Myeloproliferative Neoplasms Cases Research Articles

Evaluation of the Incidence of Hematologic Malignant Neoplasms Among Breast Cancer Survivors in France

Breast cancer survivors are at an increased risk of developing certain types of hematologic malignant neoplasm after diagnosis. To estimate the incidence of various types of hematologic malignant neoplasm in breast cancer survivors, both in absolute terms and in association with the general population. This nationwide cohort study conducted in France used data from the French National Health Data System, a database that contains all of French residents' health-related expenses. All French women aged 20 to 85 years with an incident breast cancer diagnosis between July 1, 2006, and December 31, 2015, were included (n = 439 704) and followed up until hematologic malignant neoplasm occurrence, death, loss of follow-up, or December 31, 2016, whichever came first. Comparisons were made with all French women in the general population who were registered in the French general health insurance program each year from January 1, 2007, and December 31, 2016. Data analysis was performed from January 23, 2018, to May 25, 2018. Main outcomes were incident hematologic malignant neoplasm cases occurring at least 6 months after breast cancer diagnosis. The various types of hematologic malignant neoplasm considered were acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), multiple myeloma (MM), Hodgkin lymphoma or non-Hodgkin lymphoma (HL/NHL), and acute lymphoblastic leukemia or lymphocytic lymphoma (ALL/LL). Incidence of these various types was estimated among breast cancer survivors and compared with the incidence in women in the general population. The 439 704 women in the study had a median (interquartile range [IQR]) age of 59 (50-69) years and were followed up for a median (IQR) duration of 5 (2.8-7.5) years. Overall, 3046 cases of hematologic malignant neoplasm occurred: 509 cases (16.7%) of AML (crude incidence rate [CIR] per 100 000 person-years, 24.5; 95% CI, 22.4-26.8), 832 cases (27.3%) of MDS (CIR, 40.1; 95% CI, 37.4-42.9), and 267 cases (8.8%) of MPN (CIR, 12.8; 95% CI, 11.4-14.5). Lymphoid neoplasm cases included 420 cases (13.8%) of MM (CIR, 20.3; 95% CI, 18.4-22.3), 912 cases (29.9%) of HL/NHL (CIR, 44.4; 95% CI, 41.1-50.0), and 106 cases (3.5%) of ALL/LL (CIR, 5.1; 95% CI, 4.2-6.2). Compared with the general population, breast cancer survivors had statistically significantly higher incidence of AML (standardized incidence rate ratio [SIRR], 2.8; 95% CI, 2.5-3.2) and MDS (SIRR, 5.0; 95% CI, 4.4-5.7) and, to a lesser extent, MM (SIRR, 1.5; 95% CI, 1.3-1.7]) and ALL/LL (SIRR, 2.0; 95% CI, 1.3-3.0). The finding that AML and MDS still occur among breast cancer survivors today, and that ALL/LL and MM may also be of concern, merits the continuous monitoring of hematologic malignant neoplasms and the thorough investigations into their underlying mechanisms.

Clinicopathological spectrum of BCR-ABL-Negative myeloproliferative neoplasms with correlation with janus-associated kinase 2 mutation

Abstract Background: Non chronic myelogenous leukemia (non-CML)/BCR-ABL-negative myeloproliferative neoplasms (MPNs) include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) (apart from chronic neutrophilic leukemia and chronic eosinophilic leukemia, which are rare). They are uncommon clonal disorders of adults, with an incidence ranging from 0.5 to 3/100,000 persons, BCR-ABL negative, and characterized by the activation of Janus-associated kinase 2 (JAK2). Very few studies have been reported from India. Aims and Objectives: The aims and objectives of this study were to analyze the clinicopathological spectrum and to determine the frequency of JAK2 mutation in patients of non-CML/BCR-ABL negative MPNs. Materials and Methods: Clinical and morphological features and frequency of JAK2 mutation in patients with PV, ET, and PMF were studied at a tertiary care hospital. The material was retrieved from the hematopathology records and reviewed. Results: JAK2V617F mutation was found in 10 of 14 cases (71%) of MPNs, 100% in PV, 50% in ET, and 71% of idiopathic myelofibrosis. The presence of JAK2V617F mutation was associated with a higher hemoglobin level (P < 0.05), a higher TLC (P < 0.05), and higher age (P < 0.05). Results showed that there are morphologic differences, and megakaryocytic morphology represents a useful clue for the differential diagnosis of these three BCR-ABL-negative MPN subtypes. Conclusion: The JAK2 V617F mutation was detected in 71% of patients with MPN disorders. Peripheral blood mutation screening for JAK2 V617F should be incorporated into the initial evaluation of patients suspected to have MPNs. Differences in megakaryocytic morphology provide the histomorphological hallmark of BCR-ABL-negative MPN subtypes.

