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M.S. Tsao
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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: 7
- 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): A. Vaishnavi, S.B. Keysar, A.T. Le, D.L. Aisner, L. Heasley, A. Jimeno, 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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ORAL21.02 - Landscape and Functional Significance of KRAS Co-Mutations in Lung Adenocarcinoma (LUAC) (ID 3224)
10:56 - 11:07 | Author(s): F. Skoulidis, L. Byers, P. Tong, L. Diao, W. Denning, J. Gudikote, Y. Fan, V. Papadimitrakopoulou, J.G. Izzo, C. Behrens, H. Kadara, E.R. Parra Cuentas, J. Rodriguez-Canales, D.L. Gibbons, J.N. Weinstein, L. Girard, J. Minna, J. Wang, I.I. Wistuba, J.V. Heymach
- Abstract
- Presentation
Background:
The biological heterogeneity of KRAS-mutant LUAC represents a major impediment to the successful implementation of targeted therapeutic strategies for this clinically challenging group of lung cancer patients. Through integrative, multi-platform analysis of large scale omics data we recently identified three major subsets of KRAS-mutant LUAC defined on the basis of co-occurring genomic alterations in STK11/LKB1 (KL subgroup), TP53 (KP) and CDKN2A/B (KC), the latter coupled with low expression of the TTF1 transcription factor. We further demonstrated subset-specific molecular dependencies, patterns of immune system engagement and therapeutic vulnerabilities. Here, we extend these findings through comprehensive analysis of a wide panel of KRAS co-mutations and assess the impact of key co-mutations on facets of the malignant phenotype including flux through the MAPK and PI3K/AKT pathways and heterotypic interactions with the host immune system.
Methods:
Our datasets consisted of 431 tumors from TCGA (122 KRAS-mutant), 41 additional chemo-naive KRAS-mutant LUACs (PROSPECT dataset) and 36 platinum-refractory KRAS-mutant LUACs from the BATTLE-2 clinical trial. Significant KRAS co-mutations were identified on the basis of a P value threshold of ≤0.05 (Fisher’s exact test) coupled with a baseline prevalence of ≥3%. RNASeq data were downloaded directly from the TCGA site. Expression profiling of PROSPECT tumors was performed using the Illumina Human WG-6 v3 BeadChip Array whereas BATTLE-2 tumors were profiled using the GeneChipâHuman Gene 1.0 ST Array from Affymetrix. Generation of MAPK and PI3K proteomic scores, based on Reverse Phase Protein Array (RPPA) data, has been previously reported.
Results:
Our analysis identified somatic mutations in 31 genes as significantly co-mutated with KRAS in LUAC samples. Among them, co-mutations in STK11/LKB1 (P=0.00011) and ATM (P=0.0004) predominated. Somatic mutations in ERBB4 (P=0.0059), encoding a member of the ErbB family of receptor tyrosine kinases and MAP3K4 (P=0.0017) were also enriched in KRAS-mutant LUAC. We assessed the impact of KRAS co-mutations on the amplitude and directionality of signaling downstream of mutant KRAS using the proteomic “MAPK score“ and “PI3K score” as surrogates of effector pathway activation. Interestingly, co-mutations in ERBB4 were associated with significantly suppressed flux through the MAPK pathway (P=0.0024, t-test). Somatic mutations in other genes, including CAMSAP2, were associated with suppressed signaling through both the MAPK (P=0.00876, t-test) and PI3K-AKT (P=0.0032, t-test) cascades. Finally, within KRAS-mutant tumors, co-mutations in NLRC5, a master transcriptional regulator of MHC Class I molecules were associated with reduced mRNA expression of several of its classical target genes. In addition, low mRNA expression of NLRC5 correlated strongly with reduced expression of key components of the antigen presentation pathway across multiple independent datasets of chemotherapy naïve and platinum refractory KRAS-mutant tumors and cell lines. Thus, in addition to cell autonomous effects, co-mutations can also impinge on the reciprocal relationship between malignant cells and their immune microenvironment.
Conclusion:
Our work identifies a compendium of KRAS co-mutations that impact classical and emerging cancer hallmarks, including evasion of the host immune response. Systematic interrogation of the functional impact of prevalent KRAS co-mutations is essential for the development of personalized treatment approaches for this heterogeneous group of tumors.
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ORAL21.03 - KEAP1-Mutations and NFE2L2-Mutations in Patients with Non-Small Cell Lung Cancer (NSCLC) (ID 2792)
11:07 - 11:18 | Author(s): R. Frank, M. Scheffler, S. Michels, A. Eisert, R.N. Fischer, K. König, S. Merkelbach-Bruse, M.H. Serke, Y. Ko, U. Gerigk, T. Geist, L.C. Heukamp, R. Büttner, J. Wolf
- Abstract
- Presentation
Background:
Mutations in genes of the KEAP1-NFE2L2 pathway in patients with NSCLC are associated with an increased tumor growth, resistance towards cytostatic drugs and reduced survival rates. KEAP1 suppresses NFE2L2 under physiological conditions. Oxidative stress or electrophiles cause NFE2L2 to stabilize and translocate to the nucleus, resulting in transcription of various cytoprotective genes. Mutations in KEAP1 and NFE2L2 are described for diverse tumor entities and often cause an increased level of NFE2L2 leading to resistance of cancer cells against anti-cancer drugs and irradiation. This study was performed to characterize KEAP1-mutated and NFE2L2-mutated NSCLC clinically and genetically.
Methods:
Tumor tissue collected from 446 patients within a regional screening network was analysed for KEAP1 mutations and NFE2L2 mutations using next-generation sequencing (NGS). Clinical, pathological and genetic characteristics of these patients are described and compared with a control group of patients without KEAP1 mutation and without NFE2L2 mutation.
Results:
So far, we identified 33 patients with KEAP1 mutations. Among these we found 34 different mutations, of which the majority was not previously described. KEAP1 mutations were not restricted to a special exon. In 30 patients (90.9%), additional driver aberrations in KRAS, EGFR, FGFR1, FGFR3, STK11, ALK, DDR2, HRAS, BRAF, PIK3CA, PTEN, NFE2L2, EP300, TSC1, CREBBP, NRAS, MET and Her2 could be detected, as well as mutations and polymorphisms in TP53. KEAP1 mutations occurred in both genders (male/female ratio 3/1), in squamous-cell carcinoma (36.4%) and adenocarcinoma (60.6%) and were significantly associated with smoking. We also identified 26 patients with NFE2L2 mutations. Among these we found 15 different mutations, of which W24R and E79K were the most common. In 20 patients (76.9%) additional driver aberrations were detected. NFE2L2 mutations occurred in squamous-cell carcinoma (69.2%) and adenocarcinoma (23.1%) and were significantly associated with smoking as well. NFE2L2 mutations also occurred in both genders with 61.5% male and 38.5% female. Two patients had both a KEAP1 mutation and a NFE2L2 mutation.
Conclusion:
Our data suggest a role of KEAP1-mutations and NFE2L2-mutations as a cofactor in addition to classical driver mutations underlying the malignant phenotype of lung cancer cells. So far, this is the largest cohort of patients with KEAP1-mutations and NFE2L2-mutations analysed and described. Further survival and treatment analyses will reveal the role of these mutations for the outcome of these patients.
