Epitope mapping of spontaneous autoantibodies to anaplastic lymphoma kinase (ALK) in non-small cell lung cancer.

The anaplastic lymphoma kinase (ALK) is recognized by the immune system as a tumor antigen, and preclinical evidence suggests that ALK-rearranged NSCLCs can also be successfully targeted immunologically using vaccine-based approaches. In contrast to ALK-rearranged lymphomas, the frequency and clinical significance of spontaneous ALK immune responses in patients with ALK-rearranged NSCLCs are largely unknown. We developed an enzyme-linked immunosorbent assay (ELISA) to measure anti-ALK antibody levels and mapped specific peptide epitope sequences within the ALK cytoplasmic domain in patients with non-small cell lung cancer. The ELISA method showed good correlation with ALK antibody titers measured with a standard immunocytochemical approach. Strong anti-ALK antibody responses were detected in 9 of 53 (17.0%) ALK-positive NSCLC patients and in 0 of 38 (0%) ALK-negative NSCLC patients (P<0.01), and the mean antibody levels were significantly higher in ALK-positive than in ALK-negative NSCLC patients (P=0.02). Across individual patients, autoantibodies recognized different epitopes in the ALK cytoplasmic domain, most of which clustered outside the tyrosine kinase domain. Whether the presence of high ALK autoantibody levels confers a more favorable prognosis in this patient population warrants further investigation.

Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer.

IMPORTANCE: Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. OBJECTIVE: To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation. DESIGN, SETTING, AND PARTICIPANTS: Patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute-designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting. MAIN OUTCOMES AND MEASURES: Plasma ddPCR assay sensitivity, specificity, and TAT. RESULTS: Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRAS G12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically. CONCLUSIONS AND RELEVANCE: Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.

Clinical and Molecular Characteristics of NF1-Mutant Lung Cancer.

PURPOSE: NF1 is a tumor suppressor that negatively regulates Ras signaling. NF1 mutations occur in lung cancer, but their clinical significance is unknown. We evaluated clinical and molecular characteristics of NF1 mutant lung cancers with comparison to tumors with KRAS mutations. EXPERIMENTAL DESIGN: Between July 2013 and October 2014, 591 non-small cell lung cancer (NSCLC) tumors underwent targeted next-generation sequencing in a 275 gene panel that evaluates gene mutations and genomic rearrangements. NF1 and KRAS cohorts were identified, with subsequent clinical and genomic analysis. RESULTS: Among 591 patients, 60 had NF1 mutations (10%) and 141 (24%) had KRAS mutations. 15 NF1 mutations (25%) occurred with other oncogenic mutations [BRAF (2); ERBB2 (2); KRAS (9); HRAS (1); NRAS (1)]. There were 72 unique NF1 variants. NF1 tumor pathology was diverse, including both adenocarcinoma (36, 60%) and squamous cell carcinoma (10, 17%). In contrast, KRAS mutations occurred predominantly in adenocarcinoma (136, 96%). Both mutations were common in former/current smokers. Among NF1 tumors without concurrent oncogenic alterations, TP53 mutations/2-copy deletions occurred more often (33, 65%) than with KRAS mutation (46, 35%; P < 0.001). No difference between cohorts was seen with other tumor suppressors. CONCLUSIONS: NF1 mutations define a unique population of NSCLC. NF1 and KRAS mutations present in similar patient populations, but NF1 mutations occur more often with other oncogenic alterations and TP53 mutations. Therapeutic strategies targeting KRAS activation, including inhibitors of MAP kinase signaling, may warrant investigation in NF1 mutant tumors. Tumor-suppressor inactivation patterns may help further define novel treatment strategies. Clin Cancer Res; 22(13); 3148-56. ©2016 AACR.

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.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.

PURPOSE: Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). EXPERIMENTAL DESIGN: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. RESULTS: NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. CONCLUSIONS: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.

Long-term Benefit of PD-L1 Blockade in Lung Cancer Associated with JAK3 Activation.

PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.

Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.

PURPOSE: Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype. EXPERIMENTAL DESIGN: ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib. RESULTS: We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression. CONCLUSIONS: Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression.