Increased Frequency of Mutations in the Gene Responsible for Familial Mediterranean Fever (MEFV) in a Cohort of Patients with Chronic Idiopathic Neutropenia

Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients. Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms. Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or "atypical" FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (P<0.0001, Fisher's exact test). NGS analysis confirmed the mutational pattern of NIRCA and specifically showed: (a) one patient with heterozygous I720M (ATC>ATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels. Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. Disclosures No relevant conflicts of interest to declare.

Follow-up Next Generation Sequencing (NGS) Analyses on Clonal Chronic Idiopathic Neutropenia (CIN) Patients: Insights in the Natural History of the Disease

Background: We have previously performed NGS analysis of genes that are recurrently mutated in myeloid malignancies in a cohort of patients with the diagnosis of chronic idiopathic neutropenia (CIN) according to previously reported criteria that largely overlap with those proposed for idiopathic cytopenia/neutropenia of undetermined significance (ICUS-N). We have thus estimated for the first time the frequency of clonal hematopoiesis in patients with CIN/ICUS-N (11.54%) and found that clonal CIN patients have a significantly higher risk of developing a myeloid neoplasm than those with no evidence of clonality (non-clonal). 1 However more longitudinal follow-up NGS studies are required for the tracking of clonal evolution and delineation of clonal CIN natural history. Aims: To conduct longitudinal follow-up NGS analyses in order to assess clonal evolution and associate the clinical significance of detected clonal aberrations with the risk of transforming to myeloid malignancy in clonal CIN clinical outcome. Methods: Genomic DNA was extracted from patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted next generation sequencing (NGS) on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies. Results: Follow-up analysis by NGS was performed in 16 clonal CIN patients (Figure 1). (Out of these 16 patients, follow-up NGS data has already been published in 9 patients, however additional timepoints were tested in 3 of them). 1 The median time between the first and subsequent analysis was 28.5 months (range 8-164 months). Ten of these patients carried the initial somatic mutations with only subtle changes in the size of clone as estimated by the variant allele frequency (VAF); the patients displayed absence of additional mutations and did not develop myeloid malignancy (Figure 1A-C, E-G, I, K, L, P). Two patients acquired a second mutation at follow-up. One of them still displayed stable disease course (Figure 1D) whereas the second eventually progressed to CMML (Figure 1H). The analysis also revealed that one patient lost the initial detected mutation at follow-up after 98 months (Figure 1J). Two patients who progressed to MDS/MPN and AML respectively, displayed a notable clonal expansion with additional mutations at the time of progression (Figure 1M and Figure 1N, respectively). Specifically, the patient who progressed to MDS/MPN acquired a mutation in JAK2 and ASXL1 while the patient who progressed to AML acquired the typical NPM1 p.L287fs mutation. The patient who developed MDS with multilineage dysplasia, carrying three mutations in DNMT3A and IDH1, showed a moderate increase in the VAF of these mutations at first follow-up (Figure 1O). The patient progressed to acute lymphoblastic leukemia (2 nd follow-up) with acquisition of additional truncating mutation in ETV6. Following treatment (3 rd and 4 th follow-up) mutation in ETV6 was lost, however the three mutations in DNMT3A and IDH1 persisted and their clone size increased. Conclusions: In the majority of patients tested for clonal evolution over time, most mutant clones appeared to be remarkably stable, with minimal VAF change, no acquisition of new molecular alterations and no progression to overt myeloid malignancy. Two CIN patients who transformed to a myeloid malignancy displayed a clonal expansion as was reflected by the increase of VAF and the development of additional mutations whereas in the third patient only a modest VAF increase was identified before malignant transformation. Finally in the patient bearing 4 mutations no progression to overt malignancy was observed after 12 months of follow-up. This ongoing study of sequential NGS analysis of CIN patients is anticipated to contribute to the better understanding and enrich further the knowledge on the natural history of this rare disease. References: Tsaknakis G, Galli A, Papadakis S et al. Incidence and Prognosis of Clonal Hematopoiesis in patients with Chronic Idiopathic Neutropenia. Blood. 2021 Jun 24:blood.2021010815. doi: 10.1182/blood.2021010815. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Late Onset and Long Lasting Idiopathic and Autoimmune Neutropenia As Epiphenomena of Immune Dysregulation: Preliminary Data Study from the Italian Neutropenia Registry

