Bruton's Tyrosine Kinase Gene Research Articles

A novel Bruton tyrosine kinase gene variation was found in an adult with X‐linked agammaglobulinemia during blood cross‐matching prior to surgical operation

To investigate the underlying molecular mechanism of the patient's ABO typing discrepancy. ABO typing discrepancy was frequently seen in patients due to different causes. In this study, ABO typing discrepancy was found in a 24-year-old man with arthralgia, whose forward ABO grouping was O and reverse ABO grouping was AB. Primary immunodeficiency disease was speculated in this patient, especially X-linked agammaglobulinemia (XLA). Immunoglobulins of all isotypes were detected using a specific protein analyser. Lymphocyte subgroups were analysed by flow cytometry. All 19 exons and boundaries of BTK gene were amplified by polymerase chain reaction (PCR), and all PCR products were sequenced by a DNA analyser. BTK protein in the leukocytes and platelets was detected by Western blot. No B lymphocytes could be detected in the peripheral blood of the patient. A novel BTK gene variation, c.817G>T, in the exon 9 of BTK gene was discovered. No BTK protein expression could be detected in the leukocytes and platelets of the patient. XLA could be occasionally discovered by ABO typing discrepancy in some cases because of the deficiency of reciprocal IgM anti-A and/or anti-B antibodies in the serum of the patient. Humoral immunodeficiency is one of the causes of ABO typing discrepancy.

Treatment for X-Linked Agammaglobulinemia with Allogeneic Umbilical Cord Blood Stem Cell Transplantation from HLA-Matched Sibling Confirmed Healthy through Prenatal Diagnosis

Background X-linked agammaglobulinemia (XLA) is a rare congenital humoral immunodeficiency characterized by marked reduction in all classes of serum immunoglobulins and the near absence of mature CD19+ B-cells which was caused by mutations in Bruton's tyrosine kinase (BTK). We herein describe the first case of XLA transplanted from HLA-matched sibling allogeneic umbilical cord blood donor.Method The patient was an 8-year-old boy with XLA who previously had life-threatening infectious episodes and diagnosed by direct sequencing of BTK gene mutations (c.992-923delGAN(NM-000061)) when he was 3 years old. He was treated with the regular replacement of immunoglobulin G (IgG) products. Taking into consideration significant infectious complications and the prompt availability of HLA-matched sibling umbilical cord blood, he underwent HLA-matched sibling UCBT. The FC conditioning regimen consisting of Flu (30mg/m2/d×4 days) and CTX (30mg/kg/d×4 days) was provided for the patient. The source of stem cells was umbilical cord blood of his younger brother who was confirmed completely healthy through prenatal diagnosis with villi at the fetus period of 4 months. The number of infused total nucleated and CD34+ cells was 6.51×108/L and 2.67×106/L, respectively. He received a combination of CsA, short-term MTX and MMF for GVHD prophylaxis.Result Hematopoietic engraftment was rapidly achieved, with an absolute neutrophil count of >0.5×109/L on day 15 and platelet count of >20×109/L on day 20. The patient was discharged on day 45 and has been followed as an outpatient without any infectious episodes for more than 180 days. No expected toxicities were observed. Regarding immune constitution, CD19+ cells, IgA and IgM, which were undetectable before transplantation, started to increase in number 10 days after transplantation and continued to increase for more than 3 months. The patient did not exhibit any GVHD manifestations. He is currently alive and doing well at six months post-transplant, and he is free of any infection episodes.Conclusion The above results provide strong evidence that HLA-matched sibling UCBT may be safe and technically feasible for the treatment of XLA when an appropriately matched related or unrelated donor is unavailable. The umbilical cord blood could be considered being frozen, especially in the era of “the second child” in China.Keywords X-linked agammaglobulinemia; HLA-matched sibling donor; allogeneic umbilical cord blood stem cell transplantation; immune recovery. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Bruton’s Tyrosine Kinase Deficiency in Patients with Presumed X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a fully penetrant X-linked recessive disorder characterized by the early onset of recurrent bacterial infections, profound hypogammaglobulinemia and a marked decrease in the number of B-lymphocytes [1]. The gene defective in XLA has been identified as a non-receptor proteine tyrosine kinase, BTK (Bruton´s tyrosine kinase) [2,3]. Over 600 mutations have been described in the BTK gene and they are spreaded throughout the gene [4]. Mutational analysis has revealed that there is considerable heterogeneity in the clinical spectrum of XLA, with atypical individuals having small numbers of mature B lymphocytes and some immnoglobulin production [5]. Analysis of Btk protein expression in primary cells of XLA patients has shown that the large majority of individuals do not express protein regardless of their mutation [6]. As it is yet known Btk is expressed not only in B cells but also in granulocytes and monocytes. In the present work we applied a flow cytometric test employing whole blood to detect Btk expression in monocytes to identify XLA patients.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%–92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14–21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

X-linked agammaglobulinemia in children

X-linked agammaglobulinemia (XLA) is one of the most common types of primary immunodeficiency disease in children, and is an antibody deficiency disease which is seen in men.Most XLA patients carry mutations in Bruton tyrosine kinase (BTK) gene, they typically present with very low numbers of peripheral B cells and a profound deficiency of all immunoglobulin isotypes.XLA is characterized by recurrent bacterial infections within 2 years, sometimes life-threatening.The prognosis of XLA has been improved by the treatment of gammaglobulin that allow normal concentrations of serum IgG. Key words: X-linked agammaglobulinemia; Bruton tyrosine kinase gene; Mutation

Integrated ATAC-Seq and Chemosensitivity Profiling Identifies Rational Drug Combinations in Ibrutinib-Treated CLL Patients

▪Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and accumulation of malignant B lymphocytes in the blood, bone marrow, spleen, and lymph nodes. This process is associated with constitutively activated B cell receptor (BCR) signaling, and interference with BCR signaling provides therapeutic benefit. Specifically, the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib prevents BTK tyrosine phosphorylation and thereby interferes with pathways downstream of BCR. It has shown high clinical response rates in patients with relapsed and refractory CLL, including patients with adverse cytogenetic profiles.Despite the high response rates achieved by ibrutinib, the drug has important limitations. Ibrutinib treatment induces a redistribution of CLL cells from protected niches to the peripheral blood, and the cellular response to ibrutinib is slow and often incomplete. Further, there is no evidence that a cure can be achieved, and drug discontinuation (e.g., due to toxicity) is associated with rapid progression. Even among patients that tolerate long-term treatment with ibrutinib, a considerable percentage develops drug resistance (e.g., due to mutations in the BTK gene), BTK independent disease progression, or Richter's transformation, indicating drug synergies with ibrutinib may increase prognosis.Recent studies have explored the combined use of ibrutinib with the proteasome inhibitor carfilzomib, the BCL2 inhibitor venetoclax, and the HDAC inhibitor abexinostat in preclinical models, which has shown promising initial results. However, these approaches were largely empirical, and little is known about the gene-regulatory effects of ibrutinib treatment in CLL cells.Here, we charted the ibrutinib-induced chromatin regulatory landscape of CLL, and in parallel mapped targetable pathways for synergistic combination therapies that could potentially improve disease control. Therefore, peripheral blood from 24 fully characterized CLL cases, including the clinical and hematological parameters, disease stage, cytogenetics (13q del, 11q del, 17p del, trisomy 12), p53 and IGHV mutation status were collected before and during therapy with ibrutinib. Chromatin accessibility was measured by ATAC-seq, a powerful assay for mapping the genome-wide regulatory landscape of cell. In addition, the ex vivo chemosensitivity to >140 drugs on paired CLL samples collected before and during ibrutinib treatment was measured using a novel method for ex vivo single-cell drug cytotoxicity profiling in primary samples (Snijder et al. submitted; NCT03096821).We bioinformatically analyzed the changes in chromatin accessibility and differential drug responses before and during ibrutinib treatment, thereby establishing a comprehensive picture of the cellular responses to the drug. As a proof of principle for this approach, ex vivo results confirmed strong clinical activity of BCL2 inhibitors with and without ibrutinib. We identified increases in chromatin accessibility and chemosensitivity in proteasome, inflammatory NF-κB/TNF signaling, CoA biosynthesis, PI3K/Akt, caffeine metabolism pathways, along with changes affecting genes such as FOXO3 and IκBa. Integration of chromatin accessibility and differential chemosensitivity data revealed robust ibrutinib-induced signatures, which we exploited to prioritize approved drugs and drug candidates for synergistic treatment of CLL cells in co-culture models using primary bone marrow stromal cells. Cell viability assays revealed that ibrutinib treatment in vivo and in vitro sensitizes CLL cells to compounds such as the proteasome inhibitor bortezomib, the JAK inhibitor ruxolitinib, the PLK1 inhibitor volasertib, and the bisphosphate zoledronate.In summary, our results show that the synergistic combination and bioinformatic integration of chromatin profiling with functional drug screening is a powerful tool to identify targetable pathways in CLL. This approach may be useful for designing personalized therapies as well as the rational planning of clinical studies. Our approach is directly transferable to other leukemic diseases in which malignant cells can be obtained for chromatin profiling and drug sensitivity analysis, thus providing a widely applicable tool for the rational development of combination therapies. [Display omitted] DisclosuresVladimer:Allcyte Gmbh: Equity Ownership. Snijder:Allcyte: Equity Ownership. Krall:Allcyte Gmbh: Equity Ownership. Hoermann:Gilead: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria; Ariad: Honoraria. Staber:Morphosys: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Gilad: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Honoraria. Superti-Furga:Allcyte GmbH: Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Functional Analyses of BTK Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, novel mutations in the Bruton's Tyrosine Kinase (BTK) have been identified in FL (Krysiak et al, Blood 2017). Additional mutations in other components of BCR signaling pathways in FL have been reported as well but functional consequences have not been fully explored.Methods: Using a combination of WES on N=82 FLand BTK resequencing in an extended FL panel we uncovered novel BTK mutations in FL. These BTK mutations are all distinct from the previously identified mutations suggesting that a full complement of BTK mutations has not yet been identified. Using crispr-Cas9-mediated BTK gene targeting in multiple lymphoma cell lines, we generated novel BTK-/- null lines. We introduced all BTK mutations by site-directed mutagenesis into a reference BTK cDNA. Subsequently, we re-introduced HA-tagged wild type and mutated BTK constructs using lentiviral transduction into the BTK-/- cell lines and also into BTK-/- DT40 cells. We measured signal transduction pathway components following BCR cross linking. The location of BTK mutations was modeled onto a 3D-model assembled from various published BTK substructures.Results: Mutations in BTK are distributed along the entire length of the gene and are targeting the PH-, SH2- and catalytic domain, with the catalytic domain carrying the majority of BTK mutations. Interestingly, occasional frame shift mutations as reported and a splice acceptor site mutation by genetic criteria suggest gene inactivation. The 3D modeling data show that all BTK mutations are on the surface of the BTK protein within the various functional domains. The quantitation of BTK protein after reconstitution into various engineered BTK-/- cell lines uncovered lower steady state levels of various mutants especially for mutations targeting the catalytic domain. PLCγ2 remains the best studied BTK substrate and various sites have been reported to be phosphorylated by BTK in vivo (PLCγ2 amino acids 753, 759 and 1217). Following BCR crosslinking in engineered BTK-/- lymphoma cell lines we found only a modest reduction in the phosphorylation of PLCγ2 residue 759 or 1217 demonstrating that these sites are under the control of various tyrosine kinases and not exclusively targeted by BTK. After BCR crosslinking in the BTK-/- cells lines we detected a substantial increase in PLCy2 residues 759 or 1217 phosphorylation over baseline and this increase was abolished by ibrutinib pre-treatment. Therefore, PLCγ2 residue 759 or 1217 phosphorylation is due to multiple BCR stimulated and ibrutinib sensitive tyrosine kinases. In engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK we did not detect major effects by the BTK mutants on the phosphorylation state of PLCγ2 residue 759 or 1217. In addition, phosphorylation of BTK at residue 223 (a major autophosphorylation site) was reduced or absent in multiple BTK mutants. Summarizing these findings, FL-associated BTK mutations do not activate the BTK kinase; instead, multiple BTK mutants are inactivating.Focusing on BCR-regulated signaling pathways, we measured ERK and AKT phosphorylation in the engineered BTK-/- lymphoma cell lines reconstituted with HA-tagged wild type and mutated BTK. Following BCR crosslinking, we detected increased p-AKT-473 phosphorylation in most BTK mutants in a total of 4 separate cell lines. In contrast, p-ERK was largely unaffected. Recent reports implicated AKT activation as a major factor in tonic BCR signaling in germinal center type DLBCL. We did not detect elevated AKT phosphorylation prior to BCR stimulation in our BTK reconstituted cell systems but an exaggerated response following BCR engagement. The effects of BTK mutations on protein-protein interactions within the BTK signalosome as well as effects on sensitivity to ibrutinib will be updated at the meeting.Conclusion : Our initial efforts at functional characterization of FL-associated BTK mutations uncovered that despite reduced stability and impaired catalytic activity most BTK mutations resulted in enhanced AKT activation following BCR crosslinking. Given the established pro-growth and pro-survival function of AKT, the data provide a clear reason why FL would select for such mutations. DisclosuresPhillips:Incyte: Other: Travel/Expenses; Pharmacyclics: Consultancy; KITE: Consultancy; Seattle Genetics: Consultancy. Malek:Abbvie: Equity Ownership; Janssen R&D: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding.

