos de trois cas

Juvenile myelomonocytic leukemia (JMML), previously known as juvenile chronic myeloid leukemia (JCML), is a rare, myelodysplastic-myeloproliferative disease typically presenting in early childhood. This disorder is difficult to distinguish from other myeloproliferative syndromes such as chronic myeloid leukemia (CML) because of the similarities in their clinical and bone marrow findings. However, because of its unique biological characteristics such as absolute monocytosis with dysplasia, absence of Philadelphia chromosome or BCR-ABL fusion protein, hypergammaglobulinemia, and raised fetal hemoglobin level, this disorder does not satisfy the criteria for inclusion in the CML or chronic myelomonocytic leukemia (CMML) group, as seen in adult patients. We describe three cases of JMML, who had very similar clinical and laboratory findings.

Juvenile myelomonocytic leukemia in 12 children

Objective To analyze the clinical and cytogenetic features of juvenile myelomonocytic leukemia(JMML), and to provide a genotype-phenotype based management. Methods Clinical data of 12 cases of JMML who were diagnosed at Department of Pediatrics, Sun Yat-sen Memorial Hospital , Sun Yat-Sen University from October 2009 to October 2015 were analyzed.Either chemotherapy management or hematopoietic stem cell transplantation (HSCT) was chosen according to the disease risk factors. Results (1) All 12 cases displayed fever and abdominal distention, 3 cases (25%) had rashes, mucocutaneous hemorrhage as well as hepatosplenomegaly and lymphadenopathy.(2)The medium WBC count, hemoglobin and platelet in peripheral blood were 39.93×109/L, 76.87 g/L, 34.63×109/L respectively, and monocytosis could be seen and median of absolute monocyte count was 1.63×109/L.(3)A markedly increased synthesis of fetal hemoglobin(HbF) could be seen in 8 cases (66.7%). (4)Chromosomal studies of bone marrow cells showed absence of Philadelphia chromosome in all cases, and monosomy 7 was found in 2 cases while other abnormal karyotypes were found in another 2 cases.(5)Analysis of genetic mutations showed 8 cases (66.7%) with JMML positive gene mutation, 1 case with PTPN11+ NF1 mutation, 7 with NRAS genes mutation(2 of them combined with CBL mutation simultaneously and progressed to blast crisis; 1 case showed KRAS+ NF1 mutation simultaneously). (6)Nine of them received chemotherapy with the protocol of A-3V [Cytarabine 100 mg/(m2·d)+ Vincristine 1.5 mg/(m2·d)+ Etoposide 100 mg/(m2·d)+ Isotretinoin 75-100 mg/(m2·d)]. Complete remission(CR) could be seen in 6 cases(66.7%). Two cases progressed and received HSCT.Three cases were treated with low-dose Cytarabine and 6-thioguanine and none obtained CR, and 1 case of them progressed to blast crisis and died of severely infection after intensive chemotherapy.(7) Four patients with high risk were treated with HSCT and remained disease free at present. Conclusions JMML is rare in clinical practice without specific features.Chromosomal studies and genetic analysis are key diagnostic procedures for JMML.The protocol A-3V is suitable for JMML and patients without high risk factors, who could obtain continuous CR after chemotherapy.Intensive chemotherapy was not recommended.HSCT were mandatory therapeutic option for the patients with high risk.Genotype-phenotype based management can further improve the therapeutic efficacy of JMML. Key words: Myelomonocytic leukemia; Juvenile; Hematopoietic stem cell transplantation; Child

Allogeneic hematopoietic stem cell transplantation for chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia

This study was purposed to explore the therapeutic efficacy and influencing factors of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with chronic myelomonocytic leukemia (CMML) and in patients with juvenile myelomonocytic leukemia (JMML). The clinical data of 3 cases of CMML and 2 cases of JMML underwent allo-HSCT were analysed in term of multiparameter. The results showed that the hematopoietic stem cells in 5 patients grafted successfully. One case of JMML died of pulmonary disease, other 4 cases survive without disease. The analysis found that the disease burden before transplant, chromosome karyotype, acute GVHD II-IV and poor risk cytogenetics all associated with the relapse rate and disease-free survival rate of CMML. The low intensity conditioning regimen was better than myeloablative conditioning regimen. Type of donor and source of stem cells did not statistically and significantly affect OS and RFS. The splenectomy before allo-HSCT as well as spleen size at time of the alloHSCT did not influence on posttransplantation outcome of JMML. However, cord blood HSCT for JMML patients delayed hematologic recovery as compared to that of bone marrow or peripheral blood HSCT. The age, GVHD, HbF level played an important role in leukemia replace. It is concluded that the allogeneic hematopoietic stem cell transplantation is a curative regimen for CMML and JMML, but there also is a serial problems to be resolved.

Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML. Disclosures: No relevant conflicts of interest to declare.

Aggressive Transformation of Juvenile Myelomonocytic Leukemia Associated with Duplication of Oncogenic KRAS due to Acquired Uniparental Disomy

A small fraction of cases of juvenile myelomonocytic leukemia (JMML) develop massive disease activation. Through genomic analysis of JMML, which developed in an individual with mosaicism for oncogenic KRAS mutation with rapid progression, we identified acquired uniparental disomy at 12p. We demonstrated that duplication of oncogenic KRAS is associated with rapid JMML progression.

Whole Exome Analysis Reveals Spectrum of Gene Mutations in Juvenile Myelomonocytic Leukemia

AbstractAbstract 170 Introduction:Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in >70% affected children in a mutually exclusive manner. To define the molecular pathogenesis of JMML, we identified the full spectrum of gene mutations in 13 cases of JMML using whole exome sequencing of paired tumor-normal DNA. We also performed target-deep sequencing of relevant mutational targets in 92 cases of JMML. Patient and Methods:We evaluated 92 children (61 boys and 31 girls) with JMML, including 7 with Noonan syndrome-associated myeloproliferative disorder, who were diagnosed at institutions throughout Japan. The median age at diagnosis was 19 months (range, 1–160 months). Karyotypic abnormalities were detected in 15 cases, including 8 with monosomy 7. Fifty-six of the 92 (61%) cases received allogeneic hematopoietic stem cell transplantation.Exome capture from paired tumor-normal (CD3-positive T cell) DNA obtained from 13 cases of JMML was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA, USA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA, USA) according to the manufacturers’ protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate germline and somatic nucleotide changes were validated by Sanger/deep sequencing. A total of 92 JMML tumor specimens were screened for mutations in RAS pathway genes (PTPN11, NRAS, KRAS, c-CBL, and NF1) and 3 newly identified genes using deep sequencing. Results:For the current exome study, 10 missense and 1 nonsense single nucleotide variations were confirmed as nonsilent somatic mutations. The average number of mutations per sample (0.79; range, 0–4) was surprisingly low compared with those reported in other human cancers. Among the 11 somatic mutations, 6 involved the known RAS pathway genes (1 NF1, 1 NRAS, 2 KRAS, and 2 PTNP11 mutations) and included 5 mutations/deletions of either NF1 (n = 2), c-CBL (n = 1), or PTPN11 (n = 2) as detected in the germline samples. Nonoverlapping RAS pathway mutations were confirmed in 11 of the 13 discovered cases of JMML (85%). Five of the 11 somatic mutations were observed in 3 non-RAS pathway genes that have never been reported in JMML cases.Deep sequencing revealed RAS pathway mutations in 80 of 92 cases (87%) in a mutually exclusive manner; PTPN11 mutations were predominant (39/92 or 42%), followed by N/KRAS (24/92 or 26%), c-CBL (11/92 or 12%), and NF1 (6/92 or 6.5%) mutations. In agreement with previous reports, the majority of c-CBL (7/11) and NF1 (5/6) mutations were bi-allelic in the affected cases, showing compound heterozygous mutations or uniparental disomy of the mutant alleles, whereas most of the PTPN11 and N/KRAS mutations were heterozygous. In contrast, the remaining 12 (13%) cases were negative for known RAS pathway mutations. In addition, the 3 newly identified genes were recurrently in 18 cases (20%). Many of these mutations had lower allele frequencies compared to the accompanying RAS pathway mutations, indicating that they were responsible for disease progression rather than the establishment of JMML. The probability of 5-year transplantation-free survival (95% confidence interval) for the latter patients was significantly inferior to that of other cases (0% vs. 19% (8–34%), p < 0.001). Conclusion:Whole exome sequencing revealed the spectrum of gene mutations in cases of JMML. Together with a very high frequency of RAS pathway mutations, the paucity of non-RAS pathway mutations is a prominent feature of JMML. Somatic mutations of 3 newly identified genes were common among recurrent secondary events presumed to be involved in tumor progression and associated with poor clinical outcomes. Our findings provide an important clue that aids in understanding the pathogenesis of JMML and will help in the development of novel diagnostics and therapeutics for this type of leukemia. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding.

