scholarly journals Novel Clinical, Laboratory, Molecular and Pathological (2018 CLMP) Criteria for the Differential Diagnosis of three Distinct JAK2, CALR and MPL Mutated Myeloproliferative Neoplasms: The Role of Driver Mutation Analysis and Bone Marrow Histology

2018 ◽  
Vol 3 (2) ◽  
Keyword(s):  
Bone Marrow ◽  
Clinical Laboratory ◽  
Early Stage ◽  
Myeloproliferative Neoplasms ◽  
Pegylated Interferon ◽  
Jak2v617f Mutation ◽  
Bone Marrow Histology ◽  
Low Dose Aspirin

The broad spectrum of JAK2V617F mutated trilinear phenotypes varies from essential thrombocythemia (ET), prodromal polycythemia vera (PV), masked PV, erythrocythemic PV, classical PV, and PV complicated by splenomegaly and myelofibrosis (MF). ET heterozygous for the JAK2V617F mutation is associated with normal life expecancy. JAK2V617F mutation load increases from low to 40% in ET, from below to above 50% in early stage PV and above 50% up to 100% in overt and advanced PV and MF. Pretreatment bone marrow morphology and cellularity distinguish JAK2V617F mutated trilinear MPN from calreticulin (CALR) and MPL mutated MPN. The morphology of clustered mature enlarged pleomorphic megakaryocytes with hyperlobulated nuclei are similar in JAK2V67F ET and PV patients. MPL515 mutated thrombocythemia is featured by monolinear proliferation of large to giant mature megakaryocytes with hyperlobulated nuclei in a normocellular or hypocellular bone marrow. CALR mutated thrombocythemia shows characteristic bone marrow features of primary dual megakaryocytic granulocytic myeloproliferation (PMGM) in a normocellular or hypercellular bone marrow without features of PV. JAK2V617F, CALR and MPL515 allele burden slowly increases to values around 50% together with the degree of splenomegaly, myelofibrosis and constitutional symptoms during life long follow-up. Natural history and life expectancy relate to the degree of splenomegaly, bone marrow fibrosis, anemia and the acquisition of epigenetic mutations at increasing age predict unfavorable outcome in JAK2V617F, CALR and MPL515 mutated MPN. Low dose aspirin in JAK2V617F mutated ET and PV and phlebotomy on top of aspirin in PV is mandatory to prevent platelet-mediated microvascular circulation disturbances. Pegylated interferon is the first line myeloreductive treatment option in prodromal and early stage JAK2V617F mutated PV and in CALR and MPL mutated thrombocythemia to postpone the use of hydroxyurea and ruxolitinib as long as possible.

A Novel Approach to the Treatment of Early Stage Hodgkin Lymphoma with a Mitoxantrone-Based Regimen Followed by Involved Field Radiotherapy: A 12 Year Follow up

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4901-4901
Author(s):  
Arlene Dawravoo ◽  
Neel B. Shah ◽  
Teresa O'Brien ◽  
Stephanie A. Gregory ◽  
Parmeswaran Venugopal
Keyword(s):  
Bone Marrow ◽  
Radiation Therapy ◽  
Early Stage ◽  
Marrow Transplant ◽  
Field Radiation ◽  
Involved Field ◽  
Experienced Grade

