Phylogenetic reconstruction of myeloproliferative neoplasm reveals very early origins and lifelong evolution

ABSTRACTMutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 31 years (range 12-54 years). Rates of clonal expansion varied substantially (<10% to >200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth and evolution drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.

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Phylogenetic reconstruction of adult blood cancer reveals early origins and lifelong evolution.

10.21203/rs.3.rs-93830/v1 ◽

2020 ◽

Author(s):

Jyoti Nangalia ◽

Nicholas Williams ◽

Joe Lee ◽

Luiza Moore ◽

E Baxter ◽

...

Keyword(s):

Clinical Presentation ◽

Myeloproliferative Neoplasms ◽

Phylogenetic Reconstruction ◽

Clonal Expansion ◽

In Utero ◽

Driver Mutation ◽

Driver Mutations ◽

New Paradigm ◽

Blood Cancer ◽

Age Related

Abstract Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Rates of clonal expansion varied substantially (<10% to >200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth trajectories drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.

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Driver Mutation Acquisition in Utero and Childhood Followed By Lifelong Clonal Evolution Underlie Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2020-143813 ◽

2020 ◽

Vol 136 (Supplement_2) ◽

pp. LBA-1-LBA-1

Author(s):

Nicholas Williams ◽

Joe Lee ◽

Luiza Moore ◽

Joanna E Baxter ◽

James Hewinson ◽

...

Keyword(s):

Somatic Mutations ◽

Clinical Presentation ◽

Myeloproliferative Neoplasms ◽

Clonal Expansion ◽

In Utero ◽

Driver Mutation ◽

Driver Mutations ◽

Patient Specific ◽

Blood Samples ◽

Driver Event

Background Recurrent mutations in cancer-associated genes drive tumour outgrowth, however, the timing of driver mutations and the dynamics of clonal expansion remain largely unknown. Philadelphia-negative myeloproliferative neoplasms (MPN) are unique cancers capturing the earliest stages of tumorigenesis through to disease evolution. Most patients harbor JAK2V617F, present as the only driver mutation or occurring in combination with driver mutations in genes such as DNMT3A or TET2. We aimed to identify the timing of driver mutations and clonal dynamics in adult MPN. Method We undertook whole-genome sequencing of individual single-cell derived hematopoietic colonies (n=952) together with targeted resequencing of longitudinal blood samples from 10 patients with MPN who presented with disease between ages 20 and 76 years. We identified 448,553 somatic mutations which were used to reconstruct phylogenetic trees of hematopoiesis, tracing blood cell lineages back to embryogenesis. We timed driver mutation acquisition, characterised the dynamics of tumour evolution and measured clonal expansion rates over the lifetime of patients. Resequencing of bulk blood samples corroborated clonal trajectories and provided population estimates. Results JAK2V617F was acquired in utero or childhood in all patients in whom JAK2V617F was the first or the only driver mutation. Earliest age estimates were within a few weeks post conception, and upper estimates of age of acquisition were between 4.1 months and 11.4 years, despite wide ranging ages of MPN presentation. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Subsequent driver mutation acquisition, including for JAK2V617F, was separated by decades. Disease latency following acquisition of JAK2V617F as a second driver event was still 12-27 years. DNMT3A mutations, commonly associated with age-related clonal hematopoiesis (CH), occurred as the first driver event, subsequent to mutated-JAK2, and as independent clones representing CH in MPN patients. DNMT3A mutations were also first acquired in utero or childhood, at the earliest 1.2 weeks post conception, and the latest 7.9 weeks of gestation to 7.8 years across 4 patients. A recurrent feature of the clonal landscape in MPN was the observation of similar genetic changes repeatedly occurring in unrelated clones within the same patient. Such 'parallel evolution' was observed for chr9p loss-of-heterozygosity, chr1q+ and mutations in myeloid cancer genes, suggesting that patient-specific factors flavour selective landscapes in MPN. Normal hematopoietic stem cells accumulated ~18 somatic mutations/year, however, mutant clones, particularly those with mutant-JAK2, acquired 1.5-5.5 excess mutations/ year and had shorter telomeres, reflecting increased cell divisions during clonal expansion. We modelled the rates of clonal expansion and found that they varied substantially, both across patients and within individuals. In one patient, an in utero acquired DNMT3A-mutated clone expanded slowly at <10%/year, taking 30 years to reach a clonal fraction of 1%, whilst a clone with mutated-JAK2, -DNMT3A and -TET2 expanded at >200%/year, doubling in size every 7 months. JAK2V617F as a single driver mutation also expanded variably across patients, highlighting that other factors, which may include germline, cytokine or stem cell differences between individuals, also influence selection for driver mutations. JAK2V617F associated clonal expansion rates in MPN were greater than that reported for JAK2-CH. Furthermore, rates of expansion in the cohort predicted time to clinical presentation, more so than age of mutation acquisition or tumour burden at diagnosis. This suggests that JAK2-mutant clonal expansion rates determine both if and when clinical manifestations occur. Driver mutations and rates of clonal expansion would have been detectable in blood one to four decades before clinical presentation. Conclusions MPN originate from driver mutation acquisition very early in life, even before birth, with life-long clonal expansion and evolution, establishing a new paradigm for blood cancer development. Early detection of mutant-JAK2 together with determination of clonal expansion rates could provide opportunities for early interventions aimed at minimising thrombotic risk and targeting the mutant clone in at risk individuals. Disclosures No relevant conflicts of interest to declare.

