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D.L. Aisner
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ED 13 - The EGF Receptor and Targeting T790M (ID 13)
- Event: WCLC 2015
- Type: Education Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:M. Boyer, M.A. Gubens
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 1a-1f
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ED13.02 - Detecting T790M in Blood and Tumor (ID 1825)
14:40 - 15:00 | Author(s): D.L. Aisner
- Abstract
- Presentation
Abstract:
Recent advances in targeted therapies and the molecular analysis of tumor samples have led to recommendations that EGFR mutation testing be implemented as standard of care in non-small cell lung cancer (NSCLC) of non-squamous type. This is in large part because of substantial benefits provided to patients treated with EGFR tyrosine kinase inhibitor (TKI) therapy, particularly those patients whose tumors are positive for activating, sensitizing mutations in EGFR. Despite these benefits, resistance to EGFR TKIs inevitably develops in all cases. The most common molecular mechanism of acquired resistance in this setting, occurring in approximately 50% of cases, is the evolution of a secondary ‘gatekeeper’ mutation which results in p.T790M (T790M). This acquired mutation results in a reduction in affinity of EGFR for the TKIs, while preserving catalytic function of the tyrosine kinase domain, The testing for T790M at the time of progression on TKI has emerged as an important clinical practice, as new-in-class EGFR TKIs demonstrate activity against this subset of resistant tumors. Because T790M initially emerges as a sub-clonal event, technical elements involved in its detection become paramount. Most importantly, achieving a high technical sensitivity to allow for detection of a sub-clonal (low alleleic frequency) phenomenon is critical for assays designed to detect this mutation. Sanger sequencing, for example, lacks the technical sensitivity to adequately identify low variant frequency events, and therefore alternate mechanisms of testing are required. Additional factors requiring major consideration in detection of T790M include potential sampling bias (particularly for the small biopsies or fine needle aspirates that are typically acquired), heterogeneity between multiple progressing lesions, cellular components of post-treatment biopsies, and technical ability to perform tumor enrichment to enhance detection. Genomic alterations resulting in T790M are uncommonly detected prior to TKI therapy, likely owing to the technical sensitivity of assays used to query for the presence of this alteration. Studies employing extraordinarily sensitive assays have demonstrated the presence of T790M as a subclonal event prior to TKI therapy in many tumors. Thus clonal selection under the pressure of TKI is a major mechanism allowing this alteration to be identified in the setting of progression on targeted therapy using less analytically sensitive assays. The secondary implication of this finding is that assays employed for detection of T790M must be adequately sensitive, but not over-sensitive to allow for the appropriate identification of what can best be considered the dominant mechanism of resistance. Others have postulated that highly sensitive detection of T790M prior to therapy could be used to determine a combination therapy approach which effectively prevents the evolutionary advantages of this sub-clone. In the uncommon instance that this alteration is identified at a high level pre-TKI therapy using standard assay approaches (<5% of cases), it can be associated with a germline alteration leading to a genetic predisposition for lung cancer. Recently, there has been great interest in the potential to monitor for the emergence of T790M alterations in the periphery, either via circulating tumor cells or circulating cell-free DNA. This approach is particularly attractive as it reduces requirements for invasive tissue sampling and can allow for a continuous monitoring approach. While the technical elements of liquid biopsy testing have been diversely applied, with very little in the way of consensus on methodology, numerous studies have demonstrated the promise of liquid biopsy approaches for both primary mutation detection as well as evaluation for T790M. In some cases, peripheral detection of T790M was demonstrated substantially before radiographic evidence of progression, a key proof-of-principle that such monitoring could be utilized as an effective approach for disease monitoring. Similar approaches for chimerism analysis or BCR-ABL1 transcript monitoring in the setting of bone marrow transplant or TKI therapy for chronic myelogenous leukemia, respectively, have been very successful. Major challenges still exist for both tissue-based and peripheral blood-based detection of T790M. Determination of the ideal level of assay technical sensitivity required for prediction of response to T790M-directed therapies will be a critical component to the implementation of these drugs in the clinic. In addition, for tissue biopsies, techniques to enhance the tumor cellularity of tested material and to avoid sample bias will need to be further refined. Liquid biopsy techniques, though demonstrating extraordinary promise, are widely divergent in terms of methodologies employed, and further study in this technological space is needed. 1. Arcila ME, Oxnard GR, Nafa K, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clinical cancer research : an official journal of the American Association for Cancer Research 2011;17:1169-1180. 2. Inukai M, Toyooka S, Ito S, et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res 2006;66:7854-7858. 3. Kim Y, Ko J, Cui Z, et al. The EGFR T790M mutation in acquired resistance to an irreversible second-generation EGFR inhibitor. Mol Cancer Ther 2012;11:784-791. 4. Majem M, Remon J. Tumor heterogeneity: evolution through space and time in EGFR mutant non small cell lung cancer patients. Transl Lung Cancer Res 2013;2:226-237. 5. Newman AM, Bratman SV, To J, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature medicine 2014;20:548-554. 6. Oxnard GR, Paweletz CP, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clinical cancer research : an official journal of the American Association for Cancer Research 2014;20:1698-1705. 7. Paweletz CP, Janne PA. Monitoring cancer through the blood. Cancer 2014;120:3859-3861. 8. Sorensen BS, Wu L, Wei W, et al. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib. Cancer 2014;120:3896-3901. 9. Tartarone A, Lerose R. Clinical approaches to treat patients with non-small cell lung cancer and epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance. Ther Adv Respir Dis 2015. 10. Watanabe M, Kawaguchi T, Isa SI, et al. Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR. Clinical cancer research : an official journal of the American Association for Cancer Research 2015.
