CML-143: BCR-ABL1 Translocation Combined with JAK2 or CALR Mutations in Russian CML Patients Undergoing TKI Therapy: Transcript Level and Mutation Allele Burden

Abstract Background and purpose: The clinical features and molecular markers of primary myelofibrosis (PMF) in Asian population have rarely been reported. We examined the clinical relevance of molecular markers in a large cohort of PMF patients in Taiwan. Methods: Bone marrow or blood samples at initial diagnosis from 145 patients consecutively diagnosed with PMF based on WHO criteria in Chang Gung Memorial Hospital-Linkou, Taiwan, were examined. EEC assay was performed in a serum free culture system. PRV-1 mRNA expression in granulocytes was measured by real-time RQ-PCR TaqMan assay. Pyrosequencing was used to detect JAK2 V617Fand its allele burden as well as 46/1 rs12343867 genotype in granulocytes. Mutational analysis of MPL (exon 10) was performed by PCR assay followed by direct sequencing. CALR (exon 9) mutations were screened by GeneScan analysis followed by sequencing for those with length changes. Ten of 20 patients progressed to secondary AML (sAML) had matched paired diagnosis and sAML samples available for comparative analysis. Results: Of the 145 patients with PMF, the median age was 64 years, 76 were male, IPSS low risk 25, Int I 23, Int II 41, and high risk 56 patients. In a median follow-up of 35.8 months (range 1.1 to 275.5 months), 20 patients progressed to sAML, 88 patients died with a median overall survival (OS) of 67.4 months. JAK2 V617F was detected in 52% (74/143) patients, CALR mutations in 30% (41/135) (type1 n=29; type 2 n=5; and others n=7), MPL mutations in 4% (5/141) (n=2/2/1 for W515L/K/A), and 11.0% of PMF patients were triple-negative. The incidence of 46/1 haplotype in 112 patients analyzed was TT 32 %, CT 36 %, and CC 32 %; C-allele frequency was significantly higher in PMF compared with 50 normal subjects (50% vs. 24%; P< 0.0001).EEC growth was detected in 48.9% (45/92) of patients examined. PRV-1 over-expression was present in 40% (28/70) of patients. Of the 10 matched paired PMF/sAML samples, 6 patients had CALR mutations with similar allele burden at both phases of disease whereas sAML evolved from a non-JAK2 V617F clone in one of the 3 patients carrying JAK2 V617F at diagnosis. Patients with EEC growth or PRV-1 over-expression were significantly associated with younger age, higher WBC and platelet counts. EEC-positive patients had higher Hb level and lower circulating blasts. JAK2 V617F was closely associated with higher WBC and platelet counts whereas patients with CALR mutations had lower WBC counts. None of these molecular markers had a correlation with constitutional symptom, IPSS, occurrence of thrombosis or risk of sAML transformation. EEC growth conferred a favorable leukemia-free survival (LFS) (P =0.019) and OS (P =0.013) compared with those without EEC. PRV-1 over-expression was associated with better OS (P =0.036). JAK2 V617F and MPL mutations did not influence LFS and OS. Allele burden of JAK2 V617F had no impact on outcomes. CALR mutations were associated with a favorable OS compared with mutation-negative patients (P =0.034). There were no difference in outcomes between type 1 and type 2 mutations of CALR. Patients with triple-negative mutations had a significantly inferior OS (P =0.020). CT genotype (46/1) was associated with shorter LFS (P =0.026). EEC growth was strongly associated with PRV-1 over-expression and JAK2 V617F mutation, whereas EEC formation and CALR mutations were mutually exclusive. In multivariate analysis, EEC growth was the most important predictor for LFS (HR 0.058; 95% CI: 0.005-0.676, P =0.023) and OS (HR 0.21; 95% CI 0.076-0.581, P =0.003) among the molecular markers; CALR mutations also held favorable OS (HR 0.245; 95% CI 0.085-0.709, P =0.009). Conclusions: Approximately 90% of PMF patients in Taiwan had JAK2 V617F, CALR, or MPL mutations, half were associated with C-allele genotype, 78% had EEC growth and /or PRV-1 over-expression. EEC growth was the most important independent factor for predicting better outcomes and CALR mutations also conferred a favorable OS. (Grant support: NSC96-2314-B-182-003, CMRPG330303, OMRPG3C0021, and MOHW103-TD-B-111-09) Disclosures No relevant conflicts of interest to declare.

