Context.—

Molecular testing is essential for the diagnostic workup of patients with advanced non–small cell lung cancers. Cytology specimens from minimally invasive procedures, such as endobronchial ultrasound–guided transbronchial needle aspiration, are often the only available samples for these patients. The implementation of molecular diagnostic testing, and in particular next-generation sequencing–based testing, on these cytologic specimens is currently an evolving field for lung cytopathology. The application of these molecular analyses on tyrosine kinase inhibitor–resistant non–small cell lung cancers raises unique technical, biologic, and clinical challenges.

Objective.—

To provide an overview of the implementation of next-generation sequencing analysis on endobronchial ultrasound–guided transbronchial needle aspiration samples to detect the molecular aberrations underneath the phenomenon of acquired resistance in patients with non–small cell lung cancers progressing while on the EGFR/ALK tyrosine kinase inhibitor treatment.

Data Sources.—

Peer-reviewed original articles, review articles, and published guidelines and expert opinion reports were reviewed, together with our single-center experience.

Conclusions.—

Next-generation sequencing analyses and the endobronchial ultrasound–guided transbronchial needle aspiration procedure may represent a valuable strategy to address the unique requirements of molecular testing on tyrosine kinase inhibitor–resistant non–small cell lung cancers.

The identification of key oncogenic events in tumor cells has allowed for the development of specific targeted therapies that have revolutionized the treatment and prognosis of patients with several types of cancer. Nowadays, the detection of molecular alterations in specific therapeutic targets represents an essential step in the clinical management of patients with non–small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR)–mutant NSCLC displays a unique disease course that is characterized by high response rates to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib.14  The c-Met/Hepatocyte growth factor receptor (HGFR) TKI crizotinib was found to be highly active in a small subset of nonsquamous NSCLC harboring anaplastic lymphoma kinase (ALK) or proto-oncogene tyrosine-protein kinase ROS1 rearrangements.5,6  Although these drugs show a high response rate—around 60% to 70%—resistance develops in almost all patients, resulting in clinical disease progression.7  The identification of the biologic underpinnings of the drug resistance is fundamental to monitoring disease evolution. Moreover, second- and third-generation anti-EGFR and ALK TKIs have both been developed to overcome specific resistance-driving genetic events.8,9  Therefore, it is important to investigate the presence of these specific molecular aberrations in all EGFR mutation–positive or ALK rearrangement–positive NSCLCs that have progressed after first-generation TKI treatment, in order to select patients who would benefit from the novel therapeutic strategies.

Molecular profiling is now a key part of the diagnostic workup for patients with advanced-stage NSCLC. The implementation of the same procedures in the setting of TKI-resistant disease raises unique challenges. From a technical standpoint, high-throughput and highly sensitive methods are required to profile the events subtending TKI acquired resistance that can be harbored by a subpopulation of tumor cells (ie, subclonal events).10  Next-generation sequencing (NGS) allows for the simultaneous detection of different types of molecular aberrations in multiple genes with high sensitivity. These technologies might be useful in analyzing the variety of molecular underpinnings of TKI resistance, while having a low DNA input requirement and a very low limit of detection.11  Moreover, ideally these molecular analyses should be performed at the time of disease progression and on samples from the site of disease progression. However, the rebiopsy procedure is not always feasible.12  The analysis of circulating tumor DNA (ctDNA; ie, “liquid biopsy”) is now recognized as an alternative noninvasive procedure,13  but the commercially available liquid biopsy assays do not always allow for testing the entire spectrum of resistance drivers.

During the last few years, there has been a growing interest in the application of molecular testing for NSCLC on cytology samples (molecular lung cytopathology) that may fill the gap between liquid biopsy and more invasive tissue rebiopsy. In this review, we will discuss a new field for molecular lung cytopathology focusing on the analysis of molecular drivers of resistance using NGS technology on cytology specimens from the endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) of NSCLC patients with thoracic progression after/on TKI therapy.

