- Email: [email protected]
12 mRNA helicases as therapeutic targets in lung cancer
Poster abstracts of the 14th Annual British Thoracic Oncology Group Conference 2016 / Lung Cancer 91, Suppl. 1 (2016) S1–S71
sions were subjected to subregional laser capture microdissection, and purified DNA submitted for next generation panel/whole exome sequencing. Results: 102 tumours were classified: 7 type 1, 23 type 2, 28 type 3 and 44 type 4. Survival modelling reveals worsening outcome across subtypes. In addition, the rate of nodal metastasis was increased across the tumour types from 1 to 4, at 0%, 9%, 21% and 30% respectively. NGS DNA sequencing is in progress. Conclusion: We find that partly invasive lung adenocarcinomas fall into two distinct groups. We can morphologically separate tumours representing early invasion within in situ precursors from invasive lesions showing surface colonisation. The former group show very limited nodal metastasis, while the latter frequently show nodal spread. Wholly invasive lesions show the highest rates of metastasis, representing the most agressive form of early adenocarcinoma. This is an essential advance in our understanding of the adenoma/carcinoma sequence. It also enables the precise selection of archival tissues for sequencing studies to reveal the genomic changes that drive invasive behaviour. Molecular characterisation of these lesions and their various components is underway. Disclosure: All authors have declared no conflicts of interest. 12
mRNA helicases as therapeutic targets in lung cancer
F. Raza, M. Das, J. Le Quesne. Toxicology Unit, MRC, Leicester, United Kingdom Introduction: The dysregulation of protein synthesis is a key driving event many malignancies, including lung cancer. The mRNA helicase eIF4A is a ubiquitous protein essential for the initial unwinding of mRNAs and is frequently upregulated in tumours. It has emerged as a promising target for small molecule inhibitor therapies. Methods: Lung adenocarcinoma cell lines were used as a model. siRNA techniques were used to specifically reduce expression levels of the eIF4A isoforms eIF4A1 and eIF4A2, and compared to the effect of the nonspecific eiF4A inhibitor hippuristanol. Measured outputs include cell growth, cell cycle, apotosis, and polysome profiles. A duplex chromogenic immunohistochemical assay was optimised and applied to tissue microarrays of lung adenocarcinoma tissue. Results: As expected, knockdown of eIF4A1 and hippuristanol treatment both reduce translation initiation and show a reduced growth phenotype with accompanying cell cycle blockade. However, eIF4A2 knockdown shows enhanced growth in cell culture and leads to greatly reduced cellular apoptosis. Duplex immunohistochemistry shows wide variance in expression of eIF4A isoforms with normal and tumour tissues; generally eIF4A1 is more highly expressed in proliferative and malignant tissues, and the relative isoform expression within individual cells is markedly variable even within tumours. Conclusion: Much time and money is being invested into inhibitors of eIF4A as novel cancer therapeutics. However we find that one isoform, eIF4A2, is pro-apoptotic and acts as a brake on cellular growth. Furthermore, the relative expression of the two isoforms varies widely between cases of lung adenocarcinoma, and they are expressed in a distribution further suggestive of roles as mediators of tumour cell plasticity with distinct functions. This suggests that treatment using nonspecific eIF4A inhibitors may have adverse effects in cases which are growth limited by higher levels of eIF4A2. Our duplex assay is of potential value as a biomarker to identify cases suitable for anti-eIF4A small molecule therapies. Disclosure: All authors have declared no conflicts of interest.
