Negative MPN Research Articles

JAK2/STAT5 Pathway Mutation Frequencies in South African BCR/ABL Negative MPN Patients.

Mutations in JAK2/STAT5 proliferation pathway genes are key in the diagnosis of myeloproliferative neoplasms (MPNBCR/ABLneg), with JAK2V617F being found in 50-97% of MPNBCR/ABLneg subtypes. Low JAK2V617F positivity at our facility suggested that our South African MPNBCR/ABLneg population may have a different mutational landscape. We aimed to determine the JAK2/STAT5 mutation frequencies associated with our local MPNBCR/ABLneg population, thus determining the relevance of these molecular tests in this group. We also investigated the haematopathological relevance of each test request, to assess testing practises. This study involved the retrospective audit of 886 patients for whom JAK2V617F mutation testing had been requested for a suspected MPN diagnosis. FBC indices, erythropoietin levels and bone marrow biopsy results were used to classify the patients. JAK2V617F negative patient DNA was tested for calreticulin (CALR) exon9, myeloproliferative leukaemia protein (MPL) codon515 and JAK2 exon12 mutations. Only 23% of the patients demonstrated JAK2V617F positivity, with an additional 29 cases of CALR/MPL mutations being detected. Mutations were only detected in patients with abnormal FBC indices, as expected, yet 37% of the test requests were not associated with abnormal parameters at the time of testing. Mutation frequencies were as follows: Polycythaemia Vera: 97% JAK2V617F/3% (JAK2, CALR, MPL) triple negative; Essential thrombocythemia: 72% JAK2V617F/23%CALR/5%triple negative; Primary Myelofibrosis: 78%JAK2V617F/16%CALR/6%triple negative. Our study demonstrated that our MPNBCR/ABLneg patients have a similar genetic landscape to other MPN populations, with >93% being able to be diagnosed by testing for the JAK2V617F and CALR exon9 mutations alone. Adoption of the WHO 2016 guidelines is recommended to guide testing practices.

THE INFLUENCE OF THE JAK2V617F MUTATION ON CLINICAL, LABORATORIAL, AND MORPHOLOGICAL VARIABLES IN BCR-ABL NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

Investigate the correlation between the presence of the JAK2V617F mutation and laboratorial, morphological and clinical findings. This is a retrospective, observational, single-center cohort-study involving adults with classic BCR-ABL negative MPN and myelofibrosis secondary to them (n = 119), in a Brazilian universitary hospital. It were evaluated hematological parameters and nonspecific inflammatory biomarkers such as LDH, CRP, ESR, and ferritin, in peripheral blood, as well as the occurrence of thrombo-hemorrhagic events or death, by consulting medical records. Patients diagnosed with ET (n = 75), and JAK2V617F -positive (n = 69; 92,0%) had significantly higher values of marrow cellularity, hemoglobin, hematocrit, neutrophil to lymphocyte ratio (N/L ratio), ferritin, also higher RBC and WBC counts. Besides a higher occurrence of thrombotic and/or hemorrhagic events (p = 0.006). A positive correlation (Spearman) was observed between the JAK2V617F mutation and thrombo-hemorrhagic events, bone marrow cellularity, RBC count, hemoglobin concentration, hematocrit, WBC count, N/L ratio, and ferritin dosage. Patients with ET positive for JAK2V617F mutation showed a “PV-like” profile with higher values of hemoglobin, hematocrit, hematimetry, LDH, and higher medullary cellularity in addition to, lower platelet values and higher incidence of thrombotic events, as suggested by previous studies. After all, JAK2V617F status is incorporated into the International Prognostic Score for Thrombosis in ET (IPSET). Besides demonstrating the clinical, epidemiological, laboratory profile, and mutational status in Brazilian patients in a university hospital, our study points to the need to reinforce the orientation about certain exams. It is essential to evaluate not only the incorporation of ESR in the routine investigation of patients in whom there is suspicion of classic MPN BCR-ABL negative, especially PV, but also the inclusion of CRP dosage in the prognostic evaluation of patients with ET and PV. There is a correlation between the JAK2V617F mutation and higher values of RBC count, WBC count, N/L ratio, and ferritin, in addition to greater cellularity in the bone marrow. A “PV-like” profile was also observed in JAK2-positive TE patients.

Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

Objective:The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. Methods:The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F–negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. Results:28.3% of JAK2 V617F–negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. Conclusion:Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

THE OUTCOME OF FATHERHOOD IN PATIENTS WITH PHILADELPHIA NEGATIVE MYELOPROLIFERATIVE NEOPLASMS, A SINGLE INSTITUTION EXPERIENCE

The aim of this retrospective study is to evaluate fertility in the Philadelphia-negative MPN male patients and the effect of treatment received on male fertility and the outcome. This is a single-center, mixed-design study (retrospective + phone interviews) conducted within the National Center for Cancer Care and Research. 120 patients were interviewed, only 19 patients (15.7%) had met the inclusion criteria. The majority of patients had lost follow-up or cannot be contacted, and 29.1% of patients had their families completed by the time of diagnosis. The treatment received includes hydroxyurea, interferon, and ruxolitinib. The mode of delivery was normal vaginal delivery in 68% of the pregnancies. The total number of conceptions was 27; three stillbirths were reported. The data showed that most MPN male patients on treatment had their offspring born normally with no delivery complications, no reported congenital anomaly or growth retardation, and no report of MPN-related cancers. Though, further studies with a larger sample size are required to fully understand the effect of medications on the outcome of fatherhood in Philadelphia negative MPN patients.

Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET).
 Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR.
 Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations.
 Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

Genetic factors rather than blast reduction determine outcomes of allogeneic HCT in BCR-ABL–negative MPN in blast phase

AbstractThere is a limited understanding of the clinical and molecular factors associated with outcomes of hematopoietic cell transplantation (HCT) in patients with BCR-ABL–negative myeloproliferative neoplasms in blast phase (MPN-BP). Using the Center for International Blood and Marrow Transplant Research database, we evaluated HCT outcomes in 177 patients with MPN-BP. Ninety-five (54%) had sufficient DNA for targeted next-generation sequencing of 49 genes clinically relevant in hematologic malignancies. At 5 years, overall survival (OS), cumulative incidence of relapse, and nonrelapse mortality of the study cohort was 18%, 61%, and 25%, respectively. In a multivariable model, poor-risk cytogenetics was associated with inferior OS (hazard ratio [HR], 1.71; 95% CI, 1.21-2.41) due to increased relapse (HR, 1.93; 95% CI, 1.32-2.82). Transplants using mobilized peripheral blood (PB) were associated with better OS (HR, 0.60; 95% CI, 0.38-0.96). No difference in outcomes was observed in patients undergoing HCT with PB/BM blasts <5% vs those with active leukemia. Among the 95 patients with molecular data, mutation of TP53, present in 23%, was the only genetic alteration associated with outcomes. In a multivariate model, TP53-mutant patients had inferior OS (HR, 1.99; 95% CI, 1.14-3.49) and increased incidence of relapse (HR, 2.59; 95% CI, 1.41-4.74). There were no differences in the spectrum of gene mutations, number of mutations, or variant allele frequency between patients undergoing HCT with PB/BM blasts <5% vs those with active leukemia. Genetic factors, namely cytogenetic alterations and TP53 mutation status, rather than degree of cytoreduction predict outcomes of HCT in MPN-BP. No meaningful benefit of conventional HCT was observed in patients with MPN-BP and mutated TP53.

Mutant specific anti calreticulin antibody (CAL2) immunohistochemistry as a screening test for calreticulin (CALR) mutation testing.

About 50 different CALR frameshift mutations have been identified in BCR-ABL1 negative MPN, all leading to the development of common new protein C terminus. Antibody targeting this terminal epitope can be useful to identify this driver mutation using immunohistochemistry. CALR mutation analysis was carried out in 51 JAK2V617F negative cases, PMF (n=22) and ET (n=29). PCR followed by fragment analysis was performed for molecular detection of CALR mutation. Bone marrow biopsy specimens of corresponding patients were subjected to IHC using mutation specific antibody CAL2. Staining pattern and intensity were observed. Staining of <2% of background nonmegakaryocytic (non- MK) cells were regarded as Pattern A, while staining of more than 2% of background nonmegakaryocytic (non-MK) was regarded as pattern B. CALR mutation was noted in 40.9% (9/22) and 41.4% (12/29) of JAK2V617F negative PMF and ET, respectively. All CALR mutated cases, irrespective of the mutation type, showed a positive IHC staining in the megakaryocytes with moderate to bright intensity. All CALR wild-type cases were negative on IHC. (Concordance rate- 100%). Pattern A was noted in 40% cases, while pattern B was noted in 60% cases. Pattern A staining had significantly higher chances of having type 1 mutation as compared to pattern B. In contrast, pattern B had a nonsignificant trend toward higher bone marrow cellularity and marrow fibrosis. CAL2 IHC detects all types of CALR mutation. This can act as a sensitive, specific, rapid, and cost-effective screening test for CALR mutation analysis.