Abstract Abstract 4901 Early stage Hodgkin Lymphoma (HL) has been conventionally treated with radiotherapy alone or abbreviated chemotherapy along with involved field radiation therapy. The role of combined modality therapy was investigated to study the favorable effects of lower doses of radiation on acute and long term toxicities without compromising response and cure rates. The goal of this study was to evaluate the role of combined chemotherapy, which incorporated mitoxantrone in place of doxorubicin and dexamethasone in place of dacarbazine, and involved field radiation therapy in patients with early stage, non-bulky (I and II) HL. The patients enrolled in this trial were originally enrolled in 1997–2002 and treated with 4 cycles of chemotherapy with mitoxantrone 6 mg/m2 IV, vinblastine 5 mg/m2 IV, bleomycin 5 units/m2 on day 1 and day 15, and dexamethasone 6 mg/m2 PO on days 1–5 and days 15–19 on a 28 day cycle (MBVD). Restaging was done following 4 cycles of chemotherapy. Subsequently patients received involved field radiation with total dose of 30 Gy at 150 cGy per fraction. Patients were followed for response, duration, toxicity and survival. Results: 15 patients were enrolled, 10 were evaluable. All completed treatment. 7 patients were stage II and 3 were stage I at diagnosis. 4 were males and 6 were females between the ages of 20 to 52 years with a median age 33.5 years. Following completion of treatment with chemotherapy and radiation, 8 patients achieved a complete response and 2 patients remained in partial remission. Duration of response in patients in follow up ranged between 9 and 149 months with a median of 75 months. 2 relapses occurred at 8 and 23 months with increase in size of the mediastinal lymph nodes. 7/10 patients experienced grade 1 toxicities which included anemia, leukopenia, neutropenia, thrombocytopenia, GI upset or cardiomyopathy. 4/10 patients experienced grade 2 toxicities. 2/10 patients had grade 3 toxicities with severe neutropenia. All patients experienced grade 1 skin changes following radiation. One patient's left ventricle ejection fraction decreased from 65% to 52% following therapy. At last follow up, 9/10 patients were alive with 9/10 patients still in remission. 1 patient underwent bone marrow transplant after relapse at 8 months and was lost to follow up. 1 patient experienced recurrence of disease at 23 months and underwent bone marrow transplant at 29 months and was in remission at 73 month follow up. Although the number of patients in this study is small, treatment appears to be well tolerated and acute toxicities less compared to standard therapy with ABVD chemotherapy followed by radiotherapy. At last follow up 9/10 are in remission, with 2 patients in remission for 12 years. No patients have experienced MDS or other long term sequelae. Confirmatory studies should be done to determine the safety of the MBVD regimen compared to standard ABVD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Importance of Identification of M-BCRABL Oncogene and JAK2V617F Mutation in Myeloproliferative Neoplasms

2014 ◽  
Vol 60 (2) ◽  
pp. 44-48
Author(s):  
Annamária Szántó ◽  
Zsuzsanna Pap ◽  
Z Pávai ◽  
I Benedek ◽  
Judit Beáta Köpeczi ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Molecular Biology ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Gene Mutations ◽  
Chronic Phase ◽  
Final Diagnosis ◽  
Jak2v617f Mutation ◽  
Myeloproliferative Disease

Abstract Background: The elucidation of the genetic background of the myeloproliferative neoplasms completely changed the management of these disorders: the presence of the Philadelphia chromosome and/or the BCR-ABL oncogene is pathognomonic for chronic myeloid leukemia and identification of JAK2 gene mutations are useful in polycytemia vera (PV), essential thrombocytemia (ET) and myelofibrosis (PMF). The aim of this study was to investigate the role of molecular biology tests in the management of myeloproliferative neoplasms. Materials and methods: We tested the blood samples of 117 patients between April 2008 and February 2013 at the Molecular Biology of UMF Târgu Mureș using RQ-PCR (for M-BCR-ABL oncogene) and/or allele-specific PCR (for JAK2V617F mutation). Results: Thirty-two patients presented the M-BCR-ABL oncogene, 16 of them were regularly tested as a follow-up of the administered therapy: the majority of chronic phase patients presented decreasing or stable values, while in case of accelerated phase and blast phase the M-BCR-ABL values increased or remained at the same level. Twenty patients were identified with the JAK2V617F mutation: 8 patients with PV, 4 with ET, 3 with PMF, 4 with unclassifiable chronic myeloproliferative disease and 1 patient with chronic myelomonocytic leukemia. There was no case of concomitant occurance of both molecular markers. Conclusions: Molecular biology testing plays an important role in the management of myeloproliferative neoplasms: identification of the molecular markers confirms the final diagnosis, excluding secondary causes of abnormal blood count parameters. Regular monitoring of MBCR- ABL expression level is useful in the follow-up of therapeutic efficiency.