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Monitoring of Calr Allele Burden in Patients with Essential Thrombocythemia Treated with Imetelstat, a Telomerase Inhibitor, Reveals Rapid and Substantial Molecular Responses

Blood ◽

10.1182/blood.v124.21.408.408 ◽

2014 ◽

Vol 124 (21) ◽

pp. 408-408

Author(s):

Gabriela M. Baerlocher ◽

Monika Haubitz ◽

Oliver G. Ottmann ◽

Olatoyosi Odenike ◽

Alexander Roeth ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Research Funding ◽

Mutant Allele ◽

Rapid Decrease ◽

Molecular Response ◽

Employment Equity ◽

Allele Burden ◽

Equity Ownership ◽

Calr Mutations

Abstract Background: In essential thrombocythemia (ET), mutations in the calreticulin gene (CALR) are found in the majority of patients that are negative for mutations in the JAK2 and MPL genes. Patients with mutated CALR have a better prognosis and lower thrombosis risk than those with mutated JAK2. Recently, decreases in the CALR mutant allele burden have been observed with interferon alpha after long-term treatment of two- and four-years, respectively (NEJM 2014, 371;2:188-9, Cassinat B. et al.). From the clinical phase II ET study with imetelstat (IT), a first in class, potent, specific inhibitor of telomerase, we reported a substantial and rapid decrease in the JAK2V617F allele burden. 7/8 patients (88%) reached a partial molecular response (MR: >50% reduction from baseline), 6 within the first 3 months and 1 after 12 months. Aims: We aimed to monitor molecular response to imetelstat therapy in ET patients with CALR mutations by serial measurements of CALR mutant allele burden. Methods: The study enrolled patients with ET who had failed or were intolerant to ≥1 prior therapy, or refused standard therapy. During the induction phase, patients were treated with IT 7.5 mg/kg or 9.4 mg/kg IV weekly until attainment of platelet (plt) count ~250-300x109/L. Maintenance phase was then commenced with dosing titrated to platelet count. CALR mutations were detected by Sanger sequencing and quantification of the allele burden was performed by fragment analysis. Results: 18 patients with ET (10 patients with JAK2V617F, 5 patients with CALR and 2 patients with MPL mutations) were enrolled and were treated in the study. 4 of the 5 CALR positive patients achieved complete hematologic responses (CR: Plts < 400 x109/L for 4 weeks) after a median of 6 weeks (range 5 to 14 weeks) and the 5th patient achieved a partial response after 19 weeks, with weekly imetelstat doses starting at 7.5 mg/kg in 2 patients and 9.4 mg/kg in 3 patients. CALR mutations consisted of three cases with type 1 (52-bp deletion; c.1092_1143del), one with type 2 (5-bp insertion; c.1154_1155insTTGTC) and one unknown mutation type (32-bp deletion; c.1092_1124del). Molecular monitoring of CALR allele burden at cycles 3, 6 and 9 demonstrated a rapid decrease in the CALR-mutated patients. 3 pts had a 35-50% reduction from baseline within 4 months and 1 pt had an 11% decrease within 8 months. One of these patients had a 48% reduction in 2 months and a second one had a deepening of response after 10 months to a 55% reduction. All 3 patients with CALR allele burden reductions of 35% or more also achieved hematologic CR. Conclusions: In 4 of 5 patients with CALR-mutated ET, IT induced a rapid CR and in 3 patients hematologic CR was associated with a substantial decrease in the allele burden of 35-50% after 4 months which is more rapid than what has so far been seen with other treatments for ET. Overall 9/13 patients with JAK2 or CALR mutations reached a >35-50% decrease of the mutant clone within 4 months of treatment with IT, providing clinical confirmation of imetelstat’s inhibition of neoplastic clonogenic cell growth in vivo. This additional evidence of reduction in the clonal burden supports IT’s potential to modify the biology of MPNs long-term. Disclosures Baerlocher: Geron Corporation: Research Funding. Odenike:Incyte Pharmaceuticals, Sanofi Aventis, Suneisis, Algeta, Spectrum Pharaceuticals: Honoraria. Roeth:Geron Corporation: Research Funding. Shih:Geron: Employment, Equity Ownership. Burington:Geron Corporation: Employment, Equity Ownership. Leibundgut:Geron Corporation: Research Funding.

