Immunophenotypic and Genetic Overlap between JMML and CMML

Abstract Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution. Methods: The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel. CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis. Results: We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance. We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining. Conclusions: Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

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Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20180853 ◽

2021 ◽

Vol 218 (2) ◽

Cited By ~ 1

Author(s):

Eleni Louka ◽

Benjamin Povinelli ◽

Alba Rodriguez-Meira ◽

Gemma Buck ◽

Wei Xiong Wen ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Childhood Leukemia ◽

Clonal Evolution ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Ras Pathway ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin−CD34+CD38−CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a “first hit,” (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

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Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia

Blood ◽

10.1182/blood-2007-06-093302 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1124-1127 ◽

Cited By ~ 27

Author(s):

Sophie Archambeault ◽

Nikki J. Flores ◽

Ayami Yoshimi ◽

Christian P. Kratz ◽

Miriam Reising ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Peripheral Blood ◽

Residual Disease ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Allele Specific ◽

Myelomonocytic Leukemia

AbstractJuvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre–hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies.

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Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

Blood ◽

10.1182/blood-2014-09-601690 ◽

2015 ◽

Vol 125 (3) ◽

pp. 516-524 ◽

Cited By ~ 43

Author(s):

Elliot Stieglitz ◽

Camille B. Troup ◽

Laura C. Gelston ◽

John Haliburton ◽

Eric D. Chow ◽

...

Keyword(s):

Hematopoietic Stem Cell Transplant ◽

Juvenile Myelomonocytic Leukemia ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Dismal Prognosis ◽

Key Points ◽

Polymerase Chain ◽

Rare Cells ◽

Digital Polymerase Chain Reaction ◽

Myelomonocytic Leukemia

Key Points Mutations in SETBP1 can be detected using droplet digital polymerase chain reaction in at least 30% of patients with JMML and are associated with a dismal prognosis. Patients harboring rare cells with mutant SETBP1 at diagnosis should be considered candidates for swift hematopoietic stem cell transplant.

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An Encouraging Successful Result of Allogeneic Hematopoietic Stem Cell Transplantation in a Boy Both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta : A Case Report

Blood ◽

10.1182/blood-2020-142710 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Fang Liu ◽

Xiaofan Zhu ◽

Wenyu Yang ◽

Ye Guo ◽

Xia Chen ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Osteogenesis Imperfecta ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Juvenile Myelomonocytic Leukemia ◽