scholarly journals Early Stage Follicular Lymphoma. Predictive Role of Minimal Residual Disease (MRD) and Impact of MRD-Driven Treatment with Radiotherapy and Rituximab on Clinical Outcome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2959-2959 ◽  
Author(s):  
Alessandro Pulsoni ◽  
Irene Della Starza ◽  
Maria Elena Tosti ◽  
Luca Vincenzo Cappelli ◽  
Giorgia Annechini ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Molecular Marker ◽  
Follicular Lymphoma ◽  
Early Stage ◽  
Tumor Burden ◽  
Stage I ◽  
Predictive Role ◽  
Qualitative Pcr

Abstract Background. In localized follicular lymphoma (FL, stage I-II), BCL2/IGH+ cells can be detected in the peripheral blood (PB) and/or bone marrow (BM) in 66.7% of cases (Pulsoni et al, BJH 2007). We hereby analyzed the prognostic impact of MRD in localized FL and explored the possibility of a MRD-guided therapeutic approach on a series of patients with a long follow-up. Methods. Between April 2000 and February 2015, 67 consecutive patients with a confirmed histologic diagnosis of stage I/II FL followed at our Center were enrolled in the study. PB and BM samples were collected at enrollment in all patients and investigated by qualitative PCR to identify the presence of a BCL2/IGH rearrangement. Paraffin-embedded lymph nodes (LN) were studied when available. Patients who proved positive at baseline were studied for MRD every 6 months. Real-Time Quantitative PCR (RQ-PCR) was retrospectively performed according to material availability. All patients were treated with involved field radiotherapy (RT) (24-30 Gy); from 2005, patients who were MRD+ after RT received rituximab (R) (375 mg/m2, 4 weekly administration). The median follow-up is 67 months (17-183); 21 patients (31%) have relapsed after a median of 37 months (17-165) from diagnosis. Results. At baseline, a clonal marker was found by qualitative PCR in 48/67 cases (72%): 36 were MBR+ (54%), 6 mcr+ (9%), 6 showed a minor BCL2 rearrangement (9%), while 19 (28%) were negative. Fifteen of the latter 19 were analyzed by RQ-PCR and 4 proved MBR+. Of the 13 available LNs, 11 showed the same molecular marker identified in the PB/BM; 2 cases, negative in the PB/BM, showed a rearrangement in the LN only. After RT, 40/42 MBR+/mcr+ patients were analyzed: 20 resulted MRD-, while 20 persisted MRD+. Regardless of the post-RT MRD status, an equal number of relapses was recorded in both groups (7 each). R treatment was administered to the 20 MRD+ patients after RT. Sixteen (80%) achieved a MRD- status after R: over time, 7/16 patients converted to MRD+ and 4 relapsed, whilst 9/16 patients (56.2%) remain persistently MRD- and none has relapsed so far. To evaluate the impact of R, we considered a series of 27 patients MRD+ after RT or who were MRD- and became MRD+ during the follow-up. Of the 19 patients who received R (1 could not be studied), 15 (79%) did not relapse, while of the 8 untreated patients (pre-2005), 6 (75%) relapsed (p=0.025). Progression-free survival (PFS) was significantly longer for R-treated patients (p=0.0412) (Fig. 1). To define the predictive role of MRD in the entire cohort regardless of post-RT treatment, we considered the 39 patients with molecular follow-up. Thirteen have relapsed: 10/13 (77%) were MRD+ in the follow-up, including the pre-relapse time point, while 3 resulted persistently MRD-. Contrariwise, of the 26/39 patients in continuous remission, 18 (69%) were persistently MRD- while 8 were MRD+ (p=0.015). PFS was significantly better for MRD- patients (p=0.0163) (Fig. 2). RQ-PCR was performed in 30 MBR+ patients: 17 (57%) showed a tumor burden ≥10-5 and 13 <10-5. Tumor burden at diagnosis predicted the MRD clearance following RT: 9/13 (69%) cases with low tumor burden resulted MRD- after RT compared to 2/17 (12%) cases with high tumor burden (p=0.0027). Contrariwise, tumor burden did not predict the occurrence of relapse. Conclusions. Early stage FL at diagnosis can have a heterogenous disease extension: 2 of our cases were truly localized, showing a molecular marker only in the LN. However, in most cases the use of combined qualitative approaches, including canonical MBR/mcr and minor rearrangements, together with RQ-PCR has allowed to identify circulating BCL2/IGH+ cells (52/67 cases: 77.6%), despite a negative BM biopsy. RT induced a MRD negativity in 50% of BCL2/IGH+ patients, but this did not impact on clinical outcome. The administration of R in MRD+ patients decreased significantly the risk of a subsequent relapse and improved PFS. Regardless of treatment, MRD positivity during the follow-up is a predictor of relapse and PFS. Tumor burden at diagnosis is associated with MRD clearance after RT. We support the use of a MRD-driven treatment with anti-CD20 monoclonal antibodies in patients with localized FL after RT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Globalisation of agrifood systems and sustainable nutrition