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ORAL21.04 - Discussant for ORAL21.01, ORAL21.02, ORAL21.03 (ID 3354)
11:18 - 11:28 | Author(s): D. Morgensztern
- Abstract
- Presentation
Abstract not provided
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ORAL21.05 - p53/KRAS Mutation Status Does Not Predict Sensitivity to Chemotherapy in NSCLC PDXs (ID 2459)
11:28 - 11:39 | Author(s): C. Mascaux, L. Dhont, P. Tomasini, N. Pham, M. Li, Y. Wang, E. Stewart, T.K. Waddell, M.S. Tsao, F. Shepherd
- Abstract
- Presentation
Background:
The LACE-Bio group assessed the prognostic and predictive values of KRAS and p53 mutations in 1543 completely resected non-small cell lung cancer (NSCLC) tumors. The predictive value of combined KRAS/p53 mutations for survival benefit from adjuvant chemotherapy was evaluated on 49 patients and chemotherapy was deleterious in this group compared to observation (HR 2.49 CI 95% [1.10 – 5.66], p=0.03). Patients with tumors harboring combined KRAS/p53 mutations had a worse outcome when treated with adjuvant chemotherapy compared patient with double wild type (WT) tumors (HR 3.03 (95% CI [1.29 – 7.15], p=0.01, interaction p=0.06). We have compared the chemo-sensitivity of patient derived xenografts (PDXs) with double p53/KRAS mutations, single p53, single KRAS mutation or double WT. 0
Methods:
Surgically resected early stage lung adenocarcinomas (ADC) were implanted into non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Fourteen lung ADC PDXs with various p53/KRAS status were revived and implanted: 11 engrafted and were expanded for comparison of treatment vs control. For each model, 6 replicates were included in treatment and control arms. Chemotherapy (cisplatin 3 mg/kg and vinorelbine 7 mg/kg intraperitoneally weekly) was initiated in the PDXs at tumor volumes of 150 mm[3].
Results:
Four models were p53/KRAS double mutant, 4 p53 mutant, 2 KRAS mutant and 1 double WT. The 4 double mutant PDXs responded to chemotherapy, 2 with reduced (SD) and 2 inhibited (PR) growth. Among the 4 PDXs with p53 mutation only, 2 responded (1 PR and 1 SD) and 2 were resistant. Among the 2 PDXs with KRAS mutation only, 1 had a complete response, but relapsed at treatment arrest and 1 achieved PR. The double WT PDX was highly sensitive to chemotherapy (PR) but also relapsed at treatment arrest.
Conclusion:
Among these 11 PDXs, the p53/KRAS mutation status did not predict chemo-sensitivity to cisplatin/vinorelbine, one of the most active adjuvant chemotherapy regimens in NSCLC. As these PDXs were molecularly profiled, we currently are investigating other biomarkers that might predict their sensitivity or resistance to chemotherapy.
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- Abstract
Background:
Hippo signaling is actively involved in adult tissue homeostasis and cell fate determination. Previous studies have linked the activation of YAP (the major downstream effector of Hippo pathway) with LKB1 deficiency. Here, we characterize the function of YAP in the progression and phenotypic plasticity of LKB1-deficient lung tumors and decipher the detailed mechanisms underlying those process.
Methods:
Through integrative studies on human lung cancer specimens and lung cancer mouse models, we investigate the distinct role of YAP on lung cancer malignant progression and phenotypic transition. Furthermore, we uncover the detailed mechanisms by cell line based works together with biochemistry and molecular biology methods.
Results:
The oncogenic role of YAP in malignant progression of Lkb1-deficient lung adenocarcinoma Using distinct lung cancer mouse models, we show that ectopic expression of YAP in Type II alveolar epithelial cells results in hyperplasia in the lung. YAP expression significantly accelerates lung adenocarcinoma (ADC) malignant progression in Kras[G12D] mice whereas YAP deletion dramatically delays the process in Lkb1[L/L]/Kras[G12D] mice. Further mechanistic investigations have revealed that the delayed progression in Lkb1-deficient ADC with YAP ablation attribute to the downregulation of the inhibitor of apoptosis protein, Survivin. The inhibitory role of YAP in phenotypic transition from adenocarcinoma to squamous cell carcinoma We have previously shown LKB1 inactivation confers lung adenocarcinoma with strong plasticity to progressively change the cell fate and transit to squamous cell carcinoma with unknown mechanism. Here, we find that ectopic YAP overexpression dramatically inhibits ADC to SCC transdifferentiation whereas knockdown of YAP conversely accelerates the transition process. YAP is initially activated by LKB1 loss in ADC, leading to ZEB2 up-regulation in ADC cells, which binds to DNp63 gene promoter to repress DNp63 transcription. During the transition process, extracellular matrix (ECM) depletion in ADC inactivates YAP, thus relieves ZEB2 mediated default repression on DNp63 transcription in ADC, leading to the initiation of squamous differentiation program. Functionally, p63 ectopic expression significantly rescues the inhibitory effect of YAP upon SCC transdifferentiation.
Conclusion:
Our findings uncover the two faces of YAP in lung tumor malignant progression and phenotypic plasticity. YAP is an essential mediator of malignant progression of Lkb1-deficient lung ADC via regulating Survivin whereas an important barrier for lung cancer transdifferentiation through ZEB2 dependent DNp63 repression. Those works shed light on the fundamental role of YAP in regulating cancer progression and lineage phenotypic transition in LKB1 deficient lung tumors, which might help future development of better therapeutic strategies.
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ORAL21.07 - Discussant for ORAL21.05, ORAL21.06 (ID 3355)
11:50 - 12:00 | Author(s): F. Grossi
- Abstract
- Presentation
Abstract not provided
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ORAL 38 - Liquid Biopsies (ID 147)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 8
- Moderators:M.S. Tsao, J. Wang
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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ORAL38.01 - A Prospective Study of Rapid Plasma Genotyping Utilizing Sequential ddPCR and NGS in Newly Diagnosed Advanced NSCLC Patients (ID 935)
16:45 - 16:56 | Author(s): A.G. Sacher, C. Paweletz, R. Alden, A. O'Connell, L. Lim, C. Raymond, P.A. Jänne, G.R. Oxnard
- Abstract
- Presentation
Background:
Plasma genotyping of cell-free DNA (cfDNA) has the potential to allow for noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. We have developed a quantitative droplet digital PCR (ddPCR)-based plasma genotyping assay capable of detecting common EGFR and KRAS mutations in NSCLC (Oxnard et al., CCR 2014). Although rapid and highly specific, this assay lacks the ability to both multiplex and detect complex genomic alterations such as rearrangements. In this prospective study, we evaluate the test characteristics of ddPCR combined with plasma next-generation gene sequencing (NGS) as a new paradigm for plasma genotyping.
Methods:
Patients with newly diagnosed advanced NSCLC were eligible. All patients were required to have a biopsy available or planned for tissue genotyping which was used for gold standard comparison. Patients underwent an initial blood draw and immediate plasma ddPCR for EGFR exon 19 del/L858R and KRAS G12X. A subset of patients additionally underwent plasma NGS using a unique probe set designed by our group to detect rearrangements and mutations in 12 genes (EGFR, KRAS, ALK, ROS1, BRAF, RET, NRAS, ERBB2, MET, MEK1, PIK3CA and p53). This plasma NGS assay utilized a novel bias corrected NGS which minimizes off-target reads (Resolution Bio) performed on a desktop MiSeq platform. Test turnaround time (TAT) was measured in business days from date of blood draw until test reporting.
Results:
120 patients with newly diagnosed advanced NSCLC have been enrolled and 94 have completed tissue and plasma genotyping. Tumor genotype included 25 EGFR exon 19/L858R mutants, 17 KRAS G12X mutants, 24 rare genotypes and 15 others. Median TAT for plasma ddPCR was 3 days (range 1-5). Specificity of plasma ddPCR was 99% for EGFR exon 19 del/L858R (68/69) and 100% for KRAS (77/77). Sensitivity of plasma ddPCR was 76% for EGFR exon 19 del/L858R (19/25) and 71% for KRAS (12/17). Plasma NGS is ongoing with testing completed on 11 patients with a known tumor genotype. 8 had a genotype detected on plasma NGS: 2 ALK rearrangements, 1 ROS1 rearrangement, 1 RET rearrangement, an EGFR G719A mutation, a KRAS G12C and a combined KRAS G12C/PIK3CA mutation - all matched the tumor genotype. Preliminary plasma NGS turnaround time ranged from 5-10 business days.
Conclusion:
Rapid plasma genotyping using sequential plasma ddPCR (1-5 day TAT) followed by plasma NGS (5-10 day TAT) represents a new paradigm for noninvasive plasma genotyping. This approach capitalizes on the use of rapid ddPCR for common targetable mutations and the ability of plasma NGS using an augmented MiSeq platform to multiplex and detect complex alterations. This new model for plasma genotyping uses testing platforms that can readily be employed in most molecular pathology laboratories allowing for widespread adoption.
