Inhibition of Related JAK/STAT Pathways with Molecular Targeted Drugs Shows Strong Synergy with Ruxolitinib in Chronic Myeloproliferative Neoplasms

Abstract Abstract 5054 Objectives: In the present study we have determined the antitumor effects, molecular mechanisms of action and potential synergies between Ruxolitinib and the molecular targeted drugs Sorafenib, KNK437, Dasatinib and Perifosine, in Philadelphia negative chronic myeloproliferative neoplasms (MPN). Materials and methods: The cytotoxic and cytostatic effects of the different compounds were determined in the JAK2V617F positive cell lines HEL and Ba/F3 JAK2V617F EPOR, and in mononuclear and bone marrow CD34 positive cells from 19 MPN patients. The effects of different drugs at the molecular level were analyzed by flow cytometry and western blot. The IC50 and the synergy combination index (CI) were determined with GrafPath Prism software and Calcusyn software respectively. Results: Ruxolitinib (IC50PV=15nM), as well as Sorafenib (IC50PV=8uM), KNK437 (IC50PV=100uM) and Perifosine (IC50PV=15uM), was able to inhibit proliferation in cell line models and in cells from MPN patients. Dasatinib (IC50PV=12nM), however, was only effective in patient samples, with similar effects in both MPN patients and control donors. Moreover, Dasatinib, KNK437 and Sorafenib showed a strong synergistic effect in combination with Ruxolitinib (CIPV<0. 3, table 1). The Western blot analysis confirmed that Sorafenib inhibited the activation of ERK and P38, and, as a consequence, of STAT5. Ruxolitinib blocked the ERK and STAT5 activation, Dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the protein JAK2, reducing its expression. Conclusions: The blockage of JAK2 related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of Ruxolitinib with inhibitors targeted against these pathways has a strong synergistic effect. This may be related to a decrease in the activation of the final common effector STAT5. Disclosures: No relevant conflicts of interest to declare.

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Ph-Negative Chronic Myeloproliferative Neoplasms – Population Analysis, a Single Center 10-years’ Experience

Blood ◽

10.1182/blood.v124.21.5556.5556 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5556-5556

Author(s):

Vasily Shuvaev ◽

Irina Martynkevich ◽

Alla Abdulkadyrova ◽

Vera Udaleva ◽

Tatyana Zamotina ◽

...

Keyword(s):

Overall Survival ◽

Risk Stratification ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Risk Group ◽

Myeloproliferative Neoplasms ◽

Population Analysis ◽

Myelogenous Leukemia ◽

Rank Test ◽

Chronic Myeloproliferative Neoplasms

Abstract Objectives and background. Nowadays chronic myeloproliferative neoplasms (MPN) other than chronic myelogenous leukemia undergo renaissance of interest. It results from advances in decryption of molecular mechanisms of pathogenesis and invention of target drugs. Epidemiological information is needed to assess potential effect and additional costs of new diagnostic and therapeutic techniques. The objective of our study was to review experience of MPN diagnostic and treatment in our center for past ten years. Methods. Our institution serves as primary hematological outpatient department for a half of Saint-Petersburg city with about 2 million inhabitants. We reviewed patients' charts to obtain information about incidence, symptoms, diagnostic test results, treatment options and relationship to prognostic factors. Statistical methods included descriptive statistics, nonparametric ANOVA for frequencies comparisons and Kaplan-Meyer method with log-rank test for survival comparisons in Statistica 7.0 package. Results. Since 2004 to 2013 there were 570 newly diagnosed MPN patients (pts) in our center. This group consisted of primary myelofibrosis (PMF) (203 pts; 126 female, 77 male; median age 63 years, range 16-83 years), essential thrombocythemia (ET) (201 pts; 146 female, 55 male; median age 58 years, range 23-78 years), polycythemia vera (PV) (166 pts; 96 female, 70 male; median age 57 years, range 20-85 years). The incidence rates were stable during study period: PMF incidence varied from 0.65 to 1.35 with mean of 1.01 new patient per 100 000 inhabitants per year; ET had incidence from 0.60 to 2.1 with mean of 1.00 and PV had incidence from 0.5 to 1.15 with mean of 0.83. The most prevalent symptoms of disease were: splenomegaly (65.5%), constitutional symptoms (fever, night sweats, weight loss) (31.0%), anemia (36.3%) thrombosis (24.1%) for PMF; fatigue (33.2%), headache and dizziness (25.6%), arthralgia (21.8%), erythromelalgia (15.8%) for ET; plethora (82.5%), headache and dizziness (52.4%), fatigue (31.3%) for PV. JAK2V617F was detected in 49.7% of PMF pts, 57.8% of ET pts and in 97.7% of PV pts. Thrombosis rates according WHO IPSET-thrombosis system risks` groups of ET and PV pts were: low-risk group 3.33% (3/90), intermediate-risk group 11.1% (13/117) and 39.4% (63/160) in high-risk group with highly significant (p<0.0001) differences between risks' groups. There were 169 lethal outcomes in the analysed group (102 PMF; 31 ET; 36 PV). Ten-years overall survival rates were 49.8% in PMF pts, 84.6% in ET pts and 78.3% in PV pts. (fig.1). Overall survival in PMF was significantly influenced by risk stratification as IPSS, DIPSS and DIPSS+. Survival curves according DIPSS+ groups are presented in fig.1. Conclusions. Patients with MPN are presented in substantial number; therefore need much finance for novel therapy introduction. Risk stratification systems has high predictive value. Innovative drugs treatment results should be evaluated in comparison with historical control. Figure1 Overall survival in PMF patients according to DIPPS+ stratification groups. Figure1. Overall survival in PMF patients according to DIPPS+ stratification groups. Disclosures No relevant conflicts of interest to declare.