13
S5
Creation of a large single-centre retrospective tumour archive
M. Das 1 , D.A. Moore 2 , M. Sereno 2 , C. Smith 2 , R. Hastings 3 , J. Le Quesne 1 . 1 Toxicology Unit, MRC, Leicester, United Kingdom; 2 Cancer Studies, University of Leicester, Leicester, United Kingdom; 3 Cancer Centre, University of Leicester, Leicester, United Kingdom Introduction: Leicester University Hospitals NHS Trust is a busy thoracic surgical centre, with around 200 lung resections for cancer being performed annually. Tissue microarrays (TMAs) enable the simultaneous application of in situ assays such as immunohiscotchemistry (IHC) and fluorescence in situ hybridisation (FISH) to many samples of tissue simultaneously. This project aims to construct a comprehensive TMA collection with accompanying clinicopathological and follow-up data on 2000 NSCLC patients from a single busy surgical centre. Methods: Generic ethical approval for retrospective tissue access was obtained. Cases (adenocarcinoma and squamous cell carcinoma) for inclusion were identified by application of specific criteria to cases in the diagnostic pathology database. Clinicopathological data are compiled from the pathology database, the National Cancer Registry, and other local databases. These include data on demographics, pathology and survival. Cases are anonymised, and a custom database using the RedCap application has been established behind the NHS firewall. For each case, archival slides are scanned, optimal tumour blocks (up to 3) identified, and TMAs containing 3×1 mm cores from different regions are constructed. TMAs are constructed in quadruplicate, and cores of tumour tissue retained for DNA extraction. Results: We have established a pipeline of TMA construction, and are accumulating cases into our physical TMA collection at a rate of around 30 cases (ie 90 cores) every 2 weeks. 400 cases so far have been incorporated. These arrays contain a generous amount of tissue to facilitate manual and automated analyses, and also enable some description of pathological heterogeneity. Conclusion: This retrospective tumour collection represents an invaluable platform for biomarker discovery, biological hypothesis testing, and hypothesis generation. In particular, it is invaluable for the application of quantitative multiplex assays. The use of digital pathological techniques has facilitated archive generation and added an extra dimension of image data. Disclosure: All authors have declared no conflicts of interest. 14
Phagocytic behaviour in lung adenocarcinoma cells: clinical implications and cellular mechanisms
H.L. Mackay, D.A. Moore, P. Muller, J. Le Quesne. Toxicology Unit, Medical Research Centre, Leicester, United Kingdom Introduction: Lung adenocarcinoma is the most common and histologically diverse form of lung cancer. “Cell-in-cell” structures arise when one viable tumour cell is found in a vacuole within another viable tumour cell. They have been identified in a variety of solid tumours. The repeated occurrence of theses structures may represent a process that confers a selective advantage to a malignant clone, via the acquisition of nutrients, horizontal gene transfer, or simple subclonal competition/extinction. However, the true biological significance and mechanisms of formation remain unknown. Methods: 100 consecutive cases of lung adenocarcinoma were dearchived and for each, 10 high-power fields (hpf) were examined. A scheme for identifying cell-in-cell appearances was formulated and used to score the tumours. In vitro coculture experiments using H1299 lung cancer cells labeled green or red (via transfection with fluorescent proteins) were also conducted. Cells were seeded on a selection of coated surfaces including gelatin, fibronectin and collagen to mimic extracellular matrix proteins. Results: Cell-in-cell structures are frequently observed in primary
sions were subjected to subregional laser capture microdissection, and purified DNA submitted for next generation panel/whole exome sequencing. Results: 102 tumours were classified: 7 type 1, 23 type 2, 28 type 3 and 44 type 4. Survival modelling reveals worsening outcome across subtypes. In addition, the rate of nodal metastasis was increased across the tumour types from 1 to 4, at 0%, 9%, 21% and 30% respectively. NGS DNA sequencing is in progress. Conclusion: We find that partly invasive lung adenocarcinomas fall into two distinct groups. We can morphologically separate tumours representing early invasion within in situ precursors from invasive lesions showing surface colonisation. The former group show very limited nodal metastasis, while the latter frequently show nodal spread. Wholly invasive lesions show the highest rates of metastasis, representing the most agressive form of early adenocarcinoma. This is an essential advance in our understanding of the adenoma/carcinoma sequence. It also enables the precise selection of archival tissues for sequencing studies to reveal the genomic changes that drive invasive behaviour. Molecular characterisation of these lesions and their various components is underway. Disclosure: All authors have declared no conflicts of interest. 12
mRNA helicases as therapeutic targets in lung cancer
F. Raza, M. Das, J. Le Quesne. Toxicology Unit, MRC, Leicester, United Kingdom Introduction: The dysregulation of protein synthesis is a key driving event many malignancies, including lung cancer. The mRNA helicase eIF4A is a ubiquitous protein essential for the initial unwinding of mRNAs and is frequently upregulated in tumours. It has emerged as a promising target for small molecule inhibitor therapies. Methods: Lung adenocarcinoma cell lines were used as a model. siRNA techniques were used to specifically reduce expression levels of the eIF4A isoforms eIF4A1 and eIF4A2, and compared to the effect of the nonspecific eiF4A inhibitor hippuristanol. Measured outputs include cell growth, cell cycle, apotosis, and polysome profiles. A duplex chromogenic immunohistochemical assay was optimised and applied to tissue microarrays of lung adenocarcinoma tissue. Results: As expected, knockdown of eIF4A1 and hippuristanol treatment both reduce translation initiation and show a reduced growth phenotype with accompanying cell cycle blockade. However, eIF4A2 knockdown shows enhanced growth in cell culture and leads to greatly reduced cellular apoptosis. Duplex immunohistochemistry shows wide variance in expression of eIF4A isoforms with normal and tumour tissues; generally eIF4A1 is more highly expressed in proliferative and malignant tissues, and the relative isoform expression within individual cells is markedly variable even within tumours. Conclusion: Much time and money is being invested into inhibitors of eIF4A as novel cancer therapeutics. However we find that one isoform, eIF4A2, is pro-apoptotic and acts as a brake on cellular growth. Furthermore, the relative expression of the two isoforms varies widely between cases of lung adenocarcinoma, and they are expressed in a distribution further suggestive of roles as mediators of tumour cell plasticity with distinct functions. This suggests that treatment using nonspecific eIF4A inhibitors may have adverse effects in cases which are growth limited by higher levels of eIF4A2. Our duplex assay is of potential value as a biomarker to identify cases suitable for anti-eIF4A small molecule therapies. Disclosure: All authors have declared no conflicts of interest.