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ORAL38.02 - Biopsy-Free Circulating Tumor DNA Assay Identifies Actionable Mutations in Lung Cancer (ID 2163)
16:56 - 17:07 | Author(s): V. Villaflor, B. Won, B. Nagy, K. Banks, R. Lanman, A. Talasaz, R. Salgia
- Abstract
- Presentation
Background:
The National Comprehensive Cancer Network (NCCN) non-small cell lung cancer guidelines recommend testing for seven genomic targets amenable to matched therapies, including point mutations and insertions/deletions (indels) in EGFR and ERBB2 (HER2), point mutations in BRAF, fusions in ALK, RET and ROS1, and amplification of the MET gene. Novel digital sequencing technology allows assessment of these biomarkers without an invasive tissue biopsy.
Methods:
We prospectively tested cell free DNA from 43 advanced non-small cell lung cancer patients using a cell-free circulating tumor DNA (ctDNA) next-generation sequencing (NGS) panel of 54 cancer genes (Guardant360). Single nucleotide variants (SNVs) in 54 genes and copy number variants (CNVs) in 3 genes (EGFR, ERBB2 and MET) were reported quantitatively as the mutant allele fraction (MAF) in cell-free DNA and the absolute copy numbers in plasma, respectively.
Results:
79% of patients had at least one ctDNA alteration detected. Five (11.6%) had sensitizing mutations in EGFR: EGFR L858R (n=1), EGFR exon 19 deletions (n=4), which may respond to first line tyrosine kinase inhibitors (TKIs) such as erlotinib and afatinib. Three patients with EGFR exon 19 deletions had concurrent T790M resistance mutations, which develop in over half of patients on early generation TKIs. MET amplification, which may respond to crizotinib, was identified in one patient. Clinical outcomes will be reported at the time of presentation. Of the 43 patients in our series, actionable findings were identified in 35 patients (81.4%), with an approved therapy in 5 (11.4%), off label therapies in 19 (44.2%), and clinical trials in 28 (65.1%).
Conclusion:
In our series of NSCLC patients with advanced disease, digital sequencing of cell-free circulating tumor DNA yielded results in approximately 80% of patients. Of these, over 80% had an actionable alteration, including 5 cases with EGFR alterations that could benefit from an approved therapy. Biopsy-free comprehensive sequencing of a patient’s cancer can empower informed treatment decisions from a simple blood draw, especially when repeat tissue biopsy is not feasible or tissue NGS is uninformative.
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ORAL38.03 - Assessing the Feasibility of Detecting ALK Fusions with qRT-PCR Assays in Cell-Free Plasma RNA (ID 1437)
17:07 - 17:18 | Author(s): E. Ordinario, M. Lee, H. Truong, D. Kuo, R. Dua, W. Liu, G. Spier, A. Begovich, X.M. Ma
- Abstract
- Presentation
Background:
Chromosomal rearrangements that result in transcript fusions have been a focus of attention in cancer as they provide attractive therapeutic targets. Identifying tumors that harbor chromosomal rearrangements by in situ hybridization assays has been a challenge in the clinic because these assays demand large quantities of tissue specimens. Cell-free nucleic acids from patient plasma may provide a non-invasive, alternative tool for detecting transcript fusions. Here, we demonstrate the feasibility of detecting ALK fusions with a qRT-PCR assay using cell-free plasma RNA (cfRNA).
Methods:
We designed a one-tube, four-channel multiplex ALK qRT-PCR assay that incorporates two strategies to detect ALK fusions. One channel employs variant-specific primers to detect >90% of the reported ALK fusions. The remaining three channels measure the expression of the 5’ and 3’ ends of the ALK gene relative to an internal reference and to each other, in theory, permitting the detection of all ALK fusions including those without knowledge of the fusion partner. To assess the performance of the multiplex ALK prototype assay, we made contrived samples blending mutant and wildtype cell line RNAs. In addition, we tested the multiplex ALK assay on NSCLC FFPET specimens (n=209). Moreover, to mimic plasma cfRNA, we made contrived samples by blending mutant cell line conditioned media with normal plasma.
Results:
Data from the cell line RNA blends demonstrate that both the variant-specific and the 5’ and 3’ differential expression successfully detect the EML4-ALK fusion-positive RNA. The variant-specific component of the assay is sensitive enough to detect at least 25pg of fusion-positive cell line RNA at a 1:4000 dilution with wildtype cell line RNA. From the NSCLC FFPET specimens, we identified 4 samples positive for the ALK fusion. These results were validated by a reference method that uses anchored-PCR to enrich ALK targets followed by NGS that employs a novel algorithm to identify potential fusion products. In addition, the multiplex ALK qRT-PCR assay detected transcript fusions in blends composed of plasma and EML4-ALK positive conditioned media at a dilution of 3:1. Lastly, we tested the multiplex ALK assay on confirmed ALK-fusion positive NSCLC plasma specimens, and were able to detect ALK fusions (7 out of 8) from as little as 750 ul of plasma.
Conclusion:
In summary, we have developed a one-tube, multiplex ALK qRT-PCR assay that exhibits performance characteristics suitable for transcript fusion detection in plasma cfRNA. Efforts are underway to further test and optomize the performance of this assay in clinical samples and to apply this multiplex qRT-PCR design concept to other transcript fusions including RET and ROS1.
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ORAL38.04 - Discussant for ORAL38.01, ORAL38.02, ORAL38.03 (ID 3565)
17:18 - 17:28 | Author(s): T. Mok
- Abstract
- Presentation
Abstract not provided
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ORAL38.05 - Dynamic Changes in EGFR Mutation Circulating Tumor DNA in Urine on Anti-EGFR Therapy (ID 2230)
17:28 - 17:39 | Author(s): H. Husain, V. Melnikova, K. Kosco, S. Hancock, E. Samuelsz, B. Woodward, S. Guerrero, C.R.T. Vibat, M.G. Erlander, E. Cohen, S. Lippman, R. Kurzrock
- Abstract
Background:
Circulating tumor DNA can be detected in urine efficiently, serially, and completely non-invasively. Utilizing a PCR enriched NGS detection platform, we sought to demonstrate the feasibility of detecting activating and resistance EGFR mutations in urinary ctDNA to understand mechanisms of resistance to targeted therapies in patients with EGFR-mutated lung adenocarcinoma.
Methods:
In a biomarker study of 46 patients enrolled, urine was collected every 3-6 weeks from patients on first line anti-EGFR TKI therapy and then daily at progression during the first week of 3rd generation anti-EGFR TKI treatment when available. Urinary ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Quantitative analysis of EGFR activating exon19del, L858R, and T790M resistance mutations was performed utilizing wild type blocker probes, PCR enrichment, and NGS detection (MiSeq). Early pharmacodynamic events within the first hours to days of anti-EGFR therapy were further studied by quantitating ctDNA mutations and comparing with the reponse or lack of response by RECIST on CT scans 6 weeks after initiation of second line therapy.
Results:
Interim analysis was conducted on 34 patients receiving first line anti-EGFR therapy with erlotinib. The average quantity of DNA obtained per patient was 830ng/70ml of urine. The sensitivity between tissue and urine for EGFR Exon19del, L858R, and T790M was 94%, 100%, and 100% respectively, and interim specificity was 94%, 100%, and 96% respectively. Analysis of longitudinal samples from patients on erlotinib revealed that the EGFR T790M mutation was detected in the urine of 17 out of 24 (71%) patients 4-15 weeks before radiographic progression on erlotinib. All 10 patients who were positive for T790M mutation by tissue were also positive by urine. Three patients were T790M tissue negative but urine was positive for T790M. Early peaks in EGFR Exon19del, L858R, and T790M ctDNA on days 1-4 of urine collected daily within the first week on next generation anti-EGFR TKI correlated with CT radiographic response or lack of response 6 weeks after first drug dosing. Figure 1
Conclusion:
We demonstrate that EGFR activating and resistance mutations can be detected in ctDNA in urine months before progression on anti-EGFR TKIs. Urinary ctDNA testing identifies additional patients who are potentially eligible for next generation anti-T790M treatment. The size of the peaks in ctDNA upon second line anti-EGFR inhibitors correlate with tumor lysis and CT radiographic response. The clinical utility of daily kinetic monitoring of ctDNA in urine after drug adminstration is being further validated in an expanded cohort.
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ORAL38.06 - Identification of Actionable Tumor Alterations in Circulating Cell-Free Tumor DNA (cf DNA) Using Digital Sequencing from NSCLC Patients (ID 1706)
17:39 - 17:50 | Author(s): P.C. Mack, D.R. Gandara, P. Lara Jr., R.A. Tsai, L. Snyder-Solis, J.W. Riess, K. Kelly, K.C. Banks, O.A. Zill, S.A. Mortimer, R.B. Lanman, A. Talasaz, H. Eltoukhy
- Abstract
- Presentation
Background:
To fully implement precision therapy in lung cancer, transition to a re-biopsy policy will be required at baseline and at progression after each line of therapy. The molecular testing paradigm is shifting toward next generation sequencing (NGS). As tissues are limited and repeat invasive biopsy introduces cost and risk, novel technologies sensitive and specific enough for multiplexed assessment in cell-free DNA (cfDNA) isolated from patient blood would represent a significant advance. Preliminary experience from investigators suggest a high degree of correlation between repeat tumor biopsy and plasma NGS. Here, we present the Guardant Health (GH) digital sequencing approach in a consecutive series of NSCLC cases.
Methods:
225 consecutive blood specimens from NSCLC patients, collected February–March 2015, were evaluated for cfDNA tumor alterations by digital sequencing using the GH panel of 68 genes. The test includes all reported fusion partners for ALK, RET, ROS1, and NTRK1 and cfDNA amplification for 16 genes. The mutant allele fraction (MAF) was calculated relative to WT in cfDNA. The test is sensitive to a single fragment of mutated cfDNA in a 10 ml blood sample and analytic specificity is >99.9999%.
Results:
Canonical EGFR activating mutations were detected in 20 cases (14 E19del, 3 L858R, 2 E20ins, 1 G719A). EGFR T790M co-occurred in 7 cases (6 E19del, 1 L858R), with EGFR amplification observed in 6 of the 20. Median age for patients with EGFR mut+ was 62.5; 18 female(90%), compared to nonEGFR-mutant cases. Four cases had driver fusions (2EML4-ALK, 2 KIF5B-RET) and five cases harbored an ERBB2 E20ins. KRAScodon 12/13 mutations were detected in 23 patients, while 3 harbored mutations in HRAS(Q61L) and NRAS(Q61L, G13R), and 6 had BRAF mutations (4 V600E, 2 G466X). All putative drivers were mutually exclusive. Mutations in signal transduction factors with confirmed gain-of-function activity included AKT1(E17K), MEK1(K57N, C121S), PIK3CA(E542K, E545K x2, H1047L, M1043V, R93W) and JAK2(V617F x2); truncating or missense mutations (>3% MAF) were observed in NF1 (6 cases), PTEN(1 case), SMAD4(4 cases) and STK11(4 cases). TP53 mutations were detected in 116/225 (51%). Evidence of gene amplification was seen in 32 cases, with 11 harboring multiple events. By function, amp events were observed for G1 cell cycle factors:11, RTKs: 17, MYC: 2; and signal transduction: 21. MAF ranged from 0.06% to 83.4% (av 5.1%; median: 9.8%), reflecting clinical and biologic diversity of patients. In a clinical subset at UC Davis, 27 patients were evaluated and alterations were detected in 18 (66.7%). Actionable findings were identified in 14 (77.8%) including 2 with EGFRL858R, 1 with EGFR E19del, and 1 interesting case with EGFR E19del at 45% MAF, EGFR amplification, and an emerging EGFR T790M clone at 0.54% MAF.
Conclusion:
In a series of NSCLC cases, high-sensitivity, high-specificity cfDNA analysis demonstrated the ability to identify somatic tumor alterations, including clinically actionable predictors, in a majority of patients via a simple blood draw, suggesting that this approach can be used for guiding therapeutic decision-making when repeat biopsy is high risk or not possible. Assuming validation, plasma cfDNA analysis may supplant invasive tumor biopsy in the near future.
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ORAL38.07 - Quantification of EGFR Mutations in Plasma of NSCLC Patients: An Early Predictor of Clinical Response to Tyrosine Kinase Inhibitors (ID 2242)
17:50 - 18:01 | Author(s): A. Marchetti, J. Palma, L. Felicioni, T. De Pas, R. Chiari, M. Del Grammastro, G. Filice, V. Ludovini, A.A. Brandes, A. Chella, F. Malorgio, F. Guglielmi, M. De Tursi, A. Santoro, L. Crinò, F. Buttitta
- Abstract
- Presentation
Background:
As DNA analytical methods have become more sensitive, attempts to develop accurate clinical tests to assess tumor mutation status by means of patient plasma samples are now being pursued. The potential to accurately quantify EGFR mutations in plasma from non-small cell lung cancer (NSCLC) patients would enable more rapid and more frequent analyses to assess disease status; however, the utility of such analyses for clinical purposes has only recently started to be explored.
Methods:
Plasma samples were obtained from 69 NSCLC patients with EGFR-mutated tumors and 21 negative control cases. EGFR mutations in plasma were analyzed by a standardized allele-specific polymerase chain reaction (PCR) test and ultra-deep next generation sequencing (NGS). A semi-quantitative index (SQI) was derived from dilutions of known EGFR mutation copy numbers. Clinical responses were evaluated by RECIST 1.1 criteria and expressed as percent tumor shrinkage.
Results:
The sensitivity and specificity of the PCR test and NGS assay in plasma versus tissue were 72% versus 100%, and 74% versus 100%, respectively. Quantitative indices by the PCR test and NGS were significantly correlated (P<0.001). EGFR testing at baseline and serially at 4–60 days during TKI therapy revealed a progressive decrease in SQI , starting from day 4, in 95% of cases. The rate of SQI decrease correlated with percent tumor shrinkage at 2 months (P<0.0001); at 14 days it was more than 50% in 70% of patients (rapid responders) (Fig.1A-B). In 2 patients with slow response (Fig.1B), an early increase in the circulating levels of the T790M mutation was observed. These patients were defined as early resistant (Fig.1C). No early T790M mutations were seen in plasma samples of rapid responders, suggesting that slow responders are more prone to develop early resistance.
Conclusion:
Quantification of EGFR mutations from plasma with a standardized PCR test is feasible. To our knowledge, this is the first study showing a strong correlation between the EGFR SQI during therapy and clinical response with relevant implications for patient management. With the strong correlation between EGFR SQI in plasma and clinical outcome, this study opens the way to prospectively design clinical trials to confirm these data and evaluate the diagnostic value of this test. Figure 1
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ORAL38.08 - Discussant for ORAL38.05, ORAL38.06, ORAL38.07 (ID 3481)
18:01 - 18:11 | Author(s): G.R. Oxnard
- Abstract
- Presentation
Abstract not provided
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.13 - Immune Related Gene Signature Reveal Potential Role for Leukocyte-Associated Immunoglobin-Like Receptor 2 (LAIR2) in Lung Cancer Regulation (ID 1243)
11:55 - 12:00 | Author(s): M.S. Tsao
- Abstract
- Presentation
Background:
Cancer development and biology is influenced by the host immune system. Emerging data indicate that the context of immune cell infiltrates within the tumor is associated with cancer prognosis. Both the activation state and type of immune cells present can provide mediators that either promote or inhibit tumor growth. While the presence of activated cytotoxic T lymphocytes (CTL) may correlate with better patient survival, the presence of tumor associated macrophages and effector lymphocytes that lack cytotoxic properties may promote tumor growth. Thus, in established tumors, a balance between pro and anti-tumor mediators are present, but as advanced tumors rarely regress without therapeutic intervention, this balance is likely skewed towards tumor-promoting inflammation. In attempts to gain insight into the immune networks that regulate tumorigenesis, we used genome wide gene expression datasets of primary lung cancer tissues to identify and functionally validate immune related genes that are associated with patient survival.
Methods:
Gene expression analysis was conducted on microarray datasets from 128 early-stage NSCLC resected tumor samples. Limiting analysis to immune-related gene sets curated by NIAID ImmPortal, we identified a minimum gene set using MAximizing R Square Algorithm (MARSA) that selected for the greatest separation between good and poor prognostic patient subgroups. The prognostic value of this gene signature was validated in nine additional independently published microarray datasets of NSCLC. From the gene signature, we functionally characterized the potential role of the soluble protein LAIR2 in immune regulation of lung cancer.
