Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms

Key Points The total number of somatic mutations was inversely correlated with survival and risk of leukemic transformation in MPN. The great majority of somatic mutations were already present at MPN diagnosis, and very few new mutations were detected during follow-up.

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Whole Exome Sequencing Reveals Clonal Evolution of Myeloproliferative Neoplasms to Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.1626.1626 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1626-1626

Author(s):

Jelena D Milosevic Feenstra ◽

Elisa Rumi ◽

Daniela Pietra ◽

Andreas Schönegger ◽

Christoph Bock ◽

...

Keyword(s):

Chromosomal Aberrations ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Clonal Evolution ◽

Chronic Phase ◽

Jak2 V617f ◽

Leukemic Transformation ◽

Whole Exome ◽

Acute Myeloid

Abstract Disease progression to acute myeloid leukemia (AML) is observed in 7% of the cases with the three classical BCR-ABL1 negative myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). According to the WHO, the presence of ≥ 20% blasts in bone marrow or peripheral blood is the diagnostic criterion for establishing leukemic transformation. PMF patients are at the highest risk, while PV and ET patients often develop secondary myelofibrosis prior to the leukemic transformation. Post-MPN AML patients have poor prognosis and clonal evolution of MPN chronic phase to AML is not well understood. Here we aimed to study the clonal evolution from MPN to AML in 7 cases, by performing whole exome sequencing (WES) on samples taken at various disease stages from individual patients. From the 7 post-MPN AML patients included in the study, 4 were diagnosed with PV and 3 with ET during chronic phase of the disease. For all 7 patients WES was performed on DNA samples from the control tissue, chronic phase and/or accelerated phase, and the leukemic phase of the disease. All variants identified by WES were validated using Sanger sequencing. In addition, tumor samples were analyzed for genomic deletions, gains and uniparental disomies (UPD) using SNP microarrays. We identified on average 16 somatic mutations (range 12-27) and 4 chromosomal aberrations (range 0-9) per patient in the leukemic stage of the disease. All patients were JAK2-V617F positive. 115 validated somatic mutations affected a total of 100 different genes. Most mutations were found in genes that were previously not linked to myeloid cancers, however, they were not recurrent. Besides JAK2-V617F, recurrent mutations were found in TP53 (N=3/7), RUNX1 (N=2/7), TET2 (N=2/7) and MPL (N=2/7). Biallelic TP53, RUNX1 and TET2 mutations were present in single patients. Known MPN and AML-related genes such as DNMT3A, SRSF2, U2AF1, IDH2, KIT, and PHF6 were mutated in single patients. We identified 25 chromosomal aberrations in 7 patients. Del5q, del6p, del7q and 9pUPD were recurrent. UPDs and trisomies of chromosomes 9, 12q, 17p and 21 were coupled with mutations in JAK2, SH2B3, TP53 and RUNX1. One patient harbored focal deletions of <1Mb on chromosomes 10 and 12, targeting TET1 and ETV6, respectively. We used variant frequencies detected by WES and copy number ratios and allelic difference values detected by microarrays at various stages of the disease to reconstruct the clonal evolution from chronic phase to AML in the 7 studied cases. Mutations with similar variant frequencies showing changes of allelic frequency in the same direction were assumed to be part of the same clone. Figure 1 illustrates an example of the proposed model for clonal evolution in Patient 6. As in this patient we had WES data from chronic, accelerated and leukemic stage of the disease, we first analyzed the clonal evolution from chronic to the accelerated phase. In the chronic phase the ~60-80% of granulocytes were derived from a single clone carrying 5 somatic mutations (JAK2, CAD, PPFIA2, SCNG, USH2A) and 2 chromosomal aberrations (1q gain and trisomy 9). At the accelerated phase of the disease we could observe that the main clone acquired somatic mutations in ADIPOQ, EYA3 and FAM123C and that there is at least one subclone (~40% of cells) appearing with mutations in OGDH, PHF6, USH2A, del7q and 9qUPD. At the leukemic stage, the clone with 9qUPD was suppressed by the outgrowing clone carrying OGDH and other mutations. We could also show that at the final leukemic stage the dominant clone acquired RUNX1 S400X mutation, amplified with a trisomy of chromosome 21, while the other RUNX1 allele mutated to Q262X. In each of the 7 studied patients the clonal evolution was unique and complex process with a few common features. Loss of TP53 is the most common genetic lesion. TET2 mutations are early events in clonal evolution of MPN and often precede the acquisition of JAK2-V617F, while RUNX1 mutations seem to be late events, leading to differentiation arrest and appearance of blasts. We demonstrated that in 6/7 studied cases the clonal evolution was a linear process, led by sequential acquisition of somatic mutations on the basis of the same clone causing the chronic disease. This finding is in line with the results of our previous study where we showed that the genetic basis of secondary AML is significantly different from de novo AML. Disclosures Kralovics: AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

