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Å. Helland
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.01 - Genomic Characterization of Large-Cell Neuroendocrine Lung Tumors (ID 1667)
11:05 - 11:16 | Author(s): Å. Helland
- Abstract
Background:
Neuroendocrine lung tumours account for 25% of all lung cancer cases, and they range from low-aggressive pulmonary carcinoids (PCA) to highly malignant small-cell lung cancer (SCLC) and large-cell neuroendocrine lung carcinoma (LCNEC). The last two are strongly associated with heavy smoking and are typically detected at a clinically advanced stage, having a poor survival. Comprehensive genomic analyses in lung neuroendocrine tumours are difficult because of limited availability of tissue. While more effort has been done in the context of SCLC, the detailed molecular features of LCNEC remain largely unknown.
Methods:
We conducted 6.0 SNP array analyses of 60 LCNEC tumours, exome sequencing of 55 tumor-normal pairs, genome sequencing of 11 tumour-normal pairs, transcriptome sequencing of 69 tumours, and expression arrays on 60 tumors. Data analyses were performed using in house developed and published pipelines.
Results:
Analyses of chromosomal gene copy number revealed amplifications of MYCL1, FGFR1, MYC, IRS2 and TTF1. We also observed deletions of CDKN2A and PTPRD. TTF1 amplifications are characteristic of lung adenocarcinoma (AD); CDKN2A deletions are frequent alterations in both AD and squamous-cell lung carcinoma (SQ); FGFR1 amplifications are found in SQ and, less frequently, in SCLC; and MYCL1 and IRS2 amplifications are frequent events in SCLC. Similar to the copy number data, we found patterns of mutations characteristic of other lung cancer subtypes: TP53 was the most frequently mutated gene (75%) followed by RB1 (27%), and inactivation of both TP53 and RB1, which is the hallmark of SCLC, occurred in 20% of the cases. Mutations in STK11 and KEAP1-NFE2L2 (frequently seen in AD and SQ) were found in 23% and 22% of the specimens, respectively. Interestingly, mutations in RB1 and STK11/KEAP1 occurred in a mutually exclusive fashion (p-value=0.016). Despite the heterogeneity observed at the mutation level, analysis of the pattern of expression of LCNEC in comparison with the other lung cancer subtypes (AD, SQ, SCLC, and PCA) points to LCNEC as being an independent entity. An average mutation rate of 10.7 mutations per megabase was detected in LCNEC, which is in line with the rate observed in other lung tumours associated with smoking. We found that, similar to SCLC, the mutation signatures associated with APOBEC family of cytidine deaminases, smoking, and age (based on Alexandrov et al 2013) were the predominant ones in LCNEC. However, the contribution of the individual SCLC and LCNEC samples to these three signatures was quite different, and we are currently exploring it.
Conclusion:
Taking into account somatic copy number and mutation data, we distinguished two well-defined groups of LCNEC: an SCLC-like group, carrying alterations in MYCL1, ISR2, and in both RB1 and TP53; and a group resembling AD and SQ, with alterations in CDKN2A, TTF1, KEAP1-NFE2L2, and STK11. Although these results suggest that LCNEC might be a mix of different lung cancer subtypes, mutation clonality and expression analyses show that they are likely to be a separate entity, sharing molecular characteristics with the other lung cancer subtypes. Their heterogeneity suggests that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ.
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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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P1.04-060 - Pathways Involved in Lung Adenocarcinomas, - Integrated Analyses on Methylation and mRNA Data (ID 2699)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
Lung cancer is one of the biggest cancer killers in the world. Despite certain recent advances, mortality is still high. Targeted therapy has increased the time to death for metastastic lung cancer, but such therapy is not available for all lung cancer patients. Targeted therapy is more often available for never smokers, due to presence of druggable driver mutations. In order to search for new putative targets of therapy, we seek to identify pathways involved different subgroups of patients and in patients with early relapse.
Methods:
A total of 190 patients undergoing surgery for lung cancer were included in the study (154 EGFR positive, 23 EGFR negative, 170 smokers and 20 non-smokers). Lung cancer tissue and clinical information was available for all patients and normal lung tissue was available for 30 of the patients. Whole genome expression array analysis (Agilent) was performed using mRNA isolated from all samples and DNA-methylation was analysed for 168 tumours and 21 matched normal lung tissue samples. R was used for statistical analyses; annHeatmap (from Heatplus) for hierarchical clustering, limma to identify differentially expressed genes, SPIA for pathway analysis and canonical correlation of methylation and mRNA-expression was performed with the CCA function from the PMA package. Pathways with an FDR<0.1 were considered significant. DAVID was used for gene ontology analysis.
Results:
Based on correlation of mRNA and methylation, different pathways were identified as predominant in specific subgroups of lung adenocarcinomas. Preliminary results indicate that genes involved in the KEGG-pathways cell cycle are more highly expressed in EGFR positive than in EGFR negative tumours in smokers. In the EGFR-negative tumours, several pathways are up-regulated: Oocyte meiosis, progesterone-mediated oocyte maturation, HTLV-1 infection, p53 signalling pathway and small cell lung cancer. For non-smoking patients, four pathways were up-regulated in EGFR-positive tumours: ECM-receptor interaction, TGF-beta signalling pathway, bile secretion and cocaine addiction. There were no pathways up-regulated in EGFR-negative compared with EGFR-positive never-smokers. This may partly be due to small numbers. Similarly, pathways dominating the tumours of patients with early relapse will be identified. Genes whose expression and methylation status were correlated were identified within smokers and non-smokers separately.
Conclusion:
Based on correlation between mRNA and methylation, specific pathways were identified activated in subgroups of lung adenocarcinomas. There are significant differences between ever-smokers and never-smokers. Survival analyses are ongoing.
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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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P2.04-011 - Whole-Genome Copy Number Analyses of NSCLC Tumors Reveal Aberrations Associated With EGFR Mutations and May Have Prognostic Impact (ID 1504)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
Knowledge about genetic alterations in Non-Small Cell Lung Cancer (NSCLC) has given us a significant insight in the biology of these tumors. It is of great clinical importance with consequences for the patients, and DNA mutations and translocations are currently targets for therapy. Aberrations in DNA copy number are frequent events in NSCLC tumors and important in tumorogenesis. In this present study we want to investigate how the copy number changes varies between different subgroups of NSCLC tumors based on the patients’ smoking status, histology or EGFR-, KRAS- and TP53 mutations. The DNA copy number data will be integrated with global mRNA expression to study the cis-associated mRNA expression changes. Last, we want to investigate whether genomic events, like specific copy number changes or the complex arm-wise aberration index (CAAI), have prognostic impact in patients with NSCLC.
Methods:
In this study we have included 200 patients with operable NSCLC tumors. Copy number data were obtained by using the Affimetrix Genome-wide human SNP array 6.0. Histopathological information, EGFR-, KRAS- and TP53 mutation status were determined and clinical information and follow-up data was obtained for all patients. The mRNA expression was determined by the Agilent 60K mRNA expression array on a subset of 117 patients. The data was analyzed by using bioinformatic tools like ASCAT and integration of the mRNA data and the survival analyses are on-going.
Results:
Preliminary results have shown that copy number aberrations are frequent events in NSCLC tumors, consistent with previous reports. We have identified that the copy number patterns differ between adenocarcinomas and squamous cell carcinomas, and between tumors from patients with different smoking history. However, the largest differences were found between the EGFR-mutated adenocarcinomas compared with EGFR wildtype tumors, where we identified a specific pattern of copy number changes in the tumors that harbour EGFR mutation. These changes were mainly located at chromosome arm 1p, 2p, 3q, 5q, 7, 12 and 13. Preliminary analyses have also identified specific copy number aberrations with prognostic significance.
Conclusion:
Copy number aberrations are frequent in NSCLC tumors and may have great impact on gene expression and give us valuable prognostic information. EGFR-mutated adenocarcinomas have a specific pattern of copy number changes, which provides new insight of the biology of these tumors.
