Anthony E. Reeve

We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential.Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases.Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT-PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse.Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P < 0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.

Wilms tumors with WT1 mutations [WT1(−)] have a stromal-predominant histology with varying extents of rhabdomyogenesis. These tumors also frequently have mutations in the beta-catenin gene (CTNNB1). We have investigated the molecular events that may explain the origins of rhabdomyogenesis in WT1(−) tumors. Of 35 Wilms tumors, we identified 12 with WT1 mutations, of which 9 carried CTNNB1 mutations. We compared WT1 wild-type tumors [WT1(+)] with WT1(−) tumors for histological features, localization of beta-catenin, Bcl-2 expression, and apoptosis using an in-situ end-labeling technique. WT1(+) tumors showed triphasic and blastemal- and epithelial predominant-histology. Expression of WT1, beta-catenin, and Bcl-2 recapitulated those of normal kidney epithelial development. Localization of beta-catenin was observed in the cytoplasm and cytoplasmic membrane of early glomerular epithelial structures. Bcl-2 is also expressed in condensing blastema and early glomerular epithelial structures which had little apoptosis. WT1(−) tumors, regardless of whether CTNNB1 mutations were detected or not, showed a stromal-rich phenotype with abundant expression of beta-catenin in the nucleus of the rhabdomyoblasts. Bcl-2 was expressed in rhabdomyoblasts, but not in blastemal cells undergoing apoptosis, suggesting that WT1 regulates Bcl-2 positively in the epithelial pathway, but negatively in the myogenic pathway. These data indicate that mutations in WT1 might alter the Wnt signaling pathway and Bcl-2 related-apoptosis. In WT1(−) tumors, the nuclear accumulation of beta-catenin and Bcl-2 expression are associated with rhabdomyogenesis, and dysregulation of Bcl-2 may be a mechanism by which the histogenesis (loss of blastemal component, muscle differentiation) may be explained.

The progression of a malignant tumour is understood to be the result of the accumulation of multiple genetic aberrations. As up to 14% of organ‐confined renal cell carcinomas will recur after surgery, tumour clones with metastatic potential must already be present in some of these localized tumours. The association of 14q LOH with high‐grade tumours and advanced tumour stage suggests an important role for the gene in tumour progression. Chromosome 14q LOH has been analysed in microdissected specimens from 130 organ‐confined (UICC TNM stage 1 and 2) clear cell renal cell carcinomas using three microsatellite markers (D14S588, D14S617, GATA136B01). Tumours were classified as 14q LOH or not on the basis of LOH at one or more of the markers. The allelic imbalance ratio was used to determine both LOH and LOH proportion and the association between LOH and mortality, tumour size, histological grade and growth kinetics, measured by quantification of nucleolar organizer regions, was analysed. 14q LOH was present in 35.4% of informative cases at marker D14S588, 24.4% at D14S617, 36.4% at GATA136B01 and 39.5% for any one of the three markers. The mean 14q LOH proportion was 0.24 (range 0.009–0.80). LOH proportion correlated significantly with tumour size, AgNOR score and histological grade. It was also significantly associated with disease‐specific mortality; (hazard ratio 1.22; 95% CI 1.02–1.45; p = 0.039). LOH proportion did not remain significant after adjusting for tumour size (hazard ratio 0.98; 95% CI 0.76–1.27; p = 0.90). These results indicate that the proportion of cells with 14q LOH in the tumour is associated with tumour aggressiveness; while this is not an independent predictor of survival, it may have some utility as a marker of latent metastatic potential. Copyright © 2002 John Wiley & Sons, Ltd.

The gene WT1 is required for the normal development and function of the urogenital tract. Constitutional mutations are associated with familial Wilms tumor and syndromes such as Denys‐Drash syndrome (DDS) characterized by nephropathy, genital anomalies and often a predisposition to Wilms tumor. We report a case of constitutional WT1 mutation in an XX female with multifocal Wilms tumor but no genital anomalies or renal dysfunction and, for the first time, review patients previously reported with this germline mutation. The mutation (1084C>T) changes the amino acid arginine at position 362 to the translation stop codon TGA (R362X) resulting in a predicted truncated protein lacking three of the four zinc finger domains necessary for correct functioning of the gene. This constitutional mutation has been reported to cause a variety of phenotypes in eleven different patients, including the classical Denys‐Drash phenotype of diffuse mesangial sclerosis which leads to early renal failure, genital anomalies in XY individuals and Wilms tumors. The absence of mesangial sclerosis and renal failure in our patient excludes DDS. Our case differs from those previously described as the normal kidney tissue shows some small subcapsular glomeruli indicating that the WT1 mutation has impaired nephron development. This patient extends the range and variation of phenotypes that may arise from a specific germline mutation in WT1.