Lung cancer is one of the leading causes of cancer deaths in both developing and developed countries. Primary lung carcinoma is the most common type of cancer. It is classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Among all lung cancer, NSCLC, which has a highly heterogeneous population, accounts for about 85% and is further subdivided into lung adenocarcinoma and lung squamous cell carcinoma. The poor survival rate of lung cancer is mainly due to lung cancer diagnosis at a late stage. Hence, early diagnosis and treatment would be an effective way to reduce the mortality rate in lung cancer patients.

Liquid biopsy is a promising tool to diagnose all the types of lung cancer and find the potential to diagnose the lung cancers which cannot be easily diagnosed with tissue biopsy. It is also helpful in circumventing tumor heterogeneity, identification of response to therapy, real-time monitoring of the tumor and to reveal the process of development of resistance in lung cancer. Liquid biopsy is a less-invasive method to easily obtain tumor derived analytes for the diagnosis of cancer in patients. Liquid biopsies are helpful for the clarification of the characteristics of lung cancer by identifying tumor cells or tumor DNA which are released into the bloodstream by the tumor cells.

The analytes of liquid biopsy which is typically detected in bodily fluids are cell free DNA (cfDNA), circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, microRNAs (miRNA), peripheral blood circulating RNA, tumor-educated platelets (TEPs) and extravascular vesicles. Among all of these, CTCs, cfDNA, ctDNA, and exosomes are the most commonly detected biomarkers in bodily fluids.

Liquid biopsy analytes in lung cancer

1. 1. Circulating tumor cells (CTCs)

The CTCs can be detected in the bodily fluids of patients with primary or metastatic lung cancer. A large number of clinical data suggested that efficient isolation of CTCs can provide early diagnosis, real time monitoring of cancer, predict the risk of metastasis and determine relapse and evolution of advanced lung cancer. Real time monitoring of treatment with liquid biopsies may provide a strong prospect for the treatment of lung cancer in future. The monitoring of CTCs in patients with early lung adenocarcinoma reflects the value of early diagnosis and the counts of CTCs increased significantly in all patients with progression of lung cancer. Also, the concentration of CTCs increases with progression of adenocarcinoma in post-operative patients as compared to patients with disease-free survival.

Clinical data has shown that for the diagnosis of early detection of lung cancer, NGS method is used for isolation of CTCs and mostly four common types of lung cancer related gene mutations are observed. These genes are Notch1, IGF2, EGFR and PTCH1 which have been found in more than 50% of lung cancer patients. In another study, 10 different metabolites were identified to have potential for the diagnosis of CTC-positive early lung cancer. Therefore, NGS and metabolomics studies of CTCs may provide new tumor markers for the early diagnosis of lung cancer.

Some studies reported that the DNA detected by CTCs may be helpful for the prediction of lung cancer progression and drug resistance. Continuous monitoring of the treatment response of lung cancer patients through CTCs analysis is of great value in the determination of precise treatments also. Reported studies have shown that CTCs detection can be used as an alternative method in patients with anaplastic lymphoma kinase (ALK) gene rearrangement in NSCLC and it can also be used to supplement the tissue based EGFR mutation detection in lung cancer from plasma in every two months to guide the precision treatment for EGFR mutation.

There are various methods which are developed to isolate CTCs in the peripheral blood. It is mainly of two categories: biological methods and physical methods. These method of isolation of CTCs may also be classified as positive selection (select CTCs only) or negative selection (separate all the blood cells except CTCs). The Cell Search® test is the first and only FDA approved test used for the detection of CTCs. Detection of CTCs in this test is based on positive enrichment type of immune-magnetic bead method. It can automatically photograph, count and analyze the stained CTCs.

1. 2. Circulating tumor DNA (ctDNA)

The CTCs can be detected in the bodily fluids of patients with primary or metastatic lung cancer. Various clinical data suggests that efficient isolation of CTCs can provide early diagnosis, real-time monitoring of cancer, predict the risk of metastasis and determine relapse and evolution of advanced lung cancer. Real-time monitoring of treatment with liquid biopsies may provide a strong prospect for the treatment of lung cancer in future. The monitoring of CTCs in patients with early lung adenocarcinoma reflects the value of early diagnosis and the counts of CTCs increased significantly in all patients with progression of lung cancer. Also, the concentration of CTCs increases with progression of adenocarcinoma in post-operative patients as compared to patients with disease-free survival.

Clinical data has shown that for the diagnosis of early detection of lung cancer, the NGS method is used for isolation of CTCs and most four common types of lung cancer related gene mutations are observed. These genes are Notch1, IGF2, EGFR and PTCH1, which have been found in more than 50% of lung cancer patients. In another study, 10 different metabolites were identified to have the potential for the diagnosis of CTC-positive early lung cancer. Therefore, NGS and metabolomics studies of CTCs may provide new tumor markers for the early diagnosis of lung cancer.

