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L. Byers
Moderator of
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MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 13
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MINI22.01 - Detecting ALK, ROS1 and RET Fusion Genes in Advanced Non-Small Cell Lung Cancer (NSCLC) Using a Novel Multiplexed NCounter-Based Assay (ID 2254)
16:45 - 18:15 | Author(s): N. Reguart, A. Gimenez-Capitan, M.A. Molina, P. Galvan, L. Pare, S. Viteri, C. Teixidó, S. Rodriguez, J. Castellví, E. Aldeguer, N. Viñolas, R. Rosell, A. Prat
- Abstract
- Presentation
Background:
Gene fusions of anaplastic lymphoma kinase (ALK), ROS1, and RET are targetable oncogenes present in approximately 9% of advanced NSCLC. Current assays for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHQ) and qRT-PCR. These tests, however, are complex and have disadvantages in terms of turnaround, sensitivity, cost and throughput. The nCounter platform allows joint detection, in a single tube, without any enzymatic reaction and in 72 hours, of multiple fusion genes by transcript-based method from formalin-fixed paraffin-embedded (FFPE) samples.
Methods:
A custom set consisting of 5´and 3´ probes and/or fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was evaluated. A panel of ALK-ROS-RET positive cell lines (H2228, H3122 [EML4-ALK], SU-DHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS], BaF3 pBABE [CD74-ROS], LC2/ad [RET]) and control fusion negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) were used for nCounter validation. To determine the minimum of tumor surface area for detection, ALK translocated cell line H2228 was tested in FFPE at increasing cell numbers (2500, 5000, 10.000, 25000, 50000) corresponding to a surface area of 0.27, 0.55, 1.1, 2.75 and 5.5 mm2, respectively, in the FFPE block. A total of 38 FFPE samples positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS (n=7) and RET (n=1) were also analyzed. Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and 4 house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative ALK fusion translocation was defined by a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively. Response to crizotinib by RECIST criteria was retrospectively collected in patients with ALK-positive NSCLC.
Results:
nCounter sensitivity to predict fusion transcripts ALK, ROS and RET in cell lines by using both methods (3’/5’ and direct reporter probes) was 100%. Results indicate that samples containing as few as 10% positive tumor cells and a 2.75 mm2 tumor surface area were sufficient for adequate gene fusion detection. The accuracy of prediction (AUC) of ALK 3’-5’ ratio score in 45 independent samples was 82.6% (95% CI 69.3-95.6) with a kappa coefficient score of 0.637. Among 28 samples ALK-FISH-positive, ALK 3’-5’ scoring was positive in 27 samples (96%). One sample was non-evaluable by ALK 3’-5’ scoring. Among the 17 samples ALK-FISH-negative, ALK 3’-5’ score was negative and positive in 10 (59%) and 7 (41%) samples, respectively. All patients with ALK-FISH-negative samples but ALK 3’-5’ score positive (n=7) were positive for ALK IHC and 5 of them were treated with crizotinib. Response assessment was available in 3 of these patients and response rate was 100%. One patient non-evaluable by FISH but positive 3’-5’ scoring also responded to crizotinib.
Conclusion:
The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening modality that might identify FISH-negative/non-evaluable NSCLC patients who could benefit from ALK inhibitors.
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MINI22.02 - Clinically Adoption of MSK-IMPACT, a Hybridization Capture-Based next Generation Sequencing Assay, for the Assessment of Lung Adenocarcinomas (ID 2881)
16:45 - 18:15 | Author(s): M.E. Arcila, A. Zehir, H. Yu, A. Drilon, B.T. Li, G.J. Riely, N. Rekhtman, O. Lin, D. Hyman, M. Berger, C.M. Rudin, M.G. Kris, M. Ladanyi
- Abstract
- Presentation
Background:
Mutation analysis plays a central role in the management of lung adenocarcinomas (LUAD). The use of multiple single gene or mutation specific assays, broadly adopted in many laboratories to detect clinically relevant genomic alterations, often leads to delays if sequentially performed, tissue exhaustion, incomplete assessment and additional biopsy procedures. Comprehensive assays using massively parallel “next-generation” sequencing (NGS) offer a distinct advantage when addressing the increased testing needs of genotype-based therapeutic approaches. Here we describe our experience with a 410 gene, clinically validated, hybrid-capture-based NGS assay applied to testing of LUAD.
Methods:
Consecutive LUAD cases submitted for routine mutation analysis within a 1 year period were reviewed. Unstained slides of formalin fixed, paraffin embedded tissue were received for each case (range 15-20 slides/case). Corresponding H&E stained slides were reviewed and cell counts were performed in a subset of cases with limited material to establish minimal tissue requirements. Testing was performed by a laboratory-developed custom hybridization-capture based assay (MSK-IMPACT) targeting all exons and selected introns of 410 key cancer genes (J Mol Diagn 17:251-264, 2015). Barcoded libraries from tumor / normal pairs were captured and sequenced on an Illumina HiSeq 2500 and analyzed with a custom analysis pipeline.
