CSF3R Mutations Research Articles

G-CSF and Its Receptor in Hematonosis

The granulocyte colony-stimulating factor (G-CSF), now referred to as CSF3, is a very important cell growth factor that supports the proliferation, survival, and differentiation of neutrophilic progenitor cells, and also is a strong immune regulator of T cells and a promising therapeutic tool in acute graft versus host disease (GVHD). G-CSF acts by binding to its receptor G-CSFR (also called CSF3R), a member of the cytokine receptor type I superfamily, which after binding with G-CSF activates the canonical Janus kinase (Jak)/signal transducer, activator of transcription (STAT)and Ras/Raf/MAP kinase pathways. G-CSF has been applied to the clinic to treat congenital and acquired neutropenia before or during courses of intensive chemotherapy. It has also been applied to mobilize hematopoietic stem cells into the peripheral blood for Auto-or allogeneic transplantation, and the priming strategies designed to enhance the sensitivity of leukemia stem cells to cytotoxic agents in protocols aimed to induce their differentiation, accompanying growth arrest, and cell death. With the rapid development of molecular genetics and clinical research, CSF3R mutations have been implicated in the progression of severe congenital neutropenia (SCN) to leukemia. Recently, CSF3R mutations have been discovered frequently in chronic neutrophilic leukemia (CNL). Such findings might provide the theoretical basis for the targeted therapy. In this review, the clinical application of G-CSF receptor in hematonosis is briefhy summarized.

Molecular characterization of atypical chronic myeloid leukemia and chronic neutrophilic leukemia

Background and objectiveAtypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) display similar clinical and haematological characteristics. The objective of the present study was to determine the mutational status of SETBP1 and CSF3R in these diseases. Patients and methodThe mutational status of SETBP1 and CSF3R was studied in 7 patients with aCML (n=3), CNL (n=1) and unclassifiable myeloproliferative neoplasms (MPN-u) (n=3). Additionally, mutations in ASXL1, SRSF2, IDH1/2, DNMT3A, and RUNX1 were also analysed. ResultsSETBP1 mutations (G870S and G872R) were detected in 2 patients with MPN-u, and one of them also presented mutations in SRSF2 (P95H) and ASXL1 (E635fs). The CNL case showed mutations in CSFR3 (T618I), SETBP1 (G870S) and SRSF2 (P95H). No patient classified as aCML had mutations in SETBP1 or CSF3R. One of the patients with mutations evolved to acute myeloid leukemia, while the other 2 had disease progression without transformation to overt leukemia. ConclusionThe knowledge of the molecular alterations involved in these rare diseases is useful in the diagnosis and may have an impact on both prognosis and therapy.

Hereditary Diffuse Leukoencephalopathy with Spheroids (HDLS) due to CSF1R mutations: Towards early disease recognition. (P2.185)

Hereditary Diffuse Leukoencephalopathy with Spheroids (HDLS) due to CSF1R mutations: Towards early disease recognition. (P2.185)

Chronic neutrophilic leukemia: novel mutations and their impact on clinical practice.

Chronic neutrophilic leukemia (CNL) is a rare BCR-ABL1-negative myeloid malignancy that is characterized by mature granulocytosis without dysgranulopoiesis. Differential diagnosis of CNL includes reactive or secondary granulocytosis and other myeloid neoplasms, such as atypical chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML). Herein, we focus on recently described mutations in CNL and their impact on diagnosis, prognosis and treatment. In 2013, membrane-proximal CSF3R mutations, most frequently CSF3RT618I, were described in CNL and aCML. Subsequent studies confirmed the presence of such mutations in nearly all patients with CNL but not in aCML. Furthermore, the majority of the patients with CSF3R-mutated CNL also expressed other mutations, such as SETBP1 and ASXL1, which might be prognostically detrimental. Laboratory studies revealed that CSF3RT618I induced JAK inhibitor-sensitive activation of JAK-STAT and CNL-like disease in mice. Case reports have indicated palliative but not disease-modifying activity of JAK inhibitor therapy in CSF3R-mutated CNL. CNL is now a morphologically and molecularly defined myeloid malignancy, and no longer a diagnosis of exclusion. The identification of CNL-specific molecular markers provides a much needed pathogenetic insight and also offers the opportunity to revise current diagnostic criteria and identify prognostic biomarkers and potential drug targets.

Diagnostic molecular pathology of lymphatic and myeloid neoplasms

Molecular pathology has been an integral part of the diagnostics of tumors of the hematopoietic system substantially longer than for solid neoplasms. In contrast to solid tumors, the primary objective of molecular pathology in hematopoietic neoplasms is not the prediction of drug efficacy but the diagnosis itself by excluding reactive proliferation and by using molecular features for tumor classification. In the case of malignant lymphomas, the most commonly applied molecular tests are those for gene rearrangements for immunoglobulin heavy chains and T-cell receptors. However, this article puts the focus on new and diagnostically relevant assays in hematopathology. Among these are mutations of MYD88 codon 265 in lymphoplasmacytic lymphomas, B-raf V600E in hairy cell leukemia and Stat3 exon 21 in indolent T-cell lymphomas. In myeloproliferative neoplasms, MPL W515, calreticulin exon9 and the BCR-ABL and JAK2 V617F junctions are the most frequently analyzed differentiation series. In myelodysplastic and myeloproliferative neoplasms, SRSF2, SETBP1 and CSF3R mutations provide important differential diagnostic information. Genes mutated in myelodysplastic syndromes (MDS) are particularly diverse but their analysis significantly improves the differential diagnostics between reactive conditions and MDS. The most frequent changes in MDS include mutations of TET2 and various genes encoding splicing factors.

Controversies in pediatric and adolescent hematology

Haematological disorders are relatively frequent during childhood and adolescence, and are encountered in both specialist paediatric haematology as well as general practice settings. These are also extremely heterogeneous, and range from common conditions like iron deficiency and immune thrombocytopenia to disorders needing complex and specialized diagnosis and care, like haemophagocytic lymphohistiocytosis in intensive care units (ICUs) and complications like graft-versus-host-disease or secondary malignancies in haematopoietic stem cell transplant (SCT) survivors. Revolutions in technology, in the fields of diagnostics, genomics or drug development, have resulted in an ever-increasing array of new laboratory tests, pharmaceutical agents and treatment modalities including gene-based, personalised and targeted therapies. While this makes paediatric and adolescent haematology a challenging and fascinating field to practice, it also presents a wide range of diagnostic options and challenges to haematopathologists, along with management and treatment dilemmas to paediatric clinical haematologists. This book, a collection of review articles, although titled ‘Controversies’, actually examines critically, dispassionately and in minute detail the most cutting-edge advances in the management of paediatric and adolescent haematological problems. Of the 11 chapters, four cover disorders of haemostasis (including neonatal and immune thrombocytopenia, haemophilia and thrombosis in paediatrics); three chapters have been devoted to malignancies (acute lymphoblastic leukaemia, myelodysplastic syndromes and myeloproliferative neoplasms) and SCT; two on disorders of erythropoiesis (iron chelation and iron deficiency) and two on haematological problems in the ICU and transitional care in haematology. A few more topics that are either important or have lately been in the limelight, like bone marrow failure including congenital dyserythropoietic anaemia, paediatric transfusion practices and childhood lymphomas and allied disorders like the autoimmune lymphoproliferation syndrome could have also been included. However, this is at best a minor objection. Each chapter is preceded by an abstract and closes with a short conclusion section that are both useful previews of the contents for the reader pressed for time. Of the individual chapters, the chapters, ‘New Advances in the Treatment of Children with Hemophilia’ and ‘Towards Personalised Medicine in Childhood Acute Lymphablastic leukaemia’ are eminently absorbing. The former discusses the latest strategies to enhance half-lives of coagulation factors including chemical conjugates, fusion proteins and innovative therapies including DNA aptamers and siRNAs, in this challenging bleeding disorder. The latter splits the field into personalised pharmacology, therapeutic targets and molecular genetics, with the last rubric also covering recent advances such as IKAROS gene deletions and CRLF2 as potential targets. The discussion of paediatric myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) is well written. It focuses on the transient abnormal myelopoiesis of Down syndrome, MDS/MPN overlap diseases including juvenile myelomonocytic leukaemia and primary and familial myelodysplasias. A brief mention is made of the pure MPNs, all of which, with the exception of chronic myeloid leukaemia (CML) and essential thrombocythemia, are rare in children. Although the CSF3R mutation in atypical CML and chronic neutrophilic leukaemia is mentioned, the major recent discovery of the calreticulin (CAL-R) possibly occurred too recently to be included in this chapter. The chapters on iron chelation potentially replacing stem cell transplants in thalassaemia and on thrombosis in paediatrics are delightful. These truly live up to the ‘Controversies’ in the title of the compendium. On the other hand, the two lengthy chapters on haematological problems in paediatric intensive care and reduced intensity conditioning in paediatric haematopoietic cell transplantation provide well-researched scholarly insights on inherently complex and difficult topics. Many chapters discuss quality of life and affordability issues, notably those on ITP (burden of treatment), iron deficiency (costs of oral-versus-intravenous medications) and paediatric thrombosis (various aspects of screening for thrombophilia) and yield important insights into these areas. The last chapter is devoted to the very important issue of transitional care in chronic haematological disorders (haemoglobinopathies, bleeding, cancer) that is often neglected in India due to competing pressures. In summary, this up-to-date book deals with both specific haematologic disorders in paediatric and adolescent practice and also haematologic perturbations in systemic disease. Aimed squarely at physicians involved in the care of haematology patients, this volume makes for a very interesting and appealing read for busy practitioners as well as final year residents for a comprehensive yet quick update.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms.

