JAK2 V617F-Negative and MPL W515K/L-Negative Essential Thrombocythemia: A High Resolution SNP Array Study

Abstract Background JAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The molecular cause of the remaining ET cases is still largely unknown. Aims We sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH). Methods We studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix). Results Only 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size. Conclusions Previous studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset. Circos plot showing the recurrent CN-LOHs Left half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links. Disclosures: No relevant conflicts of interest to declare.

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A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5215-5215

Author(s):

Munazza Rashid ◽

Rifat Zubair Ahmed ◽

Shariq Ahmed ◽

Muhammad Nadeem ◽

Nuzhat Ahmed ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Diagnostic Algorithm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Common Mutation ◽

Hypochondriac Region ◽

Exon 9 ◽

Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

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Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽

10.1182/blood.v116.21.3074.3074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3074-3074

Author(s):

Brady L Stein ◽

Donna M Williams ◽

Michael A McDevitt ◽

Christine L. O'Keefe ◽

Ophelia Rogers ◽

...

Keyword(s):

Molecular Genetics ◽

De Novo ◽

Conflicts Of Interest ◽

Single Nucleotide Polymorphism Array ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Snp Array ◽

Uniparental Disomy ◽

Jak2 V617f ◽

Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

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Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽

10.1182/blood.v118.21.4687.4687 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4687-4687

Author(s):

Yue Xu ◽

Changxin Yin ◽

Han He ◽

Lingling Shu ◽

Fuqun Wu ◽

...

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Western Countries ◽

Arms Pcr ◽

V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

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JAK2 Signaling Specifies Phenotypic Pleiotropy In Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v122.21.1608.1608 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1608-1608

Author(s):

Lily Huang ◽

Huiyu Yao ◽

Yue Ma

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Phenotypic Diversity ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Wild Type ◽

Gain Of Function ◽

Hematopoietic Stem ◽

Phenotypic Differences ◽

V617f Mutation

Abstract Myeloproliferative neoplasms (MPNs) are a phenotypically diverse group of pre-leukemic diseases characterized by overproduction of one or more of the myeloid cell lineages. Gain-of -function mutations in the Janus tyrosine kinase 2 (JAK2) are major determinants in MPNs, These include the V617F mutation and mutations in exon 12. Interestingly, MPN phenotype in patients with exon 12 mutations is distinct from that of patients with the V617F mutation. Mechanisms underlying the phenotypic differences are not well understood. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function mutations. Interestingly, three of them with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only. To determine the mechanisms underlying the phenotypic differences by different JAK2 mutants, we characterized hematopoietic progenitors and precursor subsets in these mice for their proliferation, apoptosis and differentiation. Quantification of the hematopoietic stem and progenitor population showed an increased percentage of granulocyte-monocyte progenitors (GMP) and skewing of differentiation towards the granulocytic lineage in JAK2(V617F) and JAK2(N622I) mice compared to JAK2(K539I) or wild-type JAK2 mice. Because no difference was observed in the proliferation or apoptosis of bone marrow progenitors from JAK2 mutant mice, differentiation of the common myeloid progenitors (CMP) was likely skewed towards GMP by JAK2(V617F) and JAK2(N622I). Consistent with this hypothesis, similar results were observed in colony forming assays from sorted CMP populations. In the spleen, a decrease in GMP apoptosis and an increase in apoptosis of the megakaryocyte-erythrocyte progenitors (MEP) also contributed to the skewing towards the granulocytic lineage in JAK2(N622I) mice. Similar to MPN patients, mice expressing JAK2 mutants exhibited splenomegaly. We found that JAK2 mutants caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. As a result, more differentiated precursors were expanded in the spleens of JAK2 mutants mice compared to mice expressing wild-type JAK2. Consistent with their phenotypes, the percentage of Annexin V＋7AAD－erythroblasts in JAK2(K539I) and JAK2(V617F) mice was significantly less than in JAK2(N622I) or wild-type JAK2 mice. On the other hand, both proliferation and apoptosis contribute to the differential degrees of granulocytosis among mice expressing different JAK2 mutants. In line with the different effects elicited by different JAK2 mutants in progenitor and precursor cells, signal transduction pathways were differentially activated downstream of different JAK2 mutants. In summary, our results showed that JAK2 mutants differentially skew differentiation in early stem and progenitor compartments, and also regulate apoptosis and proliferation of distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide the mechanistic basis for the phenotypic diversity observed in MPNs with different JAK2 mutants. Disclosures: No relevant conflicts of interest to declare.

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JAK2 Signaling Specifies Phenotypic Pleiotropy in Myeloproliferative Neoplasms.

Blood ◽

10.1182/blood.v120.21.2868.2868 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2868-2868

Author(s):

Lily Huang ◽

Huiyu Yao ◽

Yue Ma

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Phenotypic Diversity ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Hematological Toxicity ◽

Gain Of Function ◽

Hematopoietic Stem ◽

Erythroid Precursor ◽

Tyrosine Kinase 2

Abstract Abstract 2868 The Janus tyrosine kinase 2 (JAK2) plays an important role in hematopoiesis of multiple lineages. A gain-of-function JAK2 mutation, V617F, is the major determinant in myeloproliferative neoplasms (MPNs), a phenotypically diverse group of hematological diseases in which cells of the myelo-erythroid lineage are overproduced. JAK2 kinase inhibitors showed hematological toxicity in treating MPNs, calling for novel therapeutics that can target only the affected lineage while sparing others. This task is hindered by lack of understanding in how JAK2 signaling differentially regulates the generation of different blood cells. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function JAK2 mutations in addition to V617F (1). Surprisingly, three activating JAK2 mutants with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only (2). Quantification of the hematopoietic stem and progenitor populations in mice expressing wild-type JAK2 or JAK2 mutants showed significant granulocytic skewing by JAK2(V617F) and JAK2(N622I) both in the bone marrow and spleen. In contrast, erythroid skewing by JAK2(K539I) was observed. Consistent with these results, qualitative and quantitative differences were observed in signaling events downstream of JAK2 in stem and progenitor cells from mice expressing different JAK2 mutants. JAK2 mutants also caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. In later more differentiated compartments, JAK2(K539I) and JAK2(V617F) expanded erythroid precursor cells, including proerythroblasts and later precursors, to cause erythrocytosis, while JAK2(V617F) and JAK2(N622I) expanded myeloid precursors to cause granulocytosis. The expansion of these later compartments was at least in part due to a decrease in apoptosis. Together, our results showed that JAK2 mutants differentially skew early stem and progenitor compartments toward the erythroid or granulocytic lineage, and expand distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide mechanistic basis for the phenotypic diversity observed in mice expressing different JAK2 mutants. Our results show that differential JAK2 signaling regulates hierarchically early and late progenitor compartments to drive erythropoiesis vs. granulopoiesis. These results shed light on MPN biology and may facilitate the design of novel and more effective therapeutic agents that specifically target affected lineage without compromising other lineages. Disclosures: No relevant conflicts of interest to declare.

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Reconciling Phenotype with Genotype in the MPN: Impact of SNP Array-Based Chromosomal Analysis.

Blood ◽

10.1182/blood.v114.22.1893.1893 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1893-1893

Author(s):

Michael A McDevitt ◽

Donna Williams ◽

Brady L. Stein ◽

Christine O'Keefe ◽

Ramon V. Tiu ◽

...

Keyword(s):

