Medical Prognosis Institute (Denmark)

e20025 Background: Multiple myeloma (MM) is one of the most common hematological malignancies. It tends to be a hard-to-treat cancer since all patients eventually progress due to drug resistance. Given the current lack of lasting therapeutic benefit for MM patients, there is a need for new treatment options. We aim to address this issue by introducing a new immune-oncology drug (APO010) in MM treatment. APO010 is a hexameric FAS-ligand and acts like a cytotoxic T-lymphocyte to induce apoptosis in cancer cells and could therefore be a new effective drug for MM as these cells express CD95 (FAS-receptor). APO010 is now in a clinical trial in MM patients. However, as resistance to APO010 may develop in these patients, it is important to understand the molecular mechanism underlying this resistance as such knowledge could lead to the identification of predictive biomarkers of APO010 in MM treatment. A 160-gene predictor of APO010 sensitivity is currently used in the clinic. Methods: We developed APO010-resistant variants of four human myeloma cell lines (LP1, Raji, MOLP-8 and KMS-12-BM) by exposing the cells to gradually increasing concentrations of APO010 over a period of 6 months to 1 year. The resistant cell lines were characterized on the basis of FAS-receptor protein expression, global gene expression and pathway analysis. Results: All APO010 resistant cell lines showed significant increase in resistance to APO010 as compared to their parental controls. The protein expression of FAS-receptor was down-regulated in all resistant cell lines, completely in LP1APO and KMS-12-BMAPO and partially in RajiAPO and MOLP-8APO. Gene expression profiling of the resistant and parental lines did not show any significant alteration in FAS receptor expression between sensitive and resistant cell lines, but did reveal differences in mRNA processing. The 160-gene APO010 predictor in all cases predicted a lower sensitivity of the resistant cell line than its parental sensitive cell line. Conclusions: Based on data obtained so far points to a post-transcriptional or post-translational modification of the FAS-receptor being responsible for APO010 resistance.

IntroductionEffective predictive biomarkers for selection of patients benefiting from adjuvant platinum-based chemotherapy in non-small cell lung cancer (NSCLC) are needed. Based on a previously validated methodology, molecular profiles of predicted sensitivity in two patient cohorts are presented.MethodsThe profiles are correlations between in vitro sensitivity to cisplatin and vinorelbine and baseline mRNA expression of the 60 cell lines in the National Cancer Institute panel. An applied clinical samples filter focused the profiles to clinically relevant genes. The profiles were tested on 1) snap-frozen tumors from 133 patients with completely resected stage 1B-2 NSCLC randomized to adjuvant cisplatin and vinorelbine (ACV, n = 71) or no adjuvant treatment (OBS, n = 62) and 2) formalin-fixed paraffin-embedded (FFPE) tumors from 95 patients with completely resected stage 1A-3B NSCLC receiving adjuvant cisplatin and vinorelbine.ResultsThe combined cisplatin and vinorelbine profiles showed: 1) univariate Hazard Ratio (HR) for sensitive versus resistant of 0.265 (95% CI:0.079–0.889, p = 0.032) in the ACV cohort and a HR of 0.28 in a multivariate model (95% CI:0.08–1.04, p = 0.0573); 2) significant prediction at 3 year survival from surgery in univariate (HR = 0.138 (95% CI:0.035–0.537), p = 0.004) and multivariate analysis (HR = 0.14 (95% CI:0.030–0.6), p = 0.0081). No discrimination was found in the OBS cohort (HR = 1.328, p = 0.60). The cisplatin predictor alone had similar figures with 1) univariate HR of 0.37 (95% CI:0.12–1.15, p = 0.09) in the ACV cohort and 2) univariate HR of 0.14 (95% CI:0.03–0.59, p = 0.0076) to three years. Functional analysis on the cisplatin profile revealed a group of upregulated genes related to RNA splicing as a part of DNA damage repair and apoptosis.ConclusionsProfiles derived from snap-frozen and FFPE NSCLC tissue were prognostic and predictive in the patients that received cisplatin and vinorelbine but not in the cohort that did not receive adjuvant treatment.

ABSTRACTHistone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.