Transcriptome profiling of monocytes from XLA patients revealed the innate immune function dysregulation due to the BTK gene expression deficiency

X-linked agammaglobulinemia (XLA) is a rare genetic disorder, caused by mutations in BTK (Bruton’s Tyrosine Kinase) gene. Deep high-throughput RNA sequencing (RNA-Seq) approach was utilized to explore the possible differences in transcriptome profiles of primary monocytes in XLA patients compared with healthy subjects. Our analysis revealed the differences in expression of 1,827 protein-coding genes, 95 annotated long non-coding RNAs (lncRNAs) and 20 novel lincRNAs between XLA patients and healthy subjects. GO and KEGG pathway analysis of differentially expressed (DE) protein-coding genes showed downregulation of several innate immune-related genes and upregulation of oxidative phosphorylation and apoptosis-related genes in XLA patients compared to the healthy subjects. Moreover, the functional prediction analysis of DE lncRNAs revealed their potential role in regulating the monocytes cell cycle and apoptosis in XLA patients. Our results suggested that BTK mutations may contribute to the dysregulation of innate immune system and increase susceptibility to apoptosis in monocytes of XLA patients. This study provides significant finding on the regulation of BTK gene in monocytes and the potential for development of innovative biomarkers and therapeutic monitoring strategies to increase the quality of life in XLA patients.

