Introduction

Adult chronic idiopathic neutropenia (CIN) is a rare and heterogenous condition with a broad differential diagnosis and several possible associations, such as autoimmune diseases, lymphoproliferative syndromes, and inborn errors of immunity1. The exclusion of such forms as well as of secondary causes of neutropenia (drugs, nutrients deficiencies, endocrinopathies, chronic infections etc.) is required, along with testing for anti-neutrophil autoantibodies despite a high heterogeneity in sensitivity and specificity2. The positivity of this test identifies the subset of autoimmune neutropenia (AIN), whose clinical behavior is almost indistinguishable from CIN3. Additionally, congenital neutropenia should be considered, particularly in young adults with suggestive personal and family history as well as in those with syndromic features2. While a body of literature, as well as recent guidelines, addressed the diagnosis and management of neutropenia in childhood and infancy, the adult setting is less explored. Previous studies showed a general benign course of adult CIN with limited infectious risk, although some patients might be re-classified as myelodysplastic/lymphoproliferative neoplasms during clinical course3. Specifically, large granular lymphocyte (LGL) expansion is typically associated with autoimmune cytopenias and CIN and may be difficult to distinguish from “true” LGL leukemia4. Similarly, bone marrow failure/dysplastic features and, more recently, somatic mutations of myeloid genes5 may be present in patients with CIN, challenging the differential diagnosis with myeloid neoplasms. Starting from the results of a previous analysis of 76 CIN patients3, we aimed at evaluating the natural history of CIN by expanding the number of patients and extending the follow up in a prospective analysis. We focused on the risk of infections and possible disease evolution and on bone marrow features, including immunohistochemistal assessment of the humoral response and complement activation, and mutational status.

Patients and methods

We prospectively collected clinical and laboratory data of patients diagnosed with CIN/AIN in a tertiary referral hospital for autoimmune cytopenias in Milan, Italy, and enrolled in the prospective observational CYTOPAN study (NCT05931718) since June 1st, 2021 until  the time of writing. The study was conducted according to the Declaration of Helsinki, approved by the local Ethical Committee Milano Area 2, and patients gave informed consent. Inclusion criteria were CIN/AIN diagnosis (revised according to most recent recommendations2) and adequate follow up of at least 6 months. Exclusion criteria were diagnosis of severe congenital neutropenia, inborn errors of immunity (patients were screened for Ig levels and lymphoid subpopulations) or of hematological malignancies at the time of neutropenia diagnosis, along with secondary causes (i.e. exposure to environmental or occupational agents, e.g. radiations or benzene; Epstein-Barr virus, cytomegalovirus, human acquired immunodeficiency virus infection, hepatitis B or C virus infection; hereditary cytopenias).

At enrolment complete medical history was collected, including associated conditions and blood counts at first neutropenia episode. Thereafter, full blood counts, serum electrophoresis, liver, kidney and thyroid function were tested at baseline, after 3 months, and then every 6 months. Anti-neutrophil antibodies were tested by direct granulocytes immuno-fluorescence test (GIFT) and indirect GIFT (GIIFT).