Transfusion associated peak in Hb HPLC chromatogram – a case report

High performance liquid chromatography (HPLC) and electrophoresis are commonly used to diagnose various hemoglobinopathies. However, insufficient information about the transfusion history can lead to unexpected and confusing results. We are reporting a case of Juvenile myelomonocytic leukemia (JMML) in which HbHPLC was done to quantify fetal hemoglobin (HbF). The chromatogram showed elevated HbF along with a peak in the HbD window. A transfusion acquired peak was suspected based on the unexpectedly low percentage of HbD and was subsequently confirmed using parental HbHPLC.

Pathogenesis of aplastic anaemia and paroxysmal nocturnal haemoglobinuria: the concept of clonality in stem cell disease

Molecular technologies have been applied not only to the investigations of haematological malignancies but also bone marrow failure syndromes, including aplastic anaemia (AA), paroxysmal nocturnal haemoglobinuria (PNH) and single lineage cytopenias, often associated with T cell large granular lymphocyte leukaemia (T-LGL). One of the main diagnostic problems is the distinction of hypoplastic clonal processes consistent with the diagnosis of myelodysplasdtic syndrome (MDS) from true idiopathic AA. Due to the paucity of the marrow, pathomorpho-logical diagnosis may be difficult and metaphase cytogenetics may not yield informative results, precluding proper diagnostic case assignment. Single nucleotide polymorphisms arrays (SNP-A) do not require cell division and may help to detect clonal chromosomal abnormalities. Systematic application of this technology has shown that chromosomal defects consistent with the diagnosis of MDS can be detected in a proportion of otherwise typical AA patients, initially or throughout the clinical course. At presentation, clonal abnormalities, in particular non-recurrent/random defects may be transient and reflect oligoclonality of the stem cell compartment rather than true malignant clonal process. Using SNP-A, submicroscopic chromosomal microdeletions can be detected; we have found recurrent microdeletions involving HLA and PIG-A loci on chromosomes 6 and X, respectively. These lesions likely evolve as a result of immune selection pressure and facilitate escape of the immune-resistant haematopoietic clones. Manifest evolution of MDS in the course of AA can be expected in up to 20% of patients with AA. The most common abnormality is monosomy-7, present in most cases of MDS derived from antedescent AA. In this context, monosomy-7 is associated with a distinct mutational pattern that includes presence of CBL mutations and RUNX1, while common mutations such as TET2, U2AF1, SRSF2 and ASXL1 are frequent in primary MDS with monosomy-7 or deletion7q. In contrast, childhood cases of juvenile myelomonocytic leukemia with monosomy-7 have a similar mutational pattern as seen in AA. Whole genome sequencing technologies including exome sequencing have led to identification of new mutations, not only in MDS but also in T-LGL which is characterised by frequent somatic activating mutation in STAT3. By ultra-deep sequencing of T cell receptor (TCR) VB chains, one can also characterise TCR repertoire and identify oligoclonal T cell mediated processes. The presence of STAT3 mutations distinguish clonal processes from semi-reactive oligoclonal T cell expansions.

