Mutational Status of TET2, JAK2, and MPL in Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 418-418
Author(s):  
Luca Malcovati ◽  
Angela Brisci ◽  
Daniela Pietra ◽  
Matteo G Della Porta ◽  
Anna Gallífi ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Ringed Sideroblasts ◽  
Clonal Hematopoiesis ◽  
Myeloid Neoplasms ◽  
Mutational Status ◽  
Mutant Genes ◽  
Myelomonocytic Leukemia

Abstract Abstract 418 According to the WHO classification, myelodysplastic/myeloproliferative neoplasms include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). The best characterized of these latter conditions is the provisional entity defined as refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T); up to 60% of RARS-T patients harbor the JAK2 (V617F) mutation. Somatic mutations of TET2 have been recently described in myeloid neoplasms, where they appear to be associated with the amplification of the mutated clone at the early stages of hematopoietic differentiation [N Engl J Med. 2009 May 28;360(22):2355-7]. In order to gain a deeper insight into the pathophysiology of RARS-T, we studied a cohort of 187 patients with myeloid neoplasms and investigated the relationship between ringed sideroblasts, thrombocytosis, and mutational status of TET2, JAK2 and MPL. RARS-T was defined according to the following WHO criteria: i) refractory anemia associated with erythroid dysplasia and ringed sideroblasts ≥ 15%; ii) < 5% blasts in the bone marrow; iii) platelet count ≥ 450 × 109/L; iv) presence of large atypical megakaryocytes similar to those observed in BCR/ABL1-negative myeloproliferative neoplasms; v) absence of del(5q), t(3;3)(q21;q26) or inv(3)(q21q26). The combination of ringed sideroblasts ≥ 15% and platelet count ≥ 450 × 109/L was found in 19 subjects fulfilling the diagnostic criteria for RARS-T, while 24 patients had RARS without thrombocytosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells - but not in T-lymphocytes - from 11 out of 19 (58%) RARS-T patients. Three RARS patients, who initially had low to normal platelet counts, progressed to RARS-T, and two of them acquired JAK2 (V617F) at this time. Somatic mutations of TET2 were found in three of the 15 RARS-T patients studied, and the presence of multiple mutant genes allowed analysis of subclones in two of them. One of these patients carried the following three somatic mutations: TET2 (C1271Y), JAK2 (V617F) and MPL (W515L). Analysis of genomic DNA from circulating granulocytes showed 50% TET2 (C1271Y) mutant alleles but smaller proportions of JAK2 (V617F) and MPL (W515L) mutant alleles (5.8% and 20% respectively). We then analyzed five BFU-E grown from peripheral blood mononuclear cells obtained from this patient. All these five colonies were heterozygous for TET2 (C1271Y), while three of them were heterozygous also for MPL (W515L) and the remaining two were heterozygous also for JAK2 (V617F), clearly indicating that erythroid progenitors carrying JAK2 or MPL mutants belonged to subclones of the dominant TET2 (C1271Y) clone. A woman with the TET2 (S1612LfsX4) mutation (50% granulocyte mutant alleles) and fully clonal hematopoiesis as indicated by X-chromosome inactivation patterns, carried 28% JAK2 (V617F) mutant alleles in circulating granulocytes, indicating that granulocytes harboring JAK2 mutant alleles belonged to a subclone of the initial TET2 (S1612LfsX4) mutant clone. Over a 5-year period, in fact, the initial TET2 mutant clone was completely replaced by the TET2/JAK2 mutant subclone. In other two female patients with RARS-T and no somatic mutation of TET2, granulocytes carrying JAK2 (V617F) represented only a fraction (11 to 22%) of clonal granulocytes as determined by X-chromosome inactivation patterns (96 to 100%). Somatic mutations of TET2 were detected also in a significant proportion of patients with RARS without thrombocytosis, while no JAK2 or MPL mutation was identified in these individuals. These observations suggest that the occurrence of a TET2 mutation may represent the initial event determining clonal dominance of hematopoietic cells both in RARS and RARS-T patients, while the subsequent occurrence of JAK2 and/or MPL mutations likely generates myelodysplastic/myeloproliferative subclones in RARS-T patients. In conclusion, RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical level, and it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL or other as-yet-unknown genes on the background of clonal hematopoiesis caused by somatic mutations of TET2 or other similar (as-yet-unknown) mutant genes. Disclosures: No relevant conflicts of interest to declare.

Molecular and clinical features of refractory anemia with ringed sideroblasts associated with marked thrombocytosis

Blood ◽  
2009 ◽  
Vol 114 (17) ◽  
pp. 3538-3545 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Daniela Pietra ◽  
Emanuela Boveri ◽  
Andrea Pellagatti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Somatic Mutations ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Refractory Anemia ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasm ◽  
Normal Platelet ◽  
Cd34 Cells ◽  
Patient Groups

Abstract We studied patients with myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis. The combination of ringed sideroblasts 15% or greater and platelet count of 450 × 109/L or greater was found in 19 subjects fulfilling the diagnostic criteria for refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T), and in 3 patients with primary myelofibrosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells, but not in T lymphocytes, from 11 of 19 patients with RARS-T. Three patients with RARS, who initially had low to normal platelet counts, progressed to RARS-T, and 2 of them acquired JAK2 (V617F) at this time. In female patients with RARS-T, granulocytes carrying JAK2 (V617F) represented only a fraction of clonal granulocytes as determined by X-chromosome inactivation patterns. RARS and RARS-T patient groups both consistently showed up-regulation of ALAS2 and down-regulation of ABCB7 in CD34+ cells, but several other genes were differentially expressed, including PSIP1 (LEDGF), CXCR4, and CDC2L5. These observations suggest that RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical levels and that it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL, or other as-yet-unknown genes.

Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 854-854 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Daniela Pietra ◽  
Anna Galli ◽  
Erica Travaglino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
X Chromosome ◽  
Jak2 V617f ◽  
X Chromosome Inactivation ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Mutation Status ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasms ◽  
V617f Mutation

Abstract The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from &lt;.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

Pathway Mutations in the Splicing Machinery in Myeloid Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-14-SCI-14
Author(s):  
Seishi Ogawa
Keyword(s):  
Epigenetic Regulation ◽  
Splice Site ◽  
Rna Splicing ◽  
Myeloproliferative Neoplasms ◽  
Refractory Anemia ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasms ◽  
Mutational Hotspots ◽  
Site Recognition ◽  
Splicing Machinery

Abstract Abstract SCI-14 During the past decade, significant progress has been made in our understanding of the molecular pathogenesis of myelodysplastic syndromes (MDS) and related myeloid neoplasms, in which one of the major findings was frequent mutations of genes in epigenetic regulation, such as DNA methylation (DNMT3A, TET2, and IDH1/2) and chromatin modifications (ASXL1, EZH2, EED, and SUZ12). They are also found in comparable or even higher fractions of other myeloid neoplasms, underscoring the common impact of deregulated epigenetic regulation on myeloid leukemogenesis. On the other hand, a new class of pathway mutations has been uncovered recently that commonly involve the RNA splicing machinery (1, 2, 3). Thus, at least eight different components of the machinery, invariably engaged in the 3' splice site recognition, have been reported to be mutated in as high as 45 percent to 85 percent of cases with different subtypes of MDS and related myeloid neoplasms mostly in a mutually exclusive manner. This indicates that the 3' splice site recognition is the functional target of these mutations. There exist discrete mutational hotspots in three out of four major targets, including SF3B1, SRSF2, U2AF35, and ZRSR2, and these mutant alleles can induce abnormal RNA splicing, indicating the gain-of-function nature of the mutations (2). Splicing factor mutations are largely specific to myelodysplasia phenotypes but relatively rare in acute myeloid leukemia and myeloproliferative neoplasms (2), suggesting their primary roles in the pathogenesis of myelodysplasia. The genotype-phenotype association is especially prominent in the case of SF3B1 mutations, which were found in 76 percent to 83 percent of cases of refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), and refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) (1, 2). In this session, the updated findings on the spliceosome mutations found in myelodysplasia, including their clinical and functional aspects, will be discussed. Disclosures: No relevant conflicts of interest to declare.

Clonal Analysis of SF3B1, JAK2 and MPL in Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 172-172
Author(s):  
Ilaria Ambaglio ◽  
Anna Gallì ◽  
Daniela Pietra ◽  
Matteo G Della Porta ◽  
Marta Ubezio ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Wild Type ◽  
Mutation Status ◽  
Allele Burden ◽  
Ring Sideroblasts ◽  
Exon 10

Abstract Abstract 172 Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype. In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis. Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%). Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones. We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F). In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones. Disclosures: No relevant conflicts of interest to declare.

TET2 Mutations Are Frequent in RARS-T.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3794-3794
Author(s):  
Hadrian Szpurka ◽  
Anna M. Jankowska ◽  
Hideki Makishima ◽  
Nelli Bejanyan ◽  
Eric D. Hsi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Significant Proportion ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Common Mutation ◽  
Cell Counts ◽  
Mutational Status ◽  
Ring Sideroblasts

