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E.R. Parra
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MA04 - HER2, P53, KRAS and Other Targets in Advanced NSCLC (ID 380)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Advanced NSCLC
- Presentations: 1
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MA04.07 - Impact of Major Co-Mutations on the Immune Contexture and Response of KRAS-Mutant Lung Adenocarcinoma to Immunotherapy (ID 6343)
16:42 - 16:48 | Author(s): E.R. Parra
- Abstract
- Presentation
Background:
Activating mutations in the KRAS proto-oncogene define a prevalent and clinically heterogeneous molecular subset of lung adenocarcinoma (LUAC). We previously identified three major subgroups of KRAS-mutant LUAC on the basis of co-occurring genetic events in TP53 (KP), STK11/LKB1 (KL) and CDKN2A/B (KC) and reported that LKB1-deficient tumors exhibit a “cold” tumor immune microenvironment, with reduced expression of several immune checkpoint effector/mediator molecules, including PD-L1 (CD274). Here, we extend these findings and examine the clinical outcome of co-mutation defined KRAS subgroups to therapy with immune checkpoint inhibitors.
Methods:
We conducted a single-institution analysis of clinical and molecular data (PCR-based next generation sequencing of panels of 50, 134 or 409 genes) prospectively collected from patients enrolled into the MD Anderson Lung Cancer Moon Shot GEMINI database. KRAS-mutant LUAC were separated into KP, KL and K (wild-type for TP53 and STK11) groups. The log- rank test and Fisher’s exact test were used for comparison of progression-free survival (PFS) and objective response rate (ORR) respectively between the groups. In addition, automated IF-based enumeration of lymphocyte subsets was performed in 40 surgically resected LUAC (PROSPECT cohort) with available whole exome sequencing data.
Results:
Among 229 patients with KRAS-mutant LUAC who consented to the protocol we identified 35 patients with metastatic disease (17 KP, 6 KL, 12 K) that received immunotherapy with nivolumab (N=29), pembrolizumab (N=3), nivolumab/urelumab (N=1) and durvalumab/tremelimumab (N=2) and had robust clinical outcome data. There was no impact of different KRAS alleles (G12C/G12V/G12D) on PFS (P=0.6149, log-rank test) or ORR to immune checkpoint inhibitors (P=0.88, Fisher’s exact test, 2x3 contingency table). In contrast, co-mutation defined KRAS subgroups exhibited significantly different median PFS to immunotherapy (KP: 18 weeks, KL: 6 weeks, K: 16 weeks, P=0.0014, log-rank test). Objective responses were observed in 9/17 (52.9%) KP and 3/12 (25%) K tumors compared to 0/6 (0%) KL tumors (P=0.049, Fisher’s exact test, 2x3 contingency table). In the PROSPECT cohort of surgically resected LUACs with available whole exome sequencing data, somatic mutation in STK11 was associated with reduced intra-tumoral densities of CD3+ (P=0.0016), CD8+ (P=0.0125) and CD4+ (P=0.0036) lymphocytes.
Conclusion:
Mutations in STK11/LKB1 are associated with an inert tumor immune microenvironment and poor clinical response of KRAS-mutant LUAC to immune checkpoint blockade. The mechanism that underlies this phenotype and strategies to overcome it are under investigation. The impact of additional co-mutations on the immune profile and response of KRAS-mutant LUAC to immunotherapy is also being explored.
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OA20 - Immunotherapy and Markers (ID 401)
- Event: WCLC 2016
- Type: Oral Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:M. Früh, C.S. Baldotto
- Coordinates: 12/07/2016, 11:00 - 12:30, Stolz 2
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OA20.05 - The Influence of Neoadjuvant Chemotherapy, on Immune Response Profile in Non-Small Cell Lung Carcinomas (ID 5738)
11:35 - 11:45 | Author(s): E.R. Parra
- Abstract
- Presentation
Background:
The clinical efficacy observed with PD-1/PD-L1 inhibitors in non-small cell lung carcinoma (NSCLC) has prompted to characterize the immune response in lung tumors treated with chemotherapy. Our goal was to determine the characteristics of immune microenvironment of localized, surgically resected, NSCLCs from patients who received and did not receive neo-adjuvant chemotherapy. Using multiplex immunofluorescence (mIF) and image analysis, we investigated PD-1/PD-L1 expression, and quantified tumor infiltrating lymphocytes (TILs) and tumor associated macrophages (TAMs).
Methods:
We studied formalin-fixed and paraffin embedded (FFPE) tumor tissues from 111 stage II and III resected NSCLC, including 61 chemonaïve (adenocarcinoma, ADC=33; squamous cell carcinoma, SCC=28) and 50 chemotherapy-treated (ADC=30; SCC=20) tumors. mIF was performed using the Opal 7-color fIHC Kit™ and analyzed using the Vectra™ multispectral microscope and inForm™ Cell Analysis software (Perkin Elmer, Waltham, MA). The markers studied were grouped in two 6-antibody panels: Panel 1, AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), CD3, CD4, CD8 and CD68; and Panel 2, AE1/AE3, PD1, Granzyme B, FOXP3, CD45RO and CD57.
Results:
Positive PD-L1 expression (>5%) in malignant cells (MCs) was detected in 48% (n=53/111) of NSCLCs. Overall, chemotherapy-treated tumors showed significantly higher percentages of MCs expressing PD-L1 (median, 18.2%) than chemo-naïve cases (median, 1.8%; P=0.033). Higher densities of inflammatory cells expressing granzyme B (P=0.036), CD57 (P=0.001) and PD-1 (P=0.016) were detected in chemotherapy-treated NSCLCs compared with chemo-naïve tumors. In contrast, lower densities of FOXP3-positive regulatory T cells were detected in chemotherapy-treated tumors when compared with chemo-naïve cases (P=0.032). Following chemotherapy ADCs exhibited significantly higher levels of CD57-positive cells (P<0.0001) and lower density of FOXP3-positive cells (P=0.002) than chemo-naïve tumors. Chemotherapy-treated SCCs demonstrated higher density of PD-1-positive cells than chemo-naïve tumors (P=0.004). In chemotherapy-treated cancers, lower levels of CD4 helper T positive cells and tumor associated macrophages (TAMs) CD68-positive cells were associated with worse overall survival (OS; P=0.04 and P=0.005, respectively) in univariate analysis. In chemotherapy-treated ADC patients, lower levels of CD68-positive (P=0.010) and higher levels of FOXP3-positive cells correlated with worse OS (P=0.044).
Conclusion:
We developed a robust mIF panel of 10 markers to study inflammatory cells infiltrates in FFPE NSCLC tumor tissues. Chemotherapy-treated NSCLCs exhibited higher levels of PD-L1 expression and T cell subsets compared to chemo-naïve tumors, suggesting that chemotherapy activates specific immune response mechanisms in lung cancer. (Supported by CPRIT MIRA and UT Lung SPORE grants, and MD Anderson Moon Shot Program).
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-035 - Protein and Molecular Alterations in EMT Pathways of Lung Cancer: A Comparative Analysis between NSCLCs (ID 5537)
14:30 - 14:30 | Author(s): E.R. Parra
- Abstract
Background:
The adoption of next-generation sequencing (NGS) may help to identify single nucleotide variants (SNVs), small insertions–deletions (indels), and larger structural variations including chromosomal rearrangements. Many molecular alterations have protein-level associations that can be questioned using immunohistochemistry (IHC). The goal of our work was investigated molecular patterns of predictive biomarkers and new genes involved as potential therapeutic targets with an emphasis on protein IHC and their translational promise.
