The Biomarkers of Lupus Disease Study: A Bold Approach May Mitigate Interference of Background Immunosuppressants in Clinical Trials.

OBJECTIVE: Molecular medicine raised expectations for strategically targeted biologic agents in systemic lupus erythematosus (SLE), but clinical trial results have been disappointing and difficult to interpret. Most studies add investigational agents to various, often effective, standard therapy immunosuppressants used at baseline, with unknown treatment interactions. Eliminating polypharmacy in trials of active lupus remains controversial. We undertook the Biomarkers of Lupus Disease study to test withdrawal of immunosuppressants as a novel approach to rendering SLE trials interpretable. METHODS: In 41 patients with active, non-organ-threatening SLE flare (group A), temporary steroids were given while background immunosuppressants were withdrawn. Time to loss of disease suppression (time to disease flare) and safety were evaluated; standard therapy was immediately resumed when symptoms recurred. Immunologic impacts of standard therapy were studied at baseline by multiplex assay, enzyme-linked immunosorbent assay, and messenger RNA array in group A patients plus 62 additional patients donating a single sample (group B). RESULTS: Patients with lower or higher baseline disease activity had median times to flare of 71 or 45 days, respectively; 40 of 41 patients (98%) had disease flares by 6 months. All flares were treated and resolved within 6 weeks. No serious adverse events occurred from flare or infection. Type I interferon (IFN), Th17, and B lymphocyte stimulator pathways tracked together. Baseline immunosuppressants had distinct impacts on Th17 and B lymphocyte stimulator, depending on IFN signature. CONCLUSION: Trials in active, non-organ-threatening SLE can safely withdraw background treatments if patients who have disease flares are designated nonresponders and returned to standard therapy. Immunologic effects of standard therapy vary between IFN-defined subsets. These findings provide a strategy for minimizing or optimizing treatment combinations in lupus trials and clinical care.

Tofacitinib attenuates pathologic immune pathways in patients with psoriasis: A randomized phase 2 study.

BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. OBJECTIVE: We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. METHODS: Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. RESULTS: In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/µm(2); day 1, median of 332 pSTAT1(+) cells/µm(2); and nonlesional, median of 155 pSTAT1(+) cells/µm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. CONCLUSIONS: Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.

Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226.

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.

Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized.

BACKGROUND: Human mammary epithelial cells (HMEC) overcome two well-characterized genetic and epigenetic barriers as they progress from primary cells to fully immortalized cell lines in vitro. Finite lifespan HMEC overcome an Rb-mediated stress-associated senescence barrier (stasis), and a stringent, telomere-length dependent, barrier (agonescence or crisis, depending on p53 status). HMEC that have overcome the second senescence barrier are immortalized. METHODS: We have characterized pre-stasis, post-selection (post-stasis, with p16 silenced), and fully immortalized HMEC by transcription profiling and RT-PCR. Four pre-stasis and seven post-selection HMEC samples, along with 10 representatives of fully immortalized breast epithelial cell lines, were profiled using Affymetrix U133A/B chips and compared using both supervised and unsupervised clustering. Datasets were validated by RT-PCR for a select set of genes. Quantitative immunofluorescence was used to assess changes in transcriptional regulators associated with the gene expression changes. RESULTS: The most dramatic and uniform changes we observed were in a set of about 30 genes that are characterized as a "cancer proliferation cluster," which includes genes expressed during mitosis (CDC2, CDC25, MCM2, PLK1) and following DNA damage. The increased expression of these genes was particularly concordant in the fully immortalized lines. Additional changes were observed in IFN-regulated genes in some post-selection and fully immortalized cultures. Nuclear localization was observed for several transcriptional regulators associated with expression of these genes in post-selection and immortalized HMEC, including Rb, Myc, BRCA1, HDAC3 and SP1. CONCLUSION: Gene expression profiles and cytological changes in related transcriptional regulators indicate that immortalized HMEC resemble non-invasive breast cancers, such as ductal and lobular carcinomas in situ, and are strikingly distinct from finite-lifespan HMEC, particularly with regard to genes involved in proliferation, cell cycle regulation, chromosome structure and the DNA damage response. The comparison of HMEC profiles with lines harboring oncogenic changes (e.g. overexpression of Her-2neu, loss of p53 expression) identifies genes involved in tissue remodeling as well as proinflamatory cytokines and S100 proteins. Studies on carcinogenesis using immortalized cell lines as starting points or "normal" controls need to account for the significant pre-existing genetic and epigenetic changes inherent in such lines before results can be broadly interpreted.

