RAD21 cohesin overexpression is a prognostic and predictive marker exacerbating poor prognosis in KRAS mutant colorectal carcinomas.

BACKGROUND: RAD21 is a component of the cohesion complex and is integral to chromosome segregation and error-free DNA repair. RAD21 is functionally important in tumour progression but its role in colorectal carcinoma (CRC) is unclear. We therefore assessed its clinicopathological and prognostic significance in CRC, as well as its effect on chemosensitivity. METHODS: A retrospective observation study examined RAD21 expression in 652 CRCs using a tissue microarray approach. Correlation with clinicopathological factors including gender, tumour grade, mucinous subtype, TNM stage, disease-specific survival (DSS), BRAF and KRAS mutation status, tumour p53 immunostaining, tumour microsatellite instability and tumour CpG island methylator phenotype was performed. Colorectal cancer cell clones with stable RAD21 knockdown were generated and tested for cellular sensitivity to conventional chemotherapeutic drugs. RESULTS: RAD21 expression was significantly correlated with male gender (56.7% vs 43.3%, P=0.02), well-differentiated histology (14.4% vs 4.0%, P=0.0001), higher T-stage (36.1% vs 27.0%, P=0.01), presence of metastasis (18.8% vs 12.6%, P=0.03), and shorter DSS (hazard ratio (HR) 1.4, 95% CI 1.1 to 1.9, P=0.01) in both univariate and multivariate analysis. RAD21 expression was associated with shorter DSS in patients with KRAS mutant tumours (HR:2.6, 95% CI:1.4-4.3, P=0.001) and in patients receiving adjuvant chemoradiotherapy (HR:1.9, 95% CI:1.2-3.0, P=0.008). Colorectal cancer cells with RAD21 knockdown exhibited enhanced sensitivity to 5-fluorouracil, either alone or in combination with oxaliplatin. CONCLUSIONS: RAD21 expression in CRC is associated with aggressive disease especially in KRAS mutant tumours and resistance to chemoradiotherapy. RAD21 may be an important novel therapeutic target.

Fitness Penalties in the Evolution of Fungicide Resistance.

The evolution of resistance poses an ongoing threat to crop protection. Fungicide resistance provides a selective advantage under fungicide selection, but resistance-conferring mutations may also result in fitness penalties, resulting in an evolutionary trade-off. These penalties may result from the functional constraints of an evolving target site or from the resource allocation costs of overexpression or active transport. The extent to which such fitness penalties are present has important implications for resistance management strategies, determining whether resistance persists or declines between treatments, and for resistance risk assessments for new modes of action. Experimental results have proven variable, depending on factors such as temperature, nutrient status, osmotic or oxidative stress, and pathogen life-cycle stage. Functional genetics tools allow pathogen genetic background to be controlled, but this in turn raises the question of epistatic interactions. Combining fitness penalties under various conditions into a field-realistic scenario poses an important future challenge.

Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas.

BACKGROUND: Microsatellite instability (MSI) is seen in 10%-15% of sporadic colorectal cancers mostly in the right colon, but the precursors of cancers with MSI remain unknown. We examined whether sporadic cancers with MSI arise from pre-existing benign proliferative lesions (such as hyperplastic polyps or serrated adenomas [together denoted as "serrated polyps"]). METHODS: The frequency of benign epithelial lesions (serrated polyps and conventional adenomas) was determined by histologic review of resection specimens from individuals (n = 29) with sporadic colorectal cancer with MSI and from a matched control group (n = 29) with cancer showing microsatellite stability (MSS). MSI status, expression of mismatch repair enzyme (product of the human mut-L homologue 1 [hMLH1] gene), and hMLH1 gene promoter methylation in the benign lesions were determined. Data were analyzed by the chi-square test, by Wilcoxon's rank-sum test, and by conditional logistic regression as appropriate, and a two-sided probability less than.05 was considered to be statistically significant. RESULTS: Individuals with cancers showing MSI were more likely to harbor at least one serrated polyp than individuals with cancers showing MSS (odds ratio = 4.0; 95% confidence interval = 1.1 to 14.2; P =.03), but the frequency of conventional adenomas was the same in both groups (P =.52, Mann-Whitney test). Loss of hMLH1 protein expression was seen in lesions from 10 of 13 patients with MSI, but no loss was seen in lesions from four patients with MSS (P =.02, Fisher's exact test). Loss of hMLH1 protein expression was associated with MSI in assessable lesions. The hMLH1 promoter was methylated in all assessable serrated polyps from patients with cancers showing MSI but in none of the lesions from patients with MSS cancers. CONCLUSIONS: Some right-sided hyperplastic polyps may give rise to sporadic colorectal carcinomas with MSI. Methylation of the hMLH1 gene promoter within neoplastic cell subpopulations may be a critical step in the progression to carcinoma. The frequency with which benign lesions progress to cancer with MSI is unknown.

