Types Of Driver Mutations Research Articles

Molecular markers as possible efficacy predictors of targeted therapy for myelofibrosis: single-center study

Background. Targeted therapy is the most promising in the treatment of myelofibrosis, but it is necessary to search for the reasons limiting its effectiveness. There are known factors negatively affecting the development of myelofibrosis, but data on their negative impact in the context of targeted therapy are scarce.Aim. Assessing the impact of cytogenetic and genetic abnormalities on the course and therapy results for primary and secondary myelofibrosis during ruxolitinib therapy.Materials and methods. The prospective study included 106 patients with myelofibrosis in the chronic phase (53 (50 %) men and 53 (50 %) women) who received ruxolitinib at the Moscow City Hematology Center, S.P. Botkin City Clinical Hospital. The median age of patients was 62 (18–84) years. The median disease duration before initiation of ruxolitinib therapy was 79 (1–432) months. Before therapy, genetic studies were performed, including next-generation sequencing. The median duration of ruxolitinib therapy was 33 (1–111) months. The influence of the cytogenetic landscape, driver mutations, allele burden of JAK2 (over time) and CALR, additional mutations on the dynamics of symptoms, spleen size, achievement of hematological response, overall survival, progression-free survival, survival without blast crisis and without progression of myelofibrosis with targeted therapy was assessed.Results. The studied genetic factors did not have a significant correlation with hemogram parameters. The hematological response in patients with JAK2 and CALR mutations compared favorably with the response in the groups with the MPL mutation and triple negative status (TNS). Higher hematological response rate was obtained in the group with initially low allele burden <50 % of JAK2 or CALR. Significant differences in 5-year overall survival were found between groups of patients with TNS and JAK2 and CALR mutations (p <0.05); with CALR allele burden <50 % and ³50 % before initiation of ruxolitinib therapy (p = 0.01); the presence or absence of positive dynamics of the JAK2 allele burden during treatment (p <0.05); additional mutations assigned to different pathogenicity groups (p <0.05); with different number of pathogenic mutations (1 or ³2), the presence or absence of pathogenic mutations in the ASXL1 (p = 0.002) and SETBP1 (p = 0.00001) genes. The 5-year progression-free survival was significantly different in cohorts of patients with or without positive dynamics of the JAK2 allelic load during treatment (p <0.05); additional mutations assigned to different pathogenicity groups (p <0.05); with a different number of pathogenic mutations (1 or ³2), the presence or absence of a pathogenic mutation of the SETBP1 gene (p = 0.003). Progression-free survival did not correlate with the type of driver mutation or its absence; however, all patients with TNS died from myelofibrosis progression. Significant differences in 5-year blast crisis-free survival were observed between groups with JAK2 and MPL mutations (p = 0.001), JAK2 and TNS (p = 0.002); difference in 5-year survival without progression of fibrosis – between groups with pathogenic and benign (p = 0.031); uncertain and benign (p = 0.001) mutations.Conclusion. The study identified genetic markers associated with decreased efficacy of ruxolitinib therapy.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

Chaperon Protein GRP78 Released from MPN Cells Increase Expression of Lysyl Oxidase in Human Stroma Cell Line

Impaired function of the endoplasmic stress (ER) response causes numerous pathological conditions, including tissue fibrosis. In the present study, we aimed to determine the pathological role of ER stress response systems in myeloproliferative neoplasms (MPNs). We found an increased expression of the chaperone protein glucose-regulated protein (GRP)78, which is a central regulator of ER stress in megakaryocytes from primary myelofibrosis (PMF) (n=7) or post-essential thrombocythemia myelofibrosis(POST-ET-MF) patients (n=2) regardless of the type of driver mutation. In accord with these notions, we found that GRP78 was overexpressed in JAK2V617F-harboring cell lines (HEL and SET-2) and in UT-7/TPO cells overexpressing either type1 or type2 mutant form of calreticulin. To address the mechanism responsible for the induction of GRP78, we treated the HEL and SET-2 cells with 4-phenyl butyric acid and tauroursodeoxycholic acid both of which are well known inhibitors of ER stress, however, these inhibitors did not affect the expression levels of GRP78. These results indicated that overexpression of GRP78 shown in these cells were not a result of activation of ER stress. In contrast, ruxolitinib, an inhibitor of JAK2V617F, blocked GRP78 expression in these cells. We also found ruxolitinib blocked nuclear accumulation of the transcription factor 4 (ATF4), one of the main transcription factors which regulate GRP78. Furthermore, knockdown of ATF4 using siRNA also resulted in suppression of GRP78 levels in HEL and SET-2 cells. These results suggested that JAK2V617F induce GRP78 through activation of ATF4. We also confirmed that megakaryocytes from MF patients had statistically higher nuclear to cytoplasmic intensity of ATF4 compared to megakaryocytes found in control subjects. For the next step, we tried to find out the pathological action of GRP78 in MPN cells. Although the GRP78 usually works as chaperon protein in cytosol, some cancer cells secret GRP78 into tumor microenvironment and activates signal transduction molecules in surrounding cells. Therefore, we hypothesized that overexpressed GRP78 proteins in MPN cells may secret and modulate phenotype of bone marrow stroma cells. Initially, we confirmed the presence of GRP78 proteins in culture media from HEL and SET-2 cells. Next, we investigated whether GRP78 secreted by HEL and SET-2 cells affect phenotype of bone marrow stromal cells. For this purpose, we used human bone marrow stroma cell line, HS-5. HS-5 cells were co-cultured with HEL or SET-2 cells with trans-well system, and changes of several key molecules involved in development of fibrosis were analyzed. We found that co-culture with HEL or SET-2 cells clearly indued expression of lysyl oxidase (LOX) , which mediate cross-linking of collagen fiber and induce tissue fibrosis , in HS-5 cells. Anti-GRP78 neutralizing antibody abrogated LOX elevation, in contrast, recombinant form of GRP78 proteins induced LOX proteins in HS-5. According to the above results, we examined LOX expression in bone marrow specimens from MF patients. The areas of LOX expression cells were statistically higher in MF patients compared to control group. In addition, high power field observations detected not only in round cells but also in spindle like cells expressed LOX in MF patients, suggesting that bone marrow stromal cells in MF patients express LOX. In conclusion, we demonstrated that GRP78 was overexpressed both in MPN derived cell lines and also in primary megakaryocytes from MF patients. Our observations proposed that overexpressed GRP78 in MPN cells contribute development of myelofibrosis through secreted in microenvironment and induced expression of extracellular matrix modulating enzyme LOX in bone marrow stromal cells.

