Germline Genetic Factors Affecting Interferon Alpha Therapy in Polycythemia Vera

Introduction: Interferon alpha (IFNa) based therapies can induce sustained hematological and molecular responses (HR/MR) in Polycythemia Vera (PV) and other Myeloproliferative Neoplasms (MPN), however, not all patients respond sufficiently. While previous studies suggest that disease driving somatic mutations and genomic aberrations do not predict response to IFNa, the role of common germline polymorphisms remains elusive. We addressed the effect of germline genetic factors on PV therapy with Ropeginterferon alfa-2b (Ropeg), a novel monopegylated IFNa. We performed genome-wide association studies (GWAS) as an unbiased approach, and additionally evaluated a previously reported influence of polymorphisms at the IFNL4 locus in a large PV patient cohort. Methods: Genomic DNA was isolated from whole blood of PV patients (n=115) on Ropeg therapy, who provided consent within the PROUD-PV (NCT01949805) and CONTI-PV (NCT02218047) study programs. Patients were genotyped for >900k tagging single nucleotide polymorphisms (SNPs) across the genome on the Affymetrix SNP 6.0 array platform. Additional IFNL4 SNPs were typed using an amplicon-based next generation sequencing approach. Association analyses were performed using the PLINK toolset. JAK2-V617F mutant allele burden was quantified using a qPCR-based assay (ipsogen MutaQuant, Qiagen). MR was measured based on changes in mutant allele burden upon therapy as defined by the European LeukemiaNet (ELN) criteria. Results: To test for potential associations between germline genetic variation and response to IFNa therapy, we performed GWAS for MR data upon 12 months (M), 18M, 24M and 30M and 36M follow-up on Ropeg treatment. Genome-wide tagging SNPs were tested both for association in a case-control setup applying chi-square statistics as well as for quantitative trait association, using peripheral blood JAK2-V617F mutational burden changes from baseline as continuous variables. After Bonferroni correction for multiple testing, none of those analyses revealed a statistically significant association, suggesting the absence of strong germline predisposition factors for MR. Germline variation at the interferon-lambda (IFNL) locus was previously reported to strongly influence viral clearance during IFNa therapy of Hepatitis C (reviewed in Wack et al., Nat Immunol, 2015). While the same variants were recently reported to also affect HR during IFNa treatment in an MPN patient cohort (Lindgren et al., EJH, 2018), their potential impact on MR as surrogate marker for the size of the malignant MPN clone has not yet been evaluated. Upon testing for MR in our PV cohort, in a case-control setup we observed a statistically significant association only upon 36M follow-up (p=0.02; OR=2.51; 95%CI=[1.14-5.62]; Figure 1a). Notably, testing for change of JAK2-V617F mutational burden under Ropeg treatment as quantitative trait, we found the association to be present at formal statistical significance at all stages during follow-up (Figure 1a). For the Hepatitis C association, it is now widely acknowledged that a diplotype of two coding variants covers most of the causality, where rs368234815_TT disrupts the open reading frame (no IFNL4) while rs117648444_G results in the impaired IFNL4-S70 in contrast to the fully functional IFNL4-P70, the latter paradoxically impacting negatively on viral clearance (Terczynska-Dyla et al., Nat Commun, 2014). Similarly, in our PV cohort genotyping and phasing of this diplotype revealed a significant influence on MR in carriers of the fully functional IFNL4-P70 (p=0.01; Figure 1b; shown are patients at 36M follow-up (n=70)). Conclusions: The absence of a strong germline predisposition factor for Ropeg treatment response in our cohort implies that any PV patient may be eligible for Ropeg therapy independently of their genetic makeup. While the IFNL4 diplotype is of potential use for patient stratification, it remains to be investigated whether increase of treatment duration and/or dose adjustments can overcome the adverse effect of functional IFNL4 on Ropeg therapy. Longitudinal monitoring of JAK2-V617F mutational burden under Ropeg treatment in conjunction with determination of the IFNL4 germline genetic status may allow for optimizing patient management. Disclosures Krejcy: AOP Orphan Pharmaceuticals AG: Employment. Klade:AOP Orphan Pharmaceuticals AG: Employment. Zoerer:AOP Orphan Pharmaceuticals AG: Employment. Gisslinger:Janssen-Cilag: Honoraria; Roche Austria GmbH: Consultancy; Novartis Pharma GmbH: Consultancy, Honoraria, Research Funding; Myelopro GmbH: Consultancy; Celgene GmbH: Honoraria; Pharma Essentia: Other: Personal fees; AOP Orphan Pharmaceuticals: Consultancy, Honoraria, Research Funding. Kralovics:Novartis: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Other: Advisory board; Pharma Essentia: Honoraria; Qiagen: Honoraria; MyeloPro Diagnostics and Research: Equity Ownership.

