Finke, Christy M.

Abstract Background: Systemic mastocytosis (SM) sometimes coexists with an associated clonal hematological non mast cell lineage disease and such an occurrence is recognized by the 2001 World Health organization (WHO) classification system as SM-AHNMD (Leuk Res. 2001 25:603). However, SM-AHNMD has not been further stratified into prognostically relevant subgroups. This was the major objective of the current study, which also provides information on prevalence and relevance of detecting KITD816V and JAK2V617F mutations. 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 cases where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. SM- and AHNMD-components were classified according to WHO criteria. Results: AHNMD sub-classification: Of 342 consecutive cases of SM that were screened, 138 (40%) were identified as having SM-AHNMD (median age 65 years, range 20–87). The most frequent AHNMD was chronic myelomonocytic leukemia (SM-CMML) seen in 38 (28%) patients. 23 (17%) patients had SM associated with myelodysplastic syndrome (SM-MDS), 22 (16%) with hypereosinophilic syndrome/chronic eosinophilic leukemia (SM-HES/CEL) and 33 (24%) with another myeloproliferative neoplasm (SM-MPN). The number of cases with other SM-AHNMD was much lower and included lymphoma 8 (6%), acute leukemia 3 (2%), MDS/MPN 3 (2%), and 8 (6%) cases of multiple myeloma, primary amyloidosis or chronic lymphocytic leukemia. Of the 33 SM-MPN patients, 2 had polycythemia vera, 6 essential thrombocythemia, and 2 myelofibrosis; the remaining 23 patients had an unclassified MPN in association with SM. Clinical characteristics at presentation: The combined incidence of urticaria pigmentosa (UP) was 18%, mast cell mediator release symptoms 28% and constitutional symptoms 62%. 76 patients (57%) had palpable splenomegaly. 99 patients (72%) had anemia, 18 (13%) leukopenia (43% with SM-MDS; p<0.0001) and 76 (55%) thrombocytopenia. 70 (51%) patients had leukocytosis and 63 (55%) displayed circulating immature myeloid cells (78% with CMML; p=0.001). 59 (54%) patients had elevated serum alkaline phosphatase level: SM-CMML 70%, SM-MPN 60%, SM-HES/CEL 40%, and SM-MDS 30% (p=0.01). Where evaluated, serum tryptase was elevated in 92% of the patients (median=75 ng/mL; range=4–1360). BM blasts were ≥5% in 25 patients (22%), including 48% with SM-MDS (p=0.004). 12 of 35 patients tested carried the FIP1L1-PDGFRA mutation (all had SM-HES/CEL; p<0.0001). Disease course and prognostic factors At a median follow up of 15 months (range 0–182), 99 deaths were recorded and the combined median survival was 24 months: SM-CMML 15 months; SM-MDS 15 months, SM-MPN 30 months; and SM-HES/CEL 87 months (p=0.003). Fifteen patients (11%) transformed to acute leukemia or mast cell leukemia. Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and SM-AHNMD sub-type (p=0.007), presence of constitutional symptoms (p=0.04), thrombocytopenia (p=0.01), presence of circulating immature myeloid cells (p=0.006), and hypoalbuminemia (p<0.0001). In SM-CMML, hypoalbuminemia was associated with inferior survival (p=0.03). KIT D816V and JAK2 V617F analysis: Archived BM was available in 88 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 33%: SM-CMML 48%, SM-MDS 21%, SM-HES 8%, and SM-MPN 45% (p=0.03). In patients with SM-HES/CEL (p=0.0009) and SM-MPN (p=0.04), detection of KITD816V was associated with inferior survival. Greater than 1% (range 5–57) JAK2V617F allele burden was detected in 6 patients (9%): 4 in SM-MPN, 1 in SM-CMML and 1 in SM-MDS. Conclusions: The WHO-defined SM-AHNMD comprises prognostically diverse subcategories among which SM-CMML is the most frequent. Survival is favorable in SM-HES/CEL and significantly worse in SM-CMML and SM-MDS. Additional independent predictors of inferior survival include constitutional symptoms, thrombocytopenia, circulating immature myeloid cells, and hypoalbuminemia. In SM-HES/CEL and SM-MPN, BM KITD816V allele burden may influence survival. JAK2V617F occurs infrequently in SM-AHNMD and when it does, it clusters with SM-MPN.

