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S. Ekman
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MINI 10 - ALK and EGFR (ID 105)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T. Yap, T. Li
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 1a-1f
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MINI10.05 - ALK Rearrangements in Non-Small Cell Lung Cancer: Comprehensive Integration of Genomic, Gene Expression and Protein Analysis (ID 2731)
17:10 - 17:15 | Author(s): S. Ekman
- Abstract
- Presentation
Background:
Identification of EML4-ALK fusion proteins has revolutionized the treatment of a subgroup of non-small cell lung cancer (NSCLC) patients. Although the gene inversion is regarded as the relevant event for therapy response, the relation between gene rearrangement, mRNA and protein levels has not been evaluated in detail. Thus, the objective of this study was to comprehensively define the molecular relations induced by ALK rearrangements in a large representative Swedish NSCLC cohort incorporating genomic, gene expression and protein data, as well as corresponding clinical correlates.
Methods:
ALK protein analysis was performed on 860 NSCLC patients (551 adenocarcinoma, 224 squamous cell carcinomas, 85 large cell carcinomas/NOS) using immunohistochemistry (IHC) on tissue microarrays (TMAs), applying an established monoclonal ALK antibody (clone D5F3, Cell signaling). In parallel, ALK rearrangement was determined by fluorescent in situ hybridization (FISH, Abbott, Vysis ALK Break Apart FISH Probe Kit) on the same TMAs. A subgroup of patients was additionally analyzed utilizing gene expression microarrays (Affymetrix, n=194) or RNA-sequencing (n=202). The RNA sequencing data was also used to identify ALK gene fusions.
Results:
ALK protein expression was observed in 12/860 (1.4%). ALK rearrangement was detected in 11/860 samples (1.3%) by FISH analysis. Of 194 patients evaluated by microarray, six (3.1%) showed high ALK gene expression and of 202 patients analyzed by RNA-seq, nine (4.5%) demonstrated high ALK transcript levels. Of the 11 FISH rearranged patients, eight (73%) showed positive protein expression. High ALK gene expression was observed in all four ALK-FISH rearranged samples with matching microarray or RNA-seq data. Of five patients with positive protein expression, only three (83%) showed high gene expression levels according to gene expression microarray and RNA-seq data. RNA-seq revealed that 2/202 samples were ALK rearranged, both of which were detected by FISH and IHC. One sample that was not rearranged according to RNA-seq-data did, however, demonstrate rearrangement with FISH.
Conclusion:
The overall frequency of ALK rearrangements in this NSCLC cohort was lower than previously reported, with a significant but variable correlation on different molecular levels. It is possible that technical issues with regard to the use of TMAs, where only a fraction of the whole tumor is represented, may have hampered the results. Therefore, the FISH and IHC analysis will be complemented with assessments on whole tissue sections. The discordant results also stress the need for careful validation of these methods before they can be implemented in the clinical practice.
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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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MINI21.10 - The TORK/DNA-PK Inhibitor CC-115 Shows Combination Anti-Proliferative Effects with Erlotinib in NSCLC Cells Resistant to EGFR Inhibition (ID 641)
17:40 - 17:45 | Author(s): S. Ekman
- Abstract
Background:
In non-small cell lung cancer (NSCLC), activation of the phosphoinositide-3-kinase (PI3K)/mTOR pathway is common in tumors resistant to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). CC-115 (Celgene Corporation), an mTOR kinase inhibitor that targets both mTORC1 and mTORC2 as well as DNA-dependent protein kinase (DNA-PK), is currently under early clinical development. We evaluated CC-115 in combination with Erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.
Methods:
In the present study we investigated whether CC-115 is able to increase the therapeutic effect of the EGFR TKI Erlotinib in several different NSCLC cell lines which exhibit intermediate or high resistance to EGFR TKIs: A549, H1975, H1650, HCC95, H2122 and H23. Mechanisms of inhibition were analyzed with assays for proliferation, apoptosis, and cell cycle progression. Cell signaling activity was analyzed using phospho-specific antibodies in Western blotting. Xenograft mice studies were performed to confirm the results in vivo.
Results:
CC-115 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.07 up to 6.9 mM. The anti-proliferative efficacy of Erlotinib was increased in the NSCLC cells synergistically by combination treatment with CC-115 with combination indices down to 0.04-0.2, indicating strong synergy. The synergistic, anti-proliferative effect of the combination treatment could be explained by increased cell cycle arrest and inhibition of signaling components in the mTOR pathway, especially 4E-BP1. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of Erlotinib and CC-115.
Conclusion:
We demonstrate that the therapeutic effect of the EGFR tyrosine kinase inhibitor Erlotinib can be increased by simultaneous treatment with the mTOR kinase/DNA-PK inhibitor CC-115, justifying further clinical studies in lung cancer patients with primary or acquired resistance to EGFR TKIs.
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MS 11 - New Approaches to Combined Modality Therapy for Stage III Disease (ID 29)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Treatment of Locoregional Disease – NSCLC
- Presentations: 1
- Moderators:D. Jablons, J.P. Van Meerbeeck
- Coordinates: 9/08/2015, 14:15 - 15:45, 605+607
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MS11.04 - Overview of Current International Randomized Trials (ID 1899)
15:20 - 15:40 | Author(s): S. Ekman
- Abstract
Abstract:
Overview Stage III non-small cell lung cancer (NSCLC) is a heterogeneous disease with regard to tumor extent, prognosis and treatment options. Surgery is indicated in some patients, but the majority of patients receive radiotherapy and chemotherapy alone. The standard of care for unresectable stage III NSCLC patients with a good performance status consists of concurrent chemoradiation. The chemotherapy regimen usually consists of a platinum doublet and radiation doses of at least 60 Gy is standard. The prognosis for these patients remains dismal with a median survival time of 15-22 months and the need for improved treatment approaches is urgent. The optimal choice of chemotherapy and radiation dose and the schedules for these have been under investigation but is still not clearly defined. Radiation dose escalation studies have not resulted in better outcomes and with potentially harmful effects. Clinical studies of targeted agents in unselected patient groups, including therapies against epidermal growth factor receptor (EGFR) and angiogenic factors, have not been successful. The recent advances in molecularly targeted therapies together with technological advances in radiotherapy opens up for novel treatment strategies with potentially improved outcomes and less toxicity. This presentation will give an overview of randomized studies incorporating new approaches to combined modality therapy in stage III disease, including immune therapy with PD-1/PD-L1 inhibitors, inhibitors of EGFR and ALK as well as proton radiotherapy.
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ORAL 41 - Immune Biology, Microenvironment and Novel Targets (ID 159)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.07 - The Identification of Therapeutic Targets in Lung Cancer Based on Transcriptomic and Proteomic Characterization of Cancer-Testis Antigens (ID 1555)
19:35 - 19:46 | Author(s): S. Ekman
- Abstract
- Presentation
Background:
Most immunotherapeutic modalities are based on the concept that the immune system can attack targets that are specifically expressed in cancer cells. Cancer testis antigens (CTAs) are a group of genes with a broad expression in cancers including non-small cell lung cancer (NSCLC). In normal tissues the expression of CTAs is restricted to immune privileged organs such as testis and placenta. This limited expression in somatic tissues renders CTAs as a valuable group of genes for the exploration of potential immunotherapeutic targets. The aim of this study was to comprehensively explore the CTA repertoire in NSCLC and to try identifying new CTAs.
Methods:
RNA sequencing (RNAseq) was performed on 202 NSCLC samples from a consecutive clinical cohort of surgically resected patients. For the analysis of the comprehensive CTA expression profile in NSCLC we used Cancer Testis (CT) Database containing all genes reported as CTAs in the literature. The NSCLC transcriptome was compared to the normal transcriptome comprising of 22 paired normal lung tissues as well as to 122 samples from 32 different normal human tissues. Corresponding protein expression was evaluated by using immunohistochemistry (IHC) on tissue microarrays (TMAs) containing tumor tissue from the same patients as used in the RNA sequencing.
Results:
Of the 276 established CTAs, 155 genes (56%) were restricted to testis and placenta among normal tissues and were identified as CTAs. One third (35%) was expressed in at least one of the 202 individual NSCLC cases and 28 of these genes were previously not reported to be expressed as CTAs in NSCLC. Applying stringent analysis criteria on our RNA sequencing data set we identified 61 genes that were expressed in NSCLC and testis or placenta, but not in other normal tissues. Thus, these genes present potential new CTAs. The specific cancer/testis expression of selected genes (ZNF560, TGIF2LX, TFPI2, HMGB3, TKTL1 and STK31) from this group was confirmed on protein level using IHC. Additional analysis revealed that most CTAs were concurrently expressed in adenocarcinoma and squamous cell carcinoma. The expression of a subset of genes was histology dependent, with predominant expression in adenocarcinoma (e.g. XAGE family members) and in squamous cell carcinoma (e.g. MAGE family members).
Conclusion:
Our study provides deep sequencing mRNA expression profiles of the whole CTA repertoire in NSCLC. Several CTAs previously identified in other cancers but not analyzed in NSCLC have been identified on both mRNA and protein level. Additionally, we have identified 61 novel genes as CTAs in NSCLC that previously have not been reported as CTAs and several of these were also confirmed on protein level. This data offers the opportunity to design individual therapy options to target single CTAs or CTA clusters.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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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: 2
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-021 - Mutation Profiling by Targeted Next-Generation Sequencing for Diagnostics and Patient Cohort Screening in FFPE NSCLC Samples (ID 920)
09:30 - 09:30 | Author(s): S. Ekman
- Abstract
Background:
Recent discovery of the landscape of somatic mutations in non-small cell lung cancer (NSCLC), and introduction of new therapeutics have raised the demands for multiplex mutation assays. In exploratory research, mutation profiling has largely been performed on fresh-frozen tissue from surgical specimens. However, for patients with advanced disease the assays need to be adapted to small formalin-fixed paraffin embedded (FFPE) biopsies and cytology preparations. Targeted next-generation sequencing (NGS) techniques are now being developed to address these challenges and have now reached the point where they are more cost efficient than previously used methods, hence there is a need to optimize and validate these techniques to determine if they are robust enough to work in clinical diagnostics.