Results:
We identify a 9-gene signature that separate patients into high and low-risk subgroups for 5-year cancer-free survival (hazard ratio 10.26, 95% confidence interval 4.32-24.34, p<0.0001). The prognostic accuracy of this signature was validated in additional NSCLC datasets (total 1095 patients without adjuvant treatment). Amongst the 9-genes, the gene encoding the soluble protein LAIR2 was highly expressed within the high-risk patient subgroup. Functionally, we found that addition of recombinant LAIR2 resulted in increased NK cell expression of cytotoxicity receptors and secretion of pro-inflammatory cytokines in the presence of lung cancer cell lines.
Conclusion:
By limiting gene expression analysis to immune related genes, we identify a 9-gene prognostic immune signature in resected early stage NSCLC patients. The signature identifies a role for the soluble protein LAIR2 in the modulation of immune cell activation during lung cancer development and may suggest that LAIR2 induce a pro-inflammatory microenvironment which promote tumorigenesis and poor patient outcome.
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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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MINI14.12 - Genomic Profiling of Patient-Derived Xenografts Identify Passenger Aberrations Associated with Better Prognosis in Non-Small Cell Lung Cancer (ID 1735)
11:50 - 11:55 | Author(s): M.S. Tsao
- Abstract
Background:
Patient-derived tumor xenografts (PDXs) increasingly are being used as preclinical models to study human cancers, test novel therapeutics, and identify potential biomarkers, as they more accurately model human cancers than established tumor cell line cultures. However, uncertainty remains as to how well the genomic characteristics of patient non-small cell lung cancer (NSCLC) are recapitulated in these PDX models.
Methods:
PDXs were established by implantation of surgically resected NSCLC patient tumors into the subcutaneous or sub-renal capsule of non-obese diabetic severe combined immune deficient (NOD-SCID mice. Comprehensive genomic profiling including exome, gene copy number, DNA methylation and mRNA expression were conducted on 36 independent PDX models, their matched patient tumors and normal lung tissue. Publicly available cell line and TCGA data were used for comparison. Integrative analysis was performed to identify genomic alterations in PDXs that are associated with significant clinical outcomes in patients.
Results:
From 441 resected NSCLC tumors, 127 serially transplantable and stable PDX models were established. Among 264 NSCLC patients with at least 3-years follow-up, patients whose tumor formed stable PDXs (versus those who did not) showed significantly worse disease free (HR=3.12, 95% CI =2.02-4.83, P<0.0001) and overall survival (HR=4.08, 95% CI =2.16-7.73, P<0.0001), after multivariable adjustment for clinical pathological factors. Genomic and transcriptomic profiling of 36 PDXs showed greater similarity in somatic alterations between PDX and primary tumors than with published cell line data. In addition to known mutations, we found at least 16 non-synonymous somatic mutations in known oncogenes and tumor suppressors that have never been reported. All these mutations had higher observed variant allele frequency in PDXs compared to their matched patient tumors, suggesting that these were tumor sub-clones selected or enriched for growth in the PDXs. Tumor models characterized by a higher number of somatic alterations among 865 frequently altered genes were associated with better overall patient survival (HR=0.15, p=0.00015) compared to patients with corresponding PDXs characterized by higher alteration number; this was validated in the TCGA lung cancer dataset patients (HR=0.28, p=0.000022). These 865 genes were enriched for those encoding for proteins involved in cell adhesion and interactions with the extracellular matrix, and a quarter of the genomic alterations would putatively form neo-antigens implicating a potential role of immune response in the observed improved patient survival.
Conclusion:
PDXs are close preclinical models of patient tumors. Further investigations of passenger mutations may clarify their clinical impact on interactions between tumor cells, stroma, immune microenvironment and patient prognosis.
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.08 - Functional Role of Cancer Associated Fibroblasts in Non-Small Cell Lung Cancer Patients (ID 3134)
19:05 - 19:10 | Author(s): M.S. Tsao
- Abstract
Background:
Cancer-associated fibroblasts (CAFs) are well known to strongly influence tumor development, progression and metastasis. Their characteristics and prognostic role in non-small cell lung cancer (NSCLC) patients have been recognized. However, the functional heterogeneity of CAFs between patients and their genetic basis are less understood.
Methods:
Primary cultures of CAFs and noncancer fibroblasts were established from 28 independent resected non-small cell lung cancers and their corresponding non-neoplastic lung parenchyma. Collagen gel contraction, xCELLigence Real-Time Cell Analysis of proliferation and in vivo tumorigenicity were studied to assess the CAF activity. Percent area of desmoplasia among total tumor stroma was used to define high desmoplasia (HD) versus low desmoplasia (LD). Gene expression data on RNA extracted from contracted gels following 8 hours incubation was obtained using Illumina Human HT-12v4 Bead Chips array and was preprocessed and normalized using RMA and values were log2 transformed. Two-fold change cutoff was applied to identify differentially expressed genes in CAF-HD versus CAF-LD.
Results:
High desmoplasia correlates with higher ability to contract collagen gel, increased cell proliferation and tumor growth. Microarray gene expression analysis of the 24 CAF cell lines identified 23 genes that were differentially expressed between 12 CAF-HD versus 12 CAF-LD lines and were correlated significantly (p ≤ 0.05) with the gel contraction. 23 differentially gene expression were evaluated in gene expression microarray data (Affymetrix HG-U133 Plus 2 Array) from 181 NSCLC patients. We found 7 out of 23 differential gene expression to be significantly in concordant with the cohort of 181 NSCLC patients. Taking 7 prioritized genes, we have generated physical protein-protein interaction network by quering I2D ver. 3 and visualizing it in NAViGaTOR ver 2.3 (http://ophid.utoronto.ca/navigator). To study the degree of desmoplasia and outcome, we used the cohort of 181 NSCLC patients data set. We observed that desmoplasia appears to be associated with the time to relapse in univariable analysis. The association was far stronger in the adenocarcioma group with significance for both univariable and multivariable analysis.
Conclusion:
We provide evidence for a functional heterogeneity of CAFs in NSCLC patients based on the level of desmoplasia in tumor stroma. Furthermore, we develop desmoplasia-specific gene signature that could subgroup CAFs and contribute to their functional heterogeneity.
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MINI 36 - Imaging and Diagnostic Workup (ID 163)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Screening and Early Detection
- Presentations: 1
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MINI36.04 - Automated Measurement of Malignancy Risk of Lung Nodule Detected by Screening Computed Tomography (ID 1737)
18:45 - 18:50 | Author(s): M.S. Tsao
- Abstract
- Presentation
Background:
We have previously reported a practical predictive tool that accurately estimates the probability of malignancy for lung nodules detected at baseline screening LDCT (New Engl J Med. 2013;369:908-17). Manual measurement of nodule dimensions and generation of malignancy risk scores is time consuming and subjected to intra- and inter-observer variability. The goal of this study is to prepare a nodule malignancy risk prediction model based on automated computer generated nodule data and compare it to an established model based on radiologists’ generated data.
Methods:
Using the same published PanCan dataset (New Engl J Med. 2013;369:908-17) with the number of lung cancers updated, we prepared a logistic regression model predicting lung cancer using computer-generated imaging data from the CIRRUS Lung Screening software (Diagnostic Imaging Analysis Group, Nijmegen, The Netherlands). Ninety-one of the 2,537 baseline (first) scans were not available or could not be processed by CIRRUS. The remaining 2,446 scans were first annotated by the CIRRUS software. A human non-radiologist reader then accepted/rejected the annotated marks and manually searched the LDCT for nodules missed by CIRRUS or the study radiologist. New nodules found that were not recorded by the study radiologist were reviewed by a subspecialty trained chest radiologist with 14 years experience in lung cancer screening (JM). Nodule morphometric measurements (maximum and mean diameter, volume, mass, density) and total nodule count per scan irrespective of size were automatically generated by the CIRRUS software. The nodule type (nonsolid, part-solid, or solid), nodule description (lobulated, spiculated or well defined) and nodule location (upper versus middle or lower lobe) were manually entered. The variables were evaluated in models as untransformed and natural log transformed variables. Nonlinear relationships with lung cancer were also evaluated. Socio-demographic and clinical history predictors were not included in the model.