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Anlotinib Shows Potent Antileukemia Effects in B-Cell Acute Lymphocytic Leukemia through the Blockage of Angiogenic Related Pathways

Blood ◽

10.1182/blood-2020-141039 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 49-49

Author(s):

Qiuling Chen ◽

Yuelong Jiang ◽

Qinwei Chen ◽

Long Liu ◽

Bing Xu

Keyword(s):

B Cell ◽

Solid Tumors ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Unmet Need ◽

Lymphocytic Leukemia ◽

Advanced Lung Cancer ◽

Antitumor Effects

Acute lymphoblastic leukemia (ALL) derives from the malignant transformation of lymphoid progenitor cells with ~85% being originated from B-cell progenitors (B-ALL). Despite fairly good prognoses for most pediatric B-ALL patients, the outcome is fatal in over 50% of adult patients who have a recurrent or progressive disease and lack of effective therapeutic approaches. Therefore, novel treatment strategies with high efficacy and low toxicity are an unmet need for B-ALL patients, especially those with relapsed or refractory status. Angiogenesis is a process of new vessel formation that requires the participation of multiple proangiogenic factors (e.g., VEGF, PDGF, and FGF) and their corresponding receptors (e.g., VEGFR, PDGFR, and FGFR). Angiogenesis, a well-established feature of solid tumors, also contributes to leukemia progression and correlates with the involvement of specific sanctuary sites in ALL, highlighting that the perturbation of angiogenesis would be an attractive approach for ALL treatment. Anlotinib is an oral tyrosine kinase (TKI) inhibitor with a broad range of antitumor effects via the suppression of VEGFR, PDGFR and FGFR. Of importance, anlotinib has been approved for the treatment of advanced lung cancer in China. Here, we evaluated the antileukemia activity of anlotinib in preclinical B-ALL models and its underlying molecular mechanisms. In this study, we observed that anlotinib significantly blunted the capability of cell proliferation and arrested cell cycle at G2 phase in B-ALL cell lines. Subsequently, we found that anlotinib resulted in remarkably enhanced apoptosis in B-ALL in vitro. To assess the in vivo antileukemia potential, we established a B-ALL patient-derived xenograft (PDX) mouse model and then treated the B-ALL PDX model with anlotinib. As a result, oral administration of anlotinib pronouncedly delayed in vivo B-ALL cell growth and reduced leukemia burden with acceptable safety profiles in this model. As for the mechanism of action, the antileukemia effect of anlotinib was associated with the disruption of the role of VEGFR2, PDGFRb, and FGFR3. Moreover, we revealed that this drug blocked the PI3K/AKT/mTOR/ signaling, a pathway that is linked with angiogenesis and its proangiogenic regulators, including VEGFR2, PDGFRb, and FGFR3. In aggregate, these results indicate that anlotinib is a potent antitumor agent for the treatment of B-ALL via the inhibition of angiogenic relevant pathways, which provide a novel potential treatment intervention for patients with B-ALL who have little effective therapy options. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Anlotinib originally designed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

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Towards a Targeted Therapy:Phosphoproteomics Reveals Signaling Pathways That Are Normalized in AML Cells Following Treatment with Anti-CD44 Antibodies

Blood ◽

10.1182/blood.v126.23.1402.1402 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1402-1402

Author(s):

Heba M. Jalal El-Din ◽

Jasmeen S. Merzaban

Keyword(s):

Signaling Pathways ◽

Protein Interactions ◽

Membrane Trafficking ◽

Molecular Mechanisms ◽

Isotope Labeling ◽

Conflicts Of Interest ◽

Small Gtpases ◽

Major Pathway ◽

Hl60 Cells ◽

And Control

Abstract Acute myeloid leukemia (AML) is a clonal malignant disease characterized by a blockage in the differentiation of myeloid cells resulting in the accumulation of highly proliferating immature blast cells. With the success of All Trans Retinoic acid (ATRA) in acute promyelocytic leukemia (AML3), differentiation therapy has become a very attractive treatment option. Ligation of CD44 (a cell surface antigen) with anti-CD44 monoclonal antibodies (mAbs) is reported to reverse the blockage of differentiation and suppress the proliferation of blasts derived from most AML subtypes. However, the molecular mechanisms underlying this apparent 'normalization' (reversal) of AML cells induced by CD44 have not been fully elucidated. To expand our understanding of the cellular regulation and circuitry involved, we aimed to apply a quantitative phosphoproteomic approach using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to monitor dynamic changes of phosphorylation states in HL60 cells following treatment with CD44 mAbs. Phosphoproteomic analysis identified differentially phosphorylated proteins among CD44 mAb treated and control HL60 cells that are involved in a number of major signaling pathways as determined by the Ingenuity Pathway analysis (IPA) platform. Among others, Rho signaling emerged as a major pathway significantly changed by CD44 mAb treatment. Rho GTPases are well-recognized regulators of the actin cytoskeleton but have also been implicated in diverse cellular events such as cell polarity, microtubule dynamics, membrane trafficking, transcriptional regulation, cell growth control and development. An interesting Rho family member, PAK-2 was identified in our search. PAK-2 is a ubiquitously expressed serine/threonine protein kinase, which is a direct target for small GTPases and has been identified as a switch between cell survival and cell death signaling depending on its mode of activation. Western-blot analysis of cell lysates of CD44 mAb treated and control HL60 cells confirmed that the phosphorylation of Pak-2 was altered as early as 5 minutes following treatment. Further validation and characterization of the activation mode, phosphorylation dynamics and protein-protein interactions of PAK-2 are essential in understanding its role in AML. Disclosures No relevant conflicts of interest to declare.

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Photodynamic Therapy Enhanced the Antitumor Effects of Berberine on HeLa Cells

Open Chemistry ◽

10.1515/chem-2019-0048 ◽

2019 ◽

Vol 17 (1) ◽

pp. 413-421 ◽

Cited By ~ 3

Author(s):

Han-Qing Liu ◽

Ya-Wen An ◽

A-Zhen Hu ◽

Ming-Hua Li ◽

Guang-Hui Cui

Keyword(s):

Photodynamic Therapy ◽

Western Blot ◽

Hela Cells ◽

Mammalian Target Of Rapamycin ◽

Control Group ◽

Antitumor Effects ◽

Underlying Mechanisms ◽

And Control

AbstractIn this study we investigated the antineoplastic effects of Berberine (BBR)-mediated photodynamic therapy (PDT) on HeLa cells and its related mechanisms. The CCK-8 assay and flow cytometry were used to evaluate the proliferation and apoptosis of cells respectively. In addition, changes in protein expression levels were assessed using western blot. BBR at dose of 10 mg/kg was injected intraperitoneally to mice with tumors and PDT treatments were performed 24 hours later. In vivo imaging systems were used to evaluate the fluorescence of BBR. In vitro, PDT significantly enhanced the effects of BBR on inducing cell apoptosis and inhibiting proliferation. The in vivo results showed that the fluorescence intensity in the PDT group was decreased compared with that in the BBR group. Tumor weights and tumor size in the PDT group were less than those in the control group; however, when BBR was applied without PDT, no significant differences were observed between the BBR and control group. The results of western blot showed that PDT enhanced the inhibitory effects of BBR on the mammalian target of rapamycin (mTOR) signaling pathway, that may partly explain the potential underlying mechanisms.