Background: This study evaluated the predictive effect of a mRNA-based Drug Response Predictor DRP in the adjuvant setting of non-small cell lung cancer (NSCLC). We apply a mathematical method combining in vitro sensitivity with gene expression patterns in tumors (Knudsen et al PLoS ONE 2014 9(2): e87415.). The cisplatinum DRP has previously been tested in three independent datasets (ASCO Abstract 2015 e18502). Methods: RNA was isolated from formalin-fixed paraffin embedded tumor tissue obtained after radical surgical resection in 95 consecutive stage Ib-IIIb NSCLC patients of different histotypes. Adjuvant chemotherapy was four courses of i.v. cisplatinum and vinorelbine. Affymetrix HG-133_Plus_2 microarray method was applied. Multigene biomarker profiles for cisplatinum and vinorelbine were evaluated blinded on the samples. Primary endpoint was overall survival (OS). Results: Baseline demographics are representative of a NSCLC population. Follow-up time was in median 76.6 months (range 3.5-7 years) after surgery. Statistical analysis suggested that time-dependent analysis of OS was warranted. Using a time dependent Cox model stratifying at 3 years, the hazard ratio (HR) for OS up to 3 years was 0.50 (95% CI: 0.30-0.84, p = 0.008) and the HR for cancer-specific death was 0.46 (95% CI: 0.27-0.78, p = 0.004). Results for multivariable analyses for OS are shown in the table. The DRP remained significant in the multivariable analysis for each endpoint. Conclusions: The DRP for the combined cisplatinum and vinorelbine adjuvant treatment of resectable NSCLC offers definite prediction of effect of the two drugs since it is significant within 36 months after surgery.

e18502 Background: There are no established biomarkers for prediction of clinical platinum sensitivity. A new platinum response prediction method using multigene expression is reported. Methods: We...

Physical activity and molecular ageing presumably interact to precipitate musculoskeletal decline in humans with age. Herein, we have delineated molecular networks for these two major components of sarcopenic risk using multiple independent clinical cohorts. We generated genome-wide transcript profiles from individuals (n = 44) who then undertook 20 weeks of supervised resistance-exercise training (RET). Expectedly, our subjects exhibited a marked range of hypertrophic responses (3% to +28%), and when applying Ingenuity Pathway Analysis (IPA) up-stream analysis to ,580 genes that covaried with gain in lean mass, we identified rapamycin (mTOR) signaling associating with growth (P = 1.4610 230 ). Paradoxically, those displaying most hypertrophy exhibited an inhibited mTOR activation signature, including the striking down-regulation of 70 rRNAs. Differential analysis found networks mimicking developmental processes (activated all-transretinoic acid (ATRA, Z-score = 4.5; P = 6610 213 ) and inhibited aryl-hydrocarbon receptor signaling (AhR, Z-score = 22.3; P = 3610 27 )) with RET. Intriguingly, as ATRA and AhR gene-sets were also a feature of endurance exercise training (EET), they appear to represent ''generic'' physical activity responsive gene-networks. For age, we found that differential geneexpression methods do not produce consistent molecular differences between young versus old individuals. Instead, utilizing two independent cohorts (n = 45 and n = 52), with a continuum of subject ages (18-78 y), the first reproducible set of age-related transcripts in human muscle was identified. This analysis identified ,500 genes highly enriched in posttranscriptional processes (P = 1610 26 ) and with negligible links to the aforementioned generic exercise regulated gene-sets and some overlap with ribosomal genes. The RNA signatures from multiple compounds all targeting serotonin, DNA topoisomerase antagonism, and RXR activation were significantly related to the muscle age-related genes. Finally, a number of specific chromosomal loci, including 1q12 and 13q21, contributed by more than chance to the age-related gene list (P = 0.01-0.005), implying possible epigenetic events. We conclude that human muscle age-related molecular processes appear distinct from the processes regulated by those of physical activity.

Insulin resistance in skeletal muscle is a major risk factor for the development of type 2 diabetes in women with polycystic ovary syndrome (PCOS). In patients with type 2 diabetes, insulin resistance in skeletal muscle is associated with abnormalities in insulin signaling, fatty acid metabolism, and mitochondrial oxidative phosphorylation (OXPHOS). In PCOS patients, the molecular mechanisms of insulin resistance are, however, less well characterized. To identify biological pathways of importance for the pathogenesis of insulin resistance in PCOS, we compared gene expression in skeletal muscle of metabolically characterized PCOS patients (n = 16) and healthy control subjects (n = 13) using two different approaches for global pathway analysis: gene set enrichment analysis (GSEA 1.0) and gene map annotator and pathway profiler (GenMAPP 2.0). We demonstrate that impaired insulin-stimulated total, oxidative and nonoxidative glucose disposal in PCOS patients are associated with a consistent downregulation of OXPHOS gene expression using GSEA and GenMAPP analysis. Quantitative real-time PCR analysis validated these findings and showed that reduced levels of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha) could play a role in the downregulation of OXPHOS genes in PCOS. In these women with PCOS, the decrease in OXPHOS gene expression in skeletal muscle cannot be ascribed to obesity and diabetes. This supports the hypothesis of an early association between insulin resistance and impaired mitochondrial oxidative metabolism, which is, in part, mediated by reduced PGC-1alpha levels. These abnormalities may contribute to the increased risk of type 2 diabetes observed in women with PCOS.

This chapter contains sections titled: Microarray Studies of Brain Tumors Summary Further Reading

This chapter includes the following topics: Selection of Genes for an Array Gene Finding Selection of Regions Within Genes Selection of Primers for PCR Selection of Unique Oligomer Probes Remapping of Probes