Results:
A total of 469 specimens were received for comprehensive testing (98 cytology samples, 239 needle biopsies, 132 large biopsies/resections) of which 93% (436/469) were successfully tested. Thirty four cases (7%, 34/469) failed due to very low tumor content or low DNA yield. Cell counts for failed samples averaged 239 cells / slide (range 10-270) while all successfully tested had over 1,000 cells / slide each. Failure rate was similar for cytologies and biopsies. An average of 10 genomic alterations were detected per patient (range 1-96). The most frequently mutated genes were TP53, EGFR, KRAS, KEAP1 and STK11. Copy number gains of NKX2-1 and EGFR genes and CDKN2A loss were most common. EGFR mutations and ALK fusions were detected in 28% and 4% of cases, respectively. Among the 299 EGFR / ALK WT cases, MSK-IMPACT uncovered targetable genomic alterations that would have remained undetected through focused EGFR/ALK testing alone. These included fusions in RET (10) and ROS1 (13), mutations in ERBB2 (11) and BRAF (19) and amplifications in MET (12, unrelated to EGFR), MDM2 (26) and CDK4 (20) among others. The higher than expected rates of RET and ROS1 fusions are related to enrichment of previously tested cases known to be negative for other driver alterations.
Conclusion:
Comprehensive hybrid-capture based NGS assays such as MSK-IMPACT are an efficient testing strategy for LUAD across sample types. This upfront broad approach enables more optimal patient stratification for treatment by targeted therapeutics.
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MINI22.03 - Next Generation Immunohistochemical Stains; True Multiplex (Quadruple) Immunohistochemical Panel for Non-Small Cell Lung Carcinoma (ID 2119)
16:45 - 18:15 | Author(s): C.C. Solomides, R. O'Neill, L. Behman, T. Shingler, J. Ashworth-Sharpe, B. Kelly, E. Roberts, L. Morrison
- Abstract
- Presentation
Background:
Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung cancers, the vast majority of which are non-small cell lung carcinomas (NSCC). It is critical to further distinguish adenocarcinomas from squamous carcinomas in order to optimize the efficiency of the precision medicine analysis for the detection of active molecular targets for therapies. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin-A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin-A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung.
Methods:
Developmental reagents from Ventana Medical Systems, Inc. were leveraged for this study. Detection of CK 5/6 and p40 [BC28] was used to identify squamous cell carcinoma cells. Detection of Napsin A and TTF-1 was used to identify adenocarcinoma cells. Detection was accomplished using secondary antibody:enzyme conjugates and orthogonal chromogenic detection chemistries to simultaneously detect all 4 biomarkers. Fully automated multiplexed detection was performed on a Benchmark XT with 4 microns thick sections from formalin fixed paraffin embedded, non-small cell lung cancer specimens obtained from both the Ventana Medical Systems, Inc. tissue bank and from the Thomas Jefferson University’s Department of Pathology, Anatomy and Cell Biology laboratories. Detection of each marker in multiplex was compared to individual detections using diaminobenzidine deposition according to established Ventana Medical Systems, Inc. protocols. All detections were reviewed by a board certified pathologist.
Results:
Adenocarcinomas (7 of 7) and the adenocarcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for TTF-1 (yellow nuclear stain) and Napsin-A (pink cytoplasmic granular stain). Squamous cell carcinomas (5 of 5) and the squamous cell carcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for p40 (blue nuclear stain) and CK5/6 (brown cytoplasmic stain). The colors were clear, distinct, easily differentiated and recognizable. There was no discrepancy between the expression of the individual antibodies and the expression of the same antibodies in the multiplex setting.
Conclusion:
Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or IHC, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. This new quadriplex IHC offers the capability with a single 4 micron section to accurately diagnose primary lung adenocarcinoma, squamous cell carcinoma or adenosquamous carcinoma and while conserving tissue for additional molecular testing.
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MINI22.04 - Discussant for MINI22.01, MINI22.02, MINI22.03 (ID 3550)
16:45 - 18:15 | Author(s): J. Botling
- Abstract
- Presentation
Abstract not provided
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MINI22.05 - Quality Control Process for NGS to Minimize False Positives (ID 2989)
16:45 - 18:15 | Author(s): C.D. Morrison, J. Conroy, A. Papanicolau-Sengos, M.K. Nesline, J. Mastroianni
- Abstract
- Presentation
Background:
Next generation sequencing (NGS) has exceptional sensitivity, but at the expense of false positives. This can result in a less than optimal positive predictive value and eventually the futile treatment of patients. We have developed a unique set of quality control filters for both Ion Torrent and Illumina that minimize false positives, but have little negative impact on sensitivity. To address this paradoxical association of sensitivity and false positives, we developed a dual platform methodology of NGS using both the Ion Torrent and Illumina to solve this classical dilemma.
Methods:
A series of filters were developed to determine quality cutoffs for variant calls to minimize false positives that included the minimum quality score threshold (QUALT), minimum percent variant reads (MPVR), minimum variant reads (MVR), minimum variant reads threshold (MVRT), minimum variant allelic frequency threshold (MVAF), minimum variant reads positive predictive value (MVR-PPV), and systematic errors (SE). A parallel system of using the MiSeq and PGM to sequence all specimens within an IT systems control and a Classify Callsmatrix solution for mutational analysis was designed. Unique cohorts of patients with prior exome sequencing as part of TCGA were used as gold standard controls with matching fresh frozen and FFPE samples.
Results:
Table 1 provides the results of filters developed to maximize sensitivity versus PPV. Using our targeted sequencing panel the PGM consistently outperformed the MiSeq for the standard performance characteristics of sensitivity and PPV for both frozen and FFPE samples. Both platforms have systematic false positives that are unique and gene specific.
Table 2 provides the results for dual platform sequencing which show a marked reduction in false positives while maintaining sensitivity.Table 1 Platform Tissue VAF setting QUAL Cutoff MVRT Cutoff MVAF Cutoff Mean Sensitivity Range Sensitivity Mean PPV Range PPV PGM FF 0.2% None None None 100% 93-100% 88% 70-96% PGM FF 0.2% >99 >=20 >.035 99% 93-100% 95% 78-100% PGM FFPE 0.2% None None None 99% 93-100% 58% 2-94% PGM FFPE 0.2% >99 >=21 >.018 97% 63-100% 92% 40-100% MiSeq FF 1% None None None 97% 79-100% 49% 31-66% MiSeq FF 1% >99 >=5 >.017 95% 66-100% 82% 66-95% MiSeq FFPE 1% None None None 94% 43-100% 10% 2-37% MiSeq FFPE 1% >99 >=10 >.028 92% 39-100% 62% 6-100% Table 2 FF FF FF FF FFPE FFPE FFPE FFPE SNV(s) SNV(s) Indels Indels SNV(s) SNV(s) Indels Indels Percent VAF Percent VAF Percent VAF Percent VAF Assay Sensitivity 99.8% 2.87% 100.0% 2.90% 98.3% 3.56% 100.0% 3.60% Assay PPV 97.5% 2.87% 91.0% 2.90% 96.7% 3.56% 91.0% 3.60%
Conclusion:
Single platform NGS is plagued by false positives. Dual platform sequencing is a reliable method of diminishing false positives with minimal to no impact on sensitivity.
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MINI22.06 - The Challenge of Molecular Testing for Clinical Trials in Advanced Non-Small Cell Lung Cancer Patients: Analysis of a Prospective Database (ID 1240)
16:45 - 18:15 | Author(s): S. Lepers, A. Ottevaere, C. Oyen, L. Peeters, E.K. Verbeken, C. Dooms, K. Nackaerts, J. Vansteenkiste
- Abstract
- Presentation
Background:
Molecular testing has become important in managing advanced non-small cell lung cancer (NSCLC), both in clinical practice, as well as in clinical trials. For the latter, tissue samples often have to be analysed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process.
Methods:
We reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 (start) and October 31, 2014. The following time points were considered: T1 (request for tissue sections from the pathology lab); T2 (receipt of sections and shipment); T3 (arrival of sections in central lab (CL)); T4 (receipt of biomarker result from CL).
Results:
251 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis, as the request for central molecular testing was cancelled: insufficient tissue (n=11); exclusion criterion (n=10); patient refusal (n=2). Results for the remaining 228 patients were: failure of central biomarker analysis due to insufficient quantity of tissue (n=18), or quality of tissue (n=3, i.e. decalcification or poor fixation). Valuable results were obtained for 207 patients. In 91 of 228 (39.9%) samples sent, a biomarker of interest was documented. This led to 34 clinical trial inclusions. Other patients were no longer eligible due to loss of performance status (n=20), loss of contact (n=14), no trial slot available at the appropriate time (n=18), or exclusion criteria (n=5). The mean waiting time between signing informed consent (T1) and receiving results of the biomarker analysis (T4) was 25.1 calendar (SD 17.3) days (Table). The preparation of the unstained slides by the pathology lab took about 9.1 (SD 6.8) days, the time of the biomarker testing itself accounted for 12.8 (SD 7.3) days. For 18 of 228 (7.9%) patients, repeated sample shipments were needed because of insufficient tumor cells, their mean waiting time between informed consent and receiving the biomarker result was 62.2 (SD 38.4) days. Table: Waiting times (t) in molecular testing for 228 patients.Time interval Mean StDev Median Range Pathology lab (T2-T1) 9.1 6.8 7.0 1 - 70 Shipment (T3-T2) 1.8 1.6 1.0 0 - 17 Analysis (T4-T3) 12.8 7.3 12.0 2 - 58 Request to result (T4-T1) 25.1 17.3 22.0 7 - 184
Conclusion:
While molecular testing is important in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients.
