Jennifer C. Heng

MET Exon 14 Mutations in Non–Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression

PURPOSE Non-small-cell lung cancers (NSCLCs) harboring mutations in MET exon 14 and its flanking introns may respond to c-Met inhibitors. We sought to describe the clinical, pathologic, and genomic characteristics of patients with cancer with MET exon 14 mutations. PATIENTS AND METHODS We interrogated next-generation sequencing results from 6,376 cancers to identify those harboring MET exon 14 mutations. Clinical characteristics of MET exon 14 mutated NSCLCs were compared with those of NSCLCs with activating mutations in KRAS and EGFR. Co-occurring genomic mutations and copy number alterations were identified. c-Met immunohistochemistry and real-time polymerase chain reaction to detect exon 14 skipping were performed where sufficient tissue was available. RESULTS MET exon 14 mutations were identified in 28 of 933 nonsquamous NSCLCs (3.0%) and were not seen in other cancer types in this study. Patients with MET exon 14-mutated NSCLC were significantly older (median age, 72.5 years) than patients with EGFR-mutant (median age, 61 years; P < .001) or KRAS-mutant NSCLC (median age, 65 years; P < .001). Among patients with MET exon 14 mutations, 68% were women, and 36% were never-smokers. Stage IV MET exon 14-mutated NSCLCs were significantly more likely to have concurrent MET genomic amplification (mean ratio of MET to chromosome 7, 4.3) and strong c-Met immunohistochemical expression (mean H score, 253) than stage IA to IIIB MET exon 14-mutated NSCLCs (mean ratio of MET to chromosome 7, 1.4; P = .007; mean H score, 155; P = .002) and stage IV MET exon 14-wild-type NSCLCs (mean ratio of MET to chromosome 7, 1.2; P < .001; mean H score, 142; P < .001). A patient whose lung cancer harbored a MET exon 14 mutation with concurrent genomic amplification of the mutated MET allele experienced a major partial response to the c-Met inhibitor crizotinib. CONCLUSION MET exon 14 mutations represent a clinically unique molecular subtype of NSCLC. Prospective clinical trials with c-Met inhibitors will be necessary to validate MET exon 14 mutations as an important therapeutic target in NSCLC.

Association Between Younger Age and Targetable Genomic Alterations and Prognosis in Non–Small-Cell Lung Cancer

IMPORTANCE Non-small-cell lung cancer (NSCLC) diagnosed in young patients is rare, and the genomics and clinical characteristics of this disease are poorly understood. In contrast, the diagnosis of other cancers at a young age has been demonstrated to define unique disease biology. Herein, we report on the association of young age with targetable genomic alterations and prognosis in a cohort of 2237 patients with NSCLC. OBJECTIVE To determine the relationship between young age at diagnosis and the presence of a potentially targetable genomic alteration, disease prognosis, and natural history. DESIGN, SETTING, AND PARTICIPANTS A cohort of all 2237 patients with NSCLC who were genotyped at the Dana-Farber Cancer Institute between January 2002 and December 2014 were identified. Tumor genotype, patient characteristics, and clinical outcomes were collected and studied at a National Cancer Institute-designated comprehensive cancer center. Multivariate logistic regression was used to analyze the relationship between age and mutation status, and multivariate Cox proportional hazard models were fitted for survival analysis. MAIN OUTCOMES AND MEASURES The frequency of targetable genomic alterations by defined age categories as well as the association of these age groups with survival. Age categories used in this analysis were younger than 40, 40 to 49, 50 to 59, 60 to 69, and 70 years or older. RESULTS A cohort of 2237 patients with NSCLC was studied. Of the 2237 participants, 1939 (87%) had histologically confirmed adenocarcinoma, 269 (12%) had NSCLC not otherwise specified, and 29 (1%) had squamous histologic findings; 1396 (63%) had either stage IIIB or IV cancers; and median (range) age was 62 (20-95) years. We found that gene mutations for EGFR (P = .02) and ALK (P < .001) were associated with cancer diagnosis at a younger age, and a similar trend existed for ERBB2 (P = .15) and ROS1 (P = .10) but not BRAF V600E (P = .43). Among patients tested for all 5 targetable genomic alterations (n = 1325), younger age was associated with an increased frequency of a targetable genotype (P < .001). Those diagnosed at 50 years or younger have a 59% increased likelihood of harboring a targetable genotype. While presence of a potentially targetable genomic alteration treated with a targeted agent was associated with improved survival, the youngest and oldest age groups had similarly poor outcomes even when a targetable genotype was present. CONCLUSIONS AND RELEVANCE Younger age is associated with an increased likelihood of harboring a targetable genotype and is an underappreciated clinical biomarker in NSCLC. The survival of young patients with NCSLC is unexpectedly poor compared with other age groups, suggesting more aggressive disease biology. These findings underscore the importance of comprehensive genotyping, including next-generation sequencing, in younger patients with lung cancer.

