Shruti Sharma

Widely divergent transcriptional patterns between SLE patients of different ancestral backgrounds in sorted immune cell populations.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease of uncertain etiology. Patients from different ancestral backgrounds demonstrate differences in clinical manifestations and autoantibody profiles. We examined genome-wide transcriptional patterns in major immune cell subsets across different ancestral backgrounds. Peripheral blood was collected from African-American (AA) and European-American (EA) SLE patients and controls. CD4 T-cells, CD8 T-cells, monocytes, and B cells were purified by flow sorting, and each cell subset from each subject was run on a genome-wide expression array. Cases were compared to controls of the same ancestral background. The overlap in differentially expressed gene (DEG) lists between different cell types from the same ancestral background was modest (<10%), and only 5-8% overlap in DEG lists was observed when comparing the same cell type between different ancestral backgrounds. IFN-stimulated gene (ISG) expression was not up-regulated synchronously in all cell types from a given patient, for example a given subject could have high ISG expression in T and B cells, but not in monocytes. AA subjects demonstrated more concordance in ISG expression between cell types from the same individual, and AA patients demonstrated significant down-regulation of metabolic gene expression which was not observed in EA patients. ISG expression was significantly decreased in B cells in patients taking immunosuppressants, while ISGs in other cell types did not differ with medication use. In conclusion, gene expression was strikingly different between immune cell subsets and between ancestral backgrounds in SLE patients. These findings emphasize the critical importance of studying multiple ancestral backgrounds and multiple cell types in gene expression studies. Ancestral backgrounds which are not studied will not benefit from personalized medicine strategies in SLE.

Clinical Validation of a Next-Generation Sequencing Genomic Oncology Panel via Cross-Platform Benchmarking against Established Amplicon Sequencing Assays

Next-generation sequencing (NGS) genomic oncology profiling assays have emerged as key drivers of personalized cancer care and translational research. However, validation of these assays to meet strict clinical standards has been historically problematic because of both significant assay complexity and a scarcity of optimal validation samples. Herein, we present the clinical validation of 76 genes from a novel 1212-gene large-scale hybrid capture cancer sequencing assay (University of Chicago Medicine OncoPlus) using full-data comparisons against multiple clinical NGS amplicon-based assays to yield dramatic increases in per-sample data comparison efficiency compared with previously published validations. Using a sample set of 104 normal, solid tumor, and hematopoietic malignancy specimens, head-to-head NGS data analyses allowed for 6.8 million individual clinical base call comparisons, including 2729 previously confirmed variants, with 100% sensitivity and specificity. University of Chicago Medicine OncoPlus showed excellent performance for detection of single-nucleotide variants, insertions/deletions up to 52 bp, and FLT3 internal tandem duplications of up to 102 bp or larger. Highly concordant copy number variant and ALK/RET/ROS1 gene fusion detection were also observed. In addition to underlining the efficiency of NGS validation via full-data benchmarking against existing clinical NGS assays, this study also highlights the degree of performance similarity between hybrid capture and amplicon assays that is attainable with the application of strict quality control parameters and optimized computational analytics.

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patients clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

Clonal evolution underlying leukemia progression and Richter transformation in patients with ibrutinib-relapsed CLL

Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTKC481 , we identified BTKT316A , a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts. Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

B-Cell Lymphoma Patient-Derived Xenograft Models Enable Drug Discovery and Are a Platform for Personalized Therapy

Purpose: Patients with B-cell lymphomas often relapse after frontline therapy, and novel therapies are urgently needed to provide long-term remission. We established B-cell lymphoma patient-derived xenograft (PDX) models to assess their ability to mimic tumor biology and to identify B-cell lymphoma patient treatment options. Experimental Design: We established the PDX models from 16 patients with diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, or Burkitt lymphoma by inoculating the patient tumor cells into a human bone chip implanted into mice. We subjected the PDX models to histopathologic and phenotypical examination, sequencing, and drug efficacy analysis. Primary and acquired resistance to ibrutinib, an oral covalent inhibitor of Bruton tyrosine kinase, were investigated to elucidate the mechanisms underlying ibrutinib resistance and to identify drug treatments to overcome resistance. Results: The PDXs maintained the same biological, histopathologic, and immunophenotypical features, retained similar genetic mutations, and produced comparable drug responses with the original patient tumors. In the acquired ibrutinib-resistant PDXs, PLC-γ2, p65, and Src were downregulated; however, a PI3K signaling pathway member was upregulated. Inactivation of the PI3K pathway with the inhibitor idelalisib in combination with ibrutinib significantly inhibited the growth of the ibrutinib-resistant tumors. Furthermore, we used a PDX model derived from a clinically ibrutinib-relapsed patient to evaluate various therapeutic choices, ultimately eliminating the tumor cells in the patients peripheral blood. Conclusions: Our results demonstrate that the B-cell lymphoma PDX model is an effective system to predict and personalize therapies and address therapeutic resistance in B-cell lymphoma patients. Clin Cancer Res; 23(15); 4212–23. ©2017 AACR.

Is vascular endothelial growth factor receptor 2 (VEGFR2) involved in systemic lupus erythematosus (SLE)? A combined structural biological and genetic approach

Background The VEGFR2 directly regulates the formation of blood vessels, a process dependent upon the function of specialised cell-types including vascular endothelial cells. VEGFR2 gene polymorphisms have been correlated with vascular diseases such as Coronary Heart Disease (CHD). Aim In view of the premature atherosclerosis observed in SLE, the authors sought to clarify the structural/functional consequences of two common polymorphisms in VEGFR2 in SLE and determine whether these polymorphisms are associated with risk of SLE by influencing endothelial cells. Materials and methods Three-dimensional (3D) homology modeling of the mutation V297I was based on the 3D structure of domains D2 and D3 of VEGFR2 in complex with VEGF-C, while mutation Q472H was investigated by homology modeling on the KIT ectodomain structure. The V297I (rs2305948) and Q472H (rs1870377) single nucleotide polymorphisms (SNPs) in VEGFR2 were genotyped with Taqman technology in 250 SLE patients and 241 healthy controls from a Greek population (Cretan). The replication sample set for the rs1870377 SNP consisted of 253, 184 and 77 patients with SLE of African-, European- and Hispanic-American origin, respectively, as well as geographically/ethnically-matched controls. Results Modeling revealed that amino acid position #297 is located on the D3 Ig-like domain of the extracellular region of VEGFR2 on a surface loop and polymorphism V297I affects the efficiency of trans-autophosphorylation and cell signaling. On the other hand, position #472 is located on the surface of the D5 domain and mutation Q472H affects homotypic contacts of membrane proximal Ig-like domains. This finding prompted us to examine whether these polymorphisms contribute to SLE risk or vascular damage. No significant difference was observed in the frequency of the minor allele A of rs1870377 when SLE patients were compared with controls either in the Greek population or in the 3 replication groups analysed. No association was detected between rs2305948 SNP and SLE when tested in the Greek population. Conclusions Although structural data suggest that both VEGFR2 SNPs may contribute to SLE pathogenesis by impairing cell signaling, none of the SNPs analysed was associated with increased susceptibility to SLE. Experiments in progress address the role of these polymorphisms on the number of circulating endothelial cells and their possible influence to vascular injury observed in the disease.

THU0491 Genome-Wide Transcriptional Profiling of Isolated Immune Cell Populations from SLE Patients with Different Ancestral Backgrounds

Background Systemic lupus erythematosus (SLE) is a complex multi-system autoimmune disease of uncertain etiology. Patients from different ancestral backgrounds demonstrate differences in clinical manifestations and autoantibody profiles. Objectives In this study we examined genome-wide transcriptional patterns in major immune cell subsets across different ancestral backgrounds. Methods Peripheral blood was collected and run on 208 Illumina HumanHT-12 V4 expression BeadChip arrays. Subjects included 21 African-American (AA) and 21 European-American (EA) SLE patients, 5 AA and 5 EA controls. CD4+ T-cells, CD8+ T-cells, monocytes and B cells were purified by flow sorting. Each cell subset from each subject was run on a separate array. Differentially expressed genes (DEGs) were determined by comparing cases and controls of the same ancestral background. Results The overlap in DEG lists between different cell types from the same ancestral background was very modest (<1%). Typically between 5-10% of DEGs were shared when comparing the same cell type between different ancestral backgrounds. Global IFN-stimulated gene (ISG) expression revealed that AA subjects demonstrated more concordance across all studied cell types. Two subgroups of patients were identified based on the ISG expression profiles. One subgroup showed higher ISGs expression in all cell types, and the other subgroup had higher ISG expression only in T and B lymphocytes but not in monocytes. Conclusions We find striking differences in gene expression between different immune cell subsets and between ancestral backgrounds in SLE patients. The IFN signature is diverse, with different transcripts represented in different cell populations, and signature-positive cell subsets differed in EA vs. AA patients. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5291