Abstract 1226: Targeting BTK family in ER+ breast cancer with Ibrutinib for personalized precision medicine: a double-edged sword

Abstract Background: Metastasis is the principal cause of morbidity and mortality from breast cancer. Although current therapies are effective in early-stage of breast cancer, resistance to therapy is common leading to the development of metastasis. The commonest site of metastases is the regional lymph nodes. The presence and the number of involved lymph nodes remains the single best predictor of metastases and an important guide for the use of systemic adjuvant therapy. Identification of the underlying molecular targets of lymph node metastasis and a better understanding how to therapeutically target these will be a significant step in the goal of prevention of metastases. Experimental Design: To determine the key players associated with the development of metastasis in breast cancer, we performed RNA sequencing (Illumina Paired-end RNA-seq) analysis in a cohort of matched pairs of primary and (nodal) metastatic tumors (n=18). Gene expression profiles were analyzed using DESeq2. For targeted therapeutics, we performed orthotopic tumor implantation into mammary fad pads of athymic nude mice of breast cancer models. Results: Our results identified significant differential expression of the Bruton's tyrosine kinase (BTK) family members in nodal metastasis. Among the family members, BTK, BLK, RLK/TXK and JAK3 are expressed at very high levels in the metastatic lesions as compared to matched primary tumors. Among the 51 breast cancer cell lines analyzed, the BTK genes were significantly upregulated in luminal, but not basal cell lines. Categorized by the molecular subtypes of breast cancer, hormone receptor- positive (HR) and /or HER2 overexpressing breast cancer cell lines present higher BTK expression. Among the 51 breast cancer cell lines analyzed, the BTK genes were significantly regulated in both ER+ HER2- (T47D and ZR75.1) and ER+HER2+ (BT-474 and ZR75.30) breast cancer cell lines. Ibrutinib, a selective and irreversible inhibitor of BTK targeting the cysteine-481 residue in the active site, exhibited potent activity in preclinical models of BTK-expressing targets. Ibrutinib decreased primary tumor growth significantly in ER+HER2+ (BT-474) mouse xenograft model in a dose dependent manner (10 mg/kg vs 25 mg/kg; P< 0.05), whereas it promoted the tumor growth in a significant manner in ER+HER2- xenograft models (T47D and ZR75.1; P<0.05). The differences might be due to the differences in model systems due to the HER2 presence. Conclusion: Our data suggests that differential expression of target genes can be used to select agents as treating breast cancer. However, caution is necessary, as, in some situations, this might lead to enhancement of the growth in in vivo in subsets of breast cancer. Citation Format: Yesim Gökmen-Polar, Xiaoping Gu, Edyta Vieth, Sunil Badve. Targeting BTK family in ER+ breast cancer with Ibrutinib for personalized precision medicine: a double-edged sword [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1226. doi:10.1158/1538-7445.AM2017-1226

Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

Noninvasive Detection of Ibrutinib Resistance in Non-Hodgkin Lymphoma Using Cell-Free DNA

Background: Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), plays an emerging role in targeted therapy of several lymphoid malignancies. Due to its activity in B-cell receptor (BCR) dependent human B-cell cancers, Ibrutinib is currently approved for the treatment of patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, Ibrutinib responses can be seen in patients with follicular lymphoma (FL) and in a subset of patients with ABC- (activated B-cell-) like diffuse large B-cell lymphoma (DLBCL) (Wilson et al., Nat Med 2015; Fowler et al., ASH Annual Meeting, 2015). However, resistance mutations in BTK and other genes (e.g., PLCγ2)are a major cause of treatment failure and early identification of those aberrations remains challenging. Circulating tumor DNA (ctDNA) is an emerging biomarker with potential for disease monitoring as well as biopsy-free detection of evolving somatic aberrations. In this case study, we show that noninvasive genotyping of blood plasma allows early detection of resistance mutations duringIbrutinib therapy in a patient with relapsed/refractory follicular lymphoma associated with histological transformation.Methods: We profiled two tumor lymph node (LN) biopsy specimens and 8 plasma samples before, during, and after Ibrutinib therapy of a 67-year-old man with relapsed/refractory FL with evidence of transformation. Preceding Ibrutinib, this patient received 6 lines of lymphoma treatment over a ~3 year disease course, including Rituximab monotherapy, Benadmustine/Rituximab, and R-CHOP. Despite achieving stable disease after 12 weeks, the patient experienced significant disease progression after additional 4 months on Ibrutinib (Figure 1). We profiled these 10 serial samples using CAPP-Seq, a capture-based targeted high-throughput sequencing (HTS) method (Newman et al., Nat Med, 2014), which allows the sensitive identification of emerging somatic aberrations in circulating tumor DNA.Results: We detected the emergence of two independent BTK C481S resistance mutations in the blood plasma during Ibrutinib therapy, which were undetectable immediately before treatment. The major resistance clone harboring the BTK C481S A>T mutation (Figure 1, orange) was identified 2 months after the start of Ibrutinib, with allele frequencies as low as 0.03%. The second resistant subclone emerged 63 days later, carrying a BTK C481S somatic mutation with a substitution of C to G (Figure 1, green). Importantly, despite encoding an identical amino acid mutation, these two adjacent variants were never observed within the samectDNA molecule, demonstrating convergent evolution of independent resistantsubclones. We confirmed the existence of the two resistance mutations by tumor genotyping of a LN biopsy after clinical diagnosis of disease progression. Notably, while the minorsubclone completely disappeared under subsequentimmunochemotherapy with Rituximab/Bendamustine/Lenalidomide (RBL), the major resistance clone reappeared at the time of disease progression and the patient ultimately succumbed to his disease.Conclusions: Resistance mutations in BTK C481S have been described in tumor cells of Ibrutinib-refractory patients with CLL and MCL, with in vitro evidence of responsiveness to BH3-mimetics and SYK inhibitors. This study, to our knowledge, is the first report of its noninvasive detection in the plasma of a patient with Non-Hodgkin lymphoma, with the subsequent partial eradication of a resistant subclone. Therefore, we envision that biopsy-free genotyping will allow early detection of lymphoma patients developing Ibrutinib resistance and will be incorporated into future clinical trials involving targeted therapies. [Display omitted] DisclosuresNewman:Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Varian Medical Systems: Research Funding; Novartis: Consultancy; Roche: Consultancy; Quanticel Pharmaceuticals: Consultancy.

Characteristics, Outcome and Pattern of Mutations in Patients with Follicular Lymphoma Who Progressed on BTK Inhibitors (Ibrutinib and acalabrutinib)

Introduction: Acalabrutinib and ibrutinib are Bruton's tyrosine kinase (BTK) inhibitors, which have revolutionized the treatment of B-cell lymphoma/leukemias such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in frontline or relapsed refractory setting. Outcomes of patients with MCL and/or CLL who progressed on ibrutinib therapy are poor. Few reports have shown that ibrutinib resistant patients develop specific BTK mutations which confer resistance. We present the clinical and molecular data on patients with follicular lymphoma (FL) who have progressed on either ibrutinib based regimen or on acalabrutinib.Methods: We selected FL patients enrolled in clinical studies at our institution between 2013 and 2015 who discontinued acalabrutinib or ibrutinib for various reasons. Survival post ibrutinib discontinuation was calculated. We performed whole-exome sequencing on pre and post treatment (paired) samples of 3 FL patients (progressed on ibrutinib) using DNA extracted from formalin fixed paraffin embedded tissue samples. Library preparation and capture were performed using Agilent's SureSelect XT Human All Exon V4. Following capture the enriched libraries were pooled and sequenced in two Illumina HiSeq4000 lanes using a 75nt paired-end configuration.Results: Twenty of 55 patients discontinued treatment with BTK inhibitors; 5 came off from single agent acalabrutinib in relapsed refractory FL (RR-FL) while 15 came off from ibrutinib based regimen (4 as a single agent in RR-FL while 11 were treated with ibrutinib and rituximab in the frontline setting). These patients discontinued BTK inhibitor treatment, after a median of 2.8 months (range 0.9-22 months). Patients who received frontline ibrutinib (n=11), discontinued at a median of 2.8 months (0.9-22 months); previously treated patients discontinued BTK inhibitors at a median of 2.8 months (1.4-7.6 months). The reasons for discontinuation were as follows - 14 disease progression (3 on acalabrutinib and 11 on ibrutinib; 1 of them developed classical Hodgkin lymphoma), 2 non-responders (1 acalabrutinib and 1 ibrutinib) and 4 for other reasons (1 due to fatigue on acalabrutinib, 3 due to ibrutinib - 1 elevated liver enzymes, 1 asymptomatic atrial fibrillation another lost to follow up). Pre-treatment characteristics of the 16 patients who either progressed or were nonresponders to BTK inhibitors were - median age 59 years (range 41-75), 69% had stage 4 disease, 31% had FLIPI score of 3 or 4 and median LDH levels were 457 IU/L (range 290-754). Median follow up after discontinuation for all patients was 14.4 months (0-32 months). Eighteen patients (90%) were alive at the time of last follow up and 2 (10%) have died. Median survival (figure 1) of patients according to the causes of discontinuation was: not reached for both group of patients, those with other causes and those in progressive disease and/or non-responders.All 16 patients who progressed or not responded were treated with additional therapies. At the time of last follow up, 10/16 (62%) were responding to subsequent therapy - 5/10 (62%) achieved CR on lenalidomide-based therapies, 4/10 (40%), responded to chemoimmunotherapy and 1 patient responded to venetoclax-bendamustine and rituximab with CR. None were treated with PI3K inhibitors. WES studies showed EZH2 Y646 mutations in 2 of 3 FL patients in both pre and post treatment samples. Comparison of pre and post treatment samples on 3 patients showed multiple non-recurrent variants (2-12 per patient) in several genes in the post-treatment samples. These included DNMT3A R882H, NOTCH2 A3D and MLL E765G, all of which were noted in the post treatment sample of the patient that progressed to CHL. None of the other detected variants have been implicated in cancer. Of note, none of the samples showed BTK mutations despite adequate coverage.Conclusions: In contrast to other B-cell malignancies such as CLL and MCL, we observed that almost all FL patients who progressed on BTK inhibitors were salvageable by alternate therapies. Absence of BTK gene mutations in paired samples of 3 patients who progressed on ibrutinib suggests alternate mechanisms of drug resistance. [Display omitted] DisclosuresWang:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; BeiGene: Research Funding. Fayad:Seattle Genetics: Consultancy, Research Funding. Oki:Novartis: Research Funding. Westin:Spectrum: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees. Fowler:Gilead: Research Funding; Jannsen: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; TG Therapeutics: Consultancy.

Clinical characteristics and genetic profiles of 174 patients with X-linked agammaglobulinemia: Report from Shanghai, China (2000-2015).

