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W.A. Franklin
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MINI 01 - Pathology (ID 93)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 14
- Moderators:W.A. Franklin, A.G. Nicholson
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI01.01 - Immunohistochemical Distinction between Primary Lung Squamous Cell Carcinoma and Pulmonary Metastasis of Head and Neck Squamous Cell Carcinoma (ID 1525)
10:45 - 10:50 | Author(s): J. Ichinose, A. Shinozaki-Ushiku, K. Nagayama, J. Nitadori, M. Anraku, M. Fukayama, J. Nakajima, D. Takai
- Abstract
- Presentation
Background:
It is extremely difficult to distinguish between primary lung squamous cell carcinoma (LUSq) and pulmonary metastasis of head and neck squamous cell carcinoma (HNSq) in patients with a past history of HNSq, even by histological examination of the resected specimen. This study aimed to establish an immunohistochemical scoring system for discrimination between LUSq and pulmonary metastasis of HNSq.
Methods:
We selected genes that were expressed in a markedly different manner in LUSq and HNSq using the results of expression microarrays and chose the antibodies for four proteins (CK19, MMP3, ZNF830, PI3) that had immunohistochemical results shown in the Human Protein Atlas (http://www.proteinatlas.org) that were distinguishable between LUSq and HNSq. We constructed the tissue microarrays using the resected specimens of 39 LUSqs and 48 HNSqs as the training set and evaluated the tendency of HNSq using the 16-grade system according to the positive staining of the four antibodies. Twenty-seven of the patients with pulmonary tumors that were resected and pathologically diagnosed as squamous cell carcinoma between 1999 and 2014 had a past history of HNSq. Their pulmonary tumors and primary HNSqs were analyzed immunohistochemically as the test set. We defined LU-associated recurrence as postoperative recurrence in the thoracic cavity, mediastinum, brain, bone, and liver and defined HN-associated recurrence as recurrence in the other sites. We compared the diagnosis of our immunohistochemical scoring system to the preoperative clinical diagnosis and the pathological diagnosis according to the predictive power of HN-associated recurrence.
Results:
The sensitivity, specificity, and accuracy of our immunohistochemical scoring system were 90%, 67%, and 79%, respectively in the training set, and our system correctly diagnosed 96% of HNSq specimens in the test set. Twenty-three out of 27 pulmonary tumors in the test set were diagnosed as pulmonary metastasis of HNSq, and four were diagnosed as LUSq. Eleven of 23 patients (48%) with pulmonary metastasis of HNSq developed HN-associated recurrence (3-year HN-associated recurrence-free probability was 46%), and 10 died because of HNSq, while none of the four patients with LUSq had HN-associated recurrence. Compared with the clinical diagnosis (five LUSq, 14 pulmonary metastasis of HNSq, eight uncertain) and the pathological diagnosis (two LUSq, 17 pulmonary metastasis of HNSq, eight uncertain), our immunohistochemical scoring system could predict the risk of HN-associated recurrence more accurately. Figure 1
Conclusion:
Immunohistochemical analysis of four proteins (CK19, MMP3, ZNF830, PI3) was clinically useful for discrimination between LUSq and pulmonary metastasis of HNSq.
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MINI01.02 - Is It Possible to Distinguish between Second Primary vs Metastasis in Resectable Synchronous Nodules with the Same Histotype of Lung Cancer? (ID 2122)
10:50 - 10:55 | Author(s): G. Luciano, P. Viola, M. Al Sahaf, Z. Niwaz, H. Raubenheimer, M.E. Cufari, H. Chavan, C. Proli, M. Leung, V. Anikin, N. McGonigle, E. Beddow, G. Ladas, S. Jordan, M. Dusmet, A.G. Nicholson, E. Lim
- Abstract
- Presentation
Background:
The prognostic significance of additional lung nodules in the setting of lung cancer is important as the impact on survival is often considered for the justification of surgical selection in the management of patients with synchronous nodules. TNM 7 down staged the impact on T category but did not distinguish between second primary versus metastasis. Traditional distinctions such as the Martini criteria do not take the same histological type into account and classification continues to improve (e.g. IASLC classification of adenocarcinoma). The aim of our study is to determine if it is possible to distinguish between second primary versus metastases in patients with the same histological type and quantity any difference in survival.
Methods:
We retrospectively analysed data from a prospectively collated database at our institution. We collected all the records which included two resected nodules. The detailed pathology reports of these patients were retrieved and the histology, subtype and pTNM of tumours documented. Slides were re-reviewed to determine the histological subtypes according to the current IASLC adenocarcinoma classification. Survival was calculated using Kaplan Meier methods and adjusted survival compared using Cox regression on R (statistical software).
Results:
From April 1999 to February 2013, a total of 2925 lung cancer resection were performed. Of these, 379 (14%) operations fulfilled the inclusion criteria of multiple nodules with 316 having synchronous tumours (83.3%) and 63 having metachronous tumours (16.6%). The tumours were ipsilateral in 87.3% and the vast majority were in the same lobe (70.9%). For synchronous tumours, patients with the same histological type had a poorer 5-years survival rate compared to tumours with different histology (p=0.041). The pathologist’s designation between second primary versus intra-pulmonary metastasis distinguished between overall survival (p= 0.001) and this remained statistically significant in the tumours of the same cell type (p= 0.035). Figure 1. Survival outcomes between patients with multiple nodules classified as second primary versus intra-pulmonary metastasis Figure 1
Conclusion:
Our results suggest that distinction between second primary and intra-pulmonary metastasis remains important for staging as appreciable differences in survival were observed in patients with synchronous nodules. Survival was poorer in patients with multiple nodules of the same histologic type (compared to different histology) and within the same histological subtype it is possible for pathologists to distinguish between second primary and intra-pulmonary metastasis. As this is currently confirmed only on pathologic stage in the majority, it presently does not influence the selection for surgery.
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MINI01.03 - Pathology-Imaging Agreement in Distinguishing Separate Primary Tumours and Intrapulmonary Metastasis in Staging of Multiple Lung Cancers (ID 2659)
10:55 - 11:00 | Author(s): P. Viola, A. Devaraj, E. Lim, G. Luciano, S. Popat, A.G. Nicholson
- Abstract
- Presentation
Background:
The 7[th] TNM staging system for lung cancer recommended staging for cases with multiple nodules viewed as intrapulmonary metastases (IM) as T3 (same lobe), T4 (ipsilateral different lobe) and M1a (contralateral lobe), whilst those classified as separate primary tumours (SPTs) as T(x)NM where “x” is the number of primary tumours, either as a number or “m” for multiple. With an increase in patients presenting with multiple nodules, we sought to develop a set of criteria for c-staging on imaging and to determine the agreement between clinical and pathological staging in a cohort of resected lung cancers who had multiple nodules.
Methods:
In 2013 and 2014, there were a total of 48 consecutive cases with available imaging resected with multiple tumours, ranging from 2 to 5 nodules. Of these, one case was excluded as it was a carcinoid with background DIPNECH. These cases were classified as SPT or IM based on previously published criteria (Girard et al. Am J Surg Pathol 2009;33:1752-64). Imaging criteria were generated based on clinical experience in similar fashion with indicators of SPT being 1) Lesions of equivalent size (one not more than 100% of the other) 2) Smaller lesion is spiculated , 3) At least one lesion is subsolid, 4) Presence of field change. (For signs 1 and 2, if the lesions were in different lungs, an absence of mediastinal disease on imaging was required). Cases with at least one positive sign were classified as SPTs. The interobserver agreement between radiologists and pathologist were then generated.
Results:
Of the 47 cases, the additional nodules were not identifiable on CT in 7 cases. In the remaining 40 cases, there was agreement in 28 cases, of which 16 were SPTs and 12 were IM. Of 12 cases where there was disagreement, only 3 were SPTS and the majority were cases classified on pathology as IM. There was 70% agreement, greater than that expected by chance (p = 0.002) with a kappa value of 0.41.
Conclusion:
Moderate agreement can be achieved in terms of clinical and pathological staging of lung cancers presenting with multiple nodules using imaging and pathologic criteria. Using pathology as the gold standard, there was greater agreement in categorisation of SPTs (84% (16/19)) than IM (57% (12/21)).
