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W.A. Franklin
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MA01 - Improvement and Implementation of Lung Cancer Screening (ID 368)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Radiology/Staging/Screening
- Presentations: 1
- Moderators:M. Studnicka, C. Berg
- Coordinates: 12/05/2016, 11:00 - 12:30, Lehar 1-2
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MA01.02 - Non-Invasive LuCED® Test for Endobronchial Dysplasia, Enabling Chemoprevention Therapy with Drugs Such as Iloprost (ID 3866)
11:06 - 11:12 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
The LuCED test for early stage lung cancer detects rare abnormal cells that are exfoliated from tumors into sputum and promotes cancer case detection with >92% sensitivity and >95% specificity. Additionally, the LuCED test detects endobronchial lung dysplasia to triage patients with pre-cancer to chemoprevention therapy involving drugs such as Iloprost that show potential for reversing dysplasia. This test is complementary to lung cancer screening methods such as LDCT that do not detect dysplasia. We discuss the performance of the LuCED test for the non-invasive detection of endobronchial dysplasia.
Methods:
We analyzed 23,188 normal, 690 cancer, and 65 moderate/severe dysplasia cells from patient sputum. Each individual cell was imaged in 3D using the Cell-CT. Cells were analyzed to measure 594 3D structural biomarkers. Classifiers were developed with cytopathology as the gold standard to predict stages of carcinogenesis, from normal to dysplasia and cancer. The classifier output was binned into two diagnostic indications: 1) cancer vs. all other cell types; and 2) moderate/severe dysplasia vs. all other cell types.
Results:
Areas under ROC curves for the two diagnostic bins were both = 0.993. Thresholds were chosen to yield case specificity >95%. Using these thresholds, cell classification sensitivity of 75% was measured for cancer and dysplasia detection. Since abnormal sputum typically contains at least three abnormal cells the cancer case detection sensitivity is at least {100% x [1 – (1 - 0.75)[3]]} = 98%.Figure 1 Figure 1 shows ROC curves plus examples of cells imaged in 3D by the Cell-CT.
Conclusion:
This study demonstrates the feasibility of using the LuCED test to detect endobronchial dysplasia in the lung, achieving an estimated 98% case sensitivity and 95% case specificity. Future efforts will include testing on independent sets. Importantly, the non-invasive detection of dysplasia by LuCED testing may enable chemoprevention of lung cancer using drugs such as Iloprost.
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OA19 - Translational Research in Early Stage NSCLC (ID 402)
- Event: WCLC 2016
- Type: Oral Session
- Track: Early Stage NSCLC
- Presentations: 1
- Moderators:G. Heller, G. Goss
- Coordinates: 12/07/2016, 11:00 - 12:30, Schubert 3
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OA19.01 - A Standardized and Validation of Prognostic Gene Expression Signatures for Squamous Cell Lung Carcinoma by the SPECS Lung Consortium (ID 4329)
11:00 - 11:10 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
High-throughput gene expression profiling led to proposal of multiple expression-based prognostic signatures for squamous cell lung carcinoma (SCC), but none has been validated. A multi-institutional squamous lung cancer consortium of investigators is developing prognostic signatures through the US NCI Lung SPECS (Strategic Partnership for Evaluation of Cancer Signatures) program. Six institutions contributed tumor specimens and published/unpublished expression-based prognostic signatures for validation using standardized sample cohorts (a primary validation cohort comprising institutional cases, and additional validation cohorts from two prospective cooperative group studies) and quality controlled assessment in independent laboratory and statistical cores. Here, we report the results of the primary validation.
Methods:
Cases of primary SCC (by central pathology review) meeting clinical (Stage I-II; surgical treatment only; 3-year followup) and specimen quality criteria (Tumor cellularity >= 50%; necrosis <= 20%) were submitted. Clinical, pathological and outcome data were uploaded to a central database. Frozen tumor samples underwent centralized mRNA extraction (Qiagen Symphony), quality control (RIN >= 6.0) and microarray profiling (Affymetrix U133) in core labs. An independent statistical core assessed validation of 7 pre-existing mRNA signatures and generated new models using MCP clustering.
Results:
Among 250 cases meeting entry criteria, median age was 70 (43-92), 161 (65%) were male, and most were former (70%) or current (28%) smokers. Surgery was pneumonectomy: 5%; bilobectomy: 2%; lobectomy: 74%; sublobar: 18%. Pathologic staging was T1: 49%; T2: 50%; T3: 1%; N0: 88%; N1: 12%, and grade was G1: 4%; G2: 50%; G3: 44%. At followup, 148 (59%) were deceased. Three mRNA signatures demonstrated significant univariable association with OS and added independent prognostic value (see Figure) to a multivariable model accounting for age, sex and stage (c-index = 0.641).
Conclusion:
The validated signatures, along with two novel signatures generated from the current dataset, are currently undergoing further validation studies using two prospective co-operative group cohorts. Figure 1
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-084 - Polo-Like Kinase 1 (PLK1) Inhibition Decreases Mutational Activity in Bronchial Epithelial Cells Exposed to Tobacco Carcinogens (ID 6206)
14:30 - 14:30 | Author(s): W.A. Franklin
- Abstract
Background:
Persistence of bronchial dysplasia (BD) is associated with increased risk for the development of squamous cell carcinoma (SCC) of the lung. A comparison of persistent and regressive BD demonstrated that alterations in gene expression can distinguish these types of lesions. Using the genes that differentiate persistent and regressive BD we have identified PLK1 as a key mediator of persistence and have hypothesized that the role it plays in abrogating G2-M cell cycle arrest in the presence of mutational alterations may be central to progression of premalignant airway lesions to invasive SCC.
Methods:
Cultures of the bronchial epithelium derived BEAS-2B (B2B) cell line were exposed to airway mutagens (cigarette smoke condensate [CSC, Kentucky Reference Cigarette 3R4F] or Benzo[A]pyrene, [BaP]) alone or in the presence of 100 nM PLK1 inhibitor Volasertib. Following treatment, clones were selected by ring isolation and DNA was collected to assess mutational events as compared to untreated controls using the TruSightOne targeted next generation sequencing panel (12 megabase coverage, >4800 genes, Illumina, San Diego, CA). Modal distribution of mutation frequencies indicated that ring clones were composed of between 1 - 3 distinct clones. Mutation rates were calculated using these adjusted clone counts for standardization.
Results:
Using the untreated B2B cultures as reference sequence, CSC or BaP alone induced mean incidences of mutations of 15.7 and 60.9 per clone, respectively, although the induction of fewer mutations by CSC made accurate estimates of clonal numbers more difficult. B2B cultures treated with the same concentration and duration of mutagen in the presence of Volasertib showed mean mutational incidences of 13 and 17 per clone, respectively. The ratio of mutation rate for the CSC and BaP treated groups with versus without Volasertib were 0.83 and 0.28, respectively. All mutations detected were single nucleotide variants and characteristic patterns of base changes were noted between mutagen groups with C>A and G>T transversions being more prominent in BaP treated cultures. Ongoing analyses using whole genome sequencing will allow for more accurate enumeration of clones represented and a richer assessment of mutation rates including rearrangements and copy number variants in mutagen treated cells in the presence and absence of PLK1 inhibition.
Conclusion:
Preliminary analyses of bronchial epithelial cell cultures treated with mutagens show reduction of BaP induced mutations in the presence of PLK1 inhibition. The results suggest PLK1 overexpression in BD may promote genomic instability.
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P1.05 - Poster Session with Presenters Present (ID 457)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Early Stage NSCLC
- Presentations: 2
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.05-001 - Creation and Early Validation of Prognostic miRNA Signatures for Squamous Cell Lung Carcinoma by the SPECS Lung Consortium (ID 6088)
14:30 - 14:30 | Author(s): W.A. Franklin
- Abstract
Background:
Despite overall favorable prognosis for operable early stage non-small cell lung cancer, predicting outcome for individual patients has remained challenging. Small retrospective studies have reported potential non-coding micro(mi)RNAs that might have prognostic significance; however, these studies lacked statistical power and validation. To refine these initial findings to clinical application, the investigators have undertaken a collaborative, structured evaluation of multiple signatures putatively prognostic for lung squamous cell carcinoma (SCC) under a NCI/SPECS (Strategic Partnerships fo Evaluating Cancer Signatures) award. The study design specifies a primary validation cohort comprising institutional cases, and additional validation cohorts of Cooperative Group cases, all profiled via a common pipeline.
