Thrombotic Events in Primary Myelofibrosis: Prevalence and Prognostic Factors in 207 Consecutive Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3915-3915
Author(s):  
Michelle Elliott ◽  
Terra Lasho ◽  
Christy Finke ◽  
Animesh D. Pardanani ◽  
Ayalew Tefferi
Keyword(s):  
Platelet Count ◽  
Prognostic Factors ◽  
Mutant Allele ◽  
Primary Myelofibrosis ◽  
Hemoglobin Level ◽  
Snp Genotyping ◽  
Taqman Assay ◽  
Allele Burden ◽  
Vein Thrombosis ◽  
Jak2v617f Allele Burden

Abstract Abstract 3915 Poster Board III-851 Background Thromboembolic disease (TE) is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). There is limited information on the prevalence of TE in primary myelofibrosis (PMF) and associated prognostic factors. Methods Study patients were recruited form the Mayo Clinic database for PMF. Diagnosis was according to the 2001 WHO criteria. Clinical and laboratory information was collected at time of diagnosis and correlated with the occurrence of TE in general and arterial (AT) or venous (VT) thrombosis in particular. Leukocytosis was defined as a leukocyte count of > 10 × 109/L. Qualitative JAK2V617F analysis was done according to previously published PCR-based methods. In addition, a subset of the study patients underwent quantitative PCR for JAK2V617F and rs12343867 SNP genotyping (performed by a commercially available Taqman assay). All molecular studies were performed on stored bone marrow. Standard statistical methods were used to test significance of associations between thrombosis and other variables. Results 207 patients with PMF were studied (median age 62, range 28-87; 132 males). At presentation, the median (range) of hemoglobin (Hb), leukocytes (WBC) and platelets (plt) were 11 g/dl (6.4-15.4), 8.6 × 109/L (0.8-156.7) and 273 × 109/L (13-1916), respectively. Dupriez prognostic scores at diagnosis were low, intermediate and high in 57%, 33% and 10% of the patients, respectively. 130 (63%) patients were JAK2V617F positive. Among 73 patients in whom quantitative JAK2V617F analysis was performed, the mutation was not detected in 30 (41%) patients and the median mutant allele burden in the 43 mutation-positive patients was 28% (range 1-85). The same 73 patients had rs12343867 SNP genotyping performed that revealed C/C genotype in 14 (19%) patients, C/T in 34 (47%) and T/T in 25 (34%). At or before diagnosis, a total of 29 patients (14 %) had TE: 23 (11%) AT and 9 (4%) VT. The AT included acute coronary syndromes (ACS; n=19), transient ischemic attack/ cerebrovascular accidents (TIA/CVA; n=6) and peripheral arterial thrombosis (PAT; n=1). The VT included abdominal vein thrombosis (AVT; n=4), deep vein thrombosis/pulmonary embolism (DVT/PE; n=6) and dural sinus thrombosis (n=1). Several patients had more than one event and some both AT and VT. During follow-up, 22 (11%) patients experienced TE: 17 (8%) VT and 8 (4%) AT. The post-diagnosis VT included AVT (n=8), DVT/PE (n=13) and both DVT/PE and AVT (n=4). Post-diagnosis AT included CVA (n=4), TIA (n=1), ACS (n=2) and PAT (n=1). AT at or before diagnosis was associated with advanced age (p=0.003) but not with sex, leukocytosis, hemoglobin level, platelet count, JAK2 mutational status, JAK2 mutant allele burden or rs12343867 SNP genotype. VT at or before diagnosis also did not correlate with all these variables or age. These variables were also evaluated regarding their relationship with post-diagnosis AT, which showed significant associations with hemoglobin level (p=0.006), platelet count (p=0.04) and high JAK2V617F allele burden (0.03). The corresponding significant variables for post-diagnosis VT were leukocytosis (p=0.02) and high JAK2V617F allele burden (p=0.01). Of note, AT or VT at or before diagnosis did not predict post-diagnosis events. During multivariable analysis, only higher hemoglobin level predicted post-diagnosis AT. Conclusion The current study provides an accurate description of both arterial and venous thrombotic events in PMF. The association between higher hemoglobin and leukocyte counts at diagnosis and post-diagnosis occurrence, respectively, of arterial and venous thrombosis is similar to observations in patients with PV or ET and suggests a potential benefit from early institution of cytoreductive therapy for the appropriate patient groups. Disclosures: No relevant conflicts of interest to declare.

