Cancer Research, Statistics, and Treatment

: 2020  |  Volume : 3  |  Issue : 3  |  Page : 475--480

Concordance of epidermal growth factor receptor mutation detection in bodily fluids other than blood with tissue biopsy: A retrospective analysis

Anuradha Chougule1, Priyanka Pange1, Shrutika Kale1, Vinita Jagtap1, Kavya Nambiar1, Ankita Nikam1, Priyanka Tiwrekar1, Vaishakhi Trivedi1, Vichitra Behel1, Akhil Kapoor2, Nandini Menon2, Vijay Patil2, Vanita Noronha2, Kumar Prabhash2, SD Banavali2,  
1 Medical Oncology Molecular Laboratory, Homi Bhabha National Institute, Mumbai, India
2 Medical Oncology, Homi Bhabha National Institute, Mumbai, India

Correspondence Address:
Anuradha Chougule
Medical Oncology Molecular Laboratory,Tata Memorial Hospital Mumbai


Background: The concept of liquid biopsy as a surrogate for tumor tissue is not only restricted to blood-derived samples but also includes other malignant bodily fluids such as cerebrospinal fluid, pleural fluid, pericardial fluid, and ascites. The application of liquid biopsy in patients with metastatic lung cancer, with disease progression on epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment, is unexplored in India. Objective: We aimed to determine the presence of EGFR gene mutations in various bodily fluids of patients with EGFR-mutant progressive metastatic lung adenocarcinoma. Materials and Methods: In this retrospective study conducted at the Tata Memorial Hospital in Mumbai, India, data from 11 patients with advanced EGFR-mutant lung adenocarcinoma were analyzed. Between December 2017 and June 2018, the cell-free DNA (cfDNA) obtained from the malignant bodily fluids and plasma of 11 patients were tested for the presence of EGFR-sensitizing mutations using real-time reverse transcription-polymerase chain reaction. The status of EGFR-sensitizing mutations in plasma and other bodily fluids was compared with that of the baseline tissue biopsy. In addition, the status of the acquired T790M resistance mutation in exon 20 at disease progression was determined using plasma and other bodily fluids. Results: Of the 11 patients, at disease progression, EGFR sensitizing mutations (exon 19 deletion and exon 21 L858R insertion) could be detected in the plasma of 7 (63%) and in other bodily fluids of all the 11 (100%) patients. Thus, the concordance for EGFR-sensitizing mutation detection in the plasma and other bodily fluids with the baseline formalin-fixed paraffin-embedded tissue was 63% and 100%, respectively. In addition, the acquired T790M resistance mutation was detected in the plasma of 3 (27.3%) patients and other bodily fluids of 9 (81.8%) patients. Conclusion: The rate of detection of the T790M mutation is higher in the free DNA obtained from malignancy-involved bodily fluid supernatant as compared to the plasma cfDNA. Our results suggest that cfDNA obtained from bodily fluids other than plasma can be used for the detection of EGFR mutations and that the mutation status may be a useful predictor of the response to TKIs.

How to cite this article:
Chougule A, Pange P, Kale S, Jagtap V, Nambiar K, Nikam A, Tiwrekar P, Trivedi V, Behel V, Kapoor A, Menon N, Patil V, Noronha V, Prabhash K, Banavali S D. Concordance of epidermal growth factor receptor mutation detection in bodily fluids other than blood with tissue biopsy: A retrospective analysis.Cancer Res Stat Treat 2020;3:475-480

How to cite this URL:
Chougule A, Pange P, Kale S, Jagtap V, Nambiar K, Nikam A, Tiwrekar P, Trivedi V, Behel V, Kapoor A, Menon N, Patil V, Noronha V, Prabhash K, Banavali S D. Concordance of epidermal growth factor receptor mutation detection in bodily fluids other than blood with tissue biopsy: A retrospective analysis. Cancer Res Stat Treat [serial online] 2020 [cited 2021 Aug 5 ];3:475-480
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Full Text


Mutations in the epidermal growth factor receptor (EGFR) gene are major oncogenic drivers in non-small cell lung cancer (NSCLC). The incidence of EGFR mutations in patients with NSCLC has been reported to be 23% among Indians, 10%–15% among Caucasians, and 27%–62% among the East Asians.[1],[2] The emergence of EGFR-tyrosine kinase inhibitors (TKIs) such as gefitinib, afatinib, and erlotinib has significantly improved the outcomes and the quality of life in a subset of patients with NSCLC.[1] Most patients develop resistance to EGFR-TKIs within 10–16 months of initiation of therapy.[1],[3],[4] The development of the T790M mutation in the exon 20 of the EGFR gene is the most common mechanism of acquired resistance observed in up to 50% of the patients being treated with TKIs.[5],[6]

Malignancy in various bodily fluids such as the cerebrospinal fluid (CSF), pleural effusion, and ascites is a common complication in patients with metastatic lung cancer, observed in approximately 15% of the patients at the time of diagnosis and in 10%–50% at some point during the course of the disease.[7],[8],[9],[10] Studies have shown that early diagnosis and treatment can effectively slow the nerve function defect caused by disease progression in patients with leptomeningeal metastases.[8],[10]

A repeat tissue biopsy at the time of recurrence may be difficult in some patients.[11] Therefore, testing of the cell-free DNA (cfDNA) obtained from the plasma of patients with advanced cancers is rapidly becoming a standard practice.[12] The concept of “liquid biopsy” as an alternative to tumor tissue biopsy is not just limited to blood-derived samples, but also includes other bodily fluids such as CSF and pleural effusion.[13],[14] However, the application of other bodily fluid based-liquid biopsies in Indian patients with EGFR TKI-resistant metastatic lung cancer is unexplored.

In this study, we report the data from a small cohort of patients with progressive metastatic EGFR-mutant NSCLC. Our objective was to compare the rate of detection of EGFR-sensitizing and resistant mutations from various bodily fluids with that from the tumor tissue. We also aimed to establish the concept of circulating tumor DNA (ctDNA) derived from bodily fluids other than plasma as a surrogate biomarker in patients with metastatic lung adenocarcinoma.

 Materials and Methods

General study details

This is a retrospective analysis conducted in the Molecular Laboratory of the Department of Medical Oncology at Tata Memorial Hospital, a tertiary care oncology center in Mumbai, India, between December 2017 and June 2018. The data were obtained from the prospective audit of patients with lung cancer (study 1043), which was approved by the Institutional Ethics Committee (on October 8, 2012) and registered with the Clinical Trials Registry, India (CTRI/2013/01/003335) [Supplementary Appendix 1]. Written informed consent was provided by the patients before enrollment in the lung cancer audit. The study was conducted according to the ethical principles established by the Declaration of Helsinki and Good Clinical Practice Guidelines.


The study included 11 patients with advanced EGFR-mutant NSCLC with progressive disease. In our setup, testing for the presence of EGFR gene mutations in the formalin-fixed paraffin-embedded (FFPE) samples at baseline and plasma cfDNA at progression is a part of the routine protocol for all patients with NSCLC. We included all patients with a baseline EGFR mutation test on the tissue biopsy and for whom EGFR testing had been performed at progression on a bodily fluid sample, as well as on a plasma sample. The prerequisite for performing a molecular test for EGFR mutation detection on a tissue sample is the presence of more than 10% of tumor tissue content as determined by the hematoxylin and eosin staining. Similarly, the prerequisite for performing such a test on a cytology sample is the presence of more than 20% of tumor cell content. Our allele-specific real-time reverse transcription-polymerase chain reaction (RT-PCR) assay developed in-house for EGFR developed detection is accredited by the National Accreditation Board for Testing and Calibration Laboratories (NABL) and the College of American Pathologists (CAP).[15] As per the NCCN guidelines, the test results are generally reported within 10 days in our NABL- and CAP-accredited laboratory. However, for the 11 patients enrolled in this study, the turnaround time was <5 days.


