Qiu-Hua Li, Zhao-Zhe Liu

1Department of Oncology, The Second Hospital of Liaoning University of Chinese Medicine, Shenyang 110034, China.2Department of Oncology, General Hospital of Northern Theater Command, Shenyang 110016, China.

Abstract Lung cancer is one of the malignant tumors with the fastest increasing morbidity and mortality in the world.Although surgical techniques have been improved and new chemotherapy drugs and local treatment methods keep emerging in recent years, the overall therapeutic effect on lung cancer is unsatisfactory.The advent of immunotherapy has brought about a major revolution in treating lung cancer, especially for non-small cell lung cancer (NSCLC).However, based on the heterogeneity of cancer types, not all patients with NSCLC can benefit from immunotherapy.It is essential to find reliable biomarkers to guide clinical immunotherapy.Compared with histological biopsy, biomarker-based detection not only has the advantages of early and non-invasive detection, but also can reflect the tumor’s biological characteristics and the body’s immune status.This article reviews the research progression on immunotherapy biomarkers of peripheral blood in NSCLC, with the aim of providing references for the evaluation of immunotherapy efficacy and prognosis of patients with NSCLC.

Keywords: non-small-cell lung cancer; biomarkers; immunotherapy; review

Background

According to Global Cancer Statistics 2020, the latest release of the American Cancer Society academic journal in 2021, lung cancer will be the second most common cancer worldwide, accounting for about a tenth (11.4%) of all diagnosed cancers and one-fifth (18.0%) of cancer deaths [1].Non-small cell lung cancer (NSCLC) accounts for up to 85% of all lung cancer patients.In recent years, immune checkpoint inhibitors (ICIs) have challenged the traditional treatment of NSCLC and brought new hope to patients with advanced NSCLC.However, the objective response rates to ICIs are limited in the treatment of NSCLC, with only about 19% of patients with NSCLC responding to ICIs [2].Compared with tissue biopsy, sampling of peripheral blood is non-invasive, convenient for repeated collection, and can reflect the immune status of the tumor and host.These unique advantages have shown great clinical promise in the immunotherapy of NSCLC.This review focuses on the research progress of circulating tumor cells (CTCs), blood tumor mutation burden (bTMB), circulating tumor DNA (ctDNA), neutrophil to lymphocytic ratio (NLR) and exosomes in NSCLC [3].

Circulating tumor cells

CTCs are a group of tumor cells existing in the peripheral blood.After entering the peripheral blood, most tumor cells are engulfed or undergo apoptosis, with a few surviving tumor cells developing into metastases.The detection of CTCs, as an emerging diagnostic method, has become a research hotspot and has an important guiding role in immunotherapy and prognostic evaluation in NSCLC [4].

Tumor tissues (TTs) are heterogeneous, PD-L1 expressed on TTs is inadequately representative of host immunity and features of the tumor microenvironment.The tissue biopsies are invasive and less reproducible [5].So, detecting PD-L1 expression in CTCs can make up for the deficiency of tissue biopsy.Yu-Xin Cheng et al.obtained TTs and corresponding blood samples from 66 patients with NSCLC and successfully isolated CTCs in 89% (59/66) of patients after detecting and counting CTCs.The expression of PD-L1 in CTCs and paired TTs of these 59 patients were analyzed.The results showed that compared with the expression of PD-L1 in paired TTs, the sensitivity, specificity, concordance, positive predictive value and negative predictive value of PD-L1 expression were 88.89%, 73.91%, 80%, 72.73% and 89.47%, respectively.Cohen kappa coefficient for CTCs and paired TTs was 0.613, but PD-L1 expression was inconsistent in CTCs and paired TTs specimens in 18 retreated patients.This confirms the feasibility of clinically detecting PD-L1 expression in CTCs, while further studies are needed to validate and explore for the standardize thresholds to guide clinical decision-making [6].

