JAK2 Variant Research Articles

A newly identified 45-kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance.

Tyrosine-protein kinase (janus kinase; JAK)-signal transducer and activator of transcription (STAT) signaling plays a pivotal role in the development of myeloproliferative neoplasms (MPNs). Treatment with the potent JAK1/JAK2-specific inhibitor, ruxolitinib, significantly reduces tumor burden; however, ruxolitinib treatment does not fully eradicate the malignant clone. As the molecular basis for the disease persistence is not well understood, we set out to gain new insights by generating ruxolitinib-resistant cell lines. Surprisingly, these cells harbor a 45 kDa JAK2 variant (FERM-JAK2) consisting of the N-terminal FERM domain directly fused to the C-terminal kinase domain in 80% of sublines resistant to ruxolitinib. At the molecular level, FERM-JAK2 is able to directly bind and activate STAT5 in the absence of cytokine receptors. Furthermore, phosphorylation of activation-loop tyrosines is dispensable for FERM-JAK2-mediated STAT5 activation and cellular transformation, in contrast to JAK2-V617F. As a result, FERM-JAK2 is highly resistant to several ATP-competitive JAK2 inhibitors, whereas it is particularly sensitive to HSP90 inhibition. A murine model of FERM-JAK2 leukemogenesis showed an accelerated MPN phenotype with pronounced splenomegaly. Notably, most current protocols for the monitoring of emerging JAK variants are unable to detect FERM-JAK2, highlighting the urgent need for implementing next-generation sequencing approaches in MPN patients receiving ruxolitinib.

Familial Polycythemia Associated with JAK2 Mutation Previously Known As Variant of Unknown Significance

Introduction Mutations in the Janus Kinase 2 gene (JAK2) are commonly encountered in patients with myeloproliferative neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia. JAK2 mutations particularly V617F is associated with PV in 98% cases. Gain of function mutations in the JAK2 exon 12 are reported to be associated with isolated erythropoiesis. Atypical mutations in JAK2 including (c.3323A>G, p.N1108S) were reported in patients with MPN who tested negative for the established driver mutations including exon 14 JAK 2, exon 9 CALR and exon 10 MPL genes. Primary familial congenital polycythemia (PFCP) is a rare disease inherited in an autosomal dominant fashion caused by hypersensitivity of EPO receptor. In such cases, an EPO receptor mutation is only found in 12-15% of affected patients. Due to associated increased risk of thrombosis, bleeding, leukemic involvement and myelofibrosis in PV, bone marrow evaluation is essential in this patient population. In patients with polycythemia, the goals of treatment are to maintain appropriate level of hemoglobin/hematocrit and to reduce severity of symptoms. We present a family affected by polycythemia and leukocytosis associated with an atypical heterozygous variant of JAK2 [c.3323A>G (p.Asn1108Ser)] currently reported as benign or variant of unknown significance (VUS) in literature. Methods Case series, retrospective electronic medical record review with institutional board review exemption Results We report a case series of three Caucasian family members affected with JAK2 VUS mutation (mother and two sons). The proband is a 17-year-old male who presented to an outside facility at the age of 14 with complaints of fatigue, headache, and intense pruritis after hot showers. Initial workup was significant for marked erythrocytosis and reticulocytosis without evidence of abnormal cells on peripheral film. Bone marrow evaluation showed trilineage hematopoiesis with no morphological features of a myeloproliferative neoplasm. Genetic testing was negative for JAK2 V617F and exon 12 mutations. However, whole exome sequencing (WES) revealed a heterozygous mutation of JAK2 gene, c.3323A>G (p.Asn1108Ser). Additional investigation on proband's younger brother, who complained of similar symptoms, also revealed the same JAK2 variant. The proband's mother reported non-specific symptoms including headache, flushing, and occasional blurry vision that she previously attributed to hypertension. Her labs were significant for leukocytosis, monocytosis and thrombocytosis in the presence of normal red blood cell count and WES showed the same mutation. Bone marrow evaluation of the brother and the mother showed normal trilineage hematopoiesis and was negative for malignancy (Table 1). Proband's father is reportedly negative for the aforesaid mutation and is clinically asymptomatic. The proband and his brother are currently managed with phlebotomy every 3 to 4 weeks to maintain hematocrit below 45%, leading to improvement in symptoms. Cytoreductive therapies have been discussed but not initiated. Proband's mother is managed by an adult hematologist. Scheduled phlebotomies with close surveillance is recommended for clinical practice along with interval research sequencing. Conclusion Our case series reports a family with clinical features and laboratories suggestive of a familial form of polycythemia, WES detected the presence of an atypical JAK2 mutation: c.3323A>G, (p.Asn1108Ser). The phenotypical and clinical significance of this mutation has been considered non canonical per reports and classified as likely benign by reputable commercial genetic testing laboratories. However, other reports identify this JAK2 variant as having a significant increased prevalence in patients with MPNs and AML compared to the general population. It may result in a gain of function of the JAK2 protein and subsequent development of MPNs. Enrollment in research sequencing protocols is encouraged in such patients to closely follow the pathologic potential and spectrum of genetic variants rarely reported in literature. Additional research to determine the pathogenicity of this genetic variant is highly warranted.

Modulation of Biomarkers By BET Inhibitor, BMS-986158, Including JAK2 Variant Allele Frequency (VAF), Bone Marrow (BM) Fibrosis, and Reversal of Abnormal Cytokine Production in Intermediate- or High-Risk Myelofibrosis (MF)

