Mutation In Essential Thrombocythemia Research Articles

Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

The Impact of JAK2 V617F, CALR, and MPL Mutations as Molecular Diagnostic Markers of Myeloproliferative Neoplasms in Kurdish Patients. A Single-center Experience.

Myeloproliferative neoplasms have a high prevalence and genetic mutations play a role in their occurrence. Determination of these mutations can be valuable in the screening, diagnosis, and treatment of patients. Therefore, this study was conducted to investigate the mutation of JAK2, CALR, and MPL genes as diagnostic and prognostic biomarkers in patients with myeloproliferative neoplasms in the Kurdistan region of Iraq. This case-control study was conducted in 2021 on 223 patients with myeloproliferative neoplasm referred to Hiwa Sulaymaniyah Cancer Hospital. The data were collected from three groups of Polycythemia Vera (PV) patients (70 people), Essential Thrombocythemia (ET) (50 people), and Primary Myelofibrosis (PMF) (103 people) by sampling for JAK2, CALR, and MPL gene mutation tests and demographic and clinical information have been collected through examination. The data were analyzed by SPSS v. 23 software and descriptive and chi-square statistical tests. The study included 223myeloproliferative neoplasms (MPN) patients. JAK2 V617F mutation was detected mostly in PV patients and CALR and MPL mutations in ET and PMF patients and this mutation difference was significant in prognosis and disease diagnosis. An association between JAK 2 mutation and splenomegaly was also demonstrated. Considering the lack of a definitive diagnostic method in myeloproliferative disease, the results of this study showed that molecular studies, including JAK2 V617F, CALR, and MPL mutations and other hematological tests can be useful and effective in the diagnosis of MPN. In addition, it is necessary to pay attention to new diagnostic methods.

Hematological relevance of JAK2 V617F and calreticulin mutations in Tunisian patients with essential thrombocythemia

BackgroundThe genetic investigation of essential thrombocythemia(ET) has highlighted the presence of driver mutations in ET. Janus kinase JAK2V617F and calreticulin(CALR) mutations are the most frequent driver mutations and have significantly improved the molecular diagnosis of ET. The impact of genetic heterogeneity on clinical features has not been fully elucidated.This is the first study which aimed to determine the frequency of JAK2V617F and CALR exon9 mutations in Tunisian ET patients and to establish the correlation between hematological characteristics and mutational status.MethodsThis study included Tunisian patients suspected with ET and was conducted between September 2017 and March 2021. Genomic DNA of patients was isolated from peripheral blood samples. JAK2V617F was detected by AS‐PCR and CALR mutations were detected by PCR/direct sequencing. Clinical and hematological characteristics were also analyzed.ResultsTwo hundred and fifty ET patients were enrolled in this study. JAK2V617F mutation was found in 166/250 (66.4%) of patients, whereas CALR mutations were detected in 27/84 (32.1%) patients without JAK2V617F. Compared with JAK2V617F‐positive patients, those with CALR mutations showed lower hemoglobin level and lower leucocytes count (p = 0.007 and p = 0.004,respectively). CALR type 2 was the most frequent mutation of CALR detected in 55.55% of CALR mutated. Six of seven patients with thrombotic events harbored JAK2V617F mutation.ConclusionThe prevalence of driver mutations JAK2V617F or CALR mutations was 77.2% in Tunisian ET patients. Moreover, patients with JAK2 V617F mutation had a higher risk of thrombosis. The mutational status is necessary to improve the diagnosis and contribute to the therapeutic decisions.

