The prognostic value of cancer stem-like cell markers SOX2 and CD133 in stage III colon cancer is modified by expression of the immune-related markers FoxP3, PD-L1 and CD3.

Cancer stem-like cells are highly tumourigenic cells that can repopulate entire tumours after apparent successful treatment. Recent evidence suggests they interact with other cells in the tumour microenvironment, including immune cell subsets, to enhance their survival. The aim of this study was to determine whether the expression of immune cell markers in primary colon cancer impacts the prognostic significance of cancer stem-like cell marker expression. Immunohistochemistry was used to assess the expression of putative stem cell markers (ALDH1, CD44v6, CD133, Lgr5, SOX2) and immune cell related markers (CD3, CD8, FoxP3, PD-L1) in 104 patients with stage III colon cancer. Associations of marker expression with overall and cancer-specific survival were determined using Kaplan-Meier analysis. High SOX2 expression in the central tumour area was found to be an independent factor for poor cancer-specific survival [hazard ratio (HR) 6.19; 95% confidence interval (CI) 2.24-17.14; p=0.001]. When immune-related factors were taken into account, patients categorised as SOX2low/FoxP3high had good outcome (HR 0.164; 95%CI 0.066-0.406; p<0.0001) whereas patients categorised as SOX2high/PD-L1low had poor outcome (HR 8.992; 95%CI 3.397-23.803; p<0.0001). The prognostic value of the SOX2 cancer stem-like cell marker in colon cancer is modified by expression of immune-cell related factors FoxP3 and PD-L1.

Objective analysis of cancer stem cell marker expression using immunohistochemistry.

Analysis of immunohistochemical expression is often a subjective and semiquantitative process that can lead to the inconsistent reporting of results. To assess the effect that region selection and quantification method have on results, five different cancer stem cell markers were used in this study to compare tissue scoring with digital analysis methods that used three different tissue annotation methods. Samples of tumour and normal mucosa were used from 10 consecutive stage II colon cancer patients and stained for the putative cancer stem cell markers ALDH1, CD44v6, CD133, Lgr5 and SOX2. Tissue scoring was found to have considerably different results to digital analysis with the three different digital methods harbouring concordant results overall. However, SOX2 on normal tissue and CD133 on tumour and normal tissue produced discordant results which could be attributed to the different regions of tissue that were analysed. It is important that quantification method and selection of analysis areas are considered as part of study design to ensure that reproducible and consistent results are reported in the literature.

Can population differences in chemotherapy outcomes be inferred from differences in pharmacogenetic frequencies?

Inter-ethnic differences in drug handling and frequencies of pharmacogenetic variants are increasingly being characterized. In this study, we systematically assessed the feasibility of inferring ethnic trends in chemotherapy outcomes from inter-ethnic differences in pharmacogenetic variant frequencies. Frequencies of 51 variants and chemotherapy outcomes of East Asian and Caucasian colorectal cancer patients on standard chemotherapy regimens were summarized by meta-analyses, and variant frequencies were validated by MassARRAY analysis. Inferences of relative chemotherapy outcomes were made by considering minor allele function and population differences in their frequency. Significant population differences in genotype distributions were observed for 13/23 (60%) and 27/35 (77%) variants in the meta-analyses and validation series, respectively. Across chemotherapy regimens, East Asians had lower rates of grade 3/4 toxicity for diarrhea and stomatitis/mucositis than Caucasians, which was correctly inferred from 13/18 (72%, P=0.018) informative genetic variants. With appropriate variant selection, inferring relative population toxicity rates from population genotype differences may be relevant.

Integrating next-generation sequencing into the diagnostic testing of inherited cancer predisposition.

The clinical application of next-generation sequencing (NGS) as a diagnostic tool has become increasingly evident. The coupling of NGS technologies with new genomic sequence enrichment methods has made the sequencing of panels of target genes technically feasible, at the same time as making such an approach cost-effective for diagnostic applications. In this article, we discuss recent studies that have applied NGS in the diagnostic setting in relation to hereditary cancer.

Sequential expression of putative stem cell markers in gastric carcinogenesis.

