Emerging uses of circulating tumor DNA in advanced stage non-small cell lung cancer

Introduction

Lung cancer remains the leading cause of cancer-related death in the United States (1). The majority of patients with non-small cell lung cancer (NSCLC) are diagnosed with metastatic disease, which is generally fatal (1,2). Recent research has revealed that patients with metastatic NSCLC constitute a heterogeneous group. For a minority of patients with advanced NSCLC whose tumors harbor specific genetic aberrations detected by next generation sequencing and other molecular analyses, administration of oral tyrosine kinase inhibitors (TKIs) can result in improved clinical outcomes (3-5). As a result, epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) directed therapies are now recommended for first-line treatment of advanced NSCLC patients harboring appropriate genetic abnormalities (6). The expanding number of somatic genomic targets and recent availability of second- and third-generation TKIs specific to certain resistance mutations have prompted reflex molecular profiling of tumor tissue to become commonplace both at diagnosis and at disease progression in order to characterize these oncogenic drivers and mechanisms of resistance and help guide appropriate treatment selection.

Tissue biopsies have long been the gold standard for determining the genetic profile of a patient’s cancer, but tissue biopsies are often difficult to obtain safely (7). Furthermore, genetic testing of tumor tissues can take several weeks, ultimately delaying initiation therapy whether or not a genetic aberration is identified. Although TKIs are initially very effective in most patients whose tumors harbor a genetic aberration, the majority of patients will eventually develop resistance to these agents within a year of their initiation (8-10). The mechanism of resistance is in many cases unknown and is a matter of intensive research. Some resistance mechanisms, such as the T790M acquired resistance mutation in EGFR, are better characterized and can specifically direct future treatments with drugs such as osimertinib (11,12). Many patients, however, have less common or unknown resistance mechanisms that cannot be directly targeted with FDA approved therapies (9,10,13). Repeat molecular analysis at the time of progression is therefore essential to identify the mechanism of resistance and potentially new treatment approaches.

Tissue biopsies are challenging to obtain, sometimes associated with adverse events, and expensive. Clonal heterogeneity, whereby different clones may harbor distinct resistance mechanisms (14,15), adds further complexity; it is simply not feasible to biopsy every tumor in a given patient. Consequently, there is growing interest in analyzing tumor material and other tumor biomarkers in various bodily fluids, so-called “liquid biopsies”, as a way to more easily and, if necessary, serially detect molecular alterations during a patient’s treatment course. In addition to being easier to obtain than a tissue biopsy, liquid biopsies offer the hope of providing a more complete, integrated picture of a patient’s cancer compared with conventional tissue biopsy of a single tumor site since bodily fluids may contain tumor material released from multiple disease sites. Analysis of cancer cell derived DNA from circulating tumor DNA (ctDNA) enables real-time molecular monitoring of cancer and demonstrates great promise toward realizing personalized cancer care in NSCLC.

ctDNA consists of short, double-stranded DNA fragments that are shed into the blood stream by tumor cells undergoing apoptosis or necrosis (16). ctDNA has a relatively short half-life (approximately 2 hours) and often represents a small fraction (<1.0%) of the total cell-free DNA (cfDNA) circulating in the bloodstream. It is distinguished from normal cfDNA by the identification of somatic mutations present in the cancer genome (17). Highly sensitive genotyping assays such as digital polymerase chain reactions (PCR) (18); beads/emulsion/amplification/magnetics (BEAMing) (19), and next-generation sequencing (NGS) technologies (20) have enabled the accurate detection of rare mutant variants present in body fluids. Several studies have demonstrated that ctDNA can be readily detected in patients with NSCLC, with improved sensitivity in patients with advanced stage disease and those with a higher burden of disease (21,22). ctDNA analysis allows clinicians to non-invasively interrogate tumor specific molecular alterations without the need for a traditional tissue biopsy. ctDNA technology can be utilized for identifying actionable mutations at diagnosis, tracking genomic evolution and the development of resistance mutations to targeted therapies, and monitoring response to therapy.

Detecting mutations in ctDNA

Limited panels

The initial panels developed to characterize a patient’s tumor through liquid biopsies used digital PCR to detect hotspot mutations in the most clinically relevant genes. Sacher et al. looked prospectively at the feasibility and concordance of digital PCR to detect EGFR exon 19 deletion, L858R, T790M and KRAS G12X in the peripheral blood of patients with NSCLC compared to tissue biopsy. Plasma testing had an excellent sensitivity, with a high concordance with tissue biopsy. The authors also noted a significant advantage of plasma testing over tissue biopsy—the test turnaround time for digital PCR was a median 3 days. This compared favorably to tissue biopsy, which had a median turnaround time of 12–27 days (22).

