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C. Mascaux
Moderator of
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MINI 34 - RNA and miRNA (ID 162)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 15
- Moderators:C. Mascaux, L.(. Wang
- Coordinates: 9/09/2015, 18:30 - 20:00, 205+207
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MINI34.01 - MicroRNA Profiling of a Familial Primary Pulmonary Enteric Adenocarcinoma (ID 397)
18:30 - 18:35 | Author(s): I. Garajová, N. Funel, M. Fiorentino, V. Agostini, M. Ferracin, M. Negrini, G.L. Frassineti, C. Rolfo, G. Biasco, E. Giovannetti
- Abstract
- Presentation
Background:
Primary pulmonary enteric adenocarcinoma (PEAC) is defined as a pulmonary adenocarcinoma with a predominant component (>50%) of intestinal differentiation and tumor cells positive for at least one intestinal marker. Due to its peculiarity and rarity, the optimal clinical management of PEAC patients remains unclear. A total of thirty cases have been described in literature to date, though familial aggregation has never been reported before. This is the first study describing the histological and molecular characterization of the PEAC from a patient with a family member affected by the same tumor.
Methods:
We evaluated the molecular characteristics of proband’s PEAC applying a previously validated 47-miRNA signature and applying the predictive method to estimate tissue-of-origin probabilities. Immunohistochemical (IHC) staining (TTF-1, Napsin A, CDX2, cytokeratonins, mucins) and mutational analyses (EGFR, K-RAS, ALK) were performed on formalin-fixed, paraffin-embedded tissue of a patient affected by PEAC.
Results:
The familial aggregation of PEAC was associated with similar clinicopathological features (age at diagnosis, smoking-habit, tumor localization, multiple colon polyps), histologic findings (IHC staining negative for TTF-1 and positive for CDX2) and genetic findings (K-RAS(Gly12Asp) mutation, but no EGFR/ALK aberrations). MiRNA profiling revealed main similarities with NSCLC (75.98%), and partial overlap with pancreatic cancer (PDAC, 23.34%), but not with colorectal cancer (less than 0.5%). Notably, this PEAC shares with PDAC key miRNAs associated with tumor aggressiveness (mir-31/-126/-506/-508-3p/-514). Figure 1
Conclusion:
In conclusion, we described, for the first time, PEACs in two members of the same family, associated with clinicopathological features. Moreover, miRNA profiling resembled mostly NSCLC, with a partial overlap with PDAC’s pattern that could explain the aggressive behavior compared to most NSCLC and guide future tailored-therapy approaches.
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MINI34.02 - Prognostic Impact of Bcl-2 Depends on Tumor Histology and Expression of MALAT-1 lncRNA in NSCLC (ID 3221)
18:35 - 18:40 | Author(s): L.H. Schmidt, D. Goerlich, C. Rohde, M. Schuler, A. Huge, R. Voss, A. Faldum, C. Mueller-Tidow, W.E. Berdel, R. Wiewrodt
- Abstract
Background:
Apoptosis is a crucial pathway in tumor growth and metastatic development. Apoptotic proteins regulate the underlying molecular cascades and are thought to modulate the tumor response to chemotherapy and radiation. However, the prognostic value of the expression of apoptosis regulators in localized non-small-cell lung cancer (NSCLC) is still unclear.
Methods:
We investigated the protein expression of apoptosis regulators Bcl-2, Bcl-xl, Mcl-1, and pp32/PHAPI, and the expression of the lncRNA MALAT-1 in tumor samples from 383 NSCLC patients (median age: 65.6 years; 77.5% male; paraffin embedded tissue microarrays) For statistical analysis correlation tests, Log rank tests and Cox proportional hazard models were applied.
Results:
Tumor histology was significantly associated with the expression of Bcl-2, Bcl-xl and Mcl-1 (all p<0.001). Among the tested apoptotic markers only Bcl-2 demonstrated prognostic impact (HR=0.64, p=0.012). For NSCLC patients with non-adenocarcinoma histology, Bcl-2 expression was associated with increased overall survival (p=0.036). Besides tumor histology, prognostic impact of Bcl-2 was also found to depend on MALAT-1 lncRNA expression. Gene expression analysis of A549 adenocarcinoma cells with differential MALAT-1 lncRNA expression demonstrated an influence on the expression of Bcl-2 and its interacting proteins.
Conclusion:
Bcl-2 expression was specifically associated with superior prognosis in localized NSCLC. An interaction of Bcl-2 with MALAT-1 lncRNA expression was revealed, which merits further investigation for risk prediction in resectable NSCLC patients.
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MINI34.03 - Novel microRNA Prognostic Signature in Malignant Pleural Mesothelioma (ID 2988)
18:40 - 18:45 | Author(s): F. Grossi, C. Genova, A. Truini, S. Coco, E. Nadal, M.G. Dal Bello, I. Vanni, A. Alama, E. Rijavec, G. Barletta, F. Biello, D. Beer
- Abstract
- Presentation
Background:
Malignant pleural mesothelioma (MPM) is an aggressive tumor mainly associated with asbestos exposure. MPM patients have a poor outcome (median overall survival (mOS) <1 year), therefore novel therapeutic approaches are needed. MiRNA have been demonstrated to have a role in tumorigenesis and progression in MPM. This study aimed to identify a miRNA signature associated with poor prognosis.
Methods:
We identified 26 un-resected MPM patients split as follows: 11 long survivors (LS) OS>3 years and 15 short survivors (SS) OS<1 year. MiRNA expression in 26 FFPE biopsy and 3 normal pleura (NP) was evaluated using Agilent Human miRNA Microarray platform including 2006 miRNA. Expression data were normalized by GeneSpring software (v.12.6). Class-comparison analysis between MPM/NP and SS/LS was performed using a t-test adjusted for multiple comparisons using Benjamini-Hochberg. OS curves were estimated using the Kaplan-Meier method and compared with the log-rank test. In silico validation was performed using miRseq data from TCGA portal based upon 16 patients (LS: 8; SS: 8). Candidate miRNA were assessed by univariate analysis using Kaplan-Meier method and median as cutoff.
Results:
Patients’ characteristics: median age 67 years (53-77); 81% males, 19% females; 73% epithelioid histotype, 12% sarcomatoid, 12% biphasic and 1 unspecified MPM. No differences in age, gender and histotype were observed between LS and SS. By class-comparison analysis, 30 miRNAs were significantly up-regulated and 11 down-regulated in MPM vs NP (adjusted p-value <0.05). Fourteen miRNAs were significantly associated with outcome, in the univariate survival analysis and differentially expressed in MPM. A miRNA signature, based on the top 6 prognostic miRNAs (unfavorable, miR-1224; favorable, miR-99a, miR-125b, let-7b, let-7c and let-7i) classified patients into low- or high-risk. High-risk patients showed a significantly shorter median OS (4.1 months, 95% CI 2.2-5.9) as compared with low-risk patients (median not reached, Log-rank p<0.001). In silico validation analysis confirmed that low expression of mir-99a, miR-125b and let-7c was associated with shorter OS. Relevant pathways, such as PI3K/AKT, WNT were associated with these top miRNAs by pathway analysis.
Conclusion:
A prognostic miRNA signature was identified by profiling a cohort of un-resected MPM, underlying the clinical potential of miRNA as predictors of survival. An additional validation in a larger independent cohort of MPM is ongoing.
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- Abstract
Background:
Lung cancer is the leading cause of cancer mortality worldwide. About 30–40% of patients with lung cancer developed bone metastasis during the course of their disease, with the median survival time of 7 months approximately. Bone lesion is characterized by the imbalance of osteoblastic and osteoclastic activity induced by the tumor cells. Mesenchymal stem cells (MSCs) are the progenitor cells of osteoblast cells, and therefore it is interesting to investigate whether and how MSCs have biological alterations in the tumor microenvironment of lung cancer patients with bone lesion.
Methods:
We tested miR-139-5p and Notch1 expression during MSC osteogenic differentiation, and validated the effect of miR-139-5p in MSC osteogenesis by transfection of miR-139-5p mimic and inhibitor. We also collected blood samples of healthy donors and lung cancer with bone metastasis, and tested serum miR-139-5p expression.
Results:
We demonstrated that during MSC osteogenic differentiation in vitro, the expression of miR-139-5p increased significantly while Notch1 and its downstream factors decreased. By gain and loss of function studies we showed that miR-139-5p could positively regulate MSC osteogenic differentiation. And luciferase and western blot assays confirmed that miR-139-5p targeted Notch1 expression directly. Moreover, Notch1 knockdown by siRNA could no longer confer miR-139-5p induced MSC osteogenic differentiation. Lung cancer cell A549 and L9981 secreted factors upregulated miR-139-5p expression and meanwhile downregulated Notch1 expression in MSC, as well as impaired ALP activity, the early osteogenic marker of MSCs. More importantly, we observed that serum miR-139-5p was significantly lower in lung cancer patients with lytic bone lesion compared to healthy donors.
Conclusion:
We demonstrate for the first time, that miR-139-5p could regulate osteogenic differentiation of MSC by targeting Notch1 mediated signalling pathway. Lung cancer cells could decrease miR-139-5p expression and inhibit MSC osteogenic potential. Importantly, serum miR-139-5p is significantly lower in lung cancer patients with lytic bone lesion compared to healthy donors. Therefore, miR-139-5p might be a biomarker of bone metastasis in lung cancer patients and treatment with miR-139-5p mimic might be an interesting strategy to restore the impaired MSC osteogenic differentiation and to control bone disease in lung cancer patients with bone lesion.
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MINI34.05 - Discussant for MINI34.01, MINI34.02, MINI34.03, MINI34.04 (ID 3433)
18:50 - 19:00 | Author(s): C.J. Rivard
- Abstract
- Presentation
Abstract not provided
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MINI34.06 - MicroRNA 10b, 27a and 27b Are Upregulated in Lung Cancer Cell with Epidermal Growth Factor (EGFR) Mutation Resistant to EGFR TKI (ID 2950)
19:00 - 19:05 | Author(s): H. Montes, E.B. Reyes, J. Kevern, P.J. Van Veldhuizen, C.H. Huang
- Abstract
Background:
EGFR Tyrosine Kinase Inhibitor (TKI) is now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and would require treatment with another EGFR TKI or chemotherapy. The gate keeper mutation T790M is responsible for about 50% of resistance cases. There are other mechanisms that could confer resistance in these patients. High expression of microRNA (mir) 10 is associated with worse prognosis in resected lung cancer patients with EGFR mutation. The mir 27a and 27b is associated with increased expression of c-met which is a potential mechanism of resistance to EGFR. We explored the expression of mir 10b and 27a and 27b in lung cancer cell lines with EGFR mutation that were resistant to Erlotinib.
Methods:
Lung cancer cell lines with EGFR mutation CRL-2868 and CRL 2871 were treated with 5 nM of erlotinib for 24hours and 72 hours. The erlotinib resistant cells were then harvested and then microRNA profiling was done by real time PCR. HTB177 cell line without EGFR mutation was used as control.
Results:
We observed an increased expression of mir 10b, mir27a and mir27b in CRL-2871 compared to control HTB 177 cells. Mir 27b is upregulated in both cell lines. The increase expression of mir 10b and mir 27a were higher in CRL-2871 than the control cells 9 fold and 8 folds respectively. The mir 27b was increased 300 folds in the CRL 2868 compared with control.
Conclusion:
We observed upregulation of mir 10b, 27a and 27b in lung cancer cells lines with EGFR mutation that were resistant to Erlotinib treatment. This suggests an epigenetic mechanism of resistance other than the T790M mutation. Further research in patients with EGFR mutation resistance to EGFR TKI should be done to confirm this finding.
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MINI34.07 - A Novel microRNA Signature Associated with Cisplatin Resistance in NSCLC (ID 2709)
19:05 - 19:10 | Author(s): L. Mac Donagh, S.G. Gray, V. Young, R. Ryan, S. Nicholson, N. Leonard, K.J. O'Byrne, S.P. Finn, S. Cuffe, M.P. Barr
- Abstract
- Presentation
Background:
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that range in size from 19 to 25 nucleotides. Alteration in miRNA expression can cause them to act as either tumour suppressor or oncogenes. They have also been shown to regulate a number of processes involved in tumour biology such as metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemo- and radio-resistance in many solid tumours, including lung cancer.
Methods:
An isogenic model of cisplatin resistance was established by chronically exposing a panel of NSCLC cell lines (MOR, H460, A549, SKMES-1, H1299) to cisplatin for 12 months, generating cisplatin resistant (CisR) sublines from their corresponding age-matched parental (PT) cells. MicroRNA expression profiling was carried out using 7th generation miRCURY LNA™ microRNA arrays consisting of 1,919 miRNAs (Exiqon). MicroRNAs that were significantly increased in CisR sublines were inhibited using antagomirs (Exiqon), while those that were significantly decreased were over-expressed using pre-miRs (Ambion). Functional studies examining clonogenic survival ability, proliferation (BrdU) and apoptosis (Annexin V/PI) were subsequently carried out in the presence or absence of cisplatin. To examine the translational relevance of these microRNAs, their expression was further examined in a cohort of pre-treatment matched normal and tumour lung tissues from NSCLC patients of different histological subtypes. Validation of this miRNA signature is currently being investigated in serum samples from this same cohort of patients and normal controls.
Results:
MicroRNA profiling analysis identified ten miRNAs which were significantly altered between parental and corresponding cisplatin resistant lung cancer cell lines. Validation of these miRNAs by real-time PCR (qPCR) identified a specific 5-miR signature that was significantly altered in CisR cells relative to their parental counterparts. Modification of these microRNAs altered the response of resistant cells to the cytotoxic effects of cisplatin and decreased the clonogenic survival of CisR cells when treated with increasing doses of cisplatin (0.1µM-10μM). Significant differential expression was found between normal and tumour tissues across each histological subtype, highlighting the potential use of these microRNAs as markers of response to cisplatin therapy in NSCLC patients. Three miRNAs (miR-A, B, C) belonging to the same family were significantly altered in tumour lung tissue of adenocarcinoma and squamous cell histology compared to matched normal lung tissue. MicroRNA-D expression was significantly altered in squamous cell carcinomas while miR-E was differentially expressed in adenocarcinomas only. Data relating to the expression of this novel signature in the circulation of our NSCLC patient cohort and normal controls will be presented at WCLC 2015.
Conclusion:
We have identified and validated a novel miRNA signature associated with cisplatin resistance in a panel of cisplatin resistant cell lines and in patient lung tumours. Genetic manipulation of these specific miRNAs in vitro altered the cisplatin resistant cell response to the cytotoxic effects of cisplatin chemotherapy. The data obtained from this study may provide a basis for the potential development of a companion diagnostic for lung cancer patients who are most likely to benefit, or not, from cisplatin chemotherapy.
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- Abstract
- Presentation
Background:
Lung adenocarcinoma is one of the main pathological tissue types of lung small cell lung cancer (NSCLC). Tumor metastases are responsible for approximately 90% of lung adenocarcinoma-related death. The epithelial–mesenchymal transition (EMT) is regarded as a critical event during tumor metastasis. From our previous study, we found miR-33b might be involved in the EMT process of lung adenocarcinoma. However, the specific mechanism of miR-33b regulating EMT has not been established.
Methods:
Quantitative real time-PCR (qRT-PCR), in situ hybridization and immunohistochemistry were used to investigate expression of miR-33b and ZEB1 in paired lung adenocarcinoma tissues. MiR-33b target gene ZEB1 was investigated using luciferase reporter gene assays. Western blot, qRT-PCR, immunofluorescence were used to compare the expression levels of ZEB1, E-cad, Vim and β-catenin in A549 cells which were transfected miR-33b or miR-NC, anti-miR-33b or anti-miR-NC and in vivo. MTT was used to analysis growth curves of transfected cells with miR-33b or miR-NC, anti-miR-33b or anti-miR-NC, siRNA-NC or siRNA -ZEB1, siRNA-NC or siRNA-β-catenin. The transfected cells were subjected to Transwell migration and invasion assays.
Results:
Mean miR-33b levels were significantly lower in lung adenocarcinoma tissues compared with matched non-cancerous tissues (0.024± 0.028 vs 0.063 ± 0.074, P < 0.0001). The level of miR-33b in NSCLC was strongly correlated with lymph node metastasis (P < 0.0001). ZEB1 levels were significantly higher in lung adenocarcinoma tissues compared with matched non-cancerous tissues (0.447± 0.371vs0.084 ± 0.112, P <0.0001). The level of miR-33b in lung adenocarcinoma was negative correlated with ZEB1 (P < 0.0001,r=-0.6077). MiR-33b significantly inhibited EMT in lung adenocarcinoma metastasis in vitro and vivo (P < 0.05).MiR-33b directly targets the ZEB13[']UTR(P < 0.01).β-catenin was down regulation by overexpression of miR-33b (P < 0.01). MiR-33b overexpression and ZEB1 inhibition suppressed lung adenocarcinoma migration and invasion in vitro (P < 0.01).
Conclusion:
On the basis of our results, we propose that miR-33b suppress EMT in lung adenocarcinoma through direct targeting of ZEB1 in vitro and in vivo. Moreover, we found that Wnt/β-catenin/ZEB1 pathway regulated by miR-33b might involved in lung adenocarcinoma EMT. These findings provide new insights into the molecular functions of miR-33b as well asthe role of ZEB1 in lung adenocarcinoma metastasis.
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- Abstract
Background:
Recent evidence indicates that long non-coding RNAs (lncRNAs) play a critical role in the regulation of cellular processes, such as differentiation, proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. In our previous study, we have demonstrated that lncRNA AK126698 regulated A549 cells cisplatin resistance partly through Wnt/β-catenin signaling. However, the clinical significance of lncRNA AK126698, and its’ molecular mechanisms of controlling cancer cell proliferation and migration are still unclear.
Methods:
Expression of lncRNA AK126698 was analyzed in 55 non-small cell lung cancer (NSCLC) tissues and 3 NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of AK126698 in NSCLC cells. The effects of AK126698 on cell proliferation were evaluated by CCK-8 and colony formation assays. Apoptosis was evaluated by flow cytometry. Protein levels of AK126698 targets were determined by western blotting and fluorescent immunohistochemistry.
Results:
AK126698 expression was significantly decreased in NSCLC tumor tissues compared with normal lung tissues. Additionally, reduced AK126698 expression was associated with larger tumor size and advanced pathological staging of NSCLC patients. Ectopic expression of AK126698 impaired cell proliferation and migration and induced apoptosis in vitro. However, knockdown of AK126698 expression promoted cell migration and invasionin in vitro. In addition, the expression of FZD8 was inversely correlated with AK126698 in both NSCLC tissues and cell lines and that over-expression of AK126698 could significantly down-regulate the expression of gene downstream of FZD8, including β-catenin, CyclinD1 and c-Myc.
