Peptide delivery of a multivalent mRNA SARS-CoV-2 vaccine.

Lipid nanoparticles (LNP) have been instrumental in the success of mRNA vaccines and have opened up the field to a new wave of therapeutics. However, what is ahead beyond the LNP? The approach herein used a nanoparticle containing a blend of Spike, Membrane and Envelope antigens complexed for the first time with the RALA peptide (RALA-SME). The physicochemical characteristics and functionality of RALA-SME were assessed. With >99% encapsulation, RALA-SME was administered via intradermal injection in vivo, and all three antigen-specific IgG antibodies were highly significant. The IgG2a:IgG1 ratio were all >1.2, indicating a robust TH1 response, and this was further confirmed with the T-Cell response in mice. A complete safety panel of markers from mice were all within normal range, supported by safety data in hamsters. Vaccination of Syrian Golden hamsters with RALA-SME derivatives produced functional antibodies capable of neutralising SARS-CoV-2 from both Wuhan-Hu-1 and Omicron BA.1 lineages after two doses. Antibody levels increased over the study period and provided protection from disease-specific weight loss, with inhibition of viral migration down the respiratory tract. This peptide technology enables the flexibility to interchange and add antigens as required, which is essential for the next generation of adaptable mRNA vaccines.

Dissolving microneedle patches loaded with amphotericin B microparticles for localised and sustained intradermal delivery: Potential for enhanced treatment of cutaneous fungal infections.

Fungal infections affect millions of people globally and are often unreceptive to conventional topical or oral preparations because of low drug bioavailability at the infection site, lack of sustained therapeutic effect, and the development of drug resistance. Amphotericin B (AmB) is one of the most potent antifungal agents. It is increasingly important since fungal co-infections associated with COVID-19 are frequently reported. AmB is only administered via injections (IV) and restricted to life-threatening infections due to its nephrotoxicity and administration-related side effects. In this work, we introduce, for the first time, dissolving microneedle patches (DMP) loaded with micronised particles of AmB to achieve localised and long-acting intradermal delivery of AmB for treatment of cutaneous fungal infections. AmB was pulverised with poly (vinyl alcohol) and poly (vinyl pyrrolidone) to form micronised particles-loaded gels, which were then cast into DMP moulds to form the tips. The mean particle size of AmB in AmB DMP tips after pulverisation was 1.67 ± 0.01 µm. This is an easy way to fabricate and load microparticles into DMP, as few steps are required, and no organic solvents are needed. AmB had no covalent chemical interaction with the excipients, but the crystallinity of AmB was reduced in the tips. AmB was completely released from the tips within 4 days in vitro. AmB DMP presented inhibition of Candida albicans (CA) and the killing rate of AmB DMP against CA biofilm inside porcine skin reached 100% within 24 h. AmB DMP were able to pierce excised neonatal porcine skin at an insertion depth of 301.34 ± 46.86 µm. Ex vivo dermatokinetic and drug deposition studies showed that AmB was mainly deposited in the dermis. An in vivo dermatokinetic study revealed that the area under curve (AUC0-inf) values of AmB DMP and IV (Fungizone® bolus injection 1 mg/kg) groups were 8823.0 d∙µg/g and 33.4 d∙µg/g, respectively (264-fold higher). AmB remained at high levels (219.07 ± 102.81 µg/g or more) in the skin until 7 days after the application of AmB DMP. Pharmacokinetic and biodistribution studies showed that AmB concentration in plasma, kidney, liver, and spleen in the AmB DMP group was significantly lower than that in the IV group. Accordingly, this system addressed the systemic side effects of intravenous injection of AmB and localised the drug inside the skin for a week. This work establishes a novel, easy and effective method for long-acting and localised intradermal drug delivery.

Interleukin-7: a potential factor supporting B-cell maturation in the rheumatoid arthritis synovium.

OBJECTIVES: The exact function of interleukin-7 (IL-7) in autoimmune diseases remains unclear although it is a recognised therapeutic target for cytokine blockade. Our objective was to investigate the regulation and downstream effect of IL-7 in diseased tissue from rheumatoid arthritis (RA) patients notably with respect to its function as bone turnover regulator and tissue architecture (TA) organiser. METHODS: Synovial tissues (fresh, frozen or xed) were obtained from our tissue bank and distributed between experiments for live cell cultures, histology, immunohistochemistry or gene expression array by qPCR. RESULTS: IL-7 expression in synoviocyte cultures was up-regulated by pro-in ammatory cytokines, notably IL-6. Gene expression pro ling segregated synovial biopsies based on the presence of B/plasma cells and ectopic TA. IL-7 gene expression was associated with that of several genes whose function was to support B-cell maturation in tissue with distinct B-cell aggregates (despite the lack of IL-7-Receptor expression on B-cells) as well as with ectopic germinal-like centres. IL-7 was associated with bone turnover regulation in biopsies with diffuse in ltration. A novel relationship between the IL-7 and IL-6 axis was also highlighted in human tissue. CONCLUSIONS: Overall, IL-7 may contribute to the maintenance of the pro-in ammatory cycle perpetuating in ammation in RA synovium. We therefore propose a novel role for IL-7 as an orchestrator of TA with an impact on B-cell maturation in relation with IL-6.

Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α-TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4-37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α-TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

Nanocomposite-coated porous templates for engineered bone scaffolds: a parametric study of layer-by-layer assembly conditions.

Using the layer-by-layer (LbL) assembly technique to deposit mechanically reinforcing coatings onto porous templates is a route for fabricating engineered bone scaffold materials with a combination of high porosity, strength, and stiffness. LbL assembly involves the sequential deposition of nano- to micro-scale multilayer coatings from aqueous solutions. Here, a design of experiments (DOE) approach was used to evaluate LbL assembly of polyethyleneimine (PEI), polyacrylic acid (PAA), and nanoclay coatings onto open-cell polyurethane foam templates. The thickness of the coatings, and the porosity, elastic modulus and collapse stress of coated foam templates were most strongly affected by the pH of PAA solutions, salt concentration, and interactions between these factors. The mechanical properties of coated foams correlated with the thickness of the coatings, but were also ascribed to changes in the coating properties due to the different assembly conditions. A DOE optimization aimed to balance the trade-off between higher mechanical properties but lower porosity of foam templates with increasing coating thickness. Micromechanical modeling predicted that deposition of 116 QLs would achieve mechanical properties of cancellous bone (>0.05 GPa stiffness and >2 MPa strength) at a suitable porosity of >70%. When capped with a final layer of PAA and cross-linked via thermal treatment, the PEI/PAA/PEI/nanoclay coatings exhibited good indirect cytotoxicity with mesenchymal stem cells. The ability of LbL assembly to deposit a wide range of functional constituents within multilayer-structured coatings makes the general strategy of templated LbL assembly a powerful route for fabricating engineered tissue scaffolds that can be applied onto various porous template materials to achieve a wide range of properties, pore structures, and multifunctionality.

Preoperative chemoradiation with capecitabine, irinotecan and cetuximab in rectal cancer: significance of pre-treatment and post-resection RAS mutations.

BACKGROUND: The influence of EGFR pathway mutations on cetuximab-containing rectal cancer preoperative chemoradiation (CRT) is uncertain. METHODS: In a prospective phase II trial (EXCITE), patients with magnetic resonance imaging (MRI)-defined non-metastatic rectal adenocarinoma threatening/involving the surgical resection plane received pelvic radiotherapy with concurrent capecitabine, irinotecan and cetuximab. Resection was recommended 8 weeks later. The primary endpoint was histopathologically clear (R0) resection margin. Pre-planned retrospective DNA pyrosequencing (PS) and next generation sequencing (NGS) of KRAS, NRAS, PIK3CA and BRAF was performed on the pre-treatment biopsy and resected specimen. RESULTS: Eighty-two patients were recruited and 76 underwent surgery, with R0 resection in 67 (82%, 90%CI: 73-88%) (four patients with clinical complete response declined surgery). Twenty-four patients (30%) had an excellent clinical or pathological response (ECPR). Using NGS 24 (46%) of 52 matched biopsies/resections were discrepant: ten patients (19%) gained 13 new resection mutations compared to biopsy (12 KRAS, one PIK3CA) and 18 (35%) lost 22 mutations (15 KRAS, 7 PIK3CA). Tumours only ever testing RAS wild-type had significantly greater ECPR than tumours with either biopsy or resection RAS mutations (14/29 [48%] vs 10/51 [20%], P=0.008), with a trend towards increased overall survival (HR 0.23, 95% CI 0.05-1.03, P=0.055). CONCLUSIONS: This regimen was feasible and the primary study endpoint was met. For the first time using pre-operative rectal CRT, emergence of clinically important new resection mutations is described, likely reflecting intratumoural heterogeneity manifesting either as treatment-driven selective clonal expansion or a geographical biopsy sampling miss.

