Infection risk in patients with autoimmune cytopenias and immune dysregulation treated with mycophenolate mofetil and sirolimus.

Introduction: Autoimmune cytopenias (AICs) are a group of disorders characterized by immune-mediated destruction of blood cells. In children, they are often secondary to immune dysregulation that may require long-lasting immunosuppression. Mycophenolate mofetil and sirolimus represent two well-tolerated options to treat these disorders, often as a steroid-sparing option. However, no data are available on the infection risk for patients undergoing long-lasting treatments. Patients and methods: The rate of severe infective events was calculated in episodes per 100 persons/months at risk (p/m/r) documented by the analysis of hospitalization charts between January 2015 and July 2023 of patients treated with mycophenolate mofetil or sirolimus given for isolated AIC or AICs associated with autoimmune lymphoproliferative syndrome (ALPS)/ALPS-like syndromes in two large Italian pediatric hematology units. Results: From January 2015 to July 2023, 13 out of 96 patients treated with mycophenolate mofetil or sirolimus developed 16 severe infectious events requiring hospitalization. No patients died. Overall infection rate was 0.24 person/*100 months/risk (95% CI 0.09-0.3). Serious infectious events incidence was higher in patients with ALPS-like compared to others (0.42 versus 0.09; p = 0.006) and lower in patients who underwent mycophenolate treatment alone compared to those who started sirolimus after mycophenolate failure (0.04 versus 0.29, p = 0.03). Considering only patients who started treatment at the beginning of study period, overall cumulative hazard was 18.6% at 60 months (95% CI 3.4-31.4) with higher risk of infectious events after 5 years in ALPS-like patients (26.1%; 95% CI 3.2-43.5) compared to other AICs (4%; 95% CI 0-11.4; p = 0.041). Discussion: To the best of our knowledge, this is the first study to describe the infectious risk related to mycophenolate and sirolimus chronic treatment in patients with AICs and immune dysregulation. Our data highlight that infection rate is very low and mainly related to the underlying hematological condition. Conclusions: Mycophenolate and sirolimus represent a safe immunosuppressive therapy in AICs and immune dysregulation syndromes.

Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene.

Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.

Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte homeostasis classically due to mutation of FAS, FASL, and CASP10 genes (ALPS-FAS/CASP10). Despite recent progress, about one-third of ALPS patients does not carry classical mutations and still remains gene orphan (ALPS-U, undetermined genetic defects). The aims of the present study were to compare the clinical and immunological features of ALPS-FAS/CASP10 versus those of ALPS-U affected subjects and to deepen the genetic characteristics of this latter group. Demographical, anamnestic, biochemical data were retrieved from medical record of 46 ALPS subjects. An enlarged panel of genes (next-generation sequencing) was applied to the ALPS-U group. ALPS-U subjects showed a more complex phenotype if compared to ALPS-FAS/CASP10 group, characterized by multiorgan involvement (P = 0.001) and positivity of autoimmune markers (P = 0.02). Multilineage cytopenia was present in both groups without differences with the exception of lymphocytopenia and autoimmune neutropenia that were more frequent in ALPS-U than in the ALPS-FAS/CASP10 group (P = 0.01 and P = 0.04). First- and second-line treatments were able to control the symptoms in 100% of the ALPS-FAS/CASP10 patients, while 63% of ALPS-U needed >2 lines of treatment and remission in some cases was obtained only after target therapy. In the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic and 8 of 19 (42%) as likely pathogenic. A characteristic flow cytometry panel including CD3CD4-CD8-+TCRαß+, CD3+CD25+/CD3HLADR+, TCR αß+ B220+, and CD19+CD27+ identified the ALPS-FAS/CASP10 group. ALPS-U seems to represent a distinct entity from ALPS-FAS/CASP10; this is relevant for management and tailored treatments whenever available.

Autoimmune Neutropenia and Immune-Dysregulation in a Patient Carrying a TINF2 Variant.