Methods:
Completely resected SCC (confirmed by central pathology review) meeting clinical (Stage I-II; complete 3-year follow-up) and specimen quality criteria (Tumor cellularity ≥ 50%;necrosis ≤ 20%) were submitted by 6 institutions. Clinical, pathological and outcome data were uploaded to a central database. Lysates from 5 um sections of FFPE SSC tumor samples were run on the HTG EdgeSeq Processor (HTG Molecular Diagnostics, Tucson, AZ) using the miRNA whole transcriptome assay in which an excess of nuclease protection probes (NPPs) complimentary to each miRNA hybridize to their target. S1 nuclease then removes un-hybridized probes and RNA leaving behind only NPPs hybridized to their targets in a 1-to-1 ratio. Samples were individually barcoded (using a 16-cycle PCR reaction to add adapters and molecular barcodes), individually purified using AMPure XP beads (Beckman Coulter, Brea, CA) and quantitated using a KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA). Libraries were sequenced on the Illumina HiSeq platform (Illumina, San Diego, CA) for quantification. Standardization and normalization was provided to the project statistical core for validation of two pre-existing signatures and generation of new models (MCP clustering).
Results:
Among 224 cases with miRNA data, median age was 70 (43-92), 143 (64%) male, with 67% former (67%) and current (26%) smokers. All patients were completely resected stage I or II. . At follow-up, 59 (26%) had documented recurrence and 129 (58%) were deceased. To date, we have been unable to validate the previous models, but have created a novel signature of three miRNAs (see Figure) that is being validated in the second phase of the project using an independent, blinded multi-institutional cohort.
Conclusion:
The Squamous Lung Cancer SPECS Consortium has established well-annotated and quality-controlled resources for validation of prognostic miRNA signatures. A new candidate 3-miRNA signature has been identified for further development as a clinically useful biomarker.
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P1.05-027 - Novel Prognostic Gene Expression Signatures for Squamous Cell Lung Carcinoma: A Study by the SPECS Lung Consortium (ID 4490)
14:30 - 14:30 | Author(s): W.A. Franklin
- Abstract
Background:
A multi-institutional squamous lung cancer consortium of investigators is developing prognostic signatures through the US NCI Lung SPECS (Strategic Partnership for Evaluation of Cancer Signatures) program. Six institutions contributed tumor specimens and published/unpublished expression-based prognostic signatures for validation using standardized sample cohorts (a primary validation cohort comprising institutional cases, and additional validation cohorts from two prospective cooperative group studies) and quality controlled assessment in independent laboratory and statistical cores. Here, we report on de novo prognostic signatures derived using the pooled institutional dataset.
Methods:
Highly quality-controlled cases of primary SCC from the pooled cohort (N=249) were analyzed to generate de novo prognostic signatures from among the 147 genes comprising pre-existing signatures, and from among all profiled genes. Minimax Concave Penalty (MCP) selection and Ward’s minimum variance clustering yielded survival analyses with 2 clusters that were evaluated using Cox regression and bootstrap cross validation (bCV; 500 iterations).
Results:
Two significantly prognostic models were generated (see Figure): Pooled Model A (PMA) was the optimal 2-cluster model using probesets representing 6 genes selected from components of pre-existing signatures: CASP8, MDM2, SEL1L3, RILPL1, LRR1, COPZ2. Pooled Model B (PMB) was the optimal 2-cluster model using probesets representing 6 genes selected from among all those profiled: SSX1, DIAPH3, LOC619427, CASP8, EIF2S1, HSPA13. PMA and PMB each remained independently prognostic in multivariable analyses incorporating an a priori baseline model (age, sex, stage; c-index = 0.641).
Conclusion:
Two de novo prognostic signatures were derived using a pooled multi-institutional cohort of SCC assembled for validation of pre-existing signatures. PMA and PMB were each found to be independently prognostic, accounting for established clinical predictors. Both now move forward, along with validated pre-existing signatures, to additional assessment of discrimination, calibration and clinical usefulness using additional independent prospective US co-operative group cohorts of cases. Figure 1
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P2.06 - Poster Session with Presenters Present (ID 467)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Scientific Co-Operation/Research Groups (Clinical Trials in Progress should be submitted in this category)
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.06-032 - Oral Pioglitazone for the Chemoprevention of Lung Cancer in Current and Former Smokers (ID 4395)
14:30 - 14:30 | Author(s): W.A. Franklin
- Abstract
Background:
Clinical Trial with Data Analysis in Progress
Methods:
Subjects (n=90) were selected for the trial if they met one the following criteria: current or former smoker (> 10 pack years); biopsy proven endobronchial dysplasia; airflow obstruction (FEV1/FVC < 0.70); or at least mild sputum cytologic atypia. Fluorescent bronchoscopy was performed at trial entry with biopsy of 6 standard endobronchial sites and all other abnormally appearing areas. Subjects also had pulmonary function testings and quantitative high resolution CT scans at the start and completion of the trial. Subjects were then randomized to oral pioglitazone or placebo for 6 months, followed by a second fluorescent bronchoscopy with repeat biopsy of all the central airway areas sampled on the first bronchoscopy. The endobronchial biopsies were scored on a 1-8 scale based on WHO criteria. The primary endpoint for the study is change in maximum (worst) endobronchial histology.
Results:
Final data analysis is pending
Conclusion:
clinical trial with data analysis in progress
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-021 - Reproducibility of Comprehensive Histologic Assessment and Refining Histologic Criteria in P Staging of Multiple Tumour Nodules (ID 5365)
14:30 - 14:30 | Author(s): W.A. Franklin
- Abstract
Background:
Multiple tumor nodules (MTNs) are being encountered, with increasing frequency with the 8[th] TNM staging system recommending classification as separate primary lung cancers (SPLC) or intrapulmonary metastases (IM). Pathological staging requires assessment of morphological features, with criteria of Martini and Melamed supplanted by comprehensive histologic assessment of tumour type, predominant pattern, other histologic patterns and cytologic features. With publication of the 2015 WHO classification of lung tumours, we assessed the reproducibility of comprehensive histologic assessment and also sought to identify the most useful histological features.
Methods:
We conducted an online survey in which pathologists reviewed a sequential cohort of resected multifocal tumours to determine whether they were SPLC, IM, or a combination. Specific histological features for each nodule were entered into the database by the observing pathologist (tumour type, predominant adenocarcinoma pattern, and histological features including presence of lepidic growth, intra-alveolar cell clusters, cell size, mitotic rate, nuclear pleomorphism, nucleolar size and pleomorphism, nuclear inclusions, necrosis pattern, vascular invasion, mucin content, keratinization, clear cell change, cytoplasmic granules¸ lymphocytosis, macrophage response, acute inflammation and emperipolesis). Results were statistically analyzed for concordance with submitting diagnosis (gold standard) and among pathologists. Consistency of each feature was correlated with final determination of SPLC vs. IM status (p staging) by chi square analysis and Fisher exact test.
Results:
Seventeen pathologists evaluated 126 tumors from 48 patients. Kappa score on overall assessment of primary v. metastatic status was 0.60. There was good agreement as measured by Cohen’s Kappa (0.64, p<0.0001) between WHO histological patterns in individual cases with SPLC or IM status but proportions for histology and SPT or IM status were not identical (McNemar's test, p<0.0001) and additional histological features were assessed. There was marked variation in p values among the specific histological features. The strongest correlations (<0.05) between p staging status and histological features were with nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intra-alveolar clusters and necrosis pattern. Correlation between lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization and emperipolesis did not reach a p value of 0.05.
Conclusion:
Comprehensive histologic assessment shows good reproducibility between practicing lung pathologists. In addition to main tumour type and predominant patterns, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate appear to be useful in distinguishing between SPLC and IM.
