Circulating tumor DNA testing in advanced non-small cell lung cancer

Lung Cancer 119 (2018) 42–47

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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan

Review

Circulating tumor DNA testing in advanced non-small cell lung cancer Everett J. Moding

a,b,c

, Maximilian Diehn

a,b,c,⁎

, Heather A. Wakelee

a,d,⁎

T

a

Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA d Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Circulating tumor DNA Liquid biopsy Biomarker Non-small cell lung cancer Epidermal growth factor receptor Drug resistance

Circulating tumor DNA (ctDNA) shed from cancer cells into the peripheral blood can be non-invasively collected and tested for the presence of tumor-specific mutations. Mutations identified in ctDNA can predict responses to targeted therapies and emerging evidence suggests that changes in ctDNA levels over time can be used to monitor response to therapy and detect disease recurrence. Given the emergence of targeted therapies in advanced non-small cell lung cancer (NSCLC), liquid biopsies utilizing ctDNA testing represent a powerful approach to genotype tumors and monitor for the development of resistance. Here, we review current and potential future clinical applications of ctDNA testing for patients with advanced NSCLC.

1. Introduction

2. ctDNA testing approaches

Tumors continually shed DNA into peripheral blood due to cell turnover both when not being treated and in response to therapy [1]. Circulating tumor DNA (ctDNA), which can be non-invasively detected in the plasma samples of patients through simple blood draws, delivers a unique window into an individual patient’s tumor biology [2]. Studies in multiple tumor types have demonstrated that ctDNA testing can effectively predict the response of patients to targeted therapies [3–5]. Furthermore, emerging data suggests that ctDNA levels can be used to monitor response to local and systemic therapies [6]. Personalized and adaptive therapy continues to emerge and revolutionize the treatment of non-small cell lung cancer (NSCLC) [7]. Accurate information regarding the mutational status of a patient’s tumor is critical to guide treatment decisions. However, easily accessible tumor tissue is not always available, and a small sample of one lesion may not demonstrate the full mutational picture within a given patient [8]. In these clinical situations, ctDNA testing can provide valuable and unique insights that can help guide therapy. Here, we review ctDNA testing approaches and present an extended clinical vignette of a patient with metastatic epidermal growth factor receptor (EGFR)-mutant lung cancer who undergoes targeted therapy but ultimately develops treatment resistance. After each step in his clinical course, we review the clinical applications and interpretation of ctDNA testing and highlight promising approaches that may become available in the future.

Several approaches have been developed to analyze the presence and quantity of ctDNA ranging from single-locus amplification to whole genome sequencing (Table 1) [9]. Initial studies utilized polymerase chain reaction (PCR)-based amplification of specific cancer-associated mutations [10,11]. Allele-specific PCR provides reliable amplification of hot-spot mutations, but low sensitivity limits the application of this approach to early stage lung cancers or patients with very low burden of disease. Digital PCR improves upon conventional PCR by partitioning samples into multiple, smaller reactions allowing for absolute quantification and increased sensitivity [12,13]. Both conventional and digital PCR-based approaches test for a limited number of well-defined mutations. As a result, their applicability is limited to patients with common driver mutations. The advent of next generation sequencing (NGS) has led to the development of several additional ctDNA testing approaches. One broad approach utilizes a combination of multiplexed PCR assays to amplify a small number of regions of interest followed by NGS to identify gene mutations and quantify the fraction of mutant alleles [14–16]. This approach allows for high sensitivity but interrogates a small number of genes and cannot detect copy number variants or structural variants if the breakpoint sequence has not been previously characterized. A second approach involving hybrid capture followed by NGS maintains an extremely high level of sensitivity while allowing larger panels of variants to be identified and quantified in each sample [17,18]. Furthermore, this approach enables detection of copy number

⁎

Corresponding authors at: 875 Blake Wilbur Dr, Stanford, CA 94305, USA. E-mail addresses: [email protected] (M. Diehn), [email protected] (H.A. Wakelee).

https://doi.org/10.1016/j.lungcan.2018.02.019 Received 10 July 2017; Received in revised form 23 February 2018; Accepted 25 February 2018 0169-5002/ © 2018 Elsevier B.V. All rights reserved.

