Unclassifiable Myeloproliferative Neoplasms Research Articles

Age-Related Macular Degeneration in Patients With Chronic Myeloproliferative Neoplasms

ImportanceIt has been suggested that systemic inflammation increases the risk of age-related macular degeneration (AMD). Given that chronic immune modulation is present in patients with myeloproliferative neoplasms (MPNs), the risk of AMD in these patients may be increased.ObjectiveTo compare the risk of AMD in patients with MPNs with the risk of AMD in matched controls from the general population.Design, Setting, and ParticipantsA nationwide population-based cohort study using Danish registers was conducted of all patients in Denmark who received a diagnosis between January 1, 1994, and December 31, 2013, of essential thrombocythemia, polycythemia vera, myelofibrosis, or unclassifiable MPNs. For each patient, 10 age- and sex-matched controls were included. All patients without prior AMD were followed up from the date of diagnosis (or corresponding entry date for the controls) until the first AMD diagnosis, death or emigration, or December 31, 2013, whichever occurred first. Data analysis was performed from April 1, 2015, to October 31, 2016.Main Outcomes and MeasuresIncidence of AMD recorded in specialized hospital-based care. The rates and absolute risk of AMD were calculated. Using Cox proportional hazards regression models, smoking and risk-time adjusted hazard ratios (HRs) between patients and controls were calculated. In addition, HRs of neovascular AMD after 2006 were calculated since antivascular endothelial growth factor treatment was introduced nationwide at hospitals thereafter.ResultsA total of 7958 patients with MPNs (4279 women [53.8%] and 3679 men [46.2%]; mean [SD] age at diagnosis, 66.4 [14.3] years) were included in the study. The rate of AMD per 1000 person-years at risk was 5.2 (95% CI, 4.6-5.9) for patients with MPNs (2628 with essential thrombocythemia, 3063 with polycythemia vera, 547 with myelofibrosis, and 1720 with unclassifiable MPNs) and 4.3 (95% CI, 4.1-4.4) for the 77 445 controls, while the 10-year risk of AMD was 2.4% (95% CI, 2.1%-2.8%) for patients with MPNs and 2.3% (95% CI, 2.2%-2.4%) for the controls. The risk of AMD was increased overall for patients with MPNs (adjusted HR, 1.3; 95% CI, 1.1-1.5), with adjusted HRs for the subtypes of 1.2 (95% CI, 1.0-1.6) for essential thrombocythemia, 1.4 (95% CI, 1.2-1.7) for polycythemia vera, 1.7 (95% CI, 0.8-4.0) for myelofibrosis, and 1.5 (95% CI, 1.1-2.1) for unclassifiable MPNs. In addition, patients with MPNs had a higher risk of neovascular AMD (adjusted HR, 1.4; 95% CI, 1.2-1.6).Conclusions and RelevanceOur results suggest that patients with MPNs are at increased risk of AMD, supporting the possibility that systemic inflammation is involved in the pathogenesis of AMD.

Clinical Exome Sequencing Unravels New Disease Causing Mutations in Myeloproliferative Neoplasms (MPNs): A Pilot Study in Patients from the State of Qatar

In Qatar, a cluster of ET familial cases (25 families from 5 major tribes) has been documented. This observation represents a unique interest for the scientific community worldwide, as the cultural traditions of tribal marriages between 1stcousins is expected to lead to the preservation of a limited genetic pool (giving rise to founder effects). CES was employed to analyze a 3 probands from three unrelated families (S1, S2, S3). All 6 patients were Qatari belonging to consanguineous families with a positive family history of MPNs. CES confirmed the diagnosis in the 3 probands: two patients (S1 and S2) were classified as Essential Thrombocythemia (ET), one patient (S3) as unclassified MPNs. In the other 3 subjects (S4, S4, S6), several shared deleterious mutations and polymorphisms were uncovered. CES identified 10 novel mutations in known genes as well as 7 novel candidate genes and 2 (6%) previously reported mutations in the three probands (S1, S2, S3) who's diagnosis was confirmed . S1: a confirmed diagnosis and phenotype of ET (JAK2 and THPO), S2: a confirmed diagnosis and phenotype of ET (JAK2 and MPL) and S3: a confirmed diagnosis of U-MPNs (TERT, ATM, NUMA1 and ASXL) The mutations/variants were classified according to ACMG recommendations into 5 classes. The number of mutations/variants detected in each class is as follows: Class 1 - Previously reported as disease-causing: 6 Class 2 - Previously unreported, but of the type which can cause the disorder: 3 Class 3 - Previously unreported, may or may not be the cause of the disorder (Variant of uncertain clinical significance): 48 Class 4 - Previously unreported but likely neutral: 1 Class 5 - Previously reported as neutral: 3 CES identified a total of 61 interesting variants which occurred in 50 genes, 13 were recurrently. The vast majority of the prioritized variants were missense 50(82%); the remaining were frameshift (small insertions and deletions) 10(16%) or affecting splicing sites 1(2%). The most interesting finding is the presence of 10 novel mutations in known genes related to MPNs as well as 7 variants in novel candidate genes in the three probands who's diagnosis was confirmed via CES. This study presents novel data that contributes to understanding the high level of genetic complexity in MPNs Qatari patients. It has identified new variants in cancer-related genes potentially involved in the pathogenesis of the disease Our study of familial MPNs led to the identification of new disease-causing mutations and variants, some previously described as either germline or somatic. A clinical cohort of 3 Qatari probands suspected to have MPNs as well as three family members with a positive family history of MPNs presented at National Center for Cancer Care (NCCCR) S1) is a female who presented with high hemoglobin (Hb) level, red blood cells (RBCs) count, leukocytosis with neutrophilia, very high platelets and normal erythropoietin (EPO); subject 2 (S2) is a female who presented with normal Hb, RBCs count, slightly high WBCs and high platelets; subject 3 (S3) is a male who presented with high Hb, normal WBCs and EPO 3 family members, subject 4 -a mother presented with normal Hb level, sustained high RBCs count and slightly high WBCs count, subject 5 a daughter presented with unexplained sustained erythrocytosis, leukocytosis (absolute neutrophilia, for more than 2 years Chronic headache, normal MRI brain and no progression to cancer no Ischemic events, subject 6- healthy daughter with normal CBC indicesThe pattern of mutations identified was unique. In this study 10 novel mutations in known genes already implicated in the pathogenesis of MPNs were detected including THPO, CBL, ASXL1, MPL (2x), BCL2, MYO18B, MTOR, PDGFRB, TERT. Mutations/variants were detected in 9 genes not previously associated with MPNs suggesting potential novel gene associations. Pathogenic/likely pathogenic mutations, uncertain variants and neutral/likely neutral variants were identified using the CES approach. A complex combination of mutations was observed in our cohort of subjects. For example in subject 1 (S1), an ET patient, the following mutations were observed: THPO S184R, ITGA2 R76Q, JAK2 V617F, CBL E196G, CBFB G180S, FANCA S858R, ASXL1 T600P fs103. The following detailed analysis of each mutation reveals that all these mutations could potentially play a role in the pathogenesis of MPNs Disclosures No relevant conflicts of interest to declare.