Characteristics and prognostic analysis of patients with detected KRAS mutations in resected lung adenocarcinomas by peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp method.

Kirsten rat sarcoma virus (KRAS) gene mutations are a type of driver mutation discovered in the 1980s, but for a long time no molecular targeted drugs were available for them. Recently, sotorasib was developed as a molecular targeted drug for KRAS mutations. It is therefore necessary to identify the characteristics of patients with KRAS mutations. This was the single-institution retrospective study. Surgically resected tumors from lung adenocarcinoma patients were collected at a single institution from June 2016 to September 2019. Peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp analysis of KRAS G12X mutations was compared with analysis by therascreen KRAS RGQ kit. The association between KRAS mutation status and patient characteristics and prognosis was assessed. Among 499 lung adenocarcinomas, KRAS mutations were evaluated in 197 cases, excluding stage IV lung cancer and tumors with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. KRAS G12X mutations were detected in 59 cases (29.9%). The highest frequency by gene mutation subtype was G12V in 23 cases (39.0%), followed by G12C in 16 cases (27.1%), G12D in 12 cases (20.3%), G12S in 4 cases (6.8%) and G12A in 2 cases. For the G12C mutation, the PNA-LNA PCR clamp and therascreen methods were consistent, but for the G12D and G12S mutations, the PNA-LNA PCR clamp method showed higher detection rates. In operable tumors, G12C mutations were more frequent in males, smokers, and patients with high expression of programmed death-ligand 1 (PD-L1), and had no correlation with prognosis. By the PNA-LNA PCR clamp method, G12C mutation of surgical specimens was detected successfully. The PNA-LNA PCR clamp method is expected to be applied to the detection of druggable G12C mutations.

Pediatric Philadelphia-Negative Myeloproliferative Neoplasms in the Era of WHO Classification: A Systematic Review.

Philadelphia-negative myeloproliferative neoplasms (MPN) are most prevalent in the older population (median age at the diagnosis is above 60 years) and rarely diagnosed in pediatrics. Thus, our knowledge about the clinical presentation, mutational status, and complications of MPNs in pediatrics is limited. The literature in English (PubMed, SCOPUS, and Google Scholar) was searched for studies, reviews, case series, and case reports of patients with Philadelphia-negative MPNs (including essential thrombocythemia, polycythemia vera, primary myelofibrosis, and profibrotic myelofibrosis) in the pediatrics age group (less than 18 years). Only studies that fulfilled WHO 2008 or 2016 criteria for MPNs were included. We aimed to describe the clinical characteristics, vascular and long-term complications, types of driver mutations, and treatment approaches in pediatric patients with MPNs. We reviewed 33 articles of available published literature from 2008 to 2022 and collected data from a total of 196 patients of the pediatric population. Among the cohort of patients, 139 had essential thrombocythemia (ET), 20 had polycythemia vera (PV), and 37 had primary myelofibrosis (PMF). The median age at the time of diagnosis for each disease varied, with 8.8 years for ET, 10 years for PV, and 3.6 years for MF. There was a slight difference in gender prevalence between both gender groups and all three diseases. The presenting symptoms were not mentioned in more than 50% of studies. We found that JAK2 was the most prevalent among all mutations. Both bleeding and thrombosis were present equally in ET, with 9% of cases complicated by bleeding and 9% complicated by thrombosis. Hemorrhagic events did not occur in patients with PV; thrombosis in children with MF was also not found. The progression into AML occurred in two patients with PV and one with ET. Given the rarity of MPNs in pediatrics and their different characteristics compared with adults, we believe there is a need for unique diagnostic criteria to match the different molecular statuses in pediatrics. Based on our review, the incidence of MPN complications in pediatrics, including thrombotic events, hemorrhage, and leukemic transformation, differs from that in adults.

Circulating Tumor DNA: Less Invasive, More Representative Method to Unveil the Genomic Landscape of Newly Diagnosed Multiple Myeloma Than Bone Marrow Aspirates.

Simple SummaryThe study of malignant plasma cell DNA genomics in multiple myeloma is a hot topic, which is mainly based on one-site bone marrow aspirates. In this study, we showed that circulating tumor DNA targeted next-generation sequencing analysis revealed a more comprehensive genomic architecture than bone marrow aspirates in newly diagnosed multiple myeloma. Circulating tumor DNA mutation in the transcriptional regulation pathway and DNA repair pathway were independent predictors of progression-free survival. ctDNA alterations correlated with prognosis and therapy response in newly diagnosed multiple myeloma.Multiple myeloma (MM) is highly heterogenous and dynamic in its genomic abnormalities. Capturing a representative image of these alterations is essential in understanding the molecular pathogenesis and progression of the disease but was limited by single-site invasive bone marrow (BM) biopsy-based genomics studies. We compared the mutational landscapes of circulating tumor DNA (ctDNA) and BM in 82 patients with newly diagnosed MM. A 413-gene panel was used in the sequencing. Our results showed that more than 70% of MM patients showed one or more genes with somatic mutations and at least half of the mutated genes were shared between ctDNA and BM samples. Compared to the BM samples, ctDNA exhibited more types of driver mutations in the shared driver genes, higher numbers of uniquely mutated genes and subclonal clusters, more translocation-associated mutations, and higher frequencies of mutated genes enriched in the transcriptional regulation pathway. Multivariate Cox analysis showed that age, ctDNA mutations in the transcriptional regulation pathway and DNA repair pathway were independent predictors of progression-free survival (PFS). Our results demonstrated sequencing of ctDNA provides more thorough information on the genomic instability and is a potential representative biomarker for risk stratification and in newly diagnosed MM than bone marrow.