Abstract Background: Previous cytogenetic studies in polycythemia vera (PV) have included a relatively small number of patients (“n” ranging 10–64). In the current study (n=137), we describe cytogenetic findings at presentation and examine their relationship to clinical and laboratory features, including bone marrow JAK2V617F allele burden. Methods: The study consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom bone marrow biopsy and cytogenetic studies were performed at diagnosis. Results I: cytogenetic details At diagnosis: A total of 137 patients (median age, 64 years; 49% females) were studied at diagnosis and had adequate metaphases for interpretation. Cytogenetics were normal in 117 patients (85%) and displayed either a sole -Y abnormality in 5 patients (7% of the male patients), and other chromosomal abnormalities in 15 (11%). The latter included trisomy 8 in five patients, trisomy 9 in three patients, two patients each with del(13q), del(20q), and abnormalities of chromosome 1, and one patient each with del(3)(p13p21), dup(13)(q12q14), and del(11)(q21). At follow-up: Repeat cytogenetic studies while still in the chronic phase of the disease were performed in 19 patients at a median of 60 months (range, 8–198) from diagnosis. Of these, 4 had aquired new cytogenetic clones including 3 with normal cytogenetics at time of initial PV diagnosis. The new abnormalities included del(20q), del(5q), del(1p), chromosome 1 abnormality, and inv(3)(q21q26.2). At time of disease transformation: Leukemic transformation was documented in 3 patients of whom cytogenetic information at the time was available in 2 patients; both patients had normal results at time of initial PV diagnosis and complex cytogenetic abnormalities at time of leukemic transformation. In contrast, among 6 patients with available cytogenetic information at time of fibrotic transformation, the results were unchanged from those obtained at time of diagnosis in 5 patients. ii) Correlation between cytogenetics at diagnosis and JAK2V617F allele burden: Allele-specific, quantitative PCR analysis for JAK2V617F was performed in 71 patients using genomic DNA from archived bone marrow obtained at the time of the initial cytogenetic studies. JAK2V617F mutation was detected in 64 of the 71 (90%) patients; median mutant allele burden was 16% (range 3–80%) without significant difference among the different cytogenetic groups: normal vs. –Y vs. other cytogenetic abnormalities (p=0.72). iii) Clinical correlates and prognostic relevance of cytogenetic findings at diagnosis: Among several parameters studied for significant correlations with cytogenetic findings at diagnosis, an association was evident only for age (p=0.02); all –Y abnormalities (n=5) as well as 13 of the 15 (87%) other cytogenetic abnormalities occurred in patients ≥ 60 years of age. Stated another way, the incidence of abnormal cytogenetics (other than -Y) was 4% for patients younger than age 60 years and 15% otherwise. The presence of abnormal cytogenetics at diagnosis had no significant impact on either overall or leukemia-free survival. Conclusions: Abnormal cytogenetic findings at diagnosis are infrequent in PV, especially in patients below age 60 years. Furthermore, their clinical relevance is limited and there is not significant correlation with bone marrow JAK2V617F allele burden.

7030 Background: JAK2V617F occurs in approximately 50% of patients with essential thrombocythemia. Qualitative studies of mutation analysis have previously reported an association between JAK2V617F and advanced age, higher hemoglobin level, higher leukocyte count, and lower platelet count. A possible association with thrombotic complication has also been considered. Methods: Allele-specific, quantitative PCR analysis for JAK2V617F was performed on 176 patients with ET, using genomic DNA from archived bone marrow, which was collected within one year (n=72), between 1 and 5 years (n=64), or after 5 years (n=40) of diagnosis. Results: JAK2V617F was detected in 96 patients (55%), in whom mutant allele burden ranged from 1% to 100% (median 6.3%). Neither mutational frequency (p=0.37) nor mutant allele burden (p=0.62) was affected by the timing of bone marrow sample collection. Presence of JAK2V617F was significantly associated with higher hemoglobin level (p<0.0001), lower platelet count (p=0.001), higher leukocyte count (p=0.008), increased incidence of venous thrombosis occurring after diagnosis (p=0.02), and older age at diagnosis (p=0.03). All but age retained significance in multivariable analysis. In mutation-positive patients (n=96), JAK2V617F allele burden clustered between 1% and 22% in 94 cases, in whom it correlated directly and significantly with platelet and leukocyte counts, palpable splenomegaly at diagnosis, and venous thrombosis occurring after diagnosis. Conclusions: JAK2V617F allele burden imparts additional phenotypic effects in ET. No significant financial relationships to disclose.

