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R.C. Doebele
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MS 22 - Variety in the Oncogene (Does the Exact Mutation Matter?) (ID 40)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 4
- Moderators:R.C. Doebele, G.V.V. Scagliotti
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 4a-4f
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MS22.01 - EGFR Mutations (e.g., Exon 18 vs. 19 vs. 20 vs. 21) (ID 1945)
14:20 - 14:40 | Author(s): D.B. Costa
- Abstract
- Presentation
Abstract:
The most common epidermal growth factor receptor (EGFR) mutations identified in lung adenocarcinomas – termed classic somatic EGFR kinase domain mutations – occur as small inframe deletion (indels) mutations within exon 19 (45% of EGFR mutations, the most common delE746_A750) or the exon 21 L858R (40% of EGFR mutations) point mutation. Tumors harboring these classic EGFR mutations become addicted to EGFR’s signaling cascades and are susceptible (i.e., have a favorable therapeutic window) to inhibition by ATP-mimetic reversible (1[st] generation) EGFR tyrosine kinase inhibitors (TKIs) and C797-covalent (either wild-type specific [2[nd] generation] or mutation specific [3[rd] generation]) EGFR TKIs. EGFR-exon 19 deletions or EGFR-L858R are predictors of radiographic response and progression-free survival when gefitinib, erlotinib (1[st] generation) and afatinib (2[nd] generation) are used for patients with advanced lung adenocarcinomas. These anti-cancer compounds are approved by regulatory agencies and have revolutionized evidence-based care of advanced lung cancer. However, the palliative benefits of these drugs are limited by acquired mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation (which in turn can be inhibited by 3[rd] generation TKIs: mereletinib/AZD9291 and rociletinib. Both of these drugs are undergoing rapid development as palliative therapies for EGFR exon 19 deletion or L858R plus T790M mutated lung cancer and will soon be approved for evidence-based clinical care). The median survival of patients with EGFR-exon 19 deleted or EGFR-L858R mutated lung adenocarcinomas usually exceeds 24-36 months with a substantial portion of patients living for longer than 3 years when given sequential EGFR TKI therapy plus evidence-based cytotoxic chemotherapy. Consistently, patients with EGFR exon 19 deletion mutated lung adenocarcinomas have improved outcomes on 1[st] and 2[nd] generations EGFR TKIs than those with L858R mutated tumors (for biological and clinical reasons that remain to be elucidated). Other EGFR mutations have also been linked in preclinical models and in patients with lung adenocarcinomas to sensitivity to 1[st] and 2[nd] generation EGFR inhibitors. These include exon 18 point mutations in position G719 (G719A, C or S [3% of EGFR mutations]), inframe exon 19 insertions (1% of EGFR mutations), the exon 20 S768I mutation (<1% of EGFR mutations) and the exon 21 L861Q mutation (2% of EGFR mutations). Since most data for response to EGFR TKIs for these less frequent EGFR mutated lung adenocarcinomas comes from retrospective studies or single center experience; the true response rate, progression-free survival and overall survival of these tumors when given gefitinib, erlotinib, afatinib and 3[rd] generation EGFR TKIs is not clear. Interestingly, G719X, L858R and L861Q TKI-sensitive mutations can be commonly identified in conjunction (i.e., complex/compound mutations in >15% of cases) with other less well-defined EGFR kinase domain mutations (such as E709X, L747X, S768X, R776X, T790M, A871G, among others); and these double mutations may affect some of the single mutant pattern of response to EGFR TKIs. In the absence of formal regulatory approval for G719X, exon 19 inserted and L861Q mutated lung adenocarcinomas (groups that comprise more than 5% of all EGFR mutated tumors), the use of EGFR TKIs is often provided as “off label therapy” with clinical management similar to EGFR-exon 19 deletions or EGFR-L858R mutated lung adenocarcinomas. How often EGFR-T790M emerges as a mechanism of resistance in these tumors is unclear. The third most common and most diverse group of EGFR mutations are EGFR exon 20 insertions mutations (up to 10% of all EGFR mutations), which usually occur near the end of the C-helix within the N-lobe of the kinase, after residue M766 up to amino-acid C775, but a small subset map to the middle of the C-helix affecting amino-acids E762 to Y764. Unlike the other aforementioned EGFR mutated lung adenocarcinomas, most tumors with EGFR exon 20 insertion mutations are insensitive (i.e., do not respond radiographically or clinically) to 1[st] and 2[nd] generation EGFR TKIs; with the exception of EGFR-A763_Y764insFQEA (identical to D761_E762insEAFQ and with structural homology similar to exon 21 single mutants by inducing a N-terminal shift in the C-helix while replacing the active site residue E762 of EGFR), where responses to 1[st] and 2[nd] generation EGFR TKIs arise. Preclinical models – that mirror clinical behavior – have convincingly demonstrated that Y764_V765insHH, M766_A767insAI, A767_V769dupASV, D770_N771insNPG, D770_N771insSVD and H773_V774insH are not inhibited by clinically-achievable doses of gefitinib, erlotinib or afatinib. The structure of D770_N771insNPG (a representative EGFR TKI-insensitive exon 20 mutation at the most common insertion position D770_N771) has disclosed the amino acids inserted lock the helix in its active position but don’t alter the kinase domain TKI biding pocket (i.e., these mutants lack a therapeutic window to TKIs when compared to wild-type). Therefore, EGFR exon 20 insertion mutations affecting amino-acids Y764 to V774 should be classified as non-sensitizing to EGFR TKIs and development of mutation-specific TKIs may be hampered by the lack of therapeutic window of the kinase domain when compared to wild-type EGFR. Most EGFR exon 20 insertion mutated lung adenocarcinomas – in lieu of innovative clinical trials – should be treated with evidence-based approaches for “oncogene negative” lung adenocarcinomas. In conclusion, EGFR mutations comprise a heterogeneous group of activating oncogene mutations that have become the most clinically-relevant “driver” oncogenes for the clinical care of lung adenocarcinomas.
