Study of the Relation Between the Time Course of the JAK2V617F Allele Burden and the Clinical Evolution in Patients with Myeloproliferative Neoplasms (MPN); A Single Center Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5250-5250
Author(s):  
Marta Anna Sobas ◽  
Manuel Pérez-Encinas ◽  
Celsa Quinteiro ◽  
Teresa González ◽  
Sandra Suaréz ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Initial Increase ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Clinical Evolution ◽  
Clinical Control ◽  
Cytoreductive Treatment ◽  
The Mean ◽  
Jak2v617f Allele Burden

Abstract Introduction: The JAK2V617F mutation is frequent in MPN, however its clinical implication is still in debate. There are few publications that analyze changes in JAK2V617F allele burden. Methods: We performed a single centre study on 65 patients (19 polycythemia vera (PV), 42 essential thrombocythemia (ET) and 4 primary myelofibrosis (PMF)) all of them with at least two JAK2V617F determination separated by minimum 12 months. The JAK2 mutation was determinated in DNA from peripheral blood granulocytes (in 3 cases from bone marrow smear) by MutaScreenTMKit (IPSOGEN). Results: The mean follow-up period was 26 months (range 12–176). In 42/65 (64.6%) patients JAK2 mutation was positive and in 24/42 (57%) changes in JAK2V617F allele burden (>10%) were observed. Interestingly, in 7/18 (39%) patients who showed an increase of JAK2V617F allele burden progression was observed. In this group, one case of PMF with initial increase of JAK2V617F allele burden during transformation to acute myeloid leukemia (AML) and posterior decrease after transformation* and one case of ET with an increase during transformation to myelofibrosis (MF) and posterior conversion to JAK2V617F negativity after allo-HSCT** were observed (table1). 36/42 (85.7%) of JAK2V617F positive patients were under cytoreductive treatment: 30 with hydroxyurea (HDU), 1 anagrelide (ANA) and 5 mixed. There was no decrease of JAK2-V617F allele burden in patients without cytoreductive treatment. Data of patients with HDU treatment are presented in table 2. 3/5 of patients with mixed cytoreduction showed an increase of JAK2V617F allele burden: all of them presented bad clinical control but no data of progression were observed. None of JAK2V617F negative patients (n=23: 1 PV, 2 PMF, 20 ET) became positive for the mutation with the mean follow-up period of 19.37 months. There were no cases of transformation in this group of patients. Conclusions: According to our study, JAK2V617F allele burden can change during the follow-up. Increase (or stability) of JAK2V617F allele burden are more frequent in transformation from ET to PV or MF; in cases of progression to AML we can observe decrease, stability or initial increase with posterior decrease of JAK2V617F allele burden. Decrease of JAK2 allele burden can be related to cytoreductive treatment. Increase of JAK2V61F in patients with cytoreductive treatment can be related to transformation or bad clinical control. More prospective studies are needed to conclude whether changes in JAK2-V617F allele burden could be useful to predict transformation of MPN patients. Table1. Changes of JAK2V617F allele burden and clinical evolution in JAK2V617F positive patients. JAK2 Clinical evolution (N=42) STABILITY PROGRESSION DECREASE 6/42 (14%) 5 1 [1 PV-AML] STABILITY 18/42 (33%) 15 3 [1 PV-MF; 1 ET-MF; 1 ET-AML] INCREASE 18/42 (43%) 11 7 [3 ET-PV; 3 ET-MF (one case with posterior allo-HSCT**); 1 PMF-AML*] Table2. Changes of JAK2V617F allele burden in JAK2V617F positive patients with HDU and no cytoreductive treatment. Treatment JAK2V617F: changes of allele burden DECREASE STABILITY INCREASE Untreated - 4 2 [ET-PV] HDU newly started 2 5 [PV-MF and ET-AML after 14 and 7 years of treatment respectively] 7 [2 ET-PV; PMF-AML*] HDU already treated 3 [PV-AML] 7 6 [3 ET-MF (one case with posterior allo-HSCT**)]

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.