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The Impact of 3D Chromosomal Topology in Acute Leukemia

Blood ◽

10.1182/blood.v128.22.sci-15.sci-15 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-15-SCI-15

Author(s):

Iannis Aifantis

Keyword(s):

Survival Rate ◽

Large Scale ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Tumor Formation ◽

Cohesin Complex ◽

Self Renewal ◽

Treatment Regimens ◽

The Impact

Abstract Acute myeloid leukemia (AML) is the most common adult leukemia characterized by excessive proliferation of abnormal myeloid progenitors. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate), which can largely be attributed to limited advances in treatment regimens that, for the last decades, have relied on the use of two non-targeted cytotoxic drugs: cytarabine and anthracycline. Large-scale sequencing efforts have shed new light on genetic and epigenetic determinants of AML. Interestingly, these studies identified a frequent co-occurrence of somatic mutation between genes encoding cohesin complex subunits (such as STAG2, SMC1A, RAD21 and SMC3) and well-characterized AML oncogenic triggers, such as FLT3-ITD, TET2, and NPM1. Recent work has demonstrated an important role for the cohesin complex in normal stem/progenitor self-renewal and differentiation, gene regulation, and suppression of myeloproliferative neoplasms and AML, despite the precise mechanisms underlying these functions remaining poorly understood. It is believed that cohesin may suppress tumor formation by regulating chromatin looping at loci critical for self-renewal and myeloid progenitor differentiation. Utilizing established models of murine and human AML, this we will focus on the molecular mechanisms of cohesin-dependent myeloid tumor-suppression, with an emphasis on understanding novel treatment approaches that can exploit these functions. Using established protocols for identifying genome-wide changes in chromatin topology and gene expression, we propose undertaking an extensive characterization of cohesin-regulated chromatin changes driving AML. Furthermore, we investigate the application of targeted agents in cohesin-deficient AML whilst extensively mapping the mechanisms-of-action underlying these specific treatments. Ultimately, our work aims to generate novel, pre-clinical disease models of cohesin-mutated AML with strong mechanistic insights into the tumor-suppressive function of this complex. Disclosures No relevant conflicts of interest to declare.

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JAK2 Inhibitor Therapy in Chronic Myeloproliferative Neoplasms Ph(-): Hematologic, Clinical and Molecular Responses

Blood ◽

10.1182/blood.v120.21.5059.5059 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5059-5059

Author(s):

Ana Esther Kerguelen Fuentes ◽

Dolores Hernández-Maraver ◽

Miguel Angel ◽

Canales Albendea ◽

Ana Rodriguez de la Rua

Keyword(s):

Clinical Response ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Molecular Response ◽

Spleen Size ◽

Molecular Responses ◽

Allele Burden ◽

Jak2 Inhibitor ◽

Chronic Myeloproliferative Neoplasms ◽

Jak2 Inhibitors

Abstract Abstract 5059 JAK2 inhibitors are known to improve symptoms, to control myeloproliferation and to reduce splenomegaly in patients diagnosed with chronic myeloproliferative neoplasms (MPNs)Ph(-). However their ability to decrease the allele burden and achieve molecular responses is controversial. Objective: To evaluate hematologic, clinical and molecular responses according to the criteria of the European LeukemiaNet and European Myelofibrosis Network in 13 patients treated with JAK2 inhibitors. Material and Methods: We performed a prospective study in the Haematology Service of the Hospital La Pazbetween 1987 and 2012 in 13 patients diagnosed with NMP Ph (-) and treated with of JAK2 inhibitors: 5 secondary mylofibrosis (SFM)to homozygous polycythemia vera JAK (+), 4 SFM to essential thrombocythemias JAK (-), 2 primary myelofibrosis (one JAK (-) and one heterozygous JAK (+)) and 2 homozygous PV JAK (+) resistant to hydrea. The RT-PCR was performed at 6 or 12 months after the first determination of the allelic burden. Median follow-up was 3 months (1 – 15). A) Hematologic Response (HR): 3/5 SFM to PV(1)/TE JAK(-)(2) reached HR at 3 months of initiation of JAK2 inhibitor to 20mg/day. Molecular and clinical response were not evaluated. B) Clinical Response: Three patients had a reduction in the spleen size. Only one patient in the SFM group had a reduction in the spleen size (18 cm before the drug was commenced to 13. 7 cm) and the allele burden decrease from 55% to 23% after 5 months of therapy with JAK2 inhibitor at 25mg/12h (increase of 5mg/12h after 15 days of initiation of medication). 2/3 MFS to TE JAK(-) had a reduction from 15, 3 cm before the drug was commenced to 9 cm after 3 months of therapy with JAK2 inhibitor at 20 mg/12h. 3/3 MFP JAK(-) had a 6cm reduction in spleen size. Twenty cm splenomegaly was documented before starting JAK2 inhibitor to 15 mg/day. C) Molecular Response: 2/5 SFM to PV decreased the previous allele burden value. One patient decreased by 25% the previous allele burden value (99. 28%) at 6 months of JAK2 inhibitor. Second patient decreased by 13% the previous allele burden value (55%) at 6 months of starting JAK2 inhibitor to 25 mg/day. In 1/2 PV, the previous allele burden value (93. 17%) decreased by 11. 4% at 6 months of starting JAK2 inhibitor at 100mg/24h. D) Lack of response and disease progression: One patient with SMF secondary to JAK 2 (-) ET had dose reductions from 20 mg twice a day secondary to grade IV thrombocytopenia and renal toxicity. Patient finally developed acute leukemia. Conclusions: Our study confirms that JAK2 inhibitors reduce splenomegaly in MPNs JAK(-)and JAK(+). Prospective studies with an adequate sample size are necessary to demonstrate whether splenomegaly and symptom reductions achieved with inhibition of JAK2 could be associated to decrease the allele burden and achieve molecular responses in MPNs JAK(+). Disclosures: No relevant conflicts of interest to declare.

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The Role of CD39/CD73/Ado/A2AR Axis and HIF-1α in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-115732 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4406-4406 ◽

Cited By ~ 1

Author(s):

Yiqing Cai ◽

Lili Feng ◽

Dai Yuan ◽

Qian Wang ◽

Xin Wang

Keyword(s):

Protein Expression ◽

Western Blot ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Control Group ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Protein Expressions ◽

A2ar Agonist

Abstract Introduction: CD39/CD73/ADO/A2A system plays important roles in tumor growth and therapy. Extracellular adenosine (ADO) receptor A2A (A2AR) is the dominant receptor expressed on chronic lymphocytic leukemia (CLL) cells. ADO has been shown to protect CLL cells from spontaneous and drug-induced apoptosis through A2AR activation. Overexpression of hypoxia inducible factor-1α (HIF-1α), an important mediator controlling the expression of a wide variety of apoptotic genes, has been observed in bone marrow leukemic cells from CLL patients. However, the reciprocal action of A2AR and HIF-1α in CLL remains elusive. This study was aimed to explore the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α in CLL. Materials and Methods: Peripheral blood (PB) and bone marrow (BM) samples were collected from 30 healthy volunteers (control) and 20 patients who were diagnosed with CLL for the first time at the Hematology Department in our hospital. CD39/CD73/ADO/A2AR axis-related protein and HIF-1α protein expressions in CLL PB and BM tissues were examined by Western-blot. CD39, CD73, A2AR and HIF-1α expression on CLL cells were also determined by FACS. CLL cell line (Lym-2) was purchased from ATCC and incubated in DMEM medium supplemented with 10% FBS. The protein expressions of CD39, CD73 and A2AR on Lym-2 cells were confirmed by Western-blot. To study the impacts of A2AR activation and inactivation on CLL cells, CLL cells were treated with A2AR agonist CGS21680 or antagonist SCH58261, followed by determination of cell proliferation and HIF-1α protein expression. Results: The protein expressions of CD39, CD73, A2AR and HIF-1α were higher in CLL group than those in control group (BM: CD39 1.471 vs 0.926, CD73 1.097 vs 0.489, A2AR 1.139 vs 0.342 and HIF-1α 0.940 vs 0.362, P<0.05 Figure 1A; PB : CD39 1.809 vs 1.331, CD73 1.039 vs 0.653, A2AR 1.738 vs 1.119 and HIF-1α 1.336 vs 1.010, P<0.05 Figure 1B). Moreover, CD73 and HIF-1α protein expression was found to have relationship with disease stage. Patients who belonged to stage IV (Rai) and stage B/C (Binet) exhibited higher levels of CD73 and HIF-1α than patients who belonged to stage I to III (Rai) and stage A (Binet) . Data from FACS analysis showed that the proportion of CD39+CLL cells was higher than CD73+CLL cells and HIF-1α+CLL cells (P<0.05, Figure 2A). When compared to control group, the proportions of CD39+cells and CD73+ cells were significant higher in CLL group (Figure 2B). Western-blot results showed that Lym-2 cells expressed CD39, CD73 and A2AR simultaneously (Figure 3A). The results from CLL cells and A2AR agonist/antagonist incubation showed that when CGS21680 concentration was of ≥ 10uM, cell proliferation was promoted obviously (Figure 3B). On the contrary, SCH58261 could inhibit CLL cells proliferation when its concentration was of ≥5uM at a concentration dependent manner (Figure 3C). Moreover, HIF-1α protein expression was also affected by A2AR agonist at a concentration dependent manner(0uM 0.851, 10uM 1.116, 20uM 1.420, 50uM 1.41, Figure 3D). Conclusion: Our study showed that ADO produced by CD39 and CD73 could affect CLL cells proliferation and HIF-1α expression through A2AR-mediated mechanisms. The exploration of the molecular mechanisms underlying the relationship between A2AR activation and HIF-1α could help us to find a novel approach to CLL therapy. Disclosures: No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

