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P. Micke
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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): P. Micke
- 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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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): P. Micke
- 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.
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P2.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 207)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.01-061 - COX-2 Expression Does Not Predict Outcome of Celecoxib in Addition to Standard Chemotherapy in Advanced Non-Small Cell Lung Cancer (ID 2100)
09:30 - 09:30 | Author(s): P. Micke
- Abstract
Background:
Increased expression of cyclooxygenase-2 (COX-2) is common in non-small cell lung cancer (NSCLC), and is therefore a potential target for treatment. However, phase III trials have failed to demonstrate beneficial survival effects of adding COX-2 inhibitors to standard chemotherapy. We investigated whether COX-2 expression in tumor and stromal cells had any predictive impact on the effects of celecoxib, a selective COX-2 inhibitor.
Methods:
In a previously published multicenter phase III trial, 316 patients with NSCLC stage IIIB or IV and WHO performance status 0-2 were randomized to receive celecoxib 400 mg b.i.d. or placebo up to one year in addition to a two-drug platinum-based chemotherapy regimen. In a subset of 122 patients, archive tumor tissue was available for further analyses. Immune stainings for COX-2 expression were undertaken. Intensity and extent of positively stained cells in tumor and stroma cells were scored on a 0 to 3 scale, and the product of these scores was used as a co-variable in the predictive analysis.
Results:
An updated analysis of all 316 patients included in the original trial, and of the 122 patients with available tumor tissue showed no survival differences between the celecoxib or placebo arms (HR 0.99; 95% CI 0.79-1.24 and HR 0.89; 95% CI 0.62-1.28, respectively). Similarly, in patients with high COX-2 expression in tumor cells (n=71) or stroma cells (n=55), survival did not differ significantly between patients who received celecoxib or placebo (HR 0.96; 95% CI 0.60-1.54 and HR 0.66; 95% CI 0.38-1.16). The p-value for interaction effect between COX-2 score in tumor or stroma cells and celecoxib effect on survival was 0.48 and 0.25, respectively.
Conclusion:
In this subgroup analysis of patients with advanced NSCLC treated in a randomized trial, we could not detect any significant interaction between COX-2 expression in tumor or stroma cells and outcome of celecoxib treatment in addition to standard chemotherapy.
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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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P3.04-021 - 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): P. Micke
- 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.