Bone marrow evaluation

Bone marrow evaluation included morphology, flow-cytometry, and cytogenetics on marrow aspirate, and trephine biopsy. Bone marrow biopsies were evaluated by an expert hemopathologist. Histologic parameters were first assessed on H/E, Giemsa and Gomori stains and supplemented with a panel of antibodies for the following antigens: CD8 (clone C8, Dako Agilent), C3 (polyclonal, Merck), C4d (polyclonal, Cell Marque), IgM (polyclonal, Dako Agilent), IgG (polyclonal, Dako Agilent), and cleaved-Caspase_3 (Asp175 clone, Cell Signaling). Cellularity was considered as reduced if below the normal range according to recent literature (up to 20 years old: range 45–85%; 20–40 years-old: 40–70%; 40–60 years old: 35–65%; ≥ 60 years old: 30–60%)6. C3 and C4d were assessed as surrogates of complement activation at the tissue level, while IgM and IgG as indicators of humoral immune activation. A 4-tiered, semi quantitative scoring was provided (0, lack of staining; 1, faint, mostly focal staining; 2, moderate intensity, mostly diffuse; 3, intense staining, diffuse). The involved compartments were recorded, as observed in a serous pattern (i.e., finely granular/diffuse staining present on the non-cellular component of the vascular lumina and sinusoids) or staining red blood cells and nucleated cells of the hematopoietic tissue. Cleaved-Caspase_3 was assessed as a marker of apoptosis. Phenotyping of LGL in blood was made by flow cytometry with the following antibodies: CD2/CD3/CD4/CD7/CD8/CD16/CD19/CD26/CD45/CD56/CD57. BM staining on trephines was performed with the following antibodies: CD34/CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD30/CD57/perforin. TCRG rearrangement was assessed in selected cases, either on bone marrow aspirate or biopsy, by Sanger sequencing according to BIOMED-2 criteria.

Next generation sequencing

Next generation sequencing assay (Ion Torrents5), Ion Reporter software 5.2 (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0028321-IonReporter5.20-UG-EN.pdf) was used to evaluate the mutational status of the following genes on peripheral blood leukocytes, as by the Oncomine Myeloid Research Assay: hotspot genes (ABL1, BRAF, CBL, CSFR3, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MOL, MYD88, NPM1, NRAS, PTPN11, SETPB1, SF3B1, SRSF2, U2AF1, WT1), full genes (ASXL1, BCOR, CALR, CEBPA, ETV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53, ZRSR2), fusion genes (ABL1, ALK, BCL2, BRAF, CCDN1, CREBBP, EGFR, ETV6, FGFR1, FGFR2, FUS, HMGA2, JAK2, KMT2A, MECOM, MET, MLLT10, MLLT3, MYBL1, MYH11, NTRK3, NUP214, PDGFRA, PDGFRB, RARA, RBM15, RUNX1, TCF3, TFE3).

Statistical analysis

Numerical variables were summarized by median and range; categorical variables were described with count and relative frequency (%) of subjects in each category. Comparison of numerical variables between groups was carried out using a non-parametric approach (Mann–Whitney test or Kruskall Wallis ANOVA). Comparison of the distribution of categorical variables in different groups was performed with either Fisher’s exact test (2 × 2 tables) or the χ test (larger tables).

Results

Baseline features

In total, 131 patients were included in the analysis (Table 1). Median age at diagnosis was 55 years (IQR 41–69), and the majority were females (77%). Median absolute neutrophil count (ANC) at diagnosis was 0.8 × 109/L (interquartile range, IQR, 0.5–1.14), with moderate neutropenia (< 1 × 109/L) in 39% and severe (< 0.5 × 109/L) in 24%. Anti-neutrophil autoantibodies were found in 57 of 102 tested patients (56%). Patients with anti-neutrophil autoantibodies showed similar clinical and laboratory features of negative patients (Supplementary Table 1). A minority of patients showed splenomegaly (8%) or enlarged lymph nodes (5%), and 25% had positive non-organ specific autoantibodies (anti-nucleus 24%, anti-extractable nuclear antigen 9%, and anti-double strand DNA autoantibodies in 1% of evaluable patients, respectively). A monoclonal gammopathy of uncertain significance (MGUS) was detected in 7% of patients, and 3 and 4% showed reduced levels of immunoglobulin A (IgA) and IgM, respectively. Forty patients (31%) had concurrent autoimmune diseases, namely “secondary autoimmune neutropenia”, most frequently autoimmune thyroiditis and Sjögren’s syndrome; these patients displayed a higher prevalence of ANA positivity (32% vs 8%, p = 0.003) and the same prevalence of anti-neutrophil autoantibodies positivity (52% vs 55% in primary autoimmune neutropenia) (Supplementary Table 2).

Table 1 Main features of the 131 adult neutropenia patients included in the study.
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Clinical course and infections

History of Grade > 1 infections before diagnosis was reported by 32 (26%) patients, nine of whom (7%) had a grade 3 episode (sepsis, pneumonia, cholangitis, peritonitis, cutaneous abscess). During a median prospective follow-up of 3 years, 37 subjects (28%) experienced 54 infectious events grade > 1, for a calculated incidence of 0.09 per person-year, with 13 (10%) experiencing a grade 3 event (viral meningitis, COVID-19, urinary infections, cholangitis, appendicitis, and fever of uncertain origin) (Table 2). Only 6 patients received granulocyte colonies stimulating factor (G-CSF), and only two in a chronic fashion. Only one of these two patients also received immunosuppressive therapy because of autoimmune neutropenia (see dedicated paragraph). Presence of an LGL infiltrate on BM at diagnosis was associated with a higher rate of grade ≥ 3 infection, although not significantly (21% vs 10%). Baseline ANC < 0.5 × 109/L and somatic mutations detected by NGS did not correlate with grade ≥ 3 infections. Regarding survival, only two elderly patients died during follow up due to myocardial infarction and colorectal cancer, whilst no evolution to hematological malignancy was noted.

Table 2 Infections during the follow up.
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Bone marrow studies

Bone marrow (BM) studies revealed reduced cellularity in 31% of patients (median cellularity 40%, IQR, 25–50%), reticulin fibrosis in 16%, and a mildly expanded, mostly CD8+ lymphoid infiltrate in 82%, failing short of diagnostic features of T-cell lymphoma. Interestingly, 53 of 101 (52%) evaluable patients had some degree of LGL expansion: 21 NK-cells, 15 T-cells, and 17 mixed T/NK phenotype; only five of them had clonal T cell receptor rearrangement. Patients with LGL infiltrate displayed higher age (60 years, 23–86 versus 52, 20–93; p = 0.02), and more frequently showed severe neutropenia (35% versus 17%, p = 0.03) as compared to those without LGL expansion (Supplementary Table 3). Chromosome aberrations were found in 3 patients only: deletion of 5q, 9 and Y, respectively. The evaluation of immune deposits on bone marrow trephine by immune-histochemistry was performed in 57 patients and showed IgM and IgG deposition in almost all samples. Semiquantitative scores for each patient are reported in Supplementary Table 4. IgM deposits were the most represented (score 3 in 21, 37%) and were mostly serous but deposition was also noted on myeloid precursors (21, 37%); IgG positivity was milder (score 3 in 5, 8%), although with the same pattern as for IgM (22, 38%); finally apoptotic activity by caspase evaluation was almost negative. C3 and C4d deposits were also observed in > 80% of samples, again with a prevailing serum pattern (Fig. 1). Deposits of C3 and C4 on precursors were noted in 6 and 11 samples, respectively; score > 2 for C3 and C4, were observed in 10 and 5 patients, respectively. Interestingly, patients with negative C3 deposition displayed significantly higher neutrophil levels at baseline (median 0.9 × 109/L, range 0.3–1.4, versus 0.5 × 109/L, 0.05–1.6, p < 0.01).

Fig. 1
figure 1

Immuno-histochemistry with anti-IgG, anti-IgM, and anti-C3 showed abundant deposits of Ig and complement within the bone marrow, with a prevalent serous pattern and some activation on precursor membranes.

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NGS analysis

Thirty-six patients underwent NGS analysis on peripheral blood leukocytes, that identified at least one somatic mutation in 7 (19%), including DNMT3A, TET2, IDH1, and ETV6-CHIC2. Variant allele frequency (VAF) ranged from 4.6 to 18.8% (Fig. 2A). Median age was significantly higher in patients with somatic mutations versus those without (68 years, 48–81 versus 51 years, 20–72, p = 0.002). Reduced BM cellularity did not correlate with presence of LGL infiltrate, NGS mutational status or anti-neutrophil antibodies positivity. As shown in Fig. 2B, CIN is marked by an interplay among autoimmune features, autoantibody positivity, T-LGL infiltrate, and clonal hematopoiesis.

