Robyn L. Ward

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Germline epimutation of MLH1 in individuals with multiple cancers

Epigenetic silencing can mimic genetic mutation by abolishing expression of a gene. We hypothesized that an epimutation could occur in any gene as a germline event that predisposes to disease and looked for examples in tumor suppressor genes in individuals with cancer. Here we report two individuals with soma-wide, allele-specific and mosaic hypermethylation of the DNA mismatch repair gene MLH1. Both individuals lack evidence of genetic mutation in any mismatch repair gene but have had multiple primary tumors that show mismatch repair deficiency, and both meet clinical criteria for hereditary nonpolyposis colorectal cancer. The epimutation was also present in spermatozoa of one of the individuals, indicating a germline defect and the potential for transmission to offspring. Germline epimutation provides a mechanism for phenocopying of genetic disease. The mosaicism and nonmendelian inheritance that are characteristic of epigenetic states could produce patterns of disease risk that resemble those of polygenic or complex traits.

Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among ≈12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as α-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-β superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.

Microsatellite instability and the clinicopathological features of sporadic colorectal cancer

BACKGROUND AND AIMS In this study, we prospectively examined the clinical significance of the microsatellite instability (MSI) phenotype in sporadic colorectal cancer, and investigated methods for effective identification of these tumours in routine pathology practice. METHODS DNA was extracted from 310 tumours collected from 302 consecutive individuals undergoing curative surgery for sporadic colorectal cancer. Microsatellite status was determined by polymerase chain reaction amplification using standard markers, while immunostaining was used to examine expression of MLH1, MSH2, and p53. RESULTS Eleven per cent of tumours showed high level instability (MSI-H), 6.8% had low level instability (MSI-L), and the remainder were stable. MSI-H tumours were significantly more likely to be of high histopathological grade, have a mucinous phenotype, and to harbour increased numbers of intraepithelial lymphocytes. They were also more likely to be right sided, occur in women, and be associated with improved overall survival. In total, 25 (8%) tumours showed loss of staining for MLH1 and a further three tumours showed absence of staining for MSH2. The positive and negative predictive value of immunohistochemistry in the detection of MSI-H tumours was greater than 95%. CONCLUSIONS We conclude that the MSI-H phenotype constitutes a pathologically and clinically distinct subtype of sporadic colorectal cancer. Immunohistochemical staining for MLH1 and MSH2 represents an inexpensive and accurate means of identifying such tumours.

HerMES: The SPIRE confusion limit

We report on the sensitivity of SPIRE photometers on the Herschel Space Observatory. Specifically, we measure the confusion noise from observations taken during the Science Demonstration Phase of the Herschel Multi-tiered Extragalactic Survey. Confusion noise is defined to be the spatial variation of the sky intensity in the limit of infinite integration time, and is found to be consistent among the different fields in our survey at the level of 5.8, 6.3 and 6.8 mJy/beam at 250, 350 and 500 microns, respectively. These results, together with the measured instrument noise, may be used to estimate the integration time required for confusion-limited maps, and provide a noise estimate for maps obtained by SPIRE.

Adverse Prognostic Effect of Methylation in Colorectal Cancer Is Reversed by Microsatellite Instability

PURPOSE DNA methylation is an important biologic event in colorectal cancer and in some cases is associated with the development of microsatellite instability (MSI). In this study, we sought to determine the prognostic significance of DNA methylation, both in univariate analysis and in concert with other clinicopathologic factors known to influence outcome. PATIENTS AND METHODS Fresh tissue (625 cancers) was obtained from 605 individuals (age range, 29 to 99 years) undergoing curative surgery for colorectal cancer at one institution during a period of 8 years. Clinicopathologic details were recorded for all tumors, including stage, grade, type, vascular space invasion, and clinical follow-up to 5 years. Microsatellite status was assessed using standard markers. Methylation of p16 and hMLH1 promoters was determined by methylation-specific polymerase chain reaction (PCR), whereas methylation at methylated-in-tumor loci (MINT)1, MINT2, MINT12, and MINT31 loci were assessed by bisulfite-PCR. RESULTS Patients with microsatellite unstable tumors (12%) had better disease-specific survival than those with microsatellite stable (MSS) tumors (univariate analysis: hazard ratio [HR], 0.53; 95% CI, 0.27 to 1.0). Overall survival of individuals with MSS tumors was influenced by three independently significant factors: tumor stage (HR, 7.3; 95% CI, 5.1 to 10.4), heavy tumor methylation (HR, 2.1; 95% CI, 1.1 to 4.0), and vascular space invasion (HR, 1.9; 95% CI, 1.3 to 2.9). In MSS tumors, methylation at any single site was not independently predictive of survival. Neither methylation nor microsatellite status predicted a favorable response to chemotherapy. CONCLUSION DNA methylation is associated with a worse outcome in colorectal cancer, but this adverse prognostic influence is lost in those methylated tumors showing MSI. The mechanisms of these events warrant additional investigation.

HerMES : SPIRE galaxy number counts at 250, 350, and 500 μm

Emission at far-infrared wavelengths makes up a significant fraction of the total light detected from galaxies over the age of Universe. Herschel provides an opportunity for studying galaxies at the peak wavelength of their emission. Our aim is to provide a benchmark for models of galaxy population evolution and to test pre-existing models of galaxies. With the Herschel Multi-tiered Extra-galactic survey, HerMES, we have observed a number of fields of different areas and sensitivity using the SPIRE instrument on Herschel. We have determined the number counts of galaxies down to ~20 mJy. Our constraints from directly counting galaxies are consistent with, though more precise than, estimates from the BLAST fluctuation analysis. We have found a steep rise in the Euclidean normalised counts <100 mJy. We have directly resolved ~15% of the infrared extra-galactic background at the wavelength near where it peaks.

The relationship between hypomethylation and CpG island methylation in colorectal neoplasia

Tumors are often characterized by an imbalance in cytosine methylation as manifested both by hypermethylation of CpG islands and by genome hypomethylation. These epigenetic changes were assessed in colorectal neoplasia to determine whether they arose through a common mechanism or indeed were distinct and unrelated phenomena. Fresh representative samples of adenomas, hyperplastic polyps, colorectal cancers, and normal mucosa were used in this study. Global methylation levels were measured by analyzing the methyl-accepting capacity of DNA. Methylation of p16, hMLH1, and MINT 1, 2, 12, and 31 were assessed by bisulfite polymerase chain reaction. Microsatellite status was determined by polymerase chain reaction using six markers and hMLH1 and proliferating cell nuclear antigen expression was assessed by immunohistochemistry. Normal colonic mucosa had a higher endogenous 5-methyl cytosine content than all proliferative lesions of the colon (P < 0.001). The extent of demethylation in hyperplastic polyps and adenomas was significantly related to its proliferative rate. Right-sided hyperplastic polyps were more likely to be methylated than adenomas (odds ratio, 2.3; confidence interval, 1.1 to 4.6). There was no relationship between the level of global hypomethylation and hypermethylation. Some hyperplastic colorectal polyps have a propensity to develop dense CpG island methylation. Hypermethylation and hypomethylation contribute separately to the process of carcinogenesis.

Hypomethylation of L1 retrotransposons in colorectal cancer and adjacent normal tissue

Background and aimsMalignant cells often exhibit perturbations in the pattern of cytosine methylation. Hypermethylation of CpG islands has been extensively documented, but genome-wide hypomethylation is also a common feature of malignant cells. The bulk of cytosine methylation in the mammalian genome occurs on repetitive elements. This study analysed the methylation status of L1 retrotransposons in colorectal cancer.Patients and methodsMethylation-sensitive Southern blotting was used to determine L1 promoter methylation in colon tumours, adjacent normal tissue, and normal colonic mucosa from healthy individuals.ResultsHypomethylation of L1 promoter sequences was detected in all tumours but was also detected in the histologically normal colonic mucosa of 6 of 19 cancer patients, even at a considerable distance from the tumour. L1 hypomethylation was not detected in matched normal peripheral blood, lymph node or smooth muscle tissue from cancer patients or in the colonic mucosa of 14 healthy individuals. We also assayed for the total proportion of methylated CpG in normal bowel specimens from normal and colon cancer patients. Normal mucosa from cancer patients exhibited lower levels of genomic methylation than the mucosa from healthy individuals, and levels were significantly lower in those patients exhibiting L1 promoter hypomethylation.ConclusionThese results suggest that genomic hypomethylation is an early event in tumourigenesis. Progressive demethylation of L1 promoter sequences could lead to disturbance of normal gene expression and facilitate the process of neoplastic progression.

Serrated and non-serrated polyps of the colorectum: their prevalence in an unselected case series and correlation of BRAF mutation analysis with the diagnosis of sessile serrated adenoma

Aims: To determine the prevalence of colorectal polyps of different types in an unselected population, and to correlate the morphological diagnoses with BRAF mutation analysis. Methods: Cases of colorectal polyps diagnosed at endoscopy were retrieved from the files of Southern.IML Pathology. All slides were reviewed and the lesions classified histologically. A diagnosis of sessile serrated adenoma was made even if the characteristic features were present only focally. If there was more than one polyp of a particular type in any patient, one lesion was chosen at random so that the results represent the number of patients with each type of polyp rather than the total number of polyps. A proportion of the polyps was subjected to BRAF mutation analysis. Results: A total of 1479 patients were identified. Non-serrated (“conventional”) adenomas were found in 964 patients (65%), hyperplastic polyps in 437 (30%), sessile serrated adenomas in 57 (3.9%), traditional serrated adenomas in 11 (0.7%) and mixed hyperplastic adenomatous polyps in 10 (0.7%). BRAF V600E mutation analysis was performed in 148 selected cases; mutations were found in 44/49 (90%) of lesions diagnosed as sessile serrated adenoma, in 10/34 (29%) of hyperplastic polyps of microvesicular type, in 4/11 (36%) of traditional serrated adenomas, in 10/10 (100%) of mixed hyperplastic adenomatous polyps, and in 2/42 (5%) of “conventional” adenomas. Conclusions: Sessile serrated adenomas are encountered commonly in routine endoscopy practice. The histological diagnosis correlates strongly with the presence of BRAF mutation.