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- Abstract
Background:
Lung cancer is the leading cause of cancer mortality worldwide, and metastatic spread of cancer to distant organs is the main reason for lung cancer deaths. They spread to different distant organs, exhibit an outstandingly different situation of clinical characteristics and will be medically and surgically incurable. Thus, there is a clear need for a reliable and efficient in vitro culture model to enable transition to invasion and journey to distant organs of these critical steps in cancer metastatic progression.
Methods:
Here we report a biomimetic multi-organ microfluidic chip system more closely reconstituting the structural tissue arrangements, functional complexity and dynamic mechanical microenvironments and reproducing survival, growth, transition to invasion and journey to distant multi organs in lung cancer metastasis. To reconstitute the actual growth conditions of lung cancer in vivo, we created a thin, porous, flexible membrane, integrated microfluidic chip emulating the in vivo tissue structure and enabling heterotypic cell interactions, while maintaining cell compart-mentalization. The human bronchial epithelial cells and stromal cells were cultured on opposite sides of the membrane. Once the cells were grown to confluence, air was introduced into the epithelial compartment to create an air-liquid interface and more precisely mimic the lining of the lung air space. Then lung cancer cells were cultured on the human epithelial compartment to mimic lung cancer formation and the multi organ chambers were linked with side channels that supply lung cancer cells to the brain, bone or liver cells chamber to mimic lung cancer metastasis. In addition, the system provided analyzing cell physiology and visualizing complex cell behaviors in a more physiologically relevant context.
Results:
A biomimetic multi-organ microfluidic chip system was created. The quick formation of lung cancer cells that grow away from their natural margins and then attack adjacent components and spread to other organs were observed at all times and the cells characterizations were also detected accurately and effectively. In this multi-organ pathogenesis system, it might be possible to provide an ultrahigh level of reproducibility, authenticity and sensitivity.
Conclusion:
This microdevice provides a proof of principlefor this novel biomimetic strategy that is inspired by the integrated chemical, biological, and mechanical structures and functions of the living multi organs. This versatile system enables direct visualization and quantitative analysis of diverse biological processes of the intact lung cancer metastasis in ways that have not been possible in traditional cell culture or animal models.
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MINI22.08 - Development of a Protein Viewer for Displaying Variants of Unknown Significance in Relation to Actionable Mutations and Protein Domains (ID 2917)
16:45 - 18:15 | Author(s): A. Papanicolau-Sengos, M. Qin, L. Wei, J. Wang, M.K. Nesline, C. Hoeflich, K. Lahrs, J. Mastroianni, C.D. Morrison
- Abstract
- Presentation
Background:
Next-generation sequencing (NGS) can be used to interrogate multiple areas of the tumor genome. Several hot-spot panels have been developed to identify variants amenable to targeted therapies and enrollment into clinical trials. Variants of unknown significance (VUS) in the vicinity of hot-spots are routinely discovered. To better understand these obscure VUS, we built a Protein Viewer that displays the relationship of known actionable variant(s) to the VUS.
Methods:
We developed a web-based protein viewer that can be deployed across multiple browsers. The tool supports the visual representation of 23 genes which are interrogated by our NGS platform. We used the longest mRNA transcript (hg19) to define the protein domains. All actionable variants as reported by an knowledge database were included, with the selected VUS differentially highlighted. VUS is defined as a non-actionable variant that is not reported in dbSNP.
Results:
Approximately 50% of all stage III and IV lung cancer patients tested by our NGS platform have one or more VUS. After the variant information is loaded in the Protein Viewer, a two-dimensional image of the full length protein with actionable variants and VUS is displayed (Figure 1). The Viewer is utilized at RPCI to present cases at our molecular tumor board for quick visualization and discussion. Figure 1 Figure 1: Protein Viewer with a PIK3CA VUS harboring a Q546H (pink) in a lung adenocarcinoma. Top panel with PIK3CA exons 2-21 boundaries (vertical lines) with protein domains (blue rectangles along axis). Bottom panel with the zoom feature which allows more discreet visualization of the VUS, a neighboring Q546K actionable variant (green), and additional actionable variants for ovarian cancer (green rectangles).