A JAK2 Germline Genetic Variation Is Associated with Shortened Survival in Primary Myelofibrosis: Clinical and JAK2V617F Allele Burden Correlates of rs12343867 SNP Genotype in 132 Cases.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2895-2895 ◽  
Author(s):  
Ayalew Tefferi ◽  
Terra Lasho ◽  
Christy Finke ◽  
Kebede Hussein ◽  
Michelle Elliott ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Cox Regression ◽  
Conflicts Of Interest ◽  
Multivariable Analysis ◽  
Primary Myelofibrosis ◽  
Taqman Assay ◽  
Snp Analysis ◽  
Allele Burden ◽  
Mutational Status ◽  
Jak2v617f Allele Burden

Abstract Abstract 2895 Poster Board II-871 Background: Recent studies have reported a significant association between JAK2V617F acquisition and a specific JAK2 germline haplotype; rs12343867 (located at intron 14 of the JAK2 gene) is one of several SNPs that tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with primary myelofibrosis (PMF) in order to study the clinical and laboratory correlates of the particular JAK2 haplotype and its effect on survival. Methods: Study patients were recruited form the Mayo Clinic database for PMF. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results: 132 patients with PMF were studied (median age 62, range 28-82; 82 males). Risk distribution according to the International Prognostic Scoring System (IPSS) for PMF were low in 39 (29%) patients, intermediate-1 in 41 (31%), intermediate-2 in 25 (20%) and high in 27 (20%). 77 (58%) patients were JAK2V617F positive; median mutant allele burden was 26% (range 1-85) and 21 patients displayed > 50% mutant allele burden. The rs12343867 genotype distributions were C/C 22%, C/T 44% and T/T 34% . The corresponding figures in JAK2V617F positive/negative cases were 31%/9%, 38%/53%, 31%/38% (p=0.01). Among the 21 patients with > 50% JAK2V617F allele burden, 14 (67%) displayed the C/C allele, 2 C/T and 5 T/T. The specific SNP genotype was not significantly affected by age (p=0.33), sex (p=0.46), leukocyte count (p=0.39), platelet count (p=0.09), or IPSS risk category (p=0.63). Patients were followed for a median of 54 months and during this period 73 (55%) deaths were documented. The T/T SNP genotype was significantly associated with shortened survival, compared to either C/C or C/T (p=0.001; Figure). Multivariable analysis showed this association to be independent of IPSS, JAK2V617F mutational status, age or sex. The adverse prognostic effect of the T/T genotype was also apparent when JAK2V617F negative (p=0.01) or positive (p=0.06) cases were analyzed separately. Conversely, JAK2 mutational status or allele burden did not affect survival in patients with T/T or C/C allele. Conclusion: The current study illustrates the non-random haplotype distribution of JAK2V617F in patients with PMF. More importantly, a non-JAK2 haplotype, tagged by the rs12343867 T allele, was associated with inferior survival that is not accounted for by IPSS or JAK2V617F mutational status. These findings, together with previous observations regarding shortened survival associated with low JAK2V617F allele burden, suggest the presence of molecular events in PMF that are more aggressive than JAK2V617F and not necessarily linked to the JAK2 haplotype. Disclosures: No relevant conflicts of interest to declare.

Clinical correlates of JAK2V617F allele burden in essential thrombocythemia

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7030-7030
Author(s):  
J. Kittur ◽  
R. A. Knudson ◽  
T. L. Lasho ◽  
C. M. Finke ◽  
N. Gangat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Venous Thrombosis ◽  
Essential Thrombocythemia ◽  
Mutant Allele ◽  
Leukocyte Count ◽  
Hemoglobin Level ◽  
Thrombotic Complication ◽  
Allele Burden ◽  
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.

The Clinical Phenotype of Patients with Essential Thrombocythemia Harboring MPL 515W>L/K Mutation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 678-678 ◽  
Author(s):  
Alessandro M. Vannucchi ◽  
Elisabetta Antonioli ◽  
Alessandro Pancrazzi ◽  
Paola Guglielmelli ◽  
Simonetta Di Lollo ◽  
...  
Keyword(s):  
Platelet Count ◽  
Essential Thrombocythemia ◽  
Mutant Allele ◽  
Clinical Phenotype ◽  
Primary Myelofibrosis ◽  
Allele Burden ◽  
Long Term Stability ◽  
Blood Smears ◽  
Significant Difference ◽  
Systemic Symptoms

