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M. Noguchi
Moderator of
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MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 10
- Moderators:E. Brambilla, M. Noguchi
- Coordinates: 12/05/2016, 14:20 - 15:50, Stolz 2
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MA02.01 - Extracellular Vescicle miRNAs Regulate Gene Expression in Local Lung Adenocarcinoma Endothelial Cells (ID 4655)
14:20 - 14:26 | Author(s): J. Lawson, C. Dickman, R. Towle, J. Jabalee, S. Lam, C. Garnis
- Abstract
- Presentation
Background:
Extracellular vesicles are small vesicles released from all cell types which can be used as a form of cell to cell communication. Recently these extracellular vesicles have been shown to play a key role in cancer development, growth, progression and angiogenesis. These extracellular vesicles are loaded with functional mRNAs, miRNAs and proteins which can be transferred from one cell to another. Extracellular vesicles have been known to enter neighboring cells including the surrounding stroma, and even enter biofluids. Our research shows that miRNAs transferred from lung adenocarcinoma cells through extracellular vesicles influence gene expression in endothelial cells and enhance their ability to form new blood vessels.
Methods:
Using 5 lung adenocarcinoma cell lines (H1395, H1437, H2073, H2228 and H2347) we isolated extracellular vesicles using differential ultracentrifugation. RNA was extracted from the extracellular vesicles as well as the cells from which they were derived and profiled for 742 miRNAs using the miRCURY LNA[TM] Universal RT miRNA PCR system (Exiqon) to identify miRNAs that were enriched by at least 4-fold in the extracellular vesicles. Tube formation assays were conducted on a commonly used endothelial cell line HMEC-1.
Results:
We found an enrichment of a select set of miRNAs within lung adenocarcinoma extracellular vesicles. These miRNAs have previously been identified as tumor suppressors: miR-142-3p, miR-143-3p, miR-144-3p, miR-145-5p, miR-150-5p, miR-223-3p, miR-451a, miR-486-5p, miR-605-5p in various cancer types. When extracellular vesicles are isolated from miR-143 and miR-145 over expressing adenocarcinoma lines they contain an increase in their over expressed miRNAs. When these miRNA enriched exosomes were incubated with HMEC-1 cells, we observed an increase in their ability to form new blood vessels and a decrease in the expression of CAMK1D in the endothelial cells. miR-143-3p and miR-145-5p were also found to be enriched in serum samples draining directly from lung adenocarcinoma tumors compared to arterial serum.
Conclusion:
Extracellular vesicles originating from lung adenocarcinoma cells can enter into endothelial cells and increase their ability to form new blood vessels through extracellular vesicle transfer of miR-145/miR-143 suggesting that this form of communication increases angiogenesis within lung adenocarcinoma tumors.
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MA02.02 - A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC (ID 5948)
14:26 - 14:32 | Author(s): L. Mac Donagh, S.G. Gray, S. Cuffe, S.P. Finn, N. Fitzgerald, V. Young, R. Ryan, S. Nicholson, N. Leonard, K. O’byrne, M.P. Barr
- Abstract
Background:
MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer.
Methods:
MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x10[3 ]cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and cisplatin resistance was investigated relative to cisplatin sensitive controls.
Results:
Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.
Conclusion:
A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.
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MA02.03 - Expression of Oncofetal miRNAs Inactivates NFIB, a Developmental Transcription Factor Linked to Tumour Aggressiveness in Lung Adenocarcinoma (ID 5224)
14:32 - 14:38 | Author(s): D.D. Becker-Santos, B.C. Minatel, K.M. Lonergan, K.L. Thu, J.C. English, V.D. Martinez, C.E. Macaulay, W.W. Lockwood, W.P. Robinson, I. Jurisica, S. Lam, W.L. Lam
- Abstract
- Presentation
Background:
Fetal and tumour development share striking similarities, such as intense cell proliferation, angiogenesis, increased cell motility, and immune evasion. Molecular regulators, including microRNAs (miRNAs), play important roles in both fetal lung development and in the malignant transformation of adult lung cells. Consequently, investigation of lung tumour biology in the context of lung development may reveal key regulatory mechanisms that tumours hijack from normal development, which potentially play critical roles in the pathology of lung cancer.
