Background. Wt1 expression is thoroughly studied in acute myeloblastic leukemia and widely used for disease response monitoring.Its role in MPN is less known. Aim. The aim of the study was to reveal the incidence of elevated Wt1 expression in PMF and secondary MF, as well as to find out clinical significance during ruxolitinib therapy. Patients and methods. 38 pts were included ( Primary Mf-31, post PV- and post ET Mf- 8, males - 12, females- 26). Wt1 expression in peripheral blood was studied by qPCR at diagnosis and during ruxolitinib treatment using Quiagen kit. Dynamics of Wt1 expression was studied in 20 pts treated by ruxolininib. Spleen size was measured in cm below costal margin. Results. Wt1 increased expression was found in 35/38 pts. Correlation of Wt1 expression and DIPSS was not found. The relation between blast cell and Wt1 expression was studied by dividing pts in 3 subgroups according to the number of blast cells in peripheral blood- 0, 1-2, >2. Wt1 expression was lowest in the 1st group, and the highest in the 3rd group((p<.05). Ruxolitinib treatment resulted in the decrease of spleen size and parallel decrease of Wt1 level( p<.05 -Fig1).Pts with transformation to acute leukemia(5) had higher level of Wt1 than before transformation. Conclusion. Wt1 expression is elevated in the majority of pts with PMF as well as in secondary MF. Correlation was found with blast level and spleen size. Wt1 could be used for monitoring efficacy of ruxolitinib therapy Disclosures Konopleva: Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.
Background: In Chuvash polycythemia (CP) (Problemi Gematologii I Perelivaniya Krovi 1974, 10:30), impaired degradation of hypoxia inducible factor (HIF)-1α and HIF-2α from a homozygous germline VHLR200W mutation leads to augmented hypoxic responses during normoxia (Nat Genet 2002, 32:614). In addition to elevated hematocrit, CP is marked by leg varices, benign vertebral hemangiomas, decreased systemic blood pressure, increased systolic pulmonary artery pressure, and by the defining phenotypes of thrombosis and early mortality (Blood 2004, 103:3924; Haematologica 2012, 97:193). There is no effective therapy. While phlebotomy has been recommended for idiopathic polycythemia by the British Committee for Standards in Haematology (Br J Haematol 2005, 130:174) and is administered to some CP patients, its benefits are unknown. Phlebotomy-induced iron deficiency inhibits PHD2 enzyme, the principal negative regulator of HIFs, which further augments hypoxic responses. This affects the transcription of many genes (BCMD 2014, 52:35). Hypoxia-regulated IRAK1 is augmented in inflammation and may promote thrombosis (Circ Res. 2013, 112:103). Methods: 165 patients with CP were enrolled in a registry between 2001 and 2009 after providing written informed consent. Survival analysis was used to examine the predictors of new thrombosis and death during the follow-up period. mRNA from peripheral blood mononuclear cells (PBMCs) was profiled by Affymetrix Human Exon 1.0 ST Array in 42 of the subjects. Results: The median age at enrollment was 35 years and 90 participants were females, 25 had a history of one thrombosis, 5 of two thromboses and 3 of three thromboses. In the year prior to study entry, 72 had received phlebotomy therapy (Table 1). In July 2015 the median follow-up was 9.0 years (range 1-14.5). During this follow-up period, 30 (18.2%) participants had one new thrombosis, 6 (3.6%) had two new thromboses and 17 (10.3%) died. The median age of death was 55 years (range 16-76) and deaths were related to thrombotic cerebrovascular accident (n = 4), myocardial infarction (n = 4), mesenteric or portal vein thrombosis (n = 3), other major thromboembolic events (n = 2) and trauma or unknown cause (n = 2). Baseline characteristics of older age, prior thrombosis, pentoxifylline treatment, smoking and splenomegaly were independently associated with greater thrombosis risk during follow-up (P < 0.003). After adjustment for these variables, the estimated probability of new thrombosis at 10 years was 26% in those receiving phlebotomies compared to 12% in those not phlebotomized (log rank P = 0.014) (Figure 1). There was also a trend for increased risk of death with phlebotomy: estimated probability 8.7% versus 3.7% (P = 0.15). Examination of gene transcripts affecting thrombosis by logistic regression identified 12 protective and 16 risk genes at 5% false discovery rate. Upregulation of two mRNAs was of singular significance: 1) IL1RAP, a proximal signaling adaptor of IRAK1 (Immunity 1997, 7: 837) and 2) THBS1, encoding thrombospondin1 (Blood 2015, 125: 399). Both genes have known roles in thrombosis promotion and we previously reported that THBS1 is upregulated in CP (BCMD 2014, 52:35). Further analysis revealed a further upregulation of THBS1 in patients with baseline history of phlebotomy (β=0.41, P=0.046). Conclusion: These findings underscore a high rate of thrombosis and death in patients with CP and reveal a potential role of increased IRAK1/IL1RAP signaling in these complications. They raise the possibility that phlebotomy therapy has a detrimental rather than beneficial effect, possibly contributed to by increased THBS1 expression. Table 1. Baseline characteristics by phlebotomy in the year prior to enrollment. Results in median (interquartile range) or n (%); four without phlebotomy data. No phlebotomy N=89 Received phlebotomy N=72 Age (years) 32 (18-48) 37 (26-49) 0.08 Female gender, n (%) 52 (58%) 34 (47%) 0.16 Smoking, n (%) 18 (20%) 24 (33%) 0.060 History of thrombosis, n (%) 20 (23%) 12 (17%) 0.4 Splenomegaly, n (%) 2 (2.3%) 2 (2.8%) 0.8 ASA treatment, n (%) 27 (30%) 36 (50%) 0.011 Pentoxifylline, n (%) 7 (7.9%) 17 (23.6%) 0.005 BMI (kg/m2) 20.4 (18.3-22.9) 21.6 (19.9-24.6) 0.010 Systolic BP (mm Hg) 109 (100-123) 118 (105-124) 0.6 Diastolic BP (mm Hg) 76 (68-84) 78 (71-83) 0.8 Hemoglobin (g/dL) 18.1 (16.4-21.0) 17.9 (16.0-19.8) 0.5 WBC (per uL) 5.7 (4.6-7.0) 5.5 (4.6-6.7) 0.9 Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.
We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi , 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi, 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. Klampfl T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013 Dec 19;369(25):2379-90. Tefferi A. et al. The prognostic advantage of calreticulin mutations in myelofibrosis might be confined to type 1 or type 1-like CALR variants. Blood. 2014 Oct 9;124(15):2465-6. Disclosures Zaritskey: Janssen: Consultancy; Novartis: Consultancy.
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