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Development of a Murine Model for Leukemic Transformation of Myeloproliferative Neoplasms for Preclinical Therapeutic Studies

Blood ◽

10.1182/blood.v120.21.808.808 ◽

2012 ◽

Vol 120 (21) ◽

pp. 808-808 ◽

Cited By ~ 1

Author(s):

Raajit K. Rampal ◽

Suveg Pandey ◽

Omar Abdel-Wahab ◽

Jennifer J Tsai ◽

Taghi Manshouri ◽

...

Keyword(s):

Murine Model ◽

Research Funding ◽

Somatic Mutations ◽

Myeloproliferative Neoplasms ◽

Survival Curve ◽

Philadelphia Chromosome ◽

In Vivo Studies ◽

Leukemic Transformation

Abstract Abstract 808 A subset of patients with Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) (Polycythemia Vera (PV), Essential Thrombocytosis (ET), and Primary Myelofibrosis (PMF)) subsequently transform to acute myeloid leukemia (AML). Leukemic transformation (LT) after MPN occurs in as many as 23% of PMF patients within 10 years of diagnosis, and in 4–8% of PV and ET patients in the first 18 years after diagnosis. The development of AML after an antecedent MPN is associated with a dismal clinical outcome, and is associated with a poor response to conventional anti-leukemic therapies. Although somatic mutations in the JAK-STAT signaling pathway, including in JAK2 and MPL, occur in the majority of MPN patients, the somatic mutations that drive LT from a pre-existing MPN have not been fully delineated. Recent candidate mutational studies have identified recurrent somatic mutations in a subset of known leukemogenic disease alleles at the time of transformation from MPN to AML, including mutations in TP53, IDH1/2, TET2 and SRSF2 as well as deletions in IKZF1. However, the functional contribution of these specific genetic events to LT has not been delineated, and genetically accurate models of transformation of Philadelphia-chromosome negative MPN to AML have not been reported to date. In order to develop a genetically accurate murine model of LT, we have modeled expression of JAK2V617F mutation in combination with TP53 loss in vivo to further our understanding of progression from MPN to AML and to use this preclinical model of LT to test novel therapies. Bone marrow (BM) cells from C57/Bl6 Tp53−/− and littermate control mice were infected with JAK2V617F-IRES-GFP retrovirus, followed by transplantation of transduced cells into lethally irradiated congenic recipients. Of note, transplantation of JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells was associated with impaired survival; 50% of mice injected with JAK2V617F/Tp53−/− cells died by day 100, whereas all mice injected with JAK2V617F positive cells survived 100 days or longer (p=0.011) (figure 1). Mice injected with JAK2V617F/Tp53−/− cells presented with significant leukocytosis, with a mean WBC of 38.4 in mice engrafted with JAK2V617F/Tp53−/− cells compared with 11.4 in JAK2V617F/Tp53 wildtype mice. At the time of sacrifice, all mice engrafted with JAK2V617F/Tp53−/− cells had increased numbers of blasts in the peripheral blood and bone marrow, as assessed by morphologic evaluation and flow cytometric analysis which noted CD117 expression on leukemic blasts. BM cells from mice engrafted with JAK2V617F/Tp53−/− cells were characterized by increased serial replating (>10 platings), which was not observed in plating studies with JAK2V617F positive cells. In addition, we noted that the disease from JAK2V617F/Tp53−/− cells, but not JAK2V617F positive cells, was transplantable into secondary recipients consistent with increased self-renewal in vivo. We have begun testing the efficacy of novel therapies in this murine model, using both in vitro assays and in vivo studies in secondary transplantation studies. Treatment with the JAK kinase inhibitors INCB18424 and CYT 387 resulted in dose-dependent inhibition of colony formation in vitro. The combination of INCB18424 and Decitabine (which has demonstrated clinical efficacy in post-MPN-AML) is associated with synergistic inhibitory effects in vitro. Based on these results, we are performing in vivo studies with INCB18424, Decitabine, and INCB18424 + Decitabine, and results from these preclinical therapeutic studies will be presented in detail. Taken together, our data demonstrate that expression of JAK2V617F plus Tp53 loss, a genoptype commonly seen in patients who transform to AML after MPN, efficiently models LT in vivo. This model can now be utilized to examine the mechanisms of leukemic transformation, including assessment of the leukemic cell of origin in transformed disease. In addition this model can be utilized to test novel therapeutic strategies in a preclinical setting, which can be used to inform clinical trials in this poor-risk hematologic malignancy. Figure 1: Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Figure 1:. Survival curve of mice transplanted with JAK2V617F in presence and absence of Tp53 Disclosures: Verstovsek: Incyte Corporation: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Celgene: Research Funding; SBIO: Research Funding; Lilly Oncology: Research Funding; Bristol-Myers: Research Funding; Geron Corp.: Research Funding; Gilead: Research Funding; YM Biosciences: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Infinity Pharmaceuticals: Research Funding.

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Diffuse large B-cell lymphoma genotyping on the liquid biopsy

Blood ◽

10.1182/blood-2016-05-719641 ◽

2017 ◽

Vol 129 (14) ◽

pp. 1947-1957 ◽

Cited By ~ 70

Author(s):

Davide Rossi ◽

Fary Diop ◽

Elisa Spaccarotella ◽

Sara Monti ◽

Manuela Zanni ◽

...

Keyword(s):

B Cell ◽

Liquid Biopsy ◽

Somatic Mutations ◽

Cell Lymphoma ◽

Clonal Evolution ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Key Points ◽

Diagnostic Biopsy ◽

Large B Cell

Key Points Plasma cfDNA genotyping is as accurate as genotyping of the diagnostic biopsy in detecting clonal somatic mutations in DLBCL. Plasma cfDNA genotyping is a real-time, noninvasive tool that can be used to track clonal evolution in DLBCL.

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Driver somatic mutations identify distinct disease entities within myeloid neoplasms with myelodysplasia

Blood ◽

10.1182/blood-2014-03-560227 ◽

2014 ◽

Vol 124 (9) ◽

pp. 1513-1521 ◽

Cited By ~ 158

Author(s):

Luca Malcovati ◽

Elli Papaemmanuil ◽

Ilaria Ambaglio ◽

Chiara Elena ◽

Anna Gallì ◽

...

Keyword(s):

Somatic Mutations ◽

Clinical Decision Making ◽

Myeloproliferative Neoplasms ◽

Clinical Decision ◽

Driver Mutations ◽

Myeloid Neoplasms ◽

Key Points ◽

Distinct Disease ◽

Disease Entities

Key Points Different driver mutations have distinct effects on phenotype of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Accounting for driver mutations may allow a classification of these disorders that is considerably relevant for clinical decision-making.

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Treatment outcomes following leukemic transformation in Philadelphia-negative myeloproliferative neoplasms

Blood ◽

10.1182/blood-2012-10-464248 ◽

2013 ◽

Vol 121 (14) ◽

pp. 2725-2733 ◽

Cited By ~ 66

Author(s):

James A. Kennedy ◽

Eshetu G. Atenafu ◽

Hans A. Messner ◽

Kenneth J. Craddock ◽

Joseph M. Brandwein ◽

...