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Analysis Of Molecular Responses and Chromosomal Aberrations In Patients With Polycythemia Vera Treated With Peg-Proline-Interferon Alpha-2b (AOP2014/P1101) In The Peginvera Study

Blood ◽

10.1182/blood.v122.21.1589.1589 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1589-1589 ◽

Cited By ~ 1

Author(s):

Nicole C.C. Them ◽

Heinz Gisslinger ◽

Veronika Buxhofer-Ausch ◽

Richard Greil ◽

Josef Thaler ◽

...

Keyword(s):

Chromosomal Aberrations ◽

Research Funding ◽

Clonal Evolution ◽

Single Gene ◽

Jak2 V617f ◽

Molecular Response ◽

Molecular Responses ◽

Mutational Burden ◽

Ifna Therapy

Abstract Polycythemia vera (PV) is one of the most common type of BCR-ABL negative myeloproliferative neoplasms. It is characterized by elevated erythrocyte mass, variable presence of thrombocytosis and leukocytosis, predisposition to secondary myelofibrosis, thrombosis, bleeding and leukemic transformation. Most patients carry the JAK2-V617F mutation (up to 95%) or the less common JAK2 exon 12 mutations (around 3%). The current treatments include phlebotomy, low-dose aspirin, hydroxyurea, interferon alpha (IFNa) and bone marrow transplantation. However, PV is still lacking a curative treatment, with the exception of cases with successful bone marrow transplantation (in the spent phase) and few reports of complete clinical and molecular remission using IFNa. Measuring mutant JAK2 burden offers an opportunity to evaluate efficacy of therapy on the molecular level. We have previously reported sound clinical and molecular responses of PV patients treated with a new, once every 14 days formulation of peg-proline-IFNa-2b (AOP2014/P1101) in the Phase I/II clinical study PEGINVERA. The rational of the cytogenetic evaluation in this study is to investigate if chromosomal aberrations have an influence on the clinical course of PV patients, treated with AOP2014/P1101. It might be that cytogenetically complex patients have lower response rates to IFNa therapy. Furthermore, cytogenetic lesions may serve as additional markers to evaluate the response in parallel to JAK2 mutational burden analysis. Results of JAK2 mutational burden as well as high-resolution SNP array-based cytogenetic analysis, in 45 patients, treated with AOP2014/P1101, are presented here. Genome-wide human SNP 6.0 Affymetrix arrays were performed for the baseline sample (at the start of IFNa treatment) and latest follow-up sample. Mutant JAK2-V617F burden was determined by allele specific-PCR and quantitative PCR. For JAK2 exon 12 mutations a fragment analysis-based assay was used. Molecular response, defined by at least 10% decrease in mutant JAK2 burden, was observed in 73% of patients. The median follow-up time of patients was 500 days. The median follow-up time of molecular responding patients was 633 days, for partial responders 959 days and for non-responding patients 168 days. At least one chromosomal aberration was present in 69% of patients, of which chromosome 9p uniparental disomies (9pUPDs) were the most prevalent ones. Molecular non-responding patients did not have recurrent specific cytogenetic lesions or more chromosomal aberrations than responding patients. Molecular responses analyzed by JAK2 mutational burden correlated well with cytogenetic changes. A complete cytogenetic remission with around 3% residual JAK2-V617F burden could be achieved in 3 patients, all showing chromosome 9p UPDs at baseline. Interestingly, one patient had in addition to the 9p UPD a chromosome 14q UPD and another one trisomies of chromosome 8 and 9. This indicates that IFNa therapy is not restricted to mutant JAK2 clones but is also able to target other aberrant clones with common genetic changes found in MPN. Cytogenetic lesions found in follow-up samples that were not detected at baseline may indicate clonal evolution during IFNa therapy. These emerging clones might be responsible for acquisition of IFNa resistance and/or acceleration of disease progression. We found 3 such patients in our study, 1 with molecular response that acquired a small clone with a deletion of chromosome Y, 1 with partial molecular response where a single gene deletion on chromosome 10p (USP6NL) was detected and 1 that had no molecular response which showed a single gene gain on chromosome 3q (FXR1) as well as a single gene deletion on chromosome 7p (NXPH1). Additional follow-up samples will be necessary to assess the impact of the clonal evolution in these patients. Not only acquired somatic changes (including large chromosomal aberrations and point mutations) but also germline variants might influence IFNa response or resistance. Since we did not observe any difference in cytogentic lesions between molecular responding and non-responding patients, we suspect that in some cases germline variants are likely to influence the outcome of IFNa therapy. Further characterization of AOP2014/P1101 treated patients and later follow-up samples will help to better understand the clonal evolution and molecular responses during long-term IFNa treatment. Disclosures: Them: AOP Orphan Pharmaceuticals AG: Research Funding. Gisslinger:AOP Orphan Pharmaceuticals AG: Research Funding. Buxhofer-Ausch:AOP Orphan Pharmaceuticals AG: Research Funding. Greil:AOP Orphan Pharmaceuticals AG: Research Funding. Thaler:AOP Orphan Pharmaceuticals AG: Research Funding. Schloegl:AOP Orphan Pharmaceuticals AG: Research Funding. Gastl:AOP Orphan Pharmaceuticals AG: Research Funding. Berg:AOP Orphan Pharmaceuticals AG: Research Funding. Bagienski:AOP Orphan Pharmaceuticals AG: Research Funding. Zahriychuk:AOP Orphan Pharmaceuticals AG: Employment. Klade:AOP Orphan Pharmaceuticals AG: Employment. Kralovics:AOP Orphan Pharmaceuticals AG: Research Funding.

