Low JAK2V617F Research Articles

CD133 Marks a Stem Cell Population That Drives Human Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm of alleged stem cell origin. To define the characteristics of malignant PMF stem cells previous studies have focused on the isolation and xenotransplantation of circulating and/or splenic, PMF patient - derived CD34+ stem/progenitor cells. Despite the reported engraftment of CD34+ cell pool, former analyses failed to reproduce major PMF parameters attributed to abnormal human myeloid cell differentiation. The focus of our work was to identify the stem cell population responsible for initiation and development of PMF. To assess the presence of malignant stem cells we analyzed peripheral blood of 30 PMF patients for expression of LT-HSC antigen CD133. To exclude committed myeloid and lymphoid circulating progenitors we performed lineage depletion of PBMCs and isolated CD133+ and/or CD34+ stem cells. Variable CD133+/CD34 ± and CD133-/CD34+ stem cell fractions from 15 PMF patients were assessed for their clonogenic potential in semisolid media and for reproduction of PMF morbidity in a xenotransplantation mouse model. JAK2V617F mutation was used as a genetic marker to track clonal evolution both in vitro and in vivo. In patients' PBMC we detected the consistent presence of a CD133+ population ranging from 0.3% to >30%, which varies in the expression of CD34. CD133 marks overlapping but also distinct cell populations as compared to CD34. To determine the differentiation potential of disparate stem cell populations, CD133+CD34+, CD133-CD34+ and CD133+CD34- cells were subfractionated from PB of 7 patients and assessed for clonogenic capacity. Strikingly, CD133+CD34+ cells exhibited multipotent, bipotent, and unipotent myeloid (including erythroid) and endothelial-like output, whereas CD133-CD34+ cells gave rise predominantly to lineage-restricted granulocyte/monocyte (GM) progenitors or endothelial-like progenitors. Thus, in contrast to circulating CD133-/CD34+ cells in PMF patients, CD133+ cells have a broader and more robust differentiation capacity to all myeloid cell types, including megakaryocyte /erythrocyte lineages. Four JAK2V617F+ patient samples were used to assess mutation burden at the single-cell level from representative colony types. Obtained results demonstrate an early acquisition of JAK2V617F mutation in the primitive CD133+ stem cell compartments, but also revealed an unexpected variability in the genotypes of emerging progenitors. Homozygous JAK2617F/617F progenitors were detectable in all analyzed patient samples, even if a relative low JAK2V617F burden (30%) was determined from the initial pool of CD133+ cells. A disproportionately high incidence of a homozygous JAK2V617F genotype was observed in erythroid progenitors, indicating a skewing for this lineage. Homozygosity was additionally detected in megakaryocytic and multipotent progenitors. In vivo xenotransplantation experiments of various subfractions confirm the origin of multipotent JAK2V617F+ progenitors from CD133+/CD34± stem cells. Transplantation of PMF patient-derived CD133+/CD34± stem cells in immuno-compromised mice induces abnormal human JAK2V617F+ erythroid, megakaryocytic, and monocytic differentiation, splenomegaly, bone marrow/splenic fibrosis and anemia, reproducing many aspects of PMF development. Our data provide the first evidence for the existence of a CD133+ LT-HSC population responsible for development of PMF. It is for the first time demonstrated that JAK2V617F mutation in PMF occurs at the level of a multipotent stem cell, from which all abnormal myeloid cells emanate during evolution of the disease. Identification of the stem cell compartment involved in the triggering and progression of PMF provides the basis to elucidate the nature of the complex niche interactions in myeloproliferative neoplasms. Disclosures:No relevant conflicts of interest to declare.

Clinical Implications for Patients with Low JAK2V617F Allele Burden

AbstractAbstract 1745In 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 MethodsA 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 FindingsAmong 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 AspectsThe 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. DiscussionIn 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.

A Novel Case of Co-Inheritance of Rare Hemoglobin Variant; Hb Rancho Mirage [βCd 143 (CAC→GAC) His→Asp] with Hb D-Punjab

AbstractAbstract 4759 IntroductionCo-inheritance of Hb Rancho Mirage and Hb D-Punjab produces a clinically significant condition through increasing oxygen affinity that causes a compensatory erythrocytosis. We report a first case of co-inheritance of Rancho Mirage with Hb D-Punjab which differs in its clinical presentation from that described in the literature. Case presentationA 59-year-old Pakistani male was admitted to hospital because of chest tightness. The imaging studies showed no marked organomegaly or masses. Laboratory investigations revealed erythrocytosis with high hematocrit and borderline MCV and MCH. Arterial blood gas testing (ABG) showed low pO2 and JAK2 V617F mutation was not detected. Erythropoietin level was normal. Hemoglobin analysis through high performance liquid chromatography (HPLC) revealed two hemoglobin variants; Hb D-Punjab (32.5%) and the remaining Hb comprised of a fast moving variant. The variant was faster than Hb F expressing at a considerably high level. There was no detectable Hb A. (figure 1, HPLC shows both variants)At the molecular level, the β globin gene was amplified and sequenced using specific primers. DNA analysis demonstrated the presence of two mutations in Exon 3. The first one was a rare mutation in βCd 143 (CAC→GAC) causing a substitution of His→Asp that was identified as Hb-Rancho Mirage. It is speculated that the replacement of the ‘His‘ residue with that of ‘Asp' would unequivocally alter the oxygen binding characteristics of the heme pocket. The other mutation was the common variant defined as Hb D-Punjab βCd 121 (GAA→CAA). (figure 2, DNA sequencing shows both variants). The patient had undergone regular venesection to alleviate erythrocytosis. DiscussionThe co-inheritance of Hb Rancho Mirage with Hb D-Punjab has never been reported previously. Both mutations exist in the β globin gene giving rise to concomitant beta chain variants. Carriers of Hb Rancho Mirage are anticipated with mild anemia. However, coexistence of both abnormal hemoglobins showed functional defects manifested as polycythemia, likely due to the disruption of the 2,3 DPG binding site which is actively involved in the allosteric equilibrium during oxygen transport.The current case depicts the first occurrence of a double heterozygosity for two beta chain variants affecting exon 3 of the β globin gene with a relatively mild to moderate phenotype. Family studies are recommended to understand further the structure and function relationship of this rare hemoglobin variant in association with a more common Hb D- Punjab. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thrombosis History and Relationship with Low Thrombocytosis, Leukocytosis, and Jak2 V617F Mutation In A Cohort of 977 Patients with Essential Thrombocythemia (ET): Preliminary Report of the Registro Italiano Trombocitemia(RIT),

Abstract 3836▪▪This icon denotes a clinically relevant abstract Background.In essential thrombocythemia (ET) patients history of thrombosis and age over 60 y are validated risk factors for thrombosis; the thrombocytosis is a constitutive abnormality associated with both thrombotic and hemorrhagic risk; JAK2 V617F mutation, leukocytosis, and male gender are candidate thrombotic risk factors. Aims.To evaluate in ET patients clinical-biological characteristics at diagnosis and their relationship with thrombosis occurrence before and after diagnosis. Methods.A cohort of ET patients of the Registro Italiano Trombocitemia (RIT) was retrospectively analysed. This preliminary report in detail considers data on thrombosis history. Results.A total of 977 patients, 387 males and 590 females, presented at diagnosis: median age 55 y, median PLT count 783 × 109/L, median WBC count 8.8 × 109/L, median Hb 14.1 g/dL, bone marrow fibrosis (grade >0) in 33% of cases, history of thrombosis in 189 cases (19.3%). The patients at high risk (age over 60 y and/or history of thrombosis) were 511 (52 %). The thrombosis history in univariate analysis was significantly related to: age over 60 y (p 0.001), male gender (p 0.045), low thrombocytosis (PLT <783 x109/L, p 0.001), leukocytosis (WBC >8.8 x109/L, p 0.01), and JAK2 V617F mutation (p 0.006). No relationship was found with the bone marrow fibrosis.The 977 patients, when both PLT count and WBC count at diagnosis were considered, were subdivided in four groups: group 1: 202 pts with low thrombocytosis and leukocytosis; group 2: 270 pts with low thrombocytosis and no leukocytosis; group 3: 272 pts with high thrombocytosis and leukocytosis; group 4: 197 pts with high thrombocytosis and no leukocytosis. In those patients, the thrombosis history was: 26.7% in the group 1(both risk factors); 24.1% in the group 2 (PLT risk factor); 20.2% in the group 3 (WBC risk factor); 7.6% in the group 4 (no risk factors). The rate of thrombosis history in patients with one or two of these risk factors was significantly (p 0.001) higher than in patients with no risk factors. The thrombotic events during the follow up (4088 pt-y) are still object of analysis. Conclusion.In this cohort of ET patients the rate of thrombosis history in univariate analysis was related to age over 60 y. Moreover, a significant relationship was found with PLT count at diagnosis below the median value (783 x109/L), WBC count at diagnosis over the median value (8.8 x109/L), JAK2 V617F mutation, and male gender. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact of EZH2 and ASXL1 Mutation in Myelofibrosis

Abstract▪2811▪This icon denotes a clinically relevant abstractAmong chronic myeloproliferative neoplasms (MPN) myelofibrosis (MF) has the worst prognosis. The International Prognostic Score System-IPSS reliably discriminates four categories of patients (pts) with different survival (OS); however, the possibility to identify pts with very high-risk disease, based on intrinsic characteristics of the underlying disease, would be particularly useful for therapeutic choices that include early stem cell transplantation. Several studies have addressed the prognostic value of molecular abnormalities harbored by MF pts, but a part from a negative role of low JAK2V617F allele burden on survival independently reported by the Mayo Clinic and our own group, none of them impacted significantly on survival. Here we have genotyped at diagnosis 370 pts with primary MF (PMF) and 148 with post-polycythemia vera/essential thrombocythemia MF (PPV/PET-MF) for mutations of EZH2 and ASXL1. EZH2 is the catalytic component of polycomb repressive complex 2 involved in trimethylation of H3 lysine 27, while ASXL1 is a member of enhancer of trithorax and polycomb group required for repression of HOX genes through deubiquitination of histone H2A. All 20 EZH2 exons were screened by high-resolution melting followed by direct sequencing whereas direct sequencing was used for ASXL1 exon 12 genotype. Pts median FU was 39 mo (1–340); 128 pts died (25.9%), 81 (18.3%) because of leukemia (AL). Among PMF pts survival differed significantly according to the IPSS category and was also predicted by the lowest V617F quartile.EZH2 mutation. We found 22 PMF (6%), 1 PPV-MF (1.2%) and 6 PET-MF (9.4%) mutated pts, harboring 25 different mutations, 20 exonic (heterozygous in 83%), 5 intronic. Concurrent JAK2 V617F, MPL W515L/K, IDH1/2, TET2, CBL and ASXL1 mutations were found in 41.4%, 0%, 0%, 6.9%, 3.4% and 20.7% of EZH2 -mutated pts. In multivariate analysis EZH2 mutation was associated with leukocytosis (12.3±13.2 vs 18.7±11.5×10e9/L) in PMF pts, but no additional clinical/hematological correlation was discovered. EZH2 mut pts clustered preferentially in the IPSS high-risk category (52.6%; P=.002). More EZH2 mut than wt PMF pts died (51.9% vs 24.4%; P<.001); median OS was shorter in EZH2 mut pts (32 vs 137 mo, P=.001). Leukemia occurred in 31.8% of EZH2 mutated and 17.6% of wt pts; leukemia-free survival (LFS) was shorter in EZH2 mut PMF pts (25.7 vs 63.6 mo; P=.028).ASXL1 mutation. In a total number of 334 evaluated patients we found 38 different mutations in 49 pts (14.7%), 35 PMF (15%), 5 PPV-MF (8.6 %) and 9 PET-MF (21.4%). Mutations were frameshift, non sense and missense mutations, heterozygous in 46 of 49 cases. Co-expression of JAK2 V617F, MPL W515L/K and EZH2 mutation was found in 55.1%, 4.1% and 12.2%, respectively. Co-espression of ASXL1 and IDH1/2, TET2 or CBL was observed in two, one and one of the evaluable subjects, respectively. ASXL1 mutation was associated with leucocytosis (P=.006), a blast count >1% (43.8% vs 16.6%, P<.0001) and constitutional symptoms (P=.027). ASXL1 mutated pts clustered preferentially in the IPSS high-risk category (46.4%; P<.0001). OS was significantly shortened in ASXL1 mutated PMF patients (median= 87.6 mo vs 264; P=.004); leukemia occurred in 34.9% of ASXL1 mutated and 15.2% of wt pts (P=.002). LFS was shorter in ASXL1 mutated PMF patients [137 mo than wt 264 mo, (P=.01)].According to EZH2 and ASXL1 mutational status, median OS was significantly shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (34 vs 116 vs 198 vs 272 mo, respectively. P<.0001). On multivariate analysis, OS was predicted by IPSS score (P<.001), ASXL1 (P=.016) and EZH2 mutational status (P=.011). Leukemia-free survival (LFS) was shorter in double EZH2/ASXL1 mutated PMF pts compared with single EZH2 or ASXL1 mutated or WT patients (25 vs 153 vs 138 vs 264 mo; P=.002), but the low number of cases (n=6) prevented multivariate analysis.No impact of EZH2 or ASXL1 mutations on OS or LFS was seen in PPV/PET-MF.Overall, these results showed that mutations in genes involved in epigenetic gene regulation such as EZH2 and ASXL1 negatively impact on OS and LFS in pts with PMF, suggesting that screening for these mutations might be useful for risk stratification and treatment decisions. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria.