In recent years, the knowledge about the immune-mediated impairment of bone marrow precursors in immune-dysregulation and autoimmune disorders has increased. In addition, immune-dysregulation, secondary to marrow failure, has been reported as being, in some cases, the most evident and early sign of the disease and making the diagnosis of both groups of disorders challenging. Dyskeratosis congenita is a disorder characterized by premature telomere erosion, typically showing marrow failure, nail dystrophy and leukoplakia, although incomplete genetic penetrance and phenotypes with immune-dysregulation features have been described. We report on a previously healthy 17-year-old girl, with a cousin successfully treated for acute lymphoblastic leukemia, who presented with leukopenia and neutropenia. The diagnostic work-up showed positive anti-neutrophil antibodies, leading to the diagnosis of autoimmune neutropenia, a slightly low NK count and high TCR-αß+-double-negative T-cells. A next-generation sequencing (NGS) analysis showed the 734C>A variant on exon 6 of the TINF2 gene, leading to the p.Ser245Tyr. The telomere length was short on the lymphocytes and granulocytes, suggesting the diagnosis of an atypical telomeropathy showing with immune-dysregulation. This case underlines the importance of an accurate diagnostic work-up of patients with immune-dysregulation, who should undergo NGS or whole exome sequencing to identify specific disorders that deserve targeted follow-up and treatment.

Mutational analysis of ribosomal proteins in a cohort of pediatric patients with T-cell acute lymphoblastic leukemia reveals Q123R, a novel mutation in RPL10.

T-cell acute lymphoblastic leukemia (T-ALL) is a subtype of ALL involving the malignant expansion of T-cell progenitors. It is driven by a number of different possible genetic lesions, including mutations in genes encoding for ribosomal proteins (RPs). These are structural constituents of ribosomes, ubiquitous effectors of protein synthesis. Albeit the R98S mutation in RPL10, recurring with a higher frequency among RP mutations, has been extensively studied, less is known about the contribution of mutations occurring in other RPs. Alterations affecting translational machinery may not be well tolerated by cells, and there may be a selective pressure that determines the emergence of mutations with a compensatory effect. To explore this hypothesis, we sequenced the exomes of a cohort of 37 pediatric patients affected by T-ALL, and analyzed them to explore the co-occurrence of mutations in genes involved in ribosome biogenesis (including RPs) and translational control, and in known T-ALL driver genes. We found that some of the mutations in these sub-classes of genes tend to cluster together in different patients, indicating that their co-occurrence may confer some kind of advantage to leukemia cells. In addition, our sequencing highlighted the presence of a novel mutation in RPL10, namely the Q123R, which we found associated with a defect in protein synthesis. Our findings indicate that genetic alterations involving ribosome biogenesis and translational control should be carefully considered in the context of precision medicine in T-ALL.

Underlying Inborn Errors of Immunity in Patients With Evans Syndrome and Multilineage Cytopenias: A Single-Centre Analysis.

Background: Evans syndrome (ES) is a rare disorder classically defined as the simultaneous or sequential presence of autoimmune haemolytic anaemia and immune thrombocytopenia, but it has also been described as the presence of at least two autoimmune cytopenias. Recent reports have shown that ES is often a manifestation of an underlying inborn error of immunity (IEI) that can benefit from specific treatments. Aims: The aim of this study is to investigate the clinical and immunological characteristics and the underlying genetic background of a single-centre cohort of patients with ES. Methods: Data were obtained from a retrospective chart review of patients with a diagnosis of ES followed in our centre. Genetic studies were performed with NGS analysis of 315 genes related to both haematological and immunological disorders, in particular IEI. Results: Between 1985 and 2020, 40 patients (23 men, 17 women) with a median age at onset of 6 years (range 0-16) were studied. ES was concomitant and sequential in 18 (45%) and 22 (55%) patients, respectively. Nine of the 40 (8%) patients had a positive family history of autoimmunity. Other abnormal immunological features and signs of lymphoproliferation were present in 24/40 (60%) and 27/40 (67%) of cases, respectively. Seventeen out of 40 (42%) children fit the ALPS diagnostic criteria. The remaining 21 (42%) and 2 (5%) were classified as having an ALPS-like and an idiopathic disease, respectively. Eighteen patients (45%) were found to have an underlying genetic defect on genes FAS, CASP10, TNFSF13B, LRBA, CTLA4, STAT3, IKBGK, CARD11, ADA2, and LIG4. No significant differences were noted between patients with or without variant and between subjects with classical ES and the ones with other forms of multilineage cytopenias. Conclusions: This study shows that nearly half of patients with ES have a genetic background being in most cases secondary to IEI, and therefore, a molecular evaluation should be offered to all patients.

Targeted NGS Yields Plentiful Ultra-Rare Variants in Inborn Errors of Immunity Patients.

