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M. Kohonen-Corish
Moderator of
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MO20 - Preclinical Therapeutic Models II (ID 93)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 10
- Moderators:P. Waring, M. Kohonen-Corish
- Coordinates: 10/30/2013, 10:30 - 12:00, Bayside Gallery B, Level 1
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MO20.01 - Protein Kinase C iota is required for maintenance of a tumor initiating cell phenotype in lung squamous cell carcinoma (ID 2644)
10:30 - 12:00 | Author(s): H.J. Ross, V. Justilien, K. Hill, M. Walsh, A.P. Fields
- Abstract
- Presentation
Background
We discovered that PKCι is an oncogene in non-small cell lung cancer (NSCLC), elucidated a major oncogenic PKCι signaling mechanism, and identified therapeutic agents that target oncogenic PKCι signaling. We have shown that PKCι signaling is genetically activated in approximately 70% of lung squamous cell carcinomas (LSCCs) through tumor-specific amplification of the PKCι gene, PRKCI. More recently, we have investigated the role of PKCι in bronchio-alveolar stem cells (BASCs), which are putative lung tumor-initiating cells (TICs). We demonstrated that PKCι is required for Kras-mediated transformation of BASCs in a mouse model of Kras-mediated lung adenocarcinoma. We hypothesize that PKCι plays a critical role in the development and maintenance of the TIC phenotype in LSCC by activating cell autonomous proliferative signaling mechanisms.Methods
We isolated “oncospheres” from four human LSCC cell lines (H1703, H1299, Calu-1, and ChagoK1) grown in non-adherent culture in defined stem cell medium using established protocols. Lentiviral shRNA techniques were used to genetically knock down expression of PKCι to assess the effect of PKCι depletion on the TIC phenotype. Non-target (NT) and PKCι RNAi TICs were assessed for the ability to grow as non-adherent oncospheres, to clonally expand, express stem marker genes, form colonies in soft agar, and initiate tumors in immune deficient mice. The effect of the selective and potent PKCι signaling inhibitor auranofin on TIC behavior and PKCι signaling activity was assessed as was the mTOR inhibitor, rapamycin.Results
LSCC oncospheres exhibited characteristics of cancer stem or tumor-initiating cells including the ability to redifferentiate into bulk tumor cells when returned to adherent culture. Oncosphere cells express elevated levels of stem genes, clonally expand, exhibit enhanced transformed growth, and efficiently initiate and maintain lung orthotopic tumors and metastases. Biochemical studies indicate that the oncogenic PKCι-Rac1-Ect2-MMP10 signaling axis is activated in LSCC TICs. To assess the role of PKCι in TIC growth, we knocked down PKCι in TIC cultures derived from the four LSCC cell lines described above. Whereas TICs expressing NT RNAi grew efficiently as anchorage-independent colonies in soft agar and clonally expanded, PKCι RNAi TICs were severely impaired in soft agar growth, clonal expansion, and tumorigenicity in vivo. Treatment of TICs with the potent and selective PKCι inhibitor auranofin (ANF) likewise led to inhibition of PKCι signaling, TIC growth, clonal expansion, and tumorigenicity. Combined inhibition of PKCι and mTOR with ANF plus rapamycin was synergistic against TIC proliferation in vitro.Conclusion
Our data demonstrate that PKCι signaling is activated in LSCC TICs and that PKCι signaling is important for maintaining the TIC phenotype. We showed that the selective PKCι inhibitor ANF potently inhibits LSCC TIC behavior. Taken together, our data support the targeting of LSCC TICs through selective inhibition of PKCι for treatment of patients with LSCC. Based on these and earlier results showing synergistic tumor inhibition with combined PKCι and mTOR inhibition, a phase I clinical trial of auranofin with the mTOR inhibitor sirolimus has been instituted as maintenance therapy for LSCC patients who have completed initial chemotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.02 - Proteomic analysis identifies baseline PI3K/Akt pathway activation and treatment-induced supppression of mTOR signaling as determinants of response to MEK inhibition (ID 2845)
10:30 - 12:00 | Author(s): K.A. Gold, L.A. Byers, D. Xia, Y. Fan, L. Diao, P. Groth, J. Paul, J. Wang, U. Giri, J. Gudikote, H.T. Tran, K.R. Coombes, J.D. Minna, J.V. Heymach, N. Liu
- Abstract
- Presentation
Background
Inhibition of MEK is a promising treatment strategy for non-small cell lung cancer (NSCLC). MEK inhibitors are being investigated for KRAS mutant disease, but KRAS alone is not predictive of efficacy, and other predictors of response and resistance are not known. The downstream effects of MEK inhibition have not been fully described. Here, we report broad proteomic analysis of NSCLC cell lines before and after treatment with MEK inhibitor BAY86-9766.Methods
We treated 109 NSCLC cell lines with BAY86-9766. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3[rd] of those tested. Proteomic analysis for regular and phospho-proteins was performed by reverse phase protein array. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines.Results
Increased activation of the PI3 kinase pathway at baseline correlated with resistance to MEK inhibition, with resistant cell lines showing higher baseline levels of pAkt (S437), pAkt (T308), pPDK1, and p4E-BP1 (S65), and lower baseline levels of PTEN (all p<0.05). Cell lines with increased MEK phosphorylation at baseline were more sensitive to MEK inhibition (p=0.048). BAY86-9766 was very effective at reducing pERK (p=1.65x10[-35]) but this modulation was not significantly different between sensitive and resistant cell lines (p=0.64). Increased phosphorylation of MEK was seen with treatment (1.66x10[-16]). mTOR signaling was suppressed by MEK inhibition, with decreased phospho-p70S6K, pS6 (S235/236), and pS6 (S240/S244) and increased eIF4E following treatment (all p<0.02). These effects were significantly more pronounced in sensitive vs resistant cell lines (all p<0.01). Higher levels of LKB1 total protein, pAMPK, and pTSC2 were also seen following treatment (all p<0.02).Conclusion
We have performed broad proteomic analysis of NSCLC cell lines treated with MEK inhibitor BAY86-9766. Baseline activation of the PI3K/Akt pathway predicts for resistance to MEK inhibition. Sensitive cell lines, but not resistant cell lines, show suppression of mTOR activity with treatment with BAY86-9766. The effects of MEK inhibition of mTOR may be modulated by p90RSK through an LKB1 dependent pathway. This suggests a basis for combining targeted agents to overcome resistance, such as combinations of MEK inhibitors with PI3K inhibitors or mTOR inhibitors.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.03 - Development and characterization of a panel of GDC-0980 resistant NSCLC cell lines (ID 2798)
10:30 - 12:00 | Author(s): S. Heavey, M. Barr, K. O'Byrne, K. Gately
- Abstract
- Presentation
Background
The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Multiple novel agents, targeting different regulators within the pathway are currently under development. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which demonstrated excellent downstream inhibition of the PI3K pathway in vitro, with the strongest effects being observed in lung, breast and prostate cancer cell lines. There are 12 clinical trials ongoing for this drug, with Phase I studies in solid tumours and Phase II studies in endometrial carcinoma, renal cell carcinoma, prostate cancer and breast cancer. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. Multiple mechanisms of resistance to GDC-0980 may develop while a patient is being treated with this drug. The aim of this project is to develop four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to predict which mechanisms of resistance may occur in patients. This will allow us to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.Methods
H460, A549, H1975 and SKMES-1 cells were treated with a dose response curve of GDC-0980 and BrdU proliferation assays determined IC50 values for each cell line. Each cell line was then cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Each month, BrdU proliferation assay were carried out in order to track the development of resistance to the drug. When a log fold difference between the parent and resistant IC50s was observed, the cells were deemed to be resistant. Matched parent and resistant cells were then screened for a panel of mutations. Cells lines were also screened for gene alterations using a human cancer drug resistance PCR array. Identified genes of interest were validated at the RNA and protein level by PCR and Western blot, respectively.Results
All four cell lines exhibited a dose-dependent decrease in proliferation when treated with GDC-0980. H1975 cells (adenocarcinoma; PIK3CA mutant) were most sensitive to GDC-0980, however they developed resistance to the drug more rapidly than the other 3 cell lines. Results from mutational analysis and investigation of the gene and protein expression of each of the 4 pairs of parent and resistant cell lines will be presented.Conclusion
While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly. As such, understanding the mechanisms involved in the development of resistance to this drug will be crucial so that we may design optimal treatment strategies. Specific conclusions regarding the mechanisms of resistance in this panel of cell lines will be drawn based on identified genes and proteins of interest.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.04 - Divergent activity of afatinib (AFAT) and cetuximab (CET) in patient-derived xenograft (PDX) models of acquired erlotinib resistance. (ID 1458)
10:30 - 12:00 | Author(s): P. Mack, N. Goodwin, W. Holland, K. Kelly, T. Li, P. Lara, D. Gandara
- Abstract
- Presentation
Background
The combination of AFAT and CET has demonstrated remarkable clinical activity in patients with acquired resistance to erlotinib. Preclinical modeling in genetically engineered mice and cell lines predicted activity in cases where erlotinib resistance was mediated by the EGFR T790M gatekeeper mutation. However, in the clinic, patients lacking T790M-positive tumors showed equivalent benefit from this combination, suggesting alternative mechanisms of synergy. We explored the individual and combined molecular and growth inhibitory activity of these agents in PDX models derived from NSCLC patient tumors with distinct mechanisms of acquired resistance to erlotinib. These models were developed by the UC Davis - Jackson Laboratories Consortium, which has xenotransplanted over 170 NSCLC models using the nod/scid/IL2Rgamma chain-null (NSG) mouse.Methods
EGFR-mutant PDX models LG0703 (T790M-negative) and LG1049 (T790M-positive) were established from tumor biopsies from patients who progressed following durable responses to erlotinib. Both patients were subsequently treated with AFAT+CET, with the LG0703 donor patient exhibiting a prolonged response and the LG1049 donor patient exhibiting a transient response followed by rapid progression. Excised tumors from passage 1 PDXs were fragmented and implanted into treatment cohorts. When tumors reached 300mm[3], mice were randomized to erlotinib (50 mg/kg qd po), AFAT (20 mg/kg qd po), CET (10 mg/kg twice weekly iv), AFAT-CET, or vehicle control (n per arm = 12) for 3 weeks followed by a 75-day monitoring period. In a parallel cohort, tumor pharmacodynamic changes in signal transduction mediators and RTKs were assessed after 6 and 24h treatment exposures using kinase arrays (R&D systems) and immunoblotting.Results
In LG0703, AFAT, CET and AFAT-CET resulted in complete tumor response (CR) during the 21-day treatment period. After cessation of treatment, mice treated with CET or AFAT-CET remained in complete remission; whereas AFAT-treated mice progressed within 2 weeks. Clinical activity in this model was associated with complete blockade of EGFR and Her2 phosphorylation. Substantial down-regulation of AKT1, AKT2, ERK1, p38a, RSK1 and p70S6K phosphorylation was evident within 6h of treatment. In contrast, the T790M-postive LG1049 model demonstrated only modest clinical benefit from AFAT, with no single-agent CET activity, and no CET-mediated synergy with AFAT. No treatments were able to ablate EGFR phosphorylation or downstream signal transduction, and compensatory induction of EGFR, HER2, ERK1 and p38 were noted after 24h of drug exposure.Conclusion
In these PDX models derived from patients with EGFR-activating mutant cancer with acquired resistance to erlotinib, treatment with AFAT+CET recapitulated the clinical experience of the donor patients receiving this combination. In the LG0703 model, both the AFAT-CET combination as well as single-agent CET resulted in complete tumor regression associated with total ablation of EGFR phosphorylation and subsequent blockade of multiple signal transduction pathways. In the LG1049 model, AFAT prompted limited but statistically significant tumor delay, with no additional benefit from CET. These experiments demonstrate the considerable potential of this PDX resource to assess therapeutic strategies in models representing individual patients. Supported by BJALCF.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.05 - DISCUSSANT (ID 3907)
10:30 - 12:00 | Author(s): C.G. Ferreira
- Abstract
- Presentation
Abstract not provided
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MO20.06 - Histone deacetylase inhibition downregulates thymidylate synthase (TS) expression and enhances pemetrexed-induced cytotoxicity in NSCLC models (ID 2010)
10:30 - 12:00 | Author(s): D. Trisciuoglio, M. Desideri, M. Di Martile, C. Gabellini, T. De Luca, S. Vari, F. Cognetti, A. Eramo, R. De Maria, M. Milella, D. Del Bufalo
- Abstract
- Presentation
Background
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM’s molecular targets) expression. Recently, TS expression has been found to be regulated by histone acetylation status, thus raising the interesting hypothesis that histone deacetylase inhibitors (HDACi) may sensitize NSCLC cells to PEM cytotoxicity.Methods
Molecular and functional effects of single and combined HDAC inhibition and PEM exposure were assessed in NSCLC cell lines (A549, H1299, H1650, Calu-1) and patient-derived lung cancer stem cells (L-CSC). Pharmacologic interactions were assessed by conservative isobologram analysis using the Chou-Talalay method and the Calcusyn software. TS expression was studied by WB analysis and real-time PCR. Apoposis induction was assessed by flow cytometry and WB. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells, detection of acidic vesicle organelles (AVO) formation and WB. In vivo experiments were conducted in xenograft models established by i.m. injection of NSCLC cells into 6-8 week-old male athymic mice (nu/nu).Results
In NSCLC cell lines and L-CSC, the HDACi ITF2357 dose-dependently inhibited cell growth (IC~50~: <1-20 mM), induced histone H3 acetylation, and downregulated TS expression at the mRNA and protein levels. Combined HDAC inhibition and PEM exposure was then tested using three different administration schedules: simultaneous exposure to both drugs, ITF2357 followed by PEM, and the reverse sequence. Simultaneous PEM/ITF2357 treatment resulted in antagonistic growth inhibitory interactions (combination index – CI >1) in all cell lines tested, while ITF2357 followed by PEM had additive effects in A549 cells and slightly synergistic effects in H1299 and Calu-1 cells; conversely, PEM followed by ITF2357 had strikingly synergistic effects (CI <<1) in all NSCLC cell lines, as well as in the L-CSC143. Most notably, only the ITF2357 followed by PEM sequence synergistically induced apoptosis, resulting in approximately 50% Annexin V-positive cells; apoptosis was only partially rescued by caspase inhibition by z-VAD-fmk, which led us to investigate autophagy as an alternative mechanism of combination-induced cell death. Indeed, ITF2357, and to a significantly greater extent PEM followed by ITF2357, induced autophagy as evidenced by AVO formation, LC3BII processing, p62 downregulation, and Beclin1 induction. Most importantly, autophagy induction was instrumental to the cytotoxic interaction between PEM and ITF2357, as Beclin1 silencing by shRNA completely reversed their growth inhibitory synergism and prevented both autophagy and apoptosis induction. The synergistic cytotoxic interaction between PEM and ITF2357 was at least partly due to ITF2357 ability to prevent PEM-induced TS upregulation, as TS silencing by siRNA further enhanced apoptosis induction by single and combined PEM/ITF2357 exposure. Finally, both H1650 and H1299 xenografts had a robust response to sequential PEM/ITF2357 administration in vivo, resulting in an approximately doubled mice survival in the H1650 model.Conclusion
Overall, our data indicate that HDAC inhibition by ITF2357 downregulates TS expression and synergistically potentiates apoptosis and autophagy induction following PEM exposure, supporting the clinical investigation of sequential PEM/ITF2357 schedules for the treatment of advanced NSCLC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.07 - Identification of New Chemotherapeutic Strategies in Mesothelioma and Non-Small Cell Lung Cancer Using a Drug-Induced Apoptosis Assay (MiCK Assay) (ID 3158)
10:30 - 12:00 | Author(s): D.A. Wigle, J. Molina, A. Hallquist, C. Presant
- Abstract
- Presentation
Background
Given limited progress in developing novel chemotherapies for mesothelioma and multi-negative NSCLC, new technology is needed to identify promising drug strategies. A drug-induced apoptosis assay has been developed that has been applied in acute myelocytic leukemia, ovarian cancer, and a variety of solid tumors including breast cancer (Cancer Research 2012; 72:3901). We explored the use of the MiCK assay in mesothelioma and NSCLC tumor specimens.Methods
Fresh tumor specimens from resected tissue or malignant effusions were processed in a central laboratory. Cell separation techniques were used to prepare >95% tumor cell suspensions for the MiCK assay (as described in Cancer 2012; 118: 4877). Over 48 hours in short term culture, optical techniques based on Mie light scattering measured apoptosis in control wells and test wells containing different chemotherapy drugs or combinations. Significant apoptosis gave results over 1.0 kinetic units (KU). Drugs or combinations producing the highest KU +/- 1 SD compared to other drugs were defined as best regimens. Differences of over 0.57 KU correlated with clinically significant better responses.Results
15 specimens have been submitted with 9 successfully assayed to date. Mean numbers of drugs or combinations assayed successfully were 32 in mesothelioma and 20 in NSCLC. New treatment strategies in individual patients with mesothelioma were: epirubicin 5.0 and 9 KU, idarubicin 4.0 KU, pemetrexed+doxorubicin 4.9 and 4.6 KU, ifosfamide 3.4 and 2.2 KU, bendamustine 4.0 KU, dactinomycin 4.7 and 3.4 KU, vinorelbine 4.7 KU, asacytidine 3.8 KU, bortezomib 3.0 KU, doxorubicin 3.9 KU, cyclophosphamide+doxorubicin+vincristine 3.0 KU and cisplatin+irinotecan 3.1 KU. New treatment strategies in individual patients with NSCLC were: doxorubicin 2.0, 1.7 and 1.7 KU, epirubicin 1.6 KU, 5-fluorouracil+leucovorin 1.3 KU, and cyclophosphamide+doxorubicin+vincristine 2.3 KU. In class differences in drug activity were apparent in individual patients: cisplatin>carboplatin, epirubicin>doxorubicin, and docetaxel>paclitaxel. In one mesothelioma patient with paired specimens from malignant effusion and solid tumor the most active (pemetrexed+doxorubicin) and least active regimens (cisplatin+paclitaxel) were concordant. In mesothelioma, the most active chemotherapy regimens in individual patients were epirubicin, idarubicin, pemetrexed+doxorubicin, vinorelbine, cisplatin+etoposide, cisplatin+irinotecan, cyclophosphamide+doxorubicin+vincristine, and dactinomycin. In NSCLC, the most active regimens were doxorubicin, cisplatin, docetaxel, irinotecan, and cyclophosphamide+doxorubicin+vincristine.Conclusion
Use of the MiCK assay in mesothelioma and NSCLC can identify unexpected new leads for innovative therapeutic strategies for individual patients, and for candidate enrollment in phase II and III studies. The MiCK assay may play a role in designing precision therapeutics for patients with mesothelioma and NSCLC. Marked differences between patients in individual drug activities, and discordant in-class drug effectiveness indicate the need for individualized patient tumor testing of drug-induced apoptosis. Since use of the MiCK assay has correlated with improved clinical outcomes in prior studies, clinical trials of drugs with unexpected activity may be warranted in mesothelioma and NSCLC patients.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.08 - Activation of CD1d-restricted NKT cells may inhibit cancer cell repopulation between cycles of chemotherapy through modulating immune responses in murine mesothelioma (ID 650)
10:30 - 12:00 | Author(s): L. Wu, Z. Yun, L. De La Maza, J. Yu, Y. Zhao, M. De Perrot
- Abstract
- Presentation
Background
Considerable evidence has shown that cancer cell repopulation during the intervals of chemotherapy is a neglected factor of treatment failure. The efficacy of cancer treatment may be improved if this process could be effectively controlled. It has been demonstrated that the number of invariant natural killer T cells (iNKT) increased during the development of murine mesothelioma models. NKT cells specifically recognize the glycolipid α-galactosylceramide (KRN7000, KRN) through CD1d molecule resulting in their activation and expansion. Our goal is to study the impact of NKT cell activation by KRN on cancer cell repopulation between cycles of chemotherapy in murine mesothelioma model.Methods
Tumor-bearing mice were treated with chemotherapy once weekly, and KRN was followed after each cycle of chemotherapy. Both WT and CD1dKO mice were used to evaluate the effect on tumor growth. Cancer cell proliferation and apoptosis was evaluated by Ki67 and TUNEL immunohistochemistry, respectively. The proportion of CD4[+] and CD8[+] T cells and their activation in the tumor, spleen, draining lymph node and peripheral blood from tumor-bearing mice were determined by using flow cytometry, and gene expression of activated T cell-related cytokines and cytolytic enzymes were quantified by RT-PCR. NKT were recognized specifically by CD1d-tetramer staining.Results
In WT mice, tumor growth delay was achieved by chemotherapy alone, and this effect was improved when combined with KRN. Cancer cell repopulation between cycles of chemotherapy was significantly inhibited by KRN, whereas apoptosis changed inversely. KRN following chemotherapy resulted in an increase of IFN-γ production in the draining lymph node, blood and spleen. Strikingly, the percentage of ICOS+CD4 T cells, Th17 and Tc17 cells increased in splenocytes. NKT expansion was observed in both peripheral blood and lymphoid organs. Gene expression of immune-associated cytokines was somewhat upregulated after NKT cell activation during the intervals of chemotherapy. In KO mice, however, Cis alone or Cis+KRN was less effective than in WT mice. KRN alone had little effect in both animals.Conclusion
NKT activation between cycles of chemotherapy can improve the efficacy of treatment through modulating anti-tumor immunity against cancer cell repopulation. KRN may be a promising agent for mesothelioma immunotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.09 - Systemic blockade of CTLA-4 signaling can improve the abscopal effect induced by local radiotherapy in a murine mesothelioma model (ID 651)
10:30 - 12:00 | Author(s): L. Wu, Z. Yun, L. De La Maza, M. Wu, J. Yu, Y. Zhao, M. De Perrot
- Abstract
- Presentation
Background
Radiotherapy can induce direct cancer cell death and systemic anti-tumor immunity known as abscopal effect. For malignant pleural mesothelioma (MPM), postoperative hemithoracic radiation is important to control recurrence and metastasis for the resectable patients. We hypothesized that the abscopal effect also exists in malignant mesothelioma, and removal of the immunosuppressive checkpoints was able to enhance this effect induced by local radiotherapy.Methods