Simple Radical Polymerization of Poly(Alginate-Graft-N-Isopropylacrylamide) Injectable Thermoresponsive Hydrogel with the Potential for Localized and Sustained Delivery of Stem Cells and Bioactive Molecules.

In this study, thermoresponsive copolymers that are fully injectable, biocompatible, and biodegradable and are synthesized via graft copolymerization of poly(N-isopropylacrylamide) onto alginate using a free-radical reaction are presented. This new synthesis method does not involve multisteps or associated toxicity issues, and has the potential to reduce scale-up difficulties. Chemical and physical analyses verify the resultant graft copolymer structure. The lower critical solution temperature, which is a characteristic of sol-gel transition, is observed at 32 °C. The degradation properties indicate suitable degradation kinetics for drug delivery and bone tissue engineering applications. The synthesized P(Alg-g-NIPAAm) hydrogel is noncytotoxic with both human osteosarcoma (MG63) cells and porcine bone marrow derived mesenchymal stem cells (pBMSCs). pBMSCs encapsulated in the P(Alg-g-NIPAAm) hydrogel remain viable, show uniform distribution within the injected hydrogel, and undergo osteogenic and chondrogenic differentiation under appropriate culture conditions. Furthermore, for the first time, this work will explore the influence of alginate viscosity on the viscoelastic properties of the resulting copolymer hydrogels, which influences the rate of medical device formation and subsequent drug release. Together the results of this study indicate that the newly synthesized P(Alg-g-NIPAAm) hydrogel has potential to serve as a versatile and improved injectable platform for drug delivery and bone tissue engineering applications.

RALA complexed α-TCP nanoparticle delivery to mesenchymal stem cells induces bone formation in tissue engineered constructs in vitro and in vivo.

A range of bone regeneration strategies, from growth factor delivery and/or mesenchymal stem cell (MSC) transplantation to endochondral tissue engineering, have been developed in recent years. Despite their tremendous promise, the clinical translation and future use of many of these strategies is being hampered by concerns such as off target effects associated with growth factor delivery. Therefore the overall objective of this study was to investigate the influence of alpha-tricalcium phosphate (α-TCP) nanoparticle delivery into MSCs using an amphipathic cell penetrating peptide RALA, on osteogenesis in vitro and both intramembranous and endochondral bone formation in vivo. RALA complexed α-TCP nanoparticle delivery to MSCs resulted in an increased expression of bone morphogenetic protein-2 (BMP-2) and an upregulation in a number of key osteogenic genes. When α-TCP stimulated MSCs were encapsulated into alginate hydrogels, enhanced mineralization of the engineered construct was observed over a 28 day culture period. Furthermore, the in vivo bone forming potential of RALA complexed α-TCP nanoparticle delivery to MSCs was found to be comparable to growth factor delivery. Recognizing the potential and limitations associated with endochondral bone tissue engineering strategies, we then sought to explore how α-TCP nanoparticle delivery to MSCs influences early mineralization of engineered cartilage templates in vitro and their subsequent ossification in vivo. Despite accelerating mineralization of engineered cartilage templates in vitro, RALA complexed α-TCP nanoparticle delivery did not enhance endochondral bone formation in vivo. Therefore the potential of RALA complexed α-TCP nanoparticle delivery appears to be as an alternative to growth factor delivery as a single stage strategy for promoting bone generation.

Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and "BRCA-Like" Status, in Both Blood and Tumour DNA.

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies.

HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials.

HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. One suggested anti-EGFR resistance mechanism in colorectal cancer (CRC) is aberrant MEK-AKT pathway activation through HER2 up-regulation. We assessed HER2-amplification/overexpression in stage II-III and IV CRC patients, assessing relationships to KRAS/BRAF and outcome. Pathological material was obtained from 1914 patients in the QUASAR stage II-III trial and 1342 patients in stage IV trials (FOCUS and PICCOLO). Tissue microarrays were created for HER2 immunohistochemistry. HER2-amplification was assessed using FISH and copy number variation. KRAS/BRAF mutation status was assessed by pyrosequencing. Progression-free survival (PFS) and overall survival (OS) data were obtained for FOCUS/PICCOLO and recurrence and mortality for QUASAR; 29/1342 (2.2%) stage IV and 25/1914 (1.3%) stage II-III tumours showed HER2 protein overexpression. Of the HER2-overexpressing cases, 27/28 (96.4%) stage IV tumours and 20/24 (83.3%) stage II-III tumours demonstrated HER2 amplification by FISH; 41/47 (87.2%) also showed copy number gains. HER2-overexpression was associated with KRAS/BRAF wild-type (WT) status at all stages: in 5.2% WT versus 1.0% mutated tumours (p < 0.0001) in stage IV and 2.1% versus 0.2% in stage II-III tumours (p = 0.01), respectively. HER2 was not associated with OS or PFS. At stage II-III, there was no significant correlation between HER2 overexpression and 5FU/FA response. A higher proportion of HER2-overexpressing cases experienced recurrence, but the difference was not significant. HER2-amplification/overexpression is identifiable by immunohistochemistry, occurring infrequently in stage II-III CRC, rising in stage IV and further in KRAS/BRAF WT tumours. The value of HER2-targeted therapy in patients with HER2-amplified CRC must be tested in a clinical trial. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERß1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERß1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERß1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells. CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERß1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERß1 may not be the most important miR-92 target in breast cancer.

A mobile phone application for the collection of opinion data for forest planning purposes.

The last 30 years has seen an increase in environmental, socio-economic, and recreational objectives being considered throughout the forest planning process. In the Finnish context these are considered mainly at the regional level potentially missing out on more local issues and problems. Such local information would be most efficiently collected with a participatory GIS approach. A mobile participatory GIS application called Tienoo was developed as a tool for collecting location-specific opinions of recreational and aesthetical characteristics of forests and forest management. The application also contains information the user can access regarding the practical details of the area, for instance about the recreational infrastructure. The application was tested in Ruunaa National Hiking Area, North Karelia, Eastern Finland. Through this application it is possible to continuously collect geolocated preference information. As a result, the collected opinions have details which can be located in both time and space. This allows for the possibility to monitor the changes in opinions when the stands are treated, and it also allows for easily analyzing the effect of time of year on the opinions. It is also possible to analyze the effect of the spatial location and the forest characteristics on the opinions using GIS analysis.

Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial.

BACKGROUND: Therapeutic antibodies targeting EGFR have activity in advanced colorectal cancer, but results from clinical trials are inconsistent and the population in which most benefit is derived is uncertain. Our aim was to assess the addition of panitumumab to irinotecan in pretreated advanced colorectal cancer. METHODS: In this open-label, randomised trial, we enrolled patients who had advanced colorectal cancer progressing after fluoropyrimidine treatment with or without oxaliplatin from 60 centres in the UK. From December, 2006 until June, 2008, molecularly unselected patients were recruited to a three-arm design including irinotecan (control), irinotecan plus ciclosporin, and irinotecan plus panitumumab (IrPan) groups. From June 10, 2008, in response to new data, the trial was amended to a prospectively stratified design, restricting panitumumab randomisation to patients with KRAS wild-type tumours; the results of the comparison between the irinotcan and IrPan groups are reported here. We used a computer-generated randomisation sequence (stratified by previous EGFR targeted therapy and then minimised by centre, WHO performance status, previous oxaliplatin, previous bevacizumab, previous dose modifications, and best previous response) to randomly allocate patients to either irinotecan or IrPan. Patients in both groups received 350 mg/m(2) intravenous irinotecan every 3 weeks (300 mg/m(2) if aged ≥70 years or a performance status of 2); patients in the IrPan group also received intravenous panitumumab 9 mg/kg every 3 weeks. The primary endpoint was overall survival in KRAS wild-type patients who had not received previous EGFR targeted therapy, analysed by intention to treat. Tumour DNA was pyrosequenced for KRASc.146, BRAF, NRAS, and PIK3CA mutations, and predefined molecular subgroups were analysed for interaction with the effect of panitumumab. This study is registered, number ISRCTN93248876. RESULTS: Between Dec 4, 2006, and Aug 31, 2010, 1198 patients were enrolled, of whom 460 were included in the primary population of patients with KRASc.12-13,61 wild-type tumours and no previous EGFR targeted therapy. 230 patients were randomly allocated to irinotecan and 230 to IrPan. There was no difference in overall survival between groups (HR 1·01, 95% CI 0·83-1·23; p=0·91), but individuals in the IrPan group had longer progression-free survival (0·78, 0·64-0·95; p=0·015) and a greater number of responses (79 [34%] patients vs 27 [12%]; p<0·0001) than did individuals in the irinotecan group. Grade 3 or worse diarrhoea (64 [29%] of 219 patients vs 39 [18%] of 218 patients), skin toxicity (41 [19%] vs none), lethargy (45 [21]% vs 24 [11%]), infection (42 [19%] vs 22 [10%]) and haematological toxicity (48 [22%] vs 27 [12%]) were reported more commonly in the IrPan group than in the irinotecan group. We recorded five treatment-related deaths, two in the IrPan group and three in the irinotecan group. INTERPRETATION: Adding panitumumab to irinotecan did not improve the overall survival of patients with wild-type KRAS tumours. Further refinement of molecular selection is needed for substantial benefits to be derived from EGFR targeting agents. FUNDING: Cancer Research UK, Amgen Inc.

Mutation detection by clonal sequencing of PCR amplicons and grouped read typing is applicable to clinical diagnostics.

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.

Intra-tumoral heterogeneity of KRAS and BRAF mutation status in patients with advanced colorectal cancer (aCRC) and cost-effectiveness of multiple sample testing.

KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE) tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor). We also investigated the utility and efficiency of genotyping a 'DNA cocktail' prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1%) showed intratumoral heterogeneity; 5 (7.2%) at KRAS codons 12, 13 and 2 (2.9%) at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in 'DNA cocktail' samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a 'DNA cocktail' from two or more tumor blocks, improves mutation detection at minimal extra cost.

Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer.

PURPOSE: It is uncertain whether modest benefits from adjuvant chemotherapy in stage II colorectal cancer justify the toxicity, cost, and inconvenience. We investigated the usefulness of defective mismatch repair (dMMR), BRAF, and KRAS mutations in predicting tumor recurrence and sensitivity to chemotherapy. PATIENTS AND METHODS: Immunohistochemistry for dMMR and pyrosequencing for KRAS/BRAF were performed for 1,913 patients randomly assigned between fluorouracil and folinic acid chemotherapy and no chemotherapy in the Quick and Simple and Reliable (QUASAR) trial. RESULTS: Twenty-six percent of 695 right-sided colon, 3% of 685 left-sided colon, and 1% of 407 rectal tumors were dMMR. Similarly, 17% of right colon, 2% of left colon, and 2% of rectal tumors were BRAF mutant. KRAS mutant tumors were more evenly distributed: 40% right colon, 28% left colon, and 36% rectal tumors. Recurrence rate for dMMR tumors was half that for MMR-proficient tumors (11% [25 of 218] v 26% [438 of 1,695] recurred; risk ratio [RR], 0.53; 95% CI, 0.40 to 0.70; P < .001). Risk of recurrence was also significantly higher for KRAS mutant than KRAS wild-type tumors (28% [150 of 542] v 21% [219 of 1,041]; RR, 1.40; 95% CI, 1.12 to 1.74; P = .002) but did not differ significantly between BRAF mutant and wild-type tumors (P = .36). No marker predicted benefit from chemotherapy with efficacy not differing significantly by MMR, KRAS, or BRAF status. The prognostic value of MMR and KRAS was similar in the presence and absence of chemotherapy. CONCLUSION: MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial.

