Btk Gene Research Articles

Pleiotropic Action of Novel Bruton's Tyrosine Kinase Inhibitor BGB-3111 in Mantle Cell Lymphoma.

Bruton's tyrosine kinase (BTK) is a key mediator of BCR-dependent cell growth signaling and a clinically effective therapeutic target in mantle cell lymphoma (MCL). The molecular impact of BTK inhibition remains unclear particularly in hematopoietic malignancies. We analyzed the molecular mechanisms of BTK inhibition with the novel inhibitor BGB-3111 (zanubrutinib) in MCL models. The efficacy of BGB-3111 was investigated using growth proliferation/cell viability and apoptosis assays in MCL cell lines and patient-derived xenograft (PDX) MCL cells. The activity and mechanisms of BGB-3111 were further confirmed using a cell line xenograft model, an MCL PDX mouse model, and a human phosphokinase profiler array and reverse phase protein array. Finally, the mechanisms related to resistance to BTK inhibition were analyzed by creating cell lines with low levels of BTK using CRISPR/Cas 9 genome editing. We found that inhibition of BTK leads to suppression of tumor growth, which was mediated via potent suppression of AKT/mTOR, apoptosis, and metabolic stress. Moreover, targeted disruption of the BTK gene in MCL cells resulted in resistance to BTK inhibition and the emergence of novel survival mechanisms. Our studies suggest a general efficacy of BTK inhibition in MCL and potential drug resistance mechanism via alternative signaling pathways.

Vedolizumab treatment in a patient with X-linked agammaglobulinemia, is it safe and efficient?

Çekiç Ş, Özgür T, Karalı Y, Özkan T, Kılıç SŞ. Vedolizumab treatment in a patient with X-linked agammaglobulinemia, is it safe and efficient? Turk J Pediatr 2019; 61: 937-940. The loss of inflammatory regulation resulting from the absence of B-lymphocytes leads to a risk for autoimmune and autoinflammatory complications. There is no data on the use of Vedolizumab in patients with X-linked agammaglobulinemia (XLA) as well as children with another primary immunodeficiency (PID) diseases. A 4-year-old boy was admitted to our clinic with a history of recurrent respiratory tract infections. He was diagnosed with XLA based on extremely low immunoglobulins, very low level of B cells, genetic mutation of BTK gene, and family history. At the age of 8, he suffered from intermittent fever attacks, abdominal pain, weakness, oral aft, and weight loss. His clinical and laboratory features were consistent with inflammatory bowel disease. Histopathological examination of the biopsy material obtained from terminal ileum, colon and cecum showed Crohn`s disease. Initially, he was treated with prednisolone and infliximab. Because of the lack of response, infliximab treatment was switched to adalimumab. Terminal ileum was resected to relieve obstruction complication. Although he had been treated with adalimumab, a significant improvement was not observed. Vedolizumab (Entyvio™), a humanized monoclonal antibody α4β7 integrin-receptor antagonist, was commenced. After treatment with vedolizumab, his fever and abdominal pain attacks reduced, his total daily calorie intake increased and weight gain improved. He began to walk again and continued his school education properly. No side effects were observed in 18 months. This is the first immunocompromised child treated with vedolizumab. The symptoms of the patient receded and no side effect were seen during the treatment.

Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. DisclosuresZucca:Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

Validation of Clinical Prognostic Models and Integration of Genetic Biomarkers of Drug Resistance in CLL Patients Treated with Ibrutinib

