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Aim: To demonstrate that MSI-WES is an accurate testing method for microsatellite instability (MSI). Materials & methods: Microsatellite-based indels were counted in the variant call-formatted whole exome sequencing (WES) data of 441 gastric cancer cases using Unix-based algorithms, and the counts expressed as a fraction of the genome sequenced to obtain next-generation sequencing-based MSI indices. Results: The next-generation sequencing-based MSI indices showed a near-perfect concordance with PCR-based MSI status, and moderate to good correlations with the molecular targets of MSI index, MLH1 expression and MLH1 methylation status, at a level comparable to the strengths of correlation between PCR-based MSI status and molecular targets of MSI index/ MLH1 expression and methylation. Conclusion: MSI-WES is a valid, adequate and sensitive approach for testing MSI in cancer.

Recent advances in whole‐slide imaging (WSI) technology have led to the development of a myriad of computer vision and artificial intelligence‐based diagnostic, prognostic, and predictive algorithms. Computational Pathology (CPath) offers an integrated solution to utilise information embedded in pathology WSIs beyond what can be obtained through visual assessment. For automated analysis of WSIs and validation of machine learning (ML) models, annotations at the slide, tissue, and cellular levels are required. The annotation of important visual constructs in pathology images is an important component of CPath projects. Improper annotations can result in algorithms that are hard to interpret and can potentially produce inaccurate and inconsistent results. Despite the crucial role of annotations in CPath projects, there are no well‐defined guidelines or best practices on how annotations should be carried out. In this paper, we address this shortcoming by presenting the experience and best practices acquired during the execution of a large‐scale annotation exercise involving a multidisciplinary team of pathologists, ML experts, and researchers as part of the Pathology image data Lake for Analytics, Knowledge and Education (PathLAKE) consortium. We present a real‐world case study along with examples of different types of annotations, diagnostic algorithm, annotation data dictionary, and annotation constructs. The analyses reported in this work highlight best practice recommendations that can be used as annotation guidelines over the lifecycle of a CPath project.

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. 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. 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. Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.

AimsWe sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation.MethodsFor MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested.ResultsAll the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases.Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data.ConclusionWe describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.

AimsGene dosage can have a major impact on cell biology, although, hitherto, it has been difficult to study using in vitro models. We sought to refine and accelerate the development of ‘gene dosage’ models through using CRISPR/Cas9 (a gene editing technology) for sequential knockout of gene alleles.MethodsOur method involved (1) using Cas9 nuclease mRNA rather than expression plasmids, (2) using a fluorescently labelled FAM-6 tracr complexed with guide RNA and (3) using high-resolution melting (HRM) analysis to screen for mutations. HCT116 cells, wild-type for TP53, were transfected with different molarities of FAM-6 tracr-labelled and guide RNA targeting different exons of TP53 and selected by fluorescence-activated cell sorting. Single-cell colonies were then isolated, expanded and tested for mutation in the targeted region by PCR/HRM.ResultsOut of 32 clones tested, 12 have shown aberrant melting by HRM, giving a targeting efficiency of 37.5%. One clone was sequenced and a heterozygous mutation found — in this case comprising a single base deletion in exon 3. mRNA sequencing confirmed the mutation was expressed, and western blotting for p53 showed the presence of both wild-type and truncated protein bands. Changes in expression of MDM-2 isoforms suggested a functional effect of the induced TP53 mutation.ConclusionsWe have developed an in vitro model to study TP53 gene dosage effects. The protocol is efficient and applicable to any gene. Importantly, we have used Cas9 mRNA and labelled tracr/guide RNA to isolate likely mutated cells and HRM for rapid mutation detection.