Abstract A 515W>L/K mutation in MPL (MPLmut) has been described in 5–10% of patients (pts) with myelofibrosis (Pikman Y, PloS Med 2006), possibly associated with JAK2617V>F allele. Among 217 subjects with primary myelofibrosis, the 18 MPLmut pts presented a more severe anemic phenotype than MPL wild-type (MPLWT) pts (Guglielmelli P, BJH 2007). MPL 515W>L mutation has been reported also in 4 pts with essential thrombocythemia (ET) (1%) (Pardanani A, Blood 2006). We have collected 13 MPLmut pts in an unselected series of 273 ET pts according to WHO criteria (4.7% of total) to evaluate whether MPL mutation associated with unique clinical characteristics and eventually whether MPLmut ET pts should be re-classified as having WHO pre-fibrotic myelofibrosis. A novel quantitative real-time PCR assay for 515W>L and 515W>K allele in granulocyte DNA has been designed; detection limit was 0.01% for W>L allele and 0.1% for W>K allele. Six pts were 515W>L and seven were 515W>K; 1 pt with W>L and 4 pts with W>K allele had only mutant allele. Mean mutant allele burden was 44(+/−25)% and 59(+/−21)% for W>L and W>K, respectively. Seven MPLmut pts (53%) also harbored JAK2617V>F allele, as compared to 164/260 of MPLWT (63%); they were 2/6 pts with 515W>L and 5/7 with 515W>K. Mean 617V>F allele burden was significantly lower in MPLmut (11+/− 9%) than MPLWT pts (24+/− 17%; P=0.03). There was no difference in age or gender, but median disease duration was longer in MPLmut pts (110 vs 57 months, P=0.001). At diagnosis, platelet count was significantly higher in MPLmut pts (1,113+/− 438x109/L vs 864+/− 302x109/L; P=0.02), while hemoglobin, serum ferritin, LDH level, and leukocyte count were not statistically different. Frequency of pts presenting endogenous erythroid colonies or PRV-1 over-expression was similar among MPLmut (37% and 44%, respectively) or MPLWT pts (48% and 43%). There was no difference in splenomegaly, systemic symptoms, major thrombosis (30% vs 21%) or hemorrhages (8% vs 6%) between MPLmut and MPLWT pts. However, pts with microvessel disease were significantly more frequent among MPLmut (77% vs 34%, P=0.002). Bone marrow (BM) biopsy at diagnosis of MPLmut pts was reviewed in a blinded fashion among 30 random biopsies from MPLWT ET pts. There was no significant difference in total cellularity, erythoid or myeloid lineage beteween MPLWT and MPLmut pts. Megakaryocyte hyperplasia was prominent in MPLmut pts, with Mks being either scattered or in loose clusters similarly to MPLWT pts. However, in addition to typical large Mks, MPLmut pts displayed a discrete number of small-size vWF and/or CD61-pos Mks. BM fibrosis quantification (revised EUMNET criteria) revealed grade 0–1 fibrosis in all but one MPLmut pts, who presented grade 1–2. Finally, there was no evidence of leukoerythroblastosis in blood smears. One pt died after 11 yrs of major thrombosis, none evolved to myelofibrosis. Overall, these data indicate that prevalence of MPL mutation in ET may be higher (5%) than previously reported, but the mutation per se does not associate with a unique clinical phenotype, a part for a higher platelet count and greater occurrence of microvessel symptoms. Increased number of small-size Mks was observed in BM biopsy, but the whole hystologic pattern, as well as long-term stability of disease, did not support alternative diagnosis of pre-fibrotic myelofibrosis.

Influence of the Jak2V617F Mutational Load at Diagnosis on Major Clinical Aspects in Patients with Polycythemia Vera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5-5 ◽  
Author(s):  
Alessandro M. Vannucchi ◽  
Elisabetta Antoniolim ◽  
Paola Guglielmelli ◽  
Alessandro Pancrazzi ◽  
Costanza Bogani ◽  
...  
Keyword(s):  
Platelet Count ◽  
Polycythemia Vera ◽  
Mutant Allele ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Clinical Aspects ◽  
Inverse Regression ◽  
Vascular Events ◽  
Allele Burden ◽  
Tyrosine Kinase 2