Introduction:We previously reported a prognostic scoring system in CLL using pre-treatment factors in patients treated with ibrutinib [Ahn et al, 2016 ASH Annual Meeting]. Here we present long-term follow-up results and validation of the prognostic models in a large independent cohort of patients. We also determine the incidence of resistance-conferring mutations in BTK and PLCG2 genes in different clinical risk groups.Methods and Patients:The discovery cohort comprised 84 CLL patients on a phase II study with either TP53 aberration (deletion 17p or TP53 mutation) or age ≥65 years (NCT01500733). The validation cohort comprised 607 patients pooled from four phase II and III studies for ibrutinib in treatment-naïve or relapsed/refractory CLL (NCT01105247; NCT01578707; NCT01722487; NCT01744691). All patients received single-agent ibrutinib 420mg once daily. We used Cox regression models to identify independent predictors of PFS, Kaplan-Meier method to estimate probabilities of PFS, log-rank test to compare PFS, and Cochran-Armitage trend test to compare the incidence of mutation among subgroups. We used R version 3.5.0 or SAS® version 9.3 for statistical analyses. For biomarker correlation, we tested cellular DNA or cell-free DNA collected from patients in the discovery cohort with the targeted sequencing of BTK and PLCG2 genes.Result:At a median follow-up of 5.2 years, 28 (33.3%) of 84 patients in the discovery cohort progressed or died. 52 (61.9%) patients had treatment-naïve CLL. Independent factors of PFS on univariate analysis were; TP53 aberration, prior treatment, and β-2 microglobulin (B2M) >4mg/L (P<0.05 for all tests). Unmutated IGHV and advanced Rai stage (III/IV) showed a trend toward inferior outcome without reaching statistical significance. Because higher levels of B2M were associated with relapsed/refractory CLL, we performed two multivariate Cox regression models to assess B2M and prior treatment status separately. Risk groups were determined by the presence of TP53 aberration, advanced Rai stage, and B2M >4mg/L for Model 1, and TP53 aberration, advanced Rai stage, and relapsed/refractory CLL for Model 2 (Table 1). The high-risk group had all three adverse risk factors; the intermediate-risk group had two risk factors; and the low-risk group, none or one. The median PFS of the high-risk group was 38.9 months for Model 1 and 38.4 months for Model 2, and was significantly shorter than those of intermediate and low-risk groups.In the validation cohort, 254 (41.8%) of 607 patients progressed or died at a median follow-up of 4.2 years. 167 (27.5%) patients had treatment-naïve CLL. Both models showed statistically significant differences in PFS by risk groups (Table 1). For the high-risk group, 4-year PFS was 30.2% in Model 1 and 30.5% in Model 2, which were inferior to those of intermediate (53.4 and 52.4%) and low-risk groups (68.7 and 73.7%). Model 1 classified 20% of patients and Model 2 classified 28% of patients to the high-risk group.BTK and PLCG2 mutations are common genetic drivers of ibrutinib resistance in CLL. To determine whether the incidence of these mutations correlates with prognostic risk groups, we performed targeted sequencing of BTK and PLCG2 of samples collected from patients in the discovery cohort. We used cell-free DNA for patients who received long-term ibrutinib (≥3 years) and had low circulating tumor burden, and cellular DNA, for samples collected within 3 years on ibrutinib or at progression. Of 84 patients, 69 (82.1%) were tested at least once, and 37 (44.0%) were tested at least twice. The frequency of testing was similar across the risk groups by two models (P>0.05). The cumulative incidences of mutations at 5 years in the low-, intermediate-, and high-risk groups were: 21.4%, 44.8% and 50%, respectively, by Model 1 (P=0.02); and 22.6%, 41.4% and 66.7%, respectively, by Model 2 (P=0.01).Conclusion:We developed and validated prognostic models to predict the risk of disease progression or death in CLL patients treated with ibrutinib. Risk groups classified by three commonly available pre-treatment factors showed statistically significant differences in PFS. The clinically-defined high-risk disease was linked to higher propensity to develop clonal evolution with BTK and/or PLCG2 mutations, which heralded ibrutinib resistance. DisclosuresAlbitar:Neogenomics Laboratories: Employment. Ma:Neogenomics Laboratories: Employment. Ipe:Pharmacyclics, an AbbVie Company: Employment, Other: Travel; AbbVie: Equity Ownership. Tsao:Pharmacyclics LLC, an AbbVie Company: Employment. Cheng:Pharmacyclics LLC, an AbbVie Company: Employment. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Spatial Convergent Clonal Evolution Leading to Ibrutinib Resistance and Disease Progression in Chronic Lymphocytic Leukemia

Introduction: Bruton tyrosine kinase inhibitor ibrutinib has altered the therapeutic landscape of chronic lymphocytic leukemia (CLL) with remarkable responses in relapsed or refractory CLL. Despite mostly durable responses, approximately 20% of patients experience disease progression with, in many cases, BTK or phospholipase Cg2 (PLCG2) resistance mutations predating the clinical progression by up to 15 months. Longitudinal studies have shed some light on the temporal aspects of clonal evolution processes leading to ibrutinib resistance, however, the spatial heterogeneity within the various environmental niches has not been described in detail yet.Here, we report the case of a CLL patient in which we are able to document, for the first time, an example of ibrutinib driven spatial convergent evolution leading to disease progression. The patient developed ibrutinib resistance after 21 months of treatment with simultaneous lymphadenomegaly and lymphocytosis. Sanger sequencing revealed a canonical BTK p.C481S mutation in peripherial blood and a PLCG2 p.D993H mutation in the lymph node with the absence of the BTK p.C481S mutation present in the peripherial blood suggesting convergent evolution in terms of the BTK and PLCG2 variants with both commonly associated with ibrutinib resistance. The patient was subsequently treated with venetoclax but due to further progression despite of high-dose salvage chemotherapy and autologous stem cell transplantation the patient died.Methods: Serial samples at ten different timepoints during the disease course were analysed. Genomic DNA was isolated from peripheral blood mononuclear cells and native lymph node tissue. Circulating cell-free DNA (ccfDNA) was isolated from peripheral blood samples. The fraction of tumor cells was determined by flow cytometry. During the Sanger sequencing BTK exons 11, 15, 16, and PLCG2 exon 12, 19, 20, 24, 27 and 30 were examined. Targeted ultra-deep next-generation sequencing (NGS) analysis of the BTK and PLCG2 genes was performed on a MiSeq platform (Illumina). Abundances of the PLCG2 p.P993H and BTK p.C481S variants were quantified using custom assays on a QX200 droplet digital PCR system (ddPCR, BioRad).Results: To dissect this spatial heterogeneity, BTK and PLCG2 mutations were screened by NGS which confirmed the Sanger finding and in addition to the dominant PLCG2 p.D993H variant, identified the BTK p.C481S mutation as a minor clone in the lymph node, with the BTK p.C481S mutation remaining the exclusive for the peripheral blood. Next, we tested the presence of these mutations in the circulating ccfDNA with ddPCR. Notably, both mutations were identified in the ccfDNA with a VAF of 1%. The ddPCR analysis revealed that both variants were absent in the pre-ibrutinib peripheral blood sample, however their emergence predated the clinical progression by 15 months. We observed a gradual expansion of these variants in the sequential samples, demonstrating clonal selection under the selective pressure of ibrutinib. Of note, the ddPCR assay identified the PLCG2 p.D993H mutation as a minor clone (VAF: 0.2%), previously unseen in the peripheral blood by NGS at the time of ibrutinib relapse. Intriguingly, the two subclones displayed differential sensitivity to the venetoclax therapy. In the peripheral blood, we observed a reduction of the BTK p.C481S positive subclone accompanied by expansion of the subclone harbouring PLCG2 p.D993H which was previously dominant in the lymph node. With these dynamic changes in the subclonal architecture, the disease progressed and ultimately led to death of the patient.Conclusions: Clonal evolution is a major driving force of disease progression in CLL. Several studies have traced the effect of treatment on temporal evolutionary trajectories in the context of standard and targeted therapies and identified subclonal heterogeneity and active clonal selection. However, the spatial aspect of subclonal dynamics has not been appreciated until recently. Here, we presented a unique case of CLL, developing ibrutinib resistance via multiple routes simultaneously, with the observed spatial heterogeneity leading to progression and aggressive transformation of the disease. This case highlights the importance of genetic profiling of multiple affected sites as investigations restricted to peripheral blood may underestimate the repertoire of clinically relevant genetic alterations. DisclosuresNo relevant conflicts of interest to declare.

Molecular genetic test system for diagnosis of x-linked agammaglobulinemia

Aim. Primary immunodeficiencies are disorders in which immune system is missing or does not function normally due to genetic disorders. X-linked agammaglobulinemia (XLA) is a rare genetic disorder in which don’t generate of mature B cells, which manifests as a complete or near-complete lack of proteins called gamma globulins, including antibodies, in their bloodstream. The BTK protein is the key regulator of β-cell maturation, so mutations in the BTK gene lead to hereditary agammaglobulinemia. Detection of mutations of the BTK gene is important for confirmation of the diagnosis in patients with XLA. Methods. DNA and RNA extraction, RT-PCR, direct Sanger sequencing of PCR fragments. Results. An analysis of blood samples from patients with XLA using a proposed test system was performed. It has been shown that the proposed test system allows for the initial screening of mutations in the BTK gene. Conclusions. The test system for the detection of mutations in patients with X-linked agammaglobulinemia, based on using of reverse transcriptase PCR and direct sequencing, was developed and tested on clinical samples. This system is proposed for the initial screening of BTK gene mutations inUkraine. Keywords: X-linked agammaglobulinemia, XLA, BTK, mutation, RT-PCR, primary immunodeficiency.

