Houman Khalili

Dissecting the Genetic Complexity of the Association between Human Leukocyte Antigens and Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an inflammatory disease with a complex genetic component. An association between RA and the human leukocyte antigen (HLA) complex has long been observed in many different populations, and most studies have focused on a direct role for the HLA-DRB1 shared epitope in disease susceptibility. We have performed an extensive haplotype analysis, using 54 markers distributed across the entire HLA complex, in a set of 469 multicase families with RA. The results show that, in addition to associations with the DRB1 alleles, at least two additional genetic effects are present within the major histocompatibility complex. One of these lies within a 497-kb region in the central portion of the HLA complex, an interval that excludes DRB1. This genetic risk factor is present on a segment of a highly conserved ancestral A1-B8-DRB1*03 (8.1) haplotype. Additional risk genes may also be present in the HLA class I region in a subset of DRB1*0404 haplotypes. These data emphasize the importance of defining haplotypes when trying to understand the HLA associations with disease, and they clearly demonstrate that such associations with RA are complex and cannot be completely explained by the DRB1 locus.

Genome-Wide Association Scan Identifies Candidate Polymorphisms Associated with Differential Response to Anti-TNF Treatment in Rheumatoid Arthritis

The prediction of response (or non-response) to anti-TNF treatment for rheumatoid arthritis (RA) is a pressing clinical problem. We conducted a genome-wide association study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning anti-TNF therapy as part of Autoimmune Biomarkers Collaborative Network (ABCoN [Autoimmune Bio-markers Collaborative Network]) patient cohort. Response to therapy was determined by the change in Disease Activity Score (DAS28) observed after 14 wks. We used a two-part analysis that treated the change in DAS28 as a continuous trait and then incorporated it into a dichotomous trait of “good responder” and “nonresponder” by European League Against Rheumatism (EULAR) criteria.We corrected for multiple tests by permutation, and adjusted for potential population stratification using EIGENSTRAT. Multiple single nucleotide polymorphism (SNP) markers showed significant associations near or within loci including: the v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene on chromosome 20; the type I interferon gene IFNk on chromosome 9; and in a locus on chromosome 7 that includes the paraoxonase I (PON1) gene. An SNP in the IL10 promoter (rs1800896) that was previously reported as associated with anti-TNF response was weakly associated with response in this cohort. Replications of these results in independent and larger data sets clearly are required. We provide a reference list of candidate SNPs (P < 0.01) that can be investigated in future pharmacogenomic studies.

Peripheral blood gene expression profiling in rheumatoid arthritis

We carried out gene expression profiling of peripheral blood mononuclear cells (PBMCs) in 29 patients with active rheumatoid arthritis (RA) and 21 control subjects using Affymetrix U95Av2 arrays. Using cluster analysis, we observed a significant alteration in the expression pattern of 81 genes (P<0.001) in the PBMCs of RA patients compared with controls. Many of these genes correlated with differences in monocyte counts between the two study populations, and we show that a large fraction of these genes are specifically expressed at high levels in monocytes. In addition, a logistic regression analysis was performed to identify genes that performed best in the categorization of RA and control samples. Glutaminyl cyclase, IL1RA, S100A12 (also known as calgranulin or EN-RAGE) and Grb2-associated binding protein (GAB2) were among the top discriminators. Along with previous data, the overexpression of S100A12 in RA patients emphasizes the likely importance of RAGE pathways in disease pathogenesis. The altered expression of GAB2, an intracellular adaptor molecule involved in regulating phosphatase function, is of particular interest given the recent identification of the intracellular phosphatase PTPN22 as a risk gene for RA. These data suggest that a detailed study of gene expression patterns in peripheral blood can provide insight into disease pathogenesis. However, it is also clear that substantially larger sample sizes will be required in order to evaluate fully gene expression profiling as a means of identifying disease subsets, or defining biomarkers of outcome and response to therapy in RA.

The autoimmunity associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.

Paraneoplastic Syndrome Secondary to Treatment Emergent Neuroendocrine Tumor in Metastatic Castration-resistant Prostate Cancer: A Unique Case

Treatment-emergent neuroendocrine prostate cancer has become increasingly more common owing to the development of second-generation anti-hormonal therapy. It is characterized by a morphology similar to small-cell carcinoma, a low prostate-specific antigen, and visceral metastasis. This case features a paraneoplastic Cushing syndrome, initial resistance to androgen receptortargeted therapy, a continuously rising prostatespecific antigen, and a lack of disease spread to visceral organs. RNA sequencing revealed a gene expression profile consistent with a neuroendocrine tumor and identified the potential therapeutic targets such as Aurora kinase A and EZH2. Regardless of PSA levels and the extent of metastatic disease, patients with initial resistance to androgen receptor-directed and new-onset paraneoplastic syndrome should raise a high suspicion of neuroendocrine prostate cancer.

Abstract P2-07-09: Integrative analysis of miRNA and mRNA expression in metastatic versus non-metastatic triple negative breast cancer

Background: Triple Negative Breast Cancer (TNBC) is a subset of breast cancer that is difficult to treat clinically and is characterized by being estrogen receptor (ER) negative, progesterone receptor (PR) negative, and does not overexpress human epidermal growth factor receptor 2 (HER2). Patients with TNBC tend to have a worse prognosis than other breast cancer subtypes. Methods: We obtained fifteen TNBC sample FFPE tissue blocks with corresponding plasma samples. All samples were from primary tumors; seven samples having metastasized, four samples that had not metastasized and four samples with unknown metastatic status. The total RNA was isolated from FFPE blocks using the RecoverAll Total Nucleic Acid Isolation Protocol. miRNA from plasma was isolated using Ambion9s mirVANA kit. The plasma and tissue miRNAs were evaluated using the QuantStudio qPCR platform, capturing ˜750 miRNAs. The mRNA was processed using the TruSeq RNA Access kit and sequenced on the Illumina NextSeq platform. Analysis of the miRNA and mRNA individually was performed using limma and DESeq2 packages, respectively. Gene enrichment analysis of the mRNA expression was done using the GAGE package on KEGG pathways while the integrative analysis was done with sparse Canonical Correlation Analysis (sCCA) using the PMA pacakge. Results: Analysis of plasma miRNA had four miRNAs with a significant difference in raw p-value (p Conclusions: One of the circulating plasma miRNAs, miR483-3p, has been found to promote tumorigenesis, while miR581f and miR766 have not been reported in cancer to date. Further investigation into these miRNA could provide a feasible biomarker. The downregulation of immune pathways observed within the metastatic TNBC subjects implies immune evasion is of particular importance for metastasis and a targeted immunotherapy may be a viable treatment option. The integrative analysis of the miRNA and mRNA showed an enrichment in pathways previously linked to increased proliferation and chemoresistance, with an increased signal compared to either miRNA or mRNA alone. Citation Format: Shih AJ, Korsunsky I, Guttadauria B, Bhuiya T, Liew A, Khalili H, Gregersen PK, Lee AT. Integrative analysis of miRNA and mRNA expression in metastatic versus non-metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-09.