The JAK2 46/1 Haplotype Analysis In Essential Thrombocythaemia and Polycythaemia Rubra Vera Reveals That CC Genotype Is Associated with a Higher JAK2V617F and c-MPL W515 Allele Burden

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1977-1977
Author(s):  
Shameem Mahmood ◽  
Louise Mellish ◽  
Nicholas Lea ◽  
Austin G Kulasekararaj ◽  
Atiyeh Abdallah ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Association Studies ◽  
Statistical Significance ◽  
Jak2 V617f ◽  
Tagging Snp ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Significant Difference ◽  
Jak2v617f Allele Burden

Abstract Abstract 1977 First 2 authors contributed equally. Background: Genomic-wide association studies have identified the germline 46/1 haplotype as a predisposing allele associated with JAK2V617F positive myeloproliferative neoplasms (MPN). The present study analysed data on 856 JAK2V617F positive patients, 326 of which had complete clinical data. Aims: To evaluate the JAK2 46/1 haplotype frequencies, JAK2V617F allele burden, c-MPL 515 mutation and risk of transformation. Methods: Genomic DNA from whole peripheral blood or bone marrow patient samples was analysed as follows: JAK2V617F allele burden by Q-PCR, JAK2 exon 12 mutations by Q-PCR and PCR fragment analysis, MPL W515 L and K mutations by allele specific PCR. The 46/1 JAK2 mutation susceptibility haplotype (46/1) tagging SNP rs12343867 (susceptibility allele C) were analysed by pyrosequencing. Results: The allele frequency for the 46/1 tag SNP rs1234867 in the 856 patients was calculated for the total JAK2V617F cohort (0.48) and the clinical entities ET (0.34) and PRV (0.44) confirming that the 46/1 haplotype is greatly over represented in JAK2V617F MPD patients as compared to published the control population (Wellcome Trust Case Control Consortium (WTCCC) (0.24). The Analysis of the 856 patients demonstrated that JAK2V617F and c-MPL W515L/K mutations co-existed in 16 patients(1.9%), the incidence of c-MPL W515L being twice as common as the c-MPL W515K mutations. There was no correlation between these mutations and age or 46/1 haplotype status. The JAK2V617F allele burden (AB) was lower in the c-MPL mutant patients, the average JAK2AB 31%. 3 out 4 c-MPL patients for which clinical information was available had a diagnosis of ET. No JAK2 exon 12 mutations were found in any of the 859 JAK2V617F positive samples suggesting that co-existing JAK2 exon 14 and exon 12 mutations are extremely rare. The genotypic data in ET patients showed: C/C 12%, C/T 44%, T/T 44% and their respective JAK2V617 allele burden (AB) were 46%, 32%, 29%. The genotype data in PRV patients: C/C 18%, C/T 53%, T/T 28.6% and their respective AB were 47%, 31% and 39%. The median AB was 32% (n=121) for ET and 37% (n=103) for PRV. Within a cohort of 255 patients (ET=138, PRV=117) 4% of ET and 6% of PRV patients transformed to acute myeloid leukaemia or myelofibrosis with no predominant haplotype association. In the ET patients, the median AB was 35%, there was no significant difference in the JAK2 V617F AB between those who transformed or not (p=0.45). Interestingly, on the whole ET group C/C genotype patients were more likely to have an allele burden >50% (p=0.058). In the PRV patients, the median AB was 48%. Again, the C/C genotype, PRV patients were more likely to have an AB>50% (p=0.06), although not reaching statistical significance. Conclusions: The 46/1 haplotype in both clinical entities ET and PRV demonstrated a higher allele burden in the C/C genotype in comparison to the other genotypes. No predominant haplotype predicted the risk of transformation to a more aggressive disease such as MF or AML. The analysis also showed that c-MPL W515K/L mutations can co-exist with JAK2V617F. The c-MPL W515K/L mutations did not exhibit a positive correlation with a preferential 46/1, but was associated with a lower allele burden. No co-existing exon 12 and exon 14 mutations were found, suggesting the rarity of this occurrence. Disclosures: No relevant conflicts of interest to declare.

