To the best of our knowledge, here we report the first study using mucin 1- and EpCAM-based immunomagnetic enrichment, followed by real-time RT-PCR analysis of KRT19, MUC1, EPCAM, CEACAM5 and BIRC5, as a way to detect and evaluate the prognostic and predictive effect of CTCs in PB of colorectal cancer patients.
As treatment has become more effective for colorectal cancer, decision making has also become more complicated. Five classes of drugs are currently available for treatment and therefore selection and monitoring of therapy have become more difficult
. Standard practise is to change treatment after several weeks or months of therapy if there is evidence of progression. However, after initiation of systemic treatment, current methodologies do not often allow for an accurate and early assessment of clinical benefit. Thus, patients may be either treated for prolonged periods with an inactive therapy or a potentially active therapy may be discontinued prematurely
. Therefore, we have evaluated whether CTC status, before and during treatment, are indicative of treatment benefit in advance of radiographic changes.
In the present study, we report the results of an in-house immunomagnetic/real-time RT-PCR assay for the detection and characterization of CTCs in colorectal cancer patients. Assay development and validation (addressing typical technical concerns, such as specificity, contaminants, efficiency, sensitivity and sample quality), are extensively explained elsewhere
[7–9]. By applying this methodology, our results show a correlation between CTC assessments and radiographic determinations of disease progression in patients receiving chemotherapy for metastatic colorectal cancer. This correlation applies not only for CTC results obtained during the treatment, but also to CTC assessments obtained as far in advance as 6 months before imaging. Moreover, fluctuations in CTC levels during the course of treatment were also associated with tumor responsiveness determined by radiographic imaging. Patients with an increase in the number of positive markers, during the course of therapy, had 20.57 times the odds of radiographic disease progression compared with patients who had no CTCs or a decrease in CTC positive markers. Therefore, our findings suggest that serial CTC assessments taken before and during the first 3 months of treatment, used in conjunction with imaging, could help to confirm evidence of tumor response or decide doubtful findings.
Analysis of CTCs in blood of cancer patients can be performed by several rare cell detection techniques and promising results with potential clinical relevance have been obtained. One of the largest published studies on CTC detection in metastatic colorectal cancer, involving 430 patients, was performed by Cohen et al.
. Patients had their blood collected before treatment and at four different points during treatment schedule and CTCs were detected using the CellSearch® System. Patients were stratified according to favourable (< 3 CTCs per 7.5 ml of blood) or unfavourable (≥ 3 CTCs per 7.5 ml of blood) CTCs counts. The study showed that, at all time points, median PFS and OS rates were twice as high for patients in the favourable group as compared to those in the unfavourable group. The study also found a significant prognostic correlation between patient grouping by image response and CTCs, meaning that if patients presented elevated CTCs after therapy, they were more likely to have a worse prognosis. An update of this study, 1 year later and enclosing extended follow-up times, reported a pronounced PFS and OS difference between favourable and unfavourable groups, mostly in patients receiving first-line therapy
Our study, even if using another CTC detection method and a smaller cohort of patients, confirms not only the previous findings by Cohen et al., but gives also extra information about CTC phenotypes. To date, a variety of research methods have been developed to isolate and enumerate CTCs. The existing CTC detection assays rely on various properties of CTCs, with each one having unique advantages and limitations. However, no enrichment or detection method has yet proven to be the golden standard and continuing efforts are needed to improve the reliability of CTC detection techniques. Even if the CellSearch® System has been validated via multicenter studies and is the only FDA cleared device for enumeration of CTCs, its use presents some limitations that may be crucial for elucidating and optimizing the use of CTCs in cancer management. By simply enumerating tumor cells, the CellSearch® System is missing information regarding the enormous biological and clinical contributions that CTCs can provide
. Therefore the future of rare tumor cell analysis relies also in the development of sensitive and relatively inexpensive assays capable of generating CTC molecular profiles, which may allow for the identification of subgroups of patients who would benefit from a specific therapy.
Nevertheless, limitations of this work must be considered. The study population was relatively small which may influence the interpretation of the results. However, small well-designed studies are of great value once that they can provide results quickly and becoming part of a preliminary selection in order to further design larger confirmatory studies. Patients also had flexibility regarding the exact dates of blood draws and computed tomography scans. However, the time frames for data analysis were well defined and this flexibility reflects the everyday in clinical practice.