Allogeneic Bone ◽

Hematopoietic Stem ◽

Myelomonocytic Leukemia

Background: Juvenile myelomonocytic leukemia (JMML) is a rare hematologic malignancy in young children that is classified as a myelodysplastic/myeloproliferative neoplasm and not only characterized by young age, hepatosplenomegaly, thrombocytopenia and monocytosis, but also by molecular aberrations in the RAS-RAF-MEK-ERK signaling pathway and GM-CSF-hypersensitivity. Most children with JMML experience an aggressive clinical course and the only curative treatment option for these children is stem cell transplantation (SCT). Osteogenesis imperfect (OI) is also an orphan inherited monogenic bone fragility disorder that usually is caused by mutations in one of the two genes coding for collagen type I alpha chains, COL1A1 or COL1A2. A common issue associated with the molecular abnormality is a disturbance in bone matrix synthesis and homeostasis inducing bone fragility. In very early life, this can lead to multiple fractures and progressive bone deformities. Current multidisciplinary management could only improve quality of life for patients, including physical therapy, drug treatment and orthopaedic surgery. Innovative therapies, such as progenitor and mesenchymal stem cell or bone marrow transplantation, targeting the specific altered pathway rather than the symptoms, may develop new curative treatments. Here we report a 3-year-old boy who suffered from both JMML and OI, was successfully transplanted and kept presenting an encouraging outcome up to now. Aims:To investigate the possible efficacy and safety of Allogeneic Hematopoietic Stem Cell Transplantation in a boy both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta. Methods:A 3-year-old boy presented with fatigue, fever, petechia and rash in Aug 2019, accompanying with loss of appetite, joint pain and severe hepatosplenomegaly. The boy had a special appearance of short stature and blue sclerae, meanwhile he suffered intermittent eczema and bone fracture twice since he was 2 years old. Similar characteristics were also positive in his grandmother, father and father's sister. The blood cell counts revealed anemia, thrombocytopenia and leukocytosis especially monocytosis. Bone marrow aspirate showed excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor in vitro. Somatic mutation of gene NF1, PTPN11 and COL1A1 were identified by Next Generation Sequencing.Therefore, the little boy was diagnosed with two rare diseases of Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta at the same time. After 4 courses of hypomethylating agents therapy, the boy underwent haploidentical allogeneic bone marrow stem cell transplantation combined with allogeneic single umbilical cord blood transplant in May 2020. The myeloablative conditioning regimen was composed of Decitabine (20mg/m2/day, days -13 to -9), Cyclophosphamide (25mg/kg/day, days -8 and -7), Busulfan(100mg/m2/day, days -6 to -3), Fludarabine (40mg/m2/day, days -6 to -2) and Cytarabine (100mg/m2/day, days -6 to -2). Post-Cyclophosphamide (50mg/kg/day, days +3 and +4), tacrolimus and mycophenolate mofetil were used for prophylaxis of graft-versus-host disease (GVHD). Results:The number of infused TNCs from haplo-bone marrow and cord blood unit was 41.4×10^8/kg and 9.72×10^7/kg, respectively, while the number of infused CD34+ cells was 11.84×10^6/kg and 2.33×10^5/kg, respectively. The boy achieved sustained engraftment of both neutrophils and platelets at 16 days and 24 days, respectively, with complete haplo-donor chimerism of confirmed at 14 days. He developed grade III acute GVHD (skin, gut and liver) and recovered at 39 days after transplant. Clinical symptoms such as rash, joint pain and hepatosplenomegaly got complete remission, and the mutated genes like NF1, PTPN11 and COL1A1 all disappeared at 30 days. At the time of this report, the boy was alive with negative MRD and good quality of life with a follow-up of 3 months after HCT. Conclusion:To our knowledge, this is the first report that a child both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta was cured by allogeneic hematopoietic stem cell transplantation.Our experience suggests that allogeneic bone marrow transplantation may be a novel safe and effective therapeutic strategy for OI patients. Disclosures No relevant conflicts of interest to declare.

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Feasibility and Outcome of Hematopoietic Stem Cell Transplantation Combined with Decitabine for Children with Juvenile Myelomonocytic Leukemia

Blood ◽

10.1182/blood.v128.22.5869.5869 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5869-5869

Author(s):

Huaying Liu ◽

Chunfu Li ◽

Yuelin He

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Complete Remission ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Juvenile Myelomonocytic Leukemia ◽

Unrelated Donor ◽

Hematopoietic Stem ◽

Myelomonocytic Leukemia

Abstract Background Juvenile myelomonocytic leukemia(JMML) is a rare clonalmyelodysplastic/myeloproliferative disorder that occurs during infancy and early childhood with poor prognosis. Chemotherapy has not been found to be dffective, and Hematopoietic stem cell transplantation(HSCT) is currently the only curative treatment for JMML. Relapse and engraftment failure are the major causes of HSCT failure in JMML. Patients and method We report the outcomes of 4 patients with JMML who received HSCT combined with Decitabine between 2014-2015. Patient median age was 2 years(range,1-3years), and 3 were boys and 1 girl. Decitabine was given before and after the HSCT for one time(20mg/m2.d X 5d、10mg/m2 .d X 5d). Before HSCT, all the patients received mild chemotherapy(three or four course). The bone marrow evaluations of all the patients before HSCT were complete remission(CR). Two patients received human leukocyte antigen(HLA)-matched HSCT from unrelated donors, and two patients received haploidentical HSCT from parents followed by unrelated cord blood transplantation(UCB). Conditioning regimen of Unrelated donor-PBSCT was Busulfan+fludarabine+Thiotepa+Thymoglobuline, and the conditioning of haplo-HSCT was Busulfan+fludarabine+Cytarabine+Thymoglobuline. The number of nucleated cells of HLA-matched HSCT was 8×108/kg. The number of nucleated cells of Haplo-HSCT was 47.2×108/kg、61.26×108/kg , respectively, and the number of nucleated cells of UCB was 7.23×107/kg、9.4×107/kg, respectively. GVHD prophylaxis was based on post-transplant high-dose cyclophosphamide(PTCy, 50mg/kg on days +3 and +4) combined with mycophenolate plus cyclosporine A or tacrlimus. Results: The median follow-up was 21 months(range,11-27 months). The overall survival(OS) and the Disease free survival(DFS) both were 100%, All the patients got 100% engraftment(Unrelated-donor stem cell engrafted and Haploidentical-donor stem cell engrafted in 2 and 2 patients , respectively). None of the patients developed relaps, the bone marrow evaluations were complete remission(CR) after HSCT. The most common toxicities were infection with neutropenia(100%, n=4), The cumulative incidences of acute GVHD gradesII-III and CMV infection were 50% and 75% respectively. Conclusion: The combination of decitabine and HSCT shows encouraging results with highly effective and less toxicity for JMML. The futhuer study should be developed in the future. Disclosures No relevant conflicts of interest to declare.