Identification of Homozygous JAK2V617F Mutation in Unfractioned Blood Samples in a Substantial Proportion of Myeloproliferative Disorders: Long Term Follow-Up Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4644-4644
Author(s):  
Irina Panovska ◽  
Nadica Matevska ◽  
Martin Ivanovski ◽  
Sanja Trajkova ◽  
Aleksandar Stojanovik ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Jak2v617f Mutation ◽  
Study Group ◽  
Hematopoietic Progenitors ◽  
Blood Samples ◽  
Allele Burden ◽  
Long Term Follow Up ◽  
Thrombotic Complications

Abstract The discovery of the activating V617F mutation in the JAK2 tyrosine kinase gene in patients with myloproliferative disorders (MPD) provides a major breakthrough in the understanding of the pathogenesis, proving clonality and securing diagnosis of these diseases. The JAK2 V617F allele is an acquired somatic disease allele that arises in hematopoietic progenitors and confers a selective growth advantage. The mutation has been traced to a primitive stem cell that is capable of erythroid and myeloid differentiation. The data for the potential involvement of the B and T lymphocytes with the JAK2V617F mutation are still inconsistent. We present the results from the study designed the evaluate the prevalence of the JAK2V617F mutation in MPDs patients in our population and to investigate whether MPD patients that carry the JAK2V617F mutation differ in clinical course and outcome from JAK2V617F negative MPD patients. The study group consisted of 64 living MPD patients diagnosed according to standard WHO criteria for diagnosis of MPD (26 patients were diagnosed as polycythemia vera (PV), 34 as essential thrombocythemia (ET), 6 as idiopatic myelofibrosis (MF) and 8 were classified as atypical MPD) with the median follow-up of 7,4 years. DNA samples were obtained from unfractionated blood samples and the frequency of V617F JAK2 mutation was analyzed by allele-specific PCR assay. The mutant allele burden in mutation positive samples was analyzed by DNA sequencing. Our results showed that the JAK2 V617F mutation was present in 79% of patients with PV (36% were homozygous for the mutation), 58% with ET (11% homozygous), 69% with MF (28% homozygous) and in 33% of patients with atypical MPD. The mutant JAK2V617F allele burden was greater than 95% in two PV patients, which in the presence of 27% lymphocytes in the peripheral blood of the patients indicate lymphocytes involvement with the mutation. The high frequency of observed homozygosity for JAK2V617F in our study group was probably due to the long disease duration (median follow-up 11,4 years) and favors the theory of a time dependent increase in clonal dominance. Correlations of clinical and laboratory features at diagnosis and subsequent follow-up, including incidence of thrombo-hemorrhagic events, disease transformation and survival of JAK2V617F-positive and JAK2V617F-negative patients did not reveal significant differences except for the incidence of thrombotic complications. The JAK2V617F positive group had a higher incidence of thrombotic complications (30%) compared with the JAK2-V617F-negative group (14%, P< 0.05). Although we observed a disparity in the incidence of the JAK2V617F mutation in different MPD entities in our population with respect to the expected frequency of JAK2V617F from the literature, our results confirm the diagnostic significance of the JAK2V617F mutation in MPDs and support the notion that patients with this mutation should be classified in a new entity of MPDs. Identification of homozygous JAK2V617F mutation in unfractionated blood samples in a substantial proportion of long term follow-up MPD patients, together with the identification of the mutant JAK2V617F allele burden greater than 95% in two PV patients, suggests that acquisition of the JAK2V617F mutation arises in early multilineage hematopoietic progenitors and warrants further investigation.

Cytoreductive Therapeutic Approach in the Essential Thrombocythemia (ET) Patients of the Registro Italiano Trombocitemia (RIT): Preliminary Data

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5248-5248
Author(s):  
Luigi Gugliotta ◽  
Alessia Tieghi ◽  
Anna Candoni ◽  
Monia Lunghi ◽  
Gianluca Gaidano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Therapeutic Approach ◽  
Jak2 V617f ◽  
Bone Marrow Biopsies ◽  
Thrombotic Risk ◽  
Significant Difference ◽  
Cytoreductive Treatment ◽  
Wbc Count