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Synergic Crosstalk between Inflammation, Oxidative Stress, and Genomic Alterations in BCR–ABL-Negative Myeloproliferative Neoplasm

Antioxidants ◽

10.3390/antiox9111037 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1037

Author(s):

Alessandro Allegra ◽

Giovanni Pioggia ◽

Alessandro Tonacci ◽

Marco Casciaro ◽

Caterina Musolino ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cell ◽

Chronic Inflammation ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Tumor Stage ◽

Driver Mutations ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Neoplasms ◽

Inflammatory State

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have recently been revealed to be related to chronic inflammation, oxidative stress, and the accumulation of reactive oxygen species. It has been proposed that MPNs represent a human inflammation model for tumor advancement, in which long-lasting inflammation serves as the driving element from early tumor stage (over polycythemia vera) to the later myelofibrotic cancer stage. It has been theorized that the starting event for acquired stem cell alteration may occur after a chronic inflammation stimulus with consequent myelopoietic drive, producing a genetic stem cell insult. When this occurs, the clone itself constantly produces inflammatory components in the bone marrow; these elements further cause clonal expansion. In BCR–ABL1-negative MPNs, the driver mutations include JAK 2, MPL, and CALR. Transcriptomic studies of hematopoietic stem cells from subjects with driver mutations have demonstrated the upregulation of inflammation-related genes capable of provoking the development of an inflammatory state. The possibility of acting on the inflammatory state as a therapeutic approach in MPNs appears promising, in which an intervention operating on the pathways that control the synthesis of cytokines and oxidative stress could be effective in reducing the possibility of leukemic progression and onset of complications.

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Clonal Hematopoiesis and Mutations of Myeloproliferative Neoplasms

Cancers ◽

10.3390/cancers12082100 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2100

Author(s):

Lasse Kjær

Keyword(s):

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Clinical Intervention ◽

Driver Mutation ◽

Response Monitoring ◽

Driver Mutations ◽

Clonal Hematopoiesis ◽

Disease Biology ◽

Background Population ◽

The Impact

Myeloproliferative neoplasms (MPNs) are associated with the fewest number of mutations among known cancers. The mutations propelling these malignancies are phenotypic drivers providing an important implement for diagnosis, treatment response monitoring, and gaining insight into the disease biology. The phenotypic drivers of Philadelphia chromosome negative MPN include mutations in JAK2, CALR, and MPL. The most prevalent driver mutation JAK2V617F can cause disease entities such as essential thrombocythemia (ET) and polycythemia vera (PV). The divergent development is considered to be influenced by the acquisition order of the phenotypic driver mutation relative to other MPN-related mutations such as TET2 and DNMT3A. Advances in molecular biology revealed emergence of clonal hematopoiesis (CH) to be inevitable with aging and associated with risk factors beyond the development of blood cancers. In addition to its well-established role in thrombosis, the JAK2V617F mutation is particularly connected to the risk of developing cardiovascular disease (CVD), a pertinent issue, as deep molecular screening has revealed the prevalence of the mutation to be much higher in the background population than previously anticipated. Recent findings suggest a profound under-diagnosis of MPNs, and considering the impact of CVD on society, this calls for early detection of phenotypic driver mutations and clinical intervention.

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Profile of Myeloproliferative Neoplasms in Kuwait: Population-Based Study

Blood ◽

10.1182/blood-2019-128536 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5373-5373

Author(s):

Mazyad Jamal Almazyad ◽

Aisha S Alwehaib ◽

Salem Alshemmari

Keyword(s):

High Risk ◽

Similar Pattern ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Population Based ◽

Driver Mutation ◽

Driver Mutations ◽

Positive Finding ◽

Population Based Study ◽

Arms Pcr

Introduction Myeloproliferative neoplasms (MPNs) are a group of hematopoietic disorders of stem-cell origin, characterized by mutations that disrupt hematopoietic signal-transduction pathways. The Middle East lacks an MPN registry representative of the disease in our area. Here we report on the epidemiology of these neoplasms in our area, including phenotype, clinical features and relevant outcomes. Methods This population-based study reports various demographic characteristics and clinical attributes of all suspected and confirmed MPN patients from all over Kuwait referred to the research hematology lab at Kuwait University & cytogenetic lab in Kuwait Cancer Control Centre (KCCC) during the period from 2007 to 2018. Molecular determination of the patients' driver mutation status currently relies on ARMS-PCR. Confirming a diagnosis follows the WHO criteria, and its refinements, for the diagnosis of MPNs. Data entry and analysis was performed using SPSS (v.22) software. Results Most patients are ≥ 40 years old (79.8%), with a median age of 55 years. Gender distribution is almost equal, with ethnic categorization as Kuwaiti and Non-Kuwaiti showing a similar pattern. ET is the most common diagnosis (40.1%), followed by PRV (32.3%). JAK2 V617F mutation is reported positive in 89.7% of cases, followed by CALR in 8.0% of MPNs. The incidence of MPNs ranged from 0.5 to 2.1 per 100,000 in 2007 through 2018. The lowest rate was recorded in 2007 (0.511) and the highest was observed in 2011 and 2016 (2.417 and 2.101, respectively). The increase in 2011 is likely due to the introduction of a more sensitive technique using ARMS-PCR for the diagnosis of MPNs, whereas the increase in 2017 may be explained by the publication of WHO 2016 modified criteria. Moreover, throughout the years, the distribution of MPNs in different age groups showed similar pattern, with the highest incidence in patients aged ≥ 60. Driver mutations can fit with a general increase in incidence from 2007-2017, which may be attributed to increased awareness among treating physicians asking suspected cases to screen for MPNs using molecular techniques.One hundred and twenty-four (18.5%) cases were documented to have a prior history of thrombosis, with roughly equal distribution between arterial and venous sites. A large proportion (89.5%) of the thrombotic events occurred in those who are ≥ 40 years old, with most events being associated with ET (34.7%) and PRV (33.1%). Almost one-third of cases of thrombosis were associated with undetermined MPN diagnosis. Participating patients were categorized as either low or high risk for thrombotic events, with the latter being defined as age ≥ 55 years and the presence of a previous thrombotic event. The results demonstrate that a total of 46 cases were defined as high risk, most of them being associated with ET (20 cases) and PRV (19 cases). A statistically significant association was reported between gender and site of occurrence of thrombotic events, with males having more arterial thromboses, and females were documented to have more venous thromboses. Conclusion JAK2 V617F driver mutation is the most common positive finding in the participating patients. Roughly one-fifth of the participants encountered thrombotic events, and the site of thrombosis is associated with gender, demonstrating statistical significance. These results should warrant a more thorough evaluation of MPNs in Kuwait to provide a better understanding of its epidemiology. This can be achieved through optimized documentation of patients' data, and testing for additional novel driver mutations and transformation; as well as encourage physicians in primary care centers to refer suspected cases for molecular diagnosis. Disclosures No relevant conflicts of interest to declare.

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MYB insufficiency disrupts proteostasis in hematopoietic stem cells leading to age-related neoplasia

10.1101/2021.09.04.458970 ◽

2021 ◽

Author(s):

M.L. Clarke ◽

R.B. Lemma ◽

D.S. Walton ◽

G. Volpe ◽

B. Noyvert ◽

...

Keyword(s):

Myeloproliferative Neoplasms ◽

Later Life ◽

Driver Mutations ◽

Myb Transcription Factor ◽

Hematopoietic Stem ◽

Myb Gene ◽

Age Related ◽

Disease Occurrence ◽

Age Profile ◽

Coding Variants

ABSTRACTThe Myb transcription factor plays critical roles in normal and malignant hematopoiesis. Acquired genetic dysregulation of Myb, which plays a central role in hematopoietic stem cell (HSC) gene regulation, is involved in the etiology of a number of leukemias. Also, inherited non-coding variants of the Myb gene are a factor in susceptibility to many hematological conditions, including myeloproliferative neoplasms (MPN), but the mechanisms by which variations in Myb levels predispose to disease, including age-dependency in disease occurrence, are completely unknown. Here, we address these key points by showing that Myb insufficiency in mice leads in later life to MPN, myelodysplasia, and leukemia, mirroring the age profile of equivalent human diseases. This age-dependence is intrinsic to HSC, involving progressive accumulation of subtle changes. Interestingly, and linking to previous studies showing the importance of proteostasis to the maintenance of normal HSC, we observed altered proteosomal activity in young Myb-insufficient mice and later elevated ribosome activity. We propose that these alterations collectively cause an imbalance in proteostasis, potentially creating a cellular milieu favoring disease initiation by driver mutations.

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Spectrum of Myeloid Mutations in Patients with Elevated Hemoglobin

Blood ◽

10.1182/blood-2021-148230 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2577-2577

Author(s):

Pratibha Bhai ◽

Benjamin Chin-Yee ◽

Ian Cheong ◽

Maxim Matyashin ◽

Michael A. Levy ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Prognostic Significance ◽