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency. XLA patients typically present with very low numbers of peripheral B cells and a profound deficiency of all immunoglobulin isotypes. Most XLA patients carry mutations in Bruton tyrosine kinase (BTK) gene.The genetic background and clinical features of 174 Chinese patients with XLA were investigated. The relationship between specific BTK gene mutations and severity of clinical manifestations was also examined. Mutations were graded from mild to severe based on structural and functional prediction through bioinformatics analysis.One hundred twenty-seven mutations were identified in 142 patients from 124 families, including 45 novel mutations and 82 recurrent mutations that were distributed over the entire BTK gene sequence. Variation in phenotypes was observed, and there was a tendency of association between genotype and age of disease onset.This report constitutes the largest group of patients with BTK mutations in China. A genotype–phenotype correlation was observed in this study. Early diagnosis of congenital agammaglobulinemia should be based on clinical symptoms, family history, and molecular analysis of the BTK gene.

X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

A novel BTK gene mutation, c.82delC (p.Arg28 Alafs*5), in a Korean family with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a hereditary humoral immunodeficiency that results from Bruton’s tyrosine kinase (BTK) gene mutations. These mutations cause defects in B-cell development, resulting in the virtual absence of these lymphocytes from the peripheral circulation. Consequently, this absence leads to a profound deficiency of lg all isotypes, and an increased susceptibility to encapsulated bacterial infections. A 15-month-old Korean boy presented with recurrent sinusitis and otitis media after 6 months of age, and had a family history of 2 maternal uncles with XLA. Laboratory tests revealed a profound deficiency of Ig isotypes, and a decreased count of CD19+ B cells in the peripheral circulation. Based on his family history and our laboratory test results, he was diagnosed with XLA. We performed BTK gene analysis of peripheral blood samples obtained from family members to confirm the diagnosis. Mutational analysis revealed a novel hemizygous frameshift mutation (c.82delC, p.Arg28Alafs*5), in the BTK gene. His mother and maternal grandmother were heterozygous carriers of this mutation and his two maternal uncles were hemizygous at the same position. After XLA diagnosis, intravenous immunoglobulin (400 mg/kg, monthly) treatment was initiated; recurrent sinusitis and otitis media were subsequently brought under control. To our knowledge, this is the first reported case of a Korean pedigree with a novel mutation in the BTK gene.

BET Protein Bromodomain Antagonist-Based Combinations Exert Synergistic Pre-Clinical Activity Against Ibrutinib-Sensitive or -Resistant Human Mantle Cell Lymphoma (MCL) Cells

Mantle Cell Lymphoma (MCL) exhibits genetic alterations in p53, CDK4, CDKN2A, MYC, BCL2, B cell receptor (BCR) and NFkB signaling, which confer a cell autonomous, pro-growth and pro-survival advantage on MCL cells. Treatment with the Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib inhibits BCR and NFκB activity, mediating growth inhibition and apoptosis of MCL cells. Although ibrutinib induces impressive clinical responses in patients with MCL, long-term durable remissions and cure remain elusive. Ibrutinib-resistant MCL cells exhibit mutations in TRAF2/3 and MAP3K14 (NIK), activating the alternative NFκB signaling. Additionally, a cysteine-to-serine C481S mutation in BTK at the binding site of ibrutinib, which confers resistance to ibrutinib, has also been documented in MCL that relapsed while on ibrutinib. These observations suggest that ibrutinib-resistant MCL cells remain dependent for growth and survival on the NFκB-activated gene-expressions. In the ibrutinib-refractory primary MCL cells, mutations in MLL2, CREBBP, PIM1 and ERB4 were also detected. Our findings have shown that the BET (bromodomain and extra-terminal) protein BRD4 antagonists (BA), e.g., JQ1, transcriptionally repress the levels of MCL-relevant oncogenes BCL-2, c-Myc, cyclin D1, CDK6 and PIM1, while inducing the levels of HEXIM1, p21 and p27 in the MCL cells. Consistent with the reported findings that BRD4 is essential for the transcriptional activity of NFkB, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Collectively, this resulted in growth arrest and apoptosis of the cultured (MO2058, Mino, Z138 and JeKo-1) and primary MCL (pMCL) cells. We also demonstrate that compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibits pBTK, BTK, pPLCγ2, pAKT, PIM1 and nuclear RelA levels, as well as synergistically induced apoptosis of the cultured and pMCL cells with combination indices (CI) of less than 1.0 by the median dose effect isobologram analysis. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental MCL Mino or MO2058 cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR (>13-fold resistant) and MO2058/IR cells (>8-fold resistant), respectively. Sequencing of the BTK gene did not reveal any mutation in the BTK gene (e.g. C481S) in the ibrutinib-resistant cells. Notably, compared to Mino, Mino/IR cells expressed higher protein levels of BTK, AKT, CDK6, PIM1, XIAP, Bcl-xL and BCL2 levels, while simultaneously displaying lower NOXA, PUMA and p21 levels. Importantly, treatment with JQ1 inhibited the suspension culture growth of Mino/IR cells. JQ1 treatment also induced similar level of apoptosis in Mino/IR, as compared to Mino cells. JQ1-induced apoptosis of Mino/IR cells was associated with a reduction in the nuclear levels of RelA, as well as inhibition of the mRNA levels of BTK, CDK6, and BCL2. In contrast, JQ1 treatment induced the mRNA expression of BCL2L11 and HEXIM1. JQ1 treatment also markedly attenuated the protein levels of BTK, c-Myc, PIM and CDK6 in Mino/IR cells; however, a more modest inhibition of Bcl-xL, BCL2, and XIAP protein levels, and a lack of effect on the protein levels of AKT was observed. Furthermore, pre-treatment of Mino/IR cells with JQ1 enhanced their sensitivity to ibrutinib. Next, we determined that co-treatment with JQ1 and the BCL2 antagonist ABT-199, palbociclib (a CDK4 and CDK6 inhibitor) or AZD1208 (a PIM1 & 2 kinase inhibitor) was synergistically lethal against Mino and Mino/IR cells (CI < 1.0). Co-treatment with JQ1 and ABT199, AZD1208 or palbociclib was also synergistically lethal against pMCL cells, but not against the normal B or hematopoietic cells. These findings demonstrate that treatment with the BA JQ1 sensitizes MCL cells to not only ibrutinib but also to BCL2, PIM1 & 2 and CDK4/6 antagonists. Furthermore, co-treatment with BA and BCL2, PIM1 & 2 or CDK4/6 antagonist are promising combination therapies for testing against ibrutinib-sensitive or ibrutinib-resistant MCL cells. DisclosuresWang:Pharmacyclics, Janssen, Celgene, Oncopep, Kite, Juno: Research Funding; Janssen: Honoraria.