Summary Survival prediction in essential thrombocythaemia (ET) and polycythaemia vera (PV) is currently based on clinically‐derived variables; we examined the possibility of integrating genetic information for predicting survival. To this end, 906 molecularly‐annotated patients (416 Mayo Clinic; 490 University of Florence, Italy), including 502 ET and 404 PV, were recruited. Multivariable analysis identified spliceosome mutations to adversely affect overall ( SF3B1 , SRSF2 in ET and SRSF2 in PV) and myelofibrosis‐free ( U2AF1 , SF3B1 in ET) survival; TP53 mutations predicted leukaemic transformation in ET; “adverse” mutations occurred in 51 (10%) ET and 8 (2%) PV patients. We confirmed the independent survival effect of adverse mutations [hazard ratio (HR) 2·4, 95% CI 1·6–3·5], age >60 years (6·6, 4·6–9·7), male sex (1·8, 1·3–2·4) and leukocytosis ≥11 × 10 9 /l (1·6, 1·1–2·2), in ET, and adverse mutations (7·8, 3·1–17·0), age >67 years (5·4, 3·6–8·1), leukocytosis ≥15 × 10 9 /l (2·8, 1·8–4·2) and thrombosis history (2·0, 1·4–2·9), in PV. HR‐based risk point allocation allowed development of three‐tiered mutation‐enhanced international prognostic systems (MIPSS) which were validated in both cohorts and performance was shown to be superior to conventional scoring systems. Spliceosome mutations enhance survival prediction in ET and PV and identify patients at risk for fibrotic progression. TP53 mutations predict leukaemic transformation in ET.

Abstract Background : Approximately 25% of patients with primary myelofibrosis (PMF) harbor calreticulin (CALR) mutations, which have been associated with longer survival (Klampf et al. NEJM 2013). More than 80% of CALR mutated patients harbor one of two mutation variants: type 1, a 52-bp deletion (p.L367fs*46) or type 2, a 5-bp TTGTC insertion (p.K385fs*47). Recent studies have suggested phenotypic and prognostic differences between these two variants (Tefferi et al. Blood 2014, Leukemia 2014 and AJH 2014). Furthermore, data are emerging that suggest functionally-relevant structural differences between type 1 and type 2 CALR variants, including a higher alpha-helix content of the mutant C-terminus in type 2, compared to type 1 (Eder-Azanza et al. Leukemia 2014). Objectives : We used statistical models to calculate helix propensity for thirty-one unique amino acid sequences that were altered by CALR mutations and used the results to subclassify non-type 1/2 CALR mutations into “type 1-like” and “type 2-like” variants. Subsequently, we examined the prognostic relevance of these subgroups. Methods : Calculation of helix propensity, which is the percentage of residues that are predicted to be involved in the formation of an alpha-helix, was performed using AGADIR, which is a statistical approximation algorithm (Munoz et al. Biopolymers 1997). The helix tendency calculations were performed using conditions of pH 7.0, 5 and 25 °C, an ionic strength of 0.1 M and no N- or C-terminal protection. Results : 532 PMF patients were screened for JAK2, CALR and MPL mutations; the respective mutational frequencies were 58%, 24.6% and 7.3%. Among the 131 CALR-mutated cases, 98 (74.8%) harbored type 1, 15 (11.5%) type 2 and 18 (13.7%) other variants. Based on predicted helix propensity scale, the “other” CALR mutations were subclassified as type 1-like (n=12) or type 2-like (n=6) and respectively grouped with type 1 and type 2 variants, for purposes of phenotypic and prognostic comparisons. The AGADIR-derived predicted helix propensity scale was 29.69 for wild-type CALR and 8.6 or 34.17 for type 1 and type 2 mutant CALR, respectively; accordingly, CALR variants with values that are close to or above the value for wild-type CALR were classified as “type 2-like” (range 26.47-36.12) and those with values close to or below the value for type 1 as “type 1-like” (range 2.11-17.3). Comparison of “type 1/type 1-like” (n=110) and “type 2/type 2-like” (n=21) CALR mutations showed the latter to be associated with higher DIPSS-plus score (p=0.01), EZH2 mutations (p<0.01), leukocyte count >25 x 10(9)/L (p<0.01), higher circulating blast percentage (p=0.02) and palpable spleen size >10 cm (p<0.01). Comparison of “type 1/type 1-like” CALR and JAK2 mutations (n=309) showed the former to be associated with younger age, higher platelet count, lower transfusion need, higher hemoglobin level, lower leukocyte count and lower DIPSS-plus score (p<0.01 for all comparisons). None