Laboratory Data ◽

Gene Mutations ◽

Jak2 V617f ◽

Driver Mutations ◽

Genetic Mutations ◽

World Population ◽

Age Related

Abstract Background: JAK2 V617F and exon 12 mutations are the characteristic driver mutations in polycythemia vera (PV), identified in more than 95% of patients. In addition, other genetic mutations have previously been described in JAK2-positive PV that appear to have prognostic significance (Tefferi et al., Blood 2016). The incidence of other driver mutations in unselected patients referred for elevated hemoglobin is less well studied. This study aims to characterize the genetic mutational landscape in a real-world population of patients referred for elevated hemoglobin using a targeted Next-Generation Sequencing (NGS)-based assay. Methods: We reviewed all patients referred for elevated hemoglobin levels (>160 g/L in females or >165 g/L in males) between 2018 and 2020 to hematology clinics at London Health Sciences Centre in Southwestern Ontario, Canada who underwent testing for genetic variants using the NGS-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA). This assay targets 40 key genes with diagnostic and prognostic implications in several myeloid malignancies (17 full genes and 23 genes with clinically relevant "hotspot" regions) and a panel of 29 fusion driver genes (>600 fusion partners). Patient demographics, laboratory data and final diagnosis were extracted from the electronic medical record. For all patients with genetic mutations, clinical diagnosis was confirmed by three independent reviewers. Results: A total of 529 patients underwent genetic testing for elevated hemoglobin levels: 389 (73.5%) were males (mean age 58; range 18-95) and 140 (26.5%) were female (mean age 60; range 24-85). JAK2 mutations were detected in 10.9% (58/529) of patients and a diagnosis of PV was confirmed. The majority of JAK2-mutated PV patients (n=57) were positive for JAK2 V617F, while one patient had an exon 12 mutation. Additional single myeloid mutations were detected in 34.5% (20/58) of JAK2-positive patients and involved the following genes: TET2 (11; 19%), DNMT3A (2; 3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%) and ZRSR2 (1; 1.7%) (Figure 1A). JAK2 mutations were not detected in 89.0% (471/529) of our cohort. A diagnosis of PV was confirmed in 2 JAK2-negative patients based on clinical features and myeloid mutations were detected in both: SRSF2 and TET2 gene mutations in 1 patient and SRSF2, IDH2, ASXL1 gene mutations in the other patient. Three JAK2-negative patients tested positive for the BCR-ABL fusion and were diagnosed with chronic myeloid leukemia. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and myeloid mutations were found in 6% (28/466) of these cases. Mutations were detected in DNMT3A (12; 2.6%), TET2 (5; 1.1%), ASXL1 (5; 1.1%), TP53 (2; 0.4%), NF1 (2; 0.4%), KIT (1; 0.2%), U2AF1 (1; 0.2%) (Figure 1B). All patients with JAK2-negative secondary erythrocytosis had only one myeloid gene mutation detected. Conclusion: Additional myeloid mutations other than JAK2 mutations are frequently identified in patients referred for erythrocytosis, with the highest frequencies observed in the TET2, DNMT3A and ASXL1 genes. The spectrum of myeloid mutations and overall incidence in JAK2-negative patients with secondary erythrocytosis is similar to the reported incidence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) (Jaiswal et al., NEJM 2014), and suggests that these may represent incidental age-related mutations. By contrast, among the JAK2-positive patients, 34.5% had at least one additional myeloid mutation supporting a pathogenic role in these patients with myeloproliferative neoplasms. While concomitant myeloid mutations in patients with PV are well-described, further research is required to elucidate the significance of variants identified in JAK2-negative patients classified as secondary erythrocytosis in order to determine whether these mutations contribute to clinical phenotype or represent background CHIP. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Spliceosome Mutations Are Common in MPN-Associated Myelofibrosis with RBC-Transfusion-Dependence and Correlate with Response to Pomalidomide

Blood ◽

10.1182/blood-2018-99-119402 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3037-3037

Author(s):

