Pathology - Research Publications
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ItemSomatic mutations of the coding microsatellites within the beta-2-microglobulin gene in mismatch repair-deficient colorectal cancers and adenomas(SPRINGER, 2018-01)In colorectal cancers (CRCs) with tumour mismatch repair (MMR) deficiency, genes involved in the host immune response that contain microsatellites in their coding regions, including beta-2-microglobulin (B2M), can acquire mutations that may alter the immune response, tumour progression and prognosis. We screened the coding microsatellites within B2M for somatic mutations in MMR-deficient CRCs and adenomas to determine associations with tumour subtypes, clinicopathological features and survival. Incident MMR-deficient CRCs from Australasian Colorectal Cancer Family Registry (ACCFR) and the Melbourne Collaborative Cohort Study participants (n = 144) and 63 adenomas from 41 MMR gene mutation carriers from the ACCFR were screened for somatic mutations within five coding microsatellites of B2M. Hazard ratios (HR) and 95% confidence intervals (CI) for overall survival by B2M mutation status were estimated using Cox regression, adjusting for age at CRC diagnosis, sex, AJCC stage and grade. B2M mutations occurred in 30 (20.8%) of the 144 MMR-deficient CRCs (29% of the MLH1-methylated, 17% of the Lynch syndrome and 9% of the suspected Lynch CRCs). No B2M mutations were identified in the 63 adenomas tested. B2M mutations differed by site, stage, grade and lymphocytic infiltration although none reached statistical significance (p > 0.05). The HR for overall survival for B2M mutated CRC was 0.65 (95% CI 0.29-1.48) compared with B2M wild-type. We observed differences in B2M mutation status in MMR-deficient CRC by tumour subtypes, site, stage, grade, immune infiltrate and for overall survival that warrant further investigation in larger studies before B2M mutation status can be considered to have clinical utility.
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ItemHi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2(BMC, 2013-11-08)BACKGROUND: Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. METHODS: Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. RESULTS: We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. CONCLUSIONS: These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.
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ItemCross-platform compatibility of Hi-Plex, a streamlined approach for targeted massively parallel sequencing(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013-11-15)Although per-base sequencing costs have decreased during recent years, library preparation for targeted massively parallel sequencing remains constrained by high reagent cost, limited design flexibility, and protocol complexity. To address these limitations, we previously developed Hi-Plex, a polymerase chain reaction (PCR) massively parallel sequencing strategy for screening panels of genomic target regions. Here, we demonstrate that Hi-Plex applied with hybrid adapters can generate a library suitable for sequencing with both the Ion Torrent and the TruSeq chemistries and that adjusting primer concentrations improves coverage uniformity. These results expand Hi-Plex capabilities as an accurate, affordable, flexible, and rapid approach for various genetic screening applications.
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ItemNo Preview AvailableA high-plex PCR approach for massively parallel sequencing(FUTURE SCI LTD, 2013-08)Current methods for targeted massively parallel sequencing (MPS) have several drawbacks, including limited design flexibility, expense, and protocol complexity, which restrict their application to settings involving modest target size and requiring low cost and high throughput. To address this, we have developed Hi-Plex, a PCR-MPS strategy intended for high-throughput screening of multiple genomic target regions that integrates simple, automated primer design software to control product size. Featuring permissive thermocycling conditions and clamp bias reduction, our protocol is simple, cost- and time-effective, uses readily available reagents, does not require expensive instrumentation, and requires minimal optimization. In a 60-plex assay targeting the breast cancer predisposition genes PALB2 and XRCC2, we applied Hi-Plex to 100 ng LCL-derived DNA, and 100 ng and 25 ng FFPE tumor-derived DNA. Altogether, at least 86.94% of the human genome-mapped reads were on target, and 100% of targeted amplicons were represented within 25-fold of the mean. Using 25 ng FFPE-derived DNA, 95.14% of mapped reads were on-target and relative representation ranged from 10.1-fold lower to 5.8-fold higher than the mean. These results were obtained using only the initial automatically-designed primers present in equal concentration. Hi-Plex represents a powerful new approach for screening panels of genomic target regions.