Inborn errors of immunity (IEI) include a large group of inherited diseases sharing either poor, dysregulated, or absent and/or acquired function in one or more components of the immune system. Next-generation sequencing (NGS) has driven a rapid increase in the recognition of such defects, though the wide heterogeneity of genetically diverse but phenotypically overlapping diseases has often prevented the molecular characterization of the most complex patients. Two hundred and seventy-two patients were submitted to three successive NGS-based gene panels composed of 58, 146, and 312 genes. Along with pathogenic and likely pathogenic causative gene variants, accounting for the corresponding disorders (37/272 patients, 13.6%), a number of either rare (probably) damaging variants in genes unrelated to patients' phenotype, variants of unknown significance (VUS) in genes consistent with their clinics, or apparently inconsistent benign, likely benign, or VUS variants were also detected. Finally, a remarkable amount of yet unreported variants of unknown significance were also found, often recurring in our dataset. The NGS approach demonstrated an expected IEI diagnostic rate. However, defining the appropriate list of genes for these panels may not be straightforward, and the application of unbiased approaches should be taken into consideration, especially when patients show atypical clinical pictures.

Genetic screening of children with marrow failure. The role of primary Immunodeficiencies.

The differential diagnosis of marrow failure (MF) is crucial in the diagnostic work-up, since genetic forms require specific care. We retrospectively studied all patients with single/multi-lineage MF evaluated in a single-center to identify the type and incidence of underlying molecular defects. The diepoxybutane test was used to screen Fanconi Anemia. Other congenital MFs have been searched using Sanger and/or Next Generation Sequencing analysis, depending on the available tools over the years. Between 2009-2019, 97 patients (aged 0-32 years-median 5) with single-lineage (29%) or multilineage (68%) MF were evaluated. Fifty-three (54%) and 28 (29%) were diagnosed with acquired and congenital MF, respectively. The remaining 16 (17%), with trilinear (n=9) and monolinear (n=7) MF, were found to have an underlying primary immunodeficiency (PID) and showed clinical and biochemical signs of immune-dysregulation in 10/16 (62%) and in 14/16 (87%) of cases, respectively. Clinical signs were also found in 22/53 (41%) and 8/28 (28%) patients with idiopathic and classical cMF, respectively. Eight out of 16 PIDs patients were successfully transplanted, four received immunosuppression, two did not require treatment, and the remaining two died. We show that patients with single/multi-lineage MF may have underlying PIDs in a considerable number of cases and that MF may represent a relevant clinical sign in patients with PIDs, thus widening their clinical phenotype. An accurate immunological work-up should be performed in all patients with MF, and PID-related genes should be considered when screening MF in order to identify disorders that may receive targeted treatments and/or appropriate conditioning regimens before transplant.

Late-onset and long-lasting autoimmune neutropenia: an analysis from the Italian Neutropenia Registry.

Primary autoimmune neutropenia (pAN) is typified by onset in early infancy and a mild/moderate phenotype that resolves within 3 years of diagnosis. In contrast, secondary AN is classically an adult disease associated with malignancy, autoimmunity, immunodeficiency, viral infection, or drugs. This study describes a cohort of 79 children from the Italian Registry who, although resembling pAN, did not fully match the criteria for pAN because neutropenia either appeared after age 5 years (LO-Np) or lasted longer than 3 years (LL-Np). These 2 categories compared with classical pAN showed a far inferior rate of resolution (P < .001), lower severity of neutropenia (P = .03), leukopenia (P < .001), lymphopenia (P < .001) with low B+ (P = .001), increased need of granulocyte colony-stimulating factor (P = .04), and increased frequency of autoimmunity over the disease course (P < .001). A paired comparison between LO-Np and LL-Np suggested that LO-Np had a lower rate of resolution (P < .001) and lower white blood cell (P < .001) and lymphocyte (P < .001) values, higher occurrence of apthae (P = .008), and a stronger association with autoimmune diseases/markers (P = .001) than LL-Np, thus suggesting a more pronounced autoimmune signature for LO-Np. A next-generation sequencing panel applied in a small subgroup of LO-Np and LL-Np patients identified variants related to immune dysregulations. Overall, these findings indicate that there are important differences among pAN LL-Np and LO-Np. Forms rising after 3 years of age, with low tendency to resolution, require tight monitoring and extensive immune investigations aimed to early identify underlying immunologic disease.

FAS-mediated apoptosis impairment in patients with ALPS/ALPS-like phenotype carrying variants on CASP10 gene.

