p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer.

Mutations of the TP53 and Ki-ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki-ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3-144 months) whether or not p53 protein expression, TP53 mutation and K-ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5-8 of the TP53 gene and in codons 12 and 13 of the K-ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki-ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P = 0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours (P = 0.010). The presence or absence of mutations in Ki-ras did not correlate with prognosis (P = 0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR) = 2.16, 95% confidence interval (CI) 1.12-4.11, P = 0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.

A colorectal cancer classification system that associates cellular phenotype and responses to therapy.

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor-targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Antitumor activities of ATP-competitive inhibitors of mTOR in colon cancer cells

Le cancer colorectal est la 3ème cause de décès liée au cancer dans l'Europe de l'Ouest et nécessite une prise en charge pluridisciplinaire. Les thérapies anticancéreuses récentes développées visent à inhiber les voies de signalisation cellulaires responsables de la prolifération des cellules tumorales. L'inhibition de la voie de signalisation cellulaire mTOR, est une stratégie prometteuse. En effet, mTOR est souvent suractivé dans les cellules du cancer colorectal et régule la croissance, la prolifération et la survie cellulaire. De nombreuses études récentes ont démontrés l'importance de l'activité de mTOR dans le développement du cancer colorectal et l'efficacité anti-tumorale des inhibiteurs allostériques de mTOR, telle que la rapamycine. Récemment, une nouvelle classe d'inhibiteur de mTOR, notamment PP242 et NVP-BEZ235, agissant comme inhibiteur ATP- compétitif a été développée. L'efficacité de ces inhibiteurs n'a pas été démontrée dans le contexte du cancer colorectal. Dans cette étude, nous avons comparé l'effet de PP242, un inhibiteur ATP-compétitif de mTOR et NVP-BEZ235, un inhibiteur dual de PI3K/mTOR par rapport à la rapamycine. Nous avons étudié, in vitro, leur effet sur la croissance, la prolifération et la survie cellulaire sur des lignées cellulaires du cancer du colon (LS174, SW480 et DLD-1) et, in vivo, sur la croissance de xénogreffes dans un modèle murin. Nous avons émis l'hypothèse que l'effet des ces nouveaux inhibiteurs seraient plus importants qu'avec la rapamycine. Nous avons observé que le PP242 et le NVP-BEZ235 réduisent significativement et de façon plus marquée que la rapamycine la croissance, la prolifération et la survie cellulaire des cellules LS174T et DLD-1. Ces inhibiteurs réduisent également la prolifération et la survie cellulaire des cellules SW480 alors que celles-ci étaient résistantes à la rapamycine. Nous avons également observé que les inhibiteurs PP242 et NVP-BEZ235 réduisaient la croissance des xénogreffes avec les lignées cellulaires LS174 et SW480. Finalement, nous avons remarqué que l'effet anti-tumoral des inhibiteurs ATP-compétitifs de mTOR était potentialisé par l'U0126, un inhibiteur de MEK/MAPK, souvent activé dans les voies de signalisation cellulaire du cancer colorectal. En conclusion, nous avons observé que les inhibiteurs ATP-compétitifs de mTOR bloquent la croissance de cellules tumorales du cancer colorectal in vitro et in vivo. Ces résultats démontrent que ces inhibiteurs représentent une option thérapeutique prometteuse dans le traitement du cancer colorectal et méritent d'être évalués dans des études cliniques.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

COLOX: a new blood-based test for colorectal cancer (CRC)screening

BACKGROUND: The objective is to develop a cost-effective, reliable and non invasive screening test able to detect early CRCs and adenomas. This is done on a nucleic acids multigene assay performed on peripheral blood mononuclear cells (PBMCs). METHODS: A colonoscopy-controlled study was conducted on 179 subjects. 92 subjects (21 CRC, 30 adenoma >1 cm and 41 controls) were used as training set to generate a signature. Other 48 subjects kept blinded (controls, CRC and polyps) were used as a test set. To determine organ and disease specificity 38 subjects were used: 24 with inflammatory bowel disease (IBD),14 with other cancers (OC). Blood samples were taken and PBMCs were purified. After the RNA extraction, multiplex RT-qPCR was applied on 92 different candidate biomarkers. After different univariate and multivariate analysis 60 biomarkers with significant p-values (<0.01) were selected. 2 distinct biomarker signatures are used to separate patients without lesion from those with CRC or with adenoma, named COLOX CRC and COLOX POL. COLOX performances were validated using random resampling method, bootstrap. RESULTS: COLOX CRC and POL tests successfully separate patients without lesions from those with CRC (Se 67%, Sp 93%, AUC 0.87), and from those with adenoma > 1cm (Se 63%, Sp 83%, AUC 0.77). 6/24 patients in the IBD group and 1/14 patients in the OC group have a positive COLOX CRC. CONCLUSION: The two COLOX tests demonstrated a high Se and Sp to detect the presence of CRCs and adenomas > 1 cm. A prospective, multicenter, pivotal study is underway in order to confirm these promising results in a larger cohort.

Combined radioimmunotherapy and radiotherapy of liver metastases from colorectal cancer: a feasibility study.

BACKGROUND: A combination of radioimmunotherapy (RIT) and radiotherapy (RT) should allow one to increase the dose of radiation targeting a particular tumour without the concomitant increase of toxic side effects. This might be obtained if the dose limiting side effect of each individual radiation therapy concerned different organs. METHODS: Six patients with limited liver metastatic disease from colorectal cancer were treated with 6.9 GBq (range 4.7 to 8.4 GBq) 131I-labelled anti-CEA MAb F(ab')2 fragments combined with 20 Gy RT to the liver. Both treatments were given in close association, according to timing schedules evaluated in animals that gave the best results. RESULTS: Reversible bone marrow and liver toxicity was observed in 6 and 5 patients, respectively. Three patients who first received 20 Gy RT to the liver, showed a significant platelet drop upon completion of RT. Repeat computerized tomography (CT) after 2 months showed a minor response in 1 patient and stable disease in 3 patients. CONCLUSION: The study shows potential ways of combining RIT and RT, suggesting that this combination is feasible for the treatment of liver metastases.

Risk factors for young-onset colorectal cancer.

PURPOSE: We investigated risk factors for colorectal cancer in early-onset cancers, to provide quantitative estimates for major selected risk factors. METHODS: We analyzed data from three Italian and Swiss case-control studies conducted between 1985 and 2009, including 329 colorectal cancer cases and 1,361 controls aged ≤45 years. We computed odds ratios (ORs) from unconditional logistic regression models, adjusted for major confounding factors. RESULTS: The OR of young-onset colorectal cancer was 4.50 for family history of colorectal cancer in first-degree relatives, the association being higher in subjects with affected siblings (OR 11.68) than parents (OR 3.75). The ORs of young-onset colorectal cancer were 1.56 for ≥14 drinks/week of alcohol, 1.56 for the highest tertile of processed meat, 0.40 for vegetables, 0.75 for fruit, and 0.78 for fish intake. Among micronutrients, the ORs were 0.52 for β-carotene, 0.68 for vitamin C, 0.38 for vitamin E, and 0.59 for folate. No significant associations emerged for physical activity, overweight, and diabetes. CONCLUSIONS: This study-the largest on young-onset colorectal cancer-confirms that several recognized risk factors for colorectal cancer are also relevant determinants of young-onset colorectal cancer. Family history of colorectal cancer in particular is a stronger risk factor in young subjects, as compared to middle age and elderly ones.

Two Signatures For A New Blood-Based Test For Colorectal Cancer Screening

Background: Detection rates for adenoma and early colorectal cancer (CRC) are unsatisfactory due to low compliance towards invasive screening procedures such as colonoscopy. There is a large unmet screening need calling for an accurate, non-invasive and cost-effective test to screen for early neoplastic and pre-neoplastic lesions. Our goal is to identify effective biomarker combinations to develop a screening test aimed at detecting precancerous lesions and early CRC stages, based on a multigene assay performed on peripheral blood mononuclear cells (PBMC).Methods: A pilot study was conducted on 92 subjects. Colonoscopy revealed 21 CRC, 30 adenomas larger than 1 cm and 41 healthy controls. A panel of 103 biomarkers was selected by two approaches: a candidate gene approach based on literature review and whole transcriptome analysis of a subset of this cohort by Illumina TAG profiling. Blood samples were taken from each patient and PBMC purified. Total RNA was extracted and the 103 biomarkers were tested by multiplex RT-qPCR on the cohort. Different univariate and multivariate statistical methods were applied on the PCR data and 60 biomarkers, with significant p-value (< 0.01) for most of the methods, were selected.Results: The 60 biomarkers are involved in several different biological functions, such as cell adhesion, cell motility, cell signaling, cell proliferation, development and cancer. Two distinct molecular signatures derived from the biomarker combinations were established based on penalized logistic regression to separate patients without lesion from those with CRC or adenoma. These signatures were validated using bootstrapping method, leading to a separation of patients without lesion from those with CRC (Se 67%, Sp 93%, AUC 0.87) and from those with adenoma larger than 1cm (Se 63%, Sp 83%, AUC 0.77). In addition, the organ and disease specificity of these signatures was confirmed by means of patients with other cancer types and inflammatory bowel diseases.Conclusions: The two defined biomarker combinations effectively detect the presence of CRC and adenomas larger than 1 cm with high sensitivity and specificity. A prospective, multicentric, pivotal study is underway in order to validate these results in a larger cohort.