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorder which lead to erythrocytosis, thrombocytosis and leukocytosis. MPN with BCR-ABL positive is chronic myeloid leukemia (CML) while BCR-ABL negative MPN includes polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, Formalin-Fixed Paraffin-Embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive cross-sectional study. Forty FFPE marrow specimens were retrieved from the year 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 3 out of 4 in PV, 0/2 in ET, 4/4 in PMF, 4/10 in non-classified MPN and 1/20 in CML. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay, which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

Clinical Exome Sequencing unravels new disease-causing mutations in the myeloproliferative neoplasms: A pilot study in patients from the state of Qatar

Clinical Exome Sequencing (CES) has increasingly become a popular diagnostic tool in patients suffering from genetic disorders that are clinically and genetically complicated. Myeloproliferative Neoplasms (MPNs) is an example of a heterogeneous disorder. In Qatar, familial cases of MPNs are more frequently seen than described in the literature. In this study, we aimed to use CES to classify six Qatari subjects that were suspected of clinical diagnosis of MPNs, according to the WHO 2008 diagnostic criteria for hematologic malignancies, and identify variants that can potentially explain the phenotypic diversity of MPNs. We sequenced six Qatari subjects using CES, of whom, three probands were unrelated families and three members were from the same family, all probands come from consanguineous families, and had a positive family history of MPNs. CES identified 61 variants in 50 genes; of which, 13 were recurrently mutated in our patients. Ten novel variants were identified in ten known genes related to MPNs and seven variants were identified in seven novel candidate genes. The genotype of the six subjects was due to a combination of different variants in different genes. This study serves as a pilot study to investigate the complexity of the genotype of patients with MPNS in Qatar, and serves as a guide for further well-controlled genetic epidemiological studies for patients with MPNs. CES is a powerful tool to be used in the genetic clinics for differential and definitive diagnosis of patients with MPNs.

Pan-Myeloid Leukemia Analysis: Machine Learning-Based Approach to Predict Phenotype and Clinical Outcomes Using Mutation Data

Pan-Myeloid Leukemia Analysis: Machine Learning-Based Approach to Predict Phenotype and Clinical Outcomes Using Mutation Data

Skewed ratio between type 1 and type 2 calreticulin mutations in essential thrombocytosis patients with concomitant Janus kinase 2 V617F mutation

Detection of somatic mutations in cardinal driver genes is a strong argument for diagnosis in classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Driver mutations in Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL), are generally considered mutually exclusive, but several reports have suggested that they coexist in a small subgroup of patients. In this study, we retrospectively searched for CALR mutations in 136 suspected MPN patients with low allelic burden (≤5%) JAK2 V617F. Fifteen patients with concomitant JAK2 V617F and CALR mutations were identified, of whom 10 were diagnosed with essential thrombocytosis (ET). More than 50 different indel mutations in exon 9 of CALR have been reported, with type 1 (52 bp deletion) and type 2 (5 bp insertion) accounting for more than 80% of CALR-mutated MPN cases. Type 1 is generally considered the most common mutation, but, interestingly, our double-mutated ET patients seem to have an inversed ratio between type 1 and type 2 CALR mutations. Our findings support the possibility of coexisting JAK2 V617F and CALR mutations and stress the importance of further molecular screening in MPN patients with low allele frequencies of JAK2 V617F.

Bone Marrow Morphology in Myeloproliferative Neoplasms – Relevance in the Molecular Era

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders. The mutation BCR-ABL is sine qua non for diagnosis of chronic myeloid leukemia and JAK2 V617F is present in majority of cases of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Recent 2016 classification consigns importance to BM morphology as mutations are not universal. Identification of specific features would aid diagnosis of JAK2V617F negative MPN. This study aims to assess utility of peripheral blood and bone marrow morphology in myeloproliferative neoplasms in correlation with BCR-ABL and JAK2V617F. Methods: A retrospective 7 years study of peripheral blood, bone marrow aspirate (BMA) and bone marrow biopsy (BMB) of 44 cases of myeloproliferative neoplasms with BCR-ABL and JAK2 V617F mutation analysis. Results: Forty four cases of MPNs including 24 (54.5%) cases of CML, 7 (15.9% ) cases of PV, 5 (11.3%) cases of MF, 3 (6.8%) cases of ET, and 5 (11.3%) cases of MPN-U. MPV and LDH were significantly higher in MF in comparison to PV and ET. Higher haemoglobin (Hb) and platelets were seen in PV and ET. Dwarf MKs with hypolobated, cloud like nuclei in CML (fig 1), large MKs with staghorn nuclei were seen in ET. normal sized MKs with cloud like hypolobated forms in PV(fig2) and cloud like atypical dysmorphic nuclei with dense clustering were seen in MF. Conclusion: Hb, mean platelet volume (MPV), and LDH in conjunction with MK morphology aids in the diagnosis of MPNs. These findings will enable accurate diagnosis in JAK2 negative MPNs and benefit low resource center.

Abstract 3093: Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident

Abstract Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) are a unique group of hematological malignancies including Polycytemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) characterized by impaired function and structure of bone marrow. MPN driver mutations are usually found in Janus Kinase 2 (JAK2), Thrombopoietin Receptor (MPL) and Calreticulin (CALR) genes, however, 10-15% of MPN cases are triple negative (TN) for these mutations. A previous study showed a lower rate of JAK2 V617F mutations in PMF patients exposed to sublethal doses of ionizing radiation (IR) from the Chernobyl accident in Ukraine. We hypothesized a lower rate of the usual driver mutations in IR-exposed MPN patients. To examine whether there are distinct driver mutations, 281 Ukrainian IR-exposed and unexposed MPN patients, were enrolled in the study. Their records were reviewed for classification by the WHO 2016 MPN criteria. Genomic DNA was obtained from the peripheral blood leukocytes of 281 MPN patients to identify JAK2 V617F, MPL W515, and type 1- and 2-like CALR mutations by allele-specific PCR, Sanger Sequencing and RT-PCR, respectively. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed in 30 PMF patients by high-density Affymetrix CytoScan HD oligo-SNP microarray platform. Whole exome sequencing was used to identify additional genetic variants in these 30 PMF patients. Statistical significance for categorical variables and continuous variables were evaluated by Fisher's exact test and Wilcoxon's rank sum test using Statistical Analysis R, version 3.4.2. Clinical features of exposed and unexposed MPN patients were similar. More PMF IR-exposed patients were transfusion dependent (32.4%) than PMF unexposed patients (14.1%) (p = 0.04). JAK2 V617F was detected in 58% of IR-exposed and in 74% of unexposed MPN patients (p = 0.007). JAK2 V617F was also less frequent in IR-exposed PV patients, but not statistically significant. Type 1-like CALR mutation was detected in 12% of exposed and 3% of unexposed patients (p = 0.033). Overall, exposed patients were TN in 28% of IR-patients versus 16% of unexposed patients (p = 0.027). Among other genetic variants, ATM S1691R mutation with cnLOH at 11q22.3 was identified in one TN IR-exposed PMF patient. Previously the mutation was reported, but not in MPN patients. Missense mutations in EZH2 at 7q36.1 and SUZ12 at 17q11.2 with copy number loss were also identified in TN IR-exposed PMF patients. Our results confirm a lower rate of JAK2 V617F and higher rate of TN cases among IR-exposed MPN patients versus unexposed. We also demonstrated a higher frequency of type 1-like CALR mutation in IR-exposed MPN patients and new potential MPN driver mutations. Thus, IR-exposed MPN patients represent a disease group with distinct genomic characteristics worthy of further study. Citation Format: Larysa Poluben, Maneka Puligandla, Donna Neuberg, Christine R. Bryke, Nancy Hsu, Sergiy Klymenko, Olga Mishcheniuk, Oleksandr Shumeiko, Steven Balk, Xin Yuan, Olga Voznesensky, German Pihan, Miriam Adam, Ernest Fraenkel, Paula G. Fraenkel. Genomic characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3093.

