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Table of Contents
REAL WORLD DATA
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 197-203

Outcomes with liquid biopsy to determine the EGFR mutation status in poor performance status, biopsy-ineligible, advanced NSCLC patients


1 Department of Medical Oncology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India
2 Director, Molecular Biology and Cytogenetics, Oncquest Laboratory, New Delhi, India

Date of Web Publication20-Dec-2019

Correspondence Address:
Avinash Pandey
Department of Medical Oncology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CRST.CRST_60_19

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  Abstract 


Background: Outcomes based on liquid biopsy in poor performance, older, biopsy-ineligible advanced non-small cell lung cancer (NSCLC) patients are unknown.
Aim: The aim of the study was to evaluate the outcomes of patients treated on the basis of liquid biopsy to determine the epidermal growth factor receptor (EGFR) status.
Objectives: The objective was to evaluate the progression-free survival (PFS) and overall survival (OS) of patients treated based on EGFR mutation status, determined on a liquid biopsy.
Materials and Methods: This was a retrospective audit of prospectively maintained database of patients who underwent liquid biopsy EGFR mutation testing between July 2017 and January 2019 and received treatment based on that report. The primary end point was PFS defined as duration between the date of sample collection and disease progression or death, whereas OS was calculated as the interval between the date of sample collection and death. Kaplan–Meier survival was used for the above two end points.
Results: Out of 28 patients, 11 (39%) were EGFR mutation positive, and 17 (61%) were EGFR mutation negative. The median age was 62 years, with 15 out of the 28 being females (54%). Nine (82%) and 13 (76%) patients had performance status (PS) 2 or 3 in the EGFR mutation-positive and EGFR mutation-negative cohorts, respectively. In the EGFR mutation-positive cohort, 4 (36%) were exon 19, 6 (54%) were exon 21, and 1 (9%) was T790M positive. After receiving tyrosine kinase inhibitors or physician's choice of chemotherapy in EGFR mutation-positive and negative cohorts, the response rates were 63% and 12%, respectively. The median follow-up was 14 months (range: 10–17 months). The median PFS was 8 months versus 2 months (P = 0.002), whereas the median OS was 17 months versus 5 months (P = 0.004) in the EGFR mutation-positive and EGFR mutation-negative cohorts, respectively.
Conclusion: In patients with advanced NSCLC in whom a tissue biopsy is not feasible, especially in older, poor PS patients, treatment based on the results of liquid biopsy gives comparable outcomes to those of a tissue-based biopsy.

Keywords: cfDNA, ctDNA, epidermal growth factor receptor, non-small cell lung cancer, poor performance status


How to cite this article:
Pandey A, Dutt S, Singh A, Kumar A, Singh S. Outcomes with liquid biopsy to determine the EGFR mutation status in poor performance status, biopsy-ineligible, advanced NSCLC patients. Cancer Res Stat Treat 2019;2:197-203

How to cite this URL:
Pandey A, Dutt S, Singh A, Kumar A, Singh S. Outcomes with liquid biopsy to determine the EGFR mutation status in poor performance status, biopsy-ineligible, advanced NSCLC patients. Cancer Res Stat Treat [serial online] 2019 [cited 2020 May 30];2:197-203. Available from: http://www.crstonline.com/text.asp?2019/2/2/197/273678




  Introduction Top


Tissue-based molecular testing to identify sensitive targetable mutations in advanced metastatic non-small cell lung cancer (NSCLC) has been the holy grail of personalized precision therapy.[1] Getting adequate tissue from an invasive tumor biopsy for sequential testing such as morphology, immunohistochemistry, molecular profiling, and next-generation sequencing (NGS) is theoretically ideal but practically difficult, especially in tier two to three cities in India where facilities and expertise for image-guided interventional procedures are scarce.[2],[3] Moreover, in patients with poor performance status (PS), significant respiratory distress, and who are frail and older, doing an invasive tissue biopsy is a major challenge.[4] It is not uncommon to come across patients who refuse an invasive tissue biopsy after cytological confirmation of malignancy done in peripheral centers, hence denying the clinician the critical tissue needed to detect the sensitive mutations through molecular profiling.

