The Role of Liquid Biopsy Biomarkers in Lung Cancer: Current and Future Applications

Abstract

Given the growing incidence of cancer worldwide, successful detection and screening of these diseases are crucial to lowering the risk of devastating effects. Among all cancers, lung cancer is the leading cancer-related cause of death. Currently, tissue biopsy is the gold standard to diagnose lung cancer and, while effective, this procedure is invasive and has several risks due to its surgical nature. Liquid biopsy is emerging as an alternative procedure to address these limitations, revolutionizing the field by enabling a non-invasive diagnosis that is less expensive, faster, and has lower risks associated. Specifically, liquid biopsy enables via blood samples a snapshot of the biological variables that affect the tumor growth and progression, contributing to lung cancer detection as well as to the analysis of prognosis and therapy response over the course of the disease. This is attained thanks to the identification of several biomarkers such as circulating tumor cells or cell-free DNA (approved by the US Food and Drug Administration) that are related to lung cancer progression. While the use of these biomarkers for assessing the effectiveness of therapy over time has translated into clinical practice, the use of them for cancer detection and the initial diagnosis is yet to be implemented in the clinical routine. Regarding the latter, this work highlights the use of several biomarkers in combination, to boost performance and the need for strong validation of the biomarkers proven prognostic in research settings.

Abbreviations and Acronyms

• cfDNA – Circulating cell-free DNA
• ctDNA – Circulating tumor DNA
• CTC – Circulating Tumor Cells
• ELISA – Enzyme-linked immunosorbent assay
• NSCLC – Non-small cell lung cancer
• SCLC – Small cell lung cancer
• TAAbs – Serum tumor-associated autoantibodies

Introduction

Lung tumors are the leading cause of death in cancer-related illnesses¹. As malignant tumor cells accumulate uncontrollably in the lung, they spread out to the lymph nodes and potentially to other organs such as the brain. Recently, there have been increasing causes of lung cancer due to exogenous factors related to unhealthy lifestyles such as smoking tobacco, which contains several carcinogenic substances². While it is the second most common type of cancer, reaching 1 in 15 men and 1 in 17 women, most patients who develop lung cancer are not diagnosed until they reach a metastatic stage¹. At this stage, prognoses are poor and the usual five-year survival rate for cancers is slim³. This indicates that the later stage of lung cancer has very few effective treatment interventions and explains the aforementioned high mortality associated with lung cancer, which translates to about 25% of all cancer deaths, or a death toll of approximately 1.6 million people annually⁴. All this highlights the need for earlier diagnosis and personalized treatment plans for individuals who develop this disease.

Imaging techniques, like computed tomography scans and X-rays, may locate potential tumors but are not able to differentiate between cancerous and noncancerous cells. Thus, tissue biopsy has been and still is the gold standard technique in clinical practice to diagnose lung cancer³. While effective, this procedure is however invasive and has several risks associated due to its surgical nature (e.g., inflammation, infection, bleeding, etc.). Given these risks, liquid biopsy is emerging as an alternative to conventional tissue biopsy, revolutionizing the field by enabling a non-invasive diagnosis that is less expensive, faster, and has lower risks associated². In brief, liquid biopsy consists of a blood test, usually sampled from the arm, that analyzes the fragments of cells that have broken off or entered the bloodstream as a result of cancer progression. The vast amount of malignant tumor cells can invade the blood vessels quickly. The liquid biopsy functions with specific biomarkers in combination to collect the DNA sequencing from the blood vessels of the bundle of tumor cells².

The non-invasive and inexpensive nature of liquid biopsy enables easy and continuous monitoring by repeated sampling, in contrast to conventional tissue biopsy, changing the paradigm of cancer screening and individualized therapy planning. This review paper further delves into the specifics of biomarkers used in liquid biopsy and this method’s capabilities of early diagnosis and the extent of treatment progression.