Phase II Trial of SGI-110 (Guadecitabine) in Philadelphia Negative Myeloproliferative Neoplasms

Phase II Trial of SGI-110 (Guadecitabine) in Philadelphia Negative Myeloproliferative Neoplasms

Coexistence of Myeloid and Lymphoid Neoplasms: A Single-Center Experience.

The coexistence of a myeloid and a lymphoid neoplasm in the same patient is a rare finding. We retrospectively searched the records of the Hematology Division of the Second Department of Internal Medicine and Research Institute at Attikon University General Hospital of Athens from 2003 to 2018. Nine cases have been identified in a total of 244 BCR-/ABL1- negative MPN and 25 MDS/MPN patients and 1062 LPD patients referred to our institution between 2003 and 2018. Each case is distinct in the diversity of myeloid and lymphoid entities, the chronological occurrence of the two neoplasms, and the patient clinical course. All of them exhibit myeloproliferative (6 JAK2 V617F-positive cases) and lymphoproliferative features, with 1 monoclonal B-cell lymphocytosis (MBL), 3 B-chronic lymphocytic leukemias (B-CLL), 3 B-non-Hodgkin lymphomas (B-NHL), 1 multiple myeloma (MM), and 1 light and heavy deposition disease (LHCDD), while in three cases myelodysplasia is also present. The challenges in identifying and dealing with these rare situations in everyday clinical practice are depicted in this article.

JAK2 (V617F) positively regulates PD-L1 mRNA expression via STAT3/5 activation in MNP (PV and ET) patients

Abstract Background Escalated PD-L1 expression is reported in many cancer types initiating an immune escape mechanism. This led to the development of checkpoint inhibitors against PD-1/PD-L1. However, the mechanisms underlying escalated production of PD-L1 in many cancers is not clear yet. Methods In this study, we studied PD-L1 mRNA expression levels in concert to JAK2 (V617F) mutation in a group of 72 MPN patients (38 PV and 33 of ET). Clinical and demographical characters were noted carefully. Results Confirmed MPN patients were screened for JAK2 (V617F) mutation by tetra-primer ARMS-PCR. We next quantified JAK2 (V617F) with ASO-PCR. The patients were also screened for BCR/ABL1 fusion gene transcripts to clarify Ph negative MPN status. The mRNA expression levels of PD-L1, STAT3/5 in all MPN patients were evaluated and it was identified that PD-L1, STAT3 but not STAT5 mRNA levels were significantly high in JAK2 (V617F) patients compared to JAK2 (WT) ones. It was also identified that PD-L1, STAT3/5 mRNA expression was upregulated in MPN patients with more JAK2 (V617F) percentage/allele burden than those MPN patients with less JAK2 (V617F) percentage/allele burden. Finally, we evaluated correlation of JAK2 (V617F) percentage with PD-L1, STAT3/5 mRNA expression levels and discovered that PD-L1 and STAT3 are in strong and direct correlation with JAK2 (V617F) burden while STAT5 was found to be moderately correlated. In addition, we identified strong coexpression of PD-L1 and STAT3 and as expected STAT5 moderately coexisted with PD-L1. Conclusion In summary,this study shows that increased PD-L1 expression accompanies JAK2 (V617F) mutation. The increased expression of PD-L1 may be caused by excessive activation of STAT3/5 which are regulators of PD-L1. The study finds that PD-L1 expression is mainly mediated by JAK2 (V617F) via STAT3 and thatSTAT5 only plays a minor role.The study supports the concept of using PD-L1/STAT3/5 axes as targets for developing checkpoint inhibitors.

Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics

Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations.Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them.Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045).Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs.Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Programmed Cell Death Receptor (PD-1), PD-1 Ligand (PD-L1) Expression in Philadelphia Chromosome Negative Myeloid Neoplasms

Introduction : PD-1 and PD-L1 pathway inhibitor therapy has been extensively explored in the field of oncology treatment recently, however there is no information so far about its usefulness in myeloid neoplasms (MPN). We have previously reported on PD-1 and PD-L1 expression in Philadelphia chromosome negative myeloid neoplasm (MPN), but now that we have accumulated more cases , we wish to report the final results.Materials and Methods: 63 patients with Philadelphia chromosome negative MPN in total were studied which included 16 MF (14 PMF, 2 post-PV-MF, 2 post-ET-MF), 29 ET, 18 PV, and 25 normal volunteer controls.Flow CytometryStudies: Leukocytes were obtained from whole blood after RBC lysis using RBC lysis buffer (Qiagen). To quantify surface bound PD-1 and PDL-1, the leukocytes were incubated with CD279-APC and CD274-PE, as well as CD4-FITC, CD8-FITC, CD14-PCP, or CD34-FITC (BD Biosciences) and were subjected to flow cytometric analysis.Results: PD-1 and PD-L1 positive cells were expressed as a percentage in gated CD4bright, CD8+, CD14+, or CD34+ cells. We found that 1) there was no difference in PD-1 or PD-L1 levels among different groups of MPN, but there was a significant difference when they were grouped of PV, ET and MF as MPN and compared with controls in all the immune cells including CD4+, CD8+, CD14 + and,CD34+ cells.(Representative of CD4+ shown inFig(1a). 2) High expression levels (more than 50% cell positivity) was found on the CD34+ cells with PD-1 and PD-L1 expression of 20% and 26% in MPN patients respectively ( Fig 1b,1c) . 3) We also found that there was no correlation between of the PD-1 or , PD-L1 expression levels with clinical features including such as WBC counts , platelet counts, and hemoglobin levels, , or presence or absence of the JAK2 , MPL, or CALR gene mutations or splenomegalyConclusion: We have shown elevated increased expression levels of PD1 and PDL-1 on the immune cells and high expression ( more than 50%) in the CD34+ cells , suggest that PD-1 or , PD-L1 pathway inhibitor therapy maybe worthwhile in Philadelphia chromosome negative Ph(-) MPN .. [Display omitted] DisclosuresWang:celgene: Other: clinical trial.

Monocytic Myeloid-Derived Suppressor Cells (Mo-MDSC) Are Increased but Not M1 or M2 TAM Macrophages in the Bone Marrow of Patients with BCR-ABL Negative Myeloid Neoplasm (MPN)

Abstract Introduction. Evidences indicate that megakaryocyte/platelet lineage is crucial in BCR-ABL negative MPN, especially in myelofibrosis (MF) induction. Monocyte/Macrophage lineage were also reported to play a significant role . These Monocyte /Macrophage studies were on the blood monocytes such as have increased the intermediate forms of monocytes (CD14+ CD16+) and Substance P in MF , but in the bone marrow these cell lineages were not studied. Tumor-associated macrophages (TAM) have been classified into two subsets. as the M1 subset (CD86+ ) which produces Th1 cytokines such as interferon (IFN)-γ and pro-inflammatory cytokines. They are involved in the host defense against different pathogens and play a role in the anti-tumor immunity. The M2 subset, that secretes IL-10 and other iimmunosuppresive cytokines and are identified as CD206+ (in vitro induced by IL4) or CD163+(IL10). These M2 macrophages are associated with tumor progression and metastasis. Myeloid suppressor cells (MDSC ) also were found abundantly in the tumor matrix. We previously reported an increase in MDSC in the peripheral blood of BCR-ABL(-) MPN, therefore, these cells were also studied. Material and Methods: Patient and sample preparation: Peripheral blood, bone marrow (BM) aspirate and biopsy were obtained and used in isolation for mononuclear cells (MNCs). 11 MPN patients consisting of 5 ET, 1 PV , 2 PMF, 1 ET-MF, and 2 PV-MF were studied along with 5 controls ,including 4 questionable spike on protein electrophoresis with normal bone marrow exminations and one with leukemoid reaction. Flow cytometric quantification of monocytes, TAMs and MDSCs MNCs from peripheral blood or BM (aspirates and biopsy) were incubated (1X106/ml) with fluorophore-conjugated monoclonal antibodies against the following markers (CD14, HLA-DR, CD86, CD163, and CD206; BD Bioscience, Inc.) The raw flow data were analyzed using FlowJo (v 7.6.2).Thecell populations were gated and identified with the following immunophenotypes: CD14+HLA-DR-/low as the Mo-MDSCs , CD14+ CD86+as M1 macrophage, and CD14+ CD206+ or CD14+CD163+ as M2 TAMs . TAM or Mo-MDSC cells were presented in MFIs, as well as their percentages of total gated MNCs. Results: 1) Mo-MDSC were significantly elevated in MPN compared to that of controls both in the bone marrow aspirates and in the biopsy specimens (Fig1); 2) No difference was identified in M1 macrophages between MPN and controls in the percentage of total gated cells or by the MFI (data,not shown); 3) No difference was noted in M2 macrophages between MPN and controls in the percentage of total gated cells (Fig 2a) or by the MFI (Fig 2b). Conclusions: Mo-MDSC are increased in the bone marrow aspirates and biopsy specimens of patients with BCR-ABL(-) MPN. No difference was noted in the M1 and M2 TAM macrophages between the MPN and control. Download : Download high-res image (102KB) Download : Download full-size image Disclosures Wang: Celgen: Research Funding.

Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T): 2017 update on diagnosis, risk-stratification, and management.

Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). MDS-RS is a lower risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥ 450 × 10(9)/L and large atypical megakaryocytes (similar to BCR-ABL1 negative MPN). Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F, ASXL1, DNMT3A, SETBP1, and TET2 mutations; with ASXL1/SETBP1 mutations adversely impacting survival. Cytogenetic abnormalities are uncommon in both diseases. Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-International Prognostic Scoring System (R-IPSS). Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia. Both diseases have a low risk of leukemic TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain.

Refractory anemia with ring sideroblasts and RARS with thrombocytosis.

Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). RARS is a lower risk myelodysplastic syndrome (MDS) with dysplasia limited to the erythroid lineage, <5% bone marrow (BM) blasts and ≥15% BM RS. RARS-T is a provisional entity in the MDS/MPN (myeloproliferative neoplasm) overlap syndromes, with diagnostic features of RARS, along with a platelet count ≥450 × 10(9)/L and large atypical megakaryocytes similar to those observed in BCR-ABL1 negative MPN. Mutations and Karyotype: Mutations in the SF3B1 gene are seen in ≥80% of patients with RARS and RARS-T, and strongly correlate with the presence of BM RS; RARS-T patients have additional mutations such as, JAK2V617F (∼60%), MPL (<5%), and CALR (<5%). Cytogenetic abnormalities are uncommon in both RARS and RARS-T. Most patients with RARS are stratified into lower risk groups by the International Prognostic Scoring System (IPSS) for MDS and the revised IPSS. Disease outcome in RARS-T is better than that of RARS, but worse than that of essential thrombocytosis. Both RARS and RARS-T have a low risk of leukemic transformation. Anemia and iron overload are complications in both diseases and are managed similar to lower risk MDS. Aspirin therapy is reasonable in RARS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain. Case reports of RARS-T therapy with lenalidomide warrant additional studies.

Therapeutic Strategies in Patients with Atypical CML (aCML) and Unclassified MDS/MPN (MDS/MPN-U). a Single Center Report

Introduction: The WHO defined overlapping MDS/MPN group includes two rare entities: BCR-ABL negative aCML and MDS/MPN-U, defined by at least 3 months persistent WBC >13G/l or platelets >450G/l with at least 1 cytopenia, significant dysplasia (>10%) in at least 1 lineage, no circulating monocytosis (<1G/l or less <10% WBC) and <20% peripheral and/or BM blasts (Wang et al. Blood 2014). aCML is distinguished from MDS/MPN-U by >10% immature circulating precursors. Median overall survival (OS) in aCML and MDS/MPN-U ranges from 12 to 22 months, without any standard treatment clearly defined. We previously found Azacitidine (AZA) to yield responses in BCR-ABL negative MPN including a small subset of MDS/MPN patients (Thépot el al. Blood 2011).Methods: 20 patients, with WHO defined aCML or MDS/MPN-U, were diagnosed in our center from November 2003 to January 2014. Their characteristics and response to treatments administered, including AZA (using IWG 2006 criteria for MDS and evolution of MPN characteristics, such as WBC and splenomegaly (SM) were analyzed.Results: Median age was 73.5 years (range 54-89), M/F 9/11, WHO diagnosis aCML (N=13) and MDS/MPN-U (N=7). Median WBC was 23.4G/l (13.2-96.4), median circulating myeloid precursors were 13.5% (4-66), median ANC was 13.1G/l (3.8-33.8), median Hb level was 10.6g/dl (7.2-14.5) and median platelets 125G/l (27-900). Median BM blast count was 3% (2-12). Five patients (25%) had SM at diagnosis (4 aCML; 1 MDS/MPN-U). Karyotype was normal in 9 and abnormal in 11: (isolated +8 (N=5), del20q (N=3), del5q (N=1), t(3 ;5);+8 (N=1) and tetrasomy 8 (N=1)). Of 14 patients analyzed, only 3 (21.4%) were JAK2-V617F mutated (2 aCML; 1 MDS/MPN-U). Median OS of the entire cohort was 26 months and median OS of aCML and MDS/MPN-U patients was 22 months and 32 months respectively (p=0.71). Time to disease progression (defined as the appearance of marrow blasts >5%, or of a new cytopenia (ANC<1.5G/l; Hb<10g/dl or transfusion dependency, Plt<100G/l) was 24 months. 11 patients (55%) progressed, including 4 to AML. At diagnosis, 8 patients received hydroxyurea (HU), 2 patients AZA because of transfusion dependent anemia and excess of blasts and 10 patients best supportive care only (BSC). At disease progression, 7/11 patients received AZA and 4 other therapies (intensive chemotherapy (IC), n=1, HU alone, n=1, BSC, n=2). HU yielded no significant ORR in the 9 patients treated except for control of WBC. The patient receiving IC had treatment failure. Six of the 9 patients treated with AZA also needed concomitant HU. Median BM blast count at AZA onset was 12% (5 aCML, 3 MDS/MPN-U, 1 AML) and median number of AZA cycles was 6 (3-11) with an ORR according to IWG 2006 criteria of 22.2% (2/9 patients) with 1 CR and 1 mCR+HI-P. In those 2 patients, WBC normalized despite HU discontinuation and a third patient experienced a significant decrease of SM while HU could be also discontinued. In this cohort of aCML and MDS/MPN-U, only 1/7 patients with chromosome 8 abnormalities responded to AZA. Median OS of patients receiving AZA was 12 months. All patients treated with AZA died from disease progression at the closing date of our study.Conclusions: Treatment options for aCML and MDS/MPN-U, other than supportive care and HU are currently mostly limited to investigational agents. The role, if any, of hypomethylating agents, in these diseases, is not established. In this single center cohort, 2 marrow responses (1 CR and 1 mCR+HI-P) with AZA were observed, yielding similar modest ORR as reported for proliferative CMML treated by AZA (Drummond et al. Leukemia 2014). Due to the rarity of these diseases, and their genetic heterogeneity, multicenter prospective trials are needed to assess any potential clinical benefit of hypomethylating agents. DisclosuresNo relevant conflicts of interest to declare.

Targeted High-Throughput Sequencing For The Detection Of Mutations Associated With Myeloproliferative Neoplasms

BackgroundMyeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid cell lineages. Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are the classical types of Philadelphia negative MPN. The JAK2 V617F missense mutation is found in 95% of patients with PV and in 50-60% of patients with PMF or ET. Molecular screening for JAK2 V617F mutation in suspected MPN patients by pyrosequencing has become routine method in diagnostic services. With the introduction of new methods of screening, a growing number of mutations have been described in a range of genes that code for receptor structure proteins (MPL, JAK2 exon 12), intracellular signalling proteins (CBL, SH2B3, NF1, SOCS1/3), proteins involved in leukemic progression (TP53, NRAS/KRAS, NF1, IDH1/2), or epigenetic modifications (EZH2, ASXL1, TET2). Molecular genetic screening for these targets in JAK2 V617F negative cases can be laborious, time-consuming and expensive, but the advent of benchtop-class high-throughput sequencers and custom target enrichment platforms gives the opportunity to test all relevant mutations in multiple genes in a single assay. Aims: To evaluate the Ion Torrent PGM high-throughput sequencer and custom AmpliSeq capture platform for the screening of most relevant MPN associated mutations in JAK2 V617F negative cases and validate candidate mutations using pyrosequencing. Methods32 DNA samples (30 JAK2 V617F negative by pyrosequencing, 1 positive, 1 not tested) from patients with MPN disorders (thrombocytosis, neutrophilia or polycythemia) were screened (with appropriate ethical consent) for mutations using the Ion Torrent PGM sequencing platform. AmpliSeq multiplex primers were used for the target capture and the panel was designed via the online portal (v1.2), targeting 23 regions of 13 genes associated with MPN. The workflow involved library preparation and multiplex sample pooling following qPCR quantification (16 samples per run), emulsion PCR template preparation, Ion 318 chip loading and sequencing. Alignment and variant calling was via the Torrent Server 3.6.2 plugins, using custom coverage and hot-spot files to define the regions of interest. Variant calls were visualised using IGV and UCSC, and functionally annotated using CONDEL, Mutation Assessor and PROVEAN. Results31 of the 32 samples were successfully sequenced, with a mean depth of 1049 reads and the FASTQC plugin indicated good quality sequencing metrics. All JAK2 V617F negative samples were called V617F negative by the PGM. The V671F positive control sample was called at a frequency of 18.1% (19% by pyrosequencing). The untested sample was called positive (10%) for JAK2 V617F and this was validated using pyrosequencing (11%). No MPL or JAK2 exon 12 mutations were detected. Novel SNPs were seen in CBL (Chr11:119149062; G>A; intron 8-9), JAK2 (Chr9:5072649; T>C; intron 13-14), SOCS3 (Chr17:76354148; C>G; 3'UTR) and SH2B3 [(Chr12: 111856076; C>T; p.R43C) and (Chr12: 111856105; T>G; p.H52Q); both in exon 2]. Of these, the SH2B3 variants were flagged as deleterious (H52Q) and highly deleterious (R43C: SIFT=0; PPH2=0.998). Five samples (4 het, 1 hom) had a SNP in IDH1 associated with poor outcome in AML (rs11554137; MAF=0.05) and a further 6 samples had variants flagged as deleterious by at least one of the annotation platforms: one in SH2B3 [p.186I (rs183913232 )] and five in TET2 [2 samples with p.G355D (rs61744960); 2 with p.L34F (rs111948941) and one p.Y867H (COSM327337)]. Additionally, one sample with a TET2 L34F also had a COSMIC annotated JAK2splice site variant (c.1864+14C>T; COSM88203). SummaryThis study shows the ability of the Ion Torrent PGM and AmpliSeq platforms to analyse qualitatively and quantitatively multiple samples for multiple genes with good accuracy and specificity (no false positives for V617F), making this qualitative and quantitative method more time and cost-effective than traditional sequencing techniques. Additionally, several potentially disease-associated variants were detected, which would have been missed by conventional MPN screening strategies and we are proposing to roll this approach out as a diagnostic screening in our MPN clinic in the very near future. Disclosures:Milojkovic:BMS: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Novartis: Honoraria. Apperley:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Pfizer, Ariad: Honoraria (not direct from company), Honoraria (not direct from company) Other.

TET2 Mutations in Ph-Negative-Myeloproliferative Neoplasms: Identification of Three Novel Mutations and Relationship with Clinical and Laboratory Findings

High-throughput DNA sequence analysis was used to screen for TET2 mutations in peripheral blood derived DNA from 97 patients with BCR-ABL-negative-myeloproliferative neoplasms (MPNs). Overall six mutations in the coding region of the gene were identified in 7 patients with an overall mutational frequency of 7.2%. In polycythemia vera patients (n = 25) were identified 2 mutations (8%); in those with essential thrombocythemia (n = 55) 2 mutations (3.6%); in those with unclassifiable MPN (n = 8) 3 mutations (37.5%). No primary myelofibrosis patiens (n = 6) harboured TET2 mutations. Three unreported mutations were identified (p.P177fs, p.C1298del, p.P411del) the first two in patients with unclassifiable MPN, the last in a patient with essential thrombocythemia. On multivariate analysis the diagnosis of an unclassifiable MPN was significantly related to the presence of TET2 mutations (p = 0.02; OR: 2.81; 95% CI 1.11 - 7.06). We conclude that TET2 mutations occur in both JAK2V617F-positive and -negative MPN and are more frequent in MPN-U patients. This could represent the biological link between the different classes of myeloid malignancies.