Genomic Instability ◽

Copy Number ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Snp Array ◽

Uniparental Disomy ◽

Disease Classification ◽

Single Mutation ◽

Dependent Manner ◽

Disease Evolution

Abstract Abstract 1893 Poster Board I-916 The diagnosis and accurate prognostication of the myeloproliferative neoplasms (MPN) is complicated by phenotypic mimicry and variable rates of disease evolution. The somatic mutation of JAK2 (JAK2V617F), identified in more than 90% of PV and in 50% of ET and PMF patients, is associated with acquired uniparental disomy (aUPD) on chromosome 9p, generating copy number-neutral loss of heterozygosity (CN-LOH) and results in homozygosity of JAK2V617F in roughly one third of JAK2V617F-positive patients. The molecular events upstream of the mitotic recombination that leads to somatic CN-LOH are essentially unknown. Intriguingly, JAK2V617F itself has been implicated in the generation of genomic instability (Plo et al 2008). Although these observations provide a rationale for how a single mutation could give rise to different clinical pathologies and downstream genomic instability in MPN, the JAK2V617F allele burden (AB) has not been systematically correlated with high resolution evaluation of amplifications, deletions, and CN-LOH in a large MPN cohort. We performed single nucleotide polymorphism arrays (SNP-A), a powerful karyotyping tool with the unique ability to detect CN-LOH on neutrophil DNA from 90 MPN patients. Published copy number variants (CNVs) and those identified in our internal cohort of 995 healthy controls were excluded as well as germline, non-clonal CN-LOH regions based on size criteria. Genomic results were correlated with the quantitative JAK2V617F AB, clinical phenotypes within and between disease classes, and in 21 paired longitudinal samples. SNP-A detected aUPD and/or chromosomal gains or losses in 81% of MPN patients. The prevalence of genomic lesions was lowest in ET compared to PV or PMF (40% of ET cases were lesion free), and lower still in JAK2V617F-negative ET (62% lesion free). aUPD was the most common genomic lesion, occurring in 59% of the MPN cases, and involving every chromosome except chromosomes 18 and 23. As expected, aUPD most commonly involved 9p (38 cases), encompassing JAK2. aUPD encompassing the TET2 gene at 4q24, recently implicated in MPN and other myeloid disorders, was seen in 3 of the 90 cases, with deletions spanning this gene in 2 additional cases. Five cases of aUPD at 2q were observed, and other recurrent regions were limited to 3 cases or fewer. The prevalence of chromosomal losses alone was far lower than UPD (22%), most commonly involved chromosome regions 20q, 13q and 17q, and was a feature primarily of PMF. The prevalence of aUPD in our MPN series of 59% was higher than in MDS (20%, p<0.001), MDS/MPN (35%, p=0.0074), and sAML (23%, p=0.0004) using the same arrays and analytical methods. Independent of MPN classification category, JAK2V617F positive patients had a higher prevalence of UPD than did JAK2V617F negative patients, although when excluding 9p UPD (24/59 vs 11/31) this was not significant. However, homozygous JAK2V617F patients had twice the prevalence of aUPD (again excluding 9P UPD) than did the heterozygous patients (8/25 vs 5/33), and twice the prevalence of multiple aUPD within individual cases. Of the 21 patients studied longitudinally over 5 years (range 2–9), 3 patients with chromosomal lesions present on the first sample were not present in the second: 9p UPD resolved in one PV patient who transformed to PMF, 1p UPD resolved in a JAK2V617F-negative ET patient, and a 20q deletion resolved in a heterozygous JAK2V617F-positive ET patient who transitioned to PV. Additional UPD regions occurred in only 1/8 heterozygous JAK2V617F positive MPN patients, while 5/11 JAK2V617F homozygous patients developed additional aUPD. In contrast, the acquisition of deletions occurred primarily in JAK2V617F heterozygous ET patients, all of whom had transitioned to PV or PMF. We conclude that aUPD is increased in the MPN compared to other myeloid disorders, and associates with the presence of JAK2 V617F in a gene dose-dependent manner. We hypothesize that aberrant JAK2 signaling contributes to the development of UPD but not chromosomal losses or gains. Furthermore, comparison of deletion, amplification, and UPD events in ET and PV relative to PMF suggest that PMF bears a closer genomic resemblance to MDS than to ET or PV. Thus SNP-A provides unique insight into MPN disease classification (PMF), disease progression, and genomic instability mechanisms. Disclosures: No relevant conflicts of interest to declare.

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High-Resolution SNP-Array Profiling Discloses Novel Genomic Aberrations in BCR/ABL-Negative Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽

10.1182/blood.v112.11.2794.2794 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2794-2794

Author(s):

Frank Stegelmann ◽

Lars Bullinger ◽

Martin Griesshammer ◽

Susanne Kuhn ◽

Stefanie Schauer ◽

...

Keyword(s):