Peripheral blood circulating tumor DNA (ctDNA/liquid biopsy) for epidermal growth factor receptor (EGFR) mutation has shown good concordance with tissue-based biopsy, with sensitivity reaching up to 70% with droplet digital polymerase chain reaction (ddPCR).[5],[6] It is reasonable and convenient to opt for a non-invasive liquid biopsy in the above cohort of patients where invasive tissue biopsy is not feasible and comes with a high probability for technical failure with complications such as pneumothorax, pulmonary hemorrhage, or air embolism.[3],[7] To the best of our knowledge, no literature exists yet on the clinical utility and outcomes of patients treated upfront with tyrosine kinase inhibitors (TKIs) solely based on the ctDNA EGFR report in the absence of tissue-based molecular testing. We report a retrospective audit of outcomes of therapy based solely on liquid biopsy for EGFR testing in older, frail, poor PS, and biopsy-ineligible advanced metastatic NSCLC.


  Materials and Methods Top


General study details

Our institute is one of the apex tertiary referral cancer centers in Eastern India. We have prospectively maintained a database in the Department of Medical Oncology of advanced metastatic NSCLC patients who underwent peripheral blood sampling for ctDNA for EGFR mutations in the absence of tissue biopsy or samples with inadequate tissue for molecular testing between July 1, 2017, and January 31, 2019. Age, sex, PS, and site and extent of metastasis were noted. With background knowledge of higher probability of EGFR mutation positivity in non-smokers and in order to improve the diagnostic yield with reduced cost, we offered ctDNA peripheral blood sampling to an enriched population of non-smoking metastatic NSCLC patients only. All patients had cytological confirmation of malignancy either on pleural fluid or fine-needle aspiration cytology. All patients underwent contrast-enhanced computed tomography (CT) scan of the chest and abdomen for staging. Bone scan and magnetic resonance imaging of the brain were done if patients were symptomatic or if there was a high index of clinical suspicion. Positron emission tomography scan was neither done at baseline nor at subsequent response evaluation in any metastatic patient, as we do not have this facility at our institute.

All patients were discussed in the multidisciplinary tumor board comprising surgical oncologists, radiation oncologists, medical oncologists, chest medicine physicians, radiologists, and pathologists. After decision to send peripheral blood sample for ctDNA EGFR mutation analysis was made, further therapy decision to offer TKI or chemotherapy depending on reports of the above test was made by the medical oncology team. Written informed consent was obtained from all patients, explaining the surrogate role of ctDNA in the absence of tissue-based molecular profiling, its limitations, and the type of varied treatment they will receive depending on the result of that report. Patients were categorized into four groups based on the reason for undergoing peripheral blood ctDNA instead of regular invasive tissue biopsy: (a) patient refused an invasive procedure, (b) patient requested biopsy but received only cytology/technical issues, (c) biopsy not feasible due to patient-related issues/poor PS, and (d) inadequate tissue. The study was conducted in accordance with the Declaration of Helsinki and the Indian Council of Medical Research guidelines for ethical conduct.

Specimen collection and DNA extraction

The sample collected for ctDNA from peripheral blood was outsourced to a National Accreditation Board for Hospitals and Healthcare Providers-accredited laboratory which adheres to strict sample processing and procedures, mandated as per the College of American Pathologists guidelines. ddPCR was used for detecting EGFR mutation in peripheral blood. A volume of 10–12 ml of peripheral whole blood was collected from each patient in PAXgene tubes (Cat No. 768165, BD) and was centrifuged at 3000 rpm for 10 min at 4°C first, and then 4700 rpm for 10 min at 4°C to isolate the plasma, which was then stored at −80°C until ctDNA extraction. Plasma ctDNA was extracted from 4 ml plasma from each patient with the QIAamp Circulating Nucleic Acid Kit (Cat No. 55114, Qiagen, Hilden, Germany) following the manufacturer's instructions. The concentration of extracted DNA was measured by Qubit fluorometer (Cat No. Q32856, Thermo Scientific, USA). Eluted DNA was used to detect EGFR mutations immediately.