Conclusion:
Understanding the relationship of VUS to protein domains and proximity to previously known actionable sites is a potentially powerful way to evaluate and determine whether a patient might be a candidate for targeted therapy. Because the exact effect of the VUS on the function of the protein is still impossible to discern (tyrosine kinase inhibitor sensitivity/ resistance/no effect), the next generation of protein viewers should incorporate 3D and protein folding/domain interaction prediction capabilities.
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MINI22.09 - Discussant for MINI22.05, MINI22.06, MINI22.07, MINI22.08 (ID 3534)
16:45 - 18:15 | Author(s): P. Mazzone
- Abstract
- Presentation
Abstract not provided
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MINI22.10 - A New Approach to Large Scale Proteomic Profiling to Uncover Tumor Phenotypes (ID 2166)
16:45 - 18:15 | Author(s): R. Ostroff, K. Delisle, W.A. Franklin, L. Gold, D.T. Merrick, S. Williams, Y.E. Miller
- Abstract
- Presentation
Background:
Genomic profiling is a powerful method for identifying mutations that drive tumors and matching patients to targeted therapies. However, this may only be a transient solution and resistance commonly emerges as the mechanism of targeted inhibition is overcome. Proteomic profiling of the tumor provides a dynamic tool to survey altered protein expression and deregulated pathways, which in turn may implicate specific treatments or identify novel therapeutic targets. Mass spectrometry offers highly multiplexed proteomic measurements, but extensive sample pre-processing and low sample throughput can lead to extended analysis times of weeks or months. Thus a need exists for a high throughput, sensitive and quantitative platform for proteomic analysis.
Methods:
We used the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV, to analyze protein lysates from 63 lung tumor samples. The assay does not require sample pre-fractionation, and this study (which generated over 142,000 protein measurements) represents less than one day of SOMAscan throughput. The study consisted of matched tumor/non-tumor protein lysates prepared from 18 squamous cell carcinoma and 45 adenocarcinoma fresh-frozen resected specimens, 86% of which were Stage I/II. The paired log~2~ tumor/non-tumor ratio was calculated and hierarchical clustering heat maps and dendrograms were constructed to identify related protein regions and tumor phenotypes.
Results:
Common proteomic changes and unique tumor phenotypic groups were identified by unbiased clustering algorithms. Large, consistent tumor/non-tumor differences of at least 4-fold were observed for 35 proteins in at least 20 (32%) of the tumors. Some of these proteins were more than 100-fold higher in individual tumors. The two most commonly elevated proteins were thrombospondin 2 and MMP12, which were increased in 81% and 61% of the tumors, respectively. We have previously reported higher levels of MMP12 in the serum of lung cancer patients, and the current data supports a tumor-associated origin for circulated MMP12. A second analysis identified sub-phenotypes of tumors clustered by common protein alterations independent of histological classification or mutation status. Many of these tumor subsets had increased expression of known oncology drug targets.
Conclusion:
Broad, unbiased high-throughput proteomic profiling of tumor tissue may reveal individual phenotypes that hold the potential to respond to targeted therapies and to monitor therapeutic efficacy throughout treatment. Measuring proteins complements mutation analysis by enabling therapeutic selection beyond driver mutation targets, including immune modulator therapies, repurposing existing drugs and enriching clinical trials with likely responders. While genomics is a fixed snapshot, blood- and tissue-based serial proteomic measurements respond to change and can lead to the personalized adaptation of treatment and identification of novel therapeutic targets.
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MINI22.11 - A Clinical Platform for Examining Mechanism-Driven Chemotherapeutic Agents (ID 724)
16:45 - 18:15 | Author(s): C.P. Erkmen, E. Dmitrovsky, V.A. Memoli, K.H. Dragnev
- Abstract
- Presentation
Background:
There is a clinical need to establish whether those pathways activated in vitro and in animal models, are also activated in human lung cancer. We established a window-of-opportunity clinical trial platform in lung cancer where novel agents are administered in the preoperative period. Intratumoral drug concentrations are correlated with molecular marker changes. Our four completed window-of-opportunity clinical trials established that optimal intratumoral drug concentrations are needed for the desired pharmacodynamic effects, providing direction for optimal dose and schedule. To further evaluate the value of this window-of-opportunity platform, we investigate the impact on standard postoperative outcome measures.
Methods:
39 consecutive patients enrolled under the window-of-opportunity platform were matched to 39 contemporary patients undergoing the same operation by the same surgeon. Co-morbidities and stage of lung cancer and postoperative complications were compared using univariate and multivariate analysis. Wilcoxon Scores (Rank Sums) for variable data elements and Fisher’s Exact Test was used for analysis.
