The Role of Epigenetics in Circulating Tumour Cells – What is Its Clinical Utilities?

Emily Le:
Hi everyone, I'm Emily Le from the Molecular Diagnostics Europe event. I'm really pleased to have the opportunity to speak with Evi Lianidou professor of Analytical Chemistry, Clinical Chemistry, in the Analysis of Circulating Tumor Cells lab, Department of Chemistry from University of Athens about epigenetic alterations in CTCs and corresponding ctDNA. She will be giving a talk in the molecular characterization of CTC session at the Sixth Annual Molecular Diagnostics Europe Conference on Wednesday, May 23 in Lisbon.

Evi, thank you for joining us.

Evi Lianidou:
Thank you too. It's my pleasure.

Emily Le:
Can you outline what you consider to be some of the greatest obstacles in Circulating Tumor Cell Analysis?

Evi Lianidou:
For me, the main obstacles in CTC analysis for routine use are the following. Well, first of all, CTCs are circulating in low numbers, especially in early stages of cancer, so this is one analytical challenge. Secondly, they are highly heterogeneous even within the same patient. So there is a limited number of studies that have clearly shown this and that perform at the single cell level. Third, it is not possible to isolate and detect CTCs with exactly the same methodology in all types of cancer. Another obstacle is that CTC methodologies usually require extensive analytical and clinical validation steps before their use in clinical practice and also there are a lot of pre-analytical issues and this applies actually to all liquid biopsy methodologies like evaluation of stability, standardization of transportation of peripheral blood samples prior to analysis. All this has to be standardized and this is the main reason for lack of external quality assurance programs for CTC analysis. This is all excluding one system that is FDA cleared, and that is the CellSearch up to now.

Emily Le:
What is your vision for the role of epigenetics in CTCs for clinical utilities?

Evi Lianidou:
Epigenetic changes are very important generally in cancer development, we already know that, since tumor metastasis suppressor genes can be silenced through DNA methylation of their promoters, and this can affect virtually every step in human progression. During the last years, DNA methylation is gaining ground as a potential biomarker for diagnosis, staging, prognosis, and monitoring of response to therapy. So DNA methylation in combination with liquid biopsy has a great potential to be used as a screening and or as a diagnostic tool in a non-invasive and cost effective way. And especially analysis, we already have FDA clearance assay for DNA methylation in plasma, and this is on Septin 9 promoter, for colorectal cancer. But in circulating tumor cells, this is also very very promising, as it can give important information on the molecular and biological nature of the cells. Epigenetic silencing of tumor and metastasis suppressor genes can be very important because it plays a key role in the survival and regulation of the metastatic potential of these genes. And our group was the first to demonstrate that tumor suppressor and metastasis suppressor genes are epigenetically silenced in CTCs and corresponding ctDNA.

And epigenetic silence is of critical therapeutic targets, especially could also affect treatment efficacy. A nice example on that is epigenetic silencing of estrogen receptors in patients with breast cancer. In our recent study in clinical cancer research, we evaluated for the first time ESR -1 methylation in CTCs, in paired ctDNA in primary tumors of breast cancer patients and we report that estrogen receptor methylation in CTCs, in paired plasma is highly concordant. We also found that estrogen receptor methylation in CTCs was associated with lack of response to everolimus/exemestane treatments and we believe that this epigenetic biomarker should be further evaluated as a potential liquid-based biomarker for the follow up of breast cancer patients treated with everolimus/exemestane combinations.

Emily Le:
So up unto now, we still have not completely understood the complex biology of CTCs and what it means to the patient when CTCs are detected. However, we continue to see more and more emerging technologies out there to detect CTCs. Can you comment on that?

Evi Lianidou:
I would say that these are actually two different questions and concerning your first question, what it means to the patient when CTCs are detected, I would like to say that nowadays the detection and molecular characterization of CTCs is one of the most active and hot areas of liquid biopsy research. There is considerable interest in the development of reliable robust assays for CTC analysis and molecular characterization. And this is important since these are well defined targets for understanding tumor cell dissemination. The clinical importance of CTCs enumeration in metastatic breast, colorectal and prostate cancer has already been shown and is FDA cleared more than a decade ago. But simple enumeration of CTCs is not enough to take full advantage of this important source of biomarkers. So many different groups so far, even by using completely different experimental approaches have clearly shown that understanding CTCs biology could be a key issue in favor of cancer patients. And there are a lot of biomarkers that can only be checked in CTCs. I would like to give three important examples.

First of all, in early breast cancer patients, the expression of HER-2 in CTCs of patients which have HER-2 negative breast cancer when combined with trastuzumab and it has been shown that this could lead to a better progression free survival and this is now evaluated in clinical trials. This is one example in breast cancer.

In castration-resistant metastatic prostate cancer, it has already been clearly shown that the expression of the Androgen receptors splice variants and especially AR-V7 in CTCs is related to response to Abiraterone Enzalutamide treatment.

And the third is, as an example, that in many types of cancer, the expression of PD-L1 in CTCs could be an indication for immunotherapy. And we have also recently shown that in head and neck cancer, this is important since this is indicating a worse prognosis for patients with PD-L1 positive CTCs.

So in conclusion, molecular characterization of CTCs can give a lot of information and an early indication of the response of patients to specific treatments.

Now, concerning your second question on what we continue to see more and more emerging technologies out there to detect the CTCs. This is important. There are a lot of liquid biopsy technologies, and the main question is whether CTCs and ctDNA composition is representative of the patient's tumor, and in most studies so far, this is based on the isolation of these cells through epithelial properties and most likely EpCAM or cytokeratin expression.

However, it is now known that EMT or epithelial mesenchymal transition can play a severe role in the isolation of CTCs through EpCAM because in some cases some cells can be missed when only EpCAM-based isolation protocols are used. For this reason, there are a lot of alternative technologies nowadays, and these technologies are mainly based on size and filtration, microfluidics and use of different antibodies. And all these methodologies need extensive validation in different types of cancer before being applied in clinical practice. It is important to use the correct isolation system in each type of cancer before moving down to molecular characterization of these cells. And towards this direction, the European Consortium CANCER ID that is coordinated by Professor Klaus Pantel in Hamburg and Leon Terstappen in the Netherlands together with many academic centers, including ours and many pharmaceutical and diagnostic companies is working very hard.

Emily Le:
What are some of the most exciting new studies and emerging technologies that you have seen recently in CTC field?

Evi Lianidou:
So, the CTC field is very active and thus it is very hard for me to fully follow in terms of publications, but the number of publications in this field is rising exponentially during the last years, and there are numerous exciting new studies that come up, and these are really hard to select and name them one by one. In general, immunological, molecular, and functional assays have already revealed a high heterogeneity in CTCs, and they offer a promising approach for therapeutic monitoring and discovery of new therapeutic targets.

I would point out that some issues like single cell analysis of CTCs is an important area now since it has revealed a degree of heterogeneity within each individual patient and across different patients. So studying the biology of CTCs at the single cell level could give important information on the metastatic process and respond to specific therapies. A nice example was a recent connection of early detection of ESR1 mutations in single CTCs of patients with breast cancer with the emergence of endocrine resistance. So, these findings could have a severe impact on the choice of further therapy.

Another issue is studying of CTC clusters. It is also very interesting since these clusters may present one of the key mechanisms indicating the metastatic process. There are a lot of information on these clusters and it seems that understanding the biology of these clusters is critical to assess the unified scheme employed by cancer and to devise strategies to overcome key pathways responsible for these for the improvement of metastatic potential of these clusters.

One more issue could be that single CTC analysis or CTC analysis combined with the NGS technologies is very important because we can elucidate further the association between CTCs and ctDNA and also isolation of viable CTCs and ex-vivo cultures of them is also important, because in this way we can get a very important information on the metastatic process.

Emily Le:
So, what advice do you have for other scientists in this field to move liquid biopsy forward?

Evi Lianidou:
Yes. There are many people entering the field now and they find this fascinating. This is great, and I would suggest first of all to use before analyzing clinical samples, that each group should analytically validate the protocols and the methodologies that are going to be used in clinical samples in terms of sensitivity and specificity and use quality control steps and quality control in each step used in this analysis.

Second, to use state-of-the-art instrumentation and technologies and follow intergrated liquid biopsy approaches and that means to perform analysis at the same time, not only in CTCs, but in circulating tumor DNA as well and possibly in exosomes or extracellular vesicles isolated from the same patient, and perform analysis at the DNA mutation, gene expression level, epigenetic level. So a comprehensive and all-inclusive information for each patient can be actually combined and this is very important to reveal mechanism that leads to resistance to specific therapies, let's say.

Third, liquid biopsy is very promising for the early detection of cancer. You can use liquid biopsy not only for detection of mechanisms that are indicative of resistance in the metastatic setting, but also for early detection. And there are also some important papers, especially some indications in the field of circulating tumor DNA. And I think that there's a lot of work to be done towards this direction and it's very promising.

And the fourth suggestion is that beyond blood, I will say that other body fluids can be analyzed in a minimally invasive way, not only peripheral blood, not for CTCs, but also for ctDNA like saliva, urine, and this is also a potential source of liquid biopsy biomarkers. For me this area deserves also a lot of attention.

Emily Le:
And lastly, what are you looking forward to the most at this Molecular Diagnostic Europe Conference in Lisbon this May?

Evi Lianidou:
I think that you have put together a very nice panel of speakers, very interesting topics, so I look forward to this meeting and I strongly believe that there will be a lot of fruitful discussions and a lot of collaborations will come up at this Congress.

Emily Le:
Evi, thank you so much for your time and insights today.

Evi Lianidou:
My pleasure. Thank you.

Emily Le:
That was Evi Lianidou, professor of Analytical Chemistry, Clinical Chemistry in the Analysis of Circulating Tumors Cells Lab from University of Athens. She will be speaking in the Circulating Tumor Cells track at the Molecular Diagnostic Europe Conference this May in Lisbon. If you would like to hear her in person, go to moleculardxeurope.com for registration information and enter the key code "Podcast." I'm Emily Le. Thank you for listening.