Transvaginal specimen extraction in colorectal surgery: current state of the art.

Aim  Background The expected benefit of transvaginal specimen extraction is reduced incision-related morbidity. Objectives A systematic review of transvaginal specimen extraction in colorectal surgery was carried out to assess this expectation. Method  Search strategy The following keywords, in various combinations, were searched: NOSE (natural orifices specimen extraction), colorectal, colon surgery, transvaginal, right hemicolectomy, left hemicolectomy, low anterior resection, sigmoidectomy, ileocaecal resection, proctocolectomy, colon cancer, sigmoid diverticulitis and inflammatory bowel diseases. Selection criteria Selection criteria included large bowel resection with transvaginal specimen extraction, laparoscopic approach, human studies and English language. Exclusion criteria were experimental studies and laparotomic approach or local excision. All articles published up to February 2011 were included. Results  Twenty-three articles (including a total of 130 patients) fulfilled the search criteria. The primary diagnosis was colorectal cancer in 51% (67) of patients, endometriosis in 46% (60) of patients and other conditions in the remaining patients. A concurrent gynaecological procedure was performed in 17% (22) of patients. One case of conversion to laparotomy was reported. In two patients, transvaginal extraction failed. In left- and right-sided resections, the rate of severe complications was 3.7% and 2%, respectively. Two significant complications, one of pelvic seroma and one of rectovaginal fistula, were likely to have been related to transvaginal extraction. The degree of follow up was specified in only one study. Harvested nodes and negative margins were adequate and reported in 70% of oncological cases. Conclusion  Vaginal extraction of a colorectal surgery specimen shows potential benefit, particularly when associated with a gynaecological procedure. Data from prospective randomized trials are needed to support the routine use of this technique.

Fatigue and weight loss predict survival on circadian chemotherapy for metastatic colorectal cancer.

BACKGROUND: Chemotherapy-induced neutropenia has been associated with prolonged survival selectively in patients on a conventional schedule (combined 5-fluorouracil, leucovorin, and oxaliplatin [FOLFOX2]) but not on a chronomodulated schedule of the same drugs administered at specific circadian times (chronoFLO4). The authors hypothesized that the early occurrence of chemotherapy-induced symptoms correlated with circadian disruption would selectively hinder the efficacy of chronotherapy. METHODS: Fatigue and weight loss (FWL) were considered to be associated with circadian disruption based on previous data. Patients with metastatic colorectal cancer (nâeuro0/00=âeuro0/00543) from an international phase 3 trial comparing FOLFOX2 with chronoFLO4 were categorized into 4 subgroups according to the occurrence of FWL or other clinically relevant toxicities during the initial 2 courses of chemotherapy. Multivariate Cox models were used to assess the role of toxicity on the time to progression (TTP) and overall survival (OS). RESULTS: The proportions of patients in the 4 subgroups were comparable in both treatment arms (Pâeuro0/00=âeuro0/00.77). No toxicity was associated with TTP or OS on FOLFOX2. The median OS on FOLFOX2 ranged from 16.4 (95% confidence limits [CL], 7.2-25.6 months) to 19.8 months (95% CL, 17.7-22.0 months) according to toxicity subgroup (Pâeuro0/00=âeuro0/00.45). Conversely, FWL, but no other toxicity, independently predicted for significantly shorter TTP (Pâeuro0/00<âeuro0/00.0001) and OS (Pâeuro0/00=âeuro0/00.001) on chronoFLO4. The median OS on chronoFLO4 was 13.8 months (95% CL, 10.4-17.2 months) or 21.1 months (95% CL, 19.0-23.1 months) according to presence or absence of chemotherapy-induced FWL, respectively. CONCLUSIONS: Early onset chemotherapy-induced FWL was an independent predictor of poor TTP and OS only on chronotherapy. Dynamic monitoring to detect early chemotherapy-induced circadian disruption could allow the optimization of rapid chronotherapy and concomitant improvements in safety and efficacy.

Colorectal cancer metastasis: the DNA repair inhibitor Dbait increases sensitivity to hyperthermia and improves efficacy of radiofrequency ablation.

PURPOSE: To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS: The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS: When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION: Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.

High constant incidence of second primary colorectal cancer.

Patients who had a colorectal cancer have a 1.5- to 2-fold excess risk of a second colorectal cancer as compared to the general population, the excess being higher at younger age at diagnosis. To further investigate the risk and the age-relation of the incidence of second primary colorectal cancer, we considered 9,389 first colon and rectal cancers registered in the Vaud Cancer Registry, Switzerland, between 1974 and 2008, and followed-up to the end of 2008 for a total of 44,113 person-years. There were 136 second colorectal cancers versus 90.5 expected, corresponding to a standardized incidence ratio (SIR) of 1.5 (95% confidence interval, CI, 1.3-1.8). The SIRs were not heterogeneous between men and women, and in strata of calendar year at diagnosis, duration of follow-up, and subsite. However, the SIR was 7.5 (95% CI 4.2-12.4) for subjects diagnosed below age 50 and declined thereafter to reach 1.0 (95% CI 0.6-1.6) at age 80 or over. Consequently, the incidence of second primary colorectal cancer was stable, and exceedingly high, around 300-400/100,000 between age 30-39 and 70 or over. This age pattern is consistent with the existence of a single mutational event in a population of highly susceptible individuals.

hTERT methylation is necessary but not sufficient for telomerase activity in colorectal cells

Colorectal cancers exhibit a high telomerase activity, usually correlated with the hypermethylation of the promoter of its hTERT catalytic subunit. Although telomerase is not expressed in normal tissue, certain proliferative somatic cells such as intestinal crypt cells have demonstrated telomerase activity. The aim of this study was to determine whether a correlation exists between telomerase activity, levels of hTERT methylation and telomere length in tumoral and normal colorectal tissues. Tumor, transitional and normal tissues were obtained from 11 patients with a colorectal cancer. After bisulfite modification of genomic DNA, hTERT promoter methylation was analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA). Telomerase activity and telomere length were measured by a fluorescent-telomeric repeat amplification protocol assay and by Southern blotting, respectively. A significant increase of hTERT methylation and telomerase activity, and a reduction of the mean telomere length were observed in the tumor tissues compared to the transitional and normal mucosa. In the transitional and normal mucosa, telomerase activity was significantly lower than that in tumor tissues, even with high levels of hTERT methylation. Nevertheless, hTERT promoter methylation was not linearly correlated to telomerase activity. These data indicate that hTERT promoter methylation is a necessary event for hTERT expression, as is telomerase activity. However, methylation is not sufficient for hTERT activation, particularly in normal colorectal cells.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Transketolase-like 1 expression is modulated during Colorectal cancer progression and metastasis formation

Background Transketolase-like 1 (TKTL1) induces glucose degradation through anaerobic pathways, even in presence of oxygen, favoring the malignant aerobic glycolytic phenotype characteristic of tumor cells. As TKTL1 appears to be a valid biomarker for cancer prognosis, the aim of the current study was to correlate its expression with tumor stage, probability of tumor recurrence and survival, in a series of colorectal cancer patients. Methodolody/Principal Findings Tumor tissues from 63 patients diagnosed with colorectal cancer at different stages of progression were analyzed for TKTL1 by immunohistochemistry. Staining was quantified by computational image analysis, and correlations between enzyme expression, local growth, lymph-node involvement and metastasis were assessed. The highest values for TKTL1 expression were detected in the group of stage III tumors, which showed significant differences from the other groups (Kruskal-Wallis test, P = 0.000008). Deeper analyses of T, N and M classifications revealed a weak correlation between local tumor growth and enzyme expression (Mann-Whitney test, P = 0.029), a significant association of the enzyme expression with lymph-node involvement (Mann-Whitney test, P = 0.0014) and a significant decrease in TKTL1 expression associated with metastasis (Mann-Whitney test, P = 0.0004). Conclusions/Significance To our knowledge, few studies have explored the association between variations in TKTL1 expression in the primary tumor and metastasis formation. Here we report downregulation of enzyme expression when metastasis appears, and a correlation between enzyme expression and regional lymph-node involvement in colon cancer. This finding may improve our understanding of metastasis and lead to new and more efficient therapies against cancer.