of these associations was evident during comparison of “type 2/type 2-like” CALR with JAK2 mutations. Survival was similar between patients with type 1 and “type 1-like” (p=0.8) and between type 2 and “type 2-like” (p=0.63) CALR mutations. In contrast, survival was significantly shorter in patients with type 2 (HR 2.4, 95% CI 1.2-4.8) and “type 2-like” (HR 3.2, 95% CI 1.0-10.6), when compared to those with type 1 CALR mutations. Survival was also significantly shorter with “type 2/type 2-like” vs “type 1/type 1-like” CALR mutations (p=0.003; HR 2.5, 95% CI 1.4-4.5) and the difference remained significant when analysis was adjusted for age (p=0.047), ASXL1 (p=0.003) or EZH2 (p=0.001) mutations. Similarly, compared to JAK2-mutated cases (n=309), survival was longer in patients with “type 1/type 1-like” (HR 0.4, 95% CI 0.3-0.5) but not in those with “type 2/type 2-like” (HR 0.9, 95% CI 0.5-1.6) CALR mutations; the difference in survival between JAK2 and “type 1/type 1-like” CALR mutated cases remained significant (P<0.01) when analysis was adjusted for age, ASXL1 or EZH2 mutations or DIPSS-plus score. Conclusions : CALR mutations in PMF might be subclassified into type 1-like and type 2-like variants, based on predicted helical propensity of their mutant C-terminus. The favorable impact of CALR mutations in PMF might be restricted to type 1 or “type 1-like” variants. Disclosures No relevant conflicts of interest to declare.

Abstract Background : Momelotinib is a JAK1 and JAK2 inhibitor that is currently being evaluated in phase-3 clinical trials for the treatment of myelofibrosis (MF) (NCT01969838, NCT02101268). In a phase-1/2 clinical trial of patients with MF, the drug was shown to improve anemia, reduce spleen size and alleviate symptoms (Leukemia 2013;27:1322). In the current study, we considered 100 consecutive patients from the Mayo Clinic who received momelotinib therapy, in order to identify molecular and cytogenetic predictors of treatment response and survival from the time of study entry. Methods : The study patients constituted part of a phase-1/2 clinical trial (NCT00935987) using momelotinib in MF. Details of the protocol, including inclusion and exclusion criteria were previously published (Leukemia 2013;27:1322). Response was determined by the 2006 response criteria published by the IWG-MRT. Data analysis and interpretation was carried out independent of any influence from the sponsor. Results : 100 patients with MF (median age 66 years; 58% males) were treated at the Mayo Clinic between 11/20/09 and 11/10/10. MF type was primary in 64% of the patients and post-polycythemia vera/essential thrombocythemia in 36%. DIPSS-plus risk distribution was 63% high, 36% intermediate-2 and 1% intermediate-1. Red cell transfusion dependency was documented in 49% and palpable spleen size of >5 cm (median 19 cm) in 92%. All patients were annotated for JAK2/CALR/MPL mutations with respective frequencies of 73%, 16% and 7%. Frequencies for other mutations included 42% for ASXL1 (84 evaluable) and 18% for SRSF2(78 evaluable). All patients were evaluable for karyotype, which was unfavorable in 17%. Previous treatment included other JAK inhibitors in 21 patients, thalidomide/lenalidomide/pomalidomide in 31 and hydroxyurea or other cytoreductive agents in 55. Momelotinib was initiated at 100 mg/day in 3 patients, 150 mg/day in 21, 150 mg twice-daily in 20, 200 mg/day in 3, 300 mg/day in 47 and 400 mg/day in 6. The maximum momelotinib dose was 150 mg/day in 6 patients, 150 mg twice-daily in 20, 300 mg/day in 68 and 400 mg/day in 6. Treatment duration was <6 months in 12 patients, 6-12 months in 22, 12-24 months in 19 and >24 months in 47. Genetic correlates of response to momelotinib : Overall response rate was 57% and included no complete remissions (0%), one (1%) partial remission and 57 (57%) clinical improvements (CI). Response rates for spleen/liver and anemia were 43% (91 evaluable) and 44% (68 evaluable), respectively. Transfusion independency was achieved in 25 (51%) of 49 evaluable cases. Spleen/liver response rate was significantly affected by JAK2/CALR/MPL mutational status (34% vs 73% vs 60%, respectively, and 50% in triple-negative; p=0.04), CALR mutated vs unmutated status (73% vs 37%; p=0.009), and unmutated vs mutated ASXL1 (53% vs 29%; p=0.035). Furthermore, all 8 (100%) patients who were CALR+/ASXL1- responded vs 6 (25%) of 24 CALR-/ASXL1+ vs 3 (50%) of 