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J. Jen



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    MO05 - Prognostic and Predictive Biomarkers II (ID 95)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO05.01 - Validation of gene expression biomarker panels in non-small cell lung cancer (ID 2928)

      16:15 - 16:20  |  Author(s): J. Jen

      • Abstract
      • Presentation
      • Slides

      Background
      Many studies in the literature have suggested that gene expression biomarkers may guide patient classification and clinical management in NSCLC. Despite minimal external validation and no clinical trial evidence, gene expression biomarker panels have been proposed as tools for making treatment decisions. Recent controversy surrounding the validity of such data and its potential applicability to clinical practice led us to perform an external validation study of published gene expression biomarker panels.

      Methods
      We performed gene expression profiling for a total of 209 patients with both Affymetrix whole transcriptome U133Plus2 arrays in addition to a NSCLC-specific array constructed by our group for assessment of mRNA expression in frozen tumor specimens of NSCLC. Clinical outcome data were collected and analyzed for correlations of gene expression with disease-free and overall survival. Cox proportional hazard models were used to test significance of individual genes and for gene sets defined by each panel. Panels tested included those previously published from Michigan, Mayo Clinic, Taiwan, Toronto, and UCSF.

      Results
      Expression profiling data were generated for a total of 209 patients with NSCLC. This included U133Plus2 arrays of 242 tumor samples and 105 matched surrounding normal lung tissue, as well as 111 tumor profiles using the NSCLC-specific array. There were 98 women and 111 men in the study cohort, with 120 patients having Stage I NSCLC (57.4%), 38 with Stage II (18.2%), 50 with Stage III (23.9%), and one patient with Stage IV disease (0.5%). Mean follow-up time after surgical resection was 62.4 ± 48 months. Seventy-four patients (35.1%) developed post-resection recurrence after a mean of 53.3 ± 49.3 months, of which 62 patients died (83.8%). Known clinical predictors such as TNM stage, histology, and tumor grade were predictive of survival. Although many genes within the published biomarker panels were significantly correlated with disease-free and overall survival, none provided additive prognostic value beyond standard clinical predictors.

      Conclusion
      Although a number of individual gene expression biomarkers have prognostic significance in univariate models, published biomarker panels perform poorly in external validation studies such as this. The additive prognostic value beyond standard, known clinical predictors in the TNM staging system casts doubt as to whether such information will be useful in clinical practice. Despite the success of gene expression biomarkers for molecular subtyping in other cancers, our data suggests that this information has a low likelihood of clinical translation in NSCLC for unselected patients.

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    MO10 - Molecular Pathology II (ID 127)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO10.08 - Genomic alterations in pulmonary carcinoid tumors (ID 3162)

      16:55 - 17:00  |  Author(s): J. Jen

      • Abstract
      • Presentation
      • Slides

      Background
      Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults and comprise 30% of all carcinoid malignancies. They are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors, and are characterized by neuroendocrine differentiation and the potential to metastasize. Relatively little is known about bronchopulmonary carcinoid tumorigenesis, and understanding of these tumors has yet to benefit from the insight of genomic studies. This unfortunately has translated into relatively limited treatment options for these patients and no recent advances in therapy. We aimed to characterize genomic alterations in pulmonary carcinoid tumors under the hypothesis that a better molecular understanding may lead to improved therapeutic approaches and patient outcomes.

      Methods
      We characterized genomic alterations in pulmonary carcinoid tumors using whole genome, exome, and RNA sequencing, in addition to mRNA expression and SNP genotyping from specimens of normal lung, typical and atypical carcinoid, and SCLC. Fresh-frozen specimens from 54 patients with primary lung neuroendocrine tumors were obtained from our lung specimen registry and clinical data collected. This included a total of 31 typical and 11 atypical carcinoid tumors with associated normal tissue, and 12 SCLC. Whole transcriptome mRNA expression profiling and SNP genotyping for evaluating copy number variation was performed using Illumina array platforms. For a subset of tumors, whole genome sequencing was performed through Complete Genomics, and exome and RNA sequencing performed through BGI and the Mayo Clinic Genomics Facility. These data were correlated with the histologic subtype, stage and survival data available from this cohort of patients.

      Results
      Gene expression clearly identified distinct profiles differentiating carcinoid tumors from SCLC, though not between typical and atypical carcinoids. Copy number variations (CNV) were widely prevalent in SCLC, less frequent in AC, while TC had the lowest frequency of CNV. Validated sequencing data from exome and WGS platforms revealed a number of novel mutations for pulmonary carcinoid tumors, including ADNP, BRIP1, cyclin B3, CREBL2, GLI3, HERC1, IRAK3, NEDD4L, PRRX2, and ZDBF2, among others. RNA sequencing data did not reveal any novel fusions from analysis to date. Despite a low overall mutation frequency versus other forms of lung cancer, each carcinoid tumor had at least one potential driver mutation, suggesting possible targeted therapy opportunities for a disease where currently none exist.

