Implication de MEK1 et MEK2 dans l'initiation et la progression du cancer colorectal

Une dérégulation de la voie de signalisation Ras/Raf/MEK/ERK1/2 est observée dans plus de 30% des cancers et des mutations activatrices de RAS sont observées dans 30% à 50% des adénomes colorectaux. À la suite d’une analyse extensive de biopsies de tumeurs colorectales humaines par micromatrices tissulaires (TMA), nous avons observé que 44% des tissus cancéreux exprimaient MEK1/2 phosphorylés, contre 10% des tissus normaux. L'analyse des TMA a également révélé que 79% des tumeurs arboraient un marquage nucléaire de MEK1/2 phosphorylés, contre 4 % pour les tissus normaux. Bien que la voie MEK/ERK1/2 soit fréquemment activée dans les cancers, le rôle précis des isoformes de MEK1 et de MEK2 n'a jamais été clairement établie. De même, l'impact de cette localisation nucléaire aberrante de phospho-MEK1/2, dans l'initiation et la progression des cancers colorectaux, est inconnu. Lors d'un premier projet, nous avons démontré, que l’expression de MEK1 ou MEK2 activé est suffisante pour transformer in vitro des cellules intestinales épithéliales de rat (IEC-6). L'expression des mutants actifs de MEK1 ou MEK2 est suffisante pour induire une dérégulation de la prolifération cellulaire et engendrer la formation d'adénocarcinomes invasifs dans un modèle de greffe orthotopique du côlon chez la souris. Nous avons également démontré que l'inhibition de MEK2 par shRNA supprime complètement la prolifération des lignées humaines de cancer du côlon, alors que la suppression de MEK1 a peu d'effet sur la capacité de prolifération. Le deuxième projet, nous a permis d'observer que l'expression d'un mutant nucléaire de MEK1 dans les cellules IEC-6 transforme drastiquement les cellules. Une augmentation de prolifération, une résistance à l'anoikose, un dérèglement du cycle cellulaire, de l'instabilité chromosomique (CIN), de la tétra/aneuploïdie sont observés. La caractérisation des mécanismes responsables de cette localisation aberrante de MEK1/2 phosphorylés, a permis d'identifier la protéine Sef, un régulateur de la localisation cytoplasmique de MEK/ERK1/2. Nous avons démontré que l'expression d'une forme oncogénique de Ras (H-RasV12) inhibe l'expression de Sef, engendrant alors une accumulation nucléaire de MEK1/2 activés. Plus encore, la réexpression de Sef restaure la localisation cytoplasmique de MEK1/2 et renverse les propriétés tumorigéniques ainsi que l'aneuploïdie induite par Ras activé. Un troisième projet, visant la caractérisation des mécanismes associés à la CIN et à l'aneuploïde engendrés par l'activation aberrante de la voie de Ras-ERK1/2, a permis d'observer que l'hyperactivation de ERK1/2 induit des anomalies mitotiques menant à la binucléation. Une localisation erronée et une surexpression de la kinase Aurora A, de même que des protéines de passage du complexe chromosomique (CPC), Aurora B, Survivine et INCENP, sont observées. L'inhibition partielle de l'activation de ERK1/2 par de faible dose de PD184352, un inhibiteur de MEK1/2, est suffisante pour renverser la surexpression de ces régulateurs mitotiques, de même que corriger les anomalies de la mitose et réduire la tétra/aneuploïdie engendrée par Ras oncogénique. Ainsi, nous avons démontré, pour la première fois, que la voie des MAP kinases ERK1/2 est impliquée dans la CIN, la tétraploïdie et l'aneuploïdie. Nos résultats suggèrent que la perte de Sef est un événement oncogénique précoce, qui contribue à la localisation nucléaire aberrante de MEK1/2 qui est observée dans les tumeurs colorectales. Cette localisation anormale de MEK1/2 est associée à l'initiation de la transformation, la progression tumorale et la CIN, via l'activité soutenue de ERK1/2. Ces informations sont capitales et démontrent l’importance de la voie de signalisation Ras/Raf/MEK/ERK1/2 dans le processus de tumorigénèse colorectale.

