Activation of the hypoxia pathway in breast cancer tissue and patient survival are inversely associated with tumor ascorbate levels.

BACKGROUND: The transcription factor hypoxia inducible factor (HIF) -1 drives tumor growth and metastasis and is associated with poor prognosis in breast cancer. Ascorbate can moderate HIF-1 activity in vitro and is associated with HIF pathway activation in a number of cancer types, but whether tissue ascorbate levels influence the HIF pathway in breast cancer is unknown. In this study we investigated the association between tumor ascorbate levels and HIF-1 activation and patient survival in human breast cancer. METHODS: In a retrospective analysis of human breast cancer tissue, we analysed primary tumor and adjacent uninvolved tissue from 52 women with invasive ductal carcinoma. We measured HIF-1α, HIF-1 gene targets CAIX, BNIP-3 and VEGF, and ascorbate content. Patient clinical outcomes were evaluated against these parameters. RESULTS: HIF-1 pathway proteins were upregulated in tumor tissue and increased HIF-1 activation was associated with higher tumor grade and stage, with increased vascular invasion and necrosis, and with decreased disease-free and disease-specific survival. Grade 1 tumors had higher ascorbate levels than did grade 2 or 3 tumors. Higher ascorbate levels were associated with less tumor necrosis, with lower HIF-1 pathway activity and with increased disease-free and disease-specific survival. CONCLUSIONS: Our findings indicate that there is a direct correlation between intracellular ascorbate levels, activation of the HIF-1 pathway and patient survival in breast cancer. This is consistent with the known capacity of ascorbate to stimulate the activity of the regulatory HIF hydroxylases and suggests that optimisation of tumor ascorbate could have clinical benefit via modulation of the hypoxic response.

Phosphohistone H3 outperforms Ki67 as a marker of outcome for breast cancer patients.

AIMS: The proliferation marker Ki67 has been extensively investigated as a prognostic factor in breast cancer, but has not gained widespread clinical acceptance. Phosphohistone H3 is a new immunohistochemical marker for quantifying mitoses; however, there is limited information on its prognostic value in breast cancer. In this study, we performed a head-to-head comparison of Ki67 and phosphohistone H3 to establish the marker with the greatest prognostic value. METHODS AND RESULTS: Tissue microarrays from 108 breast cancer patients were immunohistochemically stained for Ki67 and phosphohistone H3. Our results showed that phosphohistone H3 had a greater prognostic value than Ki67 in a multivariable model that adjusted for traditional prognostic variables in breast cancer. Phosphohistone H3 staining was a stronger predictor of survival at 5 years after diagnosis [hazard ratio (HR) 4.35, P < 10(-5) ] than Ki67 (HR 2.44, P = 0.004), and better separated the risk of death in patients aged >45 years. Importantly, phosphohistone H3 consistently showed strong unequivocal staining, in contrast to the variable staining intensities associated with Ki67. CONCLUSIONS: Our study suggests that phosphohistone H3 staining is a stronger and more robust prognostic indicator than Ki67 staining in breast cancer patients, and has the potential for use in routine diagnostic laboratories.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 µg ascorbate/100 mg tissue, range 0.8 - 20.4 µg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.

Ascorbate content of clinical glioma tissues is related to tumour grade and to global levels of 5-hydroxymethyl cytosine.

Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten-eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy.

RNAscope compatibility with image analysis platforms for the quantification of tissue-based colorectal cancer biomarkers in archival formalin-fixed paraffin-embedded tissue.

RNAscope®, has emerged as an important in-situ hybridisation method to validate mRNA expression within single cells whilst preserving tissue morphology in histological samples. The aim of this research was to compare the utility of various open-source and commercial image analysis methods, to quantify mRNA transcripts identified by RNAscope within formalin fixed paraffin embedded (FFPE) histological samples and cell monolayer preparations. Examination of MLH1 expression from 10 histological FFPE colorectal cancer specimens using four image analysis tools (Colour Deconvolution, SpotStudio, WEKA and the LEICA RNA-ISH algorithm) showed the WEKA tool as having the greatest level of agreement with manual quantification. Comparing image analysis methods to qRT-PCR for quantifying MLH1, GFI1 and TNFRSF11A expression within two colorectal cell lines results suggest that these image analysis methods perform at a similar level to qRT-PCR. Furthermore, we describe the strengths and limitations for each image analysis method when used in combination with RNAscope assays. Our study concludes that there are several freely available and commercial image analysis tools that enable reliable RNA in situ expression analysis, however operators need to consider factors, such as expected expression levels of target genes, software usability and functionality.