BACKGROUND: Gastric carcinogenesis has been well documented in the step-wise histopathological model, known as Correa pathway. Several biomarkers including CD44, Musashi-1 and CD133 have been reported as putative stem cell (PSC) markers. METHODS: We investigated expression of PSC markers CD44, Musashi-1 and CD133 in relation to gastric carcinogenesis and prognosis and chemoresponse. Immunohistochemistry staining was performed in gastric cancer (GC) clinical specimens representing different steps of the Correa pathway. Gastric cancer samples taken before and after neoadjuvant chemotherapy with docetaxel, cisplatin and capecitabine (DCX) were also evaluated for PSC marker expression. RESULTS: We showed that the expression of three PSC markers was significantly elevated in GC relative to normal gastric mucosa (P<0.001 for each marker). Precancerous lesions, including intestinal metaplasia and dysplasia, demonstrated increased expression of CD44 and Musashi-1. CD133 was predominantly expressed along the border between intramucosal carcinoma and connective tissue at later stages. High CD44 and CD133 expression showed prognostic value for worse patient survival (P=0.014 and P=0.019, respectively). A small number of tumours with high expression of CD44 and CD133 showed pathological response to DCX-based neoadjuvant chemotherapy. CONCLUSION: CD44 and Musashi-1 are frequently expressed in both premalignant gastric lesions and invasive GC, whereas CD133 expression is restricted mainly to neoplastic tissues.

Meat consumption and cooking practices and the risk of colorectal cancer.

BACKGROUND/OBJECTIVES: The association between meat consumption and the risk of colorectal cancer (CRC) has been controversial. One of the difficulties in determining this association has been measurement of different attributes of meat consumption, including cooking methods and level of doneness. SUBJECTS/METHODS: We investigated the association between meat consumption and cooking practices and the risk of CRC in a population-based case-control study in the Western Australian Bowel Health Study. From July 2005 to February 2007, 567 incident CRC cases and 713 controls, who were frequency matched to cases for age- and sex, completed questionnaires on lifestyle and meat consumption. Estimated odds ratios (ORs) comparing meat consumption quartile groups were obtained from multivariate logistic regression models. RESULTS: The amount of red baked meat consumed had a statistically significant inverse trend of association with CRC (Q4 OR=0.73 95% confidence interval 0.53-1.01). When frequency was multiplied by serving size and by doneness, the association remained protective but was no longer statistically significant. The protective trends for red pan-fried meat were also borderline statistically significant. There were no other statistically significant or meaningful associations with any of the types of meat cooked by any method and the risk of CRC. CONCLUSIONS: Our data do not support the hypothesis that meat consumption is a risk factor for CRC.

KRAS mutation and microsatellite instability in endometrial adenocarcinomas showing MELF-type myometrial invasion.

BACKGROUND: Some uterine endometrioid adenocarcinomas exhibit a distinctive morphological phenotype characterised by the formation of microcystic, elongated and fragmented (MELF) glands. Immunohistochemical studies have suggested that MELF-type changes represent an epithelial-mesenchymal transition which has been associated with KRAS activation in various tumours. AIMS: To investigate the molecular characteristics of endometrial tumours showing MELF, with particular reference to the frequencies of KRAS and BRAF mutations and of microsatellite instability (MSI). METHODS: MSI, and KRAS and BRAF mutation status, were assessed in 33 low-grade endometrial adenocarcinomas showing MELF features and the results compared with 33 control cases exhibiting a 'conventional' pattern of myometrial invasion. Standard histological parameters were also reviewed. RESULTS: Tumours with a MELF pattern of myometrial invasion showed more frequent vascular invasion and focal mucinous differentiation. KRAS mutations were more frequent in MELF positive than MELF negative tumours (45% vs 30%), but this difference was not statistically significant. BRAF mutations were not identified in any of the cases. MSI was identified in 20% of cases overall but did not correlate with the MELF phenotype. CONCLUSIONS: Mutations in KRAS and BRAF genes are not directly implicated in the development of a MELF pattern of invasion in endometrial carcinoma. However, RAS-associated signalling pathways could be activated through other genetic or epigenetic mechanisms. The characterisation of such alterations may become increasingly important as novel therapies are developed that target mediators involved in tumour invasion.

