Surgery (RMH) - Research Publications
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ItemEvaluation of the transferability of survival calculators for stage II/III colon cancer across healthcare systems(WILEY, 2019-07-01)Adjuvant! Online Inc (A!O), the Memorial Sloan Kettering Cancer Center (MSKCC), MD Anderson (MDA) and Mayo Clinic (MC) provide calculators to predict survival probabilities for patients with resected early-stage colon cancer, trained on data from United States (US) patient cohorts or patients enrolled in international clinical trials. Limited data exist on the transferability of calculators across healthcare systems. Calculator transferability to Australian community practice was evaluated for 1,401 stage II/III patients. Calibration and discrimination were assessed for overall (OS), cancer-specific (CSS) or recurrence-free survival (RFS). The US patient cohort-based calculators, A!O, MSKCC and MDA, significantly overestimated risks of recurrence and death in Australian patients, with 5-year OS, CSS and RFS prediction differences of -6.5% to -9.9%, -9.1% to -14.4% and - 3.8% to -6.8%, respectively (p < 0.001). Significant heterogeneity in calibration was observed for subgroups by tumor stage and treatment, age, gender, tumor location, ECOG and ASA score. Calibration appeared acceptable for the clinical trial patient-based MC calculator, but restricted tool applicability (stage III patients, ≥12 examined lymph nodes, receiving adjuvant treatment) limited the sample size. Compared to AJCC 7th edition tumor staging, calculators showed improved discrimination for OS, but no improvement for CSS and RFS. In conclusion, deficiencies in calibration limited transferability of US patient cohort-based survival calculators for early-stage colon cancer to the setting of Australian community practice. Our results demonstrate the utility for multi-feature survival calculators to improve OS predictions but highlight the importance for performance assessment of tools prior to implementation in an external health care setting.
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ItemA mutant BRAF V600E-specific immunohistochemical assay: correlation with molecular mutation status and clinical outcome in colorectal cancer(SPRINGER, 2015-03)The B-type Raf kinase (BRAF) V600E mutation is a well-established biomarker for poor prognosis in metastatic colorectal cancer (mCRC) and is a highly attractive drug target. A barrier to the development of new therapies targeting BRAF V600E in mCRC is the low prevalence of mutations (approximately 10 %) and the current need for access to sequencing-based technologies which are not routinely available outside of large cancer centres. Availability of a standardised immunohistochemistry (IHC) test, more suited to routine pathology practice, would provide much broader access to patient identification. We sought to evaluate the accuracy and clinical utility of a recently developed BRAF V600E IHC method as a prognostic biomarker in a large cohort of community-based CRC patients. Archival tumour samples from 505 patients with stage I-IV CRC were immunohistochemically tested with two antibodies, pBR1 for total BRAF and VE1 for BRAF V600E. Cases were assessed by two blinded pathologists, and results were compared to BRAF V600E mutation status determined using DNA sequencing. Discordant cases were retested with a BRAF V600E SNaPshot assay. BRAF mutation status was correlated with overall survival (OS) in stage IV CRC. By DNA sequencing and IHC, 505 and 477 patients were respectively evaluable. Out of 477 patients, 56 (11. 7 %) had BRAF V600E mutations detected by sequencing and 63 (13.2 %) by IHC. Using DNA sequencing results as the reference, sensitivity and specificity for IHC were 98.2 % (55/56) and 98.1 % (413/421), respectively. IHC had a positive predictive value (PPV) of 87.3 % (55/63) and a negative predictive value (NPV) of 99.8 % (413/414). Compared to DNA sequencing plus retesting of available discordant cases by SNaPshot assay, IHC using the VE1 antibody had a 100 % sensitivity (59/59), specificity (416/416), NPV (416/416) and PPV (59/59). Stage IV CRC patients with BRAF V600E protein detected by IHC exhibited a significantly shorter overall survival (hazard ratio = 2.20, 95 % CI 1.26-3.83, p = 0.005), consistent with other published series. Immunohistochemistry using the BRAF V600E VE1 antibody is an accurate diagnostic assay in CRC. The test provides a simple, clinically applicable method of testing for the BRAF V600E mutation in routine practice.
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ItemDifferent APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/β-catenin signalling thresholds for tumourigenesis(NATURE PUBLISHING GROUP, 2013-09-26)Biallelic protein-truncating mutations in the adenomatous polyposis coli (APC) gene are prevalent in sporadic colorectal cancer (CRC). Mutations may not be fully inactivating, instead producing WNT/β-catenin signalling levels 'just-right' for tumourigenesis. However, the spectrum of optimal APC genotypes accounting for both hits, and the influence of clinicopathological features on genotype selection remain undefined. We analysed 630 sporadic CRCs for APC mutations and loss of heterozygosity (LOH) using sequencing and single-nucleotide polymorphism microarrays, respectively. Truncating APC mutations and/or LOH were detected in 75% of CRCs. Most truncating mutations occurred within a mutation cluster region (MCR; codons 1282-1581) leaving 1-3 intact 20 amino-acid repeats (20AARs) and abolishing all Ser-Ala-Met-Pro (SAMP) repeats. Cancers commonly had one MCR mutation plus either LOH or another mutation 5' to the MCR. LOH was associated with mutations leaving 1 intact 20AAR. MCR mutations leaving 1 vs 2-3 intact 20AARs were associated with 5' mutations disrupting or leaving intact the armadillo-repeat domain, respectively. Cancers with three hits had an over-representation of mutations upstream of codon 184, in the alternatively spliced region of exon 9, and 3' to the MCR. Microsatellite unstable cancers showed hyper-mutation at MCR mono- and di-nucleotide repeats, leaving 2-3 intact 20AARs. Proximal and distal cancers exhibited different preferred APC genotypes, leaving a total of 2 or 3 and 0 to 2 intact 20AARs, respectively. In conclusion, APC genotypes in sporadic CRCs demonstrate 'fine-tuned' interdependence of hits by type and location, consistent with selection for particular residual levels of WNT/β-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.
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ItemAnalysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion(BMC, 2013-01-03)BACKGROUND: Prevalence of colorectal cancer (CRC) in the British Bangladeshi population (BAN) is low compared to British Caucasians (CAU). Genetic background may influence mutations and disease features. METHODS: We characterized the clinicopathological features of BAN CRCs and interrogated their genomes using mutation profiling and high-density single nucleotide polymorphism (SNP) arrays and compared findings to CAU CRCs. RESULTS: Age of onset of BAN CRC was significantly lower than for CAU patients (p=3.0 x 10-5) and this difference was not due to Lynch syndrome or the polyposis syndromes. KRAS mutations in BAN microsatellite stable (MSS) CRCs were comparatively rare (5.4%) compared to CAU MSS CRCs (25%; p=0.04), which correlates with the high percentage of mucinous histotype observed (31%) in the BAN samples. No BRAF mutations was seen in our BAN MSS CRCs (CAU CRCs, 12%; p=0.08). Array data revealed similar patterns of gains (chromosome 7 and 8q), losses (8p, 17p and 18q) and LOH (4q, 17p and 18q) in BAN and CAU CRCs. A small deletion on chromosome 16p13.2 involving the alternative splicing factor RBFOX1 only was found in significantly more BAN (50%) than CAU CRCs (15%) cases (p=0.04). Focal deletions targeting the 5' end of the gene were also identified. Novel RBFOX1 mutations were found in CRC cell lines and tumours; mRNA and protein expression was reduced in tumours. CONCLUSIONS: KRAS mutations were rare in BAN MSS CRC and a mucinous histotype common. Loss of RBFOX1 may explain the anomalous splicing activity associated with CRC.