Clinical Implications of Variant ALK FISH Rearrangement Patterns

Introduction: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non–small-cell lung cancer (NSCLC), identifying patients who can gain dramatic benefit from ALK kinase inhibitors. Assay interpretation can be technically challenging, and either splitting of the 5′ and 3′ probes or loss of the 5′ probe constitute rearrangement. We hypothesized that there may be clinical differences depending on rearrangement pattern on FISH. Methods: An IRB-approved database of NSCLC patients at Dana-Farber Cancer Institute was queried for ALK rearrangement. Clinical characteristics and response to crizotinib were reviewed. Immunohistochemistry (IHC) and targeted next-generation sequencing (NGS) were obtained when available. Results: Of 1614 NSCLC patients with ALK testing, 82 patients (5.1%) had ALK rearrangement by FISH: 30 patients with split signals, 25 patients with 5′ deletion, and 27 patients with details unavailable. Patients with 5′ deletion were older (p = 0.01) and tended to have more extensive smoking histories (p = 0.08). IHC was positive for ALK rearrangement in all 27 patients with FISH split signals, whereas three of 21 patients with FISH 5′ deletion had negative IHC (p = 0.05). Targeted NGS on two of three cases with discordant FISH and IHC results did not identify ALK rearrangement, instead finding driver mutations in EGFR and KRAS. Patients with 5′ deletion treated with crizotinib had a smaller magnitude of tumor response (p = 0.03). Conclusions: Patients with 5′ deletion on ALK FISH harbor features less typical of ALK-rearranged tumors, potentially indicating that some cases with this variant are false positives. Corroborative testing with IHC or NGS may be beneficial.

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study

Purpose:Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care.Methods:One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences.Results:At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach.Conclusion:The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017

Discrimination of germline EGFR T790M mutations in plasma cell-free DNA allows study of prevalence across 31,414 cancer patients

Purpose: Plasma cell-free DNA (cfDNA) analysis is increasingly used clinically for cancer genotyping, but may lead to incidental identification of germline-risk alleles. We studied EGFR T790M mutations in non–small cell lung cancer (NSCLC) toward the aim of discriminating germline and cancer-derived variants within cfDNA. Experimental Design: Patients with EGFR-mutant NSCLC, some with known germline EGFR T790M, underwent plasma genotyping. Separately, deidentified genomic data and buffy coat specimens from a clinical plasma next-generation sequencing (NGS) laboratory were reviewed and tested. Results: In patients with germline T790M mutations, the T790M allelic fraction (AF) in cfDNA approximates 50%, higher than that of EGFR driver mutations. Review of plasma NGS results reveals three groups of variants: a low-AF tumor group, a heterozygous group (∼50% AF), and a homozygous group (∼100% AF). As the EGFR driver mutation AF increases, the distribution of the heterozygous group changes, suggesting increased copy number variation from increased tumor content. Excluding cases with high copy number variation, mutations can be differentiated into somatic variants and incidentally identified germline variants. We then developed a bioinformatic algorithm to distinguish germline and somatic mutations; blinded validation in 21 cases confirmed a 100% positive predictive value for predicting germline T790M. Querying a database of 31,414 patients with plasma NGS, we identified 48 with germline T790M, 43 with nonsquamous NSCLC (P < 0.0001). Conclusions: With appropriate bioinformatics, plasma genotyping can accurately predict the presence of incidentally detected germline risk alleles. This finding in patients indicates a need for genetic counseling and confirmatory germline testing. Clin Cancer Res; 23(23); 7351–9. ©2017 AACR.

Abstract 2417: The INHERIT EGFR study: Investigating hereditary risk from T790M.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: The EGFR T790M mutation, commonly associated with acquired resistance to EGFR kinase inhibitors, has also been described rarely as a germline mutation in association with familial lung cancer. In collaboration with the Addario Lung Cancer Medical Institute (www.ALCMI.net), we initiated a prospective trial to identify patients and families carrying germline EGFR mutations in order to characterize their lung cancer risk. Preliminary data: In a prior study (GR Oxnard et al, JTO, 2012), it was identified that patients with lung cancers found to harbor EGFR T790M at diagnosis have a 50% chance of carrying an underlying germline T790M mutation. This suggests that by focusing on patients already identified to carry T790M in their cancer, it is possible to enrich for a germline mutation that may be otherwise too rare to study prospectively. Subject eligibility: The following groups are eligible: (1) Patients with a cancer (lung or other) harboring an EGFR T790M mutation on tumor genotyping; lung cancers that acquired EGFR T790M only following treatment with an EGFR kinase inhibitor are ineligible. (2) First-degree relatives of patients found to carry a germline EGFR mutation. (3) Patients who already are known to carry a germline EGFR mutation on prior testing. Subjects are referred to cohort 1 based upon genotyping results from academic centers, oncology practices, or partnering commercial laboratories (e.g. Response Genetics, Los Angeles, CA). Trial design: Subjects may present at a participating cancer center or may participate remotely through a study website (www.Dana-Farber.org/T790Mstudy). Eligible patients submit a saliva and/or blood specimen for central testing in a CLIA lab. After counseling, patients carrying germline EGFR mutations have the option of inviting first-degree relatives to participate. Genetic counseling is coordinated at the participating center or offered over the phone. Chest CT scans are collected from germline carriers and analyzed centrally to study nodule prevalence and characteristics. Patients are then clinically followed for 2 years. Funding: Supported by grants from the Conquer Cancer Foundation of ASCO and the Bonnie J. Addario Lung Cancer Foundation. Citation Format: Geoffrey R. Oxnard, Elizabeth J. Root, Alicia Sable-Hunt, Irene Rainville, Suzanne E. Dahlberg, Jennifer C. Heng, David M. Jackman, Pasi A. Janne, Judy E. Garber. The INHERIT EGFR study: Investigating hereditary risk from T790M. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2013-2417

Abstract B104: Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA)

Introduction: Genomic analysis of cfDNA is emerging as a powerful tool for noninvasive characterization of advanced cancer. Some cfDNA assays are quantitative, allowing analysis of genomic subpopulations. Here, we sought to use quantitative cfDNA analysis to distinguish somatic and germline variants, gaining insight into cancer biology as well as inherited risk without needing paired germline DNA. To do this, we studied EGFR mutations in cfDNA aiming to distinguish those with somatic T790M, generally acquired after resistance to targeted therapy, from those with germline T790M, a rare allele associated with inherited lung cancer risk. Methods & Results: We first explored an institutional cohort of lung cancer patients undergoing cfDNA genotyping using droplet digital PCR (ddPCR). We identified 64 patients positive for T790M and a concurrent EGFR driver mutation by ddPCR. For the vast majority of cases, the mutant allelic fraction (MAF) of T790M was lower than the MAF of the driver mutation, resulting in a T790M/driver ratio of 0.001-1. For two cases, T790M was the predominant mutation, with T790M/driver ratios of 4.2 and 54.6; MAF of T790M was 49% and 53% for these cases. Under an IRB-approved protocol (NCT01754025), we performed sequencing of DNA extracted from PBMCs which confirmed germline T790M in both. We then submitted cfDNA from 3 patients with lung cancer and germline T790M for blinded NGS using the 68-gene Guardant360 assay. In each, NGS identified over 90 coding and non-coding variants. T790M was identified at a high MAF (56%, 50%, 49%) in the range of other recognized SNPs, while an EGFR driver mutation (L858R or L861Q) was identified at a lower level (23%, 4%, 1%) in the range of coding TP53 mutations. Each case was treated with a third-generation EGFR kinase inhibitor targeting T790M. NGS of post-treatment cfDNA demonstrated the expected response in the EGFR driver mutation (0.2%, 0%, 0%) but minimal change in the high MAF T790M (50%, 49%, 49%), consistent with persistent shed of germline DNA on therapy. To broadly screen for germline T790M carriers, we queried a cohort of 1082 lung cancer patients who had undergone NGS of cfDNA using Guardant360. 74 were positive for EGFR T790M (median MAF 3%, range 0.2%-51%). 58 also harbored a second EGFR driver mutation (median MAF 5%), with 23 also having EGFR amplification (median MAF 16%). Four cases were identified with high MAF T790M in the expected range of SNPs (51%, 49%, 49%, 48%), but no driver EGFR mutations were identified at this high MAF. These 4 cases have been referred for germline testing on a prospective study of germline T790M (NCT01754025). Conclusions: We have identified that quantitative cfDNA analysis with NGS can identify germline mutations through differentiation of high MAF germline variants from lower MAF somatic variants. In this cohort, 4 of 74 cases (5%) positive for T790M using NGS of cfDNA were consistent with a germline mutation. This ability to differentiate germline from somatic variants differentiates NGS of cfDNA from NGS of tumor specimens, where these distinctions are challenging without paired germline DNA. Diagnostic labs performing plasma NGS will need to be vigilant to identify these potential germline alterations, and will need strategies for reporting these to providers and patients as appropriate. Citation Format: Geoffrey R. Oxnard, Adrian G. Sacher, Ryan S. Alden, Nora B. Feeney, Jennifer C. Heng, Rebecca J. Nagy, Richard B. Lanman, Cloud P. Paweletz, Pasi A. Janne. Differentiating somatic and germline variants using targeted next-generation sequencing (NGS) of cell-free plasma DNA (cfDNA). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B104.

Abstract 4250: 5′ deletion on ALK break-apart FISH and risk of false positive results: Table 1.

Purpose: Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), identifying patients (pts) who may benefit from ALK tyrosine kinase inhibitors (TKIs). A case is considered positive for rearrangement either based on split 3′ and 5′ signals or loss of the 5′ signal (5′ deletion). We hypothesized that 5′ deletion on FISH may not be completely specific for presence of rearrangement, making the assay vulnerable to false positive results. Methods: An institutional IRB-approved database of NSCLC pts was queried for those with ALK-rearrangements. Clinical characteristics and response to ALK TKIs were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were obtained to further characterize ALK status. Results: Of 1614 NSCLC pts with ALK testing, 97 (6.0%) patients had abnormal ALK FISH: 30 with split signals, 24 with 5′ deletion, 3 with 3′ deletion, 1 with atypical rearrangement, and 39 with details unavailable. Pts with 5′ deletion were older (p = 0.01), had a more extensive smoking history (p = 0.03), and were more likely to harbor mutations in EGFR or KRAS (2 cases vs 0). Pts with 5′ deletion treated with ALK TKIs had a trend toward less tumor shrinkage (p = 0.07). ALK IHC and FISH were discordant in 3 of 21 pts with 5′ deletion and 0 of 26 pts with split signals (p = 0.03). NGS was concordant with ALK IHC, but not FISH, in 2 of 7 samples with 5′ deletions. All three testing modalities were concordant in 3 samples with split signals. Conclusions: NSCLC pts with 5′ deletion on ALK FISH have clinical and molecular features less typical of ALK-rearranged tumors, suggesting an unappreciated risk of false positives with this assay. While the majority of patients with 5′ deletion on ALK FISH harbor a true rearrangement and respond to ALK TKI, confirming these cases with IHC or NGS could reduce the false positive rate. Citation Format: Xin Gao, Lynette M. Sholl, Mizuki Nishino, Jennifer Heng, Pasi A. Janne, Geoffrey R. Oxnard. 5′ deletion on ALK break-apart FISH and risk of false positive results. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4250. doi:10.1158/1538-7445.AM2015-4250