Lysosomal Disruption Selectively Targets Leukemia Cells and Leukemia Stem Cells Through A Mechanism Related to Increased Reactive Oxygen Species Production

Abstract 61To identify new therapeutic strategies for AML, we compiled and screened an in-house library of on-patent and off-patent drugs to identify agents cytotoxic to leukemia cells. From this screen, we identified mefloquine, an off-patent drug indicated for the treatment and prophylaxis of malaria. In secondary assays, mefloquine decreased the viability of 9/10 human and murine leukemia cell lines (EC50 3.25–8.0 μM). Moreover, it reduced the viability of 4/5 primary AML samples, but was not cytotoxic to normal hematopoietic cells (EC50>31 μM). Importantly, mefloquine reduced the clonogenic growth of primary AML samples, but not normal hematopoietic cells, and completely inhibited engraftment of primary AML cells into immune deficient mice. Finally, systemic treatment with oral mefloquine (50 mg/kg/day) decreased leukemic burden without evidence of toxicity in 4 mouse models of leukemia, including mice engrafted with primary AML cells. Thus, mefloquine effectively targets leukemic cells, including leukemia stem cells, at concentrations that appear pharmacologically achievable and are not toxic to normal hematopoietic cells.To identify the mechanisms of mefloquine-mediated cell death in AML cells, we performed a binary drug combination screen, hypothesizing that drugs that synergized with mefloquine may share overlapping mechanism of action. From this combination screen of 550 drugs, we identified 18 that reproducibly synergized with mefloquine as measured by the Excess over Bliss additivism score, including 3 members of the artemisinin class of anti-malarials: artemisinin, artesunate and artenimol. Strikingly, 10/18 synergistic compounds, including the artemisinins, were known generators of reactive oxygen species (ROS). Therefore we tested mefloquine’s ability to increase ROS in leukemic cells. Mefloquine increased ROS production in leukemia cells in a dose- and time-dependent manner. Co-treatment with ROS scavengers α-tocopherol and N-acetyl-cysteine abrogated mefloquine-induced ROS production and cell death, indicating that ROS production was functionally important for mefloquine-mediated cell death. Moreover, the artemisinins induced ROS as single agents, and synergistically increased ROS when combined with mefloquine.To identify cellular target(s) of mefloquine’s anti-leukemic effects, we performed a yeast genome-wide functional screen to identify heterozygous gene deletions that rendered yeast more sensitive to mefloquine. 21/37 genes whose depletion conferred >4-fold sensitivity to mefloquine were associated with function of the yeast vacuole, equivalent to the mammalian lysosome. Consistent with these data, fluorescent confocal microscopy demonstrated that mefloquine and artesunate disrupted lysosomes. Cell death after mefloquine and artesunate treatment was caspase-independent and associated with increased incorporation of monodancylcadaverin in autophagosomes, consistent with the effect of these drugs on the lysosomes. To further explore the anti-leukemic activity of lysosomal disruption, we evaluated the anti-leukemic effects of the known lysosomal disrupter L-leucine-leucine methyl ether (LeuLeuOMe). Similar to mefloquine and artesunate, LeuLeuOMe induced cell death in leukemia cells, increased ROS production, and disrupted the lysosomes. Highlighting the potential clinical utility of lysosomal disrupters for the treatment of leukemia, a patient with relapsed/refractory juvenile myelomonocytic leukemia self-administered artemisinin. The artemisinin cleared the circulating blasts from the circulating blasts and the patient proceeded to allotransplant.Finally, to investigate the basis of leukemic cell hypersensitivity to lysosomal disruption, we assessed lysosomal characteristics of primary AML and normal hematopoietic cells. By gene expression analysis, AML patient samples had higher mRNA levels of the lysosomal cathepsins A, B, C, D, H, L, S and Z, compared to CD34+ normal hematopoietic cells, and cathepsins C, D and Z were significantly over-expressed in the LSC compartment, compared to normal HSCs.In summary, our data demonstrate that lysosomal disruption preferentially targets AML cells and AML stem cells through a mechanism related to increased ROS production. Thus, this work highlights lysosomal disruption as a novel therapeutic strategy for AML. Disclosures:Off Label Use: This study includes a case report of off-label use of the anti-malarial artemisinin in the treatment of a case of juvenile myelomonocytic leukemia.