Autoimmune lymphoproliferative syndrome (ALPS) is a congenital disorder that results in an apoptosis impairment of lymphocytes, leading to chronic lymphoproliferation and autoimmunity, mainly autoimmune cytopenias. FAS gene defects are often responsible for the disease, the phenotype of which can vary from asymptomatic/mild forms to severe disease. More rarely, defects are associated to  other genes involved in apoptosis pathway, such as CASP10. Few data are available on CASP10-mutated patients. To date, two CASP10 mutations have been recognized as pathogenic (I406L and L258F) and others have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l). In this study, we evaluated apoptosis function in patients with an ALPS/ALPS-like phenotype carrying CASP10 variants. Molecular findings were obtained by next generation sequencing analysis of genes involved in immune dysregulation syndromes. Functional studies were performed after inducing apoptosis by FAS-ligand/TRIAL stimulation and analysing cell death and the function of CASP10, CASP8 and PARP proteins. We identified 6 patients with an ALPS (n = 2) or ALPS-like (n = 4) phenotype, carrying I406L (n = 1),V410l (n = 2),Y446C (n = 1) heterozygous CASP10 variants or the L522l polymorphisms (n = 2) associated with another polymorphic homozygote variant on CASP8 or a compound heterozygous mutation on TNFRSF13C. Apoptosis was impaired in all patients showing that such variants may play a role in the development of clinical phenotype.

Clinical heterogeneity in a family with DKC1 mutation, dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome in first cousins.

Dyskeratosis congenita (DC) is an inherited bone marrow failure disorder characterized by mucocutaneous features (skin pigmentation, nail dystrophy and oral leukoplakia), pulmonary fibrosis, hematologic and solid malignancies. Its severe form, recognized as Hoyeraal-Hreidarsson syndrome (HHS), also includes cerebellar hypoplasia, microcephaly, developmental delay and prenatal growth retardation. In literature phenotypic variability among DC patients sharing the same mutation is wellknown. To our knowledge this report describes for the first time a family of DC patients, characterized by a member with features of classic DC and another one with some features of HHS, both with the same mutation in DKC1. Our family confirms again that one mutation can be associated with different phenotypes and different hematological manifestations. It's possible to speculate that there are likely to be patients who do not clinically fit neatly into either classical DC or HHS, but whose clinical features are due to mutations in DKC1 or in genes responsible for autosomal DC/HHS.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.

Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disease characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3/million inhabitants/year, in Europe, but higher in East Asia. Survival in severe aplastic anemia (SAA) has markedly improved in the past 2 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. In SAA hematopoietic stem cell transplant (HSCT) from a matched sibling donor (MSD) is the treatment of choice. If a MSD is not available, the options include immunosuppressive therapy (IST) or unrelated donor HSCT. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary, evidence-based document issued by a group of pediatric hematologists was discussed, modified and approved during a series of "Consensus Conferences" according to procedures previously validated by the AIEOP Board. The guidelines highlight the importance of referring pediatric patients with AA to pediatric centers with long experience in diagnosis, differential diagnosis, management, supportive care and follow-up of AA.

Etiology, clinical outcome, and laboratory features in children with neutropenia: analysis of 104 cases.

BACKGROUND: Neutropenia is not uncommon in childhood. The aim of our study was to analyze the underlying causes of neutropenia and to evaluate its clinical significance in a series of children referred to our center. METHODS: One hundred and four consecutive children with neutropenia were enrolled in this study. Clinical and laboratory features were analyzed. RESULTS: The majority of patients (63.5%) showed chronic neutropenia. Among all chronic forms, the most frequent was chronic idiopathic neutropenia (CIN), followed by autoimmune neutropenia (AIN). Congenital neutropenia was identified in 6 patients. Acute neutropenia was mainly due to infections. Overall, at the time of first detection, neutropenia was more frequently severe or moderate. One-third of our patients who presented with severe neutropenia were ultimately diagnosed with a post-infectious acute form. Conversely, nearly half patients with CIN, AIN, or congenital neutropenia showed moderate/mild neutropenia at onset. Among patients with AIN and CIN, nearly half recovered between 7 months and 46 months and approximately one-fourth experienced infectious episodes during follow-up. No significant difference was noticed in terms of mean ANC between patients with and without remission, neither between patients with and without infections. CONCLUSIONS: Our study confirms the great etiological heterogeneity of neutropenia in children. We could not demonstrate a correlation between ANC level at onset and the underlying disorder, nor a correlation between mean ANC and duration of neutropenia or infectious episodes during follow-up. Neutropenia remains a disease of concern to pediatricians, requiring several laboratory investigations, prolonged follow-up, and, in few cases, advanced molecular methods.

Poikiloderma with neutropenia: a case report and review of the literature.

Poikiloderma with neutropenia (PN, OMIM 604173) is a rare autosomal-recessive genodermatosis. Mutations in the C16orf57 gene have been recently identified as the cause. Here we describe a new case of PN in a white patient, review the literature, and point out the attention on importance of differential diagnosis.