Abstract Background: the Registro Italiano Trombocitemia, that is a GIMEMA project, has been activated to registry Italian Essential Thrombocythemia (ET) patients, to improve the diagnosis appropriateness (WHO criteria), to verify the prognostic value of the clinical and biological parameters, to evaluate the compliance to the therapeutical Italian guidelines (1), and to create a network for activation of new studies. Objective: this analysis is mainly devoted to describe the ET patients registered in the RIT and to evaluate the therapeutic approach adopted in the 102 participating hematological centers. Material and methods: two thousand and fifteen ET patients have been registered after the written informed consent was obtained, and data validation by various expert panels is in progress. This preliminary report considers 1785 patients, diagnosed mainly (1078, 60.4%) since the publication in the year 2004 of the ET therapy Italian guidelines (1). Results: the patients, 678 (38%) males and 1107 (62%) females, showed at diagnosis: age 60.3 ± 16.8 years with higher values in males than in females (61.7 ± 15.3 vs. 59.4 ± 17.7, p&lt;0.05), being the patients below 40 years 14% and those over 70 years 33% of cases; PLT count (109/L) 846 ± 309 with lower values in males than in females (813 ± 261 vs. 866 ± 334, p&lt;0.002), and with values 1001–1500 and over 1500 in 16% and 4% of cases, respectively; WBC count (109/L) 9.1 ± 2.9, without difference by sex, and with values 12–15 in 10% and over 15 in 3% of cases; Hgb (g/dL) 14.2 ± 1.6 with higher values in males than in females (14.8 ± 1.5 vs. 13.8 ± 1.5, p&lt;0.001), and with values over 16.5 in 8.5% of males and 2.7% of females, respectively; splenomegaly in 488 (27%), echo-documented in 324 cases (18%); history of hemorrhage and thrombosis in 90 (5%) and 325 (19%) of cases, respectively; disease-related symptoms in 41% and general thrombotic risk factors in 93% of cases, respectively. The WHO 2001 diagnostic criteria were reported for 33% of cases observed before the year 2004 and for 53 % of cases observed since the year 2004. Detailed data at diagnosis were reported as follows: bone marrow biopsy in 1087 cases (61%) with a frequency of 51% and 68% before and since the year 2004, respectively; bcr-abl study in 1045 cases (59%); cytogenetics in 828 cases (46%) with karyotype abnormalities in 27 patients (3%). The JAK2 V617F mutation, searched in 574 cases (32%), was observed in 320 of them (56%). The patient follow-up was 4.5 ± 4.5 years with a total of 5245 pt-yrs. During the follow-up the hemorrhagic events were 5.7% (1.3/100 pt-yrs), being the major events 1.9% (0.4/100 pt-yrs); the thrombotic complications were 14.9 % (3.3/100 pt-yrs), resulting the major arterial 9.4% (2.1/100 pt-yrs), the major venous 3.5% (0.8/100 pt-yrs) and the minor thrombosis 2% (0.4/100 pt-yrs). An antiplatelet treatment, almost always with low dose aspirin, was performed in 75% of the patients, without significant difference in the cases diagnosed before and since the 2004. A cytoreductive treatment was done with use of Hydroxyurea (HU, 64%), Interferon alpha (IFN, 16%), Anagrelide (ANA, 15%), Busulfan (BUS, 4%), and Pipobroman (PIPO, 2 %). In the ET patients diagnosed since the year 2004 respect those diagnosed before, it was observed a decrease in the use of all the cytoreductive drugs, particularly BUS (−62%), IFN ((−62%), and ANA ((−68%). The use of the cytoreductive drugs was related to the patient mean age (years): BUS (76), PIPO (72), HU (67), ANA (53), IFN (48). In the patients diagnosed since the 2004 as compared with those before 2004, the mean age of the treated patients increased for BUS (from 69 to 81 yrs, p&lt;0.001) and for HU (from 64 to 69 yrs, p&lt;0.001) while it decreased for IFN (from 49 to 46 yrs, p&lt;0.05). Conclusion: in the analyzed patients of the ET Italian registry the diagnosis appropriateness resulted improved in the cases observed since the year 2004 respect those observed before, with an increase of bone marrow biopsies from 51% to 68% of patients. Moreover, in accord with the ET therapy Italian guidelines, the use of the cytoreductive drugs was less frequent in the patients diagnosed since the year 2004 than before (particularly for BUS, IFN, and ANA) and the more safe molecules IFN and ANA were preferentially deserved to the younger patients.

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.

JAK2V617F Complete Molecular Remission in Long-Term Follow-up of Patients with Polycythemia Vera and Essential Thrombocythemia Treated with Ruxolitinib

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3185-3185
Author(s):  
Lisa Pieri ◽  
Alessandro Pancrazzi ◽  
Annalisa Pacilli ◽  
Claudia Rabuzzi ◽  
Giada Rotunno ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Research Funding ◽  
Jak2v617f Mutation ◽  
Prolonged Treatment ◽  
Molecular Response ◽  
Molecular Remission ◽  
Allele Burden ◽  
Jak2v617f Allele Burden