Apoptosis in v-myc transformation of myelomonocytic cells and its modulation by CSF-1.

c-myc is a proto-oncogene essential for cell growth. When activated, its expression can lead to uncontrolled cell proliferation, transformation and tumorigenesis. The cell line tEMmyc4 is a murine myelomonocytic cell line that was established following transformation by v-myc. It has a high level of v-myc expression and constitutively expresses endogenous CSF-1, the monocytic growth and viability factor. Under growth restricting conditions (high cell density serum deprivation, heat shock, or dexamethasone addition) cells of this line were found to undergo cell death through apoptosis. The induction of apoptosis by dexamethasone was associated with a decrease in constitutive CSF-1 expression without significant change in v-myc expression. Exogenous CSF-1 rescued these cells from dexamethasone induced-apoptosis. In vivo studies showed that tEMmyc4 cells were tumorigenic in syngeneic animals despite exhibiting some spontaneous apoptosis within the tumour mass. Co-administration of dexamethasone with the tumour cells significantly inhibited tumor development and the administration of dexamethasone to mice with established tumors resulted in tumor regression in all mice. This regression was associated with a high level of apoptosis and necrosis in the tumors. This study shows a correlation between the in vitro and in vivo induction of apoptosis and indicates that cancer cells bearing activated oncogenes may be more sensitive to apoptosis induction by chemotherapeutic agents.

Developmentally-regulated expression of murine K-ras isoforms.

The products (p21) of the three mammalian H-, N- and K-ras genes play important roles in intracellular signal transduction, linking membrane receptor kinases to the nuclear pathway through raf and mitogen activated protein kinase. They are involved in the regulation of proliferation and differentiation, and activating mutations of these genes are commonly associated with human cancers. Two p21 proteins are encoded by the K-ras gene (p21K-rasA and p21K-rasB) due to alternative splicing of the last exon. While the four p21ras proteins are highly homologous, their sequences diverge significantly at the C-termini, to which distinct biochemical and perhaps even functional differences may be ascribed. However, H-, N- and K-rasB appear to be ubiquitously expressed, with little evidence of tissue-specific or developmental regulation. In contrast, we now demonstrate that the expression of K-rasA is strikingly different. K-rasA is induced during differentiation of pluripotent embryonal stem cells in vitro. Its expression during early embryogenesis is limited temporally and spatially in a tissue-specific distribution which is largely maintained as an adult. This suggests a distinct biological role for p21K-rasA.

Phase I clinical trial of the chimeric monoclonal antibody (c30.6) in patients with metastatic colorectal cancer.

The murine antibody 30.6 recognizes an antigen that is expressed on a high proportion of colorectal carcinomas and their metastases. We report the results of single-dose escalation studies of the chimeric 30.6 (c30.6) monoclonal antibody in metastatic colorectal cancer, to evaluate its safety, pharmacokinetics, and biodistribution. Recombinant c30.6 (IgG1kappa) antibody was secreted from Chinese hamster ovary cells and purified by a multistep chromatography process. Seventeen patients with metastatic colorectal cancer were enrolled in this dose escalation study. The first four patients were treated with 3 mg of 123I-labeled c30.6, whereas the next 13 received a single dose of unlabeled antibody (maximum dose, 50 mg/m2). The most frequent side effect was a novel syndrome of severe burning and erythema of the face, chest, neck, ears, palms, soles, and genitalia. The frequency of this syndrome was markedly reduced in those patients premedicated with high doses of histamine receptor 1 and histamine receptor 2 blockers. Other side effects were mild and predictable. Biodistribution studies showed a rapid and intensive hepatic uptake. At the 50 mg/m2 level the half-life and maximum serum concentration were 81 +/- 15 h and 7.9 microg/ml, respectively. One patient developed a low-level human anti-c30.6 response. Tumor response was assessed by computed tomography, positron emission tomography scanning, and serial carcinoembryonic antigen measurements. There were no partial responses, although positron emission tomography scanning demonstrated some reduction in tumor activity in three individuals. The chimerized c30.6 antibody is not immunogenic in humans and appears worthy of further study. It does, however, produce a unique profile of side effects that can be well controlled with premedication.