Methods:
131 pairs of non-small cell lung cancer (NSCLC) tumour and non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Genes controlled by the oncofetal miRNAs identified were first investigated by miRNA-Data-Integration-Portal (mirDIP) prediction, followed by luciferase-reporter assays. Associations between patient survival and mRNA expression of oncofetal miRNA-gene targets were evaluated in independent samples (>1,400 cases) across multiple NSCLC cohorts. Immunohistochemical analysis of oncofetal miRNA targets was performed on 96 lung adenocarcinoma (LUAD) specimens.
Results:
We describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, and identified numerous miRNAs that recapitulate their fetal expression patterns in NSCLC. Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, was identified as being frequently targeted by these oncofetal miRNAs. Overexpression of the oncofetal miRNA miR-92b-3p, significantly reduced NFIB levels in vitro. Concordantly, analysis of NFIB expression in multiple NSCLC cohorts revealed its frequent underexpression in tumours (~40-70%). This is in contrast with its recurrent oncogenic overexpression recently reported in SCLC. Low expression of NFIB was significantly associated with poorer survival in LUAD patients but not in squamous cell carcinoma patients, consistent with the functional role of NFIB in distal lung cell differentiation (i.e., precursor cells of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumours, comprising several well-known lung differentiation markers (e.g., TTF-1, SFTPB, ABCA3). The underexpression of NFIB protein was ultimately validated in LUAD specimens, which also revealed that tumours presenting lower levels of this transcription factor are associated with higher grade, biologically more aggressive LUAD (invasive mucinous, micropapillary and solid subtypes).
Conclusion:
This work has revealed a prominent mechanism for the downregulation of NFIB, a transcription factor essential for lung differentiation, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Restoration of NFIB expression, specifically in LUAD, has the potential to induce lung cell differentiation and thereby reduce tumour aggressiveness.
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MA02.04 - Discussant for MA02.01, MA02.02, MA02.03 (ID 6958)
14:38 - 14:50 | Author(s): L.M. Montuenga
- Abstract
- Presentation
Abstract not provided
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MA02.05 - Distinct Angiogenic microRNA-mRNA Expression Profiles among Subtypes of Lung Adenocarcinoma (ID 5464)
14:50 - 14:56 | Author(s): M. Giordano, L. Boldrini, A. Servadio, M. Lucchi, F. Melfi, A. Mussi, G. Fontanini
- Abstract
- Presentation
Background:
Non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers and adenocarcinoma (ADC) represents the most common histological type with a heterogeneous pattern of growth classified as lepidic, acinar, papillary, solid, and micropapillary. For ADC there are restricted available therapeutic options except for patients that could benefit from target therapy. A valuable therapeutic strategy is represented by angiogenesis inhibitors such as bevacizumab that has been approved for the treatment of NSCLC patients. However, there are concerns about its treatment-related toxicity and the identification of new reliable biomarkers to stratify patients who can really benefit from antiangiogenic drugs is urgently needed. Using miRNA target prediction tools, we selected and investigated the expression level of a panel of miRNAs togheter with their mRNA target involved in the angiogenesis pathway.
Methods:
We designed a custom codeset including probes for six genes (VEGF-A, FLT1, KDR, FLT4, PDGFRa and PDGFRb) and sixteen miRNAs. The expression analysis was performed by the nCounter System® (NanoString Technologies) directly on RNA, enriched of small RNA, purified from the formalin-fixed and paraffin-embedded tumor tissues of 80 ADC patients. Of these 25 were predominatly lepidic (31.25%), 24 were predominatly solid (30%), 20 were predominatly acinar (16%), 11 were predominatly papillary (13.75%).
Results:
Comparing the expression levels of mRNAs with the different histological ADC subtypes we found a significant higher expression of VEGF-A in papillary than in other subtypes (p=0.02). In contrast PDGFRa and PDGFRb were upregulated in lepidic and downregulated in papillary subtypes (both p=0.03). Among 16 miRNAs that target the angiogenic mRNA, 6 were significantly downregulated in papillary compared to other groups.
Conclusion:
Our data suggest a distinct angiogenic miRNA-mRNA expression profile among the subtypes of ADC. The higher level of VEGF-A in papillary than in lepidic subtypes could represent a useful biomarker to stratify patients who can effectively treated with bevacizumab, which is directed against VEGF. Moreover, the regulation of angiogenic mRNA factors by miRNAs could provide a novel therapeutic approach based on their expression pattern specific for distinct ADC subtypes. Further studies are nedeed in a larger cohort of patients to confirm our results and to investigate whether different rates of response to treatment are observed among patients stratified according to the proposed biomarkers.