Abstract A006: Modeling the impact of driver mutations on local tumor heterogeneity

Abstract Tumors consist of an accumulation of somatic mutations, some of which are “passenger” mutations which do not contribute to positive selection of cancer cells and others of which are “driver” mutations which confer a growth advantage, the benefit of which may vary depending on the type of mutation, to tumor cells. As genomic sequencing to identify driver mutations is typically only done on small samples of a tumor, identification of these driver mutations may be complicated by intra-tumor spatial heterogeneity. With this in mind, we decided to create a basic model of different classes of driver mutations to investigate the impact of the timing and type of these mutations on tumor heterogeneity. Using the PhysiCell framework as a foundation, a platform developed for creating multicellular cancer models, we built a model to identify spatial trends specific to certain classes of driver mutations and distinguish them from passenger and “hitchhiker” (heritable mutations with an increased frequency due to their relation with a driver mutation) mutations. We model cells within a single, solid, non-cancer-specific tumor grown from a single cell, allowing these cells to mutate and daughter cells to potentially gain broad types of driver mutations at each division. We define three “driver” mutation classes in this model: an increase in the reproduction rate of tumor cells (defined as fitness), a decrease in the death rate of tumor cells (defined as survivability), and an increase in the motility rate of tumor cells. To investigate the distribution of common mutations, we define “common mutations” as those appearing in at least 5% of the tumor cells at end-point, when the tumor has reached 10,000 cells. To investigate spatial heterogeneity within this model, we split the tumor into 9 distinct spatial regions at the end-point of each simulation. We use these regions to assess the distribution of common, driver, and passenger mutations and quantify spatial heterogeneity. We find that in tumors with driver mutations that increase fitness or motility, we require on average fewer samples to detect all the common mutations than within tumors containing no driver mutations. On the other hand, we find that tumors containing driver mutations increasing only survivability tend to yield similar sample heterogeneity to tumors without driver mutations. These results suggest that in addition to the timing of a mutation, the type of driver mutation can have an impact on the spatial heterogeneity within a tumor which may inform biopsy strategies in clinical settings. In future work, this model could be used to observe the relationship between spatial tumor heterogeneity and the prevalence of different classes of driver mutations within different microenvironments through the implementation of hypoxic or therapeutic agent gradients. Our model could be compared with results of clinical tissue sampling data to further understanding of the impact of spatial sampling and driver mutation type on the discovery of driver mutations in different tumors and microenvironments. Citation Format: David Oh, Steph Owen, Luka Opasic, Jacob Scott. Modeling the impact of driver mutations on local tumor heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A006.

Prognostic Impact of Baseline Liver Metastasis in ALK Fusion-Positive Metastatic Lung Cancer: A Retrospective Review.

Akhil KapoorIntroduction The prognosis of anaplastic lymphoma kinase (ALK) fusion-positive metastatic non-small cell lung cancer (mNSCLC) patients has improved drastically since the introduction of targeted therapies. Apart from age, performance status, and type of driver mutation in a mNSCLC, prognosis also depends on baseline metastatic sites number as well as location with liver metastases being a poor prognostic factor. However, the clinical and prognostic association of baseline liver metastases in ALK fusion-positive mNSCLC is not well known. Material and Methods We performed a retrospective analysis of ALK fusion-positive mNSCLC patients to assess prognostic impact of liver metastases. Records were obtained from lung cancer audit database and electronic medical records. Patients were started on either chemotherapy, ALK-directed tyrosine kinase inhibitors, or given best supportive care as per the clinical scenario. Radiological response was assessed every 2 to 3 months or earlier at clinical suspicion of progressive disease. Adverse events were evaluated as per Common Terminology Criteria for Adverse Events v4.02. Results A total of 441 patients were screened, out of which 76 had baseline liver metastases. Median age was 49 years with 64.5% males. Median progression-free survival (mPFS) was 14.2 months (95% confidence interval [CI] 8.9-19.4) in patients with baseline liver metastases. In patients who received first-line ALK inhibitor therapy versus who received first-line chemotherapy, mPFS was significantly better in the ALK-directed therapy subgroup, 15.3 months (95% CI 11.7-18.9) versus 5.9 months (95% CI 2.7-9.1), respectively (hazard ratio [HR] 0.3 [95% CI 0.17-0.54]; p < 0.001). Median overall survival (mOS) was 27.6 months (95% CI 17.4-37.7) in patients with baseline liver metastases which was not statistically significant from patients without baseline liver metastases which was 32.3 months (95% CI 28.8-35.7) (HR 1.32 [95% CI 0.91-1.9]; p = 0.22). Use of ALK-directed therapy in patients with baseline liver metastases resulted in better OS, mOS not reached versus 15.7 months (95% CI 2.7-28.8) in the chemotherapy group (HR 0.33 [95% CI 0.16-0.67]; p < 0.001). Conclusion In patients with ALK fusion-positive mNSCLC, baseline liver metastases was not found to be an independent prognostic factor. However, the use of ALK-directed therapy resulted in a significantly better PFS and OS as compared with chemotherapy in patients with baseline liver metastases. This underscores the importance of the use of ALK-directed therapy whenever feasible in this group of patients.

Targets in MPNs and potential therapeutics.