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Impact of Underlying Mutational Profile, and Changes during Treatment, in MPN Patients Treated with JAK Inhibitors

Blood ◽

10.1182/blood.v126.23.353.353 ◽

2015 ◽

Vol 126 (23) ◽

pp. 353-353

Author(s):

Paola Guglielmelli ◽

Annalisa Pacilli ◽

Giada Rotunno ◽

Francesca Gesullo ◽

Alessandro Pancrazzi ◽

...

Keyword(s):

Platelet Count ◽

Clinical Response ◽

Mutational Analysis ◽

Jak2 V617f ◽

Symptomatic Improvement ◽

Driver Mutations ◽

Jak Inhibitors ◽

Advisory Committees ◽

Allele Burden ◽

Calr Mutations

Abstract Background. Most patients (pts) with MPN derive clinical benefits from treatment with JAK inhibitors (JAKi) but predictors of response or loss of response have not been clearly identified. Aims. To address the impact of underlying mutational profile of driver mutations (JAK2V617F, CALR, MPL), including other genes of JAK/STAT pathway, and "subclonal" (epigenetic, spliceosome) mutations, and their modifications under treatment, on the response to JAKi. Methods. 100 patients with WHO2008-diagnosis of MPN, 39 PV, 14 ET, 64 MF (37 PMF, 27 PPV/PET-MF), were included. 25 (12PV, 13 MF) had received hydroxyurea (HU), 75 (20 PV, 7 ET, 48 MF) a JAKi (67 ruxolitinib, 8 fedratinib). Response criteria were as by IWG-MRT (Blood 2013; 121:4778; Blood 2013; 122:1395). Mutations in 22 genes (JAK1, JAK2, JAK3, EZH2, ASXL1, TET2, IDH1, IDH2, CBL, SRSF2, DNMT3A, NFE2, SOCS1, SOCS2, SOCS3, SH2B3, STAT1, STAT3, STAT5A, STAT5B, SF3B1, U2AF1) were analyzed in blood DNA at baseline (bl) and at the latest available sample by deep sequencing with Ion Torrent-PGM. CALR mutations were analyzed by capillary electrophoresis; JAK2 V617F allele burden was measured by RT-qPCR assays. The nonparametric Wilcoxon rank-sum test, Kaplan-Meier method and Cox regression, were used for statistical analysis. Results. A. Impact of baseline mutational profile. PV and ET:At bl, 17/40 (42.5%) pts had subclonal mutations. Spleen volume reduction (SVR) by IWG-MRT criteria was obtained in 100% of pts without subclonal mutations compared to 66% of those with >1 mutations (P=0.04). Presence of NFE2 mutations uniformly predicted for lack of SVR (P=0.03). Loss of SVR was predicted by ASXL1 mut (67% vs 12% in un-mutated; P<0.05). No correlation of mutations with symptomatic improvement, normalization of leukocyte and platelet count, and control of hematocrit to <45% in PV pts was noticed. A trend to lower pruritus responses were seen in PV pts harboring >1 subclonal mutations (P=0.06). JAK2 V617F homozygous pts were more prone to have platelet count control (81%) compared to heterozygous (30%; P<0.04). MF:At