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Table 1 Comparison of ctDNA testing approaches. Test

Description

Allele-specific PCR

Amplicon-based NGS

0.1–1% [10,11] Amplification and quantification of pre-selected variants 0.01–0.1% [12,13] Amplication of pre-selected variants after partitioning into multiple reactions to increase sensitivity Deep sequencing of PCR 0.01–2% [14,15] amplicons

Capture-based NGS

Deep sequencing of hybrid captured DNA molecules

0.00025–0.01% [17,18]

Whole Exome NGS Whole Genome NGS

Deep sequencing the exome

5–10% [19]

Deep sequencing of the genome

1–10% [20,21]

Digital PCR

Detection Limit

Variants Detected

Advantages

Disadvantages

Cost

Well-defined SNVs and Indels

– Lowest cost

Well-defined SNVs and Indels

– High sensitivity – Lower cost

– Small number of variants tested $ per sample – Lower sensitivity – Small number of variants tested $ per sample

SNVs and Indels

– High sensitivity – Less expensive than other NGS-based methods – Highest sensitivity – Broadly applicable

$$ – Fewer variants tested per sample than other NGS-based methods – Less comprehensive than whole $$-$$$ exome and genome NGS

– – – –

– – – –

SNVs, Indels, SCNAs, and recurrent SVs SNVs, Indels, SCNAs, and SVs SNVs, Indels, SCNAs, and SVs

Entire exome analyzed Broadly applicable Entire genome analyzed Broadly applicable

Expensive Low sensitivity Expensive Low sensitivity

$$$$ $$-$$$

Abbreviations: PCR = polymerase chain reaction, NGS = next generation sequencing, SNVs = single nucleotide variations, Indels = insertions or deletions, SCNAs = somatic copy number alterations, SVs = structural variants.

a patient’s tumor. Several approaches have been demonstrated to effectively detect activating mutations of the EGFR gene from plasma samples. The cobas EGFR Mutation Test v2 is a real-time PCR based assay that was originally approved by the FDA to test for EGFR mutations in formalin-fixed paraffin-embedded specimens [29]. The application to plasma samples was validated as part of the ENSURE clinical trial, which compared first line erlotinib versus gemcitabine and cisplatin [26]. The FDA noted in their approval that the test may benefit patients who are unable to provide a tumor specimen for EGFR testing. Although the cobas EGFR Mutation Test v2 can detect multiple mutations in exons 18–21, including L861Q, G719X, and S768I, it is currently only approved as an indication for erlotinib therapy when exon 19 deletions and L858R substitution mutations are detected. In 76.7% of patients with exon 19 deletion or L858R mutations detected from tissue samples, the same mutation was detected in the plasma, suggesting that plasma samples can substitute for tissue biopsy in the majority but not all patients. Thus, the FDA recommends that negative plasma samples should prompt additional tissue sampling. Several additional randomized trials comparing EGFR TKIs to chemotherapy have demonstrated the ability of allele-specific PCR ctDNA testing to predict outcomes to treatment. The IPASS study compared first-line gefitinib versus carboplatin and paclitaxel in a Japanese cohort of patients with advanced lung adenocarcinoma. Plasma ctDNA testing led to a high rate of false negatives (56.9%) when using tissue as a reference, but progression-free survival was significantly longer in patients with positive ctDNA testing who received gefitinib compared with chemotherapy [30]. Similarly, in European patients with EGFR mutations detected with ctDNA testing on the EURTAC trial, erlotinib treatment was associated with a longer progression-free survival compared with combination chemotherapy [31]. Afatinib improved progression-free survival compared with platinum doublet chemotherapy on the LUXLung 3/6 trials for patients with EGFR-mutant ctDNA [32]. Finally, the FASTACT-2 study compared erlotinib versus placebo after 6 cycle of gemcitabine and platinum chemotherapy and demonstrated a significant PFS benefit for erlotinib in patients positive for EGFR mutations but not in patients negative for EGFR mutations by ctDNA testing [33]. Several additional clinical studies have demonstrated the concordance of ctDNA testing for EGFR mutations with tumor biopsies and the ability of ctDNA testing to predict responses to EGFR TKIs [5,34–39].