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MS22.02 - ALK, ROS1, and RET - Does the Partner Gene Matter? (ID 1946)
14:40 - 15:00 | Author(s): C. Lovly, M. Childress
- Abstract
- Presentation
Abstract:
Chromosomal rearrangements involving the ALK, ROS1, RET, and NTRK1 tyrosine kinases with several different gene fusion partners have been identified as therapeutically actionable genomic alterations in collectively up to 10% of non-small cell lung cancer (NSCLC) [1-4]. Notably, these kinase fusions have also been detected in several other epithelial, hematologic, neural, and mesenchymal malignancies, underscoring the importance of understanding fusion kinase biology in order to develop the most effective therapeutic strategies. In fact, numerous studies have now shown that tumors which harbor ALK, ROS1, RET, or NTRK1 fusions exhibit a dependency on the activated tyrosine kinase for proliferation and survival. This dependency, or ‘oncogene addiction’, makes the cancer highly sensitive to small molecule tyrosine kinase inhibitors (TKIs). In particular, ALK serves as the paradigm for therapeutically targetable kinase fusions in NSCLC. Crizotinib was the first ALK TKI to be approved for treatment of patients with ALK fusion positive (ALK+) NSCLC. Several other ALK TKIs, including ceritinib, alectinib, X-396, brigatinib, ASP3026, and PF-06463922 are also being developed for the treatment of ALK+ malignancies. These ‘next-generation’ ALK TKIs typically have more on-target efficacy against the ALK kinase domain and are able to overcome some of the crizotinib resistance mutations which have been observed clinically. While much emphasis has been placed on the study of the tyrosine kinase portion of ALK, ROS1, RET, and NTRK1 fusions, less is known about the 5’ gene fusion partners. However, the biology of the 5’ gene fusion partner is essential for driving the expression and function of the kinase fusion. Numerous different 5’ gene partners have been identified for each of the kinase fusions in NSCLC (Table 1). For example, EML4 is the most common fusion partner for ALK in NSCLC; however, KIF5B, TFG, KLC1, PTPN3, STRN, and SQSTM1 have also been identified as ALK partner genes in this disease. To add to the complexity, more than 10 different EML4-ALK fusions have been detected in NSCLC, varying by the extent of the EML4 gene which is fused to ALK. Likewise, numerous gene fusion partners have been described for ROS1, RET, and NTRK1 fusions in lung cancer (Table 1). Although the fusion partners can vary, they share three basic features. First, the promoter of the 5’ fusion partner dictates the expression of the fusion. Second, most fusion partners contribute an oligomerization domain, which can aid in auto-activation of the kinase [5]; although, this has not been verified for all fusion partners. The most common oligomerization domain found in the fusion partners is the coiled-coil domain. EML4-ALK homodimerizes by virtue of a coiled-coil domain in EML4. Disruption of this domain abrogates the ability of EML4-ALK to transform cells [5]. Furthermore, the extent of oligomerization may be important for transformation; some fusions dimerize, trimerize [6], or form tetramers [7]. Lastly, the 5’ gene fusion partner also determines subcellular localization of the fusion, and this can have significant effects on the interaction of the kinase fusion with other cellular proteins, influencing activation, signaling, function, and degradation of the fusion. For example, a thorough structural analysis of the most common EML4-ALK variants found in lung cancer revealed differences in the variant’s function, localization, and sensitivity to HSP90 inhibitors in clinical use [6]. Additionally, for some fusions, subcellular localization controls fusion activation, as is the case for MSN-ALK which congregates at the plasma membrane [8]. While most ALK fusions appear pan-cytoplasmic, others like RANBP2-ALK (perinuclear) and NPM-ALK (nuclear, nucleolar, and cytoplasmic) have different localization, the effects of which have yet to be investigated [9]. Very little is known about how signaling downstream of an ALK fusion may differ from that of a ROS1 or RET fusion in lung cancer. In addition, how different gene fusion partners may affect downstream signaling from a specific kinase fusion also remains an open question. One provocative study of various ALK fusions found in anaplastic large cell lymphoma demonstrated that the fusions were differentially able to activate PI3K and JAK-STAT signaling [10]. Furthermore, the ability of the different ALK fusions to activate PI3K kinase activity correlated with the fusion’s transendothelial migration properties. Overall, this study supports the hypothesis that the specific fusion gene partner defines the activity, signaling specificity, and phenotypic properties of the kinase fusion. Notably, the most commonly employed clinical diagnostics used to detect kinase fusions, including immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), will not specify which fusion partner is present within a tumor. However, as more sophisticated next-generation sequencing technologies come to the forefront of clinical diagnostics, clinicians will not only know that a tyrosine kinase fusion is present, but also to which specific gene partner the kinase is fused At present, there is very little data, all retrospective, to address the question of how a different fusion partner may affect clinical outcomes and disease responsiveness to targeted therapies. This is largely because the trials have used methods, such as IHC and FISH, to define eligibility criteria. In-depth contextual studies in pre-clinical models of lung cancer and in clinical trials in patients with kinase fusion positive disease are lacking; however, further analysis of this issue will allow us to refine the treatment of fusion positive lung cancer on a more personalized level in order to more effectively inhibit tumor growth and understand potential therapeutic resistance mechanisms. References 1. Kwak, E.L., et al., Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med, 2010. 363(18): p. 1693-703. 2. Shaw, A.T., et al., Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med, 2014. 371(21): p. 1963-71. 3. Drilon, A., et al., Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discov, 2013. 3(6): p. 630-5. 4. Vaishnavi, A., et al., Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat Med, 2013. 19(11): p. 1469-72. 5. Soda, M., et al., Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature, 2007. 448(7153): p. 561-6. 6. Richards, M.W., et al., Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain. Biochem J, 2015. 467(3): p. 529-36. 7. Zhao, X., et al., Structure of the Bcr-Abl oncoprotein oligomerization domain. Nat Struct Biol, 2002. 9(2): p. 117-20. 8. Tort, F., et al., Molecular characterization of a new ALK translocation involving moesin (MSN-ALK) in anaplastic large cell lymphoma. Lab Invest, 2001. 81(3): p. 419-26. 9. Chiarle, R., et al., The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer, 2008. 8(1): p. 11-23. 10. Armstrong, F., et al., Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells. Oncogene, 2004. 23(36): p. 6071-82.Table 1: Spectrum of tyrosine kinase fusions detected to date in NSCLC
Kinase Gene Fusion partner ALK EML4 KIF5B KLC1 PTPN3 SQSTM1 STRN TFG NTRK1 CD74 MPRIP ROS1 CCDC6 CD74 CLTC EZR FIG GOPC LIMA LRIG3 MSN SDC4 SLC34A2 TPM3 RET CCDC6 CUX1 KIAA1468 KIF5B NCOA TRIM33
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MS22.03 - MET - Gene Amplification vs. Overexpression vs. Exon 14 Skipping (ID 1947)
15:00 - 15:20 | Author(s): R. Salgia
- Abstract
- Presentation
Abstract not provided
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MS22.04 - KRAS - Are All KRAS Mutations the Same? (ID 1948)
15:20 - 15:40 | Author(s): T.E. Stinchcombe
- Abstract
- Presentation
Abstract:
KRAS mutations are the most commonly detected mutation in non-small cell lung cancer (NSCLC). KRAS mutations encode proteins containing a single amino acid substitution and in NSCLC most mutations are in codons 12 and 13. KRAS mutant proteins are constitutively activated leading to stimulus independent activation of the RAF-MEK-ERK pathway. KRAS mutations are associated with a history of tobacco use, and are more common in adenocarcinoma than in squamous histology. Patients with history of never smoking have a higher rate of transition mutations, but the biological and clinical significance is unknown.[1]KRAS mutations are mutually exclusive with EGFR mutations and ALK and ROS1 rearrangements. The benefits of testing for KRAS mutations are to eliminate the need for further molecular testing and to enroll patients in trials investigating KRAS directed therapy. KRAS mutational status is predictive of benefit of anti-EGFR monoclonal antibodies in advanced colorectal cancer (CRC), and the benefit is restricted to patients with KRAS wild-type CRC. However, patients with metastatic CRC with KRAS G13D mutations have better prognosis and benefit from monoclonal antibodies demonstrating that the specific KRAS mutation may have clinical implications.[2]KRAS mutational status is not predictive of benefit of cetuximab in advanced NSCLC.