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.
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.
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.
CD44 and Musashi-1 are frequently expressed in both premalignant gastric lesions and invasive GC, whereas CD133 expression is restricted mainly to neoplastic tissues.
CD44; CD133; Musashi-1; Correa pathway; gastric cancer
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).
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.
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).
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.
colorectal cancer; metastatic; chemotherapy; predictive factors
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.
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].
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.
colorectal cancer; fluorouracil; predictive; prognostic; thymidylate synthase
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.
RUNX3; colorectal cancer; tissue arrays; prognosis; Wnt
5‐fluorouracil; colorectal cancer; microsatellite instability; adjuvant chemotherapy
BRAF mutation; methylation; colorectal tumours; microsatellite instability
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 γ-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.
CIMP; GGH; promoter methylation; colorectal cancer
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.
colon cancer; health services; mayo regime; toxicity; 5-fluorouracil
The CpG-island methylator phenotype (CIMP+) in colorectal cancer (CRC) is characterised by frequent hypermethylation of promoter regions in tumour suppressor genes. Low level methylation of some CpG islands is also seen in the normal colonic mucosa and increases with age; however, it is still unclear what other factors regulate this phenomenon. The first aim of our study was to determine whether the level of promoter methylation is elevated in the normal colonic mucosa of patients with CIMP+ tumours. The second aim was to investigate whether common, functional polymorphisms in genes involved in methyl group metabolism are associated with the level of methylation in this tissue. CpG islands within the ERα, MYOD, P16(INK4A), MLH1, APC, P14(ARF), DAPK and TIMP3 genes were quantitatively evaluated for methylation in normal colonic mucosa from a large series of CRC patients using the MethyLight assay. Genotyping was carried out for polymorphisms in the MTHFR, TS, MS, MTHFD1 and DNMT3b genes. Methylation of ERα and MYOD in normal colonic mucosa increased with age and was higher in female subjects. Methylation of P16(INK4A), MLH1, TIMP3 and DAPK in normal mucosa occurred at a lower level than ERα and MYOD but also increased with age and was significantly higher in patients with CIMP+ tumours. The DNMT3b C46359T polymorphism was associated with significantly less methylation of MYOD and MLH1 and with trends for lower methylation in each of the other CpG islands examined. Our results demonstrate that age, gender and genetic factors can influence the methylation level of CpG islands in gene promoter regions of normal colonic mucosa. Further work is required to determine whether such methylation is associated with the development of CIMP+ CRC.
ERα; MYOD; promoter methylation; DNMT3b; polymorphism; ageing
Human papilloma viruses (HPVs) are accepted as being carcinogenic in human cervical and anogenital cancers. The suspicion that HPVs may also have a role in human breast cancer is based on the identification of HPVs in human breast tumours and the immortalisation of normal human breast cells by HPV types 16 and 18. For this investigation, DNA that had been previously extracted and fresh frozen at −70°C from 50 unselected invasive ductal breast cancer specimens were screened by polymerase chain reaction (PCR) for HPV type 16, 18 and 33 gene sequences. We show that HPV 18 gene sequences are present in DNA extracted from breast tumours in Australian women. Overall, 24 (48%) of the 50 samples were HPV positive. Overall no correlations with tumour grade, patient survival, steroid receptor status, ERB-2, p53 expression and mutation were observed. Human papilloma viruses may have a role in human breast cancer. We speculate that HPVs may be transmitted by hand from the female perineum to the breast.
human papilloma virus; human breast cancer; grade of tumour; patient mortality; hormone receptor status; abnormal p53 protein expression; p53 mutations and ERB-2 expression
molecular analysis; Kirsten ras gene; colorectal cancer
Background and aims: A subgroup of colorectal cancers (CRC) referred to as the CpG island methylator phenotype (CIMP+) shows simultaneous methylation of multiple CpG islands. The clinicopathological and molecular characteristics of this phenotype remain uncertain however.
Methods: We analysed methylation of CpG islands in the p16 and MDR1 genes and MINT-2 clone in 275 stage II/III CRCs.
Results: Concurrent methylation of two or more CpG islands was observed in 32% of cases and was considered to represent CIMP+. These were often poorly differentiated, had less TP53 mutations, and originated frequently in the proximal or higher stage CRC compared with CIMP− tumours (p<0.05 for each). CIMP+ had no prognostic significance in stage II or stage III CRC treated by surgery alone. hMLH1 methylated tumours comprised the majority (81%) of cases with microsatellite instability, were frequently observed in older female patients, were often poorly differentiated or CIMP+, and contained wild-type K-ras (p<0.05 for each). Females who were heterozygous or homozygous for the C677T MTHFR polymorphism were at increased risk of developing CIMP+ CRC (odds ratio 2.17, 95% confidence interval 1.03–4.57; p=0.037).
Conclusions: These observations made in a relatively large unselected series of CRC support the notion that CIMP+ characterises a subgroup of tumours with distinctive phenotypic features.
colorectal cancer; CpG island methylator phenotype; hypermethylation
Background: The enzyme methylenetetrahydrofolate reductase (MTHFR) catalyses the formation of folate intermediates that are vital to methylation reactions. A polymorphic variant (TT) has been linked to reduced levels of plasma folate, aberrant DNA methylation in leucocytes, and increased risk of colorectal cancer (CRC) under conditions of low folate intake. The cystathionine beta-synthase (CBS) enzyme reduces homocysteine levels and thus may protect against CRC. The CBS gene has a variant, 844ins68, that has been linked with increased activity. These variants may be involved in the development of the subgroup of CRC displaying aberrant DNA methylation and frequently associated with microsatellite instability (MSI).
