The Cancer Research UK Stratified Medicine Programme as a model for delivering personalised cancer care.

Genomic screening is routinely used to guide the treatment of cancer patients in many countries. However, several multi-layered factors make this effort difficult to deliver within a clinically relevant timeframe. Here we share the learnings from the CRUK-funded Stratified Medicine Programme for advanced NSCLC patients, which could be useful to better plan future studies.

GSK3ß-SCFFBXW7α mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover.

IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3ß (Glycogen Synthase Kinase 3ß) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.

Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice.

INTRODUCTION: This study assessed KRAS mutation detection and functional characteristics across 13 distinct technologies and assays available in clinical practice, in a blinded manner. METHODS: Five distinct KRAS-mutant cell lines were used to study five clinically relevant KRAS mutations: p.G12C, p.G12D, p.G12V, p.G13D and p.Q61H. 50 cell line admixtures with low (50 and 100) mutant KRAS allele copies at 20%, 10%, 5%, 1% and 0.5% frequency were processed using quantitative PCR (qPCR) (n=3), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) (n=2), next-generation sequencing (NGS) (n=6), digital PCR (n=1) and Sanger capillary sequencing (n=1) assays. Important performance differences were revealed, particularly assay sensitivity and turnaround time. RESULTS: Overall 406/728 data points across all 13 technologies were identified correctly. Successful genotyping of admixtures ranged from 0% (Sanger sequencing) to 100% (NGS). 5/6 NGS platforms reported similar allelic frequency for each sample. One NGS assay detected mutations down to a frequency of 0.5% and correctly identified all 56 samples (Oncomine Focus Assay, Thermo Fisher Scientific). One qPCR (Idylla, Biocartis) and MALDI-TOF (UltraSEEK, Agena Bioscience) assay identified 96% (all 100 copies and 23/25 at 50 copies input) and 92% (23/25 at 100 copies and 23/25 at 50 copies input) of samples, respectively. The digital PCR assay (KRAS PrimePCR ddPCR, Bio-Rad Laboratories) identified 60% (100 copies) and 52% (50 copies) of samples correctly. Turnaround time from sample to results ranged from ~2 hours (Idylla CE-IVD) to 2 days (TruSight Tumor 15 and Sentosa CE-IVD), to 2 weeks for certain NGS assays; the level of required expertise ranged from minimal (Idylla CE-IVD) to high for some technologies. DISCUSSION: This comprehensive parallel assessment used high molecular weight cell line DNA as a model system to address key questions for a laboratory when implementing routine KRAS testing. As most of the technologies are available for additional molecular biomarkers, this study may be informative for other applications.

Genome-wide Identification of IRF1 Binding Sites Reveals Extensive Occupancy at Cell Death Associated Genes.

IRF1 is a transcription factor involved in interferon signaling and has been shown to harbor tumor suppressor activity. In order to comprehensively identify pathways regulated by IRF1, we used chromatin immunoprecipitation followed by massive-parallel sequencing (ChIP-seq) to evaluate the gene targets of IRF1 genome-wide. We identified 17,416 total binding events in breast cancer cells. Functional categorization of the binding sites after IFN-gamma (interferon-gamma) treatment determined that 'apoptosis' or 'cell death' is the most enriched target process. Motif discovery analysis of the chromosomal regions bound by IRF1 identified a number of unique motifs correlated with apoptosis, DNA damage and immune processes. Analysis of GEO transcriptome data from IRF1-transduced cells or IFN-gamma treated fibroblasts indicates that IRF1-bound targets in IFN-treated cells are associated with a positive transcriptional response. Many of the enriched target genes from the expression analysis are associated with apoptosis. Importantly, this data indicates that a significant function of IRF1 is the regulation of anti-cancer apoptotic pathways and this reinforces IRF1's role as a tumor suppressor.

Identification of Sp1 and GC-boxes as transcriptional regulators of mouse Dag1 gene promoter.

Dystroglycan is a widely expressed adhesion complex that anchors cells to the basement membrane and is involved in embryonic development and differentiation. Dystroglycan expression is frequently reduced in human dystrophies and malignancies, and its molecular functions are not completely understood. Several posttranslational mechanisms have been identified that regulate dystroglycan expression and/or function, while little is known about how expression of the corresponding Dag1 gene is regulated. This study aimed to clone the Dag1 gene promoter and to characterize its regulatory elements. Analysis of the mouse Dag1 gene 5'-flanking region revealed a TATA and CAAT box-lacking promoter including a GC-rich region. Transfection studies with serially deleted promoter constructs allowed us to identify a minimal promoter region containing three Specificity protein 1 (Sp1) sites and an E-box. Sp1 binding was confirmed by chromatin immunoprecipitation assay, and Sp1 downregulation reduced dystroglycan expression in muscle cells. Treatment with 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased Dag1 mRNA expression levels in myoblasts, and methylation decreased promoter activity in vitro. Furthermore, Dag1 gene promoter methylation was reduced while its expression increased during differentiation of C(2)C(12) myoblast cells in myotubes. In conclusion, for the first time we have characterized the activity of the mouse Dag1 gene promoter, confirming a complex regulation by Sp1 transcription factor, DNA methylation, and histone acetylation, which might be relevant for a better understanding of the physiopathology of the dystroglycan complex.

Static electromagnetic fields generated by corrosion currents inhibit human osteoblast differentiation.

STUDY DESIGN: Human osteoblast cultures were exposed to a very low intensity static magnetic fields (SMF) to investigate its effects on osteoblast growth and differentiation. OBJECTIVE: Analysis of the effects of periprosthetic SMF on the growth and differentiation of human osteoblast cell cultures in vitro. SUMMARY OF BACKGROUND DATA: The effects of pulsed electromagnetic fields (PEMF) on cell proliferation, especially in human osteoblast-like cells is well described, whereas few data are available on the effects of SMF on osteoblast cell culture. We previously demonstrated that the proliferation of human osteoblast cultures is reduced when cells are exposed to a continuous low intensity SMF comparable to the one that occurs around metal devices (Ti spinal implant) because of the generation of electric currents between the screw (Ti6Al4V) and the rod (Ti). METHODS: Primary osteoblastic cells were isolated from a human femoral head. Osteoblast cultures were exposed to SMF and alkaline phosphatase activity was evaluated in the osteoblast cell cultures at different time points. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression levels of osteocalcin, Runx2, and collagen I genes. RESULTS: The SMF-treated cells showed a progressive increase in the alkaline phosphatase activity which, however, remained always lower than the one observed in the control group at each observation time (72 hours, 7 and 14 days). RT-PCR demonstrated that Runx2 and collagen I mRNA were downregulated following SMF stimulation, whereas no change in osteocalcin mRNA was observed. CONCLUSION: Continuous low-intensity electromagnetic field comparable to the one that generates around metal devices because of the generation of corrosion currents inhibits osteoblasts differentiation pattern and might contribute at least in part to a decrease in periprosthetic bone formation occurring in vivo.

Periprosthetic electrochemical corrosion of titanium and titanium-based alloys as a cause of spinal fusion failure.

STUDY DESIGN: Posterior lumbar spine implants retrieved from patients affected by periprosthetic osteolysis were analyzed to identify corrosion and to investigate the electromagnetic fields (EMF) generated by corrosion currents and their effect on human osteoblasts proliferation. OBJECTIVE.: Analysis of retrieved instrumentation to better understand periprosthetic osteolysis and correlation of this information with clinical factors. SUMMARY OF BACKGROUND DATA: Recent studies have pointed out that mechanically assisted crevice corrosion represents the initial failure of spinal implants, resulting in the local decrease in pH, which leads to osteolysis. METHODS: Electrochemical analysis was performed to characterize the corrosion currents and the EMF generated around the implants retrieved. Human primary osteoblasts cultures were used to determine the effect of continued EMF stimulation on cell growth. Cultures were exposed to the EMF stimulation for 48 hours, 72 hours, 7 days, and 14 days. RESULTS: During the electrochemical corrosion tests both the screws and the bar showed a passivation current of 0312 and 0.05 muA/cm, respectively. Osteoblasts exposed to an EMF of 12.1 x 10 T displayed a decreased proliferation rate. At each observation time, there were differences in cell numbers between the unexposed cells and the exposed cells. CONCLUSION: Aseptic periprosthetic bone loss can be due in part to the generation of electric and electromagnetic phenomena generated around metal devices, which inhibit osteoblasts growth and might hamper periprosthetic bone formation. This mechanism is of clinical significance and should be more deeply evaluated.

Expression of dystroglycan correlates with tumor grade and predicts survival in renal cell carcinoma.

The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor aggressiveness. In this study expression of the DG subunit was evaluated by immunostaining in a series of renal epithelial cancers and its relation with traditional prognostic indicators and with the clinical outcome of the patients was evaluated. alphaDG expression was undetectable in a significant fraction of tumors (54%). In renal cell carcinomas (RCC) loss of alpha-DG staining correlated with higher tumor grade (p = 0.02) but not with tumor stage nor tumor size. In clear cell RCC patients loss of alphaDG staining correlated with an increased risk of recurrence (p = 0.002 by log-rank test) and death (p = 0.004) also when patients with lower grade or stage tumors were analyzed separately. In a multivariate analysis loss of DG staining confirmed to be and independent predictor of shorter disease-free (p = 0.001; RR = 4.9) and overall (p = 0.009; RR = 4.9) survival stronger than tumor grade and size. These findings demonstrate that loss of alphaDG expression, which correspond to loss of a functional DG complex, is a frequent event in human renal tumorigenesis and is an independent predictor of early recurrence and death for patients with clear cell RCC.

Aberrant expression of alpha-dystroglycan in cervical and vulvar cancer.

OBJECTIVES: Cervical and vulvar cancers develop through well-defined precursor lesions but their exact pathogenesis is still unknown. The dystroglycan complex is a transmembrane glycoprotein that forms a continuous link from the extracellular matrix to the actin cytoskeleton. Deregulated expression of dystroglycan has been reported in human malignancies and related to tumor differentiation and aggressiveness. In this study, expression of dystroglycan was evaluated in the multistep cervical and vulvar tumorigenesis. METHODS: Expression of the dystroglycan complex was evaluated by immunostaining in lesions representing different stages of vulvar and cervical tumorigenesis using a monoclonal antibody which recognizes carbohydratic epitopes on the alpha-dystroglycan subunit. RESULTS: alpha-dystroglycan was constantly detected in normal cervical epithelium with a mean percentage of positive cells higher than 80%. A progressive significant reduction in the mean percentage of positive cells was observed in low (67%) and high grade SIL (14%) and in invasive carcinomas (2.6%) of the cervix. In cancers, no differences were observed in terms of percentage of positive cells when cases were stratified according with either tumor grade or stage. A progressive significant reduction in the mean percentage of positive cells was also observed from normal vulvar epithelium (90%) to VIN1 (66%), VIN2 (28%) and invasive vulvar carcinomas (22%). No significant decrease in the alpha-dystroglycan staining was observed in squamous cell hyperplasia lesions (85%) while lichen sclerosus displayed a percentage of positive cells (47%) significantly lower than normal epithelium. CONCLUSIONS: Detection of alpha-dystroglycan is frequently lost in human cervical and vulvar tumorigenesis and further studies are warranted to verify whether evaluation of this molecule might serve as marker of risk progression of preneoplastic lesions and to better understand its significance in terms of cancer development.