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 (VEGF(121), VEGF(165), VEGF(189), 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 (VEGF(121), VEGF(165), VEGF(189)) 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 (VEGF(121), VEGF(165), 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 VEGF(189) 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 VEGF(189).

Molecular quantification and mapping of lymph-node micrometastases in cervical cancer.

BACKGROUND: A proportion of patients with cancer and lymph nodes negative on histology will develop recurrence. Reverse-transcriptase PCR (RT-PCR) is a highly sensitive method for detection of lymph-node micrometastases, but accurate quantitative assessment has been difficult. METHODS: We studied primary tumours and 156 lymph nodes from 32 patients with cervical cancer (stage IA2, IB1, and IB2) and 32 lymph nodes from nine patients with benign disease. A fully quantitative, real-time RT-PCR assay was used to document absolute copy numbers of the epithelial marker cytokeratin 19. Primers and probe were designed not to amplify either of the two cytokeratin 19 pseudogenes. FINDINGS: All primary tumours and histologically involved lymph nodes (six) had more than 106 copies of cytokeratin 19 mRNA per microg total RNA. Expression of cytokeratin 19 (up to 1.1 x 10(5) copies per microg RNA) was detected in 66 (44%) of 150 histologically uninvolved lymph nodes, and in nodes from 16 of 32 patients with cervical cancer. 15 of these 16 patients with evidence of micrometastases had the highest cytokeratin 19 transcription level in a first lymph-node drainage station (three obturator, six internal, and six external iliac node). Transcription of cytokeratin 19 was found at a low level in just one of 32 lymph nodes obtained from nine patients with benign disease. Median copy number of cytokeratin 19 transcription was significantly higher (>10(3) copies) in association with adverse prognostic features. INTERPRETATION: The results suggest that about 50% of early-stage cervical cancers shed tumour cells to the pelvic lymph nodes. The amount of cytokeratin 19 expression was related to clinicopathological features. Further studies are required to document the clinical implications of molecular micrometastases.

Elevated levels of RanBP7 mRNA in colorectal carcinoma are associated with increased proliferation and are similar to the transcription pattern of the proto-oncogene c-myc.

We have used suppression subtractive hybridisation, "in silico" cloning and reverse Northern dot blot analysis to identify significant up-regulation of RanBP7 transcription in a human colorectal carcinoma. Quantitative RT-PCR analyses using the Taqman system demonstrated that RanBP7 mRNA levels were elevated in 47/75 colorectal tumours. There was no significant difference in 17 matched normal and tumour pairs and reduced levels in 11. Since RanBP7 specifies a key member of nuclear transport receptors responsible for the nuclear import of histone H1 and ribosomal proteins, we investigated whether this up-regulation might be proliferation-associated. RanBP7 mRNA copy numbers were significantly correlated with those of proliferating cell nuclear antigen in both normal and cancer tissue. Interestingly, the transcription pattern of the proto-oncogene c-myc showed a similar correlation with PCNA mRNA. Our results highlight the need for the careful interpretation of quantitative data that compare mRNA levels in normal and cancer tissue.

Transcription of the inositol polyphosphate 1-phosphatase gene (INPP1) is upregulated in human colorectal cancer.

We have used suppression subtractive hybridization to demonstrate significant overexpression of the inositol polyphosphate 1-phosphatase gene (INPP1) in colorectal cancer compared with matched normal colon epithelium. Its gene product catalyses the hydrolysis of inositol 1,3,4-trisphosphate and inositol 1, 4-bisphosphate, a key molecule in the phosphoinositide metabolic and signaling pathways. Following confirmation of the differential expression by reverse Northern dot blot analysis, fully quantitative Taqman reverse transcriptase-polymerase chain reaction assays showed that its transcription was upregulated in 42/49 colorectal tumors. There was no significant difference in four tumors and reduced transcription was observed in three. This is the first study to report the upregulation of the INPP1 gene in a human cancer and should facilitate further studies looking at the role of phosphatidylinositol signaling reactions in human colorectal cancer.

Cytokeratin 20 is not a tissue-specific marker for the detection of malignant epithelial cells in the blood of colorectal cancer patients.

BACKGROUND: The clinical benefit of using reverse transcription polymerase chain reaction (RT-PCR)-based assays to detect circulating tumour cells during post-operative surveillance of cancer patients remains unclear. Cytokeratin 20 has been proposed as a tissue-specific marker for the detection of micrometastases in the blood of colorectal cancer patients. However, recent reports have challenged its specificity, and hence the validity of its use. AIMS: The aim of this study was to evaluate the tissue-specificity of ck20 mRNA transcription and its use for detecting circulating colon epithelial cells. PATIENTS AND METHODS: RNA was isolated from the peripheral blood of 51 colorectal cancer patients, four patients with benign gastrointestinal disease and 42 healthy controls. In addition, it was prepared from 32 colorectal cancers, from a pituitary cancer, from normal kidney, liver, fibroblasts, keratinocytes and from 24 lymph nodes obtained from eight patients with benign gastrointestinal disease. Real-time RT-PCR assays were used to quantitative and compare ck20 transcription. RESULTS: Significant levels of ck20 mRNA were detected in all 42 blood samples from healthy volunteers and in all pre- and post-operative blood samples from colorectal cancer patients regardless of the presence of metastatic disease. It was also detected in all other mRNA samples analysed. CONCLUSION: The lack of colon tissue-specificity renders ck20 useless as a marker for the post-operative surveillance of colorectal cancer patients.

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.