Studies have shown that patients with PD-L1 expression at baseline have poorer PFS than those with negative PD-L1 expression in CTCs or those with negative PD-L1 expression in TTs [6].The patients with negative PD-L1 expression on CTCs achieved clinical benefit after treatment with ICI, whereas the patients with PD-L1 expression on CTCs developed disease progression.The persistent positive expression of PD-L1 on CTCs may also reflect a mechanism of therapeutic escape [7].The study by Alama et al.included 89 patients with NSCLC who received nivolumab and had blood sampling before treatment, at the time of the first and second treatments.Response assessments indicated that patients with a lower-median number of CTCs at baseline were found to have significantly longer survival than those with a higher number [8].Tamminga also observed that high levels of CTCs were associated with poor patient outcomes in patients with advanced NSCLC.These results suggested that the expression of PD-L1 on CTCs in peripheral blood could be a prognostic marker for NSCLC immunotherapy.Its presence was associated with a lower response rate, poorer progression-free-survival (PFS) and overall survival (OS) [9].

Blood tumor mutation burden

TMB is defined as the total number of detected somatic gene coding errors, base substitutions, gene insertions or deletions errors per million bases [10].The greater the number of somatic mutations, the more neoantigens are present on the surface of tumor cells, which increases immunogenicity and makes tumors more sensitive to ICIs treatment [11].Multiple studies have demonstrated bTMB as a prognostic biomarker in NSCLC patients undergoing ICIs treatment [12–14].Gandara et al.showed that bTMB was a biomarker for predicting PFS in NSCLC patients receiving Atezolizumab, in which patients with bTMB ≥16 mut/MB had a better prognosis.This study firstly demonstrated that TMB could be measured accurately and repeatedly in peripheral blood.Recently, studies showed that bTMB had a higher success rate in achieving an effective TMB score (809/1001; 81%) than tTMB (460/735; 63%), and bTMB ≥20 mut/MB was selected as the optimal threshold for clinical benefit in ICIs treatment [15].Further experiments are needed to determine the bTMB threshold for clinical benefit from ICIs treatment in patients so as to provide the basis for the individual treatment of NSCLC patients.In summary, the measurement of bTMB is a feasible method for predicting the patient responses to ICIs in the immunotherapy of NSCLC.

Circulating tumor DNA

ctDNA is the extracellular DNA that is released into the circulatory system after tumor somatic DNA has been shed or when cells undergo apoptosis.ctDNA is able to assess tumor heterogeneity and is helpful for the classification of tumor molecular subtypes with different genomic signatures [16, 17].Peripheral blood ctDNA testing holds promise as a non-invasive approach to cancer classification and diagnosis [18, 19].

The application of ctDNA has been widely studied in the treatment and prognosis prediction of NSCLC.ctDNA was used to predict survival in lung cancer patients treated with the anti-PD-L1 drug Durvalumab.It was shown that a reduction in ctDNA variant allele frequency in early therapy was a predictor of long-term benefits from immunotherapy [20].It was possible to utilize early changes in ctDNA in the clinic to aid clinical treatment decisions.By analyzing 255 samples from 40 patients with stage I-III lung cancer and 54 healthy adults, Chaudhuri et al.found that ctDNA could be detected in blood samples from 94% of relapsed patients after their first treatment.In 72% of the patients, ctDNA detection was 5.2 months earlier than imaging after treatment, and ctDNA mutational signatures in 53% of patients were associated with clinical benefit from tyrosine-kinase inhibitor or immune checkpoint blockade therapy [21].A number of studies also demonstrated that ctDNA was of great value in the diagnosis, treatment and prognosis prediction of lung cancer [22–25].