Philadelphia-negative classical Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hemopathies that emerge in the hematopoietic stem cell (HSC) compartment. MPN driver mutations are restricted to specific exons (14 and 12) of Janus kinase 2 (JAK2), thrombopoietin receptor (MPL/TPOR) and calreticulin (CALR) genes, are involved directly in clonal myeloproliferation and generate the MPN phenotype. As a result, an increased number of fully functional erythrocytes, platelets and leukocytes is observed in the peripheral blood. Nevertheless, the complexity and heterogeneity of MPN clinical phenotypes cannot be solely explained by the type of driver mutation. Other factors, such as additional somatic mutations affecting epigenetic regulators or spliceosomes components, mutant allele burdens and modifiers of signaling by driver mutants, clonal architecture and the order of mutation acquisition, signaling events that occur downstream of a driver mutation, the presence of specific germ-line variants, the interaction of the neoplastic clone with bone marrow microenvironment and chronic inflammation, all can modulate the disease phenotype, influence the MPN clinical course and therefore, might be useful therapeutic targets.

Myeloproliferative Neoplasms (MPN) Clonal Evolution Landscape and Its Impact on Patients' Prognosis

▪Introduction:Although myeloproliferative neoplasms (MPN) are driven by three mutually exclusive driver mutations (JAK2, CALR and MPL), targeted deep sequencing studies identified multiple additional somatic mutations potentially impacting MPN evolution. Presence of a high molecular risk (HMR: ASXL1, EZH2, SRSF2 and IDH1/2) or a TP53 mutations has been associated with adverse prognosis. However, to date, the effect of clonal evolution (CEv) on MPN patients' outcome has not been evaluated, as most of the studies assessed mutational-based prognosis stratification from single baseline molecular genotyping. The objective of our study was to describe the clinical and molecular characteristics of patients with CEv in a large cohort of MPN patients and analyze its impact on patients' outcome.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. From this large retrospective cohort, we included in this study 446 patients who had at least 2 molecular analyses during the chronic phase of MPN, performed at diagnosis and/or during follow-up using next generation sequencing (NGS), targeting a panel of 36 genes involved in myeloid malignancies. Significant variants were retained with a sensitivity of 1%. CEv was defined as the acquisition of a new additional non-driver mutation between baseline and subsequent NGS evaluation. Statistical analyses were performed using the STATA software (STATA 17.0 for Mac Corporation, College Station, TX).Results:The 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, essential thrombocythemia and primary myelofibrosis (MF) respectively.With a median interval of 1.6 years [IQR 1.0 - 2.8] between the first and the second NGS analysis in the whole cohort, CEv occurred in 128 patients (29%). Patients with CEv were significantly older compared to patients without CEv (n=318) (p=0.03). MPN diagnosis, the type of driver mutation and complete blood counts at MPN diagnosis did not differ between the 2 groups. Eighty-one (63%) and 198 (62%) patients with or without CEv respectively had at least one additional non-driver mutation at baseline NGS (p=0.59), while the rate of HMR (n=25 (20%) versus n=79 (25%)) or TP53 (n=7 (5%) versus n=20 (6%)) mutations at baseline NGS did not differ between the 2 groups.Thirty six out of 128 (28%) of patients with CEv had more than 1 acquired mutation. Most recurrently acquired mutations involved the epigenetic regulators TET2 and DNMT3A that were mutated in respectively 33% and 25% of patients with CEv (Figure 1A). Moreover, 38% of CEv patients acquired HMR (ASXL1 (14%), EZH2 (6%), SRSF2 (3%), IDH1/2 (2%)) or TP53 (13%) mutations. After a median follow up of 10.8 years [IQR 6.6 - 17.2] in the whole cohort representing a total of 5635 patient years, 32 (7%) patients died, and 11 (2.5%) and 11 (2.5%) patients with at least 2 NGS performed during MPN chronic phase transformed respectively into secondary MF or myelodysplastic syndrome / acute myeloid leukemia (MDS/AML). Interestingly, CEv (HR 11.27, 95%CI [5.09; 24.96], p<0.001) (Figure 1B), age at MPN diagnosis (HR 1.11, 95%CI [1.07; 1.15], p<0.001) and the presence of HMR mutations at baseline NGS (HR 4.48, 95%CI [2.05; 9.77], p <0.001) independently adversely impacted OS in a COX regression multivariate analysis. CEv also independently adversely impacted MDS/AML free survival (HR 13.15, 95%CI [3.88; 44.47], p<0.001) and secondary MF free survival (HR 21.13, 95%CI [6.18; 72.20], p<0.001) in a COX regression multivariate analysis.Conclusion:Our study on a large retrospective clinically and biologically annotated real-life cohort of MPN patients with long-term follow up shows that CEv independently adversely impacts OS, MDS/AML and secondary MF free survivals. CEv occurred in a clinically relevant proportion of MPN patients (28%) and was associated with patients' age. Acquired mutations mainly involved epigenetic regulators, HMR and TP53 genes. These results suggest that serial molecular monitoring using NGS could be routinely implemented in MPN patients follow up, to assess more accurately disease evolution and potentially update therapeutic management. [Display omitted] DisclosuresRaffoux: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ABBVIE: Consultancy; ASTELLAS: Consultancy. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; 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; PharmaEssentia: Other: Personal fees; 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. Benajiba: Gilead: Research Funding; Pfizer: Research Funding.