Clinico-Pathological Characteristics of RAS Associated Autoimmune Lymphoproliferative Syndrome Like Disease (RALD), and RAS Mutated Juvenile Myelomonocytic Leukemia (JMML)

Abstract 5169Autoimmune lympho proliferative syndrome (ALPS) is a disease characterized by dysfunction of the FAS-mediated apoptotic pathway, currently categorized as Type Ia, germline TNFRSF6/FAS mutation; Type Ib, germline FAS ligand mutation; Type Is, somatic TNFRSF6/FAS mutation; and Type II, germline Caspase 10 mutation. Patients exhibit lymphadenopathy, hepatosplenomegaly, and autoimmune diseases such as immune cytopenia and hyper-γ-globulinemia. An additional subclassification has been proposed that includes Types III, which has been defined as that with no known mutation but with a defect in FAS-mediated apoptosis. Juvenile myelomonocytic leukemia (JMML) is a chronic leukemia in children. Patients show lymphadenopathy, hepatosplenomegaly, leukocytosis associated with monocytosis, anemia, thrombocytopenia. About 80% of patients with JMML have been shown to have a genetic abnormality in their leukemia cells including mutations of NF1, RAS family, CBL, or PTPN11. HSCT is currently considered the only curative treatment. However, some patients have been shown to achieve remission without HSCT. Interestingly, there are several cases of JMML reported simultaneously having clinical and laboratory findings compatible with autoimmune disease. Germline RAS pathway mutations cause Costello (HRAS), Noonan (PTPN11, KRAS, and SOS1), and cardio-facio-cutaneous (CFC) syndromes (KRAS, BRAF, MEK1, and MEK2). Patients with Costello and Noonan syndromes have an increased propensity to develop solid and hematopoietic tumors, respectively.We have recently reported a unique disease which lies between ALPS and JMML. The patients were characterized by prominent autoimmune cytopenia, lymphoadenopathy/splenomegaly and moderate monocytosis resembling to ALPS and JMML, but they did not satisfy the diagnostic criteria for ALPS or JMML. KRAS mutation in hemopoietic precursors including T and myeloid linage is the common molecular feature, and we have recently proposed these cases to be defined under a new disease entity of RAS associated ALPS like disease (RALD). These patients were characterized by activated T cells resistant to IL-2 depletion but not to FAS-dependent apoptosis associated with the decreased expression of pro-apoptotic protein BIM.Since somatic RAS mutation has been identified in classical JMML and RALD, it is tempting to speculate that the developmental stage dependent RAS mutation of hemopoietic stem/precursors may determine the clinical features of JMML and RALD. Clinico-pathological characteristics of three RALD cases and three JMML cases cured without SCT will be presented and discussed. Disclosures:No relevant conflicts of interest to declare.

非血縁者間同種骨髄移植を施行し良好な経過をとっているNF-1に合併した若年性骨髄単球性白血病 (JMML) の2例

非血縁者間同種骨髄移植を施行し良好な経過をとっているNF-1に合併した若年性骨髄単球性白血病 (JMML) の2例

Juvenile Myelomonocytic Leukemia Presenting With Coexistent Cytomegalovirus Infection—A Case Report

We report a case of juvenile myelomonocytic leukemia (JMML) with coexistent cytomegalovirus (CMV) infection in a 10-month-old child that caused initial diagnostic dilemma. The patient presented with fever, anemia, lymphadenopathy, and hepatosplenomegaly. The peripheral blood smear and bone marrow aspirate examination showed monocytosis, leukoerythroblastosis, myeloid hyperplasia, and increased blasts. Serologic test for CMV was positive and thus the hematologic picture was attributed to CMV infection and gancyclovir was started. The patient, however, did not improve clinically. A repeat peripheral smear and marrow showed persistence of the above picture and a diagnosis of JMML was made. Viral infections in young children may present with hematologic features overlapping with JMML and simultaneous occurrence of both may cause diagnostic dilemma.