Neutrophil to lymphocyte ratio

Chronic inflammation in the tumor microenvironment is an important factor in mediating tumorigenesis and progression.Because of its accessibility and non-invasive properties, NLR is now considered as an ideal indicator for evaluating systemic inflammatory responses [26].It has shown great potential in the diagnosis and prognostic evaluation of NSCLC.NLR in peripheral blood can be used as a biomarker for diagnosing NSCLC.Studies have shown that NLR values are higher in NSCLC patients than in patients with benign pulmonary nodules (P & Lt; 0.001), NLR values are positively correlated with NSCLC stage, and NLR is significantly associated with NSCLC differentiation (P & Lt; 0.05) [27].Zhu et al.included 210 lung cancer patients and 261 health examinees.According to the White Blood Cell count, the lung cancer patients and health examinees were divided into low-level group, a middle-level group and a high-level group.In both the lung cancer group and the control group, NLR values in the serum of patients with lung cancer were higher than those of healthy adults (P & Lt; 0.001) as the level of leukocytes increased [28].According to the above research, NLR can be used as a biomarker for early screening, clinical diagnosis and staging of NSCLC.

Multiple studies showed that elevated NLR in NSCLC patients was associated with poor prognosis and that patients with lower NLR tended to have longer PFS and OS [29–31].Each 5-unit increase in the NLR increased a patient’s risk of death by 11%, and an increase in the NLR was associated with cachexia and severe weight loss in patients and had the potential to result in a reduction in serum 25 (OH) D concentrations [26, 30].NLR also showed great potential in predicting the efficacy of immunotherapy.A large retrospective multicenter study including 187 patients with NSCLC who received nivolumab showed that an NLR < 5 was associated with prolonged PFS and OS, which was also confirmed by a study by Kartolo et al.[32, 33].These findings inferred that when NLR > 5, the choice of other treatment methods might bring greater benefit to patients.

Exosomes

Exosomes mainly affect gene expression and signaling pathways in recipient cells through the transfer of miRNAs, which have potential applications in the immunotherapy of cancer[34].Most patients with NSCLC are at the advanced stage of the disease and have high mortality.Early diagnosis is helpful in improving the prognosis.In 43 patients with stage I and II lung cancer, 90.7% of patients had a higher similarity of plasma exosomes to lung cancer cell exosomes than the healthy controls, and this similarity was proportional to cancer progression [35].Bone marrow Mesenchymal stem cells (BMSCs) are an integral part of the tumor microenvironment and play an essential role in the development of cancer.The hypoxic environment in tumors affects both tumor cells and interstitial cells.The relationship between the exosomes derived from BMSCs and the cancer cells in the hypoxic region was elucidated.Mir-193a-3p, Mir-210-3p and miR-5100 in the exosomes derived from the hypoxic BMSCs enhanced the invasion of the adjacent cancer cells.The combination of these three microRNA in exosomes had a higher diagnostic accuracy for lung cancer patients with or without metastasis, compared with the application of a single microRNA for diagnosis.Also, the plasma exosome miR-193a-3p could distinguish cancer patients from non-tumor controls [36].Studies by Salehi demonstrated that specific miRNAs in exosomes could be used for early diagnosis and prognosis prediction of lung cancer[37].In addition, exosome protein profiling is of great clinical value in judging the stage and histological subtype of lung cancer, indicating its clinical promise as a biomarker [38].As a kind of liquid biopsy, exosome detection can be used in the early diagnosis and auxiliary staging of lung cancer, and it has great potential for clinical application.

In recent years, the role of exosomes has also aroused great interest in the treatment of lung cancer.Exosomes derived from lung cancer cells can modulate the anti-tumor immune response and drug resistance in the course of cancer development.Therapeutic delivery exosomes are characterized by natural cell-to-cell communication, good biocompatibility, low immunogenicity, low toxicity, longer blood circulation capacity, biodegradability, and the ability to cross multiple biological barriers.Therapeutic delivery exosomes will play an important role in the precision treatment of cancer [39].

