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P.P. Massion
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.09 - A DLL3-Targeted ADC Effectively Targets Pulmonary Neuroendocrine Tumor-Initiating Cells to Result in Sustained Tumor Regressions (ID 2533)
17:30 - 17:35 | Author(s): P.P. Massion
- Abstract
- Presentation
Background:
Pulmonary neuroendocrine tumors such as small cell lung cancer (SCLC) and large cell neuroendocrine cancer (LCNEC) remain among the most deadly malignancies and are increasing in incidence. Patient-derived xenograft (PDX) tumors provide excellent models to study tumor biology and discover tumor-initiating cell (TIC) populations. Novel therapies that target and eradicate TIC represent a promising strategy to improve survival. An effectively targeted antibody-drug conjugate (ADC) carrying a cell-cycle independent toxin should result in significant anti-tumor activity and eliminate TIC.
Methods:
Whole transcriptome sequencing was performed using TIC isolated by fluorescence-activated cell sorting from SCLC and LCNEC PDX tumors. Quantitative RT-PCR, microarray analysis of PDX tumors and cell lines, and mining of publically available transcriptome and proteome datasets were executed to validate that prospective targets, such as Delta-like protein 3 (DLL3), were highly expressed in neuroendocrine tumors, but limited in their expression in normal tissues. DLL3-specific monoclonal antibodies were generated and used to determine protein expression by immunohistochemistry, flow cytometry and ELISA. Select DLL3-specific antibodies were conjugated to a cell-cycle independent pyrrolobenzodiazepine (PBD) dimer toxin and evaluated for their ability to internalize and mediate cell killing. Finally, established SCLC and LCNEC PDX tumors were treated in vivo with a lead anti-DLL3 ADC (i.e. SC16LD6.5). Limiting dilution assay (LDA) serial transplantation experiments were executed to assess the impact of SC16LD6.5 on TIC.
Results:
Elevated expression of DLL3 mRNA was discovered in TIC of SCLC and LCNEC PDX tumors and confirmed in additional distinct primary SCLC and LCNEC tumor samples and PDX tumors. In contrast, little to no mRNA expression was detected in vital organs and other normal tissues outside of the brain. DLL3-specific antibodies confirmed protein expression on the cell surface in both primary SCLC and LCNEC tumors and in PDX tumors initiated from patients with these diseases, whereas protein was scarce in normal tissues. SC16LD6.5 rapidly internalizes and localizes to late endosomes, and treatment of 10 SCLC and 2 LCNEC PDX tumor models resulted in significant and durable tumor regression with a median time to progression benefit of 75 days versus 16 days with standard-of-care (SOC: SCLC, cisplatin/etoposide; LCNEC, cisplatin). During the course of these in vivo studies, many mice were cured as tumors often did not recur despite being followed for 120+ days post-randomization and treatment. LDA experiments executed using tumors actively responding to SC16LD6.5 provided further functional evidence that the common lack of tumor recurrence following treatment resulted from effective targeting of DLL3-expressing TIC. In vivo efficacy strongly correlated with DLL3 protein expression, and responses were observed in PDX tumor models initiated from patients with both limited and extensive stage disease and independent of their sensitivity to SOC.
Conclusion:
The DLL3-targeted ADC, SC16LD6.5, effectively targets and eradicates TIC in SCLC and LCNEC PDX tumors. SC16LD6.5 (i.e. rovalpituzumab teserine) is currently concluding Phase 1b trials and is a promising first-in-class therapeutic for the treatment of high grade pulmonary neuroendocrine tumors.
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ORAL 07 - Lung Cancer Pathogenesis (ID 91)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:J. Sage, E. Brambilla
- Coordinates: 9/07/2015, 10:45 - 12:15, 102+104+106
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ORAL07.02 - Metabolic Reprogramming in the Airway Epithelium of Individuals at High Risk for Lung Cancer (ID 2493)
10:56 - 11:07 | Author(s): P.P. Massion
- Abstract
- Presentation
Background:
What defines the high risk airway epithelium for lung cancer remains a major challenge. Airway epithelium is prone to assault by the risk factors and considered to be the primary cell type involved in the field cancerization. Transcriptomic aberrations in the airway epithelium of individuals at risk for lung cancer have been reported earlier. However, very limited information exists about proteomic alterations in the airway epithelium. We investigated the molecular underpinnings of risk from proteomic alterations in the cytologically normal airway epithelium from individuals at risk for developing lung cancer.
Methods:
Bronchial brushings specimens were collected from individuals categorized as low, medium and high risk groups based on Bach risk model. Shotgun proteomic profiling data were acquired by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified using a combination of database search tools and candidate proteins were selected based on Jonckheere-Terpstra trend analysis. Pathway analysis was performed using WebGestalt. In vitro model of human bronchial epithelial cell line treated with cigarette smoke condensate (CSC) was used for metabolic flux experiments by gas chromatography mass spectrometry (GC MS) analyses.
Results:
We identified 2901 proteins in bronchial epithelial cells from risk stratified individuals. Jonckheere-Terpstra trend test resulted significantly altered expression of 315 proteins (trend p <0.05) with 238 up and 77 down trends. KEGG pathway analysis with the 315 proteins revealed very early events of possible metabolic reprogramming in the cytologically normal bronchial epithelium of individuals at high risk for lung cancer development. Fourteen enzymes of the glycolytic pathway, TCA cycle, pentose phosphate pathway, and glycogenolysis were over expressed. Six of these fourteen enzymes, PYGB, PFKP, PFKL, PKM2, IDH1, and IDH2 were rate limiting enzymes. In in vitro culture of human bronchial epithelial cells treated with CSC, lactate production and glucose consumption were increased suggesting Warburg effect and metabolic reprogramming. Evidence of glutamine metabolism through reductive carboxylation in CSC treated cells was obtained from the metabolic flux analyses of cells from this in vitro model. Contribution of labeled carbon from [U-[13]C5]-glutamine to TCA cycle in CSC treated cells were more than untreated control cells and there was strong M+5 citrate labeling in CSC-treated cells.
Conclusion:
Shotgun proteomic analysis of cytologically normal bronchial epithelial cells in individuals at increasing risk for lung cancer revealed over expression of carbohydrate metabolic enzymes in high risk individuals suggesting possible metabolic reprogramming. The altered profile of metabolic enzymes may provide a signature of lung cancer risk assessment and serve as the basis of patient selection for surveillance programs and chemoprevention.
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ORAL 24 - CT Detected Nodules - Predicting Biological Outcome (ID 122)
- Event: WCLC 2015
- Type: Oral Session
- Track: Screening and Early Detection
- Presentations: 1
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ORAL24.07 - Behavior Differences of Screen-Detected Lung Cancers in the CT Arm of the National Lung Screening Trial (NLST) (ID 587)
11:50 - 12:01 | Author(s): P.P. Massion
- Abstract
Background:
Lung cancer screening identifies cancers with heterogeneous behaviors. In addition to screen-detected incidence lung cancers, screening also identifies prevalence cancers at the baseline screen and interval lung cancers diagnosed following a negative screen at any time point prior to the next screening round. To date, few studies have performed a comprehensive analyses comparing prevalence and interval lung cancers and screen-detected lung cancers based on sequence of screening results in the NLST.
Methods:
The entire CT arm of the NLST was reconstructed according to baseline and follow-up screening results (positive vs. negative screen). Lung cancers immediately following a positive baseline (T0), and prior to the T1 screen, formed the prevalence cancers (PC); interval cancers (IC) were defined as lung cancers diagnosed following a negative screen at any point prior to the next screening round. Two screen-detected lung cancer (SDLC) cohorts were identified based on one (SDLC1) or two (SDLC2) prior positive screens and two screen-detected lung cancer cohorts following one (SDLC3) or two (SDLC4) prior negative screens. Differences in patient characteristics, progression-free survival (PFS), and overall survival (OS) were assessed.
