Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

Purpose: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials.

Experimental Design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments.

Results: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments.

Conclusions: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

Development and independent validation of a prognostic assay for stage ii colon cancer using formalin-fixed paraffin-embedded tissue

Purpose: Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. Patients and Methods: A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. Results: The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P <.001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P <.001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P <.001). Conclusion: This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples. © 2011 by American Society of Clinical Oncology.

A CFTR Potentiator in Patients with Cystic Fibrosis and the G551D Mutation

Background:

Increasing the activity of defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is a potential treatment for cystic fibrosis.

Methods:

We conducted a randomized, double-blind, placebo-controlled trial to evaluate ivacaftor (VX-770), a CFTR potentiator, in subjects 12 years of age or older with cystic fibrosis and at least one G551D-CFTR mutation. Subjects were randomly assigned to receive 150 mg of ivacaftor every 12 hours (84 subjects, of whom 83 received at least one dose) or placebo (83, of whom 78 received at least one dose) for 48 weeks. The primary end point was the estimated mean change from baseline through week 24 in the percent of predicted forced expiratory volume in 1 second (FEV1).

Results:

The change from baseline through week 24 in the percent of predicted FEV1 was greater by 10.6 percentage points in the ivacaftor group than in the placebo group (P < 0.001). Effects on pulmonary function were noted by 2 weeks, and a significant treatment effect was maintained through week 48. Subjects receiving ivacaftor were 55% less likely to have a pulmonary exacerbation than were patients receiving placebo, through week 48 (P < 0.001). In addition, through week 48, subjects in the ivacaftor group scored 8.6 points higher than did subjects in the placebo group on the respiratory-symptoms domain of the Cystic Fibrosis Questionnaire-revised instrument (a 100-point scale, with higher numbers indicating a lower effect of symptoms on the patient's quality of life) (P < 0.001). By 48 weeks, patients treated with ivacaftor had gained, on average, 2.7 kg more weight than had patients receiving placebo (P < 0.001). The change from baseline through week 48 in the concentration of sweat chloride, a measure of CFTR activity, with ivacaftor as compared with placebo was -48.1 mmol per liter (P < 0.001). The incidence of adverse events was similar with ivacaftor and placebo, with a lower proportion of serious adverse events with ivacaftor than with placebo (24% vs. 42%).

Conclusions:

Ivacaftor was associated with improvements in lung function at 2 weeks that were sustained through 48 weeks. Substantial improvements were also observed in the risk of pulmonary exacerbations, patient-reported respiratory symptoms, weight, and concentration of sweat chloride.

Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages

The Burkholderia cepacia complex (Bcc) is a group of opportunistic bacteria chronically infecting the airways of patients with cystic fibrosis (CF). Several laboratories have shown that Bcc members, in particular B. cenocepacia, survive within a membrane-bound vacuole inside phagocytic and epithelial cells. We have previously demonstrated that intracellular B. cenocepacia causes a delay in phagosomal maturation, as revealed by impaired acidification and slow accumulation of the late phagolysosomal marker LAMP-1. In this study, we demonstrate that uninfected cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages or normal macrophages treated with a CFTR-specific drug inhibitor display normal acidification. However, after ingestion of B. cenocepacia, acidification and phagolysosomal fusion of the bacteria-containing vacuoles occur in a lower percentage of CFTR-negative macrophages than CFTR-positive cells, suggesting that loss of CFTR function contributes to enhance bacterial intracellular survival. The CFTR-associated phagosomal maturation defect was absent in macrophages exposed to heat-inactivated B. cenocepacia and macrophages infected with a non-CF pathogen such as Salmonella enterica, an intracellular pathogen that once internalized rapidly traffics to acidic compartments that acquire lysosomal markers. These results suggest that not only a defective CFTR but also viable B. cenocepacia are required for the altered trafficking phenotype. We conclude that CFTR may play a role in the mechanism of clearance of the intracellular infection, as we have shown before that B. cenocepacia cells localized to the lysosome lose cell envelope integrity. Therefore, the prolonged maturation arrest of the vacuoles containing B. cenocepacia within cftr(-/-) macrophages could be a contributing factor in the persistence of the bacteria within CF patients.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2(-/-)CX3CR1(GFP/GFP) Mice

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17~60% of 12-month, and 30~100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (~800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1ß, IL-12 and TNF-a under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon

Colon-residing bacteria, such as vancomycin-resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit®-based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso-tetra(N-methyl-4-pyridyl)porphine tetra-tosylate (TMP), or 5-aminolevulinic acid hexyl-ester (h-ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen-releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6-h period. For TMP and h-ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon-residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.

AXL Is a Key Regulator of Inherent and Chemotherapy-Induced Invasion and Predicts a Poor Clinical Outcome in Early-Stage Colon Cancer

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse.

Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression.

Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues.

Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed.

Engineering plasmonic nanorod arrays for colon cancer marker detection

Engineering plasmonic nanomaterials or nanostructures towards ultrasensitive biosensing for disease markers or pathogens is of high importance. Here we demonstrate a systematic approach to tailor effective plasmonic nanorod arrays by combining both comprehensive numerical discrete dipole approximations (DDA) simulation and transmission spectroscopy experiments. The results indicate that 200×50 nm nanorod arrays with 300×500 nm period provide the highest FOM of 2.4 and a sensitivity of 310 nm/RIU. Furthermore, we demonstrate the use of nanorod arrays for the detection of single nucleotide polymorphism in codon 12 of the K-ras gene that are frequently occurring in early stages of colon cancer, with a sensitivity down to 10 nM in the presence of 100-fold higher concentration of the homozygous genotypes. Our work shows significant potential of nanorod arrays towards point-of-care applications in diagnosis and clinical studies.

Experimental Autoimmune Uveoretinitis (EAU)-Related Tissue Damage and Angiogenesis Is Reduced in CCL2−/−CX3CR1gfp/gfp Mice

Purpose: To investigate the roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in experimental autoimmune uveoretinitis (EAU)-mediated retinal tissue damage and angiogenesis.

Methods: The C57BL/6J wild-type (WT) and CCL2−/−CX3CR1gfp/gfp (double knockout [DKO]) mice were immunized with IRBP1-20. Retinal inflammation and tissue damage were evaluated clinically and histologically at different days postimmunization (p.i.). Retinal neovascular membranes were evaluated by confocal microscopy of retinal flat mounts, and immune cell infiltration by flow cytometry.

Results: At day 25 p.i., DKO mice had lower clinical and histological scores and fewer CD45highCD11b+ infiltrating cells compared with WT mice. The F4/80+macrophages constitute 40% and 21% and CD11b+Gr-1+Ly6G+ neutrophils constitute 10% and 22% of retinal infiltrating cells in WT and DKO mice, respectively. At the late stages of EAU (day 60–90 p.i.), DKO and WT mice had similar levels of inflammatory score. However, less structural damage and reduced angiogenesis were detected in DKO mice. Neutrophils were rarely detected in the inflamed retina in both WT and DKO mice. Macrophages and myeloid-derived suppressor cells (MDSCs) accounted for 8% and 3% in DKO EAU retina, and 19% and 10% in WT EAU retina; 71% of infiltrating cells were T/B-lymphocytes in DKO EAU retina and 50% in WT EAU retina.

Conclusions: Experimental autoimmune uveoretinitis–mediated retinal tissue damage and angiogenesis is reduced in CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation is dominated by neutrophils at the acute stage and lymphocytes at the chronic stage in these mice. Our results suggest that CCR2+ and CX3CR1+monocytes are both involved in tissue damage and angiogenesis in EAU.