2016 ◽  
Vol 76 (1) ◽  
pp. 12-21 ◽  
Author(s):  
Matin Qaim
Keyword(s):  
Developing Countries ◽  
Production Systems ◽  
Dietary Diversity ◽  
Early Stage ◽  
Overweight And Obesity ◽  
Agricultural Technologies ◽  
Rural And Urban ◽  
Access To Food

The globalisation of agrifood systems is a mega-trend with potentially profound nutritional implications. This paper describes various facets of this globalisation process and reviews studies on nutritional effects with a particular focus on developing countries. Results show that global trade and technological change in agriculture have substantially improved food security in recent decades, although intensified production systems have also contributed to environmental problems in some regions. New agricultural technologies and policies need to place more emphasis on promoting dietary diversity and reducing environmental externalities. Globalising agrifood systems also involve changing supply-chain structures, with a rapid rise of modern retailing, new food safety and food quality standards, and higher levels of vertical integration. Studies show that emerging high-value supply chains can contribute to income growth in the small farm sector and improved access to food for rural and urban populations. However, there is also evidence that the retail revolution in developing countries, with its growing role of supermarkets and processed foods, can contribute to overweight and obesity among consumers. The multi-faceted linkages between changing agrifood systems and nutrition are a new field of interdisciplinary research, combining agricultural, nutritional, economics and social sciences perspectives. The number of studies on specific aspects is still limited, so the evidence is not yet conclusive. A review at this early stage can help to better understand important relationships and encourage follow-up work.

scholarly journals The role of bone marrow edema on autologous matrix induced chondrogenesis for the treatment of osteochondral lesions of the talus

2017 ◽  
Vol 2 (3) ◽  
pp. 2473011417S0001
Author(s):  
Riccardo D’Ambrosi ◽  
Camilla Maccario ◽  
Federico Giuseppe Usuelli
Keyword(s):  
Bone Marrow ◽  
Repeated Measures ◽  
Bone Marrow Edema ◽  
Lesion Size ◽  
Osteochondral Lesions ◽  
Significant Difference ◽  
T1 And T2 ◽  
Marrow Edema

Category: Ankle, Arthroscopy, Basic Sciences/Biologics Introduction/Purpose: to assess the functional and radiological outcomes after AT-AMIC® (arthroscopic talus autologous matrix induced chondrogenesis) in 2 groups: patients with and without bone marrow edema (BME). Methods: Thirty-seven patients of which 24 without edema (GNE) and 13 with edema (GE) were evaluated. All patients were treated with AT-AMIC® repair for osteochondral talar lesion. MRI and CT-scan evaluations, as well as clinical evaluations measured by the VAS score for pain, AOFAS and SF-12 were performed preoperatively (T0) and at 6 (T1), 12 (T2), and 24 (T3) months postoperatively. Results: GNE consisted of 24 patients while GE consisted of 13 patients. In both groups we found a significant difference for clinical and radiological parameters with ANOVA for repeated measures through four time points(p<0.001). In GNE, AOFAS improved significantly at each follow-up(p<0.05); while CT and MRI showed a significant decrease between T1 and T2 and T2 and T3(p<0.05). In GE, AOFAS improved significantly between T0 and T1 and T2 and T3(p<0.05); CT decreased between T1 and T2(p<0.05), while MRI showed a reduction at each follow-up(p<0.05). Lesion size was significantly higher both in MRI and CT in GE in respect to GNE(p<0.05). In the GNE no patients presented edema at T3, while in GE only 23.08% of the patients presented edema at T3. Conclusion: The study revealed that osteochondral lesions of the talus were characterized by bigger size both in MRI and CT in patients with edema. We conclude that AT-AMIC® can be considered a safe and reliable procedure that allows effective healing, regardless of edema and more than half of patients did not present edema six months after surgery.

