Chasing down the triple-negative myeloproliferative neoplasms: Implications for molecular diagnostics

Introduction: Mutations in Janus kinase 2, calreticulin and thrombopoietin receptor genes have been identified in the genetic background of Philadelphia chromosome negative, “classic” myeloproliferative neoplasms. Aim: The aim of the authors was to identify driver mutations in a large myeloproliferative cohort of 949 patients. Method: A complex array of molecular techniques (qualitative and quantitative allele-specific polymerase chain reactions, fragment analyzes, high resolution melting and Sanger sequencing) was applied. Results: All 354 patients with polycythemia vera carried Janus kinase 2 mutations (V617F 98.6%, exon 12: 1.4%). In essential thrombocythemia (n = 468), the frequency of V617F was 61.3% (n = 287), that of calreticulin 25.2% (n = 118), and that of thrombopoietin receptor mutations 2.1% (n = 10), while 11.3% (n = 53) were triple-negative. Similar distribution was observed in primary myelofibrosis (n = 127): 58.3% (n = 74) V617F, 23.6% (n = 30) calreticulin, 6.3% (n = 8) thrombopoietin receptor mutation positive and 11.8% (n = 15) triple-negative. Conclusions: The recent discovery of calreticulin gene mutations led to definite molecular diagnostics in around 90% of clonal myeloproliferative cases. Orv. Hetil., 2014, 155(52), 2074–2081.

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Mutations in Triple-Negative Patients with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-128764 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5395-5395

Author(s):

Maria Carolina Costa Melo Svidnicki ◽

Paula De Melo Campos ◽

Moisés Alves Ferreira Filho ◽

Caio Augusto Leme Fujiura ◽

Tetsuichi Yoshizato ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Triple Negative ◽

Myeloproliferative Neoplasms ◽

Biological Significance ◽

Primary Myelofibrosis ◽

Prognostic Impact ◽

Hematopoietic Stem ◽

Missense Variants ◽

Myeloid Neoplasms ◽

Equity Ownership

Background Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). JAK2, MPL, and CALR mutations are considered as "driver mutations" and are directly implicated in the disease pathogenesis by activation of JAK/STAT signaling. However, some patients do not harbor any of these mutations. Since such triple-negative MPNs are very rare, no specific molecular markers were established to use for a precise differential diagnosis yet. So far, the introduction of next generation sequencing (NGS) technologies in research of myeloid neoplasms has provided valuable contributions on the identification of new molecular biomarkers, establishing more accurate risk rating and selection of more specific therapeutic interventions. This study aimed to identify, through targeted deep sequencing, specific genetic variants in patients with triple-negative MPNs. Methods We performed NGS targeted sequencing in 18 Brazilian triple-negative patients (11 MF and 7 ET). The median age at diagnosis was 64 years for primary myelofibrosis (range 42-78), and 52 years for essential thrombocythemia (range 19-79). In 14 cases, we used the Illumina TruSight Myeloid Panel covering 54 genes and in 4 cases we used a custom Sure Select Agilent panel containing more than 300 genes previously reported to be related to myeloid neoplasm. The inclusion criteria for variant filtering was quality score>30, read count>50, minor allele frequency<0.05, frameshift, nonsense, splice site and 5`UTR variants, and missense variants described as deleterious for at least three prediction softwares. Results Possible pathogenic mutations were identified in 33 genes by Illumina and/or Agilent panels. Frameshift/nonsense or missense variants previously described as pathogenic correspond to 11 variants (Table 1). Out of these, mutations in TET2 were the most frequently identified (in 9/18 (50%) of the cases). In three MF patients with TET2 mutations no other considered pathogenic mutation was identified, indicating a possible role of TET2 as a driver gene. According to previous reports, the frequency of TET2 mutations in triple-negative MPNs patients were only 7%. Phenotypically, in our triple-negative MPNs, 6/11 (54.5%) MF and 3/7 (42.9%) ET patients harbored TET2 mutations. Clinically, the adverse prognostic impact of TET2 mutations in MPN had not been consistently shown by previous studies. In addition, mutations in SF3B1, CEBPA, and KMT2A genes were the second most frequent ones detected in 2/18 each (11%) of the patients, some of which were concomitant with TET2 mutations, suggesting additional clonal advantage due to these genetic events. Other potentially pathogenic variants were also detected is genes that have been reported to be related to other myeloid neoplasms (KMT2A, CDKN2A, TERT, DIS3, ZFPM1, PCDHA8, SAMD9, SAMD9L, DCLRE1C,ERBB3, SDHA, PCDHA6, SVEP1, MAP2K1 and EP300). Conclusions We have characterized the genomic alterations in 18 Brazilian patients with MPN triple-negative for either JAK2, CALR or MPL main mutations. Using a sensitive NGS platform, we identified significantly more frequent mutations in TET2 gene (in as many as a half of the cases) compared to JAK2, MPL, CALR mutation-positive MPN cases. We also uncovered mutations in genes not previously related with in MPN. Our novel findings call for further studies validating the frequencies, biological significance, and prognostic impacts of somatic mutations in triple-negative MPNs. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding.