INTRODUCTION: Idiopathic neutropenia (IN) of childhood is a benign, self-limiting disorder usually occurring in the first 3 years of life that differs from primary autoimmune neutropenia (AIN) because anti neutrophil antibodies are not detected on repeated indirect testing over time(1). Chronic idiopathic neutropenia (CIN) is a well characterized disorder of elderly with a peculiar immunological pattern(2). Pediatric AIN patients, with atypical features represented by longer disease duration (Long Lasting) or diagnosed later (Late Onset), were recently shown by our group to display a different immune-hematological profile vs typical primary AIN(3). Atypical pediatric IN subjects, i.e. diagnosed later or with longer disease duration, though occasionally reported, were never systematically investigated. In the present study we analyzed a cohort of young patients with atypical IN and AIN, diagnosed after the age of 3 years or with longer disease duration, aiming to identify an immunological signature that might predict their different outcome from classical AIN and IN of infancy. PURPOSE OF THE STUDY: to analyze a cohort of patients affected with AIN and IN rising > 3 years of age and with duration >12 months or rising <3 years of age but persisting over 36 months. MATERIALS AND METHODS: Clinical, immunological and genetic data (NGS panel of 160 immunodeficiency/disimmunity genes) of eligible patients were collected from the database of the Italian Neutropenia Registry. RESULTS: From 2005 to 2020, data from 46 patients (24F, 24 autoimmune and 22 Idiopathic Neutropenia) were retrieved. Median age at onset was 11.2 years (IQR13.2-16.7), median follow-up was 4.3 years (IQR 3.2-6.8). Neither autoimmunity nor additional cytopenias to neutropenia were present at onset. Cumulative incidence at 5 years of autoimmune manifestations (thyroiditis, arthritis, vitiligo, recurrent skin rash) was 11.8% (CI 95% 4.3-30). Throughout the follow up, infections occurred in 32/46 (70%) subjects and in only 16% were severe (meningitis, recurrent pneumonia, sepsis and pericarditis). Infections sites were upper respiratory (in 84% of subjects mouth and gums (47%), and skin (40%) ear (25%), lung (19%) and urinary tract (12%). Fever of unknown origin was detected in 40% of patients. Recurrent infections (more than 3/y) involved mouth (85%) and ear (88%). White Blood Cell, Absolute Neutrophil and Lymphocyte counts retrieved at the beginning of the study, in the middle and at the end of follow up are shown in Table 1. Leucocytes and Lymphocytes at the end of follow up were significantly lower than values seen at diagnosis (p < 0.001) whereas neutrophil count remained stable (p=ns). One third of the cohort had lower values than normal of CD19+ and CD3-CD56+CD16+ cells. B switched memory CD27+/IgD-/IgM- (in 94%) were lower, while marginal zone B lymphocytes CD27+/IgD+/IgM+ (62%) and Tγδ cells (70%) were increased than the normal population according to a pattern comparable to that described in chronic idiopathic neutropenia (CIN). Immunoglobulin serum levels (IgA, IgG and IgM) were below the normal value in 7.5% of the population, but specularly a small subset (7.5%) of IN showed an increase of IgM was seen, similar to what already descibed in adult CIN. The genetic study carried out in 32 patients showed in 5 (16%) pathogenic variants: of immunodeficit/dysregulation (2 TACI, 1 TINF2, 1 CARD11) and in 9 cases (28%) VUS in genes of the same groups (1CARD11, 4CASP10,1DDX41/SOCS1, 1TSR2/DCLRE1c, 1PIK3D, 1TERT/TACI). CONCLUSIONS: Atypical IN and AIN (of longer duration or occurring in advanced pediatric age) are different from those appearing in early infancy. They seem to display dysimmune/autoimmune features that is some case is proven to rely on a genetic dysimmune background. They might represent an anticipation of more complex autoimmune disorders which may fully manifest later in life. Interstingly these patients seem to share some features with CIN of adults. Definitive conclusions will be drawn by applying more comprehensive genetic nalysis (WES and WGS) on larger group of subjects. References: 1. Farruggia et al, Am J Hematol. 2019, 94:216-222 2. Mavroudi I et al, Clin Immunol. 2017 Oct;183:75-84 3. Fioredda F et al, Blood Adv 2020 Nov 24;4:5644-5649 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Incidence and Prognosis of Clonal Hematopoiesis in patients with Chronic Idiopathic Neutropenia