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Monitoring Molecular Response, Minimal Residual Disease and Clonal Structures in Polycythemia Vera Patients Treated with Interferon Alpha

Blood ◽

10.1182/blood.v128.22.1944.1944 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1944-1944

Author(s):

Roland Jäger ◽

Edith Bogner ◽

Kerstin Trenker ◽

Michael Gurbisz ◽

Bettina Gisslinger ◽

...

Keyword(s):

Polycythemia Vera ◽

Minimal Residual Disease ◽

Interferon Alpha ◽

Research Funding ◽

Mutant Allele ◽

Residual Disease ◽

Jak2 V617f ◽

Mutational Burden ◽

Paired Analysis ◽

Minimal Residual

Abstract Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) primarily characterized by an elevated red blood cell count, consequently to clonal expansion of myeloid progenitor cells driven almost exclusively by a mutation of a specific single nucleotide in JAK2 exon 14 (JAK2-V617F). JAK2-V617F mutational burden therefore represents a surrogate measure for the size of the "liquid tumor" underlying PV, i.e. the amount of malignant cells in the peripheral blood. We previously reported on sustained molecular responses (MR) in PV patients on interferon alpha (IFNa) based therapies. Monitoring MR is crucial for understanding the therapeutic potential of IFNa and can be an important parameter in future PV patient care. Thus, highly sensitive laboratory techniques providing most accurate quantification at lowest limit of detection (LOD) are required. Here we evaluate four different methods for JAK2-V617F mutational burden monitoring, which are digital droplet PCR (ddPCR), next-generation sequencing (NGS), quantitative PCR (qPCR) and allele-specific PCR (AS-PCR). Moreover, using an NGS-based approach we investigate the patients' clonal composition by targeted re-sequencing involving 54 genes in longitudinal patient samples. To determine the characteristics of the different methods for burden quantification, we benchmarked a ddPCR assay (Assay ID dHsaCP2000061/dHsaCP2000062, Bio-Rad, Hercules, CA) using serial dilutions of JAK2-V617F positive in JAK2-V617F negative gDNA. The assay showed an LOD of 0.01% JAK2-V617F with a false positivity rate of 0 mutation-positive droplets in 8 independent measurements of healthy donor gDNA. Next we quantified the JAK2-V617F burden in baseline and follow-up (FU) gDNAs from a PV patient cohort on IFNa-based therapy (n=51) using ddPCR, a qPCR-based assay (ipsogen MutaQuant, Qiagen, Hilden, Germany), an NGS-based approach (TruSight Myeloid Sequencing Panel, Illumina, San Diego, CA) and an AS-PCR assay described previously (Kralovics et al, Blood, 2006). Covering the full spectrum of allelic burden values, correlations with ddPCR-derived values were best for NGS (R2=0.998) followed by qPCR (R2=0.976) and AS-PCR (R2=0.951). While mutational burden values >10% JAK2-V617F showed high concordance between methods, JAK2-V617F burdens <10% exhibited significant deviations from the benchmarked ddPCR assay, suggesting that ddPCR is most suitable for monitoring residual disease in PV. Therefore, applying ddPCR, we next re-evaluated two PV patients with baseline JAK2-V617F burdens of 46% and 28%, respectively, both previously classified as complete MR based on JAK2-V617F undetectable by AS-PCR. For those two patients, ddPCR revealed an actual residual disease of 3.02% and 2.39% mutant allele burden at 138 and 68 weeks post initiation of IFNa therapy, respectively. Further monitoring of these patients showed a sustained decrease in mutant allele burden to 1.53% and 0.20% at weeks 217 and 171, respectively. In addition to JAK2-V617F burden quantification, our NGS approach allowed for quantitative evaluation of mutations in 54 genes implicated in myeloid diseases. After somatic variant calling, paired analysis of baseline and FU samples (n=96) from 48 PV patients revealed an average of 2.8 variants per patient in a total of 17 genes. Besides JAK2, recurrently affected genes were TET2, PHF6, ASXL1 and CEBPA. The paired analysis allowed for tracking changes in clonal structures upon therapy. Most notably, TET2-JAK2 double-positive clones present in two patients showed mutant burden decreases of 13% and 59% respectively, arguing against an implication of TET2 mutations in IFNa resistance. Furthermore, PHF6 positive clones present in three patients showed ambiguity upon therapy, shrinking concordantly with the JAK2 positive clone in two patients while expanding as a JAK2-independent clone in the third patient. In summary, while larger studies will be required to assign statistical significance to mutations implicated in IFNa-based therapies, targeted re-sequencing of serial samples in MPN patient cohorts has the potential to deconvolute the impact of the patients' clonal architecture on therapeutic success. Moreover, as medications such as IFNa-based drugs have been shown to induce deep MR in a substantial fraction of PV patients, the use of highly accurate assays such as ddPCR will be crucial for monitoring minimal residual disease. Disclosures Klade: AOP Orphan: Employment. Gisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Hoermann:Novartis: Honoraria; Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

