Cases Of Idiopathic Myelofibrosis Research Articles

Analysis of JAK2V617F mutation in Jordanian patients with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are heterogeneous clonal bone marrow stem cell disorders and include polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (IMF) neoplasia. In 2005, the JAK2(V617F) mutation was identified in Philadelphia chromosome-negative patients. The aim of this study was to sequence coding exons 12 and 14 of the JAK2 gene in Jordanian patients with MPN. Both exons 12 and 14 of the JAK2 gene were amplified using polymerase chain reaction from DNA extracted from 68 blood and bone marrow samples belonging to 57 MPN patients and subjected to DNA sequencing. JAK2(V617F) mutations were detected in 26 of 57 Jordanian patients (45%) with different MPNs. JAK2(V617F) was identified in 70%, 31%, and 14% of PV, ET, and IMF cases, respectively. Five men diagnosed with PV were homozygous for JAK2(V617F), whereas the other 21 patients were heterozygous for the mutation. Neither the JAK2(V617F) mutation nor any DNA polymorphism in exon 12 or exon 14 of the JAK2 gene was detected among the 40 leukemic patients. A rare single nucleotide polymorphism, c.1860C→T (rs375442615), was detected in one patient with ET. This study is the first molecular investigation of the JAK2 gene in Jordan. We successfully identified the JAK2(V617F) mutation in Jordanian patients with Philadelphia chromosome-negative MPNs. Our results provide a basis for the early detection of this mutation and simplify the diagnostic workup for these disorders at the molecular level.

Epigenomic Profiling of Myeloproliferative Diseases Reveal Idiopathic Myelofibrosis as An Epigenetically Distinct Subgroup and Highlights the Epigenetic Effects of Jak2V617F Mutation

AbstractAbstract 627Myeloproliferative diseases (MPD) are clonal hematologic disorders that present with increased numbers of functional, mature, terminally differentiated myeloid elements. Even though genetic, biochemical and functional studies have provided important insights into the pathogenesis of MPDs, the role of epigenetic changes in disease pathobiology is not well elucidated. We performed the HELP assay to study genome-wide methylation patterns in cases of Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Idiopathic Myelofibrosis (IMF) and compared it with normal matched controls. The HELP assay uses differential methylation specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG methylation of 25636 loci. Analysis of 26 MPD neutrophil samples comprising 9 cases of ET, 6 cases of PV and 11 Cases of IMF was performed and compared to normal healthy controls. Unsupervised clustering based on global methylation profiles showed that IMF cases formed a distinct epigenetic cluster, while PV and ET cases were more similar to the normal controls. Further analysis of epigenetic differences between these groups showed that PV and ET samples were characterized by aberrant hypermethylation when compared to controls and had 143 genes that were uniformly hypermethylated. These genes are involved in pathways regulated by the NF-Kb and HNF-4alpha transcription factors. IMF on the other hand was characterized by both aberrantly hyper (n=162) and hypomethylated (n=95) loci when compared to controls. The pathways affected by hypomethylated genes were involved in cytokine cell signaling and MAP kinases. We subsequently validated these observations in an independent set of 8 IMF cases compared to 8 age matched controls. Methylation profiles obtained from whole blood by the HELP assay were able to clearly separate IMF cases from controls demonstrating the validity of our observations. These changes were quantified on a whole genome level by the Luminometric methylation assay (LUMA) that also revealed significantly more hypomethylation in IMF when compared to PV and ET cases (49% hypomethylation in IMF vs. 38% in PV/ET, p<0.05). Selected changes were validated by Mass Array sequencing.We next wanted to determine the epigenomic effects of Jak2V617F mutation and compared methylation profiles of MPD cases with and without the mutation. We observed that cases with Jak2 mutation had a higher number of differentially hypomethylated loci. This striking difference was seen by the LUMA assay also with 67% hypomethylation seen in mutant cases when compared to 48% in those without the mutation (p=0.02). This observation was validated in vitro in a cell line (FDCP) that expressed wither WT or mutant Jak2 kinase. We observed that expression of the mutant kinase led to global hypomethylation. This observation builds on recent data demonstrating nuclear binding of the mutant Jak2 kinase and its effects on the histone epigenetic machinery.In conclusion, we report that MPDs are characterized by various novel epigenetic alterations that affect important functional pathways and IMF is grossly epigenetically distinct from ET and PV. The Jak2 mutation also affects the methylome and leads to global hypomethylation that potentially contributes to the genomic instability and disease pathobiology. Disclosures:No relevant conflicts of interest to declare.

Unrelated stem cell transplant for infantile idiopathic myelofibrosis

Idiopathic myelofibrosis (IMF) is a rare disease in children that can present during infancy and have a protracted course. The only known curative approach for this disease in adult patients is allogeneic stem cell transplant. We present two cases of IMF during infancy that did not resolve with supportive care measures. Both patients underwent unrelated stem cell transplant with complete resolution of their hematologic manifestations and resolution of the bone marrow fibrosis.

Cutaneous extramedullary hematopoiesis in a patient with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder and some cases of IM have extramedullary hematopoiesis. Extramedullary hematopoiesis is commonly seen in the liver, spleen and lymph nodes, but cutaneous extramedullary hematopoiesis (CEH) is very rare in cases of IM. We report a case of CEH in a 65-year-old Japanese woman with IM. This patient had many hard brownish nodules on her chest, abdomen and scalp. Histopathological examination of the nodule on her chest showed the existence of various stages of immature erythrocytes, leukocytes and megakaryocytes indicating that these hard nodules showed extramedullary hematopoiesis in the dermis. The proliferation of these immature cells in the dermis plays an important role in the pathogenesis of CEH. CEH is a very rare manifestation of IM and progresses slowly.

The incidence of JAK2 V617F mutation in bcr/abl-negative chronic myeloproliferative disorders: assessment by two different detection methods

A recurrent specific JAK2 V617F mutation has been reported in bcr/abl-negative chronic myeloproliferative diseases (cMPD), including polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The mutation is detectable in a variable proportion of neoplastic clones, depending on the molecular methods employed. In this study, we attempted to establish the JAK2 V617F mutation frequency in two partially overlapping cMPD patient series by two different PCR-based techniques. Using an allele-specific polymerase chain reaction assay (AS-PCR), the JAK2 V617F mutation was detected in 124/158 (78.5%) cMPD patients; in particular, 90.2, 72.1, 63.2 and 50% of PV, ET, IMF and unclassified (U)-MPD cases, respectively, showed the mutation. Employing a semiquantitative 5′ fluorogenic TaqMan assay, the JAK2 V617F mutation was identified in a much larger percentage of cMPDs patients (118/127: 92.9%). Rates of mutation in PV, ET, IMF and U-MPD cases were 95.9, 85.7, 91.7 and 92.9%, respectively. Furthermore, a statistically higher percentage of JAK2 mutated alleles was detected by TaqMan assay in PV (68 ± 3.5, mean value ± SEM) and IMF (64 ± 9.3) cases as compared with ET (35 ± 5.4). Finally, a significant correlation between JAK2 V617F mutational status and hematocrit (Ht), white blood cell and platelet counts in PV patients, and Ht values in ET cases, was observed by AS-PCR. Overall, these data indicate that TaqMan technology significantly improved sensitivity in detecting the JAK2 mutation in cMPD patients and may be worth of further evaluations as a clinically useful tool for detection of small amounts of mutated clones.

Quantitative Evaluation of Bone Marrow Angiogenesis in Idiopathic Myelofibrosis

Bone marrow angiogenesis in patients with idiopathic myelofibrosis (IM) was evaluated by using a quantitative morphometric analysis test system. IM specimens had a significantly higher degree of bone marrow microvessel density (MVD) compared with control specimens. The higher degree of angiogenesis was associated with increased expression of basic fibroblast growth factor in megakaryocytes and endothelial cells in the IM bone marrow specimens. Increased vascular endothelial growth factor expression was seen in some cases of the prefibrotic form of IM. Morphometric quantitative measurements revealed that the MVD was reduced progressively in 4 cases of IM after allogeneic stem cell transplantation. These findings suggest that angiogenesis likely has an important role in the pathogenesis of IM.

A Case of Idiopathic Myelofibrosis Associated with Acromegaly Patient

Acromegaly is a chronic condition resulting from the excessive secretion of growth hormone and insulin like growth factor 1, generally from pituitary adenoma. Although there have been several reports suggesting the possible association of hematologic malignancies with acromegaly, myelofibrosis with acromegaly is very rare. Here we report 54-year-old male patient with myelofibrosis accompanied with acromegaly. We treated this patient with low dose thalidomide (50 mg/day) and prednisone (30 mg/day). We reported this case with literature review. (J Kor Soc Endocrinol 21:328~332, 2006)

JAK2 Mutation Screening and Chromosome Analysis Are Necessary for a Comprehensive Diagnostic Work up in CMPD: A Study on 469 Cases.

The diagnosis of BCR-ABL negative chronic myeloproliferative disorders (CMPD) is still a challenge for the morphologist and clinician, mainly because of overlapping phenotypes of essential thrombocythemia (ET), polycythemia vera (PV), idiopathic myelofibrosis (IMF) and non-malignant reactive phenotypes. Recently, a new mutation in JAK2 leading to V617F exchange in exon 12 was described in these entities. Therefore, we developed a rapid and easy LightCycler based melting curve assay and screened 469 patients with various malignancies for the respective JAK2 mutation using cDNA prepared from mononucleated cells. All cases tested positive were confirmed by sequencing and without any exception all mutations were G to T exchanges at nucleotide 1849. In total, 61 cases with PV were analysed. 56 (91.8%) were mutated (see table). A karyotype was available in 36 cases. Of the JAK2- cases 4 had normal karyotypes and one 20q-. Of the JAK2+ cases 21 had normal karyotype; four (7.1%) had +9, one +8, one +22, one 20q-, and three showed a complex aberrant karyotype. Of the 47 analyzed ET 26 (55%) were mutated. Cytogenetics was available in 20 cases (9 JAK2-, 11 JAK2+). All JAK2- cases all had normal karyotypes. Of the JAK2+ 10 had a normal karyotype and one a t(9;13)(p24;q22) involving the JAK2 locus. Of the 25 IMF cases 14 (56%) were mutated. Karyotype was available in 17 cases (7 JAK2-, 10 JAK2+). All 7 JAK2- had normal karyotype. Of the 10 JAK2+ 6 had normal karyotype and four were aberrant (+9:n=2; 20q-: n=2). In addition, 2/7 cases (28.6%) with CMML were JAK2+. Furthermore, we analysed 89 BCR-ABL negative MPS that were not further specified. V617F was detectable in 43 patients (48.3%). At next 128 AML were analyzed (84 novo AML, 11 t-AML after a previous malignancy, 15 secondary to MDS, and 16 secondary to MPS (PV:n=7, ET:n=5, OMF:n=2). No V617F was detected in all cases with t-AML and s-AML after MDS. In contrast, in de novo AML 6/84 (7.1%) were JAK2+, of which 4 were proven homozygous. Surprisingly, 4 of these six cases had a +9. In AML secondary to a previous CMPD 11/16 (68.8 %) were JAK2+. Of these 2 had +9, one a normal karyotype and 12 a complex aberrant karyotype. These data suggest that acquisition of +9 may lead to progress or blast crisis in JAK2 mutated MPS and supports the gain of function mechanism of the mutation.In conclusion, more than half of all BCR-ABL-negative MPS harbour a V617F mutation. This helps in the differential diagnosis of MPS versus reactive disorders. V617F is more frequently associated with aberrant karyotype than wildtype JAK2. In addition, there is an increasing level of aberrant cytogenetics and mutation rate with respect to incidence and status from ET<IMF<PV indicating that these diseases might be overlapping or even a continuum. It demonstrates that the present classification is artificial and a new classification of “JAK2” positive diseases may be more adequate. In addition, the same mutation was observed in most cases of AML secondary to CMPD. It was also found in few cases with de novo AML and correlated with +9. JAK2V617F is a new and promising target for therapy as well as for molecular monitoring of therapy response in CMPD.Distribution of JAK2 mutation in various malignanciesETIMFPVMPS*CMMLde novo AMLAML after MPStotal4725618978416mutated (n=)261456432611mutated (% of all)55.0 %56.0 %91.8 %48.3 %28.6 %7.1 %68.8 %homozygous (n=)393319147homozygous (% of all)6.4 %36.0 %54.1 %21.3 %14.3 %4.8 %43.8 %*not further specified

Osteosclerosis in advanced chronic idiopathic myelofibrosis is associated with endothelial overexpression of osteoprotegerin