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MINI01.04 - Discussant for MINI01.01, MINI01.02, MINI01.03 (ID 3296)
11:00 - 11:10 | Author(s): N. Rekhtman
- Abstract
- Presentation
Abstract not provided
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MINI01.05 - Local Diagnostic Practices for Advanced Non-Small-Cell Lung Cancer in Europe and Japan: ASSESS Study (ID 2629)
11:10 - 11:15 | Author(s): N. Normanno, K. Hagiwara, B. Han, S. Tjulandin, C. Grohé, T. Yokoi, A. Morabito, S. Novello, E. Arriola, O. Molinier, R. McCormack, M. Ratcliffe, M. Reck
- Abstract
- Presentation
Background:
ASSESS (a large, multicentre, non-interventional, diagnostic study; NCT01785888) evaluated local diagnostic practices for patients with advanced non-small-cell lung cancer (aNSCLC) in Europe/Japan.
Methods:
Eligible patients: local/metastatic aNSCLC; chemotherapy-naïve, newly diagnosed/recurrent disease after resection; ineligible for curative treatment. We report diagnostic assessments and epidermal growth factor receptor (EGFR) mutation test turnaround times (secondary endpoints) associated with tissue/cytology samples from patients in Europe/Japan.
Results:
1311 patients enrolled (300 Japan). Immunohistochemistry (IHC) was used to confirm pathological diagnosis in 727/960 (76%) and 142/146 (97%) patients in Europe and Japan, respectively (where data were available); the following markers were assessed using IHC: TTF-1 (Europe 96% and Japan 79%); p65 (4% and 8%); and p40 (9% and 24%). EGFR mutation tests were not performed on samples from 110 patients and tested samples from 17 patients did not yield results. The most common reason for not testing was insufficient material provided (Europe 60% [47/78 responses]; Japan 56% [5/9 responses]). The percentages of neoplastic cells in samples (data available: Europe n=281; Japan n=20) were: <20% tumour cells: Europe 15% vs Japan 35%; 20–50% tumour cells: 23% vs 45%; >50% tumour cells: 61% vs 20%. Considering sampling methodologies, the most common sampling sites (data available: Europe n=996; Japan n=291) were the lung parenchyma (Europe 73%; Japan 79%) or lymph nodes (Europe 9%; Japan 9%); the most common sample collection method was bronchoscopy (Europe 39%; Japan 68%; Table 1). Median EGFR mutation test turnaround time was longer in Europe (11 days) versus Japan (8 days; Table 2). Mutation test success rates for Europe and Japan were 98.3% and 99.6%, respectively.
Conclusion:
Diagnostic assessments, sampling methodologies and EGFR mutation testing practices vary between and within Europe and Japan; further understanding of local practices will drive improvements and enable more patients to receive appropriate personalised treatment. Figure 1 Figure 2
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MINI01.06 - Pathological Second-Predominant Component Predicts Recurrence in Lung Adenocarcinoma (ID 1070)
11:15 - 11:20 | Author(s): M. Ito, Y. Miyata, Y. Tsutani, T. Mimura, S. Murakami, H. Ito, H. Nakayama, M. Okada
- Abstract
- Presentation
Background:
Lung adenocarcinoma is pathologically subdivided according to its predominant component. Approximately 50–70% of invasive adenocarcinomas are diagnosed as adenocarcinomas of either papillary or acinar predominant subtype. The prognostic difference between these subtypes has not been revealed, and these 2 similar subtypes may further be classified. This study aimed to investigate whether the pathological second-predominant component that follows the most predominant component predict recurrence in adenocarcinoma.
Methods:
We retrospectively reviewed 347 consecutive cN0 lung adenocarcinoma cases resected between April 2006 and December 2010 at Hiroshima University Hospital and Kanagawa Cancer Center. We further classified papillary and acinar predominant adenocarcinomas into either the papillary/acinar-lepidic type (Pap/Aci-Lep type) or the papillary/acinar-nonlepidic type (Pap/Aci-NonLep type). Tumor recurrence and the frequency of each invasion status such as lymphatic, vascular, and pleural invasion were compared between Pap/Aci-Lep type and Pap/Aci-NonLep type adenocarcinomas. In addition, we estimated the correlation between the radiological and pathological characteristics of these subtypes. Whole-tumor size, ground-glass opacity (GGO) ratio, solid size, and tumor disappearance ratio (TDR) on high-resolution computed tomography and maximum standardized uptake value (SUVmax) on positron emission tomography (CT) were measured as radiological parameters.
Results:
Papillary (n = 70) and acinar predominant adenocarcinomas (n = 61) were subdivided into the Pap/Aci-Lep type (n = 72) and Pp/Aci-NonLep type (n = 59). Compared with the Pap/Aci-NonLep type, the Pap/Aci-Lep type showed a significantly higher disease-free survival rate (5-year DFS: 89.4% vs 70.6%, p = 0.0374) and fewer cases of lymphatic invasion (15.3% vs 30.5%, p = 0.037), vascular invasion (15.3% vs 33.9%, p = 0.013), and pleural invasion (9.72% vs 25.4%, p = 0.031). Furthermore, radiological findings significantly differ between the Pap/Aci-Lep and Pap/Aci-NonLep types as follows: GGO ratio (μ ± 1 ´ SD: 34.4% ± 25.2% vs 3.81% ± 18.0%, p < 0.01), CT solid size (μ ± 1 ´ SD: 1.35 ± 0.65 cm vs 1.73 ± 0.55 cm, p = 0.015), TDR (μ ± 1 ´ SD: 41.8% ± 26.7% vs 17.5% ± 22.6%, p < 0.01), and SUVmax (μ ± 1 ´ SD: 2.37 ± 2.15 vs 3.96 ± 3.06, p < 0.01). Significant recurrence-free survival and prevalences of lymphatic and vascular invasion were observed between the lepidic predominant type (n = 109) and Pap/Aci-Lep type.
Conclusion:
The pathological second-predominant component allows for subclassification of papillary and acinar predominant adenocarcinomas with prognostic significance. Pathological features of these subtypes can be represented on clinical imaging. Not only the most predominant component but also the second-predominant component should be given clinical and pathological attention in order to predict malignant potential or decide indication for adjuvant therapy.
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MINI01.07 - Comparison of Grading Systems Based on Histologic Patterns and Mitotic Activity to Predict Recurrence in Stage I Lung Adenocarcinoma (ADC) (ID 3030)
11:20 - 11:25 | Author(s): K.S. Tan, K. Kadota, A. Moreira, P.S. Adusumilli, W.D. Travis
- Abstract
- Presentation
Background:
An established grading system for lung adenocarcinoma does not exist but is greatly needed. The histologic classification proposed by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS) has been shown to define prognostically significant subgroups of lung adenocarcinoma (ADC). Since then, various grading systems based on histologic patterns have emerged as promising methods to further discriminate patient risk of clinical outcomes. The aim of this work is to quantitatively assess the discrimination properties of a set of grading systems proposed in recent years to identify the best grading scale(s) independent of other clinical factors to predict recurrence.
Methods:
We considered five grading systems: (1) single predominant pattern as six subtypes; (2) as three grades of low (lepidic), intermediate (acinar, papillary) and high (micropapillary, solid); (3) two most predominant grades; (4) predominant grade with mitotic grade; and (5) predominant grade with cribriform pattern and mitotic activity criteria. We evaluated the performance of each grading system with the concordance predictive estimate (CPE). The CPE represents the probability that for any pair of patients, the patient with the better predicted outcome from the Cox model had the longer survival time. CPE > 0.80 demonstrates strong performance. To compare the performance of the grading systems, we determined the significance of the differences between the CPEs. Five-year recurrence-free probability (RFP) was derived using the Kaplan-Meier method.
Results:
We applied the grading systems to a uniform large cohort of stage I lung ADC (N=909). The scale based on the single predominant pattern as five subtypes yielded a CPE of 0.63 (95% CI, 0.59-0.67), indicating moderate discrimination. Our analysis showed that grading systems (1), (2), and (3) were not significantly different from each other, suggesting that identifying finer subtypes and second predominant pattern may not improve discrimination. Grading system (4) [CPE, 0.67; 95% CI, 0.63-0.71] yielded a significantly higher CPE than (1), (2) and (3) [p<0.01]. Grading system (5) [CPE, 0.67; 95% CI, 0.63-0.71] was significantly better than (1), (2) and (3) but not (4) [p=0.776]. The lack of improvement in discrimination with the inclusion of cribriform between (4) and (5) can be attributed to the significant relationship between cribriform pattern and mitoses. As the proportion of cribriform pattern increased, the amount of mitotic activity also increased (p<0.001). Under (2), the 5-year RFP of the intermediate grade was 0.81. The addition of cribriform and mitotic counts further classified the intermediate (acinar, papillary) grade such that those with <10% cribriform and low mitotic count had 5-year RFP of 0.89, while the 5-year RFP for the other combinations are between 0.73-0.75.
Conclusion:
Grading systems based on histologic patterns and mitotic activity out-perform those with only histologic pattern. This comparison study suggests that proposed grading systems (4) or (5) provide valuable information in discriminating patients with different risks of disease-recurrence in patients with lung ADC.
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MINI01.08 - Survival Differences of Adenocarcinoma Lung Tumors with Squamous Cell Carcinoma or Neuroendocrine Profiles by Gene Expression Subtyping (ID 384)
11:25 - 11:30 | Author(s): G. Mayhew, N. Hayes, C. Perou, M. Lai-Goldman, H. Faruki
- Abstract
- Presentation
Background:
Gene expression profiling can provide valuable information beyond the morphologic diagnosis. A previously validated 52-gene Lung Subtype Panel (LSP) for differentiating lung tumors into Adenocarcinoma (AD), Squamous Cell Carcinoma(SQ), and Neuroendocrine (NE) was explored in several publically available lung tumor datasets, including the TCGA RNAseq dataset.
Methods:
The LSP 3-class nearest centroid predictor developed in array data was applied to AD and SQ samples in TCGA (RNAseq, n=1,160), the Director’s Challenge (Affy array, n=442), and Tomida et al. (Agilent array, n=117) datasets. Each sample was predicted as AD, SQ, or NE. Kaplan Meier plots and log rank tests were used to assess and compare 5-year overall survival in two gene expression groups, AD predicted AD (AD-AD) and AD predicted SQ or NE (AD-notAD). Cox models were used to assess survival differences while controlling for T stage, N stage, and proliferation (as measured by the PAM50 score). The distribution of samples among the AD subtypes (Terminal Respiratory Unit(TRU), Proximal Proliferative(PP), and Proximal Inflammatory(PI)) was investigated.
Results:
The predictor confirmed AD in 80% of the AD samples. AD samples were called SQ and NE by the LSP in 8% and 12% of cases, respectively. The AD-notAD group (AD by histology and SQ or NE by gene expression LSP) had worse survival than the AD-AD group (AD by both histology and LSP) in each data set (logrank p-value in TCGA, Director’s Challenge, and Tomida were 1.17e-06, 0.0009, and 0.0001, respectively). Pooling the 3 data sets and using a stratified cox model that allowed for different baseline hazards in each study, the hazard ratio comparing AD-notAD to AD-AD was 2.14 (95% CI 1.70-2.70). When we fit the model adjusting for T stage, N stage, and proliferation score, the HR was 1.70 (95% CI 1.31-2.20). Adenosubtype profiling of AD-notAD samples indicated that tumors were overwhelmingly of the PP or PI gene expression subtypes (209/213).
Conclusion:
Gene expression tumor subtyping may provide valuable clinical information identifying a subset of AD samples with poor prognosis. Poor prognosis adenocarcinoma samples belong to the PI and PP expression subtypes, and demonstrate elevated proliferation scores. This subset of AD tumors may be less responsive to standard adenocarcinoma management.
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MINI01.09 - Discussant for MINI01.05, MINI01.06, MINI01.07, MINI01.08 (ID 3297)
11:30 - 11:40 | Author(s): E. Thunnissen
- Abstract
- Presentation
Abstract not provided
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MINI01.10 - Analysis of the Status of Lymphocyte Infiltration in Patients with NSCLC (ID 2292)
11:40 - 11:45 | Author(s): E.A. Richardet, M.E. Richardet, P.A. Hernandez, E. Pets, M. Cortes, M. Molina, A.A. Riso, C. Di Tada, L.P. Acosta, P. Companys, M. Paradelo
- Abstract
- Presentation
Background:
Current evidence highlights the potential role of tumor-infiltrating lymphocytes (TILS) as a prognostic factor in many types of tumors; in non-small cell lung cancer (NSCLC), this relationship is not well determined. TILs are being studied with different methods such as immunohistochemistry and optical microscopy. The primary endpoint is to identify TILS in patients with NSCLC, classified as present or absent, and its relation to progression-free survival (PFS) and overall survival (OS). Our secondary endpoint is to establish the relationship between the TILS and treatment received.
Methods:
Retrospective and analytical case study of Instituto Oncológico de Córdoba, from 2004 to 2014. 166 patients with stage IIIB and IV NSCLC were analyzed. TILS are descriptively classified as present or absent. Survival curves were calculated using the Kaplan-Meier method.
Results:
59% of patients had adenocarcinoma and 41% squamous cell carcinoma. 70% were men. 82% were smokers. 58% of patients with squamous histology and 65% with adenocarcinoma, showed TILS. In relation to first-line chemotherapy, 63,8% of patients received carboplatin-paclitaxel (CP) and 36,2% gemcitabine-cisplatin (GC). Patients with adenocarcinom with TILS present had higher PFS and OS; 8.86 and 13.43 months respectively, compared to patients with absent, 3.78 and 7.9 months. These differences were statistically significant (PFS: p = 0.000002 and OS: p = 0.003). The patients with squamous cell carcinoma with TILS had 6.78 and 12 months PFS and OS respectively. Those who had infiltrated absent had a PFS of 3.96 months and OS of 6.37 months. These differences were also statistically significant (PFS: p = 0.003 OS p = 0.001).
Conclusion:
Our study shows that patients whose pathological samples presented inflammatory infiltrate had higher OS and PFS. The presence of TILS could be used as an important prognostic factor in this patient population.
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MINI01.11 - Transcriptome Sequencing of Tumor vs. Surrounding Non-Malignant Lung Tissue in Non-Small Cell Lung Cancer (ID 1765)
11:45 - 11:50 | Author(s): K. Reynders, E. Wauters, J. Vansteenkiste, H. Decaluwé, P. De Leyn, K. Nackaerts, S. Peeters, C. Dooms, W. Janssens, D. Lambrechts, D. De Ruysscher
- Abstract
Background:
Both the response and the therapeutic ratio of targeted agents in NSCLC may depend on the expression of the target molecules in the tumor and the surrounding non-malignant lung tissue. We therefore performed transcriptome analysis and investigated correlations with histology, gender, age, CRP level and smoking status as well as evaluated the differential pathway expression in primary resected NSCLC and the surrounding non-malignant lung of the same patient.
Methods:
Transcriptome sequencing was performed on the primary tumor and distant lung tissue of the same patient from resection specimens of NSCLC patients. Differential gene expression between different conditions was identified using the statistical algorithms Cufflinks, EdgeR and DeSeq. Differential expression with P-values <0.05 after Benjamini-Hochberg correction was considered significant. Pathway analysis for overall tumor versus distant lung tissue was performed with the PANTHER gene classification platform using the Cufflinks, DeSeq and EdgeR differentially expressed gene sets as input.
Results:
Twenty-five patients were studied, 19 males and 6 females, with a median age of 69 years. Ten were current smokers, 14 former smokers (>4 weeks before surgery) and 1 non-smoker. Eleven patients had squamous cell carcinoma, 14 adenocarcinoma. A heat map with the results for the most commonly targeted genes in NSCLC is represented in figure 1. When compared to distant lung tissue, PD-L1 was downregulated in tumor tissue of adenocarcinoma and active smokers, but not in squamous cell carcinoma or ex-smokers. Internal control of tumor tissue of squamous vs. adenocarcinoma and ex-smokers vs. active smokers shows an important trend towards a higher expression of PD-L1 in squamous cell carcinoma and ex-smokers in both Cufflinks and EdgeR algorithms. Additional pathway analysis revealed 188 differentially regulated pathways. The most notable were downregulation of VEGF signaling, angiogenesis and B and T cell activation in tumor tissue when compared to distant lung tissue. Figure 1
Conclusion:
Our first results show a higher expression of PD-L1 in squamous tumors than in adenocarcinoma and a higher expression in tumors of ex-smokers than in those of active smokers. This may have consequences for the therapeutic ratio with anti-PD-L1 treatment. Downregulation of VEGFR-genes in tumor tissue was observed across almost all conditions. We will make this data more complete by adding methylation data as well as immunohistochemistry for protein localization.