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Kleine-Levin syndrome is associated with birth difficulties and genetic variants in the TRANK1 gene loci

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005753118 ◽

2021 ◽

Vol 118 (12) ◽

pp. e2005753118

Author(s):

Aditya Ambati ◽

Ryan Hillary ◽

Smaranda Leu-Semenescu ◽

Hanna M. Ollila ◽

Ling Lin ◽

...

Keyword(s):

Bipolar Disorder ◽

Genome Wide Association Study ◽

Perinatal Outcomes ◽

Case Control ◽

Risk Scores ◽

Circadian Regulation ◽

Genetic Associations ◽

Imaging Studies ◽

Genome Wide

Kleine-Levin syndrome (KLS) is a rare disorder characterized by severe episodic hypersomnia, with cognitive impairment accompanied by apathy or disinhibition. Pathophysiology is unknown, although imaging studies indicate decreased activity in hypothalamic/thalamic areas during episodes. Familial occurrence is increased, and risk is associated with reports of a difficult birth. We conducted a worldwide case−control genome-wide association study in 673 KLS cases collected over 14 y, and ethnically matched 15,341 control individuals. We found a strong genome-wide significant association (rs71947865, Odds Ratio [OR] = 1.48, P = 8.6 × 10−9) within the 3′region of TRANK1 gene locus, previously associated with bipolar disorder and schizophrenia. Strikingly, KLS cases with rs71947865 variant had significantly increased reports of a difficult birth. As perinatal outcomes have dramatically improved over the last 40 y, we further stratified our sample by birth years and found that recent cases had a significantly reduced rs71947865 association. While the rs71947865 association did not replicate in the entire follow-up sample of 171 KLS cases, rs71947865 was significantly associated with KLS in the subset follow-up sample of 59 KLS cases who reported birth difficulties (OR = 1.54, P = 0.01). Genetic liability of KLS as explained by polygenic risk scores was increased (pseudo R2 = 0.15; P < 2.0 × 10−22 at P = 0.5 threshold) in the follow-up sample. Pathway analysis of genetic associations identified enrichment of circadian regulation pathway genes in KLS cases. Our results suggest links between KLS, circadian regulation, and bipolar disorder, and indicate that the TRANK1 polymorphisms in conjunction with reported birth difficulties may predispose to KLS.

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

Blood ◽

10.1182/blood.v118.21.3838.3838 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3838-3838 ◽

Cited By ~ 9

Author(s):

Animesh Pardanani ◽

Jason Gotlib ◽

Catriona Jamieson ◽

Jorge E. Cortes ◽

Moshe Talpaz ◽

...

Keyword(s):

Platelet Count ◽

Research Funding ◽

Jak2 V617f ◽

Intermediate Risk ◽

Spleen Size ◽

Safety And Efficacy ◽

Allele Burden ◽

Jak2v617f Allele Burden

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

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High Percentage of JAK2 exon 12 Mutation in Polycythemia Vera and Essential Thrombocythemia Among Populations of Qatar

Blood ◽

10.1182/blood.v120.21.5064.5064 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5064-5064

Author(s):

Mohamed A. Yassin ◽

Hanadi Rafii El-Ayoubi ◽

Nader Al-Dewik

Keyword(s):