EBUS-TBNA SAMPLES FOR NGS

EBUS-TBNA is a procedure that couples real-time evaluation and sampling of lymph nodes and parabronchial lesions during bronchoscopy. This technology has revolutionized the diagnostic approach to lung cancer, giving the possibility of specimen procurement for the complete workup of patients with advanced NSCLC, including diagnosis, staging, and molecular analysis, with a minimally invasive procedure.14  EBUS-TBNA is now recommended as the first diagnostic procedure for patients with locally advanced lung cancer, based on its high sensitivity but low morbidity compared to more invasive procedures, such as mediastinoscopy.15  Recently, the American College of Chest Physicians (CHEST) published updated guidelines and expert panel reports on the technical aspects of this procedure.16  Briefly, the fundamental steps of EBUS-TBNA include the ultrasound-guided identification of the target lesion, the puncture of this lesion with a 21- or 22-gauge needle (at least 3 separate needle passes), and the aspiration of the material. This material can be smeared on a slide for immediate evaluation and/or conventional cytology stains, and placed in fixative solution for formalin-fixed, paraffin-embedded (FFPE) cell block preparation.17,18  Although slight differences in procedural details seem to have minimal effects on the diagnostic yield of EBUS-TBNA, minimal standards and a trained multidisciplinary team with specific expertise are required to maximize the efficacy of this procedure.16  Cytopathologists and cytotechnologists play an important role in assessing the adequacy of the specimens by rapid on-site evaluation and by handling these specimens according to standardized procedures to optimize the material for the subsequent analyses. The rapid on-site evaluation procedure is usually performed on an air-dried and Diff-Quik–stained smear, and it has been historically used to increase the diagnostic yield of TBNA, reducing the need for additional diagnostic intervention.16  With the advent of molecular marker testing as a mandatory standard of care for patients with nonsquamous NSCLC, cytopathologists should be trained to evaluate “on site” not only the diagnostic adequacy of the aspirated material but to perform an immediate quantitative and qualitative triage to accommodate molecular testing requests. The current “on site” question for cytopathologists is not only “is the sample adequate for the diagnosis?” but “is the material adequate and sufficient (ie, adequate overall cellularity and tumor cell content) for the diagnosis and subsequent molecular profiling?” After the initial triage, the processing and the use of these samples should aim to preserve material for molecular analyses. However, the lack of standardized preanalytic procedures across different laboratories, including various preparation techniques, fixatives, and stains, has complicated the validation of molecular assays on these samples.19,20  Several studies addressed the feasibility of the molecular testing implementation on EBUS-TBNA samples. Fourteen studies have been reviewed in the CHEST guideline that reported a high rate of adequacy and reproducibility of EBUS-TBNA providing tissue for molecular markers.16  Recently, we have also shown that after the standardization of technical procedures, EBUS-TBNA represents a reliable and robust method for routine molecular diagnosis, with results equivalent to those previously obtained in a large series of surgical samples.21 

The implementation of molecular diagnostics on cytologic specimens is currently one of the topics of lung cytopathology, given that these specimens may represent the only available samples in most patients with advanced NSCLC. Indeed, during the last few years, the decrease in material obtained for the diagnosis as a result of minimally invasive procedures paralleled the increasing number of molecular markers to be tested. Besides the recommended upfront testing of EGFR mutation and ALK and ROS1 gene rearrangements for all patients with advanced-stage NSCLC, additional therapeutic targets have been recently introduced in clinical practice or are under investigation, including MET, RET, BRAF, and HER2.22  In this setting, NGS represents a cost-effective strategy to screen multiple genes with a low DNA input requirement.11,23  Next-generation sequencing technologies are being integrated into the routine workflow of a growing number of molecular diagnostic laboratories. However, the commercially available NGS assays have been validated on FFPE tissue, and they cannot be automatically used on cytologic specimens, even on FFPE cell blocks, without proper validation procedures.24,25  Most series reporting the implementation of NGS analysis in pulmonary cytopathology described specimens obtained from different sites and different procedures, including both computed tomography–guided and EBUS-guided fine-needle aspiration.19,20,2631  However, given that a similar NGS success rate has been shown among different techniques,19,20  data about the viability of NGS on EBUS-TBNA samples can be inferred from these studies. Overall, the rate of success of NGS technology on cytologic samples ranged from 49% to 100%.25  This rate was mainly affected by the DNA yield of samples.32  Different clinical NGS platforms may require different minimum amounts of input DNA, and samples with DNA yields below these thresholds are usually not subjected to the analysis. These thresholds are largely determined by gene panel size and the enrichment capture strategy. Amplicon-based methods usually require lower amounts of input DNA compared with hybridization-capture methods, and they can represent a valid approach for testing small cytology specimens.25  Indeed, these thresholds have been established according to validation procedures on FFPE tissue samples. Roy-Chowdhuri et al19  have recently reported on the improvement of the NGS success rate of cytology samples (from 43% to 85% and from 74% to 96% for smear and cell block, respectively) by validating an NGS workflow with a DNA input lower than the manufacturer's recommendation. Despite the intrinsic variability of preanalytic factors in cytopathology, no significant differences have been reported in NGS success rates with different stains and fixatives.30  Moreover, both cytology smears and cell blocks have been used for NGS analysis.32  Although cell block preparation is often preferred for molecular testing, avoiding the sacrifice of the smeared slides, direct nonfixed smear could ideally provide higher DNA quantity and quality for NGS analysis.24  Direct comparisons of NGS performance and results between cytology and FFPE tissue samples from the same lesions have revealed high concordance rates in both NGS quality metrics and molecular aberration calls.29,30,33  Overall, these data demonstrated the technical feasibility of the application of NGS analysis on cytology specimens, including those obtained with the EBUS-TBNA procedure. The Papanicolaou Society of Cytopathology Molecular has endorsed NGS-based molecular testing in cytopathology in the recent consensus recommendations for ancillary testing of lung specimens.32  These recommendations highlight the opportunities and limitations of the clinical implication of this high-throughput and highly sensitive technology. Beside the technical validity of NGS methods on EBUS-TBNA, the clinical validity and utility of these analyses need to be interpreted in the clinical context of their application.

CLINICAL-BIOLOGIC UNDERPINNINGS OF ACQUIRED RESISTANCE TO ANTI-EGFR AND ALK TKIs

During the last few years, our knowledge about the biology and treatment of NSCLC has undergone a dramatic improvement. Nowadays, treatment decisions based on both molecular aberrations in druggable genes and the ability to detect them has led to a revolutionary change in the clinical course of NSCLC. From unselected lung cancer treatment based on chemotherapy, we have moved to an “oncogene addiction” disease concept, in which treatment strategies are based on the molecular profile of the tumor of each patient.3436  The activating EGFR mutations and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene were the first druggable alterations identified in NSCLC that implicate a high sensitivity to anti-EGFR and ALK TKIs, respectively.3537  EGFR exon 19 deletions and L858R mutation in exon 21 of EGFR are detected in about 10% to 12% of white and 30% to 40% of Asian patients with advanced NSCLC, respectively, and they represent the most frequently identified EGFR alterations conferring sensitivity to first- or second-generation EGFR TKIs.38  These agents, such as afatinib, gefitinib, and erlotinib, show a dramatic antitumor activity compared with standard chemotherapy in a first-line setting, with improvements in progression-free survival (PFS) and quality of life, and a superior safety profile.1,2,37  ALK molecular alterations, such as mutation, gene amplification, or chromosomal rearrangements, can be detected in about 5% to 7% of NSCLCs, leading to the expression of a potent oncogenic driver.39,40  In patients with ALK translocation, different randomized trials have demonstrated improved survival and response rates for patients receiving different TKIs such as crizotinib, ceritinib, alectinib, and lorlatinib.8  Despite the initial high efficacy of these therapies, the activity of these targeted agents is limited by the development of acquired resistance.41  Jackman et al7  described formal criteria for acquired resistance to EGFR TKIs in lung cancer. Although comparable criteria have not been established for ALK-positive NSCLC in clinical practice, the approach can be considered similar.

During the last few years, different mechanisms of TKI resistance have been reported. These mechanisms can be broadly separated into 3 categories: secondary mutation in the target gene, activation of downstream or alternative pathways, and phenotypic transformation.41  The understanding of the biologic bases of this acquired resistance is fundamental to monitoring the disease course of patients and to developing alternative treatment strategies. In patients with sensitizing EGFR mutation–positive NSCLC, the presence of the secondary mutation T790M in the exon 20 of the EGFR gene represents the most common mechanism of resistance, accounting for 50% to 60% of the cases.42,43  Third-generation EGFR TKIs (eg, osimertinib, rociletinib, olmutinib) have been shown to be very active in this selected group of patients, improving survival,9  and in 2015 the US Food and Drug Administration (FDA) granted accelerated approval to osimertinib for the treatment of patients with metastatic EGFR T790M mutation–positive NSCLC who have progressed on or after EGFR TKI therapy.44  For this reason, molecular testing of T790M EGFR mutation is now recommended in all of these patients.13  In addition to the gatekeeper EGFR T790M mutation, activation of alternative pathways is the second most common resistance mechanism to EGFR-TKIs. MET oncogene amplification and aberrations of ERBB2 (amplification or mutation) have been reported in about 7% and 10% of acquired resistance, respectively.45,46  Moreover, both of these alternative pathways have been described as acquired resistance mechanisms in patients treated with the third-generation inhibitor osimertinib.45  Activation of EGFR downstream pathways has been detected in patients with acquired EGFR-TKI resistance, including activating BRAF (1% of cases)47  and PIK3CA (5% of cases)42  mutations. The phenotypic change represents a unique mechanism of resistance to therapy. The transformation from adenocarcinoma to small cell lung cancer has been reported in up to 3% of patients with acquired resistance to EGFR TKIs.43  However, the frequency of this phenomenon could be underestimated, given that the detection of this lineage change clearly required the presence of rebiopsy sample to be evaluated (Figure 1). Although the biologic mechanism of this phenotypic switch has not been elucidated, a common cell of origin can be postulated. Indeed, in our experience, common founder genetic events can be identified in both morphologic components (Figure 1).

Figure 1

Comparison between endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) samples at diagnosis and progression from a patient with EGFR-mutated lung adenocarcinoma and clinical resistance to EGFR-tyrosine kinase inhibitors (TKIs), revealing neuroendocrine phenotypic transformation. Representative micrographs of diagnostic EBUS-TBNA cytology samples with tumor cell clusters (hematoxylin-eosin–stained, alcohol-fixed smears [A], and hematoxylin-eosin–stained cell block [B]), displaying TTF-1 positivity (C), favors lung adenocarcinoma, but no immunoreactivity for neuroendocrine markers, including chromogranin A (D) and synaptophysin (E). Mutational analysis of this sample revealed the presence of EGFR-activating exon 19 deletion. Cytology samples (Diff-Quik [F] and hematoxylin-eosin–stained cell block [G]) from a second EBUS-TBNA procedure performed at progression during EGFR TKI therapy (ie, acquired resistance) showed morphologic features of small cell lung cancer. Immunohistochemical analysis confirmed the phenotypic transformation. Tumor cells were positive for TTF-1 (H) and for neuroendocrine markers chromogranin A (I; focal immunoreactivity) and synaptophysin (J). Mutational analysis of this sample revealed the presence of the same EGFR-activating exon 19 deletion as in the primary tumor (original magnifications ×200 [A, B, D, E, G, H, and J] and ×400 [C, F, and I]).

Figure 1

Comparison between endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) samples at diagnosis and progression from a patient with EGFR-mutated lung adenocarcinoma and clinical resistance to EGFR-tyrosine kinase inhibitors (TKIs), revealing neuroendocrine phenotypic transformation. Representative micrographs of diagnostic EBUS-TBNA cytology samples with tumor cell clusters (hematoxylin-eosin–stained, alcohol-fixed smears [A], and hematoxylin-eosin–stained cell block [B]), displaying TTF-1 positivity (C), favors lung adenocarcinoma, but no immunoreactivity for neuroendocrine markers, including chromogranin A (D) and synaptophysin (E). Mutational analysis of this sample revealed the presence of EGFR-activating exon 19 deletion. Cytology samples (Diff-Quik [F] and hematoxylin-eosin–stained cell block [G]) from a second EBUS-TBNA procedure performed at progression during EGFR TKI therapy (ie, acquired resistance) showed morphologic features of small cell lung cancer. Immunohistochemical analysis confirmed the phenotypic transformation. Tumor cells were positive for TTF-1 (H) and for neuroendocrine markers chromogranin A (I; focal immunoreactivity) and synaptophysin (J). Mutational analysis of this sample revealed the presence of the same EGFR-activating exon 19 deletion as in the primary tumor (original magnifications ×200 [A, B, D, E, G, H, and J] and ×400 [C, F, and I]).

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The clinical course of patients with ALK fusion gene–positive NSCLC mirrors that of those with EGFR-mutated NSCLC. After the initial remarkable response to ALK TKIs, these patients developed acquired resistance to these drugs. The mechanisms of this resistance are similar to those described above, including secondary ALK aberrations (mutation and amplification), activation of ALK-independent pathways, and lineage changes.48  Secondary mutations in ALK genes represent the most common mechanism of acquired resistance, accounting for 20% to 30% and nearly 60% of patients who progress on crizotinib and second-generation ALK TKIs, respectively.4951  Several secondary ALK mutations have been described, and each shows a unique profile of sensitivity/resistance to different ALK TKIs. ALK mutations identified at the time of resistance include L1196M, G1269A, C1156Y, L1152R; G1202R, F1174C with crizotinib, F1174V after ceritinib, V1180L, I1171T, and I1171N after alectinib.8,48  For this reason, the accurate profile of ALK mutations is fundamental to driving treatment decision in these patients. ALK gene amplification has been reported as a resistance mechanism only in patients treated with crizotinib.

The biologic phenomenon of acquired resistance represents the proof of principle of the continuous evolution process of tumors, in which advantageous new or subclonal molecular aberrations become drivers of tumor growth under specific environmental conditions (ie, treatment).10  With the advent of next-generation TKIs that are effective against tumors with acquired resistance to first-generation agents, new compound resistance mutations have been reported. For example, the compound T790M/C797S mutations identified in NSCLC treated with third-generation anti-EGFR confers resistance to all available EGFR TKIs.52,53  Compound ALK C1156Y/I1171N and E1210K/D1203N mutations have been described in patients with sequential progression (and sequential acquired resistance) to crizotinib/ceritinib/alectinib and crizotinib/brigatinib, respectively.51  Moreover, loss of T790M EGFR mutation has been described as a resistance mechanism to third-generation inhibitors.52,5456  These findings may open a new therapeutic window for these patients based on treatment rechallenge with the first-generation TKIs.42,57  Indeed, given tumor heterogeneity, a significant subset of cancer cells remains sensitive to these targeted agents even after progression.58  This complex parallel evolution of NSCLC and targeted therapies underlines the importance of serial analysis of the biologic underpinnings of acquired resistance at the time of each progression. The continuous monitoring of the polyclonal resistance by NGS-based assays could offer different therapeutic chances and have an impact on patient survival and quality of life.

ANALYSES OF RESISTANCE-DRIVING MOLECULAR ABERRATIONS USING NGS ON EBUS-TBNA SAMPLES

Molecular testing in the setting of acquired resistance poses unique challenges related to the samples and methods to be used. Tumor heterogeneity may represent an important pitfall in the detection of molecular drivers of acquired resistance, given that these dynamic changes are usually present in a minority of the tumor cell population.9  For this reason, representative tumor samples and highly sensitive technologies are required to optimize the diagnostic yield of these tests. The most routinely used assays include real-time polymerase chain reaction–based methods, such as the cobas EGFR Mutation Test (Roche Diagnostics, Rotkreuz, Switzerland) and the therascreen EGFR amplification refractory mutation system (ARMS) assay (Qiagen, Manchester, United Kingdom).59  Both these technologies display a limit of detection less than or equal to 5% that accommodates the sensitivity recommended by the guidelines of the College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology on molecular testing of lung cancer, endorsed by the American Society of Clinical Oncology.60  Moreover, the cobas EGFR Mutation Test v2 (Roche Diagnostics) is indicated by the FDA as being a companion diagnostic to select for osimertinib treatment the NSCLC patients who harbor the T790M EGFR mutation.9  However, the implementation of NGS assays in these cases may aid in simultaneously looking for the different molecular aberrations that subtend the mechanism of resistance.6164  Indeed, some of the genetic aberrations depicted in the previous section (eg, genetic alterations of MET) are already therapeutic targets of drugs under clinical evaluation for the patients with disease that progresses after TKI therapy.65,66  Therefore, a comprehensive NGS panel covering the composite spectrum of resistance-driving molecular aberrations may have a strong impact on the clinical management of these patients.

The procurement of suitable samples for these analyses is far from trivial. Current guidelines strongly encourage a tissue rebiopsy in patients with NSCLC with EGFR-sensitizing mutations or ALK translocations at the time of progression after TKIs, and liquid biopsy for ctDNA analysis is considered a valid alternative when repeated tissue biopsy is not feasible.13  In the last few years, many studies have been published that aim to demonstrate the validity and the pros67  and cons68  of either the former or latter method, especially for the detection of EGFR T790M mutation. What is the role for cytopathology, between the growing implementation of liquid biopsy and the gold standard tissue rebiopsy for the management of NSCLC patients with acquired TKI resistance? Most studies in the current literature reporting molecular analyses on TKI-resistant NSCLC already comprised cytology specimens under the umbrella name of “rebiopsy.”43,6972  These studies described a similar rate of valid molecular testing and detection of EGFR T790M mutation between tissue biopsies and cytology specimens, with only slightly inferior results for body fluid–based analyses (pleural effusion, cerebrospinal fluid).69,70,73  However, the failure rates of samples obtained from EBUS-TBNA were similar to those reported for tissue biopsy samples.69,70  Indeed, these cytology specimens offer the opportunity to overcome some of the cons that have been reported with tissue rebiopsy. The rapid on-site evaluation of EBUS-TBNA samples may reduce the inadequacy rates reported with tissue.74  Moreover, given that the EBUS-TBNA procedure allows for sampling multiple areas of a lesion, these samples may be more representative of tumor heterogeneity and for the detection of the subclonal TKI resistance–associated genetic aberrations.75  Above all, tissue rebiopsy is still a more invasive procedure with a higher rate of complications, including pneumothorax and pulmonary hemorrhage, compared with EBUS-TBNA.76  For this reason, tissue biopsy may not always be feasible, and aspiration cytology specimens may represent the only available “tissue rebiopsy.”

Although the use of EBUS-TBNA is increasing in the routine workup of NSCLC patients with acquired TKI resistance and thoracic disease progression, few studies have addressed this topic specifically. Recently, Izumo et al77  described a consecutive series of 53 patients who underwent EBUS-guided rebiopsy of NSCLC after EGFR-TKI treatment, including 9 EBUS-TBNA and 44 EBUS-guided sheath transbronchial biopsy procedures without severe complications. They achieved adequate samples for EGFR mutation analysis using a scorpion ARMS method in 100% and 75% of EBUS-TBNA and EBUS-guided sheath specimens, respectively. In our institution, we are increasingly required to test resistance-related molecular aberrations on EBUS-TBNA samples from patients with progressing NSCLC. Using the validated procedures implemented for the upfront NGS-based molecular testing, we were able to obtain clinically meaningful results from all of these samples, including the detection of T790M EGFR mutation after negative liquid biopsy results (Figure 2).

Figure 2

Next-generation sequencing (NGS) analysis of endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) sample at the progression from a patient with EGFR-mutated lung adenocarcinoma and clinical resistance to EGFR-tyrosine kinase inhibitors (TKIs) revealing T790M EGFR mutation. Representative micrographs of hematoxylin-eosin–stained cell block preparation showing rare clusters and isolated tumor cells (A), with TTF-1 immunoreactivity (B), favors lung adenocarcinoma (original magnification ×200). The DNA extracted from this sample was subjected to NGS analysis using the CE-IVD Oncomine Solid Tumour kit on an Ion Torrent Personal Genome Machine and Ion Reporter Software platform (Thermo Fisher Scientific, Carlsbad, California). Quality metrics showed a mean depth of 1.413×. Together with the known “primary” sensitizing exon 21 EGFR mutation L858R (C; reads aligned to the reference genome as provided by the Integrative Genomics Viewer, IGV v.2.3, Broad Institute software, Cambridge, Massachusetts), “secondary” mutation at position 790 (D; T790M, reads aligned to the reference genome as provided by the Integrative Genomics Viewer, IGV v.2.3) of EGFR exon 20 was identified, which conferred resistance to first- and second-generation EGFR-TKIs.

Figure 2

Next-generation sequencing (NGS) analysis of endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) sample at the progression from a patient with EGFR-mutated lung adenocarcinoma and clinical resistance to EGFR-tyrosine kinase inhibitors (TKIs) revealing T790M EGFR mutation. Representative micrographs of hematoxylin-eosin–stained cell block preparation showing rare clusters and isolated tumor cells (A), with TTF-1 immunoreactivity (B), favors lung adenocarcinoma (original magnification ×200). The DNA extracted from this sample was subjected to NGS analysis using the CE-IVD Oncomine Solid Tumour kit on an Ion Torrent Personal Genome Machine and Ion Reporter Software platform (Thermo Fisher Scientific, Carlsbad, California). Quality metrics showed a mean depth of 1.413×. Together with the known “primary” sensitizing exon 21 EGFR mutation L858R (C; reads aligned to the reference genome as provided by the Integrative Genomics Viewer, IGV v.2.3, Broad Institute software, Cambridge, Massachusetts), “secondary” mutation at position 790 (D; T790M, reads aligned to the reference genome as provided by the Integrative Genomics Viewer, IGV v.2.3) of EGFR exon 20 was identified, which conferred resistance to first- and second-generation EGFR-TKIs.

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On the other hand, liquid biopsy allows for the detection of molecular biomarkers in body fluids with a minimally invasive/noninvasive procedure and faster turnaround time.74  The “circulating” nature of the biomarkers detected may theoretically capture the molecular heterogeneity of different tumor sites better than a single-site rebiopsy.67  Recently, many studies have reported on the technical and clinical validity of liquid biopsy analyzing the molecular alterations beneath TKI resistance, particularly the detection of EGFR T790M mutation in plasma ctDNA.69,71,7882  Moreover, different NGS platforms have already been implemented for ctDNA analysis.83,84  Although in the future liquid biopsy–based analyses might become the dominant platform for molecular testing in NSCLC, the analysis of ctDNA is not yet an everyday practice.68,85  The lack of robust preanalytic and analytic protocols of the different available technologies and the still high cost of NGS-based liquid biopsy testing represent the major constraints.86  Moreover, a discordant mutational status may be encountered,9  and a tissue rebiopsy is still recommended in patients with T790M-negative liquid biopsy results, to achieve a complete evaluation of progressing lung tumor.13  Indeed, the currently available liquid biopsy platforms may not be able to detect the entire spectrum of TKI resistance mechanisms. Above all, the histologic transformation still requires tissue for the morphologic and ancillary analyses that can elucidate the phenotypic changes in tumor cells.42,87  Formalin-fixed and paraffin-embedded cell block preparations obtained from material collected during EBUS-TBNA represent a proxy for histologic sample, and they are perfectly suitable for morphologic evaluation and to accommodate the necessary ancillary “in situ” tests (Figure 1, C through E, and H through J).

In our experience, NGS analyses and EBUS-TBNA samples may represent a valuable platform to address the unique requirement of the clinical context of TKI acquired resistance, and it might be set as an important step in the clinical management of these patients (Figure 3). However, the implementation of this platform in the acquired resistance setting may be hampered by the same preanalytic or analytic factors as for the “primary” analysis. Indeed, the use of cytology samples for molecular analysis is still debated, particularly in clinical trial enrollment that is often precluded if only cytology material is available. Therefore, additional studies are warranted to ascertain the technical and clinical validity and utility of this NGS/EBUS-TBNA sample platform to drive the clinical management of patients with NSCLC undergoing systemic progression after/on TKI therapy.

Figure 3

Comprehensive algorithms for the clinical management of non–small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs). A, Proposed algorithm for EGFR TKI-resistant NSCLC with systemic progression, highlighting the potential role of next-generation sequencing (NGS) analysis on endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) sample as rebiopsy (gray boxes, red arrows). B, Proposed algorithm for ALK TKI-resistant NSCLC with systemic progression, highlighting the potential role of NGS analysis on EBUS-TBNA sample as rebiopsy (gray box, red arrows).

Figure 3

Comprehensive algorithms for the clinical management of non–small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs). A, Proposed algorithm for EGFR TKI-resistant NSCLC with systemic progression, highlighting the potential role of next-generation sequencing (NGS) analysis on endobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) sample as rebiopsy (gray boxes, red arrows). B, Proposed algorithm for ALK TKI-resistant NSCLC with systemic progression, highlighting the potential role of NGS analysis on EBUS-TBNA sample as rebiopsy (gray box, red arrows).

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Author notes

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the V Molecular Cytopathology: Focus on Next Generation Sequencing in Cytopathology meeting; October 18, 2016; Napoli, Italy.