A profile of prognostic and molecular factors in European and Maori breast cancer patients.

BACKGROUND: New Zealand Maori have a poorer outcome from breast cancer than non-Maori, yet prognostic data are sparse. The objective of this study was to quantify levels of prognostic factors in a cohort of self-declared Maori and European breast cancer patients from Christchurch, New Zealand. METHODS AND RESULTS: Clinicopathological and survival data from 337 consecutive breast cancer patients (27 Maori, 310 European) were evaluated. Fewer tumours were high grade in Maori women than European women (p = 0.027). No significant ethnic differences were detected for node status, tumour type, tumour size, human epidermal growth factor receptor, oestrogen and progesterone receptor (ER/PR) status, or survival.In addition, tumour and serum samples from a sub-cohort of 14 Maori matched to 14 NZ European patients were analyzed by immunohistochemistry and enzyme linked immunosorbent assay for molecular prognostic factors. Significant correlations were detected between increased grade and increased levels of hypoxia inducible factor-1 (HIF-1α), glucose transporter-1 (GLUT-1), microvessel density (MVD) and cytokeratins CK5/6 (p < 0.05). High nodal status correlated with reduced carbonic anhydrase IX (CA-IX). Negative ER/PR status correlated with increased GLUT-1, CA-IX and MVD. Within the molecular factors, increased HIF-1α correlated with raised GLUT-1, MVD and CK5/6, and CK5/6 with GLUT-1 and MVD (p < 0.05). The small number of patients in this sub-cohort limited discrimination of ethnic differences. CONCLUSIONS: In this Christchurch cohort of breast cancer patients, Maori women were no more likely than European women to have pathological or molecular factors predictive of poor prognosis. These data contrast with data from the North Island NZ, and suggest potential regional differences.

APC Mutations Are Not Confined to Hotspot Regions in Early-Onset Colorectal Cancer.

While overall colorectal cancer (CRC) cases have been declining worldwide there has been an increase in the incidence of the disease among patients under 50 years of age. Mutation of the APC gene is a common early event in CRC but is reported at lower rates in early-onset colorectal cancer (EOCRC) than in older patients. Here we investigate the APC mutation status of a cohort of EOCRC patients in New Zealand using a novel sequencing approach targeting regions of the gene encompassing the vast majority of known APC mutations. Using this strategy we find a higher rate (72%) of APC mutation than previously reported in EOCRC with mutations being spread throughout the gene rather than clustered in hotspots as seen with sporadic mutations in older patients. The rate of mutations falling within hotspots was similar to those previously seen in EOCRC and as such our study has implications for sequencing strategies for EOCRC patients. Overall there were low rates of both loss of heterozygosity and microsatellite instability whereas a relatively high rate (40%) of APC promoter methylation was found, possibly reflecting increasing exposure of young people to pro-oncogenic lifestyle factors.

Low Vitamin C Status in Patients with Cancer Is Associated with Patient and Tumor Characteristics.

Vitamin C (ascorbate) acts as an antioxidant and enzyme cofactor, and plays a vital role in human health. Vitamin C status can be affected by illness, with low levels being associated with disease due to accelerated turnover. However, robust data on the ascorbate status of patients with cancer are sparse. This study aimed to accurately measure ascorbate concentrations in plasma from patients with cancer, and determine associations with patient or tumor characteristics. We recruited 150 fasting patients with cancer (of 199 total recruited) from two cohorts, either prior to cancer surgery or during cancer chemo- or immunotherapy. A significant number of patients with cancer had inadequate plasma ascorbate concentrations. Low plasma status was more prevalent in patients undergoing cancer therapy. Ascorbate status was higher in women than in men, and exercising patients had higher levels than sedentary patients. Our study may prompt increased vigilance of ascorbate status in cancer patients.

Vitamin C Administration by Intravenous Infusion Increases Tumor Ascorbate Content in Patients With Colon Cancer: A Clinical Intervention Study.

The use of high dose ascorbate infusions in cancer patients is widespread, but without evidence of efficacy. Several mechanisms whereby ascorbate could affect tumor progression have been proposed, including: (i) the localized generation of cytotoxic quantities of H2O2; (ii) ascorbate-dependent activation of the 2-oxoglutarate-dependent dioxygenases that control the hypoxia-inducible factors (HIFs) and that are responsible for the demethylation of DNA and histones; (iii) increased oxidative stress induced by dehydroascorbic acid. We hypothesize that the dysfunctional vasculature of solid tumors results in compromised delivery of ascorbate to poorly perfused regions of the tumor and that this ascorbate deficit acts as an additional driver of the hypoxic response via upregulation of HIFs. Using a randomized "therapeutic window of opportunity" clinical study design we aimed to determine whether ascorbate infusions affected tumor ascorbate content and tumor biology. Patients with colon cancer were randomized to receive infusions of up to 1 g/kg ascorbate for 4 days before surgical resection (n = 9) or to not receive infusions (n = 6). Ascorbate was measured in plasma, erythrocytes, tumor and histologically normal mucosa at diagnostic colonoscopy and at surgery. Protein markers of tumor hypoxia or DNA damage were monitored in resected tissue. Plasma ascorbate reached millimolar levels following infusion and returned to micromolar levels over 24 h. Pre-infusion plasma ascorbate increased from 38 ± 10 µM to 241 ± 33 µM (p < 0.0001) over 4 days and erythrocyte ascorbate from 18 ± 20 µM to 2509 ± 1016 µM (p < 0.005). Tumor ascorbate increased from 15 ± 6 to 28 ± 6 mg/100 g tissue (p < 0.0001) and normal tissue from 14 ± 6 to 21 ± 4 mg/100 g (p < 0.001). A gradient of lower ascorbate was evident towards the tumor centre in both control and infusion samples. Lower expression of hypoxia-associated proteins was seen in post-infusion tumors compared with controls. There were no significant adverse events and quality of life was unaffected by ascorbate infusion. This is the first clinical study to demonstrate that tumor ascorbate levels increase following infusion, even in regions of poor diffusion, and that this could modify tumor biology. CLINICAL TRIAL REGISTRATION: ANZCTR Trial ID ACTRN12615001277538 (https://www.anzctr.org.au/).

Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34-5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53ß transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

The Δ133p53ß isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53ß isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53ß expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53ß as a highly accurate prognostic factor for aggressive prostate cancer.

Caspase recruitment domain-containing protein 15 mutations in patients with colorectal cancer.

The caspase recruitment domain-containing protein 15 (CARD15) plays a crucial role in mediating the innate immune response. Mutations within this protein have been shown to be independent risk factors for the development of Crohn's disease in Caucasians. As Crohn's disease patients are at increased risk of developing sporadic colorectal cancer, it is conceivable that genetic variability within CARD15 may also play a role in determining susceptibility to this gastrointestinal malignancy in individuals without Crohn's disease. This hypothesis is supported by the findings of two case-control studies that found the frequencies of CARD15 mutations were significantly elevated in Polish and Greek colorectal cancer patients. Given the results of these previous studies, we examined whether the high incidence of sporadic colorectal cancer observed in New Zealand Caucasians was due to mutations within CARD15. To answer this question, we genotyped 133 colorectal cancer patients and 201 Caucasian controls for R702W, G908R, 1007fs, and P268S. Chi(2) Testing found that the combined frequency of R702W, G908R, and 1007fs was significantly elevated in colorectal cancer patients compared with controls (P = 0.001; odds ratio, 2.8; 95% confidence interval, 1.5-5.4), but no association was detected between tumor behavior or age of disease onset and CARD15 mutations in our colorectal cancer cohort. This study is the first to explore the link between CARD15 mutations and colorectal cancer in New Zealand Caucasians. Our results strongly suggest that CARD15 influences susceptibility to colorectal cancer, but we have found no evidence to indicate that CARD15 mutations predict the clinicopathologic characteristics of this disease.

The rs11515 Polymorphism Is More Frequent and Associated With Aggressive Breast Tumors with Increased ANRIL and Decreased p16 (INK4a) Expression.