BCR/ABL1-negative, triple-negative, myeloproliferative neoplasm with a hitherto undescribed, isolated, SH2B3 (LNK) gene mutation: A case report

The myeloproliferative neoplasms (MPNs) are chronic myeloid cancers (clonal hematopoietic disorders) that are characterized by the overproduction of terminally differentiated (mature) blood cells, and that may evolve into acute myeloid leukemia (AML). The literature indicates that three known driver mutations currently exist in BCR/ABL1-negative MPNs: JAK2, MPL and CALR. A small percentage of BCR/ABL1-negative MPN cases lack mutations in all three of these genes and are thus referred to as triple- negative. This case report is of a 48-year-old female with non-chronic myeloid leukemia (non-CML) MPN with increases in all three cell lineages in the bone marrow. No abnormalities were detected in chromosome analysis and FISH studies. Her myeloid molecular profile analyzed by targeted next generation sequencing (NGS) found a frameshift mutation in the SH2B3 (LNK) gene, but no mutations in any other genes typically mutated in MPNs. A few other cases of triple-negative, non-CML MPNs with SH2B3 (LNK) gene mutations have been reported, but we are adding a case of an MPN with a hitherto undescribed frameshift SH2B3 (LNK) mutation between the DD and PH domains of the gene. Currently, the literature is not clear whether SH2B3 (LNK) mutation is a driver mutation or not. More investigation is needed to clarify the significance of SH2B3 (LNK) gene mutations especially in the pathogenesis of MPNs lacking major driver gene alterations.

Coexistence of JAK2 or CALR mutation is a rare but clinically important event in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors.

There are 7 designated conditions under the category of myeloproliferative neoplasms (MPN), including chronic myelogenous leukemia (CML) and classical MPN, that is, polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF). Recently, reports about Philadelphia and JAK2 V617F-positive MPN cases have been described in literature. The coexistence of different molecular defects may change the clinical and laboratory manifestation of MPN and may result in an inappropriate interpretation of the response to treatment with tyrosine kinase inhibitors in CML patients. The morphological, cytogenetic, and molecular genetic data from a retrospective analysis of 592 adult patients aged 18-86years diagnosed with CML were analyzed. In 5 CML patients, the presence of JAK2 V617F or CALR mutation was confirmed. Three of 4 TKI-treated patients did not reach complete hematologic response due to the persistence of thrombocytosis and/or splenomegaly. In all of them (in 3 with JAK2 V617F mutation and 1 with CALR mutation), thrombocytosis was present at the time when complete cytogenetic response was documented. In 3 out of 4 reported CML patients, thrombocytosis and/or splenomegaly were still present even at the time when deep molecular response was reached. In our opinion, a detailed evaluation and appropriate interpretation of clinical and laboratory data in such a category of patients seem to be extremely important, especially when a decision about the TKI change due to therapy failure is considered.

Molecular genetics of BCR-ABL1 negative myeloproliferative neoplasms in India.

Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance.