Fig. 2
figure 2

(A) Results of next generation sequencing (NGS) evaluation on 36 patients. The histogram columns show the frequency of mutations (left vertical axis), while median VAF is reported by dark blue dots (right vertical axis). (B) Patient disposition according to laboratory associations (LGL infiltrate, anti-neutrophil autoantibodies and mutations as by NGS).

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Two peculiar clinical cases

A 59-year-old Caucasian female was referred  by her rheumatologist in August 2022 due to persistent severe isolated neutropenia (0.15 × 109/L) with recurrent hospital admissions (3 in 1 year) due to fever of unknown origin and pneumonia. Physical examination and CT scan showed no features of connective tissues diseases, nor lymphoproliferation. BM evaluation displayed reduced cellularity (20%), granulocytic dysplasia < 10%, absence of fibrosis, and a 22% lymphoid infiltrate with LGL features and polyclonal T-cell receptor. Marked IgM, IgG, and C3 and C4 deposits were noted by immune histochemistry. NGS myeloid panel was negative. By analyzing clinical history, a 5 year-long steroid treatment and 6-month cyclosporine therapy were noted, given to the purpose of “rising neutrophil counts”. Interestingly, infectious episodes had all occurred after immunosuppressive therapy start. Thereafter, steroid treatment was progressively tapered and then reinstituted at lower doses (prednisone 2.5 mg day) due to adrenal insufficiency syndrome. G-CSF thrice a week was started with response, along with cotrimoxazole and acyclovir prophylaxis, to prevent recurrent infections, and the patient is now infection-free in the past 12 months.

A 42-year-old Hispanic male followed by rheumatologists for rheumatoid arthritis treated with hydroxychloroquine and low dose methotrexate was admitted to the Hematology ward in January 2023 due to severe sepsis by methicillin sensitive Staphylococcus aureus and severe neutropenia. CT scan was unremarkable, and BM trephine displayed reduced age-corrected cellularity (30%) and a 12% T-cell LGL-like infiltrate, oligoclonal at TCR analysis. Methotrexate was stopped, and the patient was treated with intravenous antibiotics and G-CSF pulses with sepsis remission. At discharge, severe chronic neutropenia persisted, and the patient was put on clinical follow-up and referred to the hepatologist due to liver enzyme elevation that turned out to be of metabolic origin. One year later, he was readmitted to the Internal medicine ward due to fungal pneumonia and treated with anti-fungal and G-CSF with response. At discharge, chronic G-CSF stimulation thrice a week was established, and the patient is well with himself with normal neutrophil counts at last follow up.

Discussion

We conducted a prospective analysis of 131 patients with CIN diagnosed at a referral center according to recent guidelines2.

Despite a median neutrophil count below 1 × 109/L, only 1/3 of subjects experienced a grade > 1 infection, and 10% of patients only a grade 3 one, thus confirming the generally benign behavior of CIN. Such relatively low rate did not correlate with severity of neutropenia, nor with the positivity of anti-neutrophil antibodies, as shown in previous studies3,5. On the other hand, the severity of infections was associated with the presence of an LGL infiltrate, as highlighted by the two peculiar cases. The latter had experienced frequent grade > 3 infections with need of hospitalization, wide spectrum antibiotics and chronic G-CSF; both did not meet LGL leukemia criteria and did not respond to cyclosporine. Notably, one of the two started steroids for “autoimmune neutropenia” with worsening of infectious tendency, which on the contrary improved after steroids discontinuation. Our data and those from the literature3,7 suggest avoiding steroids and cytotoxic immunosuppressants in CIN patients, unless a clear diagnosis of associated autoimmune/lymphoproliferative diagnosis is established, since efficacy is limited, and toxicity may be high.