bl, 33/64 (51.5%) pts had subclonal mutations. No correlation of mutations with symptomatic improvement, normalization of leukocyte and platelet count was found. SVR was not predicted by a high molecular risk status (Leukemia 2013;27:1861; Blood 2014;123:2157); however, harboring >1 of the 22 mutations was negatively associated with SVR (P=0.02) (HR 1.9, 95% CI 1.0-4.6). No correlation of JAK2 V617F or CALR mutations, or the allele burden, on SVR or loss of SVR was discovered. B. Changes during treatment. PV and ET:follow-up mutational analysis was performed at median treatment duration of 2.9y HU, 4.7y ruxolitinib, 1.7y fedratinib in 50 pts for driver mutation and 40 pts for subclonal genes. Of HU pts (n=12), 33% each had JAK2 V617F allele burden stable, increased and reduced by >10%. Median reduction of allele burden was -18.3%. 1 pt acquired 2 novel subclonal mutations (ASXL1, EZH2) and 2 showed increased allele burden of ASXL1 and TET2 by >10%. Among JAKi pts (n=30), 20 pts (66.7%) had JAK2 V617F allele burden reduction by a median of -33.3% (-13.6 to -100%), stable in 9. Degree of allele burden reduction was positively correlated with length of JAKi treatment (P=0.04). 1 pt acquired novel ASXL1, 2 pts had increase (ASXL1, EZH2) and 6 pts reduction of >10% of allele burden (4 TET2, 1 DNMT3A, 1 SH2B3). No correlation was seen with clinical response over time. MF: follow-up mutational analysis was performed at median treatment duration of 2.4y HU, 1.9y ruxolitinib, 1.4y fedratinib. HU pts (n=13): of the 7 JAK2 V617F mut, 4 increased and 1 decreased the allele burden, and 1 of 3 CALR mutated reduced allele burden, by >10%. 1 pt acquired CBL mutation, other mutations (n=7) were stable. Among JAKi treated pts, of the 41 JAK2 V617F mutated, 51% showed a median allele burden reduction of -14.8% (-10.5% to -53.9%). Of 10 pts with subclonal mutations at baseline, 4 clones (3 ASXL1 and 1 NFE2) increased and 2 (EZH2, ASXL1) decreased by >10%. Only 1 pt acquired a novel mutation in EZH2. No correlation was seen with clinical response over time. Discussion. These data suggest that only minimal influence on clinical response is provided by driver mutations and their allele burden, or subclonal mutations, in MPN patients receiving JAKi. The clinical relevance of different clone fluctuations over treatment with conventional therapy and JAKi remains to be addressed. Disclosures Vannucchi: Baxalta: Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Calreticulin Mutation Is Associated with Milder Disease in Patients with Post Essential Thrombocythemia Myelofibrosis (PET-MF) Compared with JAK2V617F Mutation: A Study from the AGIMM Group

Blood ◽

10.1182/blood.v124.21.3179.3179 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3179-3179

Author(s):

Paola Guglielmelli ◽

Giada Rotunno ◽

Giada Brogi ◽

Annalisa Pacilli ◽

Costanza Bogani ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Allele Burden ◽

Risk Of Death ◽

Kaplan Meier ◽

Significant Difference ◽

Calr Mutations ◽

The Impact ◽

Calr Mutation

Abstract Background: Mutations in the gene calreticulin (CALR) were recently discovered in 60-80% of patients (pts) with primary myelofibrosis (PMF) and essential thrombocythemia (ET) who were un-mutated for JAK2V617F and MPLW515. CALR mutated PMF pts had better overall survival (OS) compared with JAK2V617F or MPLW515 mutated while in ET CALR mutations were associated with lower incidence of thrombosis although the effect on survival was not significant. Conversely, there is no information concerning the impact of CALRmutation on disease phenotype and prognosis in post-essential thrombocythemia myelofibrosis (PET-MF). Aims: The aim of the study was to assess whether CALR mutational status and/or allele burden had clinical and/or prognostic relevance in PET-MF compared with JAK2, MPLmutated or triple-negative (TN) pts. Methods: ET and PET-MF were diagnosed by 2008 WHO and IWG-MRT criteria respectively; all pts provided an informed consent. Genotyping for CALR, JAK2V617F and MPLW515 was performed in granulocytes using allele specific RTQ-PCR (JAK2, MPL), capillary electrophoresis and direct sequencing (CALR, MPL). The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 147 PET-MF pts from 4 Italian centres was collected. Pts median age was 63y. Median follow up from PET diagnosis was 3.2y (0.07-18.8y) and the median time from ET to PET diagnosis was 11.6y (0.9-30.6y). Death occurred in 38 pts (26%) and 14 pts (9.5%) developed acute leukemia (AML). The median OS in the entire series calculated from PET-MF diagnosis was 10.9y (7.1-14.7y). Frequency of mutations was: CALR 16%, JAK2V617F 77%; MPLW515 4.3%; TN 2.8%. The frequency of CALR mutations in PET-MF patients was superimposable to that observed in a control group of 576 ET patients from our Institution (15.5%) and slightly lower compared with other series (20-25%). Type of CALRmutations was: 59.6% type 1, 23.1%, type 2, 17.3% others, significantly different (P=0.023) from ET: 46% type 1, 38% type 2, 16% others. Median CALR allele burden in PET-MF was 56% (20%-100%) with no significant differences in the CALR mutation subtypes (57.5% in type 1, 47.5% in type 2 and 45.0% in others); however, the median mutant allele burden of CALR-mutated PET-MF patients was significantly higher than in ET patients (33%, range 2%-52%; n=100) (P<0.03) suggesting a role for mutated allele accumulation in evolution to PET-MF. Similarly, the median V617F allele burden in JAK2 mutated patients was 50.5% (range 5-100%) significantly greater than the value (24%; range, 1-87%) (P=0.02) in ET pts, confirming previous data that evolution to PET-MF is associated with accumulation of mutated JAK2allele. We then compared hematological and clinical characteristics of the patients who were categorized according to their JAK2V617F, MPLW515 and CALR mutation status. There was no statistically significant difference among the unique patient mutational groups regarding age, hemoglobin, leukocyte and platelet count, peripheral blasts, LDH, circulating CD34+ cells, abnormal karyotype, grade of bone marrow fibrosis and cellularity, and pruritus. However, JAK2+ pts showed an increased rate of large (>10 cm) splenomegaly (28.6% vs 14% in CALR+, 7.1 in MPL+ and 25% in TN pts; P=0.02) and constitutional symptoms (50% vs 18.8% in CALR+, 45% in MPL+ and 12.5% in TN pts; P=0.002). The interval from ET to PET-MF was significantly longer in CALR+ pts (14.5y) compared with JAK2+ (10.2y) and TN patients (11.0y; P=0.04 for both) and similar to MPL+ (14y). There was a reduced rate of death (13.5%) in CALR+ compared with JAK2+ (30.6%), MPL+ (21.4%) and TN (66.7%) pts (P=0.005), although Kaplan Meier estimates did not reach a statistically significant difference. Finally, there were less AML transformation in CALR+ pts (1.9%) compared with JAK2+ (13.9%), MPL+(7.1%) and TN (22.2%) (P=0.04). Conclusion: These results show that CALR mutation is associated with delayed transformation of ET to PET-MF, a milder disease in terms of splenomegaly and symptom burden and a reduced risk of death compared with JAK2V617F PET-MF pts and more in general with MPL mutated and TN pts.In addition, similar to findings in primary MF and unlike in ET, PET-MF is characterized by prevalence of type 1 CALR mutations. Disclosures No relevant conflicts of interest to declare.

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Updated antitumor activity and safety of FPA144, an ADCC-enhanced, FGFR2b isoform-specific monoclonal antibody, in patients with FGFR2b+ gastric cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.4067 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 4067-4067 ◽

Cited By ~ 8

Author(s):

