ALBERT

Description: Background: Calreticulin (CALR) mutations are one of the major driver mutations in BCL-ABL1-negative myeloproliferative neoplasm (MPN) and are frequently detected in JAK2/MPL-unmutated essential thrombocythemia and primary myelofibrosis. Mutant CALR activates JAK-STAT signaling through an MPL-dependent mechanism to mediate pathogenic thrombopoiesis in MPNs. Although JAK inhibitors such as ruxolitinib can provide important clinical benefits to MPN patients including those harboring CALR mutations, JAK inhibition does not preferentially target the MPN clone and acquired resistance develops over time. We aimed to characterize the mechanisms of acquired resistance to JAK inhibitors in CALR-mutated hematopoietic cells and to screen for novel therapeutic approaches specifically target CALR-mutant cells in this study. Methods: UT-7/TPO-derived cell lines expressing wild-type and type 1 and type 2 mutant CALR (CALRdel52 and CALRins5) were kindly provided by Drs. Komatsu and Araki. JAK2-inhibitor-resistant cells were generated by co-cultured with ruxolitinib and fedratinib (TG101348, a JAK2-selective inhibitor). JAK-STAT signaling was evaluated by Western blot on CALR-wild-type and mutated cells exposed to JAK2 inhibitor compared to untreated cells. For the detection of acquired secondary mutations in CALR-mutated cells exposed to JAK2 inhibitor, whole exome sequencing (WES) was performed using the BGISEQ-500 Sequencing platform (BGI, Shenzhen, China) with the 2 x 100 bp paired-end protocol. Genome Analysis Toolkit was used for variation detection. Reads were aligned to human reference genome hg19 using BWA version 0.7.15. Targeted resequencing was performed on leukocytes from patients with MPN who had been treated with ruxolitinib. Screening with chemical libraries/novel compounds will be conducted on UT7/TPO-CALR cell lines. Results: Compared to the parental cells, ruxolitinib-resistant UT7/TPO-CALR mutant cell lines have developed significant cross resistance to other JAK inhibitor as shown in the cell viability study. Signalling downstream of JAK2 in all 3 inhibitor-naïve UT-7/TPO/CALR parental cell lines was inhibited by acute treatment of ruxolitinib as shown on Western blot. Whereas, constitutive JAK2 activation was observed in all 3 inhibitor-resistant UT-7/TPO/CALR cell lines. No change in the expression of Epo and MPL receptors in these cell lines was found. Interestingly, constitutive JAK3 activation was also seen in inhibitor-resistant UT-7/TPO/CALR cells in comparison with parental cells. These findings indicated the presence of transphosphorylation by JAK3 in inhibitor-resistant UT-7/TPO/CALR cell lines. In addition, the results of WES identified several acquired secondary mutations in 3 inhibitor-resistant UT-7/TPO/CALR cell lines including SH2B1, ABCC1, HOXB3 and KRTAP4-5. No acquired secondary mutation was identified in CALR and other genes involved in JAK-STAT signaling. Acquired secondary mutation will be screened in primary MPN patients' samples treated with JAK inhibitor. Type II JAK inhibitor such as BBT-594 has been shown to inhibit JAK activation and signaling in JAK-persistent/resistant cells. Conclusions: Our results confirmed that the in vitro efficacy of JAK2 inhibition on CALR-mutant cells. Our data also suggested that JAK2 transphosphorylation and acquired secondary mutations could be underlying mechanisms for acquired resistance to JAK inhibitors in CALR-mutated cells. Novel therapeutics approaches should be developed to overcome acquired resistance in CALR-mutated cells. Disclosures No relevant conflicts of interest to declare.