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Childhood Myelodysplastic Syndrome: Focus on the Approach to Diagnosis and Treatment of Juvenile Myelomonocytic Leukemia

Hematology ◽

10.1182/asheducation-2010.1.357 ◽

2010 ◽

Vol 2010 (1) ◽

pp. 357-362 ◽

Cited By ~ 23

Author(s):

Mignon L. Loh

Keyword(s):

Germ Line ◽

Myeloproliferative Neoplasms ◽

Standard Of Care ◽

Mitogen Activated Protein Kinase ◽

Juvenile Myelomonocytic Leukemia ◽

Cell Transplant ◽

Current Standard ◽

Hematopoietic Stem ◽

Mitogen Activated Protein ◽

Myelomonocytic Leukemia

AbstractExpansion of myeloid blasts with suppression of normal hematopoiesis is a hallmark of acute myeloid leukemia (AML). In contrast, myeloproliferative neoplasms (MPNs) are clonal disorders characterized by overproliferation of one or more lineages that retain the ability to differentiate. Juvenile myelomonocytic leukemia (JMML) is an aggressive MPN of childhood that is clinically characterized by the overproduction of monocytic cells that can infiltrate organs, including the spleen, liver, gastrointestinal tract, and lung. Major progress in understanding the pathogenesis of JMML has been achieved by mapping out the genetic lesions that occur in patients. The spectrum of mutations described thus far in JMML occur in genes that encode proteins that signal through the Ras/mitogen-activated protein kinase (MAPK) pathways, thus providing potential new opportunities for both diagnosis and therapy. These genes include NF1, NRAS, KRAS, PTPN11, and, most recently, CBL. While the current standard of care for patients with JMML relies on allogeneic hematopoietic stem-cell transplant, relapse is the most frequent cause of treatment failure. Rarely, spontaneous resolution of this disorder can occur but is unpredictable. This review is focused on the genetic abnormalities that occur in JMML, with particular attention to germ-line predisposition syndromes associated with the disorder. Current approaches to therapy are also discussed.

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Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia

Blood ◽

10.1182/blood-2020-134494 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Tadaaki Inano ◽

Marito Araki ◽

Soji Morishita ◽

Misa Imai ◽

Yoshihiko Kihara ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Genomic Dna ◽

Research Funding ◽

Somatic Mutations ◽

Triple Negative ◽

Jak2 V617f ◽

Sequencing Analysis ◽

Hematopoietic Stem ◽

Otsuka Pharmaceutical

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.

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Clonal Evolution of Acute Myeloid Leukemia Following Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.1528.1528 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1528-1528 ◽

Cited By ~ 1

Author(s):

Matt Christopher ◽

Allegra A. Petti ◽

Christopher Miller ◽

Lukas D. Wartman ◽

Jacqueline E. Payton ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Clonal Evolution ◽

Driver Mutations ◽

Cell Transplant ◽

Immune Recognition ◽

Hematopoietic Stem ◽

Bone Marrow Biopsies ◽

Extramedullary Relapse ◽

New Mutations

Abstract The evolutionary model of cancer progression suggests that malignant clones survive initial therapy and acquire new mutations, ultimately resulting in therapy-resistant disease. Indeed, previous genomic analyses of AML relapsed after chemotherapy demonstrated clonal evolution, defined as the acquisition of new mutations including new putative driver mutations. Allogeneic hematopoietic stem cell transplant (HSCT) is a potent therapy that provides benefit in part through an immune-mediated Graft-vs-Leukemia (GVL) effect. We and others have hypothesized that the distinctive nature of the therapeutic bottleneck imposed by HSCT may result in distinct patterns of clonal evolution compared to that seen after treatment with chemotherapy alone. To test this, we performed enhanced exome sequencing of matched diagnosis and relapse tumors to identify somatic mutations in 15 patients who relapsed after matched related or unrelated donor HSCT, including 6 patients with isolated extramedullary relapse. For comparison, we analyzed 20 cases of AML relapsed after chemotherapy alone. In all 35 relapse samples, "new" somatic variants were identified. These were defined as being present in the relapse sample at a variant allele frequency (VAF) >5% and undetectable in the diagnosis sample (no variant reads with at least 40x coverage at that position). Significantly more new variants were observed post HSCT compared with post chemo (new variants post HSCT, 16.8 ±3.5 vs. post chemo, 7.3 ± 1.9, p<.01), an effect that was most striking in the extramedullary relapses (22.0 ± 4.7 new variants). When normalized for time-to-relapse, however, this difference was not significant (new variants per year post HSCT, 9.4 ± 2.1 vs. post chemo, 16.5 ± 7.2, p=NS), suggesting that the rate at which new variants are acquired is not different in relapses after HSCT vs. chemotherapy alone. At relapse, 13/15 of the post-HSCT cases had acquired previously undetected driver mutations. Of note, there were no mutations that were recurrently associated with relapse after HSCT. In addition, no mutations or copy number changes were observed in MHC genes or in genes involved with antigen presentation, suggesting that these mechanisms may not play a major role in relapse after HSCT. Similarly, no mutations were found that were associated specifically with extramedullary relapse compared to bone marrow relapse. All six extramedullary relapses showed clear evidence of new subclonal populations, and bone marrow biopsies performed at the time of extramedullary relapse showed no evidence of leukemic variants, raising the possibility that extramedullary AML may arise from small numbers of blasts that escape therapy and evolve independently outside the bone marrow. To assess whether leukemic variants could be detected in remission samples from transplanted patients before relapse, 15-25 leukemic variants were selected from each case and amplicon-based sequencing (providing >2500x coverage on average) was performed on remission samples taken before and after transplant. In 3 of 8 evaluable cases, AML-associated variants were detectable at low levels (i.e. 1 cell in 250) in remission samples obtained after transplant; in two of these patients, multiple variants were detected within 2 months of relapse. In the third patient, a single TET2 mutation was detectable more than 2 years before relapse. Finally, in 6 relapse cases (4 post HSCT and 2 post chemotherapy) we observed subclones with driver mutations that were cleared after therapy only to be replaced at the time of relapse by a different subclone with a distinct mutation in the same driver gene (Table 1). This suggests that in some cases, relapse may rely upon--and select for--tumor-specific progression events. In summary, our findings suggest that the clonal evolution of AML relapsing after HSCT is similar to that of AML relapsing after chemotherapy alone. No evidence was found for mutations specifically affecting immune recognition, although the possibility remains that epigenetic changes or host factors may influence the GvL effect after HSCT. Evolutionary complexity is a hallmark of AML, and may play a key role in its propensity for relapse despite the treatment modality used. Disclosures No relevant conflicts of interest to declare.

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Pediatric myelodysplastic syndrome

Russian Journal of Pediatric Hematology and Oncology ◽

10.17650/2311-1267-2018-5-3-23-35 ◽

2018 ◽

Vol 5 (3) ◽

pp. 23-35

Author(s):

B. V. Afanasyev ◽

L. Zubarovskaya

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Germ Line ◽

Bone Marrow Failure ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Main Method ◽

Bone Marrow Failure Syndromes ◽

Or Gene ◽

Myelomonocytic Leukemia

Pediatric myelodysplastic syndrome (MDS) are a heterogeneous group of clonal disorders often occur in the context of inherited bone marrow failure syndromes, acquired aplastic anemia or gene predisposition. Germ line syndromes predisposing individuals to develop familial MDS or acute myeloid leukemia have recently been identified – mutations in RUNX1, ANKRD, GATA2, ETV6, SRP72, DDX41. Juvenile myelomonocytic leukemia (JMML) occurs in context of inherited and somatic mutations PTPN11, KRAS, NRAS, CBL, NF1. In pathogenesis of these disorders there are a several factors – hypermethylation, clonal hematopoiesis/cytopenia of undetermined significance, disturbances of bone marrow microenvironment, telomeres, immune mechanisms. Allogeneic hematopoietic stem cell transplantation is the main method of MDS and JMML treatment but it is necessary to take into account special indications for refractory cytopenia (infections, dependence on blood transfusions) and be careful for JMML with CBL mutation.

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Investigation of New Therapeutic Compounds for Juvenile Myelomonocytic Leukemia Using Induced Pluripotent Stem Cells with Stably Activated Ras Pathway

Blood ◽

10.1182/blood-2019-131406 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4651-4651

Author(s):

Lisa Maria Kuhn ◽

Cyrill Schipp ◽

Daniel Hein ◽

Bianca Killing ◽

Nan Qin ◽

...

Keyword(s):

Stem Cells ◽

Cell Death ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Conflicts Of Interest ◽

Juvenile Myelomonocytic Leukemia ◽

Ras Signaling ◽

Hematopoietic Stem ◽

Ras Pathway ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a chronic, poor prognostic myeloid neoplasm of childhood that is characterized by malignant expansion of monocytic cells. Chemo- and radiotherapy are not effective in JMML, therefore allogeneic hematopoietic stem cell transplantation is the only therapy option for most affected children. Relapse is the most frequent cause of treatment failure and event-free-survival at five years is low (approximately 50%). Recent studies showed that in 90% of JMML patients the proliferation of monocytic tumor cells is driven by mutations in a confined set of genes (KRAS, NRAS, PTPN11, NF1 or CBL) that activate the RAS signalling pathway. Drugs specifically targeting this pathway are therefore attractive candidates for therapy of JMML patients. As in vitro models of JMML, we generated inducible pluripotent stem cells (iPSC) stably expressing wildtype or activating oncogenic versions of KRAS (G12D) or NRAS (G13D) as well as iPSCs with CRISPR interference mediated downregulated NF1 expression. Manipulation of KRAS, NRAS, and NF1 expression and activation of downstream signaling targets (MEK, ERK) of the Ras pathway were confirmed by RT-PCR and western blot analyses, respectively. After transduction iPSCs retained typical pluripotency markers and could be differentiated into CD34+ and CD45+ cells of the hematopoietic lineage. We then carried out a screen to test the response of these iPSC cell lines to experimental and clinical drugs targeting the Ras signaling pathway, as well as to other compounds suggested to be promising candidate drugs or drugs already in clinical trial for JMML. In our screen the model cell lines were resistant to all tested MEK-inhibitors, including Selumetinib and Trametinib. The broad receptor tyrosine kinase inhibitor Dovitinib and the DNA methyltransferase inhibitor Azacytidine elicited strong responses in all iPSC cell lines regardless of their KRAS, NRAS or NF1 state. This underlines their extensive, but non-targeted killing potential. In our screen, an experimental small molecule drug induced significantly more cell death in KRAS-G12D iPSCs (IC50 1.5 µM) than in comparable wildtype cells (IC50 3.3 µM, p<0.0001), which could be validated in independent assays. In addition to targeted cell death activation, the drug has been suggested to promote differentiation of hematopoietic cells, which could potentially increase its anti-tumor efficiency. Experimental studies analyzing the underlying mechanism of its differential effect on KRAS wildtype compared to KRAS-G12D cells are currently carried out and will be presented. Our results suggest, that iPSCs with RAS pathway activation due to stable expression of oncogenic KRAS or NRAS or downregulation of NF1 expression are valuable tools for preclinical testing and may identify promising novel lead compounds for JMML treatment. Disclosures No relevant conflicts of interest to declare.

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