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The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia

Blood ◽

10.1182/blood-2019-124767 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5400-5400

Author(s):

Tatiana V Makarik ◽

Adhamjon O Abdullaev ◽

Sergei M. Kulikov ◽

Elena E Nikulina ◽

Svetlana A Treglazova ◽

...

Keyword(s):

Conflicts Of Interest ◽

Cell Clone ◽

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Allele Burden ◽

Chronic Myeloproliferative Neoplasms ◽

V617f Mutation ◽

Calr Mutations

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

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Hydroxyurea Treatment In 1075 Patients with Essential Thrombocythemia and Occurrence of Extra-Hematological Adverse Events: A Preliminary Report of the Registro Italiano Trombocitemia (RIT)

Blood ◽

10.1182/blood.v116.21.1973.1973 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1973-1973 ◽

Cited By ~ 1

Author(s):

Luigi Gugliotta ◽

Cristina Papayannidis ◽

Andrea Piccin ◽

Alessia Tieghi ◽

Nicola Vianelli ◽

...

Keyword(s):

Adverse Events ◽

Essential Thrombocythemia ◽

Preliminary Analysis ◽

Preliminary Report ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Leg Ulcers ◽

Who Criteria ◽

Chronic Myeloproliferative Neoplasms ◽

Hydroxyurea Treatment

Abstract Abstract 1973 Background. Hematological and extra-hematological adverse events associated to Hydroxyurea (HU) treatment in patients with chronic myeloproliferative neoplasms (MPN) are object of particular interest in the Ph-negative MPN since they can significantly limit the HU use. Objective. To evaluate the extra-hematological adverse events associated to HU treayment in a large cohort of Essential Thrombocythemia (ET) patients. Material and Methods. One thousand and seventy-five out of the 2005 ET patients registered in the RIT were treated with HU and are the object of this report. The patients, 641 females (59.6%) and 434 males (40.4%), diagnosed according to the PVSG or WHO criteria in 54 hematological centers of the RIT, started treatment with HU as first (92%) or second (8%) line, at median age of 65 years.The mean duration of HU treatment was 3.3 years. The HU treatment was withdrawn in 221 (20.5%) patients after a mean of 3.0 years.The administered dose of HU was 0.25–3.0 g/day (median 1), and a mean cumulative dose was 1113 g. The extra-hematological adverse events (EHAEs) observed during the HU treatment were distinguished in HU related AEs (HU-EHAEs), ET related AEs (ET-EHAEs) and HU or ET unrelated AEs (U-EHAEs).Results. During the HU treatment (3587 pt-y) 378 EHAEs were reported in 207 (19.3%) patients, being the HU-EHAEs 244 (6.8/100 pt-y) in 170 (15.8%) patients. In detail, the HU-EHAEs were: dermatological in 108 (48.3%) cases (38 hyperpigmentation, 26 leg ulcers, 22 maculo-papular rash, 10 lichenoid eruptions, 5 skin cancer, 4 alopecia); gastro-intestinal in 80 (32.8%) cases (37 nausea/vomiting, 30 diarrhea, 13 gastro-intestinal intolerance); systemic in 35 (14.3%) cases (28 fever, 7 fatigue); neurological in 19 (7.8%) cases (headache); miscellanea in 2 (0.8%) cases. Conclusion. This preliminary analysis in 1075 ET patients of the Registro Italiano Trombocitemia (RIT) treated with HU shows that the extra-hematological adverse events referred to the HU (HU-EHAEs) occurred with not negligible rate (6.8/100 pt-y) and need to be object of attention in the management of ET patients. Disclosures: No relevant conflicts of interest to declare.

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Molecular Characterization Of Polycythemia Vera Based On The Relationship Of JAK2V617F and 9pUPD

Blood ◽

10.1182/blood.v122.21.1607.1607 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1607-1607

Author(s):

Linghua Wang ◽

Sabina Swierczek ◽

Kimberly Hickman ◽

Soo-Jin Kim ◽

David A. Wheeler ◽

...

Keyword(s):

Polycythemia Vera ◽

Conflicts Of Interest ◽

Molecular Subtype ◽

Myeloproliferative Neoplasms ◽

Uniparental Disomy ◽

Jak2v617f Mutation ◽

Small Subset ◽

Wild Type ◽

The Stability ◽

The Relationship

Abstract The gain-of-function mutation at codon 617 of JAK2 (JAK2V617F) is the most common somatic event observed in patients with polycythemia vera (PV), occurring in over 95% of PV patients. JAK2V617F confers cytokine hypersensitivity and cytokine-independent growth of erythroid progenitors, which are characteristic features of PV. Homozygous JAK2V617F is observed in about half of PV patients, whereas it is rarely seen in essential thrombocythemia (2-4%) and other myeloproliferative neoplasms. Homozygous JAK2V617F has been assumed to result from homozygous recombination, leading to uniparental disomy on 9p (9pUPD). It has been reported that the JAK2 46/1 (GGCC) haplotype may predispose carriers to the JAK2V617F mutation, and the JAK2V617F mutation facilities the acquisition of homozygous JAK2V617F. Challenging this view is a single study reporting 9pUPD in two PV subjects with wild-type JAK2, suggesting that in these two individuals, 9pUPD might have preceded the JAK2V617F mutation (Blood. 2011;118(24):6468-6470). However, the relationship between JAK2V617F and 9pUPD, the frequency of this new PV molecular subtype, its clinical relevance, and the stability of this genotype need to be systematically defined in a larger sample cohort. To address this, we combined whole-exome sequencing (WXS) of DNA from 31 consecutive PV patients with high-resolution SNP arrays, and further validated our findings in two additional cohorts comprising 59 PV consecutive patients collected from a single institution. In addition, we investigated the stability of each molecular subtype by using serial samples collected from 25 PV patients. We obtained an average of 125x coverage on JAK2 locus by WXS (Illumina Hiseq2000) and 2225x coverage by targeted deep sequencing using Ion PGM sequencer. Analysis of these data shows that the relationship between the JAK2 locus and 9pUPD is more complex than originally assumed. We defined 4 subgroups: 41% of patients had JAK2V617F in a heterozygous state without detectable 9pUPD (Subgroup I); 43% of patients had JAK2V617F with an allelic fraction in direct proportion to the level of 9pUPD (Subgroup II; homozygous JAK2V617F); 10% of patients harbored 9pUPD at approximately twice the level of the JAK2V617F allelic burden (Subgroup III; UPD with heterozygous JAK2V617F); and a small subset (6%) of patients exhibited trisomy of 9p, generating 3 copies of the JAK2 allele by chromosome duplication (Subgroup IV). No difference in the frequency of the JAK2 46/1 (GGCC) haplotype was found among these 4 subgroups. We found that this subtype classification was stable over time in over 60% of patients, whereas it transformed among the 9pUPD-positive subtypes in the remaining patients, indicating the outgrowth of a new PV subclone. While 2 PV patients with 9pUPD and wild-type JAK2 were previously reported (Blood. 2011;118(24):6468-6470), we now show a relative high proportion of PV patients having the novel, previously not recognized JAK2 genotype; i.e. JAK2 9pUPD with heterozygous JAK2V617F mutation. Our study will provide novel perspectives on the molecular basis of the evolution of PV and a better understanding of the roles of JAK2V617F and 9pUPD in this disease. Disclosures: No relevant conflicts of interest to declare.

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