Keyword(s):

Disease Control ◽

Treatment Outcomes ◽

Myeloproliferative Neoplasms ◽

Leukemic Transformation ◽

Curative Intent ◽

Key Points

Key Points Induction followed by allo-transplantation can achieve long-term disease control in select patients with AML arising from a Ph-MPN. In this population, transplant should be the goal in patients treated with curative intent, as induction alone provides limited benefit.

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Clinical Relevance of Clonal Hematopoiesis in the Oldest-Old Population: Analysis of the "Health and Anemia" Study

Blood ◽

10.1182/blood-2018-99-114717 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 750-750

Author(s):

Marianna Rossi ◽

Manja Meggendorfer ◽

Matteo Zampini ◽

Mauro Tettamanti ◽

Emma Riva ◽

...

Keyword(s):

Somatic Mutations ◽

Clonal Evolution ◽

Oldest Old ◽

Vascular Diseases ◽

Employment Equity ◽

Clonal Hematopoiesis ◽

Increased Risk ◽

Equity Ownership ◽

The Relationship

Abstract Background. Age-dependent clonal expansion of somatic mutations in the hematopoietic system is associated with an increased risk of hematological cancers (including myelodysplastic syndromes, MDS) and other illnesses (coronary heart disease and stroke). However, the presence of clonal hematopoiesis per se in a given individual has only limited predictive power. We hypothesized that the study of oldest-old population can define more specifically the relationship between mutations in the hematopoietic system and risk for MDS, inflammation and vascular diseases. Methods. We analyzed 1004 oldest-old subjects (median age 84.2y, range 80-105) included in the "Health and Anemia" population-based study [Haematologica 2010;95:1849]. Using peripheral blood DNA, we looked for somatic mutations in 47 genes recurrently mutated in hematologic cancers. Results. Clonal mutations were observed in 32.8% of individuals (range 1-5). The majority of variants occurred in 3 genes: DNMT3A (36.4%), TET2 (24.3%) and ASXL1 (6.5%). Mutations in splicing genes, PPM1D and TP53 were found in 7.4%, 5% and 2% of cases, respectively. The mutation frequencies increased with age, up to 50% in individuals aged over 90 years (P=.011). Clonal hematopoiesis was associated with a lower 5-y probability of survival (P=.03), and prognosis was even poorer in patients carrying ≥2 mutations (P=.002) We first focused on the relationship between clonal hematopoiesis and MDS phenotype. Carrying a somatic mutation with a variant allele frequency (VAF) ≥.10, carrying ≥2 mutations, spliceosome gene mutations and co-mutation patterns involving TET2, DNMT3A had a positive predictive value for MDS (from .85 to 1.0). The most frequent early phenotypic changes in patients who developed MDS included an increasing red blood cell distribution width (RDW) and mean corpuscular volume (MCV). Preliminary analyses suggested that the combination of mutations and non-mutational factors (RDW, MCV, after excluding iron/vitamin depletion and thalassemia) may improve the capability to capture individual risk of developing MDS with respect to molecular data alone (P=.01) We studied clonal evolution in 72 patients with multiple samples available over a period of 5y. Clonal hematopoiesis was found at baseline in 22 cases: 2 individuals acquired additional mutations during follow-up, and 5 displayed significant increase in VAF. In 9 subjects without clonal hematopoiesis, mutations were acquired during follow-up. RDW and MCV changes, induction of unexplained cytopenia and overt MDS phenotype were significantly restricted to subjects displaying clonal evolution. We hypothesized that in oldest-old populations MDS could be underdiagnosed (many patients are not considered for bone marrow aspiration because of age). Cytopenia was a common finding in our cohort (20%) the underlying cause remaining unexplained in 27% of cases. In patients with unexplained anemia, carrying a somatic mutation had a positive predictive value for persistent, progressive, multilineage cytopenia (findings consistent with a MDS phenotype) and shorter survival (from .8 to .94). On this basis, 8% of all cytopenias might be undiagnosed MDS. Finally, we investigated the association between clonal hematopoiesis with inflammatory and vascular diseases. Mutations in DNMT3A, TET2, and ASXL1 were each individually associated with risk of coronary heart disease and death, and preliminary analyses suggest that clonal hematopoiesis is also associated with increased risk of rheumatological diseases (P from .03 to.009). We identified mutations in macrophages isolated from synovial fluid of 4/17 patients with rheumatoid arthritis and from atherosclerotic plaques of 3/25 patients with carotid stenosis. Functional studies of macrophages (expression of specific chemokine and cytokine gene patterns) are ongoing. All these findings are under validation in an independent cohort of 800 subjects enrolled in the "Monzino 80-plus" study [Alzheimers Dement 2015;11:258]]. Conclusion. Clonal hematopoiesis was associated with reduced survival in an oldest-old population. Specific mutational profiles define different risks of developing MDS and inflammatory/vascular diseases. Non mutational factors, such as early changes in red blood cell indices, may improve the capability to identify patients at increased risk of developing myeloid cancers. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Bolli:Celgene: Honoraria. Vassiliou:KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Chronic Exposure to Cytoreductive Treatment Shapes Clonal Evolution in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2021-152622 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3620-3620

Author(s):

Lin-Pierre Zhao ◽

Nabih Maslah ◽

Rafael Daltro De Oliveira ◽

Emmanuelle Verger ◽

Juliette Soret-Dulphy ◽

...

Keyword(s):

Board Of Directors ◽

Mutation Rate ◽

Chronic Exposure ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Clonal Evolution ◽

Advisory Committees ◽

Cytoreductive Treatment ◽

The Impact

Abstract Introduction: Myeloproliferative neoplasms (MPN) are a heterogeneous group of chronic myeloid malignancies resulting from the combination of a driver mutated gene (JAK2, MPL or CALR) and a variety of acquired additional somatic mutations. Although next-generation sequencing (NGS) has identified high molecular risk mutations associated with adverse prognosis (Vannucchi et al., Leukemia, 2013 , Guglielmelli et al., Leukemia, 2014 ), the clonal evolution of these mutations remains poorly described. Chronic exposure to cytoreductive treatment, especially genotoxic drugs such as hydroxyurea (HU), could impact clonal evolution. A previous study suggested that Interferon-α (IFN) could limit the accumulation of cytogenetic abnormalities compared to HU (Mondello et al., Blood, 2018). The objective of our study was to describe the long-term evolution of the mutational landscape in the era of NGS in a large cohort of MPN patients. Methods: A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. This study included 1039 of them in whom a NGS molecular analysis targeting 36 myeloid genes with a sensitivity of 1% was performed at diagnosis and/or during follow-up. Patients with only one NGS (n=588), AML/MDS transformation at either the first (n=3) or the second NGS (n=2) were excluded from the analysis. Serial NGS data obtained in chronic MPN phase were thus analyzed for 446 patients. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Mutation rates per year were calculated for each gene as the difference in the number of mutations between first and last NGS divided by the time interval (in years) between both NGS. Results : Median age at MPN diagnosis in our whole cohort was 51 years [IQR 41-60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with Polycythemia Vera (PV), Essential thrombocythemia (ET) and primary myelofibrosis (MF) respectively. 279 patients (63%) had at least one additional mutation at first NGS, and respectively 27 (6%) and 104 (23%) patients had TP53 and high molecular risk mutations. Median interval between MPN diagnosis and the first NGS was 6.5 years [IQR 1.7-13] while median time between the first and the last NGS was 2.5 years [IQR 1.6-4, range 0.3-14.3]. Overall, 178 patients (39.9%) acquired an additional mutation at last NGS evaluation, most frequently involving TET2, DNMT3A, ASXL1, TP53 and NFE2 genes . To study the impact of chronic MPN therapy on clonal evolution, we focused on patients who electively received HU (n=112) or IFN (n=92) as a monotherapy, or did not receive any cytoreductive treatment (n=119) between the first and the last NGS. The remaining patients received ruxolitinib (n=44), anagrelide (n=10), vercyte (n=7) or polytherapy (n=62). At last follow-up, 74 patients receiving IFN (80.4%) and 65 (58%) treated with HU had a complete hematological response. When combining all additional mutations, the global mutation rate per year did not significantly differ between treatment groups. When analyzing individual genes, TP53 mutation rate was higher in patients treated with HU compared to the patients receiving IFN (p=0.014) or not treated (p=0.008) (Figure). MDS/AML evolution occurred in 4 patients (3.6%) treated with HU, 2 (1.7%) without cytoreductive therapy versus none of the 92 patients treated with IFN (ns). In the whole cohort, MDS/AML evolution was significantly increased in patients harboring TP53 mutations (p= 0.004). In contrast, DNMT3A mutation rate was significantly increased in patients receiving IFN compared to patients treated with HU (p=0.045) (Figure). The latest result is in line with previous observations showing that loss of DNMT3A could confer resistance to IFN in a JAK2-V617F mouse model (Stetka et al., Blood, 2020). Conclusion: Our results highlight the impact of chronic cytoreductive therapy on clonal evolution shaping in MPN. IFN limits the emergence of TP53 mutated clones compared to HU, thus potentially reducing the risk of leukemogenesis. Emergence of DNMT3A mutated clones under IFN therapy requires further exploration and could potentially play a role in therapeutic resistance. This study on a large clinically and biologically annotated cohort illustrates how serial NGS analysis may guide therapeutic options for MPN patients. Figure 1 Figure 1. Disclosures Raffoux: PFIZER: Consultancy; ASTELLAS: Consultancy; ABBVIE: Consultancy; CELGENE/BMS: Consultancy. Kiladjian: AbbVie: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; PharmaEssentia: Other: Personal fees. Benajiba: Pfizer: Research Funding; Gilead: Research Funding.

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NFE2 Mutations Impact AML Transformation and Overall Survival in Patients with Myeloproliferative Neoplasms (MPN)

Blood ◽

10.1182/blood-2020-136177 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Clemence Marcault ◽

Lin-Pierre Zhao ◽

Rafael Daltro De Oliveira ◽

Juliette Soret ◽

Nicolas Gauthier ◽

...

Keyword(s):

Overall Survival ◽

Board Of Directors ◽

Somatic Mutations ◽

Response To Therapy ◽

Age At Diagnosis ◽

Prognostic Impact ◽

Leukemic Transformation ◽

Advisory Committees ◽

Increased Risk

Introduction: MPN are a heterogeneous group of chronic hematological malignancies often resulting from a combination of a driver gene mutation (JAK2, MPL or CALR) and a variety of somatic mutations harboring diverse prognosis values. A subset of MPN patients carry somatic mutations in the hematopoietic transcription factor NFE2 (nuclear factor erythroid 2) resulting in a functionally enhanced truncated form of NFE2 (Jutzi JS et al., JEM, 2013). Moreover, epigenetically induced overexpression of NFE2 has recently been reported in the majority of MPN patients (Peeken JC et al., Blood, 2018). In transgenic murine models, NFE2 overexpression results in an MPN phenotype (thrombocytosis, leukocytosis, EPO-independent colony formation, characteristic bone marrow histology and expansion of stem and progenitor compartments) and has recently been shown to predispose to the acquisition of additional genetic abnormalities and subsequent leukemic transformation (Kaufmann KB et al., JEM, 2012) (Jutzi JS et al., Blood, 2019). However, clinical impact of NFE2 mutations in MPN patients remains unknown. The aim of this study was to evaluate the phenotypic characteristics and prognostic impact of NFE2 somatic mutations in a large mono-centric cohort of MPN patients. Methods: A total of 1243 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and May 2020. This study included 707 of them in whom a next-generation sequencing (NGS) molecular analysis targeting 36 myeloid genes was performed at diagnosis and/or during follow-up. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Statistical analyses were performed using the STATA software (STATA 15.0 Corporation, College Station, TX). Results: In our cohort, 411 patients presented with polycythemia vera (PV), 577 with essential thrombocythemia (ET), 184 with primary or pre-fibrotic myelofibrosis (PMF), 59 with unclassified MPN and 12 with MDS/MPN. Median age at diagnosis was 51 years [40-63]. 73.1% patients had a JAK2V617F mutation, 14.1% a CALR mutation and 3.3% a MPL mutation. Overall, 64 (9.05%) patients harbored a NFE2 mutation with a variant allelic frequency (VAF) ≥ 0.5% and 36 had a VAF ≥ 5%, the latest were considered as NFE2 mutated for the rest of the study as VAF <5% may refer to a minor clone without clinical relevance. NFE2 mutations were present in 7.3%, 5.3% and 3.6% of PV, PMF and ET patients respectively. No significant association between the presence of NFE2 mutation and clinical/molecular MPN characteristics (driver mutation, constitutional