High Resolution ◽

Copy Number ◽

Myeloproliferative Neoplasms ◽

Snp Array ◽

Jak2 V617f ◽

Suppressor Gene ◽

Chromosome 9 ◽

Trisomy 8 ◽

Trisomy 9 ◽

Genomic Aberrations

Abstract Although the recent identification of the gain-of-function mutations JAK2 V617F and MPL W515L/K provided new insights into the pathogenesis of myeloproliferative neoplasms (MPN), the biological origins of myelofibrosis (MF) are complex and remain poorly understood. Until today, it is unclear whether JAK2 or MPL mutations are causative lesions of bone marrow fibrosis in MPN patients (pts). Approximately 50% of the pts lack evidence of clonality such as gene mutations or an abnormal karyotype. In addition, the clinical course of pts with MF differs from primary polycythemia vera (PV) or essential thrombocythemia (ET) pts. Therefore, it is most likely that other unknown genetic events contribute to the disease phenotype. To identify novel disease-related aberrations in this subset of MPN, we applied single nucleotide polymorphism (SNP) arrays [Affymetrix 250K Nsp arrays] that allow for genome-wide screening of both DNA copy number alterations (CNAs) and copy neutral losses of heterozygosity (uniparental disomies, UPDs) at high resolution. For SNP-array mapping, DNA from granulocytes of 61 clinically well characterized MPN pts in fibrotic stage was studied [primary MF (PMF), n=47; post-ET MF, n=6; post-PV MF, n=8]. Genotypes were analyzed using CNAG 2.0 software. Data were normalized against an own set of 30 reference samples. Genomic regions recently detected as copy number polymorphisms were excluded from data analysis. CNAs were identified in 55% of PMF, 33% of post-ET MF, and 62% of post-PV MF cases. The most frequent recurrent CNAs were losses of 20q11-q13 (n=7), followed by trisomy 9 (n=5), trisomy 8 (n=4), gains of 1q (n=2), and micro-deletions in 17q11.2 (n=2; 1.3 and 2.3 Mb, respectively). Deletions in 20q and trisomy 9 were detected in all MF subgroups, whereas gains of 1q and trisomy 8 were restricted to PMF. Furthermore, 17q11.2 micro-deletions occurred in secondary MF pts (one post-ET and one post-PV). In both cases, monoallelic loss of the tumor suppressor gene NF1 was confirmed by fluorescence in-situ hybridization (FISH). In addition, 18 more non-recurrent micro-deletions ranging from 0.1 to 1.9 Mb in size were identified in 14 PMF cases and one post-PV MF pt. For instance, one PMF case exhibited a micro-deletion in 13q12.12 (1.4 Mb) encompassing TNFRSF19 (TNF-receptor superfamily member 19) that encodes a receptor which is capable of inducing apoptosis by a caspase-independent mechanism. UPDs were detectable in 27% of PMF, 17% of post-ET MF, and 62% post-PV MF pts. The most frequent UPDs included the JAK2 locus in 9p24 (n=9; 11.6 to 38 Mb), followed by UPDs in 7q31.1-q33 (24.3 Mb), 7q34-q35 (5.6 Mb), and 17q23.3-q24.2 (4.8 Mb) in two cases, each. Interestingly, all pts of the post-PV MF cohort exhibited chromosome 9 abnormalities, either by trisomy 9 (n=3) or by 9p UPD (n=5). Furthermore, all pts with trisomy 9 were heterozygously JAK2 V617F mutated, whereas all cases with 9p UPD revealed homozygous JAK2 mutations. In conclusion, our data on a large series of well defined fibrotic MPN cases demonstrate that array SNP-mapping is an excellent tool to identify known and novel genomic aberrations, thereby pinpointing to regions that harbor potential disease-related genes. Furthermore, the large number of micro-deletions in single MF cases presume a broad genetic heterogeneity and complexity of fibrotic MPN that has to be further investigated in larger pts series.

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High-Resolution SNP 6.0 Array Profiling Discloses Novel Genomic Aberrations in Polycythemia Vera and Essential Thrombocythemia.

Blood ◽

10.1182/blood.v114.22.1898.1898 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1898-1898

Author(s):

Marta Anna Sobas ◽

Teresa Gonzalez ◽

Manuel Perez-Encinas ◽

Celsa Quinteiro ◽

Jose Diaz ◽

...

Keyword(s):