Summary

The biomarkers of peripheral blood have been developed rapidly in recent years because of their high safety and reproducibility in the clinical exploration of immunotherapy, especially in NSCLC.Many clinical studies have shown that biomarkers of peripheral blood have great potential in early diagnosis, adjuvant clinical staging, accurate treatment and prognostic evaluation, while there is still a need for validation with a large number of basic and clinical trials to explore the differences in the use of these biomarkers alone or in combination, to standardize thresholds and guide clinical decision-making [40].Moreover, as much as possible, it is important to analyze lung adenocarcinoma and lung squamous cell carcinoma separately, as their pathogenesis and clinical mechanisms are distinct.Their responses to immunotherapy treatment may also differ.It is therefore important to consider these factors and explore the corresponding potential biomarkers when analyzing NSCLC.With the progress and development of scientific and technological means, detection efficiency could be further improved, and more new biomarkers could be found, which will help clinicians make more accurate treatment plans and benefit more patients with NSCLC in the future.

References

1.Sung H, Ferlay J, Siegel RL, et al.Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries.CA A Cancer J Clin2021;71(3): 209–249.Available at: http://doi.org/10.3322/caac.21660

2.Borghaei H, Paz-Ares L, et al.Nivolumab versus Docetaxel in Advanced Nonsquamous Non–Small-Cell Lung Cancer.N Engl J Med2015;373(17):1627–1639.Available at: http://doi.org/10.1056/NEJMoa1507643

3.Heitzer E, Haque IS, Roberts CES, Speicher MR.Current and future perspectives of liquid biopsies in genomics-driven oncology.Nat Rev Genet2018;20(2):71–88.Available at: http://doi.org/10.1038/s41576-018-0071-5

4.Chen Q, Ge F, Cui W, et al.Lung cancer circulating tumor cells isolated by the EpCAM-independent enrichment strategy correlate with Cytokeratin 19-derived CYFRA21-1 and pathological staging.Clinica Chimica Acta2013;419:57–61.Available at: http://doi.org/10.1016/j.cca.2013.01.015

5.Jilaveanu LB, Shuch B, Zito CR, et al.PD-L1 Expression in ClearCell.Renal Cell Carcinoma: An Analysis of Nephrectomy and Sitesof Metastases.J Cancer2014;5(3):166–172.Available at: http://doi.org/10.7150/jca.8167

6.Cheng Y, Wang T, Lv X, et al.Detection of PD-L1 Expression and Its Clinical Significance in Circulating Tumor Cells from Patients with Non-Small-Cell Lung Cancer.Cancer Manag Res2020;12: 2069–2078.Available at: http://doi.org/10.2147/CMAR.S245425

7.Nicolazzo C, Raimondi C, Mancini M, et al.Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab.Sci Rep2016;6:31726.Available at: http://doi.org/10.1038/srep31726

8.Alama A, Coco S, Genova C, et al.Prognostic Relevance of Circulating Tumor Cells and Circulating Cell-Free DNA Association in Metastatic Non-Small Cell Lung Cancer Treated with Nivolumab.J Clin Med2019;8(7):1011.Available at: http://doi.org/10.3390/jcm8071011

9.Tamminga M, de Wit S, Schuuring E, et al.Circulating tumor cells in lung cancer are prognostic and predictive for worse tumor response in both targeted- and chemotherapy.Transl Lung Cancer Res2019;8(6):854–861.Available at: http://doi.org/10.21037/tlcr.2019.11.06

10.Yarchoan M, Hopkins A, Jaffee EM.Tumor Mutational Burden and Response Rate to PD-1 Inhibition.N Engl J Med2017;377(25):2500–2501.Available at: http://doi.org/10.1056/NEJMc1713444

11.Rizvi NA, Hellmann MD, Snyder A, et al.Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer.Science2015;348(6230):124–128.Available at: http://doi.org/10.1126/science.aaa1348

12.Gandara DR, Paul SM, Kowanetz M, et al.Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab.Nat Med2018;24(9):1441–1448.Available at: http://doi.org/10.1038/s41591-018-0134-3

