Spectrum of Pathogenic Genetic Variants in a Large Cohort of North American Congenital and Cyclic Neutropenia Patients: A Report from the Severe Chronic Neutropenia International Registry

Severe congenital neutropenia (SCN) is characterized by persistent neutropenia and risk of invasive, life-threatening infection as well as transformation to hematopoietic malignancy. The closely related syndrome cyclic neutropenia is characterized by recurrent episodic neutropenia accompanied by symptoms including infection. Understanding the genetic etiology of congenital neutropenia can help to direct therapy, guide surveillance and health maintenance strategies, and contribute to our understanding of basic neutrophil biology. Although mutations in ELANE are the most frequent cause of congenital neutropenia, there is a wide and ever-growing list of additional causative variants. Additionally, there appears to be regional genetic variability. For example, mutations in HAX1 are rarely if ever observed in North America while they are more common in Europe. We undertook exome sequencing of a large cohort from the Severe Chronic Neutropenia International Registry (SCNIR) of North America in an effort to define the genetic spectrum of congenital neutropenia and aid in the discovery of new pathogenic variants. We expanded our previously reported study of whole exome sequencing to include 152 cases of chronic neutropenia, comprised of 94 cases of SCN and 58 cases of cyclic neutropenia. We selected cases in which ELANE testing was negative, or in a small minority of cases, where ELANE testing had not yet been performed. Indeed, exome sequencing only identified 7 cases (5 SCN and 2 cyclic) carrying pathogenic ELANE mutations in this cohort. In the remaining 145 cases, we analyzed exomes for the presence of variants in genes previously associated with congenital neutropenia including AK2, AP3B1, CD40LG, CLPB, CSF3R, CXCR2, CXCR4, DNAJC21, DNM2, DOCK2, EFL1, EIF2AK3, ELANE, G6PC3, GATA1, GATA2, GFI1, GINS1, HAX1, IRAK4, JAGN1, KAT6A, KRAS, LAMTOR2, LYST, MYD88, PGM3, PSTPIP1, RAB27A, RAC2, SBDS, SEC61A1, SLC37A4, SMARCD2, SRP54, STK4, TAZ, TCIRG1, TCN2, TLR8, USB1, VPS13B, VPS45, WAS, WDR1 and WIPF1. Pathogenic heterozygous mutations of CLPB that localize to the ATP-binding pocket were identified in 7 cases, making it the second most common cause of congenital neutropenia in North America. We additionally identified 4 cases with G6PC3 pathogenic variants, and one case each with pathogenic variants in JAGN1, CXCR4 (the cause of WHIM syndrome), germline homozygous CSF3R, and GFI1. Interestingly, we identify 2 unrelated individuals (one with SCN and one with cyclic) and 2 siblings with SCN all of whom possess a recently described heterozygous variant in SRP54 (p.T117del). We collected genomic DNA from the affected mother of the 2 siblings, an additional unaffected sibling, and the unaffected grandparents. Through this kinship, we can confirm the de novo appearance of this variant in the second generation and demonstrate that it tracks with disease status (Figure 1). We also identified 2 unrelated individuals with SRP54 variants affecting residue 175 also located within the GTPase domain (p.G175E or p.G175del). Both variants are absent from the gnomAD database, and studies are underway to demonstrate de novo acquisition. In summary, we have defined the spectrum of mutations present in ELANE-wildtype chronic neutropenia cases in North America. Pathogenic or likely pathogenic variants were identified in 26 out of 145 (18%) cases. The most frequently mutated genes were of CLPB, SRP54, and G6PC3, while mutations in HAX1 were not seen. Importantly, some of these mutations are associated with genetic syndromes with extra-hematopoietic findings (for example, CLPB and SRP54) that would warrant additional evaluations and targeted health maintenance. These findings emphasize the importance of sending large panels for genotyping, rather than targeted ELANE testing. Figure 1 Figure 1. Disclosures Bolyard: X4 Pharmaceuticals: Research Funding. Makaryan: Emendo Biotherapeutic: Research Funding. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Chronic Lymphocytic Leukemia, T-Cell Large Granular Lymphocytic Leukemia in a Pediatric Patient without Underlying Condition