PURPOSE: Activating mutation of the KRAS oncogene is an established predictive biomarker for resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies in advanced colorectal cancer (aCRC). We wanted to determine whether KRAS and/or BRAF mutation is also a predictive biomarker for other aCRC therapies. PATIENTS AND METHODS: The Medical Research Council Fluorouracil, Oxaliplatin and Irinotecan: Use and Sequencing (MRC FOCUS) trial compared treatment sequences including first-line fluorouracil (FU), FU/irinotecan or FU/oxaliplatin in aCRC. Tumor blocks were obtained from 711 consenting patients. DNA was extracted and KRAS codons 12, 13, and 61 and BRAF codon 600 were assessed by pyrosequencing. Mutation (mut) status was assessed first as a prognostic factor and then as a predictive biomarker for the benefit of adding irinotecan or oxaliplatin to FU. The association of BRAF-mut with loss of MLH1 was assessed by immunohistochemistry. RESULTS: Three hundred eight (43.3%) of 711 patients had KRAS-mut and 56 (7.9%) of 711 had BRAF-mut. Mutation of KRAS, BRAF, or both was present in 360 (50.6%) of 711 patients. Mutation in either KRAS or BRAF was a poor prognostic factor for overall survival (OS; hazard ratio [HR], 1.40; 95% CI, 1.20 to 1.65; P < .0001) but had minimal impact on progression-free survival (PFS; HR, 1.16; 95% CI, 1.00 to 1.36; P = .05). Mutation status did not affect the impact of irinotecan or oxaliplatin on PFS or OS. BRAF-mut was weakly associated with loss of MLH1 staining (P = .012). CONCLUSION: KRAS/BRAF mutation is associated with poor prognosis but is not a predictive biomarker for irinotecan or oxaliplatin. There is no evidence that patients with KRAS/BRAF mutated tumors are less likely to benefit from these standard chemotherapy agents.

Integrated genomic and transcriptional analysis of the in vitro evolution of telomerase-immortalized urothelial cells (TERT-NHUC).

Much progress has been made in identifying the molecular genetic alterations that occur in bladder cancer. However, in many cases the genes targeted by these alterations are not known. Telomerase immortalized human urothelial cells (TERT-NHUC) are a useful resource for in vitro studies of genes involved in urothelial transformation. When cultured under standard conditions they remain genetically stable but when cultured under low-density conditions they exhibit genetic instability and acquire chromosomal alterations. TERT-NHUC from three donors were cultured at low plating density and examined at four time-points during a culture period of 600 days. Analyses included population doubling kinetics, array-based CGH (aCGH), chromosome counts, fluorescence in situ hybridization (FISH), mutation analysis, Affymetrix gene expression analysis, Western blotting for p16, anchorage-independent growth and tumorigenicity assays. Alterations acquired during continued culture of TERT-NHUC at low density (TERT-NHUC-L) included some observed in urothelial carcinoma (UC) cell lines and primary UC. Examination of gene expression in TERT-NHUC with distinct acquired genetic aberrations may pinpoint genes targeted by these alterations. Data from an aCGH study of UC cell lines and primary tumors were examined for changes in chromosomal regions that also showed alterations in TERT-NHUC-L. Loss of a region on 2q including BOK was identified in UC cell lines and primary tumors. DNER and FRAS1 were identified as potential candidate genes, whose expression is altered independently of the acquisition of any genetic event.