Conclusion:
The results of present study indicate that lncRNA AK126698 might serve as a suppressor that regulates the pathogenesis of NSCLC through Wnt/β-catenin signaling pathway. It may provide a new target for therapeutic intervention of NSCLC.
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MINI34.10 - Discussant for MINI34.06, MINI34.07, MINI34.08, MINI34.09 (ID 3434)
19:20 - 19:30 | Author(s): G. Reid
- Abstract
- Presentation
Abstract not provided
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MINI34.11 - Identification and Functional Characterization of Non-Small Cell Lung Cancer-Associated Splice Variants and Splicing Factors (ID 2101)
19:30 - 19:35 | Author(s): F. De Miguel, M.J. Pajares, E. Martinez-Terroba, X. Morales, R.D. Sharma, A. Rouzaut, A. Rubio, L. Montuenga, R. Pio
- Abstract
- Presentation
Background:
Deregulation of alternative splicing has become a hallmark of cancer. In non-small cell lung cancer (NSCLC), the biological importance of splicing is evidenced by the identification of aberrant RNA transcripts associated with somatic mutations in genes encoding splicing factors. We have previously developed ExonPointer, an algorithm optimized to detect differential splicing cassette events from data obtained in microarrays containing probes in exons and junctions. Our present objective was to apply this technology for the identification and characterization of cancer-associated splice variants and splicing factors in NSCLC.
Methods:
We applied ExonPointer for the identification of differential splice forms in lung cancer tissues (8 adenocarcinomas, 13 squamous cell carcinomas and 1 large cell carcinoma) and matched normal lung. We validated the events by RT-PCR and used bioinformatics tools, such as DAVID and Ingenuity Pathway Analysis, for cluster enrichment analyses. siRNA knockdown of specific splice isoforms was used for functional analyses. Prognostic studies were performed by immunohistochemistry in 127 primary tissues from patients with NSCLC.
Results:
The validation rate for the top 20 differentially expressed splice events identified by ExonPointer was 70%. Gene cluster analyses using the first 250 events showed a significant enrichment of cancer-related clusters such as Cellular Growth and Proliferation, or Cell Death and Survival. Among the validated genes, we identified Extended synaptotagmin-2 (ESYT2). ESYT2 is a membrane protein that mediates fibroblast growth factor receptor-1 (FGFR1) endocytosis and actin dynamics. A significantly different pattern of ESYT2 alternative splicing was found in primary lung tumors as compared to normal lung tissue (p<0.001). In particular, an isoform containing an extra exon was overexpressed in cancer tissues, while the expression of the canonical isoform was decreased. We found a significant correlation between the splicing pattern of ESYT2 and the expression of FGFR1 in a panel of 43 lung cancer cell lines (r=-0.724, p<0.001). Using siRNA downregulation, we analyzed the implication of the ESYT2 isoforms in tumor biology and demonstrated a distinct role of the splice isoforms in actin and tubulin cytoskeleton organization. We also searched for splicing factors responsible for the splicing of ESYT2. We found that the ratio of ESYT2 isoforms correlated with the expression of the splicing factor QKI in lung cancer cell lines (r=-0.793, p<0.001). Moreover, in vitro downregulation of QKI markedly affected ESYT2 splicing. Interestingly, we found a significant enrichment of QKI targets in the list of differentially spliced genes identified by ExonPointer (p<0.001), suggesting that this factor is a critical regulator of splicing in lung cancer. Finally, we observed a significant downregulation of QKI expression in primary NSCLC compared to adjacent normal lung cancer cells (p<0.001), and an association between the nuclear expression of this factor and disease-free survival (HR=0.61; 95%CI=0.35-1.05) or overall survival (HR=0.44; 95%CI=0.21-0.94).
Conclusion:
Using a novel analytical tool we have identified new splicing variants with functional relevance in lung cancer. Moreover, changes in splicing events in lung primary tumors were found to be largely regulated by the splicing factor QKI, a potential tumor suppressor gene downregulated in NSCLC and associated with the prognosis of the disease.
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MINI34.12 - Oncofetal miRNA Expression Inactivates Nuclear Factor I/B, a Critical Regulator of Lung Development and Lung Adenocarcinoma Pathogenesis (ID 3023)
19:35 - 19:40 | Author(s): D.D. Becker-Santos, K.L. Thu, L.A. Pikor, C.E. Macaulay, W.W. Lockwood, J.C. English, W.P. Robinson, I. Jurisica, S. Lam, W.L. Lam
- Abstract
Background:
Fetal development shares many biological similarities with tumourigenesis such as high rates of cell proliferation and vasculature restructuring. Particularly in the lung, a number of genes and pathways involved in the development of this organ play important roles in the malignant transformation of adult lung cells. Despite these biological similarities, there is a paucity of information regarding how specific molecular regulators involved in fetal lung development become oncogenes in lung cancer. This is the case for micro RNAs (miRNAs), which have a pivotal role in regulating gene expression during organ development and tumourigenesis. Therefore, a better understanding of the human fetal lung miRNA-transcriptome has the potential to reveal key players in lung cancer development and progression.
Methods:
131 pairs of non-small cell lung cancer (NSCLC) tumour and adjacent non-malignant lung tissues and 15 human fetal lung tissue samples were profiled by miRNA-sequencing. Mann–Whitney U tests were performed with Benjamini-Hochberg multiple testing corrections to identify miRNAs abundantly expressed in fetal and tumour tissues but scarce in non-malignant adult lung (i.e. oncofetal miRNAs). To investigate protein-coding genes controlled by the oncofetal miRNAs identified, miRDIP was applied followed by gene reporter luciferase assay experiments. The assessment of associations between patient survival and mRNA expression of selected genes targeted by the oncofetal miRNAs was evaluated in multiple NSCLC cohorts (>1,400 tumour cases). To validate the protein expression of the most prominent gene regulated by the oncofetal miRNAs identified, immunohistochemical (IHC) analysis was performed on a lung adenocarcinoma (LUAD) tissue microarray (TMA).