Design and Application of a Novel PNA Probe for the Detection At Single Cell Level of JAK2V617F Mutation

AbstractAbstract 1761 Background:Mutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative MPN patients and its detection is now a cornerstone in the diagnostic algorithm. The method most frequently used for measuring the distribution of cell populations is based on JAK2 sequencing and Q-PCR. Therefore the chance of distinguishing the JAK2 wild type or mutated population at the single-cell level still represents a challenge.The aim of the study was to developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence. Methods:We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring the mutated (V617F) or the wild type form of JAK2. CD34+ cells were enriched from 24 PV patients (5 of them were selected for the absence of JAK2V617F), 13 PMF (10 with the mutation and 3 JAK2 wild type) and 6 ET patients (2 of them were wild type). In addition 20 BM samples were collected from healthy donors and used as control. Patients were a priori found to be either positive or negative for the JAK2V617F mutation by standard sequencing and by Q-PCR. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the human JAK2 sequence surrounding the nucleotide at position 1849, which is responsible for the V617F substitution (JAK2V617F/PNA). Slides were analyzed by fluorescence confocal microscopy. Results:The analysis revealed that among JAK2V617F PV patients the distribution pattern is fairly similar to that reported by Scott and colleagues in 2006 analyzing JAK2V617F in colonies. We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 40% to 100% in PV patients, from 15% to 80% in ET and from 25% to 100% in PMF. These findings are in agreement with previous data reporting that a variable proportion of progenitors from patients affected by JAK2V617F positive PV are capable of generating JAK2V617F negative colonies. In addition these data indicate that fluorescinated JAK2V617F/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of JAK2V617Fpositive cells collected from 3 JAK2 wild type subjects defined by sequencing and by Q-PCR, we identified a small percentage of cells positive for the JAK2V617F/PNA staining. However, this apply only to patients with PV but not to PMF and ET patients. Quantitatively, this percentage did not exceed 3% of the CD34+ cell population indicating a high level of sensitivity of the procedure since the PNA/FISH technique may detect JAK2V617F-positive cells within a CD34+ population isolated from patients reported as JAK2V617Fnegative by standard approaches. Importantly, the lack of positivity detected in CD34 positive cells from 20 healthy subjects demonstrates a high specificity of this method. Conclusions:JAK2V617F/PNA-FISH method displays high specificity and reliability in discriminating cell subpopulations harbouring the JAK2V617F mutation. In addition, it allows to analyze the CD34+ population at the single cell level, avoiding the time consuming analysis of hematopoietic colonies. In addition, this approach allows to monitor longitudinally the evolution of a defined cell population over time in MPNs and the characterization of the CD34+ compartment in patients with MPNs which still represents a challenging issue. Disclosures:No relevant conflicts of interest to declare.

Prevalence of MPL W515L/K Mutations in Taiwanese Patients With Philadelphia-negative Chronic Myeloproliferative Neoplasms

The discovery of Janus kinase 2 (JAK2)-V617F has provided important insight into the pathogenesis of Philadelphia-negative chronic myeloproliferative neoplasms (Ph-negative MPNs); however, the etiology of JAK2(V617F)-negative Ph-negative MPN remains unidentified. MPL(W515L) and MPL(W515K) (MPL(W515L/K)) are 2 gain-of-function mutations, which have been found in some Ph-negative MPN patients from Western countries. However, little is known about the incidence of these mutations in Taiwanese Ph-negative MPN patients. We determined the MPL sequence of DNA samples from 105 patients, including 88 patients with Ph-negative MPNs and 17 with myelodysplastic syndrome, using polymerase chain reaction amplification of the cytokine receptor MPL exon 10 sequence. All the patients were normal at codon 515 regardless of their JAK2 status. The MPL W515L/K mutations are rare in Taiwanese patients with Ph-negative MPNs.