Description: Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood. We have reported that activated B cells are increased in patients with essential thrombocythemia, and can facilitate platelet production mediated by cytokines, such as interleukin-1beta (IL-1β) and interleukin-6 (IL-6) regardless JAK2 V617F mutational status (Thromb Haemost. 2014, 112: 537). Recently, Calreticulin (CALR) mutations were discovered in JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. Although CALR mutations may be associated with activated JAK-STAT signaling pathway, its exact molecular pathogenesis remains elusive in MPN. Interestingly, in vitro study has shown that CALR is capable of driving B cells activation through the toll-like receptor 4 (TLR4) pathway (J Immunol.2010; 185: 4561). Here we sought to evaluate the association between CALR mutations and B cell immune profiles in ET patients. Methods: Fifty-four patients diagnosed with ET based on the 2008 WHO classification were enrolled into this study. CALR mutations were screened by high-resolution melting analysis and nucleotide sequencing. JAK2 V617F and MPL mutations were screened by allele-specific PCR and nucleotide sequencing, respectively. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) and CALR levels, B cells TLR4 expression and intracellular levels of IL-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF and plasma CALR concentrations were measured by ELISA. Forty-eight healthy adults and 17 patients with reactive thrombocytosis were used for comparison. The association between clinical, laboratory and molecular characteristics were studied. Statistical significance was defined as a two-sided p value

Description: Abstract 2906 Poster Board II-882 Background: The World Health Organization (WHO) classification system recognizes indolent SM (ISM) as an entity characterized by low systemic mast cell (MC) burden but frequent skin involvement. The WHO system also recognizes 2 provisional ISM sub-variants: smoldering SM (SSM) and bone marrow (BM) mastocytosis (BMM). The two are respectively characterized by increased systemic MC burden (presence of ≥ 2 ‘B-findings') and BM MC infiltration in the absence of skin or multiorgan visceral lesions. The prognostic relevance of this sub-classification remains unclear. Methods: The study population was drawn from a larger cohort of 342 adult SM patients (Blood. 2009 Jun 4;113(23):5727). Clinical data and BM histology were reviewed, and the diagnosis of ISM and its subclassification confirmed per the 2008 WHO proposal. The primary objectives of the study were to: (i) describe the clinical characteristics of a large cohort of ISM patients, within the context of the WHO classification; (ii) evaluate the prevalence of molecular and cytogenetic abnormalities; (iii) evaluate whether ISM subclassification is prognostically relevant on the basis of survival and risk of transformation to acute leukemia or aggressive SM (ASM). Results: (i) Clinical characteristics: 159 ISM patients were evaluated (69 males; median age 49 years, range 19-84); 22 (14%) had SSM, 36 (23%) BMM, and 101 (63%) ‘ISM-other' (ISMo). By definition, ‘B-findings' were absent in BMM patients. ‘B-findings' in SSM (and ISMo) patients was as follows: hepatosplenomegaly and/or lymphadenopathy 91% (21%), BM MC 〉30% or serum tryptase level 〉200ng/mL 68% (8%), and hypercellular BM or dysmyelopoiesis without cytopenias 50% (2%). SSM patients were older (p

Description: Introduction: Essential thrombocythemia (ET) is a classic BCL-ABL1-negative chronic myeloproliferative neoplasm (MPN), and is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. Recently, two research groups discovered a high frequency of somatic calreticulin (CALR) mutations in patients with JAK2 V617F/MPL-unmutated ET and primary myelofibrosis. The pattern of most CALR mutations in MPNs is indels in exon 9 causing one base pair reading frameshift. CALR mutations have been found to have important diagnostic and prognostic significances in patients with ET. High-resolution melting analysis (HRMA) is a well-established method for the detection of or screening for mutations. The aims of this study were to screen for CALR mutations and to determine its clinical correlation in a cohort of adult Taiwanese patients with ET, and to test for the feasibility of HRMA as a screening method for the detection of CALR exon 9 mutations. Methods: The screening for mutations in patients with hematologic neoplasms was approved by the Institutional Review Board of Mackay Memorial Hospital. 