Polycythemia Vera ◽

Essential Thrombocythemia ◽

Genomic Dna ◽

Research Funding ◽

Mutant Allele ◽

Jak2 V617f ◽

Research Fund ◽

Idiopathic Myelofibrosis ◽

Essential Thrombocytosis ◽

High Incidence

Abstract Abstract 5064 The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis. It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and Methods We enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection <1%). For exon 12 mutation screening, 80 ng of genomic DNA was amplified by specific primers designed to amplify a region of 453 bp containing the 128 bp of the exon 12 sequence of JAK2. PCR products were directly sequenced in both directions on an ABI 3730 DNA Analyzer using the BigDye Terminator Sequencing kit. Results We examined the occurrence of JAK2V617F and JAK2 exon 12 mutations in a clinical cohort of 82 patients with polycythemia vera (PV) and Essential thrombocythemia (ET) Of which 42 patients had PV aged 25 to 53, 13 (31%) females and 29 (69 %) males and V617F mutation was detected in all of them exon 12 mutation was detected in 38 (90. 47%) patients. We found 2 different exon 12 mutations:3 N542-E543del, 1 F537-K539delinsL, and among 40 ET patients aged 25 to 59, 22 (55 %) males and 18 (45%) females, 35 patients (87. 5%) were JAK2 V617F and JAK 2 exon12 positive and 5 (12. 5%) were JAK2V617F as well as exon 12 negative patients with V617F and exon 12 mutations showed significantly higher WBC and platelet counts at diagnosis than patients with exon V617F mutation alone (P =. 021 and P =. 038, respectively). We report a surprisingly high incidence of exon 12 mutations in MPN patients with PVand ET in Qatar, a result quite different from reports in the Western literature (P =. 001). Conclusion Our data suggest that exon 12 mutation of JAK2 in patients with PV and ET may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation. the importance of such associations may need further studies and evaluations. Disclosures: Yassin: Qatar National Research Fund: Patents & Royalties, Research Funding. Rafii El-Ayoubi:Qatar National Research Fund: Patents & Royalties, Research Funding. Al-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding.

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Prognostic Significance of Baseline FLT3-ITD Mutant Allele Burden in Acute Myeloid Leukemia Treated with Intensive Chemotherapy with/without Sorafenib

Blood ◽

10.1182/blood-2018-99-117692 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3983-3983 ◽

Cited By ~ 2

Author(s):

Fevzi Yalniz ◽

Hagop M. Kantarjian ◽

Tapan M. Kadia ◽

Guillermo Garcia-Manero ◽

Keyur Patel ◽

...

Keyword(s):