Abstract Most patients with polycythemia vera (PV) carry a mutation in the Janus tyrosine kinase 2 gene (JAK2V617F); in 20–30% of them, mitotic recombination leads to homozygosity. It has been suggested that phenotype may be in part dependent on the mutant allele burden and the residual amount of wild-type (wt) allele, since wt JAK2 exherted a dominant negative effect during co-transfection experiments. However, simply differentiating heterozygotes from homozygotes may be misleading, since it does not distinguish between true heterozygosity or homozygosity in a mixed normal background, nor does it provide any measure of the relative contribution of the two alleles; consistently, homozygous progenitors have been found almost invariably in PV patients. The aim of this study was to assess the influence of JAK2V617F mutant allele burden on hematologic and clinical parameters in 116 PV patients diagnosed according to the WHO criteria, for whom a blood sample, collected at, or within six months from, the diagnosis was available. To measure the ratio between mutated and wt JAK2, we employed an ARMS-PCR procedure on RNA, purified from ≥95% pure preparations of PB granulocytes; mutation-specific amplicons were resolved and quantitated using capillary electrophoresis. Detectable amount of JAK2V617F RNA in the granulocytes was found in 96 patients (83%), with a median value of JAK2V617F/JAK2WT ratio of 38% (range, 1 to 100%). JAK2V617F patients were divided into four classes based on the relative amount of mutant allele: 32 patients (28%) had JAK2V617F/JAK2WT ratio of 1–25%, 24 (21%) of 26–50%, 17 (16%) of 51–75%, and 23 (20%) had 76–100% ratio. There was no difference among these classes as concerned the relative frequency of patients, to indicate that the extent of mutant allele at diagnosis is heterogeneous and that acquisition of the highest allele burden is not necessarily a time-dependent event. The hematocrit, leukocyte count, LDH and ALP values were directly related to the relative amount of JAK2V617F RNA ( P&lt;0.0001 for all), while the MCV progressively decreased with increasing JAK2V617F/JAK2WT ratio (P&lt;0.001). There was also a trend to a lower platelet count with the highest JAK2V617F/JAK2WT ratios (P=0.067). The frequency of patients who had PRV-1 over-expression was 8% among JAK2WT, and rose to 53%, 77%, 100% and 100% in the four ratio classes, respectively (P&lt;0.0001); there was also a significant inverse regression between PRV-1 CT levels and the JAK2V617F/JAK2WT ratio (r=−0.596, P&lt;0.0001). Furthermore, the highest JAK2V617F/JAK2WT ratios at diagnosis pointed to patients more likely to have splenomegaly (relative risk (RR) 5.0 to 7.82) or presenting pruritus (RR 1.25 to 5.21) (P&lt;0.0001 for both), and predicted for an exceedingly higher RR of requiring chemotherapy for the control of disease in the follow-up, that ranged from 10.6 to 15.6 in the different ratio classes (P&lt;0.0001). The RR of presenting major thromboses was progressively higher up to 4-times in the 76–100% ratio class compared to JAK2WT patients. A multivariate analysis, including age, leukocytosis, hematocrit, platelet count, and treatment options, indicated that the JAK2V617F/JAK2WT ratio behaved as an independent risk factor for major vascular events (P= 0.027). These data support a meaningful correlation between the proportion of mutant JAK2 allele and the propensity to a more symptomatic disease in PV patients, and foresee the quantitation of JAK2 mutant allele as an approach for patient risk stratification.

SAR302503: Interim Safety, Efficacy and Long-Term Impact on JAK2 V617F Allele Burden in a Phase I/II Study in Patients with Myelofibrosis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3838-3838 ◽  
Author(s):  
Animesh Pardanani ◽  
Jason Gotlib ◽  
Catriona Jamieson ◽  
Jorge E. Cortes ◽  
Moshe Talpaz ◽  
...  
Keyword(s):  
Platelet Count ◽  
Research Funding ◽  
Jak2 V617f ◽  
Intermediate Risk ◽  
Spleen Size ◽  
Safety And Efficacy ◽  
Allele Burden ◽  
Jak2v617f Allele Burden

Abstract Abstract 3838 Background: SAR302503 (SAR503, formerly TG101348), a potent, oral JAK2-selective inhibitor was studied in a Phase I/II trial for the treatment of patients with high- or intermediate-risk primary, post-polycythemia vera (PV) and post-essential thrombocythemia (ET) myelofibrosis (MF). SAR503 was administered orally once daily in 28-day cycles. Eligibility criteria included platelet count of ≥50 × 109/L. Interim safety and efficacy data from this study up to April 2010 have been previously published (JCO 2011, 29(7):789–796). The aim of this presentation is to report updated safety and efficacy of ongoing patients as well as an analysis of the JAK2V617F allele burden in this cohort. Results: Overall, 59 subjects (median age 64 years) were treated. Forty four patients had PMF, 12 post-PV MF and 3 post-ET MF; 86% were JAK2 V617F-positive. Median palpable spleen size was 18 cm at study enrollment. Twenty eight patients were treated in the dose-escalation cohort (30–800 mg administered as a single daily dose); thirty one patients were treated at the MTD (680 mg) in the dose confirmation cohort. 43/59 patients (73%) completed 6 cycles of treatment and continued treatment on the extension study. Currently, 22 patients (37%) remain on treatment with a median number of 28.5 cycles (24–41 range) and a median of last dose of 440 mg/day. Safety: Treatment-emergent toxicities in cycle 1–6 have been previously reported; toxicities were dose-dependent and generally alleviated with dose-reduction. Five patients discontinued treatment beyond cycle 6 for treatment-related adverse events: thrombocytopenia, depression, mental status changes, creatinine elevation and subdural hematoma. For the subgroup of patients with a baseline platelet count between 50–100 × 109/L (n =13; median 73, range 51–94); the platelet count at defined times points during follow up was: cycle 3; median 50, range 21–138 (p=0.09) and cycle 6; median 47, range 13–85 (p=0.01). Despite 7 of the 13 patients being treated at ≥680 mg/day, only 2 instances of Grade 4 thrombocytopenia were noted in this group Spleen response: As previously reported, spleen responses were seen early, usually within first 3 cycles, with half or more patients in each dose level ≥240 mg/day showing a durable ≥50% decrease in palpable spleen size. Spleen size (mean, median, range, and proportion with ≥50% reduction) at the following time points was: Baseline (n=58; 18.33cm, 18cm, 4–38cm, NA) ; 6 months (n=57; 9.05cm, 9cm, 0–30cm, 54.4%;) 12 months (n=42; 8.55cm, 9cm, 0–28cm, 66.7%) 18 months (n=36; 8.03cm, 8.5cm, 0–33cm, 52.8%); 24 months (n=31; 8.10cm, 8cm, 0–30cm, 54.8%,) 30 months (n=18; 6cm, 7.5cm, 0–16cm, 61.1%,and) 36 months (n=9; 5.89cm, 3cm, 0–16cm, 66.7%). JAK2V617F allele burden: We previously reported a significant decrease in JAK2V617F allele burden at the end of cycles 6 and 12. A durable decrease was also demonstrable after 24 cycles of treatment (n =21; median 9%, range 0–100%) relative to baseline (n =51; median 20%, range 3–100%) (p=0.03). Similarly, for patients with JAK2 V617F allele burden >20% at baseline; there was a significant decrease after cycle 24 (n =12; median 21%, range 6–100%) relative to baseline (n =23; median 60%, range 23–100%) (p=0.03). Conclusions: SAR503 is safe and efficacious treatment with long term effect on spleen size and JAK2V617F allele burden in patients with high- and intermediate-risk myelofibrosis. Additional follow up information will be updated at the time of meeting. Disclosures: Jamieson: Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Gao:Sanofi-Aventis: Employment. Zhang:Sanofi-Aventis: Employment. Neumann:Sanofi-Aventis: Employment.

