Giuseppe Palermo

Investigation of single nucleotide polymorphisms and biological pathways associated with response to TNFα inhibitors in patients with rheumatoid arthritis.

Objective Recently, two genome-wide association studies identified single nucleotide polymorphisms (SNPs) significantly associated with the treatment response to tumor necrosis factor &agr; (TNF&agr;) inhibitors in patients with rheumatoid arthritis (RA). We aimed to replicate these results and identify SNPs and the possible biological pathways associated with the treatment response to TNF&agr; inhibitors. Methods TNF&agr;-naive patients with RA, who had available DNA and initiated TNF&agr; inhibitor therapy between 1999 and 2008, were identified in the DANBIO registry and genotyped using the Illumina HumanHap550K Duo array. The associations between SNPs and changes in the absolute and the relative Disease Activity Score, and European League Against Rheumatism good versus no response after 14 weeks of treatment were tested. SNP data were combined with two independent cohorts in a meta-analysis. A gene-set enrichment analysis (GSEA) was carried out to identify the biological pathways associated with the treatment response. Results After genotyping and quality control, 486 450 SNPs were analyzed in 196 Danish patients with moderate to severe RA treated with infliximab (n=142), etanercept (n=12), and adalimumab (n=42). None of the previously identified SNPs were confirmed in our dataset or in meta-analyses of available studies. Other potential SNPs were identified, but none achieved genome-wide significance. A GSEA identified the transforming growth factor &bgr;, TNF, mitogen-activated protein kinase, and mammalian target of rapamycin pathways to have a potential influence on the treatment response. Conclusion In a genome-wide association study of 196 genetically homogenous Danish patients with RA and in a meta-analysis, we found no SNPs associated with treatment response to TNF&agr; inhibitors. A GSEA suggested that the transforming growth factor &bgr;, TNF, mitogen-activated protein kinase, and mammalian target of rapamycin pathways may be associated with treatment response.

Inactivation of TP53 correlates with disease progression and low miR-34a expression in previously treated chronic lymphocytic leukemia patients

In chronic lymphocytic leukemia (CLL) patients, disruptions of the TP53 tumor suppressor pathway by 17p13 deletion (del17p), somatic TP53 mutations, or downregulation of microRNA-34a have been associated with a poor prognosis. So far, the impact of the various TP53 defects has not been evaluated in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53 mutations and 37 had del17p. In 24 (46%) of the TP53 mutated patients, no del17p was found and in 9 of the del17p patients, no TP53 mutation was identified. Based on a predicted proportion of TP53 disruption, a complete disruption of TP53 function, either by a combination of point mutations and/or del17p, was associated with a high risk for disease progression. Progression-free survival of patients with a heterozygous TP53 mutation was not significantly different from patients with a completely intact TP53 locus. In addition, only a complete loss of TP53 function correlated with low microRNA-34a expression levels. This trial was registered at www.clinicaltrials.gov as #NCT00090051.

Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2)

Primary hyperaldosteronism is a common cause of resistant hypertension. Aldosterone is produced in the adrenal by aldosterone synthase (AS, encoded by the gene CYP11B2). AS shares 93% homology to 11&bgr;-hydroxylase (encoded by the gene CYP11B1), responsible for cortisol production. This homology has hitherto impeded the development of a drug, which selectively suppresses aldosterone but not cortisol production, as a new treatment for primary hyperaldosteronism. We now report the development of RO6836191 as a potent (Ki 13 nmol/L) competitive inhibitor of AS, with in vitro selectivity >100-fold over 11&bgr;-hydroxylase. In cynomolgus monkeys challenged with synthetic adrenocorticotropic hormone, single doses of RO6836191 inhibited aldosterone synthesis without affecting the adrenocorticotropic hormone–induced rise in cortisol. In repeat-dose toxicity studies in monkeys, RO6836191 reproduced the adrenal changes of the AS−/− mouse: expansion of the zona glomerulosa; increased expression of AS (or disrupted green fluorescent protein gene in the AS−/− mouse); hypertrophy, proliferation, and apoptosis of zona glomerulosa cells. These changes in the monkey were partially reversible and partially preventable by electrolyte supplementation and treatment with an angiotensin-converting enzyme inhibitor. In healthy subjects, single doses of RO6836191, across a 360-fold dose range, reduced plasma and urine aldosterone levels with maximum suppression at a dose of 10 mg, but unchanged cortisol, on adrenocorticotropic hormone challenge, up to 360 mg, and increase in the precursors 11-deoxycorticosterone and 11-deoxycortisol only at or >90 mg. In conclusion, RO6836191 demonstrates that it is possible to suppress aldosterone production completely in humans without affecting cortisol production. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01995383.

PTK2 expression and immunochemotherapy outcome in chronic lymphocytic leukemia

Addition of rituximab (R) to fludarabine and cyclophosphamide (FC) has significantly improved patient outcomes in chronic lymphocytic leukemia (CLL). Whether baseline gene expression can identify patients who will benefit from immunochemotherapy over chemotherapy alone has not been determined. We assessed genome-wide expression of 300 pretreatment specimens from a subset of 552 patients in REACH, a study of FC or R-FC in relapsed CLL. An independent test set was derived from 282 pretreatment specimens from CLL8, a study of FC or R-FC in treatment-naïve patients. Genes specific for benefit from R-FC were determined by assessing treatment-gene interactions in Cox proportional hazards models. REACH patients with higher pretreatment protein tyrosine kinase 2 (PTK2) messenger RNA levels derived greater benefit from R-FC, with significant improvements in progression-free survival, independent of known prognostic factors in a multivariate model. Examination of PTK2 gene expression in CLL8 patients yielded similar results. Furthermore, PTK2 inhibition blunted R-dependent cell death in vitro. This retrospective analysis from 2 independent trials revealed that increased PTK2 expression is associated with improved outcomes for CLL patients treated with R-FC vs FC. PTK2 expression may be a useful biomarker for patient selection in future trials. These trials were registered at www.clinicaltrials.gov as #NCT00090051 (REACH) and #NCT00281918 (CLL8).

Motor, cognitive, and functional declines contribute to a single progressive factor in early HD

Objective: To identify an improved measure of clinical progression in early Huntington disease (HD) using data from prospective observational cohort studies and placebo group data from randomized double-blind clinical trials. Methods: We studied Unified Huntington Disease Rating Scale (UHDRS) and non-UHDRS clinical measures and brain measures of progressive atrophy in 1,668 individuals with early HD followed up prospectively for up to 30 to 36 months of longitudinal clinical follow-up. Results: The results demonstrated that a composite measure of motor, cognitive, and global functional decline best characterized clinical progression and was most strongly associated with brain measures of progressive corticostriatal atrophy. Conclusions: Use of a composite motor, cognitive, and global functional clinical outcome measure in HD provides an improved measure of clinical progression more related to measures of progressive brain atrophy and provides an opportunity for enhanced clinical trial efficiency relative to currently used individual motor, cognitive, and functional outcome measures.

Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. Various disease-related and patient-related factors have been shown to influence the course of the disease. The aim of this study was to identify novel biomarkers of significant clinical relevance. Pretreatment CD19-separated lymphocytes (n=237; discovery set) and peripheral blood mononuclear cells (n=92; validation set) from the REACH trial, a randomized phase III trial in relapsed CLL comparing rituximab plus fludarabine plus cyclophosphamide with fludarabine plus cyclophosphamide alone, underwent gene expression profiling. By using Cox regression survival analysis on the discovery set, we identified inositol polyphosphate-5-phosphatase F (INPP5F) as a prognostic factor for progression-free survival (P<0.001; hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.35–1.98) and overall survival (P<0.001; HR, 1.47; 95% CI, 1.18–1.84), regardless of adjusting for known prognostic factors. These findings were confirmed on the validation set, suggesting that INPP5F may serve as a novel, easy-to-assess future prognostic biomarker for fludarabine-based therapy in CLL.

Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease

Mutant huntingtin and neurofilament in biofluids may have prognostic potential in Huntington’s disease. Improving Huntington’s disease detection Early detection of Huntington’s disease (HD) could help the development of effective therapeutic strategies to block or delay disease progression. Byrne and colleagues now show that in blood and cerebrospinal fluid, mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations correlated with disease severity in HD patients. Computational analysis further showed that alterations in circulating NfL and mHTT concentrations may be among the earliest detectable changes in HD. Thus, the results suggest that analysis of mHTT and NfL concentrations in biofluids might be used in combination with other clinical measures for improving the accuracy and efficiency of early HD detection. Huntington’s disease (HD) is a genetic progressive neurodegenerative disorder, caused by a mutation in the HTT gene, for which there is currently no cure. The identification of sensitive indicators of disease progression and therapeutic outcome could help the development of effective strategies for treating HD. We assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF) and blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. Among HD mutation carriers, NfL concentrations in plasma and CSF correlated with all nonbiofluid measures more closely than did CSF mHTT concentration. Longitudinal analysis over 4 to 8 weeks showed that CSF mHTT, CSF NfL, and plasma NfL concentrations were highly stable within individuals. In our cohort, concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. In silico modeling indicated that mHTT and NfL concentrations in biofluids might be among the earliest detectable alterations in HD, and sample size prediction suggested that low participant numbers would be needed to incorporate these measures into clinical trials. These findings provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD and could be integrated into both clinical trials and the clinic.

F24 Design of a prospective, longitudinal, natural history study in huntington’s disease

Background With the advent of huntingtin protein-lowering therapies and the ability to measure mutant huntingtin protein (mHTT) in cerebrospinal fluid (CSF), there is a need to better understand the potential of CSF mHTT as a biomarker for predicting and measuring Huntington’s disease (HD) progression. Observational studies including HD-Clarity and UCL-CSF aim to fill this knowledge gap but CSF is only collected once every 1- or 2-years, respectively, leaving unexplored the shorter-term timecourse of CSF mHTT and associated change in biological and clinical outcome measures. Aim We are conducting a Natural History study with a primary objective to determine the relationship between CSF levels of mHTT, clinical measures of HD progression, markers of neuronal injury and brain atrophy over 15 months. Methods/Results This prospective, longitudinal, multi-site Natural History study will follow patients with early manifest HD (Stage I or II). Clinical outcome measures will include composite Unified Huntington’s Disease Rating Scale (cUHDRS), Total Functional Capacity (TFC), Total Motor Score (TMS), Symbol Digit Modalities Test (SDMT), and Stroop Word Reading (SWR) test. Other assessments include the measurement of CSF levels of mHTT, blood biomarkers and brain imaging. The utility of clinical outcome measures collected via sensors in smartphones and wrist-worn wearables will be assessed. CSF will be collected at four time points, namely, baseline, 3, 9 and 15 months. Conclusion This study will provide valuable information on the relationship between putative biomarkers, including mHTT, and clinical outcomes in HD. It is expected to aid interpretation of future clinical trials. Funded by F. Hoffmann-La Roche.

Results and evaluation of a first‐in‐human study of RG7342, an mGlu5 positive allosteric modulator, utilizing Bayesian adaptive methods

The objectives of this first‐in‐human study were to evaluate the safety and tolerability, pharmacokinetics and pharmacodynamics, and maximum tolerated dose (MTD) of single ascending oral doses of RG7342, a positive allosteric modulator (PAM) of the metabotropic glutamate receptor 5 (mGlu5) for the treatment of schizophrenia, in healthy male subjects.