6 CALR+/ASXL1+ vs 16 (43%) of 37 CALR-/ASXL1- patients (p=0.003). Spleen/liver response rates were not affected by SRSF2 mutations (p=0.93) or karyotype (p=0.41). Anemia response rate was not affected by JAK2/CALR/MPL (p=0.81), ASXL1 (p=0.16), SRSF2 (p=0.12) or CALR/ASXL1 (p=0.42) mutational status or karyotype (p=0.65). The number of informative cases for EZH2, IDH, SF3B1 and U2AF1mutations were not adequate enough to warrant similar analysis. Genetic correlates of survival from time of protocol entry : Median follow-up after the institution of protocol drug therapy was 35 months (range 1-49). During this period, 57 (57%) deaths and 12 (12%) leukemic transformations were documented. On multivariable analysis, shortened survival was predicted by the presence of ASXL1 (p=0.01; HR 2.1, 95% CI 1.2-3.8) or SRSF2 (0.01; HR 2.4, 95% CI 1.2-4.8) mutations and the absence of CALR mutations (p=0.03; HR 3.2, 95% CI 1.1-8.9). Karyotype was significant on univariate (p=0.01) but not in multivariable (p=0.6) analysis that included the aforementioned mutations. The prognostic relevance of each specific mutation was DIPSS-plus independent. Conclusions : We demonstrate significant associations between CALR and ASXL1 mutational status and spleen response to momelotinib therapy in MF. We also show that momelotinib therapy did not overcome the inferior survival associated with the presence of ASXL1 or SRSF2 mutations or the absence of CALR mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Background : The 2016 revision to the World Health Organization classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In addition, CMML is characterized by a spectrum of cytogenetic and molecular abnormalities (Patnaik Leukemia 2014). In the current study, we looked for correlations between gene expression profiles (GEP) and morphologic or molecular categories of previously untreated patients with CMML. Methods : 35 patients with CMML were included in the study. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for 28 myeloid-relevant genes (Patnaik Blood C. J.). RNA sequencing (Stranded Total RNA- Illumina, San Diego, CA), was performed on 9 normal BM controls, and 35 CMML samples (25 peripheral blood (PB) and 10 BM). The RNA-Seq data was analyzed using MAPRSeq v.2.0. Normalization and differential expression analysis was performed using edgeR 2.6.2. We selected genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 and a fold change bigger than 2. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). Results: Among the 35 study patients, 75% were males and median age was 71 years (range, 55-87). 21 (60%), 9 (25%) and 5 (15%) patients were classified as CMML-0, 1 and 2, respectively. 16 (45%) had a dysplastic while 19 (55%) had a proliferative phenotype. At a median follow-up of 16 months, 5 (14%) deaths and 4 (14%) leukemic transformations were documented. Mutational frequencies included; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. Abnormal cytogenetics (n=9, 35%) included; -Y 8%, trisomy 8 6%, monosomy 7 3%, del(13q) 3% and monosomal karyotype 3%. i) Differential GEP in PB samples: 25 PB CMML samples were analyzed. Unsupervised GEP demonstrated two unique clusters (figure one); cluster one was enriched with dysplastic CMML (90%) and cluster two with proliferative CMML (64%). Consistent with the difference in the distribution of morphologic categories, in comparison to cluster one, patients in cluster two had a higher white blood count (p=0.003), higher monocyte count (p=0.02) and higher risk prognostication by the Mayo Model (p=0.016); there was no significant difference between the two clusters in terms of age and gender distribution, hemoglobin level, neutrophil and platelet counts, PB or BM blast content, cytogenetic risk stratification, ASXL1, TET2, RAS, CBL and SETBP1 mutational status. Genes significantly upregulated (FDR<1e-5 and fold change ≥ 16x); in cluster two (proliferative CMML) in comparison to cluster one included; PRG2, CTSG, BEX1, CD34, CRISP2, while those significantly down-regulated included (FDR<1e-15 and fold change ≥ 5x); IL2RB, PRF1, GNLY, PDGFRB. IPA analysis identified the following pathways preferentially expressed in cluster two (proliferative CMML) 1) mitotic roles of polo-like kinase (KIF23, WEE-1, PLK1, PLK4), 2) Cell cycle: G2/M checkpoint regulation (CDC25C, WEE-1, AURKA, CDK1), 3) Cell cycle: chromosomal replication (CDC45, RPA3, MCM2, CDC7), and 