Clinical and functional spectrum of RAC2-related immunodeficiency.

ABSTRACT: Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Unusual Presentation of SET::NUP214-Associated Concomitant Hematological Neoplasm in a Child-Diagnostic and Treatment Struggle.

Simultaneous multilineage hematologic malignancies are uncommon and associated with poorer prognosis than single-lineage leukemia or lymphoma. Here, we describe a concomitant malignant neoplasm in a 4-year-old boy. The child presented with massive lymphoproliferative syndrome, nasal breathing difficulties, and snoring. Morphological, immunocytochemical, and flow cytometry diagnostics showed coexistence of acute myeloid leukemia (AML) and peripheral T-cell lymphoma (PTCL). Molecular examination revealed a rare t(9;9)(q34;q34)/SET::NUP214 translocation as well as common TCR clonal rearrangements in both the bone marrow and lymph nodes. The disease showed primary refractoriness to both lymphoid and myeloid high-dose chemotherapy as well as combined targeted therapy (trametinib + ruxolitinib). Hence, HSCT was performed, and the patient has since been in complete remission for over a year. This observation highlights the importance of molecular techniques for determining the united nature of complex SET::NUP214-positive malignant neoplasms arising from precursor cells with high lineage plasticity.

Comparison of disease phenotypes and mechanistic insight on causal variants in patients with DADA2.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) results in heterogeneous manifestations including systemic vasculitis and red cell aplasia. The basis of different disease phenotypes remains incompletely defined. OBJECTIVE: We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. METHODS: We analyzed the clinical features and ADA2 variants in 33 patients with DADA2. We compared the transcriptomic profile of 14 patients by bulk RNA sequencing. ADA2 variants were expressed experimentally to determine impact on protein production, trafficking, release, and enzymatic function. RESULTS: Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. CONCLUSIONS: Transcriptomic signatures of inflammation are observed in patients with different disease phenotypes, including some asymptomatic individuals. Disease-associated ADA2 variants affect protein function by multiple mechanisms, which may contribute to the clinical heterogeneity of DADA2.

Vemurafenib combined with cladribine and cytarabine results in durable remission of pediatric BRAF V600E-positive LCH.

Langerhans cell histiocytosis (LCH) is a disorder with a variety of clinical signs. The most severe forms affect risk organs (RO). The established role of the BRAF V600E mutation in LCH led to a targeted approach. However, targeted therapy cannot cure the disease, and cessation leads to quick relapses. Here, we combined cytosine-arabinoside (Ara-C) and 2'-chlorodeoxyadenosine (2-CdA) with targeted therapy to achieve stable remission. Nineteen children were enrolled in the study: 13 were RO-positive (RO+) and 6 RO-negative (RO-). Five patients received the therapy upfront, whereas the other 14 received it as a second or third line. The protocol starts with 28 days of vemurafenib (20 mg/kg), which is followed by 3 courses of Ara-C and 2-CdA (100 mg/m2 every 12 h, 6 mg/m2 per day, days 1-5) with concomitant vemurafenib therapy. After that, vemurafenib therapy was stopped, and 3 courses of mono 2-CdA followed. All patients rapidly responded to vemurafenib: the median disease activity score decreased from 13 to 2 points in the RO+ group and from 4.5 to 0 points in the RO- group on day 28. All patients except 1 received complete protocol treatment, and 15 of them did not have disease progression. The 2-year reactivation/progression-free survival (RFS) for RO+ was 76.9% with a median follow-up of 21 months and 83.3% with a median follow-up of 29 months for RO-. Overall survival is 100%. Importantly, 1 patient experienced secondary myelodysplastic syndrome after 14 months from vemurafenib cessation. Our study demonstrates that combined vemurafenib plus 2-CdA and Ara-C is effective in a cohort of children with LCH, and the toxicity is manageable. This trial is registered at www.clinicaltrials.gov as NCT03585686.

Novel SLFN14 mutation associated with macrothrombocytopenia in a patient with severe haemorrhagic syndrome.

