Marked activity of irinotecan and rapamycin combination toward colon cancer cells in vivo and in vitro is mediated through cooperative modulation of the mammalian target of rapamycin/hypoxia-inducible factor-1alpha axis.

PURPOSE: Despite recent progress, colon cancer is often resistant to combination chemotherapy, highlighting the need for development of novel therapeutic approaches. An attractive target is hypoxia-inducible factor-1alpha (HIF-1alpha), a key transcription factor with a pivotal role in tumor cell metabolism. One potential class of therapeutic agents targeting HIF-1alpha are mammalian target of rapamycin inhibitors such as rapamycin. A second class are topoisomerase I inhibitors, such as irinotecan, which are able to inhibit the accumulation of HIF-1alpha. We here investigated whether combination of rapamycin and irinotecan was active in human colon cancer models. EXPERIMENTAL DESIGN: Human metastatic tumors were xenografted in nude mice and treated with low doses of irinotecan alone, rapamycin alone, or combination of both drugs. The cellular effects of irinotecan and rapamycin were further characterized for HT-29 and HCT-116 colon cancer cells in vitro. RESULTS: In contrast to single-agent therapy, xenografted tumors treated with combination of irinotecan and rapamycin showed potent inhibition of the mammalian target of rapamycin/HIF-1alpha axis, which was accompanied by a dramatic reduction in tumor volume. In vitro experiments showed that exposure to low concentrations of the two drugs resulted in massive HT-29 cell death under hypoxic, but not normoxic, conditions, in full agreement with a cytotoxic effect mediated through HIF-1alpha rather than through induction of genotoxic lesions. HCT-116 cells were less sensitive to the combined treatment due to constitutive activation of phosphatidylinositol 3-kinase/Akt and Ras/mitogen-activated protein kinase pathways. CONCLUSION: These results identify HIF-1alpha as a promising target and provide a rationale for clinical trials of low-dose irinotecan and rapamycin combination toward metastatic colon cancer.

IR spectral imaging for histopathological characterization of xenografted human colon carcinomas.

This study aims to develop IR imaging of tumor tissues for generating an automated IR-based histology. Formalin-fixed paraffin-embedded xenografts of human colon carcinomas were analyzed. Chemometric and statistical multivariate treatments of spectral data permitted to probe the intrinsic chemical composition of tissues, directly from paraffinized sections without previous dewaxing. Reconstructed color-coded spectral images revealed a marked tumor heterogeneity. We identified three spectral clusters associated to tumoral tissues, whereas HE staining revealed only a single structure. Nine other clusters were assigned to either necrotic or host tissues. This spectral histology proved to be consistent over multiple passages of the same xenografted tumor confirming that intratumoral heterogeneity was maintained over time. In addition, developing an innovative image analysis, based on the quantification of neighboring pixels, permitted the identification of two main sequences of spectral clusters related to the tissue spatial organization. Molecular attribution of the spectral differences between the tumor clusters revealed differences of transcriptional activity within these tumor tissue subtypes. In conclusion, IR spectral imaging proves to be highly effective both for reproducible tissue subtype recognition and for tumor heterogeneity characterization. This may represent an attractive tool for routine high throughput diagnostic challenges, independent from visual morphology.

Histologic characteristics of non-microsatellite-instable colon adenomas correlate with distinct molecular patterns.

Colon carcinogenesis encompasses the stepwise accumulation of genomic aberrations correlated with the transition of aberrant crypt-adenoma-carcinoma. Recent data have revealed that, in addition to the microsatellite-instable phenotype, the chromosome instability pathway, representing four fifth of the colon carcinoma, could be involved in heterogeneous molecular alterations. Our project was aimed at determining the existence of distinct molecular subtypes in 159 non-microsatellite-instable colon polyps and their correlation with histology and dysplasia, using allelotyping, MGMT promoter gene methylation status, and K-RAS mutation analyses. Allelic imbalance, MGMT methylation, and K-RAS mutations arise in 62%, 39%, and 32% of polyps, respectively. Only 14% of polyps had no alterations. A 2-way hierarchical clustering analysis of the allelic imbalances identified subgroups of polyps according to their allelic imbalance frequency and distribution. Not only tubulovillous adenoma but also high-grade adenomas were correlated with high global allelic imbalance frequency (P = .005 and P = .003), with allelic imbalance at microsatellites targeting chromosomes 1, 6, and 9. In conclusion, the data presented in this study show that a large heterogeneity exists in the molecular patterns of alterations in precancerous colon lesions, favoring different modes of tumor initiation. Therefore, molecular alterations correlated with tubulovillous-type and high-grade dysplasia could represent targets identifying predictive factors of progression.

Evidence for various 20q status using allelotyping, CGH arrays, and quantitative PCR in distal CIN colon cancers.

The genomic aberration profile of chromosome 20q in distal CIN colon carcinomas was analysed using allelotyping and CGH arrays. Allelotyping revealed carcinomas with allelic imbalance along the full long arm, and carcinomas with fully non-aberrant 20q. Oligonucleotide-based CGH showed that among the carcinomas without allelic imbalance, 47% had in fact a gain. In this subgroup, quantitative PCR for the TOPI gene (20q12) confirmed this gain, and fluorescence in situ hybridization showed that the chromosome 20q gain resulted from tetra/polysomy instead of aneusomy. The 20q gain correlated with a high frequency of aberrations, with allelic imbalance at TP53 locus but not at APC locus, and carcinomas with a disomic 20q showed low frequency of genomic aberrations and were significantly associated to mucinous phenotype. The prognostic value of 20q amplification was not demonstrated in this study. These results indicate that on the basis of aberration frequency, chromosome 20q and TP53/APC locus status, distal CIN carcinomas harbor a high degree of genetic heterogeneity suggesting several pathways for carcinogenesis. This study also indicates that allelotyping needs to be carried out with a complementary technique, such as quantitative PCR.