Our primary aim was to determine the presence of EGFR gene mutations in various bodily fluids of patients with EGFR-mutant progressive metastatic lung adenocarcinoma. Our secondary aims were to evaluate the concordance of the results of the EGFR-testing, both for sensitizing and resistant mutations, from various bodily fluids and from plasma, with those from the baseline tumor tissue.

Study methodology

At progression, malignant bodily fluids, including pleural fluid, ascites, pericardial fluid, and CSF, were collected along with the blood samples from the 11 patients. EGFR mutation testing was performed on the extracted cfDNA samples. The results of mutation testing on cfDNA obtained from malignant bodily fluids were compared with the result of their corresponding plasma cfDNA testing.

Procedure for cell-free DNA extraction from pleural, ascitic, and pericardial fluids

The peripheral blood samples and other bodily fluids were received in the laboratory within one hour of collection. Plasma and bodily fluids (pleural, ascitic, and pericardial fluid) were centrifuged at 2000 rcf (relative centrifugal force) for 20 min at room temperature to separate the supernatant. The supernatant was transferred to a sterile 15-mL polypropylene (PP) conical tube and centrifuged at 3200 rcf for 30 min at room temperature to remove any cell pellets. cfDNA was extracted from the supernatant immediately using the QIAamp Circulating nucleic acid kit (QIAGEN catalog number 551144).[2]

Procedure for cell-free DNA extraction from the cerebrospinal fluid

CSF was centrifuged at 2000 rcf for 20 min at room temperature to separate the supernatant. The supernatant was transferred to a sterile 15-mL PP conical tube. The volume of the supernatant was made up to 2/4 mL using phosphate-buffered saline, if the sample volume received initially was less than the required volume. cfDNA extraction was performed immediately using the same extraction QIAGEN KIT mentioned as above. The quality, quantity, and size of the extracted cfDNA were analyzed using the TapeStation 4200 from Agilent.[2] For the detection of EGFR mutations from cfDNA samples, the extracted cfDNA fragments had to be 120–180 bp in size.

Method for real-time allele-specific polymerase chain reaction

Allele-specific real-time RT-PCR for EGFR mutation detection was performed as per our in-house established assay using the ROCHE II LC 480 Platform.[2]


As this was a retrospective observational study, a formal sample size calculation was not done. The status of the EGFR-sensitizing mutations determined using the FFPE tumor tissue at baseline was considered the gold standard for comparison with the mutation status determined using plasma and other bodily fluids collected at disease progression. In addition to sensitizing mutations, the presence of the additional T790M mutation for acquired resistance was also evaluated in the plasma and other bodily fluids of the patients. Among patients in whom a T790M mutation was detected in the bodily fluids at progression, only those for whom the status of the EGFR-sensitizing mutations were concordant at baseline and progression were considered to be T790M positive. Given the observational nature of the study and the small sample size, analysis has been performed using absolute numbers and percentages; no further statistical analyses were possible.


Between December 2017 and June 2018, there were 11 patients with EGFR-mutant NSCLC who had disease progression and the EGFR mutation status was determined for their malignant bodily fluid samples as well as their blood samples. As detailed in [Table 1], there were six men and five women in our study cohort. Majority of these patients were nonsmokers (8/11, 72.7%). At baseline, 7 out of the 11 patients had multiple brain lesions, 2 had additional bone metastases, 2 had only bone metastases, and 2 had single brain lesions. As determined using the baseline FFPE tumor tissues, 7/11 (63.6%) patients harbored EGFR exon 19 deletions, and 4/11 (36.4%) harbored an exon 21 L858R point mutation.{Table 1}

In 9 out of the 11 patients, malignant cells were observed on cytologic examination of the bodily fluids at disease progression; in the remaining two patients, atypical cells were observed with a suspicion of malignancy. The yield of plasma cfDNA was very low in these patients, and this could be because of the presence of only few malignant cells. More than half of the patients showed the presence of malignant cells in the CSF (6/11, 54.5%). Additionally, 3 (27.3%) patients showed malignancy in pleural effusions, 1 (9.1%) in ascitic fluid, and 1 (9.1%) in pericardial effusion.