Abstract Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by the presence of JAK2V617F mutation in >95% and 60% of patients (pts), respectively. This mutation usually affects one allele in ET while most PV pts are homozygous due to mitotic recombination. Acquisition of the JAK2V617F mutation is strongly associated with the germline 46/1 predisposition haplotype. Ruxolitinib is a JAK1/JAK2 inhibitor recently approved for myelofibrosis (MF) and under investigation in PV and ET pts intolerant or resistant to hydroxyurea. We enrolled 24 pts, 11 with PV and 13 with ET, in the phase II INCB18424-256 trial that overall included 34 PV and 39 ET pts. 21/24 pts were still on treatment at 5 years (yr), of which 19 JAK2V617F mutated. Results of the PV cohort have been reported recently (Verstovsek et al. Cancer, 2014): with a median follow up of 35 months (mo), the JAK2V617F allele burden decreased by a mean of 8%, 14%, and 22%, respectively, after 12, 24 and 36 mo. The proportion of pts who achieved a reduction ≥50 % at any time during the 1st yr, 2nd yr, and 3rd yr were 5.9%, 14.7%, and 23.5%, respectively, but no patients achieved a complete remission. In our series of pts we evaluated the JAK2V617F allele burden by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. We also analysed by next generation sequencing (NGS; Ion Torrent platform) a series of MPN-associated mutations including TET2, ASXL1, IDH1/2, LNK, CBL, SRSF2, EZH2 and MPL at baseline and at 5 yr of treatment in ruxolitinib treated pts who achieved a >25% JAK2V617F allele burden reduction at 5 yr (n=13/19). JAK2V617F allele burden decreased by a mean of 7%, 11%, and 19% at 12, 24 and 36 mo, and decreased further by a mean of 28% after 60 mo. Three (1 PV, 2 ET) of 19 pts (16%) achieved a 50% or greater allele burden reduction after 2 yr; no additional pts achieved this degree of allele burden reduction even in prolonged follow up. These 3 pts further improved their molecular response to a complete molecular response (CMR) after 5 yr of treatment. Their mean JAK2V617F allele burden was 46.6% at baseline, 28.3%, 16.3%, 8.7% and 0% after 1 yr, 2 yr, 3 yr and 5 yr, respectively. The JAK2 CMR was confirmed in at least one independent sample at 3 mo after first discovery. At this last timepoint, the PV pt was in complete haematological remission according to ELN criteria, the 2 ET pts were in partial remission due to platelet count still >400x109/L: 422x109/L and 812x109/L, respectively. BM histopathology in the 2 ET pts at 5 yr, while they were in CMR, showed still evidence of megakaryocyte hyperplasia. In the PV pt, histopathology at 5 yr is pending; evaluation at 3 yr, a time when she was in complete hematologic remission and JAK allele burden had decreased from 69 to 8%, showed normalization of cellularity, megakaryocyte and myeloid lineage compared to baseline but still slight erythroid hyperplasia. All 3 pts had normal karyotype at baseline that remained unchanged thereafter. CMR for JAK2V617F was confirmed by NGS. The 2 ET pts achieving CMR did not show any additional mutations, while the PV pts presented a TET2 Y867H mutation with an allele burden of 48.9% and 52%, respectively at baseline and 5 yr. No recurrent mutations in genes other than JAK2 were found in all other examined cases at baseline or at 5 yr. In 3 informative pts, we also analysed the proportion of JAK2V617F homozygous, heterozygous and wild type clones by the method of Hasan et al (Leukemia 2013) based on allelic discrimination of 46/1 haplotype and JAK2. We found that JAK2V617F/V617F clones were reduced by a mean of 95.5%, JAK2V617F/WT showed an uneven trend with a mean reduction of 45.54% while JAK2WT/WT conversely increased (mean 61.43%) at 5 yr, suggesting that in a subset a patients who present significant reduction of VF allele burden ruxolitinib may preferentially target the homozygous clones. Until now, complete molecular remission in PV pts has been described only in patients treated with interferon. Our data suggest that a subset of pts who present a rapid and sustained reduction of the JAK2V617F allele burden under ruxolitinib may eventually reach a condition of CMR with prolonged treatment. However, similar to findings with interferon, mutations establishing clonality, such as in TET2, may still persist in patients who eventually show the disappearance of JAK2V617F mutated subclones. Disclosures Verstovsek: Incyte: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sequential Analysis By Next Generation Sequencing of 18 Genes in Polycythemia Vera and Essential Thrombocythemia Reveals a Association Between Mutational Status and Clinical Outcome after 3-Year Follow-up