Myeloid Leukemia ◽

Research Funding ◽

Mutant Allele ◽

Optimal Cutoff ◽

Allele Burden ◽

Genetics Research ◽

Group 2 ◽

Allelic Burden ◽

Group 1

Abstract Background Internal tandem duplication (ITD) mutation of the FMS-like tyrosine kinase-3 (FLT3) receptor gene occurs in about 25% patients with acute myeloid leukemia (AML) and confers a poor prognosis. Several studies have reported that a higher mutant allelic burden is associated with a worse prognosis. Methods Adult patients with FLT3-ITD mutated AML treated at our institution from January 2001 to January 2018, who had quantified FLT3-ITD allele burden, were identified. Patients with acute promyelocytic leukemia and core-binding factor AML were excluded.Patients were assigned into 2 groups; Group 1 included patients who received idarubicin and cytarabine (IA) containing induction and Group 2 included patients who received sorafenib in addition to IA containing regimens at induction. Mutant allelic burden was expressed as the ratio of area under the peak of mutant allele to total FLT3. Relapse free survival (RFS) for patients achieving complete response (CR)/CR with incomplete recovery (CRi) was defined as time from CR/CRi to relapse or death. Overall survival (OS) was defined as time from treatment to death. Patients were censored at last follow up. Time from therapy to allogeneic hematopoietic cell transplant (AlloHCT) was handled as a time-dependent variable. The optimal cutoff of mutant FLT3-ITD allelic burden was defined as the cutoff to divide the whole cohort with the highest statistical significance. Results A total of 183 patients withFLT3-ITD mutated AML were identified including 104 (57%) in Group 1 and 79 (43%) in Group 2. Baseline characteristics are summarized in Table 1. The median age was 52 years (range, 17-64). The median allelic burden of mutant was 33% (range, 0.3% to 88%). This was comparable between the two groups (p=0.6). The CR/CRi rates following induction for Group 1 vs Group 2 were 85% vs 99%, respectively (p=0.004). Overall, 111 (61%) patients received an AlloHCT, at any time during the follow up, more frequently in Group 2 than in Group 1 (67% vs 56%, respectively, p=0.1). The median RFS for Group 1 and 2 were 12 and 45 months, respectively (p=0.02); the median OS was 17 months in Group 1 and has not been reached in Group 2 (p=0.008) (Table 2). The optimal mutant allele burden cutoff for OS and RFS in the entire cohort was 1.55% (p=0.002) confirming the adverse effect of FLT3-ITD even at low level. The cutoff was 6.9% in Group 1, with no optimal cutoff value in Group 2 (Figure 1) confirming the value of sorafenib inFLT3-ITD mutated AML. When censored for AlloHCT; the FLT3-ITD cutoff for OS and RFS was 60% and 60% in the entire group. For Group 1 the cutoff values where 59% and 7.9% for OS and RFS, respectively. For Group 2 the cutoff values were 46% and 46%, respectively (Figure 2). On multivariate analysis, AlloHCT (HR 0.52; 95%CI 0.33-0.82; p=0.005), sorafenib (HR 0.6; 95%CI 0.38-0.93; p=0.02) and white blood cell count (HR 1.005; 95%CI 1.002-1.009) were independent predictors of OS with no impact by cytogenetics, concurrent NPM1, TP53, FLT3-D835 mutations or the FLT3-ITD allelic burden. Conclusion Although a higher FLT3-ITD allele burden is associated with a worse RFS and OS in patients with FLT3-ITD mutated AML treated with IA-based chemotherapy, it is no longer prognostic when sorafenib is added to the therapeutic regimen. Addition of sorafenib to intensive chemotherapy as well as AlloHSCT are associated with a significant improvement in OS on multivariate analysis. Disclosures Kadia: Novartis: Consultancy; BMS: Research Funding; Takeda: Consultancy; Celgene: Research Funding; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy; BMS: Research Funding; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Abbvie: Consultancy. Sasaki:Otsuka Pharmaceutical: Honoraria. Daver:BMS: Research Funding; ImmunoGen: Consultancy; Karyopharm: Consultancy; Sunesis: Research Funding; Novartis: Consultancy; Otsuka: Consultancy; Karyopharm: Research Funding; Novartis: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Sunesis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; ARIAD: Research Funding; Alexion: Consultancy; Kiromic: Research Funding. DiNardo:Medimmune: Honoraria; Bayer: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Agios: Consultancy; Karyopharm: Honoraria. Pemmaraju:Affymetrix: Research Funding; SagerStrong Foundation: Research Funding; plexxikon: Research Funding; daiichi sankyo: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; abbvie: Research Funding; cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; novartis: Research Funding. Short:Takeda Oncology: Consultancy. Bose:Astellas Pharmaceuticals: Research Funding; Incyte Corporation: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; CTI BioPharma: Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding. Cortes:Daiichi Sankyo: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Arog: Research Funding; Novartis: Consultancy, Research Funding. Ravandi:Macrogenix: Honoraria, Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Sunesis: Honoraria; Macrogenix: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding, Speakers Bureau; Jazz: Honoraria; Orsenix: Honoraria; Sunesis: Honoraria; Orsenix: Honoraria; Abbvie: Research Funding; Jazz: Honoraria; Bristol-Myers Squibb: Research Funding; Abbvie: Research Funding; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria.

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Germline-Somatic Interactions in Myelofibrosis Susceptibility

Blood ◽

10.1182/blood-2021-150634 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 313-313

Author(s):

Derek W Brown ◽

Youjin Wang ◽

Andrew St. Martin ◽

Stephen R. Spellman ◽

Shu-Hong Lin ◽

...

Keyword(s):

Telomere Length ◽

Board Of Directors ◽

Research Funding ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Risk Haplotype ◽

Advisory Committees ◽

Genome Wide ◽

Increased Risk ◽

V617f Mutation

Abstract Introduction: Myelofibrosis (MF) is a rare myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, progressive bone marrow failure, and increased risk of acute myeloid leukemia. While MF arises from somatic driver mutations in JAK2, MPL, and CALR, some MPN patients may have a heritable component. To comprehensively examine the genetic etiology of MF, we performed the first integrative analysis of SNP array genotyping (using Infinium Global Screening Array), targeted long-read sequencing (using PacBio SMRT sequencing) and telomere length (TL, using qPCR assay). Methods: Our study included 937 MF patients who received an allogeneic hematopoietic cell transplant (HCT) between 2000 and 2016 and had an available pre-HCT blood sample at the Center for International Blood and Marrow Transplant Research Repository. Somatic mosaic chromosomal alterations (mCAs, including deletions, duplications, or copy-neutral losses-of-heterozygosity (CNLOH)) were called with the Mosaic Chromosomal Alteration (MoChA) algorithm using raw genotyping intensity data. A genome-wide association study (GWAS) was restricted to include 827 MF patients of European ancestry and utilized 4,135 genetically-matched healthy controls. Results: GWAS identified six independent MF susceptibility loci at genome-wide significance (P< 5×10 -8); four of which replicate prior MPN susceptibility loci [9p24.1(JAK2), 5p15.33(TERT), 3q25.33(IFT80), and 4q24(TET2)] and two novel MF loci [6p21.35(HLA-DQB1-AS1) and 17p13.1(TP53)] (Figure 1). A transcriptome-wide association analysis using whole blood GTEx data highlighted the 9p24.1 locus with increased JAK2 expression associated with elevated risk of MF (P= 2.18×10 -19). A strong colocalization statistic further indicated shared genetic component between eQTL and this JAK2 locus (HyPrColoc Posterior Probability= 0.6) (Figure 2). Based on the strong signal identified at TERT (Figure 1), we investigated the relationship between MF risk and genetically-inferred telomere length using a panel of 19 germline variants previously found to be associated with telomere length. Of the 19 telomere-length associated variants investigated, 7 were found to be associated with MF risk (binomial P= 2.31×10 -5, linear trend P= 5.48×10 -4) (Figure 3). Both Mendelian randomization and genome-wide genetic correlation analyses further indicated that increased risk of MF was associated with longer inherited telomere length. Utilizing available clinical mutation data on a subset of 185 patients, MF cases carrying the germline risk haplotype of the 9p24.1(JAK2) susceptibility locus were observed to more frequently have the JAK2 V617F mutation (71% vs 59%; P= 0.02). Targeted PacBio long-read sequencing around JAK2 provided further evidence of linkage between the germline risk allele and the JAK2 V617F mutation. Detectable autosomal mCAs were also abundant in MF cases with 67.4% having at least one mCA (compared to ~3% in the general population) and 27.6% having an mCA spanning JAK2 (mostly CNLOH) (Figure 4). In addition, using a binomial test for biased allelic imbalance, a cis relationship was identified at 9p24.1 in which the MF risk haplotype was predominantly duplicated by CNLOH (binomial P=1.36×10 -9). Regional sequencing of JAK2 further confirmed duplication of JAK2 V617F by CNLOH. Finally, we observed an inverse association between autosomal mCAs and qPCR measured telomere length (OR= 0.22, 95% CI= 0.07-0.65, P= 6.40×10 -3). These results were consistent by mCA chromosomal region and copy number state. Conclusion: Our results suggest a molecular framework for the genetic etiology of MF in which both genetically-inferred telomere length and germline variation at JAK2 are associated with increased MF risk. The 9p24.1 risk haplotype predisposes to the acquisition of a somatic JAK2 V617F mutation in cis and subsequent duplication of JAK2 V617F by mCAs (usually CNLOH). This process leads to aberrant JAK2 activity and increased clonal proliferation, accelerating telomere length shortening and increasing genomic instability in patients with MF. Figure 1 Figure 1. Disclosures Gupta: AbbVie: Consultancy, Honoraria; Constellation Pharma: Consultancy, Honoraria; Roche: Consultancy; Pfizer: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding. Lee: Janssen: Other; Incyte: Research Funding; AstraZeneca: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Syndax: Research Funding; Takeda: Research Funding; Amgen: Research Funding. Saber: Govt. COI: Other.

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Genetic factors of polygenic urolithiasis

Urologia Journal ◽

10.1177/0391560319898375 ◽

2020 ◽

Vol 87 (2) ◽

pp. 57-64

Author(s):

Filippova Tamara Vladimirovna ◽

Khafizov Кamil Faridovich ◽

Rudenko Vadim Igorevich ◽

Rapoport Leonid Mikhailovich ◽

Tsarichenko Dmitry Georgievich ◽

...

Keyword(s):

Genetic Factors ◽

Preventive Measures ◽

Association Studies ◽

Case Control ◽

International Studies ◽

Genome Wide Association Studies ◽

Case Control Studies ◽

Genome Wide ◽

Genetic Technologies ◽

High Throughput Dna Sequencing

The article summarizes the findings of Russian and international studies of the genetic aspects of polygenic urolithiasis associated with impairment of calcium metabolism. The article analyzes the genetic risk factors of polygenic nephrolithiasis that show significant association with the disease in case-control studies and Genome-Wide Association Studies (16 genes). We described the gene functions involved in concrement formation in polygenic nephrolithiasis. The modern molecular and genetic technologies (DNA microarray, high-throughput DNA sequencing, etc.) enable identification of the genetic predisposition to a specific disease, realization of the individualized treatment of the patient, and carrying out timely preventive measures among the proband’s relatives.

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Impact of the Molecular Profile of Malignant Clones on the Response to Interferon Alpha (IFNa) Therapy in JAK2V617F-Negative Essential Thrombocythemia (ET)

Blood ◽

10.1182/blood.v124.21.407.407 ◽

2014 ◽

Vol 124 (21) ◽

pp. 407-407

Author(s):

Emmanuelle Verger ◽

Bruno Cassinat ◽

Christine Dosquet ◽

Marie-Helene Schlageter ◽

Valérie Ugo ◽

...