At the time of disease progression, a repeat tissue biopsy was not feasible in any of the patients. As detailed in [Table 2], of the 11 patients harboring EGFR-sensitizing mutations (exon 19 deletion and exon 21 L858R mutation) at baseline, only 7 (63.6%) showed the presence of sensitizing mutations in the plasma sample collected at disease progression, whereas these sensitizing mutations were observed in other bodily fluids (CSF, pleural effusion, ascites, pericardial effusion) collected at disease progression of all the 11 (100%) patients. Thus, the concordance for EGFR-sensitizing mutation detection of plasma and other bodily fluids with FFPE tumor tissue as the reference standard was 64% and 100%, respectively [Figure 1].{Table 2}{Figure 1}

Out of 11 patients, the exon 20 T790M acquired resistance mutation was observed in the plasma of 3 (27.3%) patients and other bodily fluids of 9 (81.8%) patients [Figure 2]. Thus, for 3 patients (patient numbers 1, 7, and 9), the T790M mutation was detected in both the plasma as well as other bodily fluids. In the remaining 6 patients, this mutation was detected only in bodily fluids other than blood. There was no patient in whom the T790M mutation was detected in the plasma but not in the other bodily fluids.{Figure 2}

In the 2 patients with atypical cells in their cytology samples (patient numbers 3 and 4), the T790M resistance mutation was not detected; instead, the baseline EGFR-sensitizing mutation was observed. Moreover, for these 2 patients, the results of mutation testing were discordant between plasma at disease progression and FFPE tumor tissue at baseline.

Thus, our results suggest that compared to plasma cfDNA, the free DNA obtained from bodily fluid supernatants has a higher detection rate and sensitivity for tumor-specific mutations, especially the exon 20 T790M resistance mutation.

Of the nine patients with T790M mutation, seven were treated with osimertinib; these patients have had good disease control and are continuing on osimertinib to date. One patient died before therapy could be started, and one had financial constraints and therefore could not afford osimertinib.


In our small cohort of patients with EGFR-mutant lung adenocarcinoma with disease progression, the liquid biopsy samples from bodily fluids other than blood showed a 100% (11 out of 11) concordance with the baseline FFPE tumor-based testing for the detection of EGFR-sensitizing mutations. On the other hand, the concordance for EGFR-sensitizing mutation detection between liquid biopsy from plasma and FFPE tumor tissue was 63% (7 out of 11). An absolute match of all the baseline mutations was observed in other bodily-fluid-based liquid biopsies, indicating higher mutation recall potential of bodily fluids other than blood. The EGFR-TKI resistance mutation, T790M, was found in other bodily fluid samples of 9 (81.8%) patients and in the plasma samples of 3 (27.7%) patients. Overall, our findings suggest that free DNA obtained from bodily fluid supernatants has a higher detection rate and sensitivity than plasma for EGFR-sensitizing mutations (100% vs. 63%) as well as the T790M resistance mutation (81% vs. 27.7%). Therefore, cfDNA obtained from various malignant bodily fluid supernatants could be used as high-quality specimens for EGFR mutation detection in patients with progressive lung cancer.

Our observations are in line with those of other published studies. The results of Zhang et al. suggest that compared to sedimented tumor cells from malignant bodily fluids and cfDNA from plasma, free DNA obtained from bodily fluid supernatants has a higher detection rate and sensitivity for tumor-specific mutations.[13] They suggested that the free DNA from supernatants of bodily fluids could serve as alternative specimens for molecular testing and mutation detection in patients with lung cancer. Various studies in the literature also strongly advocate that because of the blood-brain barrier, DNA released by brain tumors has a low likelihood of being detected in the plasma, and hence, in patients with brain metastases, other bodily fluids especially CSF can be used to help in disease management and treatment decision making.[14],[16],[17]

In our study, the rate of detection of the T790M resistance mutation was lower in the plasma (27%) than in other malignant bodily fluids (81%). This could be because of the higher yield of cfDNA in the malignant bodily fluid than in the plasma at disease progression.

Thus, in this single-gene biomarker study, we attempted to detect EGFR mutations in various malignant bodily fluids and determine their potential as a predictor of response to the EGFR-TKI. Liquid biopsy (from bodily fluids other than plasma) has attracted much attention due to its minimally invasive nature, minimal trauma caused to the patient, and high reproducibility. It presents the possibility for the use of malignant bodily fluids as specimens for rapid and sensitive molecular detection.[9],[13],[14],[15],[16],[17] Various studies indicate that “non-blood” fluids close to metastatic sites are superior to blood for the detection of the relevant mutations and can offer valuable clinical information,[14],[16],[17]

The limitations of our study include the small sample size and the fact that the cohort comprised only those patients in whom the treating physician ordered EGFR mutation testing in bodily fluids other than plasma. Patients were not systematically enrolled, and thus there may have possibly been a selection bias. In future, a larger sample size-based study should be performed in a systematic fashion. The study should be expanded to include patients with metastatic lung adenocarcinoma with a wide spectrum of disease stages and metastatic sites; the exact metastatic sites should be correlated with the results of molecular testing, especially the status of EGFR exon 20 T790M mutation, in the malignant bodily fluids. The use of highly sensitive tests like droplet digital PCR (ddPCR) to detect the TT90M mutation in the plasma at disease progression might increase the yield.[18] However, currently, as we do not have the facility to perform ddPCR in our department, we are unable to comment on this aspect. There are some limitations associated with the use of free DNA obtained from bodily fluids. Blood samples can be collected in Streck tubes and stored for several days. This allows for convenient shipping and processing of the samples at a later time point. Whether one can transport these malignant bodily fluids in a similar manner for mutation profiling is still unclear.


There is a very high concordance for EGFR mutation detection between cfDNA from bodily fluids other than plasma and FFPE tumor tissue in patients with progressive EGFR-mutant lung adenocarcinoma. The rate of detection of the EGFR T790M resistance mutation is considerably higher in other bodily fluids than plasma. The primary observation of high mutation recall efficiency of cfDNA from “non-blood” malignant fluids is intriguing and though not practice-changing, can help generate a hypothesis for the detection of EGFR mutations, particularly in patients who progress on EGFR TKI.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 Supplementary Appendix

Supplementary Appendix 1: Protocol Title: Establishment of Registry for Collating the Data of Lung Cancer Patients Receiving Systemic Therapy with Palliative Intent in India

Co-ordinating Investigator:

Dr. Kumar Prabhash.

Prinicipal Investigators:

Dr. P. M. Parikh, Mumbai.

Dr. Vanita Noronha, Tata Memorial Hospital, Mumbai.

Dr. Rajutitus Chako, CMC Vellore.

Dr. Narayan Prasad, Narayan Hryudralaya, Bangalore.

Dr. Sachin Hingmere, Pune.

Dr. Sushil Mandania, Nagpur.


1. Brief ………………………………………………………………. 3

2. Introduction ………………………………………………………. 3

3. Objectives …………………………………………………………… 4

4. Methods:

4:1 Resource …………………………………………………… 4

4:2 Data collection …………………………………………….. 4

4:3 Treatment and follow up …………………………………. 4

4:4 Sample size.………………………………………………… 4

4:5 Confidentiality.……………………………………………... 5

5. References ………………………………………………………….. 6


The primary aim of this activity is to establish a multicentric lung cancer registry involving multiple institutes in India to record the epidemiology pattern and study the treatment outcome results in lung cancer patients in palliative setting.

The secondary aim is to improve upon the treatment outcomes and look into the prospect of developing a consensus on management of lung patients in India. It will also help in establishing a network of oncologist who may collaborate in future studies.


Lung Ca is most common cancer world-wide (~12% of all new cancers). [Figure 1] and [Figure 2].

Despite decades of research:

Five years survival rate: 5%–15%Median survival <12 months.



Deaths due to lung cancer are greater than those due to colorectal, breast and prostate cancers put together. Focus worldwide shifted to practicing personalized medicine for lung Ca, especially NSCLC.

Trend in incidence of lung cancer (GLOBOCAN 2008):

In India 47,000 males and 11,000 females suffered with lung cancer in 2008 of which 40,000 males and 10,000 females, died.[1,2] There is paucity of Indian data published on epidemiology, treatment pattern and outcome.

We anticipate that through the establishment of this lung cancer registry we would be able to understand the epidemiological distribution of the various lung cancer subtypes treatment pattern followed and outcome. This information collated herewith would also benefit the treating physicians to optimize and/or improvise treatments of the lung cancer patients. The information generated would also help us formulate treatment consensus which can be disseminated later. Eventually, other institutes from the region may join the registry.


The objectives of establishing the registry is to study the epidemiology and improve the treatment outcomes, of lung cancer. The data for this registry would be collated via an Excel based data collection form at the participating centers; and we wish to club together the data form across the centers once in a year.

The specific aims of the lung cancer registry are the following:

Registration of lung cancer patients and record their demographic, clinical and pathological features.Collation of information on clinical course of the disease, treatment and after therapy follow-up.To promote collaborative research.



Data will be collected with the help of Excel based data collection form at the participating sites of this lung cancer Registry. The collation of the data would be done on a yearly basis or as decided by the participants; the collated data may be stored at a central location as decided by the participants. Monitoring will be done regularly to ensure completion of data.

This activity is an initiative of the ICON. Each site will be provided data entry person and monitor for regular monitoring will be provided by ICON. Monitoring report will be shared with all sites. Data from individual site will remain with the site till PI agrees to share data with each other. This will be done usually every year for analysis and publication. Only ICON members will be included as PI in the study.

Data collection

This registry aims at both (i) retrospective and (ii) prospective collection of the data of lung cancer patients being treated with palliative intent.

Retrospective collection

The available data of treated lung cancer patients will be collected retrospectively. The data of all the patients who have received at least one cycle of chemotherapy will be collected. The data of patients who have come for consultation on relapse/after receiving chemotherapy during this period will not be included.

Since the data of the patients for retrospective collection would already be there with the institute and most of the patients would have progressed/died/lost to follow up, the informed consent for retrospective collection would not be taken.

Prospective collection

Obtaining of Informed consent of the lung cancer patients is kept optional (based on the Institute specific requirements). Patients would complete work up and staging in the regular OPD's. The patients will be counseled on the purpose and process of the lung cancer registry. Anyone eligible for enrolment to the registry would be invited to participate and data sheet will be filled out even if the patient does not want to register giving the reasons for refusal.

Once the patient consent (as per the institution guidelines) for the same, they will be given a unique identity which will be used to maintain confidentiality. The clinic staff will maintain the patient identification, unique I-D code as well as addresses and contact numbers of patient and at least two close relatives. The investigations/clinical procedures deemed necessary to stage and prognosticate the disease will be done as per the routine clinical practice. Additional investigations (if done) would be recorded for completion of data.

The patient's data will be entered in excel based data collection form within a week of the patients visit to the OPD. The common data set for registration of lung cancer patients will be managed by PI. The collated data would be shared with all the investigators on regular basis only after the consent of PI.

Treatment and follow up

The treatment planning, decision to intervene and final treatment given would be left to the choice of the treating doctor. The registry will record the data available on the treatment given. The details of treatment given, dose modification and side effects, during treatment, date of first diagnosis start and end of treatment, recurrence and progression, last follow up and death will be recorded.

Sample size

This is a lung cancer registry. No power calculations are required. Completeness of data may be evaluated by regular monitoring of lung cancer registry data.


Due to the confidential nature of the medical data utmost care will be taken to protect the privacy of the persons about whom information is received.


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