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Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics

Blood ◽

10.1182/blood-2018-99-118013 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1772-1772

Author(s):

Jiwon Yun ◽

Jung-Ah Kim ◽

Byungjin Hwang ◽

Hee Sue Park ◽

Kyongok Im ◽

...

Keyword(s):

Telomere Length ◽

Triple Negative ◽

Mutational Analysis ◽

Conflicts Of Interest ◽

Novel Mutation ◽

Myeloproliferative Neoplasms ◽

Molecular Characteristics ◽

Mutation Site ◽

Npm1 Mutation ◽

Molecular Features

Abstract Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations. Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them. Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045). Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs. Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere Disclosures No relevant conflicts of interest to declare.

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Targeted next-generation sequencing identified novel mutations in triple-negative myeloproliferative neoplasms

Medical Oncology ◽

10.1007/s12032-017-0944-z ◽

2017 ◽

Vol 34 (5) ◽

Cited By ~ 15

Author(s):

Yu-Cheng Chang ◽

Huan-Chau Lin ◽

Yi-Hao Chiang ◽

Caleb Gon-Shen Chen ◽

Ling Huang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Triple Negative ◽

Myeloproliferative Neoplasms ◽

Next Generation ◽

Novel Mutations ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

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Myeloproliferative Neoplasms and Myelodysplastic Syndromes: Molecular Diagnostics

Molecular Genetic Pathology ◽

10.1007/978-1-4614-4800-6_34 ◽

2012 ◽

pp. 929-944

Author(s):

Gerlinde Wernig ◽

Daniel A. Arber

Keyword(s):

Molecular Diagnostics ◽

Myelodysplastic Syndromes ◽

Myeloproliferative Neoplasms

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Triple-Negative Essential Thrombocythemia: Clinical-Pathological and Molecular Features. A Single-Center Cohort Study

Frontiers in Oncology ◽

10.3389/fonc.2021.637116 ◽

2021 ◽

Vol 11 ◽

Author(s):

Daniele Cattaneo ◽

Giorgio Alberto Croci ◽

Cristina Bucelli ◽

Silvia Tabano ◽

Marta Giulia Cannone ◽

...

Keyword(s):

Bone Marrow ◽

Essential Thrombocythemia ◽

Triple Negative ◽

Clinical Laboratory ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Molecular Profile ◽

Single Center ◽

Driver Genes ◽

Molecular Features

Lack of demonstrable mutations affecting JAK2, CALR, or MPL driver genes within the spectrum of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is currently referred to as a triple-negative genotype, which is found in about 10% of patients with essential thrombocythemia (ET) and 5–10% of those with primary myelofibrosis (PMF). Very few papers are presently available on triple-negative ET, which is basically described as an indolent disease, differently from triple-negative PMF, which is an aggressive myeloid neoplasm, with a significantly higher risk of leukemic evolution. The aim of the present study was to evaluate the bone marrow morphology and the clinical-laboratory parameters of triple-negative ET patients, as well as to determine their molecular profile using next-generation sequencing (NGS) to identify any potential clonal biomarkers. We evaluated a single-center series of 40 triple-negative ET patients, diagnosed according to the 2017 WHO classification criteria and regularly followed up at the Hematology Unit of our Institution, between January 1983 and January 2019. In all patients, NGS was performed using the Illumina Ampliseq Myeloid Panel; morphological and immunohistochemical features of the bone marrow trephine biopsies were also thoroughly reviewed. Nucleotide variants were detected in 35 out of 40 patients. In detail, 29 subjects harbored one or two variants and six cases showed three or more concomitant nucleotide changes. The most frequent sequence variants involved the TET2 gene (55.0%), followed by KIT (27.5%). Histologically, most of the cases displayed a classical ET morphology. Interestingly, prevalent megakaryocytes morphology was more frequently polymorphic with a mixture of giant megakaryocytes with hyperlobulated nuclei, normal and small sized maturing elements, and naked nuclei. Finally, in five cases a mild degree of reticulin fibrosis (MF-1) was evident together with an increase in the micro-vessel density. By means of NGS we were able to identify nucleotide variants in most cases, thus we suggest that a sizeable proportion of triple-negative ET patients do have a clonal disease. In analogy with driver genes-mutated MPNs, these observations may prevent issues arising concerning triple-negative ET treatment, especially when a cytoreductive therapy may be warranted.

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Myeloproliferative Neoplasms and Myelodysplastic Syndromes: Molecular Diagnostics

Molecular Genetic Pathology ◽

10.1007/978-1-4614-4800-6_35 ◽

2012 ◽

pp. 945-951

Author(s):

Timothy C. Greiner

Keyword(s):

Molecular Diagnostics ◽

Myelodysplastic Syndromes ◽

Myeloproliferative Neoplasms

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Mutational Landscape of Patients with Myelofibrosis That Do Not Harbor Mutations in JAK2, MPL and Calreticulin Driver Genes

Blood ◽

10.1182/blood.v126.23.4091.4091 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4091-4091

Author(s):

Giada Rotunno ◽

Paola Guglielmelli ◽

Annalisa Pacilli ◽

Tiziana Fanelli ◽

Carmela Mannarelli ◽

...