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MINI01.12 - Implementation of a Molecular Tumor Board: The Impact on Treatment Decisions for NSCLC Patients Evaluated at Dartmouth-Hitchcock in One Year (ID 2719)
11:50 - 11:55 | Author(s): L.J. Tafe, I.P. Gorlov, F. De Abreu, J.A. Lefferts, X. Liu, J.R. Pettus, J.D. Marotti, K.J. Bloch, V.A. Memoli, A.A. Suriawinata, C.E. Fadul, G.N. Schwartz, C.R. Morgan, B.M. Holderness, J.D. Peterson, G.J. Tsongalis, T.W. Miller, M.D. Chamberlin, K.H. Dragnev
- Abstract
- Presentation
Background:
Genetic profiling of tumors is a powerful approach to predict drug sensitivity and resistance. Definitive interpretation of the clinical significance of somatic mutations is possible for only a few well studied mutations. For the majority, prediction of clinical significance is challenging. We established a Molecular Tumor Board (MTB) at our Cancer Center to interpret individual patients’ tumor genetic profiles and provide treatment recommendations.
Methods:
DNA from tumor specimens was sequenced in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory to identify coding mutations in a 50-gene panel. Cases were evaluated by a MTB composed of molecular and anatomic pathologists, medical oncologists, basic research scientists, and genetic counselors.
Results:
35 cases were evaluated in 1 year by the MTB including 8 metastatic NSCLC cases. The most common reason for MTB referral was for recommendations on targeted therapies (91.9%), and for potential germline mutations. Tumors exhibited genetic heterogeneity: 71 different mutations were found across 300 genes (for NSCLC 18 mutations across 10 genes). In 18/32 of advanced/metastatic cases, MTB recommended non-standard therapy with a specific targeted agent (11 clinical trials; 7 off-label use), 4 of the 18 patients were subsequently treated with a MTB-recommended targeted therapy. The remaining 14 patients continued on current therapy because disease was stable (n=4), were treated with non-MTB-recommended standard therapy (n=4), declined conventional therapy (n=5), or died prior to receiving further therapy (n=1). For 4 out of the 8 NSCLC cases MTB recommended a BRAF inhibitor (1), RET inhibitor (1), or MET inhibitor (2). One patient received a BRAF inhibitor, 6 continued on current standard of care therapy, one declined therapy.
Conclusion:
Case evaluation by a multidisciplinary group of individuals in the context of a MTB frequently shapes treatment options and decisions. Importantly, anticipated obstacles to capitalizing on the benefits of a MTB such as access to drugs were rarely encountered in the entire cohort and in the NSCLC patients. Instead, the most commonly encountered reasons that MTB-recommended therapy was not administered stemmed from patient preferences, and genetic profiling at a very late stage of disease.
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MINI01.13 - Biologically Driven Sub-Classification of Early Lung Adenocarcinomas (ID 2418)
11:55 - 12:00 | Author(s): D.A. Moore, E. Al Dujaily, J. Le Quesne
- Abstract
- Presentation
Background:
Early lung adenocarcinomas have previously been successfully sub-classified by Noguchi et al on the basis of histopathological characteristics, in particular the pattern of growth exhibited by the tumour and the response of the adjacent stroma. A wholly in situ pattern of growth characterises preinvasive lung lesions, namely atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS). It is not uncommon for large areas of an invasive tumour to show this in situ pattern of growth. Metastases to the lung from other organs can also show areas of in situ spread, and this shows that truly invasive malignant clones of cells can grow along the alveolar surface. This study aimed to identify whether the characteristics of in situ growth in an individual tumour may give an indication of the underlying tumour biology, and be of prognostic value.
Methods:
We reviewed all small (sub 35 mm) lung adenocarcinomas resected with curative intent over a 4 year period from our thoracic surgical centre. Nodal metastasis data was also collected, which the reviewing pathologists were blinded to. All tumour sections were reviewed by 2 thoracic histopathologists, who separated these into 4 categories, based on the patterns of growth, stromal changes, cytological changes between in situ and invasive components and overall symmetry of the lepidic growth. On the basis of these appearances early lung adenocarcinomas were divided into 4 groups. Type 1 showed minimal stromal reaction analogous to Noguchi A/B tumours. Types 2 and 3 are subdivisions of mixed in situ/invasive adenocarcinomas (equivalent to the Noguchi C group). Type 2 showed marked stromal changes in the invasive component and a cytologically lower grade asymmetrical lepidic component. Type 3 showed a concentric rim of lepidic growth cytologically similar to the invasive disease. Type 4 were wholly invasive tumours. Tumour type was subsequently correlated to pathological lymph node staging.
Results:
156 tumours were included and sub-classified. Of these 12 were type 1, 30 were type 2, 46 were type 3 and 68 were type 4. The rate of nodal metastasis was increased across the tumour types from 1 to 4, at 0%, 7%, 24% and 29% respectively.
Conclusion:
We find that partly invasive lung adenocarcinomas fall into two histologically and biologically distinct groups with different potential toward nodal metastasis. We suggest the type 2 tumours represent the emergence of an invasive subclone in an in situ adenocarcinoma lacking this property. The type 3 tumours have a lepidic region at their edge which represents infiltration of normal structures by migratory malignant cells but may have no truly biologically ‘in situ’ disease. This group also shows more tendency toward metastasis. Type 4 lesions without any kind of lepidic growth have the highest rate of nodal involvement, and their destructive pattern of growth represents the most aggressive form of early tumours. Future molecular characterisation of these lesions and their various components may further inform our understanding of the pathways of tumorigenesis in lung adenocarcinoma.
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MINI01.14 - Discussant for MINI01.10, MINI01.11, MINI01.12, MINI01.13 (ID 3298)
12:00 - 12:10 | Author(s): K. Politi
- Abstract
- Presentation
Abstract not provided
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MS 25 - Lung Carcinogenesis (ID 43)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Screening and Early Detection
- Presentations: 5
- Moderators:W.A. Franklin, H. Kato
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 2a-3b
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MS25.01 - Early Airway Disease (ID 1958)
14:20 - 14:35 | Author(s): S.M. Janes
- Abstract
- Presentation
Abstract:
In my talk I will report a prospective surveillance program, longitudinally following patients with pre-invasive disease over a 10 year period. It is the largest study of its kind and demonstrates unexpectedly high rates of both local progression to invasive cancer of high grade lesions and the development of synchronous tumours elsewhere in the lung. Further I will show data identifying both gene expression and epigenetic signatures predicting progression of these lesions. These signatures may provide the biomarker strategy we require to identify those patients with lesions at high risk of progression and therefore requiring treatment. Lung cancer accounts for more deaths than breast, prostate and colon cancers combined. Over three quarters of lung cancer patients are diagnosed at a late stage when curative treatment is not possible. Initiatives are underway to detect lung cancer earlier. CT screening of high risk smokers or ex smokers is proven to save lives through increased detection of largely early stage adenocarcinomas (1, 2). Meanwhile sputum cytometry and autofluorescence bronchoscopy of high risk individuals are under investigation as screening tools for the early detection of major airway squamous cell carcinomas in several studies. Squamous carcinogenesis is initiated by pre-invasive dysplastic lesions in the central airways and therefore lends itself to bronchoscopic evaluation. Bronchial dysplasia represents the earliest stages of what is traditionally thought to be a stepwise progression towards invasive disease commencing with squamous hyperplasia and metaplasia followed by mild, moderate, severe dysplasia (SD) and carcinoma-in-situ (CIS) with lesions possessing a greater mutational burden at each stage (WHO classification) (Figure 1). With progression of the lesion there are characteristic morphological changes and increasing cytological disarray. Initial changes affect only the basal epithelium, whilst ‘full thickness’ change is seen in the more advanced CIS. Once the basement membrane has been breached, invasive squamous cell carcinoma has developed. Figure 1 Our early findings, and those of others, have challenged this traditional stepwise model. With longitudinal follow up, few low grade dysplasia lesions (LGD: hyperplasia, metaplasia mild and moderate dysplasia) are seen to progress and largely remain indolent or often regress. High grade dysplasia lesions (HGD: SD and CIS) however, more frequently persist or progress to invasive disease. Bronchial dysplastic lesional destiny is unpredictable and despite research examining the genetic and epigenetic changes that occur, as yet no robust biomarker is able to determine which lesions will continue to progress to invasive disease. Low grade lesions rapidly progressing to cancer have been reported, and these rare lesions have been found to possess a high degree of chromosomal instability including DNA copy number alterations even at a metaplastic stage, seeming to confer a committed course to cancer development. It is likely that close analysis of these rare lesions and other high grade lesions that progress will lead to greater biological insight regarding key lung cancer driver mechanisms. Autofluorescence bronchoscopy (AFB) using blue-violet excitation light has made progress in facilitating not only the detection and delineation of extent of early stage invasive cancers in the airway but also the identification of precancerous central airway lesions that are generally missed on CT. AFB detection of precancerous lesions has been shown to have sensitivity exceeding that of white light bronchoscopy (WLB) alone. The sensitivity of combining AFB with WLB improves detection of bronchial premalignant and malignant lesions up to 96.8% versus 76.3% for WLB alone, whilst corresponding negative predictive values are 97.2% versus 83.1% (3). Treatment of precancerous lesions might be expected to lead to improved survival in those patients harboring them. However our lack of knowledge of the natural history of these lesions, the appearance of new lesions, the regular occurrence of separate lung primaries and the lack of interventional studies in this area leaves the role of early intervention (both surgical and local tissue sparing procedures) under dispute. Due to this poverty of knowledge, our strategy, in keeping with previously published studies, has been the surveillance of all grades of dysplasia. These include our own, initial observations that suggest a low rate of lesion progression but high synchronous invasive cancer occurrence (4, 5). This early experience indicates patients with preinvasive disease are at a globally high risk of developing lung cancer, although not necessarily from the lesion under observation and multiple lesions both centrally and peripherally commonly develop over time. Due to the shared risk factor of tobacco smoke exposure, patients often have significant respiratory and cardiovascular co-morbidity and radical treatment of preinvasive disease may lead to insufficient lung capacity to offer curative intervention to future invasive lung cancer. 1. NCCN Clinical Practise Guidelines in Oncology. Lung Cancer Screening. Version http://www.nccn.org/professionals/physician_gls/pdf/lung_screening.pdf 2. National Lung Screening Trial Research Team, Aberle DR, Berg CD, Black WC, Church TR, Fagerstrom RM, Galen B, Gareen IF, Gatsonis C, Goldin J, Gohagan JK, Hillman B, Jaffe C, Kramer BS, Lynch D, Marcus PM, Schnall M, Sullivan DC, Sullivan D, Zylak CJ. The National Lung Screening Trial: overview and study design.Radiology. 2011 Jan;258(1):243-53. 3. Hanibuchi M, Yano S, Nishioka Y, Miyoshi T, Kondo K, Uehara H, Sone S. Autofluorescence bronchoscopy, a novel modality for the early detection of bronchial premalignant and malignant lesions. J Med Invest. 2007 Aug;54(3-4):261-6. 4. George JP, Banerjee AK, Read CA, O'Sullivan C, Falzon M, Pezzella F, Nicholson AG, Shaw P, Laurent G, Rabbitts PH. Surveillance for the detection of early lung cancer in patients with bronchial dysplasia. Thorax. 2007 Jan;62(1):43-50. 5 Auerbach O, Stout AP, Hammond EC, et al. Changes in bronchial epithelium in relation to cigarette smoking and in relation to lung cancer. N Engl J Med 1961;265:255–67.
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MS25.02 - Transcriptional Profiling of Malignant Lesions (ID 1959)
14:35 - 14:50 | Author(s): B. Gomperts
- Abstract
- Presentation
Abstract:
Epithelial cancers are thought to arise in a stepwise fashion from premalignant lesions and removal of premalignant lesions in epithelia such as colon and cervix has made a major improvement in survival in these cancers. However, premalignant lesions of the airway epithelium are poorly understood and it is not even known whether they represent a true premalignant state. This is in large part because of the heterogeneity of premalignant lesions of the airway and the fact that most of them will spontaneously resolve, even in high-risk patients. Premalignant lesions are thought to arise because of aberrant repair after injury but our understanding of the biology of normal repair after injury of the airways is limited and thus we do not know what the mechanisms are that drive aberrant repair and even less what the mechanisms are that drive the development of invasive non-small cell lung cancer. In order to increase our understanding of premalignant lesions of the airway, we laser-microdissected representative cell populations along the purported squamous cell lung cancer pathological continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We obtained sufficient mRNA to perform high throughput RNA-sequencing. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. We also identified several pathways that were upregulated in a stepwise fashion with progression of lesions. One of the pathways found to be upregulated with stepwise progression was redox regulation. Low levels of Reactive Oxygen Species (ROS) are known to be critical for cell regulation, while high levels of ROS are toxic to cells. We found that airway basal stem cells have low levels of ROS at baseline, but injury results in an increase in ROS and this flux from low to higher levels of ROS mediates proliferation of the basal cells via signaling through ROS/Nrf2/Notch1. Perturbation of this pathway at the level of Nrf2 or Notch both in vitro and in vivo results in excessive proliferation of basal cells and the formation of premalignant lesions with hyperplasia and dysplasia of the repairing airway epithelium. Our results provide much needed information about the biology of airway epithelial repair, premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of squamous cell lung cancer, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.
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MS25.03 - Peripheral Premalignancy (ID 1960)
14:50 - 15:05 | Author(s): M.S. Tsao
- Abstract
- Presentation
Abstract:
The recently published Fourth Edition of the WHO Classification of Tumours of the Lung[1] recognizes atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) as pre-invasive or premalignant precursor lesions of invasive adenocarcinoma, which arises mostly in the periphery of the lung. In the previous (Third) Edition of WHO classification, AIS was classified as bronchiolo-alveolar carcinoma (BAC), one of the subtypes of malignant adenocarcinoma.[2,3] Reclassification of AIS into the preinvasive category represents a conceptual confirmation of its role in multi-stage pathogenesis of peripheral lung adenocarcinoma.[4-7] This is consistent with the histological hallmark of lack of invasion in AIS, and its association with 100% survival after surgical resection. The neoplastic nature of these lesions are supported at the molecular level with the identification of known genomic aberrations found in invasive lung adenocarcinoma.[8,9] AIS is characterized histologically by the lepidic proliferation of neoplastic epithelium along pre-existing alveolar structures and lacking stromal, vascular or pleural invasion (Figure 1). A majority of AIS is composed of non-mucinous neoplastic cells with Clara cell and/or type-2 pneumocyte phenotype. Mucinous AIS is rare. By definition, AIS is limited to tumors that is ≤ 3 cm in greatest diameter and by TNM classification, is classified a Tis. AIS commonly shows varying degree of stromal thickening by fibrosis and chronic inflammatory cell infiltrate, with some cases showing focal or central area of fibrosis or scar. Around these areas, entrapment of the tumor cells within architecturally distorted and thickened alveolar septa give rise to morphological appearances of invasion. This remains one of the areas of diagnostic difficulty in distinguishing AIS from minimally invasive adenocarcinoma (MIA). However, limited data suggests that MIA is also associated with 100% curability by surgical resection. A majority of AAH are identified incidentally during microscopic examination of non-cancerous lung of surgically resected adenocarcinoma (Figure 2). The reported incidence in lung adenocarcinoma cases may reach up to 30%, and the reported number of lesion can reach up to 40/case, depending on the extent of sampling. They are typically ≤ 5 mm, but size is not a diagnostic criteria for its diagnosis. Histologically it is characterized by slightly thickened alveolar septa that are lined by atypical appearing cuboidal to low columnar epithelial cells with gaps in between them. These cells have similar ultrastructural features as AIS cells, mainly those of type-2 pneumocyte and/or Clara cell. A spectrum of nuclear atypia may be observed but grading has not been recommended, as they have not been demonstrated as reproducible or correlated with neoplastic progression. AAH is considered a precursor of AIS, as they may harbor KRAS or EGFR mutations. In some cases, the histological distinction between AAH and AIS can be very challenging, even though both lesions are considered cured by surgical resection. Further deep genomic analyses of AAH and AIS can provide greater insights into the multistep molecular carcinogenesis of lung adenocarcinoma and potentially novel prevention strategies for this disease. References: 1. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. IARC Press, Lyon, 2015, page 46-50. 2. World Health Organization International Histological Classification of Tumours. Histological Typing of Lung and Pleural Tumours. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Springer Verlag, Berlin, Heidelberg, New York, 1999, page 21-29. 3. WHO Classification of Tumours, Pathology and Genetics. Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Muller-Hermelink HK,, Harris CC. IARC Press: Lyon 2004, page 35-44, 73-75. 4. Miller RR, Nelems B, Evans KG, Muller NL, Ostrow DN. Glandular neoplasia of the lung. Cancer 1988;61:1009-1015. 5. Kitamura H, Kameda Y, Ito T, Hayashi H. Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma. Am J Clin Pathol 1999;111:610-22. 6. Mori M, Rao SK, Popper HH, Cagle PT, Fraire AE. Atypical adenomatous hyperplasia of the lung: A probably forerunner in the development of adenocarcinoma of the lung. Mod Pathol 2001;14:72-84. 7. Chapman AD, Kerr KM. The association between atypical adenomatous hyperplasia and primary lung cancer. Br J Cancer 2000;83:632-36. 8. Westra WH, Baas IO, Hruban RH, Askin FB, Wilson K, Offerhaus GJ, Slebos RJ. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996;56:2224. 9. Sakamoto H, Shimizu J, Horio Y, Ueda R, Takahashi T, Mitsudomi T, Yatabe Y. Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas. J Pathol 2007;212:287-94. Figure 1. Adenocarcinoma in situ Figure 1 Figure 2. Atypical Adenomatous Hyperplasia. Figure 2
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MS25.04 - Molecular Pathology of Alveolar Premalignancy (ID 1961)
15:05 - 15:20 | Author(s): M. Noguchi
- Abstract
- Presentation
Abstract:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We examined epigenetical and genetic abnormalities of very early adenocarcinoma and compared them to early but advanced adenocarcinoma. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma. Suppression of SFN expression in A549 (a human lung adenocarcinoma cell line) by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. In vivo, tumor development or metastasis to the lung was reduced in shSFN-transfected A549 cells. Moreover, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN under the control of a tissue-specific enhancer, the SPC promoter. We found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after administration of chemical carcinogen, NNK (Fig 1). Interestingly, several Tg-SPC-SFN+/- mice developed tumors without NNK. These tumor cells showed high hSFN expression. These results suggest that SFN facilitates lung tumor development and progression. SFN appears to be a novel oncogene with potential as a therapeutic target. Next, gnetic abnormality in early-stage lung adenocarcinoma was examined. Six in situ lung adenocarcinomas and nine small but invasive adenocarcinomas were examined by array-comparative genomic hybridization (array-CGH), and candidate genes of interest were screened. To examine gene abnormalities, 83 cases of various types of lung carcinoma were examined by quantitative real-time genomic PCR (qPCR) and immunohistochemistry (IHC). The results were then finally verified using another set of early-stage adenocarcinomas. Array-CGH demonstrated frequent amplification at chromosome 3q26, and among the 7 genes located in this region, we focused on the epithelial cell transforming sequence 2 (ECT2) oncogene, as ECT2 amplification was detected only in invasive adenocarcinoma, and not in in situ carcinoma. FISH and IHC analyses also detected amplification and overexpression of ECT2 in invasive adenocarcinoma (Fig 2), and this was correlated with both the Ki-67 labeling index and mitotic index. In addition, it was associated with disease-free survival and overall survival of patients with lung adenocarcinoma. These results were verified using another set of early-stage adenocarcinomas resected at another hospital. Abnormality of the ECT2 gene occurs at a relatively early stage of lung adenocarcinogenesis and would be applicable as a new biomarker for prognostication of patients with lung adenocarcinoma. Figure 1Figure 2
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MS25.05 - Premalignant Lesions: Cytokines and Microenvironment (ID 1962)
15:20 - 15:35 | Author(s): S.M. Dubinett
- Abstract
- Presentation
Abstract not provided
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Author of
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MINI 11 - Tobacco Control and Prevention (ID 108)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Prevention and Tobacco Control
- Presentations: 1
- Moderators:S.M. Janes, E. Stone, K.M. Cummings
- Coordinates: 9/07/2015, 16:45 - 18:15, 601+603
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MINI11.04 - A New Preclinical Model of Airway Progenitor Cells to Identify Responders to Iloprost-Mediated Chemoprevention (ID 1698)
16:55 - 17:00 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
Lung cancer is the leading cause of cancer related deaths worldwide. The 5-year survival rate for this cancer is only 16%. Chemoprevention can improve prognosis in these patients. However, previous attempts at lung cancer chemoprevention that were soley based on epidemiological data were ineffective. Squamous cell lung cancer develops through a series of bronchial lesions or dysplasia. Persistent dysplasia harbors similar genetic changes as the tumor and has significantly higher chance of progression. Thus, bronchial dysplasia is a risk biomarker for SCC and improvement in dysplasia grade can be used as an outcome for chemoprevention trials. The long-acting prostacyclin analogue, iloprost is the only drug that has improved dysplasia in former smokers (p = 0.006). Despite this positive outcome we have little insight into the mechanisms of iloprost function. Understanding these mechanisms would be essential to identify people who have the highest chance to benefit from iloprost treatment. We propose that this endeavor will require a preclinical model that recapitulates the human disease and is amenable to mechanistic studies.
Methods:
Airway progenitor cells are critical for the maintenance of normal airways, because of their ability to self-renew (i.e. replicate) and differentiate into all cell-types of the airway (i.e. multipotentiality). Together these properties allow progenitors to return injured tissue to normal structure and function. In dysplasia, normal bronchial epithelium is changed into one that contains increased numbers of basal cells and lacks ciliated cells. These findings led to our hypothesis that ‘airway progenitors are malfunctioning in dysplasia’. Previously we showed that Keratin (K) 5/p63-expressing basal cells are the multipotential progenitors of the airway epithelium. During in vitro culture these cells form a unique 3-deimensional structure called the rim clone, which allows them to be distinguished from non-progenitors. To investigate a role of epithelial progenitors in dysplasia, we have collected bronchial biopsies from high-risk smokers and purified rim clone forming basal progenitor cells.
Results:
We demonstrate that both self-renewal and multipotentiality of progenitors is significantly (p < 0.001 for both) decreased in dysplasia. During differentiation in vitro at the air-liquid interface, progenitors from normal biopsies generated a normal epithelium. In contrast, progenitors from dysplasia made a squamous epithelium containing only basal cells and lacking ciliated cells. Mutational analyses of paired samples from epithelial brushings and biopsy-derived progenitors identified the same somatic mutations in p53, Notch 1, Notch 3, Survivin and FGFR1. Thus, epithelial progenitor culture reflects the histologic and genetic changes of dysplasia and therefore can be used as a personalized, preclinical model. A proof of concept study where dysplastic progenitor cells were treated with iloprost resulted in decreased dysplasia in 2 out of 3 cases.
Conclusion:
Thus our data indicate that progenitor cell cultures from a patient’s dysplasia may be used to identify responders versus non-responders to iloprost, as well as other chemopreventives. Future studies could focus on identifying downstream mechanisms via which iloprost exerts its beneficial effect.
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MINI 12 - Biomarkers and Lung Nodule Management (ID 109)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:J.M. Siegfried, H.I. Pass
- Coordinates: 9/07/2015, 16:45 - 18:15, 401-404
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MINI12.02 - Clinical Utility of Chromosomal Aneusomy in High Risk Individuals (ID 1299)
16:50 - 16:55 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
In the context of CT screening in current and former smokers at high risk for lung cancer, the false positive rate is high (26% at first NLST screening; 13% with Lung-RADS criteria applied to NLST) and indeterminate nodules are frequently discovered. Noninvasive biomarkers are urgently needed to reduce false positives with screening CT and to improve risk stratification in those with indeterminate nodules. The Colorado (CO) Lung SPORE program performed a retrospective longitudinal evaluation (Pepe Phase 3 validation) to assess the potential of chromosomal aneusomy detected in sputum via fluorescence in situ hybridization (CA-FISH) as a biomarker for early detection in four nested case-control studies. Two of the cohorts (ACRIN/NLST and PLuSS) enrolled current and former smokers to investigate use of low dose CT to diagnose lung cancer. The other two were Colorado cohorts in which pulmonary clinic patients (mostly current and former smokers) were enrolled to investigate biomarkers to predict lung cancer. One of these cohorts (CO High Risk) was a COPD population and the other, still in the accrual phase, comprises patients referred for care of indeterminate lung nodules (CO Nodule).
Methods:
The cohorts were grouped into a Screening cohort (ACRIN/NLST (49 cases, 96 controls) and PLuSS (48 cases, 89 controls)) and a High Risk cohort (CO High Risk (55 cases, 59 controls) and CO Nodule (13 cases, 10 controls)). The CA-FISH assay was a 4-target panel including genomic sequences encompassing the EGFR and MYC genes, and the 5p15 and centromere 6 regions or the FGFR1 and PIK3CA genes. At the subject level, the assay was scored on a 4-category scale representing normal, probably normal, probably abnormal and abnormal. Operating characteristics (with 95% CI) of the assay were estimated for each group of cohorts overall and separately for COPD patients: sensitivity, specificity, likelihood ratio+ (LR+) and likelihood ratio- (LR-).
Results:
Using the cutoff of abnormal vs. not abnormal for CA-FISH, sensitivity and specificity for Screening subjects are 0.20 (0.13, 0.30) and 0.84 (0.78, 0.89), respectively; and for High Risk subjects are 0.67 (0.55, 0.78) and 0.94 (0.85, 0.98), respectively. Likelihood ratios for Screening subjects are LR+: 1.36 (0.81, 2.28) and LR-: 0.93 (0.83, 1.05), and for High Risk subjects are LR+: 11.66 (4.44, 30.63), and LR-: 0.34 (0.24, 0.48). Similar results were observed when only COPD subjects were analyzed.
Conclusion:
The high LR+ of sputum CA-FISH indicates that this noninvasive biomarker could be a clinically useful adjunct to CT among patients in high risk settings. Whether this same high level of LR+ will be reproducible in patients at high risk because of their indeterminate nodules remains to be seen. If so, a hypothetical patient with indeterminate nodules and a pre-test (CA-FISH) lung cancer risk of 20% would have a post-test probability of lung cancer of 78% if the CA-FISH test were positive. In the screening setting, however, the low LR+ of CA-FISH limits its clinical utility. Prospective assessment of sputum CA-FISH is ongoing in the Nodule Cohort of the CO Lung SPORE.
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MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
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MINI22.10 - A New Approach to Large Scale Proteomic Profiling to Uncover Tumor Phenotypes (ID 2166)
17:40 - 17:45 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
Genomic profiling is a powerful method for identifying mutations that drive tumors and matching patients to targeted therapies. However, this may only be a transient solution and resistance commonly emerges as the mechanism of targeted inhibition is overcome. Proteomic profiling of the tumor provides a dynamic tool to survey altered protein expression and deregulated pathways, which in turn may implicate specific treatments or identify novel therapeutic targets. Mass spectrometry offers highly multiplexed proteomic measurements, but extensive sample pre-processing and low sample throughput can lead to extended analysis times of weeks or months. Thus a need exists for a high throughput, sensitive and quantitative platform for proteomic analysis.
Methods:
We used the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV, to analyze protein lysates from 63 lung tumor samples. The assay does not require sample pre-fractionation, and this study (which generated over 142,000 protein measurements) represents less than one day of SOMAscan throughput. The study consisted of matched tumor/non-tumor protein lysates prepared from 18 squamous cell carcinoma and 45 adenocarcinoma fresh-frozen resected specimens, 86% of which were Stage I/II. The paired log~2~ tumor/non-tumor ratio was calculated and hierarchical clustering heat maps and dendrograms were constructed to identify related protein regions and tumor phenotypes.
Results:
Common proteomic changes and unique tumor phenotypic groups were identified by unbiased clustering algorithms. Large, consistent tumor/non-tumor differences of at least 4-fold were observed for 35 proteins in at least 20 (32%) of the tumors. Some of these proteins were more than 100-fold higher in individual tumors. The two most commonly elevated proteins were thrombospondin 2 and MMP12, which were increased in 81% and 61% of the tumors, respectively. We have previously reported higher levels of MMP12 in the serum of lung cancer patients, and the current data supports a tumor-associated origin for circulated MMP12. A second analysis identified sub-phenotypes of tumors clustered by common protein alterations independent of histological classification or mutation status. Many of these tumor subsets had increased expression of known oncology drug targets.
Conclusion:
Broad, unbiased high-throughput proteomic profiling of tumor tissue may reveal individual phenotypes that hold the potential to respond to targeted therapies and to monitor therapeutic efficacy throughout treatment. Measuring proteins complements mutation analysis by enabling therapeutic selection beyond driver mutation targets, including immune modulator therapies, repurposing existing drugs and enriching clinical trials with likely responders. While genomics is a fixed snapshot, blood- and tissue-based serial proteomic measurements respond to change and can lead to the personalized adaptation of treatment and identification of novel therapeutic targets.
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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): W.A. Franklin
- 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: 2
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ORAL23.02 - Pioglitazone for the Chemoprevention of Lung Cancer (ID 2419)
10:56 - 11:07 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
Prior clinical studies have shown that the oral prostacyclin agonist iloprost improves bronchial dysplasia in former smokers. Prostacyclin is a PPAR gamma agonist, and epidemiologic and pre-clinical studies suggest PPAR gamma agonists like pioglitazone may chemoprevent lung cancer. Based on these promising results, a double-blind, placebo controlled, phase II trial of pioglitazone in subjects at increased risk for lung cancer was sponsored by the Department of Veterans Affairs.
Methods:
Subjects 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 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 and then 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:
A total of 90 subjects (46 pioglitazone, 44 placebo) have been enrolled in the trial, with 76 completing both bronchoscopies. Subjects are well matched in terms of age, gender, tobacco exposure, and sputum cytology. No significant differences in lung function were observed between the treatment groups. While the investigators remain blinded in regards to treatment group, aggregate data is available. Overall, mild dysplasia or worse was seen in 26% of the initial biopsies. Similar to prior studies, current smokers exhibited more dysplasia at baseline compared to former smokers (32.4% vs. 16.6%) and also had more angiogenic squamous dysplasia (11.7% vs. 3.2%). Our primary endpoint is change in maximum histology, and histologic scores from matched biopsies in all participants showed a change of at least 1 grade in 50.2% (25.9% improved, 24.3% progressed). More histologic changes were observed in current smokers (59.2%) than former smokers (41.7%). Summary data for the non-normal biopsy pairs (ie those with a histologic score of at least 2 on baseline biopsy) showed that the majority of pairs (73.7%) changed by at least one grade. Current smokers exhibited more progression (29.3%) compared to former smokers (14.6%).
Conclusion:
The pioglitazone lung cancer chemoprevention trial is currently in progress. The treatment has been well tolerated and histologic changes were observed in many of the subjects.
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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): W.A. Franklin
- 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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ORAL 24 - CT Detected Nodules - Predicting Biological Outcome (ID 122)
- Event: WCLC 2015
- Type: Oral Session
- Track: Screening and Early Detection
- Presentations: 1
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ORAL24.05 - Reclassification of Lung Cancers Detected by CT Imaging in the American College of Radiology Imaging Network National Lung Screening Trial (ID 1454)
11:28 - 11:39 | Author(s): W.A. Franklin
- Abstract
- Presentation
Background:
The National Lung Screening Trial (NLST) found a 20% reduction in lung cancer-specific mortality using low dose CT vs chest radiography for screening. The magnitude of mortality benefit has been questioned given that a higher proportion of tumors in the CT arm were diagnosed as “bronchioloalveolar cell carcinoma”. Subsequent to the initiation of the NLST, the pathological classification of lung cancer was revised to take into account the reported favorable outcome for solitary in situ nodules <3 cm. The term “bronchioloalveolar carcinoma” (BAC) was eliminated in favor of the more explicit terms adenocarcinoma in situ (AIS), microinvasive adenocarcinoma (MIA), and invasive carcinoma with various predominant histological patterns. To better assess the impact of these recent changes in the Pathological classification of lung cancer on possible over-diagnosis in the NLST, we have reviewed the histology of lung tumors detected through the ACRIN-NLST trial and reclassified them according to the most recent WHO pathology classification.
Methods:
Histology was initially classified by the pathologists at sites where NLST participants were managed. Representative slides of 192 surgical resection specimens and 15 non-surgical biopsies from 207 patients were collected from 19 participating institutions. Digital images were prepared from 533 glass H&E stained slides using an Aperio digital slide imager. Digital images were examined by three pulmonary pathologists (WAF, DTM and JDH) and reclassified according to criteria and nomenclature of the recently published 2015 edition of the WHO classification.
Results:
There was 92% concordance between submitting and reference pathologists when cases were grouped into the broad categories of adenocarcinoma, squamous carcinoma, neuroendocrine and large cell lung carcinoma (LCLC). The WHO classification permitted a more detailed analysis of the tumors. Invasive adenocarcinoma was the largest tumor category comprising 61% (127) of all tumors and included 70 acinar tumors, 23 solid, 13 papillary, 8 micropapillary, 5 mixed mucinous/non-mucinous, 4 invasive mucinous, 3 lepidic and 1 adenocarcinoma that could not be further classified. There were 48 (23%) squamous tumors, 10 (5%) LCLC, 15 (7%) neuroendocrine tumors including 6 (3%) small cell lung carcinomas. Finally, one tumor had sarcomatoid histology and an additional tumor was classified at sclerosing pneumocytoma. On reclassification, only 5 of the 26 tumors originally referred to as BAC or as having BAC features by submitting pathologists met criteria for adenocarcinoma in situ or minimally invasive carcinoma. Twenty-one of these 26 tumors were reclassified as invasive adenocarcinoma, most frequently acinar pattern predominant (8 cases).
Conclusion:
Reclassification of tumors identified through low dose CT screening in the National Lung Screening Trial permitted a detailed analysis of histological features and should permit a more nuanced assessment of biology and prognosis of this important cohort than has been available to date. Reclassification of BAC mainly as invasive adenocarcinoma conflicts with the suggestion that much of the benefit in the NLST CT screening trial was derived from surgical removal presumably non-invasive low grade tumor. *ACRIN received funding from the National Cancer Institute through the grants U01 CA079778 and U01 CA080098.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-010 - Pilot Internet Survey of Interobserver Variability in Pathology Diagnoses of Multiple Tumor Nodules (ID 2851)
09:30 - 09:30 | Author(s): W.A. Franklin
- Abstract
Background:
The distinction between separate primary lung cancers (SPLC) or intrapulmonary metastases (IM) is of great clinical importance because of the substantial staging and prognostic implications. With the broad implementation of CT screening for lung cancer, the recognition of multiple tumor nodules is increasingly common. Currently, similarities and differences in histology between two tumors provide the most definitive distinction between SPT and IM. However, the level of agreement among pathologists regarding this question has not been tested. The IASLC Pathology Committee and the Multidisciplinary SPT Working Group has addressed this issue through a pilot online survey. This study assesses the feasibility and reports preliminary results of a web-based survey to determine interobserver variation in distinguishing SPT and IM.
Methods:
A pilot study was conducted to test whether multiple observers could assess a collection of 50 cases of multiple tumors through a digital web-based system. Five pairs of resected nodules were assembled from the University of Colorado and scanned into an image database using an Aperio AT2 slide scanner (Leica Biosystems) with a 40X objective. Reviewers were asked to review slide images, to provide a histological diagnosis according to WHO criteria, to answer questions regarding specific histological details related to each nodule and to determine whether the multiple nodules were SPT and IM. Combined results were evaluated for level of concordance on the central question of primary or metastatic status. Results were also correlated with EGFR, KRAS, ALK and TP53 mutational status.
Results:
A total 21 pulmonary pathology subspecialists completed the survey, evaluating 10 nodules from 5 patients. Ten of the reviewers were from the US, 3 from Japan, 2 from the UK, and one each from Canada, France, Germany, the Netherlands, Korea and Sweden. On the question of SPLC vs IM, 10 reviewers agreed on all cases and these determinations were regarded the histological consensus. There was 85% overall concordance with the consensus diagnosis. Most of dissenting opinions related to a single case. In all but one instance, tumors from the same individual with different histological diagnoses were designated SPLC. However, in 30% of the cases, tumors from the same individual with identical histological diagnoses were determined to be SPLC. The histological attributes regardless of WHO diagnostic category that significantly (each p>0.0001) contributed to this conclusion included lepidic growth, cell size, nuclear pleomorphism and nucleolar prominence. The mutational status of these cases was in complete agreement with the histological consensus. Mutations that distinguished SPT included KRAS, EGFR or TP53 mutation in only one member of a tumor pair or different EGFR mutations in each member of a pair. In IM, identical KRAS mutation was found in both members of a tumor pair.
Conclusion:
In this pilot study a high level of consensus was achieved in separating SPLC vs or IM. A large minority (30%) of tumor pairs with identical histological diagnoses were determined to be SPLC suggesting that histological features beyond those used for WHO classification are taken into account when determining SPT status.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-016 - Minority Exon 19 Deletions Also Have Major Response of EGFR Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer (ID 2658)
09:30 - 09:30 | Author(s): W.A. Franklin
- Abstract
Background:
This study points out an issue of PCR methods to detect exon 19 deletions. Exon 19 deletions are most important among exon 18 to 21 EGFR mutations to dictate EGFR tyrosine kinase inhibitors (EGFR-TKIs) therapy in non-small cell lung cancer (NSCLC), and exon 19 deletions and insertions have over 170 species by catalog of somatic mutation in cancer (COSMIC). PCR methods are used for clinical examination, because they are useful, rapid and cost-effective to detect EGFR mutations. Some PCR methods could detect all of exon 19 deletions and insertions, while others could not. We investigated the clinical significance of minority exon 19 deletions, which could not be detected according to the PCR methods, selected majority deletions.
Methods:
The study included a series of 73 NSCLC patients, which were treated with EGFR-TKI for recurrent disease after they had undergone surgery from 1992 to 2004. EGFR mutations were detected in 34 (47%) in 73 patients. Sixty patients were evaluable for response, and remaining 13 patients who had taken EGFR-TKI for less than one month. In 60 assessable patients, exon 19 deletions and exon 21 point mutation were detected from 19 patients and 10 patients, respectively. Patients with EGFR mutations had significantly higher response rates to EGFR-TKI than those with wild-type (p=.047), and exon 19 deletions had still rates (p=.024). In 51 samples, including 17 exon 19 deletions and 6 exon 21 mutations, four PCR methods are commonly used in Japan, were performed and compared. PCR-based methods were (1) PCR-Invader for the selected common mutations of exons 18, 19, 20 and 21, and micro capillary electrophoresis for the exhaustive detection of exon 19 deletions and insertions, (2) Peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp for the selected common mutations of exons 18, 19, 20 and 21, and direct sequence for the other mutations, (3) Cycleave PCR for the selected common mutations of exons 18, 20 and 21, and fragment analysis with micro capillary electrophoresis for the exhaustive detection of exon 19 deletions and insertions, (4) Scorpion Amplification Refractory Mutation System (ARMS) for the selected 29 mutations including 19 species of exons 19 deletions and insertions.
Results:
All four methods detected 6 exon 21 mutations as L858R point mutation. However, in exon 19 deletions and insertions including over 170 species, only micro capillary electrophoresis detected all 17 exon 19 deletions. PNA-LNA PCR clamp and direct sequence missed one 9 bp short deletion “L747-E749 del”, which had complete response on EGFR-TKI therapy. Scorpion ARMS missed one 24 bp deletion and insertion “T751-I759 del ins S”, which had stable disease for over 3 years on EGFR-TKI therapy.
Conclusion:
This study suggests micro capillary electrophoresis is necessary for the exhaustive detection of exon 19 deletions and insertions, and may identify tumors responsive to EGFR-TKIs therapy, especially those with small or unusual deletions.
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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): W.A. Franklin
- 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.
