Pre-trial inter-laboratory analytical validation of the FOCUS4 personalised therapy trial.

INTRODUCTION: Molecular characterisation of tumours is increasing personalisation of cancer therapy, tailored to an individual and their cancer. FOCUS4 is a molecularly stratified clinical trial for patients with advanced colorectal cancer. During an initial 16-week period of standard first-line chemotherapy, tumour tissue will undergo several molecular assays, with the results used for cohort allocation, then randomisation. Laboratories in Leeds and Cardiff will perform the molecular testing. The results of a rigorous pre-trial inter-laboratory analytical validation are presented and discussed. METHODS: Wales Cancer Bank supplied FFPE tumour blocks from 97 mCRC patients with consent for use in further research. Both laboratories processed each sample according to an agreed definitive FOCUS4 laboratory protocol, reporting results directly to the MRC Trial Management Group for independent cross-referencing. RESULTS: Pyrosequencing analysis of mutation status at KRAS codons12/13/61/146, NRAS codons12/13/61, BRAF codon600 and PIK3CA codons542/545/546/1047, generated highly concordant results. Two samples gave discrepant results; in one a PIK3CA mutation was detected only in Leeds, and in the other, a PIK3CA mutation was only detected in Cardiff. pTEN and mismatch repair (MMR) protein expression was assessed by immunohistochemistry (IHC) resulting in 6/97 discordant results for pTEN and 5/388 for MMR, resolved upon joint review. Tumour heterogeneity was likely responsible for pyrosequencing discrepancies. The presence of signet-ring cells, necrosis, mucin, edge-effects and over-counterstaining influenced IHC discrepancies. CONCLUSIONS: Pre-trial assay analytical validation is essential to ensure appropriate selection of patients for targeted therapies. This is feasible for both mutation testing and immunohistochemical assays and must be built into the workup of such trials. TRIAL REGISTRATION NUMBER: ISRCTN90061564

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

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

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

Role of a conserved gluatmine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase

The pentaheme cytochrome c nitrite reductase (NrfA) of Escherichia coli is responsible for nitrite reduction during anaerobic respiration when nitrate is scarce. The NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a glutamine (Q263) positioned 8 Å from the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion. Mutation of this glutamine to the more usual calcium ligand, glutamate, results in an increase in the Km for nitrite by around 10-fold, while Vmax is unaltered. Protein film voltammetry showed that lower potentials were required to detect activity from NrfA Q263E when compared with native enzyme, consistent with the introduction of a negative charge into the vicinity of the active site heme. EPR and MCD spectroscopic studies revealed the high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme. Comparison of the X-ray crystal structures of the as-prepared, oxidized native and mutant enzymes showed an increased bond distance between the active site heme Fe(III) iron and the distal ligand in the latter as well as changes to the structure and mobility of the active site water molecule network. These results suggest that an important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand

Kinetic and thermodynamic resolution of the interactions between sulfite and the pentahaem cytochrome NrfA from Escherichia coli

NrfA is a pentahaem cytochrome present in a wide-range of ?-, d- and e-proteobacteria. Its nitrite and nitric oxide reductase activities have been studied extensively and contribute to respiratory nitrite ammonification and nitric oxide detoxification respectively. Sulfite is a third substrate for NrfA that may be encountered in the micro-oxic environments where nrfA is expressed. Consequently, we have performed quantitative kinetic and thermodynamic studies of the interactions between sulfite and Escherichia coli NrfA to provide a biochemical framework from which to consider their possible cellular consequences. A combination of voltammetric, spectroscopic and crystallographic analyses define dissociation constants for sulfite binding to NrfA in oxidized (~54 µM), semi-reduced (~145 µM) and reduced (~180 µM) states that are comparable with each other, and the Km (~70 µM) for sulfite reduction at pH 7. Under comparable conditions Km values of ~22 and ~300 µM describe nitrite and nitric oxide reduction respectively, whereas the affinities of nitrate and thiocyanate for NrfA fall more than 50-fold on enzyme reduction. These results are discussed in terms of the nature of sulfite co-ordination within the active site of NrfA and their implications for the cellular activity of NrfA

LTCcovid International living report on COVID-19 and long-term care

This “live report” has been compiled collaboratively by researchers on Long-Term Care all over the world. It aims to: Provide an overview of Long-Term Care systems around the world Assess how the people who use and provide long-term care have been affected by the COVID-19 pandemic Describe the measures adopted to mitigate the impacts of the pandemic in the long-term care sector Compile actions and reforms that countries are adopting to strengthen their care systems and be better prepared for future pandemics and shocks. This report does not seek to provide detailed or comprehensive information for each country, but instead aims to summarize key reports and articles and point the reader towards those. It builds on the country reports previously published in this website, as well as other more recent sources. It is being developed collaboratively, by answering a list of questions for as many countries as possible and updating as new information and research become available

Associations between AI-assisted tumor amphiregulin and epiregulin IHC and outcomes from anti-EGFR therapy in the routine management of metastatic colorectal cancer.

PurposeHigh tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG immunohistochemistry (IHC) was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis.Experimental designPatients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival FFPE tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS).Results494 of 541 patients (91.3%) had adequate tissue for analysis. 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n=360; 80.2%) was associated with significantly prolonged PFS (median: 8.5 vs 4.4 months; HR 0.73; 95% CI, 0.56-0.95; p=0.02) and OS (median: 16.4 vs 8.9 months; HR 0.66 [0.50-0.86]; p=0.002). The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection.ConclusionsHigh tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice

Associations between AI-assisted tumor amphiregulin and epiregulin IHC and outcomes from anti-EGFR therapy in the routine management of metastatic colorectal cancer.

High tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG immunohistochemistry (IHC) was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis. Patients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival FFPE tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS). 494 of 541 patients (91.3%) had adequate tissue for analysis. 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n=360; 80.2%) was associated with significantly prolonged PFS (median: 8.5 vs 4.4 months; HR 0.73; 95% CI, 0.56-0.95; p=0.02) and OS (median: 16.4 vs 8.9 months; HR 0.66 [0.50-0.86]; p=0.002). The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection. High tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice