RAS screening in colorectal cancer: a comprehensive analysis of the results from the UK NEQAS colorectal cancer external quality assurance schemes (2009–2016)

Evidence strongly indicates that extended RAS testing should be undertaken in mCRC patients, prior to prescribing anti-EGFR therapies. With more laboratories implementing testing, the requirement for External Quality Assurance schemes increases, thus ensuring high standards of molecular analysis. Data was analysed from 15 United Kingdom National External Quality Assessment Service (UK NEQAS) for Molecular Genetics Colorectal cancer external quality assurance (EQA) schemes, delivered between 2009 and 2016. Laboratories were provided annually with nine colorectal tumour samples for genotyping. Information on methodology and extent of testing coverage was requested, and scores given for genotyping, interpretation and clerical accuracy. There has been a sixfold increase in laboratory participation (18 in 2009 to 108 in 2016). For RAS genotyping, fewer laboratories now use Roche cobas®, pyrosequencing and Sanger sequencing, with more moving to next generation sequencing (NGS). NGS is the most commonly employed technology for BRAF and PIK3CA mutation screening. KRAS genotyping errors were seen in ≤10% laboratories, until the 2014–2015 scheme, when there was an increase to 16.7%, corresponding to a large increase in scheme participants. NRAS genotyping errors peaked at 25.6% in the first 2015–2016 scheme but subsequently dropped to below 5%. Interpretation and clerical accuracy scores have been consistently good throughout. Within this EQA scheme, we have observed that the quality of molecular analysis for colorectal cancer has continued to improve, despite changes in the required targets, the volume of testing and the technologies employed. It is reassuring to know that laboratories clearly recognise the importance of participating in EQA schemes

Targeted Next-Generation Sequencing Validates the Use of Diagnostic Biopsies as a Suitable Alternative to Resection Material for Mutation Screening in Colorectal Cancer

Background: Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens. Methods: We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples. DNA was extracted and analysed using the TruSight tumour kit, allowing the interrogation of 26 cancer driver genes. Samples were run on an Illumina MiSeq. Mutations were validated using quick-multiplex-consensus (QMC)-polymerase chain reaction (PCR) in conjunction with high resolution melting (HRM). The paired biopsy and resection tumour samples were assessed for presence or absence of mutations, mutant allele frequency ratios, and allelic imbalance status. Results: A total of 81 mutations were detected, in ten of the 26 genes in the TruSight kit. Two of the 25 paired cases were wild-type across all genes. The mutational profiles, allelic imbalance status, and mutant allele frequency ratios of the paired biopsy and resection samples were highly concordant (88.75–98.85%), with all but three (3.7%) of the mutations identified in the resection specimens also being present in the biopsy specimens. All 81 mutations were confirmed by QMC-PCR and HRM analysis, although four low-level mutations required a co-amplification at lower denaturation temperature (COLD)-PCR protocol to enrich for the mutant alleles. Conclusions: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy

Investigating the poor outcomes of BRAF - mutant advanced colorectal cancer: Analysis from 2530 patients in randomised clinical trials

Background: To improve strategies for the treatment of BRAF-mutant advanced colorectal cancer (aCRC) patients we examined individual data from patients treated with chemotherapy alone in three randomised trials to identify points on the treatment pathway where outcomes differ from BRAF wild-types. Patients and Methods: 2530 aCRC patients were assessed from three randomised trials. End-points were progression free survival (PFS), response rate (RR), disease control rate (DCR), post-progression survival (P-PS) and overall survival (OS). Treatments included first-line oxaliplatin/fluorouracil (OxFU), and second-line irinotecan. Clinicians were unaware of BRAF-status Results 231 patients (9.1%) had BRAF-mutant tumours. BRAF-mutation conferred significantly worse survival independent of associated clinicopathological factors known to be prognostic. Compared with wild-type, BRAF-mutant patients treated with first-line OxFU had similar DCR (59.2% vs 72%; adjusted OR=0.76,p=0.24) and PFS (5.7 vs 6.3 months; adjusted HR=1.14, p=0.26). Following progression on first-line chemotherapy, BRAF-mutant patients had a markedly shorter P-PS (4.2 vs 9.2 months, adjusted HR=1.69,p6 months; OS=24.0 months), however 36.5% progressed rapidly through first-line chemotherapy and thereafter, with OS=4.7 months. Conclusions BRAF-mutant aCRC confers a markedly worse prognosis independent of associated clinicopathological features. Chemotherapy provides meaningful improvements in outcome throughout treatment lines. Post-progression survival is markedly worse and vigilance is required to ensure appropriate delivery of treatment after first-line progression

5D gravity and the discrepant G measurements

It is shown that 5D Kaluza-Klein theory stabilized by an external bulk scalar field may solve the discrepant laboratory G measurements. This is achieved by an effective coupling between gravitation and the geomagnetic field. Experimental considerations are also addressed.Comment: 13 pages, to be published in: Proceedings of the 18th Course of the School on Cosmology and Gravitation: The gravitational Constant. Generalized gravitational theories and experiments (30 April-10 May 2003, Erice). Ed. by G. T. Gillies, V. N. Melnikov and V. de Sabbata, (Kluwer), 13pp. (in print) (2003

Traditional Roles in a Non‐Traditional Setting: Genetic Counseling in Precision Oncology

Next generation sequencing technology is increasingly utilized in oncology with the goal of targeting therapeutics to improve response and reduce side effects. Interpretation of tumor mutations requires sequencing of paired germline DNA, raising questions about incidental germline findings. We describe our experiences as part of a research team implementing a protocol for whole genome sequencing (WGS) of tumors and paired germline DNA known as the Michigan Oncology Sequencing project (MI‐ONCOSEQ) that includes options for receiving incidental germline findings. Genetic counselors (GCs) discuss options for return of results with patients during the informed consent process and document family histories. GCs also review germline findings and actively participate in the multi‐disciplinary Precision Medicine Tumor Board (PMTB), providing clinical context for interpretation of germline results and making recommendations about disclosure of germline findings. GCs have encountered ethical and counseling challenges with participants, described here. Although GCs have not been traditionally involved in molecular testing of tumors, our experiences with MI‐ONCOSEQ demonstrate that GCs have important applicable skills to contribute to multi‐disciplinary care teams implementing precision oncology. Broader use of WGS in oncology treatment decision making and American College of Medical Genetics and Genomics (ACMG) recommendations for active interrogation of germline tissue in tumor‐normal dyads suggests that GCs will have future opportunities in this area outside of research settings.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147078/1/jgc40655.pd

Confirmation that somatic mutations of beta-2 microglobulin correlate with a lack of recurrence in a subset of stage II mismatch repair deficient colorectal cancers from the QUASAR trial

Aims: Beta2‐Microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair‐deficient colorectal cancer (dMMR CRC) with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. Methods: Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Results: Deleterious B2M mutations were detected in 39/121 (32%) dMMR tumours. Five contained missense B2M‐variants of unknown significance, so were excluded from further analyses. With median follow‐up 7.4 years, none of the 39 B2M‐mutant tumours recurred, compared with 14/77 (18%) B2M‐wildtype tumours (p=0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87% and 71% respectively. Significantly (p<0.0001) fewer 3/104 (2.9%) of the 108 pMMR CRCs demonstrated deleterious B2M mutations. One pMMR tumour, containing a frameshift mutation, later recurred. Conclusion: B2M mutations were detected in nearly one third of dMMR cancers, none of which recurred. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC. The mechanism of protection against recurrence and whether this protection extends to stage III disease remains unclear

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

Comparing mutation calls in fixed tumour samples between the Affymetrix OncoScan® Array and PCR based next-generation sequencing

Background: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. Methods: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. Results: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5–10%) than product claims indicated. Conclusions: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results

Validation of the modified Fresno Test: assessing physical therapists' evidence based practice knowledge and skills

<p>Abstract</p> <p>Background</p> <p>Health care educators need valid and reliable tools to assess evidence based practice (EBP) knowledge and skills. Such instruments have yet to be developed for use among physical therapists. The Fresno Test (FT) has been validated only among general practitioners and occupational therapists and does not assess integration of research evidence with patient perspectives and clinical expertise. The purpose of this study was to develop and validate a modified FT to assess EBP knowledge and skills relevant to physical therapist (PT) practice.</p> <p>Methods</p> <p>The FT was modified to include PT-specific content and two new questions to assess integration of patient perspectives and clinical expertise with research evidence. An expert panel reviewed the test for content validity. A cross-sectional cohort representing three training levels (EBP-novice students, EBP-trained students, EBP-expert faculty) completed the test. Two blinded raters, not involved in test development, independently scored each test. Construct validity was assessed through analysis of variance for linear trends among known groups. Inter and intra-rater reliability, internal consistency, item discrimination index, item total correlation, and difficulty were analyzed.</p> <p>Results</p> <p>Among 108 participants (31 EBP-novice students, 50 EBP-trained students, and 27 EBP-expert faculty), there was a statistically significant (p < 0.0001) difference in total score corresponding to training level. Total score reliability and psychometric properties of items modified for discipline-specific content were excellent [inter-rater (ICC (2,1)] = 0.91); intra-rater (ICC (2,1)] = 0.95, 0.96)]. Cronbach's α was 0.78. Of the two new items, only one had strong psychometric properties.</p> <p>Conclusions</p> <p>The 13-item modified FT presented here is a valid, reliable assessment of physical therapists' EBP knowledge and skills. One new item assesses integration of patient perspective as part of the EBP model. Educators and researchers may use the 13-item modified FT to evaluate PT EBP curricula and physical therapists' EBP knowledge and skills.</p

In-depth clinical and biological exploration of DNA Damage Immune Response (DDIR) as a biomarker for oxaliplatin use in colorectal cancer

PURPOSE: The DNA Damage Immune Response (DDIR) assay was developed in breast cancer (BC) based on biology associated with deficiencies in homologous recombination and Fanconi Anemia (HR/FA) pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and oesophageal cancers. In colorectal cancer (CRC) there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in CRC and characterised the biology in DDIR-positive CRC. METHODS: Samples and clinical data were assessed according to DDIR status from patients who received either 5FU or FOLFOX within the FOCUS trial (n=361, stage 4), or neo-adjuvant FOLFOX in the FOxTROT trial (n=97, stage 2/3). Whole transcriptome, mutation and immunohistochemistry data of these samples were used to interrogate the biology of DDIR in CRC. RESULTS: Contrary to our hypothesis, DDIR negative patients displayed a trend towards improved outcome for oxaliplatin-based chemotherapy compared to DDIR positive patients. DDIR positivity was associated with Microsatellite Instability (MSI) and Colorectal Molecular Subtype 1 (CMS1). Refinement of the DDIR signature, based on overlapping interferon-related chemokine signalling associated with DDIR positivity across CRC and BC cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in CRC. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in CRC. However, data presented here suggests the potential of the DDIR assay in identifying immune-rich tumours that may benefit from immune checkpoint blockade, beyond current use of MSI status