Results:
Since there were no differences in patient characteristics and outcomes between SDLC1 and SDLC2 and between SDLC3 and SDLC4, the four screen-detected cancer case groups were combined into two combined SDLC case groups (SDLC1/SDLC2 and SDLC3/SDLC4). The lung cancer-specific death rate was higher for SDLC3/SDLC4 compared to SDLC1/SDLC2 lung cancers (136.6/1,000 person-years vs. 71.3/1,000 person-years, P < 0.001). PFS and OS were significantly lower for SDLC3/SDLC4 than SDLC1/SDLC2 (P < 0.004; P < 0.002, respectively). Overall, PFS and OS were highest in SDLC1/SDLC2 and lowest in the interval cancers (Figure 1); PFS and OS for the prevalence cancers were intermediate between SDLC1/SDLC2 and SDLC3/SDLC4. All findings were consistent when stratified by stage and histology. Multivariable Cox proportional models revealed that the SDLC3/SDLC4 case groups were associated with significantly poorer PFS (HR=1.72; 95% CI 1.19-2.48) and OS (HR=1.62; 95% CI 1.08-2.45) compared to SDLC1/2 lung cancers (HR=1.00). Figure 1
Conclusion:
This post hoc analysis reveals novel insight to the heterogeneity of lung cancers diagnosed in a screening population. As with interval cancers diagnosed following a negative screen, lung tumors that arise in a lung environment ostensibly free of lung nodules are likely more rapidly growing and aggressive which results in significantly poorer outcomes. Additional research will be needed to understand the potential translational implications of these findings and to reveal biological differences of screen-detected tumors.
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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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P1.04-047 - The Inhibitory Effects of CDK4 and MDM2 on Migration and Invasion in Human Non-Small Cell Lung Cancer Cells (ID 2833)
09:30 - 09:30 | Author(s): P.P. Massion
- Abstract
Background:
Cyclin-dependent kinase 4 (CDK4)/RB and mouse double minute 2 (MDM2)/p53 are the two main regulators of the tumor-suppressor pathways that control cellular responses to potentially oncogenic stimuli. CDK4 inhibits RB by triggering its phosphorylation, leading to releasing the G1-S restriction point. MDM2 inhibits the transcription activity of p53 by blocking the transfer of p53 from cytoplasm to nucleus and by accelerating ubiquitination of p53. Our preliminary SNP microarray analysis using lung specimens from non-invasive tumor (adenocarcinoma in situ) and invasive tumor (lepidic predominant adenocarcinoma) showed the amplification of chromosome 12q13–15, including CDK4 and MDM2 gene regions. The aim of the present study was to determine the mechanistic implications of CDK4 and MDM2 in lung adenocarcinoma migration and invasion.
Methods:
Using siRNAs specific for CDK4 and MDM2, the expressions of CDK4 and MDM2 were knocked down in the human non-small cell lung cancer cell lines A549, H460, H1299, SK-Lu-1 and H23, which harbor wild-type RB yet contain other aberrations in p53 (wild-type in A549, H460, absent in H1299, and mutated in SK-Lu-1, H23). Cell proliferation (AlamarBlue staining), mobility (scratch assay), and invasion (transwell-matrigel chamber system) were investigated.
Results:
The knockdown of CDK4 (5.5, 18.5, 2.2, 22.8 and 8.3% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) significantly inhibited cell proliferation in H23 and SK-Lu-1, and decreased cell migration in SK-Lu-1 and H460. It also repressed cell invasion in H460, SK-Lu-1 and A549. The decreased expression of MDM2 (43.4, 69.6, 6.4, 27.3, 8.7% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) dramatically inhibited cell proliferation in H1299, SK-Lu-1 and H23, and diminished cell migration in H23, A549 and SK-Lu-1. It also hindered cell invasion in H460 and H23.
Conclusion:
These findings suggest CDK4 and/or MDM2 pathways may play critical roles in cell proliferation, mobility and invasion, and furthermore, the targeting CDK4 and/or MDM2 may provide therapeutic benefit to lung cancer patients.
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P1.06 - Poster Session/ Screening and Early Detection (ID 218)
- Event: WCLC 2015
- Type: Poster
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.06-023 - Addition of Low Dose Computed Tomography Image-Features Improves Diagnostic Accuracy for Indeterminate Pulmonary Nodules (ID 1019)
09:30 - 09:30 | Author(s): P.P. Massion
- Abstract
Background:
Lung cancer is the leading cause of cancer related deaths world-wide. While low dose computed tomography (LDCT) screening of the high risk patient population was recently shown to decrease deaths from lung cancer by 20%, LDCT also resulted in 18% over-diagnosis [c.f. Patz-E.-F.-JAMA-2003] with a positive predictive value of only 52.9% when a suspicious LDCT finding led to a biopsy [c.f. Church-T.-NEJM-368-2003]. We tested whether combining novel image-features (IF) with routinely collected baseline-features (BF) can improve the accuracy of diagnosing suspicious findings on baseline LDCT.
Methods:
This exploratory case-control study included N=123 (66-cancer, 57-no-cancer) high risk subjects with at least one suspicious finding (nodule >= 8mm [c.f. Lung-RADS-ACR-2014]) on baseline LDCT screening at Vanderbilt University on a VCT Discovery (GE-Healthcare, UK) or a Brilliance iCT 128 SP (Philips, Amsterdam) system. The cohort was randomly divided into a separate training-set (N=55, 32-cancer, 23-no-cancer) and a test-set (N=68, 34-cancer, 34-no-cancer). All model training and leave-one-out cross-validation were strictly restricted to the training-set. Performance was evaluated on the unseen test-set. Definitive lung cancer or no-cancer diagnosis, smoking history and at least 6 baseline-features (BF6) viz. age, family-history, pack-smoking-years, body-mass-index, nodule-location, nodule-size were recorded for all subjects. Baseline lung cancer predictions were generated by (a) using the Gould-model [c.f. Gould,M.-Chest-2007] and (b) fitting an Elastic-Net Regularized Generalized Linear Model (GLMnet [c.f. Zou-H.-Journ-Royal-Stats-Soc-B-2005]) to BF6. The final baseline model (“GLMnet:BF”) effectively utilized 4 baseline-features with the coefficients for age and body-mass-index shrunk to zero. New LDCT specific information was extracted by computing 589 intensity, shape, surface and texture features (IF589) [c.f. Aerts-H.-Nat-Comm-S2014, Way-T.-Med-Phys-2009] from a 3D volume-of-interest (VOI) encompassing a rough Graph-cuts [c.f. Li-K.-IEEE-PAMI-2006] segmentation for each suspicious nodule. A GLMnet was fit to all 595 features (BF6 and IF589) yielding a final enhanced model (“GLMnet:BF+IF”), which contained 12 features after GLMnet shrinkage : 10 IF related to VOI energy, nodule shape and surface statistics and image intensity variability and 2 BF (family-history, nodule-location).
Results:
Baseline AUC increase by 7.4% from 0.81 (Gould-model) and 0.80 (GLMnet:BF) to 0.87 (GLMnet:BF+IF). At 88% sensitivity, false positive rate reduced by 60% from 56% (Gould-model) and 44% (GLMnet:BF) to 18% (GLMnet:BF+IF); accuracy improved from 65% (Gould-model) and 71% (GLMnet:BF) to 84% (GLMnet:BF+IF). Fig.1 below shows more details: Figure 1
Conclusion:
This initial exploratory analysis showed that image-features extracted from suspicious LDCT findings may help reduce the number of unnecessary biopsies. Additional validation studies are warranted to determine the value of this structural imaging-based approach.
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P1.07 - Poster Session/ Small Cell Lung Cancer (ID 221)
- Event: WCLC 2015
- Type: Poster
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.07-002 - FAK Inhibition by PF228 Has Anti-Tumoral Effects Associated with Inhibition of Histone 3 and Aurora Kinases A/B Phosphorylation in SCLC (ID 2844)
09:30 - 09:30 | Author(s): P.P. Massion
- Abstract
Background:
Lung cancer is the most common cancer and the leading cause of cancer-related death worldwide. Small cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is the most aggressive histologic type, with a five-year overall survival as low as 5%. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase, which regulates integrin and growth factor signaling pathways involved in cell proliferation, survival, migration, and invasion. FAK is overexpressed and/or activated in many cancers,including SCLC. We hypothesized that FAK may represent a good target for therapeutic intervention in SCLC and tested the changes of cell phenotype and signaling events following FAK inhibition, by using PF-573,228 (PF-228) in SCLC cell lines.
Methods:
Two SCLC cell lines growing in suspension (NCI-H82 and NCI-H146), an adherent SCLC cell line (NCI-H196), and a mixed morphology SCLC cell line (NCI-H446) were treated with increasing concentrations of PF-228. Cell proliferation was evaluated by WST-1 assay, cell cycle by flow cytometry following propidium iodide (PI) and bromodeoxyuridine (BrdU) staining, and apoptosis by flow cytometry after intracellular caspase 3 staining. FAK expression/activity and signaling events downstream of FAK were evaluated by Western blotting (WB).