Clinical Implications for Patients with Low JAK2V617F Allele Burden

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1745-1745
Author(s):  
Marguerite Vignon ◽  
Dorota Jeziorowska ◽  
Pierre Hirsch ◽  
Ollivier Legrand ◽  
Nicole Casadevall ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Kinase Domain ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Vein Thrombosis ◽  
Myeloid Neoplasms ◽  
Exon 10 ◽  
Jak2v617f Allele Burden

Abstract Abstract 1745 In classical Philadelphia-negative myeloproliferative neoplasms (MPN), JAK2V617F is considered as a driver mutation when the threshold of 1% JAK2V617F/JAK2total allele burden is reached. However a lower ratio is sometimes detected with highly sensitive assays. We investigated the clinical significance of such minor clones by describing the characteristics of 27 patients with a low JAK2V617F allele burden ranging from 0.1% to 0.99%. Material and Methods A commercially available quantitative ASO-PCR assay of 0.1% sensitivity (MutaQuant® kit, Ipsogen) was used. Two thousand five hundred consecutive blood samples were sent to our lab for JAK2V617F mutation between 2009 and 2012. Total blood DNA was extracted by an automated standardized procedure (Qiasymphony®, Qiagen). All samples were tested in duplicate. The 27 samples of our cohort were controlled using a second assay of 0.01% sensitivity (Larsen et al, BJH 2007). Thirty samples from healthy donors were also tested. High resolution melting curve (HRM) analysis of JAK2 exon 14 ruled out the possibility of an additional mutation hampering the annealing of a primer. Patients with a known classical MPN clinical phenotype were also tested for JAK2 exons 12–17 (entire pseudo-kinase domain) or for MPL exon 10 depending on the context. Results Laboratory Findings Among the 2500 samples, 735 (29.4%) were positive above 1%, 27 (1.1%) had low JAK2V617F allele burden ranging from 0.12 to 0.99%. The patient with the lowest ratio (0.12%) was not confirmed by the second assay and therefore was excluded from the study. This allowed the median to settle at 0.40%. No associated mutations were found in the JAK2 pseudo-kinase domain in patients with polycythemia vera (PV) and in MPL exon 10 in patients with essential thrombocytosis (ET) and primary myelofibrosis (PMF). Healthy patients were all tested JAK2V617F negative. Clinical Aspects The cohort included 19 men and 7 women ranging from 28 to 95 years of age (median 63 years old). Two patients had secondary acute myeloid leukaemia following JAK2V617F positive MPN indicating the presence of residual JAK2V617F cells and the negativity of the myeloblastic population. Thirteen patients (50%) had a classical MPN with a median ratio of 0.36%: 7 ET, 5 PV and 1 PMF according to WHO 2008 criteria. However a bone marrow biopsy was available for only two patients (1 ET, 1 PMF). None of them had received pegylated interferon alpha-2a. Four patients had a prior history of thrombosis: two strokes, one pulmonary embolism, two portal vein thrombosis (PVT). For one PV patient, a 6 months follow-up blood and bone marrow sample confirmed a low allele burden in the same range (0.4%) and in vitro Epo-independant erythroid colonies were observed. Five patients had other chronic myeloid neoplasms (two myelodysplastic/myeloproliferative neoplasms, one chronic eosinophilic leukaemia, one chronic myeloid leukaemia, one refractory anaemia with ring sideroblasts). Among these five, four had an abnormal karyotype. We did not observe any thrombotic event in these patients. We cannot conclude on hematological diagnosis for the last six patients: four patients were screened for JAK2 mutation because of PVT. One patient had chronic polycythemia in a context of alcohol and tobacco abuse. One patient had homozygous hemochromatosis with a normal haemoglobin level in spite of repeated phlebotomies. Discussion In this single centre study low JAK2V617F allele burden represented 1% of all samples sent for JAK2V617F study and 3.5% of JAK2V617F positive patients. Seventeen patients (65%) had classical MPN or splanchnic vein thrombosis. To our knowledge PV patients with such low JAK2V617F allele burden have not been reported in the absence of associated JAK2 pseudo-kinase domain mutation. A larger screen for cooperating mutations responsible for the PV phenotype is under process. In the context of other chronic myeloid neoplasms, the JAK2V617F mutation is thought to belong to a more complex clonal architecture mostly implicating chromatin remodeling genes. Here, the presence of a JAK2 mutation could argue in favour of clonal haematopoiesis. In conclusion the clinical phenotype of low JAK2V617F patients overlaps with classical JAK2V617F MPN. The technical implications might be challenging for molecular diagnostic platforms. More data are needed to further characterize these patients. Disclosures: No relevant conflicts of interest to declare.

