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Promise Of Liquid Biopsies Coming To Light

By Deborah Borfitz 

September 21, 2022 | Ongoing collaboration between the fields of immunotherapy and liquid biopsy has opened vast opportunities to improve the care of cancer patients, including the prevention of full-blown disease or relapse. It was also the focus of several talks at the recent Next Generation Dx Summit, including a keynote on the long-hoped-for promise of liquid biopsy approaches for diagnosing and treating solid tumors aptly delivered by Klaus Pantel, Ph.D., professor of medicine and director and chairman of the Institute of Tumor Biology at University Hospital Hamburg, Eppendorf. 

Predicting response or resistance to immune checkpoint inhibition therapy has been the focus of his group for some time, says Pantel, citing the 30-year quest to apply immunotherapy before patients reach the end stage of their disease when drugs have the least chance of succeeding. One key objective has been to utilize immunotherapeutic drugs as adjuvant therapy, alongside surgery or chemotherapy, to address minimal residual disease (MRD). 

The tools exist to detect the small number of cancer cells left in the body after treatment, he says, and “the molecular presence of micro-metastasis before conventional imaging procedures can do that.” Cancer patients can therefore be targeted for treatment when their disease is still potentially curable, and immunotherapy has enough time to act. 

In December 2020, Nature Milestones put liquid biopsies on the list of key discoveries in cancer over the past 20 years, notes Pantel. These are tests designed to diagnose or analyze tumors using only a fluid sample, typically blood in a search of shed tumor cells and DNA—and, most importantly in later disease stages, their source. 

The primary tumor is the key source initially, but later if metastasis has occurred it might also be the lymph nodes or distant organs, creating a complex mix of circulating tumor cells (CTCs) as well as circulating tumor DNA (ctDNA), microRNA (miRNA), and various proteins, he explains. But if the blood can reveal micro-metastatic deposits, and those cells can be characterized, the information will be critical to the “right use of immunotherapies.” 

Pantel’s hypothesis is that liquid biopsy approaches, be they focused on CTCs, or a tumor-derived molecule being shed into the bloodstream, are complementary and their developers will all be rewarded handsomely once the tests are accepted in clinical practice. The technical challenge is the low blood concentration of CTCs and ctDNA, which is usually below 1% or even 0.1% in patients at the MRD stage. “We need very sensitive methods and have to make sure [they] aren’t operating at the lowest level of detection; otherwise, we get very unstable results,” he says.  

Harmonization Efforts 

The two key complementary analytes are CTCs and ctDNA, says Pantel, although for immunotherapies CTCs have the advantage of looking at the expression of relevant proteins on the cell surface, such as MHC (major histocompatibility complex) or checkpoint inhibitors such as PD-L1. “In my view, immunotherapies offer a great chance for CTC characterization to be integrated into clinical trials.”  

The European Liquid Biopsy Society (ELBS), comprising almost 70 institutions from academia and industry, was established to introduce liquid biopsies into clinical practice, he continues. Among its goals is to ensure liquid biopsy methods get harmonized, which in the context of immunotherapies is not to say that different applications aren’t needed for patients with metastatic disease versus those with MRD. 

ELBS, a founding member of the International Liquid Biopsy Standardization Alliance, co-authored a 2020 white paper on global harmonization efforts (Critical Reviews in Oncology/Hematology, DOI: 10.1016/j.critrevonc.2020.103112) with groups from the U.S. It was an initiative of the Foundation for the National Institutes of Health, Pantel says. 

Technical validation of liquid biopsy assays is ongoing, he says, citing a trio of Clinical Chemistry papers specific to ctDNA (DOI: 10.1373/clinchem.2019.306837), miRNA (DOI: 10.1373/clinchem.2019.303271), and CTCs (DOI: 10.1093/clinchem/hvaa322) that discuss the technical challenges to harmonization. 

Among the working groups of the ELBS are ones for addressing dissemination and education, determining the kinds of clinical studies that are reasonable and the technologies that should be included, validation of technologies and experiments, and how liquid biopsies factor into regulatory and reimbursement schemas, says Pantel. 