Abstract 2230: Integrative genomic analysis of refractory metastatic cancer

Abstract Metastatic cancer refractory to treatment has a dismal prognosis. While genetic mechanisms of primary tumors and to a lesser extent of metastatic cancers have been studied on large cohorts, it is still not well understood if metastatic tumors refractory to systemic treatment contain specific signatures at genomic or transcriptional levels. Molecular characterization of metastatic cancers at terminal stage is indispensable for understanding the mechanisms of resistance to treatment and for prediction of tumor aggressiveness at earlier stages. Here, we present a large pan-cancer cohort of 914 tumors resistant to systemic therapies. Comprehensive clinical information is available for all the patients and includes tumor subtype, biopsy site, treatment, age, overall survival, etc. Biopsies of tumor metastases were taken after the diagnosed resistance. For this cohort we performed whole exome sequencing of a subset of 427 tumors with a median depth of 123X and matching blood, and transcriptome sequencing of all 914 tumors. We will discuss the variation in this cohort along 3 axes. Firstly we will describe the somatic mutations and indels by types of driver mutations, mutational burden, level of subclonality and mutational signatures. Additionally we will elaborate on the genomic instability and quantity of somatic copy number alterations (SCNAs). Secondly, we will present the annotation of germline DNA, frequency and classification of pathogenic variants predisposing to cancer. Germline variants are analyzed in the context of somatic genome focusing on the detection of the second hit by a mutation or a SCNA. Thirdly, we will present results of the analysis of differentially expressed genes and transcripts, activation of oncogenic and treatment resistance pathways, and fusion genes reported by multiple tools. The aforementioned genetic parameters are fed into a multi-omics model for identification of the key factors associated with the clinical features in the cohort. Furthermore this cohort is compared to primary tumors and to another cohort of metastatic tumors in order to unravel genetic markers of refractory disease. To this aim, we harmonized DNA- and RNA-derived variables from The Cancer Genome Atlas (TCGA) and MET500 (Robinson et al., Nature 2017). Such integrative analysis unravels genetic hallmarks of aggressive and treatment-resistant metastatic cancers highlighting the importance of multi-level tumor profiling to inform on therapeutic strategies. Citation Format: Julien Viot, Yoann Pradat, Ismael Padioleau, Andrey Yurchenko, Loic Verlingues, Rebecca Clodion, Stefan Michiels, Marc Deloger, Gerome Jules-Clément, Yohann Loriot, Benjamin Besse, Fabrice Andre, Paul-Henry Cournedes, Daniel Gautheret, Sergey Nikolaev. Integrative genomic analysis of refractory metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2230.

Genomic Profiling of Blast Phase in Philadelphia-Negative Myeloproliferative Neoplasms

Background Classic Ph-negative myeloproliferative neoplasms (MPN) include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Clonal evolution can lead MPN patients in chronic phase (CP) to developacute myeloid leukemia (AML), called blast phase (BP), a rare and late event in the natural history of MPN.Aim To evaluate differences in clinical, cytogenetic and molecular features and outcome in 92 patients with BP MPN, according to MPN diagnosis and mutational profile.Methods We identified in our database all patients affected with ET, PV and PMF who developed AML according to 2016 WHO criteria and for whom at least one DNA sample was available to define the mutational status of the 3 MPN driver genes (JAK2, CALR, MPL). Target sequencing was performed using the Illumina TruSight Myeloid Sequencing Panel kit on the HiSeq 2000 platform. For each patient circulating granulocytes or CD34+ cells were analyzed as neoplastic tissue and CD3+ T cells as control tissue; data were analyzed with the pipeline for somatic variant calling implemented at CeMM. Treatment used in BP was classified as palliation (supportive care or low intensity chemotherapy) or induction chemotherapy (de novo AML-like therapy).Results We retrospectively identified 92 patients who progressed to BP, with a known molecular profile: 29 ET (18 JAK2 V617F , 9 CALR, 1 MPL mutated and 1 triple-negative), 39 PV (all JAK2 V617F mutated) and 24 PMF patients (18 JAK2 V617F, 1 CALR, 2 MPL mutated and 3 triple-negative).Median age at BP was 70.8 years (range 46.3-86); age at evolution was not significantly different (P 0.182) in the 3 diagnostic categories (ET, PV, PMF).The complete blood count at leukemic evolution was not influenced by the initial diagnosis.Median time to leukemic evolution was 110 months (range 3.6-367.6) and it was shorter in PMF compared to ET (29.8 vs 153.4 months, P < 0.001) and PV (29.8 vs 129.7 months, P < 0.001). Moreover, time to leukemic evolution was shorter in JAK2 V617F positive ET compared to CALR mutated ET (117.2 vs 210.9 months, P 0.027), but not statistically different in JAK2 mutated compared to CALR mutated PMF (P 0.312).At the time of BP, 35 out of 49 patients (71%) for whom cytogenetic analysis was available showed an abnormal karyotype (22 patients with complex karyotype or high risk aberrations).JAK2 mutated MPN can evolve into JAK2 wild type AML (8 of 32 patients with blast DNA available), whereas CALR and MPL mutations were identified also in AML blasts in all patients for which DNA was available (3 for CALR and 3 for MPL mutated AML).We performed targeted re-sequencing in 53 patients, in 23 studying both the CP and the BP sample. Overall, we identified 176 variants in 39 different genes (68% were missense mutations); the most commonly involved pathways were DNA methylation (22%), transcriptional regulation (20%) and chromatine regulation (18%). The most frequently involved genes were TET2, TP53, RUNX1 and ASXL1 . In the 23 paired cases, we observed a statistically significant increase in the median number of mutations in the BP, compared to CP [4 (range 1-10) vs 2 (range 0-9); P 0.002]. This was observed in JAK2 mutated patients (P 0.006) and in those with PV (P 0.008), but not in subjects with TE or PMF. In our paired-sample cohort, BP evolution was associated with the acquisition of mutations (e.g. IDH1) and/or an increase in mutation occurrence or variant allele frequency (VAF) of genes implicated in leukemogenesis including TET2 (P 0.026), RUNX1 (P 0.003), ETV6 (P 0.068) and TP53 (P 0.003), the latter known to be involved also in JAK2 mutated MPN evolving into JAK2 wild type AML. In addition, VAFs suggested an heterogeneous clonal architecture and evolution of somatic mutations in these patients.Outcome was dismal, with a median OS of 3.8 months (range 3.1-5.3), independently from MPN diagnosis (P 0.603) and treatment during BP (5.6 months with induction chemotherapy vs 3.5 months with palliation; P 0.171).Conclusion Clinical phenotype and outcome of BP is not influenced neither by the diagnosis in CP nor by the type of driver mutation; likewise the outcome is poor irrespective of treatment. PMF patients have a shorter time to BP than ET and PV patients; in ET JAK2 V617F mutation is associated with a shorter time to BP compared to CALR mutation. TP53, RUNX1, TET2 and ETV6 are more frequently involved in leukemic evolution and the number of mutations increases from CP to BP. DisclosuresNo relevant conflicts of interest to declare.

