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Year : 2017  |  Volume : 54  |  Issue : 5  |  Page : 25--30

Liquid biopsy: A potential and promising diagnostic tool for advanced stage non-small cell lung cancer patients

DC Doval1, R Deshpande2, B Dhabhar3, KG Babu4, K Prabhash5, R Chopra6, PV Sripada7, C Deshmukh8, M Suryavanshi9,  
1 Senior Consultant Director of Medical Oncology, Rajiv Gandhi Cancer Institute & Research Centre, Delhi, India
2 Surgical Oncologist, Asian Cancer Institute, Mumbai, India
3 Senior Consultant, Medical Oncology, Fortis Hospital, Mulund, Mumbai, India
4 Associate Professor in Medical Oncology, Kidwai Memorial Institute of Oncology, Bangalore and Consultant Medical Oncologist HCG Hospitals, Bangalore, India
5 Professor, Department of Medical Oncology, Tata Memorial Hospital, Mumbai, India
6 Director & Head, Department of Oncology & Hematology, Artemis Health Institute, Gurgaon, Haryana, India
7 Senior Consultant, Department of Oncology, Apollo Health City, Apollo Hospitals, Hyderabad, India
8 Consultant Oncologist, Dinanath Mangeshkar Hospital, Pune, India
9 Consultant Molecular Pathologist, Rajiv Gandhi Cancer Institute & Research Centre, Delhi, India

Correspondence Address:
Dr. D C Doval
Senior Consultant Director of Medical Oncology, Rajiv Gandhi Cancer Institute & Research Centre, Delhi


More than 50% of non-small cell lung cancer (NSCLC) cases harbor an actionable mutation, and molecular testing at different intervals can help in personalized and targeted treatment. Core tissue biopsy and needle biopsy done at the time of diagnosis/disease progression are interventional, time-consuming and can affect the patients adversely. Noninterventional biomarker testing by liquid biopsy promises to revolutionize advanced stage cancer screening. The present report was formulated based on an expert panel meeting of renowned oncologists who gave their opinions for minimally invasive liquid biopsy to detect targetable molecular biomarkers in advanced NSCLC cases. An exhaustive literature search was done to support their recommendations. Clinical utility of minimally invasive liquid biopsy, for detection of molecular biomarkers in advanced stage NSCLC patients, was broadly discussed by the key opinion leaders.

How to cite this article:
Doval D C, Deshpande R, Dhabhar B, Babu K G, Prabhash K, Chopra R, Sripada P V, Deshmukh C, Suryavanshi M. Liquid biopsy: A potential and promising diagnostic tool for advanced stage non-small cell lung cancer patients.Indian J Cancer 2017;54:25-30

How to cite this URL:
Doval D C, Deshpande R, Dhabhar B, Babu K G, Prabhash K, Chopra R, Sripada P V, Deshmukh C, Suryavanshi M. Liquid biopsy: A potential and promising diagnostic tool for advanced stage non-small cell lung cancer patients. Indian J Cancer [serial online] 2017 [cited 2020 Jul 15 ];54:25-30
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Full Text


The GLOBOCAN 2012 has reported that lung cancer constituted 6.9% of all new cancer cases in India and accounted for approximately 10% of total cancer-related deaths in both males and females.[1] Mortality data form India report approximately 3–10 deaths/100,000 population.[2] Globally, lung cancer is the leading cause of cancer-related deaths in which non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancers.[3]

The most common driver mutations in NSCLC patients are epidermal growth factor receptor (EGFR) (10%–30%); fibroblast growth factor receptor 1 (FGFR1) (20%); echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) (3%–7%); human epidermal growth factor receptor 2 (HER2) (2%–5%); MET proto-oncogene, MET (4% in de novo and 20% in patients with acquired resistance to EGFR tyrosine kinase inhibitors [TKIs]); Kirsten rat sarcoma viral oncogene homolog (KRAS) (15%-30%); BRAF (1%–3%); ROS proto-oncogene 1, ROS1 (1%); and ret proto-oncogene, RET (1%).[4],[5],[6],[7] In India, approximately 26%–31% of the NSCLC patients harbor EGFR mutation.[8],[9] Molecular testing for EGFR and ALK has become the standard of care before the initiation of targeted treatment with TKIs.[10],[11]