      Conclusion
      Despite a low overall mutation frequency and an absence of frequently recurring mutations from the tumors sequenced to date, targeted therapy opportunities may exist through mutation profiling in broncopulmonary carcinoid tumors.

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    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O04.03 - Oncogene Mutations and Novel Transcript Fusions in Lung Adenocarcinoma from Never Smokers (ID 2939)

      10:50 - 11:00  |  Author(s): J. Jen

      • Abstract
      • Presentation
      • Slides

      Background
      Lung adenocarcinoma from never smoker represents a unique disease entity in that they often involve females of younger age and have a distinct mutation spectrum compared to those of smoker population. Mutations from the tumors of these patients often involve oncogenes that can be targeted for therapy by small molecule kinase inhibitors. We surveyed for tumor specific genetic changes in lung adenocarcinomas from never smokers for common oncogene mutations and transcript fusions.

      Methods
      We first developed a multiplex assay detecting187 mutations in 10 actionable oncogenes frequently affected in lung cancer. We used this assay to examine 89 lung adenocarcinomas from never smokers identified through the Mayo Clinic Epidemiology and Genetics of Lung Cancer Program. NextGen sequencing (RNASeq) was used to identify transcript fusions affecting either a known kinase or an oncogene in 20 of 89 tumors. RT-PCR, FISH and IHC were used to verify the novel fusion identified in this study.

      Results
      Sixty-four tumors had mutation in at least one of the tested oncogenes involving EGFR (49 cases, 55%), k-RAS (5 cases, 6%), MET (9 cases, 10%), BRAF (4 cases, 5%), PIK3CA (2 cases, 2%), and ERBB2 (4 cases, 5%). RNAseq identified five transcript fusions among the 20 tested tumors, involving known fusions of EZR- ROS1 or KIF5B-RET and three novel fusions involving SND1-BRAF, EML4-BIRC6, and GMEB2-TERT genes. We used RT-PCR to confirm the presence of the SND1-BRAF fusion transcript that involved exons 1-9 of SND1 with exon 2 to 3’ end of the BRAF on chromosome 7. Screening all 89 tumors by RT-PCR identified a total of three tumors with the identical fusion. Interestingly, two of these three tumors with a BRAF fusion also had a concurrent mutation in EGFR gene (S768I) and a third tumor had an additional mutation in the ERBB2 gene (M774_A775ins). Four additional samples were positive for EML4-ALK fusion by IHC and FISH.

      Conclusion
      In our study of a primarily Caucasian population, a majority of lung adenocarcinomas from never smokers (70/89, or 78.6%) carry at least one genetic mutation in a targetable gene. For the first time, we report the presence of a transcript fusion involving SND1-BRAF in lung adenocarcinoma and that these fusions are present in tumors also having EGFR or ERBB2 mutations. Combined together, activation of BRAF by either point mutation or transcript fusion is one of the most frequent events in our study accounting for 7/89 (8%) cases. These findings support a rapid and targeted gene mutation testing strategy for lung adenocarcinoma from never smokers, as the knowledge of these mutations can be readily used to augment therapeutic management.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-003 - DNA methylation and expression patterns in adenocarcinomas of the lung of never-smokers with discordant methylation and expression patterns of homeobox related genes (ID 856)

      10:04 - 10:21  |  Author(s): J. Jen

      • Abstract

      Background
      Lung cancer occurs in a significant number of never-smokers. Epigenetic changes in lung cancer potentially represent important diagnostic, prognostic and therapeutic targets. Accordingly, we sought to determine if there are differences in DNA methylation between lung adenocarcinomas and adjacent non-malignant lung tissue in never-smokers.

      Methods
      Using the Illumina Infinium HumanMethylation27 BeadChip we compared the DNA methylation profiles of 28 adenocarcinomas of the lung of never-smokers with their paired adjacent non-malignant lung tissue. The β value represents each methylation data point as the ratio of fluorescent signals between the methylated sites and the sum of methylated and unmethylated sites, which ranges continuously from 0 (unmethylated) to 1 (fully methylated). We used the Mann Whitney U test to compare β values between groups using JMP (SAS Institute Inc. Cary, NC USA). Then, using the Illumina Human WG DASL beadchip, we correlated differential methylation changes with gene expression changes from the same 28 samples. We validated our findings with 24 samples of lung adenocarcinomas and paired non-malignant tissue from The Cancer Genome Atlas (TCGA). Database for Annotation, Visualization, and Integrated Discovery was used to determine gene enrichment.

      Results
      We observed a distinct separation in methylation profiles between tumor and adjacent nonmalignant lung tissue using principal component analysis. Tumors were generally hypomethylated (β=0.15) when compared to adjacent non-malignant tissue (β=0.17; p=0.02). There were 1906 differentially methylated (Bonferroni corrected p value <0.05) CpG sites between tumor and adjacent non-malignant lung tissue. Of these sites, 1198 were within classically defined CpG islands where tumors (median β=0.38) were hypermethylated compared to adjacent non-malignant tissue (median β=0.22; p<0.0001), and 708 sites were outside of CpG islands where tumors (β=0.49) were hypomethylated compared to adjacent non-malignant tissue (β=0.56; p<0.0001). When compared to a dataset of 24 lung adenocarcinomas and paired non-malignant tissue from TCGA, 1841 of these 1906 (96.6%) sites were also differentially methylated with the same direction of change between tumor and non-tumor lung tissue. We matched 1483 genes with the differentially methylated CpG sites and found that these genes were enriched with the terms glycoprotein, signal, plasma membrane part, homeobox in addition to a few other terms. There were significant differences in expression of 376 genes (Bonferroni corrected p<0.05) of the 1483 (25.4%) differentially methylated CpG sites. There was an inverse correlation between methylation and gene expression in 80% of these genes. Genes that were not significantly differentially expressed and were hypermethylated within CpG sites were enriched for homeobox genes (false discovery rate = 1.2E-27).

      Conclusion
      The methylation profiles of lung adenocarcinomas of never-smokers and their adjacent non-malignant lung tissue are significantly different. Differential methylation of a CpG site was associated with altered gene expression in approximately 25% of cases. Despite the differential methylation of homeobox genes, no significant changes in expression of these genes were detected.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-018 - Outcomes of lung adenocarcinoma patients with signet ring cell tumors: a three-way evaluation (ID 2884)

      09:30 - 09:30  |  Author(s): J. Jen

      • Abstract

      Background
      Pathologically, signet ring cells (SRC) describe singly dispersed tumor cells with intracytoplasmic mucin vacuoles, which eccentrically displace and compress the nucleus. SRCs are traditionally associated with adenocarcinoma of the gastrointestinal tract and are rare in lung adenocarcinoma (LACA). Patients with primary LACA with SRC features (SRC+) have been associated with poor clinical outcome and ALK gene rearrangement (ALK+). However, the impact of SRC+ on clinical outcome is not well delineated. We systematically studied LACA survival outcomes for the impact of SRC status.

      Methods
      Three distinct groups of surgically treated patients with LACA (n=763) that were followed for ≥5 years were reviewed: never smokers (n=266), 2006-2007 cohort (n=222), and smokers enriched for various degrees of lepidic growth pattern (LGP, n=275). Two pulmonary pathologists reviewed all cases; SRC+ tumors were defined as having >10% SRCs, agreed by both pathologists. SRC+ tumors were TTF1+, and generally cytoplasmic mucin+ and CDX2-. ALK immunostain was performed on all SRC+ cases, and ALK status was confirmed by FISH for cases with any degree of immunoreactivity. Impact of SRC+ on patients’ survival outcomes (overall and disease-free, OS and DFS) were analyzed using Cox models (by hazard ratio, HR) separately for the three groups, with careful evaluation of known prognostic factors: age at diagnosis; gender; smoking status; lung cancer history; tumor subtype; grade and stage; and treatment (surgery, chemotherapy and/or radiation).

      Results
      In the total of 763 patients (61% women, mean age at diagnosis 68 years), 53 (7%) were SRC+. In never smokers (73% women), 9% were SRC+; 33% of the SRC+ were ALK+ vs. 5% among the SRC- cases (p<0.0001). In the 2006-2007 cohort (55% women), 9% were SRC+; in LGP-smokers (54% women), 3% were SRC+. Across all three groups, SRC+ tumors were more likely to occur in men and have higher stage. Univariate analysis showed SRC+ never smokers had shorter survival: median DFS was 2.4 years (vs. 5.2 in SRC- never smokers, p=0.0004), and median OS was 3.7 years (vs. 7.6, p=0.0064). However, multivariate analysis did not confirm a significant impact of SRC+ on survival. In contrast, for the other two groups, crude 5-year survival was 6%-27% decreased in SRC+ cases compared to SRC- cases (none reached statistical significance); however, multivariate analysis revealed a 2-fold higher mortality (HR=2.30, 95% CI=1.01-5.27, p=0.048) for smokers with SRC+ tumors.

      Conclusion
      Based on results from three patient groups, we confirmed that SRC+ is significantly associated with ALK+. Worse survival in patients with SRC+ tumors was observed in never smokers by univariate analysis. A potential negative impact of SRC+ tumors on OS in LGP-smokers was only uncovered after adjusting for known prognostic factors. These results need to be furthered confirmed.