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2808-2808
Author(s):  
Damien Luque Paz ◽  
Aurelie Chauveau ◽  
Caroline Buors ◽  
Jean-Christophe Ianotto ◽  
Francoise Boyer ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Poor Prognostic Factor ◽  
Disease Evolution ◽  
Allele Burden ◽  
V617f Mutation

Abstract Introduction Myeloproliferative neoplasms (MPN) are molecularly characterized by driver mutations of JAK2, MPL or CALR. Other somatic mutations may occur in epigenetic modifiers or oncogenes. Some of them have been shown to confer a poor prognosis in primary myelofibrosis, but their impact is less known in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). In this study, we investigated the mutational profile using NGS technology in 50 JAK2 V617F positive cases of MPN (27 PV and 23 ET) collected at the time of diagnosis and after a 3 year follow-up (3y). Patients and Methods All patients were JAK2 V617F positive and already included in the prospective cohort JAKSUIVI. All exons of JAK2, MPL, LNK, CBL, NRAS, NF1, TET2, ASXL1, IDH1 and 2, DNMT3A, SUZ12, EZH2, SF3B1, SRSF2, TP53, IKZF1 and SETBP1 were covered by an AmpliseqTM custom design and sequenced on a PGM instrument (Life Technologies). CALR exon 9 mutations were screened using fragment analysis. Hotspots that mutated recurrently in MPN with no sequencing NGS coverage were screened by Sanger sequencing and HRM. A somatic validation was performed for some mutations using DNA derived from the nails. The increase of a mutation between diagnosis and follow-up has been defined as a relative increase of twenty percent of the allele burden. An aggravation of the disease at 3y was defined by the presence of at least one of the following criteria: leukocytosis &gt;12G/L or immature granulocytes &gt;2% or erythroblasts &gt;1%; anemia or thrombocytopenia not related to treatment toxicity; development or progressive splenomegaly; thrombocytosis on cytoreductive therapy; inadequate control of the patient's condition using the treatment (defined by at least one treatment change for reasons other than an adverse event). Results As expected, the JAK2 V617F mutation was found in all patients with the use of NGS. In addition, we found 27 other mutations in 10 genes out of the 18 genes studied by NGS (mean 0.54 mutations per patient). Overall, 29 of 50 patients had only the JAK2 V617F mutation and no other mutation in any of the genes analysed. No CALR mutation was detected. Nine mutations that were not previously described in myeloid malignancies were found. The genes involved in the epigenetic regulation were those most frequently mutated: TET2, ASXL1, IDH1, IDH2 and DNMT3A. In particular, TET2 mutations were the most frequent and occurred in 20% of cases. There was no difference in the number or in the presence of mutations between PV and ET. At 3y, 4 mutations appeared in 4 patients and 15 out of 50 patients (9 PV and 6 ET) were affected by an allele burden increase of at least one mutation. At 3y, 24/50 patients suffered an aggravation of the disease as defined by the primary outcome criterion (16 PV and 8 ET). The presence of a mutation (JAK2 V617Fomitted) at the time of the diagnosis was significantly associated with the aggravation of the disease (p=0.025). Retaining only mutations with an allele burden greater than 20%, the association with disease aggravation is more significant (p=0.011). Moreover, a mutation of ASXL1, IDH1/2 or SRSF2, which is a poor prognostic factor in primary myelofibrosis, was found in 8 patients, all having presented an aggravation of their disease (p=0.001). Only 4 patients had more than one somatic mutation other than JAK2 V617F and all of them also had an aggravation at 3y (p=0.046). In this cohort, appearance of a mutation at 3y was not associated with the course of the disease. Conversely, the increase of allele burden of at least one mutation was associated with an aggravation (p=0.019). Discussion and conclusion Despite the short follow-up and the limited number of patients, this study suggests that the presence of additional mutations at the time of the diagnosis in PV and TE is correlated to a poorer disease evolution. The increase of mutation allele burden, which reflects clonal evolution, also seems to be associated with the course of the disease. These results argue for a clinical interest in large mutation screening by NGS at the time of the diagnosis and during follow-up in ET and PV. Disclosures Ugo: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: ASH travel.