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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): D.L. Aisner
- 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 13 - Genetic Alterations and Testing (ID 120)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- 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): D.L. Aisner
- 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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MINI13.06 - Mutation Prevalence for Oncogenic Drivers in Lung Adenocarcinoma (ID 3279)
11:15 - 11:20 | Author(s): D.L. Aisner
- Abstract
- Presentation
Background:
Identification of mutations which drive pulmonary adenocarcinomas (ADC) has rapidly moved from the research arena to clinical practice. The prevalence of these mutations has been suggested by a multitude of studies but here we describe the prevalence of mutations from a large study of patients with advanced ADC treated in the international phase III study INSPIRE (Lancet Oncology 2015) with all testing performed in one CLIA-certified laboratory under standardized conditions.
Methods:
Mutation testing was performed on 412 adenocarcinoma specimens using SNaPshot® methodology. Mutations were examined in the AKT, EGFR, KRAS, BRAF, NRAS, PIK3CA, TP53, PTEN, CTNNB1, and MEK1 genes. The relative frequencies of genetic alterations were calculated based on the total number of adequate specimens and specific consent for testing.
Results:
Of the 412 adenocarcinoma specimens tested, 372 (90.3%) had evaluable results from mutation testing. A single mutation was detected in 157 (42.2%) specimens, whereas mutations in two genes were detected in an additional 20 (5.4%). The overall prevalence of mutations for each specific gene was as follows: KRAS (34.2%), EGFR (12.2%), TP53 (4.9%), PTEN (2.8%), PIK3CA (2.2%), CTNNB1 (2.2%), NRAS (1.8%), BRAF (1.2%), MEK1 (0.3%), and AKT (0%). Figure 1 Evaluation of smoking status identified a substantially higher percentage of KRAS mutations in ex-light smokers and current smokers (38.2% and 40.5%) combined compared to never smokers (7.6%, p<0.0001) , and a lower proportion of EGFR mutations in ex-light and current smokers (10.9% and 4.9%) combined compared to never smokers (39.7%, p<0.0001). Patients ≥70 years old had a higher proportion of both NRAS (7.1% vs. 0.7%, p=0.009) and TP53 mutations (12.5% vs. 3.3%, p=0.010). In addition, males had a lower incidence of EGFR mutation (8.6% vs. 19.0%, p=0.007) as compared to females. Patients from North America, Europe, and Australia/New Zealand demonstrated lower rates of mutation in CTNNB1 (1.4% vs. 8.6%, p=0.030) and PIK3CA (1.4% vs. 8.3%, p=0.032) compared to patients from Central/South America, South Africa and India. Finally, among specimens with two mutations, combinations involving KRAS were the most prevalent (70%, 14/20) followed by TP53 (50%, 10/20).
Conclusion:
These results demonstrate the wide spectrum of mutations that can be detected in adenocarcinoma specimens, with high prevalence rates in the EGFR and KRAS genes. Most patients had only one identified driver mutation. The study revealed age and geographical associations in some mutations. The clinical relevance of the studied mutations in relation to chemotherapy and the human EGFR antibody, Necitumumab, will be studied.
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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): D.L. Aisner
- 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): D.L. Aisner
- 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): D.L. Aisner
- 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): D.L. Aisner
- 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): D.L. Aisner
- 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): D.L. Aisner
- 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.