Lung Cancer Progression and Liquid Biopsy

Small cells lung cancer (SCLC) and non-small cells lung cancer (NSCLC) are the two main types of lung cancer.  SCLC is characterized by relatively smaller and round-shaped tumor cells that quickly grow and invade other regions of tissues⁵. NSCLC consists of larger tumor clusters, whose size results in a slower spread rate to other organs⁶. Despite these differences in growth rates, both types are mostly diagnosed at metastasis⁷. While there is evidence supporting the use of liquid biopsy for both types (8, 9), the collected clinical data is more centered on NSCLC, since this is the most common of the two types of lung cancer accounting for about 85% of all lung cases⁴.   

Liquid Biopsy has the potential to provide information concerning predictive markers and acquired resistance mechanisms in a shorter period compared to the state-of-the-art use of retrospective evaluation in tissue. There are several liquid biopsy-derived biomarkers used to detect and treat lung cancer. Biomarkers applied to liquid biopsy are biological molecules found in circulating blood or body fluids that signal a disease condition based on normal or abnormal cells. These biomarkers help identify patients who have the possibility of having malignant tumor cells that can lead to lung cancer. The three main biomarkers are Circulating tumor cells (CTC), Serum tumor-associated autoantibodies (TAAbs), and Circulating Tumor DNA (ctDNA)⁸. These biomarkers are connected to cancer progression as they give an analysis of genomic and proteomic data.

• Circulating Tumor Cells (CTCs): As the primary tumor sites grow, they eject tumor cells that migrate, either isolated as single cells or in clusters, to the bloodstream and invade distant sites to proliferate. These circulating tumor cells, or CTCs, can be measured using DNA, RNA, or protein-based molecular profiling, and become an effective biomarker to diagnose cancer and assess progression. Cell Search Technology, the first actionable testing for detection approved U.S. Food and Drug Administration (FDA), demonstrated that CTCs were proven effective for the detection of metastatic prostate, breast, and colorectal cancers⁹. However, for the early diagnosis of cancer cells, there are low concentrations of CTCs in blood for cancer-specific surface markers that have low identification rates⁷. To avoid this issue, enrichment of technology developed CTCs into CTC-based Folate Receptor Positive Circulating Tumor Cells (another diagnostic biomarker) that increased the sensitivity but is still limited to adenocarcinoma cancer, a type of NSCLC which has a strong association with smoking, developing in the glands that line the organs⁸. Folate Receptor Positive Circulating Tumor Cells use novel ligand-targeted polymerase chain reaction techniques to distinguish lung benign or malignant tumors in NSCLC⁹. 
  
• Serum Tumor-Associated Autoantibodies (TAAbs): Another biomarker is TAAbs, autoantibodies against overexpressed and mutated cells, which could help diagnose cancer at an early stage. The rationale for the use of this biomarker for early diagnosis is explained three-fold¹⁰. The first reason is that the immunosurveillance’s occurrence in the early immuno-editing phase indicates that autoantibodies are detectable earlier than CTCs. From using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis or enzyme-linked immunosorbent assay (ELISA), a proteomic technique is developed that enables to screen abundance of antigens instantaneously by opening immuno-proteomic to exhibit a better diagnostic value¹¹ . The TAAbs have been used as the diagnostic marker to evaluate such types of cancer as breast, NSCLC, colon, prostate, and pancreatic¹¹ . The second reason is that despite low tumor mass stabled blood, TAAbs are present at high titers. The credibility of TAAbs’ is proved as the tumor cells were easily detectable in the lung nodules, and as such, TAAbs have been approved by China Food and Drug Administration¹⁰. A combination of assays like ELISA for TAAbs establishes the panel for the multiplex detection of antibody biomarkers. Hence, TAAbs was able to overcome the heterogeneity of tumor cell proteomes which was one of the restrictions that most protein markers were not able to pass the clinical trials for prognosis¹¹ . However, TAAs may become neo-antigenic if they exhibit post-translational changes, abnormal location, or overexpression in cancer¹¹. An essential component of immunoproteomics is the pattern of formation of tumor-associated autoantigens and their particular autoantibodies in the course of carcinogenesis. As a result of immune suppression or tolerance brought on by tumor cells during the immunosurveillance’s escape and equilibrium steps, the majority of autoantibodies seen in cancer have low titers and are inefficient at activating effector functions¹¹. The last reason is that TAAbs enhance the detection of proteins that undergo alterations during tumorigenesis¹¹. Therefore, the personalized treatment of TAAbs and autoantibodies is used to associate the related vaccines which are targeted for specific immunotherapy against cancer¹¹.
  