symptoms, splenomegaly, blood counts, cytogenetic and other molecular features) was observed using a logistic regression association model. Median follow-up was 103.8 months, IQR [47.2; 175.6]. In terms of response to therapy, 52.8% of patients achieved complete response, complete hematological response or clinical improvement in NFE2 mutated vs 61.7% in non-mutated patients (p= 0.026). Interestingly, presence of a NFE2 mutation (HR 9.92, 95%CI[3.21; 30.64], p< 0.001), age at diagnosis (HR 1.09, 95%CI[1.05; 1.12], p< 0.001), PMF subtype (HR 6.92, 95%CI[2.81; 17.06], p < 0.001) and high-risk mutations (ASXL1, EZH2, SRSF2, IDH1/2 and U2AF1) (HR 2.45, 95%CI[1.14; 5.28], p=0.021) were independently associated with AML/MDS transformation free survival (TFS) in a COX regression multivariate analysis (Figure A). Presence of a NFE2 mutation was also independently associated with overall survival (OS) (HR 9.37, 95%CI [4.18; 21.03], p<0.001) (Figure B). Median TFS were 216.1 months and not reached, while median OS were 144.2 months and not reached for NFE2 mutated and non-mutated patients, respectively. No difference was observed in terms of thrombo-hemorrhagic events (HR 0.73; 95%CI [0.10; 5.21], p=0.752) and secondary myelofibrosis free survivals (HR 0.67; 95%CI [0.09; 4.87], p=0.693). Conclusion: In this retrospective study we show that presence of NFE2 mutations with a VAF ≥5% is independently associated with an increased risk of leukemic transformation and shorter overall survival. These findings are in line with recently reported animal models and suggest that NFE2 mutations screening should be routinely performed in MPN patients. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

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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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Genomics of MPN progression

Hematology ◽

10.1182/hematology.2020000129 ◽

2020 ◽

Vol 2020 (1) ◽

pp. 440-449

Author(s):

Anand A. Patel ◽

Olatoyosi Odenike

Keyword(s):

Myeloproliferative Neoplasms ◽

Clonal Evolution ◽

Philadelphia Chromosome ◽

Prognostic Information ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Heterogenous Group ◽

Calr Mutations ◽

Molecular Landscape ◽

Therapeutic Decision Making

Abstract The Philadelphia chromosome–negative (Ph−) myeloproliferative neoplasms (MPNs) are a heterogenous group of hematopoietic stem cell diseases characterized by activated JAK/STAT signaling and a variable propensity toward myelofibrotic and leukemic transformation. Acquisition of somatic mutations in addition to the canonical JAK2, MPL, and CALR mutations found in MPNs is an important catalyst in the clonal evolution and progression of these disorders. In recent years, our increasing understanding of the molecular landscape of Ph− MPNs has generated important prognostic information that informs our approach to risk stratification and therapeutic decision-making. This review will focus on the critical impact of genomics on our approach to management of advanced Ph− MPNs.

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