Long-term serial analysis of X-chromosome inactivation patterns and JAK2 V617F mutant levels in patients with essential thrombocythemia show that minor mutant-positive clones can remain stable for many years

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1241-1243 ◽  
Author(s):  
Rosemary E. Gale ◽  
Anthony J.R. Allen ◽  
Michael J. Nash ◽  
David C. Linch
Keyword(s):  
X Chromosome ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
X Chromosome Inactivation ◽  
Janus Kinase ◽  
Chromosome Inactivation ◽  
Jak2 Mutation ◽  
Clone Size ◽  
Cell Patterns

Abstract Essential thrombocythemia (ET) is heterogeneous with respect to natural history, X-chromosome inactivation patterns (XCIPs), and presence of the V617F mutation in Janus kinase 2 (JAK2). We studied 111 patients with ET; 39% were JAK2 mutant positive, and clone size (percentage mutant JAK2) was concordant with XCIP when constitutive T-cell patterns were taken into account. JAK2 mutant clones were present in both clonal and polyclonal cases as determined by XCIP, and the former had higher mutant JAK2 levels (median 26% versus 16%; P = .001). No change was observed in serial XCIP analysis of 14 polyclonal patients over a median follow-up of 61 months. Furthermore, 18 of 19 mutant-positive patients showed no significant change in mutant JAK2 level over a median follow-up of 47 months. These results suggest that, in many cases of ET, a small stable clone containing a JAK2 mutation can be maintained as a subpopulation for many years.

Similar Rate of Thrombosis in Essential Thrombocythemia and Polycythemia Vera Patients after Stratification for JAK2 V617F Allele Burden.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1745-1745
Author(s):  
Alessandra Carobbio ◽  
Guido Finazzi ◽  
Elisabetta Antonioli ◽  
Paola Guglielmelli ◽  
Alessandro M. Vannucchi ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Vascular Complications ◽  
Jak2 V617f ◽  
Similar Rate ◽  
Wild Type ◽  
Allele Burden ◽  
Real Time Quantitative Pcr ◽  
Venous Thromboembolic

Abstract Patients with Essential Thrombocythemia (ET) can be categorized as either JAK2 V617F mutated (V617F+) or wild type (V617F−). Mutated patients display multiple features resembling Polycythemia Vera (PV), with significantly higher hemoglobin level and neutrophil counts, lower platelet count, more pronounced bone marrow erythropoiesis and granulopoiesis and higher tendency to transform in PV. Presence of the mutation and/or allele burden has been variably associated with the rate of vascular complications in ET and PV, but a direct comparison between the two disorders under this respect has not been performed. To tackle this issue, we compared the rate of major thrombosis in 867 ET patients (57% were JAK2 V617F+) with that in 415 PV patients (all V617F+). The median follow-up was 4.9 (0 – 39) and 3.8 (0 – 26) years in ET and PV, respectively. High risk ET patients (age ≥ 60 years and/or previous thrombosis) received Hydroxyurea whereas the vast majority of low-risk remained untreated. PV patients were treated according to the current risk-stratified recommendations. Thrombotic episodes were recorded over time and calculated as rates % per patient/year (pt/yr). After adjusting for age, the thrombosis-free survival curves of JAK2 V617F+ and V617F− ET patients were superimposable until 10 years after the diagnosis, then they diverged so that the actuarial probability of major thrombosis in mutated ET patients reached that of PV (48% vs 55%, test for trend p=0.05). We found that JAK2 V617F+ allele burden measured by real-time quantitative PCR influenced these rates in a comparable way in both ET and PV. Actually, in JAK2 wild type ET (n=376, 43%) the rate was 1.4% pt/yr. In ET patients with JAK2 V617F+ allele burden ranging from 1 to 25% (N=190; 49%) the rate was 1.9 % pt/yr compared to 1.2 in PV patients (N=64, 19%); in the group with 26–50% the rate was 2.0 % pt/yr in ET (N=177; 45%) and 3.0 in PV patients (N=118, 36%); in cases of V617F+ allele burden greater than 50% the rate was 3.8 % pt/yr in ET (N=23; 6%) and 2.9 in PV patients (N=147, 45%). In conclusion, from this retrospective analysis, we conclude that in patients with ET harboring JAK2 V617F mutation the rate of stroke, myocardial infarction and venous thromboembolic complications is similar to that of PV patients and increases in dependence of V617F allele burden, supporting the hypothesis that ET and PV may be viewed as a continuum also in terms of vascular complications