Onima Chowdhury ◽

Jennifer O'Sullivan ◽

Nikolas Barkas ◽

Gemma Buck ◽

Angela Hamblin ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasm ◽

Unmet Need ◽

Driver Mutation ◽

Driver Mutations ◽

Polycythaemia Vera ◽

Advisory Committees ◽

Epigenetic Regulators ◽

Rbc Transfusion

Abstract Background Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterised by the frequent presence of driver mutations in genes causing activation of JAK2 signalling pathways (JAK2, CALR and MPL). Additional mutations affecting epigenetic regulators and splicing machinery are common. Anaemia with RBC-transfusion-dependence is common in patients with advanced myelofibrosis and represents a major unmet need. The RESUME study assessed the rates of RBC-transfusion independence (TI) after therapy with Pomalidomide (POM) vs placebo in persons with MPN-associated myelofibrosis and RBC-transfusion dependence. 16% of patients in both the POM and placebo arms became TI. Aims The genetic landscape of strictly confirmed transfusion dependent MF is not fully characterised. Our aim was to analyse the genetics of transfusion-dependent myelofibrosis patients in the RESUME trial and correlate with clinical characteristics, outcome and therapy response. Methods DNA samples were available from 207 of 252 patients and analysed by targeted re-sequencing using a Fluidigm Access Array gene panel followed by next generation sequencing. The panel included JAK2, CALR, MPL, TET2, ASXL1, EZH2, DNMT3A, IDH1/2, CBL, IKZF1, U2AF1, CHEK2, TP53, SF3B1, SRSF2, SH2B3, BARD1, DAP3, HRAS, IRF4, KRAS, KIT, Mir662, NFE2, POLG, SCRIB, SETBP1, TCF12 and VPS45. Results 97% (95-99%) of subjects had a mutation in ≥1 targeted gene. 2 mutations were detected in 41% (34-48%) and ≥3 in 27% (21-33%). 7 had no detectable mutation. Mutations in JAK2V617F, CALR and MPL were identified in 66% (59-72%), 14% (8-19%) and 7% (4-11%) of subjects (Figure 1), with no driver mutation in 27 patients (13%; 9-18%) (triple-negative). 68% (61-74%) had additional non-driver mutations. 42% (35-48%) (N=86) had spliceosome mutations (U2AF1 [21%]; SF3B1 [11%]; SRSF2 [8%]; ZRSR2; [6%]). More spliceosome mutations were detected in men than women (47% [39-55%] vs 27% [15-40%]; p=0.009). Spliceosome mutations were mutually exclusive in 83 subjects and were less common in subjects with prior polycythaemia vera (17% [5-37%]) compared with prior essential thrombocythaemia (39% [22-58%]) and primary MF (46% [38-54%]; p=0.024). Mutations in epigenetic regulators (ASXL1, 28%; TET2, 8%; DNMT3A 5%; EZH2 4%) were detected at similar rates to those previously reported. High molecular risk (HMR) mutations (ASXL1, EZH2, IDH1/2, SRSF2) were detected in 36% [29-43%] of subjects. Only 10 of 105 subjects with an epigenetic regulator gene mutation had ≥1 related mutation. Subjects with JAK2V617F were significantly more likely than subjects with a CALR mutation to have: (1) ≥1 additional mutation (72% [64-79%] vs. 35% [18-54%], p=0.0001); (2) a spliceosome mutation (44% [36-53%] vs. 17% [6-36%], p=0.07), in particular a U2AF1 mutation (24% [17-32%] vs. 0%; p=0.004) and (3) a HMR mutation (38% [30-47%] vs. 21% [8-40%]; p=0.07). Survival at 1.5 years was 62% (55-67%) and was not significantly associated with the presence or number of mutations in this uniformly high-risk cohort. Survival in subjects without an SF3B1 mutation was better than those SF3B1-mutated (80% [56-91%]) vs. 59% [52-65%]; p=0.07). Driver mutation status did not influence the probability of achieving red blood cell (RBC) TI, regardless of therapy. Additional non-driver mutations were more often detected in those failing to achieve RBC-TI than those achieving RBC-TI (70% [63-77%] vs 56% [40-71%], p=0.07). Furthermore, those with additional non-driver mutations were less likely to achieve ≥50% reduction in RBC transfusions (24% [17-32%] vs. 39% [27-51%]; p=0.03). A significant correlation persisted in subjects receiving POM but not in those receiving placebo. There was also a significant correlation between U2AF1 mutations and RBC-TI in POM treated subjects compared with controls; U2AF1-mutated subjects were less likely to achieve RBC-TI (3% [1, 17%]) than U2AF1-unmutated subjects (25% [17, 34%], p=0.008). No other mutations were significantly correlated with response. Conclusion We found a high incidence of spliceosome mutations in persons with MPN-associated MF and RBC-transfusion-dependence. Mutation of U2AF1 correlated with response in subjects receiving POM but not in those receiving placebo. Incorporation of mutation profiling into clinical trial design may help to inform subgroups of patients that may benefit from the intervention. Disclosures Devos: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy. Gisslinger:Shire: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan Pharmaceuticals AG: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Honoraria. Kiladjian:Celgene: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mesa:Celgene: Research Funding; UT Health San Antonio - Mays Cancer Center: Employment; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding; Novartis: Consultancy; Promedior: Research Funding; Genentech: Research Funding. Ribrag:argenX: Research Funding; BMS: Consultancy, Honoraria, Other: travel; NanoString Technologies: Consultancy, Honoraria; pharmamar: Other: travel; Infinity: Consultancy, Honoraria; MSD: Honoraria; Amgen: Research Funding; Gilead: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Roche: Honoraria, Other: travel; epizyme: Consultancy, Honoraria; Incyte Corporation: Consultancy. Schiller:Celator/Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Research Funding. Vannucchi:Celgene: Membership on an entity's Board of Directors or advisory committees; ITALFARMACO: 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, Speakers Bureau. Reiser:Celgene: Employment. Zhong:Celgene: Employment. Mead:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Cell Therapeutics: Consultancy; Celgene: Research Funding; Elstar: Research Funding; Evotek: Research Funding.