Background MF, driven by JAK/STAT pathway activation, results in an aberrant stem cell niche, leading to abnormal cytokine production, an inflammatory BM microenvironment, splenomegaly, and an array of other symptoms. Bromodomain and extra terminal (BET) proteins play a role in cancer cell proliferation, survival, and oncogenic progression. In combination with Janus kinase inhibitors (JAKi), ruxolitinib (RUX) or fedratinib (FED), BET inhibitors (BETi) have been shown to reduce inflammatory signals and disease burden in preclinical models of MF. BMS-986158 is an orally bioavailable, potent, and selective small-molecule BETi (Hilton et al. ESMO 2018. Abstr 1429). BMS-986158 in combination with RUX or FED is being evaluated in the phase 1b/2 CA011-023 (NCT04817007) study in patients (pts) with first-line (1L) and second-line (2L) MF, respectively. Clinical observations, including spleen volume reduction (SVR), were previously presented (Ayala et al. EHA 2023. Presentation S213). In this analysis, exploratory biomarkers were assessed for potential early disease-modifying activities of BMS-986158. These included: measurement of longitudinal JAK2 VAF, which may be indicative of mutation clonal burden; evaluation of the BM microenvironment, including fibrosis, megakaryocyte clusters, and other morphological features; and circulatory cytokines and other factors associated with MF (Hasselbalch. Cytokine Growth Factor Rev. 2013). Methods In dose escalation, RUX-naïve pts (1L MF) received BMS-986158 2.0, 3.0, or 3.75 mg QD 5d on/2d off over 28-day cycles and RUX 15 mg BID; pts relapsed, refractory or intolerant to prior RUX treatment (2L MF) received BMS-986158 0.5, 0.75, or 1.25 mg QD 5d on/2d off over 28-day cycles and FED 400 mg QD. Longitudinal assessment of JAK2 VAF was performed on multiple sample types, including isolated circulatory CD34+ cells, CD66b+ cells, and whole blood, using the MLL next-generation sequencing targeted exosome gene panel. Variants and indels were called using Pisces and Pindel, followed by annotation to COSMIC, ClinVar, gnomAd, and dbNSFP databases. BM assessments were performed on BM biopsies at baseline and post-treatment at the end of cycles 3 (optional), 6, and 12; fibrosis scores were determined by independent pathologist assessment (prognostic scoring system DIPSS PLUS-WHO 2017) and digital analysis of megakaryocyte clustering, reticulin pattern, and overall cellularity. The modulation of MF-associated circulatory cytokines and growth factors (79 soluble factors [SF] in total) was evaluated by multiple Rules-Based Medicine (RBM) custom MAP-HMP panels; aggregated data were analyzed. Results JAK2 VAF reduction was observed post-treatment with BMS-986158+RUX in 6/7 (86%) 1L MF pts with available data ( Figure A). The maximum VAF reduction from baseline was 52%, where > 25% VAF reduction looks to be associated with improved spleen response (6/6 pts [100%] achieved SVR35). In 2L MF pts treated with BMS-986158+FED, JAK2 VAF reduction was observed in 1/4 (25%) pts. Similar degree of changes in JAK2 VAFwere observed between CD34+ isolated cells and whole blood samples. In addition, BM fibrosis was either stabilized (3/9; 33%) or had ≥ 1 grade reduction (5/9; 56%) with BMS-986158+RUX at 24 weeks compared with baseline in 1L MF. Digital analysis further suggests potential normalized cellularity and decreased megakaryocyte clusters and reticulin levels in fibrosis-reduced samples. BM fibrosis reduction was also observed with BMS-986158+FED in 2L MF (3/8 [38%] stabilized and 2/8 [25%] reduced fibrosis). Lastly, sustained reduction of inflammatory cytokines downstream of NF-κB pathway was observed, including TNFα, IL-1ra, and VEGF ( Figure B). In addition, increased levels of metabolic adipokine/hormone SF (eg, leptin, erythropoietin) and reduction of SF specific to BETi+JAKi combination (eg, DKK1, CD27, TIMP3; Mascarenhas et al. J Clin Oncol. 2023) were observed as expected. Conclusions Preliminary data suggest combination treatment with BMS-986158 and JAKi in MF may modulate JAK2 VAF, BM microenvironment, circulatory cytokines, and other SF, providing evidence of early disease modifying potential of these drug combinations.

Loss of Heterozygosity of Chromosome 9p and Progression in MPNs

Introduction Copy-neutral loss of heterozygosity (LOH) of chromosome 9p, concomitant with mutated JAK2, can occur in myeloproliferative neoplasms (MPNs), and may be the most common chromosomal aberration in patients with polycythemia vera (PV). The prevalence of LOH of 9p varies amongst the different MPNs and is highest in PV where it has been reported to be between 30-70%. The reported prevalence of LOH of 9p is 20-50% and 10-20% in patients with myelofibrosis (MF) and essential thrombocythemia (ET), respectively. A hallmark feature of MPNs is the tendency to progress to more aggressive disease phenotypes (i.e., PV/ET to MF to accelerated- or blast-phase MPN) through the acquisition or loss of cytogenetic and molecular clonal markers. Although loss of LOH of 9p likely contributes to progression in MPNs, its pathologic role is still incompletely defined. Methods We conducted a retrospective review of patients at a single-center, tertiary-care institution who were diagnosed with an MPN, either chronic, accelerated or blast-phase (MPN-AP/BP), from January 1 st, 2017 until July 1 st, 2023. All patients underwent a microarray-based chromosome analysis using the I Scan System with the Global Diversity Array-8 (GDA) v1.0 Array BeadChip. Mutational profiling was done using the NextGENe software (SoftGenetics LLC) and MiSeq Reporter, and variant annotation and filtration was performed using VarSeq (Golden Helix). Results Fifty-two patients with MF were identified. Twenty-eight patients (53.8%) were found to have LOH of 9p, 15 of whom had secondary MF (7 with preceding ET and 8 with preceding PV). Eight of 9 patients (88.9%) with PV-MF harbored LOH of 9p, vs. 6 of 13 (46.2%) with ET-MF. The median cell fraction affected was 70% (range, 5-95%) and the median JAK2 variant allele frequency (VAF) was 75% (range, 16-97%). Eleven of the 24 patients without LOH of 9p harbored JAK2 mutations, at a median VAF of 40%. Nine patients (32.2%) with LOH of 9p had abnormal cytogenetics by conventional karyotyping, compared to 8 (33.3%) without LOH of 9p. Eleven patients (39.3%) with LOH of 9p were MIPSS-70 v2 high or very high risk, compared to 10 (41.7%) patients without LOH of 9p. Nine patients (32.1%) with LOH of 9p were DIPSS-plus intermediate-2 or high-risk, compared with 9 (37.5%) patients without LOH of 9p. Twenty patients with MPN-AP/BP were identified, of whom 11 had MPN-AP and 9 had MPN-BP. Four patients (20%), all with MPN-BP, had LOH of 9p. Median JAK2 VAF in these patients was 50% (range, 38-80%), vs. 4% (range, 3-48%) in the 6 patients with JAK2 without LOH of 9p. Two of 19 patients (10.5%) with ET were found to have LOH of 9p, with a corresponding JAK2 VAF of 70% and 20%. Twelve of the 17 patients (70.6%) who did not have LOH of 9p had a JAK2 mutation, at a median VAF of 27% (range, 4-44%). One patient with LOH of 9p had a thrombotic event, vs. 4 of 17 patients (23.5%) without LOH of 9p. Three of 9 patients (33%) with PV were found to have a LOH of 9p, with a median corresponding JAK2 VAF of 61% (range, 55-81%). Median JAK2 VAF for the other 6 patients (all with a JAK2 mutation) was 34% (range, 26-46%). One patient with LOH of 9p had a thrombotic event vs. 3 of 6 patients (50%) without LOH of 9p. Discussion The highest prevalence of LOH of 9p in patients with an MPN was seen in those with MF (>50%), followed by PV, MPN-AP/BP and ET. These results are generally in line with those previously reported, though the prevalence in PV was lower than other studies (likely due to small sample size). The prevalence of LOH of 9p in ET-MF was more than four times higher than in ET, suggesting that LOH of 9p may be an acquired cytogenetic aberration involved in ET progression. LOH of 9p was seen in almost 90% of PV-MF vs. 33% of PV patients, further supporting that LOH of 9p may be implicated in MPN progression. Notably, LOH-9p was seen in far fewer patients with accelerated- or blast-phase disease. It is likely that clonal evolution of MPNs to MPN-AP/BP is driven more so by the acquisition of high-risk mutations (e.g., TP53, ASXL1, U2AF1) and less by changes such as LOH of 9p and increased JAK2 VAF. In summary, our data largely confirms previous reports regarding the prevalence of LOH of 9p in MPNs while suggesting a role for the gain or loss of LOH of 9p in the progression of these hematologic cancers.

BMS-986158, a Potent BET Inhibitor, in Combination with Ruxolitinib or Fedratinib in Patients (pts) with Intermediate- or High-Risk Myelofibrosis (MF): Updated Results from a Phase 1/2 Study

Introduction Bromodomain and extra-terminal (BET) inhibitors in combination with Janus kinase inhibitors (JAKi) have demonstrated clinical benefits in pts with MF. BMS-986158 is an orally bioavailable, potent, and selective small molecule BET inhibitor with a dose-proportional pharmacokinetic profile with linear increases in exposure, which has shown time- and dose-dependent modulation of BET target gene expression. BMS-986158 in combination with JAKi ruxolitinib (RUX) or fedratinib (FED) is being evaluated in pts with MF in the CA011-023 study (NCT04817007). Previous analyses showed that BMS-986158+RUX in first-line (1L; RUX-naïve) MF and BMS-986158+FED in second-line (2L; relapsed, refractory, or intolerant to prior RUX treatment) MF was well tolerated, with most pts remaining on treatment, and had promising preliminary efficacy (Ayala R et al. EHA 2023. S213). Updated results with longer follow-up will be presented. Methods Eligible pts had primary or secondary MF and were either RUX-naïve (1L) or relapsed, refractory, or intolerant to RUX (2L), with splenomegaly (spleen volume [SV] ≥ 450 cm 3), ECOG PS ≤ 2, and Dynamic International Prognostic Scoring System risk scores of intermediate-1 with symptoms, intermediate-2, or high. In dose escalation, pts with 1L MF received BMS-986158 2.0, 3.0, or 3.75 mg QD 5d on/2d off + RUX 15 mg BID; pts with 2L MF received BMS-986158 0.5, 0.75, 1.0, or 1.25 mg QD 5d on/2d off (alternative schedule for 1.0-mg dose: 5d on/2d off, 3 wks on/1 wk off) + FED 400 mg QD. Primary objectives in dose escalation were safety, tolerability, and determination of the maximum tolerated dose and/or recommended phase 2 dose (RP2D) of BMS-986158+RUX and BMS-986158+FED. Secondary objectives included spleen volume reduction (SVR) from baseline and response rate (SVR35) at wk 24. Assessment of JAK2 variant allele frequency (VAF) was an exploratory objective. JAK2V617 VAF was measured longitudinally in peripheral blood CD34+ stem cells using next-generation sequencing. Results As of May 18, 2023, 16 pts with 1L MF received BMS-986158+RUX (median age 66 y [range, 36-81]) and 24 pts with 2L MF received BMS-986158+FED (median age 68 y [range, 34-81]). At baseline, median SV was 1504 cm 3 (range, 524-4379) in pts treated with BMS-986158+RUX and 2222 cm 3 (range, 581-6598) in pts treated with BMS-986158+FED. Any grade (G) treatment-related adverse events (TRAEs) were reported in 15 (94%) pts treated with BMS-986158+RUX; G 3/4 TRAEs were thrombocytopenia (n = 7, 44%), neutropenia (n = 2, 13%), anemia, leukopenia, herpes zoster, hyperbilirubinemia, and hypertension (n = 1 each, 6%). Any G TRAEs were reported in 18 (75%) pts treated with BMS-986158+FED; G 3/4 TRAEs were thrombocytopenia and anemia (n = 6 each, 25%), hyperbilirubinemia (n=2, 8%), leukocytosis, and diarrhea (n = 1 each, 4%). No TRAEs led to discontinuation of BMS-986158+RUX; 2 TRAEs (G4 thrombocytopenia and G3 hyperbilirubinemia) led to discontinuation of BMS-986158+FED. SVR was observed at wk 12 in all evaluable pts in both treatment regimens and continued to deepen at wk 24 (Figure). Median SV at wk 24 was 603 cm 3 (range, 170-2532) and 1826 cm 3 (range, 1079-5288) in evaluable pts with 1L MF and 2L MF, respectively. In pts with 1L MF, 8/11 (73%) treated with BMS-986158 (2.0, 3.0 or 3.75 mg)+RUX met SVR35 at wk 12; 9/10 (90%) pts met SVR35 at wk 24. In pts with 2L MF, 7/12 (58%) pts treated with BMS-986158 (0.5, 0.75, 1.0, or 1.25 mg)+FED met SVR35 at wk 12; 3/7 (43%) pts met SVR35 at wk 24. At the time of reporting, 32/40 (80%) pts remained on treatment: 14/16 (88%) receiving BMS-986158+RUX and 18/24 (75%) receiving BMS-986158+FED. Available analysis of JAK2 VAF showed reductions in the frequency of JAK2V617F with BMS-986158+RUX (52% max. reduction by C10, n = 7) and BMS-986158+FED (29% max. reduction by C4, n = 4). Conclusions These updated analyses of study CA011-023 show that BMS-986158+RUX in 1L MF and BMS-986158+FED in 2L MF continue to be well tolerated, with most patients remaining on treatment. TRAEs were mostly low-grade and transient. Thrombocytopenia, an on-target effect, was manageable and did not result in clinically significant bleeding. Both treatment regimens produced early and deep SVR by wk 12, which continued and deepened at wk 24 and beyond. The reductions observed in JAK2 VAF provide promising preliminary data of potential disease modification. Dose expansion with BMS-986158+RUX in 1L MF has opened and is actively enrolling patients.

JAK2 Mutations Are Rare and Diverse in Myelodysplastic Syndromes: Case Series and Review of the Literature

To investigate and characterize JAK2 mutations in myelodysplastic syndrome (MDS), we present three cases with diverse JAK2 mutations and review the literature. The institutional SoftPath software was used to find MDS cases between January 2020 and April 2022. The cases with a diagnosis of a myelodysplastic/myeloproliferative overlap syndrome including MDS/MPN with ring sideroblasts and thrombocytosis were excluded. The cases with molecular data by next generation sequencing looking for gene aberrations commonly seen in myeloid neoplasms were reviewed for the detection of JAK2 mutations including variants. A literature review on the identification, characterization, and significance of JAK2 mutations in MDS was performed. Among 107 cases of the MDS reviewed, a JAK2 mutation was present in three cases, representing 2.8% of the overall cases. A JAK2 V617F mutation was found in one case representing slightly less than 1% of all the MDS cases. In addition, we found JAK2 R564L and JAK2 I670V point mutation variants to be associated with a myelodysplastic phenotype. JAK2 mutations in MDS are rare and represent less than 3% of cases. It appears that JAK2 variant mutations in MDS are diverse and further studies are needed to understand their role in the phenotype and prognosis of the disease.

BMS-986158, a Potent BET Inhibitor, As Monotherapy and in Combination with Ruxolitinib or Fedratinib in Intermediate- or High-Risk Myelofibrosis: First Results from a Phase 1/2 Study

Background Bromodomain and extraterminal (BET) proteins play a role in cancer-cell proliferation, survival, and oncogenic progression, providing a rationale for using inhibitors of BET as anticancer drugs. BET inhibitors (BETi) alone and in combination with Janus kinase (JAK) inhibitors have been demonstrated to reduce inflammatory signals and disease burden in mouse myelofibrosis (MF) models, and combined inhibition of BET-mediated pathways and the JAK-STAT pathway has shown additional benefits for patients (pts) with MF. BMS-986158 is an orally bioavailable, potent, and selective small-molecule BETi that has demonstrated a dose-proportional pharmacokinetic profile with linear increases in exposure, as well as time- and dose-dependent modulation of BET target gene expression (Hilton J et al. Ann Oncol 2018;29(suppl 8):1429). BMS-986158 is being evaluated in pts with MF, alone and in combination with the JAK inhibitors ruxolitinib (RUX) or fedratinib (FED), in the CA011-023 study (NCT04817007). Here, we present initial safety and efficacy data from the dose-escalation parts (1A and 1B) of this study. Methods CA011-023 comprises a dose-escalation phase with BMS-986158 + RUX in RUX-naive pts (Part 1A) or BMS-986158 + FED in pts with previous RUX treatment (Part 1B), and a dose-expansion phase at the Recommended Phase 2 Dose (RP2D) evaluating BMS-986158 + RUX (Part 2A) or BMS-986158 ± FED (Part 2B). Eligible pts had primary or secondary MF, splenomegaly (spleen volume ≥ 450 cm3), ECOG performance status ≤ 2, and Dynamic International Prognostic Scoring System risk scores of intermediate-1 with symptoms, intermediate-2, or high. Planned dose levels for pts in Part 1A are BMS-986158 2.0 mg, 3.0 mg, and 4.5 mg once daily (QD) 5 days on/2 days off and RUX 15 mg twice daily. For pts in Part 1B, planned dose levels are BMS-986158 0.5 mg, 0.75 mg, 1.25 mg, and 2.0 mg QD 5 days on/2 days off and FED 400 mg QD. Primary objectives in the dose-escalation phase are assessment of safety and tolerability and determination of the maximum tolerated dose and/or RP2D of BMS-986158 in combination with RUX or FED. Secondary objectives include spleen volume reduction (SVR) from baseline. Assessment of pharmacodynamic markers, such as JAK2 variant allele, is an exploratory objective. Results As of May 31, 2022, 13 pts were treated, 6 with BMS-986158 + RUX (median 66 years [range, 36-81]; 100% male) and 7 with BMS-986158 + FED (median 62 years [range, 37-77]; 57% male). Grade 1/2 treatment-related adverse events (TRAEs) were reported in 3 (50%) pts in the BMS-986158 + RUX group and in 2 (29%) pts in the BMS-986158 + FED group. Grade 3 TRAEs considered to be related to the combination treatment reported in the BMS-986158 + RUX group were thrombocytopenia (2 pts [33%]), and neutropenia and hypertension (1 pt each [17%]). Grade 3 TRAEs considered to be related to the combination treatment in the BMS-986158 + FED group were anemia (2 pts [29%]) and thrombocytopenia (1 pt [14%]). No grade 4/5 TRAEs were reported for either combination. One dose-limiting toxicity (DLT; grade 3 thrombocytopenia leading to dose reduction of BMS-986158) was reported at the 2.0 mg dose level of BMS-986158 + RUX on cycle 1 day 19. No DLTs have been reported in pts treated with BMS-986158 + FED. No serious TRAEs or TRAEs leading to treatment discontinuation have been reported. Importantly, SVR was observed at Week 12 in all evaluable pts across both treatment groups and continued to deepen at Week 24 in evaluable pts. In the BMS-986158 + RUX group, 4/5 (80%) pts met SVR35 at week 12, and 2/2 (100%) pts met SVR35 at week 24. In the BMS-986158 + FED group 0/3 (0%) pts met SVR35 at week 12, and 1/2 (50%) pts met SVR35 at week 24 (Table 1). Pts with post-baseline spleen volume by MRI in Part 1B were treated at lower BMS-986158 dose levels (0.5 mg and 0.75 mg 5 days on/2 days off) compared to Part 1A (2 mg 5 days on/2 days off). Analysis of JAK2 variant allele frequency and determination of RP2D are ongoing. Conclusions Most TRAEs were grade 1/2 in severity, while grade 3 TRAEs were successfully managed with dose delays or modifications without leading to treatment discontinuation. Initial results demonstrate that BMS-986158 in combination with RUX or FED produced robust SVR in pts with MF, with responses that deepened beyond Week 12 under continued treatment. Enrollment is continuing in the dose-escalation cohorts to establish RP2D for both combinations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EP042: Highly sensitive blocker displacement amplification-based qPCR approach in detecting low level JAK2 variant

JAK2 exon 14 c.1849G>T, p.V617F (NM_004972.3) variant is commonly found in Myeloproliferative neoplasms (MPN); approximately 100% of polycythemia vera and 50% of essential thrombocytosis and primary myelofibrosis cases have this pathogenic variant. The pathogenic nature of JAK2 V617F in these disorders have been well-established in the past. The National Comprehensive Cancer Network (NCCN) guidelines for cancer care has indicated molecular testing using blood samples for JAK2 V617F as well as other mutant alleles is needed for MPN diagnosis.

JAK2 Variant Signaling: Genetic, Hematologic and Immune Implication in Chronic Myeloproliferative Neoplasms.

The JAK2V617F variant constitutes a genetic alteration of higher frequency in BCR/ABL1 negative chronic myeloproliferative neoplasms, which is caused by a substitution of a G ˃ T at position 1849 and results in the substitution of valine with phenylalanine at codon 617 of the polypeptide chain. Clinical, morphological and molecular genetic features define the diagnosis criteria of polycythemia vera, essential thrombocythemia and primary myelofibrosis. Currently, JAK2V617F is associated with clonal hematopoiesis, genomic instability, dysregulations in hemostasis and immune response. JAK2V617F clones induce an inflammatory immune response and lead to a process of immunothrombosis. Recent research has shown great interest in trying to understand the mechanisms associated with JAK2V617F signaling and activation of cellular and molecular responses that progressively contribute to the development of inflammatory and vascular conditions in association with chronic myeloproliferative neoplasms. Thus, the aim of this review is to describe the main genetic, hematological and immunological findings that are linked to JAK2 variant signaling in chronic myeloproliferative neoplasms.

Pathogenic "germline" variants associated with myeloproliferative disorders in apparently normal individuals: Inherited or acquired genetic alterations?