Results:
While PF-228 did not modify total FAK expression, it decreased the phosphorylation of FAK (Tyr 397) in a dose dependent manner in all tested SCLC cell lines. Inhibition of FAK activity by PF-228 significantly decreased cell proliferation, induced cell cycle arrest in G2/M phases,decrease DNA replication and increased apoptosis in all tested cell lines proportionally to the dose. Regarding signaling events, we observed that inhibition of FAK activity induced the inhibition of phosphorylation of histone-3 (Ser 10) and Aurora Kinase A (Thr288) and B (Thr232). Figure 1
Conclusion:
These results show that FAK activity is required for proliferation, cell cycle progression, and survival in SCLC cell lines, suggesting that this pathway is central to SCLC biology. The antitumoral effects of PF-228 may occur through (1) the inhibition of histone-3 phosphorylation mediated by the inhibition of Aurora kinase B and leading to cell cycle arrest in G2/M phase and (2) the inhibition of Aurora kinase A leading to decreased DNA replication.
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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: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-069 - Exportin-5 (XPO5) in Lung Adenocarcinoma: A New Biomarker of Invasion in Pathology Specimens (ID 1283)
09:30 - 09:30 | Author(s): P.P. Massion
- Abstract
Background:
The WHO/IASLC classification of lung adenocarcinoma (LADC) emphasizes the distinction of adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) from their invasive counterparts. The distinction between lepidic-pattern lesions, in particular AIS/MIA and lepidic-predominant adenocarcinoma (LPA), is difficult in small biopsies and cytology specimens. Currently, there are no biomarkers of lung invasion in this setting.
Methods:
The WHO/IASLC classification of LADC was used for all components of this study. Gene expression (GE) data from 58 LADC samples including 33 samples of AIS/MIA and LPA identified two predominant clusters of 553 differentially expressed genes (p<0.01, FDR<0.06). The 317 genes upregulated in LPA localized to 6 regions on chromosomes 1, 2, 6 and 17 (Gene Set Enrichment Analysis). Expression data was compared to copy number (CN) data of AIS/MIA and LPA pooled from a re-annotated Cancer Genome Atlas data set along with prior annotated Affy 6.0 SNP array data (total 1086 LADC samples including 43 AIS/MIA and 26 LPA). Two regions (6p and 17q) contained genes with increased expression and CN increase in LPA. The XPO5 gene at 6p21 was selected for further study. Immunohistochemistry (IHC) for the XPO5 protein product Exportin-5 (XPO5, Sigma-Aldrich, St. Louis, USA) was performed on 686 lung cancers (NSCLC), on tissue microarrays and read independently by two pathologists. Nuclear (N) and cytoplasmic (C) positivity was scored for intensity (0-3) and percentage; an H-score was calculated for each (0-300, N-score and C-score). A total score (T-score) was calculated from the sum of the N-and C-scores (0 to 600). Statistical analysis was performed using the independent-samples Kruskal-Wallis test and pairwise analysis. Cox regression was used for survival analysis (continuous variable and quartile regressions), as well as Kaplan-Meier curves, logrank statistic.
Results:
XPO5 at 6p21 showed upregulation in LPAs by CN, GE and IHC. High XPO5 IHC T-scores correlated with CN, with a median T-score of 300 in tumors with CN gain vs. 50 in tumors without gain. High T-scores were seen in the following invasive patterns of NSCLCs as compared to AIS/MIA: acinar-ADC, solid-ADC, papillary-ADC, large cell carcinoma and squamous cell carcinoma; mean T-scores ranged from 144.7-251.4 in these groups vs. 48.3 and 72.1 in AIS and MIA, respectively. Importantly, T-scores correlated with overall survival for all-stage (n=686) and stage I (n=307) analyses, with higher scores predicting inferior survival. While IHC scores did not show statistically significant staining in LPA as compared to AIS/MIA, a qualitative difference was noted in some cases with acquisition of cytoplasmic positivity in the invasive component of LPAs.
Conclusion:
XPO5 is a candidate biomarker of invasion in LADC. GE and CN data along with IHC staining patterns in 686 NSCLC samples show upregulation of XPO5 in invasive tumors and in tumors with poor survival. In addition to its application in small biopsies, this marker may be of particular use in cytology specimens, where there is significant morphologic overlap between lepidic-pattern tumors and well-differentiated invasive patterns of LADC.