13.Ma Y, Li Q, Du Y, et al.Blood Tumor Mutational Burden as a Predictive Biomarker in Patients With Advanced Non-Small Cell Lung Cancer (NSCLC).Front Oncol2021;11:1576.Available at: http://doi.org/10.3389/fonc.2021.640761

14.Chen X, Fang L, Zhu Y, et al.Blood tumor mutation burden can predict the clinical response to immune checkpoint inhibitors in advanced non-small cell lung cancer patients.Cancer Immunol Immunother2021;70(12):3513–3524.Available at: http://doi.org/10.1007/s00262-021-02943-2

15.Si H, Kuziora M, Quinn KJ, et al.A Blood-based Assay for Assessment of Tumor Mutational Burden in First-line Metastatic NSCLC Treatment: Results from the MYSTIC Study.Clin CancerRes2020;27(6):1631–1640.Available at: http://doi.org/10.1158/1078-0432.CCR-20-3771

16.Ulz P, Belic J, Graf R, et al.Whole-genome plasma sequencing reveals focal amplifications as a driving force in metastatic prostate cancer.Nat Commun2016;7:12008.Available at: http://doi.org/10.1038/ncomms12008

17.Scherer F, Kurtz DM, Newman AM, et al.Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA.Sci Transl Med2016;8(364): 364ra155.Available at: http://doi.org/10.1126/scitranslmed.aai8545

18.Ulz P, Thallinger GG, Auer M, et al.Inferring expressed genes by whole-genome sequencing of plasma DNA.Nat Genet2016;48(10):1273–1278.Available at: http://doi.org/10.1038/ng.3648

19.Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J.Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin.Cell2016;164(1–2):57–68.Available at: http://doi.org/10.1016/j.cell.2015.11.050

20.Raja R, Kuziora M, Brohawn PZ, et al.Early Reduction in ctDNA Predicts Survival in Patients with Lung and Bladder Cancer Treated with Durvalumab.Clin Cancer Res2018;24(24): 6212–6222.Available at: http://doi.org/10.1158/1078-0432.CCR-18-0386

21.Chaudhuri AA, Chabon JJ, Lovejoy AF, et al.Early Detection of Molecular Residual Disease in Localized Lung Cancer by Circulating Tumor DNA Profiling.Cancer Discov2017;7(12): 1394–1403.Available at: http://doi.org/10.1158/2159-8290.CD-17-0716

22.Abbosh C, Birkbak NJ, Wilson GA, et al.Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.Nature2017;545(7655):446–451.Available at: http://doi.org/10.1038/nature22364

23.Chabon JJ, Hamilton EG, Kurtz DM ,et al.Integrating genomic features for non-invasive early lung cancer detection.Nature2020;580(7802):245–251.Available at: http://doi.org/10.1038/s41586-020-2140-0

24.Chen K, Zhao H, Shi Y, et al.Perioperative Dynamic Changes in Circulating Tumor DNA in Patients with Lung Cancer (DYNAMIC).Clin Cancer Res2019;25(23):7058–7067.Available at: http://doi.org/10.1158/1078-0432.CCR-19-1213

25.Horn L, Whisenant JG, Wakelee H, et al.Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer.J Thoracic Oncol2019;14(11): 1901–1911.Available at: http://doi.org/10.1016/j.jtho.2019.08.003

26.Barker T, Fulde G, Moulton B, Nadauld LD, Rhodes T.An elevated neutrophil-to-lymphocyte ratio associates with weight loss and cachexia in cancer.Sci Rep2020;10(1):7535.Available at: http://doi.org/10.1038/s41598-020-64282-z

27.Chen J, Wu J, Lv X, Yang Q, Chen J, Zhang D.The value of red blood cell distribution width, neutrophil-to-lymphocyte ratio, and hemoglobin-to-red blood cell distribution width ratio in the progression of non-small cell lung cancer.Chalmers J, editor.PLoS One2020;15(8):e0237947.Available at: http://doi.org/10.1371/journal.pone.0237947