Clinical and genetic profiles of patients with X-linked agammaglobulinemia from southeast Turkey: Novel mutations in BTK gene

BackgroundX-linked agammaglobulinemia (XLA) is characterized by absent or severely reduced B cells, low or undetectable immunoglobulin levels, and clinically by extracellular bacterial infections which mainly compromise the respiratory tract. We aimed to analyze the clinical, immunological and genetic characteristics of 22 male children with XLA. MethodsTwenty-two children with XLA from 12 unrelated families were enrolled in this study. Clinical and demographic features of patients, serum immunoglobulin levels, percentage of B cells and BTK gene mutations were reviewed retrospectively. ResultsWe identified 12 different mutations in 22 patients from 12 unrelated families. The most frequent type of mutation was premature stop codon (33.3%). Ten mutations had been reported previously including three missense mutations (c.1774T>C, c.1684C>T, c.83G>T), three premature stop codons (c.1558C>T, c.1573C>T, c.753G>A), two splice-site (c.683-1G>A, c.1567-12_1567-9delTTTG) and two small nucleotide deletions (c.902-904_delAAG, c.179_181delAGA). Two novel mutations of the BTK gene were also presented and included one splice-site mutation (c.391+1G>C) and one premature stop codon mutation (c.1243_1243delG). Six out of 12 mutations of the BTK gene were located in the SH1 domain, two in the PH domain, two in the SH3 domain and two in the SH2 domain. Three patients had a history of severe infection before diagnosis. We did not identify any correlation between severity of clinical symptoms and the genotype. ConclusionsOur results show that mutations in southeast Turkey could be different from those in the rest of the world and molecular genetic tests are an important tool for early confirmed diagnosis of XLA.

Pregnancy, child bearing and prevention of giving birth to the affected children in patients with primary immunodeficiency disease; a case-series

BackgroundPatients with primary immunodeficiency disease (PID) who survive to adulthood and willing to have a child mostly are worried whether their disease affects their fertility and/or pregnancy and also if their child would be predisposed to PID.Case presentationWe report the outcome of conception, pregnancy and their management in 9 families with definite diagnosis of PID. A chronic granulomatous disease subject with an uneventful pregnancy developed fungal sacral osteomyelitis few weeks after delivery. A pregnant common variable immunodeficiency disease (CVID) patient with idiopathic thrombocytopenia had platelet count dropped before delivery. A sever neutropenic mother who refused to get IFNγ delivered two healthy children. A CVID case intolerant to IVIg with eclampsia and PTE delivered a baby. Another CVID female gave birth to a baby without being on any treatment since she was not diagnosed with immunodeficiency disease at that time. A healthy girl was implanted via preimplantation gender selection in a family who owned a Wiskott Aldrich-affected son. A family who had two children with Ataxia Telangiectasia used donated oocyte for their 3rd child. Prenatal genetic diagnosis was used to screen the fetus for the impaired BTK and CVID genes detected in sibling and father respectively in 2 separate families.ConclusionPregnancy in PID patients is more complex than normal population. Because, not only it has the chance of being inherited by the offspring, but also there are some risks for the mother if she has any kind of immunity component defects. So consultation with a clinical geneticist is crucial to choose the best available approach. They also should be observed and followed by a clinical immunologist to take the best possible safe care.

Clinical and genetic features of the patients with X‐Linked agammaglobulinemia from Turkey: Single‐centre experience

X-linked agammaglobulinemia is a primary immunodeficiency disorder resulting from BTK gene mutations. There are many studies in the literature suggesting contradictory ideas about phenotype-genotype correlation. The aim of this study was to identify the mutations and clinical findings of patients with XLA in Turkey, to determine long-term complications related to the disease and to analyse the phenotype-genotype correlation. Thirty-two patients with XLA diagnosed between 1985 and 2016 in Pediatric Immunology Department of Hacettepe University Ihsan Dogramaci Children's Hospital were investigated. A clinical survey including clinical features of the patients was completed, and thirty-two patients from 26 different families were included in the study. Getting early diagnosis and regular assessment with imaging techniques seem to be the most important issues for improving the health status of the patients with XLA. Early molecular analysis gives chance for definitive diagnosis and genetic counselling, but not for predicting the clinical severity and prognosis.