Clinical Applications 

Liquid biopsies have four clinical applications, according to Pantel. One is for the diagnosis of cancer, and another early detection of response and follow-up. They are also useful for real-time monitoring of tumor evolution as tumor cells change under the pressure of natural selection and therapy. Finally, liquid biopsies can detect MRD. 

“In many different tumor types there is a clear correlation between the CTC counts at initial diagnosis and unfavorable diagnosis,” Pantel says. Tumors that shed a lot of cells into the circulation of patients have a greater chance to progress and develop metastasis. 

CTCs can be used as “an enrichment tool to study a high-risk population,” he adds. This is reflected in some cancer staging manuals, although not yet in clinical practice.   

Pantel says he is most excited about the use of liquid biopsies to do analysis and monitoring of dynamic changes to tumor burden over time, including micro-metastatic deposits that can grow and establish MRD. “If we can now detect this disease level... this would be a fantastic time point for immunotherapies” that aim to prevent full-blown metastatic relapse. 

The question arose if MRD or micro-metastatic disease could be detected at follow-up, especially if cells were sitting in organs rather than circulating in the blood. The answer is yes, based on a 2018 study of breast cancer patients two years after adjuvant chemotherapy showing the cells were going back into the bloodstream (Nature Reviews Clinical Oncology, DOI: 10.1038/nrclinonc.2017.174).  CTCs were seen in about 20% of patients and contributing to metastatic progression, Pantel points out. 

The same is true for ctDNA, he continues, referencing one paper where it was detected during follow-up blood analysis (Precision Medicine and Imaging, DOI: 10.1158/1078-0432.CCR-18-3663). It bodes well for patient outcomes that ctDNA had a lead time in predicting metastatic relapse of between 258 and 721 days, months shorter than with conventional imaging. 

Pantel says his group has also done an analysis of ctDNA, together with Inivata’s personalized, multi-tumor liquid biopsy assay (RaDaR), to show that ctDNA levels were high in patients with distant metastasis. However, it was “not so good” for detecting local metastasis, suggesting that distant metastasis may be shedding more ctDNA into circulation than recurrence at a nearby location. 

Multiple Markers 

In metastatic patients, liquid biopsy can be used to monitor CTCs and ctDNA concentration to “get an idea about tumor burden changes” as well as characterize those analytes for a read on what type of immunotherapy is indicated, adds Pantel. 

“There are many drugs and druggable mutations, but there are also alterations that are not engraved in the DNA but require the analysis of RNA or proteins, such as variants of the antigen receptor or the PSMA [prostate-specific membrane antigen], HER2, or PD-L1,” he continues. “It is not enough to just analyze the DNA.” 

Among the clinical trials underway at the Institute of Tumor Biology are ones searching for DNA and CTCs in the blood of patients with non-small cell lung cancer (NSCLC) and melanoma, to reduce tumor burden as well as understand the quality of the CTCs, Pantel says. “We look at markers relevant to immunotherapy, such as MHC-1 [major histocompatibility complex class I] expression, PD-L1 expression, and tumor burden on ctDNA.” 

Even the “micro-signatures” (miRNA) in the blood of NSCLC patients has been correlated with the outcome of immunotherapy (Precision Oncology, DOI: 10.1038/s41698-022-00262-y), says Pantel. 

He expects that “a composite biomarker will win in the end,” and references collaborative work with 2018 Nobel Prize winner Tasuku Honjo, a Japanese physician-scientist and immunologist best known for his identification of programmed cell death protein 1. 

Circulating cell-free (cf) miRNAs is also of interest to Pantel and his team, he adds, since they have many roles affecting immunology and immunotherapy, are easily located, and encased in exosomes that protect them from free-floating protein. 

One currently ongoing study is looking at MRD detection in melanoma patients undergoing immune checkpoint inhibition therapy, he shares. Participants include 110 stage 3 patients on adjuvant therapy and 120 stage 4 (metastasis) patients. It is producing a “dense collection of blood samples” so investigators can see which changes are related to clinical outcomes (pending relapse) versus those that are incidental. Much less information comes from studies looking for changes at the three-, six-, and 12-month time points, which tend to be a “bit erratic.”  