Results:
We describe for the first time a comprehensive, unbiased characterization of miRNA expression in human fetal lung tissue by miRNA sequencing. Through comparison to a large cohort of NSCLC, we identified numerous miRNAs that recapitulate their fetal expression patterns in lung cancer. Strikingly, assessment of the genes potentially regulated by these oncofetal miRNAs led us to identify Nuclear Factor I/B (NFIB), a transcription factor essential for lung development, as being frequently targeted by oncofetal miRNAs. Concordantly, analysis of NFIB expression using RNA-sequencing data for multiple NSCLC cohorts revealed its frequent underexpression in tumours (>60%). Remarkably, 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. cells that are the precursors of LUAD). Furthermore, an NFIB-related gene signature was identified in LUAD tumours and included several well-known lung differentiation markers (TTF-1, ABCA3, GPR116, SFTPB). Finally, the underexpression of NFIB protein was validated on a LUAD TMA, 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 a crucial gene for lung development, which we found to be associated with aggressive phenotypes of LUAD and consequently, poor patient survival. Elucidating the specific role of NFIB in lung cancer biology will likely lead to the identification of targetable tumour vulnerabilities.
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MINI34.13 - Long Noncoding RNA BC070487 Represses ZNFX1 during Tobacco-Induced Lung Carcinogenesis (ID 2912)
19:40 - 19:45 | Author(s): S. Xi, J. Shan, H. Xu, Z. Xiao, Y. Xiong, S. Oyetunji1, L. Mercedes, M. Zhang, J.A. Hong, D.S. Schrump
- Abstract
- Presentation
Background:
Limited information exists regarding regulation of gene expression by long noncoding RNAs (lncRNAs) during initiation and progression of tobacco-associated lung cancers. In the present study, an in-vitro model system was used to examine the effects of cigarette smoke on lncRNA expression in human respiratory epithelia and lung cancer cells.
Methods:
Micro-array and qRT-PCR techniques were used to assess lncRNA and gene expression profiles in normal human small airway epithelial cells (SAEC) and immortalized human bronchial epithelial cells (HBEC) cultured in the presence or absence of cigarette smoke condensate (CSC). Quantitative chromatin immunoprecipitation (ChIP), methyl-DNA immunoprecipitation (MeDIP), and cross-link immunoprecipitation (CLIP) techniques were used to assess promoter occupancy, DNA methylation, and interaction of lncRNAs with target proteins. Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assays were used to identify a regulatory sequence for lncRNA BC070487.
Results:
Micro-array analysis demonstrated that under relevant exposure conditions, CSC consistently mediated a 2.5 fold increase in BC070487 expression, and 3.6 fold decrease in its immediate downstream target gene, ZNFX1, in SAEC. qRT-PCR experiments confirmed a 4-7 fold up-regulation of BC070487, with a 4-8 fold down-regulation of ZNFX1 in SAEC and HBEC, as well as Calu-6 and H841 lung cancer cells following CSC exposure. BC070487 expression correlated inversely with expression of ZNFX1 (BC070487: 1.38-10.31 fold up vs ZNFX1: 2.26-26.29 fold down) in primary lung cancers relative to adjacent normal lung parenchyma. Overexpression or depletion of BC070487 inhibited or enhanced expression of ZNFX1, respectively in normal respiratory epithelia and lung cancer cells. CSC as well as BC070487-mediated repression of ZNFX1 coincided with increased occupancy of EZH2, SUZ12, BMI1, and increased levels of H3K27Me3, with decreased H3K4Me3 within the ZNFX1 promoter region. CSC as well as BC070487 overexpression enhanced binding of BC070487 with EZH2, SUZ12 and BMI1 proteins in SAEC and Calu-6 cells. CSC exposure significantly increased DNA methylation in one of three CpG islands spanning the regulatory elements of ZNFX1. In addition, CSC enhanced binding of BC070487 with DNMT3A and DNMT3B with site-specific de novo DNA methylation of ZNFX1 in SAEC and Calu-6 cells. FAIRE assays identified a 800 bp regulatory sequence for BC070487; CSC-mediated activation of BC070487 coincided with increased levels of H3K4Me3, H3K27Ac, H3K36Me3, Sp1, and Ago proteins, with decreased levels of H3K27Me3 and H3K9Me3 within this regulatory region. miR-31, an oncomiR specifically induced by CSC, modulated the transcriptional activity of BC070487 via remodeling the distribution of histone modification marks (Up: H3K4Me3, H3K27Ac, H3K36Me3; Down: H3K27Me3 and H3K9Me3) in the regulatory region of BC070487. Overexpression or depletion of BC070487 increased or inhibited proliferation and invasion of lung cancer cells, and similarly modulated growth of SAEC and HBEC cells. In contrast, ZNFX1 overexpression or depletion decreased or enhanced growth of normal respiratory epithelial cells and lung cancer cells. BC070487 overexpression enhanced growth of lung cancer cells xenografts in athymic nude mice, while forced expression of ZNFX1 arrested growth of these xenografts.
Conclusion:
Collectively, these data suggest that CSC-mediated up-regulation of BC070487 suppresses ZNFX1 to promote pulmonary carcinogenesis.
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MINI34.14 - Biological Role of Prognostic MicroRNAs (MiRNAs) in Squamous Lung Cancer Cell Lines (ID 1171)
19:45 - 19:50 | Author(s): M. Filipska, M. Skrzypski, J.J. Bigda, J. Jassem
- Abstract
- Presentation
Background:
The benefit from postoperative chemotherapy in non-small cell lung cancer (NSCLC) is modest and there are no reliable tools allowing for individual selection of high-risk patients for this management. We earlier demonstrated that overexpression of three miRNAs: miR-662, -192 and -192* may correlate with the risk of distant relapse in squamous cell lung cancer (SCC) patients undergoing pulmonary resection (Skrzypski et al. 2014, Br J Cancer). However, the role of these miRNAs in maintaining aggressive phenotype and/or chemoresistance of SCC is unknown. The aim of this study was to assess the biological role of three abovementioned miRNAs in SCC cells in vitro.