Background Large granular lymphocytic (LGL) leukemia is a rare hematological malignancy in children. The two types of LGL leukemia that have been described are T-cell and Natural Killer cell leukemia. It is most commonly diagnosed in older adults, average age of 60-year-old. About 20 cases of LGL leukemia have been reported in children and young adults. All the patients in the reported cases had immune dysregulation conditions, such as chronic graft versus host disease, common variable immunodeficiency disorder, Crohn's disease and autoimmune hemolytic anemia. Here we report a case of T-cell LGL leukemia in a 11-year-old boy without underlying condition who presented with chronic neutropenia associated with gingival hypertrophy, recurrent skin abscesses, aphthous ulcers, clubbing of nails and low bone density. Methods Multi-institution collaboration and literature review. Case Description 11-year-old male with two years history of episodic gum bleeding with gingival hypertrophy, skin abscesses, aphthous ulcers, chronic neutropenia and lymphocytosis presented to our clinic for further evaluation. Initial workup demonstrated moderate to severe neutropenia (absolute neutrophil count between 400/ul to 800/ul) with low segmented neutrophils of 2-4% and high lymphocytes of more than 80%, but normal white blood cell count, hemoglobin for age and platelet count. Peripheral blood smear showed several variant lymphocytes with cytoplasmic blebs and no immature cells present. Expansion of T-cell large granular lymphocytes were detected in peripheral blood by flow cytometry. Due to new symptom of lower back pain, a lumbar Magnetic Resonance Imaging was performed. Results showed low bone density with mild compression deformity of L1 and abnormal heterogeneous marrow signal with heterogeneous contrast enhancement. The abnormal bone marrow signal promoted the investigation of bone marrow aspiration and biopsy. Flow cytometry detected forty-five percent of lymphocytes with immuno-phenotype of CD3+, CD8+, CD57+, CD16+, CD7+ and CD5-. The morphology of minimal cytoplasm and mature chromatin along with immunophenotype were consistent with clonal T-cell large granular lymphocytic proliferation/leukemia. Further cytogenetic tests showed TCR gamma and beta genes rearrangement, STAT3 N647I mutation with normal male karyotype. A peripheral blood congenital neutropenia panel, which included a total of 18 genes, found a heterozygous mutation c 279 G>A in the Gata2 gene; a variant of uncertain significance. Next generation sequencing showed somatic mutations of TRGV10, TRGV8 TRGJ1, TNFAIP3 and STAT3. However, there was no germline mutations detected in sample from skin biopsy. Comprehensive evaluation by immunology, rheumatology and gastroenterology failed to detect any underlying conditions. Conclusion Due to rarity of LGL leukemia in pediatrics, standard of care guidelines are currently unavailable. Extrapolated from limited literature, two management options are considered: watch and wait approach versus early initiation of immunosuppressant chemotherapy. Improved diagnostics can aide management strategies in this patient population. Disclosures No relevant conflicts of interest to declare.

ELANE Neutropenia Beyond the Classification into Cyclic Neutropenia and Severe Congenital Neutropenia: The Inconstant Time Clock