Abstract 37: An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome

Abstract Introduction: Activating Janus kinase 2 (JAK2) mutations have been described in myeloproliferative neoplasms (MPNs) since 2005. Since its initial discovery, the JAK2 V617F mutation has been described in more than 95% of patients with polycythemia vera (PV) and in 50-60% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Subsequent efforts were made to identify molecular mutations in MPN cases lacking the V617F mutation and further mutations in the exon 12 domain of the JAK2 were identified in PV patients. However, there have been only a few studies evaluating the presence of these JAK2 exon 12 mutations in PMF patients. Case Report: A 60-year-old Malay gentleman with no known prior medical history of note presented at our Emergency Department with a 1-week history of bipedal edema and exertional dyspnea. Physical examination revealed bibasal coarse crepitations with moderate hepatosplenomegaly. Laboratory investigations revealed normochromic, normocytic anemia, thrombocytopenia, hypoalbuminemia, and elevated creatinine levels. A spot urine sample showed a protein to creatinine ratio of 93 mg/mmol with an estimated total urinary protein (TUP) of 0.8 g/day. Further workup of his renal impairment, which included an autoimmune screen, hepatitis, and humman immunodeficiency tests, were all unremarkable. A myeloma screen revealed elevated immunoglobulin G levels of 29.18 g/L but with no monoclonal bands detected. Transthoracic echocardiography showed mild pulmonary hypertension with a pulmonary artery systolic pressure (PASP) of 45 mmHg, but the ejection fraction was normal at 65%. He was also noted incidentally to have a prolonged prothrombin time (PT), activated partial thromboplastic time (APTT), and thrombin clotting time. Fibrinogen levels were normal and a lupus anticoagulant was not detected. Bone marrow aspiration and biopsy results were consistent with myelofibrosis and diffuse coarse reticulin fibers of grade 4 were reported. Quantitative BCR-ABL messenger ribonucleic acid (mRNA), JAK2 V617F, and calreticulin (CALR) mutations were negative. However, a JAK2 H538QK539L (mutation leading to a substitution of glutamine for histidine at position 538 and leucine for lysine at position 539) in exon 12 was detected. Incidentally, iron stores were absent on the bone marrow aspirate sample. Ultrasound (US) and computed tomography (CT) scans showed the presence of hepatosplenomegaly with portal hypertension with no evidence of any venous thrombosis. A nonobstructive left renal calculi was seen but no other renal abnormality was detected. He was started on low-salt diet, fluid restriction, diuretic, and iron supplements. Within 1 month, his hemoglobin increased from 7.7g/dL to 14 g/dL with a rise in hematocrit to 52.1%. His iron supplements were stopped and he required 3 sessions of venesection in total to control his hematocrit. Subsequently, he was commenced on hydroxyurea for cytoreduction. His spleen reduced in size and his proteinuria normalized after 10 months. However, he developed worsening anemia and hydroxyurea had to be stopped. On follow-up, his hemoglobin increased to 12 g/dL with a baseline platelet count of > 100 X 109/L. Throughout this period, his bipedal edema remained chronic but he was not consistent with his renal follow-ups. Within 12 months, his proteinuria started increasing and is now within the nephrotic range. Discussion: We describe here a patient who was diagnosed with a JAK2 V617F negative PMF. Subsequent analysis revealed that he was harboring a JAK2 exon12 mutation instead. This was associated with a deranged coagulation profile and proteinuria which responded to hydroxyurea. This patient was never diagnosed with PV previously and thus did not meet the criteria for post-PV myelofibrosis. Also, we believe that this is the first case of a JAK2 exon12 mutation occurring in a myelofibrotic patient. Given the recent description of MPN-related glomerulopathy and the response of this patient to hydroxyurea, we believe further investigations into the associations of triple-negative (JAK2 V617F, myeloproliferative leukemia virus oncogene (MPL) and CALR negative) PMF with JAK2 exon12 mutations and the associated organ involvements may increase our knowledge of the pathogenesis of this rare condition. Citation Format: Moon Ley Tung, Hersharan Kaur Sran. An unusual case of myelofibrosis with a JAK2 H538QK539L mutation associated with nephrotic syndrome [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 37.

Whole-genome analysis reveals unexpected dynamics of mutant subclone development in a patient with JAK2-V617F-positive chronic myeloid leukemia

We report here the first use of whole-genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML), who presented in chronic phase (CP) with doubly marked BCR-ABL1+/JAK2V617F-mutant cells and, over a 9-year period, progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS revealed that the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1, and RREB1 as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only six additional coding somatic mutations, despite retention by the hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells revealed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2 of 101 cases of myeloproliferative neoplasms, but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.

Progenitor genotyping reveals a complex clonal architecture in a subset of CALR-mutated myeloproliferative neoplasms.

The identification of acquired CALR mutations in patients with essential thrombocythaemia (ET) or myelofibrosis (MF) has meant that disease-initiating mutations can now be detected in about 90% of all patients with a myeloproliferative neoplasm (MPN). Here, we show that only those CALR mutations that cause a +1 frameshift, thereby altering the carboxy-terminus of calreticulin, promote cytokine independence invitro; in-frame deletions were not functional, and are unlikely to be the pathogenetic mutation underlying some MPN cases. Expression of the thrombopoietin receptor, MPL, was also necessary for factor-independence. Although the CALR mutations are considered to occur only in JAK2 V617F-negative cases and in a heterozygous state, progenitor genotyping revealed that this is not always true. Notably, CALR mutation-positive MPNs can be polyclonal: in one case, two distinct CALR mutation-positive subpopulations could be identified; in another, separate populations of JAK2 V617F-positive and CALR-mutated cells were present. Mitotic recombination involving chromosome 19 in a third instance resulted in the emergence of a CALR mutation-homozygous subclone. Collectively, our studies demonstrate that occasional patients with CALR mutation-positive ET or MF carry other MPN-initiating genetic mutations (including JAK2 V617F), acquire "secondary mutations" before or after the CALR mutation, or evolve over time to being CALR mutation-homozygous.

Loss of p53 induces leukemic transformation in a murine model of Jak2 V617F-driven polycythemia vera.

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53 mutation. Here we show that retroviral overexpression of Jak2 V617F mutant into wild-type p53 murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit+Sca1+Lin- (KSL) and CD34-CD16/32-c-kit+Sca1-Lin- (megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71+) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1+) could not recapitulate the disease. Interestingly, recipients transplanted with CD71+ cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71+ cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53 loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML

CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation.Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells.After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. DisclosuresNo relevant conflicts of interest to declare.

Cerebrovascular events as presenting manifestations of Myeloproliferative Neoplasm

AimTo determine the incidence and main characteristics of cerebrovascular events as the presenting manifestations of myeloproliferative neoplasm (MPN). MethodsThe Hematology in Lyon (HEMILY) registry is a prospective database (763 patients) of all cases of MPN diagnosed since 2005 in the Rhône-Alpes district of France. The MPN cases were divided into four groups: polycythemia vera (PV); essential thrombocythemia (ET); myelofibrosis (MF); and atypical MPN. The ischemic stroke subtype was classified according to TOAST criteria. ResultsA stroke history revealed MPN in 35 (4.3%) patients: 22 (63%) had an ischemic stroke; eight (23%) had a transient ischemic attack; four (11%) had cerebral venous thrombosis; and one (3%) had hemorrhagic stroke. All patients had hemoglobin and/or platelet count abnormalities. In addition, 12 (34%) patients had PV, 21 (60%) had ET, one (3%) had MF and one (3%) had atypical/unclassified MPN. The JAK2 V617F mutation was found in 83% of patients. In 18 (51%) patients, an additional mechanism of stroke was present (atherosclerosis in 10 patients, atrial fibrillation in one patient and dissection in another). The median NIHSS score at entry was 2, and the median modified Rankin Scale score at 3 months was 0. Compared with the general MPN population, stroke–MPN patients presented with significantly higher levels of hemoglobin (P<0.001) and were more frequently positive for the JAK2 V617F mutation (P=0.044). ConclusionStroke revealing MPN is rare. However, careful attention should still be paid to blood counts even in patients with obvious stroke etiologies, as early diagnosis permits prompt treatment and decreases the risk of recurrence, thus limiting morbidity and mortality.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting

IntroductionRecently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60–80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. MethodsWe examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). ResultsAmong 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. ConclusionOur novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN.

Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms

AbstractWe conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10−89), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10−32), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10−14), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10−9). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B. All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm.

Tissue microarray technique is applicable to bone marrow biopsies of myeloproliferative neoplasms.

Tissue microarray (TMA) technique is an established high-throughput method to analyze multiple tissue specimens in parallel. However, in order to obtain reliable results from immunohistochemical analyses of TMA blocks, cell composition of TMA spots must correspond to whole tissue sections (WTS) particularly in tissues with a heterogeneous cell composition as it is the case in myeloproliferative neoplasms (MPN). The aim of this study was to validate TMA of bone marrow biopsies from MPN patients. TMAs of MPN bone marrow biopsies (ET: n=26, PV: n=26, and PMF: n=29) were compiled in triplicatesand MPN-specific histological parameters were assessed. Results of TMA spots were compared with WTS' results using the intra-class correlation coefficient (ICC). Immunohistochemical NFE2 and calreticulin stainings of the TMA with quantitative evaluation were performed. TMA construction was technically successful with a loss of 10% of all spots. ICC calculation revealed high to moderate correlations of TMA with WTS, especially the parameters that are typically affected in MPN tissue, e.g. cellularity of hematopoiesis (ICC 0.62-0.89), number of megakaryocytes (ICC 0.50-0.71), micro-vessel density (ICC 0.56-0.91), or grade of myelofibrosis (ICC 0.56-0.89). Results of NFE2 and calreticulin immunohistochemistry of MPN TMAs are consistent with previously published data. Overall, our results show moderate to good correlation between histological data of WTS and TMA spots illustrating that the TMA technique is applicable to bone marrow biopsies of MPN patients. However, TMA construction in triplicates is necessary to reach sufficient correlation. MPN TMAs can be applied for serial immunohistochemical surveys of archived tissues to assess the mutation status or to further sub-classify MPN cases.