Genetic Predisposition for Chemotherapy-Induced Neuropathy in Multiple Myeloma

INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med 363(12):1117-1127, 2010 4. Pardanani A, Gotlib JR, Jamieson C, et al: Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol 29:789796, 2011 5. Santos FP, Kantarjian HM, Jain N, et al: Phase 2 study of CEP-701, an orally available JAK2 inhibitor, in patients with primary or post-polycythemia vera/ essential thrombocythemia myelofibrosis. Blood 115:1131-1136, 2010 6. Verstovsek S: Therapeutic potential of Janus-activated kinase-2 inhibitors for the management of myelofibrosis. Clin Cancer Res 16:1988-1996, 2010 7. Barosi G, Bergamaschi G, Marchetti M, et al: JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis. Blood 110:4030-4036, 2007 8. Guglielmelli P, Barosi G, Specchia G, et al: Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele. Blood 114:1477-1483, 2009 9. Tefferi A, Lasho TL, Huang J, et al: Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival. Leukemia 22:756-761, 2008 10. Mesa RA: How I treat symptomatic splenomegaly in patients with myelofibrosis. Blood 113:5394-5400, 2009 11. Steensma DP, Mesa RA, Li CY, et al: Etanercept, a soluble tumor necrosis factor receptor, palliates constitutional symptoms in patients with myelofibrosis with myeloid metaplasia: Results of a pilot study. Blood 99: 2252-2254, 2002 12. Jedidi A, Marty C, Oligo C, et al: Selective reduction of JAK2V617Fdependent cell growth by siRNA/shRNA and its reversal by cytokines. Blood 114:1842-1851, 2009 13. Mesa RA, Kantarjian H, Tefferi A, et al: Functional assessment of performance status in patients with myelofibrosis (MF): Utility and feasibility of the 6-minute walk test (6MWT). J Clin Oncol 27:376s, 2009 (suppl; abstr 7083)

Janus-Activated Kinase 2 Inhibitors: A New Era of Targeted Therapies Providing Significant Clinical Benefit for Philadelphia Chromosome–Negative Myeloproliferative Neoplasms

Myelofibrosis (MF; either primary or postpolycythemia vera/ essential thrombocythemia) is a chronic and debilitating myeloproliferative neoplasm (MPN) for which there is no well-accepted therapeutic standard of care. Clinical signs of this disease, cytopenias (primarily anemia), splenomegaly, and constitutional symptoms (eg, hypercatabolic state, fatigue, night sweats, fever, bone pain) create significant treatment challenges. Common treatments are palliative, are frequently ineffective, and do not alter the natural course of MF. No agent has yet been approved by the US Food and Drug Administration for therapy of MF. Starting in 2005, mutations that directly or indirectly led to deregulated activation of nonreceptor tyrosine kinase (TK) Janusactivated kinase 2 (JAK2) have been implicated in the pathogenesis of MPN, including JAK2V617F and JAK2 exon 12 mutations, thrombopoietin receptor mutations, and mutations in the adapter protein LNK. These mutations activate the JAK2–signal transducer and activator of transcription (STAT) intracellular signaling pathways, which lead to increased cellular proliferation and resistance to apoptosis. These discoveries spurred the development of molecularly targeted agents (JAK2 inhibitors) as therapy for MPNs. The results of several studies of JAK2 inhibitors in MF (INCB018424, TG101348, and CEP701) have been reported in peer-reviewed journals. JAK2 inhibitors in clinical development exhibit differential inhibitory activity against the JAK family members, and some exhibit effects on other receptor kinases and therefore are not selective for JAK2 TK. For example, INCB018424 inhibits JAK1, whereas CEP701 and TG101348 inhibit FLT3. JAK2 inhibitors are small molecules that act by competing with adenosine triphosphate for the adenosine triphosphate–binding catalytic site in the TK domain. The V617F mutation locates outside the TK domain of JAK2. Therefore, the current JAK2 inhibitors target both wild-type and mutated JAK2 indiscriminately. This could explain why these drugs are active in patients with both wild-type and mutated JAK2. However, targeting the wild-type JAK2 is expected to lead to myelosuppression as a result of the exquisite signaling through JAK2 of thrombopoietin and erythropoietin receptors in normal hematopoiesis. This probably explains the reported therapy-related anemia and thrombocytopenia observed with JAK2 inhibitors in clinical trials. Some common features have emerged from current clinical studies. The primary therapeutic benefit with JAK2 inhibitors appears to be a reduction in splenomegaly (and hepatomegaly in those with enlarged livers) with significant improvement in the quality of life. In an intent-to-treat analysis, responses are seen in patients with and without JAK2V617F mutation; at 6 months, a 50% or greater reduction in splenomegaly (“Clinical Improvement” by the International Working Group for Myelofibrosis Research and Treatment response criteria) was seen in 52% of patients treated with optimal dose/schedule of INCB018424 and in 45% of patients treated with the maximumtolerated dose of TG101348. This benefit is usually seen within the first 2 months of therapy and is durable beyond 1 year of therapy. No significant improvement in bone marrow fibrosis has been reported so far, and the effect on the JAK2V617F allelic burden varied among studies. CEP-701 caused no significant change in the allelic burden in patients with clinical response. In 34 evaluable patients, INCB018424 suppressed allelic burden by 13% after 12 cycles of therapy. Similarly, TG101348 suppressed allelic burden in the study cohort (median 20% at baseline decreased to 19% after 12 cycles; P .01) but more so in the patients with baseline allelic burden of more than 20% (median 60% at baseline decreased to 32% after 12 cycles; P .002). Assessment of the clinical significance of these findings is confounded by at least two issues. First, there are no standardized, reference tests to measure JAK2V617F allelic burden in patient samples; different studies use different tests with different sensitivity and specificity, different controls, and different cells (mononuclear cells v granulocytes) as a source material. Second, the clinical relevance of JAK2V617F allelic burden in MF is not clear, and the significance of its modification during therapy is therefore unknown. One study reported that patients with MF and high allelic burden had higher hemoglobin and WBC counts, developed massive splenomegaly, and had higher risk of leukemic transformation. Two other studies reported that patients with MF and low JAK2V617F allelic burden had lower hemoglobin levels, a low incidence of splenomegaly, and worse survival either resulting JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L S VOLUME 29 NUMBER 7 MARCH 1 2011

