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Next-Generation Sequencing from Liquid Biopsies in Lung Cancer Patients: Advances in Comprehensive Biomarker Testing
EDITORIAL
Next-Generation Sequencing from Liquid Biopsies in Lung Cancer Patients: Advances in Comprehensive Biomarker Testing Lynette M. Sholl, MD*
Despite the overwhelming enthusiasm for the promises of immunotherapy in treating lung cancer, tumor genetic biomarker testing for other targeted therapies remains a central tenet of lung cancer management. As the list of therapeutically relevant biomarkers grows, so does the need for adequate tumor sampling. For many patients with lung cancer, however, an ample tumor biopsy can be obtained only by uncomfortable and potentially dangerous invasive procedures. In some cases, a tumor biopsy specimen containing a handful of cells may be sufficient to make a morphologic diagnosis but inadequate for complex biomarker testing. The field of molecular diagnostics has seen major advances in the last decade, to the benefit of both physicians and patients. Improvements in wet-lab chemistry and dry-lab informatics have enabled successful application of massively multiplexed genomic analysis to a variety of challenging specimen types. The application of next-generation sequencing (NGS) technologies for detection of tumor-specific genomic alterations within the blood of patients with cancer is arguably one of the most potentially impactful advances in recent years. Dying cells release their DNA into the circulation in the form of short, nucleosome-bound strands that are protected, for a short time, from degradation. These circulating DNAs offer a unique snapshot of the patient’s somatic tissue genetic makeup, including any mutations present within neoplastic cells. In patients with cancer, circulating tumor DNA (ctDNA) represents a widely variable proportion of circulating free DNA (cfDNA). Given the availability of highly sensitive and reliable molecular techniques, this previously untapped resource, which is accessible through a routine blood draw, is now open for interrogation. We can now use the patient’s blood to perform genetic characterization of tumors at diagnosis and relapse and can see potential for use of ctDNA in cancer monitoring and even screening. Despite this promise, a host of unanswered questions persists about the reliability and applicability of ctDNAbased cancer diagnostics. How does ctDNA testing compare with routine diagnostic testing of tumor tissue, Journal of Thoracic Oncology
Vol. 12 No. 10: 1464-1466
which is the current accepted standard? In what setting can mutational profiling of ctDNA replace that of tumor tissue? What is the role for NGS- based approaches in ctDNA? Is this type of testing practically within reach of patients and oncologists across diverse clinical settings? In this month’s issue of the Journal of Thoracic Oncology, Müller et al. describe a systematic, three-way concordance study of tissue-based single-gene assays with a 39-gene targeted NGS panel for tissue (termed NEOplus) and optimized for ctDNA (termed NEOliquid) in 98 patients with nonsquamous NSCLC.1 When examining targeted regions for mutations in EGFR, BRAF, KRAS, and tumor protein p53 gene (TP53) and rearrangement status for ALK receptor tyrosine kinase gene (ALK) and ROS1, the authors demonstrate 100% concordance between routine, stand-alone clinical assays and NEOplus NGS. Further, the more comprehensive NEOplus assay detected variants not queried by the routine single-gene assays, including in EGFR, MET proto-oncogene receptor tyrosine kinase (MET), and erb-b2 receptor tyrosine kinase 2 gene (ERBB2). This finding is consistent with prior reports validating the clinical use of NGS-based assays and indeed demonstrates the benefits of a more comprehensive testing approach over routine single-gene assays.2 It further bolsters arguments for laboratories to embrace NGS technologies and for payers to reimburse this type of highly multiplexed testing to enhance the detection of predictive biomarkers in lung cancer.
*Corresponding author. Brigham and Women’s Hospital and Department of Pathology, Harvard Medical School, Boston, Massachusetts. Disclosure: The author declares no conflict of interest. Address for correspondence: Lynette M. Sholl, MD, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. E-mail: lmsholl@ bwh.harvard.edu ª 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 http://dx.doi.org/10.1016/j.jtho.2017.08.004
October 2017
In 82 patients with paired plasma specimens, the authors demonstrated 97% concordance between routine diagnostic testing of tissue and NEOliquid NGS. Importantly, the specificity of NEOliquid was 100%; an essentially perfect specificity should be a prerequisite for any plasma-based assay so that the clinician can act on a positive result with confidence. ctDNA testing is limited by a lower clinical sensitivity—in this report 70.8%—relative to tissue testing. The utility of plasmabased testing is limited to patients with sufficient shed of ctDNA into the circulation to be detectable with currently available techniques. NEOliquid NGS detected known tumor variants only in patients with metastatic (M1b) disease, which is in keeping with prior reports that tumor volume and metastatic burden correlate with degree of DNA shed.3 Indeed, the authors describe a fourfold increase in the mean total cfDNA concentration in the blood specimens of patients with M1b disease relative to those with M0 disease. Highlighting the sensitivity challenge for blood-based testing, the authors show that ctDNA variants are present at a very small fraction of total cfDNA—nearly half of the variants detected were present at the level of 0.1 and 1% variant allele fraction. This type of observation should guide any laboratory embarking on the development and validation of ctDNA assays, as it demands a level of assay sensitivity that is generally not available when using routine single-gene or NGS assays designed for tissue testing. Similar to NEOplus NGS in tissue, NEOliquid detected clinically important tumor variants not captured by routine single-gene testing as a result of the broader panel of targets covered. These findings argue then, that well-designed NGS panels applied to cfDNA can generate reliable, clinically actionable results in most patients with M1b disease. The risk of uninformative ctDNA testing because of the lower clinical sensitivity must, however, be clearly communicated to the patient, particularly when the patient may be under obligation to pay out of pocket for molecular testing. That said, the option to undergo blood-based testing is likely to be significantly more attractive than repeated invasive biopsies to obtain tumor tissue, particularly in the relapse setting. Guidelines emerging from a variety of professional societies, including the College of American Pathologists, Association of Molecular Pathologists, and International Association for the Study of Lung Cancer, endorse the use of ctDNA testing in two settings in particular at this time: (1) at the time of relapse after a targeted therapy (e.g., EGFR tyrosine kinase inhibitors) to detect molecular resistance mechanisms and (2) after the diagnosis of lung adenocarcinoma when the tumor tissue is insufficient or unavailable for molecular testing (Neal Lindeman, Brigham and Women’s Hospital, 2017,
Next Gen Sequencing from ctDNA
1465
personal communication). In either setting, the patient should be prepared for the possible need for repeat biopsy if ctDNA testing is uninformative. The development of clinically relevant, affordable, and easily implemented NGS for ctDNA testing is essential for the advancement of blood-based diagnostics in patients with solid tumors. Access to ctDNA testing increases the likelihood that patients with suboptimal tissue specimens can get informative molecular testing and thus gain access to targeted therapies. Beyond individual genes, NGS-based analysis of mutational signatures predictive of an immunogenic tumor—including tumor mutational burden and microsatellite instability—may also potentially be applied to ctDNA, with implications for patient selection for immunotherapy. Dissemination of ctDNA NGS, potentially in the form of commercial kits with onboard bioinformatics solutions for use with widely available sequencing instruments, should be a priority for clinicians and laboratory scientists alike. Similarly, guidelines and resources for validation of ctDNA NGS are essential to permit efficient and appropriate deployment of this technology in laboratories across the world. Validation and implementation of ctDNA testing requires a coordinated effort between clinicians and molecular diagnostic laboratories because careful specimen handling is paramount to success of ctDNA testing. Because tissue-blood concordance testing is complex and may require consented protocols, national or international efforts are needed to establish standards for use in validation and proficiency testing. Although it is too early to speculate on the role of routine ctDNA monitoring in clinical care of patients with lung cancer, it is already clear that tumor variants can appear in the blood long before radiographic relapse becomes apparent.4 The clinical implications ctDNA monitoring for cancer relapse have not yet been established; however, if outcomes can be improved by modulating therapy according to the appearance or disappearance of specific ctDNA variants, then monitoring is likely to become an important component of management of patients with solid tumors, much as bcrabl transcript monitoring is central to the management of patients with chronic myelogenous leukemia. By extension, and technology permitting, a blood-based screening test in a high-risk patient population may lead to detection of cancer before it becomes clinically apparent. In either scenario, the cost of testing must be carefully managed to make this type of testing palatable to hospitals, payers, and society at large. Access to reliable and affordable ctDNA testing will promote the democratization of rational therapy for all patients with cancer, regardless of means. This is an essential step toward improving global outcomes for patients with lung cancer.
1466 Sholl
References 1. Müller JN, Falk M, Talwar J, et al. Concordance between comprehensive cancer genome profiling in plasma and tumor specimens. J Thorac Oncol. 2017;12:1503–1511. 2. Sholl LM, Do K, Shivdasani P, et al. Institutional implementation of clinical tumor profiling on an unselected cancer population. JCI Insight. 2016;1:e87062.
Journal of Thoracic Oncology
Vol. 12 No. 10
3. Sacher AG, Paweletz C, Dahlberg SE, et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol. 2016;2:1014–1022. 4. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545:446–451.
Next-Generation Sequencing from Liquid Biopsies in Lung Cancer Patients: Advances in Comprehensive Biomarker Testing Lynette M. Sholl, MD*
Despite the overwhelming enthusiasm for the promises of immunotherapy in treating lung cancer, tumor genetic biomarker testing for other targeted therapies remains a central tenet of lung cancer management. As the list of therapeutically relevant biomarkers grows, so does the need for adequate tumor sampling. For many patients with lung cancer, however, an ample tumor biopsy can be obtained only by uncomfortable and potentially dangerous invasive procedures. In some cases, a tumor biopsy specimen containing a handful of cells may be sufficient to make a morphologic diagnosis but inadequate for complex biomarker testing. The field of molecular diagnostics has seen major advances in the last decade, to the benefit of both physicians and patients. Improvements in wet-lab chemistry and dry-lab informatics have enabled successful application of massively multiplexed genomic analysis to a variety of challenging specimen types. The application of next-generation sequencing (NGS) technologies for detection of tumor-specific genomic alterations within the blood of patients with cancer is arguably one of the most potentially impactful advances in recent years. Dying cells release their DNA into the circulation in the form of short, nucleosome-bound strands that are protected, for a short time, from degradation. These circulating DNAs offer a unique snapshot of the patient’s somatic tissue genetic makeup, including any mutations present within neoplastic cells. In patients with cancer, circulating tumor DNA (ctDNA) represents a widely variable proportion of circulating free DNA (cfDNA). Given the availability of highly sensitive and reliable molecular techniques, this previously untapped resource, which is accessible through a routine blood draw, is now open for interrogation. We can now use the patient’s blood to perform genetic characterization of tumors at diagnosis and relapse and can see potential for use of ctDNA in cancer monitoring and even screening. Despite this promise, a host of unanswered questions persists about the reliability and applicability of ctDNAbased cancer diagnostics. How does ctDNA testing compare with routine diagnostic testing of tumor tissue, Journal of Thoracic Oncology
Vol. 12 No. 10: 1464-1466
which is the current accepted standard? In what setting can mutational profiling of ctDNA replace that of tumor tissue? What is the role for NGS- based approaches in ctDNA? Is this type of testing practically within reach of patients and oncologists across diverse clinical settings? In this month’s issue of the Journal of Thoracic Oncology, Müller et al. describe a systematic, three-way concordance study of tissue-based single-gene assays with a 39-gene targeted NGS panel for tissue (termed NEOplus) and optimized for ctDNA (termed NEOliquid) in 98 patients with nonsquamous NSCLC.1 When examining targeted regions for mutations in EGFR, BRAF, KRAS, and tumor protein p53 gene (TP53) and rearrangement status for ALK receptor tyrosine kinase gene (ALK) and ROS1, the authors demonstrate 100% concordance between routine, stand-alone clinical assays and NEOplus NGS. Further, the more comprehensive NEOplus assay detected variants not queried by the routine single-gene assays, including in EGFR, MET proto-oncogene receptor tyrosine kinase (MET), and erb-b2 receptor tyrosine kinase 2 gene (ERBB2). This finding is consistent with prior reports validating the clinical use of NGS-based assays and indeed demonstrates the benefits of a more comprehensive testing approach over routine single-gene assays.2 It further bolsters arguments for laboratories to embrace NGS technologies and for payers to reimburse this type of highly multiplexed testing to enhance the detection of predictive biomarkers in lung cancer.
*Corresponding author. Brigham and Women’s Hospital and Department of Pathology, Harvard Medical School, Boston, Massachusetts. Disclosure: The author declares no conflict of interest. Address for correspondence: Lynette M. Sholl, MD, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. E-mail: lmsholl@ bwh.harvard.edu ª 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 http://dx.doi.org/10.1016/j.jtho.2017.08.004
October 2017
In 82 patients with paired plasma specimens, the authors demonstrated 97% concordance between routine diagnostic testing of tissue and NEOliquid NGS. Importantly, the specificity of NEOliquid was 100%; an essentially perfect specificity should be a prerequisite for any plasma-based assay so that the clinician can act on a positive result with confidence. ctDNA testing is limited by a lower clinical sensitivity—in this report 70.8%—relative to tissue testing. The utility of plasmabased testing is limited to patients with sufficient shed of ctDNA into the circulation to be detectable with currently available techniques. NEOliquid NGS detected known tumor variants only in patients with metastatic (M1b) disease, which is in keeping with prior reports that tumor volume and metastatic burden correlate with degree of DNA shed.3 Indeed, the authors describe a fourfold increase in the mean total cfDNA concentration in the blood specimens of patients with M1b disease relative to those with M0 disease. Highlighting the sensitivity challenge for blood-based testing, the authors show that ctDNA variants are present at a very small fraction of total cfDNA—nearly half of the variants detected were present at the level of 0.1 and 1% variant allele fraction. This type of observation should guide any laboratory embarking on the development and validation of ctDNA assays, as it demands a level of assay sensitivity that is generally not available when using routine single-gene or NGS assays designed for tissue testing. Similar to NEOplus NGS in tissue, NEOliquid detected clinically important tumor variants not captured by routine single-gene testing as a result of the broader panel of targets covered. These findings argue then, that well-designed NGS panels applied to cfDNA can generate reliable, clinically actionable results in most patients with M1b disease. The risk of uninformative ctDNA testing because of the lower clinical sensitivity must, however, be clearly communicated to the patient, particularly when the patient may be under obligation to pay out of pocket for molecular testing. That said, the option to undergo blood-based testing is likely to be significantly more attractive than repeated invasive biopsies to obtain tumor tissue, particularly in the relapse setting. Guidelines emerging from a variety of professional societies, including the College of American Pathologists, Association of Molecular Pathologists, and International Association for the Study of Lung Cancer, endorse the use of ctDNA testing in two settings in particular at this time: (1) at the time of relapse after a targeted therapy (e.g., EGFR tyrosine kinase inhibitors) to detect molecular resistance mechanisms and (2) after the diagnosis of lung adenocarcinoma when the tumor tissue is insufficient or unavailable for molecular testing (Neal Lindeman, Brigham and Women’s Hospital, 2017,
Next Gen Sequencing from ctDNA
1465
personal communication). In either setting, the patient should be prepared for the possible need for repeat biopsy if ctDNA testing is uninformative. The development of clinically relevant, affordable, and easily implemented NGS for ctDNA testing is essential for the advancement of blood-based diagnostics in patients with solid tumors. Access to ctDNA testing increases the likelihood that patients with suboptimal tissue specimens can get informative molecular testing and thus gain access to targeted therapies. Beyond individual genes, NGS-based analysis of mutational signatures predictive of an immunogenic tumor—including tumor mutational burden and microsatellite instability—may also potentially be applied to ctDNA, with implications for patient selection for immunotherapy. Dissemination of ctDNA NGS, potentially in the form of commercial kits with onboard bioinformatics solutions for use with widely available sequencing instruments, should be a priority for clinicians and laboratory scientists alike. Similarly, guidelines and resources for validation of ctDNA NGS are essential to permit efficient and appropriate deployment of this technology in laboratories across the world. Validation and implementation of ctDNA testing requires a coordinated effort between clinicians and molecular diagnostic laboratories because careful specimen handling is paramount to success of ctDNA testing. Because tissue-blood concordance testing is complex and may require consented protocols, national or international efforts are needed to establish standards for use in validation and proficiency testing. Although it is too early to speculate on the role of routine ctDNA monitoring in clinical care of patients with lung cancer, it is already clear that tumor variants can appear in the blood long before radiographic relapse becomes apparent.4 The clinical implications ctDNA monitoring for cancer relapse have not yet been established; however, if outcomes can be improved by modulating therapy according to the appearance or disappearance of specific ctDNA variants, then monitoring is likely to become an important component of management of patients with solid tumors, much as bcrabl transcript monitoring is central to the management of patients with chronic myelogenous leukemia. By extension, and technology permitting, a blood-based screening test in a high-risk patient population may lead to detection of cancer before it becomes clinically apparent. In either scenario, the cost of testing must be carefully managed to make this type of testing palatable to hospitals, payers, and society at large. Access to reliable and affordable ctDNA testing will promote the democratization of rational therapy for all patients with cancer, regardless of means. This is an essential step toward improving global outcomes for patients with lung cancer.
1466 Sholl
References 1. Müller JN, Falk M, Talwar J, et al. Concordance between comprehensive cancer genome profiling in plasma and tumor specimens. J Thorac Oncol. 2017;12:1503–1511. 2. Sholl LM, Do K, Shivdasani P, et al. Institutional implementation of clinical tumor profiling on an unselected cancer population. JCI Insight. 2016;1:e87062.
Journal of Thoracic Oncology
Vol. 12 No. 10
3. Sacher AG, Paweletz C, Dahlberg SE, et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol. 2016;2:1014–1022. 4. Abbosh C, Birkbak NJ, Wilson GA, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017;545:446–451.