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

Non-Driver Mutations and Gene Sequence Variants Identified By Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia

Background:Driver mutations in myeloproliferative neoplasms involve mutations in the JAK2, MPL, and CALR genes. However, incorporation of next generation sequencing (NGS) testing into routine clinical practice may identify for mutations or variants in additional hematologic malignancy-associated genes, the roles of which have not been extensively elucidated in patients with polycythemia vera (PV) or essential thrombocythemia (ET). The purpose of this study is to characterize the clinical characteristics of ET/PV patients harboring additional non-driver mutations or gene variants.Methods:Eligible cases included patients with PV/ET diagnosed and treated at MD Anderson Cancer Center from January 2012 to January 2017. NGS sequencing of 28 genes known to be mutated in hematologic malignancies was performed on samples from 240 ET/PV patients. Non-driver mutations detected by the NGS platform were classified based on the allele frequency recorded in our database in conjunction with currently available and periodically updated public reference databases of somatic mutations or variants of unclear significance.Results:A total of 110 ET and 130 PV cases had NGS performed on samples collected at the time of initial referral. Among them, 21 (19%) ET cases and 28 (21.5%) PV cases had ≥1 non-driver somatic mutation detected. The distribution of non-driver somatic mutations in ET and PV are shown in Figure 1A. On grouping non-driver genes into functional pathways, the most frequent mutational targets were in genes associated with epigenetic regulation (PV, 22; ET, 25), mainly DNA methylation (PV, 18; ET, 20) followed by epigenetic modifier (PV, 4; ET, 5). In subjects with PV, TET2 (n = 12) was the most frequently mutated gene, followed by DNMT3A (n = 7), IDH1/2 (n = 5), and ASXL1 (n = 4). A similar pattern was noted in ET, with TET2 mutations (n = 8) being most frequent followed by DNMT3A (n = 7), and ASXL1 (n = 5). Older patients were more likely to display non-driver mutations in both ET (p < 0.001) and PV (p = 0.02). The combined prevalence of non-driver somatic mutations was 27% and 32% in PV and ET, respectively. Over a short median follow up of 3.9 (range, 1.0-37.1) months among PV patients with non-driver somatic mutations, no leukemic transformations occurred. Similarly, no leukemic transformations were observed in ET patients with additional non-driver somatic mutations over a median follow up of 3.8 (range, 1.1-36) months. Overall survival (OS) was significantly longer in patients without additional mutations in epigenetic modifiers (Figure 1C). The prognostic relevance of non-driver somatic mutations on OS was not determined due the small sample size and limited follow up duration. Of 13 (12%) triple-negative ET patients without non-driver somatic mutations, nine had non-synonymous gene sequence variants; the most common being in DNA methylation genes namely TET2 &IDH1/2 genes (Figure 1B).Conclusion:The spectrum of non-driver somatic mutations in our ET and PV study cohorts was dominated by mutations in the TET2, DNMT3A, ASXL1 genes, and were correlated with age (Buscarlet Blood, 2017). A larger sample size and longer follow-up is needed to determine the impact of non-driver somatic mutations on transformation-free and overall survival. Germline TET2 variants or their combinations with other variants/non-driver mutations may constitute low penetrance predisposition factors for myeloid neoplasms and their functional relevance warrants further exploration. [Display omitted] DisclosuresBose:Incyte Corporation: Honoraria. Kantarjian:Amgen: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding. Verstovsek:Astrazeneca: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Lilly Oncology: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Bristol Myers Squibb: Research Funding; Lilly Oncology: Research Funding; CTI BioPharma Corp: Research Funding; Pfizer: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Bristol Myers Squibb: Research Funding; Astrazeneca: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Promedior: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

▪Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naïve T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo .We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway.In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. DisclosuresMascarenhas:Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj:Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

MPL exon 10 mutations other than canonical MPL W515L/K mutations identified by in-house MPL exon 10 direct sequencing in essential thrombocythemia.

MPL exon 10 mutations are one of the driver mutations in essential thrombocythemia (ET) or myelofibrosis (MF). We have established an in-house MPL mutation analysis system, covering the entire region of MPL exon 10 by direct sequencing. Since 2009, MPL exon 10 mutation analysis has been performed for diagnosis of myeloproliferative neoplasms (MPN) without JAK2 V617F or CALR exon 9 mutations. So far, 11 cases of MPL exon 10 mutation have been found in 51 patients with suspected MPN. In patients with ET, we detected five non-canonical MPL mutations including one novel mutation, MPL R514_P518delinsK, and one canonical MPL W515L mutation. Notably, three ET patients without canonical MPL mutations had thrombotic events. Meanwhile, in primary or secondary MF, only canonical MPL W515L/K mutations were found. Further cases need to be examined to elucidate the full MPL mutation profile in MPN. However, our data indicate that analysis of the whole of MPL exon 10 is warranted for the diagnosis of MPL mutations, especially in ET, and that the use of Japanese commercial laboratory tests that only detect canonical MPL W515L/K mutations may miss a significant percentage of MPL exon 10 mutations, which could delay the administration of anti-thrombotic therapy.

Thrombotic Risk Detection in Patients with Polycythemia Vera: The Predictive Role of DNMT3A/TET2/ASXL1 Mutations.

The development of thrombotic events is common among patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). We studied the influence of pathogenic mutations frequently associated with myeloid malignancies on thrombotic events using next-generation sequencing (NGS) in an initial cohort of 68 patients with myeloproliferative neoplasms (MPN). As expected, the presence of mutations in DNMT3A, TET2, and ASXL1 (DTA genes) was positively associated with age for the whole cohort (p = 0.025, OR: 1.047, 95% CI: 1.006–1.090). Also, while not related with events in the whole cohort, DTA mutations were strongly associated with the development of vascular events in PV patients (p = 0.028). To confirm the possible association between the presence of DTA mutation and thrombotic events, we performed a case-control study on 55 age-matched patients with PV (including 12 PV patients from the initial cohort, 25 with event vs. 30 no event). In the age-matched case-control PV cohort, the presence of ≥1 DTA mutation significantly increased the risk of a thrombotic event (OR: 6.333, p = 0.0024). Specifically, mutations in TET2 were associated with thrombotic events in the PV case-control cohort (OR: 3.56, 95% CI: 1.15–11.83, p = 0.031). Our results suggest that pathogenic DTA mutations, and particularly TET2 mutations, may be an independent risk factor for thrombosis in patients with PV. However, the predictive value of TET2 and DTA mutations in ET and PMF was inconclusive and should be determined in a larger cohort.

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491

AbstractMutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

JAK2-negative acute monocytic leukemia with TET2 mutation in essential thrombocythemia with JAK2 mutation with literature review

Essential thrombocythemia (ET) is an indolent myeloproliferative neoplasm (MPN) with a transformation to acute myeloid leukemia in <5% of patients. A 79-year-old man with JAK2V617F-positive ET exhibited leukocytosis with an increase in monoblastic cells, leading to a diagnosis of acute monoblastic and monocytic leukemia. Leukemic cells carried a TET2 mutation but not JAK2V617F mutation. We concluded that the TET2 mutation occurred in MPN-initiating cells and overcame JAK2-mutated cells. The absence of a JAK2 mutation in the leukemic cells in this case suggests the leukemia emerged from a JAK2-negative MPN cell clone carrying the TET2 mutation.

Landscape of TP53 Mutations in MPN

Landscape of TP53 Mutations in MPN

Platelet Count Predicts Co-occurrence of Driver Mutations in Essential Thrombocythemia and Myelofibrosis with a Low JAK2 Allele Burden

Platelet Count Predicts Co-occurrence of Driver Mutations in Essential Thrombocythemia and Myelofibrosis with a Low JAK2 Allele Burden

MPL Y252H anMd PL F126fs mutations in essential thrombocythemia: Case series and review of literature.

Essential thrombocythemia (ET) is a clonal bone marrow disease, characterized by increased production of platelets along with other clinical and bone marrow findings. Most patients with ET will have a somatic mutation in one of the known gene locations of JAK2, CALR, or MPL that can upregulate the JAK-STAT pathway. MPL mutation is present in 5% of cases with the most common mutations being W515L and W515K. In this report we describe 2 cases of patients with clinical and laboratory picture of ET. One patient carried MPLY252H mutation which is previously unreported in the adult population but has been shown to be a gain-of-function mutation. The other patient carried MPL F126fs mutation which is not known to be of clinical importance and has not been previously reported.

Abstract B072: Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms

Abstract Somatic mutations in the CALR gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. CALR mutations are 1-bp frameshift mutations that result in the formation of a novel C-terminus with an identical 36-amino acid (aa) sequence that is shared by all CALR+ MPN patients. We hypothesized that the recurrence and uniformity of the novel C-terminus mark the mutated (mut)-CALR an attractive candidate as an MPN-specific tumor neoantigen that might elicit anti-tumor immune responses across patients who express this mutation. To assess the immunogenicity of mut-CALR neopeptide, we interrogated the binding affinity of epitopes from the altered C-terminus using in silico peptide binding prediction algorithms. The analysis showed that multiple epitopes could bind to class I and II human leukocyte antigens (HLA). We investigated whether we could elicit T-cell responses against mut-CALR neopeptide from CALR+ MPN patients. Peripheral blood mononuclear cells (PBMCs) from 18 MPN patients with ET (7), MF (4) or MF arising from ET (7) were stimulated in vitro with pooled overlapping long peptides (OLPs; 14-15 aa) spanning the mutated or wild type (WT) CALR C-terminus and T-cell responses were assessed by enzyme-linked immunospot assay (ELISPOT). There was a significant increase in interferon (IFN)-γ production upon stimulation with mut-CALR OLPs, whereas the corresponding WT sequence did not induce a T-cell response. Next, we evaluated whether mut-CALR-specific T-cells were undergoing exhaustion. Examination of the expression of checkpoint receptors on T-cells ex vivo determined that T-cells from peripheral blood of CALR+ MPN patients exhibited increased expression of multiple cell surface inhibitory molecules compared to healthy donor T-cells, including programmed cell death (PD)-1 and cytotoxic T lymphocyte-associated antigen (CTLA)-4. In order to address whether PD-1 and CTLA-4 overexpression suppressed mut-CALR-specific T-cell responses in CALR+ MPN patients, PD-1 or CTLA-4 signaling on T-cells was blocked through addition of monoclonal blocking antibodies. T-cell responses against mut-CALR peptides were recovered in 3 CALR+ MPN patients that were previously nonresponsive to mut-CALR stimulation. To gain further insights into checkpoint receptor-mediated T-cell suppression in CALR+ MPN patients, we investigated mut-CALR-specific T-cell responses in a CALR+ MPN patient receiving PD-1 inhibitor pembrolizumab, in our ongoing clinical trial (NCT03065400). We monitored the changes in the frequencies of peripheral blood T-cells in this patient before and after pembrolizumab treatment by both flow cytometry and T-cell receptor (TCR) sequencing. Prior to therapy, this patient had low proportion of T-cells (4.33% of PBMCs) and 32.3% of the T-cells expressed PD-1. After 2 cycles of pembrolizumab, the T-cell proportion greatly increased (26% of PBMCs) and remained high when measured after 6 cycles (18% of PBMCs). Sequencing of TCR Vβ chains of peripheral blood T-cells revealed that T-cells exhibited greater clonality after pembrolizumab treatment. Furthermore, we evaluated changes in mut-CALR-specific T-cell responses in this CALR+ MPN patient prior to and during pembrolizumab administration. At baseline, peripheral blood T-cells from this patient did not respond to stimulation with mut-CALR peptides. After pembrolizumab treatment, however, mut-CALR-specific T-cell responses were evident, as shown by increased IFN-γ production. Together, these results establish mut-CALR as a novel MPN-specific tumor shared-neoantigen and provide a strong rationale to study its immunogenic properties to inform the design of novel immunotherapies targeting mut-CALR in MPN patients carrying the mutation. Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, John P. Finnigan, John Mascarenhas, Ronald Hoffman, Camelia Iancu-Rubin, Nina Bhardwaj. Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B072.

Haploinsufficiency of CALR Confers Hematopoietic Stem Cells (HSCs) with a Clonal Advantage over Wild-Type Cells, and, in Setting of Myeloproliferative Neoplasms, Compensates for the Functions of HSCs Impaired By the Calr Mutation

CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice.In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice.First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells.Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice.Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET.Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, “defect” and “recovery” states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Lenalidomide in Myelodysplastic/Myeloproliferative Neoplasms with Ring Sideroblasts

