Liquid Biopsy in Non-Small Cell Lung Cancer—Current Status and Future Outlook

A Narrative Review

Elisabeth Smolle; Valentin Taucher; Jörg Lindenmann; Martin Pichler; Freyja-Maria Smolle-Juettner


Transl Lung Cancer Res. 2021;10(5):2237-2251. 

In This Article


Targeted treatment modalities are quickly emerging in the clinical management of non-small cell lung cancer (NSCLC). Lung cancer is the leading cause of cancer-related deaths worldwide. For both men and women, lung cancer is the most frequently diagnosed cancer entity worldwide (11.6% of all cancers) and accounts for 18.4% of total cancer-related deaths, according to data from 2018.[1] The American Cancer Society estimates, that 228,820 new cases and 135,720 deaths from lung cancer are to be expected in the United States for the year 2020.[2] Increasing knowledge has been gained within the last decade about the molecular abnormalities in lung cancer, defining disease subsets based on molecular properties.[3]

For several years, providing tailored treatment options, depending on certain molecular characteristics of the disease, has been the standard in everyday clinical practice. It is common, however, that over the course of the disease, resistance to targeted treatment is acquired. Thus, current research focuses on the establishment of next-generation therapeutics, potent enough to overpower mechanisms of drug resistance.[4–11] Genetic information from tumors is used to predict the response to treatment with certain targeted therapeutics, e.g., epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs).

The approval of first-generation EGFR TKIs consequently resulted in the development of second- and third-generation TKIs like osimertinib, having its specific point of impact against certain mutant forms of EGFR. Several advantages are provided by this novel group of treatment agents: common EGFR activating mutations are specifically and effectively addressed; inhibition of the EGFR protein harboring the T790M mutation is provided (this mutation is responsible for treatment failure of EGFR TKIs of the first or second generation); as well as the impact against wild-type (WT) EGFR is relatively low, which reduces treatment toxicity and adverse effects.[12] Another example is the ever-expanding group of anaplastic lymphoma kinase (ALK) targeting drugs, which provide valid treatment modalities for each subject having developed resistance to crizotinib, a first-generation ALK TKI.[12] These next generation ALK TKIs (alectinib, ceritinib, brigatinib, ensartinib and lorlatinib) have the potential to bind and inhibit mutant forms of ALK. Of note, all these novel ALK TKIs have different binding affinities, depending on specific resistance mutations, so as a method to find tailored treatment modalities for each individual, exact identification of these resistance mutations is mandatory.[4] Since the evolution of the tumor microbiology over the course of the disease may lead to a change in mutations, allowing for additional therapeutic options, repeated tissue biopsies have been advocated. However, this approach comes with a considerable risk for the respective patient, and depending on performance status cannot be applied to each individual.[4] For instance, computed tomography (CT)-guided lung biopsy has a 5% rate of major complications.[13]

Until today, there is only one predictive tumor biomarker which is routinely tested to outline patients suitable for first line immunotherapy, i.e., programmed cell death 1 ligand 1 (PD-L1) as evaluated by means of immunohistochemistry from tissue sections.[14] PD-L1 is thus routinely assessed in many pathology laboratories throughout the world, however the assessment can sometimes be challenging due to biologic or technical limitations.[15] PD-L1 expression is found in malignant-, but also in immune-cells, rendering a careful assessment of the PD-L1 status even more complicated.[16,17] Moreover, there is a considerable intratumoral heterogeneity regarding PD-L1 expression, and small biopsies may not be representative of the whole tumor.[17]

Tumor mutational burden (TMB) in biopsies of cancer tissue has been outlined as a new biomarker, especially for outlining NSCLC patients for treatment with immune checkpoint inhibitors, like nivolumab and ipilimumab.[18] However, until today cross laboratory technical standards and validation of TMB analysis are still lacking, making the implementation of TMB into everyday clinical practice difficult.[19] The prediction of therapeutic response based on PD-L1 immunohistochemistry from tissue biopsies, or else, TMB assessment, is not always possible due to a very small amount of tumor tissue or a minor proportion of cancer cells in the biopsy specimen. Furthermore, in a few patients with a relatively high TMB and high PD-L1 expression, immunohistochemistry might still be false negative.[17]

As an alternative, liquid biopsy is an emerging diagnostic tool, already used in clinical routine in lung cancer patients in some specialized treatment facilities. Originally, the term liquid biopsy defined circulating tumor cells (CTCs), but today also comprises circulating cell-free tumor DNA (cfDNA) as well as exosomes.[3] Liquid biopsies are utilized either as a method for the diagnosis of lung cancer, or as a tool to monitor treatment response or for the detection of minimal residual disease after curative surgery.[3]

A variety of liquid biopsy platforms have been established in order to outline mechanisms of drug resistance that have developed over time. The liquid biopsy approach has been recommended by the new College of American Pathologists (CAP)/International Association for the Study of Lung Cancer (IASLC)/Association for Molecular Pathology (AMP) guideline for the molecular testing of NSCLC patients.[20] Of note, liquid biopsy cannot substitute for an initial diagnostic tissue biopsy. Only in rare cases where tissue cannot be obtained via tissue biopsy, liquid biopsy may serve as a tool to acquire the histologic diagnosis.[21] In some cases, tissue biopsy material is small, which prevents the pathologist to carry out all the necessary molecular tests, so the acquisition of new tissue for conducting further analyses would be urgently required. Liquid biopsy is the alternative option in this scenario as well. Moreover, liquid biopsy is more cost-effective when compared to conventional tissue biopsy.[22] It has also been found that molecular properties of CTCs provide a more accurate picture of the actual systemic tumor load, furthermore reflecting more accurately the heterogeneity within a given tumor specimen, as well as tumor biology of metastases, which cannot be covered with single-site biopsy only.[23,24]

When making liquid biopsy techniques widely available in clinical routine, methods of sample collection, storage and shipping have to be optimized and standardized across treatment centers. In a recent study,[25] the cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes have been analyzed. These tubes allow for the shipping of whole blood at ambient temperature, while the integrity of cfDNA is still provided, preventing the dilution of cancer-derived DNA with WT DNA from the genome. According to this report, collection of whole blood from healthy individuals in cfDNA BCTs, followed by storing for a time period of five days or less, at room temperature, did not compromise DNA quality and mutation background levels. Mutant circulating tumor DNA (ctDNA) in the blood obtained from patients with colorectal cancer, and acquired using cfDNA BCTs, remained stable over a time period of three days of storage at room temperature. Still, as a consequence of storage at ≤10 °C and at 40 °C for a longer time period, levels of healthy DNA from the genome, and an unusually large cell plasma interface, along with reduced plasma volumes, were observed.[25] Thus, correct handling and storing, a preferably short storage time and quick sample analysis are key factors to achieve the maximum diagnostic benefit from liquid biopsy.

In this review article, we aim to elucidate the pros and cons of liquid biopsy, as well as current clinical relevance, use in everyday practice and limitations.

We present the following article in accordance with the Narrative Review reporting checklist (available at