Risk factors associated with beta-amyloid(1-42) immunotherapy in preimmunization gene expression patterns of blood cells.

BACKGROUND: A phase 2a, double-blind, placebo-controlled, multicenter study was conducted to evaluate safety, tolerability, and pilot efficacy of immunization with beta-amyloid((1-42)) in patients with Alzheimer disease. Six immunizations were planned but were halted when meningoencephalitis was recognized as an adverse event in 6% of immunized patients. OBJECTIVE: To identify biomarkers associated with both the risk of meningoencephalitis and antibody responsiveness. PARTICIPANTS: One hundred fifty-three patients with mild to moderate Alzheimer disease.Main Outcome Measure Association between response to immunization and preimmunization expression levels of 8239 messenger RNA transcripts expressed in peripheral blood mononuclear cells that had been collected at the screening visit. RESULTS: Expression patterns of genes related to apoptosis and proinflammatory pathways (tumor necrosis factor pathway in particular) were identified as biomarkers of risk for the development of meningoencephalitis. Expression patterns of genes related to protein synthesis, protein trafficking, DNA recombination, DNA repair, and cell cycle were strongly associated with IgG response to immunization. CONCLUSIONS: Candidate biomarkers associated with risk of immunotherapy-related meningoencephalitis were detected in blood collected prior to treatment. In addition, a different set of biomarkers were identified that were associated with the desired outcome of IgG response.

Clinical pharmacogenomics and transcriptional profiling in early phase oncology clinical trials.

Microarray-based expression profiling studies in the field of oncology have demonstrated encouraging correlations between tumor transcriptional profiles and eventual patient outcomes. These findings have fueled great interest in the application of transcriptional profiling to samples available from real-time clinical trials, and clinical pharmacogenomic objectives utilizing transcriptional profiling strategies are becoming increasingly incorporated into clinical trial study designs. Over the last few years several retrospective studies based on the profiling of archival tumor tissues suggest that transcriptional analysis of oncology samples may provide general prognosis measures, and in some cases may even predict response to specific therapies. Recently the FDA released a voluntary genomic data guidance meant to assist both regulatory agencies and pharmaceutical companies alike in evaluating the potential benefit of implementing expression profiling studies during the preclinical and clinical phases of drug development. Despite the great promise afforded by this technology, the ultimate benefit of applying transcriptional profiling in prospective clinical trials has yet to be realized because a number of practical impediments to this process exist. The multi-fold purpose of the current review is to highlight the increasing evidence from studies that have identified transcriptional signatures in archived tumors prognostic of patient outcome, to describe some of the drivers for the implementation of transcriptional profiling strategies in real-time drug development, to discuss the use of transcriptional profiling in the context of increasingly complex translational medicine strategies, and to highlight the practical issues and potential approaches involved in the successful application of clinical pharmacogenomic objectives during real-time clinical trials. Strategic implementation of transcriptional profiling in early oncology clinical trials can provide an opportunity to identify predictive markers of clinical response and eventually provide a substantial step forward towards the era of personalized medicine.

From differential gene expression to differential gene function and back.

Following the decoding of several plant and animal genomes, the identification of all corresponding transcripts and proteins and understanding how their expression corresponds to physiological and pathological states is the obvious next step. Nucleic acid quantification methods have become increasingly high-throughput and relatively low-cost, and moving ahead, combinations of technologies monitoring differential gene expression and those defining differential cellular function will yield maximum benefit in furthering biology and for drug target identification and validation.: