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M.W. Geraci
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MINI 23 - Lung Cancer Risk: Genetic Susceptibility and Airway Biology (ID 135)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:P.E. Postmus, R. Young
- Coordinates: 9/08/2015, 16:45 - 18:15, 401-404
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MINI23.10 - Subtraction of Allelic Fractions (Delta-θ): A Sensitive Metric to Detect Chromosomal Alterations in Heterogeneous Premalignant Specimens (ID 2434)
17:35 - 17:40 | Author(s): M.W. Geraci
- Abstract
- Presentation
Background:
Lung squamous carcinoma is believed to arise from premalignant bronchial epithelial dysplasia, which demonstrates progressive histologic changes leading up to invasive cancer. However, only a small subset of these lesions progress to carcinoma. Recent studies have shown that somatic chromosomal alterations (SCAs) status is a better biomarker than premalignant histology alone. Single-nucleotide polymorphism microarray (SNP array) has been frequently used to delineate these genomic alterations across the whole genome. However, the cellular heterogeneity, from clinical samples such as endobronchial specimens, is a basic obstacle to perform sensitive and accurate detection of SCAs.
Methods:
We used: 1) a lung cancer cell line (NCI-H1395) and its matched lymphoblastoid (NCI-BL1395) cell line; 2) frozen lung tissues containing different percentage of invasive cancer cells surgically resected from a patient; and 3) biopsies and brushings obtained at the visually concerning areas during bronchoscopy. The histology of the clinical samples were graded by the study pathologist. Genomic DNA was isolated from each sample, quantified, and labeled for Illumina SNP array (HumanOmni 2.5-Quad BeadChip). Data analysis and visualizations were performed using Partek Genomic Suite 6.6 software.
Results:
Our study focused on the detection of SCAs by the comparison of genomic DNAs from cancer/premalignant cells (subject) to blood/normal cells (reference) from the same individual. We tested a B allele frequency metric, the subtraction of allelic fractions (delta-θ), on a standardized mixture of genomic DNAs from a lung cancer cell line and its matched lymphoblastoid cell line. Delta-θ proved to be a sensitive parameter to clearly delineate SCAs present in the tumor cell line even with a large proportion of normal cells (up to 90%). To explore the utility of using delta-θ for heterogeneous samples, we used clinical lung cancer specimens with known cancer cell content. In comparison to the other publicly available analytical metrics/algorithms (conventional Log R Ratio plot, mirrored B Allele Frequency plot, and GAP algorithm), delta-θ performed as well or better (with lower computational power needed), and enabled the detection of SCAs even in highly heterogeneous clinical samples (<30% tumor cell content). In addition, we completed a study using a number of bronchial biopsies and brushings with histologic grade ranging from normal to squamous cell carcinoma. SCAs were rarely detected in those of low to mild dysplasia, while they were detected in approximately 25% of moderate or severe dysplasia, and in all carcinoma in situ (CIS) and squamous cell carcinoma specimens. Longitudinal, repeated samplings from a high risk patient who persistently showed high grade dysplasia across the bronchus, revealed that delta-θ could identify SCAs continuously across the whole genome. The fact this individual had highly overlapping SCAs between different bronchial locations indicates genomic field cancerization may occur, along with the histological field effect in premalignant epithelium.
Conclusion:
In SNP microarray studies, delta-θ is a highly sensitive metric for detecting SCAs even in heterogeneous dysplastic bronchial specimens. SNP array may be a powerful tool to understand premalignant genetic alterations and field cancerization.
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ORAL 23 - Prevention and Cancer Risk (ID 121)
- Event: WCLC 2015
- Type: Oral Session
- Track: Prevention and Tobacco Control
- Presentations: 1
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ORAL23.03 - Role of Inflammatory Infiltrates in Promoting Persistence or Regression of Bronchial Dysplasia (ID 3026)
11:07 - 11:18 | Author(s): M.W. Geraci
- Abstract
- Presentation
Background:
Inflammatory infiltrates show differing capacities to eliminate malignant cells. This capacity is related to the polarization of key inflammatory cells in tumor infiltrates. A pathway analysis of genes that are differentially expressed between persistent and regressive bronchial dysplasia (BD) identified 13 pathways associated with persistence of which 8 were related to inflammation. We have hypothesized that differences in inflammatory infiltrate polarization may contribute to lung carcinogenesis and have employed gene expression and in situ analyses to characterize differences in inflammatory infiltrates related to persistence and regression of pre-malignant BD.
Methods:
Normalized gene expression levels (Affymetrix Hu 1.0) of selected genes related to inflammatory cell polarization features were analyzed to find differences associated with follow-up histology for BD. Validational analyses of these relationships were undertaken in studies of baseline biopsies selected to represent persistent (n=43) and regressive BD (n=39). These biopsies were analyzed by quantitative immunohistochemistry and dual immunofluorescence studies to characterize the overall proportion of subsets of T-lymphocytes and macrophages in each of the groups. Image analysis tools (Aperio) were used to characterize the density of inflammatory cell subsets in the stromal and epithelial compartments of biopsy tissue within defined areas.
Results:
Analysis of expression levels for a subset of inflammatory cell related genes assessed in a global gene expression analysis indicated significantly higher levels of expression of macrophage M1 markers HLA-DRA (p=0.01) and inducible nitric oxide synthetase (iNOS; p=0.02) and T-helper lymphocyte marker CD4 (p=0.04) in regressive BD compared to persistent BD. There was also a trend toward higher expression of cytotoxic T-lymphocyte marker CD8 in regressive BD (p=0.25). Expression of B-lymphocyte and neutrophil markers were not different between regressive and persistent BD. CD68 immunohistochemical stains (IHC) demonstrated a trend toward an increase in macrophages per area of combined dysplastic epithelium and underlying stroma with a mean increase in IHC positivity of 1.75-fold in regressive versus persistent BD (p=0.08). CD4 and CD8 IHC showed 1.36- and 1.19-fold increases, respectively, in regressive BD but these changes were not statistically significant (p=0.36 and p=0.43 respectively). Dual immunofluorescence was undertaken to determine if polarization specific subsets of macrophages correlated with regression or persistence of BD. Analysis of a preliminary subset of regressive (n=3) and persistent (n=3) BD demonstrates a wide range of M1 to M2 ratios (range = 0.84 – 4.82 for ratio of HLA-DRA-CD68 dual positive M1 to CD206-CD68 dual positive M2 macrophages per high power field, 400X). Additional analyses of macrophages are ongoing to determine if the polarization status is related to regression or persistence of BD, and analysis of markers of T-helper lymphocyte subsets are planned.
Conclusion:
Gene expression analyses indicate that increased expression of markers of M1 macrophages and T-helper lymphocytes are associated with regression, and in situ analyses suggest that differences in the amount of inflammatory cell subsets may be related to outcome in BD. These studies could have implications for predicting the behavior of premalignant disease and manipulating inflammatory activity in preventing progression of BD to invasive lung cancer.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-052 - Next Generation Exome Sequencing of Archival Lung Cancer Resection Specimens (ID 2159)
09:30 - 09:30 | Author(s): M.W. Geraci
- Abstract
Background:
Genetic testing of non-small cell lung cancer has grown rapidly in recent years to accommodate expansion of the number of agents with molecular targets. Whole exome sequencing (WES) has been proposed as a method to comprehensively assess tumor mutation status that could replace current piecemeal approaches to predictive testing. The feasibility of WES for formalin fixed paraffin embedded (FFPE) clinical samples has recently been documented. However, several issues remain to be resolved before this platform can be adopted for routine clinical use. The purpose of the present study is to evaluate tissue coring as a method for obtaining DNA from FFPE tumor tissue, to assess the gene coverage of libraries prepared from FFPE, to determine how best to identify specific validated treatment targets, and to determine mutation load in clinical samples.
Methods:
We extracted DNA from 0.6 mm tissue cores selected both from tumor rich regions of paraffin blocks and normal lung tissue. DNA quality was assessed by Bioanalyzer and Qbit testing. A sequencing library was prepared using the Agilent Sure Select XT5 (v5) library kit. DNA was sequenced using an Illumina Hiseq 2500 ultrahigh throughput sequencing system. We used two flow cells for each of 4 samples to obtain a high level of coverage and to determine the effect of reducing coverage on mutation detection by computational methods. We used the DNA from non-tumoral regions to identify genomic polymorphisms and to then compile lists of mutations that were suspected of have a deleterious effect on the host. As a control, we tested DNA from each tumor by a clinically validated multiplexed panel (Illumina True Site panel). We compared our sequencing results with the TCGA database for the respective tumors.
Results:
DNA yield was 13 and 17 micrograms for the SCC and adenocarcinoma respectively. After shearing to 200 base pairs and library preparation, excellent quality DNA was obtained for sequencing. All of the mutations detected by Miseq analysis were detected by WES. Several mutations identified by WES have not been documented in TCGA. The mutations of the two tumors are sumarized below, including mutation load.WES Mutations SCC Adenocarcinoma Nonsynonomous SNV 247 51 Stopgain SNV 16 1 Fs deletion 10 1 Non-fs substitution 9 7 Fs insertion 2 2 Non-fs deletion 1 3 Non-fs insertion 1 0 Stoploss SNV 1 0 Splice region abnormality 9 0 Not present in TCGA 37 7 Present in TCGA 265 59 Mutations detected by Miseq TP53 (p.G245R) EGFR exon19 del CTNNB1 (p.S45C) Total (Mutation Load) 302 66
Conclusion:
This study confirms that WES is feasible on FFPE tissue and that the two tumors sequenced fall into the two categories, high and low mutation loads. The mutations identified include several that have not previously been reported. All mutations identified by high coverage clinical platforms were also detected by WES. WES may be suitbable for clinical application.
