Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

NGS better discriminates true MRD positivity for the risk stratification of childhood ALL treated on an MRD-based protocol.

We compared minimal/measurable residual disease (MRD) levels evaluated by routinely used real-time quantitative polymerase chain reaction (qPCR) patient-specific assays and by next-generation sequencing (NGS) approach in 780 immunoglobulin (IG) and T-cell receptor (TR) markers in 432 children with B-cell precursor acute lymphoblastic leukemia treated on the AIEOP-BFM ALL 2009 protocol. Our aim was to compare the MRD-based risk stratification at the end of induction. The results were concordant in 639 of 780 (81.9%) of these markers; 37 of 780 (4.7%) markers were detected only by NGS. In 104 of 780 (13.3%) markers positive only by qPCR, a large fraction (23/104; 22.1%) was detected also by NGS, however, owing to the presence of identical IG/TR rearrangements in unrelated samples, we classified those as nonspecific/false-positive. Risk group stratification based on the MRD results by qPCR and NGS at the end of induction was concordant in 76% of the patients; 19% of the patients would be assigned to a lower risk group by NGS, largely owing to the elimination of false-positive qPCR results, and 5% of patients would be assigned to a higher risk group by NGS. NGS MRD is highly concordant with qPCR while providing more specific results and can be an alternative in the front line of MRD evaluation in forthcoming MRD-based protocols.

Molecular Screening in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma: Anaplastic Lymphoma Kinase Analysis, Next-Generation Sequencing Fusion Gene Detection, and T-Cell Receptor Immunoprofiling.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.

Functional studies associate novel DUOX2 gene variants detected in heterozygosity to Crohn's disease.

PURPOSE: Crohn's disease is a chronic gastrointestinal inflammatory disease with possible extraintestinal symptoms. There are predisposing genetic factors and even monogenic variants of the disorder. One of the possible genetic factors are variants of the DUOX2 gene. The protein product of the DUOX2 gene is a dual oxidase enzyme producing H2O2 in the bowel. Reduced H2O2 levels impact mucosal homeostasis and contribute to the development of inflammatory bowel disease. Thus far, only 19 patients with IBD with the DUOX2 variants have been described. METHODS: Here we present a case report of an adolescent female diagnosed at eleven years of age with IBD that was subsequently reclassified as Crohn's disease. She was treated with immunosuppressants and biological therapy but experienced additional complications. Her peripheral blood lymphocyte DNA was studied using massive parallel sequencing. Detected variants were functionally studied. RESULTS: Whole exome sequencing found two novel DUOX2 gene variants: a de novo variant c.3646C>T; p.R1216W and a maternally inherited variant c.3391G>A; p.A1131T which were initially classified as variants of unknown significance. However, follow-up functional studies demonstrated that both DUOX2 variants led to impaired H2O2 generation, which led to their reclassification to the likely pathogenic class according to the ACMG.net. Therefore, we conclude that these variants are causative for the disease. CONCLUSIONS: Identifying novel variants in patients with Crohn's disease and their families is important for precision medicine approaches and understanding of the pathogenesis of likely "monogenic" rare forms of inflammatory bowel disease.

Evans syndrome caused by a deleterious mutation affecting the adaptor protein SASH3.

Increasing evidence suggests multilineage cytopenias (also known as Evans syndrome) may be caused by inborn errors of immunity (IEI) with immune dysregulation. We studied a patient with autoimmune haemolytic anaemia and immune thrombocytopenia and identified a germline mutation in SASH3 (c.862C>T;p.Arg288Ter), indicating a recently identified IEI. Immunohistochemistry performed after clinically indicated splenectomy revealed severe hypoplasia/absence of germinal centres. The autoimmune phenotype was associated with an increased CD21low T-bet+ CD11c+ subset along with decreased regulatory T cells, impaired T-cell proliferation and T-cell exhaustion. The younger brother carries the same SASH3 mutation and shares immunophenotypic features but is currently clinical asymptomatic, indicating heterogeneity of SASH3 deficiency.

Early-onset pulmonary and cutaneous vasculitis driven by constitutively active SRC-family kinase HCK.

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

Therapeutic options for CTLA-4 insufficiency.

BACKGROUND: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness. OBJECTIVE: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level. METHODS: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated. RESULTS: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed. CONCLUSION: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency.

Complex genetic and histopathological study of 15 patient-derived xenografts of aggressive lymphomas.

Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

Mutual alteration of NOD2-associated Blau syndrome and IFNγR1 deficiency.

Blau syndrome (BS) is an auto-inflammatory granulomatous disease that possibly involves abnormal response to interferon gamma (IFNγ) due to exaggerated nucleotide-binding oligomerization domain containing 2 (NOD2) activity. Mendelian susceptibility to mycobacterial diseases (MSMD) is an infectious granulomatous disease that is caused by impaired production of or response to IFNγ. We report a mother and daughter who are both heterozygous for NOD2c.2264C˃T variant and dominant-negative IFNGR1818del4 mutation. The 17-year-old patient displayed an altered form of BS and milder form of MSMD, whereas the 44-year-old mother was completely asymptomatic. This experiment of nature supports the notion that IFNγ is an important driver of at least some BS manifestations and that elucidation of its involvement in the disease immunopathogenesis may identify novel therapeutic targets.

Utility of Ruxolitinib in a Child with Chronic Mucocutaneous Candidiasis Caused by a Novel STAT1 Gain-of-Function Mutation.

PURPOSE: Signal transducer and activator of transcription 1 gain-of-function (STAT1 GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC). We aim to report the effect of oral ruxolitinib, the Janus kinase (JAK) family tyrosine kinase inhibitor, on clinical and immune status of a 12-year-old boy with severe CMC due to a novel STAT1 GOF mutation. METHODS: Clinical features and laboratory data were analyzed, particularly lymphocyte subsets, ex vivo IFNγ- and IFNα-induced STAT1, 3, 5 phosphorylation dynamics during the course of JAK1/2 inhibition therapy, and Th17-related, STAT1- and STAT3-inducible gene expression before and during the treatment. Sanger sequencing was used to detect the STAT1 mutation. Literature review of ruxolitinib in treatment of CMC is appended. RESULTS: A novel STAT1 GOF mutation (c.617T > C; p.L206P), detected in a child with recalcitrant CMC, was shown to be reversible in vitro with ruxolitinib. Major clinical improvement was achieved after 8 weeks of ruxolitinib treatment, while sustained suppression of IFNγ- and IFNα-induced phosphorylation of STAT1, STAT3, and STAT5, as well as increased STAT3-inducible and Th17-related gene expression, was demonstrated ex vivo. Clinical relapse and spike of all monitored phosphorylated STAT activity was registered shortly after unplanned withdrawal, decreasing again after ruxolitinib reintroduction. No increase of peripheral CD4+ IL17+ T cells was detected after 4 months of therapy. No adverse effects were noted. CONCLUSION: JAK1/2 inhibition with ruxolitinib represents a viable option for treatment of refractory CMC, if HSCT is not considered. However, long-term administration is necessary, as the effect is not sustained after treatment discontinuation.

Revising pathogenesis of AP1S1-related MEDNIK syndrome: a missense variant in the AP1S1 gene as a causal genetic lesion.

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269 T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269 T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269 T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. KEY MESSAGES: A missense AP1S1 c.269 T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein.

Biologic and clinical features of childhood gamma delta T-ALL: identification of STAG2/LMO2 γδ T-ALL as an extremely high risk leukemia in the very young.

PURPOSE: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease. METHODS: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts. RESULTS: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping resulting in deregulation of gene expression associated with T-cell differentiation. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine. CONCLUSION: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL. SUPPORT: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

Quality Control for IG /TR Marker Identification and MRD Analysis.

Selection of the proper target is crucial for clinically relevant monitoring of minimal residual disease (MRD) in patients with acute lymphoblastic leukemia using the quantitation of clonal-specific immunoreceptor (immunoglobulin/T cell receptor) gene rearrangements. Consequently, correct interpretation of the results of the entire analysis is of utmost importance. Here we present an overview of the quality control measures that need to be implemented into the process of marker identification, selection, and subsequent quantitation of the MRD level.

Novel XIAP mutation causing enhanced spontaneous apoptosis and disturbed NOD2 signalling in a patient with atypical adult-onset Crohn's disease.

X-linked inhibitor of apoptosis (XIAP) is the most potent human inhibitor of apoptosis, and is also involved in NOD2-dependent NFκB and MAPK signalling cascade activation. The absence or defective function of XIAP leads to the development of a rare and severe primary immunodeficiency known as X-linked lymphoproliferative syndrome type 2 (XLP-2), which is characterized by a triad of clinical manifestations, including a high incidence of haemophagocytic lymphohistiocytosis (HLH), lymphoproliferation and inflammatory bowel disease (IBD), usually with very early onset. Here, we present a novel XIAP mutation identified in a patient with atypical adult-onset IBD complicated by relapsing HLH, splenomegaly and sarcoid-like disease. The c.266delA mutation in the XIAP gene creates a premature stop codon, and causes a severe reduction in XIAP protein expression. The mutation is also associated with impaired spontaneous and staurosporine- and PMA-induced apoptosis accompanied by significantly increased expression of pro-apoptotic genes. We also confirmed the negative impact of this particular XIAP mutation on NOD2-dependent NFκB and MAPK activation, while NOD2-independent activation was found to be unaffected. Moreover, we assume that the mutation has an impact on the overproduction of IL-12 and IFNγ, the shift towards the Th1 immune response and increased numbers of central memory and effector memory CD4+ and CD8+ T cells. All these changes contribute to immune dysregulation and the clinical manifestation of XLP-2.

Concurrent TP53 and CDKN2A Gene Aberrations in Newly Diagnosed Mantle Cell Lymphoma Correlate with Chemoresistance and Call for Innovative Upfront Therapy.

Mantle cell lymphoma (MCL) is a subtype of B-cell lymphoma with a large number of recurrent cytogenetic/molecular aberrations. Approximately 5-10% of patients do not respond to frontline immunochemotherapy. Despite many useful prognostic indexes, a reliable marker of chemoresistance is not available. We evaluated the prognostic impact of seven recurrent gene aberrations including tumor suppressor protein P53 (TP53) and cyclin dependent kinase inhibitor 2A (CDKN2A) in the cohort of 126 newly diagnosed consecutive MCL patients with bone marrow involvement ≥5% using fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS). In contrast to TP53, no pathologic mutations of CDKN2A were detected by NGS. CDKN2A deletions were found exclusively in the context of other gene aberrations suggesting it represents a later event (after translocation t(11;14) and aberrations of TP53, or ataxia telangiectasia mutated (ATM)). Concurrent deletion of CDKN2A and aberration of TP53 (deletion and/or mutation) represented the most significant predictor of short EFS (median 3 months) and OS (median 10 months). Concurrent aberration of TP53 and CDKN2A is a new, simple, and relevant index of chemoresistance in MCL. Patients with concurrent aberration of TP53 and CDKN2A should be offered innovative anti-lymphoma therapy and upfront consolidation with allogeneic stem cell transplantation.

Preclinical Evaluation of a Novel SHIP1 Phosphatase Activator for Inhibition of PI3K Signaling in Malignant B Cells.

PURPOSE: PI3K signaling is a common feature of B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL), and PI3K inhibitors have been introduced into the clinic. However, there remains a clear need to develop new strategies to target PI3K signaling. PI3K activity is countered by Src homology domain 2-containing inositol-5'-phosphatase 1 (SHIP1) and, here, we have characterized the activity of a novel SHIP1 activator, AQX-435, in preclinical models of B-cell malignancies. EXPERIMENTAL DESIGN: In vitro activity of AQX-435 was evaluated using primary CLL cells and DLBCL-derived cell lines. In vivo activity of AQX-435, alone or in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, was assessed using DLBCL cell line and patient-derived xenograft models. RESULTS: Pharmacologic activation of SHIP1 using AQX-435 was sufficient to inhibit anti-IgM-induced PI3K-mediated signaling, including induction of AKT phosphorylation and MYC expression, without effects on upstream SYK phosphorylation. AQX-435 also cooperated with the BTK inhibitor ibrutinib to enhance inhibition of anti-IgM-induced AKT phosphorylation. AQX-435 induced caspase-dependent apoptosis of CLL cells preferentially as compared with normal B cells, and overcame in vitro survival-promoting effects of microenvironmental stimuli. Finally, AQX-435 reduced AKT phosphorylation and growth of DLBCL in vivo and cooperated with ibrutinib for tumor growth inhibition. CONCLUSIONS: Our results using AQX-435 demonstrate that SHIP1 activation may be an effective novel therapeutic strategy for treatment of B-cell neoplasms, alone or in combination with ibrutinib.

EuroFlow Standardized Approach to Diagnostic Immunopheneotyping of Severe PID in Newborns and Young Children.

The EuroFlow PID consortium developed a set of flow cytometry tests for evaluation of patients with suspicion of primary immunodeficiency (PID). In this technical report we evaluate the performance of the SCID-RTE tube that explores the presence of recent thymic emigrants (RTE) together with T-cell activation status and maturation stages and discuss its applicability in the context of the broader EuroFlow PID flow cytometry testing algorithm for diagnostic orientation of PID of the lymphoid system. We have analyzed peripheral blood cells of 26 patients diagnosed between birth and 2 years of age with a genetically defined primary immunodeficiency disorder: 15 severe combined immunodeficiency (SCID) patients had disease-causing mutations in RAG1 or RAG2 (n = 4, two of them presented with Omenn syndrome), IL2RG (n = 4, one of them with confirmed maternal engraftment), NHEJ1 (n = 1), CD3E (n = 1), ADA (n = 1), JAK3 (n = 3, two of them with maternal engraftment) and DCLRE1C (n = 1) and 11 other PID patients had diverse molecular defects [ZAP70 (n = 1), WAS (n = 2), PNP (n = 1), FOXP3 (n = 1), del22q11.2 (DiGeorge n = 4), CDC42 (n = 1) and FAS (n = 1)]. In addition, 44 healthy controls in the same age group were analyzed using the SCID-RTE tube in four EuroFlow laboratories using a standardized 8-color approach. RTE were defined as CD62L+CD45RO-HLA-DR-CD31+ and the activation status was assessed by the expression of HLA-DR+. Naïve CD8+ T-lymphocytes and naïve CD4+ T-lymphocytes were defined as CD62L+CD45RO-HLA-DR-. With the SCID-RTE tube, we identified patients with PID by low levels or absence of RTE in comparison to controls as well as low levels of naïve CD4+ and naïve CD8+ lymphocytes. These parameters yielded 100% sensitivity for SCID. All SCID patients had absence of RTE, including the patients with confirmed maternal engraftment or oligoclonally expanded T-cells characteristic for Omenn syndrome. Another dominant finding was the increased numbers of activated CD4+HLA-DR+ and CD8+HLA-DR+ lymphocytes. Therefore, the EuroFlow SCID-RTE tube together with the previously published PIDOT tube form a sensitive and complete cytometric diagnostic test suitable for patients suspected of severe PID (SCID or CID) as well as for children identified via newborn screening programs for SCID with low or absent T-cell receptor excision circles (TRECs).

Screening of monogenic autoimmune diabetes among children with type 1 diabetes and multiple autoimmune diseases: is it worth doing?

Background Paediatric type 1 diabetes (T1D) and rare syndromes of monogenic multi-organ autoimmunity share basic features such as full insulin dependency and the presence of circulating beta-cell autoantibodies. However, the aetiopathogenesis, natural course and treatment of these conditions differ; therefore, monogenic multi-organ autoimmunity requires early recognition. We aimed to search for these monogenic conditions among a large cohort of children with T1D. Methods Of 519 children with T1D followed-up in a single centre, 18 had multiple additional autoimmune conditions - either autoimmune thyroid disease (AITD) and coeliac disease (CD) or at least one additional organ-specific autoimmune condition in addition to AITD or CD. These 18 children were tested by direct Sanger sequencing (four patients with a suggestive phenotype of immune dysregulation, polyendocrinopathy, enteropathy, X-linked [IPEX] or signal transducer and activator of transcription 3 [STAT3]- and cytotoxic T-lymphocyte protein 4 [CTLA4]-associated syndromes) or by whole-exome sequencing (WES) focused on autoimmune regulator (AIRE), forkhead box protein 3 (FOXP3), CTLA4, STAT3, signal transducer and activator of transcription 1 (STAT1), lipopolysaccharide-responsive and beige-like anchor protein (LRBA) and interleukin-2 receptor subunit α (IL2RA) genes. In addition, we assessed their T1D genetic risk score (T1D-GRS). Results We identified novel variants in FOXP3, STAT3 and CTLA4 in four cases. All patients had a severe phenotype suggestive of a single gene defect. No variants were identified in the remaining 14 patients. T1D-GRS varied among the entire cohort; four patients had scores below the 25th centile including two genetically confirmed cases. Conclusions A monogenic cause of autoimmune diabetes was confirmed only in four patients. Genetic screening for monogenic autoimmunity in children with a milder phenotype and a combination of AITD and CD is unlikely to identify a monogenic cause. In addition, the T1D-GRS varied among individual T1D patients.