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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: 3
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-030 - Are Experienced Physicians More Likely to Acquire Adequate Tissue Material for EGFR-Testing? (ID 2405)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
Epidermal Growth Factor Receptor (EGFR) mutation testing is now recommended practice for non-squamous non-small cell lung cancer. The patients with activating EGFR mutations are eligible for targeted personalized treatment offering better survival and quality of life, often with low toxicity. However, little is known about how the physician’s level of experience influences the quality of samples taken for EGFR-analysis and if complicated interventions result in more inadequate samples. We therefore performed a retrospective analysis on correlation between doctors’ experience and tissue quality at a moderately-sized community hospital.
Methods:
The Norwegian Lung Cancer Group (NLCG) recommended EGFR- testing of all patients with non-small cell lung carcinoma from June 2010. In March 2013 squamous cell carcinomas were excluded. Basic demographic data, sample type, test results and procedure related complications were recorded for the period June 2010 to December 2013, and the level of experience (measured as inexperienced physicians having less than 10 procedures per year) of the involved physicians was recorded.
Results:
Material was sent for EGFR analysis for 256 of the 304 eligible patients diagnosed in the period. For a total of 34 patients (13%) the first biopsy was not analyzed at department of molecular pathology due to inadequate tumor material. The tissue collected by experienced physicians was sufficient for EGFR analyses in 91-97.2% (median 93%), compared to 50-90% (median 86.7%) for the less experienced physicians. For image supervised biopsies, non-analyzable samples were more frequent when puncturing small (<3 cm) peripheral tumors than when taken from large central tumors. Of 14 image guided biopsies that were returned because of inadequate tumor tissue, only two had complications: one with bleeding and the other with pneumothorax. Both tumors were peripheral. Of three bronchoscopical biopsies that were returned due to inadequate tumor tissue, one was complicated by major bleeding, in another the patient was very restless during the procedure. The last was uncomplicated.
Conclusion:
Our results show that the quality of image guided biopsies taken by more experienced physicians is better than those taken by doctors with less experience. For small peripheral tumors, the frequency of non-analyzable samples were higher than for large central tumors taken by image guided biopsy.
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P3.04-056 - Some Lung Cancer Patients End up without an EGFR-Mutation Analysis (ID 2394)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
Lung cancer patients with activating mutations in the EGFR-gene are eligible for targeted therapy with tyrosine kinase inhibitors, and clinicians strive for acquiring enough tumor tissue for the needed diagnostic analyses. However, not all patients have an EGFR-test, and little is known about the subsequent steps for patients with inadequate first biopsy.
Methods:
Data on the diagnostic work-up on all NSCLC patients eligible for EGFR-testing was collected at a medium-sized Norwegian hospital for the period June 2010 to December 2013. For samples without successful EGFR-mutation results, we recorded possible explanations.
Results:
Material was sent for EGFR analysis for 256 of the 304 eligible patients diagnosed in the period. For a total of 34 patients (13%) the first biopsy was not analyzed at the department of molecular pathology due to inadequate tumor material. Of these 34 patients, 23 (65%) had no new sample submitted for analysis. 13 of the 23 (57%) were in stage IV, and of these, three did not want active treatment and one was not a candidate for active treatment because of poor general condition. One patient was not re-biopsied due to rapid disease progression. Eigth patients were for no obvious reason never considered for re-biopsies, including a younger patient who had brain metastases at the time of diagnosis, but who lived for 19 months after diagnosis. One of the 11 sent was diagnosed with activating EGFR mutation in the second sample sent for analyses. For patients with rejected samples, EGFR results were available after 17 - 69 days (median 38) from rejection of the first sampling.
Conclusion:
For 65% of the rejected samples, no new samples were submitted for analysis. 57% of the patients with no new sample taken, were in stage IV. When a new biopsy was planned, our study shows that EGFR results from the new sampling were available after median 38 days. For this patient group, with poor prognosis and often rapid disease progression, one should strive for a new sampling and a quicker turn-around time.