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MA02.06 - Discussant for MA02.05 (ID 6957)
14:56 - 15:08 | Author(s): R. Rosell
- Abstract
- Presentation
Abstract not provided
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- Abstract
- Presentation
Background:
Non-small-cell lung cancer (NSCLC) is one of the most common and high mortality rate carcinoma in China which biomarkers for diagnosis are limited. Therefore, novel biomarkers and methods with increased specificity for diagnosis are explored and required. For now, liquid biopsy for lung cancer oncogenes and next generation sequencing technique are extensive employed in NSCLC. However, increasing evidence illustrates that exosomal microRNAs in circulating fluids provide a promising way as biomarkers for noninvasive cancer diagnosis. Exosomes are 30–150 nm particles which are released from cells into the extracellular environment and stable miRNAs have been identified in plasma exosomes, which play important role in cell communication. Furthermore, exosomal miRNAs present different profiles between patients with cancer and healthy individuals. Whether exosomal miRNAs could benefit NSCLC patient diagnosis remains to be explored.
Methods:
Blood samples were collected from 40 NSCLC patients and 24 healthy volunteers matched with age, gender and blood collection time. Plasma exosomes were accessed by 110,000×g ultracentrifugation and visualized by NS300 equipment. The raw data of exosomal miRNA profiles of NSCLC patients and healthy individuals were generated by NGS around 400× read depth and its expression were measured by Taqman probe quantitive PCR
Results:
In the present study, we revealed that nearly half of exosome RNA was miRNA and NSCLS patients expressed a set of exosomal miRNAs with specificity compared with healthy volunteers. We demonstrated that miR-126-5p and miR-21-3p were down-regulated in NSCLC patients. In addition, we showed that the expression level of miR-124-3p and miR-99a-3p in NSCLC patients was higher than that of healthy individuals. Furthermore, we found miR-99a-3p was clinical stages related in NSCLC patient plasma and miR-375-3p was a potential biomarker for diagnosis and prognosis in NSCLC.
Conclusion:
Exosomal miRNAs in plasma could indicate the progress of NSCLC and a combination of the explored miRNA could serve as a promising biomarker for NSCLS diagnosis and prognosis.
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MA02.08 - Deregulation of Cis-Acting Long Non-Coding RNAs in Non-Small Cell Lung Cancer (ID 6303)
15:14 - 15:20 | Author(s): A.P. Sage, G. Stewart, D.A. Rowbotham, K.S.S. Enfield, V.D. Martinez, S. Lam, W.L. Lam
- Abstract
- Presentation
Background:
Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 17%. Targeted therapeutics have been developed against drivers of the lung adenocarcinoma (AC) subtype, but are relevant only to the proportion of patients harbouring these genetic aberrations, emphasizing the need to explore alternative mechanisms of AC development. Natural antisense transcripts (NATs) are long non-coding RNA (lncRNA) products expressed from the opposite strand of coding mRNAs. NATs can function in cis or trans to regulate the transcriptional activity of their cognate gene partner in either a positive or negative fashion. Here we take a novel approach to identify cis- NATs deregulated in lung AC, and explore the function of these genes with regards to their protein coding partner genes.
Methods:
We performed RNA-sequencing on a set of 36 lung AC and matched non-malignant lung tissues. A sign-rank test was used to identify NATs and partner genes with significantly altered expression between tumor and matched normal tissues. These findings were validated in an external dataset of 50 lung AC tumors with matched non-malignant tissue obtained from The Cancer Genome Atlas (TCGA). Survival analysis was performed using a Cox Proportional hazard model, as well as the log-rank method.
Results:
Analysis of Illumina Hi-seq data from TCGA revealed the majority (79%) of deregulated sense-antisense partnerships observed in AC displayed concordant regulation. However, several discordant cis-NAT pairs were identified including an antisense to OPA INTERACTING PROTEIN 5 (OIP5), a gene required for chromatin segregation, as well as an antisense to HIGH MOBILITY GROUP A1 (HMGA1) a gene involved in the metastatic progression of many cancer types. Both the antisense to OIP5 (OIP5-AS1) as well as the antisense to HMGA1, (HMGA1-AS1) were significantly underexpressed in AC, while we find the overlapping protein coding partner genes to be significantly overexpressed, suggesting that these genes may negatively regulate their sense counterparts. In addition both OIP5 and HMGA1 are significantly associated with 5-year survival. Patients with higher expression levels of either of these genes had a significantly shorter overall survival time than patients with low expression levels, highlighting the potential clinical importance of these genes.
Conclusion:
This study characterizes the landscape of antisense expression in AC and highlights novel mechanisms of oncogene regulation through natural antisense transcripts. Characterizing these oncogene regulatory mechanisms could uncover therapeutic intervention points and further our understanding of AC biology.
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MA02.09 - Long Non-Coding RNA Expression from Pseudogene Loci as a Novel Mechanism of Cancer Gene Regulation (ID 6287)
15:20 - 15:26 | Author(s): G. Stewart, K.S.S. Enfield, V.D. Martinez, E.A. Marshall, S. Lam, W.L. Lam
- Abstract
- Presentation
Background:
The advent of next generation sequencing has lead to the discovery of the functional importance of non-coding RNAs (ncRNAs) in a wide variety of cellular processes, and these genes can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional parent genes but retain high sequence homology. Long non-coding RNAs (lncRNAs) have been shown to regulate protein-coding genes; however, complex folding patterns make lncRNA function difficult to predict. Several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. The biological impact of this mechanism has not been investigated in lung adenocarcinoma (LUAD). We hypothesize that expression changes in lncRNAs expressed from pseudogene loci can affect the expression of corresponding protein-coding parent genes in trans, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis.
Methods:
We analysed RNA-seq data from 50 LUAD with matched non-malignant tissue obtained from the TCGA for both protein-coding and non-coding gene expression. Significantly differentially expressed lncRNAs located within pseudogene loci were identified by sign-rank test (p<0.001). Mann Whitney U-tests were used to identify lncRNA-parent gene pairs which significantly correlated expression, and survival analysis was performed using a Cox proportional hazard model.
Results:
Our analysis has identified 172 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD. Remarkably, many of these lncRNAs were expressed from the loci of pseudogenes related to known cancer genes. One of these lncRNAs, CTD-2583A14.8, was expressed from a pseudogene to ubiquitin-conjugating enzyme E2C (UBE2C), which regulates tumor growth, apoptosis, and angiogenesis through phospho-ERK1/2. We find CTD-2583A14.8 as well as the UBE2C parent gene to be significantly upregulated in LUAD tumours compared to matched normal tissue. Furthermore, tumours with higher levels of CTD-2583A14.8 have significantly higher levels of UBE2C expression than tumours with low levels of CTD-2583A14.8, indicating that CTD-2583A14.8 may positively regulate UBE2C in trans.
Conclusion:
Here we show expression of lncRNAs within pseudogene loci is deregulated in LUAD, and can correlate with the expression of their protein-coding counterparts. Many of these genes associated with this putative lncRNA-pseudogene-protein-coding axis have previously been implicated in cancer. Therefore, this represents an alternative mechanism of cancer gene deregulation, and may represent novel therapeutic intervention points for the treatment of LUAD.
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MA02.10 - Discussant for MA02.07, MA02.08, MA02.09 (ID 7108)
15:26 - 15:38 | Author(s): B. Zaric
- Abstract
- Presentation
Abstract not provided
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Author of
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MTE11 - The Clinical Impact of the 2015 WHO Classification of Lung Tumors (Ticketed Session) (ID 305)
- Event: WCLC 2016
- Type: Meet the Expert Session (Ticketed Session)
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/06/2016, 07:30 - 08:30, Schubert 2
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MTE11.01 - The Clinical Impact of the 2015 WHO Classification of Lung Tumors (ID 6558)
07:30 - 08:00 | Author(s): M. Noguchi
- Abstract
- Presentation
Abstract:
