Barry Iacopetta

Kirsten ras mutations in patients with colorectal cancer: The 'RASCAL II' study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Tumor-Infiltrating FOXP3+ T Regulatory Cells Show Strong Prognostic Significance in Colorectal Cancer

PURPOSE To determine the prognostic significance of FOXP3(+) lymphocyte (Treg) density in colorectal cancer compared with conventional histopathologic features and with CD8(+) and CD45RO(+) lymphocyte densities. PATIENTS AND METHODS Tissue microarrays and immunohistochemistry were used to assess the densities of CD8(+), CD45RO(+), and FOXP3(+) lymphocytes in tumor tissue and normal colonic mucosa from 967 stage II and stage III colorectal cancers. These were evaluated for associations with histopathologic features and patient survival. RESULTS FOXP3(+) Treg density was higher in tumor tissue compared with normal colonic mucosa, whereas CD8(+) and CD45RO(+) cell densities were lower. FOXP3(+) Tregs were not associated with any histopathologic features, with the exception of tumor stage. Multivariate analysis showed that stage, vascular invasion, and FOXP3(+) Treg density in normal and tumor tissue were independent prognostic indicators, but not CD8(+) and CD45RO(+). High FOXP3(+) Treg density in normal mucosa was associated with worse prognosis (hazard ratio [HR] = 1.51; 95% CI, 1.07 to 2.13; P = .019). In contrast, a high density of FOXP3(+) Tregs in tumor tissue was associated with improved survival (HR = 0.54; 95% CI, 0.38 to 0.77; P = .001). CONCLUSION FOXP3(+) Treg density in normal and tumor tissue had stronger prognostic significance in colorectal cancer compared with CD8(+) and CD45RO(+) lymphocytes. The finding of improved survival associated with a high density of tumor-infiltrating FOXP3(+) Tregs in colorectal cancer contrasts with several other solid cancer types. The inclusion of FOXP3(+) Treg density may help to improve the prognostication of early-stage colorectal cancer.

Are there two sides to colorectal cancer

Colorectal carcinomas (CRC) that arise proximal (right) or distal (left) to the splenic flexure exhibit differences in incidence according to geographic region, age and gender. Together with observations that tumours in the hereditary cancer syndromes HNPCC and FAP occur predominantly in the right and left colon, respectively, the existence of 2 categories of CRC based on site of origin in the large bowel was proposed more than a decade ago. Differences between normal right and left colonic segments that could favour progression through different tumourigenic pathways are summarized in this review. Accumulating evidence suggests that the risk of CRC conferred by various environmental and genetic factors is different for proximal and distal tumours. Right‐ and left‐sided tumours also exhibit different sensitivities to fluorouracil‐based chemotherapy. Such differences are probably related to the molecular characteristics of the tumours, with the microsatellite instability and CpG island methylator phenotypes being associated with right‐sided tumours and chromosomal instability with left‐sided tumours. Future molecular‐based classification systems for CRC that rely upon distinctive gene expression patterns may allow a clearer discrimination of subgroups than that provided by tumour site alone. Until then however, the existence of 2 broadly different groups of cancer defined by site of origin in the colon should be considered in the design of future epidemiologic studies as well as in the design of new clinical trials aimed at testing novel adjuvant therapies.

A polymorphism in the enhancer region of the thymidylate synthase promoter influences the survival of colorectal cancer patients treated with 5-fluorouracil

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.

Characterisation of colorectal cancers showing hypermethylation at multiple CpG islands

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.

PIK3CA mutations in breast cancer are associated with poor outcome

SummaryThe phosphatidylinositol-3-kinase (PI3K)–AKT signaling pathway is considered to play an important role in tumorigenesis. Frequent somatic mutations in the PI3K subunit p110α (PIK3CA) occur in a variety of cancer types. We screened 250 primary human breast tumors for mutations in PIK3CA in order to determine associations with pathological features and with patient outcome. The frequency of PIK3CA mutations in the C2, helical and kinase domains was 35% (88/250). Mutations were associated with larger tumor size (p=0.004) and positive estrogen receptor status (p=0.008). Patients with PIK3CA mutations showed significantly worse survival (p=0.004), particularly those with positive estrogen receptor expression or non-amplified erbB2 (both p=0.002). PIK3CA mutation was an independent factor for worse survival in breast cancer patients with non-amplified erbB2 (RR=2.6, 95%CI [1.2–5.5], p=0.016).

