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.
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.
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.
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.
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.
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.
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).
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.
Although much progress has been made in the understanding of the ontogeny and function of dendritic cells (DCs), the transcriptional regulation of the lineage commitment and functional specialization ...of DCs in vivo remains poorly understood. We made a comprehensive comparative analysis of CD8(+), CD103(+), CD11b(+) and plasmacytoid DC subsets, as well as macrophage DC precursors and common DC precursors, across the entire immune system. Here we characterized candidate transcriptional activators involved in the commitment of myeloid progenitor cells to the DC lineage and predicted regulators of DC functional diversity in tissues. We identified a molecular signature that distinguished tissue DCs from macrophages. We also identified a transcriptional program expressed specifically during the steady-state migration of tissue DCs to the draining lymph nodes that may control tolerance to self tissue antigens.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions, and for ...drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media, are now available, making comparison of data between studies difficult. Here, we investigated the impact of two common differentiation media on phenotypic, transcriptomic, and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH, Ussing chamber) specifically showed that ATP12A and CFTR function were altered, impacting pH and transepithelial ion transport in CF hAECs. Importantly, the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications.
Reirradiation of thoracic malignancies is a treatment challenge, with concerns for toxicity and the inability to deliver definitive doses. Intensity modulated proton therapy (IMPT) may allow safe ...delivery of a higher dose of radiation to the tumor while minimizing toxicities.
Between 2011 and 2016, 27 patients who received IMPT for reirradiation of thoracic malignancies with definitive intent were retrospectively analyzed. Patients were included if they received a prior thoracic radiation course. All doses were recalculated to an equivalent dose in 2-Gy fractions (EQD2). Patients received IMPT to a median dose of 66 EQD2 Gy (range, 43.2-84 Gy) for recurrence of thoracic cancer (93%) or sequentially after a course of thoracic stereotactic ablative radiation therapy (7%).
Twenty-two patients (81%) were treated for non-small cell lung cancer. The median time to reirradiation was 29.5 months. At a median follow-up for all patients of 11.2 months (25.9 surviving patients), the median overall survival was 18.0 months, with a 1-year overall survival of 54%. Four patients (15%) experienced an in-field local failure (LF), with a 1-year freedom from LF rate of 78%. The 1-year freedom from locoregional failure and 1-year progression-free survival rates were 61% and 51%, respectively. Patients who received 66 EQD2 Gy or higher had improved 1-year freedom from LF (100% vs 49%; P = .013), 1-year freedom from locoregional failure (84% vs 23%; P = .035), and 1-year progression-free survival (76% vs 14%; P = .050). Reirradiation was well tolerated, with only 2 patients (7%) experiencing late grade 3 pulmonary toxicity, and none with grade 3 or higher esophagitis. There were no grade 4-5 toxicities.
These data represent the largest series of patients treated with IMPT for definitive reirradiation of thoracic cancers. They demonstrate that IMPT provided durable local control with minimal toxicity and suggest that higher doses may improve outcomes.
Statement of problem In badly damaged teeth and teeth with short clinical crown heights, the placement of foundation restorations has been advocated to permit the development of retention and ...resistance form. However, there is little information on the effect of these foundation restorations on the clinical performance of the definitive restoration. Purpose The purpose of this study was to evaluate the load fatigue performance of teeth restored with posts and cores, with varying tooth heights, and to compare them with similar groups having no posts and cores. A secondary purpose was to determine whether a critical tooth height existed at which the placement of a foundation restoration resulted in no significant difference in the load fatigue performance. Material and methods Three test groups (n=10) with prepared tooth heights of 2, 3, and 4 mm were tested. These were compared with another 3 groups with similar tooth heights that were restored with prefabricated titanium posts and core heights of 4, 3, and 2 mm, respectively. Cast complete crowns were then fabricated and cemented with zinc phosphate cement. A fatigue load of 58.8 N was applied at an angle of 135 degrees to the long axis of each crown-tooth specimen. The number of cycles to preliminary failure was determined. Significant differences in cycles to preliminary failure were assessed with 1-way ANOVA, followed by Tukey HSD tests (α=.05). Results The group with the greatest preparation height (4 mm) and a 2-mm post-retained foundation had the highest number of cycles to preliminary failure (437,701), while the group with the shortest preparation height and no foundation had the lowest number of cycles (53,806). The Tukey HSD multiple comparison tests showed that for all 3 tooth heights, groups with foundation restorations had a significantly higher number of cycles to preliminary failure than those without foundation restorations. Conclusions For a given tooth height, teeth restored with foundation restorations had a significantly better load fatigue performance than those with no foundation restoration.
Abstract
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.
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Background: Identification of targetable genomic alterations in patients (pts) with NSCLC can have a profound impact on treatment and clinical outcomes. Given the complexity and ...cost of comprehensive genomic testing, clinical characteristics enriching for targetable genomic alterations are of interest. We hypothesized that young adults with NSCLC would have a higher prevalence of targetable genomics alterations compared to the general NSCLC population. Methods: An institutional database of pts with NSCLC was reviewed in an IRB-approved fashion to identify subjects with age < 40 at diagnosis. Clinical characteristics and risk factors were reviewed. Tumor genotyping for alterations in EGFR, KRAS, ALK, BRAF, HER2, and ROS1was pursued as part of an institution-wide genomics protocol. Targeted next-generation sequencing (NGS) of wild-type cases is underway. Results: From 2032 subjects with NSCLC, we identified 70 diagnosed at an age < 40 (3.4%). Pt characteristics: median age 35 (range 20-39); 63% never-smokers, 33% with ≥10 pack-years; 74% adeno, 9% squam, 14% undifferentiated, 3% neuroendocrine; 19% had a family history of lung cancer; 61% stage IV at diagnosis. Median survival from date of advanced disease was 15.8 months. Genotyping was performed on 51 pts with adeno or undifferentiated histology: 14 with EGFR mutations (27%), 5 with KRAS mutations (10%), 8 with ALK rearrangements (16%), 0 with BRAF mutations, 1 with a HER2 insertion (2%), 1 with a ROS1 rearrangement (2%). Compared to a reference prevalence from the Lung Cancer Mutation Consortium (Kris et al, ASCO, 2011), KRAS mutations were less common (p=0.01) and ALK rearrangements were more common (p<0.01). NGS of 2 cases to date has identified one pt with a novel 21 base-pair insertion mutation in FGFR2, not present in germline tissue. Conclusions: 47% (CI: 33%-60%) of pts diagnosed with NSCLC under age 40 harbor a targetable alteration in EGFR, ALK, HER2, or ROS1. These patients may be enriched for targetable genotypes and deserving of a unique treatment approach, and additionally represent an attractive population for genomic discovery.Supported in part by the Bonnie J. Addario Lung Cancer Foundation and the Conquer Cancer Foundation of ASCO.
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