[3] The frequency and distribution of KRAS mutation subtype differs significantly among different cancer types. KRAS mutations can activate multiple downstream signaling pathways and activation of signaling pathways may be cancer-specific. The implication is that the success and failures of targeted agents against KRAS pathway in other cancers may not be relevant for the development of KRAS pathway targeting agents in NSCLC. A target therapy is not currently available for KRAS mutant NSCLC, and the recent focus has been on the development of MEK inhibitors. A randomized phase II trial of docetaxel alone or with selumetinib revealed that patients assigned to the selumetinib arm experienced a statistically significant higher objective response rate (ORR) (37% vs. 0%, p<0.0001) and longer progression-free survival (PFS) (hazard ratio of 0.58, 80% CI, 0.42-0.79, p=0.014; median 5.3 and 2.1 months respectively) and a numerically longer overall survival (OS) (HR of 0.80, 80% CI, 5.6 to 1.14, p=0.21; median 9.4 and 5.2 months, respectively).[4] A phase II trial compared trametinib to docetaxel in patients with KRAS mutant NSCLC. The ORR was same in the two treatment arms (12%), and the PFS similar (HR of 1.14; 95% CI, 0.75 to 1.75; p=0.5197).[5,6] Trametinib was also investigated in two separate phase IB/II trials in combination with docetaxel or pemetrexed; patients with both KRAS mutant and wild-type NSCLC were enrolled. Patients with KRAS mutant and wild-type NSCLC had similar ORR and PFS raising the question if KRAS mutations are predictive of MEK inhibitor benefit. In a subset analysis of the trial of trametinib and docetaxel patients with KRAS G12C mutations (n=8) had an ORR of 40% and a disease control rate of 80%.[7] This subset analysis is hypothesis generating and illustrates the need to collect the specific KRAS mutations in trials of novel agents. The prognostic and predictive value of KRAS mutations was investigated in a pooled analysis of resected patients enrolled in adjuvant chemotherapy trials.[8] In the observation cohort no difference OS based KRAS mutational status or subtype was observed, and KRAS mutation status and mutation subtype was not prognostic. In the adjuvant chemotherapy cohort no significant OS benefit was observed among patients with KRAS wild-type and KRAS codon 12 mutant NSCLC; a detrimental effect of adjuvant chemotherapy on OS was observed among the 24 patients with KRAS codon 13 mutant NSCLC (HR of 5.78; 95% CI, 2.06-16.2; p<0.001; interaction p=0.002). This observation needs to be prospectively validated in a larger sample before being used to make decisions about the adjuvant chemotherapy. Preclinical data suggest that the presence or absence other mutations other than KRAS may impact the efficacy of selumetinib.[9]KRAS mutations are frequently found in patients with a significant smoking history, and tobacco related NSCLC is associated high rate of mutations.[10] Thus, the potential impact of concurrent mutations or molecular alterations should be considered in future investigations. 1. Dogan S, Shen R, Ang DC, et al: Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-mutant cancers. Clin Cancer Res 18:6169-77, 2012 2. De Roock W, Jonker DJ, Di Nicolantonio F, et al: Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304:1812-20, 2010 3. O'Byrne KJ, Gatzemeier U, Bondarenko I, et al: Molecular biomarkers in non-small-cell lung cancer: a retrospective analysis of data from the phase 3 FLEX study. Lancet Oncol 12:795-805, 2011 4. Janne PA, Shaw AT, Pereira JR, et al: Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study. Lancet Oncol 14:38-47, 2013 5. Blumenschein GR, Smit EF, Planchard D, et al: MEK114653: A randomized, multicenter, phase II study to assess efficacy and safety of trametinib (T) compared with docetaxel (D) in KRAS-mutant advanced non–small cell lung cancer (NSCLC). Journal of Clinical Oncology 31:abstract 8029, 2013 6. Kelly K, Mazieres J, Leighl NB, et al: Oral MEK1/MEK2 inhibitor trametinib (GSK1120212) in combination with pemetrexed for KRAS-mutant and wild-type (WT) advanced non-small cell lung cancer (NSCLC): A phase I/Ib trial. Journal of Clinical Oncology 31:abstract 8027, 2013 7. Gandara DR, Hiret S, Blumenschein GR, et al: Oral MEK1/MEK2 inhibitor trametinib (GSK1120212) in combination with docetaxel in KRAS-mutant and wild-type (WT) advanced non-small cell lung cancer (NSCLC): A phase I/Ib trial. Journal of Clinical Oncology 31:abstract 8028, 2013 8. Shepherd FA, Domerg C, Hainaut P, et al: Pooled analysis of the prognostic and predictive effects of KRAS mutation status and KRAS mutation subtype in early-stage resected non-small-cell lung cancer in four trials of adjuvant chemotherapy. J Clin Oncol 31:2173-81, 2013 9. Chen Z, Cheng K, Walton Z, et al: A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response. Nature 483:613-7, 2012 10. Lawrence MS, Stojanov P, Polak P, et al: Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499:214-8, 2013
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ORAL 42 - Drug Resistance (ID 160)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 7
- Moderators:R.C. Doebele, J.V. DeGregori
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 4a-4f
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ORAL42.01 - ALK-Rearranged NSCLC Adaptive Cell Plasticity with Early Onset TGFb2 Mediated Precision Drug Escape through PRC-2 Epigenetic Reprogramming (ID 3111)
18:30 - 18:41 | Author(s): P.C. Ma, L. Yin, W. Zhang, I. Shi, X. Wu, J. Phillips, H. Choi, H. Makishima, D. Lindner, Y. Feng, F. Almeida, J.P. Maciejewski, Y. Saunthararajah, Z. Zhang
- Abstract
Background:
ALK-tyrosine kinase inhibitor (ALKi) is currently the standard precision therapy for advanced ALK(2p23)-rearranged (ALK+) non-small cell lung cancer (NSCLC), often with impressive primary responses. Nonetheless, acquired clinical resistance even in excellent/complete responders still develops ultimately with time; thus hampering long term benefits. Classic tumor rebiopsy studies that deciphered drug-resistance mechanisms focused on the “late phase” resistance at time of clinical progression in treated ALK+ NSCLC. These studies identified diverse pattern of drug-resistance mechanisms, including numerous non-dominant secondary drug-resistant ALK kinase mutations (e.g. C1156Y and L1196M), bypass signaling pathways (e.g. EGFR, KIT signaling), ALK gene amplification, and overexpression of microenvironmental factors (e.g. EGF, TGF-α, HGF). The mechanisms underlying the initial and early emergence of drug-resistance under precision therapy are poorly understood.
Methods:
EML4-ALK(+) H3122 and patient-derived ALKi acquired resistant biopsied-lung tumor tissue cells were used to investigate drug-escape mechanisms. Stem cell transcription factors QPCR array and RNA-sequencing profiling were performed on H3122 cells under ALKi up to day 14, compared with untreated and drug-washout controls. MTS cell viability assays using ALKis, in vitro and in vivo tissues QPCR assays, as well as in vivo xenograft IHC analyses were also performed. Patient-derived bronchoscopic biopsied NSCLC tissues (Ma0083) during ALKi resistance was procured and propagated in cell culture in accordance with approved institutional protocols.
Results:
We identified that H3122 cells displayed cell plasticity and can escape ALKi’s (TAE-684, crizotinib) remarkably early after precision therapy initiation, with augmented prosurvival signaling via upregulated autocrine TGFβ2 signaling, but not TGFβ1 or β3, as early as day 14 post-treatment. We validated using both in vitro and in vivo models the upregulated cascade of tumoral TGFβ2-HOXB3-mitochondrial priming during adaptive drug-escape. The early onset drug-resistant cells were marked by reversible autocrine TGFβ2-mediated transcriptome reprogramming with reversibly enhanced EMT-ness and cancer stemness. Moreover, RNA-seq findings strongly suggest a “reverse Warburg” cell state during adaptive drug-escape. The adaptive cellular plasticity was verified also in patient-derived bronchoscopic biopsied NSCLC tissues (Ma0083) with ALKi resistance. Interestingly, inhibiting mitochondrial priming using dual BCL-2/BCL-xL BH3-mimetics ABT-263 was effective to suppress early drug-escape, but not with the BCL-2-specific agent ABT-199, suggesting BCL-xL is a key target. Importantly, we also identified upregulated HOXB3 expression correlated with the early adaptive drug-resistance cell state, emerged through dynamic remodeling of EZH2/UTX in the polycomb repressive complex-2 (PRC-2). Deregulated EZH2/UTX epigenetic balance impacted the poised chromatin state of HOXB3 promoter H3K27me3/H3K4me3 histone marks. Early drug-escape cell state was correlated with suppressed EZH2 expression, at mRNA and also protein levels, in both in vitro and in vivo models. Finally, our results showed that specific EZH2 inhibitor GSK126 promoted ALKi drug-resistance, while UTX inhibitor GSK-J4 eradicated ALKi adaptive drug-resistance.
Conclusion:
Our study findings provide novel insights into the initial emergence and evolution of ALK precision drug-resistance and highlighted the significance of understanding the role of adaptive tumor cell plasticity in the early drug-escape process with important therapeutic implications. Therapeutic modulation of the coordinated EZH2/UTX balance in the PRC-2 complex can profoundly impact ALKi drug treatment outcome.
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ORAL42.02 - Qualitative and Quantitative Heterogeniety in Acquiring Resistance to EGFR Kinase Inhibitors in Lung Cancer (ID 572)
18:41 - 18:52 | Author(s): K. Suda, I. Murakami, K. Sakai, H. Mizuuchi, K. Sato, K. Tomizawa, K. Nishio, T. Mitsudomi
- Abstract
- Presentation
Background:
Acquisition of resistance to EGFR- tyrosine kinase inhibitors (TKIs) is one of important issues in lung cancer researches. Several resistance mechanisms have been identified. However, inter-tumor heterogeneity in acquisition of resistance to EGFR-TKIs is currently unclear.
Methods:
Eleven autopsied patients who developed acquired resistance to EGFR-TKI monotherapy were included in this study. All patients harbored activating EGFR mutations (exon 19 deletion or L858R mutation), and developed acquired resistance to EGFR-TKI after initial response to the drug. Details of patient characteristics are summarized in Table 1. The resistance mechanisms of seven patients have been reported in our previous analyses (Suda K, et al. Clin Cancer Res 2010, and Suda K, et al. APLCC 2014). In this study, we analyzed acquired resistance mechanisms in twenty-eight tumor samples obtained from the four additional patients using target sequencing technique by next-generation sequencer.
Results:
Among eleven patients, four developed T790M EGFR secondary mutation in all TKI-refractory lesions. One patient developed MET amplification in all TKI-refractory lesions. Three patients harbored both TKI-refractory lesions with T790M mutation and those with MET amplification. The other three patients showed respective resistance mechanisms (Table 1).Table 1. Summary of resistant mechanisms in eleven patients.
In the target sequence analysis, allele count data were further analyzed in tumor samples with T790M mutation, and we observed diverse T790M/activating EGFR mutation allele ratio ranging from 2 – 51%. In the analysis for time to treatment failure (TTF), we observed longer TTF in patients who developed single resistance mechanism compared with those who developed multiple resistance mechanisms (Fig. 1; p = 0.055). Figure 1Pt. ID Age/Sex Pack-Year Resistant Mechanisms TTF (m) C1 57/F 0 T790M or MET 13.8 C2 48/F 0 T790M or MET 11.0 C3 58/M 34 MET 14.5 C4 75/M 0 T790M 43.9 C5 93/F 0 T790M 14.8 C6 62/M 26 T790M 9.1 P1 86/F 0 T790M 10.8 P2 72/M 27 T790M or MET 3.8 P3 89/F 0 EGFR loss with MET or Unknown 9.0 P4 84/F 0 Unknown 22.6 A1 76/F 0 SCLC transformation or T790M 5.0
Conclusion:
In this study, we observed qualitative heterogeneity and quantitative heterogeneity of T790M allele ratio in acquisition of resistance to EGFR-TKIs in lung cancers. Qualitative heterogeneity in resistance mechanisms would have a correlation with TTF of EGFR-TKIs.
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ORAL42.03 - Discussant for ORAL42.01, ORAL42.02 (ID 3441)
18:52 - 19:02 | Author(s): L. Heasley
- Abstract
- Presentation
Abstract not provided
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ORAL42.04 - Rictor Alterations Elicit Mechanisms of Survival Advantage and Resistance to Targeted Therapy in Non-Small Cell Lung Cancer (NCSLC) (ID 2991)
19:02 - 19:13 | Author(s): D. Ruder, V. Papadimitrakopoulou, L. Shen, R. Herbst, L. Girard, J. Wang, G.M. Frampton, V. Miller, J. Minna, W.K. Hong, I.I. Wistuba, J.G. Izzo
- Abstract
- Presentation
Background:
Rictor (RPTOR independent companion of MTOR, complex 2) is a highly conserved protein and is a critical component for assembly and functionality of the mTORC2 complex. Alterations of the PI3K/mTOR/AKT pathway are hallmark of many cancer types, underscoring the potential important role of Rictor. The goal of our current study was to characterize the functional consequences of genomic alterations of Rictor in advanced refractory NSCLC. Our preliminary data suggest that Rictor alterations have the potential to, not only signal canonically (via activation of AKT), but also provide cancer cells with alternate, more advantageous oncogenic signaling via non-canonical mechanisms.
Methods:
We correlated genomic data (DNA next generation sequencing (NGS), Foundation Medicine, Inc) gene expression profiling, and clinical outcome in the context of the ongoing BATTLE-2 clinical trial of targeted therapies in chemo-refractory NSCLC(198 cases). We further (1) surveyed early stage NSCLC cases(230 cases) in The Cancer Genome Atlas (TCGA) database to perform two-way hierarchical clustering comparing gene expression profiling in amplified vs diploid cases; (2) utilized a single-nucleotide polymorphism array to select Rictor amplified and diploid NSCLC cell lines; (3) assessed Rictor protein and RNA expression by Western blot and qRT-PCR, respectively; (4) performed Rictor knockdown (siRNA), and (5) performed drug sensitivity to targeted therapies by MTS assay.
Results:
In the Battle-2 cases, we identified 15% of Rictor alterations (9% gene amplifications, 6.6% mutations, non-concomitant). Among the mutations, 1 was mapped to an N-terminal phosphorylation site, while all others are of unknown significance to date. Rictor alterations were significantly associated with lack of 8-week disease control in the AKTi+MEKi therapeutic arm. In the TCGA we found: (1) 10% Rictor amplifications and 3% mutations; (2) significant correlation between amplification and elevated Rictor gene expression; (3) a putative functional gene expression signature associated with Rictor amplification. In diploid cell lines we found concordance between AKT phosphorylation and activation of other downstream mTORC2 targets (i.e. SGK1 and PKCα), but in Rictor amplified cell lines we witnessed a discordant activation of these pathways. Furthermore, following Rictor knockdown in our amplified cell lines, a significant reduction of colony formation, migratory, and invasive potential was seen in a pathway-differential manner. Thus, suggesting that Rictor amplifications may provide survival advantage in select cancer cells by tipping the signaling balance toward a non-canonical oncogenic pathway (AKT-independent[I1] ).Also in a differential pathway manner, Rictor gene amplification and overexpression contributed to resistance to a number of targeted therapies
Conclusion:
Rictor alterations may constitute a potential novel mechanism of targeted therapy resistance via the activation of non-canonical signaling pathways. These alterations could define new molecular NSCLC subtypes with distinct biology that expose unique avenues for therapeutic implication. Ongoing studies are exploring therapeutic vulnerabilities, non-canonical signaling and Rictor mutations.
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ORAL42.05 - <em>SMARCA4</em>/BRG1 Is a Biomarker for Predicting Efficacy of Cisplatin-Based Chemotherapy in Non-Small Cell Lung Cancer (NSCLC) (ID 849)
19:13 - 19:24 | Author(s): E.H. Bell, A.R. Chakraborty, X. Mo, Z. Liu, K. Shilo, S. Kirste, P. Stegmaier, M. McNulty, N. Karachaliou, R. Rosell, G. Bepler, D.P. Carbone, A. Chakravarti
- Abstract
Background:
Adjuvant platinum-based chemotherapy remains a primary treatment of non-small-cell lung cancer (NSCLC); however, identification of predictive biomarkers is critically needed to improve the selection of patients who derive the most benefit. In this study, we hypothesized that decreased expression of SMARCA4/BRG1, a known regulator of transcription and DNA repair, is a predictive biomarker of increased sensitivity to platinum-based therapies in NSCLC. Moreover, this study also sought to confirm the prognostic role of SMARCA4/BRG1 in NSCLC.
Methods:
The prognostic value of SMARCA4 expression levels was tested using a microarray dataset from the Director’s Challenge Lung Study (n=440). Its predictive significance was determined using a gene expression microarray dataset (n=133) from the JBR.10 trial, and RT-PCR data from 69 patients enrolled on the MADe-IT trial and 33 platinum-treated patients from an institutional cohort.