Molecular markers of response and toxicity to FOLFOX chemotherapy in metastatic colorectal cancer.

BACKGROUND: To investigate three genetic alterations (TP53 mutation, Kras mutation and microsatellite instability (MSI)) and three polymorphisms (methylene tetrahydrofolate reductase (MTHFR) C677T, excision repair cross complementing group 1 (ERCC1)-118 and X-ray repair cross complementing group 1 (XRCC1)-399) for their ability to predict response, survival and toxicity to FOLFOX first line chemotherapy in the treatment of metastatic colorectal cancer (mCRC). METHODS: Tumour tissues from 118 mCRC patients who underwent FOLFOX treatment from three successive phase II trials were evaluated for mutations in TP53 (exons 5-8) and Kras (codons 12 and 13) and for MSI using PCR-based analysis. Genotyping for common single nucleotide polymorphisms in the MTHFR (codon 677), ERCC1 (codon 118) and XRCC1 (codon 399) genes was also carried out using PCR techniques. These genetic markers were correlated with clinical response, survival and toxicity to treatment. RESULTS: Patients with the T allele of ERCC1-118 showed significantly worse progression-free survival in univariate analysis (HR=2.62; 95% CI=1.14-6.02; P=0.02). None of the genetic alterations or polymorphisms showed significant association with clinical response to FOLFOX. The MTHFR, ERCC1 and XRCC1 polymorphisms showed no associations with overall haematological, gastrointestinal or neurological toxicity to FOLFOX, although MTHFR 677 TT genotype patients showed a significantly higher incidence of grade 3 or 4 diarrhoea (26%) compared with CC or CT genotype patients (6%, P=0.02). CONCLUSIONS: The ERCC1-118 and MTHFR C677T polymorphisms were associated with progression and severe diarrhoea, respectively, after FOLFOX treatment in mCRC. Although our findings require confirmation in large prospective studies, they reinforce the concept that individual genetic variation may allow personalized selection of chemotherapy to optimize clinical outcomes.

Matrix metalloproteinase-2 gene variants and abdominal aortic aneurysm.

OBJECTIVE: To investigate associations between two polymorphisms of the matrix metalloproteinase-2 gene (MMP2) and the incidence and progression of abdominal aortic aneurysm (AAA). METHODS: Cases and controls were recruited from a trial of screening for AAAs. The association between two variants of MMP2 (-1360C>T, and +649C>T) in men with AAA (n=678) and in controls (n=659) was examined using multivariate analyses. The association with AAA expansion (n=638) was also assessed. RESULTS: In multivariate analyses with adjustments for multiple testing, no association between either SNP and AAA presence or expansion was detected. CONCLUSION: MMP2 -1360C>T and +649C>T variants are not risk factors for AAA.

The expression of RUNX3 in colorectal cancer is associated with disease stage and patient outcome.

RUNX3 is believed to have tumour suppressor properties in several cancer types. Inactivation of RUNX3 has been shown to occur by methylation-induced transcriptional silencing and by mislocalization of the protein to the cytoplasm. The aim of this study was to examine the clinical significance of RUNX3 expression in a large series of colorectal cancers using immunohistochemistry and tissue arrays. With advancing tumour stage, expression of RUNX3 in the nucleus decreased, whereas expression restricted to the cytoplasmic compartment increased. Nuclear RUNX3 expression was associated with significantly better patient survival compared to tumours in which the expression of RUNX3 was restricted to the cytoplasm (P=0.025). These results support a role for RUNX3 as a tumour suppressor in colorectal cancer.

Polymorphisms of the matrix metalloproteinase 9 gene and abdominal aortic aneurysm.