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A Cure for Paroxysmal Nocturnal Hemoglobinuria Using Molecular Targeted Therapy Specific to a Driver Mutation

Blood ◽

10.1182/blood.v126.23.1215.1215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1215-1215 ◽

Cited By ~ 1

Author(s):

Takamasa Katagiri ◽

Ryo Tominaga ◽

Keisuke Kataoka ◽

Akio Maeda ◽

Hiroshi Gomyo ◽

...

Keyword(s):

Targeted Therapy ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Molecular Targeted Therapy ◽

Clonal Expansion ◽

Driver Mutation ◽

Driver Mutations ◽

Hematopoietic Progenitor Cell ◽

Complete Disappearance ◽

Molecular Response ◽

Hematopoietic Stem

Abstract Background: The clonal expansion of PIGA mutant hematopoietic stem cells (HSCs) can be induced by secondary driver mutations in genes such as HMGA2 and JAK2 in some patients with paroxysmal nocturnal hemoglobinuria (PNH). Theoretically, this type of PNH may be cured by molecular targeted therapy if the therapy is specific for the driver mutations and can eliminate PIGA mutant HSCs that acquired a proliferative advantage. However, this theory has not been proven because of the lack of an appropriate targeted therapy for known driver mutations responsible for clonal expansion of PIGA mutant HSCs. We recently treated a case of PNH complicated by chronic myeloid leukemia (CML) with nilotinib and observed a complete molecular response of CML followed by a complete disappearance of glycosylphosphatidylinositol-anchored protein-deficient (GPI-AP-, PNH-type) cells after 19 months of treatment. Case report: The patient, a 27-year-old Japanese woman, developed severe anemia with leukocytosis and thrombocytosis in 2013. The laboratory findings on admission were as follows: white blood cell count of 18.7x109/L with 1.7% stab neutrophils, 5.4% segmented neutrophils, 9.3% basophils, 1.3% promyelocytes, 5.0% myelocytes, 3.0% metamyelocytes, 18.0% lymphocytes and 0.7% blasts; hemoglobin (Hb)=6.0 g/dL, platelets=1,000x109/L, 20.0% reticulocytes, total/direct bilirubin=1.9/0.3 mg/dL, LDH=1963 IU/L and haptoglobin <10 mg/dL. A high-sensitivity flow cytometry analysis of the patient's peripheral blood at diagnosis revealed that 99.2% of granulocytes, 75.7% of erythrocytes and 99.3% of monocytes were GPI-AP-, while no T cells, B cells or NK cells had the PNH-phenotype (Figure 1A). This GPI-AP- cell distribution pattern was in sharp contrast to that of a patient with typical PNH who showed various percentages of GPI-AP- cells in all lineages of leukocytes and erythrocytes (Figure 1B). A fluorescent in situ hybridization analysis showed that 98.0% of the patient's granulocytes were BCR-ABL gene-fusion positive. Deep sequencing of leukocytes obtained at diagnosis showed a G279T (Q93H) mutation in exon 4 of the PIGA gene. The patient was diagnosed with PNH complicated by CML in the chronic phase, and was treated with nilotinib at 400 mg/day. The percentage of GPI-AP- cells rapidly decreased in response to nilotinib, with 0.02% GPI-AP- granulocytes after six months of nilotinib therapy when the patient's BCR-ABL mRNA decreased to 0.007%. BCR-ABL mRNA decreased to less than 0.0035% 15 months