ABSTRACT

Introduction

The yield of adding plasma-based next-generation sequencing (NGS) to tissue NGS for the detection of actionable aberrations (AAs) has been reported; however, additional studies are needed to determine utility in the clinical setting. In this retrospective data review, we present our real world data on the utilization of liquid biopsies in the routine management of NCSLC patients, in a community setting.

Methods

We conducted a retrospective review of 279 consecutive patients with non-small cell lung cancer (NSCLC) in the community setting, who had liquid biopsies performed between the years 2014 and 2019 as part of routine clinical management.

Results

Over a period of 5 years, 337 liquid biopsy samples, taken from 279 patients were sent for plasma NGS testing. The median age at diagnosis was 73 years (range 36–93 y, SD 10.4 y), 141, (51%) were men and 138 (49%) were women. The majority were White or Caucasian (80% versus 8% Black or African American versus 12% Multiracial or unknown race) and had a history of smoking (79%). Excluding synonymous mutations and variants of unknown significance, 254 AAs were detected in 106 patients. Commonly detected AAs were EGFR (n = 127, 50%), KRAS (n = 61, 24%), BRAF (n = 24, 9.5%), and MET (n = 23, 9%). Tissue NGS detected AAs in 45 patients, with EGFR (n = 28, 57%) and KRAS (n = 10, 20%) being the most common AAs. Concordance agreement between plasma and tissue NGS modalities was detected in 39 of 45, 87% patients and was demonstrated most commonly in EGFR (n = 25) and KRAS (n = 11). In 44 of 106 (42%) of patients, for whom tissue NGS was not performed, additional precision treatment was guided by the AA detected through liquid biopsy.

Conclusions

Integration of liquid biopsy into the routine management of patients with non-small cell lung cancer demonstrated AA detection in 44 additional patients, which comprise a 42% increase in AA detection rate, when tissue NGS was not performed.

INTRODUCTION

Nonsmall cell lung cancer (NSCLC), the most common type of lung cancer, remains an important contributor to global burden of disease and leading cause of cancer mortality.[1] The introduction of precision medicine with targeted therapies directed toward driver genetic aberrations has changed the treatment paradigm in NSCLC and has suggested possible benefits small observational studies, when compared with standard chemotherapy.[24] The challenge remains in effectively extracting tumor DNA and expeditiously detecting actionable aberrations (AA) to guide treatment decisions.

Tissue biopsy is considered the gold standard for harvesting tumor DNA. Recent advancements in cancer research have introduced liquid biopsy as a surrogate modality.[58] Liquid biopsy describes the extraction of circulating tumor DNA (ctDNA) from patients' plasma and analyzing it using different techniques, namely, next-generation sequencing (NGS), to identify AA that impart tumorigeneses or install resistance to therapy. Liquid biopsy has been shown to facilitate diagnosis, prognosis, disease monitoring, and targeted therapy in several studies.[912] The TRACERx study[6] detected cancerous ctDNA in plasma as early as 6 months before cancer diagnosis, making liquid biopsy a potential screening modality in patients who are yet to show clinical or radiologic signs of disease. Furthermore, as a minimally invasive modality, liquid biopsy carries substantially lower risk to patients when compared with tissue biopsy, and liquid biopsy has potential to serve as a surrogate option for AA detection when tissue biopsy is contraindicated or not performed.[8,11]

Conversely, liquid biopsy has important disadvantages, namely test sensitivity that reaches 85% at most[12] and cost. CtDNA is found in rare concentrations in the plasma and false-negative results are prevalent, particularly when tumor burden is low. Liquid biopsy carries high cost and variable insurance coverage, requiring careful patient selection.

METHODS

In this retrospective data analysis, we included 279 patients with NSCLC who had liquid biopsy ordered between November 2014 and July 2019, as part of routine clinical management. The study was approved by Mount Sinai Medical Center institutional review board committee. Plasma NGS was performed using the Guardant360 platform, and tissue NGS was performed using two different vendors, Caris Life Sciences and Foundation Medicine, chosen at the discretion of the physician. Both modalities targeted the following AAs, fusions and translocations: EGFR, MET, BRCA1, BRCA2, KRAS, RET, ERBB2, BRAF, ROS1, ALK, and NTRK2.

Liquid biopsy and ctDNA Isolation

A 10-mL blood samples were collected in Streck tubes and shipped to Guardant Health. The plasma was isolated by centrifugation of blood by 1600g for 10 minutes at 48°C. Using the QIAamp circulating nucleic acid kit (Qiagen), ctDNA was then extracted, concentrated, and size selected using Agencourt AMPure XP beads (Beckman Coulter), and quantified by Qubit fluorometer (Life Technologies).

ctDNA Sequencing

After isolation and oligo-nucleotide indexing, ctDNA digital sequencing library was performed. The library was amplified and enriched for target genes. After sequencing, algorithmic reconstruction of the digitized sequencing signals were used to reconstruct and subsequently stored in a secure database for analysis.

Statistical Analysis

Simple descriptive statistics was used to describe findings. The results of the genetic aberrations were reported in the form of a bar graph. Microsoft Excel for Mac, version 16.42, was used for statistical calculation.

RESULTS

Over a period of 5 years, 337 plasma samples taken from 279 patients were sent for ctDNA NGS testing. The median age at diagnosis was 73 years (range 36–93 y, SD 10.4 y), 51% were men and 49% were women. The majority were White or Caucasian (80% versus 8% Black or African American versus 12% Multiracial or unknown race) and had a history of smoking (79%).

Liquid Biopsy Utility in the Detection of AAs

Excluding synonymous mutations and variants of unknown significance, 705 somatic aberrations were detected in 239 of 337 (71%) of plasma samples and in 201 of 279 (72%) of patients. Of the aberrations, 254 of 705 (36%) were identified as therapeutically actionable. These were detected in 139 of 239 (58%) of samples, taken from 106 of 201 (53%) of patients.

Genomic Patterns of Actionable Aberrations (AAs) Detected Through Liquid Biopsy

An overall 106 patients had 254 AA identified by plasma NGS testing through liquid biopsy. Commonly detected AA were EGFR (n = 127, 50%), KRAS (n = 61, 24%), BRAF (n = 24, 9.5%), MET (n = 23, 9%), RET (n = 5, 1.9%), BRCA1 (n = 5, 1.9%), BRCA2 (n = 4, 1.6%), ERBB2 (n = 4, 1.6%), and ALK (n = 1, 0.4%). None of the patients had aberrations in ROS1 or NTRK2. One patient had microsatellite instability-high cells (see Figure 1).

Figure 1

Inclusion and exclusion criteria. Over a period of 5 years, 337 liquid biopsies were ordered for 279 patients, and sent for plasma NGS testing via the Guardant360 commercial platform. After excluding synonymous mutations and VUS, 254 aberrations were detected in 106 patients, which comprise a 58% AA detection rate through liquid biopsy and plasma genotyping. NSCLC: nonsmall cell lung cancer; VUS: variant of unknown significance; AA: actionable aberrations; NGS: next-generation sequencing.

Figure 1

Inclusion and exclusion criteria. Over a period of 5 years, 337 liquid biopsies were ordered for 279 patients, and sent for plasma NGS testing via the Guardant360 commercial platform. After excluding synonymous mutations and VUS, 254 aberrations were detected in 106 patients, which comprise a 58% AA detection rate through liquid biopsy and plasma genotyping. NSCLC: nonsmall cell lung cancer; VUS: variant of unknown significance; AA: actionable aberrations; NGS: next-generation sequencing.

Genomic Patterns of AAs Detected Through Tissue Biopsy

An overall 62 of 106 of patients had tissue biopsy performed in addition to their liquid biopsy, with 49 AA detected in 45 (73%) of the patients. (see Figure 2). Commonly detected AA were EGFR (n = 28, 57%), KRAS (n = 10, 20%), BRCA2 (n = 3, 6%), MET (n = 3, 6%), BRAF (n = 2, 4%), BRCA1 (n = 1, 2%), RET (n = 1, 2%) and ROS1 (n = 1, 2%) (see Figure 3).

Figure 2

Study design. Consort diagram of 106 patients with AA detected on plasma genotyping through liquid biopsy, 62 patients underwent tissue biopsy and 45 of them had 49 AA detected on tissue NGS. In 44 patients tissue biopsy was not performed owing to insufficient tissue or technically difficult surgical approach. Concordance agreement was observed in 39 patients between AA detected on plasma and tissue NGS. In 6 patients, the AA detected on plasma and tissue genotyping were discordant. AA: actionable aberrations; NGS: next-generation sequencing.

Figure 2

Study design. Consort diagram of 106 patients with AA detected on plasma genotyping through liquid biopsy, 62 patients underwent tissue biopsy and 45 of them had 49 AA detected on tissue NGS. In 44 patients tissue biopsy was not performed owing to insufficient tissue or technically difficult surgical approach. Concordance agreement was observed in 39 patients between AA detected on plasma and tissue NGS. In 6 patients, the AA detected on plasma and tissue genotyping were discordant. AA: actionable aberrations; NGS: next-generation sequencing.

Figure 3

Common AAs detected on plasma and tissues NGS. Overall, 254 AA were detected in 106 patients via plasma NGS and 49 AA were detected in 45 of 106 patients with tissue testing. Commonly detected AA were EGFR (n = 127, 50%), KRAS (n = 61, 24%), BRAF (n = 24, 9.5%), and MET (n = 23, 9%). Similarly, tissue NGS frequently detected EGFR (n = 28, 57%), KRAS (n = 10, 20%), BRAF (n = 3, 6%), and MET (n = 3, 6%). Concordance agreement between tissue and plasma NGS modalities was detected in 39 of 45, 87% patients and was demonstrated most commonly in EGFR (n = 25) and KRAS (n = 11). AA: actionable aberrations; NGS: next-generation sequencing.

Figure 3

Common AAs detected on plasma and tissues NGS. Overall, 254 AA were detected in 106 patients via plasma NGS and 49 AA were detected in 45 of 106 patients with tissue testing. Commonly detected AA were EGFR (n = 127, 50%), KRAS (n = 61, 24%), BRAF (n = 24, 9.5%), and MET (n = 23, 9%). Similarly, tissue NGS frequently detected EGFR (n = 28, 57%), KRAS (n = 10, 20%), BRAF (n = 3, 6%), and MET (n = 3, 6%). Concordance agreement between tissue and plasma NGS modalities was detected in 39 of 45, 87% patients and was demonstrated most commonly in EGFR (n = 25) and KRAS (n = 11). AA: actionable aberrations; NGS: next-generation sequencing.

The three commonest co-occurring aberrations detected on liquid biopsy were EGFR and BRAF (n = 14), EGFR and MET (n = 13), and MET and BRAF (n = 10). The commonest mutually exclusive aberrations were KRAS and EGFR. In 44 of 106 (42%) of patients, tissue NGS was not performed because of insufficient tissue or technically difficult biopsy and any precision treatment was determined by their liquid biopsy NGS results.

Concordance Agreement in AA Between Tissue NGS and Plasma Testing

We evaluated whether consistent AAs were found on liquid and tissue biopsies in same patients who underwent both modalities (Figure 3). Concordant AAs were found in 39 of 45 (87%) of patients who underwent both tissues and plasma testing and was demonstrated in EGFR (n = 25, 64%), KRAS (n = 11, 28%), BRAF (n = 2, 5%), and MET (n = 1, 3%). In the remaining 6 of 45 (13%) of patients, tissue NGS was discordant from their plasma NGS results.

DISCUSSION

In this retrospective study, we present real-world data from the community setting, assessing AA detection rates and genomic patterns of ctDNA between liquid biopsy and tissue biopsy genotyping modalities. Our study shows that integrating liquid biopsy into the clinical practice of patients with NSCLC increased AA detection rate in 44 patients who had no tissue NGS testing and no other means of assessing the genomic makeup of their tumor. In those patients, plasma sampling via liquid biopsy offered additional actionable targets and increased their potential therapeutic arsenal. Neither treatment assignment nor survival analysis was evaluated in this study; however, a literature review shows no consensus on survival benefits upon routine use of broad-based genomic sequencing in large populations of patients with NSCLC in the community.[1315] The reasons for the lack of consensus are multifactorial, ranging from difficulties in identifying AA in fairly low tumor burden, the scarcity of robust response to treatments, access to treatment and to clinical trials, and variable insurance coverage.

In congruency with literature, our study demonstrated that EGFR and KRAS were the most commonly identified AAs on both plasma and tissue NGS testing. Mutations in EGFR identified by liquid biopsy testing were most frequently co-occurring with aberrations in BRAF and MET and were mutually exclusive with KRAS. These data are consistent with previously published literature.[1417] An overall concordance of 87% was observed in AA detection rate when comparing between plasma and tissue NGS.

This study is limited by its retrospective nature. The information regarding tissue NGS and mortality was captured through review of the electronic medical records, which may be incomplete. The liquid biopsy information was obtained from the Guardant360 database. Detection bias may be introduced if providers choose to order liquid biopsies in selected cases depending on specific characteristics (eg, insurance coverage and nonsmoking status). Evaluating whether clinicians had access to the genomic NGS data and offered patients informed therapy was not routinely documented, limiting the understanding of clinician decision-making with broad-based genomic-sequencing results.

CONCLUSION

Although tissue biopsy remains the gold standard in cancer management, our experience advocates for using liquid biopsy alongside tissue biopsy, as means of increasing AA detection rate and enhancing the delivery of precision medicine to patients with NSCLC. Certainly, more powered studies are needed to assess whether incremental benefit exists between tissue NGS and liquid biopsy; however, our results cautiously display a role for using liquid biopsy as part of routine clinical management.

Acknowledgment

The authors thank Leylah Dubrowsky, PhD (Guardant Health, Genetics Department) for assistance with data analysis and interpretation. She did not receive compensation for her role in this study.

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Competing Interests

Source of Support: None. Conflict of Interest: Dr. Estelamari Rodriguez serves on the speaker's bureau for Genentech and advisory board for Astra Zeneca. The other authors have no conflict of interest to declare.