Chromosome position 9p21 encodes three-tumor suppressors p16(INK4a), p14(ARF), and p15(INK4b) and the long non-coding RNA ANRIL (antisense non-coding RNA in the INK4 locus). The rs11515 single-nucleotide polymorphism in the p16 (INK4a) /p14 (ARF) 3'-untranslated region is associated with glioblastoma, melanoma, and other cancers. This study investigated the frequency and effect of rs11515 genotypes in breast cancer. Genomic DNA samples from 400 women (200 with and 200 without a diagnosis of breast cancer) were genotyped for the rs11515 major (C) and minor (G) alleles. The rs11515 polymorphism was also investigated in 108 heart tissues to test for tissue-specific effects. Four 9p21 transcripts, p16 (INK4a) , p14 (ARF) , p15 (INK4b) , and ANRIL were measured in breast tumors and myocardium using quantitative PCR. Heterozygotes (CG genotype) were more frequent in women with breast cancer compared to the control population (P = 0.0039). In those with breast cancer, the CG genotype was associated with an older age (P = 0.016) and increased lymph node involvement (P = 0.007) compared to homozygotes for the major allele (CC genotype). In breast tumors, the CG genotype had higher ANRIL (P = 0.031) and lower p16 (INK4a) (P = 0.006) expression compared to the CC genotype. The CG genotype was not associated with altered 9p21 transcripts in heart tissue. In breast cancer, the rs11515 CG genotype is more frequent and associated with a more aggressive tumor that could be due to increased ANRIL and reduced p16 (INK4a) expression. The absence of association between rs11515 genotypes and 9p21 transcripts in heart tissue suggests this polymorphism has tissue- or disease-specific functions.

Sustaining a biobank through a series of earthquake swarms: lessons learned from our New Zealand experience.

In the early hours of September 4, 2010, the city of Christchurch in New Zealand was awakened by a major magnitude 7.1 earthquake event that was the start of a series of earthquake swarms. By January 2012, the city had sustained over 10,000 earthquakes and aftershocks, including 4 major events. New Zealand is positioned along the geological Pacific Rim of Fire and is subject to volcanic and seismic movements. However, this series of earthquakes arose from a previously undetected fault that had been dormant for over 10,000 years. The impact on the city, businesses, and people of Christchurch has been profound. Sustaining our cancer biobank through this period has been extremely challenging, as our city's infrastructure including utilities, telecommunication, and transport facilities were fractured, buildings collapsed, and a National State of Emergency was declared. What had not been anticipated was that this impact would continue to be felt up to the present time. After each major earthquake event, the immediate focus of our response was to ensure the safety of all personnel. The secondary response was to ensure the continued preservation of stored specimens. Our third response was to reestablish operational processes without endangering staff. Our responses have been reviewed and lessons formulated that can be incorporated into biobank emergency response plans. They include operational aspects of equipment restraint, cryostorage, staff trauma, specimen relocation, legislation, and management of the repair processes. Emergency response planning for a biobank is a "best practice" standard. Future-proofing a biobank from a significant natural disaster such as a series of earthquake swarms is limited. However, lessons learned from our experience may help to mitigate the impact of future events within our global community.

Immunohistochemical analysis of cancer stem cell markers in invasive breast carcinoma and associated ductal carcinoma in situ: relationships with markers of tumor hypoxia and microvascularity.

We performed immunohistochemical analysis of 3 cancer stem cell-related markers (CD44(+)/CD24(-/low), aldehyde dehydrogenase [ALDH]-1, CD133) in 94 invasive ductal carcinomas and assessed relationships with markers of hypoxia (carbonic anhydrase IX [CAIX]), tumor microvessel density (CD31), and clinicopathologic variables. Overall, 10% of tumors were CD44(+)/CD24(-/low), 13% were ALDH-1(+), 25% were CD133(+), 35% were immunonegative, and 1 tumor was immunopositive for all 3 markers. Associated ductal carcinoma in situ (DCIS) was present in 48% of tumors. Marker immunopositivity was detected in DCIS in 13% (CD44(+)/CD24(-/low)), 7% (ALDH-1(+)), and 32% (CD133(+)) of these tumors and was more likely present in DCIS when also detected in the invasive compartment (P = .03, P = .001, and P = .009, respectively). CD44(+)/CD24(-/low) cells were more common in progesterone receptor-negative tumors (P < .01), and ALDH-1(+) cells were more common in estrogen receptor-negative tumors (P < .01). CD133(+) cells were more common in patients younger than 50 years (P < .05) and in high grade (P < .01), localized (P < .05), and estrogen receptor-negative (P < .001), progesterone receptor-negative (P = .02), and triple-negative breast cancers (P < .001). CD44(+)/CD24(-/low) (P = .06) and CD133(+) (P = .02) tumor cells were more common in CAIX(+) versus CAIX(-) tumors, whereas ALDH-1(+) tumors had a higher mean microvessel density than did ALDH-1(-) tumors (P = .002). No significant relationships were observed between the markers studied and survival for 5 years. Our study demonstrated the presence of cancer stem cell marker-positive tumor cells in DCIS as well as invasive breast cancer and showed that CD44(+)/CD24(-/low) and CD133(+) cells were more frequently observed in hypoxic regions of tumor, whereas ALDH-1(+) cells more commonly colocalized to tumors with high microvessel density.

