Molecular characterization and prognostic significance of circulating tumor cells in patients with non-small cell lung cancer

Introduction

Circulating tumor cells (CTCs) are rare epithelial cells that can be found in the peripheral blood of cancer patients (1). Over the last few years several methods have been developed with the aim of detecting CTCs, though the CellSearch^® (CS, Veridex LLC, Raritan, NJ, USA) and the isolation by size of epithelial tumor cells (ISET, RareCell Diagnostics, Paris, France) represented up to now the most commonly used techniques (2-4).

Recently, Hodgkinson et al. demonstrated that CTCs isolated from small cell lung cancer patients can form tumors in immunocompromised mice with preserved morphological and genetic characteristics, providing evidence for their tumorigenicity (5). Furthermore, Chinese researchers demonstrated that CTCs collected from an individual patient, regardless of the cancer subtypes, exhibit reproducible copy number variation (CNVs) patterns similar to those of the metastatic tumor of the same patient (6).

An increasing body of evidence suggests that CTCs may play a role in non-small cell lung cancer (NSCLC) for diagnosis, biological characterization, disease monitoring and prognostic purposes (7-9). CTCs could be particularly relevant in this clinical setting considering that with standard procedures physicians have difficulty in obtaining an adequate tumor tissue for a comprehensive histopathological-molecular analysis and that patients are not always suitable to receive a re-biopsy, now highly recommended to identify the mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs). In the current review, we will focus on the molecular characterization and prognostic significance of CTCs in NSCLC patients.

Molecular characterization of CTCs

CTCs can be characterized by immunolabeling and molecular analysis such as reverse transcription polymerase chain reaction (RT-PCR), reverse transcription quantitative PCR (RT-qPCR), fluorescent in situ hybridization (FISH), microarray or sequencing (10). In particular in NSCLC patients, especially with adenocarcinoma, molecular characterization of CTCs may permit the assessment of druggable alterations such as EGFR mutations or anaplastic lymphoma kinase (ALK) translocation. Maheswaran et al. performed EGFR mutational analysis on DNA recovered from CTCs using the Scorpion amplification refractory mutation system (SARMS) technology and compared the results with those obtained from concurrently isolated free plasma DNA and from the original tumor-biopsy specimens (11). The expected EGFR activating mutations were observed in CTCs in 11 out of 12 patients (92%) and in free plasma DNA in 4 out of 12 patients (33%). In addition T790M, the main mechanism of acquired resistance to TKIs, was detected in CTCs of patients who had a response to TKIs (33%) and who had clinical progression (64%). The possibility to detect T790M has become clinically relevant in NSCLC patients considering the recent development of specific anti-T790M drugs, such as osimertinib (12). Sundaresan et al. evaluated the presence of T790M mutation in tumor biopsies, CTCs and circulating tumor DNA (ctDNA) of 40 EGFR-mutant NSCLC patients with progressive disease during TKI therapy (13). For all three analytic platforms the overall T790M mutation-positive rate was approximately 50%, with concordance among them ranging from 57% to 74%. However, the combination of CTC and ctDNA analysis permitted to identify the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate. Punnoose et al. conducted a molecular characterization of CTCs and ctDNA in 41 NSCLC patients (14). In this study mutations detected in CTCs, ctDNA and in matched tumor samples were strongly concordant, even though it was observed a greater sensitivity in ctDNA than in CTCs. Breitenbuecher et al. developed a novel highly sensitive and specific assay based on real-time PCR and melting curve analysis to identify the presence of activating EGFR mutations in blood cell fractions enriched in CTC (15). They achieved a 100% detection rate in a pilot cohort of 8 patients with EGFR positive NSCLC. Ran et al. employed magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level (16). Then, they performed a laser cell microdissection to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 deletion, L858R and T790M mutation detection by PCR sequencing. Using this method EGFR mutations were correctly identified in 85% of the CTCs for L858R, 55% for exon 19 deletion and 45% for T790M. In addition, Italian researchers analyzed EGFR mutations by next generation sequencing (NGS) in CTC-enriched samples of 37 advanced NSCLC patients (17). They demonstrated that the CS system coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTCs. Several studies showed also the ability to detect ALK rearrangements in CTCs (18-20). Ilie et al. reported that ALK status can be determined in CTCs isolated from patients with lung adenocarcinoma using a dual immunocytochemistry with an anti-ALK antibody-FISH assay (18). Researchers of the Institute Gustave Roussy (Villejuif, France) evaluated the feasibility to detect ALK rearrangement in CTCs of 32 patients with metastatic NSCLC (18/32 ALK positive) using a FISH method optimized for filters (19). Using a cut-off value of 4 ALK-rearranged CTCs per 1 mL of blood, ALK rearrangements were found in all of the ALK-positive patients and variations in ALK-rearranged CTCs levels were detected in patients being treated with crizotinib. Tan et al. reported high concordance (>90%) of ALK rearrangements assessed by ALK FISH testing in CTCs and tumor tissue samples of 14 ALK positive NSCLC patients (20). We monitored the presence of ALK positive CTCs, using the CS assay and a customized test with an anti EML4-ALK antibody (clone 5A4 abCam), in an ALK positive NSCLC patient during his targeted treatment (21). In this case we observed a correlation between the levels of ALK positive CTCs and the clinical response to crizotinib therapy or the development of resistance to the drug.

Recently the so-called immune checkpoint inhibitors, demonstrated to be able to interrupt inhibitory immune signals and to restore antitumor immune responses due to their interaction with the programmed cell death protein 1 (PD-1), the programmed death-ligand 1 (PD-L1) and the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in several tumors, including NSCLC (22,23). Among these new drugs, pembrolizumab, a humanized IgG4 PD-1 blocking antibody, received approval by the main regulatory authorities for the treatment of NSCLC patients whose tumors express PD-L1. Preliminary experiences have already demonstrated the feasibility of assessing the PD-L1 status in CTCs (24-26).

Prognostic significance of CTCs in NSCLC

CTCs can be detected at all stages of disease in NSCLC patients, though Krebs et al. found that CTC count was higher in NSCLC patients with stage IV compared to patients with stage III (27). The same authors indicated that the presence of >5 CTCs at baseline is predictive of poor prognosis in NSCLC patients receiving standard chemotherapy regimens. Hofman et al. evaluated the prognostic relevance of CTCs detected both by ISET and CS in 210 patients undergoing radical surgery for NSCLC (28). The authors demonstrated a significantly shorter disease free survival (DFS) in patients with preoperative detectable CTCs and suggested that ISET and CS should be considered as complementary methods. Recently, Chinniah et al. reported the results of a CTC monitoring conducted in 48 patients with locally advanced NSCLC treated with chemoradiotherapy (29). At a median follow-up of 10.9 months, 22 patients (46%) experienced a disease recurrence; moreover, 15 out of the 20 evaluable patients showed elevated CTC counts after treatment and two-thirds of them demonstrated a rise in CTCs counts an average of 6 months before radiographic evidence of recurrence. Muinelo-Romay et al. investigated the prognostic significance of CTCs count in 43 advanced NSCLC patients (30). The analysis showed the presence of CTCs in 41.9% of patients at baseline (with 23.2% of them having >5 CTCs); moreover, patients with CTCs >5 at baseline as well as patients presenting increased levels of CTCs during the treatment reported worse progression free survival (PFS) and overall survival (OS). CTCs were assessed also in patients with relapsed NSCLC enrolled in a phase II study of pertuzumab plus erlotinib (14). The authors reported a significant correlation either between higher baseline CTC counts and response to treatment or between decreases in CTC counts and radiographic response evaluated using both by 2[18F]fluoro-2-deoxy-D-glucose positron emission tomographic (FDG-PET) and computed tomographic (CT) imaging. Xu et al. detected CTCs in 47/66 (71.2%) NSCLC patients with the CS system, but they didn’t find any CTC in the control group including healthy volunteers and patients with benign lung disease (31). In addition the authors reported a statistically significant correlation between CTC changes after two courses of chemotherapy and disease progression. Qi et al. investigated the presence of CTCs in 100 patients with locally advanced squamous cell lung cancer (32). In the univariate analysis, CTC count >5 at baseline and CTC count >5 at both time points (before and after one cycle of chemotherapy) were significantly associated with a poor PFS and OS outcome. Other Chinese researchers evaluated the prognostic significance of CTC count in 46 patients with advanced NSCLC (33). In this experience CTCs, which were measured at baseline in all patients and before every chemotherapy cycle in 23 patients, were found in 40 patients (87%). The authors also reported a relationship between a CTC count of >8 at a baseline and a worse prognosis. Recently Milaki et al. showed in a large clinical trial that the detection of CK19mRNA+ CTCs before and after chemotherapy is an adverse prognostic factor in patients with stage IIIB/IV NSCLC (34). Finally, also two meta-analyses explored the prognostic role of CTCs count in lung cancer patients (35,36). In the first meta-analysis, that included data extracted from 27 articles (12 containing survival outcomes) published between the year of 1997 and 2012, both pre and post-treatment CTCs detection in peripheral blood were associated with poor prognosis (35). The second meta-analysis, including data of more than 1,500 NSCLC patients enrolled in 20 studies, confirmed that the presence of CTCs has a negative prognostic significance in terms of OS and PFS (36).

Conclusions

A real time liquid biopsy could be extremely useful for individualized therapy of advanced NSCLC, considering that tissue sampling in lung cancer presents different issues compared to other tumors and that analysis of CTCs/ctDNA, which originated from all potential lesions, could overcome the disadvantages of single site biopsy (37). Moreover, in these patients molecular characterization of CTCs may permit the assessment of druggable genetic abnormalities or the discovery of a molecular disease evolution, while the enumeration of CTCs could become a prognostic biomarker, as well as has already been proved in metastatic breast, colorectal and prostate cancer. On the other hand further advances, regarding in particular standardization of CTCs detection methods, are still needed to improve the use of CTCs in lung oncology practice.

Acknowledgements

None.

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

References

1. Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin. Cancer Res 2004;10:6897-904. [Crossref] [PubMed]
2. Truini A, Alama A, Dal Bello MG, et al. Clinical applications of circulating tumor cells in lung cancer patients by CellSearch system. Front Oncol 2014;4:242. [Crossref] [PubMed]
3. Vona G, Sabile A, Louha M, et al. Isolation by size of epithelial tumor cells: a new method for the immunomorphological and molecular characterization of circulating tumor cells. Am J Pathol 2000;156:57-63. [Crossref] [PubMed]
4. Farace F, Massard C, Vimond N, et al. A direct comparison of CellSearch and ISET for circulating tumor-cell detection in patients with metastatic carcinomas. Br J Cancer 2011;105:847-53. [Crossref] [PubMed]
5. Hodgkinson CL, Morrow C J, Li Y, et al. Tumorigenicity and genetic profiling of circulating tumor cells in small cell lung cancer. Nat Med 2014;20:897-903. [Crossref] [PubMed]
6. Ni X, Zhuo M, Duan J, et al. Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients. Proc Natl Acad Sci USA 2013;110:21083-8. [Crossref] [PubMed]
7. Alama A, Truini A, Coco S, et al. Prognostic and predictive relevance of circulating tumor cells in patients with non small cell lung cancer. Drug Discov Today 2014;19:1671-6. [Crossref] [PubMed]
8. Tartarone A, Rossi E, Lerose R, et al. Possible applications of circulating tumor cells in patients with non small cell lung cancer. Lung Cancer 2017;107:59-64. [Crossref] [PubMed]
9. Wong MP. Circulating tumor cells as lung biomarkers. J Thorac Dis 2012;4:631-4. [PubMed]
10. Lowes LE, Allan Al. Recent advances in the molecular characterization of circulating tumor cells. Cancers 2014;6:595-624. [Crossref] [PubMed]
11. Maheswaran S, Sequist LV, Nagrath S, et al. Detection of mutation in EGFR in circulating lung-cancer cells. N Engl J Med 2008;359:366-77. [Crossref] [PubMed]
12. Mok TS, Wu YL, Ahn MJ, et al. Osimertinib or platinum-pemetrexed in EGFR T790M-positive lung cancer. N Engl J Med 2017;376:629-40. [Crossref] [PubMed]
13. Sundaresan TK, Sequist LV, Heymach JV, et al. Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses. Clin Cancer Res 2016;22:1103-10. [Crossref] [PubMed]
14. Punnoose EA, Atwal S, Liu W, et al. Evaluation of circulating tumor cells and circulating tumor DNA in non small cell lung cancer: association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib. Clin Cancer Res 2012;18:2391-401. [Crossref] [PubMed]
15. Breitenbuecher F, Hoffart S, Worm K, et al. Development of a highly sensitive and specific method for detection of circulating tumor cells harboring somatic mutations in non small cell lung cancer patients. PLoS One 2014;9:e85350. [Crossref] [PubMed]
16. Ran R, Li L, Wang M, et al. Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood. Anal Bioanal Chem 2013;405:7377-82. [Crossref] [PubMed]
17. Marchetti A, Del Grammastro M, Felicioni L, et al. Assessment of EGFR mutations in circulating tumor cell preparations from NSCLC patients by next generation sequencing: toward a real-time liquid biopsy for treatment. PLoS One 2014;9:e103883. [Crossref] [PubMed]
