Lung Cancer
Comprehensive Genomic Profiling Facilitates Implementation of the National Comprehensive Cancer Network Guidelines for Lung Cancer Biomarker Testing and Identifies Patients Who May Benefit From Enrollment in Mechanism-Driven Clinical Trials JAMES H. SUH,a ADRIENNE JOHNSON,a LEE ALBACKER,a KAI WANG,a,b JULIANN CHMIELECKI,a GARRETT FRAMPTON,a LAURIE GAY,a JULIA A. ELVIN,a JO-ANNE VERGILIO,a SIRAJ ALI,a VINCENT A. MILLER,a PHILIP J. STEPHENS,a JEFFREY S. ROSSa a
Foundation Medicine, Inc., Cambridge, Massachusetts, USA; bZhejiang Cancer Hospital, Hangzhou, People’s Republic of China
Disclosures of potential conflicts of interest may be found at the end of this article.
Key Words. Non-small cell lung cancer x Comprehensive genomic profiling x National Comprehensive Cancer Network guidelines x Clinical trials
ABSTRACT Background. The National Comprehensive Cancer Network (NCCN) guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for EGFR, BRAF, ERBB2, and MET mutations; ALK, ROS1, and RET rearrangements; and MET amplification. We investigated the feasibility and utility of comprehensive genomic profiling (CGP), a hybrid capture-based next-generation sequencing (NGS) test, in clinical practice. Methods. CGP was performed to a mean coverage depth of 5763 on 6,832 consecutive cases of NSCLC (2012–2015). Genomic alterations (GAs) (point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were recorded. We also evaluated lung adenocarcinoma (AD) cases without GAs, involving these eight genes. Results. The median age of the patients was 64 years (range: 13–88 years) and 53% were female. Among the patients
studied, 4,876 (71%) harbored at least one GA involving EGFR (20%), ALK (4.1%), BRAF (5.7%), ERBB2 (6.0%), MET (5.6%), ROS1 (1.5%), RET (2.4%), or KRAS (32%). In the remaining cohort of lung AD without these known drivers, 273 cancerrelated genes were altered in at least 0.1% of cases, including STK11 (21%), NF1 (13%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), NRAS (2.3%), BRCA1 (1.7%), MAP2K1 (1.2%), HRAS (0.7%), NTRK1 (0.7%), and NTRK3 (0.2%). Conclusion. CGP is practical and facilitates implementation of the NCCN guidelines for NSCLC by enabling simultaneous detection of GAs involving all seven driver oncogenes and KRAS. Furthermore, without additional tissue use or cost, CGP identifies patients with “pan-negative” lung AD who may benefit from enrollment in mechanism-driven clinical trials. The Oncologist 2016;21:684–691
Implications for Practice: National Comprehensive Cancer Network guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for several genomic alterations (GAs). The feasibility and utility of comprehensive genomic profiling were studied in NSCLC and in lung adenocarcinoma (AD) without GAs. Of patients with NSCLC, 71% harbored at least one GA to a gene listed in the guidelines or KRAS; 273 cancer-related genes were altered in at least 0.1% of the AD cases. Although logistical and administrative hurdles limit the widespread use of next-generation sequencing, the data confirm the feasibility and potential utility of comprehensive genomic profiling in clinical practice.
INTRODUCTION Lung cancer is the leading cause of cancer mortality worldwide, accounting for more than 158,000 deaths annually in the U.S., with approximately 85% being of non-small cell lung cancer (NSCLC) histology [1, 2]. Because of the discovery of activating epidermal growth factor receptor (EGFR) point mutations and small insertions and deletions (indels), in 2003, and
anaplastic lymphoma kinase (ALK) rearrangements, in 2007, as well as commercial availability of FDA-approved EGFR and ALK tyrosine kinase inhibitors (TKIs), the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) issued joint guidelines in 2013, endorsed by the
Correspondence: James H. Suh, M.D., Foundation Medicine, Inc., 150 Second Street, Cambridge, Massachusetts 02142, USA.Telephone: 617-4182200, ext. 7402; E-Mail: [email protected] Received January 28, 2016; accepted for publication March 30, 2016; published Online First on May 5, 2016. ©AlphaMed Press 1083-7159/2016/$20.00/0 http://dx.doi.org/10.1634/theoncologist.2016-0030
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Table 1. National Comprehensive Cancer Network guidelines for non-small cell lung cancer, version 4.2016
Genomic alterations EGFR mutations ALK rearrangements HER2 mutations BRAF V600E mutations MET amplification and exon 14 skipping mutations ROS1 rearrangements RET rearrangements
Available targeted agents with activity against driver event in lung cancera Erlotinib, gefitinib, afatinib Crizotinib, ceritinib, alectinib Trastuzumab, afatinib Vemurafenib, dabrafenib with or without trametinib Crizotinib Crizotinib Cabozantinib
a
Indicates recommended use in the National Comprehensive Cancer Network Drugs and Biologics Compendium. Bold indicates approval (EGFR, ALK, and ROS1 inhibitors) or breakthrough therapy designation (BRAF inhibitors) by the FDA.
American Society of Clinical Oncology (ASCO) in 2014, that recommend routine testing for both genomic alterations (GAs) in patients with advanced-stage disease [3–6]. The most recent version of the National Comprehensive Cancer Network (NCCN) guidelines for management of NSCLC also recommends testing for BRAF and ERBB2 mutations, MET amplification and exon 14 skipping mutations, and ROS1 and RET rearrangements. Targeted therapies with approval or breakthrough therapy designation by the FDA (i.e., dabrafenib with or without trametinib) have shown benefit in patients whose tumors harbor relevant GAs (Table 1) [7–15]. Therefore, molecular testing of NSCLC, particularly adenocarcinoma (AD), is now the standard of care for patients who need systemic therapy [5–7]. Whereas the 2013 CAP/IASLC/ AMP guidelines endorsed identification of EGFR mutations by polymerase chain reaction (PCR) and ALK rearrangements by fluorescent in situ hybridization (FISH) based on the FDAapproved companion diagnostic kits at the time, the rapid maturation of clinical massively parallel or next-generation sequencing (NGS) technologies, as well as the need to identify GAs in seven different oncogenes, enabled the NCCN panel to state that EGFR and ALK testing should be conducted using broad molecular profiling methods [7]. Despite the enthusiasm for NGS, it is important to recognize that similar to other laboratory techniques such as immunohistochemistry or FISH, test performance can vary widely. Many NGS platforms are hotspot tests that are limited to the detection of point mutations and some indels, and cannot identify copy number alterations or rearrangements. Hotspot tests may also struggle with detection of point mutations and indels in small specimens with low tumor purity, leading to higher false-negative rates [16, 17]. These limitations are particularly problematic for NSCLC testing, because ALK, ROS1, and RET rearrangements and MET amplification cannot be identified using hotspot NGS platforms. In contrast, hybrid capture-based NGS platforms, also known as comprehensive genomic profiling (CGP) assays, can diagnose all four types of DNA alterations seen in cancer—point mutations, small indels, copy number changes, and rearrangements—in hundreds of cancer-related genes with high sensitivity and specificity [18, 19]. CGP provides an accurate and efficient alternative to multiplex or hotspot testing that is
attractive in the setting of NSCLC because itcan identify all seven GAs included in the NCCN guidelines as well as additional GAs that may qualify patients for enrollment in mechanism-driven clinical trials or have been associated with response to targeted therapy [20–24]. Last, the majority of patients with NSCLC are diagnosed by using small biopsy or cytology specimens for which there is a limited amount of available material for molecular testing [25, 26]. Our aim is to describe our series of all NSCLC cases submitted to Foundation Medicine for CGP over a 33-month period to demonstrate its clinical utility.
METHODS Genomic profiling was performed in a Clinical Laboratory Improvement Amendments-certified, CAP-accredited reference laboratory (Foundation Medicine) for 6,832 clinical NSCLC samples consecutively submitted between September 2012 and May 2015. At least 50 ng of DNA per sample was extracted from 40 mm of clinical formalin-fixed, paraffinembedded tumor samples and was analyzed by hybridization capture of 3,320 exons from 236 (gene set 1) or 315 (gene set 2) cancer-related genes and introns of 19 or 28 genes commonly rearranged in cancer. Specimens were sequenced to high, uniform coverage (mean: 5763) on Illumina HiSeq2000 or HiSeq2500 instruments (San Diego, CA, http://www.illumina. com), as previously described [18]. A total of 3,060 samples (45%) were analyzed using gene set 1 and the remainder (3,772; 55%) were analyzed using gene set 2. GAs (i.e., point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were determined and then reported for each patient sample. GAs involving the remaining genes in the panel were evaluated for the lung AD samples lacking these eight known drivers.The complete lists of genes for set 1 and set 2 are included in the supplemental material (supplemental online Table 1). The test has been validated to detect point mutations at $10% mutant allele frequency with $99% sensitivity, and indels at $20% mutant allele frequency with $95% sensitivity, with a false discovery rate of,1% [18].Turnaroundtime is routinely 7–10 days from sample receipt. Local site permissions to use clinical samples were obtained for this study. In addition to patient age, sex, and histologic subtype, the tissue biopsy site was recorded for each case.
RESULTS The median age of patients in this series was 64 years (range: 13–88 years), and 53% were female. Regarding histologic subtype, 5,380 cases (79%) were lung AD, 1,345 (20%) were nonsmall cell carcinoma, not otherwise specified (NSCLC-NOS), 72 (1%) were adenosquamous carcinoma (ADSQ), and 35 (0.5%) were large cell carcinoma (LCC).The difference in the numbers of AD (75%) and ADSQ (68%) cases with at least one GA in the genes listed by NCCN or KRAS versus NSCLC-NOS (57%) and LCC (37%) was statistically significant (p , .0001). Of these specimens, 50% were obtained from extrapulmonary metastatic sites. GAs involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS were identified in 4,876 cases (71%). As seen in Table 2 and Figure 1, 1,342 cases (20%) harbored EGFR alterations, 280 (4.1%) harbored ALK alterations, 388 (5.7%) harbored BRAF alterations, 408 (6.0%) harbored ERBB2 alterations, 383 (5.6%) harbored MET alterations, 100 (1.5%) harbored ROS1
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Table 2. Genomic alterations involving NCCN guideline genes and KRAS in 6,832 cases of non-small cell lung carcinoma % Cases Gene
Total cases
Point mutation
EGFR ALK BRAF ERBB2 MET ROS1 RET KRAS
1,342 280 388 408 383 100 166 2,178
12 0.3 5.1 0.8 1.7 0.1 0.2 31
a
Small indel
b
10 0 0.06 2.6 1.4 0 0 0.04
Amplificationc
Rearrangementc
Alld
% GAs described in NCCN guidelinese
5.9 0.1 0.4 3.0 3.1 0.03 0.3 3.5
0 3.9 0.2 0 0 1.3 1.9 0
20 4.1 5.7 6.0 5.6 1.5 2.4 32
15b 3.9c 2.1a 3.5b 5.3b 1.3c 1.9c N/A
a
GAs that can be detected using hotspot NGS tests. GAs that can be detected occasionally using hotspot NGS tests. GAs that cannot be detected using hotspot NGS tests. d Some cases have multiple GAs involving the same gene. e We estimate that a hotspot NGS test without supplemental FISH assays would miss up to half of the NCCN GAs in this table. Abbreviations: GA, genomic alteration; indel, insertion and deletion; N/A, not applicable; NCCN, National Comprehensive Cancer Network. b c
Last, there were 1,339 cases of lung AD without GAs involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS. In this cohort, GAs involving previously known lung AD oncogenes NF1 (13%), PIK3CA (5.4%), NRAS (2.3%), MAP2K1 (1.2%), and HRAS (0.7%) were detected. A total of 273 genes harbored GAs in at least 0.1% of cases, many of which are associated with potential benefit from targeted therapies or allow enrollment in mechanism-driven clinical trials, including STK11 (21%), MYC (9.8%), RICTOR (6.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), BRCA1 (1.7%), NTRK1 (0.7%), and NTRK3 (0.2%) (Table 3).
DISCUSSION
Figure 1. Genomic alterations (GAs) involving National Comprehensive Cancer Network guideline genes and KRAS in 6,832 cases of non-small cell lung carcinoma. Some 5.4% of cases have GAs involving more than one of these genes.
alterations, 166 (2.4%) harbored RET alterations, and 2,178 (32%) harbored KRAS alterations. With respect to the particular alterations specified by the CAP/IASLC/AMP and NCCN guidelines, 15% of cases in this series were positive for known sensitizing EGFR point mutations and indels, 3.9% were positive for ALK rearrangements, 2.1% were positive for BRAF V600E point mutations, 3.5% were positive for ERBB2 point mutations and indels, 3.1% were positive for MET amplification, 2.8% were positive for MET exon 14 skipping mutations, 1.3% were positive for ROS1 rearrangements, and 1.9% were positive for RET rearrangements (Table 2). In addition, CGP identified multiple, additional, potentially targetable GAs involving the same driver oncogenes, including 401 cases (5.9%) with EGFR amplification, 203 cases (3.0%) with BRAF non-V600E point mutations, 12 cases (0.2%) with BRAF rearrangement, and 208 cases (3.0%) with ERBB2 amplification. EGFR T790M resistance mutations were identified in 221 cases (3.2%), and ALK resistance mutations were identified in 9 cases (0.1%). www.TheOncologist.com
Despite recent advances in CT screening for lung cancer, the majority of patients continue to present with advancedstage disease and testing for EGFR point mutations and indels and ALK rearrangements is now the standard of care for selection of appropriate first-line therapy [1–7]. Since crizotinib received regular FDA approval in March 2016 for patients with NSCLC with ROS1 rearrangements, and osimertinib received FDA breakthrough therapy designation in late 2015 for patients with NSCLC with EGFR T790M resistance mutations, ROS1 and EGFR T790M testing are also emerging as the standard of care [27]. Because of these developments and ongoing clinical trials evaluating more than 900 agents against 150 or so molecularly defined targets, a growing number of academic medical centers and commercial laboratories have begun to offer NGS testing to identify alterations in other driver oncogenes that are included in the NCCN guidelines, such as BRAF, ERBB2, MET, and RET. However, they often use single-gene PCR and FISH assays with limited sensitivity compared with NGS or hotspot NGS platforms that are incapable of detecting ALK, ROS1, and RET rearrangements and MET amplification [17]. In particular, hotspot NGS platforms rely on multiple supplemental single-gene FISH assays that consume tissue, leaving testing incomplete in 30%–84% of cases if additional biopsies are not performed, as seen in the Memorial Sloan-Kettering Cancer Center and Lung Cancer Mutation Consortium experiences [16, 28]. ©AlphaMed Press 2016
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Table 3. Selection of cancer-related genes altered in 1,339 cases of “pan-negative” lung adenocarcinoma and mechanism-driven clinical trials Gene
% Cases
Targeted therapies
Relevant clinical trial examples
STK11
21
mTOR inhibitors
NF1
13
MEK inhibitors
NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02443337 – Phase II NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02419417 – Phase I NCT02391480 – Phase I NCT02431260 – Phase I/II NCT02187783 – Phase II NCT02450539 – Phase II NCT01037790 – Phase II NCT01470209 – Phase I NCT02117167 – Phase II NCT02069158 – Phase Ib NCT02154490 – Phase II/III NCT01306045 – Phase II NCT01862081 – Phase I NCT02583542 – Phase Ib/IIa NCT02412371 – Phase III NCT02264990 – Phase III NCT02106546 – Phase III NCT01306045 – Phase II NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02219711 – Phase I
MYC
9.8
Bromodomain inhibitors
CDK4, CCND1
8.3
CDK4/6 inhibitors
RICTOR
6.4
mTOR inhibitors
PIK3CA
5.4
BRCA1/2
4.2
mTOR inhibitors PI3K inhibitors Dual mTOR/PI3K inhibitors AKT inhibitors PARP inhibitors
NRAS, HRAS
3.0
MEK inhibitors
MAP2K1 NTRK1, NTRK3
1.2 0.9
MEK inhibitors NTRK1 inhibitors
The purpose of this study is to demonstrate that our CGP assay, based on a hybrid-capture approach, can diagnose simultaneously all four types of DNA alterations that occur within the eight best-characterized NSCLC driver oncogenes (EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, KRAS) as well as dozens of other genes that are linked to clinical trials. Over a 33-month period, we analyzed lung tumor samples from 6,832 patients with primarily advanced-stage disease. Of the patients in our series, 15% harbored EGFR alterations that are known to be sensitizing to FDA-approved EGFR TKIs according to the NCCN guidelines, and 3.9% harbored ALK rearrangements that predict response to FDA-approved ALK TKIs (Table 2). The frequencies for both alterations are somewhat higher than those identified within The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset but are within the ranges that have been reported in previous studies of NSCLC [28–32]. In particular, the TCGA dataset consisted mostly of patients with early-stage disease who underwent surgical resection, and the minimum percent tumor nuclei needed for whole genome sequencing was 60%, which may have created a bias for poorly differentiated tumors that are known to harbor fewer EGFR mutations and targetable alterations in general. For
example, the prevalence of mutations was similar in AD and ADSQ but lower in NSCLC-NOS and LCC. Although geographic differences persist, it is estimated that at least 75% of patients with advanced-stage lung AD or NSCLC-NOS are currently being tested for EGFR mutations [33], which represents significant progress since the 2013 CAP/ IASLC/AMP guidelines were introduced [5]. These guidelines are undergoing revision to align more closely with the NCCN guidelines, which also recommend testing for five additional genes. In our series, we identified BRAF V600E point mutations in 2.1%, ERBB2 point mutations and indels in 3.5%, MET amplification in 3.1%, MET exon 14 skipping mutations in 2.8%, ROS1 rearrangements in 1.3%, and RET rearrangements in 1.9% of patients (Table 2). Our findings are similar to those reported in previous studies and the COSMIC database [28–32, 34]. It is important to note that ERBB2 and MET amplification, and, more recently, RET rearrangement, have been linked to potential mechanisms of resistance to anti-EGFR targeted therapies [35].Therefore, the true prevalence of EGFR mutations and ALK rearrangements may be slightly lower in the setting of patients with newly diagnosed NSCLC because our cohort includes many patients who have progressed on first-line TKIs.
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Figure 2. A 45-year-old male ex-smoker (30 pack-years) with stage IV lung adenocarcinoma and NTRK1 rearrangement was enrolled in a phase I clinical trial of entrectinib. The upper panel demonstrates partial response on chest CT scan at 4 weeks (Response Evaluation Criteria in Solid Tumors:47%)andlowerpaneldemonstratescompleteresponseof15–20brainmetastases5 monthsafterinitiationoftargetedtherapy.Todate, the patient has continued to be treated with entrectinib for more than 6 months. Reprinted with permission [42].
Another significant advantage of CGP is the ability to identify new GAs in any given disease type. For example, in addition to known sensitizing EGFR mutations, 4.9% of patients in this study harbored other EGFR alterations that may predict response to EGFR-targeted therapies such as T790M inhibitors, 3.6% of patients harbored BRAF alterations other than BRAF V600E mutation, and 3.5% of patients harbored ERBB2 alterations other than ERBB2 mutation and indel. Other GAs, such as ALK mutations, have been linked to resistance to ALK TKIs [36–39]. In total, 45% of patients in our series harbored potentially targetable alterations in the seven genes included in the NCCN guidelines; 33% of patients harbored GAs specified by NCCN and 32% harbored KRAS alterations. GAs in more than one of these genes were present in 5.4% of patients, and were likely attributable to acquired resistance mechanisms and occasional de novo cases. In recent studies, up to 20% of patients with EGFR exon 19 deletion and 35% of patients with ALK rearrangement tested negative by single-gene PCR and FISH assays, respectively, and 26% of patients who tested negative by hotspot and FISH assays www.TheOncologist.com
harbored a genomic alteration listed by NCCN, providing further evidence of the value of CGP [16, 40, 41]. Overall, we estimate that a hotspot NGS test without supplemental FISH assays would miss up to half of the NCCN GAs that were detected by CGP in this study. Beyond the NCCN guidelines, CGP detects additional GAs that may qualify patients for enrollment in mechanism-driven clinical trials. In the cohort of patients with lung AD (n 5 1,339) without alterations in EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS, GAs involving 273 genes with at least 0.1% frequency were identified, including STK11 (21%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), BRCA1 (1.7%), NTRK1 (0.7%), and NTRK3 (0.2%) (Table 3). In particular, GAs involving NF1 (13%), NRAS (2.3%), MAP2K1 (1.2%), and HRAS (0.7%) have been reported previously by TCGA as likely lung AD driver oncogenes [29]. More recently, NRG rearrangements have been discovered in patients with NSCLC and are seen most often in invasive mucinous adenocarcinoma, a subtype of lung AD that is known to harbor frequent KRAS mutations as well as ALK and ROS1 ©AlphaMed Press 2016
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Figure 3. An 18-year-old male never-smoker with “pan-negative”stage IV lung adenocarcinoma and RICTOR amplification was enrolled in a phase I clinical trial of CC-223, a dual mTORC1/2 inhibitor, and had stable disease for 12 months. Following disease progression and failure of immunotherapy, he was enrolled in a phase I clinical trial of MLN0128, another dual mTORC1/2 inhibitor, with ongoing stable disease. Reprinted with permission [43]. Abbreviations: ab, abdomen; carbo/pem/bev, carboplatin/pemetrexed/bevacizumab; dx, diagnosis; dz, disease; immunoRx, immunotherapy; NGS, next-generation sequencing; PBMC, peripheral blood mononuclear cell; POD, postoperative day; PR, partial response; pt, patient.
rearrangements [22]. Perhaps most significantly, patients with NSCLC with NTRK1 rearrangement and RICTOR amplification have been shown to respond to matched targeted therapies (Figs. 2, 3) [21, 42, 43]. One of the primary challenges for CGP and other tissuebased assays is the availability of sufficient DNA from small biopsy and cytology specimens, especially if immunohistochemistry and other molecular assays are performed on these samples. Because many academic and community laboratories now perform EGFR and ALK testing, there is growing concern that other GAs cannot be identified if both tests are negative without performing a repeated biopsy.Therefore, optimization of tissue acquisition procedures by interventional radiologists and endoscopists, and implementation of careful tissue preservation protocols by pathologists are critical for all patients who may need biomarker testing [44–46]. Internal quality assurance data from one large academic medical center showed that the success rate for small biopsy and cytology specimens dropped from 86% to 53% if formalin-fixed, paraffin-embedded blocks were refaced three or more times (unpublished data). Although some investigators recommend dividing small samples into multiple blocks, this practice may preclude extraction of sufficient DNA for NGS unless blocks are melted and recombined. For rapid on-site evaluation, the key step is to limit the number of slides created for diagnosis and place extra needle passes into the cell block container. Despite these challenges, the full range of surgical pathology and cytology specimens, including recent and archival core needle biopsies, fine-needle aspirates, and effusion cytologies, are routinely tested successfully using CGP [47]. An important limitation of this study is the lack of reliable clinical stage data; such information was not provided in the submitted paperwork in at least 25% of cases. However, half of all specimens tested were obtained from extrapulmonary
metastatic sites, representing mostly patients with stage IV NSCLC. Specimens obtained from lung tissue represented a mixture of intrapulmonary metastases, biopsies performed in patients with stage IV disease, and archived early-stage surgical resections that were retrieved at the time of recurrence or metastasis. At this time, we do not encourage reflex testing for early-stage disease. The vast majority of physicians who order our test are medical oncologists who treat patients with advanced-stage disease rather than surgeons or pathologists and, in our experience, tests are usually performed after multiple lines of chemotherapy have failed in these patients.
CONCLUSION In summary, hybrid capture-based NGS, or CGP, is practical and facilitates implementation of the NCCN guidelines for lung cancer biomarker testing by enabling simultaneous detection of GAs for all seven driver oncogenes (EGFR, ALK, BRAF, ERBB2, MET, ROS1, and RET) listed by NCCN, and KRAS. Our study demonstrates that the majority of patients for whom biomarker testing is currently indicated harbor alterations involving these eight genes, enabling oncologists and patients with advanced-stage disease to choose optimal therapies, ideally for first-line treatment as well as in the context of resistance. CGP also identifies patients with other GAs in “pannegative” lung AD who may benefit from enrollment in mechanism-driven clinical trials that are currently evaluating hundreds of targeted therapies directed against GAs in more than 150 distinct cancer-related genes. As Pazdur and colleagues at the FDA point out, “finding patients whose tumors harbor low-frequency variants (e.g., ,1%) in even common malignant conditions such as NSCLC, and enrolling these patients in clinical trials, may be infeasible without the widespread adoption of multiplex NGS screening” [47]. Although logistical and administrative hurdles limit the widespread use
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of NGS, our data confirm the feasibility and potential utility of CGP in clinical practice. As of December 2015, at least one prominent regional payer (Priority Health, Grand Rapids, MI, http://www.priorityhealth.com) covers CGP across multiple tumor types, a major national payer now covers CGP for all patients with stage IIIB and IV NSCLC, and Palmetto GBA covers CGP for a subset of Medicare patients with NSCLC who have tested negative for EGFR and ALK, further demonstrating the growing value of this approach for patient care.
AUTHOR CONTRIBUTIONS Conception/Design: James H. Suh, Jeffrey S. Ross Collection and/or assembly of data: James H. Suh, Adrienne Johnson, Lee Albacker, Kai Wang, Julia A. Elvin, Jo-Anne Vergilio, Jeffrey S. Ross Data analysis and interpretation: James H. Suh, Adrienne Johnson, Lee Albacker, Kai Wang, Garret Frampton, Laurie Gay, Julia A. Elvin, Jo-Anne Vergilio, Jeffrey S. Ross
Manuscript writing: James H. Suh, Laurie Gay Final approval of manuscript: Juliann Chmielecki, Siraj Ali, Vincent A. Miller, Philip J. Stephens, Jeffrey S. Ross
DISCLOSURES James H. Suh: Foundation Medicine, Inc. (E, OI), Daiichi Sankyo (C/A), Genentech (H); Adrienne Johnson: Foundation Medicine, Inc. (E, OI); Lee Albacker: Foundation Medicine, Inc. (E, OI); Kai Wang: Foundation Medicine, Inc. (E, OI); Juliann Chmielecki: Foundation Medicine, Inc. (E, OI); Garret Frampton: Foundation Medicine, Inc. (E, OI); Laurie Gay: Foundation Medicine, Inc. (E, OI); Julia A. Elvin: Foundation Medicine, Inc. (E, OI); Jo-Anne Vergilio: Foundation Medicine, Inc. (E, OI); Siraj Ali: Foundation Medicine, Inc. (E, IP, OI); Vincent A. Miller: Foundation Medicine, Inc. (E, OI), Memorial Sloan Kettering Cancer Center (IP); Philip J. Stephens: Foundation Medicine, Inc. (E, OI); Jeffrey S. Ross: Foundation Medicine, Inc. (E, OI, RF). (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/ inventor/patent holder; (SAB) Scientific advisory board
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25. Travis WD, Rekhtman N. Pathological diagnosis and classification of lung cancer in small biopsies and cytology: Strategic management of tissue for molecular testing. Semin Respir Crit Care Med 2011; 32:22–31.
15. Drilon A, Wang L, Hasanovic A et al. Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discov 2013;3: 630–635.
26. Aisner DL, Marshall CB. Molecular pathology of non-small cell lung cancer: A practical guide. Am J Clin Pathol 2012;138:332–346.
16. Drilon A,Wang L, Arcila ME et al. Broad, hybrid capture-based next-generation sequencing identifies actionable genomic alterations in lung adenocarcinomas otherwise negative for such alterations by other genomic testing approaches. Clin Cancer Res 2015;21:3631–3639. 17. Khoo C, Rogers T-M, Fellowes A et al. Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond. Transl Lung Cancer Res 2015;4:126–141. 18. Frampton GM, Fichtenholtz A, Otto GA et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol 2013;31: 1023–1031. 19. Cheng DT, Mitchell TN, Zehir A et al. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J Mol Diagn 2015;17:251–264. 20. Ross JS, Wang K, Chmielecki J et al. The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int J Cancer 2016; 138:881–890. 21. Vaishnavi A, Capelletti M, Le AT et al. Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat Med 2013;19:1469–1472. 22. Fernandez-Cuesta L, Plenker D, Osada H et al. CD74-NRG1 fusions in lung adenocarcinoma. Cancer Discov 2014;4:415–422.
27. Shaw AT, Ou S-HI, Bang Y-J et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med 2014;371:1963–1971. 28. Sholl LM, Aisner DL, Varella-Garcia M et al. Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: The Lung Cancer Mutation Consortium Experience. J Thorac Oncol 2015;10:768–777. 29. Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma [published correction appears in Nature 2014;514(7521):262]. Nature 2014;511:543–550. 30. Arcila ME, Chaft JE, Nafa K et al. Prevalence, clinicopathologic associations, and molecular spectrum of ERBB2 (HER2) tyrosine kinase mutations in lung adenocarcinomas. Clin Cancer Res 2012;18: 4910–4918. 31. Li C, Sun Y, Fang R et al. Lung adenocarcinomas with HER2-activating mutations are associated with distinct clinical features and HER2/EGFR copy number gains. J Thorac Oncol 2012;7: 85–89. 32. Dogan S, Shen R, Ang DC et al. Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: Higher susceptibility of women to smoking-related KRAS-mutant cancers. Clin Cancer Res 2012;18:6169–6177. 33. Spicer J, Tischer B, Peters M. EGFR mutation testing and oncologist treatment choice in advanced NSCLC: Global trends and differences. Ann Oncol 2015;26(suppl 1):i60.
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39. KatayamaR, Friboulet L, Koike S et al.Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib. Clin Cancer Res 2014;20:5686–5696.
44. Yarmus L, Akulian J, Gilbert C et al. Optimizing endobronchial ultrasound for molecular analysis. How many passes are needed? Ann Am Thorac Soc 2013;10:636–643.
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41. Schrock AB, Frampton GM, Herndon D et al. Comprehensive genomic profiling identifies frequent drug sensitive EGFR exon 19 deletions in NSCLC not identified by prior molecular testing. Clin Cancer Res 2016 [Epub ahead of print]. 42. Farago AF, Le LP, Zheng Z et al. Durable clinical response to entrectinib in NTRK1-rearranged nonsmall cell lung cancer. J Thorac Oncol 2015;10: 1670–1674. 43. Cheng H, Zou Y, Ross JS et al. RICTOR amplification defines a novel subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors. Cancer Discov 2015;5: 1262–1270.
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See http://www.TheOncologist.com for supplemental material available online.
For Further Reading: Benjamin P. Levy, Marc D. Chioda, Dana Herndon et al. Molecular Testing for Treatment of Metastatic Non-Small Cell Lung Cancer: How to Implement Evidence-Based Recommendations. The Oncologist 2015;20:1175–1181. Implications for Practice: Although several professional societies have incorporated biomarker testing recommendations into clinical practice guidelines for the diagnosis and management of non-small cell lung cancer (NSCLC), health care providers still face considerable challenges when establishing and implementing these standards. Developing and instituting protocols to ensure that all appropriate patients are tested for molecular biomarkers requires communication among the various specialists involved in the care of patients with NSCLC. This report provides insights into key challenges and recommendations for molecular testing of patients with metastatic NSCLC, summarized from a multidisciplinary team of experts spanning academic, community, and integrated health systems.
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Comprehensive Genomic Profiling Facilitates Implementation of the National Comprehensive Cancer Network Guidelines for Lung Cancer Biomarker Testing and Identifies Patients Who May Benefit From Enrollment in Mechanism-Driven Clinical Trials JAMES H. SUH,a ADRIENNE JOHNSON,a LEE ALBACKER,a KAI WANG,a,b JULIANN CHMIELECKI,a GARRETT FRAMPTON,a LAURIE GAY,a JULIA A. ELVIN,a JO-ANNE VERGILIO,a SIRAJ ALI,a VINCENT A. MILLER,a PHILIP J. STEPHENS,a JEFFREY S. ROSSa a
Foundation Medicine, Inc., Cambridge, Massachusetts, USA; bZhejiang Cancer Hospital, Hangzhou, People’s Republic of China
Disclosures of potential conflicts of interest may be found at the end of this article.
Key Words. Non-small cell lung cancer x Comprehensive genomic profiling x National Comprehensive Cancer Network guidelines x Clinical trials
ABSTRACT Background. The National Comprehensive Cancer Network (NCCN) guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for EGFR, BRAF, ERBB2, and MET mutations; ALK, ROS1, and RET rearrangements; and MET amplification. We investigated the feasibility and utility of comprehensive genomic profiling (CGP), a hybrid capture-based next-generation sequencing (NGS) test, in clinical practice. Methods. CGP was performed to a mean coverage depth of 5763 on 6,832 consecutive cases of NSCLC (2012–2015). Genomic alterations (GAs) (point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were recorded. We also evaluated lung adenocarcinoma (AD) cases without GAs, involving these eight genes. Results. The median age of the patients was 64 years (range: 13–88 years) and 53% were female. Among the patients
studied, 4,876 (71%) harbored at least one GA involving EGFR (20%), ALK (4.1%), BRAF (5.7%), ERBB2 (6.0%), MET (5.6%), ROS1 (1.5%), RET (2.4%), or KRAS (32%). In the remaining cohort of lung AD without these known drivers, 273 cancerrelated genes were altered in at least 0.1% of cases, including STK11 (21%), NF1 (13%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), NRAS (2.3%), BRCA1 (1.7%), MAP2K1 (1.2%), HRAS (0.7%), NTRK1 (0.7%), and NTRK3 (0.2%). Conclusion. CGP is practical and facilitates implementation of the NCCN guidelines for NSCLC by enabling simultaneous detection of GAs involving all seven driver oncogenes and KRAS. Furthermore, without additional tissue use or cost, CGP identifies patients with “pan-negative” lung AD who may benefit from enrollment in mechanism-driven clinical trials. The Oncologist 2016;21:684–691
Implications for Practice: National Comprehensive Cancer Network guidelines for patients with metastatic non-small cell lung cancer (NSCLC) recommend testing for several genomic alterations (GAs). The feasibility and utility of comprehensive genomic profiling were studied in NSCLC and in lung adenocarcinoma (AD) without GAs. Of patients with NSCLC, 71% harbored at least one GA to a gene listed in the guidelines or KRAS; 273 cancer-related genes were altered in at least 0.1% of the AD cases. Although logistical and administrative hurdles limit the widespread use of next-generation sequencing, the data confirm the feasibility and potential utility of comprehensive genomic profiling in clinical practice.
INTRODUCTION Lung cancer is the leading cause of cancer mortality worldwide, accounting for more than 158,000 deaths annually in the U.S., with approximately 85% being of non-small cell lung cancer (NSCLC) histology [1, 2]. Because of the discovery of activating epidermal growth factor receptor (EGFR) point mutations and small insertions and deletions (indels), in 2003, and
anaplastic lymphoma kinase (ALK) rearrangements, in 2007, as well as commercial availability of FDA-approved EGFR and ALK tyrosine kinase inhibitors (TKIs), the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) issued joint guidelines in 2013, endorsed by the
Correspondence: James H. Suh, M.D., Foundation Medicine, Inc., 150 Second Street, Cambridge, Massachusetts 02142, USA.Telephone: 617-4182200, ext. 7402; E-Mail: [email protected] Received January 28, 2016; accepted for publication March 30, 2016; published Online First on May 5, 2016. ©AlphaMed Press 1083-7159/2016/$20.00/0 http://dx.doi.org/10.1634/theoncologist.2016-0030
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Table 1. National Comprehensive Cancer Network guidelines for non-small cell lung cancer, version 4.2016
Genomic alterations EGFR mutations ALK rearrangements HER2 mutations BRAF V600E mutations MET amplification and exon 14 skipping mutations ROS1 rearrangements RET rearrangements
Available targeted agents with activity against driver event in lung cancera Erlotinib, gefitinib, afatinib Crizotinib, ceritinib, alectinib Trastuzumab, afatinib Vemurafenib, dabrafenib with or without trametinib Crizotinib Crizotinib Cabozantinib
a
Indicates recommended use in the National Comprehensive Cancer Network Drugs and Biologics Compendium. Bold indicates approval (EGFR, ALK, and ROS1 inhibitors) or breakthrough therapy designation (BRAF inhibitors) by the FDA.
American Society of Clinical Oncology (ASCO) in 2014, that recommend routine testing for both genomic alterations (GAs) in patients with advanced-stage disease [3–6]. The most recent version of the National Comprehensive Cancer Network (NCCN) guidelines for management of NSCLC also recommends testing for BRAF and ERBB2 mutations, MET amplification and exon 14 skipping mutations, and ROS1 and RET rearrangements. Targeted therapies with approval or breakthrough therapy designation by the FDA (i.e., dabrafenib with or without trametinib) have shown benefit in patients whose tumors harbor relevant GAs (Table 1) [7–15]. Therefore, molecular testing of NSCLC, particularly adenocarcinoma (AD), is now the standard of care for patients who need systemic therapy [5–7]. Whereas the 2013 CAP/IASLC/ AMP guidelines endorsed identification of EGFR mutations by polymerase chain reaction (PCR) and ALK rearrangements by fluorescent in situ hybridization (FISH) based on the FDAapproved companion diagnostic kits at the time, the rapid maturation of clinical massively parallel or next-generation sequencing (NGS) technologies, as well as the need to identify GAs in seven different oncogenes, enabled the NCCN panel to state that EGFR and ALK testing should be conducted using broad molecular profiling methods [7]. Despite the enthusiasm for NGS, it is important to recognize that similar to other laboratory techniques such as immunohistochemistry or FISH, test performance can vary widely. Many NGS platforms are hotspot tests that are limited to the detection of point mutations and some indels, and cannot identify copy number alterations or rearrangements. Hotspot tests may also struggle with detection of point mutations and indels in small specimens with low tumor purity, leading to higher false-negative rates [16, 17]. These limitations are particularly problematic for NSCLC testing, because ALK, ROS1, and RET rearrangements and MET amplification cannot be identified using hotspot NGS platforms. In contrast, hybrid capture-based NGS platforms, also known as comprehensive genomic profiling (CGP) assays, can diagnose all four types of DNA alterations seen in cancer—point mutations, small indels, copy number changes, and rearrangements—in hundreds of cancer-related genes with high sensitivity and specificity [18, 19]. CGP provides an accurate and efficient alternative to multiplex or hotspot testing that is
attractive in the setting of NSCLC because itcan identify all seven GAs included in the NCCN guidelines as well as additional GAs that may qualify patients for enrollment in mechanism-driven clinical trials or have been associated with response to targeted therapy [20–24]. Last, the majority of patients with NSCLC are diagnosed by using small biopsy or cytology specimens for which there is a limited amount of available material for molecular testing [25, 26]. Our aim is to describe our series of all NSCLC cases submitted to Foundation Medicine for CGP over a 33-month period to demonstrate its clinical utility.
METHODS Genomic profiling was performed in a Clinical Laboratory Improvement Amendments-certified, CAP-accredited reference laboratory (Foundation Medicine) for 6,832 clinical NSCLC samples consecutively submitted between September 2012 and May 2015. At least 50 ng of DNA per sample was extracted from 40 mm of clinical formalin-fixed, paraffinembedded tumor samples and was analyzed by hybridization capture of 3,320 exons from 236 (gene set 1) or 315 (gene set 2) cancer-related genes and introns of 19 or 28 genes commonly rearranged in cancer. Specimens were sequenced to high, uniform coverage (mean: 5763) on Illumina HiSeq2000 or HiSeq2500 instruments (San Diego, CA, http://www.illumina. com), as previously described [18]. A total of 3,060 samples (45%) were analyzed using gene set 1 and the remainder (3,772; 55%) were analyzed using gene set 2. GAs (i.e., point mutations, small indels, copy number changes, and rearrangements) involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, and KRAS were determined and then reported for each patient sample. GAs involving the remaining genes in the panel were evaluated for the lung AD samples lacking these eight known drivers.The complete lists of genes for set 1 and set 2 are included in the supplemental material (supplemental online Table 1). The test has been validated to detect point mutations at $10% mutant allele frequency with $99% sensitivity, and indels at $20% mutant allele frequency with $95% sensitivity, with a false discovery rate of,1% [18].Turnaroundtime is routinely 7–10 days from sample receipt. Local site permissions to use clinical samples were obtained for this study. In addition to patient age, sex, and histologic subtype, the tissue biopsy site was recorded for each case.
RESULTS The median age of patients in this series was 64 years (range: 13–88 years), and 53% were female. Regarding histologic subtype, 5,380 cases (79%) were lung AD, 1,345 (20%) were nonsmall cell carcinoma, not otherwise specified (NSCLC-NOS), 72 (1%) were adenosquamous carcinoma (ADSQ), and 35 (0.5%) were large cell carcinoma (LCC).The difference in the numbers of AD (75%) and ADSQ (68%) cases with at least one GA in the genes listed by NCCN or KRAS versus NSCLC-NOS (57%) and LCC (37%) was statistically significant (p , .0001). Of these specimens, 50% were obtained from extrapulmonary metastatic sites. GAs involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS were identified in 4,876 cases (71%). As seen in Table 2 and Figure 1, 1,342 cases (20%) harbored EGFR alterations, 280 (4.1%) harbored ALK alterations, 388 (5.7%) harbored BRAF alterations, 408 (6.0%) harbored ERBB2 alterations, 383 (5.6%) harbored MET alterations, 100 (1.5%) harbored ROS1
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Table 2. Genomic alterations involving NCCN guideline genes and KRAS in 6,832 cases of non-small cell lung carcinoma % Cases Gene
Total cases
Point mutation
EGFR ALK BRAF ERBB2 MET ROS1 RET KRAS
1,342 280 388 408 383 100 166 2,178
12 0.3 5.1 0.8 1.7 0.1 0.2 31
a
Small indel
b
10 0 0.06 2.6 1.4 0 0 0.04
Amplificationc
Rearrangementc
Alld
% GAs described in NCCN guidelinese
5.9 0.1 0.4 3.0 3.1 0.03 0.3 3.5
0 3.9 0.2 0 0 1.3 1.9 0
20 4.1 5.7 6.0 5.6 1.5 2.4 32
15b 3.9c 2.1a 3.5b 5.3b 1.3c 1.9c N/A
a
GAs that can be detected using hotspot NGS tests. GAs that can be detected occasionally using hotspot NGS tests. GAs that cannot be detected using hotspot NGS tests. d Some cases have multiple GAs involving the same gene. e We estimate that a hotspot NGS test without supplemental FISH assays would miss up to half of the NCCN GAs in this table. Abbreviations: GA, genomic alteration; indel, insertion and deletion; N/A, not applicable; NCCN, National Comprehensive Cancer Network. b c
Last, there were 1,339 cases of lung AD without GAs involving EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS. In this cohort, GAs involving previously known lung AD oncogenes NF1 (13%), PIK3CA (5.4%), NRAS (2.3%), MAP2K1 (1.2%), and HRAS (0.7%) were detected. A total of 273 genes harbored GAs in at least 0.1% of cases, many of which are associated with potential benefit from targeted therapies or allow enrollment in mechanism-driven clinical trials, including STK11 (21%), MYC (9.8%), RICTOR (6.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), BRCA1 (1.7%), NTRK1 (0.7%), and NTRK3 (0.2%) (Table 3).
DISCUSSION
Figure 1. Genomic alterations (GAs) involving National Comprehensive Cancer Network guideline genes and KRAS in 6,832 cases of non-small cell lung carcinoma. Some 5.4% of cases have GAs involving more than one of these genes.
alterations, 166 (2.4%) harbored RET alterations, and 2,178 (32%) harbored KRAS alterations. With respect to the particular alterations specified by the CAP/IASLC/AMP and NCCN guidelines, 15% of cases in this series were positive for known sensitizing EGFR point mutations and indels, 3.9% were positive for ALK rearrangements, 2.1% were positive for BRAF V600E point mutations, 3.5% were positive for ERBB2 point mutations and indels, 3.1% were positive for MET amplification, 2.8% were positive for MET exon 14 skipping mutations, 1.3% were positive for ROS1 rearrangements, and 1.9% were positive for RET rearrangements (Table 2). In addition, CGP identified multiple, additional, potentially targetable GAs involving the same driver oncogenes, including 401 cases (5.9%) with EGFR amplification, 203 cases (3.0%) with BRAF non-V600E point mutations, 12 cases (0.2%) with BRAF rearrangement, and 208 cases (3.0%) with ERBB2 amplification. EGFR T790M resistance mutations were identified in 221 cases (3.2%), and ALK resistance mutations were identified in 9 cases (0.1%). www.TheOncologist.com
Despite recent advances in CT screening for lung cancer, the majority of patients continue to present with advancedstage disease and testing for EGFR point mutations and indels and ALK rearrangements is now the standard of care for selection of appropriate first-line therapy [1–7]. Since crizotinib received regular FDA approval in March 2016 for patients with NSCLC with ROS1 rearrangements, and osimertinib received FDA breakthrough therapy designation in late 2015 for patients with NSCLC with EGFR T790M resistance mutations, ROS1 and EGFR T790M testing are also emerging as the standard of care [27]. Because of these developments and ongoing clinical trials evaluating more than 900 agents against 150 or so molecularly defined targets, a growing number of academic medical centers and commercial laboratories have begun to offer NGS testing to identify alterations in other driver oncogenes that are included in the NCCN guidelines, such as BRAF, ERBB2, MET, and RET. However, they often use single-gene PCR and FISH assays with limited sensitivity compared with NGS or hotspot NGS platforms that are incapable of detecting ALK, ROS1, and RET rearrangements and MET amplification [17]. In particular, hotspot NGS platforms rely on multiple supplemental single-gene FISH assays that consume tissue, leaving testing incomplete in 30%–84% of cases if additional biopsies are not performed, as seen in the Memorial Sloan-Kettering Cancer Center and Lung Cancer Mutation Consortium experiences [16, 28]. ©AlphaMed Press 2016
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Table 3. Selection of cancer-related genes altered in 1,339 cases of “pan-negative” lung adenocarcinoma and mechanism-driven clinical trials Gene
% Cases
Targeted therapies
Relevant clinical trial examples
STK11
21
mTOR inhibitors
NF1
13
MEK inhibitors
NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02443337 – Phase II NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02419417 – Phase I NCT02391480 – Phase I NCT02431260 – Phase I/II NCT02187783 – Phase II NCT02450539 – Phase II NCT01037790 – Phase II NCT01470209 – Phase I NCT02117167 – Phase II NCT02069158 – Phase Ib NCT02154490 – Phase II/III NCT01306045 – Phase II NCT01862081 – Phase I NCT02583542 – Phase Ib/IIa NCT02412371 – Phase III NCT02264990 – Phase III NCT02106546 – Phase III NCT01306045 – Phase II NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02583542 – Phase Ib/IIa NCT02117167 – Phase II NCT02457793 – Phase Ib NCT02219711 – Phase I
MYC
9.8
Bromodomain inhibitors
CDK4, CCND1
8.3
CDK4/6 inhibitors
RICTOR
6.4
mTOR inhibitors
PIK3CA
5.4
BRCA1/2
4.2
mTOR inhibitors PI3K inhibitors Dual mTOR/PI3K inhibitors AKT inhibitors PARP inhibitors
NRAS, HRAS
3.0
MEK inhibitors
MAP2K1 NTRK1, NTRK3
1.2 0.9
MEK inhibitors NTRK1 inhibitors
The purpose of this study is to demonstrate that our CGP assay, based on a hybrid-capture approach, can diagnose simultaneously all four types of DNA alterations that occur within the eight best-characterized NSCLC driver oncogenes (EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, KRAS) as well as dozens of other genes that are linked to clinical trials. Over a 33-month period, we analyzed lung tumor samples from 6,832 patients with primarily advanced-stage disease. Of the patients in our series, 15% harbored EGFR alterations that are known to be sensitizing to FDA-approved EGFR TKIs according to the NCCN guidelines, and 3.9% harbored ALK rearrangements that predict response to FDA-approved ALK TKIs (Table 2). The frequencies for both alterations are somewhat higher than those identified within The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset but are within the ranges that have been reported in previous studies of NSCLC [28–32]. In particular, the TCGA dataset consisted mostly of patients with early-stage disease who underwent surgical resection, and the minimum percent tumor nuclei needed for whole genome sequencing was 60%, which may have created a bias for poorly differentiated tumors that are known to harbor fewer EGFR mutations and targetable alterations in general. For
example, the prevalence of mutations was similar in AD and ADSQ but lower in NSCLC-NOS and LCC. Although geographic differences persist, it is estimated that at least 75% of patients with advanced-stage lung AD or NSCLC-NOS are currently being tested for EGFR mutations [33], which represents significant progress since the 2013 CAP/ IASLC/AMP guidelines were introduced [5]. These guidelines are undergoing revision to align more closely with the NCCN guidelines, which also recommend testing for five additional genes. In our series, we identified BRAF V600E point mutations in 2.1%, ERBB2 point mutations and indels in 3.5%, MET amplification in 3.1%, MET exon 14 skipping mutations in 2.8%, ROS1 rearrangements in 1.3%, and RET rearrangements in 1.9% of patients (Table 2). Our findings are similar to those reported in previous studies and the COSMIC database [28–32, 34]. It is important to note that ERBB2 and MET amplification, and, more recently, RET rearrangement, have been linked to potential mechanisms of resistance to anti-EGFR targeted therapies [35].Therefore, the true prevalence of EGFR mutations and ALK rearrangements may be slightly lower in the setting of patients with newly diagnosed NSCLC because our cohort includes many patients who have progressed on first-line TKIs.
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Figure 2. A 45-year-old male ex-smoker (30 pack-years) with stage IV lung adenocarcinoma and NTRK1 rearrangement was enrolled in a phase I clinical trial of entrectinib. The upper panel demonstrates partial response on chest CT scan at 4 weeks (Response Evaluation Criteria in Solid Tumors:47%)andlowerpaneldemonstratescompleteresponseof15–20brainmetastases5 monthsafterinitiationoftargetedtherapy.Todate, the patient has continued to be treated with entrectinib for more than 6 months. Reprinted with permission [42].
Another significant advantage of CGP is the ability to identify new GAs in any given disease type. For example, in addition to known sensitizing EGFR mutations, 4.9% of patients in this study harbored other EGFR alterations that may predict response to EGFR-targeted therapies such as T790M inhibitors, 3.6% of patients harbored BRAF alterations other than BRAF V600E mutation, and 3.5% of patients harbored ERBB2 alterations other than ERBB2 mutation and indel. Other GAs, such as ALK mutations, have been linked to resistance to ALK TKIs [36–39]. In total, 45% of patients in our series harbored potentially targetable alterations in the seven genes included in the NCCN guidelines; 33% of patients harbored GAs specified by NCCN and 32% harbored KRAS alterations. GAs in more than one of these genes were present in 5.4% of patients, and were likely attributable to acquired resistance mechanisms and occasional de novo cases. In recent studies, up to 20% of patients with EGFR exon 19 deletion and 35% of patients with ALK rearrangement tested negative by single-gene PCR and FISH assays, respectively, and 26% of patients who tested negative by hotspot and FISH assays www.TheOncologist.com
harbored a genomic alteration listed by NCCN, providing further evidence of the value of CGP [16, 40, 41]. Overall, we estimate that a hotspot NGS test without supplemental FISH assays would miss up to half of the NCCN GAs that were detected by CGP in this study. Beyond the NCCN guidelines, CGP detects additional GAs that may qualify patients for enrollment in mechanism-driven clinical trials. In the cohort of patients with lung AD (n 5 1,339) without alterations in EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS, GAs involving 273 genes with at least 0.1% frequency were identified, including STK11 (21%), MYC (9.8%), RICTOR (6.4%), PIK3CA (5.4%), CDK4 (4.3%), CCND1 (4.0%), BRCA2 (2.5%), BRCA1 (1.7%), NTRK1 (0.7%), and NTRK3 (0.2%) (Table 3). In particular, GAs involving NF1 (13%), NRAS (2.3%), MAP2K1 (1.2%), and HRAS (0.7%) have been reported previously by TCGA as likely lung AD driver oncogenes [29]. More recently, NRG rearrangements have been discovered in patients with NSCLC and are seen most often in invasive mucinous adenocarcinoma, a subtype of lung AD that is known to harbor frequent KRAS mutations as well as ALK and ROS1 ©AlphaMed Press 2016
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Figure 3. An 18-year-old male never-smoker with “pan-negative”stage IV lung adenocarcinoma and RICTOR amplification was enrolled in a phase I clinical trial of CC-223, a dual mTORC1/2 inhibitor, and had stable disease for 12 months. Following disease progression and failure of immunotherapy, he was enrolled in a phase I clinical trial of MLN0128, another dual mTORC1/2 inhibitor, with ongoing stable disease. Reprinted with permission [43]. Abbreviations: ab, abdomen; carbo/pem/bev, carboplatin/pemetrexed/bevacizumab; dx, diagnosis; dz, disease; immunoRx, immunotherapy; NGS, next-generation sequencing; PBMC, peripheral blood mononuclear cell; POD, postoperative day; PR, partial response; pt, patient.
rearrangements [22]. Perhaps most significantly, patients with NSCLC with NTRK1 rearrangement and RICTOR amplification have been shown to respond to matched targeted therapies (Figs. 2, 3) [21, 42, 43]. One of the primary challenges for CGP and other tissuebased assays is the availability of sufficient DNA from small biopsy and cytology specimens, especially if immunohistochemistry and other molecular assays are performed on these samples. Because many academic and community laboratories now perform EGFR and ALK testing, there is growing concern that other GAs cannot be identified if both tests are negative without performing a repeated biopsy.Therefore, optimization of tissue acquisition procedures by interventional radiologists and endoscopists, and implementation of careful tissue preservation protocols by pathologists are critical for all patients who may need biomarker testing [44–46]. Internal quality assurance data from one large academic medical center showed that the success rate for small biopsy and cytology specimens dropped from 86% to 53% if formalin-fixed, paraffin-embedded blocks were refaced three or more times (unpublished data). Although some investigators recommend dividing small samples into multiple blocks, this practice may preclude extraction of sufficient DNA for NGS unless blocks are melted and recombined. For rapid on-site evaluation, the key step is to limit the number of slides created for diagnosis and place extra needle passes into the cell block container. Despite these challenges, the full range of surgical pathology and cytology specimens, including recent and archival core needle biopsies, fine-needle aspirates, and effusion cytologies, are routinely tested successfully using CGP [47]. An important limitation of this study is the lack of reliable clinical stage data; such information was not provided in the submitted paperwork in at least 25% of cases. However, half of all specimens tested were obtained from extrapulmonary
metastatic sites, representing mostly patients with stage IV NSCLC. Specimens obtained from lung tissue represented a mixture of intrapulmonary metastases, biopsies performed in patients with stage IV disease, and archived early-stage surgical resections that were retrieved at the time of recurrence or metastasis. At this time, we do not encourage reflex testing for early-stage disease. The vast majority of physicians who order our test are medical oncologists who treat patients with advanced-stage disease rather than surgeons or pathologists and, in our experience, tests are usually performed after multiple lines of chemotherapy have failed in these patients.
CONCLUSION In summary, hybrid capture-based NGS, or CGP, is practical and facilitates implementation of the NCCN guidelines for lung cancer biomarker testing by enabling simultaneous detection of GAs for all seven driver oncogenes (EGFR, ALK, BRAF, ERBB2, MET, ROS1, and RET) listed by NCCN, and KRAS. Our study demonstrates that the majority of patients for whom biomarker testing is currently indicated harbor alterations involving these eight genes, enabling oncologists and patients with advanced-stage disease to choose optimal therapies, ideally for first-line treatment as well as in the context of resistance. CGP also identifies patients with other GAs in “pannegative” lung AD who may benefit from enrollment in mechanism-driven clinical trials that are currently evaluating hundreds of targeted therapies directed against GAs in more than 150 distinct cancer-related genes. As Pazdur and colleagues at the FDA point out, “finding patients whose tumors harbor low-frequency variants (e.g., ,1%) in even common malignant conditions such as NSCLC, and enrolling these patients in clinical trials, may be infeasible without the widespread adoption of multiplex NGS screening” [47]. Although logistical and administrative hurdles limit the widespread use
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of NGS, our data confirm the feasibility and potential utility of CGP in clinical practice. As of December 2015, at least one prominent regional payer (Priority Health, Grand Rapids, MI, http://www.priorityhealth.com) covers CGP across multiple tumor types, a major national payer now covers CGP for all patients with stage IIIB and IV NSCLC, and Palmetto GBA covers CGP for a subset of Medicare patients with NSCLC who have tested negative for EGFR and ALK, further demonstrating the growing value of this approach for patient care.
AUTHOR CONTRIBUTIONS Conception/Design: James H. Suh, Jeffrey S. Ross Collection and/or assembly of data: James H. Suh, Adrienne Johnson, Lee Albacker, Kai Wang, Julia A. Elvin, Jo-Anne Vergilio, Jeffrey S. Ross Data analysis and interpretation: James H. Suh, Adrienne Johnson, Lee Albacker, Kai Wang, Garret Frampton, Laurie Gay, Julia A. Elvin, Jo-Anne Vergilio, Jeffrey S. Ross
Manuscript writing: James H. Suh, Laurie Gay Final approval of manuscript: Juliann Chmielecki, Siraj Ali, Vincent A. Miller, Philip J. Stephens, Jeffrey S. Ross
DISCLOSURES James H. Suh: Foundation Medicine, Inc. (E, OI), Daiichi Sankyo (C/A), Genentech (H); Adrienne Johnson: Foundation Medicine, Inc. (E, OI); Lee Albacker: Foundation Medicine, Inc. (E, OI); Kai Wang: Foundation Medicine, Inc. (E, OI); Juliann Chmielecki: Foundation Medicine, Inc. (E, OI); Garret Frampton: Foundation Medicine, Inc. (E, OI); Laurie Gay: Foundation Medicine, Inc. (E, OI); Julia A. Elvin: Foundation Medicine, Inc. (E, OI); Jo-Anne Vergilio: Foundation Medicine, Inc. (E, OI); Siraj Ali: Foundation Medicine, Inc. (E, IP, OI); Vincent A. Miller: Foundation Medicine, Inc. (E, OI), Memorial Sloan Kettering Cancer Center (IP); Philip J. Stephens: Foundation Medicine, Inc. (E, OI); Jeffrey S. Ross: Foundation Medicine, Inc. (E, OI, RF). (C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/ inventor/patent holder; (SAB) Scientific advisory board
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See http://www.TheOncologist.com for supplemental material available online.
For Further Reading: Benjamin P. Levy, Marc D. Chioda, Dana Herndon et al. Molecular Testing for Treatment of Metastatic Non-Small Cell Lung Cancer: How to Implement Evidence-Based Recommendations. The Oncologist 2015;20:1175–1181. Implications for Practice: Although several professional societies have incorporated biomarker testing recommendations into clinical practice guidelines for the diagnosis and management of non-small cell lung cancer (NSCLC), health care providers still face considerable challenges when establishing and implementing these standards. Developing and instituting protocols to ensure that all appropriate patients are tested for molecular biomarkers requires communication among the various specialists involved in the care of patients with NSCLC. This report provides insights into key challenges and recommendations for molecular testing of patients with metastatic NSCLC, summarized from a multidisciplinary team of experts spanning academic, community, and integrated health systems.
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