Keyword(s):

Board Of Directors ◽

Triple Negative ◽

Cox Regression ◽

Myeloproliferative Neoplasms ◽

Leukemic Transformation ◽

Driver Genes ◽

Advisory Committees ◽

Molecular Landscape ◽

Splicing Machinery

Abstract Background. In primary myelofibrosis (PMF), mutations of JAK2, CALR and MPL driver genes can be detected in about 60%, 20% and 5% of the patients (pts), respectively. Therefore, about 10% of the pts lack any of the 3 driver mutations and are operationally called "triple negative" (TN). TN pts present a higher risk of developing anemia and thrombocytopenia, suffer from reduced overall survival compared to other genotypes, particularly to CALR type1/type1-like mutations, and may be at higher risk of leukemic transformation (Blood 2014; 124:1062; Leukemia 2014; 28:1472; Blood 2014;124:2465). Aims The aim of the study was to analyse the molecular landscape of TN PMF pts by genotyping a set of myeloproliferative-neoplasms associated genes whose mutated status was shown to be prognostically relevant in previous studies (Leukemia 2013;27:1861; Blood 2014;123:2220; Leukemia 2014;28:1804;Leukemia 2014;28:1494). Methods. Diagnosis ofPMF was made according to WHO2008 criteria. All pts provided informed written consent. Previously published methods were used to screen mutations involving JAK2, MPL and CALR. A deep sequencing custom panel was designed to genotype the following genes: EZH2, ASXL1, IDH1/2, SRSF2, TP53, TET2, RUNX1, CBL, NRAS, KRAS, DNMT3A, SF3B1, IKZF1, NFE2, SH2B3. Analysis was performed using the Ion torrent PGM platform. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results. We analysed 28 TN PMF collected at the time of diagnosis. Median age was 66.7y, 57% were male. Median follow up was 2.1y (0.3-14.5). Overall, 8 patients (28.6%) progressed to AML. Death occurred in 20 pts (71.4%) after a median follow up of 2.3y (1.4-3.3y); progression to leukemia was the cause of death in 5 pts (25%). Overall, 22 pts (78.6%) presented at least one mutation in any of the 15 genes of the panel; 14 pts (50%) presented at least 2 mutations in different genes. The frequency of mutated genes was: SRSF2 39.3%, ASXL1 28.6%, EZH2 21.4%, NRAS 21.4%, TET2 10.7%, CBL 10.7%, IDH 3.6%, DNMT3A 3.6%, SH2B3 3.6%, U2AF1 3.6%. Twenty pts (71.4%) were classified as high molecular risk (HMR: ie, any mutated gene of ASXL1, EZH2, SRSF2, IDH1/2), a proportion significantly higher (P<0.01) than among JAK2 V617F (31.5%), CALR Type1/1-like (22.0%) and CALR Type2-type2-like (5.0%) (Leukemia 2013; 27:1861). Mutated genes were grouped into 3 different pathways: epigenetic regulation (ASXL1, EZH2, TET2, IDH), splicing machinery (SRSF2, U2AF1) and leukemic transformation (NRAS, DNMT3A, SH2B3, CBL). The most frequently mutated pathway was the epigenetic one with mutations in 14 pts (63.6%) of which 3 pts (21.4%) had 2 or more mutated genes of the pathway; 12 pts (54.5%) presented mutations in the splicing machinery and of these 8 pts (66.7%) had 2 or more mutated genes of the pathway; genes involved in leukemic transformation were mutated in 11 pts (50%) and 10 of 11 (90.9%) had 2 or more mutated genes. In three cases (13.6%) all 3 pathways were concurrently involved. Among the mutated genes, SRSF2 was associated with shorter survival [1.9y (1.6-2.2)] compared to pts with un-mutated SRSF2 [3.2y (0.9-5.4y)] (HR 2.3, 95%CI 0.9-6.4). SRSF2 mutations were also associated with shorter leukemia free survival (LFS): LFS not reached in un-mutated pts compared to 2.2y (1.8-2.7y) for mutated pts, with a HR=4.5 (95%CI 10.3-19.9). We also found that in pts with grade 1 bone marrow fibrosis the splicing and the leukemic pathway were more frequently mutated compared to grade 2-3 fibrosis (57% vs 28.5% and 50% vs 28.5% respectively). Conclusions. "Triple-negative" pts with PMF present high rate of mutations of MPN-associated genes, most of them are classified as "high molecular risk" and harbor >2 mutations. Mutated SRSF2 was particularly associated with shorter LFS. Such complex molecular landscape might help to explain the negative outcome of TN PMF pts. Disclosures Vannucchi: Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau.

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