Myeloproliferative syndromes (MPS) are hematologic malignancies due to the expansion of an abnormal hematopoietic stem cell. They include chronic myeloid leukemia (CML) and non-CML MPS such as polycythemia vera, essential thrombocythemia and primary myelofibrosis. The latter are distinguished by somatic pathogenic variants affecting JAK2, CALR, or MPL genes. Apparent germline pathogenic variants have been reported in the general population. Here, we found that two gnomAD data-sets report more homozygotes than expected for the JAK2 c.1849G > T(Val617Phe) variant. We propose that somatic gene conversion can explain the presence of those unexpected homozygotes in normal populations. Consistently, homozygous individuals are older than 65 years. We also found a lower-than-expected frequency of the JAK2 variant in younger individuals suggesting that somatic mutation can underlie its presence in (at least some) heterozygotes. Regarding pathogenic variants in MPL and CALR, they are also present in the gnomAD data-sets explored. However, we cannot conclude that such seemingly germline variants are in fact somatic alterations. These results suggest that apparently normal individuals bearing MPS-related variants can be subclinical/undiagnosed MPS cases of somatic origin. It would be interesting to assess the hematologic phenotype of such individuals and the presence of the relevant variants in other tissues.

Stiffer Spleen Predicts Higher Bone Marrow Fibrosis and Higher JAK2 Allele Burden in Patients With Myeloproliferative Neoplasms.

A total of 63 myeloproliferative neoplasms [MPN; 9 polycythemia vera (PV), 32 essential thrombocythemia (ET), and 22 myelofibrosis (MF)] underwent spleen stiffness (SS) measurement by vibration-controlled transient elastography equipped with a novel spleen-dedicated module. Higher SS values significantly correlated with grade 2-3 bone marrow (BM) fibrosis (p=0.035), with hemoglobin level <10 g/dl (p=0.014) and with white blood cells ≥10,000/μl (p=0.008). Median SS was significantly higher in MF patients compared to ET and PV (p=0.015). SS also correlated with higher JAK2 variant allele frequency (p=0.02). This study identifies SS as a potential noninvasive tool that reflects BM fibrosis and the mutational burden in MPN.

Revisiting Diagnostic performances of serum erythropoïetin level and JAK2 mutation for polycythemias: analysis of a cohort of 1090 patients with red cell mass measurement.

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses

AbstractAlthough somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

JAK2 Variants Associated with Secondary-AML from MPN

BackgroundCanonical JAK2 V617F and exon 12 mutations in myeloid neoplasms are well described. There are limited reports of other rare JAK2 variants of potential clinical relevance. We sought to survey JAK2 variants in a large cohort of MPN and AML patients and determine the likelihood of contribution to disease pathogenesis.MethodsWe retrospectively analyzed the JAK2 variant profile and mutational landscape of MPN and AML. Targeted, amplicon-based, next-generation sequencing of the coding region of JAK2 and 27 other leukemia-related genes was performed on DNA extracted from diagnostic bone marrow samples. The study population was classified into three cohorts: chronic myeloproliferative neoplasm-only (MPN); MPN transformed to AML (MPN>>AML); and AML-only, excluding MPN>>AML patients (AML).ResultsOf 3,376 MPN and AML patients, 2,154 patients underwent JAK2 sequencing. Non-V617F/non-exon 12 JAK2 sequence variants were identified in 114 (5.3%, Fig 1A). This included 35 unique JAK2 variants including several previously undescribed variants located within all functional domains (Fig 1B). Ten variants have been previously reported in cases of hematologic proliferation or shown to be activating alterations. Sixteen of 114 JAK2 variants occurred alone without somatic mutations in the remaining 27 somatic genes tested. JAK2 variants were detected at a significantly higher frequency in the MPN>>AML cohort (15.3%) compared with MPN (4.6%, p<0.001) and AML (5.2%, p<0.001) cohorts. Every detected variant occurred at higher-than-expected frequency in MPN and AML patients than in the general population, particularly L393V, R1063H, and N1108S (p<0.01 for each). Each missense variant was analyzed using 3 separate predictive computational platforms (FATHMM-MKL, MutationTaster, and DANN) to determine whether an amino acid substitution was likely to exert a functional effect on JAK2 . All variants were predicted to have functional consequence, and most were predicted to have a functional consequence (62.9%) by all 3 programs. The presence of a JAK2 variant in addition to JAK2 V617F (n=13) in myelofibrosis was associated with an increased cumulative risk of transformation from MPN to AML (p=0.003, Fig 1C). Some JAK2 variants, and variants in specific protein regions, were associated with increased frequency of chromosomal abnormalities. For example, L393V and N1108S variants were significantly associated with chromosome number alterations compared to other JAK2 variants (43.2% vs. 22.9%, p=0.002). Additionally, co-mutation analysis with the other 27 genes tested showed that 5/16 (31.3%) patients with JAK2 N1108S also had a TP53 mutation vs. 10/98 (10.2%) among other JAK2 variants (p=0.04).ConclusionsFull exonic gene sequencing of JAK2 identified several JAK2 variants of potential clinical relevance. JAK2 variants may represent an important and potentially overlooked component of MPN/AML pathogenesis, evidenced by the prevalence of variants previously shown to have JAK2 activating effects, the frequencies of specific variants in MPN/AML compared to the general population, and the predicted functional effect of variant amino acid substitutions. Specific JAK2 variants detected in MPN patients may be meaningful predictors for transformation to AML. [Display omitted] DisclosuresDiNardo:Agios: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Bose:Incyte Corporation: Honoraria. Pemmaraju:affymetrix: Research Funding; LFB: Consultancy, Honoraria; cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria; novartis: Consultancy, Honoraria, Research Funding; roche diagnostics: Consultancy, Honoraria; abbvie: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; BMS: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Sun Pharma: Research Funding. Kantarjian:Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Novartis: Research Funding. Verstovsek:Galena BioPharma: Research Funding; Lilly Oncology: Research Funding; Seattle Genetics: Research Funding; NS Pharma: Research Funding; CTI BioPharma Corp: Research Funding; Bristol Myers Squibb: Research Funding; Astrazeneca: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Lilly Oncology: Research Funding; Gilead: Research Funding; Galena BioPharma: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Blueprint Medicines Corp: Research Funding; Roche: Research Funding; Astrazeneca: Research Funding; Bristol Myers Squibb: Research Funding; CTI BioPharma Corp: Research Funding; Incyte: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

Novel Recurrent Germline JAK2 G571S Variant in Childhood Acute B-Lymphoblastic Leukemia: A Double Hit One Pathway Scenario