variants and recurrent structural variants. Finally, several groups have demonstrated deep sequencing of the whole exome [19] or genome [20,21] can provide comprehensive profiling of ctDNA. Although feasible, these approaches are limited to application in patients with advanced disease due to the high costs per sample and relatively low sensitivity. Currently, the cobas EGFR Mutation Test v2 (Roche Molecular Systems, Inc) is the only liquid biopsy approved by the FDA, and we review the application of this test below. Multiple commercial laboratories offer laboratory developed tests (LDTs) that are regulated under the Clinical Laboratory Improvement Amendments (CLIA) program, including both PCR-based and NGS-based approaches. A detailed description of the many available LDTs is beyond the scope of this review. 3. Testing for EGFR mutations 3.1. Sample case: part 1 A 54-year-old never-smoking man presents with chronic cough and worsening shortness of breath and is found to have a right lower lobe mass along with multiple small bilateral pulmonary nodules on CT and three subcentimeter enhancing brain lesions on MRI. He undergoes two CT-guided biopsies with a non-diagnostic first sample and the second sample showing sparse malignant cells consistent with lung adenocarcinoma. There was insufficient tissue for molecular testing. 3.2. ctDNA testing for the diagnosis of EGFR mutations EGFR gene mutations have been reported in 43% of lung adenocarcinomas in never smokers and 11% of lung adenocarcinomas in smokers in a population of patients from the United States [22]. The rates of EGFR mutations vary by ethnicity and location, with the highest reported rates of EGFR mutations occurring in Asian populations [23]. Multiple randomized controlled trials have demonstrated improved progression-free survival with the EGFR tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib, and afatinib compared with chemotherapy for EGFR-mutant metastatic NSCLC [24–28]. As a result, EGFR TKIs are currently recommended as first-line treatment for EGFRmutant metastatic NSCLC. Tissue sampling remains the gold standard for molecular testing of tumors, but clinical situations such as the example above often arise when inadequate tissue is available for testing or the risk of tumor biopsy is too high. In these situations, non-invasive genotyping using ctDNA can provide valuable information about the mutational status of

3.3. Sample case: part 2 The patient’s plasma is collected and sent for ctDNA EGFR testing 43

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(beads, emulsion, amplification, and magnetics) was associated with a favorable response to osimertinib that was similar to detection of the T790M mutation in tissue samples [50]. Of note, 31% of patients who were negative for the T790M mutation on tumor biopsy had detectable a T790M mutation in the plasma, demonstrating that a single tumor biopsy may not be fully representative of the molecular heterogeneity within a patient. Cell free DNA is cleared from the plasma through both the liver and kidneys [51], and ctDNA testing of urine samples has also been shown to detect EGFR mutations [52]. When analyzing both plasma and urine samples, one study detected T790M mutant DNA in 93% of the patients with the mutation [53].

using a focused assay. Initial testing returns negative for EGFR activating mutations. The patient’s plasma is sent for ctDNA testing of a broader panel of genes that returns positive for the EGFR L858R mutation. 3.4. Sensitivity and specificity of EGFR ctDNA testing The ability of ctDNA testing to detect EGFR mutations depends upon the level of ctDNA in the blood, which can vary amongst patients and at different time points. The half-life of ctDNA in plasma has been reported to be less than 2 h [13], leading to rapid clearance of mutant fragments. The volume and location of disease can impact the ability to detect EGFR mutations. Tumor burden is a major determinant of ctDNA concentration and patients with more extensive disease tend to have higher levels of ctDNA [17,40]. Additionally, the presence of extrathoracic disease (M1b) compared with intrathoracic disease (M1a or M0) appears to improve the likelihood of detecting EGFR mutations in plasma, although this may largely reflect differences in total body tumor burden [41]. Current commercially available ctDNA tests prioritize specificity over sensitivity and false negatives are more common than false positives [42,43]. For example, the cobas EGFR Mutation Test v2 has been reported to have a sensitivity of 84.3% and a specificity of 97.0% for EGFR activation mutations [44]. As a result, patients with a positive ctDNA test who have had negative results in the past should generally be considered positive for the EGFR mutation. As mentioned above, digital PCR-based and NGS-based approaches can achieve lower detection limits than conventional PCR and may enable detection of EGFR mutations in more patients. Furthermore, platforms that test for multiple common mutations seen in non-small cell lung cancer can help to interpret negative EGFR testing. For example, if patients are found to be negative for EGFR mutations but positive for a KRAS mutation in the same sample, the negative predictive value is significantly higher than if an EGFR mutation alone is not found.