13
S5
Creation of a large single-centre retrospective tumour archive
M. Das 1 , D.A. Moore 2 , M. Sereno 2 , C. Smith 2 , R. Hastings 3 , J. Le Quesne 1 . 1 Toxicology Unit, MRC, Leicester, United Kingdom; 2 Cancer Studies, University of Leicester, Leicester, United Kingdom; 3 Cancer Centre, University of Leicester, Leicester, United Kingdom Introduction: Leicester University Hospitals NHS Trust is a busy thoracic surgical centre, with around 200 lung resections for cancer being performed annually. Tissue microarrays (TMAs) enable the simultaneous application of in situ assays such as immunohiscotchemistry (IHC) and fluorescence in situ hybridisation (FISH) to many samples of tissue simultaneously. This project aims to construct a comprehensive TMA collection with accompanying clinicopathological and follow-up data on 2000 NSCLC patients from a single busy surgical centre. Methods: Generic ethical approval for retrospective tissue access was obtained. Cases (adenocarcinoma and squamous cell carcinoma) for inclusion were identified by application of specific criteria to cases in the diagnostic pathology database. Clinicopathological data are compiled from the pathology database, the National Cancer Registry, and other local databases. These include data on demographics, pathology and survival. Cases are anonymised, and a custom database using the RedCap application has been established behind the NHS firewall. For each case, archival slides are scanned, optimal tumour blocks (up to 3) identified, and TMAs containing 3×1 mm cores from different regions are constructed. TMAs are constructed in quadruplicate, and cores of tumour tissue retained for DNA extraction. Results: We have established a pipeline of TMA construction, and are accumulating cases into our physical TMA collection at a rate of around 30 cases (ie 90 cores) every 2 weeks. 400 cases so far have been incorporated. These arrays contain a generous amount of tissue to facilitate manual and automated analyses, and also enable some description of pathological heterogeneity. Conclusion: This retrospective tumour collection represents an invaluable platform for biomarker discovery, biological hypothesis testing, and hypothesis generation. In particular, it is invaluable for the application of quantitative multiplex assays. The use of digital pathological techniques has facilitated archive generation and added an extra dimension of image data. Disclosure: All authors have declared no conflicts of interest. 14
Phagocytic behaviour in lung adenocarcinoma cells: clinical implications and cellular mechanisms
H.L. Mackay, D.A. Moore, P. Muller, J. Le Quesne. Toxicology Unit, Medical Research Centre, Leicester, United Kingdom Introduction: Lung adenocarcinoma is the most common and histologically diverse form of lung cancer. “Cell-in-cell” structures arise when one viable tumour cell is found in a vacuole within another viable tumour cell. They have been identified in a variety of solid tumours. The repeated occurrence of theses structures may represent a process that confers a selective advantage to a malignant clone, via the acquisition of nutrients, horizontal gene transfer, or simple subclonal competition/extinction. However, the true biological significance and mechanisms of formation remain unknown. Methods: 100 consecutive cases of lung adenocarcinoma were dearchived and for each, 10 high-power fields (hpf) were examined. A scheme for identifying cell-in-cell appearances was formulated and used to score the tumours. In vitro coculture experiments using H1299 lung cancer cells labeled green or red (via transfection with fluorescent proteins) were also conducted. Cells were seeded on a selection of coated surfaces including gelatin, fibronectin and collagen to mimic extracellular matrix proteins. Results: Cell-in-cell structures are frequently observed in primary