Abstract Abstract 3794 Poster Board III-730 Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Since JAK2 V617F and MPL W515L mutations are present in a significant proportion of RARS-T patients, many investigators consider this entity to be more closely related to classical MPN. However, a significant minority of patients with RARS-T do not display either JAK2 V617F or MPL W515L mutations. We have studied a cohort of patients with RARS-T (N=20) characterized by the presence of ring sideroblasts, reticulin fibrosis and thrombocytosis (>450×109/L), that lack obvious causes of secondary thrombocytosis. While 8/20 patients harbored the JAK2 V617F, and 3/20 the MPL W515L mutations, the molecular pathogenesis for the remaining 9 patients was unexplained. Activation of JAK2 and MPL is associated with aberrant phospho-STAT5. Cases positive only for phospho-STAT5 may harbor other related, so far unidentified mutations. Many groups have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), MDS/MPN, some typical MDS, and secondary acute myeloid leukemia (sAML). Overlapping microdeletions and UPD on 4q24 pointed towards possible mutations in the TET2 gene; such mutations were subsequently found in myeloid malignancies, most significantly MPN and MDS/MPN. Based on these findings, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we evaluated the mutational status of TET2 in RARS-T patients. SNP-A allowed detection of copy neutral loss of heterozygosity (CN-LOH), such as UPD9p, which is associated with the JAK2 V617F mutation, and UPD1p, associated with MPL W515L. SNP-A facilitated detection of previously cryptic lesions; 11/20 patients showed an abnormal SNP-A-based karyotype (only 3 of these defects were also detected by MC). The new lesions seen by SNP-A included various UPD, such as, 1p, 2p, 3q, 6p, 8p, 9p and 10p. The presence of UPD9p/1p was consistent with homozygous JAK2 V617F/MPL W515L mutations. Likely, duplication of mutated alleles constituted a further permissive event during clinical evolution. However, none of the patients showed a somatic LOH at 4q24, suggesting that biallelic TET2 mutations were not involved in the pathogenesis of RARS-T. Simultaneously, lack of UPD11q suggested that CBL mutations were absent. Indeed, Cbl ring finger domain mutational screening revealed no mutations. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10). However, 4 patients demonstrated abnormal megakaryocytic STAT5 phosphorylation, despite the absence of both JAK2 V617F and MPL W515L mutations. Within this group, a monoallelic TET2 mutation, delC 1480Sfs, was identified. In addition, we found a group of 5 patients without either JAK2 V617F or MPL W515L mutations, and also without association of the aberrant phospho-STAT5 staining. One of these patients had a monoallelic TET2 V1718L mutation; interestingly, another patient's specimen showed two novel non-synonymous SNPs: Y867H and P1723S. In total, 2/19 (11%) patients harbored TET2 mutations. These findings indicate involvement of TET2 mutations in RARS-T pathogenesis. RARS-T cases with MPN-associated mutations may not show obligatory phospho-STAT5 staining. The majority of patients were characterized by lack of splenomegaly, decreased white blood cell counts, increased thrombocytosis, and a normal karyotype. In summary, the majority of RARS-T patients harbor JAK2 V617F and MPL W515L mutations that strongly activate STAT5 phosphorylation. We described herein the third most common mutation in RARS-T, which can occur with or without abnormal STAT5 activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies

Blood ◽  
2009 ◽  
Vol 114 (1) ◽  
pp. 144-147 ◽  
Author(s):  
Omar Abdel-Wahab ◽  
Ann Mullally ◽  
Cyrus Hedvat ◽  
Guillermo Garcia-Manero ◽  
Jay Patel ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Promoter Hypermethylation ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Myeloid Malignancies ◽  
Mutational Status ◽  
Myelomonocytic Leukemia

Abstract Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2 mutations, we evaluated the mutational status of TET1, TET2, and TET3 in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2 in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2 mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1 or TET3 mutations or TET2 promoter hypermethylation in MPNs. TET2 mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P = .029). These data indicate that TET2 mutations are observed in different myeloid malignancies and may be important in AML prognosis.

scholarly journals In-Frame Exon 9 CALR Deletions Co-Occur with Other Alterations in the JAK-STAT Pathway in Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4588-4588 ◽  
Author(s):  
Yongbao Wang ◽  
Albert K Ho ◽  
Qiulu Pan ◽  
Frederick Karl Racke ◽  
Dan Jones
Keyword(s):  
Mutation Analysis ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Point Mutations ◽  
Jak2 V617f ◽  
Illumina Miseq ◽  
Primary Myelofibrosis ◽  
Ensembl Database ◽  
Stat Pathway

Abstract Introduction: Mutations in the chaperone gene calreticulin (CALR) have been recently identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF), and are essentially mutually exclusive with JAK2 or MPL mutations. Normal and mutant CALR proteins may differentially affect the subcellular trafficking of JAK-STAT signaling components. CALR mutations previously reported in ET and PMF have been +1 frameshift (fs) mutations localized to exon (E) 9 that generate a novel C-terminal protein sequence with a shift from acidic to basic residues. CALR E9 in-frame (IF) deletions have been recently rarely reported as polymorphisms such as TMP_ESP_19_13054686_13054688 and TMP_ESP_19_13054650_13054658 (Ensembl database entries). We sought to determine the frequency and associated clinical features of CALR with E9 IF alterations in samples submitted for suspicion of a myeloproliferative neoplasm (sMPN). We also assessed whether CALR IF alterations are differentially associated with +1fs mutations or with JAK2 V617For other somatic mutations in MPN-associated genes. Materials and Methods: CALR mutation analysis of E9 was performed on genomic DNA extracted from blood, bone marrow (BM) aspirate or fixed BM biopsy sections using a Sanger sequencing assay with an analytic sensitivity of at least 15%. E9 IF cases were further assessed and mutations quantified by an Ion torrent sequencing panel assessing CALR, CSF3R, JAK2 and MPL, a second panel containing ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 and an Illumina MiSeq extended panel with 20 additional MPN-associated genes. These assays had a sensitivity of approximately 5%. JAK2 V617Fmutations were quantitated using a pyrosequencing assay with an analytic sensitivity of 1%. Results: We assessed CALR E9 mutation status in 733 sMPN samples that were negative for JAK2 V617F mutation. 148 (20.1%) had typical +1fs mutations (95 type 1 and variants, 53 type 2 and variants); 2 (0.3%) had point mutations (E381A and D7373M); 7 (1.0%) had IF deletions including E381_A382>A, D397_D400>D (n =4), D400_K401>D and E405_V409>V. All E9 IF deletions were present at ~50% of reads. Clinical diagnoses were cytopenia/BM fibrosis, ET, thrombocytosis/anemia, and sMPN unspecified. Mutation analysis for 27 additional MPN-associated genes revealed mutations in 5/7 (71.4%) IF deletion cases including in MPL (W515L,40%; D163Y,12%), CSF3R (A470T 46%), ASXL1 (D954fs*26, 45%) and ZRSR2 (S449_R450dup, 27%). No additional mutations were found in the 2 cases with non-synonymous CALR point mutations/SNPs. In a parallel set of 76 MPN samples that had JAK2 V617F at varying levels, we noted 1 E9 IF deletion (D397_D400>D) in a sMPN case with 21.6% JAK2 V617F, and a typical +1fs mutation (K385fs*47) in a case with low (4.2%) JAK2 V617F. All other JAK2 V617F cases had no E9 CALR alterations. Conclusions: CALR E9 in-frame deletions occur in up to 1% of sMPN samples and involve a variety of codons in the acidic domain. Therefore, sizing assays without DNA sequencing are not sufficient to unequivocally distinguish IF deletions from the characteristic +1 frameshift somatic mutations associated with ET and PMF. Given their level, these CALR IF deletions are likely germline sequence variants but are associated with a high frequency of somatic mutations in other MPN-associated genes but not with CALR +1fs mutations. Their co-occurrence with pathogenic somatic mutations in JAK2, MPL and CSF3R affecting the JAK-STAT pathway raises the possibility for a contributory role of altered CALR proteins produced by these E9 deletions in the pathogenesis of MPN. Disclosures Wang: Quest Diagnostics: Employment. Ho:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

Myelodysplastic/Myeloproliferative Neoplasms

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 264-272 ◽  
Author(s):  
Mario Cazzola ◽  
Luca Malcovati ◽  
Rosangela Invernizzi
Keyword(s):  
Clinical Laboratory ◽  
Myeloproliferative Neoplasms ◽  
Chronic Myelomonocytic Leukemia ◽  
World Health ◽  
Refractory Anemia ◽  
Molecular Diagnostic ◽  
Juvenile Myelomonocytic Leukemia ◽  
Lymphoid Tissues ◽  
Ring Sideroblasts ◽  
Myelomonocytic Leukemia

Abstract According to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, myelodysplastic/myeloproliferative neoplasms are clonal myeloid neoplasms that have some clinical, laboratory, or morphologic findings that support a diagnosis of myelodysplastic syndrome, and other findings that are more consistent with myeloproliferative neoplasms. These disorders include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable. The best characterized of these latter unclassifiable conditions is the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis. This article focuses on myelodysplastic/myeloproliferative neoplasms of adulthood, with particular emphasis on chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts associated with marked thrombocytosis. Recent studies have partly clarified the molecular basis of these disorders, laying the groundwork for the development of molecular diagnostic and prognostic tools. It is hoped that these advances will soon translate into improved therapeutic approaches.

Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 178-178
Author(s):  
Roland Jäger ◽  
Damla Olcaydu ◽  
Tiina Berg ◽  
Bettina Gisslinger ◽  
Heinz Gisslinger ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Clonal Heterogeneity ◽  
Chromosome 1P ◽  
Clonal Variability ◽  
Genomic Regions ◽  
Chromosome 13Q

Abstract A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.

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