Background: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ring sideroblasts and thrombosis (MDS/MPN-RS-T), include the dysplastic features of Refractory Anemia with Ring Sideroblasts (RARS) and the myeloproliferative features of essential thrombocythemia (ET) and are characterized by a high rate (50%) of JAK2 V617F mutations or rarely by the presence of mutations in exon 10 of the MPL (myeloproliferative leukemia) gene or in exon 9 of the calreticulin gene. We previously demonstrated that RARS-T differed from RARS and ET from a clinical, biological and prognostic point of view and that the presence of high rates of splicing factor 3B subunit 1 (SF3B1) mutations in RARS-T and the absence of these mutations in ET strengthened the hypothesis that RARS-T was a distinct entity. We also reported in a large series of patients (n=200) that the risk of thrombosis was higher in RARS-T than in RARS patients without a high platelet count, leading clinicians to reduce the platelet count in almost 33% of RARS-T patients. However, due to the presence of anemia, the use of the cytoreductive agents usually used in ET is limited since they worsen the cytopenia. This explains why it is difficult to treat patients who harbor both anemia and thrombocytosis. Lenalidomide, an immunomodulatory agent frequently used in low-risk MDS, has very rarely been tested in MDS/MPN-RS-T patients, with conflicting results. Objective : To test the effect of Lenalidomide on MDS/MPN-RS-T Patients: Here, we report our experience using lenalidomide in two women who presented with JAK2 V617F negative MDS/MPN-RS-T. Results: The first patient was a 78 year-old woman, with a complete blood count (CBC) showing hemoglobin 80 g/L, platelet count 686 G/L and leukocytes 6 G/L. The second was a 58 year-old woman whom initial CBC showed hemoglobin 113 g/L, platelet count 1,600 G/L, leukocytes 8.2 G/L. In both cases the bone marrow aspirates showed erythroid hyperplasia with myelodysplasic features and 64% and 24% of ring sideroblasts respectively, associated with marked dysmegakaryopoiesis, leading to the diagnosis of MDS/MPN-RS-T. No excess of blast cells was noted. Bone marrow cytogenetics showed in both cases a normal 46XX karyotype. A Lys700Glu SF3B1 mutation was noted for one patient (not tested for the second), without the JAK2 V617F or MPL exon10 or CALR mutations. In the first patient, blood transfusions were initiated for one year, however, due to an increase in dependence on red blood cell transfusions, treatment with lenalidomide (5 mg daily 21/28days) was started and led to a decrease in the platelet count from 686 to 220 G/L associated with an regular improvement in hemoglobin levels from 80 to 100 g/L within the first 28 weeks of lenalidomide (only 6 units of red blood cells were transfused along this period). Finally, no transfusion was required in the subsequent 47 weeks since the hemoglobin level was above 90 g/L. However, due to a low hemoglobin level, red blood cell transfusions were reinitiated, but with a lower frequency (2 units of red blood cells every 2 months), in association with lenalidomide. Altogether, over the 20 months of treatment with lenalidomide, the red blood cell requirement was drastically reduced. However, grade IV neutropenia was observed (granulocytes 0.5 G/L) but without any infectious disease, leading to stop the treatment. In the second patient, a watch-and-wait strategy was initiated, but due to an increase in the platelet count > 2,000 G/L, hydroxyurea (500 mg/day) was started, worsening the anemia to 108 g/l then 84 g/l leading to the use of erythropoietin once/week. Hydroxyurea was then stopped due to its adverse effects on hemoglobin, leading to the increase in the platelet count to 3,622 G/L. A treatment with lenalidomide (5 mg daily 21/28days) was then started and secondarily improved to 10 mg daily 21/28days since very well tolerated. A marked decrease in the platelet count was noted, from 3,622 to 985 G/L whereas hemoglobin level rised up from 92 to 115 g/L. Off note, for this patient erythropoietin treatment was maintained every 2 weeks in combination with lenalidomide. Conclusion: This two-cases experience confirms the partial efficacy of lenalidomide in MDS/MPN-RS-T in normalizing (case 1) or markedly reducing (case 2) the platelet count and allowing of independence from blood transfusion (at least for 10 months in case 1). Disclosures No relevant conflicts of interest to declare.