Results:
In the Director's challenge study, low expression of SMARCA4 was found to be associated with poor overall survival compared to high and intermediate expression (P = 0.006). Upon multivariate analysis, compared to high, low SMARCA4 expression predicted an increased risk of death and confirmed its prognostic significance (HR=1.75; P=0.002). In the JBR.10 trial, improved five-year disease-specific survival was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine (HR 0.1, P= 0.001 (low); HR 1.1 , P= 0.762 (high)). An interaction test showed significance (P=0.007). In addition, a trend toward improved progression-free survival was noted only in patients with low SMARCA4 receiving a carboplatin- versus a non-carboplatin-based regimen in the MADe-IT trial. Figure 1 Fig1. Low SMARCA4 correlates with improved disease-specific survival with adjuvant cisplatin-based chemotherapy in the JBR.10 trial.
Conclusion:
Although decreased expression of SMARCA4/BRG1 is significantly associated with worse prognosis, it is a novel significant predictive biomarker for increased sensitivity to platinum-based chemotherapy in NSCLC patients.
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ORAL42.06 - Cancer Stem Cells: Targeting Aldehyde Dehydrogenase 1 (ALDH1) as a Novel Strategy in Cisplatin Resistant Non-Small Cell Lung Cancer? (ID 2724)
19:24 - 19:35 | Author(s): L. Mac Donagh, S.G. Gray, K.J. O'Byrne, S. Cuffe, S.P. Finn, M.P. Barr
- Abstract
- Presentation
Background:
Cisplatin is the backbone of chemotherapeutic treatment of lung cancer. Unfortunately the development of resistance has become a major challenge in the use of this cytotoxic drug. Understanding the mechanisms underlying this resistance phenotype may potentially result in the development of novel agents that may enhance the sensitivity of cisplatin chemotherapy in the clinical setting. The root of this resistance is hypothesized to be due to the presence of a rare cancer stem cell (CSC) population within the tumour that can reform a heterogenic tumour, resulting in recurrence and resistance following cisplatin chemotherapy.
Methods:
An isogenic model of cisplatin resistance was established by chronically exposing a panel of NSCLC cell lines (H460, SKMES, H1299) to cisplatin for 12months, thereby creating cisplatin resistant (CisR) sublines and their corresponding age-matched parental (PT) cells. To identify a CSC population within the resistant sublines, PT and CisR cell lines representing the three classifications of NSCLC were stained for ALDH1 using the Aldefluor kit (Stemcell Technologies). ALDH1 positive (+ve) and negative (-ve) subpopulations were isolated and their functional characteristics assessed. Proliferation and survival of ALDH1+ve fractions in response to cisplatin was assessed using BrdU and clonogenic survival assays relative to ALDH1-ve cells. ALDH1 subpopulations were examined for asymmetric division and expression of the human embryonic stem cell markers Nanog, Oct-4, Sox-2, Klf-4 and c-Myc and CD133. To confirm that this ALDH1+ve population is associated with cisplatin treatment, PT and CisR cells were chronically exposed to high dose cisplatin for 2 weeks and stained for ALDH1 and re-assessed for stemness qualities. Apoptosis and clonogenic survival of PT and CisR cells was assessed in response to selective inhibition of ALDH1 using diethylaminobenzaldehyde (DEAB) in combination with cisplatin. Xenograft studies in NOD/SCID mice are currently under investigation to examine the tumourigenic potential of isolated subpopulations of ALDH1.
Results:
A significant ALDH1+ve population was detected in CisR sublines, but not in their PT counterparts. Characterisation of the ALDH1+ve subpopulation confirmed enhanced expression of stemness markers, increased resistance and clonogenic survival in response to cisplatin compared to their ALDH1-ve counterparts, and the ability to asymmetrically divide. Chronic cisplatin treatment of the PT cell lines for 2 weeks increased resistance to cisplatin, increased stemness marker expression and induced the emergence of an ALDH1+ve population. Chronic high dose cisplatin treatment significantly expanded the ALDH1+ve population in the CisR cell lines. Importantly, inhibition of ALDH1 activity, with DEAB, decreased the mean cell viability, clonogenic survival capacity and increased cisplatin-induced apoptosis of the CisR cells when used in combination with cisplatin, an effect not seen in the PT cells.
Conclusion:
In this study, we have demonstrated the existence of a putative CSC population within our model of isogenic cisplatin resistant cell lines and suggest a role for ALDH1 inhibition as a potential therapeutic strategy in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin. Further studies will focus on re-purposing of FDA-approved ALDH1 inhibitor, Disulfiram (Antabuse), used in the treatment of chronic alcoholism as a potential combination therapy to prime chemoresistant cells to cisplatin.
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ORAL42.07 - Discussant for ORAL42.04, ORAL42.05, ORAL42.06 (ID 3442)
19:35 - 19:45 | Author(s): S. Yano
- Abstract
- Presentation
Abstract not provided
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Author of
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MINI 04 - Clinical Care of Lung Cancer (ID 102)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:L. Gaspar, V. Westeel
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 2c-3c
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MINI04.08 - Malignant Pleural Effusions Are Predictive of Peritoneal Carcinomatosis in Patients with Advanced EGFR Positive Non-Small Cell Lung Cancer (ID 3191)
17:25 - 17:30 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
Lung cancer is the most frequent cause of cancer death and metastatic disease at the time of initial diagnosis is common. Peritoneal carcinomatosis (PC) from lung cancer is a rare clinical event with a reported incidence of 1.2% (Satoh et al. 2001). However, there are limited data on what factors predict peritoneal progression in lung cancer. Over the last decade, molecular analysis of NSCLC has provided more detailed classification of patterns of metastatic spread. It has also been shown that oncogene-addicted subsets of NSCLC have different patterns of metastatic spread (Doebele et al. 2012). We investigated whether certain baseline patterns of metastatic spread in patients with advanced EGFR mutation positive (EGFR+) NSCLC can predict subsequent PC.
Methods:
We identified 156 patients with EGFR+ (Exon 19 or L858R) mutations from 2009 - 2014, of which 139 had metastatic NSCLC. 11 patients developed PC. This was defined as the presence of biopsy-proven adenocarcinoma from peritoneal fluid or radiographic patterns consistent with omental metastases. We identified areas of metastatic disease in predefined sites (brain, liver, lung, adrenal, soft tissue and pleura) at the time of diagnosis or metastatic recurrence. We noted if patients developed T790M, a resistance mutation to targeted therapy, in EGFR+ patients. A Fisher-Exact test was used to determine statistical significance between metastatic site and subsequent PC.
Results:Table 1 - Sites of metastasis and presence of T790M mutation in patients with PC and without PC
The presence of a pleural effusion was universal in all 11 EGFR+ patients who subsequently developed PC and this finding was statistically significant (P = 0.0001). 9 out of 11 patients with PC were identified to have a T790M mutation, a finding that was statistically significant (P = 0.0001). Except one patient, all EGFR+ patients developed PC following targeted tyrosine kinase therapy.Metastatic site / mutation PC No PC P value Lung 9.1% 38.6% P = 0.06 Liver 18.2% 15.8% P = 0.689 Bone 36.4% 46.8% P = 0.549 Brain 0% 23.7% P = 0.3570 Adrenal 0% 6.4% P = 0.123 Soft tissue 9.1% 2.2% P = 0.265 Pleural effusion 100% 26.6% P = 0.0001 T790M mutation 81.1% 34.5% P = 0.0001
Conclusion:
The presence of a malignant effusion is highly predictive of developing PC in patients with EGFR+ NSCLC. Although the underlying mechanism of PC is not entirely clear, it may be related to serosal communication with subsequent micrometastatic seeding of the peritoneal cavity. The T790M mutation, the most common acquired resistance mechanism to EGFR kinase inhibitors, was significantly more prevalent in the group that developed PC, although it remains unclear whether this mutation has any causative effect on spread to the peritoneum.