Keyword(s):

Interferon Alpha ◽

Mutant Allele ◽

Molecular Response ◽

Fragment Analysis ◽

Blood Samples ◽

Off Label ◽

Allele Burden ◽

Ifna Therapy ◽

Calr Mutation

Abstract Background: The majority of ET patients (pts) without JAK2 or MPL mutations present somatic mutations in the calreticulin gene (CALR). However, a series of mutations in other genes involved in the epigenome, the splicing machinery or leukemic transformation have been described in ET, but contrary to MF, their impact has not been clearly assessed. We have previously shown that IFNa may considerably reduce the JAK2-mutated clones, and we also observed that TET2 mutated clones are resistant to IFNa therapy in JAK2+TET2+ pts with polycythemia vera. As little is known about the IFNa response of clones harboring mutations other than JAK2V617F in ET, we took advantage of a cohort of pts without JAK2 mutation but CALRmutated and treated with IFNa (according to international and local guidelines) to assess the dynamics of the different MPN clones during therapy. Aims: 1) To determine, using a Next Generation Sequencing (NGS) approach, the molecular pattern of mutations in genes previously shown to have a prognostic impact in MPNs in a series of CALR-mutated ET pts; 2) To study the evolution of these mutational patterns during IFNa therapy. Methods: JAK2 and MPL-negative ET pts followed in our department fulfilling the following criteria were included in the study: presence of a CALR mutation; availability of at least 3 sequential blood samples including one taken before IFNa; IFNa therapy for at least 3 months; informed consent for molecular analysis. Total DNA was extracted from blood samples (Qiagen blood DNA mini kits) for molecular analyses. Mutations in TET2, ASXL1, EZH2, SRSF2, IDH1, IDH2, SH2B3 and CSF3R were searched through a NGS approach on a MiSeq instrument using a TruSeq custom amplicon approach (Illumina). CALRgene mutation detection was done by direct Sanger sequencing of exon 9, and mutant allele burden (%CALR) was estimated by fragment analysis (GeneMapper software , Life technologies) with a sensitivity of about 1%. Both sequencing and fragment analysis were performed on a 3500xL DX Genetic Analyser (Life technologies). Molecular response was defined as complete (CMR) when CALR mutation was no longer detectable, partial (PMR) when %CALR was decreased by >50%, minor (mMR) when %CALR was decreased by 25 to 49%, and non responders if %CALR was reduced by less than 25%. Results: Among 238 ET pts without JAK2 or MPL mutations, we identified 52 pts (22%) treated with peg-IFNa-2a, of whom 24 fulfilled the inclusion criteria. Median age was 52 years (range 31-68), 67% were women, median follow up was 12 years (range : 1.5 – 29) and median IFN treatment duration was 30 months (range: 12 – 102). 23/24 (96%) pts achieved complete or partial hematological response to IFNa. CALR mutations (present in all patients, quantifiable in all but 1 because of 1bp difference between mutated and WT) were of type 1 in 10 (42%), type 2 in 8 (33%) and of neither type in 6 (25%) pts, respectively. In addition to CALR, a second mutation was found in 8 pts (33%) by NGS, affecting ASXL1 (n=2), TET2 (n=2), IDH2 (n=2), CSF3R (n=1) and SH2B3 (n=1) genes. Comparison of sequential samples showed that the %CALR decreased from a median of 43% (range: 8-57) to 26% (range: 0-49) in the last sample (p= 0.018). In details, %CALR decreased in 13 (57%) pts, including 1 CMR, 7 PMR and 5 mMR. Interestingly, molecular response to IFNa of CALR+clones appeared poorer in pts with additional mutations (vs. pts with CALR mutation alone): 50% were non responders (vs. 40%), %CALR even increased during follow up in 25% (vs. 7%), and the median decrease in %CALR was 32% (vs. 45%). Dynamics of clones harboring additional mutated genes showed that, in contrast to CALR, mutant allele burden did not vary in 6/8 pts. However, an IDH2 mutation decreased from 9% to 1% in 1 pt, and a TET2 mutation increased from 1% to 21% in another (while %CALR remained unchanged in both pts, at 44% and 19% respectively). Of note, after 12 years of median follow-up, no emergence of new mutation was observed in any patient. Conclusion: In this cohort of JAK2V617F-negative ET, 96% of pts achieved hematological response with IFNa therapy. However, molecular profiling suggests that the mutational pattern found in malignant clones modulates the molecular response to IFNa: 1) Existence of more than 1 mutation induces poorer molecular response; 2) Clones with CALR mutation alone are sensitive to IFNa; 3) Clones harboring mutations in genes other than CALR seem less responsive to IFNa therapy. Disclosures Off Label Use: Interferon alpha was used off-label in selected ET patients according to local and international guidelines (Barbui et al., J Clin Oncol. 2011 Feb 20;29(6):761-70).

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