X-linked Agammaglobulinemia - Is it Really Rare?

Background: X-Linked Agammaglobulinemia is a disease of the immune system in which there is defective development of the B lymphocytes due to which the production of gammaglobulins is markedly reduced; which results in immunodeficiency and high vulnerability to contract fatal infections. This is the reason for which a patient with XLA presents with history of recurrent infections. XLA is known to be caused due to a mutation in the Btk gene. Btk gene mutation observed on gene mapping markedly reduced levels of B lymphocytes and immunoglobulins; are the confirmatory laboratory findings for the diagnosis of XLA. As there is immunodeficiency in the patient, this condition is treated with intravenous immunoglobulin therapy. In this article, two cases with XLA have been described. Over a period of one month, two cases of XLA were admitted at the hospital. This is to pay attention to the fact that XLA is a rare condition, accounting to 1 in 200,000 live births. In view of its rarity, the two cases have been reported ahead in the article. Methods: Two patients who were a known case of X-Linked Agammaglonulinemia were reviewed in detail. Their records were reviewed and appropriate clinical data collected. The serum levels of immunoglobulins and B lymphocytes of these patients were thoroughly evaluated. BTK gene screening was also analysed to confirm the diagnosis of XLA. Results: Two patients who were previously diagnosed with XLA came to our hospital with complaints of recurrent respiratory tract infections. One patient, a 12 years old boy admitted with complaints of cough with expectoration and fever, was found to have low levels of B lymphocytes, IgM and IgA. His mother and elder brother were already diagnosed with XLA. The second patient , a 10 years old boy who got admitted at the hospital with fever and cough, has low levels of B lymphocytes, IgM and IgA, Genescreening showed BTK gene mutation. His mother is a known case of XLA confirmed with BTK genemutation on screening. Conclusion: The two cases mentioned above represent X-Linked Agammaglobulinemia, which is a rare disease with an occurrence rate of 1 in 200,000 live births. These two cases were reported at our hospital within duration of two weeks. Paying attention to the fact of the rarity of this condition, the admission of two patients with a rare disease as XLA, within such a short duration of time, claims the consideration of these cases to be reported. Implications on detecting female carriers in the family, counselling of the family members, and early diagnosis and treatment of affected males in the family ; are all important aspects associated with the diagnosis of XLA in a patient.

Neurodegenerative changes detected by neuroimaging in a patient with contiguous X-chromosome deletion syndrome encompassing BTK and TIMM8A genes.

IntroductionIn this study we describe a patient with gross deletion containing the BTK and TIMM8A genes. Mutations in these genes are responsible for X-linked agammaglobulinemia and Mohr-Tranebjaerg syndrome, respectively. X linked agammaglobulinemia is a rare primary immunodeficiency characterized by low levels of B lymphocytes and recurrent microbial infections, whereas, Mohr-Tranebjaerg syndrome is a progressive neurodegenerative disorder with early onset of sensorineural deafness.Material and methodsFor neuroimaging, the magnetic resonance imaging and magnetic resonance spectroscopy of the brain were performed. Microarray analysis was performed to establish the extent of deletion.ResultsThe first clinical symptoms observed in our patient at the age of 6 months were connected with primary humoral immunodeficiency, whereas clinical signs of MTS emerged in the third year of live. Interestingly, the loss of speech ability was not accompanied by hearing failure. Neuroimaging of the brain suggested leukodystrophy. Molecular tests revealed contiguous X-chromosome deletion syndrome encompassing BTK (from exons 6 through 19) and TIMM8A genes. The loss of the patient’s DNA fragment was accurately localized from 100 601 727 to 100 617 576 bp on chromosome’s loci Xq22.1.ConclusionsWe diagnosed XLA-MTS in the first Polish patient on the basis of particular molecular methods. We detected neurodegenerative changes in MRI and MR spectroscopy in this patient. Our results provide further insight into this rare syndrome.

Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in theBTKgene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.

Large BTK gene mutation in a child with X-linked agammaglobulinemia and polyarthritis

Large BTK gene mutation in a child with X-linked agammaglobulinemia and polyarthritis

Case report of new mutation of BTK gene of 1 child with X-linked agammaglobulinemia

Case report of new mutation of BTK gene of 1 child with X-linked agammaglobulinemia

A Novel Bruton’s Tyrosine Kinase Mutation in Russian Patient with X-Linked Agammaglobulinemia

X-Linked Agammaglobulinemia (XLA) is the major primary immunodeficiency in which the body is unable to produce the antibodies responsible for the defense against bacteria and viruses. The patient (a 6-month-old boy) was born at term to non-consanguineous parents. Both parents and older sister are clinically healthy. At 3, 5 months of age he presented acute viral infection with glue ear. At 4 month—serous meningitis, at 5 month—laryngotracheitis and serous meningitis. The levels of immunoglobulins were decreased for Ig A and IgG isotypes. The virtual lack of CD19+ B-lymphocytes was defined. Additionally there was found a complete absence of KREC (kappa-deleting recombination excision circle) in dried blood spot. The molecular diagnostics of coding region of the BTK gene was performed. DNA sequencing analysis of patient showed a 13-bp deletion in exon 2 (c.64_76delCCTCTAAACTTCA), leading to occurrence of frameshift and premature termination codon (p.Pro22fsTer28). This mutation was not described earlier. The mother and the sister of proband showed heterozygosity at the same position. Prenatal diagnostic testing has become available to this family for next pregnancy.

RNA Interference Screen Implicates TNFAIP3 and FOXO1 in MALT1 Inhibition Resistance

Recent studies have identified small molecule inhibitors of the paracaspase activity of MALT1, a protease and scaffolding protein involved in the B-cell receptor (BCR) signaling pathway, that are effective killing lymphomas in vitro and in vivo in xenograft models of Activated B-cell like Diffuse Large B-cell Lymphoma (ABC-DLBCL). ABC-DLBCL is characterized by constitutive NF-κB activity. This activation has been attributed to mutations in various protein components of the B-cell receptor (BCR) as well as Toll-like receptor (TLR) pathways. However, not all ABC-DLBCL cell lines and primary patient samples were equally sensitive to MALT1 inhibitors in vitro.In order to discover genetic modifiers of response to MALT1 inhibition we used an shRNA library screening approach. MALT1 inhibition sensitive cell line HBL-1 was infected with DECIPHER barcoded shRNA library Module 1 and cells were treated with vehicle or 300 nM of MALT1 inhibitor MI-2 for 22 days. At this time cells were harvested and genomic DNA extracted. PCR was used to amplify barcodes and gel purified bands were extracted and sequenced. Cellecta's Deconvoluter software was used to quantify the number of reads per shRNA, reads were normalized to total number of reads and fold change between vehicle and MI-2 treated cells was calculated. Among the top positively and negatively enriched hairpins, we found a significant number of genes involved in the BCR pathway including: positively regulated shRNAs against TNFAIP3 and FOXO1 and negatively regulated hairpins against BTK, CD79B and PI3K genes PIK3C2A and PIK3C2D. Interestingly, TNFAIP3 and FOXO1 are negative regulators of the BCR pathway while BTK, CD79B and PI3K genes are positive regulators of this pathway.In order to validate these results and given the abundance of inhibitors of different proteins in the BCR pathway, we run a focused combination screen using MALT1 inhibitor MI-2 and inhibitors against other proteins in the pathway in 4 MALT1 sensitive cell lines. Combinations with PI3K inhibitors were most synergistic (combination index (CI) ranging 0.12-0.67), while BTK and PKC inhibitors showed an additive effect (CI ranging 0.7-0.9). These results were confirmed using a second MALT1 inhibitor, mepazine. In order to characterize the molecular mechanism by which MALT1 inhibition cooperates with PI3K, we focused on the FDA approved drug Idelalisib. In vitro treatment of cells with MI-2 and Idelalisib showed that effect on cell growth was a combination of decreased proliferation and increased apoptosis. Moreover, we found a decrease in AKT phosphorylation followed by a decrease in FOXO1 T24 phosphorylation and an accumulation of FOXO1 protein. This result correlates with our finding that FOXO1 knockdown favors MALT1 inhibition resistance. In vivo treatment of TMD8 xenografts with a combination of MI-2 and Idelalisib showed a stronger effect than either drug used as a single agent or vehicle, confirming the increased efficacy of the combination over either drug alone.In summary, we have used an shRNA library screening in order to determine which proteins and pathways cooperate with MALT1 inhibition to kill ABC-DLBCL and to evaluate combinatorial treatments in an unbiased manner. This same approach has pointed out TNFAIP3 and FOXO1 as possible biomarkers of response. This is especially interesting since these two proteins are mutated in a proportion of ABC-DLBCL patients and could affect response to treatment not only against MALT1 inhibitors but potentially any BCR targeted therapy. DisclosuresMelnick:Janssen: Research Funding.