Methods:
Here we have developed and evaluated Haloplex gene panels in comparison to pyrosequencing and quantitative PCR(qPCR), i.e. the current standard methods for molecular diagnostics of solid tumours in Sweden. The target enrichment was focused on short DNA fragments and included independent capture of complementary strands, “two strand capture”, to address fragmentation and base damage induced by formalin fixation. The panels include all exons of 18-32 genes (for lung cancer and other solid tumors respectively) with known clinical relevance. Seventy-one clinical samples (NSCLC, colorectal carcinoma and melanoma), with known mutational status of hotspots in KRAS, BRAF, NRAS, PIK3CA and EGFR, were selected for analysis. DNA was prepared from FFPE tissues and used for library preparation using the panels and subsequently sequenced on an Illumina MiSeq instrument.
Results:
A complete concordance was seen between the previously defined pyrosequencing and qPCR genotypes and the corresponding variants detected using the gene panels. Both point mutations and smaller indels (<25bp) could be detected by this technique using an in-house bioinformatic pipeline. False positive FFPE-induced mutation artefacts could reliably be identified by the two-strand filter. The technical sensitivity of mutation detection was determined to 2%, and we have decided to use a 5% variant allele frequency threshold for clinical reporting. In addition, clonality and subclonality could be discovered in patients with complex tumour disease (mixed or multiple tumour lesions) by analysis of the mutation patterns. An extended 85 gene panel has also been designed to screen for mutations in NSCLC patient cohorts for clinical molecular research.
Conclusion:
We believe that the established lung cancer gene panels for targeted enrichment and NGS can replace pyrosequencing and qPCR for molecular diagnostics in NSCLC, and will be useful for screening of unselected population-based prospective and retrospective lung cancer patient cohorts in clinical research.
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P3.04-076 - The Crux of Molecular Prognostications in NSCLC: An Optimized Biomarker Panel Fails to Outperform Clinical Parameters (ID 2586)
09:30 - 09:30 | Author(s): S. Ekman
- Abstract
Background:
The best known prognostic factors for non-small cell lung cancer (NSCLC) patients are age, tumor stage and performance status. Numerous proteins have been analyzed to improve the traditional prognostication. Even though some proteins have shown prognostic value, the performance is not sufficient to be introduced in the clinical routine. The aim of this study was to generate a prognostic classifier based on proteins that previously have shown reproducible prognostic value and represent different aspects of tumorigenesis.
Methods:
The selection of proteins was based on literature search, meta-analysis of gene expression data sets and availability of reliable antibodies towards these proteins. Finally, five proteins (Ki67, EZH2, SLC2A1, TTF1 and CADM1) were chosen and analyzed by immunohistochemistry on tissue microarrays comprising NSCLC tissue patients (n=673), divided into a training and a validation cohort. For each patient, one score was obtained for each of the five antibodies, integrating the staining intensity and the fraction of stained tumor cells. Analyses were performed using all possible combinations of proteins and tested with or without clinical parameters. The C-index was used to develop the best prediction model on a training cohort (n=326) and the model was subsequently validated in the validation cohort (n=347).
Results:
All five proteins showed a significant prognostic impact in the univariate and the multivariate Cox analyses. Using a combination of the protein scores, the model was then fitted to provide the best prognostic performance (C-index=0.60). This did, however, not outperform the use of clinical parameters alone (C-index=0.62). The same was true when the analyses were performed separately for the adenocarcinoma (C-index=0.60) and the squamous cell carcinoma subgroup, respectively (C-index=0.60). More importantly, the addition of protein data to the clinical information (C-index=0.62) did not improve the prognostic value of the clinical parameters alone (C-index=0.60). To substantiate the results of our test cohort, we transferred the best prognostic model for all NSCLC, only adenocarcinomas and only squamous cell carcinomas respectively to a validation cohort. Again, all proteins showed prognostic relevance in the univariate analysis but did not perform better, alone or in combination, than the clinical parameters.
Conclusion:
Here we have performed a comprehensive analysis in order to obtain the best survival prediction model by using clinical parameters and the expression of five proteins. Although we chose strict criteria for protein marker selection, the prognostic power of these proteins was inferior to the traditional clinical parameters. Our findings question the general concept of using protein markers for prognostication in NSCLC but stress the value of careful assessment of traditional parameters in clinical practice.