The null oncogene hypothesis and protection from cancer.

Tumour progression involves the inactivation of tumour suppressor genes and the activation of proto-oncogenes. Inactivation of both copies of a tumour suppressor gene is required for carcinogenesis, while germline deletion or inactivation of one copy results in an increase in the risk of cancer and is responsible for many of the known hereditary cancer syndromes. In contrast, activation of only one copy of a proto-oncogene is required for carcinogenesis. Germline deletion or inactivation of one copy of a proto-oncogene halves the risk of activation at this locus. We propose that studies of high risk cancer patients will show such "null oncogene" mutations.

Production of a functional single-chain Fv fragment from the pan-leukocyte antibody WM65 using splicing by asymmetric PCR.

A method of assembly of a single-chain Fv fragment is described, whereby asymmetric polymerase chain reactions (APCR) and primer extension were used to join immunoglobulin heavy and light chain variable region genes via a linker sequence. In this procedure heavy and light chain genes, together with a linker gene containing complementary sequences, were amplified by APCR to generate single-stranded products. The single stranded heavy or light chain genes were hybridized to the relevant single-stranded link product, and extended to produce double-stranded heavy-link and link-light genes. These genes then underwent another round of APCR, resulting in two single-stranded genes (heavy-link and link-light) containing extensive overlapping sequences. Hybridization and extension of these two single-stranded products allowed the formation of the complete heavy chain-link-light chain double-stranded product. Using this method, a functional single-chain Fv fragment based on the pan-leukocyte antibody WM65 was expressed and purified from E. coli. This method of immunoglobulin gene assembly by polymerase chain reaction (PCR) offers an alternative to the current methods of the genetic engineering of antibody fragments.

Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both ß-catenin dependent and independent Wnt signalling.

PURPOSE: Wnt signalling has been implicated in breast cancer, and in particular aberrant ß-catenin-independent Wnt signalling has been associated with breast cancer metastasis and Tamoxifen resistance. Despite Wnt pathway involvement in many human cancers, attempts to target the pathway therapeutically have been disappointing. The recent discovery that the receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a novel Wnt receptor provides a potential new therapeutic and diagnostic target. METHODS: To clarify the role of ROR2 in breast cancer, we investigated its expression via ROR2 immunohistochemistry in a clinical cohort of breast cancer patients, and via in vitro studies incorporating both overexpression and knock-down of ROR2. RESULTS: ROR2 was expressed in the majority of breast cancer patients (87%), including those classed as triple negative. Breast cancer patients expressing ROR2 had a significantly shorter overall survival than those lacking ROR2 expression (P < 0.05). Overexpression of ROR2 in the mammary epithelial cell line, MCF10A, increased both ß-catenin-dependent and ß-catenin-independent targets and decreased cell adhesion. Knock-down of ROR2 in the breast cancer cell lines, MDA-MB-453 and HCC1143, decreased both ß-catenin-dependent and ß-catenin-independent targets and increased cell adhesion. Treatment of ROR2-expressing breast cancer cells with the novel berberine derivative, NAX53, significantly inhibited cell proliferation and migration. CONCLUSIONS: This is the first study to report the expression of ROR2 in breast cancer. Breast cancer patients expressing ROR2 had a significantly worse prognosis than those lacking ROR2. ROR2 may regulate both ß-catenin-dependent and ß-catenin-independent Wnt signalling pathways, and represents a potential diagnostic and therapeutic target.