Methods:
In the search for appropriate in vitro model we screened by reverse transcription quantitative PCR 11 NSCLC cell lines (H1703, H520, H662, SK-MES-1, A549, H23, H1975, HCC 827, PC9, H460 and Calu-6) for miRNA expression profiles and analyzed their phenotypic features including migration, invasion and clonogenic potential. H520 and H1703 SCC cell lines were transfected with locked nucleic acid miRNA inhibitors. The sensitivity of transfected and wild type cells to cisplatin and etoposide was compared using cytotoxic MTT test. Soft agar colony formation assay was performed to assess the clonogenic potential of transfected vs. non-transfected cells.
Results:
Among the analyzed NSCLC cell lines, H520 cells showed natural overexpression of miR-662, -192 and -192*. These cells were resistant to both chemotherapeutics and exhibited an ability to form colonies in soft agar. The inhibition of miR-662 and miR-192 sensitized these cells to etoposide (p=0.004 and 0.016, respectively) but not to cisplatin. Similar results were obtained for H1703 cells (p=0.002 and 0.02, respectively). H520 cells treated with miR-192 and miR-662 inhibitors, compared to negative control, also demonstrated reduced number of colonies in soft agar (p<0.001).
Conclusion:
We developed and optimized a cellular model for in vitro studies of biological role of three prognostic miRNAs in SCC. Genes regulated by miR-662 and/or miR-192 may be involved in maintaining the chemoresistance and clonogenic potential of SCC, and these features may be inhibited in a miR-dependent specific manner. Further studies may elucidate predictive value of these genes and a possibility of their targeting with novel therapeutics.
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MINI34.15 - Discussant for MINI34.11, MINI34.12, MINI34.13, MINI34.14 (ID 3435)
19:50 - 20:00 | Author(s): D. Beer
- Abstract
- Presentation
Abstract not provided
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ED 02 - Molecular Testing Around the World (Genomics in Clinic (Timelines/Bioinformatics), Testing Platforms & Algorithms (NGS, Targeted Panels, FISH, IHC), Cost Considerations, Strategies for Identifying Rare Genomic Subsets in Clinical Trials) (ID 2)
- Event: WCLC 2015
- Type: Education Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:M. Ladanyi, G. De Lima Lopes
- Coordinates: 9/07/2015, 14:15 - 15:45, Four Seasons Ballroom F1+F2
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ED02.03 - Europe (ID 1776)
15:00 - 15:20 | Author(s): C. Mascaux
- Abstract
- Presentation
Abstract:
Introduction Identification of molecular targets is now essential for the diagnosis, classification, selection and treatment monitoring of an increasing number of cancers. The analysis of these biomarkers must therefore be accessible to all patients, regardless of the healthcare facility where they are treated. Despite a decrease, lung cancer remains the leading cause of cancer-related deaths worldwide, often detected at an advanced stage with association with a poor prognosis. Recently, genetic alterations involved in adenocarcinomas have been discovered; some of these tumors harbor a driver mutation involving EGFR, KRAS, HER2, BRAF or PI3KCA gene or a rearrangement (ALK, ROS1, RET genes). Nearly 40% of NSCLC harbor one of those genetic abnormalities and several targeted therapies have been approved in USA and in Europe, such as erlotinib and gefitinib and crizotinib for NSCLC with EGFR mutation or ALK translocation, respectively. As many other targeted therapies are ongoing in lung cancer, molecular testing becomes a huge challenge worldwide, raising both financial and organizational issues.The French INCa molecular testing network: In 2006, the French National Cancer Institute (INCa) and the French Health Ministry have created a national network of 28 regional genetics platforms throughout the French territory, in order to offer molecular tests to all patients regardless of the institution where they are treated, i.e. university hospitals, cancer centers, hospital centers or private institutions, to enable that each innovative test could be rapidly implemented after new targeted therapies become available, and to guarantee the quality of the tests. The 28 regional platforms include a pathology laboratory in charge with the samples monitoring, and several laboratories with complementary expertise in molecular testing of hematological malignancies and solid tumors; as the tests are free of charge for the patients and the institutions, initial funds were given to all INCa genetics platforms to buy equipment (€4.7 million) and to hire non-medical personnel. The majority of these were technicians (58.32 FTE) and engineers (20.5 FTE); in addition, genetics platforms are financed on a basis of quarterly and annual activity reports to adjust budget and allocation. This initial funding was followed by the allocation of €4 million in annual funding for the centres and staff from the French Ministry of Health. The data sent in those reports are the number of tests performed each year, the number of patients undergoing tests, the percentage of patients found to have a molecular abnormality, the percentage of non-contributive results and the origin of requests. The INCa also set up a quality-assurance program with mandatory external quality evaluations, and publishes guidelines available on the Inca website. The markers studied are predictive markers that determine access to targeted therapy, markers guiding the diagnostic process, markers that contribute to establishing a diagnosis, prognostic markers guiding patient treatment strategy, and markers allowing the monitoring of residual diseases. Molecular genetics platforms do not have to perform all molecular tests: they must ensure that patients in their region have access to these tests via a referral platform. Tests concerning a large number of patients are carried out by all or almost all platforms (BCR-ABL quantification, KRAS and EGFR mutations, JAK2 mutations, MSI tests). For tests concerning a small number of patients, some platforms perform regional or national referral activity (ABL mutation screening in CML, cKIT and PDGFRA mutations in GIST, NMYC amplification in neuroblastomas, chromosomal abnormalities in sarcomas). Whereas the initial program included only EGFR and KRAS mutations’ detection in lung cancers and KRAS and BRAF mutations in colorectal cancers, a new program for detection of emerging biomarkers was set up in 2011; for lung non-squamous cell carcinoma patients at advanced stages those biomarkers were EGFR, K-RAS, HER2, BRAF and PI3KCA mutations and ALK translocations, and BRAF and KIT mutations in metastatic melanomas. In 2008, 1,269 EGFR activating mutation tests were performed, versus 2,667 in 2009, and 21,995 and 8,696 regarding EGFR mutation and ALK rearrangement, respectively in 2012. In 2013, 23,336 lung cancer samples were tested for EGFR mutations (10% were mutated), 18,861 for ALK rearrangement (3.5% rearranged), 22,9858 for KRAS mutations (27% mutated), 20,100 for BRAF mutation (2% mutated), 17,843 for HER2 mutation (0.7% mutated) and 17,375 for PI3KCA mutations (2.4% mutated).The German Network Genomic Medicine (NGM) Lung Cancer: The NGM is a health care provider network offering centralized high-quality next generation sequencing -based multiplex genotyping for lung cancer patients. Since NGS based genotyping is not reimbursed in Germany, the AOK Rheinland/Hamburg, one of the largest German public health insurances has contracted with the NGM for reimbursement of NGS-based multiplex genotyping of lung cancer in April 2014. In 2014, 4,500 lung cancer patients were gentotyped, representing nearly 10% of stage IV NSCLC patients in Germany.The Cancer Research UK (CRUK) Stratified medicine programme 1 and 2: There is no to date any national policy in UK for molecular testing for lung cancer patients, and a great variation exists regarding providers, funding and access; most of the time, the decision of referring for molecular testing depends on the clinician in reference to NICE clinical guidance. Nearly 7,300 UK patients were tested for EGFR mutations in 2010-2011 and depending on the units, ALK testing is performed either by IHC and /or FISH. In 2011, the Cancer Research UK (CRUK) Stratified Medicine Programme 1 and 2 has set-up a collaborative network of 26 hospitals and 8 CRUK Experimental Cancer Medicine Centres to provide genetic testing in lung, bowel, breast cancers and melanoma. The next plan for the NHS of England is to increase testing activity, to equitable access, to improve the quality assurance procedures and the cost values.References: 1. Collection Reports and summaries, collective volume edited by INCa, Boulogne-Billancourt, sept 2010 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/Molecular-genetic-testing-for-equal-access-to-targeted-therapies-in-France-in-2011 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/The-French-national-network-of-28-hospital-molecular-genetics-platforms-summary-of-the-activity-in-2009 http://www.e-cancer.fr/Expertises-et-publications/Catalogue-des-publications/Molecular-genetic-tests-for-access-to-targeted-therapies-in-France-in-2012 2. Nowak, F. et al. Tumour molecular profiling for deciding therapy—the French initiative. Nat. Rev. Clin. Oncol. 9, 479–486 (2012) 3. Nowak, F. et al. Europe Does It Better: Molecular Testing across a National Health Care System—The French Example; 2013 ASCO EDUCATIONAL BOOK | asco.org/edbook 4. Kostenko, A. et al. The network genomic medicine cost reimbursement model for implementation of comprehensive lung cancer genotyping in clinical routine. J Clin Oncol 33, 2015. ASCO Abstract e12556 5. CR UK Stratified Medicine Programme 1 and 2. http://www.cancerresearchuk.org/funding-for-researchers/how-we-deliver-research/our-research-partnerships/stratified-medicine-programme
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MINI 05 - EGFR Mutant Lung Cancer 1 (ID 103)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:Y. Yatabe, R. Perez-Soler
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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MINI05.13 - Treatment of EGFR/ALK-Driven Non-Small Cell Lung Cancer (NSCLC) Brain Metastases: Impact of First-Line Whole Brain Radiotherapy on Outcome (ID 1251)
17:55 - 18:00 | Author(s): C. Mascaux
- Abstract
- Presentation
Background:
Brain metastases (mets) in EGFR/ALK-driven NSCLC are common, and frequently pose treatment dilemmas. Effective systemic therapy with tyrosine kinase inhibitors (TKIs) controls extracranial disease in up to 70% of patients, but often radiotherapy is required for intracranial control. As whole brain radiation (WBRT) may be associated with neurocognitive toxicity, we aimed to evaluate the impact of molecularly targeted therapy and stereotactic radiotherapy (SRS) for EGFR/ALK-driven NSCLC on intracranial disease control with and without WBRT.
Methods:
This retrospective analysis included patients treated with EGFR/ALK-positive NSCLC at Princess Margaret Cancer Centre from 1998-2015, with brain mets at lung cancer diagnosis or during treatment/follow-up. Demographic data were collected from electronic patient records. Time to intracranial progression (TTIP) and overall survival (OS) were calculated from date of diagnosis of brain mets, using the cumulative incidence function and Kaplan-Meier methods respectively; differences between groups were tested with Gray’s or log-rank test.
Results:
From 1998-2015, 162 patients with brain mets from EGFR/ALK-driven NSCLC were identified: 138 in the EGFR cohort, 23 in the ALK cohort and one included in both cohorts for analysis, whose tumour carries both an EGFR mutation and ALK rearrangement. Table 1 contains clinical characteristics and treatment details. In the EGFR cohort, initial brain mets treatment consisted of systemic therapy alone in 19 patients (17 TKI, 2 chemotherapy), SRS +/- surgery in 27 patients and WBRT +/- SRS/surgery in 88 patients. 1-year intracranial progression rates were 26%, 32% and 12%, respectively, and median TTIP was 18, 16 and 40 months [p=0.12]. Median OS was 26, 27 and 34 months respectively [p=0.49]. In the ALK cohort, initial brain mets treatment consisted of systemic therapy alone in 4 patients (1 TKI, 3 chemotherapy), SRS/surgery alone for 4 patients and WBRT +/- SRS/surgery for 15 patients. 1-year intracranial progression rates were 50%, 50% and 13%, respectively, and median TTIP was 18, 14 and 69 months [p=0.028]. Median OS was 35 months, not reached and 51 months, respectively [p=0.75]. Multivariable analysis for the whole group showed that age [p=0.021], number of brain mets [p=0.012] and extracranial control [p=0.008] were significantly associated with OS, but not WBRT [p=0.61].
Conclusion:
In this cohort of patients with brain mets from EGFR/ALK-driven NSCLC, patients treated with WBRT trended to longer TTIP. Although not statistically significant, our data also show a trend towards longer survival in patients who received WBRT. These observations require further validation in this patient population.EGFR (N=139) ALK (N=24) Median Age (Range) 59(29-86) 53(31-77) Female Sex 93(67%) 15(62%) Ethnicity Asian Caucasian Other 58(42%) 63(45%) 18(13%) 7(29%) 13(54%) 4(17%) Smoking Never Smoker Former/Current Smoker Unknown 108(77%) 30(22%) 1(1%) 19(79%) 5(21%) 0 ECOG PS (Diagnosis) 0 1 2-4 66(48%) 67(48%) 6(4%) 7(29%) 14(58%) 3(13%) Brain Mets at Stage IV diagnosis 93(67%) 13(52%) Number of Brain Mets 1 2-4 5+ N/A 32(23%) 39(28%) 62(45%) 6(4%) 9(38%) 6(24%) 9(38%) 0 Symptomatic Brain Mets No Yes 78(56%) 61(44%) 16(67%) 8(33%) Initial Brain Mets treatment WBRT WBRT+SRS/Surgery SRS+/-Surgery Systemic Therapy None 71(51%) 17(12%) 27(19%) 19(14%) 5(4%) 13(54%) 3(12%) 4(17%) 4(17%) 0
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MINI 23 - Lung Cancer Risk: Genetic Susceptibility and Airway Biology (ID 135)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:P.E. Postmus, R. Young
- Coordinates: 9/08/2015, 16:45 - 18:15, 401-404
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MINI23.15 - Discussant for MINI23.12, MINI23.13, MINI23.14 (ID 3426)
18:05 - 18:15 | Author(s): C. Mascaux
- Abstract
- Presentation
Abstract not provided
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ORAL 21 - Biology - Moving Beyond the Oncogene to Oncogene-Modifying Genes (ID 118)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:A. Katz, M.S. Tsao
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL21.05 - p53/KRAS Mutation Status Does Not Predict Sensitivity to Chemotherapy in NSCLC PDXs (ID 2459)
11:28 - 11:39 | Author(s): C. Mascaux
- Abstract
- Presentation
Background:
The LACE-Bio group assessed the prognostic and predictive values of KRAS and p53 mutations in 1543 completely resected non-small cell lung cancer (NSCLC) tumors. The predictive value of combined KRAS/p53 mutations for survival benefit from adjuvant chemotherapy was evaluated on 49 patients and chemotherapy was deleterious in this group compared to observation (HR 2.49 CI 95% [1.10 – 5.66], p=0.03). Patients with tumors harboring combined KRAS/p53 mutations had a worse outcome when treated with adjuvant chemotherapy compared patient with double wild type (WT) tumors (HR 3.03 (95% CI [1.29 – 7.15], p=0.01, interaction p=0.06). We have compared the chemo-sensitivity of patient derived xenografts (PDXs) with double p53/KRAS mutations, single p53, single KRAS mutation or double WT. 0
Methods:
Surgically resected early stage lung adenocarcinomas (ADC) were implanted into non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Fourteen lung ADC PDXs with various p53/KRAS status were revived and implanted: 11 engrafted and were expanded for comparison of treatment vs control. For each model, 6 replicates were included in treatment and control arms. Chemotherapy (cisplatin 3 mg/kg and vinorelbine 7 mg/kg intraperitoneally weekly) was initiated in the PDXs at tumor volumes of 150 mm[3].
Results:
Four models were p53/KRAS double mutant, 4 p53 mutant, 2 KRAS mutant and 1 double WT. The 4 double mutant PDXs responded to chemotherapy, 2 with reduced (SD) and 2 inhibited (PR) growth. Among the 4 PDXs with p53 mutation only, 2 responded (1 PR and 1 SD) and 2 were resistant. Among the 2 PDXs with KRAS mutation only, 1 had a complete response, but relapsed at treatment arrest and 1 achieved PR. The double WT PDX was highly sensitive to chemotherapy (PR) but also relapsed at treatment arrest.
Conclusion:
Among these 11 PDXs, the p53/KRAS mutation status did not predict chemo-sensitivity to cisplatin/vinorelbine, one of the most active adjuvant chemotherapy regimens in NSCLC. As these PDXs were molecularly profiled, we currently are investigating other biomarkers that might predict their sensitivity or resistance to chemotherapy.
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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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P2.04-026 - Second Generation EGFR TKIs Inhibit Tumor Growth in a Chemo-Resistant Squamous Cell Lung Cancer Patient Derived Xenograft Model (ID 1265)
09:30 - 09:30 | Author(s): C. Mascaux
- Abstract
Background:
In clinical trials testing the efficacy of first generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), occasional responses were observed in patients with lung squamous cell carcinomas (LSCC) (Shepherd FA, et al. New Engl J Med 2005) and survival benefit was confirmed for erlotinib in a subset analysis of male, ever-smokers with LSCC (Clark GM, et al. Clin Lung Cancer 2006). Currently, the LUX-Lung 8 phase III clinical trial is comparing afatinib versus erlotinib in the second-line setting for LSCC after cisplatin-based chemotherapy (Goss GD, et al. ESMO 2014). Preclinical data indicate that high EGFR protein expression may be predictive of response to erlotinib in EGFR wild type LSCC (Cranston et al, AACR 2013). Herein we assessed and compared the anti-tumor efficacy of different EGFR inhibitors in chemo-resistant squamous cell lung cancer patient derived xenograft (PDX) models with high EGFR expression and EGFR amplification.
Methods:
The cryopreserved PDX model established from a resected early stage LSCC was revived in non-obese diabetic severe combined immunodeficient mice (NOD SCID), expanded and subsequently treated with chemotherapy (cisplatin 3 mg/kg and vinorelbine 7 mg/kg intraperitoneally [IP]), cetuximab 20 mg/kg IP, and daily oral schedules were followed for erlotinib 50 mg/kg, afatinib 20 mg/kg, dacomitinib 3 mg/kg. For each model, 6 mice were used in each of the different treatment and the control arms. Treatment was initiated in the PDXs at a tumor average volume of 150 mm[3].
Results:
The PDX was derived from a 57 year old male, smoker, following right pneumonectomy for a stage IIIB (T4N1M0) LSCC. This patient received adjuvant cisplatin/vinorelbine, but relapsed three weeks after the end of cycle 4 and died a week later. The tumor had a high EGFR expression by immunohistochemistry (H score = 300) and EGFR amplification (clusters) by fluorescent in situ hybridization. The PDX was EGFR wild type by Illumina exome sequencing and OncoCarta[TM ]MassArray mutation screen (Sequenom), also was refractory to cisplatin/vinorelbine. Reduced growth rate (stable disease, SD) was obtained with erlotinib and cetuximab. Treatment with afatinib and dacomitinib resulted in tumor growth inhibition (partial response, PR). The PDX developed resistance to dacomitinib after 100 days of treatment, but continued to be inhibited by afatinib after 215 days of treatment.
Conclusion:
This study shows the efficacy of second generation especially afatinib irreversible EGFR TKIs in a chemoresistant LSCC PDX, with high wild type EGFR expression and EGFR amplification. Our results lend further support to the LUX-Lung 8 trial, and also the use of PDX to model therapeutic responses in lung cancer.