Calreticulin in Essential Thrombocythemia: StressINg OUT the Megakaryocyte Nucleus.

Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control processes. The identification of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis that are mutually exclusive with the JAK2 V617F mutation has stirred an intensive research interest about the molecular functions of CALR and its mutants in myeloproliferative neoplasms (MPNs) and its diagnostic/prognostic value. The recently characterized protein–protein interaction of CALR mutants and MPL receptor has advanced our knowledge on the functional role of CALR mutants in thrombocythemia but it has also uncovered limitations of the current established research models. Human cell lines and mouse models provide useful information but they lack the advantages provided by ex vivo primary cultures of physiologically relevant to the disease cell types [i.e., megakaryocytes (MKs), platelets]. The results from gene expression and chromatin occupancy analysis have focused on the JAK-STAT pathway activated in both JAK2 V617F- and CALR-mutated MPN patient groups, although a more complete analysis is needed to be performed in MKs. Stress related processes seem to be affected in CALR mutant ET-MKs, but the precise mechanism is not known yet. Herein, we describe a culture method for human MKs from peripheral blood progenitors, which could help further toward an unbiased characterization of the role of CALR in ET and MK differentiation.

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis

Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. After choosing MeSH keywords, including essential thrombocythemia, JAK2V617F, calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. The frequency of JAK2V617F and CALR shows heterogeneity in Caucasian population [JAK2V617 I 2%=84.3, P<0.001, 95% CI 0.56 (0.51-0.61)], [CALR I 2%=96.1, P<0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2V617F and CALR was 0.57 (95% CI 0.53-0.61), I 2%=79.3 and 0.22 (95% CI 0.16-0.27), I 2%=94, respectively. JAK2V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P<0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2V617F. Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2V617F mutation. Therefore, detection of mutation in CALR and JAK2V617F may contribute to diagnosis and prognosis of ET patients.

Polycythemia vera and essential thrombocythemia: 2017 update on diagnosis, risk-stratification, and management.

Polycythemia Vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms respectively characterized by erythrocytosis and thrombocytosis; other disease features include leukocytosis, splenomegaly, thrombosis, bleeding, microcirculatory symptoms, pruritus, and risk of leukemic or fibrotic transformation. PV is defined by a JAK2 mutation, whose absence, combined with normal or increased serum erythropoietin level, makes the diagnosis unlikely. JAK2, CALR, and MPL mutations are the mutually exclusive "driver" mutations in ET with respective incidences of 55%, 25%, and 3%; approximately 17% are triple-negative. However, the same molecular markers might also be present in prefibrotic myelofibrosis, whose morphological distinction from ET is prognostically relevant. Median survivals are approximately 14 years for PV and 20 years for ET; the corresponding values for younger patients (age <60 years) are 24 and 33 years. Life-expectancy in ET is inferior to the control population. Driver mutational status has not been shown to affect survival in ET whereas the presence of JAK2/MPL mutations has been associated with higher risk of arterial thrombosis and that of MPL with higher risk of fibrotic progression. Risk factors for overall survival in both ET and PV include advanced age, leukocytosis and thrombosis. Leukemic transformation rates at 20 years are estimated at <10% for PV and 5% for ET; fibrotic transformation rates are slightly higher. Most recently, ASXL1, SRSF2, and IDH2 mutations have been associated with inferior overall, leukemia-free or fibrosis-free survival in PV; similarly adverse mutations in ET included SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2. Current risk stratification in PV and ET is designed to estimate the likelihood of recurrent thrombosis. Accordingly, PV includes two risk categories: high-risk (age >60 years or thrombosis history) and low-risk (absence of both risk factors). In ET, risk stratification includes four categories: very low risk (age ≤60 years, no thrombosis history, JAK2/MPL un-mutated), low risk (age ≤60 years, no thrombosis history, JAK2/MPL mutated), intermediate risk (age >60 years, no thrombosis history, JAK2/MPL un-mutated), and high risk (thrombosis history or age >60 years with JAK2/MPL mutation). In addition, presence of extreme thrombocytosis (platelets >1000 × 10(9)/L) might be associated with acquired von Willebrand syndrome (AvWS) and, therefore, risk of bleeding. The main goal of therapy in PV and ET is to prevent thrombohemorrhagic complications. All patients with PV require phlebotomy to keep hematocrit below 45% and once-daily aspirin (81 mg). In addition, high-risk patients with PV require cytoreductive therapy. Very low risk ET patients might not require any form of therapy while low-risk patients require at least once-daily aspirin therapy. Cytoreductive therapy is also recommended for high-risk ET patients but it is not mandatory for intermediate-risk patients. First-line drug of choice for cytoreductive therapy, in both ET and PV, is hydroxyurea and second-line drugs of choice are interferon-α and busulfan. We currently do not recommend treatment with ruxolutinib or other JAK2 inhibitors in PV or ET, unless in the presence of severe and protracted pruritus or marked splenomegaly that is not responding to the aforementioned drugs. Screening for AvWS is recommended before administrating aspirin, in the presence of extreme thrombocytosis. Am. J. Hematol. 92:95-108, 2017. © 2016 Wiley Periodicals, Inc.

