Mir-183 Over-Expression: A Potential Biomarker for Juvenile Myelomonocytic Leukemia (JMML).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2558-2558
Author(s):  
Y. Lucy Liu ◽  
Yan Yan ◽  
Shelly Y. Lensing ◽  
Todd Cooper ◽  
Peter D. Emanuel
Keyword(s):  
Peripheral Blood ◽  
Robust Regression ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Potential Biomarker ◽  
Median Level ◽  
Myelomonocytic Leukemia

Abstract Abstract 2558 Juvenile myelomonocytic leukemia (JMML) is a rare disease of early childhood with a predilection for the monocyte/macrophage lineage. The pathogenesis of JMML is linked to dysregulated signal transduction through the NF1/RAS signaling pathway that is partially caused by genetic mutation of Ras, PTPN11, and c-CBL, or loss-of heterozygosity of Nf1. The hallmark of JMML is that JMML cells are selectively hypersensitive to GM-CSF in vitro. We previously reported that protein deficiencies of PTEN, CREB, and Egr-1 were frequently observed in JMML (67–87%). Recent research indicated that CREB was regulated by miR-34b, and Egr-1 was targeted by miR-183. We hypothesized that microRNAs may play an important role in contributing to the deficiency of these proteins. Using relative-quantitative real-time PCR, we evaluated the expression levels of miR-34b and miR-183 in mononuclear cells from 47 JMML patients. We found that the median level of miR-183 was significantly higher in JMML in comparison to normal controls (median=13.8 vs 4.2, p<0.001); but the median level of miR-34b was only slightly higher in JMML subjects, and not significantly so, compared to normal individuals (median=1.4 vs 1.0, p>0.05). This suggests that miR-34b does not play a significant role in JMML. Since extreme monocyte accumulation is one of the critical characteristics of JMML, we analyzed the correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood. Strikingly, there was a significant correlation between the expression level of miR-183 and the monocyte percentage in the peripheral blood from 34 patients who had available data (p<0.05). Based on a robust regression analysis, for every unit increase in the square root of RQ miR-183, the monocyte percentage significantly increased by 0.73% (SE=0.32%, p=0.023). This is the first evidence suggesting that microRNAs may contribute to the pathogenesis of JMML. miR-183 may also serve as an important biomarker that can be directly and quantitatively linked to significant clinical parameters in JMML. It also may ultimately provide a target for JMML therapy. Disclosures: No relevant conflicts of interest to declare.

CREB Deficiency: A Potential Critical Factor for Selective GM-CSF Hypersensitivity in Juvenile Myelomonocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2604-2604
Author(s):  
Y. Lucy Liu ◽  
Priyangi A Malaviarachchi ◽  
Shelly Y. Lensing ◽  
Robert P. Castleberry ◽  
Peter Dean Emanuel
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Protein ◽  
Gm Csf ◽  
Myelomonocytic Leukemia ◽  
Normal Controls

Abstract Abstract 2604 Poster Board II-580 Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative neoplasm (MDS/MPN) of infancy and early childhood. The pathogenesis of JMML has been linked to dysregulated signal transduction through the NF1/RAS signaling pathway and PTPN11. This dysregulation results in JMML cells demonstrating selective hypersensitivity to GM-CSF in in vitro dose-response assays. Since JMML hematopoietic progenitor cells are selectively hypersensitive to (rather than independent of) GM-CSF, it is rational to hypothesize that the function of the GM-CSF receptor in JMML patients is not constitutively over-active unless stimulated by the cytokine. We previously reported that PTEN is deficient in JMML patients. PTEN expression is up-regulated by Egr-1, which is one of the targets of the cAMP-response-element-binding protein (CREB). CREB, as a transcriptional factor, is expressed ubiquitously and bound to the cAMP-response-element (CRE) of the Egr-1 promoter. After phosphorylation at serine 133, CREB selectively activates the transcription of Egr-1 in response to GM-CSF stimulation in hematopoietic cells. We evaluated the CREB protein level in peripheral blood or bone marrow samples collected from 26 JMML patients. Mononuclear cells (MNCs) were isolated and lysed in lysis buffer at a density of 107/100μl. Protein levels of CREB were evaluated by ELISA and Western-blot. We found that 22/26 (85%) of subjects were substantially CREB deficient while they had constitutively high activity of MAP kinase (Erk-1/2). In comparison to normal controls (n=7), the median level of total CREB protein by ELISA was significantly lower in JMML subjects (0.62 vs 8.85 ng/mg BSA in normal controls; p=0.006). The mechanism that causes CREB deficiency in JMML is under further investigation and further results may be available to present at the meeting. This is the first evidence that CREB, a critical component downstream of the GM-CSF receptor, is highly deficient in the majority of JMML cases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Establishment of Stable Cell Lines Representing Juvenile Myelomonocytic Leukemia with SHP2 Mutations