4) BRCA1 in DNA damage response (BARD1, RBBP8, PLK1, RAD51). Whereas in the cluster one (dysplastic CMML), preferentially expressed pathways included; 1) T-cell receptor signalling (CD247 PRKCQ, CD4, PLCG1, CD28) and 2) STAT3 signalling (SOCS3, MAP3K9). ii) Differential GEP in BM samples: 10 BM CMML samples were analyzed along with 10 normal controls. Samples and controls clustered separately and within the CMML samples two clusters were identified (figure two). These clusters had equal representations of dysplastic (n=5) and proliferative (n=5) CMML sub-types. Additional samples are being processed. Conclusions: Differential gene expression profiling in patients with CMML identifies two unique clusters, clinically demarcated, at least in PB samples, by "proliferative" and "dysplastic" CMML subtypes. The proliferative cluster is enriched in genes involved in cell cycle regulation and DNA damage response; whereas the dysplastic cluster is enriched in T-cell receptor and STAT3 signalling. The prognostic and therapeutic impact of these gene clusters remains to be assessed in a larger group of informative patients. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Al-Kali: Novartis: Research Funding; Celgene: Research Funding.

Abstract Background : Two previous studies have reported significant but inconsistent associations between the presence of JAK2V617F in primary myelofibrosis (PMF) and older age at diagnosis, risk of thrombosis, higher leukocyte count, and inferior survival (Tefferi, et al. BJH2005;131:320, Campbell, et al. Blood2006;107:2098). The clinical relevance of V617F allele burden in PMF has not been previously studied. Methods : Diagnosis of PMF was based on the World Health Organization criteria and study eligibility included the availability of bone marrow-derived DNA that was collected either at time of diagnosis or within one year of diagnosis. Quantitative allele specific PCR was utilized to meaure V617F allele burden. Results I. V617F-positive vs. V617F-negative comparisons: A total of 199 patients (60% males; median age 61 years) were suitable for analysis of comparisons between mutation-positive and mutation-negative disease. The Dupriez prognostic scoring system (PSS) risk distributions were 61% low-risk, 31% intermediate-risk, and 8% high-risk. Hypercatabolic symptoms were present in 27% of the patients and ≥1% peripheral blood (PB) blasts in 37%. At a median follow-up of 23 months (range 0–266), 57 patients (29%) had died, 17 (9%) developed leukemic transformation (LT) and 10 (5%) experienced major thrombosis. V617F mutational frequency was 58%. Univariate analysis identified older age (p=0.0007), platelet count ≥ 100 x 109/L (p=0.05), and PB blast percentage < 3% (p=0.001) as being associated with a positive mutational status; all three variables sustained their significance during multivariable analysis. The presence of the mutation did not affect the incidence of thrombosis (p=0.78), overall survival (p=0.22) or leukemia-free survival (p=0.5). Results II. Clinical correlates of V617F allele burden: Quantitative measurement of V617F allele burden was performed in 129 patients that were divided into four groups: V617F-negative (n=53) and V617F-positive with mutant allele burden in the lower quartile (n=19), middle quartiles (n=38), or upper quartile (n=19) range (median and range of V617F allele burden ratio was 29% and 1% to 74%). Kaplan-Meier plots revealed significantly shortened overall (Figure; p = 0.0008) and leukemia-free (p = 0.01) survival for the lower quartile allele burden group; survival significance was sustained in a multivariable analysis that included the Dupriez PSS. Lower quartile allele burden was also associated with lower leukocyte count (p = 0.003) and presence of hypercatabolic symptoms (p=0.05). Thrombosis incidence was not affected by allele burden. Conclusions: In PMF, patients with a low V617F allele burden, compared to those with either undetectable (i.e. wild-type) or higher allele burden, display significantly shorter overall and leukemia-free survival. In contrast, the presence or absence of the mutation, by itself, does not result in distinct groups that differ significantly in terms of survival, LT, or incidence of thrombosis. These data suggest that a low V617F allele burden in PMF is a surrogate for the development of dominant V617F-negative subclones that are more likely to undergo LT. Figure Figure