Advanced chronic idiopathic myelofibrosis (IMF) with osteosclerosis and increase and thickening of bone trabeculae is typically contrasted by the absence or sparse presence of osteoclasts. Because osteoclast formation can be inhibited by osteoprotegerin (OPG) we investigated OPG expression in IMF with severe fibrosis and osteosclerosis, which expressed significantly higher (up to 71-fold) OPG mRNA levels when compared with prefibrotic cellular IMF and control cases. The receptor activator of nuclear factor kappaB ligand (RANKL), a positive regulator of osteoclast differentiation and putative antagonist of OPG was overexpressed by up to 34-fold exclusively in advanced IMF. Case-specific calculation of the RANKL/OPG ratio in advanced IMF showed a wide range without significant differences when compared with the prefibrotic IMF and non-neoplastic haematopoiesis. Immunohistochemical detection of OPG protein revealed strong labelling of endothelial cells within proliferating vessels in fibrotic IMF and heterogeneously labelled megakaryocytes, and fibroblasts. Osteosclerosis and impaired osteoclast function in IMF appears to be associated with upregulated endothelial OPG expression but concomitant reduction of the antagonist RANKL could not be demonstrated. We conclude that osteosclerosis in IMF is associated with increased endothelial OPG expression without concomitant RANKL downregulation.

Aberrant expression of transforming growth factor β‐1 (TGFβ‐1) per se does not discriminate fibrotic from non‐fibrotic chronic myeloproliferative disorders

Transforming growth factor beta-1 (TGF beta-1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGF beta-1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGF beta-1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGF beta-1 expression in IMF and other Ph(-) CMPD, total bone marrow cells as well as laser-microdissected megakaryocytes were quantitatively analysed by real-time RT-PCR. OPG mRNA expression in fibrotic IMF was correlated with TGF beta-1 mRNA expression in a case-specific manner. Both OPG and TGF beta-1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGF beta-1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non-neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGF beta-1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGF beta-1 protein was predominantly expressed by the myeloid lineage in Ph(-) CMPD and non-neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGF beta-1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGF beta-1 expression occurs in megakaryocytes as well as myeloid cells in Ph(-) CMPD. TGF beta-1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non-fibrotic Ph(-) CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGF beta-1 expression.

Evaluation of Mast Cells in Myeloproliferative Disorders and Myelodysplastic Syndromes

Mast cells may be increased as a reactive mastocytosis in various hematologic disorders and malignant neoplasms, as well as in systemic mast cell disease (SMCD). There are no statistical differences in mast cell numbers in reactive mastocytosis and SMCD; however, SMCD usually reveals dyspoietic mast cells and other dyspoietic bone marrow elements. In addition, SMCD is frequently (45%) associated with myeloproliferative disorders (MPDs) (17%) and myelodysplastic syndromes (MDSs) (28%). Thus, it has been suggested that SMCD may represent one aspect of a hematologic disorder that involves multiple bone marrow lineages. To perform a systematic evaluation of MPDs and MDSs without SMCD for dyspoietic mast cells. A total of 55 MPDs or MDSs were reviewed, including 20 cytogenetically proven chronic myeloid leukemias, 6 essential thrombocythemias, 2 polycythemia veras, 21 cytogenetically proven MDSs, and 6 chronic myelomonocytic leukemias. Cases of idiopathic myelofibrosis were not included due to lack of spicules. The bone marrow aspirates were reviewed for an increase in mast cells (1+ to 3+), dyspoietic features within mast cells (decreased cytoplasmic granularity, uneven granule distribution), and a predominance of fusiform mast cells. All cases, except 2 MDSs, had evaluable bone marrow spicules. Of interest, the MPDs were significantly more associated with increased and dyspoietic mast cells (57% and 61%, respectively) than were the MDSs (11% and 4%, respectively). The 2 polycythemia veras and 6 chronic myelomonocytic leukemias did not reveal increased or dyspoietic mast cells. These findings indicate that MPDs (chronic myeloid leukemia and essential thrombocythemia) frequently contain neoplastic mast cells as the spectrum of abnormal bone marrow cells. This feature, in conjunction with other parameters, may possibly be useful in the differential diagnosis of MPDs and MDSs. Our findings, compared with the previously reported findings in SMCD, suggest that SMCD may be more closely related to MPDs than to MDSs.