Aims We previously described the quick multiplex consensus PCR (QMC-PCR) as a method for rapid mutation screening in low-quality template. QMC-PCR has two-stages: a prediagnostic multiplex (PDM) reaction followed by a single specific diagnostic reaction with high-resolution melting (HRM) analysis. We aimed to develop QMC-PCRx in which second stage was multiplexed to allow testing of multiple targets. Methods The PDM reaction was retained without change. For the second stage, in silico design was used to identify targets amenable to a multiplex specific diagnostic reaction and multiplex HRM (mHRM) analysis. Following optimisation, 17 colorectal cancers were tested for mutation in five hotspots. For QMC-PCR, each target was tested individually. For QMC-PCRx, the targets were tested in the following combinations (i) KRAS exon 3/ PIK3CA exon 20/ PTEN exon 3 in triplex and (ii) PTEN exon 7/ NRAS exon 2 in duplex. The degree of agreement between the novel QMC-PCRx and the standard QMC-PCR was tested by the percentage concordance. Results Optimisation of mHRM showed that peaks needed to be separated (without overlap) and the optimal number was three targets per test. Our experimental design produced distinct and widely separated peaks for the individual targets although one of the primers needed a GC-tail. A total of 85 individual targets were tested; this required 85 second-stage PCR/HRM tests by QMC-PCR versus 34 second-stage tests by QMC-PCRx. The percentage concordance between the singleplex and multiplex methodologies was 100%. Conclusions A multiplexed analysis using HRM is possible without loss of diagnostic accuracy. The novel QMC-PCRx protocol can significantly reduce workload and costs of mutation screening.

Currently, short DNA segments of sub-100 bp can be sequenced either directly by next-generation sequencing and pyrosequencing, which are expensive, or indirectly, via Sanger sequencing combined with the cumbersome and failure-prone plasmid cloning. To circumvent these issues, we have generated a novel sequencing-purposed PCR assay using long-tailed primers (squirrel primers) to Sanger sequence directly sub-100 bp genomic amplicons. Squirrel primers, 40-65 nt in length, were used to amplify 51-93 bp long genomic sequences of KRAS exons 2 and 3, BRAF exon 15, PI3K catalytic subunit alpha exon 20, and phosphatase and tensin homolog exon 3 from colorectal cancer (CRC) cell lines and preamplified clinical CRC samples with known mutation status by PCR. Following this, a short second pair of primers that bind at the 5' region of the long tails was used for sequencing on the 3130 × l ABI Prism Genetic Analyzer. The sequencing data were analyzed via FinchTV software. High-quality sequencing data were obtained from 51 to 93 bp long genomic sequences with our novel PCR assay, with capture of all of the target sequences in all of the samples in both the forward and reverse directions and confirmation of the mutation status of the CRC samples. Whereas the sequencing quality was independent of the template type, it showed a squirrel primer tail length-dependent pattern. Our novel PCR assay for direct and targeted Sanger sequencing of short genomic segments has potential applications in focused molecular/genetic profiling of cancer in research and diagnostics fields in which fragmented DNA, such as circulating tumor DNA and archival tissue DNA, are used as starting templates.

High Resolution Melting HRM measures DNA melting patterns to detect mutations Hitherto HRM has not been used to report the nature of the mutation We reasoned that in conditions with a minor allele e g allelic loss and somatic mutation CO amplification at Lower Denaturation temperature COLD PCR could change HRM patterns allowing i detection of Loss of Heterozygosity LOH and ii discrimination of somatic mutations from germline variants For LOH we tested paired tumour normal samples from cases of colorectal cancer CRC which were heterozygous for the SMAD SNP rs and case which was homozygous wild type To discriminate somatic from germline mutation we tested tumour from CRCs with somatic mutation in codon POLE paired tumour normal tissue from CRCs heterozygous for POLESNP rs and matched normal samples wild type All samples underwent both the standard and COLD PCR followed by HRM analysis In tumour samples which were heterozygous for the SMAD SNP HRM patterns changed following COLD PCR suggesting LOH Changes in HRM patterns were also seen in tumours with POLE codon mutation suggesting somatic rather than germline mutation No change was seen in the tumour and matched normal samples heterozygous for POLESNP rs We conclude that combining COLD PCR with HRM COLD HRM allows inference of LOH and somatic sequence mutation without the requirement for matched normal DNA

Mutation in KRAS, BRAF and PIK3CA are commonly present in colorectal cancer (CRC). KRAS and BRAF form part of the mitogen-activated protein kinase (MAPK) signalling pathway and KRAS lies immediately upstream of BRAF. Consequently, KRAS and BRAF mutations are mutually exclusive with mutation in either one causing activation of MAPK signalling.1 PIK3CA forms part of the phosphatidylinositide‐3‐kinase PI3K signalling pathway, and several studies have shown a significant association of KRAS and PIK3CA mutation in several cancer types including CRC.1–3 At face value, there would appear to be positive co-selection of MAPK and PI3K signalling activation implying synergy between these two pathways. If this were true, then a similar positive association would be expected between mutation of BRAF and PIK3CA. To date, less consistent or no association of BRAF and PIK3CA mutation has been reported.1 3 4 However, it is well established that BRAF mutation is strongly associated with microsatellite instability (MSI) in CRC. We therefore sought to test the association between mutation of BRAF and PIK3CA …