Treatment-Related Infections and Risk Factors in Patients with Myeloproliferative Neoplasms Treated with Ruxolitinib

▪Ruxolitinib is an orally available JAK1/2 inhibitor FDA approved for the treatment of myelofibrosis and polycythemia vera. Evidence suggest the effect of ruxolitinib on IL-1, IL-6, and TNF-α, among other immune mediators, may predispose patients to both opportunistic and non-opportunistic infections. Identification of patients at risk for infection while on a JAK inhibitor may impact clinical management. Authors sought to determine the cumulative incidence of ruxolitinib-related infections in the clinical practice setting and evaluated patient factors in order to characterize the types, onset, and severity of infections.This IRB approved, single-center, retrospective study at Memorial Sloan Kettering Cancer Center included myeloproliferative neoplasm (MPN) patients treated with ruxolitinib who were ≥18 years of age between 12/1/11 and 12/31/15. Patients on ruxolitinib for less than seven days, receiving ruxolitinib therapy for a non-MPN diagnosis, or had a manifestation of acute leukemia were excluded. Patients who met inclusion criteria were censored at time of splenectomy and hematopoietic stem cell transplant. Patients were identified through electronic medical records and an internal pharmacy database. Cumulative incidence of treatment-related infection was estimated and association with factors was assessed using competing risk regression analysis. Death without an infection was considered a competing risk event.A total of 44 patients were included in this analysis. The median age of this cohort was 66.5 years, with a slightly predominant male population (57%). There were 19 (43%) primary and 19 (43%) secondary myelofibrosis patients, 4 (9%) with polycythemia vera, and 2 (5%) patients with an unclassifiable MPN. For myelofibrosis patients, according to the Dynamic International Prognostic Scoring System (DIPSS), 2 (5%) were low risk, 3 (7%) were intermediate-1, 16 (36%) were intermediate-2, and 7 (16%) were high; remaining patients did not have available DIPSS scores at the time of evaluation. The median number of prior lines of therapy was 1.5 (range 0-4); median total daily dose of ruxolitinib was 10 mg. A majority of patients (70%) were not on concomitant immunosuppressive therapy during ruxolitinib treatment or strong CYP3A4 inhibitors (95%) that may have increased exposure to ruxolitinib.A total of 20 documented infections were identified throughout the evaluation period; median follow-up among survivors was 16 months (range 3-51). Cumulative incidences of treatment related infection were 33% by year 1 and 56% by year 2 of ruxolitinib treatment. Of these infections, four were CTCAEv4.0 grade 1 (one asymptomatic upper respiratory tract infection (URI) occurred after discontinuation was censored), seven were grade 2, and eight were grade 3. One grade 5 infection, septic shock of unknown origin occurred. Only one opportunistic pulmonary tuberculosis infection occurred 13 months post ruxolitinib treatment. The remainder of infections varied, including URIs(n=9), pneumonia, diverticulitis, osteomyelitis, clostridium difficile, proctitis, catheter-associated UTI, dental abscess, herpes zoster activation of the skin, and oral herpes simplex reactivation (all n=1)). One patient required viral prophylaxis for recurrent herpes simplex infections.With the limited number of events, no factors were found to be significantly associated with increased risk of infection but a trend toward increased risk of ruxolitinib-related infection was observed with baseline renal impairment. (Table 1).Conclusion: This data suggests a higher incidence of ruxolitinib associated infections observed in clinical practice compared to rates described in trials of ruxolitinib therapy. This may be attributable to differences between populations in the COMFORT and RESPONSE trials and patients in the clinical practice setting. Generally, infections were non-life threatening and managed with appropriate supportive care. Plasma values of ruxolitinib metabolites increase as renal function declines, correlation of infection risk via increased metabolites exposure warrants further exploration. Routine antimicrobial prophylaxis for all patients receiving ruxolitinib therapy is likely not warranted. However larger studies are needed to confirm these observations. [Display omitted] [Display omitted] DisclosuresMauro:Pfizer: Consultancy; Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy.

Cytopenia of Unknown Cause Post Allogeneic Stem Cell Transplant As a Predictor of Clinical Outcome