Methods:
We studied 212 formalin fixed and paraffin embedded tissues: 8 high-grade and 20 low-grade neuroendocrine carcinomas(NEC), 102 adenocarcinomas(ADC), 65 squamous cell carcinomas (SCC) and 17 large cell carcinomas(LCC), placed in tissue microarrays(TMAs). EGFR,P53,KRAS,ALK,ERBB2,PTEN,BRAF,VEGF,CD24 and CD44 were examined using IHC and Aperio system. DNA extracted(QIAmp) from a subset was used to analyze several variants including EGFR, ERBB2, PIK3CA, MMP2, SNAI, VGFA, VIM, ZEB1, AXL, CD44, CD276, and CDH1, using the TruSeq Custom Amplicon assay on the Illumina MiSeq System, resulting data set for 80 LC were analyzed using the Variant-Studio software and correlated them with the clinocopathological data.
Results:
The median age of the patients was 64 yrs (minimum-maximum, 24-88yrs). The population included 98(44.5%) women; 32(14,5%) never smokers and 100(45,5%) former smokers; only 2(1%) Asians. Our image analysis showed that the median IHC protein expressions were similar between low and high-grade NEC, but those were different compared to others (P<0.05). Indeed, EGFR, EBB2, P53 and BRAF IHC expression were significantly lower in NEC group compared to other subtypes (P<0.05). Overall, LCC have lower protein expression than ADC and SCC, specially P53 and VEFG. We detected several drivers mutation including EGFR 22%(19/80), ERBB2 2%(5/80), immune regulated genes CD276 6.8%(17/80) and CTLA 15.4%(39/80). We observed also EMT gene mutations as CD44 30.7%(78/80), MMP2 2.8%(7/80), VGFA 2%(5/80), CDH1 2.4%(6/80), SNAI 2.8%(7/80), VIM 1.2%(3/80), and ZEB1 4%(12/80). Interestingly, a significant higher AXL and CD44 gene expression was found in ADC and SCC specimens compared to NEC(P=0.001 and P=0.04,respectively) Similar to the protein expression in overall low gene expressions was also observed in LCC compared to others.
Conclusion:
We detected different patterns of protein and gene alteration in LC with predominant low expression in NEC. Furthermore, the high expression of EMT genes as AXL and CD44 observed among ADC and SCC can be a evidence that those genes might be a distinctive RTK in these tumor than in NEC tumor suggesting that targeting these genes will be benefit as anti-cancer treatment.
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P2.01-061 - Image Analysis-Based Expression of Nine Immune Checkpoints Identifies Distinct Immunoprofiling Patterns in Non-Small Cell Lung Carcinomas (ID 5548)
14:30 - 14:30 | Author(s): E.R. Parra
- Abstract
Background:
The understanding of the co-expression of immune checkpoints in non-small cell lung carcinoma (NSCLC) is important to potentially design combinatorial immunotherapy approaches in this disease. We examined the expression of a panel of immune checkpoints markers by immunohistochemistry (IHC) and quantitative image analysis in a large cohort of surgically resected NSCLCs, and correlated those findings with patients’ clinicopathological features and tumors’ inflammatory cells infiltrate and molecular characteristics.
Methods:
We studied 225 formalin fixed and paraffin embedded (FFPE) tumor tissues from stage I-III NSCLCs, including 123 adenocarcinomas (ADC) and 83 squamous cell carcinomas (SCC), placed in tissue microarrays (TMAs). Nine immune checkpoints markers, 4 (PD-L1, B7-H3, B7-H4, IDO1) expressed predominantly in malignant cells (MCs), and 5 (ICOS, VISTA, TIM3, LAG3 and OX40) expressed mostly in stromal tumor associated inflammatory cells (TAICs). All IHC markers were examined using quantitative image analysis system (Aperio).
