Liquid Biopsy: A New Strategy for Future Directions in Lung Cancer Treatment

Abstract

The gold standard for cancer diagnosis has always been based on radiological imaging followed by surgical tissue biopsies for molecular testing and pathological examination and surgical resection to remove the tumoral mass when possible. However, the resulting information is a limited snapshot in space and time, which poorly reflects clonal heterogeneity or tumor evolution and metastasis. Over a decade since its inception, the ability to use non-invasive methods such as a liquid biopsy to analyze tumor biomarkers has transformed the vision of future cancer care into a better patient experience thanks to real-time monitoring and early diagnosis. The liquid biopsy essay is an effective tool for detecting cancers at an early stage, when there are very few tumor-derived materials circulating in the bloodstream, being a very sensitive technique. For this reason, liquid biopsy is particularly suitable for early-stage diagnosis (stage I or II) of lung cancer whose diagnosis often occurs in the final stages of the disease as well as monitoring cancer progression and driving target therapies.

1. Introduction

1.1 An early opportunity to catch cancer

A cancer patient’s torturous journey begins with formulating the diagnosis. The detection of tumor mass, the stage, and the molecular profile are all clues that lead to the proper treatment. The advent of new technologies allows us to deepen our knowledge of molecular data useful to physicians to guide them toward specific therapies.

Cancer is the World’s second biggest killer after heart disease, in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Currently, 90% of cancer patients do not die from the primary tumor but are killed by its distant metastases. Current treatment of patients with metastatic cancer is generally driven by the molecular characteristics of the primary tumor. Detection and monitoring of the disease are carried out with tissue sampling in a common and invasive difficult way for patients with solid tumors. Recently, sequential peripheral blood tests have been introduced as a non-invasive technique, resulting in the use of liquid biopsy [1]. Liquid biopsy refers to a test, usually carried out from blood samples, to analyze tumor molecular biomarkers that can diagnose cancer and inform clinical decision-making [1].

Here, we will explore the possible molecular biomarkers that can be used:

• The circulating tumor cells (CTCs) are released from both primary and metastatic tumor sites into the bloodstream. Tumor cells are recognized by the shape and/or physical elements such as size, density, electric charges and deformability and biological characteristics, cell surface protein expression and viability [1]. CTCs have a short half-life, between 1 and 2.5 hours and the process by which the CTCs are released into the bloodstream is not yet well understood. Although their role in metastasis remains poorly undigested, it is highly probable that CTCs are the precursors of the different metastatic populations [2] even if there are less than 10 CTCs per 7.5 ml of blood [3]. For this reason, CTCs require detection and enrichment processes being caught through positive (which relies on antibodies capturing the surface tumor antigen expressed on the CTCs) or negative (removing the other blood components using size filtration) selection. CTCs can be analyzed in a multidimensional characterization, at protein, DNA, and RNA level.

• The Circulating Tumor DNA (CtDNA) is the fragmented tumoral DNA that can be detected in the bloodstream, derived from apoptotic and necrotic tumor cells. During the normal apoptosis processes (programmed cell death) or necrosis (cell trauma—premature death), the cell undergoes a series of morphological changes, and the chromatin is condensed and degraded into small fragments, approximately 200 bp in length, and released circulating in the blood. These DNA fragments are also known as cell-free DNA (cf-DNA) and can be acquired by isolating DNA from plasma or serum. Nowadays, next generation sequencing (NGS)-based analyses and digital PCR (dPCR)-based methods are the most frequently used to detect ctDNA.

The analytes mentioned above are the most studied as they are detected more easily alone or in combination, but exosomes, RNA, extracellular vesicles and, last but not least, methylation must also be counted among others.

To date, the gold standard for cancer analysis in clinical practice is tissue biopsy. This implies that, despite the advantages of liquid biopsy that immediately appear very clear, it is necessary to demonstrate that this non-invasive approach is actually better than the current one. The greatest limitation of all tissue biopsies is the lack of representativeness of tumor heterogeneity and plasticity. Tumors are highly heterogeneous, even down to the single cell level, and their characteristics change over time and under treatment pressure. The recovery of the sample through tissue biopsy is a highly invasive practice for the patient and is not easily repeatable over time, thus making the information of the data obsolete over time. For its part, the liquid biopsy can be defined as minimally invasive and allows the monitoring of the evolution of the tumor characteristics over time thanks to the possibility of being able to repeat the practice of blood sampling and in a sequential way also during the course of treatment (Figure 1).

Figure 1.

Liquid biopsy versus tumor biopsy for clinical-trial recruitment [4].

Even if the initial approach leads us to think of using the liquid biopsy rather than the tissue biopsy, we should start thinking of starting to collect different data deriving from both approaches to really have clear and comprehensive information that guides cancer treatment.

1.2 The potential use of liquid biopsy through the patient’s journey

Thanks to the sensitivity and specificity achievable with high-depth sequencing, liquid biopsy can be used for the early diagnosis of cancer, and in the next future also as a screening in healthy people. To justify this use, however, it is necessary to demonstrate that the tests are better than those currently in use. Indeed, some studies have shown that ctDNA analysis was able to diagnose lung cancer in the early stages (stage I or II) [5], as well as the detection of some mutations (i.e. TP53 or KRAS genes) which is possible many years earlier, when the individual is still asymptomatic, compared to the time of the classic diagnosis [6].

Additionally, liquid biopsy can be extremely useful in detecting residual molecular disease (MRD) after treatment. In fact, it is very common that after specific medical treatments the current radiological imaging techniques are not able to detect the MRD [7] responsible for relapse.

However, before liquid biopsy enters the clinical routine, several clinical trials are needed for normalization and experimentation and would help answer several unsuspended questions. In fact, clinical trials allow facing challenges such as [8]:

• reproducibility and sensitivity.

• distinguish positive results from negative results.

• biomarker efficacy.

A recent study [9] shows that at the moment the analyses of cfDNA are mainly included in clinical trials for cancers that have a higher incidence and mortality such as lung, breast, and colon cancer. Additionally, the study found some main drawbacks that were common among several trials that risk demoralizing and/or confusing the patient:

• The lack of consensus on terminology

• The false-positive rate: normal cells may have tumor-related mutations

• Lack of a standardized protocol for evaluating cfDNA: hinders the routine use of liquid biopsy in laboratories as an ordinary test

• Lack of numerous cohorts that allow the standardization of the data

1.3 The liquid biopsy for non-small cell lung cancer

Lung cancer is the leading cause of cancer death in industrialized countries. In the USA it represents the leading cause of death in men and has now passed breast cancer in females leading to first place in mortality. For non-small cell lung cancer (NSCLC), treatment decisions follow the assessment of the staging of pathological node tumor metastases (pTNM). The more advanced the clinical stage, the more this is associated with the risk of death. However, it is estimated that only 40% of patients have stage 2–3 and have a minimal residual disease (MRD), and this means that the remaining 60% are likely to be over-treated with the possibility of giving rise to high toxicity risks. Somatic molecular alterations in NSCLC can lead to oncogene activation through multiple genetic mechanisms (point mutations, insertions, deletions, gene rearrangements, etc.) and the treatment of cancer has thus evolved from broad chemotherapeutic approaches to therapies targeted against specific molecular abnormalities that drive tumor growth. A robust and accurate assessment of molecular alterations within tumor cells is mandatory in routine clinical practice to determine which patients are suitable for these targeted therapies.

The TRACERx study (Tracking Cancer Evolution through Therapy) is a British national observational study for patients with NSCLC who have undergone surgery. Through this study, they try to evaluate the natural history of the evolution of the disease in order to understand the biology of MRD when it is impossible to access a tissue biopsy again. By monitoring 30 tumor variants, they were able to identify cases of disease recurrence by detecting MRD prior to clinical surveillance. They are currently looking to implement over 200 variants and limit of detection (LOD) of the technique in well over 1000 plasma samples. Therefore, thanks to this pioneering study it is possible to use two approaches:

• the first allows using the ctDNA as a biomarker after the surgery of a patient affected by NSCLC in two temporal points in order to detect the MRD and to enroll the patient in combined therapy;

• the second approach is MRD surveillance to identify those patients who already have a relapse in order to intervene immediately with the therapy in a time.

Historically, the first clinical application of liquid biopsy in advanced NSCLC was the detection of EGFR mutations (Figure 2). From these pioneering studies, the scientists moved on to the analysis of next-generation sequencing (NGS) which allowed expanding the investigation to other driver mutations as well, that could provide prognostic and predictive information [10].

Figure 2.

Timeline of the development of liquid biopsy [10].

The first methods used were those of RT-PCR (real-time PCR) capable of detecting EGFR mutations and at the moment the only one approved by the FDA for the detection of the T790M resistance mutation of the EGFR gene [11]. Indeed, in 2016, the Food and Drug Administration (FDA) approved the liquid biopsy test for patients with NSCLC to verify EGFR-targeted therapy, while a European consortium from the European Liquid Biopsy Academy (ELBA) using biomarkers such as ctDNA, CTC, exosomes, and tumor educated platelets (TEP) [12].

Currently, ESMO guidelines recommend testing at least EGFR mutations, BRAF mutations, ALK fusions, ROS-1 fusions, MET exon 14 skipping mutations, RET rearrangements and PD-L1 expression levels in non-squamous advanced NSCLC [13]. This panel could be further implemented considering KRAS mutations, HER2 mutations, MET amplification, and NTRK rearrangements [13].

2. Conclusion

In conclusion, we can state that the liquid biopsy is significantly helping the management of patients with lung cancer by crossing the threshold of the use of off-label drugs in therapeutic pathways [14], but we must be aware that liquid biopsy cannot replace the PDL-1 expression assay for investigation of the tumor immune microenvironment, as well as cytological analysis of tissue biopsy. Therefore, in order to obtain the most complete treatment possible, we must consider liquid biopsy not as a competitive approach to the already existing ones, but as another valid mutation detection option.

Declaration

The authors have no conflicts of interest.