Approximately 64% of lung adenocarcinomas harbor an actionable mutation while 40% of patients with squamous cell lung carcinoma are found to have genetic alterations.[12],[13] Genotyping of the tumor can help to decide the best therapeutic first-line option, and it can also indicate the development of resistance to TKI therapies. The NSCLC patients with activating EGFR mutations can be treated with anti-EGFR agents. In EGFR-nonmutated patients, chemotherapy and immunotherapy are the therapeutic options.[6] Further, Veldore et al. suggested that 8%–10% of EGFR mutation-negative NSCLC patients show molecular abnormalities in EML4-ALK, ROS1-ALK, and KIP4-ALK and may derive benefit from targeted therapy. The authors also suggested a potential role of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA), vascular EGFR 2 (VEGFR2), RET, and FGFR2 as therapeutic targets in EGFR nonmutated lung cancer patients which, however, requires further clinical validation.[14] In addition, molecular profiling can help to identify resistant mutations in patients who have progressed after first-line precision treatment. EGFR-T790M is the most abundant secondary mutation responsible for resistance to first-generation (gefitinib and erlotinib) and second-generation (afatinib) EGFR-TKIs and is reportedly present in approximately 60% of NSCLC patients that had progressed on after treatment with EGFR-TKIs.[15],[16],[17] The other mechanisms responsible for resistance include activation of receptor tyrosine kinases such as erb-b2 receptor tyrosine kinase 2 (ERBB2), MET, FGFR1, and ALK and several other signaling pathways.[18] Tumors reportedly become resistant to these inhibitors within 10–16 months of treatment.[19],[20],[21],[22] A recent study with osimertinib (third-generation TKI) showed an overall response rate of 60% in T790M-positive cancer patients who have progressed after frontline anti-EGFR therapy.[23] Thus, molecular testing for resistant mutations can provide personalized precision treatment options for advanced stage NSCLC patients.

Fresh tissue biopsy taken at the time of disease progression is currently the most common source of tumor specimen used by physicians to detect resistant mutations. Molecular analysis of tissue biopsy can be done with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), but since only 30% of NSCLC patients present with a resectable stage at diagnosis, histologic evaluation is not always possible.[24],[25] Fine-needle aspiration cytology (FNAC) can also be used for diagnosis of advanced stage NSCLC patients but has several procedural limitations owing to progressive disease and tumor heterogeneity. Moreover, tissue obtained with this technique is insufficient to perform additional tests, and thus, misinterpretation/over interpretation of sample can lead to false negative/positive results.[26] Stereotactic needle biopsy is another technique which is minimally invasive and well tolerated but brings in the risk of lung injury (pneumothorax).[27] Physicians face multiple challenges with this technique, and in the recent past, liquid biopsy has emerged as a new strategy for tumor genotyping. It is based on the use of surrogate sources of DNA such as blood, serum, plasma, and urine samples, which often contain circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cell-free RNA (cfRNA), and circulating tumor cells (CTCs).[28] These DNA fragments are released from the cancer cells into the bloodstream either spontaneously or passively (when the capacity of phagocytes to eliminate cancer cells is exceeded by an increase in tumor volume), and as a result, the apoptotic and necrotic fragments get into the blood circulation.[29],[30] Liquid biopsy provides an alternative to tissue biopsy and addresses the limitations of the latter and the concerns of the physicians. Liquid biopsy enables real-time monitoring of cancer progression and evolving genetic changes within the tumor.[31] In India, applications of molecular testing is improving with around 80% of medical oncologists currently opting for biomarker testing at primary biopsy.[32] Recently, the US Food and Drug Administration (FDA) has approved ctDNA-based liquid biopsy (cobas EGFR Mutation Test v2) for identifying patients with metastatic NSCLC eligible for targeted therapy with erlotinib (EGFR exon 21 L858R substitution mutations) and osimertinib (EGFR T790M mutation).[33]

The current article discusses the expert opinion on the potential applicability and limitations of liquid biopsy and explores the possibilities of substituting the standard tissue biopsy, especially in patients with advanced NSCLC resistant to frontline targeted therapy.