Regulation of B-Cell Receptor Signalling By the Tumour Microenvironment in Chronic Lymphocytic Leukemia (CLL) and Its Impact on Adhesion and miRNA Expression

B-cell receptor (BCR) mediated signalling is pivotal to the pathogenesis of chronic lymphocytic leukaemia (CLL), which is broadly split into two major subsets based on the immunoglobulin heavy chain variable (IGHV) gene mutational status. Progressive disease is characterised by unmutated IGHV genes (U-CLL) and more indolent disease by mutated IGHV genes (M-CLL). Antigen/autoantigen engagement is crucial for BCR-driven CLL cell survival, disease progression and resistance to therapy. Drugs that target this pathway are revolutionising the treatment of this disease. However, these inhibitors are not curative and some patients develop resistance to ibrutinib following mutation of the BTK or PLCγ2 genes or for as yet unknown reasons. We have shown that IL-4 pre-treatment induced sIgM expression especially in cases with unmutated IGHV genes, augmented anti-IgM induced signalling and compromised the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit BCR-mediated signalling. Previous reports highlighted a role of miR-155 (Cui Blood 2014) and miR150 (Mraz Blood 2014) in regulating BCR signalling in CLL cells by targeting SHIP1 (negative regulator of BCR signalling) and GAB1 and FOXP1 (positive regulators of BCR signalling) respectively. In this study we investigated the effect of IL-4 on expression of these miRNA and demonstrated that IL-4 augments and inhibits expression of miR155 and miR150 respectively (Figure 1), but had no effect on the house keeping control gene RNU6, demonstrating a mechanism whereby IL-4 augments BCR signalling. Furthermore, since BCR engagement can promote adhesion in CLL cells, we investigated the effect of IL-4 alone and in combination with anti-IgM on integrin expression such as CD49d, CD49e and CD29 and on adhesion. IL-4 pre-treatment augmented CD49d (α4) and CD44 expression and promoted anti-IgM as well as PMA mediated adhesion of CD5\\CD19+ CLL cells to fibronectin. Interestingly in contrast to greater sIgM increases by IL-4 in U-CLL, IL-4 promoted greater anti-IgM-induced adhesion largely in the M-CLL subset. This indicates that IL-4 may have distinct effects in the two subsets.Given our results, we hypothesised that dual inhibition of IL-4 and BCR signalling may promote greater responses in patients. At ASH 2015 we demonstrated that cerdulatinib, a dual JAK/SYK inhibitor, now in phase II clinical trials in CLL, can inhibit IL-4 and anti-IgM/anti-IgD induced signalling at concentrations achievable in patients (Cmin 1μM). We have now extended these studies and shown that cerdulatinib treatment prevented IL-4-induced effects such as increased sIgM expression and subsequent downstream signalling, and prevented downmodulation of CXCR4 and to a greater extent than the specific JAK3 inhibitor tofacitinib. Furthermore, inhibition of JAK1/3 by cerdulatinib also reduced IL-6, IL-10, IL-15, IL-21 and IFNγ-induced signalling, which have all been shown to play a role in CLL biology. More importantly, cerdulatinib killed CLL cells under basal conditions, particularly in cases with progressive disease. However, CLL cells within the patient lymph nodes likely receive signals from the BCR, IL-4 and possibly CD40L, which results in the elevation of pro-survival proteins Mcl-1 and Bcl-XL, providing protection from basal and ibrutinib or venetoclax induced killing. Following simultaneous treatment with bead-immobilised anti-IgM, 10ng/ml IL-4 and 300ng/ml CD40L, cerdulatinib was still able to induce a modest amount of apoptosis, however in combination with venetoclax there was a synergistic increase in killing. Together these data provide evidence of how IL-4 increases BCR signalling by directly modulating miRNA expression, but also additional effects on adhesion which may subsequently retain the tumour cells within the lymph nodes. JAK inhibition may prevent these effects. Therefore, these findings indicate that JAK inhibition in combination with BCR kinase inhibition maybe of value for the treatment of CLL. [Display omitted] DisclosuresConley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Davies:Bayer: Research Funding; Karyopharma: Honoraria, Research Funding; GSK: Research Funding; Janssen: Honoraria; Pfizer: Research Funding; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel to scientific conferences, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel, accommodation, expenses, Research Funding. Johnson:Celldex Therapeutics: Research Funding. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Coffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Steele:Portola Pharmaceuticals: Honoraria.