Reduced Frequency of CD4+CD25++CD127low/-FOXP3+ Regulatory T Cells in Patients with Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2809-2809
Author(s):  
Margherita Massa ◽  
Rita Campanelli ◽  
Gabriela Fois ◽  
Laura Villani ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
T Cells ◽  
Platelet Count ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Predictive Value ◽  
Primary Myelofibrosis ◽  
Jak2v617f Mutation ◽  
Allele Burden ◽  
Reduced Frequency ◽  
Cd34 Cells

Abstract Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cell, categorized among the Ph-negative myeloproliferative neoplasms (MPNs). Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. However, imbalanced immune responses that lead to chronic inflammation has been hypothesized to play an important role in the pathogenesis of the disease (Hasselbalch HC et al. Leuk Res 2013;37:214-20). Study design. In the attempt to explain the mechanism of immune dysregulation, and to explore how it could influences the disease phenotype, we examined the frequency of circulating CD4+CD25++CD127low/-FOXP3+ regulatory T cells (Tregs), as measured by cytofluorimetric technique, in 193 consecutive patients with PMF and 16 healthy controls (HCs). Results. The percentage of Treg cells between PMF patients and HCs was significantly different [median, 0.87% (range 0% to 10.2%) vs. 2.09% (range 0.54% to 6.5%); Mann-Whitney U test, P<0.001]. Based on the distribution of Tregs in HCs, we established a cut-off point of 1.0% for comparing different groups. By using this cut-off, 106 PMF patients (54.9%) had reduced values of Tregs. We also compared the patients who were on hydroxyurea at the time of study with those patients who were not on the drug. There were no differences between the two groups. Considering the whole population of patients, there was no difference in Treg frequency according to sex, age, spleen size, platelet count, circulating CD34+ cells frequency, degree of bone marrow fibrosis, IWG prognostic score. However, marginally significant association between lower frequency of Tregs and lower value of hemoglobin was evidenced (r=0.14; P=0.06). By contrast, significant differences in Tregs percentage was revealed among the different disease genotypes: Tregs were lower in patients with CALR or MPL mutation than in those with JAK2V617F mutation [median, 0.72% (range 0% to 6.7%), vs. 1% (range 0% to 10.2%); Kruskal Wallis ANOVA, P=0.01)]. Unmutated JAK2V617F genotype was the only significant factor for lower frequency of Tregs in peripheral blood at a linear regression model considering the level of hemoglobin as a covariate. In patients with a JAK2V617F genotype (n=111), no significant association was shown between the percentage of Tregs and hematological or clinical parameters. By contrast, significant difference was found between patients with JAK2V617F allele burden greater or lower than 50%. In patients with low-V617F (<50% allele burden), the median Tregs frequency was 1.11% (range 0.01% to 3.7%) while in patients with high-V617F (≥50% allele burden), the median Tregs frequency was 1.93% (range 0-10.2%) (P=0.05). In patients with JAK2V617F unmutated genotype (n=50), a strong association was shown between Tregs and hemoglobin, documenting that patients with lower frequency of Tregs had the most severe anemia (r=0.60; P<0.001). In these patients, a Tregs percentage lower than 1% predicted a hemoglobin lower than 100 g/L with a positive predictive value of 37%, and a negative predictive value of 100%; thus, no patients with a hemoglobin lower than 10 g/dl had a value of Tregs higher than 1%. Tregs were also inversely correlated with age (R= -0.47; P=0.001), and a lower percentage of Tregs was associated with higher value of white blood cells (r=-0.42; P=0.03), higher value of circulating CD34+ cells (R=-0.43; P=0.03), and lower platelet count (R= 0.33; P=0.034). Conclusions. Collectively, these results indicate that reduced frequency of circulating Tregs represents a hallmark of PMF. Since Tregs play an important role in modulating innate and adaptive immune responses, low Tregs frequency is pivotal for the inflammatory/autoimmune unbalancing of the disease. We documented that the frequency of circulating Tregs was modulated by the disease genotype, having patients with JAK2V617F mutation and high allele burden the highest frequency of Tregs. In patients with unmutated JAK2V617F genotype, the severity of Treg depletion was associated with the severity of the disease. The greatest influence of Treg reduction was exerted on the severity of anemia. These findings support the development of new strategies to increase the number of Tregs in highly symptomatic PMF patients. Disclosures No relevant conflicts of interest to declare.