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34+ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34+ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Current strategies in the diagnosis and management of chronic neutrophilic leukemia

Objective To review the implications for diagnosis, pathogenesis and potential for new therapeutic option for chronic neutrophilic leukemia (CNL). Data sources Data cited in this review were obtained mainly from PubMed and Medline from 1993 to 2013 and highly regarded older publications were also included. The terms “chronic neutrophilic leukemia” and “diagnosis” were used for the literature search. Study selection We identified, retrieved and reviewed the information on the clinical and laboratory features, the new genetic findings, prognosis and disease evolution and management of CNL. Results The discovery of high-frequency granulocyte-colony stimulating factor receptor (CSF3R) mutations in CNL identifies a new major diagnostic criterion, and lends more specificity to the World Health Organization (WHO) diagnostic criteria for CNL, which are variably applied in routine clinical practice. Conclusions In patients for whom the cause of neutrophilia is not easily discerned, the incorporation of CSF3R mutation testing can be a useful point-of-care diagnostic to evaluate the presence of a clonal myeloid disorder, as well as providing the potential for genetically informed therapy. The oncogenic CSF3R mutations are molecular markers of sensitivity to inhibitors of the SRC family-TNK2 and JAK kinases and may provide a new avenue for therapy.

Co-Acquisition of RUNX1 and CSF3R Mutations Transforms Hematopoietic Progenitor Cells of CN Patients into More Primitive Highly Proliferative Blasts: Evidence in CN Patients and in a Mouse Model

Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a high risk of evolving into leukemia or myelodysplastic syndrome (MDS). Recently we demonstrated a very high frequency of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia or MDS (Skokowa, et al. Blood 2014). We proposed a novel molecular pathway of leukemogenesis: mutations in the cytokine receptor (G-CSFR) in combination with the second mutations in the hematopoietic transcription fator (RUNX1).In the majority of CN patients, CSF3R mutations were acquired prior to RUNX1 mutations. CSF3R mutations alone are unable to induce leukemia in CN patients or in mice expressing a transgenic d715 G-CSFR. Co-acquisition of RUNX1 mutations is an essential step in the leukemogenic transformation in CN. To characterize the expression signature of hematopoietic cells of CN/AML patients carrying CSF3R mutations prior to and after acquisition of RUNX1 mutations, we analyzed expression profiles of CD34+ hematopoietic cells of CN patient who developed AML. This patient acquired CSF3R mutation (p. Q718*) five years and RUNX1 mutation (p. R139G) 16 months prior to leukemia. We compared expression profiles of CD34+ cells harbouring CSF3R mutation only, or both CSF3R and RUNX1 mutations. Co-acquisition of RUNX1 and CSF3R mutations led to marked reduction of the expression of hematopoietic growth factors such as IL6 and NAMPT, inhibitors of cytokine signaling SOCS3, as well as of components of neutrophil granules OLFM4, DEFA4, MMP8, SLPI, CRISP3 and CTSG. At the same time expression levels of pro-proliferative downstream effectors of G-CSF such as STAT5A, STAT5B, SMAD1 and cyclin A1 (CCNA1) were dramatically elevated. Moreover, genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD109, HPGDS, PDLIM1, MLLT11 and FLT3 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations. Intriguingly, elevated expression of DNTT, BAALC, CD109 and FLT3 was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests rapid transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after acquisition of RUNX1mutation.To elucidate the role of cooperative CSF3R and RUNX1 mutations on the clonogenic capacity and myeloid differentiation of hematopoietic progenitors, we performed functional studies in mice. We transduced lineage negative (lin-) bone marrow hematopoietic progenitor cells of WT or transgenic d715 G-CSFR mice with lentiviral expression constructs containing either WT or mutated forms of RUNX1 cDNA. We used two different mutants of RUNX1 by introduction of mutations at amino acid positions 139 and 174. Acquired RUNX1 mutations in these amino acids were presented with high frequency in our cohort of CN/AML patients and in most of the cases were associated with acquired CSF3R mutations. We found that similar to the effect of CSF3R mutations, lin-hematopoietic cells of WT mice transduced with mutated RUNX1 alone did not show elevated clonogenic capacity in replating experiments. Interestingly, transduction of WT cells with RUNX1 mutants resulted in severely reduced numbers of CFU-G colonies but unaffected CFU-M and BFU-E colonies. Intriguingly, transduction of lin- hematopoietic cells from transgenic d715 G-CSFR mice with RUNX1 mutants resulted in a markedly elevated clonogenic capacity in replating experiments, as compared to cells transduced with WT RUNX1 or control vector: numbers of colonies after second replating were 7 and 8 times higher in RUNX1-R139G and RUNX1-R174X mutants, respectively, in comparison to RUNX1 WT transduced cells. Moreover, granulocytic differentiation of lin- cells from d715 G-CSFR mice transduced with RUNX1-R139G mutant was severely diminished, in comparison to cells transduced with WT RUNX1, as revealed by 5-fold reduction of CFU-G colonies.Taken together, co-acquisition of RUNX1 and CSF3R mutations shifted the hematopoietic differentiation program towards more primitive hematopoietic progenitors with elevated proliferative capacity and reduced myeloid differentiation, which ultimately lead to leukemia. DisclosuresNo relevant conflicts of interest to declare.