Minimal residual disease and normalization of the bone marrow after long-term treatment with alpha-interferon2b in polycythemia vera. A report on molecular response patterns in seven patients in sustained complete hematological remission

Polycythemia vera (PV) is characterized by the presence of the JAK2V617F mutation in virtually all patients. Several studies have shown that the JAK2V617F mutational load decreases during treatment with alpha-interferon 2. We report on molecular and histomorphological bone marrow responses in seven PV patients with profound molecular responses during and after long-term treatment with alpha-interferon 2b. All patients obtained a major molecular response (MMR). Subsequently all patients discontinued alpha-interferon and sustained complete hematological remission with a follow-up period of median 10 months (range 4–30 months). Complete normalization of the bone marrow was seen in three of five patients. Long term treatment with IFN2b is able to induce 'minimal residual disease' with very low JAK2 V617F allele burden and may induce profound, and in some patients total, regression of histomorphological bone marrow features of PV. Finally, hematological remissions and major molecular responses can be sustained after discontinuation of long-term treatment with IFN2b.

Normal Serum-Erythropoietin (S-epo) Level at Diagnosis of Polycythemia Vera (PV) Correlates with LowJAK2V617F Mutant Allele Burden and Indicates Mild Phenotype.

Abstract 4978A low serum-erythropoietin (S-epo) level is a minor criterion of the World Health Organization (WHO) recommendations for diagnosing polycythemia vera (PV) even though previous studies indicate that a normal level does not always rule out PV. We wished to determine whether a normal S-epo level correlates with a low JAK2V617F mutant allele burden and a low phlebotomy (PHL) rate, since the relationship between S-epo level, JAK2V617F allele burden, and PHL rate heretofore has not been reported.At diagnosis, we grouped the S-epo levels of 26 PV patients (pts) as follows: low (<5 U/l), normal (5-10 U/l), and high (>10 U/l). Of the 26 pts, 4 (15.4%) had normal S-epo levels, and 22 had low S-epo levels. The diagnosis of PV in pts with normal S-epo levels was made by quantitative JAK2V617F analysis and elevated Cr51 red cell mass (RCM). All pts had a bone marrow biopsy consistent with PV.We determined the number of phlebotomies required to maintain normal hematocrit (hct) levels ('42% women, '45% men) as an assessment of disease severity during the period prior to myelosuppressive therapy. We then examined the number of phlebotomies per year (PHL/yr) in relation to S-epo level at diagnosis. Of the 26 pts, 14 were treated with PHL-only for a mean of 15 months (mos) prior to beginning other therapies including anagrelide, hydroxyurea, imatinib, and rIFNa-2b. The number of PHL/yr were grouped into 4 categories: 0 (none), 1 – 5 (low), 6 – 10 (moderate), and >10 (high). Of the 14 pts, 3 had a normal S-epo level at diagnosis, all of whom had a low PHL requirement, median 3/yr. The other 11 pts had low S-epo levels at diagnosis and a median PHL requirement of 6/yr (7 moderate and 4 low). Thus, while a low S-epo level at presentation was usually associated with moderate PHL requirement, a normal S-epo level at diagnosis was always associated with a subsequently low PHL requirement during the period of observation (mean 15 mos).Of the 26 pts, 14 had quantitative JAK2 analyses done at diagnosis. The other 12 were diagnosed prior to 2002 and quantitative JAK2 determinations were not available. Of the 14 pts, 2 had a normal S-epo level and a low JAK2V617F mutant allele burden (1-25%). These 2 pts also had a low PHL requirement. Thus, these pts who had normal S-epo levels, had JAK2V617F allele burdens in the lowest quartile, and had low PHL rates. Of the remaining 12 pts, 5 had JAK2V617F allele burdens in the 2nd (25-50%) quartile, 5 in the 3rd (50-75%) quartile, and 1 each in the 1st and 4th quartiles.We conclude (1) as previously reported, about 15% of pts with PV present with normal S-epo levels at diagnosis, suggesting a limitation of this WHO criterion, and (2) those pts with a low JAK2V617F allele burden and a normal S-epo level required fewer PHL/yr, all suggesting a more benign phenotype. This may provide a useful prognostic tool for patients with PV. DisclosuresNo relevant conflicts of interest to declare.