BACKGROUND: Increased matrix metalloproteinase (MMP) 9 activity has been implicated in the formation of abdominal aortic aneurysm (AAA). The aim was to explore the association between potentially functional variants of the MMP-9 gene and AAA. METHODS: The -1562C > T and -1811A > T variants of the MMP-9 gene were genotyped in 678 men with an AAA (at least 30 mm in diameter) and 659 control subjects (aortic diameter 19-22 mm) recruited from a population-based trial of screening for AAA. Levels of MMP-9 were measured in a random subset of 300 cases and 84 controls. The association between genetic variants (including haplotypes) and AAA was assessed by multivariable logistic regression. RESULTS: There was no association between the MMP-9-1562C > T (odds ratio (OR) 0.70 (95 per cent confidence interval (c.i.) 0.27 to 1.82)) or -1811A > T (OR 0.71 (95 per cent c.i. 0.28 to 1.85)) genotypes, or the most common haplotype (OR 0.81 (95 per cent c.i. 0.62 to 1.05)) and AAA. The serum MMP-9 concentration was higher in cases than controls, and in minor allele carriers in cases and controls, although the differences were not statistically significant. CONCLUSION: In this study, the genetic tendency to higher levels of circulating MMP-9 was not associated with AAA.

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy.

BACKGROUND: Low tumour expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) have been linked with improved outcome for colorectal cancer (CRC) patients treated with 5-fluorouracil (5-FU). It is unclear whether this occurs because such tumours have better prognosis or they are more sensitive to 5-FU treatment. PATIENTS AND METHODS: Associations between TS, DPD and TP levels, determined by tissue microarrays and immunohistochemistry, and survival was evaluated in 945 CRC patients according to treatment status. RESULTS: Low TS and DPD expression associated with worse prognosis in stage II [hazard ratio (HR) = 1.69, 95% confidence interval (CI) (1.09-2.63) and HR = 1.92 (95% CI 1.23-2.94), respectively] and stage III CRC patients treated by surgery alone [HR = 1.39 (95% CI 0.92-2.13) and HR = 1.49 (95% CI 1.02-2.17), respectively]. Low TS, DPD and TP associated with trends for better outcome in stage III patients treated with 5-FU [HR = 0.81 (95% CI 0.49-1.33), HR = 0.70 (95% CI 0.42-1.15) and HR = 0.66 (95% CI 0.39-1.12), respectively]. CONCLUSION: Low TS and DPD expression are prognostic for worse outcome in CRC patients treated by surgery alone, whereas low TS, DPD and TP expression are prognostic for better outcome in patients treated with 5-FU chemotherapy. These results provide indirect evidence that low TS, DPD and TP protein expression are predictive of good response to 5-FU chemotherapy.

Polymorphisms of the interleukin-6 gene promoter and abdominal aortic aneurysm.

BACKGROUND: Elevated levels of circulating interleukin-6 (IL-6) have been reported in patients with abdominal aortic aneurysms (AAAs). Although this implicates inflammation as a cause of AAAs, there is also evidence that the aneurysmal aorta may secrete IL-6 into the circulation as a result of aortic proteolysis. Genetic association studies are one means of trying to clarify the role of specific mediators in the causal pathway. The aim of the present study was to examine the association between variants of the IL-6 gene and AAAs. METHODS: An association study involving 677 men with screen-detected AAAs and 656 age-matched controls was performed. Three variants in the IL-6 promoter region were analysed: IL-6-174G>C (rs1800795), IL-6-572G>C (rs1800796) and IL-6-597G>A (rs1800797). Univariate regression of SNP genotype on AAA as a binary outcome was initially performed under a range of genetic models (additive, dominant and recessive). This was followed by multivariate analyses, testing the same models but including risk factors known to be associated with AAAs. All analyses and haplotype estimation were performed under a generalized linear model framework. RESULTS: IL-6-572G>C polymorphism (frequency 1.5% in cases) was identified as an independent risk factor for AAA with an odds ratio (OR) of 6.00 (95%CI: 1.22, 29.41) when applied to the recessive model. No association was seen in the additive or dominant models. In a multivariate analysis using the most common haplotype (h.111, frequency 48.7%) as a reference, h.211 (frequency 4.4%) was an independent risk factor for AAA (OR 1.56, 95%CI: 1.02, 2.39). CONCLUSION: The IL-6 572G>C polymorphism (and h.211 haplotype) is associated with AAA, however it is too rare to be an important cause of most AAAs. This does not support the concept that the elevated level of IL-6 reported in patients with AAAs is a primary cause of the aneurysmal process.