Notably, his study looks at the tumor signature as well as immune cells since both components could be important in predicting outcomes from immunotherapy treatment, he adds.  

When tumor markers are being used to detect melanoma cells, “exclusion markers” can ensure normal cells aren’t inadvertently being picked up, Pantel says. “We do whole genome identification of the DNA and then we can detect mutations relevant to therapy, for example ARAF mutations,” so CTCs provide both protein and genomic information. “We can then look at intra-patient heterogeneity... [and] that’s not so easy to do by ctDNA.”  

Cyr61 Protein 

In collaboration with Olink, Pantel’s group is now analyzing more than 1,500 proteins from a few hundred microliters of blood, he reports. “If we can do that in patients who are responders [versus] non-responders over time, we may get some signatures of proteins which are related to outcome,” he says. 

One identified circulating protein, Cyr61, has been validated for both residual disease detection and early detection of tumors in patients with breast cancer (Clinical Chemistry, DOI: 10.1093/clinchem/hvab153). Higher levels of the protein were seen in carcinomas, even in patients with ductal carcinoma in situ (the earliest form of the disease) compared to healthy—and, importantly, age-matched—controls. 

The role of Cyr61 as a biomarker across cancer types was discussed during the Q&A at the end of Patel’s keynote. In addition to breast cancer, Pantel has experimented with its use in lung, prostate, and asbestos cancers. 

Interestingly, sex differences were seen in lung cancer patients in terms of Cyr61 levels for unknown reasons, he says. And in prostate cancer patients, higher levels were seen in only about 20% of patients. His group is now looking to see if elevated Cyr61 may be indicative of a poorer prognosis.  

Brain Metastases 

To improve the sensitivity of tumor cell detection, Pantel and his team have created a new assay that uses the epithelial cell adhesion molecule (EpCAM) to catch CTCs but also integrates epidermal growth factor receptor (EGFR) and HER3 to enhance the capture rate in patients with lung cancer. The next logical question is where the CTCs come from, he says, which is what clinical collaborators want to know.  

The challenge with brain metastases is that patients shed less ctDNA and CTCs, says Pantel. “[But] if we do single-cell, exome-based sequencing of patients with [metastatic] breast cancer,” he cites as an example, “we can see they have a special signature with gains or losses of genetic material.” Those signatures could perhaps help pinpoint where the tumor cells originate. 

In a pilot study published in 2020, Pantel and his colleagues found a “nice concordance” between the expression of ALCAM (activated leukocyte cell adhesion molecule) in the brain metastasis and CTCs of patients with NSCLC (Neuro-Oncology, DOI: 10.1093/neuonc/noaa028). “Of course, that needs to be further validated, but it’s important [to ask] to what extent the circulating tumor cells reflect what is going on in the tissue.” 

Functional analysis of CTCs requires an understanding of their biology and use in cell lines for drug screening, suggests Pantel. In fact, in 2020, the development of a permanent cell line for breast cancer research was highlighted on the cover of EMBO Molecular Medicine (DOI: 10.15252/emmm.201911908). 

In the paper, Pantel says, he and his collaborators showed that the cell lines were “quite stable over years” and in immunodeficient mice and xenografts would produce metastasis in all organs that are relevant to estrogen receptor-positive breast cancer, especially bone metastasis. 

Interventional Studies Needed 

“Liquid biopsy can provide clinically relevant information for personalized therapies, in particular immunotherapies,” he concludes. To that end, assays need be validated and harmonized, and more interventional clinical studies must be done to demonstrate the clinical utility of liquid biopsy approaches.   

Pantel’s example here was in the detection of HER2 oncogenes as therapeutic targets in breast cancer. More than a decade ago, Pantel’s group showed HER2 expression of the primary tumor is not always the same on the CTCs at the time of metastatic relapse. 

They then asked if patients who had primary negative tumors, but positive CTCs, could be successfully treated with anti-HER2 therapy. Based on unpublished results comparing those treated via immunotherapy with patients receiving only standard therapy, it appears this is the case, says Pantel. “This is to my knowledge the first larger-scale randomized interventional study indicating clinical utility of CTC characterization.”

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