Temporary or long-lasting decrease of one or more blood cell lineages following allogeneic hematopoietic stem cell transplant (allo-HSCT) can lead to increased morbidity and mortality. Post-transplant cytopenia is often secondary to drug toxicity, viral infections, graft versus host disease (GVHD), or relapse. Less is known about the significance of cytopenia of unknown cause. Here we retrospectively analyzed 91 consecutive adult patients (median age 46 years; range: 19-63) who had received allo-HSCT conditioned with myeloablative fludarabine/ I.V. busulfan at our institution. Diagnoses included: acute myeloid leukemia (AML) or myelodysplatic syndrome (MDS) (n=57), acute lymphoblastic leukemia (ALL) (n=15), chronic myeloid leukemia (CML) (n=9), non-Hodgkin lymphoma (NHL) (n=5), B cell chronic lymphocytic leukemia (B-CLL) (n=2), myelofibrosis (n=1), unclassified myeloproliferative neoplasms (MPN) (n=2). Stem cell source was granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC) in 81, bone marrow in 8 and cord blood in 2 patients. Median follow up for the entire cohort was 20 months (range 1-162 months). Patients with cytopenia of unknown cause were identified if they had full donor chimerism, negative cytomegalovirus (CMV) by molecular analysis, no active infections, acute GVHD <grade II, no chronic GVHD, no drug myelotoxicity, and at least one of the following: transfusion dependence for greater than 2 weeks, absolute neutrophil count (ANC) <1.5 x109/L, hemoglobin <8.5 g/dl or platelets <50 x103/L . Of 91 patients, 28 (31%) did not have any cytopenia post transplant, 48 (53%) had cytopenia of known cause (GVHD, n=9; infection, n=11; drug, n=2; relapse, n=25; graft failure, n=1). The remaining 15 patients (16%) had cytopenia of unknown cause. Of these 15, 9 cases had neutropenia, 6 thrombocytopenia (4 requiring platelet transfusion) and 2 had anemia (1 requiring red blood cell transfusion). Only one blood lineage was decreased in 13 patients and 2 lineages in 2 patients. Median onset of cytopenia was 60 days (range: 30-360 days) and median duration of cytopenia was 14 days (range: 14-150 days). When we compared the group of patients with cytopenia of unknown cause to the one without any cytopenia, no differences in patient characteristics, donor type, CD34+ cell dose, or stem cell source were found. A log-rank analysis did not show significant differences in overall survival (OS) (p=0.08) or progression free survival (PFS) (p=0.2) between the two groups. However, patients with cytopenia of unknown cause more frequently developed subsequent acute GVHD (53% vs 25%, p=0.06) and had a significantly higher risk of death due to infection compared to patients without cytopenia (p=0.04). Our results suggest that patients with cytopenia of unknown cause after allo-HSCT may have a worse outcome possibly due to a higher risk of acute GVHD and infections. Further studies are warranted on larger multicenter or registry data to establish the prognostic role of post-transplant cytopenia. DisclosuresNo relevant conflicts of interest to declare.

Elucidation of Additional Mutations By Next-Generation Sequencing Is of Clinical Significance in Patients with Rare MPNs and MDS/MPN Overlap Syndromes

Introduction:Recently, next-generation sequencing (NGS) has revolutionized the molecular characterization and understanding of several hematologic entities, including myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS)/MPN overlap syndromes. Nevertheless, the frequency and clinical impact of the mutations detected by NGS, remain largely unclear, especially in rare MPN which were analyzed in this study.Methods:Thus, we established a novel amplicon-based NGS panel, comprising genes that are known to be recurrently mutated in MPN and/or MDS/MPN. Hot spot regions or all exons of the following 32 genes were chosen: ABL, ASXL1, BARD, CALR, CBL, CEBPA, CHEK2, CSF3R, DNMT3A, ETNK1, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NFE2, NRAS, PDGFRA, PTPN11, RUNX1, SETBP1, SF3A1, SF3B1, SH3B2 (LNK), SRSF2, TCF12, TET2, TP53, U2AF1. After establishing this panel, peripheral blood samples of 19 patients, which were diagnosed with CMML(10), aCML(2), MPNu(1), MDS/MPNu or other MPN(6), were analyzed on a MiSeq® illumina sequencer. Variants were only analyzed if the absolute coverage at each SNV site was >50 reads, and the absolute coverage of the mutant allele was 10 or more reads and its relative coverage exceeded 10%.Results:Mean coverage of the run was 1516 reads with good Phred-score quality parameters (>84% of called bases with Q-score >= 30). In 300 bidirectional cycles, a yield of nearly seven gigabases of sequencing data was reached. One out of 19 analyzed patients was excluded from analysis due to insufficient DNA quality. In 89% of the samples(16/18), mutations were detected which had not previously been known to be present in these patients. TET2 (50%, 9/18) and SETBP1 mutations were the most common (44%, 8/18). As expected, TET2 mutations were spread over the entire gene and SETBP1 mutations were restricted to the known hot spot region (exon 4, c.2602-c.2620). Additionally, CSF3R mutations were detected in 22% (4/18) of patients. Epigenetic regulator genes were also affected as EZH2 mutations were detected in 17% (3/18), ASXL1 mutations in 39% (7/18) and IDH1/2 mutations were found in 6% (1/18) of all samples, whereas DNMT3A mutations were not present. Further mutations were found in the following genes: CBL (11%), ETV6 (6%), JAK2V617F (6%), KRAS (11%), NRAS (11%), PTPN11 (6%), SH2B3 (6%) and SRSF2 (11%). Besides previously known mutations, several novel variants could be detected. All but one patient harbored more than one of these mutations.Furthermore, clinical correlates and morphologic and cytogenetic subtypes of each patient were available to associate with the NGS data of individual patients. For example, the one patient with a solitary NRAS c.35G>A (amino acid: p.G12D) mutation showed the most aggressive clinical course in our cohort with transformation to AML only 7 months after first diagnosis of CMML. Moreover, CSF3R mutations have been shown to confer sensitivity to ruxolitinib and may thus open up new avenues of treatment for our patients.Conclusion:In a cohort of unclassified MPN and rare MDS/MPN subtypes, NGS is a powerful tool to characterize samples more extensively. Our data suggests that a more comprehensive understanding of the mutational spectrum may have important clinical impact in individual patients, including diagnosis, prognosis, and more specific treatment. DisclosuresIsfort:Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel; BMS: Honoraria; Mundipharma: Other: Travel; Amgen: Other: Travel; Hexal: Other: Travel. Brümmendorf:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia.

Objective:Calreticulin (CALR) plays important roles in cell proliferation, apoptosis, and immune responses. CALR mutations were described recently in Janus kinase 2 gene (JAK2)-negative or MPL-negative primary myelofibrosis (PMF) and essential thrombocythemia (ET) patients. CALR trails JAK2 as the second most mutated gene in myeloproliferative neoplasms (MPNs). However, little is known about CALR mutation in Chinese patients with leukemia. In the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs.Materials and Methods:Polymerase chain reaction and direct sequencing were performed to analyze mutations of CALR in 305 patients with hematopoietic malignancies, including 135 acute myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, and 113 MPN patients.Results:CALR mutations were found in 10.6% (12 of 113) of samples from patients with MPNs. CALR mutations were determined in 11.3% (6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, and unclassifiable MPN, respectively.Conclusion:We showed that MPN patients carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels compared to those with mutated JAK2. However, all of the leukemia patients had negative results for CALR mutations.

Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms.