Next generation sequencing

Although limited panels using PCR are fast, they are unable to detect clinically important rearrangements such as those occurring in the ALK, RET and ROS1 genes. In addition, they limit the detection of less common resistance mutations, as genetic pathways less commonly associated with resistance may not be in a limited panel. Targeted NGS of ctDNA can detect single nucleotide variants, fusions and insertions/deletions in a more comprehensive fashion. Targeted NGS of ctDNA has been shown to provide a high degree of accuracy across multiple tumor types (23,24). A study by Paweletz et al. investigated a NGS ctDNA panel in 48 NSCLC patients and found a sensitivity of 77% across 62 known driver and resistance mutations (25). In a recent study done by our group, we found that ctDNA NGS testing in a population enriched for patients likely to have useful reporter mutations was technically successful in 102 patients, while tissue testing was technically successful in 50 patients. The most common reasons for unsuccessful tissue analysis were inaccessible tissue and inadequate DNA in the sample. ctDNA analysis was able to detect 50 driver mutations, 12 resistance mutations, and mutations in 22 additional genes that could serve as potential therapeutic targets. The concordance between ctDNA NGS testing and tissue testing of the most common mutation detected, EGFR, was 79% (26).

In our study, ctDNA NGS testing was able to detect resistance mutations in eight patients who developed progressive disease while on targeted therapy, but who were not able to have additional tissue sequencing (26). FDA-approved targeted therapies were identified for 32% of the patients who had ctDNA testing. This proportion likely understates the utility of such an approach, however, as 55% of patients had a targetable genetic aberration, including both patients with FDA approved targeted therapies and those genetic aberrations for which published data may justify off-label use. When one includes all genetic aberrations currently being evaluated in ongoing clinical trials, up to 70% of tested patients had a genetic aberration that could be targeted. Considering the heterogeneous and diverse mechanisms of resistance to targeted therapies, the theoretical benefit of a NGS approach in this setting becomes clear; by casting a wider net, we can identify targets that may be useful in clinical care.

Clinical factors may impact the sensitivity of ctDNA in NSCLC. Two separate studies have shown that ctDNA was more likely to be detected among patients with squamous cell carcinoma than adenocarcinoma (27,28). This is thought to be due to a higher rate of tumor necrosis and passive DNA release among patients with squamous cell carcinoma. A high Ki67 proliferative index as well as pre-operative 18F-fluoro-deoxyglucose (FDG) avidity has also been associated with a higher rate of ctDNA detection (28).

Concordance of plasma, urine, and tissue testing

Testing for ctDNA can be done accurately in both urine and plasma. This has been shown in multiple studies looking at the sensitivity of ctDNA in the urine and plasma to detect T790M mutations in patients with EGFR mutations that are developing resistance to a 1^st-line TKI.

In a subset analysis of a clinical trial of rociletinib, Wakelee et al. evaluated the concordance between tissue, plasma and urine testing for T790M mutation among 242 patients with NSCLC harboring an EGFR mutation. Among the 189 patients positive for T790M by plasma, 175 were also positive on tissue biopsy (81.5% concordance between plasma and tissue testing). There were similar results in the group that had ctDNA testing of the urine, with 83.8% concordance between urine and tissue testing. Regardless of how the T790M mutation was detected the overall response and duration of response for treatment with rociletinib were similar (29).

In a series of patients with EGFR mutant NSCLC being treated with osimertinib, discordant results were evaluated further. Patients who were plasma negative and tissue positive for T790M had an ORR of 69% and progression free survival (PFS) of 16.5 months, whereas patients negative for T790M in both the plasma and tissue had inferior outcomes (ORR, 25%; PFS, 2.8 months). Patients with plasma positive and tissue negative tumors had an intermediate outcome, with an ORR of 28% and PFS of 4.3 months. The relatively high sensitivity of plasma testing (30% false negative rate) implies that a significant portion of patients can avoid tissue biopsy. It remains important to emphasize, however, that patients who were plasma negative still could benefit from osimertinib therapy if tissue biopsy was positive. As such, tissue biopsies remain a vital component of our diagnostic paradigm (30).

Discordant results remain a major clinical quandary—should a patient receive the targeted therapy detected by one test if the other test indicates a lack of potential benefit? While we have emerging data for patients with EGFR mutations suggesting that there is clinical benefit to act on the positive test result regardless of its source, this problem potentially affects all targeted therapies. It is already fairly evident that test concordance according to DNA source will not be uniform. The cause of tissue/ctDNA discordance remains unclear. One potential mechanism may be increased sensitivity of ctDNA assays, as compared to tissue assays. In a study by our group, 60% of detected aberrations in ctDNA had an allelic frequency less than 4%, which is below the threshold at which DNA sequencing at our institution can detect these same genetic alterations in tumors (26). Management of such patients can be challenging, as tissue testing is considered the gold standard.

Another source of discordance between tissue and plasma may be tumor heterogeneity. Emerging data indicate that resistance to a TKI can occur in a heterogeneous fashion (15). Biopsy of a progressing lesion, for example, may reveal a different resistance genotype than a relatively indolent lesion. Given the inherent difficulty in obtaining tissue biopsies, most trials that require biopsies for enrollment do not specify that the biopsy must be from an actively growing lesion. Thus, we do not know if the tissue biopsies obtained in the Oxnard series, for example, were from the most accessible disease site (as is usually the case) or from a progressing lesion. Another potential explanation for discordant results is differential ctDNA secretion based upon the nature of the mutation. A recent study revealed that tumor subclones harboring classical oncogenic driver mutations (e.g., EGFR) had a higher rate of ctDNA secretion than mutations in genes associated with the cell cycle (31). Further research is required to clarify the origins, significance and management of discordant results.

Serial testing

The detection of mutations by ctDNA may have utility beyond guiding selection of targeted therapies for patients. In patients with known targetable mutations on appropriate therapy, serial testing of ctDNA for the mutation can help assess treatment response. A study by Marchetti et al., for example, assessed ctDNA in patients with EGFR mutant NSCLC at baseline and then serially throughout therapy. They observed that patients who experienced a greater than 50% decrease in the allelic fraction of the EGFR variant within 14 days of treatment initiation had a better response to treatment. They also noted that absence of plasma clearance of the EGFR variant at 60 days was associated with the development of T790M mutations, and thus likely resistance to 1^st and 2^nd generation TKI’s (32). Subsequent studies have confirmed that clearance of EGFR variant ctDNA is associated with improved cancer outcomes (22,33).

In a study of patients with BRAF V600 mutated malignancies receiving targeted therapies, Janku et al. found that those patients who were plasma negative/tissue positive for BRAF V600 at baseline had a longer time to treatment failure (TTF) than those patients with circulating BRAF V600 on plasma testing. They also revealed that those patients who had a reduction in BRAF V600 ctDNA with treatment had a longer TTF than those whose plasma BRAF V600 levels rose or did not change (34). Serial testing may also be useful in an individual patient; Peled et al. recently described a case in which a patient’s tumor harbored multiple mutations in EGFR. The patient was treated with osimertinib for T790M positive disease, and enjoyed an excellent response aside from a progressive liver lesion. ctDNA levels of T790M fell, but the allelic fraction of a rarer EGFR mutation (G724S) increased. A biopsy of the liver lesion confirmed this tissue was T790M negative and G724S positive. Given an overall positive response to osimertinib and the documented sensitivity of G724S to afatinib, the patient was started on combination osimertinib/afatinib. The ctDNA level of G724S decreased substantially as the patient responded (35). Finally, emerging data indicate that changes in ctDNA may precede radiographic or clinical changes (17,21). Indeed, one patient described by our group experienced ctDNA progression 3 months prior to radiographic progression (26). Whether one should use progression detected by ctDNA as an indicator to alter therapies before radiographic progression remains an open question.

Another clinical space where serial testing may be useful is among patients who have completed definitive intent therapy. In a recent series by Abbosh et al., patients with lung cancer were followed with serial ctDNA analysis from diagnosis to death. They found that detection of a ctDNA heralded relapse in 13/14 patients. In addition, they found that an increase in ctDNA while on adjuvant chemotherapy was correlated with resistance and early relapse. In contrast, decreased (and undetectable) ctDNA with adjuvant chemotherapy was associated with prolonged disease-free survival (28). Another recent cohort of 41 patients with NSCLC treated with curative intent treatment confirmed the potential prognostic role of ctDNA. The detection of ctDNA had a 100% positive predictive value for disease progression, whereas the absence of ctDNA had a 93% negative predictive value for the absence of progression (36). These data imply that serial testing for ctDNA after definitive intent therapy may allow clinicians to more accurately counsel patients.

Prognostic value of ctDNA concentrations

Although the ability to detect molecular alterations in ctDNA currently has the most clinical utility, the earliest studies examined the prognostic value of the total concentration of cfDNA. In 2004 Gautschi et al. looked at plasma and serum cfDNA in NSCLC patients before the start of chemotherapy and after 1–2 cycles of chemotherapy. Increased cfDNA level after chemotherapy was associated with poor survival (37). Cargnin et al. published a meta-analysis of studies looking at cfDNA concentrations and clinical outcomes in lung cancer patients. They included 16 studies (1,723 patients) in the overall survival (OS) analysis and 5 studies (640 patients) in the PFS analysis. A higher baseline cfDNA concentration correlated with a statistically significant increase in the risk of death (HR 1.76, P<0.001). They were not, however, able to reach consensus on a cfDNA concentration cutoff point that portends a higher risk of poor outcomes given the heterogeneity of tests and reference genes that were used to detect the ctDNA (38).

In the study done by our group, a cfDNA concentration greater than 3 ng/µL was significantly associated with decreased OS; median OS for cfDNA greater than 3 ng/µL was 24 versus 46 months for cfDNA less than 3 ng/µL (P<0.01). This result remained significant when adjusted for age, performance status, EGFR mutation status, and number of metastatic sites (26).

Role of ctDNA in immunotherapy

Immunotherapy is changing the treatment landscape for NSCLC. Currently available biomarkers for the response to immunotherapy such as programmed-death ligand 1 (PD-L1) staining have significant limitations (39). Nonsynonymous tumor mutation burden (TMB) is emerging as a more reliable biomarker of response to immunotherapy in multiple tumor types, including NSCLC (40,41). Given the limitations of tissue samples in lung cancer, there is often not enough tumor to perform clinically validated tests. If mutational burden could be assessed by sampling ctDNA, this would be a great advance towards patient selection in immunotherapy. This is particularly complex though, as the gold standard for TMB analysis is whole exome sequencing. Whole exome sequencing is not feasible using current ctDNA technology. A recent study of 23 patients with advanced NSCLC revealed that in a preliminary analysis TMB in ctDNA was associated with responses to immunotherapy (42).

Use of ctDNA in lung cancer screening

The National Lung Screening Trial found that annual low dose computed tomographic (CT) scans of the chest decrease mortality among patients at high risk for the development of lung cancer (43). False positive results on CT scans, however, represent a significant limitation of screening. There has been interest in applying cfDNA technology in order to improve the accuracy of lung cancer screening, but this has not been borne out in subsequent clinical trials. In 2009 Sozzi et al. enrolled 1,025 heavy smokers to receive annual low-dose spiral CT along with serial plasma cfDNA quantification by PCR over 5 years. Assessment of plasma DNA did not improve the accuracy of lung cancer screening—those patients who went on to develop cancer did not have higher cfDNA levels than those who did not develop cancer during the study (44). One potential limitation to using cfDNA levels to help detect early lung cancers is that cfDNA levels can be increased in patients with conditions other than lung cancer, such as liver disease, diabetes, cardiovascular disease, non-neoplastic lung diseases and infections (45,46).

Conclusions

Treatment paradigms in advanced NSCLC have changed considerably in recent years; targeted and immune directed therapies provide new hope in an otherwise lethal disease. As we find more ways to target the mutations driving tumor growth, we will need better access to tumor genetic materials that can help guide clinical decisions. ctDNA has emerged as a potential solution to this dilemma. Given the superior outcomes of patients on appropriate targeted therapy in advanced NSCLC this advance in personalized treatment holds great promise. To validate the analysis of ctDNA for more widespread use, we will need continued refinement and standardization of the technology as well as rigorous prospective clinical trials. In Figure 1, we describe how cfDNA may be applied in the management of NSCLC.

Figure 1 Potential Roles of cfDNA in NSCLC. cfDNA, cell-free tumor DNA; NSCLC, non-small cell lung cancer; CT, computed tomography.

Acknowledgements

None.

Footnote

Conflicts of Interest: C Aggarwal: Consultant Services: Roche, BMS, Eli Lilly; Research/Grant support: Takeda, Macrogenics, Roche. E Carpenter: Commercial research support from Janssen/Johnson & Johnson. RB Cohen: Consultant Services: Takeda, Zymeworks, BMS. CJ Langer: Consultant services: Bristol-Myers Squibb Company; ImClone Systems Incorporated; Pfizer Inc.; Eli Lilly and Company; AstraZeneca Pharmaceuticals LP; Novartis Pharmaceuticals Corporation; Genentech, Inc.; Bayer HealthCare Pharmaceuticals/Onyx Pharmaceuticals, Inc.; Celgene; Abbott Laboratories; Biodesix; Clariant; CarisDx; ARIAD Pharmaceuticals, Inc.; Boehringer Ingelheim Pharmaceuticals, Inc.; Synta Pharmaceuticals Corp; Clovis Research/grant support: Pfizer Inc.; Eli Lilly and Company; GlaxoSmithKline; Clovis; Merck; Nektar; Advantagene; Inovio; Ariad; Celgene; DSMC member: Amgen, Synta, Peregrine, SWOG, Incyte, Lilly CME: PIK; PER; NOCR; CCO; RTP, MLG. J Bauml: Consultative Services: Clovis, BMS, Astra Zeneca, Celgene, Merck, Genentech, Guardant Health, Boehringer Ingleheim; Research/Grant Support: Merck, Incyte, Carevive Systems, Novartis, Bayer. M Marmarelis, JC Thompson and TL Evans have no conflicts of interest to declare.

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