92 adult Taiwanese patients with ET were enrolled. Diagnosis of ET was established based on the 2008 WHO criteria. The clinical and laboratory characteristics of all patients at the time of diagnosis or referral were determined retrospectively by chart review. Genomic DNA derived from bone marrow, peripheral whole blood, and peripheral blood granulocytes or peripheral blood mononuclear cells were used for the screening. JAK2 V617F mutational status was screened by allele-specific polymerase chain reaction. CALR exon 9 mutations and MPL exon 10 mutations were screened by nucleotide sequencing on an ABI 3730 sequencer. The correlation between CALR mutational status and clinical characteristics was determined by using SPSS Statistics software (IBM, New York, USA). HRMA was performed with a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). Results: In this cohort of ET patients, 59 (64%) patients harbored the JAK2 V617F mutation and one (1%) patient harbored the MPL W515K mutation. By Sanger sequencing, 17 (18% overall and 50% in JAK2/MPL-unmutated cases) patients harbored 6 types of CALR exon 9 mutations: 5 type 1 (p.L367fs*46), 6 type 2 (p.K385fs*47), 2 type 3 (p.L367fs*48), 2 type 34 (p.K385fs*47), and 2 novel types (p.L367fs*43 and p.E369fs*50). One patient with JAK2 V617F mutation was found to harbor a single nucleotide polymorphism in CALR exon 9 (c.1142 A〉C, rs143880510). One patient with JAK2 V617F mutation also harbored a CALR type 3 mutation (p.L367fs*48), and was excluded from the correlation study. When compared with JAK2 V617F mutated ET patients, the presence of CALR mutations correlated with higher platelet count (median 1379 x10^9/L vs. 880 x10^9/L; P

Description: Background: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm characterized by increased number of mature megakaryocytes in the bone marrow and sustained thrombocytosis in the peripheral blood. ET is associated with an increased risk of hemorrhagic and thrombotic complications and leukemic transformation. In addition to somatic mutations, microRNA (miRNA) deregulation has been proposed to play roles in the molecular pathogenesis and disease phenotype in ET patients. The aims of this study were to investigate the plasma miRNA profiles in ET patients with those obtained from healthy adults (HA) and its correlation with clinical and prognostic features. Methods: ET patients seen at the MacKay Memorial Hospital were enrolled into this study. The clinical and laboratory characteristics at the time of diagnosis or referral were determined retrospectively by chart review. Total plasma miRNA was derived from bone marrow or peripheral blood in patients or HA. Mutational status of JAK2V617F,CALR and MPL were detected by Sanger sequencing and/or allele-specific PCR. Plasma miRNA profiling was carried out on PanelChip™ Analysis System (Quark Biosciences, Taiwan) using a customized miRNA panel including 165 miRNAs called mirSCAN™ PanCancer Chips 1 & 2. KEGG pathways that miRNAs were associated with were analyzed using DIANA TOOLS - mirPath v.3. Differentially expressed miRNAs were identified by student t-test (P 〈 0.05) and significantly affected miRNAs in ET were identified by a change in expression of two-fold or more compared to the expression in HA. Statistical analysis was performed using SPSS. Results: A total of 53 ET patients (median age at diagnosis 59 years; 60.4% females) were enrolled. Frequency of the 3 driver mutations was 64% for JAK2V617F, 15% CALR, 4% JAK2V617F and CALR co-mutations, and none for MPL. 17% of patients were classified as triple-negative. A total of 40 differentially expressed miRNAs were identified (Figure left axis) including 4 miRNAs (hsa-miR-940, hsa-miR-411-5p, hsa-miR-596 and hsa-miR-376c-3p) with higher expression levels. The putative target genes of these 40 differentially expressed miRNAs were enriched in signaling pathway including TGF-beta signaling pathway (p-value = 1.86E-12), Hippo signaling pathway (p-value = 1.84E-07), MAPK signaling pathway (p-value = 0.03), and FoxO signaling pathway (p-value 〈 0.001). According to the expression levels of these 40 miRNAs, samples were grouped into two clusters, i.e. low expression group and high expression group. ET patients with JAK2V617F were significantly associated with high expression of 40 miRNAs (n = 31 of 34, 91%) when compared with triple-negative ET patients (n = 5 of 9, 56%) (p = 0.03). ET patients with low expression of 40 miRNAs were significantly associated with acute leukemia transformation (n = 2 of 11, 18%) when compared with high expression group (n = 0 of 42, 0%) (p = 0.04). However, the expression levels of 40 miRNAs were not statistically correlated with other clinical features including hemogram, secondary solid cancer, myelofibrosis transformation, or thrombotic/hemorrhagic events. Conclusions: In this cohort of ET patients, distinct plasma miRNA profiles correlated with JAK2V617F mutation and leukemic transformation. Larger cohort is warranted to validate our findings. Figure Figure. Disclosures Chen: Quark Biosciences, Inc.: Employment. Kang:Quark Biosciences, Inc.: Employment. Huang:Quark Biosciences, Inc.: Employment. Lin:Quark Biosciences, Inc.: Employment. Wang:Quark Biosciences, Inc.: Employment.