after therapy; however, small populations of GPI-AP- granulocytes (0.01%) and erythrocytes (0.005%) were still detected at this time (Figure 1A). GPI-AP- cells became undetectable after 19 months of nilotinib therapy, suggesting that the BCR/ABL fusion occurred in a subclone of a PIGA mutant hematopoietic progenitor cell (HPC). The patient was continuing nilotinib as of August 2015 without any signs of an increase in the BCR-ABL mRNA copy number. Conclusions: This case indicates the BCR-ABL fusion can be a driver mutation capable of inducing the clonal expansion of PIGA mutant clones. More importantly, the origin of PNH in our case proved to be a minor HPC clone with a PIGA mutation, suggesting that PNH can be derived from an HPC with a limited life span only if a potent second hit occurs in the PIGA mutant HPC. The identification of driver mutations in patients with PNH may therefore lead to the development of targeted therapy capable of curing PNH. Disclosures No relevant conflicts of interest to declare.

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Fulminant Essential Thrombocythemia Associated with Acquired Von Willebrand Syndrome and Bleeding Episodes in a 14-year-old Girl

Hämostaseologie ◽

10.1055/s-0039-1679929 ◽

2019 ◽

Vol 39 (04) ◽

pp. 404-408 ◽

Cited By ~ 3

Author(s):

C. Schneider ◽

E. Stutz-Grunder ◽

S. Lüer ◽

P. Keller ◽

J. A. Kremer Hovinga ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Clinical Symptoms ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Driver Mutations ◽

Acute Therapy ◽

Acquired Von Willebrand Syndrome ◽

Platelet Counts ◽

Bleeding Episodes ◽

Von Willebrand

Background Essential thrombocythemia is a chronic myeloproliferative neoplasm. It is extremely rare in children below 15 years of age with an estimated annual incidence of only 0.09 per million. Usually, clinical symptoms associated with essential thrombocythemia are mild or absent. Case Here, we present the case of a 14-year-old female patient fulminantly presenting with acute symptoms comprising visual impairment, palmar and plantar stabbing pain. Blood count revealed massive thrombocytosis of 2373 × 109/L. Bone marrow morphology showed elevated numbers of mature megakaryocytes. Von Willebrand factor activity/antigen ratio was significantly reduced compatible with an acquired Von Willebrand syndrome associated with high platelet counts. Molecular analyses for driver mutations of myeloproliferative neoplasms including JAK2V617F, CALR and MPL were negative. Acute therapy comprising hyperhydration and oxygen supply complemented by acetylsalicylic acid led to amelioration of symptoms. Medication with hydroxycarbamide maintained a significant reduction of platelet counts but had to be reduced or withheld several times due to neutropenia. Repeated bleeding episodes observed in the course were clearly associated with increases in platelet counts above 1200 × 109/L explained by acquired von Willebrand syndrome. Sixteen months after diagnosis, therapy was switched to pegylated interferon and platelet counts could be stabilized without significant side effects.

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