Results:
When comparing window-of-opportunity patients to control patients, there was no difference in age, pack years of smoking, or incidence of comorbidities including diabetes, coronary artery disease, hypertension, chronic obstructive pulmonary disease, and previous cancer. There was no difference in the stage distribution, (stage I: 28 vs. 22, stage II: 5 vs. 3, stage III: 5 vs. 2 stage IV: 1 vs. 1, p=0.1642). There was also no difference in the incidence of postoperative pneumonia (4 vs. 9, p=0.2235), other infection (2 vs. 3, p=0.8208), atelectasis (2 vs. 4, p=0.6748), myocardial infarction (0 vs. 0, p=1.000), reoperation for bleeding (1 vs. 1, p=1.000), pulmonary embolism (1 vs. 2, p=1.000) or number patients experiencing any complication (14 vs. 8, p=0.131118). There was no difference in the distribution of survival at 2 years (27 vs. 30) or 5 years (10 vs. 15), p=0.2266.
Conclusion:
The window of opportunity platform does not increase the perioperative risk of complications in early stage NSCLC patients undergoing surgery. By evaluating drug effect and the potential toxicities, window-of-opportunity trials validate mechanisms established in the laboratory and facilitate bi-directional translation research.
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MINI22.12 - Molecular Characterisation of SCLC Using Both Circulating Tumour DNA and Circulating Tumour Cells Isolated from the Same Whole Blood Sample (ID 251)
16:45 - 18:15 | Author(s): D.G. Rothwell, N. Smith, D. Morris, H.S. Leong, Y. Li, L. Carter, F. Blackhall, C. Miller, C. Dive, G. Brady
- Abstract
- Presentation
Background:
Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Response rates to first line chemotherapy are generally high, but progression free survival is short due to development of chemotherapy resistance via mechanisms not well understood. Due to the difficulty in collecting tissue biopsies in SCLC, blood, which can be sampled simply and routinely, provides a means of inferring the current genetic status of a patients tumour via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). These offer a minimally invasive opportunity to study drug resistance mechanisms, evaluate tumour heterogeneity and potentially reveal new drug targets in this disease. However, accurate assessment of both CTCs and ctDNA requires all blood cells be maintained intact until samples are processed, particularly when analytes present are at very low concentrations. Here we describe and validate a blood collection protocol that does not require on-site processing, and which is amenable for analysis of both CTCs and ctDNA following storage at ambient temperature in CellSave vacutainers for up to 96 hours after blood collection.
Methods:
To evaluate the suitability of using CellSave preserved samples for circulating free DNA (cfDNA) analysis, we undertook a 20 healthy normal volunteers (HNV) study and 45 patient sample study, with parallel EDTA and CellSave bloods collected. For each sample cfDNA was isolated between 4 hours and 96 hours post-draw and cfDNA yields determined. A potential issue with using CellSave blood was that the CellSave preservative could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, the EDTA and CellSave cfDNA samples were subjected to next generation sequencing (NGS) to estimate the overall mutation burden. In addition, the utility of CellSave ctDNA for targeted NGS was also determined. Finally, SCLC-specific copy number aberrations (CNA) were analysed in ctDNA and CTCs isolated from the same CellSave blood sample from individual SCLC patients.
Results:
We demonstrate that yields of cfDNA obtained from 96-hour whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 hours of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples, thereby validating CellSave blood as a viable source of ctDNA. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA allowing direct comparison of the genetic status of patients’ tumours.
Conclusion:
In summary, we have demonstrated the suitability of whole blood CellSave samples for both CTC and ctDNA molecular analysis in SCLC. The ability to generate informative molecular profiles of both CTCs and ctDNA from a simple whole blood sample, up to 4 days post-draw represents a significant methodological improvement for clinical benefit. We posit that as minimally invasive, liquid biopsies become increasingly employed for cancer patient management, the ability to routinely and simply draw blood and ship samples to accredited biomarker assessment laboratories will greatly facilitate the delivery of personalised cancer medicines.