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P3.04-125 - Cytokine Profiles in Non-Small Cell Lung Cancer Patients Undergoing Palliative Thoracic Radiotherapy; Predictor of Response? (ID 2588)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
A majority of patients with non-small cell lung cancer are diagnosed in later stages of disease where no curative treatment is currently available. The prognosis for these patients is poor. Median survival from diagnosis in stage IV is approximately 9 months. Many patients will benefit from external radiotherapy to the thorax for alleviation of symptoms due to advanced lung cancer. Despite adequate radiotherapy, however, many tumors progress locally in the radiation field. Here, we investigate whether the kinetics of cytokines in serum can be utilized as predictors for tumor response to radiotherapy and/or predictors of lung toxicity.
Methods:
Patients with histologically confirmed non-small cell lung cancer, eligible for palliative radiotherapy to the hilus-mediastinum, were included in a randomized phase II clinical trial; the ThoRaT-study. Patients were randomised to 1 of 2 study arms undergoing thoracic radiotherapy, 3 Gy x 10, with or without the addition of erlotinib concomitant with radiotherapy treatment. Side effects were recorded and graded according to CTC version 4.0. Clinical response in the radiation field was evaluated by CT or PET-CT scans. Blood serum was sampled at different time points; prior to treatment, at mid-therapy, at the end of therapy and 6 week following treatment completion. Multiplex immunoassays were used to measure serum concentration of 52 cytokines and 9 MMPs on all collected samples. Serum samples from COPD patients were included as controls.
Results:
Cytokine analyses of serum samples are ongoing and have to date been performed on 43 non-small cell lung cancer patients. Pre-treatment and follow up CT/PET-CT scans are currently under revision. Preliminary investigations show considerable variation in cytokine patterns between the patients and between different time points for some of the cytokines. Analyses of possible predictors for radiotherapy response and toxicity, as well as comparison with normal controls are currently ongoing and will be presented.
Conclusion:
We hypothesize that pre-treatment cytokine values and/or kinetics of concentration changes may provide information on the probability of clinical response and side effects from radiotherapy.
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P3.06 - Poster Session/ Screening and Early Detection (ID 220)
- Event: WCLC 2015
- Type: Poster
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.06-011 - Unique Combination of 6 Circulating microRNAs for Early Detection of Lung Cancer (ID 2130)
09:30 - 09:30 | Author(s): Å. Helland
- Abstract
Background:
Worlwide, lung cancer is the primary cause of cancer death. Today 75% of patients are diagnosed in a locally advanced or metastatic inoperable stage, and a new tool for early detection of lung cancer is urgently needed in order to improve the outcome. Circulating microRNAs have emerged as stable, non-invasive and promising biomarkers for diagnosis, prognostication and prediction in cancer. The purpose of this study was to identify circulating microRNAs for detection of early stage lung cancer, capable of discriminating lung cancer patients from those with chronic obstructive pulmonary disease (COPD) and healthy normal individuals.
Methods:
We profiled the expression of 756 unique microRNAs in sera from 38 patients with NSCLC, 16 patients suffering from COPD and 16 healthy volunteers, to explore the potential of the microRNAs as diagnostic biomarkers. For validation of our results, we analyzed serum from an independent cohort of high-risk individuals enrolled in the IELCAP screening trial (n=161) using RT-qPCR
Results:
Focusing on microRNAs upregulated in sera from lung cancer patients, we identified a unique set of 6 microRNAs with significantly higher abundance compared with sera from COPD patients and healthy normals. Validation of the 6-miR signature demonstrated a sensitivity of 86% and specificity of 79.3%
Conclusion:
Considering their accessibility and stability, circulating microRNAs can be a diagnostic tool for clinicians in the future, and may lead to increased fraction of lung cancers diagnosed in an early curative stage. The 6-miR signature may be a basis for a screening study and can easily be implemented in the clinic to identify those who should be further examined for lung cancer