A Novel, Highly Sensitive, Rapid, Non-PCR Based Method for the Detection of the JAK2 V617F Mutation in Chronic Myeloproliferative Neoplasms.

Abstract 3892Poster Board III-828 BackgroundThe point mutation G1849T (V617F) of the JAK2 gene occurs at high frequency in several Ph-negative chronic myeloproliferative neoplasms (Ph-neg-CMNs), such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF). The molecular analysis of this mutation is mandatory in the diagnostic work up of these diseases. Several molecular diagnostic techniques are currently used but each of them present some important limitations such as a low sensitivity and the requirement of labor intensive procedures performed with expensive specialized equipment that may not always be readily available in clinical laboratories. MethodWe have developed a non-PCR method for the identification of the JAK2V617F mutation called Allele Specific (AS)-LAMP, based on the Loop mediated isothermal AMPlification (LAMP) principle (Notomi et al. NAR 2000). LAMP reaction efficiently produces, within one hour, a large amount of amplified DNA and does not require gel separation of the amplified product which is indirectly detected by measuring the loss of intensity of a light beam through the reaction solution in which suspended particles of magnesium pyrophosphate are generated as a result of the DNA amplification process. Pyrophosphate salts produce turbidity which is both visible to the naked-eye and monitorable in Real-Time turbidimetry. AS-LAMP consists of 4 primers suitable for LAMP and a self-annealed primer highly specific for the mutated target sequence. To ensure efficiency and sensitivity a Peptide Nucleic Acid (PNA) probe specific for the wild-type allele was added thus resulting in absence of normal allele amplification within the reaction time. The AS-LAMP assay was optimized on plasmid controls and on human genomic DNA extracted from the HEL and K562 cell lines, respectively carrying or not the JAK2V617F mutation. The level of sensitivity was determined by testing serial dilutions of mutant HEL DNA in K562 DNA at concentrations of 100, 10, 1, 0.5, 0.1, 0.05, 0.01 and 0%. ResultsThis simple, easy to perform and rapid AS-LAMP assay selectively detects the JAK2V617F mutated DNA down to 0.05%. Moreover, when mutant DNA is present in the range of 1%-100% in wild type DNA, we observed a linear relationship between the mutant allele burden and the amplification time. We have validated this AS-LAMP assay on DNA obtained from 87 patient samples previously analyzed by conventional Allele Specific PCR (ASO-PCR): 19 PV, 58 TE, 3 IMF, 1 post ET Acute Myeloid Leukemia (AML), 1 post PV and 1 post ET Myelofibrosis, 2 Idiopathic Erythrocytosis (IE) and 2 unclassified CMNs. All samples which proved positive by ASO-PCR resulted positive with our AS-LAMP assay (100% concordance). In addition, 6 ET and 1 IE previously found negative by ASO-PCR were found to be low-positive (<1%) with AS-LAMP. Interestingly, the molecular monitoring in one patient with post-PV MF achieving complete remission after allogenic transplantation, proved repeatedly negative by ASO-PCR but positive by AS-LAMP. Sequencing analysis after PCR amplification with PNA confirmed the presence of the JAK2V617F mutation in all these LAMP-low-positive samples. None of the negative controls, (1 AML, 2 Acute Lymphoblastic Leukemia, 2 Follicular Non Hodgkin's Lymphoma, 2 Chronic Lymphocytic Leukemia, and 1 healthy donor) gave false positive results. ConclusionsThis novel, non-PCR based allele-specific LAMP assay is rapid and reduces the risk of contamination related to post amplification manipulations. Most importantly, it is highly specific and sensitive and significantly increases our ability to detect a low JAK2V617F tumor allele burden. For all these reasons, the AS-LAMP assay can be a valid and powerful tool in the routine diagnostic work up and the molecular monitoring of these diseases. Disclosures:Minnucci:Diasorin S.p.A.: Employment. Amicarelli:Diasorin S.p.A: Employment. Salmoiraghi:Diasorin S.p.A.: Consultancy, Honoraria. Spinelli:Diasorin S.p.A: Consultancy, Honoraria. Adlerstein:Diasorin S.p.A.: Employment. Rambaldi:Diasorin S.p.A.: Consultancy, Honoraria.

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.

Abstract 2895Poster 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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele

AbstractA total of 186 patients with primary myelofibrosis (PMF) were genotyped for JAK2V617F at diagnosis aimed at analyzing the correlation of mutational status and mutated allele burden with outcome variables, including time to anemia, leukocytosis, leukopenia, thrombocytopenia, massive splenomegaly, leukemia, and with overall survival. A total of 127 JAK2V617F-mutated patients (68% of whole series) were divided in quartiles of V617F allele burden. After a median follow-up of 17.2 months, 23 patients died, 15 because of leukemia. A JAK2V617F mutated status did not impact on the rate of leukemia transformation or overall survival. Patients in the lower quartile had shorter time to anemia and leukopenia and did not progress to large splenomegaly. Furthermore, survival was significantly reduced in the lower quartile compared with upper quartiles and JAK2 wild-type patients. In multivariate analysis, factors associated with reduced survival were age, a blast count more than 1%, and a JAK2V617F burden within first quartile. Causes of death in the lower quartile were represented mainly by systemic infections. We conclude that a low JAK2V617F allele burden at diagnosis is preferentially associated with a myelodepletive rather than myeloproliferative phenotype and represents an independent factor associated with shortened survival in patients with PMF.

Validation of a Novel Clonality Assay in Patients with Clonal Hematologic Disorders

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. They are useful in the diagnosis and assessment of therapeutic response in clonal hematologic disorders, especially in absence of an identifiable somatic mutations. Skewing of X-chromosome allelic-usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in ~30% elderly women, precluding the use of this assay in elderly patient (>65 years of age). Using a quantitative, transcriptionally-based clonality assay, we did not detect clonal hematopoiesis in >200 healthy non-elderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in forty elderly women (age 65–92, mean 81.3 years), using a novel, quantitative qPCR transcriptional clonality assay. In this assay, mRNA transcribed from five X-chromosome polymorphic genes expressed in peripheral blood neutrophils is quantified by real time, allele specific RT-PCR. We did not detect clonal hematopoiesis in any of the elderly women. However, using HUMARA assay, 30% of these elderly women were detected to have monoallelic methylation of the HUMARA gene locus, consistent with previously reported literature. We concluded that our novel transcriptional clonality assay is suitable for evaluation of clonal hematopoiesis in all women including elderly women (Swierczek S, Agarwal N et al. Blood 2008, July 18 epub). Using this novel assay, we detected clonal hematopoiesis in 31 out of 32 well characterized patients with myeloproliferative disorders:polycythemia vera (all fourteen patients were clonal by our assay and all were JAK2V617F positive),essential thrombocytosis (nine out of ten patients were clonal by our assay, one out of ten patients was cMPLW515L positive, seven out of ten were JAK2 V617F positive; however one subject with low JAK2 V617F allelic burden was polyclonal by our assay), andprimary myelofibrosis (all eight patients were clonal by our assay and two of them were positive for JAK2V617F).In addition, we detected clonal hematopoiesis in 4 patients with unexplained anemia (two eventually evolved in to myelodysplastic syndrome), and in one patient with persistent leukocytosis (eventually found to be cMPLW515L positive). Using our assay we did not detect clonal hematopoiesis in 10 patients with reactive or secondary erythrocytosis, thrombocytosis or leukocytosis. We conclude that our novel transcriptional clonality assay is suitable for detection of clonal hematopoiesis in patients with clonal hematologic disorders, especially in patients lacking known somatic mutation. Studies to detect an emerging clone in milieu of polyclonal hematopoiesis (such as seen in PNH or early stages of clonal hematological disorders) by comparison of X-chromosome allelic usage ratio in myeloid cells and in T lymphocytes are underway.

Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival

The clinical relevance of JAK2V617F allele burden in primary myelofibrosis (PMF) has not been previously studied. Bone marrow-derived DNA from 199 patients with PMF was subjected to qualitative (n=199) and quantitative (n=129) analysis for V617F. Mutational frequency was 58% and median mutant allele burden ratio in V617F-positive patients was 29% (range, 1-74%). Multivariable analysis identified older age, platelet count > or =100 x 10(9) l(-1) and peripheral blood blast percentage <3% as being associated with a positive mutational status. The mere 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). The 129 patients with allele burden information 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. Kaplan-Meier plots revealed significantly shortened overall (P=0.0008) and leukemia-free (P=0.01) survival for the lower quartile, but not for upper quartile allele burden group; independent prognostic relevance was validated by multivariable analysis. We conclude that low V617F allele burden in PMF might indicate the presence of an overriding V617F-negative clone that confers a more aggressive disease phenotype.

JAK2V617F Mutation Screening as Part of the Hypercoagulable Workup in the Absence of Splanchnic Vein Thrombosis: Assessment of Value in a Series of 664 Consecutive Patients.