Murine malignant mesothelioma AB12 cells were injected subcutaneously into the right leg and flank of Balb/c mice either sequentially (primary/secondary tumors) or concurrently (local/distant tumors). Treatment was initiated on day 5 when primary (local) tumors were developed, and the immune-deficient NOD/SCID mice were used as controls. Local radiotherapy (LRT) with Gammacell-40 Irradiator was delivered to the tumor-bearing leg, whereas the rest of the body was protected with a lead chamber. CTLA-4 blockade with mAb was given 1 day after LRT. Tumor size was measured twice weekly to evaluate the anti-tumor effect. The immune responses, especially T cell activation in tumor, spleen and lymph node was determined by flow Cytometry. The expression of immune-related genes was quantified by RT-PCR, and tumor-infiltrating T cells were determined by immunofluorescent staining.Results
The growth of primary tumors was significantly inhibited by LRT alone, and addition of anti-CTLA-4 mAb enhanced the antitumor effect. Interestingly, the secondary or distant tumors grew more slowly in mice whose primary tumor was treated with LRT than those untreated mice. Some secondary tumors were completely rejected when combined with anti-CTLA4 mAb. There was no such effect on the distant tumors in the immune deficient mice. Results demonstrated that LRT resulted in more T cell infiltration into both primary and secondary tumors. Tumor-infiltrating T cells had higher levels of ICOS and IFN-γ, and proliferated more rapidly after injection of irradiated AB12 cells. More activated CD4 and CD8 T cells were observed in the the draining lymph node (dLn) and spleen, and more dendritic cells trafficked to the dLn. The gene expression of cytolytic enzymes and cytokines was upregulated as well.Conclusion
LRT on primary tumors has abscopal effect on the secondary or distant tumor, and this effect can be enhanced by systemic blockade of CTLA-4 in murine mesothelioma. This approach might be translated into clinical trials for MPM patients.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.10 - DISCUSSANT (ID 3908)
10:30 - 12:00 | Author(s): G. Reid
- Abstract
- Presentation
Abstract not provided
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Author of
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P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 2
- Moderators:
- Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P2.06-009 - Simultaneous Profiling of Multigene Mutations for the Effective And Efficient Diagnosis of Non-Small Cell Lung Carcinoma (ID 1078)
09:30 - 16:30 | Author(s): M. Kohonen-Corish
- Abstract
Background
Identification of actionable driver mutations in non-small cell lung carcinoma (NSCLC) has become increasingly important for the prioritisation of targeted therapies. Mutational analysis of formalin-fixed paraffin embedded (FFPE) tissues presents several challenges including generally limited and fragmented DNA, the need to identify a range of biologically significant mutations and a pressing need for a fast turn around time at a cost-effective way. Our aim was to determine optimal methods for quantification of DNA for mutational analysis in NSCLC and to develop a new custom assay that could perform multigene mutational analysis on the limited quantity of DNA available in the small NSCLC samples frequently submitted for testing.Methods
DNA was extracted from FFPE tissues including cytology specimens. Spectrophotometry quantification was compared with Qubit 2 Fluorometer measurements and the Sequenom SampleID assay for accurate and meaningful assessment of extracted DNA for diagnostic mutational profiling. We have previously established a diagnostic protocol for somatic mutation profiling in NSCLC using a commercial DNA mass spectrometry kit (Oncocarta v1.0) and compared it with a new custom kit “OncoFocus” developed in collaboration with Sequenom. These assays utilise target amplicons of small sizes for efficient amplification in fragmented DNA and simultaneously profile a range of actionable mutations in EGFR, KRAS, BRAF and NRAS. Preliminary verification of the “OncoFocus” assay was performed in 27 NSCLC samples, 3 lung cancer cell lines and 2 control genomic DNA samples.Results
We found spectrophotometry significantly overestimated DNA quantity particularly at low concentrations. We also studied the correlation of DNA quantities with estimated copies of DNA templates as determined by SampleID. The results suggested that a minimum of 300 ng DNA is needed to achieve the required 300 – 500 amplifiable genomic copies per reaction for the OncoCarta analysis, which remains difficult to achieve for many diagnostic NSCLC samples. We developed a more focused diagnostic panel “OncoFocus” which could be performed reliably with less DNA but which includes key actionable mutations in 159 hotspots in EGFR (n=109), KRAS (n=17), BRAF (n=15) and NRAS (n=18) requiring only 150 ng of DNA. Somatic mutations were identified in 23 samples and 3 cell lines including EGFR (n=22), KRAS (n=6) and BRAF (n=1). No false positive results were observed in 4 FFPE and 2 control samples. The whole process from the receipt of FFPE samples to issuing a report can be completed within 5 working days and the “OncoFocus” panel has increased our capacity per chip (iPLEX II) from 15 to 31 samples. The “OncoFocus” panel also results in decreased per sample testing costs.Conclusion
The Qubit fluorometer is a more reliable and accurate method to quantify DNA derived from FFPE for mutational analysis than spectrophotometry. We also conclude that DNA mass spectrometric analysis using a new custom “OncoFocus” panel is an effective and efficient test that simultaneously detects 159 mutational hotspots, in the generally lower quantity of DNA obtained from routine FFPE NSCLC samples. -
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P2.06-011 - Phosphorylated-Akt expression is a prognostic marker in early stage non-small cell lung cancer (NSCLC) (ID 1151)
09:30 - 16:30 | Author(s): M. Kohonen-Corish
- Abstract
Background
The 5-year survival for stage IB non-small cell lung cancer (NSCLC) is only 55%, but the benefit of adjuvant chemotherapy in this setting remains equivocal. Numerous prognostic markers have been examined, but none to date have moved into clinical practice. There is an urgent need to identify novel molecular markers that can select high risk patients, who may potentially benefit from adjuvant chemotherapy.Methods
We identified 471 consecutive patients with stage IB primary NSCLC according to the American Joint Commission on Cancer, (AJCC) 6[th] edition tumour-node-metastasis staging system, who underwent surgical resection between 1990 and 2008. Patients who received neoadjuvant or adjuvant treatments were excluded. Pathology reports were reviewed and pathologic characteristics were extracted. Expression of phosphorylated Akt (pAkt) in both cytoplasmic and nuclear locations was assessed by immunohistochemistry, and clinicopathologic factors were analyzed against 10-year overall survival using Kaplan-Meier and Cox proportional hazards model.Results
455 (96.6%) cancers were adequate for pAkt immunohistochemical analysis. The prevalence of pAkt expression in the cytoplasm and nucleus of the cancers was 60.7% and 43.7% respectively. Patients, whose cancers expressed higher levels of pAkt in the cytoplasm, had a trend towards longer overall survival than those with lower levels of cytoplasmic pAkt (p=0.06). Conversely, patients whose cancers expressed higher levels of pAkt in the nucleus had a poorer prognosis than those with lower levels of nuclear pAkt expression (p=0.02). Combined low cytoplasmic/high nuclear expression of pAkt was an independent predictor of overall survival [HR=2.86 (95% CI:1.35-6.04); p=0.006] when modeled with age [HR= 1.05 (95% CI: 1.03-1.07); p<0.001], extent of operation [HR= 2.11 (95% CI: 1.48-3.01); p<0.001], visceral pleural invasion [HR=1.63 (95% CI: 1.24-2.15); p<0.001], gender, tumour size, histopathologic type and grade (p>0.05).Conclusion
Levels of expression of pAkt in the cytoplasm and nucleus and visceral pleural invasion are independent prognostic factors that can help to select patients with high risk disease, who may potentially benefit from adjuvant chemotherapy.
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P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P3.06-017 - Distinctive Insulin-Like Growth Factor 1 gene copy number and protein expression in non-small cell lung cancer (ID 1753)
09:30 - 16:30 | Author(s): M. Kohonen-Corish
- Abstract
Background
The insulin-like growth factor (IGF) pathway is involved in the development and progression of many tumours and there is growing preclinical evidence that blockade of this pathway has anti-tumour effects in NSCLC. IGF receptors (IGFR) are another potential target for targeted treatment of NSCLC and a number of agents are already undergoing clinical trial. Biomarkers are needed to select patients most likely to derive clinical benefit from these agents. The downstream pathway components of IGF1R and MET activation include PI3K and AKT, which are other potential biomarkers currently being investigated in this patient cohort. IGF1R has also been implicated in acquired resistance to EGFR-TKI treatments. Only a few small retrospective studies have investigated the prognostic role of IGF1R in NSCLC and the relationship with EGFR mutations is not known.Methods
IGF1R status was evaluated by chromogenic silver in situ hybridization (ISH) and immunohistochemistry (IHC) in tissue microarray sections from a retrospective cohort of 264 surgically resected NSCLCs and results were compared to clinicopathological features and patient survival. Patients were classified as IGF1R gene amplification (either presence of tight gene clusters, IGF1R to CEN15 ratio ≥ 2, or ≥ 15 copies of IGF1R per cell in ≥ 10% of analysed cells); high polysomy (≥ 4 copies of IGF1R in ≥ 40% of tumour cells); low copy number (< 4 copies of IGF1R in < 40% of cells). Patients were also grouped as IGF1R-positve (amplification or high polysomy) or IGF1R-negative (low copy number).Results
High IGF1R gene copy number was identified in 77 cases (29.2%) in which there were 32 amplified IGF1R cases (12.1%) and 45 high-polysomy IGF1R cases (17%). Increased copy number of IGF1R was more common in squamous cell carcinomas (SCC) compared to large cell carcinomas (LCC) or adenocarcinomas (ADC) (p<0.05). There was no correlation between IGF1R gene copy number status and other clinicopathological features including patient age, gender, smoking status, tumour size, vessel, perineural or lymphatic invasion, grade or stage. IGF1R copy number alteration in primary tumours was highly correlated with IGF1R copy number status in metastatic tumours (p<0.01). High IGF1R protein expression was observed in 61/259 (23.6%) primary tumours and 14/215 (6.5%) normal adjacent bronchial mucosae. High expression of IGF1R protein was significantly associated with SCC in comparison with non-SCC primary tumours, as well as with lymphatic and vessel invasion. There was a moderate correlation between IGF1R copy number status (positive versus negative) and IGF1R protein expression (high versus low) (Cramer’s V=0.3, p-value <0.001). Both IGF1R copy number status and protein expression were not associated with patient overall survival in univariate analyses (p>0.05).Conclusion
High IGF1R gene copy number and its protein expression are frequent in NSCLC, particularly in SCC. However, alterations of IGF1R are not associated with patient prognosis. IGF1R gene copy number can readily be assessed in formalin fixed paraffin embedded tissue and warrants further investigation as a potential biomarker of targeted therapy in NSCLC.
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P3.18 - Poster Session 3 - Pathology (ID 177)
- Event: WCLC 2013
- Type: Poster Session
- Track: Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P3.18-003 - ROS1 Gene Rearrangements in Non-Small Cell Lung Carcinoma - A New Genetic Target that can be Identified by Immunohistochemistry and FISH (ID 1482)
09:30 - 16:30 | Author(s): M. Kohonen-Corish
- Abstract
Background
Targeted therapies aimed at specific molecular genetic alterations are revolutionizing cancer treatment, particularly in non-small cell lung cancer (NSCLC). ROS1 is an oncogene that encodes a transmembrane tyrosine kinase receptor that has high homology with the intracellular kinase domain of ALK. Driver mutations involving translocation of the ROS1 gene have recently been identified in NSCLC and show promise as a target for tyrosine kinase inhibitors. In this study we aimed to: (1) Investigate the incidence and clinicopathological features of NSCLCs harbouring ROS1 rearrangements in an Australian population. (2) Investigate the accuracy of immunohistochemistry (IHC) compared to FISH at identifying tumours with ROS1 rearrangements.Methods
We tested for ROS1 translocations using both a FISH breakapart probe (Zytovision and Abbott Molecular) (≥15% cells with split signals or single green 3' signal considered positive for rearrangement), and immunohistochemistry (D4D6 clone, Cell Signaling Technology). Testing was undertaken on both (1) A retrospective cohort of 316 early stage lung adenocarcinomas in tissue microarrays. (2) A prospective cohort of 42 NSCLC, selected on clinical grounds for mutation testing (eg EGFR/KRAS/ALK negative samples and young age or never/light smoker).Results
In the retrospective cohort, only 1 case was positive for ROS1 gene rearrangement by FISH (0.3% incidence). ROS1 IHC identified positive staining in 7 (2.0%) cases, including the FISH+ case. ROS1 IHC had a sensitivity of 100% and specificity of 98% for identifying ROS1 gene rearrangements. In the prospective cohort of 42 cases, 4 cases with ROS1 gene rearrangement were identified by FISH and all 4 cases showed positive ROS1 immunohistochemical staining. Of the total 5 cases with ROS1 gene rearrangement, all occurred in adenocarcinomas from female patients with an age range of 33-81 years (mean 58). Four of the five patients were non-smokers and two were of Asian ethnicity. All 5 cases were negative for ALK rearrangements and in the 4 cases where EGFR status was known, they were all wild type.Conclusion
ROS1 gene rearrangements occur in a very small percentage of lung adenocarcinomas with distinctive clinicopathological features and appear to be mutually exclusive with other driver mutations in the small number of positive cases available for evaluation. Screening with IHC may be a suitable method of reducing the number of cases requiring FISH testing.