Daniel V.T. Catenacci ◽

Sun Young Rha ◽

Yung-Jue Bang ◽

Zev A. Wainberg ◽

Joseph Chao ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Patients ◽

Phase 1 ◽

Infusion Reaction ◽

Gene Copy ◽

Allele Burden ◽

Fgfr2 Gene ◽

Study Results ◽

Gastric Cancer Patients ◽

Mutation Allele

4067 Background: FGFR2b-overexpressing gastric cancer is characterized by poor prognosis. FPA144, a humanized monoclonal IgG1 antibody that specifically binds to and blocks FGFR2b, has been engineered for enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). FPA144-001 is a two-part Phase 1 study of FPA144 monotherapy in patients with advanced solid tumors, including gastric and gastroesophageal cancers (GEJ cancers). Methods: Part 1A was a 3+3 design to assess safety and PK and to establish a recommended dose (RD) of FPA144. Patients with gastric cancer were enrolled in Part 1B to assess PK in gastric cancer. Part 2 includes 4 cohorts of gastric cancer patients with either high, moderate, low or no FGFR2b overexpression based on a centralized immunohistochemistry (IHC) assay. Here, we describe results of gastric cancer patients that highly overexpress FGFR2b (FGFR2b+ High) enrolled in Parts 1 and 2 of the study. Results: As of October 28, 2016, 18 FGFR2b+ High (IHC 3+ ≥10% tumor membrane staining) patients were enrolled in the study. 12 of these patients received the RD of 15 mg/kg every 2 weeks. Enrolled patients received a median of 3 prior treatment regimens. Fatigue (22.2%, none ≥ gr 3) and infusion reaction (16.7%, 5.6% gr 3) were the most common treatment-related AEs. Treatment-related SAEs were reported in 2 patients: Grade 2 ulcerative keratitis and Grade 3 infusion reaction. There were 5 PRs, 4 confirmed and 1 unconfirmed. Disease control (PR+SD) was 55.6%, including a confirmed ORR of 22% with median DOR of 15.4 weeks. ctDNA analysis of a responding patient revealed baseline elevated FGFR2 gene copy (165 copies in the blood, mutation allele burden 66%) that decreased after monotherapy (nadir 75 copies, mutation allele burden 38.5%) corresponding with clinical response, serum tumor markers and near complete response on PET imaging. Conclusions: The demonstration of activity and an acceptable safety profile supports further development of FPA144 in patients with FGFR2b+ tumors. FGFR2 gene amplification detected in ctDNA may provide a non-invasive diagnostic test for patient selection. Updated data will be presented. Clinical trial information: NCT02318329.

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Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

Genes ◽

10.3390/genes12040559 ◽

2021 ◽

Vol 12 (4) ◽

pp. 559

Author(s):

Tatiana V. Makarik ◽

Adhamjon O. Abdullaev ◽

Elena E. Nikulina ◽

Svetlana A. Treglazova ◽

Elena E. Stepanova ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Gene Mutations ◽

Jak2 V617f ◽

Janus Kinase ◽

Allele Burden ◽

Chronic Myeloproliferative Neoplasms ◽

Exon 9 ◽

Calr Mutations ◽

Exon 10 ◽

Proliferative Signal

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

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A Rapid, Sensitive and Specific Method for Quantifying Calr Mutant Allele Burden in Persons with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.5210.5210 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5210-5210

Author(s):

Jiao Zhou ◽

Qiumei Yao ◽

Robert Peter Gale ◽

Jinlan Li ◽

Lingdi Li ◽

...

Keyword(s):

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Mutation Screening ◽

Heterozygous Mutation ◽

Specific Method ◽

Wild Type ◽

Allele Burden ◽

Exon 9 ◽

Calr Mutations ◽

Calr Mutation

Abstract Background: CALR mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2V617F. Consequently rapid, sensitive and specific methods to detect and quantify these mutations are needed. Methods: We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2V617F negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. Results: We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. Conclusions: PCR amplification followed by fragment length analysis is a rapid, sensitive and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PV with JAK2V617F and for estimating mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 1. Standard curve for the detection of mutant allele burden. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 2. Titration analyses of sensitivity of CALR mutation screening by sequencing and fragment analyses. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Figure 3. Figure 3 Sequencing traces show heterozygous mutation of CALR. Gene scan electropherogram from PCR method and partial sequence of CALR exon 9 from sequencing method (numbering according to GenBank access number: NC_000019.9). A-P: Detected a wild type and 15 CALR mutation types by sequencing and fragment analysis methods. A: wild type, B-I: deletions, H-L: insertions, M-P: complex indels. Disclosures No relevant conflicts of interest to declare.

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