18. Ilie M, Long E, Butori C, et al. ALK-rearrangement: a comparative analysis on circulating tumour cells and tumor issue from patients with lung adenocarcinoma. Ann Oncol 2012;23:2907-13. [Crossref] [PubMed]
19. Pailler E, Adam J, Barthelemy A, et al. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non small cell lung cancer. J Clin Oncol 2013;31:2273-81. [Crossref] [PubMed]
20. Tan CL, Lim TH, Lim TK, et al. Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non small cell lung cancer. Oncotarget 2016;7:23251-62. [Crossref] [PubMed]
21. Aieta M, Facchinetti A, De Faveri S, et al. Monitoring and characterization of circulating tumor cells (CTCs) in a patient with EML4-ALK-positive non small cell lung cancer (NSCLC). Clin Lung Cancer 2016;17:e173-7. [Crossref] [PubMed]
22. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12:252-64. [Crossref] [PubMed]
23. Brahmer JR, Tykodi SS, Chow LQ, et al. Safety and activity of anti PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366:2455-65. [Crossref] [PubMed]
24. Mazel M, Jacot W, Pantel K, et al. Frequent expression of PD-L1 on circulating breast cancer cells. Mol Oncol 2015;9:1773-82. [Crossref] [PubMed]
25. Anantharaman A, Friedlander T, Lu D, et al. Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients. BMC Cancer 2016;16:744. [Crossref] [PubMed]
26. Nicolazzo C, Raimondi C, Mancini M, et al. Monitoring PD-L1 posivitive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor nivolumab. Sci Rep 2016;6:31726. [Crossref] [PubMed]
27. Krebs MG, Sloane R, Priest L, et al. Evaluation and prognostic significance of circulating tumor cells in patients with non small cell lung cancer. J Clin Oncol 2011;29:1556-63. [Crossref] [PubMed]
28. Hofman V, Ilie MI, Long E, et al. Detection of circulating tumor cell as a prognostic factor in patients undergoing radical surgery for non small cell lung carcinoma: comparison of the efficacy of the CellSearch AssayTM and the isolation by size of epithelial tumor cell method. Int J Cancer 2011;129:1651-60. [Crossref] [PubMed]
29. Chinniah C, Aguarin L, Cheng P, et al. Prospective Trial of Circulating Tumor Cells as a Biomarker for Early Detection of Recurrence in Patients with Locally Advanced Non-Small Cell Lung Cancer Treated with Chemoradiation Therapy. Int J Radiat Oncol Biol Phys 2017;98:221. [Crossref] [PubMed]
30. Muinelo-Romay L, Vieto M, Abalo A, et al. Evaluation of circulating tumor cells and related events as prognostic factors and surrogate biomarkers in advanced NSCLC patients receiving first-line systemic treatment. Cancers 2014;6:153-65. [Crossref] [PubMed]
31. Xu YH, Zhou J, Pan XF. Detecting circulating tumor cells in patients with advanced non small cell lung cancer. Genet Mol Res 2015;14:10352-8. [Crossref] [PubMed]
32. Qi Y, Wang W. Clinical significance of circulating tumor cells in squamous cell lung cancer patients. Cancer Biomark 2017;18:161-7. [Crossref] [PubMed]
33. Zhang Z, Xiao Y, Zhao J, et al. Relationship between circulating tumour cell count and prognosis following chemotherapy in patients with advanced non small cell lung cancer. Respirology 2016;21:519-25. [Crossref] [PubMed]
34. Milaki G, Messaritakis I, Koinis F, et al. Prognostic value of chemotherapy-resistant CK19mRNA-positive circulating tumor cells in patients with advanced/metastatic non-small cell lung cancer. Cancer Chemother Pharmacol 2017;80:101-8. [Crossref] [PubMed]
35. Ma XL, Xiao ZL, Liu L, et al. Meta-analysis of circulating tumor cells as a prognostic marker in lung cancer. Asian Pac J Cancer Prev 2012;13:1137-44. [Crossref] [PubMed]
36. Wang J, Wang K, Xu J, et al. Prognostic significance of circulating tumor cells in non-small-cell lung cancer patients: a meta-analysis. PLoS One 2013;8:e78070. [Crossref] [PubMed]
37. Miyamoto DT, Ting DT, Toner M, et al. Single-Cell Analysis of Circulating Tumor Cells as a Window into Tumor Heterogeneity. Cold Spring Harb Symp Quant Biol 2016;81:269-74. [Crossref] [PubMed]