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MINI22.13 - Discussant for MINI22.10, MINI22.11, MINI22.12 (ID 3480)
16:45 - 18:15 | Author(s): E. Haura
- Abstract
- Presentation
Abstract not provided
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MS 16 - Novel SCLC Therapies (ID 34)
- Event: WCLC 2015
- Type: Mini Symposium
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:C. Barrios, N. Saijo
- Coordinates: 9/08/2015, 14:15 - 15:45, Mile High Ballroom 4a-4f
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MS16.01 - PARP Inhibitors and DNA Damage (ID 1916)
14:15 - 15:45 | Author(s): L. Byers
- Abstract
- Presentation
Abstract:
A leading cause of death in small cell lung cancer (SCLC) is the rapid emergence of drug resistance following an initial phase of chemotherapy and radiation sensitivity. Currently, response rates to existing second-line regimens (e.g., topotecan and other single-agent chemotherapies) are less than 20%. Because of this overall poor response to subsequent therapy, average survival for relapsed disease ranges between 4-6 months. As such, there is a critical need for the development of novel, active therapies for SCLC. Drugs that target DNA damage response (DDR), including PARP inhibitors, have shown promising activity against SCLC in pre-clinical models and in early clinical trials. Previously, we performed proteomic profiling of a large panel of SCLC cell lines which led to the observation that PARP1, Chk1, and several other DNA repair proteins are expressed at high levels in SCLC[1]. PARP1 overexpression was confirmed in patient tumors at the protein level by immunohistochemistry and at the mRNA level. Based on this finding, several PARP inhibitors were tested in pre-clinical models of SCLC. Olaparib, rucaparib, and talazoparib (previously BMN-673) all demonstrated striking single agent activity in a majority of SCLC cell lines tested. Furthermore, the addition of a PARP inhibitor to standard chemotherapies (e.g., cisplatin, etoposide and/or topotecan) and radiation further potentiated their effect[1][,][2]. In animal models including xenografts and patient-derived xenografts (PDXs), talazoparib has demonstrated significant anti-tumor activity as a single agent, comparable or superior to cisplatin[3][,][4]. Following these observations, several clinical trials were initiated to investigate the effects of PARP inhibition in SCLC patients. The first two studies to complete enrollment investigated the use of PARP inhibitors in relapsed SCLC. In the first study, single-agent talazoparib (BMN-673) was tested in an expansion cohort of patients with platinum-sensitive SCLC relapse (NCT01286987). Preliminary data from this trial demonstrated 2/23 patients with RECIST confirmed partial responses and 3/23 with stable disease lasting more than 24 weeks (clinical benefit rate of 25%). More than half of patients treated had some tumor volume reduction as their best response[5]. In the second study, the oral alkylating drug temozolomide with or without veliparib (ABT-888) was studied in 100 patients with sensitive or refractory relapse (NCT01638546). This trial recently completed enrollment and analysis of the results are ongoing. The use of PARP inhibitors in combination with chemotherapy builds upon prior pre-clinical data in lung cancer and other malignancies supporting the notion that PARP inhibitors potentiate the effect of other DNA damaging therapies. Currently, there are two studies investigating the use of veliparib (ABT-888) in combination with standard frontline chemotherapy (NCT01642251 and NCT02289690). E2511 (NCT01642251) is a Phase I/II trial of cisplatin, etoposide, and veliparib conducted through the ECOG-ACRIN Cancer Research Group in which treatment naïve SCLC patients receive the combination for up to 4 cycles. Published results from the Phase I portion support the safety and tolerability of the combination, with partial or complete responses observed in 5/7 evaluable patients[6]. More recently, another first-line study was initiated to investigate carboplatin in combination with etoposide and veliparib which will also address the question of veliparib maintenance (NCT02289690). To date, the activity of PARP inhibitors are best established in cancers with mutations in BRCA1/2 and other DNA repair genes that result in synthetic lethality in the setting of PARP inhibition (which provides a second “hit” to the DNA repair machinery). In fact, olaparib monotherapy was FDA-approved last year for patients with advanced, BRCA-mutated ovarian cancer who have received three or more lines of chemotherapy. This was based on a trial that demonstrated a response rate of 34% and a median duration of response or 7.9 months. Studies of other PARP inhibitors have also shown striking single-agent activity with this class of drugs in a mutation-selected population. However, in SCLC, the mechanism of action and identification of potential biomarkers of response to these drugs is an area of active investigation. Likely the universal loss of RB1, with resulting dependence on E2F1, plays a role, as may the PARP-trapping effects of several of these drugs which cause direct cytotoxicity[7]. Our group has demonstrated that expression levels of several DNA repair proteins – both individually and as a “DNA repair signature” – are associated with response in pre-clinical models of lung cancer[3]. However, further validation in the clinical setting is warranted. Additional DNA damage response (DDR) targets also show significant potential as therapeutic targets in SCLC. These include checkpoint kinases that are activated in response to DNA damage