Aim: To investigate the frequencies of the TT and 844ins68 genotypes in CRC patients with MSI+ tumours compared with those with MSI− tumours and a control population.
Subjects: Patients with CRC (n=501) and healthy control subjects (n=1207) were studied. CRC cases were classified as MSI+ (n=75) or MSI− (n=426) based on deletions within the BAT-26 mononucleotide repeat.
Methods: Subjects were genotyped for MTHFR using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP) techniques, and for CBS using PCR.
Results: The MTHFR TT genotype was more frequent in older CRC patients (≥70 y) compared with equivalent aged controls (p=0.03), was associated with a significantly later age of diagnosis in patients with proximal colon tumours (p=0.02), and was almost twice as frequent in MSI+ than in MSI− tumours (p=0.05). Compared with normal controls, the 844ins68 variant of CBS was less frequent in patients with proximal tumours (p=0.02).
Conclusions: The TT genotype of MTHFR is associated with an increased risk of CRC in older populations, possibly due to age related disturbances in folate metabolism. The TT genotype appears to predispose to CRC that is MSI+. This may reflect the involvement of aberrant DNA methylation frequently associated with MSI+. The 844ins68 CBS polymorphism may protect against proximal tumours.
colorectal cancer; folate; DNA methylation; methylenetetrahydrofolate reductase; cystathionine beta-synthase
interleukin-6; polymorphism; breast cancer; prognosis
British Journal of Cancer (2002) 86, 1524–1525. DOI: 10.1038/sj/bjc/6600279 www.bjcancer.com
© 2002 Cancer Research UK
British Journal of Cancer (2002) 86, 1366. DOI: 10.1038/sj/bjc/6600230 www.bjcancer.com
© 2002 Cancer Research UK
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. © 2001 Cancer Research Campaign http://www.bjcancer.com
High levels of thymidylate synthase (TS) expression have been associated with poor survival of colorectal cancer (CRC) patients to 5-fluorouracil (5-FU)-based chemotherapy. Recent evidence suggests that a polymorphism within the enhancer region of the TS gene promoter can influence TS expression, with the triple repeat homozygote (3R/3R) being associated with significantly higher tumour TS levels than either the double repeat homozygote (2R/2R) or heterozygotes (2R/3R). In the present study we investigated whether TS genotype was associated with the degree of survival benefit from chemotherapy in 221 Dukes' C stage CRC patients. Patients with the 3R/3R polymorphism (n = 58, 26%) showed no significant long-term survival benefit from chemotherapy (RR = 0.62, 95% CI: 0.30–1.25, P = 0.18), whereas those with the 2R/2R or 2R/3R genotype (n = 163, 74%) showed significant gains in survival from this treatment (RR = 0.52, 95% CI: 0.52–0.82, P = 0.005). These results demonstrate that a polymorphism within the TS gene, probably through its effect on TS expression levels, can influence the survival benefit obtained by CRC patients from 5-FU-based chemotherapy. © 2001 Cancer Research Campaignhttp://www.bjcancer.com
colorectal cancer; adjuvant chemotherapy; thymidylate synthase; polymorphism
The important role of the p53 gene in tumour progression and cellular response to DNA damage has prompted investigation of the clinical significance of alterations to this gene. We examined both p53 overexpression and mutation of the gene in endometrial carcinoma in order to evaluate the prognostic significance of these changes. Of 122 endometrial carcinomas, 33 (27%) showed overexpression of p53 in the nucleus and 66 (54%) in the cytoplasm. Mutation in the p53 gene was found in 16 (13%) cases but showed no significant association with patient survival. Nuclear p53 overexpression was associated with poor survival (48% vs 80% alive in negative tumours 5 years post operatively, P < 0.001). In contrast, cytoplasmic p53 overexpression was associated with better survival (85% vs 55%, P < 0.001). When patients were separated into prognostic subgroups according to established clinical markers, these associations remained significant within most subgroups examined. In multivariate analysis adjusted for surgical stage, histological grade and type and vascular invasion, both nuclear p53 overexpression [hazard ratio 4.9 (95% CI 1.3-17.6). P = 0.016] and cytoplasmic overexpression [0.25 (0.06-0.98), P = 0.047] were independent prognostic factors. Immunohistochemical assessment of p53 overexpression in the nucleus and cytoplasm could provide useful prognostic information for the management of patients with endometrial cancer.
DNA extraction and PCR amplification from paraffin wax embedded bone tumour specimens present several difficulties, firstly, because of the abundant matrix they contain and, secondly, because decalcification often causes degradation of DNA. In this report, comparative studies were carried out to determine the most efficient method for DNA extraction and PCR amplification from such specimens. The results indicated that nested PCR produced appropriate strong reaction products with minimal background contamination. A method for DNA extraction from paraffin wax embedded bone tissue and a nested PCR-SSCP technique have been developed for use in such diagnostic specimens.
DNA extraction; PCR amplification; SSCP; paraffin wax embedded bone tumour specimens
Immunocytochemistry (ICC) has been used routinely to stain for p53 overexpression in a range of human tumours. The underlying assumption has been that positive staining indicates a mutation in the p53 coding sequence. Recently, however, discordancy has been observed and the accuracy of ICC as a marker of p53 gene mutation has been questioned. In this study of 109 colorectal adenocarcinomas, we compared ICC staining with p53 gene mutations detected by single-strand conformation polymorphism (SSCP) analysis. Concordancy between the two techniques was found in 69% of tumours. ICC-positive/SSCP-negative cases accounted for 20% of tumours and ICC-negative/SSCP-positive cases for the remaining 11%. These results caution against the assumption that p53 protein overexpression is always associated with a gene mutation. Epigenetic phenomena may account for a significant proportion of ICC-positive tumours.