The Natural History of CSF3R-Mutated Chronic Neutrophilic Leukemia: A Mayo Clinic Series of 14 Consecutive Cases

Activating mutations in colony stimulating factor 3 receptor gene (CSF3R) have recently been identified as specific driver mutations in chronic neutrophilic leukemia (CNL)(NEJM 2013;368:1781). We sought to outline the clinical and laboratory characteristics and natural history of this molecularly defined entity.Methods: An IRB- approved retrospective analysis of all cases of CSF3R-mutated WHO-defined CNL was undertaken. Methods of mutation analysis have been previously published. (Leukemia 2013; 27: 1870)Results: From 10/1995 to 3/2013, fourteen patients (median age 67 years; 57% male) with CSF3R-mutated CNL were evaluated at the Mayo Clinic. Most of the CSF3R mutations identified occurred in isolation: T618I (n=11), M696T (n=2) and I598I SYN (n=1), although one T618I mutant patient also carried a cytoplasmic domain mutation (2341_2342insC ). All patients were BCR/ABL negative. Five carried a SETBP1 mutation: G870D (n=2), D868N (n=2) and G872R (n=1), all of whom carried the T618I mutation. Only one of 10 tested carried a CALR mutation (E398D) and none of 13 the JAK2 V617F mutation.Apart from fatigue, most patients were asymptomatic at diagnosis and leukocytosis had been mostly an incidental laboratory finding. Ten (71%) patients had preceding chronic leukocytosis, median duration 12.5 months (range 5-84), 5 reported weight loss, which was accompanied by fevers/ sweats in 3. Five (36%) patients had prior exposures to adjuvant chest wall irradiation for breast cancer (n=2), radioiodine for thyroid disease (n=2) and methotrexate/cyclophosphamide for autoimmune disease (n=2). Five (36%) patients had palpable splenomegaly.Peripheral blood (PB) parameters, median (range) at diagnosis were: Hb 10.8 g/dl (8.5-16.0), WBC: 54 x 10(9)/L (21.7-176.2), platelets: 201 x 10(9)/L (25-476), neutrophils: 85% (78-94), immature cells (myelocytes + metamyelocytes): 6% (0-11) and monocytes: 3% (0-10). Neutrophils had toxic granulation and Dohle Bodies in 71% of cases. LDH was elevated in all patients: 354 U/L (225-496). None had a monoclonal protein.The bone marrow (BM) was hypercellular in all patients: 95% (80-100%) and granulocytic precursors were markedly increased with left-shifted (n=11) or normal maturation. BM blasts were < 5%. Reticulin fibrosis was absent (n=5), 1+ (n=5) or 1-2+ (n=1) in those with staining. Cytogenetics were normal in all but one at diagnosis.One patient was lost to follow up after diagnosis. Hydroxyurea (HU) was the most frequently used therapy (initial therapy in 10 patients) and was ultimately received by all. Although initially effective at controlling leukocytosis, clinical course of most was defined by progressive refractory mature neutrophilia with the development of red cell (n=6) and platelet transfusion (n=3) dependancy. Other therapies included IFN or PEG/IFN (n=4), hypomethylating agents (n=4), ruxolitinib (n=2), thalidomide (n=2), cladribine (n=2) and imatinib (n=1). None of these additional drugs, induced remission and they provided primarily palliative and transient benefit.One patient transformed into AML at 17 months, one received induction chemotherapy for suspected AML (PB and BM blasts at 11-13%) at 19 months and two evolved into WHO-defined CMML, at 20 and 27 months, respectively. Both patients with documented/suspected AML were both T618I and SETBP1 positive. Of the cases progressing to CMML (one with T618I, one with M696T) neither was SETBP1 positive. At last follow up, eight patients had died and median survival was 22.5 months. Progressive refractory neutrophilic leukocytosis often preceded and contributed to death. Four (29%) patients are currently alive after a median follow-up of 61 months (14.3- 69.4).Conclusion: CSF3R-mutated CNL is an aggressive disease (median survival <2 years) characterized by progressive neutrophilic leukocytosis which is only partially controlled with current drugs, including the putative targeted therapy ruxolitinib (both ruxolitinib-treated patients progressed on treatment). Patients with CNL could progress into AML or CMML; however, most succumb from complications associated with progressive treatment-refractory leukocytosis. The observation that both patients with documented/suspected AML were SETBP1-mutation positive suggests potential pathogenetic link. DisclosuresOff Label Use: Ruxolitinib, cladribine, thalidomideand inteferon. These drugs were used in a historical account of CNL and I report on these in a retrospective chart review only..