Some studies have reported that the DNA detected by CTCs may be helpful for the prediction of lung cancer progression and drug resistance. Continuous monitoring of the treatment response of lung cancer patients through CTCs analysis is of great value in the determination of precise treatments also. Reported studies have shown that CTCs detection can be used as an alternative method in patients with anaplastic lymphoma kinase (ALK) gene rearrangement in NSCLC and it can also be used to supplement the tissue based EGFR mutation detection in lung cancer from plasma every two months to guide the precision treatment for EGFR mutation.

There are various methods which are developed to isolate CTCs in the peripheral blood. It is mainly of two categories: biological methods and physical methods. These methods of isolation of CTCs may also be classified as positive selection (select CTCs only) or negative selection (separate all the blood cells except CTCs). The Cell Search® test is the first and only FDA approved test used for the detection of CTCs. Detection of CTCs in this test is based on a positive enrichment type of immune-magnetic bead method. It can automatically photograph, count, and analyze the stained CTCs.

1. 3. Exosomes

Exosomes are invagination of the cell membrane, produced by endosomes and then exosomes are formed. The fusion of multi-vesicles with the plasma membrane leads to the release of exosomes into the bloodstream. Exosomes carry various types of proteins, lipids, DNA and RNA from the cell. Surface markers mainly include CD63, CD81, CD9, TSG101, and HSP27. Exosomes are involved in angiogenesis, EMT, invasion and metastasis, immune escape, drug resistance, and have a vital role in the occurrence and development of tumors.

Recently, it has been reported that exosomal miRNAs can be used as an important medium for information exchange between lung cancer cells and other cells, and play an important role in the formation of tumors. Exosomes can also be used for the early diagnosis, prognosis, and monitoring of drug resistance of lung tumors. Moreover, inhibition of the formation and release of exosomes may be a potential novel approach for the cure of lung cancer. Exosomes also maintain the stability of nucleic acids like ctDNA and protect them from degradation. Hence, it allows for the supplementary detection of ctDNA with miRNA found in exosomes.

Studies have also confirmed that secreted miRNAs can determine the patients with lung adenocarcinoma, pulmonary granuloma and healthy individuals. It can also be used as biomarkers to identify different types of NSCLC. It has been reported that plasma exosomes containing mir-30e-3p, mir-30a-3p, mir-181-5p and mir-361-5p and, mir-15b-5p, mir-10b-5p, and mir- 320b can be used as specific biomarkers of adenocarcinoma and squamous cell carcinoma, respectively. Some studies demonstrate that development of resistance against cisplatin has been associated with formation and secretion of exosomes in plasma. While exosomes have the potential to treat lung cancer, the main limitation to using them in clinical practice is the lack of standardized isolation and detection methods.

1. 4. Other analytes of liquid biopsy for management of lung cancer

Peripheral blood-circulating RNAs called extracellular RNA, which include miRNA and lncRNA, are actively secreted by tumor cells and released into the bloodstream or other body fluids. The miRNAs control the expression of many oncogenes and tumor suppressor genes. Therefore, detection of miRNAs is of vital significance in early diagnosis of lung cancer, risk assessment and involved in the regulation of tumor chemotherapeutic drug sensitivity. The inhibition of miR-21 and miR-200b increases the sensitivity of lung tumor to gemcitabine and inhibition of miR-141 is associated with reduced growth rate of lung tumor.

IncRNA also has the potential for supplementary diagnosis of lung cancer. The lncRNA H19 was found as a biomarker as its level has been significantly increased in the plasma of lung cancer patients. The lncRNA MIR22HG has been associated with tumor suppression and developed as a new therapeutic target for lung cancer. The decrease in expression of LINC-PINT is associated with poor prognosis and predicts advanced clinical tumor stages.

With the development of second generation sequencing technology, circular RNA is differentially expressed in lung cancer patients and healthy individuals and hence, it can be used as a biomarker for diagnosis, cancer prognosis and may be a potential therapeutic target for treatment of lung cancer.

The liquid biopsy analyte TEP ITGA2B is also a promising biomarker, which can be used for the diagnosis of stage I NSCLC patients and is able to distinguish between malignant and benign pulmonary nodules.

Reference

1. Buder A et al. 2021. Somatic copy-number alterations in plasma circulating tumor DNA from advanced EGFR-mutated lung adenocarcinoma patients. Biomolecules. 11(5):618.

2. Yang D et al. 2021. Long non-coding RNA linc00665 inhibits CDKN1C expression by binding to EZH2 and affects cisplatin sensitivity of NSCLC cells. Mol Ther Nucleic Acids. 23:1053–65.

3. Zhao L et al. 2021. DNA methylome profiling of circulating tumor cells in lung cancer at single base-pair resolution. Oncogene. 40:1884–95.

4. Qian H et al. 2021. Progress and application of circulating tumor cells in non-small cell lung cancer. Mol Ther Oncolytics. 22:72–84.

5. Li W etal. 2022. Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring. Mol Cancer. 21(1):25.

6. Rijavec E et al. 2019. Liquid Biopsy in Non-Small Cell Lung Cancer: Highlights and Challenges. Cancers (Basel). 12(1):17.