A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability.

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.

Concordance between p53 protein overexpression and gene mutation in a large series of common human carcinomas

Immunohistochemical (IHC) detection of p53 protein was compared with the presence of p53 gene mutation in many colorectal (n = 100), breast (n = 92), endometrial (n = 122), and gastric (n = 116) carcinomas. Two commercially available antibodies, DO7 and CM1, were used for IHC analysis of paraffin-embedded tissue sections. Screening for gene mutations in frozen and paraffin-embedded tumor samples was carried out using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). The frequency of nuclear staining with DO7 or CM1 for each tumor type, respectively, was colorectal (36%, 23%); breast (15%, 19%); endometrial (21%, 33%); and gastric (23%,-). Overall correlation between the two antibodies for nuclear staining was 90% for the 314 tumors analyzed. Cytoplasmic staining was observed with DO7 in 7% of breast and 5% of gastric carcinomas and with CM1 in 17% of breast and 54% of endometrial carcinomas. p53 gene mutation was found in 39% of colorectal, 28% of breast, 13% of endometrial, and 25% of gastric cancers. The concordance between p53 nuclear overexpression and gene mutation (both positive or both negative) was 68% for colorectal, 79% for breast, 76% for endometrial, and 73% for gastric carcinomas. This study provides further evidence that IHC detection of p53 protein accumulation does not always indicate the presence of a gene mutation and vice versa. Discordant results were observed in approximately 20% to 30% of the tumors studied, highlighting the need for careful characterization of both p53 gene and protein alterations when assessing the relationship between p53 status and tumor behavior.

Transferrin receptors and iron uptake during erythroid cell development

Experiments were performed to determine the level of transferrin receptors and rate of transferrin-bound iron uptake by various immature erythroid cell populations. Developing erythroid cells from the rat and mouse foetal liver at various stages of gestation were studied. In addition Friend leukaemic cells grown in culture were examined. The transferrin receptor level of Friend cells was similar to that of erythroid cells from the mouse foetal liver. During erythroid cell development the transferrin receptor level increased from about 300,000 per cell at the early normoblast stage to reach a maximum of about 8000,000 per cell on intermediate normoblasts. Further maturation of intermediate normoblasts was accompanied by a decline in the number of transferrin receptors, reaching a level of 105,000 in the circulating reticulocyte. The rate of iron uptake from transferrin during erythroid cell development was found to correlate closely with the number of transferrin receptors. In each of the immature erythroid cell populations studied the rate of iron uptake was about 36 iron atoms per receptor per hour. These results indicate that the level of transferrin receptors may be the major factor which determines the rate of iron uptake during erythroid cell development.

p53 gene mutation, microsatellite instability and adjuvant chemotherapy : Impact on survival of 388 patients with Dukes' C colon carcinoma

Two common genetic alterations in colon carcinoma, p53 mutation and microsatellite instability (MSI), were investigated to determine their prognostic importance for cancer-specific survival and response to adjuvant chemotherapy in patients with Dukes’ C colon cancer. The p53 tumour suppressor gene encodes for a nuclear phosphoprotein involved in cellular response to DNA damage, while MSI is a characteristic feature of tumours with defective DNA mismatch repair. The cellular response mechanisms to DNA-damaging agents in tumours with mutant p53 or MSI may as a consequence differ, and this might translate into different outcomes following adjuvant chemotherapy. A consecutive series of 388 Dukes’ C colon carcinomas with 5-year median follow-up was analysed for p53 mutation and for MSI (in proximal/transverse carcinomas only) using polymerase chain reaction single-strand conformation polymorphism. The incidence of p53 mutation was 28% in all carcinomas while that of MSI in proximal/transverse carcinomas was 19%. One hundred and thirty-three patients (34%) received adjuvant chemotherapy (5-fluorouracil/levamisole) with curative intent. The presence of p53 mutation did not predict for survival in either the treated or untreated groups. The presence of MSI in the proximal/transverse colon carcinoma group was associated with significantly better 5-year survival: 58 versus 32% (p = 0.015, log rank test). This was largely due to better survival observed in the MSI subgroup that received adjuvant chemotherapy (p = 0.017, log rank test). Further work in prospective, randomised clinical trials investigating the effects of adjuvant therapy should consider incorporating MSI status in order to determine whether this is an independent predictive factor for survival and/or response to adjuvant chemotherapy.