Clonality in Granulocytes Detected by an Improved HUMARA Assay: A Good Prognostic Marker in Bone Marrow Failure Patients Exhibiting Chromosomal Abnormalities of Indefinite Significance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3702-3702
Author(s):  
Ken Ishiyama ◽  
Chiharu Sugimori ◽  
Hirohito Yamazaki ◽  
Akiyoshi Takami ◽  
Shinji Nakao
Keyword(s):  
Bone Marrow ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Failure ◽  
Bone Marrow Examination ◽  
Refractory Anemia ◽  
Clonal Population ◽  
Dividing Cells ◽  
Humara Assay

Abstract Some patients with aplastic anemia (AA) and approximately 40% of patients with refractory anemia (RA) of myelodysplastic syndrome exhibit karyotypic abnormalities in bone marrow dividing cells. Although some of the patients undergo evolution to acute myeloid leukemia (AML), others follow a clinical course similar to AA patients without chromosomal abnormalities. Except for several abnormalities such as −7 and 5q-, the clinical significance of such chromosomal abnormalities in bone marrow failure patients remains unclear. We recently developed a reliable HUMARA assay capable of detecting a clonal population in granulocytes which constitutes 30% or more of total granulocytes (Blood. 2003;102:1211–1216). Studying correlation between chromosomal abnormalities and the presence of clonality may help in understanding the pathogenetic role of chromosomal abnormalities in AA and RA. We thus analyzed 50 acquired AA and 28 RA female patients who were heterozygous for the HUMARA gene. Chromosomal abnormalities such as add(5)(q13), 9q–9q+ and del(7)(q14q22) were found in 8% of AA and 21% of RA patients. Clonality was detected in 38% of AA patients and 39% of RA patients. Incidence of chromosomal abnormalities in patients with clonality (27%) was higher than that in patients without clonality (4%, p<0.01). In two AA patients who respectively exhibited add(5)(q13) in 10% and +8 in 38% dividing cells, clonality was not detected and these abnormal clones became undetectable at the time of subsequent bone marrow examination. Clonality was detected in the other 2 AA patients respectively exhibiting 9q–9q+ in 40% and del(7)(q14q22) in 25% dividing cells, and in all 5 RA patients respectively exhibiting +8 in 10%, del(5)(q13q31), dup(1)(q32q12) in 90%, del(5)(q13), add(11)(q23), inv(9) in 65% and X,-X in 100% of dividing cells. None of the 50 AA patients including 2 patients with clonality and chromosomal abnormalities underwent evolution to AML during 2-year follow up while one of 28 RA patients who exhibited del(5)(q13q31) progressed to AML. The proportion of clonal granulocytes in total granulocytes estimated by the HUMARA assay remained unchanged in most patients with clonality except for the transformed one. These data indicate that the chromosomal abnormality in bone marrow dividing cells is not necessarily associated with presence of clonal granulocyte population in peripheral blood and that detection of clonality in granulcytes in bone marrow failure patients with chromosomal abnormalities of indefinite significance is useful in predicting prognosis of these patients.

Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4114-4114
Author(s):  
Yusuke Takeda ◽  
Chiaki Nakaseko ◽  
Hiroaki Tanaka ◽  
Masahiro Takeuchi ◽  
Makiko Yui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Signaling Molecules ◽  
Janus Kinase ◽  
Idiopathic Myelofibrosis ◽  
Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

High Degree of Hematological Response After Pegylated Interferon Alpha-2a Therapy In Myeloproliferative Neoplasms