• Circulating Tumor DNA (ctDNA): The last biomarker to be introduced is the circulating tumor DNA (ctDNA), which is part of circulating cell-free DNA (cfDNA). The difference between general cfDNA and ctDNA is that while cfDNA accounts for all DNA that can freely circulate in the bloodstream, ctDNA represents only the free DNA from a tumor origin. ctDNA is derived from the primary tumor into metastasis from the malignant tumor cells due to necrosis and apoptosis. Since cfDNA can be differentiated from healthy cell DNA such as DNA methylation, the DNA methylation status resembles malignant tumor cancer¹². Signals for DNA methylation are imperative because they alert the early stage of cancer. The ctDNA of cfDNA increase in concentration is proportional to the size of tumor cells. It is demonstrated to be associated with tumor response, burden, and survival outcomes as it links to the protein to be transported by vesicles¹³. A cfDNA-based machine-learning approach can be used to term lung cancer likelihood in plasma (Lung-CLiP) to discriminate early-stage lung cancer patients from risk-matched controls to minimize false positives from low-dose computerized tomography data¹⁴.

However, the limited snapshot of liquid biopsy may fail to reflect its heterogeneity. Specificity and sensitivity are the gold standard metrics when measuring the effectiveness of liquid biopsy and tissue biopsy for detecting tumor cells. The biomarkers introduced in this section can be combined to improve these sensitivity and specificity scores toward clinical translation in applications such as treatment progression and diagnosis. Connecting back to the application of liquid biopsy for SCLC, ctDNA, and CTC are the most practical biomarkers to be integrated for cancer monitoring of response and genomic profiling¹⁵. The feasibility of ctDNA allows greater consistency of prognostic stratification to some degree in a limited number of cases¹⁵. The next section delves into the applications of liquid biopsy and the biomarkers.

Applications and Degree of Implementation

Liquid biopsy can increase the sensitivity and/or selectivity of CT scans or other non-invasive imaging or radiation methods for screening lung cancer patients. Improvements in treatment progression and early diagnosis are crucial to reduce the lung cancer burden and motivate the direction of the development of liquid biopsy. Advancements are needed to enable identifying both genetic and epigenetic aberrations. Currently, liquid biopsy provides the genetic surface of both primary and some extent of metastases to systematically track genomic evolution¹⁷. This technique has already demonstrated its potential in research settings. Regarding clinical translation, while the use of liquid biopsy for treatment progression has an acceptable degree of implementation using the combination of various biomarkers mentioned above, early diagnosis is the application the liquid biopsy is developing towards using more effective combinations. As liquid biopsy requires a small amount of sample to detect the tumor cells in the body quickly, the degree of implementation is limited: the areas where liquid biopsy excels indicate that the tumor cells have already spread out to the body in the metastasized stage¹⁸.

Treatment Progression

Liquid biopsy can also be used to monitor the biological changes of disease during treatment, such as the persistence of Estimated Glomerular Filtration Rate mutations, the presence of tumor protein p53, Phosphatidylinositol-4,5-bisphosphate 3-Kinase catalytic subunit alpha mutations, Retinoblastoma-1 alterations, FAT tumor suppressor homolog 1, or ATP binding cassette sub-family B member1 mutations in EGFR-mutated patients. This data can then be used to personalize chemotherapy, immune checkpoint inhibitors (ICI), or TKI treatment¹⁹. Hence, to discuss the application of biomarkers in treatment progression, ICI must be addressed. Immunotherapy has developed ICI, which results in significant therapeutic benefits. ICI therapy is connected to the biomarkers as it causes immune-related adverse reactions, such as various organ toxicity or any structural and functional changes in organs and tissues¹³. Hence, the predictive biomarkers from the ICI response efficiently guide the treatment decision and patient selection which helps to understand the overall resistance mechanism. CTCs or ctDNA are predictive biomarkers that give appropriate responses under immunotherapy to stratify patients for treatment progression.

Those biomarkers that are well sought from the peripheral blood sample suggest how they are feasible for analysis of tumor mutation burden in profiling tumor cells. Mutations and copy number alterations are measured in ctDNA and used to predict response to access treatment. The evaluation of mutations in the CTCs regulates and monitors patient treatment in real-time, which benefits the strong prospect for the developing treatment of lung cancer in the future. Longitudinal monitoring of ctDNA mutations is used to track the emergent therapy resistance and identify resistance mechanisms. CTCs help to identify proteins with tumor cells and measure their amount, especially PD-L1 and ARV7 expression to assist the treatment selection. Enumeration of CTCs is used to monitor treatment responses which can be harvested to generate patient-derived explant (CDX) models in immune-deficient mice supporting drug and biomarker development.

Early Diagnosis

Some biomarkers, such as ctDNA and TAABs, have demonstrated potential for early diagnostic tests with trials supporting evidence²¹. Diagnostic tests are different from screening as diagnostic tests are focused on patients who do show symptoms of lung cancer while the screening is intended for people who are asymptomatic but going through a preliminary check-up. Substantial studies verified by the indirect enzyme-linked immunosorbent assay verified that TAABs do perform some early diagnosis performance but further panel approach is needed to yield higher diagnostic records²². To delve deeply, serum single or a combination of multiple antibodies are revealed to be effective tools such as the 6 TAAbs for both early and late diagnoses. However, a more advanced panel like 7 TAAbs has a higher potential to be used as early-detection biomarker⁸. According to the experiment which the 129 plasma samples of 63 patients with malignant lung nodules and 66 patients with benign nodules, the specificity, and sensitivity were achieved at 83.3% and 82.5% respectively. Currently, CTCs that are being released in the metastasis stages have promising applications to be used for early diagnosis according to future applications. As the promising application is lung cancer screening, new methods are arising for the development of CTC biomarkers. CTC separation method detects the small tumors in the early stage and diagnosis of early or advanced cancer patients. To elaborate on the specific applications of CTCs that help with screening and early diagnosis, there are several physical enrichment methods. The physical method enriches CTCs by differentiating CTCs and blood cells, including relative density, size, and electrical characteristics. Processing the peripheral blood by the enrichment process discussed above reduces the background blood cell levels, allowing remaining cell populations to be detected and analyzed. The first one is the positive enrichment of CTCs derived from the immunomagnetic bead method of the CellSearch system’s representative product. It automatically photographs, counts, and analyzes stained cells to help the screening process. Likewise, a negative enrichment method uses immunomagnetic beads of CTCs by removing the background blood cells. Immunoadsorption methods are based on microfluidic chips and the processing speed uses a CTC chip, one of the fastest four assays of biomarkers that are currently verified.

Discussion

Liquid biopsy-derived biomarkers have been used in a variety of combinations that have shown promising results in the early detection of lung cancer. Liquid biopsy has been continuously developed by complementing the limitations of tissue biopsy requiring higher risk and cost while being slower from additional diagnosis. However, the evidence of favorable results was limited outside of the research setting in real-life cases. It is presented by the significant portion of false negatives and negative plasma tests when going through the confirmation process of tissue biopsy. This also means that clinical fields need further clarification with more sensitive and specific assays that should be provided to develop the low or absent values that limited the biomarkers’ applicability. In the future, robust studies are needed to verify an appropriate combination of biomarkers with other diagnostic tools such as imaging technology to validate the role of liquid biopsy in clinical practice for screening and diagnosis. Specifically, the incorporation of multi-analyte panels into preventive health screening should be considered to target the specific patient population, and cancer types, and how the results are integrated with other clinical information. With various effective biomarkers that liquid biopsy can generate, further efforts are warranted including the transoms analysis and validation studies. Hence, technological developments will help mitigate these current problems of liquid biopsy by defining the best biomarker combinations to increase the sensitivity and specificity of tumor cells in the early diagnosis process. Contribution to personalized and preventive medicine via CTC, TAAbs, and ctDNA is evidenced²². However, future research is needed to validate the good insights of a further area of specificity and sensitivity when used on lung cancer patients. As an example, a company named GRAIL raised funds of a total of $1 billion from Google, Amazon, and Microsoft to develop liquid biopsy for cancer blood detection. It launched to execute a whole-cancer screening using blood samples for early diagnosis. This is one of the direct examples that shows contribution in developing the liquid biopsy.

Conclusion

This review paper discussed the contribution of liquid biopsy in the medical field to help detect, screen, and give therapy responses for lung cancer patients. While it has been implemented for clinical routine therapy assessment through the use of various biomarkers, liquid biopsy has not yet been implemented in clinical practice for detection in early diagnosis.