Results:
Using > median value of the immune checkpoint expressions as positive expression we observed that MCs H-score expressing PD-L1, B7-H3, B7-H4 and IDO1was higher in SCC than ADC, with 3 out of 4 markers showing statistically significant (P<0.05) differences. In contrast, density of TAICs expressing ICOS, VISTA, OX40, LAG3 and TIM3 was higher in ADC than SCC, with 3 out of 5 markers demonstrating significant (P<0.05) differences. Furthermore, we identified frequent co-expression of markers: a) 11% ADC (13/123) and 10% SCC (8/83) co-expressed 8 to 9 markers; b) 45% ADC (55/123) and 32% SCC (27/83) co-expressed 6 to 7 markers, c) 28% ADC (35/123) and 40% SCC (33/83) co-expressed 4 to 5 markers, and d) 16% ADC (20/123) and 18% SCC (15/83) co-expressed 2 to 3 markers. In ADC, higher number of TAICs expressing OX40 and lower levels of MCs expressing B7-H4 were detected in tumors with EGFR (median, 7.49 vs. 1.16, P=0.021) and KRAS (median, 6.88 vs. 0.67, P=0.033) mutation compared with wild-type tumors, respectively. Univariate analysis demonstrated that high B7-H4 and low OX40 expression in MCs and in TAICs respectively correlated with worse overall survival (OS; P=0.016 and P=0.037, respectively) in ADC patients.
Conclusion:
We detected different patterns of immune checkpoints expression in NSCLC with higher level of markers found in malignant cells of SCC and in stromal inflammatory cells of ADC. Immune checkpoints expression correlated with the outcome of NSCLC patients. Importantly, co-expression of several immune checkpoints is a frequent event in NSCLC (Supported by CPRIT MIRA and UT Lung SPORE grants).
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P2.03b - Poster Session with Presenters Present (ID 465)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.03b-034 - Clinical Relevant Oncogenic Drivers in Advanced Adenocarcinoma Discloses New Therapeutic Targets in Negative EGFR/ALK/KRAS Patients (ID 5541)
14:30 - 14:30 | Author(s): E.R. Parra
- Abstract
Background:
The mutation profile in the brazilian population with advanced lung adenocarcinoma remains largely unexplored and also their relationship to many other genes. Next Generation Sequencing(NGS) allows higher sensitivity and multiplexing for several genes for translational research
Methods:
80 lung adenocarcinoma patients were collected. DNA concentration and quality was determined by Qubit2.0fluorometer and Agilent2100Bioanalyzer. Genomic libraries were constructed using the TruSeq®Custom Amplicon v1.5) comprising 764 amplicons of 38genes on the Illumina-MiSeq®sequencing plataform.
Results:
The 7362 genetic mutation were observed with 78% of single-nuclotide variants (SNVs) and 22% insertions and deletions. The majority of the SNVs were located in inter-genic regions or introns. EGFR were mutated in 21(6%) of patients with 19 (57%) of mean expression. The most frequent EGFR-mutation was exon 19deletions, followed by L858R amino acid substitution in exon 21. KRAS was mutated in 26 (4%) of patients. ALK rearrangement was detected in 6 patients (4.8%). The stop gained mutation was present in PIK3CA,TP53,AXL,EGFR,RAB25,CDH1,CD276 and TGFB1. The AXL receptor tyrosine kinase gene showed 11 missense-mutations, of which 7 are considered possibly damaging (Polyphen)/deleterious(SIFT)(74/79) and 14 intronSNVs(49/80). CD44 showed 50 variants, however most of them have an undetermined significance. The clustering analysis demonstrated that a select group of AXL-related gene alterations was highlighted(Fig 1). Figure 1
Conclusion:
The results suggest that genomic variants in lung adenocarcinoma tissues are complex and show that NGS is an effective way to detect novel mutations in lung cancer. 58% of patients wild type by standard testing for EGFR/KRAS/ALK have genomic changes identifiable by CGP that suggest benefit from target therapy. The AXL and CD44 genes remain a relatively unexplored target, thus we intend to increase the available data for the true translational potential of target AXLand CD44 therapy in lung cancer. CGP used when standard molecular testing for adenocarcinoma is negative can reveal additional avenues of benefit from targeted therapy.