In-Frame Exon 9 CALR Deletions Co-Occur with Other Alterations in the JAK-STAT Pathway in Myeloproliferative Neoplasms

Introduction: Mutations in the chaperone gene calreticulin (CALR) have been recently identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF), and are essentially mutually exclusive with JAK2 or MPL mutations. Normal and mutant CALR proteins may differentially affect the subcellular trafficking of JAK-STAT signaling components. CALR mutations previously reported in ET and PMF have been +1 frameshift (fs) mutations localized to exon (E) 9 that generate a novel C-terminal protein sequence with a shift from acidic to basic residues. CALR E9 in-frame (IF) deletions have been recently rarely reported as polymorphisms such as TMP_ESP_19_13054686_13054688 and TMP_ESP_19_13054650_13054658 (Ensembl database entries). We sought to determine the frequency and associated clinical features of CALR with E9 IF alterations in samples submitted for suspicion of a myeloproliferative neoplasm (sMPN). We also assessed whether CALR IF alterations are differentially associated with +1fs mutations or with JAK2 V617For other somatic mutations in MPN-associated genes.Materials and Methods: CALR mutation analysis of E9 was performed on genomic DNA extracted from blood, bone marrow (BM) aspirate or fixed BM biopsy sections using a Sanger sequencing assay with an analytic sensitivity of at least 15%. E9 IF cases were further assessed and mutations quantified by an Ion torrent sequencing panel assessing CALR, CSF3R, JAK2 and MPL, a second panel containing ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 and an Illumina MiSeq extended panel with 20 additional MPN-associated genes. These assays had a sensitivity of approximately 5%. JAK2 V617Fmutations were quantitated using a pyrosequencing assay with an analytic sensitivity of 1%.Results: We assessed CALR E9 mutation status in 733 sMPN samples that were negative for JAK2 V617F mutation. 148 (20.1%) had typical +1fs mutations (95 type 1 and variants, 53 type 2 and variants); 2 (0.3%) had point mutations (E381A and D7373M); 7 (1.0%) had IF deletions including E381_A382>A, D397_D400>D (n =4), D400_K401>D and E405_V409>V. All E9 IF deletions were present at ~50% of reads. Clinical diagnoses were cytopenia/BM fibrosis, ET, thrombocytosis/anemia, and sMPN unspecified. Mutation analysis for 27 additional MPN-associated genes revealed mutations in 5/7 (71.4%) IF deletion cases including in MPL (W515L,40%; D163Y,12%), CSF3R (A470T 46%), ASXL1 (D954fs*26, 45%) and ZRSR2 (S449_R450dup, 27%). No additional mutations were found in the 2 cases with non-synonymous CALR point mutations/SNPs. In a parallel set of 76 MPN samples that had JAK2 V617F at varying levels, we noted 1 E9 IF deletion (D397_D400>D) in a sMPN case with 21.6% JAK2 V617F, and a typical +1fs mutation (K385fs*47) in a case with low (4.2%) JAK2 V617F. All other JAK2 V617F cases had no E9 CALR alterations.Conclusions: CALR E9 in-frame deletions occur in up to 1% of sMPN samples and involve a variety of codons in the acidic domain. Therefore, sizing assays without DNA sequencing are not sufficient to unequivocally distinguish IF deletions from the characteristic +1 frameshift somatic mutations associated with ET and PMF. Given their level, these CALR IF deletions are likely germline sequence variants but are associated with a high frequency of somatic mutations in other MPN-associated genes but not with CALR +1fs mutations. Their co-occurrence with pathogenic somatic mutations in JAK2, MPL and CSF3R affecting the JAK-STAT pathway raises the possibility for a contributory role of altered CALR proteins produced by these E9 deletions in the pathogenesis of MPN. DisclosuresWang:Quest Diagnostics: Employment. Ho:Quest Diagnostics: Employment. Pan:Quest Diagnostics: Employment. Racke:Quest Diagnostics: Employment. Jones:Quest Diagnostics: Employment.