We collated the expert opinions of two separate expert panel meetings which were organized by AstraZeneca on May15, 2016, in Mumbai and September 4, 2016, in New Delhi. The meetings consisted of two different groups of expert panel members comprising of Indian medical oncologists, surgical oncologists, radiation oncologists, and clinical pathologists. The panel were comprised of 21 experts (panel members listed in the Appendix) and convened to review the emerging trends in NSCLC diagnosis with a focus on the potential advantages and limitations of liquid biopsy. Based on the expert committee discussion and literature review, the manuscript was formulated and distributed among the expert panel members. The members were requested to revert with their individual comments within a month, following which all the concerns and comments were addressed by the writing group and the content was finalized.

Literature search and selection criteria

The present report was formulated after critical analysis and discussion by the expert panel members. This was supported by an exhaustive literature search in PubMed from where relevant articles were analyzed and referenced for the preparation of the manuscript. The search terms used were “liquid biopsy,” “NSCLC,” and “NSCLC” in combination with the Boolean operators “AND/OR.” Only the studies published after the year 2005 and conducted in human subjects were considered for this report.

In total, 56 research articles, systematic reviews, or meta-analyses were used for the preparation of this manuscript. The recommendations from each group are presented and the expert panel discussions on liquid biopsy in NSCLC diagnosis are reported here.

 Expert Opinion and Rationale

The panel recommends that liquid biopsy is a better diagnostic technique to perform in NSCLC patients when compared to tissue biopsy. It can be a substitute in cases where tissue biopsy is not feasible and can also be used as an alternate to secondary biopsy. Liquid biopsy also makes rebiopsy more feasible in advanced cancer patients and shows tremendous potential due to ease of performance and potential for repetition.


The panel recommends liquid biopsy to be an important diagnostic, prognostic, and therapeutic tool for real-time monitoring of NSCLC patients undergoing TKI-therapy. Liquid biopsy can be a substitute for initial core biopsy. It is even helpful in EGFR-TKI resistant NSCLC patients who might present several characteristics which could make rebiopsy difficult such as lack of appropriate site, unstable patient condition, and small tumor size generally accompanied with fibrosis. Moreover, interventional techniques such as tissue biopsy carry a high cost. Liquid biopsy can even help in detecting several other drug resistance mechanisms and identify potentially actionable mutations and genetic alterations for advanced targeted therapy. The day is not far when liquid biopsy will be a routine practice for NSCLC patients. However, final results of large population-based studies are awaited.


Conventionally and in an ideal situation, tissue biopsy is used to obtain a cancerous tissue. However, patients are not always in a clinically stable condition to undergo the traumatic procedure of tissue biopsy. Furthermore, up to 80% of NSCLC patients with advanced disease only have tissue from small biopsies or cytology, limiting the ability to perform additional tests, and as many as 31% of patients do not have accessible tissue,[34] Further, a repeat invasive biopsy in advanced cancer patients leads to increased morbidity, mortality, and related cost.[13],[19] Tissue heterogeneity is a concern, where certain actionable mutations are present only in certain tumor portion and might not be the representative of the tested sample and thus, can be missed. Even though histological evaluation of cancer cases based on tissue biopsy is considered as the gold standard for diagnosis, there remains a risk of misdiagnosis as morphology is only taken into consideration and diagnosis varies as per the expertise of the clinicians.[20] Prabhash et al., in their Indian survey report highlighted the low rebiopsy rates (25%) in progressive NSCLC patients attributing it to rapid tumor progression and poor performance status.[32] Zanwar et al. investigated several issues relating to the feasibility of rebiopsy in Indian NSCLC patients and reported that patient unwillingness (in 47% of the cases) was the biggest hindrance in performing invasive rebiopsy which was followed by technical difficulties and physician's discretion (32% of the cases).[35] Similar report was presented by Chandrasekharan et al. where physician reluctance was the commonest reason for postprogression nonbiopsy in NSCLC patients.[36] The study by Zanwar et al. also highlighted that among the patients who underwent rebiopsy, adequate tissue was not available for 41.5% of the patients.[35] Therefore, minimally invasive techniques which can provide adequate material for subsequent analysis is the need of the hour.

Liquid biopsy can easily identify several driver genes and targetable mutations in NSCLC patients and overcome issues of tumor heterogeneity. Further, it can provide real-time genetic surveillance in patients receiving targeted therapies and those who are at a higher risk for acquired TKI-resistance.[13],[19] While single tissue biopsy provides only a spatial and limited tumor snap-shot, liquid biopsy allows detection of tumor-associated mutations in the blood, assessment of patient prognosis and early detection of tumor recurrence. It can be a powerful alternative for traditional tissue biopsies in predicting both treatment response and development of acquired resistance.[37]

The source of tumor markers for liquid biopsies is ctDNA, cfDNA, cfRNA, and CTCs. The circulating nucleic acids are released from apoptotic or necrotic tumor cells and can be detected in several body fluids such as blood, urine, saliva, and cerebrospinal fluid (CSF).[16],[18] CTCs enter the bloodstream as clusters or single cells during homogeneous cancer spread. cfRNAs (enriched in exosomes) such as microRNA (miRNA), small nucleolar RNA (smoRNA), PIWI-interacting RNA (PIWI-RNA), and long noncoding RNA (lncRNA) are released by tumor cells into the bloodstream.[34] Even platelets can sequester cfRNA released by tumor cells and be an alternative source for tumor markers.[38],[32] At present, most of these biomarkers are in their advanced research stages.

A retrospective study demonstrated that molecular analysis of cfDNA had good concordance with archived tissue testing in 91% cases for BRAF mutations (κ = 0.75, 95% confidence interval [CI] 0.63–0.88), 99% cases for EGFR mutations (κ = 0.90, 95% CI 0.71–1.00), 83% cases for KRAS mutations (κ = 0.67, 95% CI 0.54–0.80), and 91% cases for PIK3CA mutations (κ = 0.65, 95% CI 0.46–0.85).[39] Further, Breitenbuecher et al. described a novel, highly sensitive and specific strategy to detect CTCs harboring somatic mutations in NSCLC patients and suggested that it can be used for mutational profiling and monitoring of treatment efficacy in patients with EGFR-positive NSCLC. Clearance of EGFR-mutant CTCs was found to be associated with treatment response and disease control.[40]

Although tumors with nearly 50 million malignant cells release sufficient DNA for ctDNA detection in blood, current detection methods such as positron emission tomography-computed tomography fail to detect tumors which are <7–10 mm in size even though they contain a large number of cancerous cells (approximately 1 billion). Thus, recent advances in cell capture and plasma isolation methods along with highly sensitive platforms give liquid biopsy a feasible introduction in clinical practice.[34]

Liquid biopsy is generally based on digital-droplet polymerase chain reaction (ddPCR) and/or next-generation sequencing (NGS) with a concordance of 70%–80% with tissue-based genotyping.[41] Other methods for liquid biopsy include quantitative PCR (qPCR) and the recently FDA approved RT-PCR.[29],[40] However, qPCR has a lower sensitivity when compared to ddPCR, particularly in samples with low EGFR mutation abundance (secondary EGFR T790M resistance mutation). On the other hand, serial use of ddPCR enables early diagnosis of acquired resistance and helps clinicians monitor one or more driver mutations over the entire treatment period.[42] This in turn helps in evaluating treatment response and tumor recurrence. Plasma ddPCR, as reported by Sacher et al., showed a median turnaround time of 3 days compared to 12 days for tissue genotyping in newly diagnosed NSCLC patients and 27 days in patients with acquired resistance. Furthermore, plasma ddPCR had a positive predictive value of 100% (95% CI: 91%–100%) for EGFR 19 del, 100% (95% CI: 85%–100%) for L858R, and 100% (95% CI: 79%–100%) for KRAS, while 79% (95% CI: 62%–91%) for T790M. The technique was also found to be highly sensitive (82% for EGFR 19 del, 74% for L858R, and 77% for T790M).[43]

NGS also provides a comprehensive detection of mutation, but takes a longer processing time, requires advanced bioinformatic analysis, and is costlier when compared to ddPCR.[44],[45] ddPCR when compared to NGS displays a greater sensitivity for the targeted set of known mutations in cancer diagnostics.[44]

Molecular and histological analysis with FISH and IHC helps in detecting EML4-ALK driver oncogenes in NSCLC during solid tissue biopsy.


EML4-ALK, being the second most important driver oncogene in NSCLC, the panelists unanimously agreed that molecular analysis with FISH/IHC is essential for characterization of the tumor samples. They also mentioned that there is a very high concordance rate (98%) of FISH and IHC in detecting EML4-ALK rearrangements. A two-tiered approach (with both IHC and FISH) was also suggested for a better tumor tissue characterization; however, this received a mixed response from the panel members.


IHC is a readily available, rapid, and relatively inexpensive testing method requiring minimal infrastructure, and hence, many NSCLC patients are initially screened by IHC. Two ALK IHC tests using the antibody clones, particularly D5F3 and 5A4 are reported to be highly accurate and reliable in predicting ALK rearrangement in NSCLC patients.[46] FISH assay using dual-labeled break-apart probes was also found to aid patient selection for treatment with crizotinib, a targeted ALK TKI. Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology has recommended ALK FISH assay using dual-labeled break-apart probe for selecting patients for ALK TKI therapy.[11]

Furthermore, the sensitivity of detecting ALK rearrangement by IHC can be improved by including an efficient polymer-based detection system and FISH. It is recommended that ALK IHC can be considered as a screening methodology to select specimens for ALK FISH testing if carefully validated.[11]

Tissue acquisition with core biopsy and FNAC helps in characterizing the molecular profile of NSCLC patients.


The panel members recommended tissue biopsy or core biopsy as the method of choice for histological assessment of tumor samples. Few panelists considered FNAC to be as good as tissue biopsy and implicated it to be the method of obtaining tumor tissues when there is an inadequate sample or inaccessible tumor site. Cell blocks, prepared from FNAC sample were agreed to provide high cellularity along with better morphological and architectural patterns. Subsequently, it was also suggested that tests initially done with FNAC must be molecularly validated to help in further tumor characterization.


Histological evaluation of cancer cases by solid tissue biopsy is considered to be the gold standard for diagnosis, but the relevant risk of misdiagnosis is quite large since morphology is only taken into consideration and there is observed variability among clinician's interpretation.[23] Furthermore, the procedure for tissue biopsy is a limiting factor (such as endoscopic biopsy and transthoracic trucut biopsy are interventional and uncomfortable to patients).[24] In maximum number of cases, the success rate of these invasive procedures varies considerably and might as well affect the diagnosis accuracy and risk of complications.[24] These factors along with the lack of accessible sites and small tissue quantity limit the possibility of performing much needed additional molecular tests.[34]

FNAC, on the other hand, is fairly accurate and can guide medical treatment of NSCLC cases, especially when interventional tissue biopsy is not feasible. It shows a good concordance rate with histological diagnosis (88%). Sareen et al., in their recent study (2017) with 550 cytological specimens from Indian lung cancer patients showed that the sensitivity of FNAC was 87% and its diagnostic yield was 90%. They also demonstrated the specificity of FNAC to be 100% with a false negative of only 12.5%.[47] Furthermore, it has also been demonstrated that the performance of FNAC, in cases of poorly-differentiated tumors, can be improved by ancillary techniques such as IHC.[26]

Core biopsy reflects tumor architecture and immunohistochemical profile accurately, helps in diagnosing benign lesions, assesses the invasive potential of cancer, and can sometimes indicate tumor subtypes. On the other hand, FNAC remains the method of choice in recurrent and metastatic cases and is relatively quick, accurate, and cost-effective tool for diagnosis and follow-up of NSCLC patients.[48] The overall positive predictive value of FNAC was close to 99% with false-negative rate of around 10% which is mainly due to sampling error and this can be addressed by rapid on-site evaluation with proper cytoblocks. IHC can be applied to cell block preparations of FNAC samples which add to its diagnostic accuracy and even enables long-term archiving of tumor tissue. Liquid-based cytology can be used to prepare cell blocks providing additional architectural information and better tissue preservation.[49]

Liquid biopsy shows tremendous potential in molecular diagnosis of NSCLC patients, but the scientific community awaits more conclusive reports.


The use of liquid biopsy to replace tissue biopsy has been a topic of debate. Although numerous retrospective studies [50],[51],[52] demonstrate earlier detection of tumor recurrence by liquid biopsy when compared with imaging, current evidences are not sufficient to conclude the superiority of liquid biopsy. Furthermore, there is enough possibility of false positive results with liquid biopsy. The panel members, therefore, mentioned that liquid biopsy will not be able to replace tissue biopsy until modern sensitive technologies become cost effective for the general population and are ubiquitously available.


In a recently published study on liquid biopsy with 58 NSCLC patients, the sensitivity and specificity of EGFR mutation detection rate in plasma was evaluated to be 74% and 100%, respectively.[50] Another study reported plasma DNA T790M mutation in 40% of EGFR-mutated, TKI-progressed NSCLC patients.[51] Further, a study by Sundaresan et. al. reported a detection rate of 47%, 50%, and 50% for T790M mutations by tumor biopsy, ctDNA, and CTCs. The authors concluded that combined use of ctDNA and CTCs can increase the detection rate of T790M mutation, although the high cost with liquid biopsy can act as a barrier for its use.[52] In fact, rapid plasma genotyping of cfDNA by ddPCR might detect EGFR T790M missed by tissue genotyping (due to tumor heterogeneity in resistant disease) with high specificity and thus, avoid repeat biopsies.[43] However, Molina-Vila et al., in their study mentioned that high sensitivity of liquid biopsy might lead to false positives and therefore, requires a careful validation.[53]

With the advent of detecting methods for circulating biomarkers (CTCs, ctDNA, cfRNA, and cfDNA), liquid biopsy has the potential to be the diagnostic method of choice in TKI-resistant NSCLC patients.[34],[54] The major advantages and disadvantages of liquid biopsy are listed in [Table 1].{Table 1}


Liquid biopsy can detect tumor-specific clinical biomarkers such as cfDNA and CTCs which can be helpful in noninvasive screening of advanced stage cancer, monitoring treatment responses, and even explaining the underlying resistance mechanisms in advanced stage NSCLC patients. Tissue biopsy can provide better histological and molecular evaluation, but performing it in advanced cancer patients is quite challenging. Detection of circulating tumor markers in blood creates scope for carrying out liquid biopsy which is repeatable and noninterventional in nature. The present report suggests the practical applicability of liquid biopsy for advanced stage NSCLC patients. Summary of the recommendations by the experts is tabulated in [Table 2].{Table 2}


The authors would like to acknowledge AstraZeneca Pharma India Ltd and Turacoz Healthcare Solutions for Medical writing and editing support.[55],[56]

Financial support and sponsorship

Financial support to authors - Nil.

The supplement issue in which this article has been published has been sponsored by AstraZeneca Pharma India Ltd.

Conflicts of interest

There are no conflicts of interest.


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