Objective:Myeloproliferative neoplasms (MPNs) share common clonal stem cells but show significant differences in their clinical courses. The aim of this retrospective study was to evaluate thrombotic and hemorrhagic complications, JAK2 status, gastrointestinal and cardiac changes, treatment modalities, and survival in MPNs in Turkish patients.Materials and Methods:Medical files of 294 patients [112 essential thrombocythemia (ET), 117 polycythemia vera (PV), 46 primary myelofibrosis, and 19 unclassified MPN cases] from 2 different universities in Turkey were examined.Results:Older age, higher leukocyte count at diagnosis, and JAK2 mutation positivity were risk factors for thrombosis. Platelet count over 1000x109/L was a risk factor for hemorrhagic episodes. Hydroxyurea treatment was not related to leukemic transformation. Median follow-up time was 50 months (quartiles: 22.2-81.75) in these patients. Patients with primary myelofibrosis had the shortest survival of 137 months when compared with 179 months for ET and 231 months for PV. Leukemic transformation, thromboembolic events, age over 60 years, and anemia were found to be the factors affecting survival.Conclusion:Thromboembolic complications are the most important preventable risk factors for morbidity and mortality in MPNs. Drug management in MPNs is done according to hemoglobin and platelet counts. Based on the current study population our results support the idea that leukocytosis and JAK2 positivity are more important risk factors for thrombosis than hemoglobin and platelet values.

BCR–JAK2 fusion in a myeloproliferative neoplasm with associated eosinophilia

Janus kinase 2 (JAK2) is located on chromosome 9 at band p24 and JAK2V617F is the most common mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN). However, rearrangement of JAK2 is a rare event. We report a case of myeloproliferative neoplasm, unclassifiable (MPN-U) with BCR-JAK2 fusion confirmed by molecular studies. Conventional chromosome analysis (CC) revealed t(9;22)(p24;q11.2) and fluorescence in situ hybridization (FISH) showed a JAK2 gene rearrangement in 88% of interphase nuclei. The BCR-JAK2 fusion was confirmed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and demonstrated two in-frame 5'BCR/3'JAK2 transcripts with BCR exon 1 juxtaposed to JAK2 exon 15 and exon 17, respectively. Our results, together with literature review, reveal BCR-JAK2 fusions as oncogenic genetic alterations that are associated with myeloid or lymphoid neoplasms and are frequently characterized by eosinophilia. Further, patients with BCR-JAK2 are candidates for JAK2 inhibitor therapy. Given the distinct clinical and pathological characteristics, we believe that hematological neoplasms harboring BCR-JAK2 should be included as an additional distinct entity to the current WHO category of "myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR", and testing for a JAK2 fusion should be pursued in neoplasms with a karyotypic 9p24 abnormality.

Coexistence of follicular lymphoma and an unclassifiable myeloproliferative neoplasm in a treatment-naïve patient: A case report.

Myeloproliferative neoplasms are associated with lymphoproliferative diseases following the administration of cytotoxic drugs or exposure to radiation, but are rare prior to therapy. The present study reports the case of a 61-year-old female with a history of transient ischemic attack. The patient, who presented with a palpable mass in the epitrochlear area of the left arm, was simultaneously diagnosed with follicular lymphoma and an unclassifiable myeloproliferative neoplasm. Excisional lymph node biopsy revealed stage I follicular lymphoma (grade 1). Laboratory findings demonstrated leukocytosis, erythrocytosis, thrombocytosis and decreased erythropoietin. Biopsy of the bone marrow revealed hypercellularity, with predominance of erythroid cells, and large polylobated megakaryocytes with increased mitotic figures, but no evidence of lymphomatous infiltration. The janus kinase 2 V617F mutation was also detected in the cells derived from the bone marrow specimen. Following local excision of the lymph node in the left epitrochlear area, radiation was delivered to the involved field, at a dose of 24 Gy in 12 fractions. The patient was started on hydroxyurea (1 g twice per day, orally) 2 weeks subsequent to radiotherapy, and was administered 500 mg twice per day as maintenance therapy. At the six-month follow-up, the white blood cell count, hemoglobin levels and platelet count had reduced, and the patient was in a healthy condition. A computed tomography scan of the neck, chest and abdomen indicated no abnormalities. To the best of our knowledge, the present study is the first case report of follicular lymphoma coexisting with an unclassifiable myeloproliferative neoplasm in a previously healthy patient. Molecular and genetic studies are required to further evaluate this infrequent disease association.

Association of TNF polymorphisms with JAK2 (V617F) myeloproliferative neoplasms in Brazilian patients

The classical chromosome Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of disorders that share clinical, hematological, and histological features. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) are elevated in patients with MPN. The aim of this study was to verify the association between the polymorphisms of TNF gene (-308G/A and -238 G/A) in BCR-ABL-negative MPN in our population. Blood samples obtained from MPN patients were genotyped for the JAK2V617F mutation and both TNF polymorphisms using PCR-RFLP. Thirty three (26.8%) patients with polycythemia vera (PV), 35 (28.7%) essential thrombocythemia (ET), 22 (17.7%) primary myelofibrosis (PMF), and 33 (26.8%) with unclassifiable MPN (MPNu) were included in the study. The JAK2 V617F mutation was detected in 94 (76.42%) patients. Were observed a significant increase on the frequency of the TNF-238 GA genotype in MPN patients compared to controls (OR=2.21, 95% CI=1.02–4.80, P<0.04). The distribution of the genotypes and allelic frequencies of TNF-308 was significantly different among the MPNs, JAK2V617F positive, PV and PMF, and controls. Our data has demonstrated that the polymorphisms on TNF-238 GA, TNF-308 GA were associated to MPN development in this population, triggered by JAK2 V617F mutation.