Identification of serosal invasion and extramural venous invasion on review of Dukes' stage B colonic carcinomas and correlation with survival.

AIMS: To determine whether serosal invasion (SI) and/or extramural venous invasion (VI) could be more frequently identified on review of Dukes' stage B colonic carcinoma resection specimens and whether the revised findings correlated with clinical outcome. METHODS AND RESULTS: The original histology slides from 82 patients with Dukes' stage B colonic carcinoma were reviewed specifically to identify tumours showing SI and/or VI. All tumours were initially reported to be negative for both pathological parameters. The selected cases included 35 patients who died of carcinoma within 5 years of surgery and 47 patients with a minimum of 5-years' postoperative survival. The review was blinded to the original histopathology reports and to clinical follow-up data. SI and/or VI were identified in 26 cases (32%). Fourteen of 18 patients with SI, 8/12 patients with VI and all four patients with both adverse histological features died of carcinoma. CONCLUSIONS: Review of routinely sampled and stained colonic carcinoma resection specimens increased the proportion of cases classified as positive for SI and/or VI. The revised assessment correlated with patient outcome. Reliable identification of these features may permit stratification of high-risk patients with Dukes' stage B colonic cancer who could benefit from adjuvant treatment.

Failure to complete adjuvant chemotherapy is associated with adverse survival in stage III colon cancer patients.

Two recent North American studies have shown that completion of 5-fluorouracil (5FU)-based adjuvant chemotherapy is a major prognostic factor for the survival of elderly stage III colon cancer patients. The aim of the present study was to confirm this finding in a population-based series from Australia. The study cohort comprised 851 stage III colon cancer patients treated by surgery alone and 461 who initiated the Mayo chemotherapy regime. One-third of patients who initiated chemotherapy failed to complete more than three cycles of treatment. Independent predictors for failure to complete were treatment in district or rural hospitals, low socioeconomic index and treatment by a low-volume surgeon. Patients who failed to complete chemotherapy showed worse cancer-specific survival compared not only to those who completed treatment (HR=2.24; 95% confidence interval (CI) (1.66-3.03), P<0.001) but also to those treated by surgery alone (HR=1.37; 95% CI (1.09-1.72), P=0.008). The current and previous studies demonstrate the importance of completing adjuvant 5-FU-based chemotherapy for colon cancer. Further prospective studies are required to identify better the physiological and socioeconomic factors responsible for failure to complete chemotherapy so that appropriate improvements in health service delivery can be made.

Population-based study of prognostic factors in stage II colonic cancer.

BACKGROUND: Adjuvant chemotherapy in stage II colorectal cancer may be considered for patients whose tumours have poor prognostic features. The aim of this study was to evaluate the prognostic significance of commonly reported clinical and pathological features of stage II colonic cancer. METHODS: A population-based observational study of all patients with stage II colonic cancer diagnosed in the state of Western Australia from 1993 to 2003 was performed. A total of 1306 patients treated by surgery alone were identified, with a median follow-up of 59 (range 0-145) months. RESULTS: Multivariable analysis revealed that the only independent prognostic factors for disease-specific survival were stage T4 (hazard ratio (HR) 1.75 (95 per cent confidence interval (c.i.) 1.32 to 2.32); P < 0.001) and vascular invasion (HR 1.63 (95 per cent c.i. 1.15 to 2.30); P < 0.001). In younger patients (aged 75 years or less), who are more likely to be considered for chemotherapy, these two features showed independent prognostic significance but with higher HR values (1.96 for stage T4 and 2.73 for vascular invasion). Stage T4 and/or the presence of vascular invasion identified a 'poor' prognostic group, comprising 26.6 per cent of younger patients and with a 5-year survival rate of 71.2 per cent. The remaining 'good' prognostic group had a survival rate of 84.3 per cent at 5 years' follow-up. CONCLUSION: This study highlights the importance of accurate pathological assessment of tumour stage and vascular invasion for the prognostic stratification of patients with stage II colonic cancer. The results provide clarification of guidelines for the management of stage II disease in relation to recommendations for chemotherapy.