1. W. Liang, Y. Zhao, W. Huang, et al. Liquid Biopsy for Early Stage Lung Cancer. Journal of Thoracic Disease, 10; 7: S876–S881 (Apr. 2018) DOI:  10.21037/jtd.2018.04.26. [↩] [↩]
2. W. Li, J.B. Liu,  L.K. Hou, et al. Liquid Biopsy in Lung Cancer: Significance in Diagnostics, Prediction, and Treatment Monitoring. Molecular Cancer (2022), 21 (1). https://doi.org/10.1186/s12943-022-01505-z. [↩] [↩] [↩]
3. L. van Dessel; J. Martens; M. Lolkema et al Fundamentals of Liquid Biopsies in Metastatic Prostate Cancer: From Characterization to Stratification. Current Opinion in Oncology (2020), 32 (5), 527–534. DOI: 10.1097/CCO.0000000000000655. [↩] [↩]
4. American cancer society. “Can Lung Cancer Be Found Early?” Cancer.org, American Cancer Society,(2019), accessed 4 March 2021. [↩] [↩]
5. N. Guibert; A. Pradines; G. Favre et al. “Current and Future Applications of Liquid Biopsy in Nonsmall Cell Lung Cancer from Early to Advanced Stages.” European Respiratory Review, 29, no. 155, (31 Mar. 2020), err.ersjournals.com/content/29/155/190052, DOI: 10.1183/16000617.0052-2019. [↩]
6. Gawel, Susan H., et al. “Current and Future Opportunities for Liquid Biopsy of Circulating Biomarkers to Aid in Early Cancer Detection.” Journal of Cancer Metastasis and Treatment, 8, no. 5, 2022, p. 26, DOI: 10.20517/2394-4722.2022.13. [↩]
7. A. Revelo; A. Martin; R. Velasquez et al. “Liquid Biopsy for Lung Cancers: An Update on Recent Developments.” Annals of Translational Medicine, 7, no. 15, (1 Aug. 2019), DOI: 10.21037/atm.2019.03.28. [↩] [↩]
8. Z. Tang; Z-G Ling.; C-M. Wang et al Serum Tumor-Associated Autoantibodies as Diagnostic Biomarkers for Lung Cancer: A Systematic Review and Meta-Analysis. PLOS ONE (2017), 12 (7), e0182117. DOI: 10.1371/journal.pone.0182117. [↩] [↩] [↩]
9. X. Chen; F. Zhou; X. Li et al Folate Receptor-Positive Circulating Tumor Cell Detected by LT-PCR-Based Method as a Diagnostic Biomarker for Non-Small-Cell Lung Cancer. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer (2015), 10 (8), 1163–1171. DOI: 10.1097/JTO.0000000000000606. [↩] [↩]
10. W. Li; Ji. Liu; J. Zhang et al. “Liquid Biopsy in Lung Cancer: Significance in Diagnostics, Prediction, and Treatment Monitoring.” Molecular Cancer, 21, no. 1, (20 Jan. 2022), DOI: 10.1186/s12943-022-01505-z. [↩] [↩]
11. C. Heo; Y. Bahk; E. Cho et al. “Tumor-Associated Autoantibodies as Diagnostic and Prognostic Biomarkers.” BMB Reports, 45, no. 12, Dec. (2012), DOI:10.5483/BMBRep.2012.45.12.236. [↩] [↩] [↩] [↩] [↩] [↩] [↩]
12. D. Capua; D. Clarke; K. Ronan et al. “The Liquid Biopsy for Lung Cancer: State of the Art, Limitations and Future Developments.” Cancers, 13, no. 16, 4 (Aug. 2021), p. 3923, DOI: 10.3390/cancers13163923. [↩]
13. N. Li; X. Hou; S. Huang et al Biomarkers Related to Immune Checkpoint Inhibitors Therapy. Biomedicine & Pharmacotherapy (2022), 147, 112470. DOI: 10.1016/j.biopha.2021.112470. [↩] [↩]
14. Q. Gao; Q. Zeng; Z. Wang et alCirculating Cell-Free DNA for Cancer Early Detection. The Innovation (2022), 3 (4), 100259. DOI: 10.1016/j.xinn.2022.100259. [↩]
15. E. Pizzutilo; M. Pedrani; A. Amatu et al. “Liquid Biopsy for Small Cell Lung Cancer Either De Novo or Transformed: Systematic Review of Different Applications and Meta-Analysis.” Cancers, 13, no. 9, May (2021), DOI: 10.3390/cancers13092265. [↩] [↩]
16. Z. Zhang; N. Ramnath; S. Nagrath et al. “Current Status of CTCs as Liquid Biopsy in Lung Cancer and Future Directions.” Frontiers in Oncology, 5, Sept. 2015, DOI:10.3389/fonc.2015.00209. [↩]
17. E. Zulato; I. Attili; A. Pavan et al Early Assessment of KRAS Mutation in CfDNA Correlates with Risk of Progression and Death in Advanced Non-Small-Cell Lung Cancer. British Journal of Cancer (2020), 123 (1), 81–91. DOI: 10.1038/s41416-020-0833-7. [↩]
18. BiochainInstituteInc. https://www.biochain.com/blog/a-tale-of-two-biopsies-liquid-biopsy-vs-tissue-biopsy/. [↩]
19. E. Crowley; F. Di Nicolantonio; F. Loupakis et al Liquid Biopsy: Monitoring Cancer-Genetics in the Blood. Nature Reviews Clinical Oncology (2013), 10 (8), 472–484. DOI: 10.1038/nrclinonc.2013.110. [↩]
20. L. Groen; J. Schalken Liquid Biopsy for Prostate and Bladder Cancer: Progress and Pitfalls. European Urology Focus (2022), 8 (4), 904–906. DOI: 10.1016/j.euf.2022.08.013. [↩]
21. A. Campos-Carrillo; J. Weitzel; P. Sahoo et al Circulating Tumor DNA as an Early Cancer Detection Tool. Pharmacology & Therapeutics (2020), 207, 107458. DOI: 10.1016/j.pharmthera.2019.107458. [↩]
22. L. Zhong; S. Coe; A. Stromberg et al Profiling Tumor-Associated Antibodies for Early Detection of Non-Small Cell Lung Cancer. Journal of Thoracic Oncology (2006), 1 (6), 513–519. DOI: 10.1016/S1556-0864(15)30352-X. [↩] [↩]