MSI-WES: a simple approach for microsatellite instability testing using whole exome sequencing.

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.

Semantic annotation for computational pathology: multidisciplinary experience and best practice recommendations.

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.

Targeted next generation sequencing reveals a common genetic pathway for colorectal cancers with chromosomal instability and those with microsatellite and chromosome stability.

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.

Prevalence, Predictive Factors, and Characteristics of Osteoporosis in Hyperthyroid Patients.

OBJECTIVE: The osteoporosis in thyroid disorder has the lowest report especially in sub-Saharan Africa. This study aims to determine the prevalence, predictive factors, and characteristics of osteoporosis in hyperthyroid patients. METHOD: Forty (40) hyperthyroid patients and healthy controls ages 21-50 years were recruited in this study. Questionnaires were administered to capture bio- and clinical data. Biochemical tests included blood, thyroid functions, intact parathyroid hormone, corrected calcium, and 25-hydroxyvitamin D tests. Bone mineral density (BMD) was also evaluated. Data were analyzed using the SPSS 21. A p value < 0.05 was regarded as significant. RESULTS: Osteoporosis was observed in 18 (45%) of study subjects, 13 (72.2%) females and 5 (27.8%) males, respectively. The BMD of the hyperthyroid patients had a negative correlation with free triiodothyronine, FT3 (r = -0.49, p = 0.005), FT4 (r = -0.33, p = 0.009), corrected calcium (r = -0.31, p = 0.039), alkaline phosphatase (r = -0.53, p < 0.001), and osteocalcin (r = -0.61, p < 0.001). Conversely, a positive association with thyroid-stimulating hormone (TSH) (r = 0.54, p < 0.001) was observed. Multiple regression showed osteocalcin (p < 0.001) and TSH (p = 0.015) as independent predictors of osteoporosis. CONCLUSION: Thyrotoxicosis is a risk factor for osteoporosis occurrence, and we recommend routine screening for this bone disease in persons over 20 years old with this disorder.

A refined method to study gene dosage changes in vitro using CRISPR/Cas9.

AIMS: Gene 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. METHODS: Our 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. RESULTS: Out 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. CONCLUSIONS: We 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.

QMC-PCRx: a novel method 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.

"Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments.

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.

Clinicopathological and molecular characteristics of Ku 70/80 expression in Nigerian breast cancer and its potential therapeutic implications.

Ku 70/80 is a regulator of the Non-Homologous End Joining (NHEJ) roles in clinicopathological features, and has prognostic significance in breast cancer (BC) in Caucasian populations. However, its significance in the Nigerian BC population, which is characterized by a higher rate of the triple-negative and basal phenotype, p53 mutation rate and BRCA1 deficiency, still needs to be investigated. We hypothesize that Ku70/80 expression shows adverse expression in Nigerian BC and, furthermore, that it is likely to have a therapeutic implication for Black BC management. This study investigated the biological, clinicopathological and prognostic significance of Ku 70/80 expression in a BC cohort from a Nigerian population. Ku 70/80 expression was determined in 188 well-characterized formalin-fixed, paraffin-embedded (FFPE) BC samples using tissue microarray and immunohistochemistry. Ku 70/80 expression was correlated with clinicopathological, molecular and prognostic characteristics of patients. Ku 70/80 was expressed in 113 (60.1%) tumors, and was positively associated with metastatic disease, triple-negative and basal phenotype, BRCA1 down regulators (MTA-1 and ID4), p-cadherin, PI3KCA and p53 expression. It inversely correlated with BRCA1, BRCA2, BARD1 and p27. Ku 70/80 was predictive of breast cancer-specific survival in multivariate analysis, but not of disease-free interval. This study demonstrated that Ku 70/80 expression is associated with triple negativity and down-regulation of the homologous recombination pathway of DNA repair. Therefore, the development of novel drugs to target KU70/80 may improve the patients' outcome in the treatment of Black BC.

Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

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.