Acute Monoblastic Leukemia and Myeloproliferative Neoplasm Observed in an Elderly Man Had the Same Clonal Origin

It is reported that fonder mutations affects the development these disorders closely. However, it is unclear whether hematopoiesis carrying these mutations could contribute to the development of the different diseases, directly. We here report a case of acute monoblastic leukemia (AMoL) followed by myeloproliferative neoplasm, unclassifiable (MPN-U), 2 years later, both derived from the same clonal hematopoiesis. [Case report and Methods] A 75-year-old male was admitted to our hospital duo to fever and fatigue. WBC count was 114.7 x 109/L with 9% blasts and 82% monocytes. Bone marrow aspiration revealed 93% of blasts and promonocytes, which resulted in the diagnosis of AMoL with normal karyotype. He received three courses of chemotherapy, which brought CR, but minor monocytosis (>1 x 109/L) persisted without proliferation of immature blasts. After 2 years from the diagnosis of AMoL, platelet count started to increase. WBC count was between 10 and 20 x 109/L without blast, and platelet count reached 500 x 109/L. Bone marrow examination (2 years from the diagnosis of AMoL) revealed normal cellularity, and less than 5% of blast with normal karyotype. Considering the leukocytosis and thrombocytosis, we evaluated his status as similar to MPN, since dysplasia was not apparent on his hematopoietic cells. Although he had previously received chemotherapy for AMoL, it was diagnosed as MPN-U. Two years after the diagnosis of MPN-U, the disease transformed into AML, not AMoL. He died of AML, four years after AMoL developed. To study clonal status of AMoL and MPN-U in this case, bone marrow mononuclear cells of AMoL (at diagnosis) and MPN-U phases, and buccal swab collected during MPN-U phase were subjected to whole exome sequencing (WES). Validation of mutations was performed by amplicon-base deep sequencing. DNA obtained from bone marrow smear during CR was tested whether mutations existed or not by deep sequencing. Mean depth of WES was 89.15, 140.18, and 98.02 for AMoL, MPN-U, and buccal mucosa samples, respectively. Clonal evolution analysis was performed using clonality inference in tumors using phylogeny. [Results] We found total 22 mutations in samples of AMoL and MPN-U phase with different variant allele frequency (VAF) by WES. When AMoL was diagnosed, 16 genes were mutated with more than 10% of VAF, including genes such as ASXL1, CBL, GATA2, NPM1 (4 bp insertion), SRSF2, and TET2. On the other hand, when MPN-U was diagnosed, there were 16 genes mutated with more than 10% of VAF, and 10 mutations out of 16 were the same mutation as found in the sample of AMoL phase including mutations in SRSF2, GATA2, and TET2. Six mutated genes found only in the sample of MPN-U phase included GNAS and JAK2 (V617F mutation). Since 10 out of 22 genes were commonly mutated in both the AMoL and MPN-U samples, we next analyzed the mutational status of these 22 genes in the CR sample using deep sequencing. It was found that the commonly mutated 10 genes in both AMoL and MPN-U were also mutated in the CR sample with high VAF (> 25%). Some gene mutations found only in AMoL, and some only in MPN-U were also present during CR phase but with low VAF (< 10%). Clonal evolution pathway generated by using deep sequence data suggested that before AMoL, there was a founder clone with mutations of TET2, SRSF2, and GATA2, although we could not analyze the sample before AMoL. Mutations in NPM1, ASXL1, CBL, and ASH1L seemed to contribute to the development of AMoL from the founder clone, and those of JAK2 and GNAS for MPN-U. It is also suggested that small amount of hematopoietic stem / progenitor cells containing the JAK2 mutation already existed in CR sample, and these developed into MPN-U probably with gaining other gene mutations. Our data prospectively showed that clonal hematological disorder would arise from a pre-malignant, clonal hematopoiesis that produced normal levels of mature hematopoietic cells. Our analysis clearly demonstrated that two different clonal hematological disorders developed from the same clonal hematopoiesis that had TET2, SRSF2, and GATA2 mutation, which were previously reported as driver mutations. To our knowledge, this is the first report to analyze the progression of AML and MPN from the identical hematopoietic stem cells by WES and deep sequencing validation to model clonal evolution and a case to give a new suggestion about the development of the myeloid malignancies. DisclosuresMiyazaki:Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Moriuchi:Celgene: Other: Research Funding to my institution; travel, accommodations, expenses, Speakers Bureau.

A Population-Based Study of Incidence of Myeloproliferative Neoplasms in Sweden Between 2000 and 2012

BackgroundThe Myeloproliferative neoplasms (MPNs) consists of the subtypes polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPN unclassifiable (MPN-U). The incidence rates of these diseases vary substantially between different reports, ranging from 1.15 to 4.99/100,000 person-years. However, in a recent metaanalysis, there was no significant difference in MPN incidence between Europe and North America and the variations in incidence may therefore reflect the quality of the cancer registers and reporting of MPNs. In addition, there is a limited number of reports on MPN incidence during more recent years. Therefore, we assessed the incidence of MPN based on the Swedish Cancer Register, a high-quality population-based cancer register between 2000 and 2012.Patients and MethodsThe Swedish Cancer Register was used to identify all patients diagnosed with an MPN between January 1st 2000 and December 31st 2012. These Swedish Cancer Registers have very high levels of quality and completeness. Between 2008 and 2012, the reporting of newly diagnosed MPN to the cancer register was >92%. Information on the Swedish population was obtained from the Human Mortality Database (www.mortality.org). Based on information from these registers, incidence rates of MPNs with 95% confidence intervals (CIs) were calculated. Confidence intervals were estimated on the log scale. In addition, the incidence rate in relation to MPN subtype, age group (18-39, 40-49, 50-59, 60-69, 70-79, and ³80 years), as well as calendar year of diagnosis was assessed.ResultsA total of 5,442 MPN patients were reported to the cancer register between 2000 and 2012. During these years, there were 1,810 incident cases of PV, 1,862 of ET, 636 of PMF, and 1,134 with MPN-U. Between January 1st 2000 and December 31st 2012, the population in Sweden increased from 8,861,426 to 9,555,893 inhabitants.The overall annual incidence rate of MPN was 5.83 (95% CI 5.68-5.99)/100,000 persons. The incidence rate of PV was 1.94 (1.85-2.03), ET 2.00 (1.91-2.09), PMF 0.68 (0.63-0.74), and MPN-U 1.22 (1.15-1.29) per 100,000 person-years. In addition, there was a strong correlation between age and incidence of MPN with incidence rates being substantially higher among the older age groups (Table).The overall incidence rate of MPNs increased during the study period, from 5.06 (4.55-5.62)/100,000 person-years in the year 2000 to 5.98 (5.45-6.55)/100,000 person-years in 2012. The incidence rate of PV was similar throughout the study period, the incidence was 2.05 (1.74-2.42)/100,000 person-years in 2000 and 2.12 (1.81-2.47)/100,000 person-years in 2012. The annual incidence rate of ET and PMF increased, from 1.62 (1.34-1.95) to 2.49 (2.15-2.87) per 100,000 persons for ET and from 0.36 (0.24-0.53) to 0.86 (0.67-1.10) per 100,000 persons for PMF between 2000 and 2012. Conversely, the incidence of MPN-U decreased, 1.03 (0.81-1.29) to 0.52 (0.38-0.71)/100,000 person-years between 2000 and 2012.Summary and ConclusionsIn this large population-based study, the incidence of MPN was higher than previously reported in both European and North American studies. As earlier lower incidence rates likely are an effect of limited coverage of cancer registers, there may be an underreporting of MPNs in many European and American countries. The increase in MPN incidence rates during the study period may reflect increasing life expectancy of the Swedish population, improved reporting to the cancer register as well as changes in the classification and diagnostic systems. Similarly, the decrease in incidence of MPN-U is also likely a result of improved diagnostics during more recent years. In conclusion, the MPN incidences rates reported here are presumably more accurate compared to earlier reports due to the high level of coverage and accuracy of the Swedish registers.Table 1Incidence rates of MPNs overall and in relation to subtype and age at diagnosisTotal number MPN diagnosed 2000-2012Incidence/100 000 person-years (95% confidence interval)All MPN5,4425.83 (5.68-5.99)SubtypePV1,8101.94 (1.85-2.03)ET1,8622.00 (1.91-2.09)PMF6360.68 (0.63-0.74)MPN-U1,1341.22 (1.15-1.29)Age at diagnosis (years)18-392260.67 (0.59-0.76)40-493612.26 (2.04-2.51)50-597694.92 (4.58-5.28)60-691,2289.54 (9.02-10.1)70-791,68018.99 (18.1-19.9)>801,17818.92 (17.87-20.03) DisclosuresLandgren:BMJ Publishing: Honoraria; Bristol-Myers Squibb: Honoraria; Medscape: Honoraria; Onyx: Honoraria; Celgene: Honoraria; International Myeloma Foundation: Research Funding; Medscape: Consultancy; BMJ Publishing: Consultancy; Onyx: Research Funding; Bristol-Myers Squibb: Consultancy; Onyx: Consultancy; Celgene: Consultancy.

Are Chronic Myeloproliferative Neoplasms Associated with Age-Related Macular Degeneration?

Background. Patients with Chronic Myeloproliferative Neoplasms (MPNs) already have increased comorbidity at time of diagnosis, and studies show association with ophthalmic manifestations. Retinal vascular symptoms including vascular occlusions and hemorrhages are present, but other manifestations of the eye have not been thoroughly investigated in these patients. Previously reported studies show signs of systemic inflammation in patients with MPN as well as in patients with Age-Related Macular Degeneration (AMD). Our hypothesis is that the presence of MPN predisposes some individuals to develop AMD and this might be explained by the degree of systemic inflammation.Objective. To describe the prevalence of Age-Related Macular Degeneration in patients with Chronic Myeloproliferative cancer at time of diagnosis compared to the general population in Denmark.Materials and Methods. We conducted a retrospective population-based matched cohort study using Danish registries. We included all patients age 18+ or older with a first listed diagnosis of MPN in the Danish National Patient Registry between 1994 and 2013. Patients with Essential Thrombocythemia (ET), Polycythemia Vera (PV), Myelofibrosis (MF), Unclassifiable MPN (MPN-U) and Chronic Myeloid Leukemia (CML) were included. To compare the prevalence of AMD with the general population we identified 10 sex-and-age matched individuals without MPN, for each corresponding patient. The controls were identified through the Danish Civil Registration System. Index date was defined as date of MPN diagnosis, and controls had to be alive at their corresponding patient's index date. We searched for all primary AMD diagnoses in the Danish National Patient Registry within a ten-year period preceding index date + 30 days. For all patients and controls, baseline characteristics, including smoking-related conditions (yes/no), were registered. We calculated number of events in all groups, including only patients' and controls' first AMD diagnosis. Prevalence of AMD at time of diagnosis was calculated using descriptive statistics.Results. We included 9679 patients (ET=2714; PV=3170; MF=600; MPN-U=1839; CML=1356) and a total of 96737 sex-and-age matched controls in the study. Mean age of included patients with MPN at time of diagnosis was: 64 years (ET); 66 (PV); 71 (MF); 70 (MPN-U) and 61 (CML) − all with comparable age-distribution in the matched control groups. There was a higher percentage of females in the ET group (65%), an equal sex-distribution in PV (females 49 %) and MPN-U (females 51 %) and a higher percentage of men in the CML group (59 %). Significant more patients with ET, PV and MPN-U had smoking related diagnoses compared to controls (p<0.05), but no differences were seen for patients with CML and MF.We found 220 MPN patients and 6 controls, who had AMD at time of diagnosis. Patients in all subgroups had a higher prevalence of AMD with 64 ET patients vs. 3 controls; 74 PV patients vs. 2 controls; 16 MF patients vs. 0 controls; 51 MPN-U patients vs. 1 control and 15 CML patients vs. 0 controls. Our results show that 2.3 % of all patients with MPN were diagnosed with AMD, including 2.6 % of Philadelphia-negative patients (ET, PV, MF and MPN-U) and 1.1 % of Philadelphia-positive CML patients. The corresponding values for controls with AMD diagnoses were: 0.006 % (Philadelphia-negative controls 0.007 % and CML controls 0 %). In summary, we found that the patients with MPN had a higher prevalence of AMD, at time of diagnosis, compared to the general population in Denmark.Conclusion. Chronic Myeloproliferative cancers are associated with Age-Related Macular Degeneration at time of diagnosis. Patients with all MPN subtypes have an increased prevalence of AMD compared to the general population. Our results show the need for further studies to establish if the association is present, not only at time of diagnosis, but also during the course of the cancer diseases. We are currently analyzing incidence rates after diagnosis, and influence of potential confounders and effect modifiers. Our preliminary results suggest that the association between AMD and MPN is also present after diagnosis. Clinical studies are needed to investigate whether the association represents a causal relationship or if confounders are accountable for the findings. DisclosuresHasselbalch:Novartis: Research Funding.

Decreased Activity of NK Cells in Myeloproliferative Neoplasms

Natural killer (NK) cells are a subset of lymphocytes that play a critical role in both innate and adaptive immune responses and provide defense against microbial infections and malignant transformation. When stimulated through their activating or cytokine receptors, NK cells rapidly produce cytokines and chemokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of hematopoietic stem cells (HSC)s. We hypothesized that NK-mediated cytokine production could regulate HSC function and that its deregulation may favor HSC malignant transformation and proliferation. In this regard, a few studies have shown that NK cells are defective in stem cell-derived diseases such as chronic myeloid leukemia (CML), for example, raising the hypothesis that immunological dysfunctions may be involved in initiating or contributing to the pathogenesis of myeloproliferative diseases. In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. Therefore, we aimed to study NK cell activity in murine and human primary cells from MPN. NK cells and receptors were quantified by flow cytometry by the use of spleen cells from a conditional knockin Jak2V617F (Jak2VF) murine model, that faithfully resemble the main clinical and laboratorial characteristics of human Polycytemia Vera (PV), and from MPN patients diagnosed and followed at the Clinical Hospital of the Medical School of Ribeirao Preto, University of Sao Paulo. For immunophenotyping of murine lymphocytes, spleen cells were isolated and stained with fluorescence-conjugated antibodies against murine Ter119, CD19, CD4, CD8, CD3, NK1.1 (for NK cells quantification) and NKG2A, NKG2D, Ly49H and Ly49D (for receptors analysis). For analysis of human lymphocytes, peripheral blood mononuclear cells were purified by density gradient centrifugation, resuspended in serum-enriched phosphate buffered saline and stained with fluorescence-labeled anti-human antibodies against the following molecules: CD45, CD19, CD3, CD16, CD56. All the stainings were performed for 20 minutes at 40 C and cells were acquired using a FACScalibur flow cytometer (BD Biosciences). Data was analyzed by the use of the Flojo software version X and the statistical analyses were performed by the use of SPSS version 20. When compared to Jak2WT controls, Jak2VF mice had lower numbers of NK cells (2.09% versus 22.7% of the spleen cells, P = 0.022) and NKG2A (0.57% versus 2.47% of NK cells, P = 0.037) and NKG2D (0.17% versus 0.46% of NK cells, P = 0.020) receptors. For the study of human NK cells, 38 patients diagnosed with MPN and who were not in use of cytoreductive therapy were included and classified according to the WHO 2008 criteria as Polycytemia Vera (PV, n = 11), Essential Thrombocytemia (ET, n = 11), Primary Myelofibrosis (PMF, n = 11) or Unclassified MPN (uMPN, n = 5). Blood samples from 34 healthy donors served as controls. JAK2V617F (JAK2VF) mutated patients had lower numbers of CD16+ CD56dim NK cells as compared to JAK2VF negative and healthy controls (0.73% versus 1.06% versus 1.54%, P = 0.038, respectively). Among the JAK2 mutated patients, those with PV and PMF presented lower numbers of NK cells than ET patients. In agreement, NK cells were reduced in JAK2VF PV patients when compared to JAK2VF negative and controls (0.39% versus 1.42% and 1.54%, P = 0.047). PMF patients had lower NK cell numbers than controls, regardless of JAK2VF mutation (0.35%, 0.49% and 1.54% for JAK2VF positive, negative and controls, respectively, P = 0.025). No significant differences were observed in ET or uMPN. In summary, NK cells were reduced among lymphocytes in MPN patients, especially in the JAK2VF mutated cases, as shown in PV patients and Jak2VF mice. In addition, the expression of NK receptors was reduced in Jak2VF mice, thus suggesting that these cells are defective in MPN. New insights into the pathogenesis of this group of diseases are of great relevance considering that MPN treatment remains non-curative with the strategies available so far despite the development of drugs that inhibit JAK2. Thus, identifying the mechanisms of NK-cell deficiency in MPN and the detailed interaction between these cells and the malignant stem cell may help to establish combination therapeutic strategies that may improve therapy responses in MPN. DisclosuresNo relevant conflicts of interest to declare.

Mutational Analysis of SH2B3 in Korean Patients With BCR-ABL1 Negative Myeloproliferative Neoplasm

Dear Editor, Src homology 2 B3 (SH2B3), previously named LNK, has been reported as a new genetic abnormality in BCR-ABL negative myeloproliferative neoplasms (MPN) [1,2,3]. In western patients, the mutational hot spot of SH2B3 is located in exon 2, within a pleckstrin homology (PH) domain. However, SH2B3 mutations in Korean MPN patients can occur in several other regions of the SH2B3 gene, including exons 7 and 8 [4]. In this study, we performed sequencing analyses of SH2B3 in Korean patients with BCR-ABL1 negative MPN and compared the results with previous studies. In total, 75 patients were enrolled in the study, comprising 32 patients with essential thrombocytosis (ET), 25 patients with polycythemia vera (PV), 10 patients with primary myelofibrosis (PMF), and eight patients with unclassifiable MPN. All patients were diagnosed between June 2007 and March 2012 at Pusan National University Hospital in Busan, Korea. The patients comprised 40 males and 35 females with a median age of 57.3 yr. This research was reviewed and approved by full committee review of the Institutional Review Board at Pusan National University Yangsan Hospital (No. 05-2014-058). We designed the primers and performed the mutation analyses by direct sequencing of the following loci: SH2B3 exons 2, 7, and 8; Janus kinase 2 (JAK2) exon 12 and V617F mutation; and casitas B-lineage lymphoma proto-oncogene (CBL) exons 8 and 9. Alterations in the CBL gene have been identified in AML, MPN, and chronic myelomonocytic leukemia patients [5]. We identified two different SH2B3 mutations (2.7%) in exon 8 (Fig. 1 and Table 1). A novel p.Q571* (c.1711C>T) mutation which results in a premature stop codon and the known p.I568T (c.1703T>C) missense mutation were identified. In addition, two patients have a known polymorphism, p.A356T (c.1606G>A) [4]. The JAK2 V617F mutation was detected in 48 of 75 patients (64.0%); however, no mutation of JAK2 exon 12 or CBL exons 8 or 9 was identified. One of the two patients with a SH2B3 mutation also harbored a JAK2 V617F mutation (Table 1). Fig. 1 Nucleotide sequence and chromatogram of SH2B3 mutations in Korean patients with BCR-ABL1 negative myeloproliferative neoplasm. (A) Nonsense mutation (c.1711C>T; p.Q571*), (B) missense mutation (c.1703T>C; p.I568T). Table 1 The characteristics of two patients with SH2B3 mutations The mutations in SH2B3 were described in approximately 6.1-25.0% of patients with chronic phase MPN in western countries [1,2,3,6]. In this study, the frequency of SH2B3 mutation was 2.7%, and the mutations were found only in exon 8. Recently, Ha et al. [4] demonstrated that mutational frequency of the SH2B3 gene was 7.1% in exons 7 and 8 in 42 Korean patients with chronic phase MPNs. They reported that three types of SH2B3 mutation accompanied by JAK2 V617F mutation, including p.Q423* located on exon 7, and p.R551W and p.I568T located on exon 8; the p.I568T mutation was also detected in this study. Including this data, mutations of SH2B3 are discovered in exon 7 and 8 in the Korean population, and no mutation was identified in exon 2 of SH2B3 gene. The SH2B3 is known to bind JAK2 and perform a critical role in negative regulation of downstream signal transduction [7]. Pardanani et al. reported the co-occurrence frequency of SH2B3 mutations and the JAK2 V617F mutation and found that SH2B3 mutations occur at similar frequencies in both JAK2 V617F negative and positive patients [6]. The effect of co-occurrence of SH2B3 and JAK2 V671F is still unknown, but an animal model study suggested that a more severe phenotype may result in cases with both mutations [8]. In this study, there were no significant differences in prognosis in patients with SH2B3 mutation according to the presence of JAK2 V617F mutation (Table 1). Therefore, the clinical significance might be investigated in a large-scale study. This study has some limitations, such as the small investigation size and the low detection sensitivity of the direct sequencing method. In addition, since we did not search the entire coding region, there is a possibility of SH2B3 mutations in other regions outside exons 2, 7, and 8. For further study of SH2B3, mutational analysis of either the entire coding region or additional exons needs to be considered. In conclusion, racial differences can cause variances not only in the prevalence but also in the mutational hot spot region of SH2B3. Our study suggests that SH2B3 mutations occur infrequently, and exon 8 in SH2B3 may be the most frequent mutational area in BCR-ABL negative MPN patients in Korea.

Current problems in the diagnosis of Philadelphia-negative myeloproliferative neoplasms in Japan

To investigate the current situation and issues regarding the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPN) in Japan, we retrospectively analyzed an accumulated cohort consisting of 1,081 patients with suspected MPN. Based on WHO2008 diagnostic criteria, we diagnosed 101 of these patients with polycythemia vera, 179 with essential thrombocythemia, 36 with primary myelofibrosis, 45 with unclassifiable MPN, and 4 with myelodysplastic syndromes. Out of 716 patients, 235 were not diagnosed with MPN despite the detection of a JAK2, CALR, or MPL mutation. Among 156 patients with undefined MPN receiving further follow-up, none underwent bone marrow examination and screening for BCR-ABL1 was not performed in 88 cases. Thus, diagnosis was not possible in these cases due to a lack of essential examinations. Since the prognosis and treatment strategy associated with MPN differ among disease types, in addition to mutation analysis, the importance of bone marrow examination and screening for BCR-ABL1 must be re-recognized.

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN.

To Treat or Not to Treat: A Novel Stratification of Myelofibrosis Patients by Likelihood of Response to JAK2 Inhibitors

Objectives: It has been suggested in recent publications that there is a higher incidence of lymphoid malignancies (LM) in patients with myeloproliferative neoplasms (MPN). We report the prevalence of LM in patients with MPN seen at our institution, their clinical characteristics and outcome. Methods: We performed a retrospective chart review of all pts with MPN evaluated at MD Anderson Cancer Center between 1966 and 2013 (N1⁄42647) for evidence of LM. In identified patients with both MPN and LM, we recorded the pathologically-confirmed diagnosis of LM, and analyzed their characteristics, treatment received, and overall outcome, using descriptive statistics. Results: A total of 24 patients with history of both MPN and LM were identified: half were male, 79% white. In 15 patients LM was diagnosed before MPN, with median of 74 months between the two diagnoses (range 2-215), and in 9 LM was diagnosed after MPN, with a median time between the two diagnoses of 97 months (range 2-438). Half of the patients are currently alive, with median follow up of 45 months from initial visit (0-149), 50 months (0-272) from diagnosis of first malignancy. Most common MPN diagnosis among 8 represented subtypes was myelofibrosis (MF) (11 patients, 45%); however, this reflected predominance of MF among our MPN patients (47% of all MPNs). Other types included essential thrombocythemia, polycythemia Vera, mastocytosis, hyper eosinophilic syndrome, Phchronic myeloid leukemia, MPN/myelodysplasic syndrome, and unclassifiable MPN. MPN disease characteristics included: splenomegaly in 42%, abnormal karyotype in 29%, JAK2 mutation in 38%, transformation to acute leukemia in one patient. History of LM did not affect the overall outcome of MPN. LM included 17 lymphoma, 4 myeloma, and 3 Hodgkin’s disease patients that were equally distributed among 8 differentMPN subtypes. Twenty one patients received standard therapy for their type of LM (3unknown) and achieved expectedoverall response; presence orhistory ofMPN did not influence choice of therapy or outcome.Conclusions: In our experience, presence of LM in patients with MPN is very rare event. Presence or history of MPN should not affect the choice of therapy for LM, since it does not have an influence on the outcome.

Molecular characterization of atypical chronic myeloid leukemia and chronic neutrophilic leukemia

Background and objectiveAtypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) display similar clinical and haematological characteristics. The objective of the present study was to determine the mutational status of SETBP1 and CSF3R in these diseases. Patients and methodThe mutational status of SETBP1 and CSF3R was studied in 7 patients with aCML (n=3), CNL (n=1) and unclassifiable myeloproliferative neoplasms (MPN-u) (n=3). Additionally, mutations in ASXL1, SRSF2, IDH1/2, DNMT3A, and RUNX1 were also analysed. ResultsSETBP1 mutations (G870S and G872R) were detected in 2 patients with MPN-u, and one of them also presented mutations in SRSF2 (P95H) and ASXL1 (E635fs). The CNL case showed mutations in CSFR3 (T618I), SETBP1 (G870S) and SRSF2 (P95H). No patient classified as aCML had mutations in SETBP1 or CSF3R. One of the patients with mutations evolved to acute myeloid leukemia, while the other 2 had disease progression without transformation to overt leukemia. ConclusionThe knowledge of the molecular alterations involved in these rare diseases is useful in the diagnosis and may have an impact on both prognosis and therapy.

Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (P = 0.006); as well as cases with an abnormal karyotype (P = 0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (P = 0.001). MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy.