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L. Fernandez-Cuesta
Moderator of
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MINI 14 - Pre-Clinical Therapy (ID 119)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 15
- Moderators:L. Fernandez-Cuesta, A.F. Gazdar
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI14.01 - EGFR-Mutated PDX in NSCLC: Molecular Fidelity and Correlation of PDX and Patient Response to EGFR Inhibition (ID 2191)
10:45 - 12:15 | Author(s): J.W. Riess, D.R. Gandara, R.A. Tsai, M. Cheng, W.S. Holland, K.D. Crawford, H. Yang, T. Li, P. Lara Jr., K. Kelly, S. Airhart, J. Graber, C. Bult, J.G. Keck, P.C. Mack
- Abstract
- Presentation
Background:
Inevitable emergence of resistance to tyrosine kinase inhibitor (TKI) therapy in EGFR-mutated NSCLC warrants development of pro-active therapeutic strategies to delay or circumvent this evolution. To model such approaches, we are employing a clinically and genomically annotated patient derived xenotransplant (PDX) resource designed to duplicate relevant known mechanisms of resistance to TKI therapy. This analysis examines molecular fidelity and correlates response between patient and PDX in EGFR-mutant NSCLC.
Methods:
Six EGFR-mutated NSCLC, 1 EGFR-TKI naïve and 5 after progressive disease on erlotinib, were implanted subcutaneously into the flank of NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as previously described (DR Gandara, Clin Lung Cancer 2015). Models were considered established when PDX growth was confirmed in passage 1 (P1); tumor growth studies were conducted in P3-P5. The donor patient tumor (PT) and the resultant PDX were analyzed for driver mutations (Response Genetics Inc., and Illumina TSCAP), copy number variants (CNV) and global RNA expression (Affymetrix arrays). Informed consent was obtained from all patients. EGFR-mutant PDX treatments included: erlotinib, afatinib, cetuximab, and afatinib+cetuximab. Patient response was graded by RECIST 1.1 and measured in PDX by tumor shrinkage from pre-treatment baseline. In select models, pharmacodynamic studies (kinase arrays; immunoblotting) were also performed.
Results:
The EGFR mutation subtypes identified in the donor PT were preserved in all PDX models (4 EGFR E19del and 2 EGFR L858R). Corresponding putative mechanisms of resistance were identical in both PT and PDX in 3 cases: EGFR T790M (2 of 5) and MET amplification (1 of 5). Of 5 post-erlotinib progression PDX models, 3 had progressive disease (PD) and 2 had transient tumor shrinkage to erlotinib. The PDX derived from an erlotinib-naïve patient (EGFR E19del) demonstrated sustained tumor shrinkage to erlotinib. Patient-PDX treatment correlations were possible in 3 post erlotinib-progression models. Two of these patients received afatinib-cetuximab: 1 with partial response (PR) and 1 with PD. The two models corresponding to these patients, when treated with afatinib-cetuximab, underwent complete regression of tumor (CR) and PD, respectively. Pharmacodynamic assessment of the responding model at 24h showed near complete diminishment of pEGFR following afatinib-cetuximab, concomitant with decreased pHer2, pERK, pAKT and p38. Erlotinib showed transient inhibition on signaling in this model at 6h, returning to baseline by 24h. In contrast, the non-responding model showed minimal effects on target inhibition and signal transduction following treatment with any EGFR inhibitor.
Conclusion:
Genomic fidelity was preserved in EGFR-mutant PDX, including putative mechanisms of resistance in the post-erlotinib progression models. The majority (3/5) of the EGFR-mutant PDXs created after erlotinib resistance demonstrated PD. In the other post-erlotinib progression models transient tumor shrinkage was noted, which may reflect PDX passaging in the absence of selective pressure of EGFR-inhibition or pharmacokinetic considerations. Overall, the PDX response to treatment reflected the corresponding patient’s clinical course. Pharmacodynamic studies of select models informed PDX response to treatment.
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MINI14.02 - TAE226, a Bis-Anilino Pyrimidine Compound, Shows Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells including T790M Mutant (ID 1646)
10:45 - 12:15 | Author(s): H. Yamamoto, H. Otani, M. Takaoka, M. Sakaguchi, J. Soh, M. Jida, T. Ueno, T. Kubo, H. Asano, K. Tsukuda, K. Kiura, S. Hatakeyama, E. Kawahara, Y. Naomoto, S. Miyoshi, S. Toyooka
- Abstract
- Presentation
Background:
TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). These tyrosine kinases are known to be overexpressed in many malignant tumors including some NSCLCs and to play an oncogenic role in cancer cells.
Methods:
We investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. Drug sensitivity of TAE226 to various NSCLC cell lines was determined by MTS assay. Interaction of TAE226 and variant EGFR proteins was evaluated by in vitro binding assay, and kinetic interaction analysis to calculate K~d~ value. Finally, the effect of TAE226 on NSCLC was investigated using a xenograft mouse model.
Results:
TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity.
Conclusion:
We showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation. Our results showed that the EGFR L858R/T790M (or delE746_A750/T790M) mutant retains the binding affinity to TAE226 comparable to that of the L858R (or delE746_A750) mutant, suggesting that TAE226, or its relatives, is promising to overcome acquired TKI resistance mediated by EGFR T790M mutation.
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- Abstract
Background:
basic fibroblast growth factor (bFGF) is an important molecule that involved with proliferation, angiogenesis, invasion and metastasis in malignant tumors. bFGF in lung adenocarcinoma,squamous cell carcinoma, breast cancer, colon cancer, malignant melanoma cells was highly expressed in the cytoplasm and cytoplasm. bFGF expression was closely related with tumor poor prognosis. FGF pathway activation is a potent driver of lung cancer. Autocrine activation of FGF signaling in NSCLC may contribute to EGFR inhibitor insensitivity. We have developed new monoclonal antibody targeting bFGF (anti-bFGF mAb) which neutralizes bFGF, blocking its ability to activate FGFR1 in treating solid tumors. The antitumor, antiangiogenesis, antimetastatic and reversal multidrug resistance (MDR) activities of anti-bFGF mAb could be investigated.
Methods:
The effect of anti-bFGF mAb on the proliferation of cancer cells was detected by CCK-8 method. Cellular apoptosis, cell cycle distribution and the expression of associated protein were analyzed by flow cytometry. The expressions of associated protein with apoptosis, metastasis, multidrug resistance, anti-bFGF mAb in suppressing cancer cells growth through the PI3K/AKT/mTOR pathway were examined by real-time fluorescence quantitative PCR and Western blotting. Preclinical pharmacokinetics of anti-bFGF mAb was measured in mice.
Results:
Anti-bFGF mAb significantly could inhibit the proliferation and induce apoptosis of lung cancer and show obvious inhibitory effects on the migration of cancer cells and the tube formation of HUVECs in vitro. Treatment of transplanted cancer with anti-bFGF mAb in vivo resulted in significant reduction in tumor size and prolonged survival time of mice. The expression of caspase-3，caspase-9, PARP, and BAX in combination group was higher than those from either agent alone. Anti-bFGF mAb suppressed the PI3K/AKT/mTOR pathway. The radiotherapy sensitization enhancement ratio of the combined treatment group increased 2.37 times by anti-bFGF mAb. bFGF, VEGF expression and MVD were significantly decreased by anti-bFGF mAb. Anti-bFGF mAb could induce down-regulated P-glycoprotein and MDR1. The main pharmacokinetic parameters of anti-bFGF mab were as follows: T1/2α 0.2 h，T1/2β 1.84h and T1/2γ 90.3h. Lung tissue was major organ for deposition of anti-bFGF mAb.
Conclusion:
Anti-bFGF mAbs display remarkable antitumor and antiangiogenic effects in vitro and in vivo. Anti-bFGF mAb is potential therapeutic candidates for lung cancer by effectively suppressing the tumor growth through inhibition of angiogenesis, proliferation, induction of apoptosis and autophagy, reversal of MDR. Further preclinical and systematical investigation on anti-bFGF mAb may help to increase efficacy and safety of molecular target treatment in lung cancer.
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MINI14.04 - In Vitro and in Vivo Evaluation of the Kinase Inhibitor, MGCD516, in TRK and RET Fusion Cancer Cells (ID 2756)
10:45 - 12:15 | Author(s): A.T. Le, A. Estrada-Bernal, S. Nelson, J. Christensen, R.C. Doebele
- Abstract
- Presentation
Background:
The paradigm of treating oncogene-selected patients with non-small cell lung cancer (NSCLC) and other malignancies using targeted kinase inhibitors has significant improved patient outcomes, specifically for patients harboring ALK, ROS1, and EGFR oncogenes. Additional oncogene targets that may benefit from this therapeutic strategy are therefore of immense interest. NTRK1 (TRKA) and RET gene fusions are recently identified oncogenes in NSCLC (and other malignancies) without approved kinase inhibitors. MGCD516 is a spectrum-selective tyrosine kinase inhibitor with activity against TRKA, RET, MET, VEGFR, PDGFR, AXL, and Eph family of receptors. In this report, we evaluated MGCD516 in vitro activity in cell lines with an NTRK1, NTRK3, or RET gene rearrangements. Additionally, we used a mouse xenograft model to assess the in vivo effects of MGCD516 on tumors harboring TRKA and RET fusions.
Methods:
Gene fusion positive cell lines, KM12 (TPM3-NTRK1), CUTO-3 (MPRIP-NTRK1), MO-91 (ETV6-NTRK3) and LC-2/Ad (CCDC6-RET) were used for the in vitro evaluation of MGCD516 inhibitory activity against these oncogenic fusion kinases. Cell lines were assessed for cell viability (MTS-base proliferation assay) and downstream signaling pathways (immunoblot analysis) upon treatment with MGCD516. For in vivo studies, xenograft models of TRKA fusion tumors (CUTO-3 and KM12) and RET fusion tumors (LC-2/Ad and a tumor biopsy from a KIF5B-RET patient) were generated in athymic nude mice. Once tumors reached ~200cm[3], a single daily dose of 5mg/kg, 10mg/kg or 20mg/kg of MGCD516 was given to mice by oral gavage. Mice in the control arm of the study were gavaged with vehicle at similar volume. Tumor size and weight measurement of mice were assessed 3 times per week.
Results:
MGCD516 had notable in vitro effects on the proliferation of cell lines with either RET fusion (LC-2/Ad), TRKA fusion (KM12 and CUTO-3) or TRKC fusion (MO-91) with low nanomolar IC~50~. Western blot analyses showed specific loss of phosphorylated CCDC6-RET or TRKA/C fusion protein and decreased activation of the AKT and MAPK signaling pathways when cells were treated with MGCD516. In mouse xenograft studies, tumors with TRKA fusion displayed dose-dependent growth inhibition at 5mg/kg and 10mg/kg daily doses of MGCD516 compared to controls. Notably, we observed tumor regression in the mice originally assigned to the vehicle control arm once we enrolled the mice on a 10mg/kg or 20mg/kg daily regimen of MGCD516. Comparable to the TRKA fusion xenografts studies, RET fusion tumors were growth inhibited with a 20mg/kg daily dose of MGCD516.
Conclusion:
The spectrum-selective tyrosine kinase inhibitor, MGCD516, demonstrates potent in vitro activity in multiple TRKA/C and RET fusion cancer cell line models and in vivo activity against TRKA and RET fusion kinase in murine xenograft models. A phase I clinical trial of MGCD516 is ongoing and the inclusion of patients with TRK and RET fusion is planned.
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MINI14.05 - Discussant for MINI14.01, MINI14.02, MINI14.03, MINI14.04 (ID 3341)
10:45 - 12:15 | Author(s): J.V. DeGregori
- Abstract
- Presentation
Abstract not provided
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- Abstract
- Presentation
Background:
High-affinity peptidomimetic or small-molecule ligands against cancer cell surface receptors have attracted wide interest as cancer-targeting agents to enhance cancer diagnosis and treatment. We have previously identified and characterized several peptide ligands specific for different integrins, such as a3b1, a4b1 and avb3, on live tumor cells using our invented high throughput one-bead one-compound (OBOC) random combinatorial libraries and “on-bead” whole-cell binding assay. The objective of this study was to select the best integrin ligand for the screening, imaging and targeted drug delivery studies in NSCLC.
Methods:
High-affinity integrin ligands coated on the surface of TentaGel resin beads were screened for the binding to a panel of NSCLC cell lines, malignant pleural effusion and peripheral blood mononuclear cells (PBMCs) from lung cancer patients using the established whole-cell binding assay. The binding affinity was determined in selected NSCLC tumors by flow cytometry using the biotinylated integrin ligands and Streptavidin-Phycoerythrin (PE). For in vivo biodistribution, mice bearing subcutaneous and intracranial NSCLC xenograft tumors were injected with LXY30-biotin-streptavidin-Cy5.5 conjugate for 6 hrs before being subjected to in vivo and ex vivo optical imaging.
Results:
Among the available integrin ligands, LXY30, a recently optimized cyclic peptide targeting α3β1integrin, bound to the majority of NSCLC cell lines tested within one hour of incubation. We further demonstrated that LXY30 bound to α3β1 integrin on the surface of lung cancer cells with high specificity by flow cytometry and entered into the cells via endocytosis by fluorescence microscopy imaging. Flow cytometry confirmed the high specificity binding of LXY30 to NSCLC cells. While sparing the PBMCs isolated from lung cancer patients, LXY30 strongly bound to tumor cells in the pleural effusion from several NSCLC patients and could capture the tumor cells spiked into PBMCs in 1: 5,000 dilution after a 2-hour incubation. Furthermore, several EGFR-mutant lung cancer cell lines have high level expression of α3β1 integrin on their surface. In nude mice bearing two representative, EGFR-mutant lung cancer H3255 (EGFR L858R) and H1975 (EGFR 858R/T790M) xenograft tumors, optical images have shown the preferential updates of LXY30-biotin-streptavidin-Cy5.5 conjugate in the subcutaneous and intracranial xenograft tumors.
Conclusion:
The rapid, sensitive and high specificity binding of LXY30 makes it an ideal candidate for screening and isolating NSCLC tumor cells in the pleural effusion and whole blood. LXY30 can be used as a cancer-targeting agent to guide in vivo delivery of imaging dye and cancer drugs to the simulated extracranial and metastatic brain tumors.
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MINI14.07 - Bavituximab Activates CD8+ TILs in a 3D Ex Vivo System of Lung Cancer Patients Derived Tumors With Negative PD-L1 Expression (ID 2162)
10:45 - 12:15 | Author(s): S. Altiok, M. Mediavilla Valera, J. Kreahling, N.L. Kallinteris, D. Noyes, T.N. Razabdouski, J. Shan, J. Hutchins, K. Menander, S.J. Antonia
- Abstract
- Presentation
Background:
Bavituximab is a chimeric monoclonal antibody that targets the membrane phospholipid phosphatidylserine (PS) exposed on endothelial cells and cancer cells in solid tumors. Bavituximab blocks PS-mediated immune suppression and activates cytotoxic T lymphocyte anti-tumor responses.
Methods:
Tissues from consented patients with adenocarcinoma of the lung were extracted at the time of surgical resection and disaggregated to characterize expression of immune checkpoint proteins such as PD-1, CTLA-4, LAG3, TIM3, BTLA and adenosine A2A receptor on both CD4+ and CD8+ tumor infiltrating cells by flow cytometry (FACS) and stained for PD-L1, CD68, and CD163 via immunohistochemistry (IHC). 3D tumor microspheres were prepared and treated ex vivo with an IgG control, F(ab)’2 version of bavituximab, bavituximab, docetaxel, anti-PD-1 or PD-L1 and combinations of bavituximab, anti-PD-1 or PD-L1 and docetaxel for 36 hours within an intact tumor microenvironment. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by treatment. In a few cases, the expanded TILs were tested in PDX models using the consented patient’s tumor.
Results:
Bavituximab induces activation of TILs in 3D ex vivo tumor microsphere models of lung cancer, as demonstrated by a significant increase in IFN-ɣ, TNF-a, and GM-CSF secretion. FACS, IHC, and NanoString gene function analysis read out assays revealed that this effect was associated with low PD-1 expression on CD8+ cells, negative PD-L1 expression in the stating biopsy tissue, and a conversion of the M2 to M1 macrophage phenotype.
Conclusion:
These data support the use of bavituximab as an immunomodulatory treatment in adenocarcinoma of the lung by enhancing the activation of CD8+ TIL derived from patients' tumors with negative PD-L1 expression; correlating with increased cytokine production by lymphoid cells and repolarization of myeloid cells from an immunosuppressive to an immune active state.
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MINI14.08 - HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells and Inhibits Lung Cancer Stem Cells (ID 2789)
10:45 - 12:15 | Author(s): R. Gomez-Casal, C. Bhattacharya, P. Basse, M. Epperly, H. Wang, D. Proia, J. Greenberger, M.A. Socinski, V. Levina
- Abstract
- Presentation
Background:
Ionizing radiation (IR) therapy is an integral component of treatment for NSCLC, however, the majority of patients succumb to this disease as the disease tends to relapse and metastasize. The failure of the therapies is associated with hypermalignant cancer-initiating cells (CICs). CICs are radiation-resistant; therefore, targeting CICs represent an important therapeutic strategy for improving the outcome of IR treatment. Ganetespib, a novel heat shock protein 90 (HSP90) inhibitor, reduces expression of multiple HSP90-dependent client oncoproteins. We evaluated both the in vitro and in vivo antitumor effects of ganetespib, in combination with IR, in human lung adenocarcinoma (AC) cells.
Methods:
The radiosentisizing activity of ganetespib, HSP90 inhibitor, was evaluated in human lung AC cells established from surgical tumor samples.
Results:
Ganetespib inhibits growth of bulk AC cells, as well as lung CICs, growing as tumor spheres. The cytotoxic effects of ganetespib G2/M cell cycle DNA repair, apoptosis, and senescence. All of these antitumor effects were both concentration- and time-dependent. At the molecular level, ganetespib inhibited pro-survival signaling in adenocarcinoma cells through decreased p-AKT expression, the downregulation of RAD51 and the upregulation of p21. Ganetespib, at low nanomolar concentrations sensitizes AC to IR treatment. Importantly, both pretreatment and post –radiation treatment (24h after IR) with ganetespib (3nM) could dramatically augment the antitumor effects of IR decreasing the survival rate of IR-treated cells. Our study suggests that ganetespib may impart radiosensitization through multiple mechanisms: such as the down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and the promotion of cellular senescence. In vivo, ganetespib was effective in reducing the tumor growth of primary T2821 tumor xenografts in mice and sensitized tumors to IR.
Conclusion:
The HSP90 inhibitor, ganetespib, potentiates the effect of IR in NSCLC and eliminates CICs. The radiosensitizing effect of ganetespib is mediated by the combinatorial inhibition of cell growth and survival pathways. Ganetespib is the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validated in a clinically relevant in vivo model. The use of ganetespib as a therapeutic warrants a further investigation in the clinical setting.
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MINI14.09 - Discussant for MINI14.06, MINI14.07, MINI14.08 (ID 3342)
10:45 - 12:15 | Author(s): R. Dziadziuszko
- Abstract
- Presentation
Abstract not provided
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- Abstract
Background:
Histone deacetylase 6 (HDAC6) is a key regulator of many signaling pathways linked to cancer. But unlike other HDACs, inhibition of HDAC6 is believed not to be associated with severe toxicity, making HDAC6 a possible cancer treatment target. Overexpression of HDAC6 has been observed in many types of cancer including NSCLC. Knockdown of HDAC6 sensitizes NSCLC cell lines to chemotherapy induced cell apoptosis. Apoptosis and autophagy are the 2 cellular processes likely to alter efficacy of a therapeutic agent. Autophagy confers resistance to chemotherapy by inhibiting apoptosis. HDAC6 controls autophagosome maturation and is essential for autophagy. CAY10603 is a potent and selective inhibitor of HDAC6. Therefore, inhibition of HDAC6 by CAY10603 could be a promising strategy to treat NSCLC by targeting both apoptosis and autophagy pathways.
Methods:
We evaluated the effect of CAY10603 alone or in combination with autophagy inhibition on cell proliferation, apoptosis and autophagy in two human NSCLC cell lines, A549 and H460. Pharmacological (chloroquine or bafilomycin-A1) or genetic (knockdown of ATG5 or Beclin1 with shRNA) approaches were utilized to block autophagy. Cell proliferation of untreated or drug-treated cells was measured by CCK8 assay. Percentage of apoptotic cells was measured using PE-conjugated Annexin V with a flow cytometer. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot.
Results:
CAY10603 inhibits NSCLC cell proliferation and induces apoptosis. CAY10603 also inhibits HDAC6 dependent basal autophagy and activates the PI3K-Akt-mTOR pathway. Meanwhile, HDAC6 independent autophagy exists in NSCLC cells and confers resistance to CAY10603. Cotreatment with chloroquine or bafilomycin-A1 promotes the autophagy inhibition, cell growth suppression and apoptosis induction of NSCLC cells compared to CAY10603 alone. Knockdown of ATG5 or Beclin1 by shRNA also increased CAY10603-induced cytotoxicity in above NSCLC cells.
Conclusion:
Our results indicate that CAY10603 may be a promising agent for the treatment of NSCLC by modulating autophagy and apoptosis pathways. Furthermore, the combination of CAY10603 with classical autophagy inhibitors represents a promising therapeutic strategy that warrants further clinical evaluation in NSCLC.
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MINI14.11 - Establishment of a Lung Cancer Patient-Derived Xenografts Panel (ID 2607)
10:45 - 12:15 | Author(s): M. Boeri, M. Moro, G. Leone, M. Tortoreto, R. Caserini, P. Gasparini, G. Bertolini, R.M. Moresco, S. Valtorta, A. Fabbri, L. Roz, G. Pelosi, U. Pastorino, G. Sozzi
- Abstract
- Presentation
Background:
Studies based on cell lines were found to be poor predictors of clinical effects and therefore in many cases translation of the results into the clinics failed. A major determinant for the poor performance of cell lines is the observation that cell lines do not reflect the whole complexity and heterogeneity of primary tumors. A growing body of work suggests that Patient-Derived Xenografts (PDX) represent a more informative cancer model, providing a faithful representation of the patient’s original tumor.
Methods:
PDX were obtained by direct implants of small tumor fragments (30mm[3]) in previously anesthetized SCID mice, and were subsequently passaged as tissue explants. PDX metabolic in vivo imaging was performed using weekly [18F]FDG-PET and coronal and 3D-reconstruction at different days. Analysis of mutations and copy number alterations of PDX and human constitutive and tumoural DNA was performed by SALSA MLPA® probe mix X050-A1 Lung Cancer (MRC Holland).
Results:
Tumor samples from 95 lung cancer patients (66 AC, 16 SCC and 13 other lung cancer histotypes (OL)) have been implanted in the flanks of SCID mice. Overall, 36 samples (37,9%) successfully grafted and were propagated for at least 3 passages in immunocompromised mice. Take rate was 34,8 % (23/66) in AC, 43,8% (7/16) in SCC and 46.1% (6/13) in OL (2 large cell carcinomas, 1 sarcomatoid carcinoma and 3 small cell carcinomas). A detailed immunohistochemical analysis of 27 PDX, at different passage in mice, confirmed that tumor histology, expression of specific markers (TTF-1, p40, Vimentin, Ki64 and Synaptophysin) and the amount of specific tumor cell subpopulations (i.e. CD133[+] Cancer Initiating Cells) were generally maintained in PDX. In vivo animal PET imaging showed that also metabolic activity of PDX was strictly correlated with parental tumor’s features, especially for tumours with a SUV~max~ level higher than 8 (R[2]=0.67, p<0.05). Mutation and copy number analyses, performed on 29 biological samples belonging to 11 different engrafted models, showed that genetic changes were maintained in PDX that well recapitulated the frequency of the major changes involved in lung cancer development (66.7% TP53; 60% CDKN2A, 40% LKB1, 40% KEAP1, 38.4% KRAS, 20% SWI/SNF, 20% PTEN, 8% ERBB2). Furthermore, we developed a freeze/thawing procedure on samples derived from PDXs that allows for 100% successfully thawing and established a large collection of more than 200 frozen PDX samples for future preclinical studies.
Conclusion:
The deep characterization of our established PDX panel confirmed that these mouse models recapitulate the parental primary tumors in terms of tumor histology, cellular and mutation pattern, metabolic activity and expression of specific markers for several passages in mice. All these data support the use of these “human in mouse” models for functional studies, highlighting the relevance of our PDX panel as a valuable platform for preclinical studies.
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MINI14.12 - Genomic Profiling of Patient-Derived Xenografts Identify Passenger Aberrations Associated with Better Prognosis in Non-Small Cell Lung Cancer (ID 1735)
10:45 - 12:15 | Author(s): N. Pham, D. Wang, S. Sakashita, G. Allo, L. Kim, N. Yanagawa, C. Zhu, M. Li, E. Kaufman, N. Moghal, G. Liu, M. Pintilie, I. Jurisica, F. Shepherd, J.D. McPherson, M.S. Tsao
- Abstract
Background:
Patient-derived tumor xenografts (PDXs) increasingly are being used as preclinical models to study human cancers, test novel therapeutics, and identify potential biomarkers, as they more accurately model human cancers than established tumor cell line cultures. However, uncertainty remains as to how well the genomic characteristics of patient non-small cell lung cancer (NSCLC) are recapitulated in these PDX models.
Methods:
PDXs were established by implantation of surgically resected NSCLC patient tumors into the subcutaneous or sub-renal capsule of non-obese diabetic severe combined immune deficient (NOD-SCID mice. Comprehensive genomic profiling including exome, gene copy number, DNA methylation and mRNA expression were conducted on 36 independent PDX models, their matched patient tumors and normal lung tissue. Publicly available cell line and TCGA data were used for comparison. Integrative analysis was performed to identify genomic alterations in PDXs that are associated with significant clinical outcomes in patients.
Results:
From 441 resected NSCLC tumors, 127 serially transplantable and stable PDX models were established. Among 264 NSCLC patients with at least 3-years follow-up, patients whose tumor formed stable PDXs (versus those who did not) showed significantly worse disease free (HR=3.12, 95% CI =2.02-4.83, P<0.0001) and overall survival (HR=4.08, 95% CI =2.16-7.73, P<0.0001), after multivariable adjustment for clinical pathological factors. Genomic and transcriptomic profiling of 36 PDXs showed greater similarity in somatic alterations between PDX and primary tumors than with published cell line data. In addition to known mutations, we found at least 16 non-synonymous somatic mutations in known oncogenes and tumor suppressors that have never been reported. All these mutations had higher observed variant allele frequency in PDXs compared to their matched patient tumors, suggesting that these were tumor sub-clones selected or enriched for growth in the PDXs. Tumor models characterized by a higher number of somatic alterations among 865 frequently altered genes were associated with better overall patient survival (HR=0.15, p=0.00015) compared to patients with corresponding PDXs characterized by higher alteration number; this was validated in the TCGA lung cancer dataset patients (HR=0.28, p=0.000022). These 865 genes were enriched for those encoding for proteins involved in cell adhesion and interactions with the extracellular matrix, and a quarter of the genomic alterations would putatively form neo-antigens implicating a potential role of immune response in the observed improved patient survival.
Conclusion:
PDXs are close preclinical models of patient tumors. Further investigations of passenger mutations may clarify their clinical impact on interactions between tumor cells, stroma, immune microenvironment and patient prognosis.
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MINI14.13 - Small Molecule Demonstrates Potent Tumor Suppression by Inhibiting the PI3K/AKT Pathway in Non-Small Cell Lung Cancer (ID 721)
10:45 - 12:15 | Author(s): G.A. Woodard, J. Crockard, C. Peto, B. Tolani, B. He, D. Jablons
- Abstract
- Presentation
Background:
The phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) pathway is frequently activated in many malignancies including non-small cell lung cancer (NSCLC). Dysregulation of this pathway leads drives oncogenic genes and imparts resistance to conventional chemotherapy. We identified a small molecule AKT pathway inhibitor as a potential lead compound.
Methods:
The AKT pathway inhibitor was tested in vitro on a panel of NSCLC cell lines A427, A549, NCI-H1703, NCI-H2170, NCI-H1650, and NCI-H1975. Cell viability was determined by MTS assay after 72 hours of drug treatment. Activation kinases in the PI3K/AKT/mTOR pathway was determined by western blot analysis.
Results:
Treatment with the PI3K/AKT pathway inhibitor caused potent concentration-dependent inhibition of cell proliferation with a half maximal inhibitory concentration (IC~50~) in the nanomolar range. Kirsten rat sarcoma (KRAS) mutant cell lines were the most sensitivity to the PI3K/AKT pathway inhibitor while epidermal growth factor receptor (EGFR) mutant cell lines were more resistant. Western blot analysis showed inhibition of AKT and mTOR phosphorylation at nanomolar concentrations.
Conclusion:
A novel small molecule AKT inhibitor inhibits growth of NSCLC cells in vitro, is potent against KRAS mutants, and shows promise as a small molecule targeted chemotherapy drug for NSCLC.
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MINI14.14 - Suppression of Lung Cancer Growth by CD26/DPP4 Inhibitor (ID 1546)
10:45 - 12:15 | Author(s): J. Jang, F. Janker, S. Arni, Y. Yamada, I. Gil-Bazo, I.D. Meester, W. Weder, W. Jungraithmayr
- Abstract
- Presentation
Background:
Lung cancer is the most prominent cause of death among cancers, accounting for 1.38 million deaths worldwide annually. In spite of improved treatment in surgery, chemo- and radiation therapy, the five year survival is poor, being 69% for stage Ia and <5% for stage IV. The cure rates of current therapies are disappointing and did not significantly prolong long term survival. Surfactant protein (SP) in lung determines not only function of the organ, but also inflammatory reaction in an infectious condition. Recently Nishioka et al. showed that stimulated SP production in the orthotopic models of human lung cancer recruits inflammatory, type I macrophages in the tumor which decreased the size of the tumor. Also, Stephan et al. found increased productions of SPs in rat by CD26/DPP4 inhibitor treatment or CD26-/- animal. In our previous work, we found the activity of CD26/DPP4 of lung cancer from patients was four times higher than normal lung tissue from same patients (n=38). Therefore, we tested if pharmacological CD26/DPP4 inhibitor (Vildagliptin) inhibits lung cancer growth in various animal models.
Methods:
Mouse lung cancer cell line (Lewis Lung Carcinoma (LLC)) and human lung adenocarcinoma cell line, H460, were used to develop syngeneic (C57BL6: n=8) or xenogeneic (CD1-nude: n=20) tumor models by sc. injection. Tumor growth was represented by wet weight of tumor mass at harvest (4 weeks). BALB/c mouse strain (n=12) was used to induce lung cancer by Urethane (1g/kg) ip. Urethane injected mice were harvested 5 months after ip. Vildagliptin treatment was given in drinking water (0.2 mg/ml: 50mg/kg day) during the experimental course. Tumor nodules were counted macroscopically under surgical microscope. For histological assessment, HE, TUNEL, immunohistochemistry (IHC) of CD31, Ki67, CD3, Nkp46, and F4/80 were performed. The expression of surfactant protein C (SP-C) was detected by western blotting.
Results:
Vildagliptin treatment significantly reduced the size of tumor developed by lung cancer cell line injection (p<0.05 for both). Tumor induced by Urethane ip. in BALB/c mice was less incident by Vildagliptin treatment (40%: 2/5 mice) than control (100%: 7/7) group. The number of tumor nodule per mouse was also significantly reduced by Vildagliptin compared to control (p<0.05). Beside tumor weight, there was no difference in HE, TUNEL stain, and IHCs of CD31, Ki-67, CD3, and Nkp46. However we found significantly increased numbers of macrophages (F4/80) in the tumors induced by lung cancer cell line injection (p<0.05 for both) along with increased expression of SP-C in lung cancer cell lines in vitro.
Conclusion:
Inhibition of CD26/DPP4 by Vildagliptin decreased lung cancer growth in the models of mouse and human lung cancer cell lines and increased infiltrating macrophages within tumors. Furthermore, there was increased expression of SP-C by Vildagliptin treatment found in lung cancer cell lines. This finding suggests that surfactant production in lung cancer is induced and potentially activates macrophages against lung cancer by CD26/DPP4 inhibitor, Vildagliptin.
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MINI14.15 - Discussant for MINI14.10, MINI14.11, MINI14.12, MINI14.13, MINI14.14 (ID 3343)
10:45 - 12:15 | Author(s): F. Cappuzzo
- Abstract
- Presentation
Abstract not provided
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Author of
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.01 - Genomic Characterization of Large-Cell Neuroendocrine Lung Tumors (ID 1667)
10:45 - 12:15 | Author(s): L. Fernandez-Cuesta
- Abstract
Background:
Neuroendocrine lung tumours account for 25% of all lung cancer cases, and they range from low-aggressive pulmonary carcinoids (PCA) to highly malignant small-cell lung cancer (SCLC) and large-cell neuroendocrine lung carcinoma (LCNEC). The last two are strongly associated with heavy smoking and are typically detected at a clinically advanced stage, having a poor survival. Comprehensive genomic analyses in lung neuroendocrine tumours are difficult because of limited availability of tissue. While more effort has been done in the context of SCLC, the detailed molecular features of LCNEC remain largely unknown.
Methods:
We conducted 6.0 SNP array analyses of 60 LCNEC tumours, exome sequencing of 55 tumor-normal pairs, genome sequencing of 11 tumour-normal pairs, transcriptome sequencing of 69 tumours, and expression arrays on 60 tumors. Data analyses were performed using in house developed and published pipelines.
Results:
Analyses of chromosomal gene copy number revealed amplifications of MYCL1, FGFR1, MYC, IRS2 and TTF1. We also observed deletions of CDKN2A and PTPRD. TTF1 amplifications are characteristic of lung adenocarcinoma (AD); CDKN2A deletions are frequent alterations in both AD and squamous-cell lung carcinoma (SQ); FGFR1 amplifications are found in SQ and, less frequently, in SCLC; and MYCL1 and IRS2 amplifications are frequent events in SCLC. Similar to the copy number data, we found patterns of mutations characteristic of other lung cancer subtypes: TP53 was the most frequently mutated gene (75%) followed by RB1 (27%), and inactivation of both TP53 and RB1, which is the hallmark of SCLC, occurred in 20% of the cases. Mutations in STK11 and KEAP1-NFE2L2 (frequently seen in AD and SQ) were found in 23% and 22% of the specimens, respectively. Interestingly, mutations in RB1 and STK11/KEAP1 occurred in a mutually exclusive fashion (p-value=0.016). Despite the heterogeneity observed at the mutation level, analysis of the pattern of expression of LCNEC in comparison with the other lung cancer subtypes (AD, SQ, SCLC, and PCA) points to LCNEC as being an independent entity. An average mutation rate of 10.7 mutations per megabase was detected in LCNEC, which is in line with the rate observed in other lung tumours associated with smoking. We found that, similar to SCLC, the mutation signatures associated with APOBEC family of cytidine deaminases, smoking, and age (based on Alexandrov et al 2013) were the predominant ones in LCNEC. However, the contribution of the individual SCLC and LCNEC samples to these three signatures was quite different, and we are currently exploring it.
Conclusion:
Taking into account somatic copy number and mutation data, we distinguished two well-defined groups of LCNEC: an SCLC-like group, carrying alterations in MYCL1, ISR2, and in both RB1 and TP53; and a group resembling AD and SQ, with alterations in CDKN2A, TTF1, KEAP1-NFE2L2, and STK11. Although these results suggest that LCNEC might be a mix of different lung cancer subtypes, mutation clonality and expression analyses show that they are likely to be a separate entity, sharing molecular characteristics with the other lung cancer subtypes. Their heterogeneity suggests that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ.