The Relationship Between Chromosomally Abnormal Hematopoiesis and the JAK2V617F Allele Burden in Patients (pts) with Ph-Negative Chronic Myeloproliferative Disorders (Ph-neg MPD)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3106-3106
Author(s):  
Xialoli Wang ◽  
Amanda Leblanc ◽  
Steven Gruenstein ◽  
Mingjiang Xu ◽  
Nathaniel Wisch ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
Marker Chromosome ◽  
Jak2 V617f ◽  
Jak2v617f Mutation ◽  
Hematopoietic Progenitor Cell ◽  
Proliferative Capacity ◽  
Allele Burden ◽  
Cd34 Cells ◽  
The Relationship ◽  
Jak2v617f Allele Burden

Abstract The JAK2V617F mutation, present in &gt;90% of pts with polycythemia vera (PV) and in approximately 50% of pts with primary myelofibrosis (PMF) and essential thrombocythemia (ET), has been identified in CD34+ cells from pts with PV and PMF, consistent with the notion that this mutation arises in an early hematopoietic progenitor cell (HPC). The relationship between the recurrent chromosomal abnormalities, occurring occurring in 8% of ET, 29% of PV and ~50% of PMF pts, to the JAK2V617F mutation is uncertain. In order to determine a relationship between the JAK2V617F allele burden and the frequency of HPC with chromosomal abnormalities, we identified 5 pts (3 PV, 1 PMF and 1 ET) who were JAK2V617F positive and had different cytogenetic abnormalities. At the time of the study Pt 1 (PMF) had +9, del(20)(q11q13) in 50% of cells and del(20)(q11q13),+der(9)t(1;9)(q21;q13) in 50% cells. Pt 2 (PV) had 85% of cells with +9. Pt 3 (PV) had 15% of cells with del(20)(q11q13). Pt 4 (ET) had 36% of cells with dup(1)(q21q31) and Pt 5 (PV) had +der(9)t(1;9)(q21;q13) in 55% cells. The JAK2V617F allele burden in granulocytes and CD34+ cells ranged from 55% to &gt;95%. HPC (BFU-E, CFU-GM and CFU-Meg) were assayed from CD34+ cells and each individual colony was plucked, split in half, and tested for both the JAK2V617F mutation and the presence of the marker cytogenetic abnormality using FISH. Among the 289 colonies studied, 82% were JAKV617F homozygous, 8% were heterozygous for JAK2V617F and 10% contained only wild type (WT) JAK2. Cytogenetically abnormal colonies, containing the marker chromosome, as detected by FISH, were observed in 80% of the JAK2V617F homozygous, 64% of JAK2V617F heterozygous and 30% of the WT JAK2 colonies. Despite 100% of the colonies being homozygous for JAK2V617F in Pt 3 only 7% of colonies exhibited del(20q). In Pt 1, with two distinct cytogenetically abnormal populations, 2% of JAK2V617F homozygous colonies contained del(20q) alone, while 40% of colonies had both del(20q) and +9p and 52% had a combination of all three abnormalities. Further analysis of Pts 2 and 5 demonstrated that 87% and 47%, respectively, of JAK2 homozygous colonies contained gain of 9/9p. Duplication (1)(q21q31), a characteristic chromosomal abnormality of Pt 4, was identified in 18% of JAK2 WT colonies while its frequency in homozygous JAK2V617F HPC was 76%. These observations suggests that an increased JAK2V617F allele burden is associated with an increased frequency of cytogenetically abnormal HPC. A small proportion of homozygous JAK2 V617F HPC are involved by del(20q) (2%–7%) indicating their low proliferative capacity. Acquisition of trisomy 9 or trisomy 9p either in the presence of del(20q) or alone, involved a much larger population of JAK2V617F homozygous HPC (61%–93%) suggesting their higher proliferative capacity. In colonies containing only WT JAK2, 12% to 26% of cells within colonies had either +9 or dup(1q) suggesting that these chromosomal abnormalities might occur independently of JAK2V617F. These studies suggest a complex relationship between the JAK2V617F allele burden and occurrence of clonal chromosomal abnormalities in Ph-neg MPD HPC which might define important biological differences and clinical outcomes in MPD.

Sign in / Sign up

Export Citation Format

Share Document