Whether CIN patients should receive continuous G-CSF treatment is a matter of debate. In our cohort, only 6 patients required G-CSF, mainly during severe infections (G3 or greater) not responding to wide spectrum antibiotics. Therefore, we advise G-CSF therapy in this setting, whilst chronic use may be considered for those with severe and recurrent episodes only. This policy in adulthood differs from that of severe congenital neutropenia, in which chronic G-CSF is advised due to the severity and frequency of infectious episodes8. Cotrimoxazole and acyclovir prophylaxis are also debated, and patient education to avoid potential infective sources as well as to seek medical attention remains pivotal.

In this study we extensively evaluated bone marrow features in adult CIN. We observed reduced bone marrow cellularity in 31% of patients, differently from what reported for other peripheral immune cytopenias (autoimmune thrombocytopenia and hemolytic anemia)9,10. This finding, in line with others5, may suggest a prevailing immune mediated attack against bone marrow precursors in CIN, similar to bone marrow failure syndromes. Consistently, a relative increase of T-lymphocytes was noted in most patients, with LGL features in 52%, mostly polyclonal and of “reactive” nature, highlighting the contribution of cell-mediated autoimmunity to disease pathogenesis. Notably, a higher, though not statistically significant, rate of grade ≥ 3 infection was observed in patients with LGL infiltrate, pinpointing a possible clinical impact. The “central” immune attack to bone marrow precursors may involve autoantibodies and complement activation, as highlighted by a significant deposition of IgG, IgM and complement fractions within the bone marrow trephines of CIN patients in our study. Interestingly, C3 positivity was associated with lower neutrophil counts as compared to negative patients, suggesting that this immune attack might in fact be pathogenic. While this is the first demonstration of Ig and complement deposits in CIN/AIN bone marrow trephines, a comparison with other cytopenic conditions will require further investigation in a dedicated study.

Anti-neutrophil autoantibodies were detected in 56% of tested patients, identifying the subset of autoimmune neutropenia. However, positive patients had no peculiar clinical nor laboratory features as compared to negative ones. In any case, current recommendations suggest testing anti-neutrophil autoantibodies in patients with less than 1 × 109/L and to re-test several times negative patients2. In the same population, in keeping with our data, recent recommendations also suggest a proper bone marrow evaluation to inform the differential diagnosis2.

As regards NGS findings, we found a 19% prevalence of somatic mutations in genes associated to myeloid malignancies, with a risk of selection bias, since only 36 patients underwent testing. If confirmed by further studies, such finding may suggest that chronic autoimmune attack against marrow precursors might represent a bottleneck for the selection of clonal hematopoiesis. Given the concomitant presence of mutations and cytopenia, these subjects may be referred as having “clonal cytopenia of unknown significance” (CCUS), whose reported 10-year cumulative risk of malignant transformation compared with isolated cytopenia without mutations is estimated as high as 95% in recent reports11. Nonetheless, none of our patients has progressed to myeloid neoplasm yet, questioning the clinical impact of NGS testing in this setting, despite the wide availability and use of myeloid panels across hematologic centers.

All in all, the positivity of autoimmune features against neutrophils and their precursors and the limited infection tendency observed during the study, as compared to the severe infections typical of congenital neutropenias, mainly confirm the immune mediated and acquired nature of the disease. While it is possible that some forms of predisposition, such as misdiagnosed inborn errors of immunity, might be present, the latter were investigated by Ig levels and lymphoid subpopulations assays in all patients, and molecular testing is not indicated in adults with isolated neutropenia as of today2.

This study has several limitations including the relatively limited number of patients, the heterogeneity in terms of age and comorbidities, and the availability of immune-histochemistry and molecular studies in a fraction of patients only.

In conclusion, our prospective experience on 131 CIN patients confirms a generally indolent behavior, with few infections, not associated with the severity of neutropenia nor with the presence of anti-neutrophil autoantibodies. The high prevalence of LGL infiltrates, associated with some infectious tendency, pinpoints the importance of bone marrow evaluation in adult CIN.