Selectable in-vivo recombination to increase antibody library size--an improved phage display vector system.

Phage display technology permits the display of libraries of random combinations of light (LC) and heavy chain (HC) antibody genes. Maximizing the size of these libraries would enable the isolation of antibodies with high affinity and specificity. In this study, the loxP/Cre system of in-vivo recombination has been employed to construct an improved vector system for the display of antibodies. In this system, the chloramphenicol acetyl transferase (CAT) gene is linked to a HC library in a donor plasmid, pUX. This CAT gene is 'silent' before recombination but active after recombination. A second acceptor phagemid, pMOX, is used for cloning the LC repertoire. Following infection with a Cre producing phage, pMOX accepts the CAT/HC library from pUX via site-specific recombination at the loxP sites. Recombinants can then be selected via chloramphenicol resistance. Using this vector system, we have generated libraries of 4x109 recombinants. Restriction analysis and Fab expression confirmed that 100% of the colonies in the library were recombinants. This system provides a stable selectable mechanism for the generation of large libraries and avoids the isolation of non-recombinants encountered with earlier in-vivo recombination systems.

Biodistribution of filamentous phage-Fab in nude mice.

In vivo panning of peptide libraries in mice has allowed the isolation of peptides which target the vasculature of specific organs. The application of this approach to phage displaying Fab fragments (phage-Fab) could lead to the isolation of antibodies which recognize novel tumor antigens. In this study, we have evaluated the biodistribution of phage-Fab in nude mice. Balb/c nude mice were injected intravenously with 10(9) TU of phage displaying the anti-colon cancer Fab c30.6. Blood samples were collected at nine time points over a period of 72 h and three groups of four mice were sacrificed at 4 min, 24 h and 72 h. Normal tissues (liver, colon, spleen, kidneys, lungs, skeletal muscle) and faeces were collected at these time points and the number of viable phage in each sample was determined. The distribution of phage in tissues was also examined by immunohistochemical analysis of paraffin-embedded tissues. Regression analysis of plasma kinetic data showed that the half-life and the volume of distribution of phage was 3.6 h and 1 ml, respectively. Phage uptake occurred predominantly in lungs, kidneys, spleen and liver. Relatively few phage were distributed to colon and muscle, and phage were eliminated from the circulation by 72 h. Immunohistochemical analysis showed phage to be mainly within the vasculature at 4 min, whereas notable phage extravasation was observed at 24 h and 72 h. In conclusion, this study provides information on the in vivo behavior of phage-Fab which will be useful in the design of in vivo panning strategies. By choosing appropriate time points for tissue collection, it may be possible to isolate novel Fabs against both intra- and extravascular targets.

Retrieval of human antibodies from phage-display libraries using enzymatic cleavage.

A combinatorial human IgG1, kappa gene library of 2 x 10(7) clones was constructed from a pericolic lymph node using the phagemid vector pComb3H. Fabs with binding activity against tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH) were isolated from this library, and one such TT binding Fab was used to further evaluate a new phagemid vector for the display of recombinant antibody fragments (MCO1). This vector was designed to incorporate a cleavage site for the enzyme Genenase I, a myc peptide tag, and an amber codon between the heavy chain cloning site and the truncated M13 phage gene III. When MCO1 phage displaying an anti-TT Fab were bound to TT on a solid substrate, elution with Genenase I at concentrations of 5-10 micrograms/ml proved as effective as acid elution in releasing bound phage. Furthermore, enzymatic elution with Genenase I was comparable to acid elution in the enrichment of a TT binding Fab from the pericolic library subcloned into the vector MCO1. Importantly, the use of enzymatic or acid elutions resulted in the retrieval of different anti-TT Fabs from this same library. We conclude that panning of phage-displayed combinatorial antibody libraries can be successfully performed using enzymatic elution, and that this offers a useful alternative to currently available phage elution techniques.

Antibody immunity to the HER-2/neu oncogenic protein in patients with colorectal cancer.