Proposal for a 10-high-power-fields scoring method for the assessment of tumor budding in colorectal cancer

Although tumor budding is linked to adverse prognosis in colorectal cancer, it remains largely unreported in daily diagnostic work due to the absence of a standardized scoring method. Our aim was to assess the inter-observer agreement of a novel 10-high-power-fields method for assessment of tumor budding at the invasive front and to confirm the prognostic value of tumor budding in our setting of colorectal cancers. Whole tissue sections of 215 colorectal cancers with full clinico-pathological and follow-up information were stained with cytokeratin AE1/AE3 antibody. Presence of buds was scored across 10-high-power fields at the invasive front by two pathologists and two additional observers were asked to score 50 cases of tumor budding randomly selected from the larger cohort. The measurements were correlated to the patient and tumor characteristics. Inter-observer agreement and correlation between observers' scores were excellent (P<0.0001; intraclass correlation coefficient=0.96). A test subgroup of 65 patients (30%) was used to define a valid cutoff score for high-grade tumor budding and the remaining 70% of the patients were entered into the analysis. High-grade budding was defined as an average of ≥10 buds across 10-high-power fields. High-grade budding was associated with a higher tumor grade (P<0.0001), higher TNM stage (P=0.0003), vascular invasion (P<0.0001), infiltrating tumor border configuration (P<0.0001) and reduced survival (P<0.0001). Multivariate analysis confirmed its independent prognostic effect (P=0.007) when adjusting for TNM stage and adjuvant therapy. Using 10-high-power fields for evaluating tumor budding has independent prognostic value and shows excellent inter-observer agreement. Like the BRE and Gleason scores in breast and prostate cancers, respectively, tumor budding could be a basis for a prognostic score in colorectal cancer.

Characterization of CDKN2A(p16) methylation and impact in colorectal cancer: systematic analysis using pyrosequencing

Background The aim of this study is to analyse CDKN2A methylation using pyrosequencing on a large cohort of colorectal cancers and corresponding non-neoplastic tissues. In a second step, the effect of methylation on clinical outcome is addressed. Methods Primary colorectal cancers and matched non-neoplastic tissues from 432 patients underwent CDKN2A methylation analysis by pyrosequencing (PyroMarkQ96). Methylation was then related to clinical outcome, microsatellite instability (MSI), and BRAF and KRAS mutation. Different amplification conditions (35 to 50 PCR cycles) using a range of 0-100% methylated DNA were tested. Results Background methylation was at most 10% with ≥35 PCR cycles. Correlation of observed and expected values was high, even at low methylation levels (0.02%, 0.6%, 2%). Accuracy of detection was optimal with 45 PCR cycles. Methylation in normal mucosa ranged from 0 to >90% in some cases. Based on the maximum value of 10% background, positivity was defined as a ≥20% difference in methylation between tumor and normal tissue, which occurred in 87 cases. CDKN2A methylation positivity was associated with MSI (p = 0.025), BRAF mutation (p < 0.0001), higher tumor grade (p < 0.0001), mucinous histology (p = 0.0209) but not with KRAS mutation. CDKN2A methylation had an independent adverse effect (p = 0.0058) on prognosis. Conclusion The non-negligible CDKN2A methylation of normal colorectal mucosa may confound the assessment of tumor-specific hypermethylation, suggesting that corresponding non-neoplastic tissue should be used as a control. CDKN2A methylation is robustly detected by pyrosequencing, even at low levels, suggesting that this unfavorable prognostic biomarker warrants investigation in prospective studies.

Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy

Puppa G, Senore C, Sheahan K, Vieth M, Lugli A, Zlobec I, Pecori S, Wang L M, Langner C, Mitomi H, Nakamura T, Watanabe M, Ueno H, Chasle J, Conley S A, Herlin P, Lauwers G Y & Risio M (2012) Histopathology Diagnostic reproducibility of tumour budding in colorectal cancer: a multicentre, multinational study using virtual microscopy Aims:  Despite the established prognostic relevance of tumour budding in colorectal cancer, the reproducibility of the methods reported for its assessment has not yet been determined, limiting its use and reporting in routine pathology practice. Methods and results:  A morphometric system within telepathology was devised to evaluate the reproducibility of the various methods published for the assessment of tumour budding in colorectal cancer. Five methods were selected to evaluate the diagnostic reproducibility among 10 investigators, using haematoxylin and eosin (H&E) and AE1-3 cytokeratin-immunostained, whole-slide digital scans from 50 pT1-pT4 colorectal cancers. The overall interobserver agreement was fair for all methods, and increased to moderate for pT1 cancers. The intraobserver agreement was also fair for all methods and moderate for pT1 cancers. Agreement was dependent on the participants' experience with tumour budding reporting and performance time. Cytokeratin immunohistochemistry detected a higher percentage of tumour budding-positive cases with all methods compared to H&E-stained slides, but did not influence agreement levels. Conclusions:  An overall fair level of diagnostic agreement for tumour budding in colorectal cancer was demonstrated, which was significantly higher in early cancer and among experienced gastrointestinal pathologists. Cytokeratin immunostaining facilitated detection of budding cancer cells, but did not result in improved interobserver agreement.

Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the "serrated pathway" characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases. Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out. Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34-0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and - deficient cancers was 87% [OR (95% CI): 0.96 (0.95-0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases. Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events "upstream" of the development of microsatellite instability may impact Cdx2 expression.

Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer

Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

Novel biomarkers for the prediction of metastasis in colorectal cancer

INTRODUCTION In patients with metastatic colorectal cancers, multimodal management and the use of biological agents such as monoclonal antibodies have had major positive effects on survival. The ability to predict which patients may be at 'high risk' of distant metastasis could have major implications on patient management. Histomorphological, immunohistochemical or molecular biomarkers are currently being investigated in order to test their potential value as predictors of metastasis. AREAS COVERED Here, the author reviews the clinical and functional data supporting the investigation of three novel promising biomarkers for the prediction of metastasis in patients with colorectal cancer: tumor budding, Raf1 kinase inhibitor protein (RKIP) and metastasis-associated in colon cancer-1 (MACC1). EXPERT OPINION The lifespan of most potential biomarkers is short as evidenced by the rare cases that have successfully made their way into daily practice such as KRAS or microsatellite instability (MSI) status. Although the three biomarkers reviewed herein have the potential to become important predictive biomarkers of metastasis, they have similar hurdles to overcome before they can be implemented into clinical management: standardization and validation in prospective patient cohorts.

Investigation of IL23 (p19, p40) and IL23R highlights nuclear IL23p19 expression as a marker of indolent tumor features and favorable outcome in colorectal cancer patients

Objective: IL23 is involved in chronic inflammation but its role in cancer progression is not fully elucidated. Here we characterize IL23 subunits p40, p19 and IL23 receptor (IL23R) in the normal-adenoma-carcinomametastasis cascade of colorectal cancers and their relationship to clinicopathological and outcome data. Method: Immunohistochemistry for IL23R, IL12p40, IL23 and IL23p19 (monoclonal) was performed on a multi-punch tissue microarray (n=213 patients). Expression differences between normal-adenomas-cancerslymph nodes were evaluated. Correlation with clinicopathological and outcome data was undertaken. Results were validated on an independent cohort (n=341 patients). Results: An increased expression from normal-adenoma-cancer was observed (p<0.0001; all) followed by a marked reduction in lymph nodes (p<0.0001; all). Cytoplasmic and/or membranous staining of all markers was unrelated to outcome. Nuclear IL23p19 staining occurred in 23.1%and was associated with smaller tumor diameter (p=0.0333), early pT (p=0.0213), early TNM (p=0.0186), absence of vascular (p=0.0124) and lymphatic invasion (p=0.01493) and favorable survival (univariate (p=0.014) and multivariable (p=0.0321) analysis). All IL23p19 positive patients were free of distant metastasis (p=0.0146). Survival and metastasis results could be validated in Cohort 2. Conclusion: The presence of nuclear IL23p19 is related to indolent tumor features and favorable outcome supporting a more ‘protective’ role of this protein in colorectal cancer progression

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer.

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

Investigation of IL-23 (p19, p40) and IL-23R identifies nuclear expression of IL-23 p19 as a favorable prognostic factor in colorectal cancer: a retrospective multicenter study of 675 patients.

IL-23 is a heterodimeric cytokine involved in inflammatory diseases; its role in cancer progression is controversial. Here we analyse the expression of IL-23 subunits (p40 and p19) and IL-23R in colorectal cancer with regard to disease progression, clinical-pathological and molecular aspects. Immunohistochemistry for IL-23p19, IL-23p40, IL-23R and CD8 was performed on a multi-punch tissue microarray of 195 colorectal cancers (cohort 1), matched normal tissue, adenoma and lymph node metastases. Results were compared with clinical-pathological features and CD8+ T-cell counts, then validated on two patient cohorts (cohort 2: n=341, cohort 3: n=139). Cytoplasmic/membranous expression of IL-23 (p19 and p40 subunits) and IL-23R, respectively were over-expressed in carcinomas versus adenomas and normal tissues (p<0.0001) but were reduced in lymph node metastases (p<0.0001). Nuclear IL-23p19 expression was observed in 23.1% and was associated with early TNM stage (p=0.0186), absence of venous (p=0.0124) and lymphatic invasion (p=0.01493), favorable survival (p=0.014) and absence of distant metastasis (p=0.0146; specificity: 100%). This unexpected cellular localization was confirmed by cell fractionation. The beneficial effect of nuclear IL-23p19 was restricted to tumours with CD8+ high counts. Results were validated on Cohorts 2/3. This multicenter study underlines the possible CD8(+)--dependency and beneficial effect of nuclear IL-23p19 on overall patient survival.

The tumor border configuration of colorectal cancer as a histomorphological prognostic indicator.

Histomorphological features of colorectal cancers (CRC) represent valuable prognostic indicators for clinical decision making. The invasive margin is a central feature for prognostication shaped by the complex processes governing tumor-host interaction. Assessment of the tumor border can be performed on standard paraffin sections and shows promise for integration into the diagnostic routine of gastrointestinal pathology. In aggressive CRC, an extensive dissection of host tissue is seen with loss of a clear tumor-host interface. This pattern, termed "infiltrative tumor border configuration" has been consistently associated with poor survival outcome and early disease recurrence of CRC-patients. In addition, infiltrative tumor growth is frequently associated with presence of adverse clinicopathological features and molecular alterations related to aggressive tumor behavior including BRAFV600 mutation. In contrast, a well-demarcated "pushing" tumor border is seen frequently in CRC-cases with low risk for nodal and distant metastasis. A pushing border is a feature frequently associated with mismatch-repair deficiency and can be used to identify patients for molecular testing. Consequently, assessment of the tumor border configuration as an additional prognostic factor is recommended by the AJCC/UICC to aid the TNM-classification. To promote the assessment of the tumor border configuration in standard practice, consensus criteria on the defining features and method of assessment need to be developed further and tested for inter-observer reproducibility. The development of a standardized quantitative scoring system may lay the basis for verification of the prognostic associations of the tumor growth pattern in multivariate analyses and clinical trials. This article provides a comprehensive review of the diagnostic features, clinicopathological associations, and molecular alterations associated with the tumor border configuration in early stage and advanced CRC.

Blood and lymphatic vessel invasion in pT1 colorectal cancer: an international concordance study

AIM This study was performed to evaluate the concordance in pathological assessments of blood and lymphatic vessel invasion (BLI) in pT1 colorectal cancers and to assess the effect of diagnostic criterion on consistency in the assessment of BLI. METHODS Forty consecutive patients undergoing surgical resection of pT1 colorectal cancers were entered into this study. H&E-stained, D2-40-stained and elastica-stained slides from the tumours were examined by 18 pathologists from seven countries. The 40 cases were divided into two cohorts with 20 cases each. In cohort 1, pathologists diagnosed BLI using criteria familiar to them; all Japanese pathologists used a criterion of BLI from the Japanese Society for Cancer of the Colon and Rectum (JSCCR). In cohort 2, all pathologists used the JSCCR diagnostic criterion. RESULTS In cohort 1, diagnostic concordance was moderate in the US/Canadian and European pathologists. There were no differences in the consistency compared with results for Japanese pathologists, and no improvement in the diagnostic concordance was found for using the JSCCR criterion. However, in cohort 2, the JSCCR criterion decreased the consistency of BLI diagnosis in the US/Canadian and European pathologists. The level of decreased consistency in the assessment of BLI was different between the US/Canadian and European pathologists. CONCLUSIONS A uniform criterion strongly influences the diagnostic consistency of BLI but may not always improve the concordance. Further study is required to achieve an objective diagnosis of BLI in colorectal cancer. The varying effects of diagnostic criterion on the pathologists from Japan, the USA/Canada and Europe might reflect varied interpretations of the criterion. Internationally accepted criterion should be developed by participants from around the world.

Adenocarcinomas of the upper third of the rectum and the rectosigmoid junction seem to have similar prognosis as colon cancers even without radiotherapy, SAKK 40/78

Purpose To investigate the prognosis of adenocarcinomas of the upper third of the rectum and the rectosigmoid-junction without radiotherapy. Methods Patients from a multicenter randomized controlled trial from 1987–1993 on adjuvant chemotherapy for R0-resected colorectal cancers with stage I–III disease were retrospectively allocated: cancers of the lower two-thirds of the rectum (11 cm or less from anal-verge, Group A, n = 205), of the upper-third of the rectum and rectosigmoid-junction (>11–20 cm from anal-verge, Group B, n = 142), and of the colon (>20 cm from anal-verge, Group C, n = 378). The total mesorectal excision (TME) technique had not been introduced yet. The adjuvant chemotherapy turned out to be ineffective. None of the patients received neoadjuvant or adjuvant radiotherapy. Results The patients had a regular follow-up (median, 8.0 years). The 5-year disease-free survival (DFS) rate was 0.54 (95%CI, 0.47–0.60) in Group A, 0.68 (95%CI, 0.60–0.75) in Group B, and 0.69 (95%CI, 0.64–0.74) in Group C. The 5-year overall survival (OS) rate was 0.64 (95%CI, 0.57–0.71) in Group A, 0.79 (95%CI, 0.71–0.85) in Group B, and 0.77 (95%CI, 0.73–0.81) in Group C. Compared with Group C, patients in Group A had a significantly worse OS (hazard ratio [HR] for death 2.10) and a worse DFS (HR for relapse/death 1.93), while patients in Group B had a similar OS (HR 1.12) and DFS (HR 1.07). Conclusions Adenocarcinomas of the upper third of the rectum and the rectosigmoid-junction seem to have similar prognosis as colon cancers. Even for surgeons not familiar with the TME technique, preoperative radiotherapy may be avoided for most rectosigmoid cancers above 11 cm from anal-verge.

VE1 immunohistochemistry predicts BRAF V600E mutation status and clinical outcome in colorectal cancer

AIM VE1 is a monoclonal antibody detecting mutant BRAFV600E protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. METHODS Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks). RESULTS Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. CONCLUSION VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

Development of colorectal cancer gene therapy

Colorectal cancer is the number two cancer killer in the United States. Although primary colorectal cancer can be resected by surgery, patients often die from metastatic disease. Liver is the most common site of metastasis for colorectal cancer. It is difficult to selectively kill metastatic colon cancer cells without damaging normal liver functions. Thus it becomes a high priority to develop a selective targeting system for the treatment of colorectal cancer liver metastasis. ^ In the current study, a gene therapy strategy that allows a therapeutic gene to selectively destroy metastatic colon cancer cells without affecting normal liver cells is developed. The APC gene is frequently mutated in colorectal cancers. These mutations activate β-catenin responsive promoters. An optimized β-catenin responsive promoter, containing TCF consensus binding sites, was engineered for this study. This TCF promoter was found to express preferentially in APC mutated/β-catenin activated colorectal cancers while maintaining a low expression level in cell lines of liver origin. A recombinant adenoviral vector AdTCF-TK, in which the TCF promoter controls expression of the herpes simplex virus thymidine kinase gene, selectively destroyed colorectal cancer cells in vitro. AdTCF-TK virus and ganciclovir treatment also inhibited the growth of solid tumour derived from the colon cancer cell line DLD-1 in nude mice. In a control experiment, the growth inhibition effect of the same virus was attenuated in a liver cancer cell line. ^ In the present study, a novel method was developed to target therapeutic gene expression to colon cancer cells at reduced liver toxicity to the patients. The same gene therapy design may also be applied to treat tumours carrying mutations in the β-catenin gene, which is a central component of the APC signal transduction pathway. In summary, the principle for a rational design of a cancer specific treatment approach is demonstrated in this study. In the future, mutations in cancer patients will be more easily identified. Using the same principle developed in this study, specific regimen can be designed to treat these patients based on the specific genetic changes found in the tumour. ^