Case Report: Whole exome sequencing identifies a novel frameshift insertion c.1325dupT (p.F442fsX2) in the tyrosine kinase domain of BTK gene in a young Indian individual with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an extremely rare inherited primary immunodeficiency characterized by recurrent bacterial infections, decrease in number of mature B cells and low serum immunoglobulins. XLA is caused by mutations in the gene encoding Bruton's tyrosine kinase. We report a case of a young Indian boy suspected to have XLA. Immunophenotyping was performed for the affected child using CD20, CD19 and CD3 antibodies. Whole exome sequencing was performed using trio-based approach. The variants were further analyzed using capillary sequencing in the trio as well as maternal grandmother. Initial immunophenotyping in the affected child showed decreased count of CD19+ B cells. To strengthen the clinical findings and confirm the diagnosis of XLA, we performed whole exome sequencing. Our analysis identified a novel frameshift insertion (c.1325dupT) in the BTK gene, which was further validated by Sanger sequencing. Our approach shows the potential in using whole exome sequencing to pinpoint the molecular lesion, enabling timely diagnosis and genetic counseling, and potentially offering prenatal genetic testing for the family.

Gastric Adenocarcinoma in a Patient with X-Linked Agammaglobulinemia and HIV: Case Report and Review of the Literature

X-linked agammaglobulinemia (XLA) is an X-linked inherited disease usually caused by a germline mutation in the BTK gene leading to Bruton’s tyrosine kinase deficiency, which results in the impaired development of B-lymphocytes and a subsequent lack of immunoglobulin production. Patients with XLA have an increased susceptibility to bacterial and viral infections, and multiple case reports have been published regarding an association between XLA and gastrointestinal (GI) malignancy. Here, we describe a case of a 25-year-old man with XLA and HIV, who developed gastric adenocarcinoma. Previously reported cases of XLA and GI malignancy are also reviewed and summarized.

X-linked agammaglobulinemia: Twenty years of single-center experience from North West India

BackgroundX-linked agammaglobulinemia (XLA) is an X-linked genetic defect in maturation of B lymphocytes that results in the absence of B lymphocytes in the peripheral blood and profound hypogammaglobulinemia. It is caused by a mutation in the BTK gene located on the X chromosome. There are no large series describing XLA from the developing world. ObjectiveTo analyze the clinical features, immunologic and genetic characteristics, and outcomes of 36 patients with XLA diagnosed and managed for a period of 2 decades. MethodsDiagnosis of XLA was made on the basis of presence of BTK gene mutation or marked reduction of B lymphocytes in peripheral blood with a family history of an affected male relative. The diagnosis was confirmed by genetic mutation studies in 28 patients with 25 unique mutations in the BTK gene. ResultsThere was a significant delay in diagnosis in most of the patients. The mean (SD) delay in the diagnosis was 4.2 (3.5) years. Point mutations were the most common mutations detected, accounting for 68% of all mutations. Deletions and insertions were also seen in a few cases. Four of the mutations are novel mutations that have not been previously reported. Seven of the 36 patients (19%) were dead at the time of analysis in the present cohort. The mean survival was 137 months (95% confidence interval, 13–163 months). ConclusionThe present study is perhaps the largest series of patients with XLA from any developing country so far.