A Heterogeneous Response Pattern to Interferon-alpha2 with Induction of a Significant Decrease in the Calreticulin Mutant Allele Burden in a Subset of Patients with Essential Thrombocythemia and Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4057-4057
Author(s):  
Sabrina Cordua ◽  
Lasse Kjaer ◽  
Morten Orebo Holmström ◽  
Niels Pallisgaard ◽  
Vibe Skov ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Allele Burden ◽  
Travel Grant ◽  
Ifn Treatment

Abstract Introduction The discovery of mutations in the calreticulin (CALR) gene in the majority of JAK2 -V617F negative patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) (Klampfl et al., 2013; Nangalia et al., 2013) has improved the diagnostic accuracy considerably, and most recently distinct clinical and hematological characteristics according to mutational status have been described (Park et al., 2015). The perspective is to personalize and optimize treatment according to the molecular and clinical landscape. This may be achieved by obtaining more information on responses in myeloproliferative neoplasms (MPN) to existing treatment strategies as assessed by the allele burden. Mutations in the CALR gene have proven to play a major role in oncogenic and immunologic processes (Lu, Weng, & Lee, 2015). In this context, it is highly relevant to explore the effectiveness of interferon-alpha2 (IFN) in reducing the CALR -mutated clone. Until now, only one paper has reported a decrease in allele burden in two patients during IFN treatment (Cassinat, Verger, & Kiladijan, 2014). The objective of this report is to expand current knowledge on this important topic by describing the mutant CALR allele burden over time in a larger group of IFN-treated patients. Method Clinical data were collected retrospectively from a single institution on all IFN-treated CALR positive MPN patients with sequential determinations of the mutant allele burden. Type 1 and type 2 mutations were initially identified by a previously published fragment analysis (Klampfl et al 2013). We have developed a Taqman qPCR assay for precise determination of the mutant allele burden of type 1 and type 2 mutations. Stored DNA was subsequently analysed to increase follow-up time. Results Twenty-one patients were included. Fifteen patients had a diagnosis of PMF; 7 of these were diagnosed with prefibrotic myelofibrosis. Six patients had ET. The type 1 and 2 mutations were found in 15 and 6 patients, respectively. Median age was 60 years (range 42-79) and the sex ratio (M/F) was 8/13. Fifteen patients (71%) were in ongoing treatment with IFN, whereas treatment was discontinued in 6 (29%) because of side effects. Median time of IFN treatment was 756 days (range 42-3927). The IFN prescribed was either subcutaneous injection of Pegasys® (median: 45 microgram (ug) per week), PegIntron® 25-50 ug per week, or Multiferon® 3 x 3 million IU per week. Median follow up time since the first CALR measurement was 756 days (range 294-2108). Fourteen patients (67%) maintained an unchanged allele burden during follow up; 1 patient (5%) presented a temporary decrease (from 39% to 27% in allele burden) but increased to the initial level within months while still on IFN treatment (presumably due to low compliance); 1 patient (5%) displayed an increase in allele burden during transformation to acute myelogenous leukemia (Figure 1); and 5 patients (24%) exhibited a marked decrease in allele burden (median decrease: 32%, range 18-45) during treatment with IFN (Figure 2). All 5 patients with decreasing allele burden (Table 1) normalized their platelet counts within a median time of 5 weeks (range 4-20) after initiating treatment with IFN. Conclusion Using a novel sensitive assay for the CALR mutant allele burden, we have demonstrated and substantiated the effectiveness of IFN to reduce the allele burden in a larger series of CALR positive patients with PMF and ET. Importantly, we report for the first time on highly heterogeneous response patterns. Our observation of one fourth of the CALR positive patients responding to treatment with IFN strongly suggests that IFN significantly influences the CALR mutational load. Further clinical and molecular studies are urgently needed to explore the mechanisms behind the heterogeneous response patterns and the clinical implications in regard to clonal evolution and disease progression in non-responding patients. We are currently analysing these issues to assess the definite role of IFN in future treatment strategies in CALR positive MPN patients. Table 1. Patients responding to interferon-alpha2 Characteristics Number/median (range) Patients 5 Age, years 53 (42-62) Sex (M/F) 1/4 Diagnosis- Essential thrombocythemia- Primary myelofibrosis- Prefibrotic myelofibrosis 221 Calreticulin mutation type- type 1- type 2 50 Duration of interferon-alpha2 treatment, days 960 (177-2790) Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Cordua: Janssen-Cilag: Other: travel grant. Off Label Use: interferon alpha2 for myeloproliferative neoplasms. Holmström:La Roche Ltd: Other: travel grant. Pallisgaard:Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Novartis: Other: travel grant, Research Funding, Speakers Bureau; Roche: Other: travel grant. Hasselbalch:Novartis: Research Funding.

scholarly journals Distinct Clinical, Laboratory, and Molecular Features of Myeloproliferative Neoplasm Patients Presenting with Splanchnic Vein Thrombosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3121-3121
Author(s):  
Joan How ◽  
Stephen T. Oh ◽  
Kathryn M. Trinkaus
Keyword(s):  
Risk Factors ◽  
Board Of Directors ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Continuous Variables ◽  
Disease Phenotype ◽  
Advisory Committees ◽  
Allele Burden ◽  
Vein Thrombosis ◽  
Cell Counts

Abstract BACKGROUND: Myeloproliferative neoplasms (MPN), including polycythemia vera (PV), essential thrombocythemia (ET), and primary meylofibrosis (PMF), are frequently associated with splanchnic vein thromboses (SVT). Risk factors for SVT in MPN patients differ from risk factors for all-cause thrombosis. This is likely due to differing disease mechanisms at play, and suggest a separate disease phenotype for MPN/SVT patients. While several studies have characterized the features of MPN patients with SVT, a direct comparison of MPN/SVT versus all MPN patients has been lacking. METHODS: We performed a retrospective, cross-sectional analysis of patients at Barnes-Jewish Hospital from 2000-2014 with MPN and SVT. Patients were identified using ICD-9 codes in the electronic medical record. 52 available patients with both MPN and SVT were included. Randomly selected 134 patients with MPNs only were used as controls. Clinical and laboratory variables were compared between the two groups. Quantitative JAK2 V617F allele burdens were available in 20 patients. As continuous variables were not normal in distribution, a Mann-Whitney U test was performed. Non-continuous variables were compared with an N-1 Chi-squared test to accommodate rare events. All p-values were corrected for multiple testing. RESULTS: MPN/SVT patients were significantly younger at time of MPN diagnosis (median age 47 vs 57 years, p=0.003). MPN/SVT patients were more likely to have splenomegaly (83% vs 30%, p=0.003), deep vein thrombosis (37% vs 15%, p=0.003), and concomitant thrombophilia (17% vs 2%, p=0.003). MPN/SVT patients had a higher proportion of females (63% vs 54%), but this finding did not reach significance. However, PV/SVT patients had a significantly higher proportion of females compared to PV alone (67% vs 37%, p=0.02). There were no significant differences in JAK2 mutation status, race, smoking status, presence of stroke or coronary artery disease risk factors. MPN/SVT patients had significantly lower hemoglobin (13.1 vs 14.6, p=0.024), hematocrit (39.3 vs 43.5, p=0.027), and platelet count (513 vs 698, p=0.003) at time of MPN diagnosis. When analysis was restricted to PV, only hemoglobin (14.6 vs 17.24, p=0.007) and hematocrit (44.3 vs 50.68, p=0.012) were significantly lower in SVT patients. No significant differences in cell counts were detected in ET and PMF patients. MPN/SVT patients had significantly lower JAK2 mutant allele burdens, with no MPN/SVT patient having an allele burden greater than 10% (p=0.019) (Figure 1). In contrast, mutant allele burdens for MPN patients ranged from 0.1 to 99.7%, with median allele burden being 36.3%. DISCUSSION: This is the first study to directly compare clinical and laboratory features of MPN patients with and without SVT. Our results confirm that MPN/SVT patients are younger, and within PV are more likely to be female. We also demonstrate that MPN/SVT patients have lower cell counts and lower JAK2 mutant allele burdens, findings not previously shown in the literature. MPN/SVT patients are more likely to have splenomegaly, concurrent thrombophilia, and additional DVT. These results indicate that MPN/SVT patients exhibit a disease phenotype distinct from MPN patients without SVTs. While the nature of this study is retrospective and causality cannot be definitively established, the findings of younger age, lower laboratory values, and lower JAK2 allele burden are consistent with the hypothesis that MPN/SVT patients present early in disease. It is possible that in MPN/SVT patients, other environmental and host factors (such as concurrent thrombophilia), in combination with early MPN disease, result in the first manifestation of SVT. These findings have important implications, as investigating the natural course of MPN/SVT patients would allow insight into MPN disease pathogenesis. These findings also suggest that SVTs in MPN patients are not solely mediated by elevated cell counts. In addition, while the presence of the JAK2 V617F mutation likely does affect thrombotic risk, the finding of lower allele burdens in MPN/SVT patients suggests that additional interactions mediate SVT development. These interactions are likely multifactorial and include both environmental and genetic factors. Of particular interest would be the presence of yet unidentified driver mutations present in MPN/SVT patients. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

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