28.Zhu X, Song H, Chen Y, Han F, Wang Q, Cui Y.Neutrophil-to-Lymphocyte Ratio and Platelet-to-Lymphocyte Ratio in Blood to Distinguish Lung Cancer Patients from Healthy Subjects.Foddis R, editor.Dis Mark2020;2020:1–5.Available at: http://doi.org/10.1155/2020/8844698

29.Simonaggio A, Elaidi R, Fournier L, et al.Variation in neutrophil to lymphocyte ratio (NLR) as predictor of outcomes in metastatic renal cell carcinoma (mRCC) and non-small cell lung cancer (mNSCLC) patients treated with nivolumab.Cancer Immunol Immunother2020;69(12):2513–2522.Available at: http://doi.org/10.1007/s00262-020-02637-1

30.Mandaliya H, Jones M, Oldmeadow C, Nordman IIC.Prognostic biomarkers in stage IV non-small cell lung cancer (NSCLC): neutrophil to lymphocyte ratio (NLR), lymphocyte to monocyte ratio (LMR), platelet to lymphocyte ratio (PLR) and advanced lung cancer inflammation index (ALI).Transl Lung Cancer Res2019;8(6):886–894.Available at: http://doi.org/10.21037/tlcr.2019.11.16

31.Sun S, Qu Y, Wen F, Yu H.Initial neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio as prognostic markers in patients with inoperable locally advanced non-small-cell lung cancer.Biomark Med2020;14(14):1341–1352.Available at: http://doi.org/10.2217/bmm-2019-0583

32.Russo A, Russano M, Franchina T, et al.Neutrophil-to-Lymphocyte Ratio (NLR), Platelet-to-Lymphocyte Ratio (PLR), and Outcomes with Nivolumab in Pretreated Non-Small Cell Lung Cancer (NSCLC): A Large Retrospective Multicenter Study.Adv Ther2020;37(3):1145–1155.Available at: http://doi.org/10.1007/s12325-020-01229-w

33.Kartolo A, Holstead R, Khalid S, et al.Serum neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio in prognosticating immunotherapy efficacy.Immunotherapy2020;12(11):785–798.Available at: http://doi.org/10.2217/imt-2020-0105

34.Kalluri R, LeBleu VS.The biology, function, and biomedical applications of exosomes.Science2020;367(6478):eaau6977.Available at: http://doi.org/10.1126/science.aau6977

35.Shin H, Oh S, Hong S, et al.Early-Stage Lung Cancer Diagnosis by Deep Learning-Based Spectroscopic Analysis of Circulating Exosomes.ACS Nano2020;14(5):5435–5444.Available at: http://doi.org/10.1021/acsnano.9b09119

36.Zhang X, Sai B, Wang F, et al.Hypoxic BMSC-derived exosomal miRNAs promote metastasis of lung cancer cells via STAT3-induced EMT.Mol Cancer2019;18(1):40.Available at: http://doi.org/10.1186/s12943-019-0959-5

37.Salehi M, Sharifi M.Exosomal miRNAs as novel cancer biomarkers: Challenges and opportunities.J Cell Physiol2018;233(9):6370–6380.Available at: http://doi.org/10.1002/jcp.26481

38.Sandfeld-Paulsen B, Jakobsen KR, B?k R, et al.Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer.J Thorac Oncol2016;11(10):1701–1710.Available at: http://doi.org/10.1016/j.jtho.2016.05.034

39.Li MY, Liu LZ, Dong M.Progress on pivotal role and application of exosome in lung cancer carcinogenesis, diagnosis, therapy and prognosis.Mol Cancer2021;20(1):22.Available at: http://doi.org/10.1186/s12943-021-01312-y

40.Tong B, Wang M.Circulating tumor cells in patients with lung cancer: developments and applications for precision medicine.Future Oncol2019;15(21):2531–2542.Available at: http://doi.org/10.2217/fon-2018-0548