Detection of EGFR mutations in plasma ctDNA by ddPCR EGFR kit

EGFR mutations in plasma samples were tested by ddPCR-based plasma genotyping for detecting EGFR exon 19 deletion, L858R, and T790M mutations. The ddPCR EGFR test was performed in a 20-μL volume containing 3.3 ng/reaction of template DNA on a Droplet Digital™ PCR (ddPCR) system (Cat No. QX200 Bio-Rad, Hercules, CA, USA). A volume of 20 μl of the reaction mix was loaded into a sample well of a DG8™ Cartridge followed by 70 μl of droplet generation oil for probes into the oil wells, according to the QX100 or QX200 Droplet Generator Instruction Manual. After droplet generation with the QX100 or QX200 Droplet Generator, the droplets were transferred into a clean 96-well plate. The plate was sealed with thePX1 PCR plate sealer, and amplification was carried out as per instructions provided by the kit manufacturer. Thresholds for detection were set manually based on results from nontemplate control wells and negative control wells containing wild-type gDNA.

The ddPCR ctDNA EGFR test is designed as a ddPCR-based diagnostic test for detecting 45 mutation sites within the exon 18–21 region of the EGFR gene using four reactions, with a sensitivity of 70% and specificity exceeding 95%. The DNA sample harboring the target DNA is fragmented into 20,000 droplets. The amplified fragments, which contain the fluorophores FAM™ or HEX™, displayed as dots (droplets), were used to calculate concentrations (copies/20 μL) based on the Poisson distribution. The clone percentage (copies/μL) can be derived from the software for both the wild type (wt) and mutant copies from the individual sample.

Patient management details

The average turnaround time for the ctDNA report was 7 days (range: 5–10 days), and patients were given symptomatic care until the availability of the report. Depending on the final ctDNA report, if patients were found to have EGFR mutations such as exon 19 deletion, exon 21 (L858R) point mutation, or T790M, they were given gefitinib or erlotinib (for the sensitizing mutations) and osimertinib (for T790M). Similarly, in the absence of such mutations, patients received standard physician's choice of therapy as per his/her discretion with respect to age and PS of patients. The follow-up was recorded by patient visits supplemented by telephonic conversations. Patients were followed every 2 weeks with complete blood hemogram, liver, and renal profile with serum electrolytes for 2 months, and thereafter monthly to monitor toxicity and ensure compliance. Response evaluation was done with contrast-enhanced CT scan of the chest and abdomen at our center reported by trained radiologists, in the third month, and thereafter, every 3 months till disease progression.

Statistics

Data were entered prospectively in the SPSS software version 17.0 sheet (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY, USA) supplemented by information from the patients' file, and response evaluation scans were updated in the above software for individual patients under the supervision of the physician. The data were retrospectively analyzed. Descriptive statistics and frequency distribution were used to analyze the demographic variables. Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) criteria were used to measure and categorize responses as complete, partial, stable, or progressive disease. Progression-free survival (PFS), which was the primary endpoint, was defined as duration in months between ctDNA report and date of progression or death; overall survival (OS) was defined as duration in months between the ctDNA report and date of death. Kaplan–Meier survival analysis[8] was used for both the above end points, and Cox regression model was used to calculate hazard ratios in the EGFR mutation-positive and EGFR mutation-negative cohorts.[9],[10] Univariate analysis with log-rank test was used to assess the impact of various factors on PFS and OS. Follow-up was calculated using reverse Kaplan–Meier method. No extramural or intramural grants or funding were used during the conduct of the study.


  Results Top


All metastatic NSCLC patients who gave blood samples for ctDNA were analyzed irrespective of the treatment received; none were lost to follow-up. Out of 28 patients who underwent peripheral blood ctDNA testing for EGFR mutation, 11 (39%) were EGFR mutation positive, whereas the remaining 17 (61%) were negative. The median age at diagnosis was 61 years (range: 41–84 years), with 13 males (46%) and 15 females (54%). Majority of patients (13; 46%) had Eastern Cooperative Oncology Group PS of 3, whereas 9 (32%) and 6 (21%) had PS of 2 and 1, respectively. In the ctDNA EGFR mutation-positive cohort, erlotinib was the most common TKI used in 6/11 (55%) patients followed by gefitinib in 4/11 (36%) patients. One patient had upfront T790M mutation for which osimertinib 80 mg once a day was started [Table 1]. Response rate in the ctDNA EGFR mutation-positive cohort treated with oral TKI was 63%, with 45% partial response and 18% complete response compared to 11.7% (partial response only) in the EGFR-negative population who received physician choice of chemotherapy [Figure 1] and [Figure 2]. All EGFR-negative patients received either chemotherapy or best supportive care, whereas none received any TKI even while waiting for ctDNA report as the turnaround time for ctDNA was 7 days only.
Table 1: Demographic and clinical characteristics and response to treatment of patients in circulating tumor DNA epidermal growth factor receptor mutation-positive and epidermal growth factor receptor mutation-negative cohort

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Figure 1: (a and b) A primary lung mass in the anterior segment of the right upper lobe, which after 3 months of therapy with erlotinib showed a partial response. (c and d) Large primary mass in superior segment of the left lower lobe and superior lingular segment of the upper lobe, showing partial response after 3 months of gefitinib

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Figure 2: (a) On computed tomography scan, a large heterogeneous mass involving the right upper lobe anterior segment of the lung with secondary metastasis in posterior segment right upper lobe, found to have T790 M mutation on ctDNA. (b) Complete response after 3 months of treatment with osimertinib

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The median follow-up was 14 months (range: 10–17 months). The median PFS was 8 months versus 2 months (P = 0.002), whereas mean OS was 17 months versus 5 months (P = 0.004) in the ctDNA EGFR mutation-positive and EGFR mutation-negative cohorts, respectively [Figure 3] and [Figure 4]. On Cox regression analysis, testing the impact of ctDNA EGFR mutation status on survival, the hazard ratio for PFS was 0.40 (95% confidence interval [CI]: 0.26–0.54) and the hazard ratio for OS was 0.39 (95% CI: 0.02–0.80), both favoring the EGFR mutation-positive cohort. Among variables such as age, sex, and PS tested by log-rank test for PFS, PS 3 patients did significantly worse than patients with PS 1 and 2 both in the ctDNA EGFR mutation-positive cohort, 6 months versus 8 months (P = 0.04), and in ctDNA EGFR mutation-negative cohort, 1 month versus 3 months (P = 0.001), respectively, while age and sex were found to be non-significant in either cohort for PFS. The toxicity data were not collected for either cohort of patients.
Figure 3: Progression-free survival in circulating tumor DNA epidermal growth factor receptor mutation-positive and epidermal growth factor receptor mutation-negative cohort treated with tyrosine kinase inhibitors and physician-choice chemotherapy, respectively

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Figure 4: Overall survival in circulating tumor DNA epidermal growth factor receptor mutation-positive and epidermal growth factor receptor mutation-negative cohort treated with tyrosine kinase inhibitors and physician-choice chemotherapy, respectively

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  Discussion Top


In the absence of tumor tissue for molecular profiling, the use of EGFR-directed oral TKIs solely based on the detection of EGFR mutation in peripheral blood ctDNA, in biopsy ineligible, older, poor PS metastatic NSCLC patients, yielded a response rate of over 60% with a median PFS of 8 months. Our study is the first to report the clinical utility and treatment outcomes of oral TKIs prescribed exclusively based on ctDNA-based EGFR molecular profiling performed using ddPCR platform in biopsy-ineligible metastatic NSCLC. About 18% of lung cancers reporting to apex tertiary cancer centers are still diagnosed on cytology alone without adequate tissue for histopathological classification, immunohistochemistry, and molecular profiling.[11] Moreover, in 18%–30% of lung cancer patients, invasive tumor biopsy to obtain adequate tissue for molecular profiling is not possible.[11],[12] Even if tumor tissue is obtained, in 20% of cases, molecular profiling cannot be done due to insufficient tumor material, poor quality DNA, no DNA, and low tumor yield.[12] The sensitivity of ctDNA for the detection of targetable EGFR mutations ranges from 60% to 90%, whereas the specificity, positive predictive value, and negative predictive value exceed 90% compared to those of tissue-based EGFR testing.[12],[13],[14] Our results using oral TKIs in EGFR mutation-positive tumors detected on ctDNA ddPCR platform in the absence of tumor biopsy are similar to those obtained in the landmark historical trials which used TKIs in EGFR mutation-positive NSCLC, where tumor tissue-based molecular profiling was done.[15],[16],[17],[18],[19],[20],[21] Hence, we propose peripheral blood ctDNA EGFR mutation analysis in cases where biopsy is difficult or tumor tissue is insufficient to perform molecular profiling and treat patients with oral TKIs rather than chemotherapy or best supportive care, in patients with EGFR mutations in metastatic NSCLC.

For liquid biopsy, several molecular profiling platforms exist such as ddPCR, Cobas® EGFR mutation test, amplification refractory mutation system (ARMS), emulsion, amplification, beads and magnetics (BEAMing), high-pressure liquid chromatography, and NGS.[22] Among these, ddPCR is rapid, cost-effective, is more accessible with less turnover time and has been prospectively validated in comparison to tissue-based tumor profiling with a potential to detect allele frequency as low as 0.4%.[23],[24],[25] Compared to ARMS PCR and NGS, ddPCR provides much better sensitivity for targetable EGFR exon 19 deletion and exon 21 L858R point mutations, 50%–60% and 70%–80%, respectively.[26],[27] Hence, we used ddPCR-based platform for EGFR mutation detection in peripheral blood ctDNA, which also had a turnaround time of 7 days.

Lung cancer is a heterogeneous and dynamic tumor where clonal evolution and drug resistance occur spontaneously.[28] Hence, it can be subjected to longitudinal molecular profiling to rapidly detect emerging drug-sensitive targetable mutations.[28],[29] ctDNA platform has emerged as an alternative to invasive biopsy for monitoring the evolution of resistance clones and predicting the response of TKIs.[30],[31] Peripheral blood ctDNA for T790M mutation without invasive biopsy in a cohort of patients receiving first-generation TKIs has proved to be a useful tool to preempt radiological progression, and osimertinib in such T790M mutation-positive patients was found to be effective for inducing durable disease control.[32] Peripheral blood ctDNA has also emerged as an alternate diagnostic tool, which has been prospectively validated with matched tumor tissue-based EGFR molecular testing with excellent concordance and diagnostic accuracy in a prospective study and meta-analysis when performed at baseline.[12],[13] Despite this, no study has yet been published where patients detected to have peripheral blood ctDNA-based EGFR mutations were treated with first-line TKIs in the absence of tissue-based molecular profiling, with reported outcomes similar to those historically reported from patients treated with biopsy-proven targetable EGFR mutation.

Our study was retrospective with a small sample size and included patients who were non-smokers and older with poor PS at baseline, where adequate tissue collection for molecular profiling was difficult. Hence, our cohort may not be a representative sample, which would otherwise be eligible for any prospective randomized clinical trial. We did not attempt any relook biopsy in the EGFR-negative cohort despite knowing that the sensitivity of ctDNA was 70% for detecting drug-sensitive mutations, hence may have missed few patients who might have harbored a sensitive clone but received only chemotherapy or best supportive care. We did not record and hence did not report the toxicity profile of TKIs or chemotherapy in our patient population. With all four patients presenting with brain metastasis in ctDNA EGFR mutation-negative cohort and four poor PS unfit patients evaluated for chemotherapy, being offered only best supportive care, the ctDNA EGFR-negative cohort had poor outcomes.


  Conclusion Top


Clinicians may offer peripheral blood ctDNA EGFR mutation testing as a diagnostic tool in biopsy ineligible, poor PS, older patients with advanced metastatic NSCLC and treat them with EGFR-directed oral TKIs if positive, without having any qualms of offering an inferior test or suboptimal therapy based on the report. ddPCR-based peripheral blood ctDNA testing is a cost-effective, rapid, and convenient platform for EGFR mutation profiling.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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