Introduction: Current studies demonstrate an involvement of germline predispositions in the development of approximately 5% of childhood leukemias (Zhang J et al.,N Engl J Med, 2015), although their actual contribution is believed to be much higher. Being able to understand tumor evolution starting from a predisposed cell, opens up a new avenue in the form of disease prevention rather than treatment. Here, we present a novel finding of a double hit - one pathway scenario, in which a rare (MAF<0.01) germline JAK2 variant (G571S), inherited paternally, and a newly described germline STAT3 variant (K370R), transmitted from the mother's side, act synergistically to induce Ph-like B-cell precursor acute lymphoblastic leukemia (BCP-ALL).Methods: WES was carried out to identify predisposing germline variants. The cooperative functionality of the di-genic candidate mutations was experimentally tested using BaF3 oncogenic transformation assays, western blot and cell cycle analyses. In addition, we used structural homology calculations to visualize the cooperative impact of both mutations.Results: Utilizing Trio-calling based on WES we identified two concomitant germline SNVs in the JAK2/STAT3 pathway in a boy affected with BCP-ALL. The JAK2 variant, rs139504737, leads to an amino acid substitution from Glycine to Serine (p.G571S) (MAF<0.01). The second variant constitutes an extremely rare and so far for leukemia undescribed missense mutation in the STAT3 gene (c.1109A>G), causing an exchange of Lysine to Arginine (p.K370R). Structural modeling of STAT3 K370R showed that K370, which is an important site for acetylation, is located in a loop adjacent to the DNA binding site of STAT3. The substitution of Lys to Arg at position 370 leads to a strengthening of the beta-sheet due to simultaneous interaction of Arg with both E455 and E442. Moreover, in contrast to Lys, Arg cannot be acetylated, leading to a constitutively non-acetylated form of STAT3. JAK2 G571S on the other hand has a unique position affecting amino acid 571, which lies adjacent to the Y570 residue that downregulates kinase activity via autophosphorylation, indicating a potential functional mechanism of the G571S mutation by inhibiting Y570-directed negative feedback.To assess the cooperative oncogenic transforming potential of both variants, BaF3 depletion assays were carried out. In BaF3/CRLF2-IL-7Rwt cells, JAK2 G571S conferred IL-3 independent growth at similar rates as the well-known oncogenic JAK2 V617F mutation. Moreover, the combination of both JAK2 G571S and STAT3 K370R further increased the growth advantage significantly starting 2 days after IL-3 withdrawal (p=0.0226). Western Blot analyses revealed increased pSTAT5 levels, as well as high pSTAT-3 levels in the double mutant cells.We further observed that STAT3 K370R alone changed the phenotype of the in-vitro culture, with an accumulation of enlarged BaF3 cells. Surprisingly, this phenotype was reversed in cells expressing both STAT3 K370R and JAK2 G571S. Cell cycle analysis showed a significant increase of aneuploid cells (G2/M) (p=0.0009), while the G-1 phase was significantly decreased (p=0.0031) in STAT3 K370R expressing BaF3 cells compared to STAT3 WT cells. Again, this phenotype was drastically reduced in cells transfected with both mutations simultaneously (G1 phase p=0.0026; G2/M p=0.0032). Western Blot analyses confirmed increased p-CDC-2, p-Cyclin/Cyclin-B1, and Cyclin-A2 levels in BaF3 cells harboring both variants, suggesting that the cell cycle arrest observed in cells expressing STAT3 K370R is rescued by JAK2 G571S expression by enabling re-entering of the M-Phase.The link between the oncogenic capacity of JAK2 G571S and leukemia could further be strengthened by the identification of an additional B-precursor ALL patient from an independent family harboring the same JAK2 G571S germline mutation. The patient belongs to a previously described DS-ALL cohort (Bercovich D. et al., Lancet, 2008). In this patient with Down syndrome, we did neither identify the STAT3 K370R mutation nor CRLF2 activation. Thus, we hypothesize that the germline JAK2 G571S in combination with the constitutional trisomy 21 act in synergy.Conclusion: Taken together, we present evidence of an oncogenic potential of germline JAK2 G571S, which is significantly increased through additional expression of STAT3 K370R in a double-hit one-pathway scenario. DisclosuresNo relevant conflicts of interest to declare.

Comparative Genomic and Expression Analysis of Chronic and Blast-Phase Cells in Patients with Myeloproliferative Neoplasms

Background. Progression to acute myeloid leukemia (sAML) occurs in 20% of myelofibrosis (MF) and 10% of polycythemia vera (PV) or essential thrombocythemia (ET). sAML has dismal outcome with median survival of <6 months. We recently reported that a restricted set of mutations predict for leukemic transformation in MPN (Vannucchi AM, Leukemia 2013; Tefferi A, Blood Adv. 2016). However, the molecular mechanisms underlying transformation to sAML are poorly characterized, in particular the relationships between the clones establishing the chronic phase and the one dominating the leukemic phase.Aim. To study clonal heterogeneity and clonal progression in the leukemic transformation of MPN we performed whole exome (WES) and trascriptome (RNAseq) sequencing of paired samples (chronic (CP)/blast phase(BP).Patients and Methods. WES and RNAseq was done on CD34+ or blast cells of 12 paired samples from 5 PMF, 3 PET-MF, 1 PPV-MF, 3 ET pts who transformed to sAML. In addition, a myeloid neoplasm-relevant 34-gene panel was used for target NGS sequencing of paired samples plus an additional 23 samples collected from MPN pts at leukemic phase (15 PMF, 3 PET-MF, 3 ET, 2 PV).Results. In the entire series, 20 pts (57%) were JAK2 V617F mutated at CP, 8 CALR (23%; 6 type-1 and 2 type-2) and 7 (8.6%) MPL mut, while 4 (11.4%) were triple-negative. JAK2 variant allele frequency (VAF) declined by ≥50% (n=3; 15%) or became undetectable (n=4; 20%) whereas 3 heterozygous pts (15%) became homozygous at the time of BP. One of the 8 CALR mut cases halved its VAF, whereas no meaningful VAF changes were observed in MPL mut pts. The most frequent mutated genes detected by NGS at BP, other than driver mutations, were ASXL1 (51.4%), RUNX1 (37.1%), TET2 (17%), SRSF2 (16%), IDH1 (14%), TP53 (14%), NRAS (14%), FLT3 (14%), U2AF1 (11%) and KRAS (11%); three (8%) cases were mutated for EZH2DNMT3A, CBL and PTPN11, 2 cases for IDH2, SH2B3, SF3B1 IKZF1, SETBP1 and ZRSR2, and 1 case for ABL1, ATV6, BRAF and ARID1A. There were also 5 CEBPA-mutated pts, 3 of which, unlike none of 27 CEBPA-mutated de novo AML, showed an in-frame 6-bp duplication polymorphism (p. P196_197insHP) reported in 20-40% of all de novo AML and <1% of healthy population (rs762459325). Compared to the CP of the 12 paired samples, 10 pts (83%) acquired novel mutations, 3 of which acquired ≥3 variants. RUNX1 and ASXL1 were those with the highest number of acquisition (n=7 and 5, respectively). Conversely, loss of variants was observed only in ASXL1 (n=3).By WES analysis, on average 60.000 variants per paired sample unique to the BP compared with CP were identified. However, no recurrent abnormalities were found in the 12 paired samples outside the above listed mutations found by target sequencing. For RNAseq analysis, transcripts were annotated according to UCSC hg19 for a total of 25,369 genes. We found large transcriptional differences between CP and BP with 129 transcripts differentially expressed (23 up- and 106 down-regulated) and 4,155 isoforms (2,120 up- and 2,035 down-regulated). Among the most abnormally expressed transcripts we selected 8 genes (5 down-regulated: LCN2, PDGFB, PRTG, CRISP3, PF4; 3 up-regulated CDKN2, SH2D1A and LIN28) for validation by QRT-PCR based on the extent of differential expression. PF4 and CDKN2 were confirmed to be down-regulated and over-expressed in BP, respectively (P<0.0001). Analysis of RNA-seq data for fusion genes revealed 10 fusion genes acquired during BP; however, only 3 of them were confirmed by Sanger sequencing (2 cases of BCR-ABL, 1 case of KMT2A- MLLT3 and 1 case of CBFB-MYH11). Pathway analysis included Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis. Six pathways were significantly deregulated in BP compared with CP: Mitotic Spindle (P<0.0001); Androgen response (P=0.004); TGF-beta signaling (P=0.04); Apoptosis (P=0.03); PI3K-AKT-MTOR signaling (P=0.04); Reactive Oxygen Species- Pathway (P=0.03).Conclusions. Comprehensive comparative analysis of genomic and RNA abnormalities acquired in the transition from CP to BP in MPN has not been previously reported. Our data indicate that BP is associated with significant changes in mutation complexity and RNA expression, overall affecting different intracellular pathways whose further characterization might help to identifying potential targets for therapy. DisclosuresNo relevant conflicts of interest to declare.