High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients

Background: Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton's tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that Ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. In order to better understand the development of these resistance mechanisms in patients with CLL, we developed a high sensitivity (HS) assay utilizing branched and locked nucleic acids (BNA and LNA, respectively). We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell free DNA (cf-DNA) from patients with CLL.Methods: Using custom BNA or LNA oligos in a wild-type blocking polymerase chain reaction, then sequencing by Sanger and NGS methods, we achieved 100x greater sensitivity than Sanger. Sanger sequencing was capable of detecting <1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. The assay is designed to cover BTK and PLCγ2 hotspots. Using this assay we tested peripheral blood samples from 44 Ibrutinib-naïve patients (Ib-) with CLL and 7 samples from CLL patients being treated with Ibrutinib (Ib+), that showed clinical evidence of disease progression. The same wild-type blocking was also used in NGS approach for confirmation. We performed wild-type blocking in a Nextera Rapid Capture Enrichment workflow for our custom 315 gene panel.Results: No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. In contrast, all (N=7) tested patients with progressive disease on Ibrutinib showed one or more mutation in BTK or PLCγ2 using the HS method. Without the HS testing only 4 patients (57%) showed a mutation in BTK or PLCγ2. Two patients showed multiple mutant clones. One patient with double mutations in PLCγ2 (R665W and L845F) also showed triple independent mutations in BTK at codon C481 with HS testing. These mutations give rise to two distinct mutant proteins C481R (TGC>CGC) and C481S (TGC>AGC and TGC>TCC). NGS analysis confirmed that the the three BTK mutations are in three independent clones A second patient showed initially a mutation in BTK (C481S), but subsequent sample showed a mutation in PLCg2 (R665W), in addition to the BTK mutation. All mutations detected in the peripheral blood cells were also detectable in cell-free DNA (cfDNA) using HS testing. However, without using HS testing, a BTK mutation was detected in cfDNA from a patient and this mutation was not detectable when cellular DNA was used.Conclusions: Our data suggests that ibrutinib-naïve patients with CLL do not have BTK or PLCγ2 mutations even when a highly sensitive assay is used. Emerging BTK or PLCγ2 mutant clones can be seen after therapy with the possibility of multiple clones emerging at the same time and may involve both BTK and PLCγ2 genes in the same patient. Furthermore, testing cfDNA is not only as informative as cellular DNA, but might show mutations earlier than cellular DNA. This may have clinical relevance in patients with lymphoma when only few lymphoma cells are in circulation. [Display omitted] DisclosuresWiestner:Pharmacyclics: Research Funding.

Prevalence of BTK mutations in male Algerian patterns with agammaglobulinemia and severe B cell lymphopenia

X linked agammaglobulinemia (XLA) is the first described primary immunodeficiency and the most common form of agammaglobulinemia. It is characterized by susceptibility to recurrent infections, profound decrease of all immunoglobulin isotypes and very low level of B lymphocytes in peripheral blood. The disorder is caused by mutations in the Bruton's Tyrosine Kinase (BTK). Nine male patients suspected to have XLA from nine unrelated families were enrolled in this study. We performed sequencing of the BTK gene in all nine patients, and in the patients' relatives when possible. The XLA diagnosis was confirmed for six patients with six different mutations; we identified a novel mutation (c.1522G>A) and five known mutations. One third of nine unrelated patients do not have mutations in BTK and thus likely suffer from autosomal recessive agammaglobulinemia in the setting of consanguinity. Our results support that the autosomal recessive agammaglobulinemia can be more common in Algeria.

