Nicholas J. Hawkins

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.

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.

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.

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.

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.

Colorectal cancer - a model for epigenetic tumorigenesis

Recent advances in basic and clinical science have driven epigenetics to the forefront of cancer research. Together with genetic changes, the disruption of epigenetic mechanisms is now established as a hallmark of cancer in humans. Colorectal cancer, long a classic model for the genetic basis of cancer, is now providing researchers with the opportunity to view epigenetic events in the context of neoplasia in humans. Knowledge of the heritable changes in gene expression that result from epigenetic events is of increasing relevance to clinical practice, particularly in terms of diagnostic and prognostic molecular markers, as well as novel therapeutic targets.

Tolerance of Whole-Genome Doubling Propagates Chromosomal Instability and Accelerates Cancer Genome Evolution

UNLABELLED The contribution of whole-genome doubling to chromosomal instability (CIN) and tumor evolution is unclear. We use long-term culture of isogenic tetraploid cells from a stable diploid colon cancer progenitor to investigate how a genome-doubling event affects genome stability over time. Rare cells that survive genome doubling demonstrate increased tolerance to chromosome aberrations. Tetraploid cells do not exhibit increased frequencies of structural or numerical CIN per chromosome. However, the tolerant phenotype in tetraploid cells, coupled with a doubling of chromosome aberrations per cell, allows chromosome abnormalities to evolve specifically in tetraploids, recapitulating chromosomal changes in genomically complex colorectal tumors. Finally, a genome-doubling event is independently predictive of poor relapse-free survival in early-stage disease in two independent cohorts in multivariate analyses [discovery data: hazard ratio (HR), 4.70, 95% confidence interval (CI), 1.04-21.37; validation data: HR, 1.59, 95% CI, 1.05-2.42]. These data highlight an important role for the tolerance of genome doubling in driving cancer genome evolution. SIGNIFICANCE Our work sheds light on the importance of whole-genome–doubling events in colorectal cancer evolution. We show that tetraploid cells undergo rapid genomic changes and recapitulate the genetic alterations seen in chromosomally unstable tumors. Furthermore, we demonstrate that a genome-doubling event is prognostic of poor relapse-free survival in this disease type.

SMAD2, SMAD3 and SMAD4 mutations in colorectal cancer.

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.

Does MSI-low exist?

Microsatellite instability is a well‐recognised phenomenon. Ten to 15% of sporadic colorectal cancers with a high level of MSI form a well defined group with distinct clinicopathological features. The set of tumours with low level of microsatellite instability (MSI‐low), though widely referred to, is not a clearly defined group. The definitions of MSI‐low have varied among groups and between different studies from the same group. Some studies have found associations between the MSI‐L phenotype and molecular features, notably a higher frequency of K‐ras mutations, and, possibly, methylation of methylguanine methyltransferase. Two recent independent studies, however, showed respectively that 68% and 79%, non‐MSI‐H cancers showed some MSI and could therefore be classed nominally as MSI‐L. There was no evidence for a qualitatively discrete MSI‐L group, but quantitative differences in the level of MSI were found. Copyright