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5055-5055
Author(s):  
Jan Samuelsson ◽  
Gerd Larfars ◽  
Eva Ottosson ◽  
Mats Merup
Keyword(s):  
Side Effects ◽  
Median Time ◽  
Response Rate ◽  
Myeloproliferative Neoplasms ◽  
Pegylated Interferon ◽  
Interferon Α ◽  
Molecular Response ◽  
Hematological Response ◽  
Ifn Therapy

Abstract Abstract 5055 Objective: To retrospectively assess hematological response rates, and other clinical and molecular variables, in MPN patients treated with pegylated interferon α-2a (Pegasys®, Roche Ltd). Responses were graded according to criteria published by the European Leukemia Net for PV and ET (Barosi G et al Blood 2009;113:4829), with the exception that mesurement of spleen size using ultrasound was not routinely performed, and the European Myelofibrosis Network for PMF (Barosi G et al Blood 2005;106:2849), respectively. Patient characteristics: The 23 patient cohort consisted of 13 PV, 5 ET, 3 PMF and 2 post-ET/PV MF pts. Thirteen pts were JAK2V617F+, 6 were JAK2V617F wt, and in 4 pts JAK2 status is unknown. Median age was 50 years (range 26–69), 13 were female and 10 male. Median time from MPN diagnosis to start of pegylated interferon α-2a (Peg-IFN) therapy was 67 months (range 0–204). Six pts had a previous thrombotic event (TIA=2, portal vein thrombosis=2, DVT lower extremity=2), and 2 pts had a previous major hemorrhage (gynecological=1, gastrointestinal=1). Eleven pts had previously received therapy with anagrelide (n=8), hydroxyurea (n=4), interferon α-2b (n=1), busulfan (n=1) or P32 (n=1), while 12 pts had not received bone marrow suppressive therapy. All PV and ET pts were on aspirin. Phlebotomies were performed in PV with the aim of keeping the hematocrit < 0.45. Peg-IFN was given at a dose of 90 μg/week in 16 pts, 135μg/week in 6, and 180μg/week in 1. Results: The overall hematological response rate (CR+PR) was 18/21 (86 %), 14 pts achieving CR and 4 PR. Two pts are too early to evaluate at the time of astract submission. One PV and 1 PMF patient were non-responders. Resonse rates were similar in PV vs ET, female vs male pts, and previously treated vs previously untreated pts. Median time of follow-up on Peg-IFN therapy is 16 months (3+ - 49+). Thirteen pts are still on therapy, 9 in CR, 2 in PR, and 2 too early to evaluate. These 13 pts have very limited or no side effects. Therapy has also been stopped according to plan after long hematological CR with molecular response in 2 pts. Therapy has been discontinued in 8 pts, in five (22 %) due to side effects (depression n=3, joint pain n=3, hair loss n=2, pruritus n=1), non-response in 2 pts, and PMF progression in 1. Serial JAK2V617F measurements are available at time of abstract submission in 4 pts, 1 achieved molecular CR, 2 PR whereas 1 patient treated for 5 months had no molecular response. Three of 4 mildly anemic MF pts normalized their hemoglobin (HgB 113 → 137, 106 → 123, and 110 → 125 respectively). In one PMF patent a clear reduction of marrow fibrosis was noted, whereas it progressed in another. No thromboembolic or bleeding complications were observed during PEG-IFN therapy. Longer follow-up, as well as additional molecular and morphological studies will be presented. Conclusions: Pegylated interferon α-2a induced a higher hematological response rate with improved tolerability, compared to our previous experience with Peg-IFN α-2b (Samuelsson et al Cancer 2006;106:2397), although the current number of patients is limited. However, the two previous publications that describes Peg-IFN α-2a therapy in larger MPN patients cohorts have observed results similar to ours (Kiladjian JJ et al Blood. 2008;112:3065, Quintás-Cardama A et al J Clin Oncol. 2009;27:5418). Molecular responses noted in a subset of patients further highlights the effect of Peg-IFN α-2a on the malignant clone in MPN:s. Peg-IFN α-2a is a valuable therapeutic alternative in patients who tolerate initial side effects, and will soon be compared to hydroxyurea in a randomized trial in high-risk PV and ET pts performed by the MPD research consortium. Disclosures: Samuelsson: Roche Sweden: Advisory board on use of recombinant erytropoetin. Off Label Use: Alpha-interferon does not have a label for use in myeloproliferative neoplasms. Merup:Roche Sweden: Received honoraria for lectures on rituximab use in lymphoma.

Pegifna Promotes Proliferation and Myeloid Differentiation In Human Hematopoietic Progenitors In Patients With Polycythemia Vera and Essential Thrombocythemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2843-2843
Author(s):  
Katherine King ◽  
Sabina Swierczek ◽  
Katie Matatall ◽  
Kimberly Hickman ◽  
Margaret A. Goodell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Pegylated Interferon ◽  
Hematopoietic Stem ◽  
Financial Compensation ◽  
Specific Differentiation ◽  
Allelic Burden

Abstract The myeloproliferative neoplasms, polycythemia vera (PV) and essential thrombocythemia (ET), are characterized by clonal hematopoiesis that is often associated with a JAK2V617F mutation, although this does not appear to be a disease-initiating event. Treatment of PV and ET with pegylated interferon-alpha (pegInfα) has been shown to lead to hematological remission, a decrease in the JAK2V617F allelic burden in many cases, and even a reversion to polyclonal hematopoiesis. Despite promising therapeutic results, the mechanism of pegInfα-induced remission remains elusive. There are several potential mechanisms through which pegInfα may be acting, which include stimulating the immune system in order to more effectively suppress the aberrant PV clones, enhancing the activation of normal hematopoietic stem cells (HSCs), or by selectively suppressing the mutant clones. It has been previously reported that PV patients on pegInfα have an increased number of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in the peripheral blood as compared to untreated or hydroxyurea treated patients (Riley Blood, 2011), which suggests that PegIFNa maybe altering immunity against the mutated clone. However, we have found that interferon treatment leads to increased proliferation of HSCs and myeloid-specific differentiation in mice (Baldridge Nature, 2010). If this finding is also true in humans, it suggests the return to polyclonality after pegInfα could also involve an increase in normal HSC proliferation. In order to address this question, we are studying the effects of pegInfα treatment on the Tregs and HSCs of PV and EV patients, when compared to hydroxyurea or untreated patients. Previously we showed that pegInfα treatment reduced the JAK2V617F allelic burden in 17 out of 32 patients. Of the 13 female patients for which clonality could be assessed, one developed polyclonal hematopoiesis with three-fold reduction of JAK2V617F allelic burden, but one developed polyclonal hematopoiesis during therapy despite no reduction in the JAK2V617F allelic burden, suggesting that pegInfα treatment is able to affect both pre-JAK2V617F clones and JAK2V617F-positive PV clones. We have now assessed changes in the HSC population in response to pegInfα treatment. Upon analysis of bone marrow samples from these same pegInfα or hydroxyurea treated patients, we found that the number of HSCs (CD45+CD34+CD38-) was increased in patients treated with pegInfα. Further we saw a decrease in the percent of quiescent HSCs in the pegInfα treated samples, measured by the percentage of cells in G0, suggesting a more actively proliferating HSC population. In agreement with these data, our RNA analysis of the HSCs showed an increase in the expression of cell cycle genes in response to short-term pegInfα treatment. In addition to this apparent increase in HSC proliferation, we also saw an increase in the number of colonies formed in methocult media from the bone marrow samples of the pegInfα treated patients, suggesting an increase in myeloid specific differentiation. When we analyzed the RNA of patients who had received long-term pegInfα treatment, we saw a transcriptional profile that was indicative of cell death. Taken together, these data suggest a model in which pegInfα treatment is allowing for a return to polyclonal hematopoiesis by inducing cell division and differentiation of normal HSCs, while suppressing the pre-JAK2V617F or JAK2V617F-positive PV and ET clones, possibly by promoting apoptosis or inducing an immune-mediated cell death. Our findings do not exclude other potential mechanisms for salutary effects of pegInfα for treatment of PV and ET (see accompanying abstract by Swierczek et al). Disclosures: Swierczek: University of Utah: No financial compensation , No financial compensation Patents & Royalties.

