The use of a simple blood test in clinical oncology for the detection of sporadic cancer is perceived as a possible goal in the near future. Improvements in molecular and genetic approaches have permitted the identification of tumour derived nucleic acids circulating in the plasma of cancer patients.7–18
The origin and release mechanisms of these nucleic acids into plasma, and the protection of DNases and RNases action, are unknown. The most common hypothesis is that they come from lysis of circulating cancer cells or micrometastases shed by the tumour as well as from tumour necrosis, apoptosis, and active release.29
Additionally, nucleic acids have been found on the surface of cancer cells30,31
and they could be shed within vesicles bound to protein or phospholipid, thus being protected against action.32,33
While circulating tumour derived DNA has been widely studied in the majority of cancer types, current knowledge of the second nucleic acid RNA is scarce. Only three experiences of mRNA detection in cancer patients and controls have been reported.23,24,34
The study by Lo and colleagues34
detected Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinomas. However, even though it is a good example of specific mRNA expression in a malignancy, the use of RNA of virus tumour associated as a positive detection marker is restricted to those uncommon tumours with a demonstrated relation to a virus, such as nasopharyngeal or cervical carcinomas. The two other original works reported detection of specific mRNA from tumour tissues in the RNA of plasma in patients with metastatic melanoma and breast carcinoma.23,24
These studies, similar to those that investigated circulating tumour cells in minimal residual disease,19–22
demonstrated that it is possible to find free tumour mRNA in the plasma of patients with cancer, which indicates that other tumours could also be studied if suitable mRNA markers were available.
The present series analysed patients with colon cancer at several stages, and also attempted to identify cases with circulating tumour mRNA using two specific epithelial markers. Our results offer evidence for mRNA of tumour origin in the plasma of colon cancer patients, with a consistent statistically significant difference compared with normal controls. Additionally, when we examined the correlation between the presence of epithelial mRNA in the plasma of these patients and their clinicopathological characteristics, we found that advanced pathological stage was significantly prevalent in cases with one or both positive epithelial markers, and vascular invasion and lymph node metastases were close to being significant. No data on clinicopathological and molecular correlations are available.
However, based on these results and previously reported findings regarding plasma tumour mRNA in metastatic melanoma,23
we hypothesise that, at least in colon cancer patients, the presence of circulating tumour mRNA could be a prognostic factor linked to micrometastases in blood. When this possibility was examined, we observed that 92% of patients showing circulating epithelial tumour cells displayed the same positive RNA markers in plasma. This is the first study to demonstrate the close relationship between circulating tumour cells and circulating tumour RNA. Additionally, two patients with no evidence of disseminated disease had a positive epithelial marker in plasma. This may be explained by minimal illegitimate levels of CK19 mRNA expression in mononuclear blood cells35
and increased levels of CEA in almost 30% of smokers.36
In the same way, we found that 4% and 20% of controls had mRNA of CEA and CK19 in plasma, respectively. Alternatively, it could be that the enrichment in nucleic acids from tumour improves the detection capability, making the plasma study method more sensitive than others in the detection of disseminated disease.
Additionally, we would like to point out that the rate of identification of circulating tumour cells in our patients was higher than expected, but several facts may explain these results: (a) we used a circulating tumour cell enrichment protocol previous to RNA extraction, (b) two markers were used in a very sensitive nested PCR, and (c) the subset of 13 patients had advanced stage of disease (eight with stage C and five with stage B2).
In conclusion, specific determination of mRNA of epithelial origin in the plasma of patients with colon cancer is a reality. Moreover, a positive correlation with pathological parameters compatible with more aggressive tumours and circulating tumour cells was observed in patients with tumour mRNA present in plasma. Further studies in other malignancies are needed to address the potential of this new molecular marker in situations such as cancer diagnosis, monitoring the outcome of disease, and eventually as a treatment response indicator.