Introduction: ELANE neutropenia represents the cause of 25-30% of the cases of congenital neutropenia. Classically, its appears in the literature as in OMIM, under two distinct entities: Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN). The delineation between the 2 entities is the "cyclicity" i.e. the periodic variation of the absolute neutrophils count (ANC), also called a 21-day time clock 1. However, it is extremely difficult to obtain enough sequential complete blood counts (CBC) at the onset of the disease, during an enough length period, while the patient is not experiencing a severe infection or receiving GCSF therapy. The purpose of our study is to analyze the ANC periodicity at the onset of the disease, prior to the initiation of GCSF in a cohort of patients with ELANE neutropenia. Methods : Available data from the patients, with ELANE class 4 and 5 variants, enrolled in the French Severe Chronic Neutropenia Registry, were analysed. The final diagnostic of CyN and SCN was performed considering all the follow up period (median 16.7 years) and based on the presence of recurrent periodic variation of ANC in the absence of GCSF therapy. CyN was defined as multiple documented ANCs >500 cells/mm 3 , with intermittent ANC variation (n=49), while SCN is defined as ANC persistently <500 cells/mm 3 (n=94). In case of irregularity (i.e. not a regular periodic pattern during all the follow up), the classification takes in consideration the majority of the follow up. A comprehensive analysis of the infectious profile is available elsewhere 2. We were focused here on the diagnostic period (roughly the 2 first months since the diagnosis). We have analysed the initial blood count of the patients and cast the patients by categories if at least 4 ANCs can be evaluated. ANC oscillations defined 4 groups: Group 1: oscillation of ANC values above and below 500 ANC/mm 3 for at least 2 cycles lead to consider the patient as Cyclic; Group 2: clear oscillation of ANC values above and below 200 ANC/mm 3 (but ever<500 ANC/mm 3) for at least 2 cycles; Group 3: no oscillation of ANC values whose level are ever below 500 ANC/mm 3; Group 4: Early GCSF treatment. Results : Among the 143 patients enrolled in this study (Table 1), 137 have at least 4 CBC evaluable during the diagnosis period, including 30 who have been treated almost front line after diagnosis of neutropenia by GCSF hampering evaluation of periodicity. Such patients were all initially considered as SCN. Among the 67 finally classified as SCN, 28 (27.38%) showed an oscillation pattern (group 1), 14 (15.48%) showed minor oscillations (group 2), while 25 (30.95%) had a persistent and severe neutropenia (group 3). Among the 40 CyN, 1 have showed minor oscillations (group 2) below 500/mm 3, while 3 had a persistent and severe neutropenia (group 3). Globally, 32 /107 patients were miss- classified at diagnosis compare to the final diagnosis. Additional data shows that many health indicators could not be deducted from the initial classification like the infections rate, the use of GCSF, the death rate, the sequels rate. Conclusions: Periodic variation of ANC despite being the criteria to define the sub type of ELANE neutropenia is difficult to evaluate at the initial presentation of the disease. In addition, cyclicity is not a permanent feature in ELANE neutropenia, some patients being cyclic only for a certain time in their life span. It results a high rate of miss classification if we compare the initial diagnostic period and all the medical history of the patients. Noteworthy, the rate of several severe complications is not so clearly different between diagnosis sub categories. We propose to consider ELANE neutropenia as a unique disease characterized by a clinical spectrum ranging from more severe forms (corresponding to SCN) to milder forms, the latter often characterized at onset by ANC fluctuations. In addition, some intermediate severity forms could be characterized by minor oscillations. References 1 Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat.Genet. 1999;23:433-436. 2 Rotulo GA, Plat G, Beaupain B et al. Recurrent bacterial infections, but not fungal infections, characterise patients with ELANE-related neutropenia: a French Severe Chronic Neutropenia Registry study. Br J Haematol. 2021 doi: 10.1111/bjh.17695 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Perturbed NK Cell Homeostasis Associated with Disease Severity in Chronic Neutropenia

Neutrophils have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow- and mass cytometry to examine NK cell repertoires in 64 patients with neutropenia and 27 healthy age- and gender-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK cell homeostasis manifested as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK cell repertoires were characterized by expression of proliferation/exhaustion markers Ki-67, Tim-3 and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67 and TOP2A, associated with apoptosis and the cell cycle, different from conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggests that neutrophils are dispensable for NK cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.

ELANE neutropenia and solid tumors: 4 cases from the French severe chronic neutropenia registry

Neutropenia related to ELANE gene mutations predisposes to infection and leukemia/ myelodysplasia, but little is known about the predisposition to cancer. Among a cohort of 149 patients, we identified four with malignant solid tumors (papillary thyroid cancer, anal squamous cell cancer, papillary renal cell carcinoma, and adrenocortical carcinoma), all after the age of 25 years. Three occurred in cyclic neutropenia while 1 occurred in severe chronic neutropenia (among 49 and 100 patients, respectively). A previous radiotherapy was identified as risk factor in one patient. Moreover, among 18 other patients that underwent hematopoietic stem-cell transplantations, none developed a cancer.