Impact of the Molecular Profile of Malignant Clones on the Response to Interferon Alpha (IFNa) Therapy in JAK2V617F-Negative Essential Thrombocythemia (ET)

Background: The majority of ET patients (pts) without JAK2 or MPL mutations present somatic mutations in the calreticulin gene (CALR). However, a series of mutations in other genes involved in the epigenome, the splicing machinery or leukemic transformation have been described in ET, but contrary to MF, their impact has not been clearly assessed. We have previously shown that IFNa may considerably reduce the JAK2-mutated clones, and we also observed that TET2 mutated clones are resistant to IFNa therapy in JAK2+TET2+ pts with polycythemia vera. As little is known about the IFNa response of clones harboring mutations other than JAK2V617F in ET, we took advantage of a cohort of pts without JAK2 mutation but CALRmutated and treated with IFNa (according to international and local guidelines) to assess the dynamics of the different MPN clones during therapy.Aims: 1) To determine, using a Next Generation Sequencing (NGS) approach, the molecular pattern of mutations in genes previously shown to have a prognostic impact in MPNs in a series of CALR-mutated ET pts; 2) To study the evolution of these mutational patterns during IFNa therapy.Methods:JAK2 and MPL-negative ET pts followed in our department fulfilling the following criteria were included in the study: presence of a CALR mutation; availability of at least 3 sequential blood samples including one taken before IFNa; IFNa therapy for at least 3 months; informed consent for molecular analysis. Total DNA was extracted from blood samples (Qiagen blood DNA mini kits) for molecular analyses. Mutations in TET2, ASXL1, EZH2, SRSF2, IDH1, IDH2, SH2B3 and CSF3R were searched through a NGS approach on a MiSeq instrument using a TruSeq custom amplicon approach (Illumina). CALRgene mutation detection was done by direct Sanger sequencing of exon 9, and mutant allele burden (%CALR) was estimated by fragment analysis (GeneMapper software , Life technologies) with a sensitivity of about 1%. Both sequencing and fragment analysis were performed on a 3500xL DX Genetic Analyser (Life technologies). Molecular response was defined as complete (CMR) when CALR mutation was no longer detectable, partial (PMR) when %CALR was decreased by >50%, minor (mMR) when %CALR was decreased by 25 to 49%, and non responders if %CALR was reduced by less than 25%.Results: Among 238 ET pts without JAK2 or MPL mutations, we identified 52 pts (22%) treated with peg-IFNa-2a, of whom 24 fulfilled the inclusion criteria. Median age was 52 years (range 31-68), 67% were women, median follow up was 12 years (range : 1.5 – 29) and median IFN treatment duration was 30 months (range: 12 – 102). 23/24 (96%) pts achieved complete or partial hematological response to IFNa. CALR mutations (present in all patients, quantifiable in all but 1 because of 1bp difference between mutated and WT) were of type 1 in 10 (42%), type 2 in 8 (33%) and of neither type in 6 (25%) pts, respectively. In addition to CALR, a second mutation was found in 8 pts (33%) by NGS, affecting ASXL1 (n=2), TET2 (n=2), IDH2 (n=2), CSF3R (n=1) and SH2B3 (n=1) genes. Comparison of sequential samples showed that the %CALR decreased from a median of 43% (range: 8-57) to 26% (range: 0-49) in the last sample (p= 0.018). In details, %CALR decreased in 13 (57%) pts, including 1 CMR, 7 PMR and 5 mMR. Interestingly, molecular response to IFNa of CALR+clones appeared poorer in pts with additional mutations (vs. pts with CALR mutation alone): 50% were non responders (vs. 40%), %CALR even increased during follow up in 25% (vs. 7%), and the median decrease in %CALR was 32% (vs. 45%). Dynamics of clones harboring additional mutated genes showed that, in contrast to CALR, mutant allele burden did not vary in 6/8 pts. However, an IDH2 mutation decreased from 9% to 1% in 1 pt, and a TET2 mutation increased from 1% to 21% in another (while %CALR remained unchanged in both pts, at 44% and 19% respectively). Of note, after 12 years of median follow-up, no emergence of new mutation was observed in any patient.Conclusion: In this cohort of JAK2V617F-negative ET, 96% of pts achieved hematological response with IFNa therapy. However, molecular profiling suggests that the mutational pattern found in malignant clones modulates the molecular response to IFNa: 1) Existence of more than 1 mutation induces poorer molecular response; 2) Clones with CALR mutation alone are sensitive to IFNa; 3) Clones harboring mutations in genes other than CALR seem less responsive to IFNa therapy. DisclosuresOff Label Use: Interferon alpha was used off-label in selected ET patients according to local and international guidelines (Barbui et al., J Clin Oncol. 2011 Feb 20;29(6):761-70).