The HER-2/neu protein is overexpressed in approximately 20% of human adenocarcinomas and is a defined tumor antigen in breast cancer. The purpose of this study was to evaluate the endogenous HER-2/neu specific antibody response in 57 patients with colorectal cancer. HER-2/neu specific antibodies, titer > or = 1:100, were detected in 14% (8/57) of patients with colorectal cancer compared to none of the normal control population (0/200). Furthermore, detection of HER-2/neu specific antibodies in the cancer population correlated significantly with HER-2/neu protein overexpression in the patients' tumor (p < 0.01). 46% of patients with HER-2/neu overexpressing tumors (6/13) and 5% of HER-2/neu negative tumors (2/44) had detectable HER-2/neu specific antibodies. The endogenous HER-2/neu antibody response in these patients was predominantly IgG or IgA.

Characterisation and clinicopathological correlates of serum anti-p53 antibodies in breast and colon cancer.

The humoral immune response to p53 was determined in 54 individuals with colon or breast cancer and 50 healthy subjects, in an attempt to better understand the origin and significance of anti-p53 serum antibodies. The presence of anti-p53 antibodies in serum was determined by enzyme-linked immunosorbent assay using purified recombinant human p53, and results were validated by immunoprecipitation of radiolabelled p53. Immunohistochemical analysis of 28 tumours was performed to detect the accumulation of p53 protein. Antibodies against p53 were significantly more common in patients with colorectal (10 of 42) and breast (2 of 12) cancer than in healthy individuals (2 of 50). They were of both the IgM (7 of 11) and IgG (4 of 11) isotypes. There was no significant difference in prevalence of serum antibodies against p53 with respect to the p53 immunohistochemical status of the tumour or to other pathological features, including the presence of lymph node and distant metastases. These findings provide indirect evidence that, rather than arising as a result of a specific immune response, anti-p53 antibodies in individuals with cancer may represent elevated levels of naturally occurring polyreactive antibodies.

Clinicopathological significance of erbB-2 expression in colorectal carcinoma.

The level of erbB-2 expression in both the tumour tissue and serum of individuals with colorectal carcinoma is unclear. This study aims to clarify expression levels and to relate these to a range of clinicopathological parameters. Overexpression of erbB-2 was detected in 25% of patient tumour tissue and an elevation in serum concentration of the extracellular domain (ECD) of erbB-2 was detected in 10% of patients. Surprisingly, an elevated serum ECD concentration did not correlate with erbB-2 overexpression within the primary tumour, and neither tumour or serum levels of the protein correlated with any clinicopathological parameters examined. These results indicate that erbB-2 overexpression in tissue and serum are not uncommon in colorectal carcinoma, but may not be useful as predictors of disease outcome.

The role of mesangial cells in glomerular pathology.

The glomerular mesangial cell has become increasingly recognized as a multifunctional cell capable of mediating glomerular disease. This article reviews recent findings regarding the biology of these cells, and the relevance that these findings may have for our understanding of glomerular pathology.

The significance of anti-sialyl-Tn antibodies in patients with colorectal and breast cancer.

The sera of 54 individuals with colorectal or breast cancer, and 50 healthy volunteers were assayed for the presence of anti-bovine submaxillary mucin antibodies using an enzyme linked immunoassay. The serum levels of these antibodies were found to be significantly lower in people with breast (p < 0.001) or colorectal cancer (p < 0.001) with respect to healthy individuals. Within the colorectal cancer group the presence of antibodies was significantly lower in those individuals with poorly differentiated tumors compared to other histological grades (p < 0.05), but did not correlate with the presence of local or distant metastases or anatomical location of the tumor (p > 0.05). No correlation was found with respect to the age of the patient and the level of anti-sialyl-Tn antibodies (p > 0.05). Competition analysis with the anti-sialyl-Tn monoclonal antibody 3C2 indicated that the activity against bovine submaxillary mucin was primarily due to specificity for the sialyl-Tn epitope of the glycoprotein. In contrast to findings with other tumor associated antigens, we could find no evidence of an increase in the level of antibodies against this epitope.

Different APC genotypes in proximal and distal sporadic colorectal cancers suggest distinct WNT/ß-catenin signalling thresholds for tumourigenesis.

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/ß-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/ß-catenin signalling, with different 'optimal' thresholds for proximal and distal cancers.

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.