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4353-4353
Author(s):  
Yuming Zhao ◽  
Yao Guo ◽  
Chunxiao He ◽  
Dengyang Zhang ◽  
Han Zhong Pei ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Juvenile Myelomonocytic Leukemia ◽  
Cytokine Signaling ◽  
Ras Signaling ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Transformed Cell ◽  
Transformed Cell Lines ◽  
Myelomonocytic Leukemia

Abstract Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

scholarly journals Secondary Mutations of JAK3 and SETBP1 in Juvenile Myelomonocytic Leukemia and Their Propagating Capacity; A Report from the AIEOP Study Group

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4625-4625
Author(s):  
Silvia Bresolin ◽  
Paola De Filippi ◽  
Francesca Vendemini ◽  
Riccardo Masetti ◽  
Franco Locatelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Hot Spot ◽  
Univariate Analysis ◽  
Driver Mutations ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Nsg Mice ◽  
Myelomonocytic Leukemia

Abstract INTRODUCTION Juvenile myelomonocytic leukemia is a rare early childhood leukemia, characterized by excessive proliferation of granulocytic and monocytic cells. About 95% of JMML patients harbor driver mutations in the RAS signaling pathway. Recently, secondary hits in SETBP1 and JAK3 have been reported in a Japanese cohort of JMML patients showing an adverse clinical outcome of patients carrying these mutations. Here we report the mutational analysis of SETBP1 and JAK3 and clinical implications in a cohort of Italian JMML patients. METHODS Samples collected at diagnosis of 65 patients with JMML were analyzed by Sanger sequencing. Mutations were found in RAS (NRAS-KRAS) 31%, PTPN11 35%, CBL 5%, whereas in 29% of patients none of the above cited mutations was present. Mutation hot spot regions of SETBP1 (SKI domain) and of JAK3 (PTK domains) were sequenced. A xenografted murine model was used to assess the in vivo competitive repopulation advantage of clones carrying mutations of JAK3 and SETBP1. Mononuclear cells from a patient with JMML at diagnosis harboring PTPN11, SETBP1 and JAK3 mutations were transplanted in NSG mice and assessed for mutational status in the bone marrow and spleen after engraftment of JMML cells. RESULTS Screening for JAK3 and SETBP1 mutations in patients revealed 9 mutations in 8 out of 65 patients at diagnosis of JMML. All of the identified secondary mutations were associated with known driver mutations, more frequent with mutated PTPN11 and RAS (p=0.036 and p= 0.01 respectively) than with CBL or in cases without known driver mutations. Seventy-five percent of secondary mutations were found in SETBP1 and only 1 patient harbored a mutation in JAK3. Remarkably one patient carried mutations in JAK3 (L857P and L857Q, both predicted to damaging protein function), PTPN11 (G503A) and SETBP1 (D868N). All variants were identified as heterozygous mutations, confirmed bi-allelic expression at the transcriptome level. The only patient carrying JAK3 as secondary mutation at E958K showed wild-type expression of JAK3 pointing to absence of a functional role at the protein level. Univariate analysis revealed association between the presence of secondary mutations and patient’s age at diagnosis, with older patients carrying JAK3 and SETBP1 mutations (p=0.0067); no other clinical and biological characteristics (i.e. WBC count, percentage of monocyte, HbF level and platelet count) being significantly associated with the presence of secondary hits in bone marrow of JMML cases. Patients with secondary mutations showed a trend to shorter survival compared to those without secondary events in JAK3 and SETBP1 (5-years OS= 0% vs 54.01%, SE=8.1; p=0.41, respectively). Interestingly, the in vivo assay using xenografted mice revealed a different propagating capacity of JAK3 clones of patients carrying JAK3 (2 different clones), SETBP1 and PTPN11 mutations. Indeed, for JAK3 only the clone with the L857Q mutation engrafted in BM and spleen of the mouse, together with SETBP1 and PTPN11 mutations. Moreover, a second mouse engrafted with mononuclear cells of the same patients showed that only cells carrying the PTPN11 mutation had engrafted. CONCLUSIONS In conclusion we identified secondary mutations in JAK3 and SETBP1 in 12% of patients of a representative cohort of Italian JMML patients, showing a trend of adverse outcome for patients carrying these mutations. These secondary events in JMML patients showed to have distinct propagating capacities upon engraftment in NSG mice pointing to a different functional impact of these mutations. Disclosures No relevant conflicts of interest to declare.