The G-protein-coupled receptor CLR is upregulated in an autocrine loop with adrenomedullin in clear cell renal cell carcinoma and associated with poor prognosis.

PURPOSE: The G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular. EXPERIMENTAL DESIGN: In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome. RESULTS: ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01). CONCLUSIONS: In human tissues ADM expression is upregulated in cancer type-specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development.

The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma-carcinoma sequence during colorectal cancer progression.

Angiogenesis is essential for tumour growth and metastasis. It is controlled by angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF)-A. Although its role has been demonstrated in many tumour types including colorectal carcinoma (CRC), the importance of the newer family members in adenoma, invasive tumour growth, and progression to a metastatic phenotype has been poorly characterized in CRC. The aim of this study was to determine the role and timing of the VEGF angiogenic switch during CRC progression. We measured the gene expression of VEGF ligands (VEGF-A, VEGF-B, VEGF-C, and VEGF-D) and their receptors (VEGFR-1, VEGFR-2, and VEGFR-3), in normal colorectal tissues (n = 20), adenomas (n = 10), and in CRC (n = 71) representing different Duke's stages using ribonuclease protection assay, semi-quantitative relative reverse transcriptase polymerase chain reaction, together with the pattern of their expression by immunohistochemistry. VEGF-A mRNA was the most abundant in colorectal tissue, followed by VEGF-B, VEGF-C, and VEGF-D. VEGF-A and VEGF-B mRNAs were significantly more abundant in adenomas (p = 0.0003 and p = 0.04 respectively) compared with normal tissues, while VEGF-A and VEGF-C were significantly increased in carcinomas compared with normal tissues (p = 0.0006 and p = 0.0009 respectively). A significantly greater amount of VEGF-C mRNA was present in carcinomas compared with adenomas (p = 0.03), whereas there was a significant reduction of VEGF-B in carcinomas compared with adenomas (p = 0.0002). VEGF-D mRNA was significantly more abundant in normal tissues than in adenomas (p = 0.0001) and carcinomas (p < 0.0001). In normal tissues distant from the primary tumour, there was a significantly greater amount of VEGF-A and VEGF-D mRNA in patients with Duke's B and Duke's C respectively, compared with Duke's A stage tumours (p = 0.04 and p = 0.01 respectively). Immunohistochemistry showed low basal levels of all ligands in histologically normal tissues and their expression in the epithelium of tumours reflected the levels of mRNA expression identified. VEGF-A and VEGF-C mRNA levels correlated significantly with tumour grade (p = 0.01 and p = 0.01 respectively) and tumour size (p = 0.001 and p = 0.01 respectively), but not with patient age, sex, presence of infiltrative margin, lymphocytic response, vascular invasion, Duke's stage, or lymph node involvement (p > 0.05). VEGF-B mRNA correlated with an infiltrative margin (p = 0.04) but no other clinicopathological variable, and expression of VEGF-D demonstrated no association with any parameter examined. VEGFR-1 was significantly correlated with tumour grade (p = 0.02), Duke's stage (p < 0.001), and lymph node involvement (p = 0.004), VEGFR-2 with lymph node involvement (p = 0.02), and VEGFR-3 did not correlate with any of the clinicopathological variables tested. These results suggest that VEGF-A and VEGF-B play a role early in tumour development at the stage of adenoma formation and that VEGF-C plays a role in advanced disease when there is more likelihood of metastatic spread. The finding of increased levels of VEGF-A and VEGF-D expression in normal tissues collected from a site distant from the primary tumour indicates changes in the surrounding tumour environment that may enhance the subsequent spread of tumour cells.