Description: Clinical phenotype in systemic mastocytosis (SM) is markedly variable, which complicates prognostication and decision making regarding the choice and timing of therapy. In a retrospective study of 342 consecutive adult patients with SM seen at the Mayo Clinic between 1976 and 2007, disease subdesignation according to the World Health Organization (WHO) proposal was indolent (ISM) in 159 (46%), with associated clonal hematologic non–mast cell lineage disease (SM-AHNMD) in 138 (40%), aggressive (ASM) in 41 (12%), and mast cell leukemia in 4 (1%). KITD816V was detected in bone marrow–derived DNA by allele-specific polymerase chain reaction (PCR) in 68% of 165 patients evaluated (ISM, 78%; ASM, 82%; SM-AHNMD, 60%; P = .03); JAK2V617F was detected in 4%, all in SM-AHNMD. Compared with those with nonindolent SM, life expectancy in ISM was superior and not significantly different from that of the age- and sex-matched US population. In addition, multivariable analysis identified advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess bone marrow blasts as independent adverse prognostic factors for survival. The current study validates the prognostic relevance of the WHO subclassification of SM and provides additional information of value in terms of both risk stratification and interpretation of clinical presentation and laboratory results.

Description: Background: Current therapy in adult systemic mastocytosis (SM) includes observation alone, topical therapy for cutaneous disease, symptomatic non-cytoreductive therapy and cytoreductive therapy. The latter involves several drugs whose individual merit in the treatment of SM has not been well characterized. Patients and Methods: Diagnosis of SM was according to the 2001 WHO criteria (World Health Organization Classification of Tumours of Hematopoietic and Lymphoid Tissues, p 1–351. Lyon, France: IARC Press; 2001). Response to cytoreductive therapy was assessed by consensus criteria (Eur J Clin Invest.2007;37:435). The study population was selected from a series of 342 adult SM patients referred to our institution and seen between 1964 and 2008. KITD816V mutation analysis was performed by DNA sequencing. Results: A total of 134 study patients received treatment that included at least one cytoreductive drug as either first-line (n=105) or second-line (n=29) therapy. Interferon-alpha with or without prednisone (n=58), hydroxyurea (n=43), imatinib mesylate (n=42) and 2-chlorodeoxyadenosine (n=26) were the most frequently used cytoreductive agents accounting for 120 of the 134 study cases; the remaining 14 patients received other cytoreductive drugs, none of which were effective. i. Interferon-alfa (IFN-a) with or without prednisone IFN-a with or without prednisone (24 and 34 patients, respectively) was given to 12 patients with indolent SM (ISM), 14 aggressive SM (ASM), and 32 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD). For all 58 patients receiving therapy, complete (CR), major (MR), and partial (PR) response/regression were achieved in 3, 7, and 14 patients, respectively, for an overall response rate (ORR) of 41%. ORR in ISM, ASM, and SM-AHNMD was 50%, 43%, and 38%, respectively. Median duration of response was 12 months (range, 1–117 months). Anemia and elevated ESR were significantly associated with inferior ORR; 29% vs. 56% (p=0.04) and 14% vs. 57% (p=0.01), respectively. BM detection of KITD816V did not appear to affect response rates (p=0.4). ii. Hydroxyurea (HU) HU was given to 2 ASM, 40 SM-AHNMD and 1 mast cell leukemia (MCL) patients. The