Multicolor Flow Cytometry and Multi-Gene Next Generation Sequencing Are Complimentary and Highly Predictive for Relapse in Acute Myeloid Leukemia Following Allogeneic Hematopoietic Stem Cell Transplant

▪Aim: Evaluation of tandem minimal residual disease (MRD) assessment using multi-gene next generation sequencing (NGS) and multi-parameter flow cytometry (MFC) in acute myeloid leukemia (AML) pts undergoing allogeneic hematopoietic stem cell transplant (allo-HCT).Methods: MRD was measured on the same bone marrow aspirate using 10-color MFC and a targeted myeloid specific 28-gene NGS panel pre and post allo-HCT in available samples from 122 consecutive pts with AML transplanted between 2014 and 2015. Any level of MRD measured by MFC in the blast compartment was regarded as positive, while somatic mutations detected above a pre-defined variant allele frequency (VAF) threshold on bulk marrow by multi-gene NGS were regarded as positive. Mutations identified on diagnostic or relapse samples were tracked throughout the disease course. FLT3-ITD and TKD mutations were detected in a stand-alone PCR based assay and VAF was not quantified.Results: NGS (HR: 2.37 (95% CI 1.06-5.28) and HR: 3.23 (95% CI 1.21-8.62)) and MFC (HR: 2.44 (95% CI 1-5.97) and HR: 4.62 (95% CI 1.32-16.09)) predicted overall survival (OS) and time-to-relapse respectively with median observation time of 12 months among survivors. MRD detection using both assays was associated with relapse, with MRD detected by MFC being the most predictive (table 1). NGS was applicable to 85% of tested pts with probable pathogenic mutations seen at diagnosis, while all pts tested at diagnosis had abnormal blasts detected by MFC. Transplant factors including donor source, conditioning intensity, stem cell source and GVHD prophylaxis were not associated with transplant outcomes while complex and monosomal karyotype were associated with OS and time-to-relapse (table 1).Pre allo-HCT concordance rate of MRD detection using the two assays was 70% (table 2). 12 (20%) pts had detectable MRD by MFC and not NGS. Five of these patients had NGS assessment at diagnosis and on manual review of NGS results 3 of these 5 had diagnostic mutations detected on pre allo-HCT samples at VAF below threshold to call mutations. Six (10%) were MFC negative but had detectable mutations by NGS, which were typically clonal hematopoiesis (CH) type mutations with VAF ranging between 3-20%, only 1/6 of these has relapsed post allo-HSCT.MRD pre allo-HCT using both MFC and NGS was associated with relapse; however, the risk was highest in pts who had pre transplant MRD detected concurrently using both techniques and cumulative incidence of relapse was lowest in those who were MRD negative using both techniques (Figure 1A & B).No significant change in mutant DNMT3A, TET2 and JAK2 variant allele frequency (VAF) was seen between assessment at diagnosis and pre transplant, while a significant reduction in NPM1 and IDH VAF was noted. For pts in CR or CRi pre allo-HCT MRD burden quantified using MFC (on blasts) was markedly lower than the corresponding VAF of residual mutations (on bulk marrow) measured in the same sample (Figure 1C) with VAF of residual mutation ranging between 10-20% suggesting that a large bulk of cells at the time of CR were derived from the abnormal clone.We tracked pathogenic mutations identified on diagnostic samples in pts who had marrow MFC and NGS MRD assessment after transplant. In pts who relapsed, multi-gene NGS detected mutations earlier than MFC although at very low allele burden (<1%) (Figure 3D). Low VAF (<2%) DNMT3A mutations were detected after transplant in 10/11 evaluable pts who had these mutations on pre transplant assessment while MFC was negative in 9/10 of those with low VAF DNMT3A mutations detected after transplant. Of the 10 pts with detectable DNMT3A mutations after transplant only 2 have relapsed and both had mutations in TP53. This suggests that the clinical significance of persistent low allelic burden DNMT3A mutations needs further clarification, as they may not be strongly predictive of relapse even if detected after transplant.Conclusion: Despite different assay sensitivity MFC and multi gene NGS had a concordance of 70% for detecting pre allo-HCT MRD in AML.Assessing pre Allo-HCT MRD with MFC and multi-gene NGS improves the ability to predict relapse and OS. MFC had more universal applicability, while NGS could identify residual CH-type mutations with greater sensitivity than MFC and identify MRD at earlier time points than MFC post-HCT. Using a more extensive gene panel is likely to improve the sensitivity of MRD detection and may improve correlation with MFC [Display omitted] [Display omitted] [Display omitted] DisclosuresLevine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Roshal:BD Biosciences: Consultancy.

Mutational analysis of JAK2, CBL, RUNX1, and NPM1 genes in familial aggregation of hematological malignancies.