Results:
Radiologists evaluation identified 8,570 pulmonary nodules of any size in 2063 individuals - 124 nodules in 119 individuals were diagnosed as cancer in follow-up. Based on CIRRUS software annotated marks that were accepted by a human reader, computer analysis identified 11,520 pulmonary nodules in 2174 individuals - 121 nodules in 115 individuals were diagnosed as cancer in follow-up. Thirty-six percent of new nodules found by CIRRUS and/or second human reader were ≥4 mm (mean±SD, 5.9± 3.5 mm). Both the computer generated imaging data model (Model-CIRRUS) and the radiologist generated data model (Model-RAD) demonstrated excellent discrimination and calibration. Their predictive performances were also similar. Comparing Model-CAD to Model-RAD, the AUCs were 0.9537 versus 0.9541, the 90[th] percentile absolute errors were 0.0008 versus 0.0007, and the Brier scores were 0.0093 versus 0.0137. Mean nodule diameter is a better risk predictor than maximum nodule diameter, nodule density or mass.
Conclusion:
The predictive performances of computer and radiologist generated data models were similar. The model can be integrated to the CIRRUS Lung Screening software to automatically generate a nodule malignancy risk score to facilitate nodule management recommendation. Supported by the Terry Fox Research Institute, The Canadian Partnership Against Cancer and the BC Cancer Foundation on behalf of the Pan-Canadian Early Detection of Lung Cancer Study Group.
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MS 25 - Lung Carcinogenesis (ID 43)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:W.A. Franklin, H. Kato
- Coordinates: 9/09/2015, 14:15 - 15:45, Mile High Ballroom 2a-3b
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MS25.03 - Peripheral Premalignancy (ID 1960)
14:50 - 15:05 | Author(s): M.S. Tsao
- Abstract
- Presentation
Abstract:
The recently published Fourth Edition of the WHO Classification of Tumours of the Lung[1] recognizes atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) as pre-invasive or premalignant precursor lesions of invasive adenocarcinoma, which arises mostly in the periphery of the lung. In the previous (Third) Edition of WHO classification, AIS was classified as bronchiolo-alveolar carcinoma (BAC), one of the subtypes of malignant adenocarcinoma.[2,3] Reclassification of AIS into the preinvasive category represents a conceptual confirmation of its role in multi-stage pathogenesis of peripheral lung adenocarcinoma.[4-7] This is consistent with the histological hallmark of lack of invasion in AIS, and its association with 100% survival after surgical resection. The neoplastic nature of these lesions are supported at the molecular level with the identification of known genomic aberrations found in invasive lung adenocarcinoma.[8,9] AIS is characterized histologically by the lepidic proliferation of neoplastic epithelium along pre-existing alveolar structures and lacking stromal, vascular or pleural invasion (Figure 1). A majority of AIS is composed of non-mucinous neoplastic cells with Clara cell and/or type-2 pneumocyte phenotype. Mucinous AIS is rare. By definition, AIS is limited to tumors that is ≤ 3 cm in greatest diameter and by TNM classification, is classified a Tis. AIS commonly shows varying degree of stromal thickening by fibrosis and chronic inflammatory cell infiltrate, with some cases showing focal or central area of fibrosis or scar. Around these areas, entrapment of the tumor cells within architecturally distorted and thickened alveolar septa give rise to morphological appearances of invasion. This remains one of the areas of diagnostic difficulty in distinguishing AIS from minimally invasive adenocarcinoma (MIA). However, limited data suggests that MIA is also associated with 100% curability by surgical resection. A majority of AAH are identified incidentally during microscopic examination of non-cancerous lung of surgically resected adenocarcinoma (Figure 2). The reported incidence in lung adenocarcinoma cases may reach up to 30%, and the reported number of lesion can reach up to 40/case, depending on the extent of sampling. They are typically ≤ 5 mm, but size is not a diagnostic criteria for its diagnosis. Histologically it is characterized by slightly thickened alveolar septa that are lined by atypical appearing cuboidal to low columnar epithelial cells with gaps in between them. These cells have similar ultrastructural features as AIS cells, mainly those of type-2 pneumocyte and/or Clara cell. A spectrum of nuclear atypia may be observed but grading has not been recommended, as they have not been demonstrated as reproducible or correlated with neoplastic progression. AAH is considered a precursor of AIS, as they may harbor KRAS or EGFR mutations. In some cases, the histological distinction between AAH and AIS can be very challenging, even though both lesions are considered cured by surgical resection. Further deep genomic analyses of AAH and AIS can provide greater insights into the multistep molecular carcinogenesis of lung adenocarcinoma and potentially novel prevention strategies for this disease. References: 1. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. IARC Press, Lyon, 2015, page 46-50. 2. World Health Organization International Histological Classification of Tumours. Histological Typing of Lung and Pleural Tumours. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Springer Verlag, Berlin, Heidelberg, New York, 1999, page 21-29. 3. WHO Classification of Tumours, Pathology and Genetics. Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Muller-Hermelink HK,, Harris CC. IARC Press: Lyon 2004, page 35-44, 73-75. 4. Miller RR, Nelems B, Evans KG, Muller NL, Ostrow DN. Glandular neoplasia of the lung. Cancer 1988;61:1009-1015. 5. Kitamura H, Kameda Y, Ito T, Hayashi H. Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma. Am J Clin Pathol 1999;111:610-22. 6. Mori M, Rao SK, Popper HH, Cagle PT, Fraire AE. Atypical adenomatous hyperplasia of the lung: A probably forerunner in the development of adenocarcinoma of the lung. Mod Pathol 2001;14:72-84. 7. Chapman AD, Kerr KM. The association between atypical adenomatous hyperplasia and primary lung cancer. Br J Cancer 2000;83:632-36. 8. Westra WH, Baas IO, Hruban RH, Askin FB, Wilson K, Offerhaus GJ, Slebos RJ. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996;56:2224. 9. Sakamoto H, Shimizu J, Horio Y, Ueda R, Takahashi T, Mitsudomi T, Yatabe Y. Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas. J Pathol 2007;212:287-94. Figure 1. Adenocarcinoma in situ Figure 1 Figure 2. Atypical Adenomatous Hyperplasia. Figure 2
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.03 - Genome-Wide Gene Copy Number Analysis by OncoScan<sup>TM</sup> FFPE Assay in 976 Resected NSCLC From LACE-Bio2 (ID 1561)
11:27 - 11:38 | Author(s): M.S. Tsao
- Abstract
- Presentation
Background:
Genome wide SNP array studies have identified systematic gene copy number aberrations (CNA) in non-small cell lung cancer (NSCLC), but their prognostic implication is unknown. This study aimed to investigate associations between CNAs and survival using the LACE-Bio bio-bank. The LACE-Bio consortium includes large clinical trials comparing adjuvant platinum-based chemotherapy to observation after complete resection of stage I-III NSCLC.
Methods:
DNA was extracted from FFPE tumor samples from 3 pivotal adjuvant chemotherapy trials (CALGB 9633, IALT, JBR.10); 1013 samples were profiled using Affymetrix OncoScan[TM] arrays with over 300,000 probes and normalized relative to a pool of normal tissues. Segmentation was performed using the CBS algorithm and minimally recurrent regions (MCR) across the series identified by CGHregions. All analyses were performed on the level of MCRs. CNAs were correlated with clinicopathological factors and adjusted for the False Discovery Rate (FDR). The primary endpoint, disease-free survival (DFS), was assessed via univariate Cox models stratified by trial and adjusted for treatment, age, sex, PS, histology, T, and N stage.