Idiopathic Myelofibrosis Associated with Ulcerative Colitis

A patient with ulcerative colitis (UC) who developed idiopathic myelofibrosis (IM) is reported. The initial diagnosis of UC was established by colonoscopy and large bowel biopsy, performed after a one-month history of abdominal pain and bloody diarrhea. The patient showed a favorable response to prednisone and mesalamine treatment and six months later he developed a new episode of UC, which was successfully controlled with treatment. However, two years later splenomegaly and anemia were observed, with aniso-poikilocytosis, tear-drop cells, immature myeloid precursors in the peripheral blood, and increased serum LDH, arising the suspicion of IM, a diagnosis that was confirmed by bone marrow biopsy. The present case represents a new association of IM with an autoimmune disease and gives support to the hypothesis of a possible immune basis of some IM cases.

Trisomy 13 is Associated with Poor Prognosis in Idiopathic Myelofibrosis with Myeloid Metaplasia

We report a case of idiopathic myelofibrosis with trisomy 13 as the sole clonal aberration, as demonstrated by metaphase cytogenetics. The clinical course was especially poor in this case, with death in blast crisis occurring within two weeks from diagnosis. The dismal outcome bears striking similarity to two previous cases of idiopathic myelofibrosis and trisomy 13 reported in the literature. Therefore trisomy 13 may be a predictor of a rapidly fatal outcome in this otherwise indolent disease. Fluorescence in situ hybridisation (FISH) with a chromosome 13 specific probe may enhance the detection of this aberration, since only 50% of cases of idiopathic myelofibrosis are karyotyped successfully using conventional techniques.

A case of idiopathic myelofibrosis with alloimmunization to HLA-identical platelet transfusions not responsive to antibody absorption over protein-a columns

A case of idiopathic myelofibrosis has been treated with HLA-identical platelet apheresis concentrates. After a short period the patient also developed refractoriness to the haploidentical siblings. Protein A columns were used to absorb the platelet antibodies with no result. Now two apheresis platelet concentrates are used every month, crossmatched negative products produce good results maintaining cell counts between 20 and 30 × 10 9/L. The immune mechanism involved is difficult to understand.

Partial trisomy 1q in idiopathic myelofibrosis

Three cases of idiopathic myelofibrosis with partial trisomy of the long arm of chromosome 1 are described. Partial trisomy 1q was the only karyotypic change detectable in unstimulated peripheral blood cell cultures of one and bone-marrow cultures of two patients at diagnosis. The extra segment from chromosome 1 was located on different karyotype sites, i.e. 1qter, 1p34 and 6p22–23; 1q21–32 was the shortest overlapping region and the only trisomic segment in one of the three patients. These findings suggest that partial trisomy 1q is a primary chromosome aberration in myelofibrosis relevant in the pathogenesis of this hematologic disorder.

Changes in the Mitogenic Activity of Platelet-Derived Growth Factor(s) in Patients with Myeloproliferative Disease

Platelet-derived growth factor (PDGF) is thought to take part in the genesis of bone marrow fibrosis that can be found in patients with myeloproliferative diseases. We evaluated platelet mitogenic activity as the difference between serum and plasma activity in 8 patients with myeloproliferative disease. We observed a trend of lower values in 2 cases of polycythemia vera and 2 cases of essential thrombocythemia, as seen by other authors. Two patients suffering from chronic myeloid leukemia were within the normal range. In contrast, our 2 cases of idiopathic myelofibrosis showed increased levels. If confirmed by further studies, this could suggest a pathogenetic relationship between increased levels of PDGF and bone marrow fibrosis, and give differential diagnostic significance to the PDGF mitogenic assay in myeloproliferative diseases.

Discriminative value of isozymes of amino acid naphthylamidase in the diagnosis of myeloid leukemias.

Isozymes of amino acid naphthylamidase (called B and C) with deviating electrophoretic mobilities were found in peripheral blood leukocytes of 25 out 25 untreated acute and chronic myeloid leukemias (AML and CML) and in 2 out of 2 cases of idiopathic myelofibrosis, while a normal pattern was found in 3 control groups and 5 cases of polycythemia vera. In the AML group, a correlation between electrophoretic mobility and the number of blast cells was found, and on remission, the B isozyme mobility was nearly normalized. Thus, deviating electrophoretic mobility of the B isozyme seemed to be valuable for diagnosis of myeloid leukemias, and in the AML group, the change in mobility might indicate the size of the leukemic clone.