A novel BTK gene mutation creates a de-novo splice site in an X-linked agammaglobulinemia patient

Bruton's tyrosine kinase (BTK), encoded by the BTK gene, is a cytoplasmic protein critical in B cell development. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA), a primary immunodeficiency with characteristically low or absent B cells and antibodies. This report describes a five year-old boy who presented with otitis externa, arthritis, reduced immunoglobulins and no B cells. Flow cytometry showed undetectable monocyte BTK expression. Sequencing revealed a novel mutation at exon 13 of the BTK gene which created a de novo splice site with a proximal 5 nucleotide loss resulting in a truncated BTK protein. The patient still suffered from ear infection despite intravenous immunoglobulin replacement therapy. In this study, mosaicism was seen only in the mother's genomic DNA. These results suggest that a combination of flow cytometry and BTK gene analysis is important for XLA diagnosis and carrier screening.

Acquired in Vitro Resistance to Ibrutinib Is Associated with Transcriptional Re-Programming and Sustained Survival Signaling in Waldenströms Macroglobulinemia and Mantle Cell Lymphoma, Independent of BTK Cys481 Mutation

Background: While B-cell receptor (BCR) signaling is essential for the development, of normal B cells, its aberrant hyper-activation results in neoplastic transformation of B-lymphocytes. Recent investigations using small molecule inhibitors validate the BCR pathway as a valuable target. Bruton’s tyrosine kinase (BTK) is one of the components of a signaling hub that transduces signals from the BCR into the cell for its activation and has been shown to be a therapeutic target. Ibrutinib (PCI-32765), an irreversible BTK inhibitor has shown clinical efficacy in CLL, mantle cell lymphoma (MCL) and Waldenströms macroglobulinemia (WM). Ibrutinib binds to cysteine-481 of the BTK protein and blocks its phosphorylation, resulting in termination of BCR-mediated activation of cells with a concomitant induction of death. Despite the clinical success of ibrutinib, a high percentage of patients achieve only partial response and eventually acquire resistance to the drug, resulting in aggressive relapse of the disease. A mutation of Cys481-Ser in BTK (ibrutinib-BTK binding site) has been reported to be one of the reasons for the development of ibrutinib resistance (IR). To understand the mechanisms resulting in acquisition of IR, we developed preclinical models of IR in WM and MCL.Materials: Ibrutinib was obtained from Pharmacyclics, CA. Validated human WM models (BCWM.1, RPCI-WM1 and MWCL.1 cell lines) and human MCL models (Jeko-1 and Maver cell lines) were used for the study.Results: BTK was constitutively phosphorylated at Y223 and Y551 in all the cell lines tested and this was inhibited by ibrutinib in a dose dependent manner. Phosphorylation of other kinases in the cascade such as SYK (Y323 and Y525/526) and PLCg2 (Y759 and Y1217) were also inhibited while AKT phosphorylation at both Ser473 and Thr308 was consistently increased in presence of ibrutinib. Treatment with ibrutinib induced cell cycle arrest in the G1 phase by 24h followed by apoptosis. Cell growth assays (MTS assay) showed that BCWM.1 was the most sensitive cell line followed by MWCL-1, RPCI-WM1, Maver and Jeko-1. Exposure of WM and MCL cells for prolonged periods of time with progressively increasing concentrations of ibrutinib resulted in outgrowth of clones (IR WM and MCL cell lines) that were resistant to apoptosis with a slow growth rate as compared to wild type parental cells. IR cells attained 2 – 20 fold resistance to ibrutinib as compared to the respective parental lines as determined by MTS assay. Sequence analysis of the BTK gene in all the cell lines revealed no mutation in IR cells at Cys481 suggesting that in an acquired IR state, resistance to ibrutinib can be developed independent of BTK Cys481 mutation. Interestingly, we found p-BTK levels to be markedly reduced in IR cells. Ibrutinib reversal experiments suggested that while a continuous presence of ibrutinib is needed for inhibition of BTK phosphorylation, a stable IR state could be maintained (for >1 month) in the absence of ibrutinib. This suggested the cells reliance on a parallel survival pathway, independent of BTK phosphorylation. Focused mRNA (Nanostring nCounter assay) and immunoblot analysis revealed significant changes in the expression profiles of several cellular elements. These included transcription factors such as PU.1, IRF4, BLIMP1, BCL-6 b-catenin as well as the phosphorylated ERK1/2, STAT1 and 3 suggesting a reprogramming of critical cellular networks, which IR tumor cells might be utilizing to overcome ibrutinib-induced cytotoxicity. Importantly, we observed that IR cells retained high levels of p-AKT and showed an increase in expression of BCL2 family members, as well as BCL-2 itself. Treatment of IR cells with ibrutinib +/- MK2206 (AKT inhibitor), or ABT-199 (BCL-2 inhibitor), synergistically induced cytotoxicity in IR cells, suggesting the importance of these parallel survival pathways (AKT/BCL2) in maintaining an IR state.Conclusion: Here we demonstrate that in the absence of BTK Cys481 mutation, an IR state is associated with reprogramming of transcriptional networks countering ibrutinib-induced toxicity by activation of AKT and BCL-2. Our current data exposes multiple vulnerabilities within IR cells, which can be therapeutically exploited to potentially delay onset of IR, by targeting alternative oncogenic mechanisms that are activated in presence of sustained BTK inhibition. DisclosuresNo relevant conflicts of interest to declare.