High Resolution ◽

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Analytical Data ◽

Snp Array ◽

Jak2v617f Mutation ◽

Molecular Material ◽

Genomic Aberrations

Abstract Abstract 1898 Poster Board I-921 Introduction: JAK2V617F mutation is detected in more than 90% of cases of polycythemia vera (PV) and in about 50% of cases of essential thrombocythemia (ET). Recently, JAK2-exon 12 and MPL mutations have been reported in myeloproliferative neoplasms (MPN). All these three mutations have a disease causing potential. There are still 50% of ET and PMF patients negative for JAK2V617F mutation. Thus, further genetic analysis to identify novel disease-related aberrations in MPN is required. Methods: We performed whole genome analysis on granulocytic DNA of 45 MPN patients (19 PV and 26 ET) using the single nucleotide polymorphism (SNP) Array 6.0 platform [Affymetrix 6.0]. Genotypes were analyzed using Genotyping Console 3.0.2. Data were normalized against a commercial and an own set of reference samples. All patients had JAK2V617F screening performed. Clinical and analytical data and results of cytogenetics study performed at diagnosis of MPN were colected from clinical reports. Results: From 45 MPN patients, 41 had normal cytogenetics at diagnosis; there were no data concerning cytogenetics study from the rest 4 patients. Using SNP-A 6.0 platform, we detected aberrations (gain or loss of the molecular material) in the following regions: 1q12, 9p1, 17q21, 4q, 3q26 and 8p12. Aberrations in a region 1q12 (n=14) were presented in 8 of PV (gain in 3 and loss in 5) and in 6 of ET (gain in 2 and loss in 4) patients. Aberrations in a region 9p1 (n=27) were observed in 11 of PV (gain in 6 and loss in 5) and in 16 of ET (gain in 10 and loss in 6) patients. Aberrations in a region 17q21 (n=26) were presented in 11 of PV (gain in 3 and loss in 8) and in 15 of ET (gain in 9 and loss in 6) patients. Aberrations in the region 4q (n=20) were detected in 8 of PV (gain in 5 and loss in 3) and in 12 of ET (gain in 9 and loss in 3) patients. Interestingly, only the gain of the molecular material was detected in a region 3q26 (n=12; 5 PV and 7 ET patients). In case of aberration in a region 8p12 (n=20), all 12 ET patients presented gain of the molecular material, whereas PV patients had gain (n=3) or loss (n=3) of the molecular material. There were no relation between the presence of these aberrations and the status of the JAK2V617F mutation, analytical data or clinical outcome of the patients. Conclusions: 1. As we know, patients with ET have low frequency of cytogenetics aberrations. Nevertheless, using SNP-A 6.0 platform [Affymetrix 6.0], it is possible to detect new genomic aberrations in these group of patients. 2. According to our results, gain in 8p12 region is especially related to ET patients. Recently has been reported that, in a 8p12 region there is localized gen INDOL1 that, may be involved in the inhibition of immune response to tumours. 3. Gain in a 3q26 region can be related with both PV and ET. In this region, there are localized two microRNA: hsa-mir-1263 and has-mir-720. 4. SNP-A 6.0 technology should not replace conventional cytogenetics in study of MPN patients. However, SNP-A 6.0 platform, as a high resolution assay, can be useful in identification of new genomic abnormalities that may be relevant for pathogenesis of MPN. Disclosures: No relevant conflicts of interest to declare.

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Long-term Use of Anagrelid in Essential Thrombocythemia Treatment of a Child Under 12 Months

Blood ◽

10.1182/blood.v120.21.5072.5072 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5072-5072

Author(s):

Pawel Laguna ◽

Anna Klukowska ◽

Katarzyna Pawelec ◽

Michal Matysiak

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Jak2 V617f ◽

Biochemical Tests ◽

Results Of Treatment ◽

Life Threatening ◽

Laboratory Investigations ◽

Chronic Myeloproliferative Disorder

Abstract Abstract 5072 Essential thrombocythemia (ET) is currently classified as a chronic myeloproliferative disorder. Anagrelide is a novel platelet lowering agent that has recently been approved for use n ET. We present the case of ET in an 8 –month- old girl, who was treated with Anagrelid for 13 months. The girl was admitted to hospital because of elevated platelet count. She had stomach pains periodically. On admission the child's condition was good but on palpation her stomach was sore and her spleen was enlarged. Laboratory investigations showed an elevated platelet count (1. 5m) and hypercalemia (6. 3mEq/l). Thrombopoetin was normal. Acesan was added to the treatment. A morphology performed after 1 month revealed further elevation of platelets (up to 2. 65m). On the basis of laboratory investigations, bone-marrow biopsy and trepanobiopsy, we eliminated an oncological disease and infectious diseases of connective tissues. On bone-marrow investigation, acquired mutation of JAK2 (V617F) tyrosine kinase was diagnosed, which confirmed ET. On account of the growing number of platelets, which was life - threatening, we decided to administer anagrelid, starting with a dose of 0. 25 mg per day. The number of platelets decreased to 1. 4m, so the dose was increased to 0. 25 mg twice daily after 3 weeks. At present, after 37 months of anagrelide treatment, the number of platelets in the child ranges between 400 and 650. The patient visits our, department for monthly check-up (morphology, biochemical tests, ECG and echocardiography). In international literature there is no information on the use of Anagrelid in ET treatment of children under 12 months old. However, on the basis of our observations and initial results of treatment, it seems that the above protocol for Anagrelid administration is safe for infants of this age. Disclosures: No relevant conflicts of interest to declare.