and facilitate S and G2 checkpoint arrest, such as Chk1 (Checkpoint kinase 1), Wee1, and ATR (Ataxia Telangiectasia and Rad3 related). Similar to PARP1, in our previous work we demonstrated elevated expression of Chk1 in SCLC[1]. SCLC may be particularly susceptible to inhibitors of Chk1 and other checkpoint kinases due to the near universal loss of TP53 in these cancers which make them dependent on other checkpoint controls in the cell cycle. Several drugs targeting these DDR proteins have entered clinical trials. For example, based on pre-clinical data demonstrating the potentiation of topoisomerase inhibitors by ATR inhibition, a Phase I/II trial of topotecan with VX970 (an ATR kinase inhibitor) has recently been initiated for SCLC (NCT02487095). Ongoing trials of PARP inhibitors and other molecules targeting DDR will help us to understand the activity of these compounds in patients with SCLC. Important questions that require further investigation include the optimal combinations of these drugs with existing therapies or other targeted inhibitors, strategies to manage associated toxicities (especially combinations with overlapping hematologic toxicities), and further development of candidate predictive biomarkers.REFERENCES 1. Byers LA, Wang J, Nilsson MB, et al. Proteomic profiling identifies dysregulated pathways in small cell lung cancer and novel therapeutic targets including PARP1. Cancer discovery 2012;2:798-811. 2. Owonikoko TK, Zhang G, Deng X, et al. Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer. Cancer medicine 2014;3:1579-94. 3. Cardnell RJ, Feng Y, Diao L, et al. Proteomic markers of DNA repair and PI3K pathway activation predict response to the PARP inhibitor BMN 673 in small cell lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2013;19:6322-8. 4. Y. Feng, R. Cardnell, L.A. Byers, B. Wang, Y. Shen. Talazoparib (BMN 673) as single agent and in combination with temozolomide or PI3K pathway inhibitors in small cell lung cancer and gastric cancer models. 26th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics (abstract) 2014. 5. Wainberg ZA, Ramanathan RK, Mina LA, Byers LA, Chugh R, Goldman JW, Sachdev JC, Matei DE, Wheler JJ, Henshaw JW, Zhang C, Gallant G, De Bono JS. Safety and antitumor activity of the PARP inhibitor BMN673 in a phase 1 trial recruiting metastatic small-cell lung cancer (SCLC) and germline BRCA-mutation carrier cancer patients. 2014 ASCO Annual Meeting; J Clin Oncol 32:5s, (suppl; abstr 7522) 2014. 6. Owonikoko TK, Dahlberg SE, Khan SA, et al. A phase 1 safety study of veliparib combined with cisplatin and etoposide in extensive stage small cell lung cancer: A trial of the ECOG-ACRIN Cancer Research Group (E2511). Lung cancer 2015;89:66-70. 7. Murai J, Huang SY, Das BB, et al. Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer research 2012;72:5588-99.
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ORAL 13 - Immunotherapy Biomarkers (ID 104)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:D. Kim, D. Grunenwald
- Coordinates: 9/07/2015, 16:45 - 18:15, Four Seasons Ballroom F3+F4
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ORAL13.07 - EMT Is Associated with an Inflammatory Tumor Microenvironment with Elevation of Immune Checkpoints and Suppressive Cytokines in Lung Cancer (ID 2134)
16:45 - 18:15 | Author(s): L. Byers
- Abstract
- Presentation
Background:
Promising results in the treatment of NSCLC have been seen with immunomodulatory agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Epithelial-mesenchymal transition (EMT) is a key process driving metastasis and drug resistance. Previously we have developed a robust EMT gene signature, highlighting differential patterns of drug responsiveness for epithelial and mesenchymal tumor cells.
Methods:
We conducted an integrated analysis of gene expression profiling from three independent large datasets, including The Cancer Genome Atlas (TCGA) of lung and two large datasets from MD Anderson Cancer Center, Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse phase protein array (RPPA), immunohistochemistry, in vivo mouse models and correlation with clinical data were performed.
Results:
EMT is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation co-existent with elevation of immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, BTLA and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Similarly, IL-6 and indoleamine 2, 3-dioxygenase (IDO) were elevated in these tumors. We demonstrate that in murine models of lung adenocarcinoma, many of these changes are recapitulated by modulation of the miR-200/ZEB1 axis, a known regulator of EMT. Furthermore, B7-H3 is found to negatively correlate with overall survival and recurrence free survival, indicating a potential new therapeutic target in lung adenocarcinoma in the future.
Conclusion:
EMT, commonly related to cancer metastasis and drug resistance, is highly associated with an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoints and that is regulated at least in part by the miR-200/ZEB1 axis. These findings suggest that EMT may have potential utility as a biomarker selecting patients more likely to benefit from immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers.
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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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ORAL21.02 - Landscape and Functional Significance of KRAS Co-Mutations in Lung Adenocarcinoma (LUAC) (ID 3224)
10:45 - 12:15 | Author(s): L. Byers
- 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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