3.7. Sample case: part 4 Initial ctDNA testing does not detect the T790M mutation, but also no EGFR activating mutations are detected. The patient was unfit to undergo biopsy and therefore a repeat ctDNA test was ordered, which was negative for T790M but positive for L858R. 3.8. Interpretation of ctDNA testing It is critical to interpret the results of ctDNA testing in the context of the full set of results. Given this patient’s known L858R activating mutation, the first test should not be interpreted as being negative for T790M. Instead, the amount of ctDNA was likely below the detection threshold of the test or the tumor was not actively shedding DNA into peripheral blood because no activating mutation was detected. The repeat testing result is more definitive given the detection of the L858R mutation, suggesting sufficient ctDNA was collected for accurate genotyping and there is not a significant population of T790M tumor cells driving the patient’s progressive disease. 4. Beyond EGFR

3.5. Sample case: part 3

4.1. Testing for other mutations

The patient is started on erlotinib with initial disease regression followed by a year of stable disease. Starting around 13 months on therapy, the patient begins to develop slow progression with growth of several lung nodules.

As mentioned above, ctDNA testing can also be used to identify mutations in genes other than EGFR. For example, BRAF mutations in NSCLC detected by ctDNA have been shown to correlate with clinical responses to targeted therapies [54]. Liquid biopsies have also been shown to be able to detect ALK rearrangements [17,55], and ongoing studies are investigating whether ctDNA approaches can replace tissue biopsies to predict response to crizotinib and other ALK TKIs. As noted above, several approaches have been developed to examine larger panels of oncogenes and tumor suppressors in order to establish a more complete genomic picture in NSCLC via plasma analysis [17,19,56–58]. These approaches are of interest given the large number of targeted therapies available or in development for patients with NSCLC.

3.6. ctDNA testing for EGFR resistance mutations The T790M mutation has been shown to confer resistance of EGFR mutant lung cancers to TKIs and arises in approximately half of all patients treated with first-line EGFR TKIs [45]. Recently, the AURA3 trial demonstrated that the third generation EGFR TKI osimertinib improved progression free survival with fewer grade 3 toxicities than platinum and pemetrexed chemotherapy for T790M positive NSCLC that had progressed on first line EGFR TKI therapy [46]. Similar to initial molecular profiling, tissue biopsy remains the gold standard approach for identification of resistance mutations. However, the cobas EGFR Mutation Test v2 described above has also been approved by the FDA to detect the T790M mutation in plasma samples as a companion diagnostic to osimertinib. Due to the imperfect sensitivity of plasma testing, patients with undetectable T790M should be reflexed to biopsy. Furthermore, ctDNA testing is unable to detect other possible reasons for disease progression that are not driven by somatic alterations. One such mechanism is small cell transformation [47], which is histologically defined. However, since RB1 loss appears to be a central mechanism in this process [48] and RB1 mutations have been observed in ctDNA from patients with EGFR TKI resistance [49], this resistance mechanism may be detectable via ctDNA analysis at least in some patients. Multiple other approaches for detecting the T790M mutation in ctDNA have also been described. Detection of the T790M mutation in cell-free plasma DNA using a digital PCR approach called BEAMing

4.2. Lung cancer diagnosis, screening, and surveillance Tissue biopsy for histological subtyping remains the gold standard for lung cancer diagnosis. However, there are often situations where tissue biopsy is deemed exceedingly risky, and patients would prefer to avoid invasive procedures. This raises the possibility that ctDNA testing could replace tissue biopsies in some settings in the future. Although a mutation detected in the plasma cannot necessarily be attributed to a lung mass seen on imaging or distinguished from metastatic disease from another primary, the mutations observed can add to the evidence of a primary lung malignancy. Furthermore, it is possible that the types of mutations detected could in the future allow non-invasive distinction between NSCLC histological subtypes. Additional information obtained from plasma including RNA profiles or epigenetic characteristics such as DNA methylation could theoretically further aide with histological classification based on liquid biopsies alone [59,60]. Nonetheless, tissue biopsies will continue to provide critical information regarding 44

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Fig. 1. Current and potential applications of ctDNA testing in non-small cell lung cancer (NSCLC). Plasma ctDNA testing is currently FDA approved for detecting EGFR driver mutations and resistance mutations in metastatic NSCLC (bold font in boxes). Preclinical and clinical studies are currently under way to investigate the ability of ctDNA testing to screen for lung cancer in high risk patients, aid in lung cancer diagnosis, monitor response to treatment such as radiation therapy, detect minimal residual disease after treatment, and surveil for disease recurrence (normal font).

similar approach was shown to be feasible in patients with stage I–III NSCLC [62]. In 94% of patients experiencing a recurrence, ctDNA was detectable in their first post-treatment blood sample. Furthermore, freedom from progression at 3 years was 93% in patients with undetectable minimal residual disease by ctDNA compared with 0% in patients with detectable ctDNA corresponding to a hazard ratio of 43.4. Treatment of many cancers has moved toward adaptive therapy based primarily on imaging response on PET/CT, and the currently enrolling radiation therapy oncology group 1106 study (NCT01507428) is investigating this approach in locally advanced NSCLC. Because DNA release into the plasma can be triggered by cell death, ctDNA kinetics may correlate with response to treatment. Therefore, ctDNA analysis could be used to optimize chemotherapy selection or guide dose escalation or de-escalation during radiation therapy.

tumor morphology and immunohistochemistry that cannot be obtained from ctDNA such as tumor grade, presence of lymphatic or vascular invasion, stromal composition, and PD-L1 expression. The ability of ctDNA testing to detect mutations relies upon active shedding of tumor DNA into the blood, and the sensitivity of any testing approach depends on the level of ctDNA in the blood at the time of sampling. Multiple ctDNA testing techniques have evolved to detect tumor-derived ctDNA making up less than or equal to 0.01% of plasma cell free DNA [6]. These approaches have been shown to detect ctDNA in early stage lung cancer patients [17,40,61], suggesting that ctDNA testing could potentially be used for screening in the future. However, high levels of specificity to avoid false positives and associated patient harm are critical, and large-scale clinical studies will be required to document that a ctDNA-based assay has clinical utility in the screening setting. Currently, surveillance for advanced NSCLC after chemoradiation therapy or surgery or during active systemic therapy relies on serial CT and PET/CT imaging. In addition to exposing patients to additional ionizing radiation, CT surveillance is often limited by post-treatment changes that can delay the identification of recurrent disease. Monitoring ctDNA may provide an alternative method for detecting disease recurrence. Remarkably, increases in ctDNA appear to occur prior to evidence of progression on imaging [17,40,62]. For example, the T790M mutation was detected in NSCLC patients undergoing erlotinib therapy up to 344 days before disease progression was clinically evident [63]. It should be noted that the ability to detect resistance mutations or recurrent disease prior to progression on imaging is variable and dependent on the sensitivity of the ctDNA assay. Despite the ability of ctDNA to detect recurrent disease before imaging, clinical trials are necessary to demonstrate that early intervention is beneficial in these patients.

5. Conclusions Circulating tumor DNA represents a unique opportunity to non-invasively genotype tumors, and clinical studies have demonstrated the applicability of this approach to NSCLC patients. Although currently only FDA approved for EGFR mutation testing, there are several commercially available ctDNA tests that can help guide clinical decision making. Beyond detecting tumor mutations, ctDNA analysis offers promise for lung cancer screening, diagnosis, stratifying treatment response, and early identification of recurrent disease (Fig. 1). Future studies will help to clarify the application of these approaches in the clinic. Conflicts of interest M.D. is a co-inventor on patent applications related to ctDNA detection methods. M.D. is a consultant for Roche Sequencing Solutions.

4.3. Response to treatment Acknowledgements Circulating tumor DNA levels have been shown to correlate with disease burden in NSCLC patients undergoing therapy with decreasing ctDNA concentration correlating with response to therapy and increasing ctDNA concentration associated with disease progression [6,17,33,63]. As a result, successful treatment could be expected to decrease ctDNA levels to undetectable levels. In stage II colon cancer, ctDNA testing postoperatively and after completion of chemotherapy has been shown to identify patients with minimal residual disease who are at a very high risk of recurrence [64]. Additionally, ctDNA testing at the completion of curative therapy in early stage breast cancer patients could predict the development of metastatic disease [65]. Recently, a

This work was supported with grants from the National Cancer Institute (M.D.; R01CA188298), the US National Institutes of Health Director’s New Innovator Award Program (M.D.; 1-DP2-CA186569), the Ludwig Institute for Cancer Research (M.D.), and the CRK Faculty Scholar Fund (M.D.). References [1] H. Schwarzenbach, D.S.B. Hoon, K. Pantel, Cell-free nucleic acids as biomarkers in cancer patients, Nat. Rev. Cancer 11 (2011) 426–437, http://dx.doi.org/10.1038/

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