Familial aggregation of hematological malignancies has been reported highlighting inherited genetic predisposition. In this study, we targeted four candidate genes: JAK2 and RUNX1 genes assuring a prominent function in hematological process and CBL and NPM1 as proto-oncogenes. Their disruption was described in several sporadic hematological malignancies. The aim of this study is to determine whether JAK2, CBL, RUNX1, and NPM1 germline genes mutations are involved in familial hematological malignancies. Using direct sequencing, we analyzed JAK2 (exons 12 and 14); CBL (exons 7, 8 and 9); NPM1 (exon 12) and the entire RUNX1 in 88 independent families belonging to Tunisian and French populations. Twenty-one sporadic acute leukemias were included in this study. We reported a heterozygous intronic c.1641 + 6T > C JAK2 variant (rs182123615) found in two independent familial cases diagnosed with gastric lymphoma and Hodgkin lymphoma. The in silico analysis suggested a potential impact on splicing, but the functional splicing minigene reporter assay on rs182123615 variant showed no aberrant transcripts. In one sporadic acute myeloblastic leukemia, we reported an insertion 846 in. TGTT in exon 12 of NPM1 gene that may impact the normal reading frame. The rs182123615 JAK2 variant was described in several contexts including myeloproliferative neoplasms and congenital erythrocytosis and was supposed to be pathogenic. Through this current study, we established the assessment of pathogenicity of rs182123615 and we classified it rather as rare polymorphism.

Chemical Mutagenesis Screen Identifies Novel Drug Resistant JAK2 Variants in V617FJAK2 Mediated MPN and Predict HSP90 Inhibitors As Possible Agents to Overcome Drug Resistance

JAK2 V617F can be identified in the majority of polycythemia vera cases and in 50% of essential thrombocythemia and idiopathic myelofibrosis patients. JAK2 inhibitors including ruxolitinib, fedratinib and lestaurtinib display clinical activity in trials for PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors emerge due to secondary resistance mutations in the targeted kinase. To identify JAK2 point mutations mediating ruxolitinib resistance and predict alternatively treatment strategies, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2 V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and fedratinib. Phospho-deficient mutant studies provided evidence that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2.However, in this study we could not identify other genetic TKI resistant JAK2 variants than FERM-JAK2. This is in remarkable contrast to BCR-ABL mediated CML, where more than 90 residues have been described as TKI resistant variants and might indicate that in case of JAK2, the residues which mediate the drug resistance might comprise the JAK2 kinase function. Therefore, we treated JAK2 V617F expressing Ba/F3 cells with a chemical mutagen (ENU) to predict point mutations in JAK2 that might cause resistance towards the ruxolitinib. Surprisingly, after ENU pretreatment the frequency of drug resistant clones increased and most of the drug resistant clones displayed point mutations in the JAK2 kinase domain. In this screen, we identified L902Q, Y931C, L983F as the most frequent mutations and L902Q+R938E, L902Q+N947Q, L902Q+E1028K and L983F+Q959H as minor mutations. All these mutations confer cross-resistance across a panel of different JAK2 kinase inhibitors except L983FJAK2. L983FJAK2 reduces the sensitivity of JAK2-dependent cells to ruxolitinib and JAK inhibitor-1 but does not reduce the sensitivity of cell proliferation against fedratinib, thereby indicated that we identified a compound specific resistant mutations which is not an ATP-competitor specific mutation.Structure based modeling studies suggest that ruxolitinib is held by numerous hydrophobic interactions with residues Leu 855, Val 863, Ala 880, Val 911, Met 929, Leu 932 and Leu 983 that line the binding pocket. The aromatic ring of Tyr 931 is close enough to the pyrimidine ring to have p-p interaction. Surprisingly, this important interaction does not seem to be critical for the orientation of the inhibitor in the binding pocket as the Y931C mutation does not lead to a different binding pose. Leu 902 does not directly interact with ruxolitinib, however, it is close to the binding pocket and its mutation to Gln with a polar side chain significantly disturbs the inhibitor binding, thus explaining the high resistance of this mutation to ruxolitinib. Mutation of Leu 983 to Phe disrupts important hydrophobic interactions (e.g. Ala 880, Val 911, Met 929) with the pyrrolopyrimidine moiety and induces aromatic-aromatic interaction between the new phenyl ring and the pyrrol and pyrazol rings. Finally, our study also provided evidence that HSP90 inhibitors 17-AAG and geldanamycin are highly potent against TKI resistant FERM-JAK2, L902Q, Y931C and L983FJAK2 through JAK2 degradation and inhibition of the JAK2-STAT5 signaling axis. Taken together, our study highlight HSP90 inhibitors as a promising therapeutic agent in drug resistant mutations against tyrosine kinase inhibitors in MPN. DisclosuresNo relevant conflicts of interest to declare.

Identification Of a Novel Mode Of Kinase Inhibitor Resistance In JAK2: JAK2 Inhibitor Resistance Is Mediated By The Generation Of 45-Kda JAK2 Variant Which Alters The Kinase Domain Structure

JAK2 V617F can be identified in the majority of cases of polycythemia vera (PV), and in 50% of cases in essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). JAK2 inhibitors including ruxolitinib (Jakavi, INCB018424), TG101348 and lestaurtinib (CEP-701) display clinical activity in clinical trials in PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors is due to emergence of secondary resistance mutations in the target kinase. So far no JAK2 drug resistant mutations have been described in patients. To identify JAK2 point mutations mediating ruxolitinib resistance, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and TG101348. JAK2 exists in an either active or inactive state, which is largely regulated by phosphorylation of tandem tyrosines located within the activation loop. Substitution of phenylalanine at tyr1007, tyr1008 or both leads to complete inactivation of V617FJAK2 and WT-JAK2.In contrast, Y to F mutation of tyr1007, tyr1008 or both in FERM-JAK2 does not prevent activation of STAT5 and transformation of Ba/F3 cells, suggesting that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2.Phospho-deficient mutant studies further provided evidence that tyr866, tyr966, and tyr972 are crucial for the activation of FERM-JAK2, whereas in V617FJAK2 and WT-JAK2 tyr972, tyr1007 and tyr1008 were critical autophosphorylation sites. Ectopic expression of FERM-JAK2 in HEK293T, NIH3T3 and Ba/F3 cells showed activation of STAT5 and transformation of Ba/F3 cells to factor independence. Interestingly, co-immunoprecipitation studies revealed that FERM-JAK2 does not bind or activate the IL-3R in contrast to V617FJAK2. Ectopic expression of FERM-JAK2, in contrast to V617FJAK2, was able to activate STAT5 in a cytokine receptor deficient cell line. In vitro binding studies revealed that FERM-JAK2 directly binds and activates STAT5. Using flag- and myc-tagged FERM-JAK2 we show that the FERM domain is sufficient for an efficient dimerization and activation of FERM-JAK2 in the absence of an cytokine receptor. Transplantation of retrovirally transduced murine bone marrow with FERM-JAK2 compared to JAK2 V617F showed an accelerated phenotype with marked increases in WBC, HCT, RBC, HB, reticulocytes, pronounced splenomegaly and loss of body weight in mice. In contrast to V617FJAK2 expressing mice, FERM-JAK2 expressing mice displayed a lethal phenotype. Taken together, we could identify a novel JAK2 variant which alters the kinase domain structure, leads to direct, cytokine receptor independent STAT5 activation, and gives rise to an accelerated, PV-like disease in mice. Disclosures:No relevant conflicts of interest to declare.