Results:
Among 976 successfully profiled samples, 414 (42%) were adenocarcinoma (ADC), 430 (44%) squamous cell carcinoma (SCC) and 132 (14%) other NSCLC; 710 (73%) were male. Across the 431 MCRs identified, patients had on average 94 (SD 69) CNAs: 51 gains and 43 losses. A gain or loss was observed in at least 10% of patients for 177 and 166 regions respectively. The most common gains (up to 48%) were on chromosomes 1p, 3q, 5p, 6p, and 22q. The most common losses (up to 40%) were on chromosomes 3p, 8p and 9p. The size of 253 of the 431 MCRs (59%) was smaller or equal to 3Mb (and 79% ≤10 Mb). Sensitivity analyses on the subset of samples with optimal quality (n=777, defined by MAPD<0.3) gave consistent results. The CNA frequency of 195 regions was significantly different with FDR≤0.05 between ADC and SCC (of which 49% regions of size ≤3Mb and 71% ≤10Mb); the most significant were more gains in 3q, 22q and 12 in SCC and more losses in 3p, 4, 5q in SCC. With a median follow-up of 5.3 years, 510 DFS events and 451 deaths were recorded. In univariate analyses for DFS, 13 regions in loci 19p11–13, 7p12, 9p21, 15q14 had a raw p-value <0.005 (FDR<0.13, the top 8 corresponded to FDR≤0.05); 9 of those 13 regions were of size ≤3Mb (12 regions ≤10Mb). In adjusted analyses, 10 of the 13 regions retained raw adjusted p-values ≤0.005 (FDR≤0.15). Losses of focal regions including CDKN2A/B and STK11 (≤3Mb) were associated with poorer DFS: the hazard ratio (HR) for a 2-fold copy number decrease in region 9p21.3 (including CDKN2A/B) was 1.50 (95% CI: 1.2–1.9, P<0.001, FDR=0.02), and the HR for a 2-fold copy number decrease in 19p13 (including STK11) was 2.4 (1.3–4.3, P=0.005, FDR=0.15). Similar results were obtained for overall survival and lung-cancer specific survival. Results of histology-specific analyses will be presented.
Conclusion:
These large-scale genome-wide analyses of gene CNA provide new candidate prognostic markers for stage I-III NSCLC.
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ORAL 21 - Biology - Moving Beyond the Oncogene to Oncogene-Modifying Genes (ID 118)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:A. Katz, M.S. Tsao
- Coordinates: 9/08/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL21.05 - p53/KRAS Mutation Status Does Not Predict Sensitivity to Chemotherapy in NSCLC PDXs (ID 2459)
11:28 - 11:39 | Author(s): M.S. Tsao
- Abstract
- Presentation
Background:
The LACE-Bio group assessed the prognostic and predictive values of KRAS and p53 mutations in 1543 completely resected non-small cell lung cancer (NSCLC) tumors. The predictive value of combined KRAS/p53 mutations for survival benefit from adjuvant chemotherapy was evaluated on 49 patients and chemotherapy was deleterious in this group compared to observation (HR 2.49 CI 95% [1.10 – 5.66], p=0.03). Patients with tumors harboring combined KRAS/p53 mutations had a worse outcome when treated with adjuvant chemotherapy compared patient with double wild type (WT) tumors (HR 3.03 (95% CI [1.29 – 7.15], p=0.01, interaction p=0.06). We have compared the chemo-sensitivity of patient derived xenografts (PDXs) with double p53/KRAS mutations, single p53, single KRAS mutation or double WT. 0
Methods:
Surgically resected early stage lung adenocarcinomas (ADC) were implanted into non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Fourteen lung ADC PDXs with various p53/KRAS status were revived and implanted: 11 engrafted and were expanded for comparison of treatment vs control. For each model, 6 replicates were included in treatment and control arms. Chemotherapy (cisplatin 3 mg/kg and vinorelbine 7 mg/kg intraperitoneally weekly) was initiated in the PDXs at tumor volumes of 150 mm[3].
Results:
Four models were p53/KRAS double mutant, 4 p53 mutant, 2 KRAS mutant and 1 double WT. The 4 double mutant PDXs responded to chemotherapy, 2 with reduced (SD) and 2 inhibited (PR) growth. Among the 4 PDXs with p53 mutation only, 2 responded (1 PR and 1 SD) and 2 were resistant. Among the 2 PDXs with KRAS mutation only, 1 had a complete response, but relapsed at treatment arrest and 1 achieved PR. The double WT PDX was highly sensitive to chemotherapy (PR) but also relapsed at treatment arrest.
Conclusion:
Among these 11 PDXs, the p53/KRAS mutation status did not predict chemo-sensitivity to cisplatin/vinorelbine, one of the most active adjuvant chemotherapy regimens in NSCLC. As these PDXs were molecularly profiled, we currently are investigating other biomarkers that might predict their sensitivity or resistance to chemotherapy.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-095 - Integrin a11b1 Regulates Cancer Stromal Stiffness and Promotes Tumorigenicity and Metastasis in Non-Small Cell Lung Cancer (ID 3084)
09:30 - 09:30 | Author(s): M.S. Tsao
- Abstract
Background:
Integrin α11β1 is a stromal cell-specific receptor for fibrillar collagens and is over-expressed in carcinoma-associated fibroblasts (CAFs) in non- small cell lung cancer (NSCLC). We have studied the direct role of stromal integrin a11 on the growth and metastasis of NSCLC cells using novel immune-compromised a11 deficient mice.
Methods:
We developed α11 non-expressing immune-deficient mice by back-crossing for at least 10 times the α11-deficient heterozygous C57BL/6J mice (+/-) to obtain a homogenous C57BL/6 background. These were subsequently bred with the BALB/c SCID mice for 7 generations, producing α11-deficient heterozygous (+/-) in SCID background. In vivo studies were done using subcutaneous tumorigenicity assay and orthotopic model to evaluate metastatic potential of integrin α11. Immunostaining were carried out using integrin α11, α-SMA, and cytokeratin. PisroSirius red staining was used to visualize the collagen fibers. Images were taken by polarized-light microscopy using parallel and perpendicular polarizer orientations on an Olympus BX51 microscope. Second Harmonic Generation (SHG) was used to visualize fibrillar collagen and atomic force microscopy was applied to measure the stiffness in tumor stroma.
Results:
The tumor growth of both primary human lung cancer (PHLC) and established NSCLC cells in α11 knockout (α11[-/-]) mice was significantly impeded compared to wild type (α11[+/+]). Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11[-/-] and α11[+/+] mice showed significant reduction in the metastatic potential of these cells in the α11[-/-] mice. Using mouse WG-6v2 Illumina Bead Chips, we identified that alpha11 expression correlates with that of a fibrillar collagen cross-linking enzyme, LOXL1, in the xenograft stroma. Fibrillar collagen was highly disorganized and had a significantly lower elastic modulus in the alpha11 knockout xenografts compared to wildtype. The results suggest a role for α11 in promoting tumor growth and metastatic progression by affecting the collagen stiffness of the tumor stroma.
Conclusion:
The integrin a11β1 signaling pathway in CAFs promotes tumor growth and metastasis of NSCLC cells. This appears closely linked to collagen cross-linking, the organization, and stiffness of fibrillar collagen matrices.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-026 - Second Generation EGFR TKIs Inhibit Tumor Growth in a Chemo-Resistant Squamous Cell Lung Cancer Patient Derived Xenograft Model (ID 1265)
09:30 - 09:30 | Author(s): M.S. Tsao
- Abstract
Background:
In clinical trials testing the efficacy of first generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), occasional responses were observed in patients with lung squamous cell carcinomas (LSCC) (Shepherd FA, et al. New Engl J Med 2005) and survival benefit was confirmed for erlotinib in a subset analysis of male, ever-smokers with LSCC (Clark GM, et al. Clin Lung Cancer 2006). Currently, the LUX-Lung 8 phase III clinical trial is comparing afatinib versus erlotinib in the second-line setting for LSCC after cisplatin-based chemotherapy (Goss GD, et al. ESMO 2014). Preclinical data indicate that high EGFR protein expression may be predictive of response to erlotinib in EGFR wild type LSCC (Cranston et al, AACR 2013). Herein we assessed and compared the anti-tumor efficacy of different EGFR inhibitors in chemo-resistant squamous cell lung cancer patient derived xenograft (PDX) models with high EGFR expression and EGFR amplification.