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ItemRefinement of the associations between risk of colorectal cancer and polymorphisms on chromosomes 1q41 and 12q13.13(OXFORD UNIV PRESS, 2012-02-15)In genome-wide association studies (GWASs) of colorectal cancer, we have identified two genomic regions in which pairs of tagging-single nucleotide polymorphisms (tagSNPs) are associated with disease; these comprise chromosomes 1q41 (rs6691170, rs6687758) and 12q13.13 (rs7163702, rs11169552). We investigated these regions further, aiming to determine whether they contain more than one independent association signal and/or to identify the SNPs most strongly associated with disease. Genotyping of additional sample sets at the original tagSNPs showed that, for both regions, the two tagSNPs were unlikely to identify a single haplotype on which the functional variation lay. Conversely, one of the pair of SNPs did not fully capture the association signal in each region. We therefore undertook more detailed analyses, using imputation, logistic regression, genealogical analysis using the GENECLUSTER program and haplotype analysis. In the 1q41 region, the SNP rs11118883 emerged as a strong candidate based on all these analyses, sufficient to account for the signals at both rs6691170 and rs6687758. rs11118883 lies within a region with strong evidence of transcriptional regulatory activity and has been associated with expression of PDGFRB mRNA. For 12q13.13, a complex situation was found: SNP rs7972465 showed stronger association than either rs11169552 or rs7136702, and GENECLUSTER found no good evidence for a two-SNP model. However, logistic regression and haplotype analyses supported a two-SNP model, in which a signal at the SNP rs706793 was added to that at rs11169552. Post-GWAS fine-mapping studies are challenging, but the use of multiple tools can assist in identifying candidate functional variants in at least some cases.
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ItemMultiple Common Susceptibility Variants near BMP Pathway Loci GREM1, BMP4, and BMP2 Explain Part of the Missing Heritability of Colorectal Cancer(PUBLIC LIBRARY SCIENCE, 2011-06)Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.
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ItemFunctional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer(PUBLIC LIBRARY SCIENCE, 2014-06-03)MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.
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ItemRelative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer(IMPACT JOURNALS LLC, 2016-06-14)Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.
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ItemDifferential RNA-seq analysis comparing APC-defective and APC-restored SW480 colorectal cancer cells(ELSEVIER SCIENCE BV, 2016-03)The adenomatous polyposis coli (APC) tumour suppressor gene is mutated in about 80% of colorectal cancers (CRC) Brannon et al. (2014) [1]. APC is a large multifunctional protein that regulates many biological functions including Wnt signalling (through the regulation of beta-catenin stability) Reya and Clevers (2005) [2], cell migration Kroboth et al. (2007), Sansom et al. (2004) [3], [4], mitosis Kaplan et al. (2001) [5], cell adhesion Faux et al. (2004), Carothers et al. (2001) [6], [7] and differentiation Sansom et al. (2004) [4]. Although the role of APC in CRC is often described as the deregulation of Wnt signalling, its other biological functions suggest that there are other factors at play that contribute to the onset of adenomas and the progression of CRC upon the truncation of APC. To identify genes and pathways that are dysregulated as a consequence of loss of function of APC, we compared the gene expression profiles of the APC mutated human CRC cell line SW480 following reintroduction of wild-type APC (SW480 + APC) or empty control vector (SW480 + vector control) Faux et al. (2004) . Here we describe the RNA-seq data derived for three biological replicates of parental SW480, SW480 + vector control and SW480 + APC cells, and present the bioinformatics pipeline used to test for differential gene expression and pathway enrichment analysis. A total of 1735 genes showed significant differential expression when APC was restored and were enriched for genes associated with cell polarity, Wnt signalling and the epithelial to mesenchymal transition. There was additional enrichment for genes involved in cell-cell adhesion, cell-matrix junctions, angiogenesis, axon morphogenesis and cell movement. The raw and analysed RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE76307. This dataset is useful for further investigations of the impact of APC mutation on the properties of colorectal cancer cells.
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ItemLong noncoding RNA LINP1: scaffolding non-homologous end joining(NATURE PUBLISHING GROUP, 2016)