BACKGROUND: Platelet-type bleeding disorder 20 (BDPLT20), as known as SLFN14-related thrombocytopenia, is a rare inherited thrombocytopenia (IT). Previously, only 5 heterozygous missense mutations in the SLFN14 gene have been reported. METHODS: A comprehensive clinical and laboratory examination of a 17-year-old female patient with macrothrombocytopenia and severe mucocutaneous bleeding was performed. Examination was carried out using standardized questionnaires to assess bleeding, high-throughput sequencing (Next Generation Sequencing), optical and fluorescence microscopy, flow cytometry with activation and analysis of intracellular calcium signaling of platelets, light transmission aggregometry and thrombus growth in the flow chamber. RESULTS: Analysis of the patient's genotype revealed a previously undescribed c.655 A > G (p.K219E) variant in the hotspot of the SLFN14 gene. Immunofluorescence and brightfield examination of platelets in the smear showed heterogeneity in cells size, including giant forms over 10 µm (normal size 1-5) in diameter, with vacuolization and diffuse distribution of ß1-tubulin and CD63. Activated platelets showed impaired contraction and shedding/internalization of GPIb. GP IIb/IIIa clustering was increased at rest and attenuated upon activation. Intracellular signalling study revealed impaired calcium mobilization upon TRAP 35.97 nM (reference range 180 ± 44) and CRP-XL 10.08 nM (56 ± 30) stimulation. Aggregation with ADP, collagen, TRAP, arachidonic acid and epinephrine was impaired in light transmission aggregometry; agglutination with ristocetin persisted. In the flow chamber with a shear rate of 400 s-1 platelet adhesion to collagen and clot growth were impaired. CONCLUSION: The revealed disorders of phenotype, cytoskeleton and intracellular signaling explain the nature of SLFN14 platelet dysfunction and the patient's severe hemorrhagic syndrome.

Efficacy of combined immunosuppression with or without eltrombopag in children with newly diagnosed aplastic anemia.

We compared the efficacy and safety of eltrombopag (ELTR) combined with immunosuppressive therapy (IST) and IST alone in treatment-naïve children with severe (SAA) and very severe (vSAA) aplastic anemia. Ninety-eight pediatric patients were randomized to receive horse antithymocyte globulin (hATG) and cyclosporin A (CsA) with (n = 49) or without (n = 49) ELTR. The primary endpoint was the overall response rate (ORR) at 4 months. After 4 months, nonresponders were crossed over to the alternative group. In all patients, the ORR in ELTR + IST and IST groups was similar (65% vs 53%; P = .218); however, the complete response (CR) rate was significantly higher in the ELTR + IST group (31% vs 12%; P = .027). In severity subgroups, the ORR was 89% vs 57% (P = .028) in favor of IST + ELTR in SAA, but it did not differ in patients with vSAA (52% vs 50%; P = .902). At 6 months after the crossover, 61% of initial ELTR(-) patients achieved a response compared with 17% of initial ELTR(+) patients (P = .016). No significant difference in ELTR + IST and IST groups was observed in the 3-year overall survival (OS) (89% vs 91%; P = .673) or the 3-year event-free survival (EFS) (53% vs 41%; P = .326). There was no unexpected toxicity related to ELTR. Adding ELTR to standard IST was well tolerated and increased the CR rate. The greatest benefit from ELTR combined with IST was observed in patients with SAA but not in those with vSAA. The second course of IST resulted in a high ORR in initial ELTR(-) patients who added ELTR and had limited efficacy among patients who received ELTR upfront. This trial was registered at Clinicaltrials.gov as #NCT03413306.

Cancer-causing MAP2K1 mutation in a mosaic patient with cardio-facio-cutaneous syndrome and immunodeficiency.

RASopathies are disorders caused by germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. These syndromes share features of developmental delay, facial dysmorphisms, and defects in various organs, as well as cancer predisposition. Somatic mutations of the same pathway are one of the primary causes of cancer. It is thought that germline cancer-causing mutations would be embryonic lethal, as a more severe phenotype was shown in Drosophila and zebrafish embryos with cancer MAP2K1 mutations than in those with RASopathy mutations. Here we report the case of a patient with RASopathy caused by a cancer-associated MAP2K1 p.Phe53Leu mutation. The postzygotic mosaic nature of this mutation could explain the patient's survival.

Single Center Experience With Pediatric Patients With GATA2 Deficiency.

GATA2 deficiency is one of the most common predisposing conditions for MDS in young individuals. It is characterized by autosomal dominant inheritance and a high rate of de novo mutations. Here we describe the clinical phenotype and hematological presentation of 10 pediatric patients with GATA2 deficiency presented to the Dmitry Rogachev Center between 2013 and 2020. All patients had been referred for neutropenia or suspected aplastic anemia. While some patients presented with an immunological phenotype, others displayed monosomy 7 and MDS. The clinical presentation with MDS in infancy and the constitutional phenotypes in our patients underline the great variability in clinical manifestation. Careful description of cohorts with GATA2 deficiency from different countries and genetic backgrounds will help to unravel the enormous heterogeneity of this recently discovered genetic disorder.

Vemurafenib provides a rapid and robust clinical response in pediatric Langerhans cell histiocytosis with the BRAF V600E mutation but does not eliminate low-level minimal residual disease per ddPCR using cell-free circulating DNA.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a disease that arises from myeloid cells that phenotypically resemble Langerhans cells (LC), which is typically driven by the BRAF V600E mutation. High-risk LCH has a poor prognosis. PROCEDURE: Fifteen children with BRAF V600E + LCH received vemurafenib between March 2016 and February 2020. The median age at LCH onset was 2 months and the median age at the start of vemurafenib treatment was 22 months. The median disease activity score (DAS) at the start of vemurafenib treatment was 12 points. RESULTS: The median duration of vemurafenib treatment was 29 months. All patients responded to treatment, with median DAS of 4 points at week 4 and 1 point at 6 months. Two patients died: 1 of hepatic failure after NSAID overdose and 1 of neutropenic sepsis. Cessation of vemurafenib resulted in relapse in 5 patients and was only possible for 1 patient. Serial measurements of BRAF V600E using cell-free circulating DNA revealed that 7 patients had persistently high mutant allele levels. CONCLUSION: Vemurafenib is effective in children with BRAF V600E + LCH. However, treatment with vemurafenib does not eradicate the disease and its long-term toxicity has not been established.

Unique Combination of Diamond-Blackfan Anemia and Lynch Syndrome in Adult Female: A Case Report.

We present an extremely rare clinical case of a 38-year-old Russian patient with multiple malignant neoplasms of the uterus and colon caused by genetically confirmed two hereditary diseases: Diamond-Blackfan anemia and Lynch syndrome. Molecular genetic research carried out by various methods (NGS, Sanger sequencing, aCGH, and MLPA) revealed a pathogenic nonsense variant in the MSH6 gene: NM_000179.2: c.742C>T, p.(Arg248Ter), as well as a new deletion of the chromosome 15's locus with the capture of 82,662,932-84,816,747 bp interval, including the complete sequence of the RPS17 gene. The lack of expediency of studying microsatellite instability in endometrial tumors using standard mononucleotide markers NR21, NR24, NR27, BAT25, BAT26 was demonstrated. The estimated prevalence of patients with combination of Diamond-Blackfan anemia and Lynch syndrome in the world is one per 480 million people.

Relative expansion of CD19-negative very-early normal B-cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR-T cell therapy: implications for flow cytometric detection of minimal residual disease.

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.

Chimerism evaluation in measurable residual disease-suspected cells isolated by flow cell sorting as a reliable tool for measurable residual disease verification in acute leukemia patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: The presence of minimal/measurable residual disease (MRD) before or after hematopoietic stem cell transplantation (HSCT) is known as a predictor of poor outcome in patients with acute myeloid (AML) or lymphoblastic (ALL) leukemia. When performed with multiparameter flow cytometry (MFC), assessment of residual leukemic cells after HSCT may be limited by therapy-induced shifts in the immunophenotype (e.g., loss of surface molecules used for therapeutic targeting). However, in such cases, questionable cells can be isolated and tested for hematopoietic chimerism to clarify their origin. METHODS: Questionable cell populations were detected during the MFC-based MRD monitoring of 52 follow-up bone marrow samples from 37 patients diagnosed with T cell neoplasms (n =14), B cell precursor ALL (n = 16), AML (n = 7). These cells (suspected leukemic or normal) were isolated by flow cell sorting and tested for hematopoietic chimerism by RTQ-PCR. RESULTS: The origin of cells was successfully identified in 96.15% of cases (n = 50), which helped to validate the results of MFC-based MRD monitoring. CONCLUSIONS: We believe that a combination of MFC, cell sorting, and chimerism testing may help confirm or disprove MRD presence in complicated cases after HSCT.

The Clinical and Genetic Spectrum of 82 Patients With RAG Deficiency Including a c.256_257delAA Founder Variant in Slavic Countries.

Background: Variants in recombination-activating genes (RAG) are common genetic causes of autosomal recessive forms of combined immunodeficiencies (CID) ranging from severe combined immunodeficiency (SCID), Omenn syndrome (OS), leaky SCID, and CID with granulomas and/or autoimmunity (CID-G/AI), and even milder presentation with antibody deficiency. Objective: We aim to estimate the incidence, clinical presentation, genetic variability, and treatment outcome with geographic distribution of patients with the RAG defects in populations inhabiting South, West, and East Slavic countries. Methods: Demographic, clinical, and laboratory data were collected from RAG-deficient patients of Slavic origin via chart review, retrospectively. Recombinase activity was determined in vitro by flow cytometry-based assay. Results: Based on the clinical and immunologic phenotype, our cohort of 82 patients from 68 families represented a wide spectrum of RAG deficiencies, including SCID (n = 20), OS (n = 37), and LS/CID (n = 25) phenotypes. Sixty-seven (81.7%) patients carried RAG1 and 15 patients (18.3%) carried RAG2 biallelic variants. We estimate that the minimal annual incidence of RAG deficiency in Slavic countries varies between 1 in 180,000 and 1 in 300,000 live births, and it may vary secondary to health care disparities in these regions. In our cohort, 70% (n = 47) of patients with RAG1 variants carried p.K86Vfs*33 (c.256_257delAA) allele, either in homozygous (n = 18, 27%) or in compound heterozygous (n = 29, 43%) form. The majority (77%) of patients with homozygous RAG1 p.K86Vfs*33 variant originated from Vistula watershed area in Central and Eastern Poland, and compound heterozygote cases were distributed among all Slavic countries except Bulgaria. Clinical and immunological presentation of homozygous RAG1 p.K86Vfs*33 cases was highly diverse (SCID, OS, and AS/CID) suggestive of strong influence of additional genetic and/or epigenetic factors in shaping the final phenotype. Conclusion: We propose that RAG1 p.K86Vfs*33 is a founder variant originating from the Vistula watershed region in Poland, which may explain a high proportion of homozygous cases from Central and Eastern Poland and the presence of the variant in all Slavs. Our studies in this cohort of RAG1 founder variants confirm that clinical and immunological phenotypes only partially depend on the underlying genetic defect. As access to HSCT is improving among RAG-deficient patients in Eastern Europe, we anticipate improvements in survival.

Risk Factors for and the Clinical Impact of Cytomegalovirus and Epstein-Barr Virus Infections in Pediatric Recipients of TCR-α/ß- and CD19-Depleted Grafts.

Alpha/beta T cell and CD19 depletion are used to improve the outcomes of hematopoietic stem cell transplantation (HSCT). We evaluated the burden of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in pediatric patients after this HSCT type. A cohort of 182 patients with malignant (n = 114) or nonmalignant (n = 68) disorders was transplanted from either matched unrelated (n = 124) or haploidentical (n = 58) donors. The cumulative incidence of CMV and EBV viremia were 51% and 33%, respectively. Acute graft-versus-host disease (GVHD) grades II to IV, D-/R+ serology, and malignant HSCT indications were associated with increased risk of CMV viremia. CMV disease developed in 10 patients (6%). The occurrence of CMV viremia was not associated with inferior outcomes. Acute GVHD grade ≥ II was the only factor significantly associated with an increased risk of EBV viremia. Rituximab significantly decreased the rate of EBV reactivation in a subgroup that received a higher B cell dose in the graft. The rate of EBV-associated disease was .5%, and EBV viremia did not affect survival. TCR-α/ß and CD19 depletion are associated with a significant rate of CMV viremia that does not affect survival. The hazard of EBV post-transplant lymphoproliferative disease (PTLD) is eliminated by the combination of CD19 depletion and rituximab.