CAL2 Monoclonal Antibody Is a Rapid and Sensitive Assay for the Detection of Calreticulin Mutations in Essential Thrombocythemia and May Provide Prognostic Informations

Background. Calreticulin (CALR) mutations are detected in the majority of JAK2 wild-type (wt) patients with Essential Thrombocythemia (ET) or Primary Myelofibrosis (MF). At variance with JAK2 and MPL point mutations, CALR mutations are highly heterogeneous, with several types of insdel reported to date, so that sequencing techniques are needed to cover the whole spectrum of mutations. CAL2 is a new monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR.Methods. We retrospectively analyzed 172 ET patients followed at our Institution from 1980 to 2015 who were tested for JAK2V617F at the time of diagnosis or during follow-up. In JAK2wt patients exon 9 CALR mutations were searched by PCR and capillary electrophoresis and MPLW515L/K by ARMS-PCR. In the same patients, bone marrow (BM) biopsies were immunostained with CAL2 and histologically reviewed for megakaryocytic features.Results. Median age at diagnosis was 54 years (range 14-87) and median time from first evidence of thrombocytosis to BM biopsy was 8.4 months (range 0-175). According to driver mutations, patients were classified as JAK2V617F-mutated (n=119, 69%), MPL-mutated (n=2, 1%), CALR-mutated (n=31, 18%) or triple-negative (n=20, 12%). As expected, CALR-mutated patients had significantly higher platelet count than JAK2V617F (799 vs 657 x 109/L, respectively; p=.019) and lower hemoglobin values (mean 13.6 vs 14.5 g/dL, respectively; p=.003). At a median follow-up of 9.8 years, incidence of thrombosis was significantly lower in CALR- than in JAK2V617F-mutated patients (3% vs 21%, respectively; p=.017), while no differences were observed in clinical presentation and long-term outcome between CALR-mutated and triple-negative patients. Concordance between molecular and immunohistochemical (IHC) detection of CALR mutations was optimal (CohenÕs kappa >0.8) but not complete, since 3 patients were positive by IHC only and 1 patient was positive by molecular only. In CAL2-positive BM samples (n=30) we defined 2 patterns (figure), characterized by staining of megakaryocytes only (pattern A, 37%) or staining of megakaryocytes and myeloid precursors (pattern B, 63%). Type B biopsies tended to have higher median cellularity (48% vs 30%; p=.22), higher median megakaryocytic number for HMF (20 vs 12.5; p=.14), higher frequency of megakaryocytic clusters (100% vs 60%; p=.014) and higher frequency of grade-1 fibrosis (81% vs 60%; p=.37) with respect to type A samples. One type A patient and 2 type B patients progressed to post-ET MF or acute leukemia. Moreover, 3 patients had a BM biopsy performed at the time of MF evolution but not at diagnosis of ET, and they displayed a type B pattern.Conclusions. CAL2 identifies CALR-mutated ET patients in a rapid, sensitive, specific and economic manner and it integrates molecular biology informations. Prognostic value of different IHC patterns should be confirmed in wider and indipendent series. [Display omitted] DisclosuresBonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding.