Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5′-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies. © 1999 Cancer Research Campaig

A model for co-expression pattern analysis of genes implicated in angiogenesis and tumour cell invasion in cervical cancer

To date, numerous genes have been identified which are involved in both tumour neovascularisation (angiogenesis) and tumour cell invasion, and most of them are also expressed to some extent under normal physiological conditions. However, little is known about how these genes co-express in these settings. This study was undertaken to quantitate mRNA levels in normal and malignant cervical tissues of nine selected genes (VEGF121, VEGF165, VEGF189, VEGF-C, eIF-4E, b-FGF, TSP-2, MMP-2 and MMP-9) implicated in the above processes using real-time quantitative RT–PCR. In addition, the Spearman's rank correlation was used to determine their co-expression patterns. The transcript levels for the different VEGF-A splice variants (VEGF121, VEGF165, VEGF189) were at least 10-fold higher in the cancer cases, with the highest levels in the primary tumours demonstrating lympho-vascular space involvement. The lymphangiogenic factor VEGF-C and MMP-9 were upregulated 130- and 80-fold respectively in cervical cancers. The highest levels of VEGF-C mRNA were found in the lymph-node positive group. The transcript levels for b-FGF were similar in normal cervical tissue and early-stage cervical cancer, however, higher levels were found in the cervical cancers with advanced stage disease. Comparing gene transcript levels between recurrent and non-recurrent cervical cancer patients revealed significant differences (P=0.038) in transcript levels for the angiogenesis inhibitor TSP-2, with the highest levels in non-recurrent cases. Co-expression pattern analysis in normal cervical tissue revealed highly significant co-expressions (P<0.0001) between TSP-2 and most other genes analysed (VEGF121, VEGF165, VEGF-C, b-FGF and MMP-2). In cervical cancer, TSP-2 appears only to be highly co-expressed with MMP-2 (P<0.0001). In contrast to normal cervical tissue, we found a highly significant co-expression (P<0.0001) between MMP-9 and VEGF189 in cervical cancer. The combined application of real-time quantitative RT–PCR and Spearman's rank correlation identifies gene transcripts which are simultaneously co-expressed. Our results revealed a significant co-expression between the angiogenesis inhibitor TSP-2 and most other genes analysed in normal cervical tissue. In cervical cancer, we found a strong upregulation of VEGF-C and MMP-9 mRNA, with a highly significant co-expression between MMP-9 and VEGF189