The incidence and prognosis of clonal hematopoiesis in patients with isolated neutropenia among patients with idiopathic cytopenia of undetermined significance (ICUS), known as ICUS-N or chronic idiopathic neutropenia (CIN) patients, is poorly defined. In the present study we sought to investigate the frequency and clinical significance of mutations of genes implicated in myeloid malignancies using next generation sequencing, in CIN patients (n=185) with a long follow-up. We found that 21/185 patients (11.35%) carried totally 25 somatic mutations in 6 genes with median variant allele frequency (VAF) 12.75%. The most frequently mutated genes were DNMT3A and TET2 involving more than 80% of patients followed by IDH1/2, SRSF2 and ZRSR2. The frequency of transformation to a myeloid malignancy was low in the total group of patients (5/185 patients; 2.70%). However, from the transformed patients four belonged to the clonal (4/21; 19.05%) and one to the non-clonal (1/164; 0.61%) group, indicating that the presence of mutation(s) confers a relative risk for transformation 31.24 (P = 0.0017). The VAF of the mutant clones in the transformed patients was higher than 10% in all cases and the genes most frequently associated with malignant transformation were the SRSF2 and IDH1. No significant differences were identified between clonal and non-clonal groups in the severity of neutropenia. Patients with clonal disease were older compared to non-clonal patients. These data contribute to the better understanding of the heterogeneous entities underlying ICUS and highlight the importance of the mutation analysis for the diagnosis and prognosis of patients with unexplained neutropenias.

Frequency and Functional Analysis of Myeloid-Derived Suppressor Cells (MDSCs) in the Peripheral Blood and Bone Marrow of Patients with Chronic Idiopathic Neutropenia (CIN)

Myeloid-derived suppressor cells (MDSCs) are myeloid cells with immunoregulatory properties characterized mainly by suppression of T-cell responses (Bizymi et al, HemaSphere 2019). They are divided in HLA-DRlow/-/CD11b+/CD33+/CD15+ polymorphonuclear (PMN-MDSCs) and HLA-DRlow/-/CD11b+/CD33+/CD14+ monocytic (M-MDSCs) subsets and they are implicated in inflammatory and malignant diseases. Chronic idiopathic neutropenia (CIN), is a (usually benign) neutrophil disorder characterized by persistent and unexplained neutropenia following a detailed clinical/laboratory investigation including anti-neutrophil antibody testing, bone marrow (BM) biopsy and karyotype (Dale & Bolyard, Curr Opin Hematol 2017). Previous studies have shown that neutropenia in CIN is associated with increased apoptosis of BM granulocytic progenitor cells due to an inflammatory BM microenvironment consisting of oligoclonal T-lymphocytes, proinflammatory monocytes and proapoptotic cytokines. The aim of the present study is to explore the possible involvement of the MDSCs in the pathophysiology of CIN by investigating their number in peripheral blood (PB) and BM in association with their functional characteristics. We have studied 100 CIN patients and 49 age- and sex-matched healthy controls. The patients fulfilled the previously described diagnostic criteria for CIN (Papadaki et al, Blood 2003) and had mean neutrophil counts 1095.67 ± 479.52 (median 1215, range 100-1700). MDSC subsets were quantitated by flow cytometry in the PB mononuclear cell (PBMC) fraction using the combination of CD33PC7/CD15PC5/HLA-DRECD/CD14PE/CD11bFITC monoclonal antibodies and the Kaluza analysis software. MDSC subsets were also studied in the BMMC fraction of 24 CIN patients and 8 healthy controls from the study population. The T-cell suppression function of patient MDSCs was evaluated in coculture experiments of immunomagnetically sorted, CFSE stained, normal CD3+ cells with immunomagnetically sorted M-MDSCs and PMN-MDSCs from 4 patients and 4 healthy donors using recombinant human IL-2 as activating factor. CFSE staining was detected in the CD3+ cells on day 0 and day 3 of coculture and analysis was performed with the Fcs Express 7 software. Statistical analysis was performed with the Statistica software. We found that the proportion of PB M-MDSCs was statistically significant lower in CIN patients (1.45% ± 1.82%) compared to controls (3.68% ± 3.12%, Mann-Whitney test, p < 0.0001) (Figure a) whereas the proportion of PB PMN-MDSCs, although lower in patients, did not differ significantly from the controls. The proportion of BM M-MDSCs did not differ significantly between CIN patients and controls whereas the proportion of BM PMN-MDSCs was statistically significant lower in patients (13.27% ± 11.27%) compared to controls (19.49% ± 4.46%; Mann-Whitney test, p = 0.0291) (Figure b). Paired analysis showed that the proportion of PMN-MDSCs were higher in the BMMC compared to PBMC fraction in both CIN patients (13.27% ± 11.27% vs 1.14% ± 1.64%, respectively; Wilcoxon test, p = 0.005) (Figure c) and healthy controls (19.49% ± 4.46% vs 9.92% ± 9.08%, respectively; Wilcoxon test, p = 0.0118). Interestingly, the proportion of increase of PMN-MDSCs (in BMMC vs PBMC fraction) was significantly higher in patients (86.71% ± 21.26%) compared to controls (55.95% ± 38.59%; Mann-Whitney test, p = 0.0357) (Figure d). The above data indicate low production of PMN-MDSCs in CIN patients compared to controls but a trend for accumulation of these cells in patients' BM. No statistically significant difference was documented in paired analysis of M-MDSCs between BMMC and PBMC fractions in either CIN patients or healthy controls. Patient PMN-MDSCs and M-MDSCs displayed normal capacity to suppress T-cell proliferation as was indicated by the T-cell generations in coculture experiments of normal CD3+ cells in the presence or absence of patient MDSCs (Figure e). In conclusion, CIN patients display low proportion of MDSCs in the PB and lower proportion of PMN-MDSC in the BM compared to normal individuals. Patient MDSCs display normal capacity to suppress T-cell activation. The low proportions of MDSCs may sustain the inflammatory process associated with CIN whereas the accumulation of PMN-MDSCs in the BM represents probably a compensatory mechanism to suppress the inflammatory processes within patients' BM microenvironment. Figure Disclosures Papadaki: Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Μελέτη του ρεπερτορίου των γονιδίων της β΄αλυσίδας του υποδοχέα των CD8⁺ T-λεμφοκυττάρων σε ασθενείς με χρόνια ιδιοπαθή ουδετεροπενία

Η χρόνια ιδιοπαθής ουδετεροπενία (Chronic Idiopathic Neutropenia, CIN) είναι διαταραχή της κοκκιώδους σειράς που χαρακτηρίζεται από χαμηλό αριθμό ουδετεροφιλων, κυριαρχεί κυρίως στις γυναίκες και συνήθως έχει καλοήθη πορεία χωρίς επιπλοκές. Σημαντικό ρόλο στην παθοφυσιολογία της νόσου έχει η παρουσία των ενεργοποιημένων Τ λεμφοκυττάρων με μυελοκατασταλτικές ιδιότητες τόσο στο αίμα όσο στο μυελό των οστών. Προηγούμενες μελέτες έχουν δείξει αυξημένο αριθμό ολιγο-/μονο-κλωνικών Τ κυτταρικών υποπληθυσμών, κυρίως των CD8⁺, στο αίμα αλλά και στο μυελό των οστών ασθενών με CIN. Στην παρούσα μελέτη χαρακτηρίστηκε συστηματικά το ρεπερτόριο του γονιδίου της β αλυσίδας του Τ κυτταρικού υποδοχέα (TRB) στα CD8⁺ κύτταρα σε 34 ασθενείς με CIN με την μέθοδο υποκλωνοποίησης και αλληλούχιση κατά Sanger των γονιδιακών αναδιατάξεων TRBV-TRBD-TRBJ. Παρατηρήθηκε έντονη επιλεκτικότητα και ολιγοκλωνικότητα. Βρέθηκαν κοινοί κλωνότυποι σε διαφορετικούς ασθενείς, υποδεικνύοντας επιλογή από κοινό αντιγόνο. Η σύγκριση των αποτελεσμάτων από την αλληλούχιση των TRB αναδιατάξεων με δημόσιες βάσεις δεδομένων για TRB αλληλουχίες δείχνουν ότι η CIN σπάνια εμφανίζει κοινά ανοσογενετικά χαρακτηριστικά με άλλες νοσολογικές οντότητες. Με βάση τα συγκεκριμένα ευρήματα υποστηρίζεται η άποψη ότι η νόσος πιθανόν αναπτύσσεται μετά από μακροπρόθεσμη έκθεση σε περιορισμένο σύνολο ειδικών αντιγόνων που σχετίζονται με CIN. Συνολικά, η παρούσα μελέτη προσφέρει τεκμηριωμένες ενδείξεις ότι το ρεπερτόριο TRB στο CIN είναι έντονα επιλεκτικό. Το εύρημα των εκπτύξεων ολιγοκλωνικών Τ-κυττάρων και των «δημόσιων» κλωνοτύπων υποδηλώνει ότι άνοσες αποκρίσεις που υποκινούνται από αντιγόνο εμπλέκονται στην παθογένεση της CIN.