Vorinostat Impairs Cellular Viability, Differentiation, Redox Homeostasis and Gene Expression in BCR-ABL-Negative Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3222-3222
Author(s):  
Bruno A Cardoso ◽  
Helio Belo ◽  
Antonio Almeida
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloproliferative Neoplasms ◽  
Hdac Inhibitors ◽  
Growth Arrest ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Expression Of Genes

Abstract Background: The classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are characterized by increased proliferation of hematopoietic precursors in the bone marrow resulting in an elevated number of terminally differentiated cells. Despite the recent description of JAK2 activating mutations and other mutations, these do not completely explain the pathophysiology and clinical heterogeneity of MPN. Epigenetic modifications, particularly histone acetylation, play pivotal roles in the pathogenesis of several hematological malignancies, and treatment of such disorders with histone deacetylase inhibitors results cell death and proliferation arrest. Importantly, epigenetic agents have proven to be effective in several hematological malignancies. Aims: HDAC inhibition has demonstrated some efficacy in patients with MPN. In order to investigate the effects of HDAC inhibitors in MPN, we analyzed the impact of Vorinostat on the cellular biology of MPN cell lines and primary bone marrow samples. Material and Methods: MPN bone marrow samples were collected at diagnosis following informed consent in the course of routine clinical laboratory tests. Mononuclear cells were isolated by gradient separation were used for culture experiments and lysed for RNA extraction. RNA extracted from MPN primary samples was used to synthetize cDNA and the transcript levels of genes associated with Apoptosis, Proliferation, Epigenetic modifications and several Signaling pathways were analyzed by quantitative-PCR. MPN primary cells and MPN derived cell lines were incubated with Vorinostat and at different time points the cells were harvest, lysed for gene expression analysis and stained with different antibodies, Annexin-V/PI and DCF-DA to analyze cellular differentiation, apoptosis and Reactive Oxygen Species (ROS) respectively. Results: We performed a targeted-genome wide screen and compared the transcript levels of a defined set of genes between normal bone marrow and MPN primary samples. We identified 9 genes (BIRC3, TNFRSF9, DLL4, IL1B, CDKN1A, FOSL1, CREL, SERPINB9 and EGR1) whose expression increased for at least 4 fold and 2 genes (HIP1 and DTX1) whose expression decreased by at least 0.5 fold in MPN patients relative to normal bone marrow samples. Interestingly, incubation of Vorinostat in MPN cell lines at physiological concentrations increases the expression of such genes, and also the expression of genes associated with apoptosis and growth arrest while decreasing the expression of genes associated with proliferation, growth arrest and JAK-STAT signaling pathway. Regarding cellular physiology, Vorinostat induces apoptosis in MPN cultured cell lines in a time- and dose-dependent manner. Furthermore, incubation of primary MPN bone marrow samples with Vorinostat induced apoptosis, blocked differentiation and also diminished ROS levels in a dose dependent manner. These effects were most marked in the monocytic lineage, a population which expresses the highest levels of ROS. Vorinostat also reduced the levels of GPA and CD61, markers of erythroid and megakaryocytic differentiation, respectively. Summary/Conclusions: Here, we show that Vorinostat incubation impairs MPN cellular differentiation and reduces ROS and cellular viability, possibly through the down-regulation of genes associated with cellular proliferation, particularly the JAK-STAT target genes, and up-regulation of genes important for apoptosis and growth arrest. Interestingly, the genes that we identified to be up-regulated in MPN primary samples relative to normal controls, are further increased by Vorinostat treatment, suggesting that these could act as potential biomarkers for Vorinostat effectiveness in the MPN context. Furthermore, these results hold therapeutic promise as Vorinostat reduced differentiation markers associated with Polycythemia Vera and Essential Thrombocytosis. The observation that Vorinostat is particularly effective against the monocytic lineage is interesting in the context of the recently described role of bone marrow monocytes in the pathogenesis of Polycythemia Vera in mouse models. Our results point towards the potential role of Vorinostat (and possibly other HDAC inhibitors) in the treatment of MPN. This potential would require clinical trials to investigate its efficacy. Disclosures Almeida: Celgene: Consultancy; Novartis: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyer Squibb: Membership on an entity's Board of Directors or advisory committees.

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