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Inhibiting Glutamine Metabolism with CB-839 Reduces Erythrocytosis in MPN Mice

Blood ◽

10.1182/blood-2021-150321 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3585-3585

Author(s):

MARC Usart ◽

Shivam Rai ◽

Nils Hansen ◽

Alexandre Guy ◽

Jan Stetka ◽

...

Keyword(s):

Bone Marrow ◽

Oxidative Phosphorylation ◽

Aminolevulinic Acid ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Metabolic Reprogramming ◽

Glutamine Metabolism ◽

Vehicle Group ◽

Cell Counts

Abstract Metabolic reprogramming is one of the hallmarks of cancer, as these rapidly dividing cells need to adapt their metabolism to cope with an increased energy demand. We have previously showed that in mouse models of myeloproliferative neoplasms (MPN), JAK2-mutant cells display metabolic alterations, including increased oxidative phosphorylation and glycolysis. The glutamine-glutamate-alphaKetoglutarate (aKG) axis, besides fueling the Krebs cycle and anabolic processes, contributes to the synthesis of the heme precursor 5-aminolevulinic acid (5-ALA), thus making glutaminolysis a potential target for MPN therapy. Here, we examined the effects of pharmacological inhibition of the conversion of glutamine to glutamate by glutaminase 1 by Telaglenastat (CB-839) in a mouse model of JAK2-V617F driven MPN. We performed competitive transplantations of bone marrow co-expressing JAK2-V617F with a GFP-reporter and wildtype mice in a 1:10 mixture into lethally irradiated mice. At 8 weeks post-transplantation, the mice developed MPN phenotype and were randomized into 4 treatment arms. They were then treated for 6 weeks with vehicle, CB-839, 3PO (6-phosphofructo-2-kinase inhibitor) and the combination of CB-839 + 3PO. We found that hemoglobin levels were significantly reduced in CB-839 treated mice and were fully normalised in mice that received a combination of CB-839 + 3PO (Fig. 1A). Other peripheral blood cell counts did not change (not shown). Hypoglycemia was noted in the vehicle group, as previously described, but an increase in blood glucose levels was seen already after one week of treatment with CB-839. 3PO alone had less impact on hypoglycemia and mice receiving CB-839 + 3PO had similar values to CB-839 alone (Fig. 1A). In line with these findings, splenomegaly was also reduced (Fig. 1A). To characterise the changes in metabolic activity, we determined the metabolic profiles of unfractionated bone marrow (BM) cells using seahorse assays. As expected, basal and maximal glycolysis and oxidative phosphorylation were higher in vehicle treated MPN cells than cells from wildtype controls. However, when BM cells from CB-839 or CB-839 + 3PO groups were examined, they displayed a strong downmodulation of both glycolysis and oxidative phosphorylation, indicating that chronic exposure to CB-839 reprograms the metabolic machinery by reducing not only the basal glycolytic and respiratory levels, but also the maximal capacity (Fig. 1B). Glutamine is the primary source of aKG needed to produce the heme precursor 5-ALA, and hemoglobin synthesis consumes the majority of ATP in erythroid progenitors. We therefore hypothesize that 5-ALA shortage caused by CB-839 limits hemoglobin production, leading to reduced catabolic activity and drop in ATP demand. To evaluate this model, we examined the stages of erythroid differentiation from vehicle or treated mice. Early and intermediate erythroid cells were enriched at the expense of later stages in CB-839 and CB-839+3PO treatment arms (Fig. 1C). The accumulation of erythroid precursors at the stage before hemoglobin is assembled supports the hypothesis that targeting glutamine usage impairs excessive erythropoiesis by limiting hemoglobinization rate. Our data show that inhibiting glutaminolysis ameliorated MPN phenotype and that the combination with the glycolytic inhibitor 3PO was able to induce complete hematological response. Since CB-839 is already in clinical trials for other cancers, our data suggest that it could also be tested for treatment of patients with polycythemia vera. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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