Introduction Microsatellite stable sporadic colorectal cancers (CRCs) can be classified as either tumours with chromosomal instability (CIN+) or tumours that are ‘Microsatellite and Chromosomal Stable’ (MACS). The CIN + tumours are aneuploid whilst MACS are near-diploid; little else is known about their differences. We compared the mutation profiles of CIN + and MACS CRCs. Method Targeted Next Generation Sequencing for mutation in 26 driver genes (TruSight-26 kit) was undertaken in 46 CIN + and 35 MACSCRCs. Tumours were compared for mutation frequency, allelic imbalance and clonal heterogeneity. Results Mutations were detected in 58% genes and, overall, mutation in driver genes was at expected frequencies. Comparison of classes revealed similar mutation frequencies in most genes and allelic imbalance atAPC and TP53. Differences were seen in mutation frequency in KRAS (41% CIN+ vs 68% MACS, p = 0.015) and GNAS (0% CIN+ vs 12% MACS, p = 0.032). Twenty percent CIN + CRCs harboured mutations only in TP53 - a profile not seen in the MACS tumours (p = 0.009). None of the differences were significant after multiple testing corrections. Conclusions The mutation profiles of CIN and MACS CRCs are similar. The events allowing aneuploidy (or forcing retention of diploidy) remain unknown.

High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner. DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19. The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process. In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI.

SUMMARY Background: Disparities in DNA template quantity across sample wells is a common occurrence in PCR experiments and a frequent cause of bogus results in research and diagnostics. High Resolution Melt (HRM) analysis, a robust genotyping technique, was shown to be highly reproducible irrespective of the reaction volume and DNA template concentration used for PCR. The aim of this study is to determine if HRM can maintain its reproducibility/reliability when varying amounts of starting DNA templates are utilized for the upstream PCR. Methods: Serial dilutions of differentially prepared DNA templates were amplified and subjected to HRM analysis using the ABI 7500 Fast Real Time PCR machine and the ABI HRM software v2.0.6. The first serial dilution set consisted of seven 1:2 serial dilutions of amplicon-enriched DNA templates from two samples of known genotypes at exon 9 of PIK3CA, while the second serial dilution set consisted of four 1:10 dilutions of two different template types from the same sample: original FFPE DNA template and amplicon-enriched DNA template. Results: HRM analysis was reproducible across two- to one thousand-fold difference in starting DNA template in the upstream PCR, so long as the starting DNA template is of ample quality and quantity to give an adequate PCR amplification. Conclusions: HRM is highly reliable in the presence of a wide variation in the amount of starting template across wells for the upstream PCR, and the factor that determines HRM reproducibility is the adequacy of the PCR amplification. (Clin. Lab. 2015;61:xx-xx. DOI: 10.7754/Clin.Lab.2014.140821)

Introduction: Sickle Cell Disease (SCD) has a high mortality rate in the environment where we practice. There is lack of contemporal autopsy studies describing causes of death among SCD patients at our centre. Methods: This is a retrospective study of SCD patients who died between January 1991 and December 2008 and that had autopsy examination to confirm the cause of death in a Nigerian teaching hospital. The clinical data, including the age, gender, Hb genotype, and the major autopsy findings and cause of death were obtained for each patient from the complete autopsy reports that included histopathological examination. Multiple causes of death were entertained. Results: A total of 52 autopsies were performed. The mean age at death was 21.3 years (range, 1-47 years) and a male/female ratio of 1.3:1. HbS+C patients lived longer than HbS patients (21.0 years Vs 24.0 years) and peak mortality was in the 2nd and 3rd decades of life. The commonest causes of death as a single entity or in combination included infections in 78% of cases, fatal thrombotic/embolic events (37%) making acute chest syndrome a leading cause of death. This was followed closely by anemia alone or in combination with acute sequestration crises in 31% of patients.Conclusion: Infections are the commonest causes of death in Nigerian SCD patients, efforts to reduce infection especially early in life through prophylaxis or vaccination will impact on the overall survival of these patientsKey words: Sickle cell disease, cause of death, mortality, autopsy