drug was used as first-line therapy in 30 patients. MR and PR were achieved in 1 and 8 SM-AHNMD patients, respectively (ORR=21%). Median duration of response was 37 months (range, 5–84 months). iii. Imatinib mesylate (IM) in patients with SM and associated eosinophilia IM was given to 22 SM patients who had associated eosinophilia (SM-eos): 2 had ISM, 2 ASM, and 18 SM-AHNMD. The drug was used as first-line therapy in 9 patients. Ten of the 22 patients with SM-eos were known to harbor the FIP1L1-PDGFRA fusion mutation and all responded to IM (ORR=100%). None of 5 FIP1L1-PDGFRA-negative SM-eos patients had a response to IM. In the remaining 7 patients with unknown mutation status, 4 patients had a response to IM (1 CR, 1 MR, and 2 PR). iv. IM in patients with SM not associated with eosinophilia IM was administered to 20 SM patients without associated eosinophilia (7 ISM, 2 ASM, 10 SM-AHNMD, and 1 MCL). Response was documented in 4 (20%) patients (1 CR, 1 MR, and 2 PR) including 1 with ISM, 2 with ASM and 1 with SM-AHNMD. Median duration of response was 19.5 months (range, 9–69 months). Two patients developed interstitial pneumonitis. v. 2-Chlorodeoxyadenosine (2-CdA) 2-CdA was given to 10 ISM, 3 ASM, and 13 SM-AHNMD patients; the drug was used as first-line therapy in 8 patients. CR, MR and PR were achieved in 1, 7, and 4 patients, respectively (ORR=46%). ORR in ISM, ASM, and SM-AHNMD was 50%, 33%, and 46%, respectively. Median duration of response was 11 months (range, 3–74 months). Presence of leukocytosis (WBC 〉 10 x 109/L), monocytosis (absolute monocyte count 〉 0.9 x 109/L) or circulating immature myeloid cells was significantly associated with inferior response; 13% vs. 61% (p=0.02); 11% vs. 69% (p=0.006) and 0% vs. 71%, (p=0.001), respectively. Response was not affected by BM detection of KITD816V (p=1.0). Conclusions: Both IFN-a and 2-CdA are reasonable treatment options, with comparable efficacy, for symptomatic SM including the subcategories of ISM, ASM and SM-AHNMD. IFN-a response was significantly better in the absence of anemia or elevated ESR. 2-CdA response was similarly better in the absence of leukocytosis, monocytosis or circulating immature myeloid cells. The therapeutic value of IM was limited to FIP1L1-PDGFRA-positive disease.

Description: Background: Systemic mastocytosis (SM) has a varied presentation ranging from indolent forms with limited morbidity over many years to the rapidly fatal mast cell leukemia (MCL). This heterogeneity complicates clinical decision making regarding choice and timing of therapy. The aim of this study was to evaluate the prognostic value of the 2001 World Health Organization (WHO) classification of SM, and to refine risk stratification within SM sub-groups to facilitate clinical decision making. Information on KITD816V and JAK2V617F mutation analysis and clinical correlates is also provided. Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those patients where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. KITD816V mutation analysis was performed by DNA sequencing. JAK2V617F was screened by allele-specific quantitative PCR analysis. Results: i. Clinical characteristics at presentation: A total of 342 SM patients were identified (154 female; median age at diagnosis=57 years; range 19 to 87 years). Per the 2001 WHO classification, 159 had indolent SM (ISM; median age=49 years), 41 aggressive SM (ASM; median age=65 years), 138 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD; median age=65 years), and 4 MCL (median age=55 years) (p-value for age