Methods:
The cryopreserved PDX model established from a resected early stage LSCC was revived in non-obese diabetic severe combined immunodeficient mice (NOD SCID), expanded and subsequently treated with chemotherapy (cisplatin 3 mg/kg and vinorelbine 7 mg/kg intraperitoneally [IP]), cetuximab 20 mg/kg IP, and daily oral schedules were followed for erlotinib 50 mg/kg, afatinib 20 mg/kg, dacomitinib 3 mg/kg. For each model, 6 mice were used in each of the different treatment and the control arms. Treatment was initiated in the PDXs at a tumor average volume of 150 mm[3].
Results:
The PDX was derived from a 57 year old male, smoker, following right pneumonectomy for a stage IIIB (T4N1M0) LSCC. This patient received adjuvant cisplatin/vinorelbine, but relapsed three weeks after the end of cycle 4 and died a week later. The tumor had a high EGFR expression by immunohistochemistry (H score = 300) and EGFR amplification (clusters) by fluorescent in situ hybridization. The PDX was EGFR wild type by Illumina exome sequencing and OncoCarta[TM ]MassArray mutation screen (Sequenom), also was refractory to cisplatin/vinorelbine. Reduced growth rate (stable disease, SD) was obtained with erlotinib and cetuximab. Treatment with afatinib and dacomitinib resulted in tumor growth inhibition (partial response, PR). The PDX developed resistance to dacomitinib after 100 days of treatment, but continued to be inhibited by afatinib after 215 days of treatment.
Conclusion:
This study shows the efficacy of second generation especially afatinib irreversible EGFR TKIs in a chemoresistant LSCC PDX, with high wild type EGFR expression and EGFR amplification. Our results lend further support to the LUX-Lung 8 trial, and also the use of PDX to model therapeutic responses in lung cancer.
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P2.04-053 - Patient-Derived Xenograft Studies Suggest FGFR1 Amplification Is Insufficient to Predict Response to FGFR Inhibitors in Lung SqCC (ID 3067)
09:30 - 09:30 | Author(s): M.S. Tsao
- Abstract
Background:
FGFR1 amplification has been reported in 16%-20% of lung squamous cell carcinoma (SqCC). Early phase clinical trials with anti-FGFR small molecule inhibitors are in progress. It remains unclear whether genomic changes involving FGFR1 is associated with a dependency in FGFR-driven oncogenic activity that could be inhibited with pharmacologic agents. We evaluated a pan-FGFR inhibitor (BGJ398) in four SqCC patient-derived xenograft (PDX) models with amplification of the FGFR1 gene.
Methods:
FGFR1 gene copy changes were assessed by fluorescence in-situ hybridization. PDX models were established by implanting surgical resected tumor fragments into the subcutaneous tissue of non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Protein and mRNA expression levels were assessed by immunohistochemistry/western blot and RT-qPCR, respectively.
Results:
FGFR1 amplification was observed in 13 of 60 (22%) SqCC patient tumors, with all amplified tumors forming PDX. PDX models with FGFR1 gene amplification displayed higher levels of mRNA and protein compared to non-amplified tumor, excluding polysomy cases. One model demonstrated an average of 50% decrease in tumor volume in the BGJ398 treated group compared to control group, 21 days post-treatment. This model also expressed high FGFR1 and high cMYC protein. BGJ398-resistant PDX models included one model with high FGFR1 but low cMYC protein levels, and two models with low FGFR1 and high cMYC protein levels.
Conclusion:
The lack of growth arrest to a pan-FGFR small molecule inhibitor in the 4 PDX models evaluated suggests that FGFR1 amplification alone was not a sufficient predictive marker for pan-FGFR1 inhibitor activity. FGFR1 protein and MYC protein are putative markers.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-066 - Overexpression of KIF23 Predicts Clinical Outcome in Primary Lung Cancer Patients (ID 850)
09:30 - 09:30 | Author(s): M.S. Tsao
- Abstract
Background:
Lung cancer is the leading cause of cancer-related mortality worldwide. To improve the survival rate, it is important to examine or analyze metastatic lymph node samples taken from advanced lung cancer patients, especially using minimally invasive techniques like endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA). We have been attempting to isolate potential molecular targets for lung cancer by analyzing expression profiles of our microarray and various types of database. Throughout these screenings, we identified kinesin family member 23 (KIF23) as a promising molecular target gene for the treatment of lung cancer. High-level expression of KIF23, a member of microtubule-dependent molecular motors that transport organelles within cells and move chromosomes during cell division, has been observed in a variety of human malignancies. The aims of the present study were to observe the expression of KIF23 in human lung cancer, examine the role of KIF23 in lung cancer cell growth and/or survival by small interfering RNA experiments, and explore its clinicopathologic significance and evaluate KIF23 expression as a prognostic marker.
Methods:
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to detect the expression of KIF23 mRNA using metastatic lymph nodes from patients with advanced lung cancer obtained by EBUS-TBNA and normal human organs. A role of KIF23 in cancer cell growth and/or survival was examined by small interfering RNA experiments. A total of 341 lung cancers were analyzed immunohistochemically on tissue microarrays to examine the expression of KIF23 protein in archival lung cancer samples and its clinicopathologic significance.
Results:
KIF23 transcript was extremely higher in the great majority of metastatic lymph nodes from advanced lung cancers with higher frequency compared with the average expression of normal lung tissues as determined by quantitative RT-PCR. KIF23 was more highly expressed only in the testis and the thymus compared to other human organs. Inhibiting KIF23 expression effectively suppressed non-small cell lung cancer (NSCLC) cell growth, and KIF23 siRNA-treated lung cancer cells more frequently exhibited large cell bodies with two or more nuclei. High-level KIF23 expression was observed in 67.7% of the 341 cases, and this only correlated with pathological T classification (P=0.0269). Lung adenocarcinoma patients with tumors displaying a high-level of KIF23 expression was also identified as an independent prognostic factor by multivariate analysis (P=0.0042). Figure 1
Conclusion:
KIF23 not only provides additional prognostic information for surgical treatment of lung cancer, but may also be a novel therapeutic target for these patients.
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P3.04-099 - Proteome Signatures with Prognostic Impact Distinguish Non-Small Cell Lung Cancer Histology Subtypes and Metabolic States (ID 1009)
09:30 - 09:30 | Author(s): M.S. Tsao
- Abstract
Background:
We showed that the ability to establish a primary tumorderived xenograft (PDX) is an independent predictor of shorter disease-free survival in early stage non-small cell lung carcinoma (NSCLC). Hence, NSCLC engraftment may select for critical, aggressive aspects of the cancer phenotype linked to disease progression. More recently we reported dramatic remodeling of NSCLC proteomes not predicted by genomics analyses, and which distinguish between the major histological subtypes of NSCLC. Herein we report details on NSCLC proteome remodeling as a major determinant of the expression of the metabolism proteome, engraftment, and related to patient outcome.
Methods:
Omics platforms were used to comprehensively characterize the genomes and proteomes of non-engrafting, engrafting, and derived PDX tumors associated with NSCLC. To facilitate proteome quantification by mass spectrometry, tumor samples were spiked with stable-isotope-labeled proteomes from a mixture of representative NSCLC cell lines as an internal standard.
Results:
Proteome remodeling in NSCLC is extensive and largely unpredicted by gene copy number variation, and not highly correlated with mRNA-based expression. Analysis of the proteomes of cognate engrafting primary and PDX tumor pairs revealed signatures comprising sets of metabolism proteins that distinguished between the major histological subtypes, and which were particularly highly recapitulated in PDX tumors. Interrogation of The Cancer Genome Atlas showed that the genes encoding the highly recapitulated metabolism protein signatures are for the most part not highly mutated in cancers. However, when the signature-encoding genes are considered as a singular polygene, then patients with mutations are recognized as having significantly different overall survival compared to patients without mutations. The proteomes of non-engrafting NSCLC tumors were generally more similar to normal lung than were engrafting tumor proteomes. Hence, proteome remodeling affects metabolic states associated with NSCLC outcome.
Conclusion:
NSCLC is characterized by significant proteome remodeling that is invisible to genomics platforms. The proteomes of engrafting and non-engrafting NSCLC primary tumors are different, suggesting the potential to develop proteome signatures as prognostic biomarkers. Moreover, proteome signatures associated with PDX engraftment and poor outcome may be a source of new drivers and targets in NSCLC.