scholarly journals JMML and RALD (Ras-associated autoimmune leukoproliferative disorder): common genetic etiology yet clinically distinct entities

Blood ◽  
2015 ◽  
Vol 125 (18) ◽  
pp. 2753-2758 ◽  
Author(s):  
Katherine R. Calvo ◽  
Susan Price ◽  
Raul C. Braylan ◽  
Joao Bosco Oliveira ◽  
Michael Lenardo ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Clinical Care ◽  
Juvenile Myelomonocytic Leukemia ◽  
Peripheral Blood Mononuclear ◽  
Long Term Follow Up ◽  
Activated Monocytes ◽  
Myelomonocytic Leukemia

Abstract Ras-associated autoimmune leukoproliferative disorder (RALD) is a chronic, nonmalignant condition that presents with persistent monocytosis and is often associated with leukocytosis, lymphoproliferation, and autoimmune phenomena. RALD has clinical and laboratory features that overlap with those of juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML), including identical somatic mutations in KRAS or NRAS genes noted in peripheral blood mononuclear cells. Long-term follow-up of these patients suggests that RALD has an indolent clinical course whereas JMML is fatal if left untreated. Immunophenotyping peripheral blood from RALD patients shows characteristic circulating activated monocytes and polyclonal CD10+ B cells. Distinguishing RALD from JMML and CMML has implications for clinical care and prognosis.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 45-45 ◽  
Author(s):  
Hui Huang ◽  
Daniel E. Bauer ◽  
Mignon L. Loh ◽  
Govind Bhagat ◽  
Alan B. Cantor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Phosphorylation ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Brdu Incorporation ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Myelomonocytic Leukemia

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.

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

Blood ◽  
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.

scholarly journals Establishment ofin VitroDrug Sensitivity Screening Assay for Chronic Lymphocytic Leukemia (CLL) Primary Patient Samples

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-42
Author(s):  
Bandana Ajay Vishwakarma ◽  
Amy Wesa
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Drug Response ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Standard Of Care ◽  
Lymphocytic Leukemia ◽  
New Drugs ◽  
Potential Biomarker

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. CLL is characterized by proliferation and accumulation of monoclonal, small, mature CD5+ B-cells in peripheral blood, bone marrow and secondary lymphoid organs. The current treatment regimens have improved overall survival but CLL patients will eventually experience relapse. The disease is still incurable making it a necessity to discover and screen for new drugs. Patient derived CLL specimens undergo spontaneous apoptosis when grown in culture and is a major limitation to screening for new therapeutic drugs. We have established anin vitroculture conditions which supports growth and proliferation of patient derived primary CLL cells. Champions has a bank of primary CLL patient samples across different Rai stage, genetic mutations and relapsed and refractory cases. Using the optimizedin vitroculture conditions, we tested three CLL human primary samples (models) for proliferation . All three models proliferated 1.5 to to 2-fold in the optimized media in 6 days, as measured using CellTiter-Glo®. We also examined the phenotypic changes in the cells after growing them in the established media. Flow cytometric analysis showed that the CLL cells mostly retained the primary phenotypic characteristic CD5+CD10-Cd19+CD20+ even after being in the culture for 6 days. Next, we screened for sensitivity of primary CLL patient samples (N=10) against known standard of care (SOC) drugs venetoclax, ibrutinib, idelasib, chlorambucil and cytarabine. Primary patient samples derived from peripheral blood mononuclear cells (PBMC) were cultured in 96 well plates in the enriched media and treated with respective drugs over a concentration range over 5 logs. Drug sensitivity was assessed using CellTiter-Glo® luminescent cell viability assay on day 3. Ourin vitroassay indicated that some, but not all patient samples were sensitive to approved standard of care drugs. A relapsed bendamustine pre-treated patient sample was sensitive to all the SOC drugs tested. In addition to drug response, whole exome sequencing and RNAseq are being conducted on these samples, to compare mutational analyses with drug responsiveness. With clinically annotated patient-derived CLL samples, WES and RNAseq plus drug response to standard of care provides a comprehensiveex vivoplatform for the preclinical testing of drug candidates, which may not only provide information on agent efficacy, but that can permit potential biomarker mining and exploration of patient selection criteria for investigational new agents in CLL. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1124-1127 ◽  
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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