Long Term Outcome of Patients with MPN-Associated Myelofibrosis Treated with Peg-Interferon-α2a, a French Intergroup of Myeloproliferative Neoplasms (FIM) Study

Myeloproliferative neoplasm (MPN)-associated myelofibrosis (MF) can occur as a de novo (Primary MF, PMF), or as secondary MF (SMF) resulting from transformation of Polycythemia Vera or Essential Thrombocythemia. In all cases, altered hematopoiesis results in abnormal blood counts (either hyperproliferation or cytopenias), splenomegaly and general symptoms related to inflammation, all of which are the intended targets of treatments.Unfortunately, few therapeutic options exist and, besides allogeneic stem cell transplantation (ASCT), none is curative. We previously reported that pegylated-interferon-a2a (Peg-Ifn-α2a ) treatment in MF was able to induce significant clinical and hematological responses (1). We now present the long-term follow-up of this prospective cohort and correlations with driver and non-driver mutations.Sixty-two MF patients (pts) treated with Peg-Ifn-α2a were included between 2006 and 2011 in a prospective observational study in 17 centers of the FIM group. Clinical and biological parameters and treatments were collected at Peg-Ifn-α2a initiation, every 3 months during two years and every 6 months thereafter. All 62 pts were genotyped for the 3 major MPN mutations (JAK2V617F, CALR, MPL), and 49 were also screened by next generation sequencing (NGS) for mutations in the whole coding sequence of 25 genes frequently mutated in chronic myeloid disorders.The median age at Peg-Ifn-α2a initiation was 64 and 70.5 years-old, in PMF (n=29) and SMF (n=33) pts, respectively. The median follow-up was 58 months (range: 9-107) after Peg-Ifn-α2a initiation and 69.6 months (range: 10-178) from MF diagnosis. Median Peg-Ifn-α2a treatment duration was 39 months (range: 6-107). At the time of analysis, 30 patients (48.4%) were still alive. The median overall survival (OS) was 7.4 years from MF diagnosis. The Lille and the DIPSS scores clearly differentiated pts in terms of OS, but median OS observed in this cohort was clearly longer than that reported in the reference cohorts used for the establishment of these prognostic scores, especially in higher risk categories: according to the Lille score (8.9 vs. 7.75 yrs for low, 5.42 vs. 2.17 yrs for intermediate and 4.46 vs. 1.08 yrs for high risks) and to the DIPSS score (6.9 vs. 4 yrs for intermediate-2 and 4.58 vs. 1.5 yrs for high-risk pts).The type of driver mutation statistically impacted survival: CALR -mutated pts had 13.5 yrs of median OS compared to 7 yrs for JAK2 -mutated pts (p<0.0001). Forty-five pts (72.6%) discontinued Peg-Ifn-α2a: 25 (55.6%) due to resistance and 20 (44.4%) due to intolerance. Patients developing intolerance to Peg-Ifn-α2a had longer median OS and leukemia-free survival (LFS) than those with resistance (p=10-5 and p=0.048, respectively). The median survival after Peg-Ifn-α2a cessation was 17 months (3-62m), but differed according to the subsequent treatment received: 22 months for those who received ruxolitinib compared to 14 months for those who received another drug (p=0.12) and 10 months for pts who underwent ASCT (p=0.003).Sequential quantification of JAK2V617F allele burden was available in 27 pts, showing a decrease of mutant allele burden of more than 50% in 10/27 pts (37%). Four pts (15%) achieved a reduction over 90% including 3 complete molecular responses. Of the 49 pts analyzed with targeted NGS, 28 (57.1%) carried at least one additional mutation. Patients who harbored at least one non-driver mutation had shorter OS (6.1 vs. not reached, p=0.06) and LFS (not reached both, p=0.026) than those with only driver mutations. The presence of “high molecular risk” mutations previously associated with poorer prognosis (in ASXL1, EZH2, SRSF2, IDH1/2) was not associated with a poorer OS or LFS than other mutations in this series of pts treated with interferon.In conclusion, this large cohort of MF pts treated with Peg-Ifn-α2a demonstrates that a long-term use of this treatment is safe, and is associated with improved OS compared to historical series as well as significant decrease in driver mutation allele burden. The presence of additional mutations remains associated with poorer prognosis. The role of interferon therapy should be discussed in pts with MF, optimal target population possibly being high-risk pts without ASCT project and with a proliferative disease. [Display omitted] DisclosuresIanotto:Novartis: Other: Grant. Laribi:MUNDIPHARMA: Research Funding; NOVARTIS: Honoraria, Research Funding; ROCHE: Research Funding; TEVA: Research Funding; HOSPIRA: Research Funding; AMGEN: Honoraria; TAKEDA: Honoraria, Research Funding. Cony-Makhoul:BMS: Speakers Bureau. Kiladjian:AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: 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.

Haploidentical Allogeneic Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in Patients with Myelofibrosis: A Multi-Institutional Experience

Haploidentical Allogeneic Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in Patients with Myelofibrosis: A Multi-Institutional Experience

Abstract PR01: Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes

Abstract Wilms’ tumor is the most common kidney cancer in children. Despite advances in care, children with metastatic, anaplastic, or relapsed disease still fare poorly. Recent studies have uncovered novel types of driver mutations, including in microRNA processing genes such as DROSHA. However, there are no ways to rationally guide therapy based on mutation, as the mechanisms by which they cause cancer remain poorly defined. Thus, here we investigated how specific types of driver mutations affect gene and protein expression. To understand how these mutations drive Wilms’ tumor formation, we first recategorized known mutations and copy number changes using a new classification schema. We found four mutation classes to be mutually exclusive with each other: microRNA processing, MYCN-activating, chromatin remodeling, and RNA splicing. These mutations were not mutually exclusive with common mutations in kidney development genes or loss-of-heterozygosity/imprinting (LOH/LOI) of chr11p15. We propose that this mutational pattern implies a “three-hit” model, whereby 11p15 LOH/LOI and mutations impairing kidney development predispose to but are often not sufficient for Wilms’ tumor formation. A third mutation then transforms the transcriptome via microRNA processing, MYCN, chromatin remodeling, or splicing. To study how these “third hits” affect gene expression, we next performed gene set enrichment analysis. As expected, we found that microRNA impairment leads to overexpression of microRNA target genes, and MYCN activation drives MYC target genes. Interestingly, we also found that loss of microRNA processing correlated with expression of oxidative phosphorylation genes, which may reveal a metabolic dependency in these tumors. In addition, mutations affecting splicing led to high levels of interferon-stimulated genes. Thus, the abnormal RNA species generated by altered splicing appear to trigger the innate immune response that normally responds to viral RNA. Finally, we measured how these mutations affect protein levels using reverse-phase protein arrays. Strikingly, Wilms’ tumors with either anaplastic histology or microRNA processing mutations express high levels of immune-related markers such as PD-1, PD-L1, phospho-Stat3, and phospho-NF-kB. In summary, many Wilms’ tumors develop a total of three types of mutations: 11p15 LOH/LOI, kidney development impairment, and a third hit that reshapes the transcriptome in an oncogenic fashion. These “third hits” may affect microRNA processing, MYCN activity, chromatin remodeling, or RNA splicing, and each type of mutation has distinct effects on gene expression. In particular, mutations in microRNA processing cause aberrant overexpression of microRNA target genes, leading to metabolic reprogramming and increased immunogenicity. As a result, some Wilms’ tumor mutations have widespread effects on the transcriptome that may be susceptible to immune checkpoint blockade. This abstract is also being presented as Poster B05. Citation Format: Kenneth S. Chen, Kavita Desai, James F. Amatruda. Three-hit model of Wilms’ tumor formation reveals immunogenic transcriptional subtypes [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR01.

Genomic aberrations in myelodysplastic syndromes and related disorders

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are heterogeneous myeloid neoplasms that frequently evolve into secondary acute myeloid leukemia (sAML). Recent progress in next-generation sequencing technologies has allowed us to discover frequent mutations throughout the coding regions of MDS, MDS/MPN, and sAML, subsequently providing information on more than 60 driver genes in these diseases. As shown by many study groups recently, such driver mutations are acquired in a gene-specific fashion. DDX41 and SAMD9/SAMD9L mutations are observed in germline cells long before MDS presentation. In blood samples from healthy elderly individuals, somatic DNMT3A, TET2, and ASXL1 mutations are detected as age-related clonal hematopoiesis and supposed to be a risk factor for hematological neoplasms. Recent reports on MDS have shown that mutations in genes such as NRAS and FLT3, designated as Type I genes, were significantly associated with leukemic evolution. Another type (Type II) of genes, including RUNX1 and GATA2, has been shown to be related to the progression from low-risk to high-risk MDS. These driver mutations are significantly concomitant during disease progression. Overall, various types of driver mutations are sequentially acquired in MDS, accounting for the heterogeneity of these disorders.

Spontaneous T-cell responses against Arginase-1 in the chronic myeloproliferative neoplasms relative to disease stage and type of driver mutation

ABSTRACTCompelling evidence supports the existence of a profound immune dysregulation in patients with chronic myeloproliferative neoplasms (MPN). Increased Arginase-1 expression has been described in MPN patients and in solid cancers. This increase contributes to an immunosuppressive tumor microenvironment in MPN patients because of L-arginine depletion by Arginase-1-expressing regulatory cells and cancer cells, which subsequently limits the activation of circulating effector cells. In the present study, we demonstrate that Arginase-1-derived peptides are recognized by T cells among peripheral mononuclear blood cells from MPN patients. We characterized the Arginase-1-specific T cells as being CD4+ and found that the magnitude of response to the Arginase-1 peptides depends on disease stage. Activation of Arginase-1-specific T cells by vaccination could be an attractive novel immunotherapeutic approach to targeting malignant and suppressive cells in MPN patients in combination with other immunotherapeutics.

Clinical Analysis of Driver Mutations in Patients with Ph Negative Myeloproliferative Neoplasms

To explore the relationship between driver mutations and clinical characteristics in patients with Philadelphia chromosome (Ph) negative myeloproliferative neoplasms (MPN), so as to provide evidence for diagno-sis and treatment of the disease. The clinical data of 410 patients with classic Ph negative MPN including 150 cases of polycythemia vera (PV), 188 cases of essential thrombocythemia (ET) and 72 cases of primary myelofibrosis (PMF) from January 2013 to December 2016 in Fujian Medical University Union Hospital were retrospectively analyzed. The PCR or DNA sequencing were used for JAK2 V617F, JAK2 exon12, CALR and MPL W515L/K mutation analyses, and follow-up information on patients was updated by direct phone call or follow-up in outpatient. Among the 410 patients with Ph negative MPN, 136 (33.2%) cases were asymptomatic at diagnosis. 389 cases were sequenced and JAK2 V617F was detected in 87.1% (122/140) of PV, 64.1% (118/184) of ET, 64.6% (42/65) of PMF; JAK2 exon 12 mutation in 1 case of PV; MPL W515L/K mutation in 1 case of ET and PMF, respectively; CALR mutation in 18(9.8%) cases of ET and 5 (7.7%) cases of PMF. JAK2 V617F mutated PV patients ocourred in older age: the white blood cell count, platelet count and incidence of splenomegaly were higher than JAK2-negative PV cases(P<0.05). Compared with JAK2 V617F mutated ET patients, CALR mutated ET cases displayed younger age, lower leukocyte count, higher platelet count and lower incidence of thrombosis; JAK2-negative ET cases had younger age, lower leukocyte count, lower hemoglobin level, higher platelet count and lower incidence of thrombosis(P<0.05). The incidence of splenomegaly in JAK2 V617F or CALR mutated PMF patients was both higher than that in JAK2-negative PMF cases, but the incidence of leukemia transformation in JAK2-negative PMF patients was higher than that in JAK2 V617F mutated cases (P<0.05). The types of driver mutations are closely related with the clinical features and prognosis in Ph- negative MPN patients.

Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis

AbstractThe 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this “real-life” study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

Prognosis in Primary Myelofibrosis Patients According to Cytogenetic and Molecular Testing Results

Introduction. The characteristics of pathological clone cells in the Primary Myelofibrosis (PMF) strongly influence on the variability of the clinical course and prognosis. The main of them are type of driver mutation in JAK2, MPL or CALR genes ("clonal markers" (CM)), cytogenetic and epigenetic alterations. It is very important to provide the physicians necessary and sufficient information about the molecular profile of the disease to select the appropriate therapy for the patient.The aim of our study was to investigate mutational diversity in PMF patients from the North-West region of the Russian Federation and to estimate overall survival (OS) depending on the type of CM, cytogenetic and epigenetic features.Materials and methods. We examined 115 patients with PMF (45 male, 70 female). Median age was 59 years (range 19-82). The detection of V617F mutation of JAK2 was carried out for all the patients. JAK2(-) samples were tested for MPL gene 515 codon mutations and 9th exon mutations in the CALR gene. All patients underwent the analysis of mutations in EZH2, ASXL1 and IDH1 genes. Karyotype research was done for 47 patients with suitable bone marrow samples. Differences of mutation/karyotype occurrence in groups were assessed using the χ2 test. Survival curves were estimated by the method of Kaplan-Meier. Differences were considered as statistically significant with p-value <0.05.Results. CM were detected in 87 patients: JAK2(+) 47% (54/115), CALR(+) 23% (26/115), MPL(+) 6% (7/115) cases. No CM were found in 24% (28/115) patients, who considered as triple-negative (TN). The median survival of TN patients was significantly shorter than in the group of patients with any CM: 4 years vs 14.5 years, p=0.005. Five-year OS in TN patients was 30.6%, JAK2(+) - 83.8% and CALR(+) - 93.5% (p=0.046) (Figure 1). Median survival in JAK2(+) patients was 14.7 years, CALR(+) - 9.8 years. In MPL(+) patients with follow-up of 4 years median survival has not been reached.Patients were divided into 2 groups based on the results of cytogenetic analysis: low-risk group (LR) included 27 patients with normal karyotype (NK) and 5 patients - with del(13)(q22), del(20)(q12), add(6)(p25), del(6)(q15) single features; high risk group (HR) consisted of 15 patients with complex abnormalities and unfavorable aberrations (+8, -7/7q-, i(17q), inv(3), -5/5q-, 12p-). Median survival in the LR group was 7.5 years, in the HR group - less than 1.5 years (p=0.025). Unfavorable karyotype frequency was distinct in groups with different CM: CALR(+) - 9.1% (1/11), JAK2(+) - 35% (7/20), TN - 54% (7/14), MPL(+) - 0% (0/2) cases but these differences were not significant (p=0.107).We detected 28 mutations of the ASXL1 gene in 26/115 patients (22.6%): 24 patients had single mutation and two patients harbored two ASXL1 mutations at once. Mutation frequency was 13% (7/54) in JAK2(+) patients, 27% (7/26) in CALR(+), 32% in TN, 29% in MPL(+) (2/7). Differences in OS depending on the ASXL1-status were statistically significant in the TN group: median survival of the ASXL1(-) patients was 3.5 years, ASXL1(+) - 1.5 years (p=0.013).Two mutations in the EZH2 gene (1.7%) were observed - each one in TN and ASXL1(+) patients. Both cases were characterized by rapid disease progression. The first patient (a 61-year-old male, complex karyotype) progressed rapidly to the blast phase from diagnosis and died after 8 months. The second patient (a 75-year-old male, NK) had severe thrombocytopenia and died from a hemorrhagic stroke 5 months from diagnosis.We also detected 8 (7%) mutations in the IDH1 gene: 5 in CALR(+), 2 in TN, 1 in JAK2(+) patients. In seven cases it was a synonymous substitution G105 and in one case it was missense mutation R132C. The patient with the R132C mutation (a 74-year-old female, CALR(+), NK) had an unfavorable disease course: progression to blast phase was observed after 9 months from diagnosis. The patient died 13 months after disease progression.Conclusion. CM and epigenetic mutations frequencies in our PMF patients is consistent with international data. Type of CM, cytogenetic aberrations and epigenetic changes can be correlated with different prognosis of PMF. The absence of any CM, unfavorable karyotype and ASXL1-status in TN patient are associated with reduced OS. The impact of ASXL1-status in patients with CM as well as EZH2 and IDH1-status on the prognosis in PMF patients requires further studies. [Display omitted] DisclosuresShuvaev:Novartis pharma: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Fominykh:BMS: Honoraria; Novartis Pharma: Honoraria.