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.11 - Adaptor Re-Programming and Acquired Resistance in RET-Fusion Positive NSCLC (ID 2891)
17:45 - 17:50 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
RET gene fusions were identified as a novel oncogenic driver of ~1-2% of non-small cell lung cancer (NSCLC) patients and clinical trials investigating the use RET TKI therapy are underway. Like all NSCLC patients treated with TKI therapies, it is expected that drug resistance will emerge in this patient population. The mechanisms that drive acquired resistance to RET TKI therapy are still unknown. The objective of this study is to advance current understanding of RET signaling in NSCLC and to identify the cellular mechanisms of acquired RET TKI resistance that will eventually emerge in RET fusion positive NSCLC patients by using in vitro models of drug resistance.
Methods:
The LC-2/ad is a lung adenocarcinoma cell line that harbors the CCDC6-RET fusion. We created three distinct ponatinib resistant (PR) LC-2/ad cell lines (PR1, PR2, PR3) derived from three different dose-escalation strategies. RET break-apart fluorescence in situ hybridization (FISH) was performed on the parental LC-2/ad and PR-derivatives. Interactions between the RET kinase domain and known adaptor signaling molecules were assessed via proximity ligation assay (PLA) in parental LC-2/ad cells and resistant lines. Formation of RET-adaptor signaling complexes were confirmed via immunoprecipitation and western blot analysis. Next-generation RNA sequencing in conjunction with a high-throughput small molecule inhibitor screen were performed to elucidate the signaling pathways that drive resistance to RET-inhibition. Pathways and candidate molecules identified by these screens were validated using siRNA knockdown and pharmacologic inhibition in the context of a cell-proliferation MTS assay. Western blot analysis was utilized to identify the downstream signaling programs responsible for proliferation and survival in the RET-inhibition resistant cell lines.
Results:
MTS cell proliferation assay confirmed that all three ponatinib resistant cell lines are significantly less sensitive to ponatinib than parental LC-2/ad cells. RET FISH analysis demonstrated that the CCDC6-RET gene was retained in the PR1 and PR2 cell lines, but lost in the PR3 cell line. RT-PCR and western blot analysis confirmed the loss of the CCDC6-RET fusion in the PR3 cell line. DNA sequencing demonstrated no RET kinase domain mutations in either the PR1 or PR2 derivatives. Further, profound changes in the RET-signaling program have emerged in the PR1 and PR2 cell lines. Using a RET-GRB7 PLA, we have demonstrated that PR1 cells no longer form RET-GRB7 signaling complexes, while PR2 cells retain RET-GRB7 complexes even in the presence of ponatinib. Next-generation RNA sequencing of the PR1 cell line revealed an increase in expression of several known EMT markers including caveolin-1, vimentin, and ADAMTS1.
Conclusion:
Like many other targeted therapeutic strategies, resistance to small molecule Ret-inhibition in RET-fusion positive lung cancer cells can be driven by multiple mechanisms. Changes in the RET-adaptor programming appear to mitigate resistance in both the PR1 and PR2 cell lines, suggesting that RET-resistant cells may have successfully undergone an oncogenic switch to rely upon another known oncogenic driver in lieu of the CCDC6-RET fusion. Further, EMT reprogramming of the LC-2/ad cell may have contributed to the resistance phenotype in the PR1 cell line.
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MINI 13 - Genetic Alterations and Testing (ID 120)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:Y. Koh, R.K. Thomas
- Coordinates: 9/08/2015, 10:45 - 12:15, 205+207
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MINI13.01 - Clinicopathological Profiles of ROS1 Positive Patients Screened by FISH (ID 1450)
10:45 - 10:50 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
ROS1 fusion variants represent an important subset of oncogenic driver mutations in approximately 0.7 – 3.4% of non-small cell lung cancers. Since the frequency of ROS1 positive lung cancer patients is relatively low, it is unclear whether there are significant clinicopathologic associations for positive cases. Thus far, ROS1 positive patients tend to be younger and never-smokers with tumors displaying adenocarcinoma histology. This study describes a further cohort of ROS1 positive lung cancer patients in an effort to identify clinicopathologic associations.
Methods:
The data represent a retrospective analysis of the clinicopathological profiles of primary and metastatic lung cancer patients tested for ROS1 gene rearrangements by break-apart (BA) FISH at the University of Colorado School of Medicine.
Results:
The cohort consisted of 452 patients enriched for triple-negative (EGFR-, KRAS- and ALK-) non-squamous cell carcinomas screened for ROS1 rearrangements using the BA FISH assay. Nineteen cases (4.2%) were identified as positive for rearrangement, the majority (68%) of which were female, with a mean cohort age of 54.9 years (range 30-79); as compared to negative cases which included 56% female patients (P= 0.1083), and had a mean cohort age of 62.9 (range 21-90) (P= 0.0058). Seventeen out of the 19 ROS1 positive tumors were classified as adenocarcinomas, one was diagnosed as adenosquamous carcinoma, and the histology on one specimen was not otherwise specified (NOS). Among 12 individuals with information on pathologic stage at diagnosis, the majority (75%) were stage IV. The prevalent FISH pattern for rearrangement was a split 5’ and 3’ signal (68%) with the remaining specimens showing primarily single 3’ signals (21%) or a mix of split and single 3’ signals (11%).
Conclusion:
The ROS1 positive tumors in this cohort were primarily classified as adenocarcinomas, diagnosed at an advanced stage, in patients significantly younger and more likely to be women, although the sample set was biased for non-squamous lesions thereby limiting the application of this information to squamous cell lung carcinoma. The higher prevalence of ROS1 positive cases in this cohort compared to unselected cohorts is best explained by the inclusion of specimens with known negative status for EGFR and KRAS mutations and ALK fusions. As such, these data are in agreement with previous descriptions of ROS1 positive cohorts. Screening for ROS1 rearrangements in lung cancer patients displaying adenocarcinoma histology and negative for EGFR, KRAS and ALK activating events should identify a higher frequency of ROS1 rearranged tumors compared to unselected approaches and facilitate this subset of patients to be treated with targeted ROS1 inhibitors.
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MINI 14 - Pre-Clinical Therapy (ID 119)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Fernandez-Cuesta, A.F. Gazdar
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI14.04 - In Vitro and in Vivo Evaluation of the Kinase Inhibitor, MGCD516, in TRK and RET Fusion Cancer Cells (ID 2756)
11:00 - 11:05 | Author(s): 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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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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MINI21.11 - A Novel Cell Line Model of EGFR Exon 20 Insertion Mutations (ID 2828)
17:45 - 17:50 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
In-frame insertions in exon 20 of EGFR are infrequent activating mutations in the tyrosine kinase domain that have decreased sensitivity to EGFR inhibitors and currently have no available targeted therapies. In vitro studies ectopically expressing some of the common insertions (3 to 21 bp between codons 762 and 770) show reduced sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients whose tumors harbor these mutations do not respond to EGFR kinase inhibitors. To date, there are no known patient-derived cell lines that harbor the EGFR exon 20 insertions that recapitulate patient insensitivity to EGFR TKIs. Here we report the isolation and characterization of a patient derived cell line with an EGFR exon20 insertion.
Methods:
The CUTO-14 cell line was derived from a malignant pleural effusion of a lung adenocarcinoma patient harboring the EGFR exon 20 insertion p.A767_V767dupASV after obtaining IRB-approved informed consent. PCR amplification of EGFR exon 20 and subsequent Sanger sequencing was performed on genomic DNA isolated from CUTO-14. H3255 (L858R) and HCC827 (exon 19 del) cell lines were used as controls because they harbor sensitizing EGFR mutations. Cell viability was evaluated by MTS proliferation assay. Phosphorylation status and signaling was analyzed by western blot and an EGFR phosphorylation array. For tumor xenograft studies, nude mice were injected with 1.5 x 10[6] cells in matrigel and evaluated weekly for tumor growth.
Results:
Genomic sequencing of CUTO-14 demonstrated that the cell line maintains the pA767_V767dupASV EGFR exon 20 insertion. CUTO-14 showed relative resistance to gefitinib inhibition compared to HCC827 and H3255 in ERK1/2 phosphorylation assays. CUTO-14 also demonstrated reduced sensitivity to gefitinib compared to HCC827 and H3255 in cell proliferation assays. Tumor formation was observed in mice after injection in nude mice.
Conclusion:
CUTO-14 cells represent a novel model for the investigation of therapeutic strategies for EGRF exon 20 insertions mutations. The cell line has the ability to develop tumors in vivo and importantly shows reduced sensitivity to EGFR TKIs mimicking the lack of response in patients with these mutations.
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MINI 30 - New Kinase Targets (ID 157)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:K. Park, M. Villalona
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F3+F4
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MINI30.08 - ROS1 Resistance to Crizotinib Is Mediated by an Activating Mutation in c-KIT (ID 2244)
19:10 - 19:15 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
Non-small cell lung cancer (NSCLC) patients with ROS1 chromosomal rearrangement benefit from treatment with the ROS1 inhibitor crizotinib with remarkable response rates and durable disease control. Similar to ALK and EGFR mutant NSCLC treated with targeted kinase inhibitors, disease progression inevitably occurs due to acquired resistance either by mutation within the kinase domain of ROS1 or via bypass signaling. However, limited data exists on the spectrum of resistance mechanisms in ROS1+ NSCLC. Here report on a novel bypass mechanism for ROS1 resistance discovered in a ROS1+ tumor sample from patient with acquired resistance to crizotinib in which an activating mutation in the KIT receptor (p.D816G) desensitize ROS1 cells to crizotinib inhibition.
Methods:
Patients with ROS1+ NSCLC treated with crizotinib who developed acquired resistance underwent biopsy of a progressing tumor. Tumor samples were analyzed for potential resistance mechanisms. Assessment of mutations within the ROS1 kinase domain was accomplished by direct sequencing of exons 35 thru exon 42 of ROS1 from genomic DNA isolated from FFPE tissue. The SNaPshot® Multiplex System was used to profile additional tumor related genes for mutations. The ROS1 rearranged cell lines, HCC78 and CUTO-2, were transduced with lentivirus to generate ectopic expression of the KIT[D816G] cDNA. Cell proliferation was assessed by an MTS assay and cellular signaling was measured by western blot analysis.
Results:
Sequencing of the patient’s post crizotinib sample showed no mutation in the ROS1 kinase domain. Additional mutational profiling by SNaPshot® revealed the acquisition of a KIT[D816G] mutation in the post-crizotinib sample that was not present in the pre-crizotinib tumor sample. HCC78 and CUTO-2 ROS1+ cell lines expressing the KIT[D816G] mutation were refractory to crizotinib by both cell proliferation assays and analysis of downstream signaling pathways. Both ROS1 and KIT activity had to be inhibited in order to suppress downstream signaling and proliferation in these cells.
Conclusion:
Activation of KIT by a gain-of-function mutation is a novel mechanism of resistance to crizotinib in ROS1 rearranged NSCLC. This bypass-signaling pathway serves as a ROS1 independent mechanism of progression, similarly to previously identified EGFR or RAS signaling pathways, and can potentially be targeted by KIT inhibitors.
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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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ORAL21.01 - Adaptive Survival Signaling in Oncogenic Fusion Kinase Addicted NSCLC (ID 864)
10:45 - 10:56 | Author(s): R.C. Doebele
- Abstract
Background:
Gene fusions involving the proto-oncogenes ALK, ROS1, RET and NTRK1 are established or potential drug targets in cancer. Although targeted kinase inhibitors induce significant tumor shrinkage, complete patient responses are rare, and it is from that residual tumor burden that drug resistant clones eventually emerge. We have previously shown a role for WT EGFR signaling in ROS1+ cancer cells and their drug resistant derivatives. We hypothesized that EGFR performs a similar role in cancer cells harboring other gene fusions.
Methods:
Fusion oncogene NSCLC cell lines were treated as described and analyzed through immunoblot analyses or fixed onto chamber slides and assayed using kinase-adaptor proximity ligation assays (PLA). FFPE from NSCLC patients treated at the University of Colorado Hospital were also analyzed using kinase-adaptor PLAs. Nu/nu mice were injected with fusion oncogene positive NSCLC cell lines, treated as described, and volumes were measured 3x/week. FFPE tumors from mice were analyzed using various immunohistochemical markers or kinase-adaptor PLAs.
Results:
Stimulation of NSCLC cells that harbor an oncogenic fusion with EGF not only increased downstream signaling, but also rapidly increased phosphorylation of the fusion kinase itself. Additionally, EGFR signaling can dictate the engagement of different downstream signaling effectors, diversifying the signaling and cell fate responses in certain cancer cells. Proximity ligation assays (PLA) were employed to visualize wild-type EGFR-GRB2 signaling complexes in NSCLC cells driven by an oncogenic fusion kinase. We observed two modes of EGFR-GRB2 complex formation, the first in unperturbed cells, and the second only when the fusion kinase was inhibited. The kinetics of the induction of EGFR-GRB2 signaling revealed EGFR can take over the signaling in these cells as quickly as 5 minutes, and this kinase inhibitor-induced rewiring can be reversed by simply washing out the drug, suggesting a preference for the fusion kinase in the signaling circuit of these cells. Analysis of fusion-positive patient samples acquired at the time of progressive disease from treatment with an oncogene targeted monotherapy revealed the presence of EGFR-GRB2 signaling complexes. Additional analyses of patient samples revealed evidence of potentially non-cell autonomous responses to these therapies that may enable the survival of cells that would otherwise be drug-sensitive. The combination of a fusion kinase inhibitor with anti-EGFR therapy provided superior blockage of EGFR and ALK signaling complexes, as well as improved reduction in tumor volume and prolonged survival in an ALK+ xenograft model.
Conclusion:
Collectively, these results demonstrate a previously unknown role for an unmutated kinase, EGFR, in modulating the oncogenic phenotype in cells addicted to oncogenic fusion kinases. The activation of the EGFR signaling pathway can quantitatively augment fusion kinase signaling, but also diversify it by regulating the engagement of alternate signaling effector proteins. This data provides evidence for a novel role for EGFR as an oncorequisite signaling partner in certain cancer cell populations that harbor an oncogenic fusion kinase. Combination therapy of a fusion kinase targeted inhibitor with anti-EGFR therapy may improve initial tumor cell killing, and delay or prevent the onset of drug resistance in these patient populations.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.01 - FISHing TRK Activation by Gene Rearrangements in Non Small Cell Lung Cancer (ID 834)
16:45 - 16:56 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
The tropomyosin-receptor kinase (TRK) family includes genes important in nervous system development, NTRK1 (N1), NTRK2 (N2) and NTRK3 (N3). Oncogenic activation was identified long ago as N1 fusions in colon cancer and numerous fusions have been recently identified affecting all family members in multiple tumor types. This study developed FISH reagents for molecular diagnosis of NTRK rearrangements and investigated their prevalence in NSCLC. The ultimate goal is to validate a clinical assay for selection of patients who may benefit from novel tyrosine kinase inhibitors (TKIs) targeting these fusion proteins.
Methods:
Three FISH break-apart (BA) probe sets (LDTs) were tailored for diagnosis of rearrangements in N1, N2 and N3 and tested in specimens with known genomic status for these genes: cell lines KM12 (N1), CUTO3 (N1), MO-91 (N3), xenograft CULC001 (N1), and clinical specimens, and used to screen resected NSCLC. The LSI NTRK1 Cen and Tel probes (Abbott Molecular) were also tested. A specimen was positive for individual rearrangement when ≥15% tumor cells had split or single 3’,5’ signals. Moreover, a 6-target, 2-color FISH probe including the 3’N1, 3’N2 and 3’N3 sequences labeled in red and the 5’N1, 5’N2 and 5’N3 sequences labeled in green (TRKombo) was designed for rapid screening of TRK rearrangements in clinical specimens.
Results:
Results were obtained in 443, 410, and 434 examined NSCLC and positive patterns were detected in 5, 5 and 1 specimens, respectively for N1, N2, and N3. These 11 positive patients had age ranging from 38y to 76y, gender 6 male:5 female, and were current (4), former (5) or never (2) smokers. Histology was predominantly adenocarcinoma (7) but also included squamous cell (3) and neuroendocrine morphology (1). Unique to the N1 assay was the observance of FISH signal fusions where the 5’N signals appeared as doublet in >20% of the NSCLC specimens, which was determined to be copy number variation due to segmental duplication. Other atypical patterns were observed for all three targets and included doublets of the FISH fusion signals (18%, 14% and 9% respectively) and gene clusters (~5% for each). Twenty specimens (pre-clinical models and clinical cases) characterized as positive by the LDT N1 and by next generation sequencing (NGS) or atypical by the LDT NTRK1 BA were blindly analyzed with the LSI NTRK1 probe set and the results were reproducible, with brighter intensity of the fluorescent signals for the LSI probe. These specimens (positive by FISH and several atypicals) are currently under investigation to characterize the sequence specific genomic rearranged region by using a custom targeted, capture-based NGS panel (NimbleGen, Roche). The TRKombo screening probe performed well in blinded experiment using validation set including pre-selected positive and negative specimens and is under testing in clinical tissue sections.
Conclusion:
N1, N2 and N3 fusions were detected by FISH in a subset of lung carcinomas including adeno, squamous and neuroendocrine tumors. Optimization of molecular panels for diagnosis of these rearrangements is relevant since they represent a sizeable number of cases across multiple tumor types and there are numerous targeted inhibitor agents under development.
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ORAL37.06 - Defining MET Copy Number Driven Lung Adenocarcinoma Molecularly and Clinically (ID 2379)
17:39 - 17:50 | Author(s): R.C. Doebele
- Abstract
- Presentation
Background:
Increases in MET copy number define an oncogenic driver state sensitive to MET inhibition (Camidge et al, ASCO 2014). However, the level at which the genomic gain is relevant remains uncertain. When testing is performed by fluorescence in situ hybridization (FISH), variable cut-points in both mean MET/cell and MET/CEP7 ratio have been used. Partially overlapping datasets from the Lung Cancer Mutation Consortium (LCMC1) and Colorado Molecular Correlates (CMOCO) Laboratory were explored for a distinct MET-copy number driven lung adenocarcinoma subtype.
Methods:
MET was assessed by FISH. Data from non-adenocarcinomas and EGFR mutant patients with acquired resistance to an EGFR inhibitor were excluded. Positivity criteria were mean MET/cell ≥5 (low ≥5-<6, intermediate ≥6-<7, high ≥7) or MET/CEP7 ≥1.8 (low ≥1.8-≤2.2, intermediate >2.2-< 5, high ≥5). MET metrics were compared by race, sex, smoking status, stage at diagnosis, number of metastatic disease sites, site of metastases, presence of other known drivers (EGFR, KRAS, ALK, ERBB2, BRAF, NRAS, ROS1 and RET), response to first line chemotherapy and overall survival using Fisher’s exact tests, chi-square tests, Spearman correlations and log-rank tests, as appropriate. Statistical significance was set at the 0.05 level without adjustment for multiple comparisons.
Results:
1164 unique adenocarcinomas were identified (60% female, 85% Caucasian, 66% ex/current smokers). MET/CEP 7 data was available on 1164 and mean MET/cell on 700. 52/1164 (4.5%) had MET/CEP7 ≥1.8 (48% female, 83% Caucasian, 69% smokers). 50/52 (98%) had ≥1 other oncogenic driver tested (25/50 (50%) positive). 113/700 (16%) had mean MET/cell ≥ 5 (57% female, 82% Caucasian, 58% smokers). 109/113 (96%) had ≥ 1 other oncogenic driver tested (73/109 (67%) positive). Among patients with ≥1 additional driver oncogene tested, alternate drivers in low, indeterminate and high categories of mean MET/cell were 44/60 (67%), 17/24 (70%) and 12/28 (43%) respectively and for MET/CEP7: 16/29 (55%), 9/18 (50%) and 0/4 (0%) respectively. MET positive with additional drivers were excluded from further analyses. Men exceeded women in MET/CEP7 (men 4% vs women 1.6%, p = 0.019) and mean MET/cell positive cases (men 9.6% vs women 5.4%, p = 0.058). 6.4% of adrenal metastasis cases were MET/CEP7 positive vs 2% all other sites, p=0.031. Mean MET/cell: 12% adrenal vs 5% other sites, p=0.082. MET/CEP7 or mean MET/cell positive and negative groups did not differ by other variables (p > 0.05).
Conclusion:
The proportion of ‘MET positive’ adenocarcinomas varies by definition and positivity cut-point. Mean MET/cell ≥5 defines nearly 4x more positives than MET/CEP7 ≥1.8 and no mean MET/cell positive category was free from overlap with other drivers. As only high MET/CEP7 had no overlap with other drivers, MET/CEP7 ≥ 5 is the clearest candidate for a pure MET-copy number driven state, however cases free from other drivers do exist at lower MET positivity levels. MET/CEP7 positive cases free from other known drivers are more likely to be male, but unlike other known oncogenic states, race and smoking status are not significant in determining positivity. MET positivity may have a specific biological phenotype, being more likely to present with adrenal metastases.
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P1.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 206)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.01-017 - Two Cases of NSCLC with EGFR Exon 20 Insertions with Major Clinical Response to Cetuximab-Containing Therapies (ID 653)
09:30 - 09:30 | Author(s): R.C. Doebele
- Abstract
Background:
Lung tumors with EGFR Exon 20 mutations, particularly insertions between the amino acids Y764 and V774, present a major challenge for treatment. These mutations are known to confer resistance to current EGFR specific tyrosine kinase inhibitors (TKI). The mechanism of this resistance is described by Yasuda et al. as a “wedge” formed by the aberrant amino acids locking the C-helix in an inward, active position. This structural aberration prevents the TKI from accessing the critical pocket within the protein and inhibiting kinase activity. Without the ability to treat these tumors with TKIs, alternate treatments need to be pursued.
Methods:
We present, as index cases, two patients with metastatic lung adenocarcinomas demonstrating TKI unresponsive insertions in exon 20. Both patients had exuberant clinical and radiographic responses to cetuximab, an EGFR specific monoclonal antibody.
Results:
The first patient is a 39 year old male never-smoker with lung adenocarcinoma. The disease had progressed prior to molecular identification of the EGFR mutation, and the patient developed bilateral lung disease and metastatic lymph node and brain lesions. An exon 20 EGFR mutation (p.N771_P772insPHGH c.2313_2314insCCCCACGGGCAC) was identified. Following 4th line therapy with combination chemiotherapy plus cetuximab, the tumor burden was dramatically decreased and the patient had markedly improved functional status with the ability to return to employment. The second patient is a 71 year old male never-smoker with lung adenocarcinoma. The disease progressed and the patient developed widely metastatic disease. An exon 20 EGFR mutation (P770_N771insNPP) was identified. The patient was treated with combination cetuximab and afatinib therapy and experienced a dramatic decrease in lung and metastatic tumor burden with improved functional status.
Conclusion:
Cetuximab-containing therapeutic regimens may be a viable therapy for what previously have been considered treatment resistant molecular insults. Additional cases of these mutations and treatment with cetuximab are needed to demonstrate that these results are reproducible and that they warrant study in prospective clinical trials.