There are many important changes in the 2015 WHO classification of lung tumours, reflecting the numerous advances in tumour genetics and therapy over the past decade.[1] Many have been in the field of adenocarcinoma, with discontinuation of the term bronchioloalveolar carcinoma and the concept of stepwise progression accepted for adenocarcinoma.[1] Adenocarcinoma in situ (AIS) is a small (less than 3 cm in diameter), pure lepidic adenocarcinoma; minimally invasive adenocarcinoma (MIA) is also a small lepidic adenocarcinoma but has small invasive areas less than 5 mm across. As both entities have a very favorable outcome, with an expected 5-year survival rate of 100%, AIS and MIA are targets for reduction surgery, and are frequently detected by low-dose CT screening. High-resolution (HR)-CT demonstrates these tumours as a pure ground glass nodule (GGN) or a part-solid nodule (PSN), being closely correlated with their pathological features.[2] Therefore, AIS and MIA can be assessed by HR-CT. However, the size of the solid component in HR-CT images does not necessarily correspond to the extent of histological invasion, since features such as alveolar collapse and fibrosis are also included in the solid part demonstrated by HR-CT.[1] Although the new WHO classification defines the histological criteria for MIA invasion, the degree of inter-observer agreement regarding the histological definition of invasion in MIA has still not been fully studied, and a consensus trial will be needed in the near future. More advanced adenocarcinoma is subdivided into five categories: lepidic, papillary, acinar, solid and micropapillary. These subtypes are diagnosed according to the predominant component and the group comprising lepidic, papillary and acinar adenocarcinomas shows a better prognosis than those with solid and micropapillary patterns. Therefore the presence of solid and/or micropapillary adenocarcinoma should be reported, even if the predominant component is lepidic, papillary or acinar adenocarcinoma. These patterns also predict response to adjuvant chemotherapy,[3] and the above changes overall have also led new proposals for both clinical and pathologic staging in the 8[th] TNM revision in terms of multiple primary tumours and measurement of tumour invasive size.[4,5] For the other three major tumour types (large cell carcinoma (LCC), squamous cell carcinoma (SQCC) and neuroendocrine (NE) tumours), the classification has evolved from mainly morphological to a more biologically based system, which allows more appropriate decisions in relation to adjuvant therapy and better defined subgroups for studies into molecular characterisation and the search for potentially treatable targets. LCC is now restricted to resected tumours that lack clear morphologic and immunohistochemical differentiation, with reclassification of those that do to solid adenocarcinoma (TTF-1 positive) and non-keratinising SQCC (P40 and/or CK5/6 positive). This has already been shown to correlate with molecular data.[6] For SQCC, classification is simplified to keratinizing, nonkeratinizing and basaloid subtypes, with the non-keratinizing tumours ideally requiring immunohistochemical confirmation. Criteria for diagnosing NE tumours remain essentially unchanged but these tumours are now grouped in one category, with further subdivision into carcinoids, and large cell neuroendocrine carcinoma and small cell carcinoma. Molecular studies based on these definitions are already identifying interesting subgroups.[7] In relation to rarer entities, the definition of pleomorphic carcinomas is also being shown to have clinical relevance in terms of correlating with potential therapies, both in relation to specific molecular abnormalities (exon 14 skipping mutations)[8] and immunoodulatory therapy with high levels of PD-L1 expression.[9] Molecular characterisation is also increasingly important in the accurate diagnosis and potential treatment of other rare tumours, such as NUT-carcinoma and inflammatory myofibroblastic tumours (ALK and ROS1/RET gene rearrangements).[10] A classification system for small biopsies and cytology is provided for the first time, with emphasis on integration of molecular testing and usage of a limited panel of immunohistochemistry when needed (table 1). The presence of such a system for the first time provides a system for consistent classification of the majority (unresectable) of lung cancer cases, both in terms of clinical management, assignment to pathways for molecular and immunomodulatory characterisations, and for assessment of the results of clinical trials that have sometimes been confounded by inaccurate subgrouping. The book also emphasises how to obtain the greatest value from small sample via efficient usage and avoidance of inappropriate testing.[1] Table 1: Classification of non-small cell lung carcinoma in small biopsies and cytology specimens when there is no morphologic evidence of differentiation
