During the past 20 years, the interest in circulating tumor cells (CTCs) has increased dramatically due to their potential as circulating biomarkers in monitoring cancer progression and treatment. However, selecting and detecting CTCs from whole blood of cancer patients in the background of normal cells is very challenging.
Since last summer, Dr. Siegfried Hauch, Director of Liquid Biopsy Site Management at QIAGEN, has presented several webinars to discuss the challenges in detection and analysis of CTCs. He has also introduced QIAGEN’s solution – the AdnaTests. During Dr. Hauch’s presentations, we have received a number of frequently asked questions. Below are the top 10 most frequently asked questions, followed by Dr. Hauch’s responses.
- 1. How early (at which stage) in cancer development is it possible to detect CTCs in patients’ blood?
CTCs are detected independent of staging. Small tumors with N0 (no nodal involvement) were found to have the same positivity rate as larger ones with nodal involvement.
2. How sensitive is the AdnaTest?
It depends on how we define sensitivity. In terms of spike recovery, we recover 2 spiked cells in 95% of the cases. In terms of clinical sensitivity, it depends on the cancer type and the stadium of the disease (primary vs. metastatic; treated vs. naïve). In chemo-naïve primary breast cancer before surgery, the positivity is about 20-30% – but up to 75% in metastatic disease under progression.
3. Can the AdnaTest system detect 1 CTC in 1ml blood?
Yes. The stated detection limit of the system is 2 cells in 5 ml of blood.
4. How can you rule out false negative results?
This cannot be ruled out completely. However, in spiking/recovery experiments using a variety of cell lines, we observe good functionality so far. Furthermore, we confirmed in trials the clinical sensitivity to be better than only EPCAM-based methods.
5. Can you discriminate between metastatic and non-metastatic patients?
There are differences, indeed – especially if we use a larger set of patient samples to profile CTCs using the AdnaTests. Different profiles have been seen at different time points in the blood samples of metastatic patients. We also found the changes were correlated with drug resistance. You can get more detailed information from the recently published article in Dr. Kasimir-Bauer’s lab. Read more here.
6. Given that there are gene expression changes in CTCs (and in all cells, in general) that start immediately and that occur over time after collection, what is the best way to fix the cells to preserve cell morphology as well as RNA integrity?
This is especially important for receiving blood samples from collaborating centers and when you want to analyze them within 72 hours post blood collection. BD ACDA-Vacutainers, in combination with the AdnaTube (not the AdnaCollect tube), will keep your cells viable and your RNA profile stabilized over 36 hours. Analysis at 72 hours is not possible yet for mRNA profiling.
7. I have tested AdnaGen tubes that are good for keeping cell integrity within 24 hours at 4°C. But if we want to study CTCs using Fluidigm C1 or other microfluidic systems, we need to keep cell morphology intact for 72 hours. Since we cannot lyse cells and keep RNA, what is the best solution in this regard?
There is no solution currently available to keep the cells viable for 72 hours. You can stabilize morphology over that time using Streck tubes or such – but in that case, the analysis of RNA profiles is not possible anymore. BD ACDA-Vacutainers, in combination with the AdnaTube (not the AdnaCollect tube), will keep your cells viable and your RNA profile stabilized over 36 hours.
8. Have you seen correlation between CTC and metastatic tumor gene expressions?
Yes, we have seen the correlation between CTC and metastatic tumor gene expressions in the CTCs of metastatic breast cancer analyzed with the AdnaTest BreastCancer. HER2 overexpression correlates with metastatic tissue.
9. In addition to CTC count and disease, how can you perform tumor-specific mutation measurements in CTCs?
Genomic DNA can be isolated from the cell lysate, too, even when the amount is less. In a pilot study, we were able to analyze KRAS codon 12 mutations from cDNA (1). You can download the article for free here.
10. How can we better determine whether the procedure of acquiring CTCs (particularly in breast cancer) influences CTC behavior or the molecular profile at the time of the assay?
Use the recommended stabilization systems in the product handbook. The molecular profile – at least using the markers provided with the kit – is conserved for up to 36 hours after the sample is transported correctly (immediate cooling to 4–8°C after draw).
Have you missed Dr. Hauch’s webinars or do you have any additional questions? Please contact us at firstname.lastname@example.org.
The AdnaTest is an open platform that allows the profiling of additional custom-selected mRNA targets for comprehensive biomarker discovery. To learn more about the AdnaTest system, click here!
The applications presented here are for research use only. Not for use in diagnostic procedures.
1. Raimondi C, Nicolazzo C, Gradilone A, et al. (2014) Circulating tumor cells: Exploring intratumor heterogeneity of colorectal cancer. Cancer Biol Ther.15, 496–503. (Link)