Background: JAK2V617F is a recurring mutation in patients with polycythemia vera (PV) (95%) and essential thrombocythemia (ET) (50%), diseases that are frequently complicated by arterial and/or venous thrombosis. JAK2V617F is also frequently identified in patients with splanchnic vein thrombosis, including in those with occult myeloproliferative disorder (MPD). We previously reported a low prevalence of JAK2V617F in thrombosis patients - only 2% of 210 patients with non-splanchnic vein thrombosis (Leukemia , 2007 Aug; 21(8):1828–9), and <1% of 136 patients with arterial stroke (J Thromb Haemost . 2007 Aug; 5(8):1784–5). Here, we expanded the analysis to 664 thrombosis patients to describe:i.JAK2V617F prevalence, andii.clinical course of patients harboring this mutation.Methods: In Phase I, we screened 439 consecutive patients who underwent thrombophilia testing at our institution in early 2005 - patients with splanchnic vein thrombosis or overt MPD were excluded. In Phase II, we identified 228 additional patients considered to be at high risk for harboring JAK2V617F, who were seen at our institution from 2004 to 2006. JAK2V617F screening (assay sensitivity <1%) was performed as described in the aforementioned references.Results: In the initial cohort of 439 patients (median age=56 years; range 17–93 years; 222 female), 136 (31%) presented with stroke, 114 (26%) with deep venous thrombosis (DVT), 95 (22%) with pulmonary embolism (PE), 50 (11%) with DVT and PE, 10 (2%) with central retinal vein occlusion (CRVO), 7 (2%) with cerebral vein thrombosis, and the remaining 27 (6%) with other thromboses. Five patients (MC1-MC5; median age=78 years) were found to harbor JAK2V617F (prevalence=1%) - none had overt clinical features of MPD and all harbored a low level of JAK2V617F in peripheral blood (median=5–2%; range=2.2–7.5%) (Table). One patient (MC2) carried the Prothrombin G20210A mutation In the subsequent cohort, we studied 197 patients (median age=39 years; range=16–84 years) with recurrent DVT and/or PE, and 28 patients (median age=27 years; range=31–49 years) with acute myocardial infarction at a young age (<50 years). One patient (MC6) who presented with recurrent idiopathic DVT was found to harbor JAK2V617F (prevalence <0.5%) (Table) - this patient had no features of underlying MPD and the mutation burden was low (9%). After a median follow up of 27 months (range=4–66 months), none of the 6 patients harboring JAK2V617F has developed overt MPD.Conclusions:1.In patients with thrombosis, JAK2V617F is infrequently detected in the absence of overt MPD and/or involvement of splanchnic veins. Thus, JAK2V617F screening cannot be recommended as part of the hypercoagulable workup in routine clinical practice in this setting.2.The risk of developing overt MPD may be quite low for patients harboring a low JAK2V617F burden at the time of thrombosis although longer follow up is needed in this regard.Furthermore, the thrombosis risk may be well controlled with standard warfarin therapy, and the role of myelosuppressive agents (eg. hydroxyurea) is unclear in these patients.Characteristics of patients harboring JAK2V617FPatientAgeSexThrombosisJAK2V617F allele burdenMPDFollow up (months)StatusMC184MPE, DVT5.3%No66AliveMC282FPE, cerebral vein5%No38AliveMC374FPE2.2%No4DeadMC478FCentral retinal vein5.2%No22AliveMC563MStroke7.5%No30AliveMC669MRecurrent DVTNoNo24Alive

Low JAK2V617F Allele Burden in Primary Myelofibrosis, Compared to Either a Higher Allele Burden or Unmutated Status, Predicts Inferior Overall and Leukemia-Free Survival.

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. BJH 2005; 131:320, Campbell, et al. Blood 2006; 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. [Display omitted]

Behavior of CD34 + cells isolated from patients with polycythemia vera in NOD/SCID mice

We investigated if polycythemia vera (PV) peripheral blood (PB) CD34+ cells contain cells capable of engrafting nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and if the JAK2V617F mutational burden of these cells alters their behavior in NOD/SCID mice. CD34+ cells isolated from patients with PV, idiopathic myelofibrosis (IM), or granulocyte colony-stimulating factor-mobilized normal donors were transplanted into sublethally irradiated NOD/SCID mice. Cells engrafted into the NOD/SCID mice were analyzed flow cytometrically using lineage-specific antibodies. Genomic DNA was extracted from granulocytes, CD34+ cells, and sorted human CD45(+) cells purified from the bone marrow cells of these mice to examine their JAK2V617F mutational burdens. Multilineage human cell engraftment was observed in mice transplanted with CD34+ cells from mobilized normal volunteers, IM patients and PV patients with high JAK2V617F burden, but not in mice receiving grafts from PV patients with low JAK2V617F burden. The differentiation program of engrafting PV CD34+ cells with high JAK2V617F burden was remarkably different than that of IM CD34+ cells. The JAK2V617F allele frequency in the human CD45+ cells isolated from the mice receiving CD34+ cells was lower than that observed in the CD34+ cell grafts, indicating the persistence of a JAK2V617F negative compartment of stem cells. We conclude that PB CD34+ cells from PV patients with high JAK2V617F burden and patients with IM contain NOD/SCID repopulating cells, and that differentiation program of IM and PV CD34+ cells are dramatically different.