REFERENCES 1. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. Lyons, France.: International Agency for Research on Cancer (IARC); 2015. 2. Kakinuma R, Noguchi M, Ashizawa K, et al. Natural History of Pulmonary Subsolid Nodules: A Prospective Multicenter Study. J Thorac Oncol. Jul 2016;11(7):1012-1028. 3. Tsao MS, Marguet S, Le Teuff G, et al. Subtype Classification of Lung Adenocarcinoma Predicts Benefit From Adjuvant Chemotherapy in Patients Undergoing Complete Resection. J Clin Oncol. Oct 20 2015;33(30):3439-3446. 4. Detterbeck FC, Nicholson AG, Franklin WA, et al. The IASLC Lung Cancer Staging Project: Summary of Proposals for Revisions of the Classification of Lung Cancers with Multiple Pulmonary Sites of Involvement in the Forthcoming Eighth Edition of the TNM Classification. J Thorac Oncol. Feb 29 2016. 5. Travis WD, Asamura H, Bankier AA, et al. The IASLC Lung Cancer Staging Project: Proposals for Coding T Categories for Subsolid Nodules and Assessment of Tumor Size in Part-Solid Tumors in the Forthcoming Eighth Edition of the TNM Classification of Lung Cancer. J Thorac Oncol. Aug 2016;11(8):1204-1223. 6. Clinical Lung Cancer Genome P, Network Genomic M. A genomics-based classification of human lung tumors. Sci Transl Med. Oct 30 2013;5(209):209ra153. 7. Rekhtman N, Pietanza MC, Hellmann MD, et al. Next-Generation Sequencing of Pulmonary Large Cell Neuroendocrine Carcinoma Reveals Small Cell Carcinoma-like and Non-Small Cell Carcinoma-like Subsets. Clin Cancer Res. Jul 15 2016;22(14):3618-3629. 8. Schrock AB, Frampton GM, Suh J, et al. Characterization of 298 Patients with Lung Cancer Harboring MET Exon 14 Skipping Alterations. J Thorac Oncol. Sep 2016;11(9):1493-1502. 9. Chang YL, Yang CY, Lin MW, Wu CT, Yang PC. High co-expression of PD-L1 and HIF-1alpha correlates with tumour necrosis in pulmonary pleomorphic carcinoma. Eur J Cancer. Jun 2016;60:125-135. 10. Antonescu CR, Suurmeijer AJ, Zhang L, et al. Molecular characterization of inflammatory myofibroblastic tumors with frequent ALK and ROS1 gene fusions and rare novel RET rearrangement. Am J Surg Pathol. Jul 2015;39(7):957-967.2015 WHO Small Biopsy/Cytology Terminology Morphology/Stains 2015 WHO Classification in resection specimens Non-small cell carcinoma, favour adenocarcinoma using IHC Morphologic adenocarcinoma patterns (lepidic, acinar, papillary, micropapillary) not present, but supported by special stains (+TTF-1) Adenocarcinoma, solid pattern (may only be a component) Non-small cell carcinoma, favour squamous cell carcinoma using IHC Morphologic squamoid features (keratinization and/or clear intercellular bridging) not present, but supported by stains ( +p40) Squamous cell carcinoma, (non-keratinizing pattern may be just one component) Non-small cell carcinoma, not otherwise specified NSCLC-NOS using IHC No clear adenocarcinoma, squamous or neuroendocrine morphology or staining pattern (IHC or mucin stains). Large cell carcinoma
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MTE11.02 - The Clinical Impact of the 2015 WHO Classification of Lung Tumors (ID 6559)
08:00 - 08:30 | Author(s): M. Noguchi
- Abstract
- Presentation
Abstract not provided
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 3
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-014 - miR-3941: A Novel microRNA That Controls IGBP1 Expression and is Associated with Malignant Progression of Lung Adenocarcinoma (ID 4429)
14:30 - 14:30 | Author(s): M. Noguchi
- Abstract
Background:
Immunoglobulin (CD79A) binding protein 1 (IGBP1) binds to PP2Ac and exerts an anti-apoptotic effect. We have already reported that IGBP1 overexpression occurs during the course of malignant progression of lung adenocarcinoma (Sakashita S et al., Pathol Int. 2011). However, the molecular mechanism of IGBP1 overexpression is still unclear. A few reports have documented mutation, hypomethylation, or amplification of IGBP1, but only one study has suggested that down-regulation of miR-34b leads to high expression of IGBP1 (L-P Chen et al. Oncogene. 2011). In this study, we have detected miR-3941 as another functional microRNA that influences the expression status of IGBP1.
Methods:
We performed microRNA array analysis using total RNA extracted from fresh specimens of invasive lung adenocarcinoma (IGBP1+) and minimally invasive adenocarcinoma (IGBP1-). We compared the results of microRNA array with microRNAs listed in TargetScan (a microRNA database) that would potentially bind to IGBP1. Using reverse transcription-quantitative PCR (RT-qPCR), we analyzed the expression levels of candidate microRNAs in frozen specimens of lung adenocarcinoma. We also validated these microRNAs by checking IGBP1 expression and cell proliferation after they had been transfected into lung adenocarcinoma cell lines (A549, PC-9) and confirmed the direct effect of the microRNAs by luciferase reporter assay.
Results:
Using microRNA array and TargetScan, we selected 6 microRNAs (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941). RT-qPCR analysis showed that these microRNAs were down-regulated in invasive adenocarcinoma (IGPB1+) relative to adjacent normal lung tissue (IGBP1-) (Fig1A). We transfected these microRNAs into lung adenocarcinoma cell lines, and all of the microRNAs suppressed IGBP1 expression. Among these microRNAs, miR-34b and miR-3941 depressed luciferase activity by targeting 3’UTR-IGBP1 in the luciferase vector (Fig1B). Figure 1
Conclusion:
We have found that miR-3941 targets IGBP1 in addition to miR-34b. Down-regulation of both microRNAs can lead to high expression of IGBP1, and this is thought to be associated with progression of lung adenocarcinoma.
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P2.01-071 - Biological Implication of Cytoplasmic ECT2 in Malignant Progression of Lung Adenocarcinoma (ID 4361)
14:30 - 14:30 | Author(s): M. Noguchi
- Abstract
Background:
Epithelial cell transforming 2 (ECT2) is a guanine nucleotide exchange factor (GEF) for Rho family GTPases including RhoA, Rac1, and Cdc42. In normal cells, ECT2 is localized in the nucleus,where it regulates dynamic processes including the cell cycle and cytokinesis. On the other hand, several studies have suggested that ECT2 signaling promotes tumor proliferation, migration, and invasion in non-small cell lung cancer. Recently, Murata et al. demonstrated that ECT2 is amplified in early invasive adenocarcinoma but not in situ adenocarcinoma (Cancer Sci, 105:490, 2014). However, the oncogenic mechanism whereby ECT2 drives cell transformation in lung adenocarcinoma is still unknown
Methods:
Cellular fractionation assay was conducted using nine lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. Immunoblotting, Immunofluorescence, and Immunohistochemistry assays were used to evaluate the expression and localization of ECT2. For ECT2 amplification, nine lung adenocarcinoma were genetically examined using Quantitative Real-Time PCR. Immunoprecipitation was used to examine the interaction between ECT2 and PKCι. And ECT2 siRNA was confirmed the effect of ECT2 on the downstream singling pathway.
Results:
In this study, we showed that ECT2 was localized predominantly in the nucleus of normal lung epithelial cells, whereas tumor cells in nine lung adenocarcinoma cell lines expressed ECT2 protein to differing degrees in their cytoplasm. Importantly, high expression of cytoplasmic ECT2 in surgically resected materials was significantly associated with poor outcome. Moreover, our data showed that overexpression of ECT2 mRNA was roughly correlated with ECT2 amplification in lung adenocarcinoma cell lines. We then investigated the mechanism underlying the cytoplasmic localization of ECT2 and its oncogenic activity in lung adenocarcinoma using the lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, LC-2/ad, and HCC827. We found that the cytoplasmic ECT2 was phosphorylated and bound to protein kinase C iota (PKCι) in the cytoplasm. We also observed that the overexpression of cytoplasmic ECT2 greatly increased its degree of phosphorylation and enhanced its interaction with PKCι, resulting in significant promotion of tumor growth through activation of the Mek1,2/Erk1,2 cytoplasmic downstream signaling pathway.
Conclusion:
These results indicate that aberrant cytoplasmic localization of ECT2 is a specific feature of lung adenocarcinoma and important for its malignant progression. This finding offers new insight into the molecular mechanism responsible for aberrant cytoplasmic localization of ECT2, which is correlated with the progression of malignancy, and highlights cytoplasmic ECT2 expression as a new prognostic biomarker in lung adenocarcinoma.
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P2.01-076 - Drebrin: A New Targetable Molecular Marker of Lung Adenocarcinoma (ID 4976)
14:30 - 14:30 | Author(s): M. Noguchi
- Abstract
Background:
Embryonic antigens, such as carcinoembryonic antigen (CEA) and alfa-fetoprotein (AFP), are routinely employed as serum and immunohistochemical tumor markers in clinical medicine. Since they are not expressed in adult human tissues, it is reasonable to conclude that embryonic antigens are extremely specific tumor markers. However, no systematic studies have yet identified clinically useful embryonic antigens for tumor diagnosis. In the present study, we developed a strategy for systematic identification of lung adenocarcinoma markers using monoclonal antibodies generated from embryonic swine tissue. Swine mRNA shows more than 80% homology with human mRNA, and embryonic swine tissue is thought to be a useful material for detection of human embryonic markers.
Methods:
In order to produce mouse monoclonal antibodies, we immunized BALB/c mice by injection of fetal swine lung nuclear fraction into the hock, and used a human lung adenocarcinoma nuclear fraction for the second immunization. We recovered lymph nodes from the mice, and fused mouse B lymphocytes with the murine myeloma cell line SP2/0 using polyethylene glycol. The resulting hybridomas were then selectively cultured. For selection of interesting hybridoma clones, we performed immunohistochemical staining using the supernatant from each one, with tissue microarray loading swine fetal and adult lung, human lung cancer and normal lung tissue.
Results:
Immunohistochemical screening of 284-clones showed that the antibodies derived from four of them were strongly reactive with the cytoplasm and cytomembrane of fetal swine lung and human lung cancer. We then focused on one clone (B246) whose antibody reacted most clearly with human lung adenocarcinoma cells . Protein microarray analysis confirmed that B246 reacted specifically with “drebrin”, one of the actin binding proteins, originally identified in neuronal cells. There are two drebrin isoforms in human tissue: drebrin E (embryonic) is abundant in the developing neurons, and drebrin A (adult) is expressed in adult brain. We then examined the association of the drebrin expression pattern with the pathological features and prognosis of lung adenocarcinoma using 200 selected cases for which formalin-fixed and paraffin-embedded samples were available. Drebrin immunohistochemistry delineated the samples into those with strong (n=85) and weak (n=115) drebrin expression. Kaplan-Meier analysis demonstrated a significant difference in disease-free survival (DFS) between the groups with strong and weak drebrin expression (p=0.033) .
Conclusion:
Drebrin is expressed specifically in lung adenocarcinoma and is associated with outcome. The present findings indicate that drebrin is a new marker of lung adenocarcinoma and indicative of prognosis.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-021 - Reproducibility of Comprehensive Histologic Assessment and Refining Histologic Criteria in P Staging of Multiple Tumour Nodules (ID 5365)
14:30 - 14:30 | Author(s): M. Noguchi
- Abstract
Background:
Multiple tumor nodules (MTNs) are being encountered, with increasing frequency with the 8[th] TNM staging system recommending classification as separate primary lung cancers (SPLC) or intrapulmonary metastases (IM). Pathological staging requires assessment of morphological features, with criteria of Martini and Melamed supplanted by comprehensive histologic assessment of tumour type, predominant pattern, other histologic patterns and cytologic features. With publication of the 2015 WHO classification of lung tumours, we assessed the reproducibility of comprehensive histologic assessment and also sought to identify the most useful histological features.
Methods:
We conducted an online survey in which pathologists reviewed a sequential cohort of resected multifocal tumours to determine whether they were SPLC, IM, or a combination. Specific histological features for each nodule were entered into the database by the observing pathologist (tumour type, predominant adenocarcinoma pattern, and histological features including presence of lepidic growth, intra-alveolar cell clusters, cell size, mitotic rate, nuclear pleomorphism, nucleolar size and pleomorphism, nuclear inclusions, necrosis pattern, vascular invasion, mucin content, keratinization, clear cell change, cytoplasmic granules¸ lymphocytosis, macrophage response, acute inflammation and emperipolesis). Results were statistically analyzed for concordance with submitting diagnosis (gold standard) and among pathologists. Consistency of each feature was correlated with final determination of SPLC vs. IM status (p staging) by chi square analysis and Fisher exact test.
Results:
Seventeen pathologists evaluated 126 tumors from 48 patients. Kappa score on overall assessment of primary v. metastatic status was 0.60. There was good agreement as measured by Cohen’s Kappa (0.64, p<0.0001) between WHO histological patterns in individual cases with SPLC or IM status but proportions for histology and SPT or IM status were not identical (McNemar's test, p<0.0001) and additional histological features were assessed. There was marked variation in p values among the specific histological features. The strongest correlations (<0.05) between p staging status and histological features were with nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intra-alveolar clusters and necrosis pattern. Correlation between lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization and emperipolesis did not reach a p value of 0.05.
Conclusion:
Comprehensive histologic assessment shows good reproducibility between practicing lung pathologists. In addition to main tumour type and predominant patterns, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate appear to be useful in distinguishing between SPLC and IM.