Molecular Aberrations of Chronic Neutrophilic Leukemia: The CSF3R and SETBP1 Mutations

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by sustained elevated neutrophil levels with <10% immature cells according to WHO criteria. Recently, oncogenic mutations affecting the granulocyte-colony stimulating factor receptor (CSF3R) were reported to be highly prevalent in CNL. SET binding protein 1(SETBP1) mutations were also reported in some CNL cases. We reviewed clinical suspected 12 “CNL” patients and performed CSF3R and SETBP1 sequencing. All the 6 who met WHO criteria for CNL carried CSF3R T618I mutation. Four of the 6 also had SETBP1 mutations and a 5th had a CALR mutation (c.1154-1155insTTGTC). Two other who met these criteria but also had monoclonal gammopathy with uncertain significance (MGUS) had no CSF3R, SETBP1 or CALR mutation. The rest 4 carried infection or tumor and thus were excluded. We conclude including CSF3R and SETBP1 mutations into WHO criteria for CNL may improve diagnostic accuracy and may have therapeutic implications. DisclosuresNo relevant conflicts of interest to declare.

CSF3R, ASXL1,SETBP1, JAK2 V617F and CALR mutations in chronic neutrophilic leukemia

To observe the CSF3R, ASXL1, SETBP1, JAK2 V617F and CALR mutations in patients with chronic neutrophilic leukemia (CNL). Twelve suspected "CNL" patients were retrospectively reviewed according the WHO criteria (2008). CSF3R,ASXL1,SETBP1 and CALR mutations were sequenced, and JAK2 V617F was tested by allele specific (AS)-PCR. 6 of 12 cases were diagnosed as CML, and all of the 6 carried. 4 of 6 patients also had ASXL1 and SETBP1 mutations and one had a CALR mutation (c.1154-1155insTTGTC). Two patients with monoclonal gammopathy with uncertain significance (MGUS) combined with CNL-like symptoms had no CSF3R, ASXL1, SETBP1, JAK2 V617F or CALR mutation. The same results were also seen in other 4 cases with secondary neutrophilic leukocytosis. CSF3R, ASXL1 and SETBP1 mutations differential diagnosis of CNL, and should be included in the diagnostic protocol so as to improve diagnostic accuracy for CNL.

Analysis for CSF3R Mutation Confirms Cutaneous Involvement by Neoplastic Neutrophils in a Patient With Chronic Neutrophilic Leukemia

Analysis for CSF3R Mutation Confirms Cutaneous Involvement by Neoplastic Neutrophils in a Patient With Chronic Neutrophilic Leukemia

Abstract 2760: CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation

Abstract We have recently discovered that a large percentage (59%) of patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML) have mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) (Maxson et al, NEJM 2013). These mutations are either point mutations in the extracellular domain (T618I, T615A) or truncations of the cytoplasmic domain. The most frequent mutation is T618I (aka T595I). The two classes of CSF3R mutations operate through distinct mechanisms that result in differential sensitivity to inhibition of tyrosine kinases downstream of CSF3R. While the mechanism of action of CSF3R truncation mutations has been well characterized-they have increased cell surface expression due to loss of negative regulatory motifs-the way in which the CSF3R point mutations activate the receptor is unclear. Consistent with a previous report, the CSF3R T618I mutation is ligand independent in both mouse bone marrow colony assays and the Ba/F3 pro-B cell line. WT CSF3R consists of two molecular weight species suggestive of differential posttranslational modification. Analysis of CSF3R point mutations T618I and T615A revealed an alteration of banding pattern of these mutations. Since extracellular threonine residues are capable of being O-glycosylated, we employed endoglycosidase digestion to reveal that the larger CSF3R species is O-glycosylated. Furthermore the T618I and T615A mutations reduce O-glycosylation of the receptor. This analysis was confirmed by direct labeling of O-glycosylation using azide-modified galactosamine, which labeled WT CSF3R, but had minimal labeling of CSF3R T618I. Receptor dimerization studies showed increased dimerization of the CSF3R T618I mutant relative to WT. Sequencing of CSF3R from an atypical CML patient samples revealed an additional mutation not previously observed in this disease, T640N. The T640N mutation (aka T617N) is a ligand-independent mutation originally found in a family with hereditary chronic neutrophilia (Plo et al, JEM 2009). Plo et al found that this mutation resides within the transmembrane domain and predicted by molecular modeling that it increased receptor dimerization through interaction of the transmembrane helices. In mouse bone marrow colony assays we confirmed the ligand-independence of the mutation and also found that it had similar potency as T618I in colony and cytokine-independent growth assays. Furthermore, like T618I, the T640N mutation conferred sensitivity to a JAK kinase inhibitor in colony assays. Surprisingly, the T640N mutation had an alteration of banding pattern very similar to T618I, suggesting that it may also have altered glycosylation status, even though the mutation resides within the transmembrane domain. Our studies demonstrate that O-glycosylation is critical for regulation of CSF3R dimerization and that the T618I and T640N mutations can activate the receptor to promote oncogenesis. Citation Format: Julia Maxson, Samuel Luty, Jason MacManiman, Samantha Savage, Melissa Abel, Swaleh Bahamadi, Brian Druker, Jeffrey Tyner. CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2014-2760

Targetable Kinase-Activating Lesions in Ph-like Acute Lymphoblastic Leukemia

BackgroundPhiladelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR–ABL1–positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults.MethodsWe performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL.ResultsPh-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6–NTRK3 fusion was sensitive to crizotinib.ConclusionsPh-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.)

ADULT LEUKODYSTROPHY: MULTI-DISCIPLINARY STRUCTURED APPROACH

<h3>Background</h3> Leukodystrophy refers to a rare group of conditions that are inherited and involve progressive destruction of previously acquired myelin. Diagnostic difficulty arises because individual disease prevalence is less than 1 in 20000, the presentation in adults (&gt;16 years) may be different compared to the larger classical paediatric experience, and a number of acquired white matter conditions (eg multiple sclerosis and vascular disease) may have to be eliminated. In order to make the process more efficient a multi-disciplinary group was established using a structured approach. Our early experience is presented together with an illustrative case. <h3>Methods</h3> The group has met since 2012. The central features of the diagnostic pathway were: family history, ethnicity, specific clinical features and MRI analysis. <h3>Results</h3> 23 adult cases were reviewed, and new diagnoses made in 12 cases to date. The diagnoses included CADASIL, hereditary diffuse leukoencephalopathy with neuroaxonal spheroids (HDLS) (CSF1R mutation), hereditary sensory neuropathy with dementia and hearing loss (DNMT1 mutation), X-linked adrenoleukodystrophy/adrenomyeloneuropathy (ABCD1 mutation). <h3>Summary</h3> A structured diagnostic approach is helpful. Most new diagnoses came from genetic testing, which looks to be more efficient than conventional investigations. A full review is given in Ahmed <i>et al</i>, JNNP, 2014. Referrals are welcome.

Inherited biallelic CSF3Rmutations in severe congenital neutropenia

AbstractSevere congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis.

Caracterización molecular de la leucemia mieloide crónica atípica y la leucemia neutrofílica crónica

Background and objectiveAtypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) display similar clinical and hematological characteristics. The objective of the present study was to determine the mutational status of SETBP1 and CSF3R in these diseases. Patients and methodThe mutational status of SETBP1 and CSF3R was studied in 7 patients with aCML (n=3), CNL (n=1) and unclassifiable myeloproliferative neoplasms (MPN-u) (n=3). Additionally, mutations in ASXL1, SRSF2, IDH1/2, DNMT3A, and RUNX1 were also analyzed. ResultsSETBP1 mutations (G870S and G872R) were detected in 2 patients with MPN-u, and one of them also presented mutations in SRSF2 (P95H) and ASXL1 (E635fs). The CNL case showed mutations in CSFR3 (T618I), SETBP1 (G870S) and SRSF2 (P95H). No patient classified as aCML had mutations in SETBP1 or CSF3R. One of the patients with mutations evolved to acute myeloid leukemia, while the other 2 had disease progression without transformation to overt leukemia. ConclusionThe knowledge of the molecular alterations involved in these rare diseases is useful in the diagnosis and may have an impact on both prognosis and therapy.