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B. Sepesi
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OA19 - Translational Research in Early Stage NSCLC (ID 402)
- Event: WCLC 2016
- Type: Oral Session
- Track: Early Stage NSCLC
- Presentations: 1
- Moderators:G. Heller, G. Goss
- Coordinates: 12/07/2016, 11:00 - 12:30, Schubert 3
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OA19.02 - Sex Differences Are Detected in the Profile of Tumor Associated Inflammatory Cells (TAICs) Are Lung Adenocarcinoma (ID 5451)
11:10 - 11:20 | Author(s): B. Sepesi
- Abstract
- Presentation
Background:
A number of studies have characterized TAICs in lung cancer and associated their levels of infiltration with patients’ outcome. There is limited information about the correlation of TAICs infiltration with clinical and pathological features of lung cancer. We investigated the association between patterns of tumor infiltrating lymphocytes and macrophages with detailed clinical and pathological features in lung adenocarcinoma.
Methods:
We studied archival tumor tissue from 93 surgically resected lung adenocarcinomas, stages I to III. Density of TAICs expressing CD3, CD4, CD8, and CD68 was evaluated using immunohistochemistry (IHC) and image analysis. TAICs density was correlated with tumor’s histological characteristics and patients’ characteristics.
Results:
We found significant differences in the TAICs infiltrate density of lung adenocarcinomas based on patients’ characteristics. Overall: a) females showed higher levels of CD8+ (P=0.01) and CD3+ (P=0.03) cell density than males; b) smaller tumors (<3cms) showed more CD4+ (P=0.01) and CD3+ (P=0.03) cells than larger tumors; and, c) tumors with solid histology pattern showed higher levels of CD8+ (P=0.03) cells than non-solid pattern. No overall significant differences on TAICs infiltrates were detected by age, tobacco exposure by pack-years and TTF-1 IHC expression score. However when TAICs density of tumors was examined by sex we found the following: a) in larger tumor (>3cms), females demonstrated higher levels of CD8+ cells (P=0.0007) than males; b) tumors from females older than the median age (63 years) showed more CD4+ (P=0.04), CD8+ (P=0.009) and CD3+ (P=0.042) cells than males; c) tumors from females with <40 pack-years of tobacco history showed significantly higher levels of CD3+ (P=0.004) and CD68+ (P=0.004) cells than males; d) tumors from females with high levels of TTF1 expression (score >150) showed higher levels of CD8+ cells(P=0.03) than males; e) females with tumors having non-solid histology pattern showed higher CD8+ (P=0.02) and CD3+ (P=0.02) cells than males. Finally, tumors expressing low levels of TTF-1 and lower CD4+ cells correlated significantly with worse overall recurrence free survival and overall survival in both males (P<0.0001) and females (P=0.0072).
Conclusion:
In lung adenocarcinoma, TAICs infiltration correlates with clinical characteristics of patients and pathological features of tumors, particularly, sex, age, size and TTF-1 expression. Compared with men, lung adenocarcinomas from females showed higher levels of TAICs, particularly at older age, larger tumor size, less exposure to tobacco, and more differentiated histological patterns. (UT Lung SPORE and MD Anderson Moon Shot Program).
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OA20 - Immunotherapy and Markers (ID 401)
- Event: WCLC 2016
- Type: Oral Session
- Track: Biology/Pathology
- Presentations: 2
- 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): B. Sepesi
- 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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OA20.06 - Prospective ImmunogenomiC PrOfiling of Non-Small Cell Lung Cancer - The ICON Project (ID 5560)
11:45 - 11:55 | Author(s): B. Sepesi
- Abstract
- Presentation
Background:
Previous attempts to define tumor and stromal immunologic environment in non-small cell lung cancer (NSCLC) utilized archival tissue. We established prospective comprehensive immonogenomic profiling protocol in NSCLC (ICON Project). The goal is to integrate immunomic, genomic, transcriptomic, proteomic, demographic, clinical, pathologic, and outcome data from 100 surgically resected early stage NSCLC.
Methods:
Tumor and normal lung tissue are collected at the time of surgery, blood samples before and after surgery. Tumor samples are processed for tumor infiltrating lymphocyte (TILs) isolation and expansion; development of patient derived xenografts (PDX), immunohistochemical immune markers, and immunopeptidome profiling. Blood samples are analyzed with flow cytometry.
Results:
57 patients with median age of 65 years (27 males) have been enrolled within 5 months, of which 33 (66%) contributed samples to the study. Four were never smokers, with others being former or current smokers. Majority (N=27) had adenocarcinoma, 4 squamous cell carcinoma, and 2 pleomorphic carcinoma. 15 patients had stage I, 11 stage II, and 7 stage III disease; 5 patients received induction chemotherapy. Median tumor size was 3.5 cm and 29 underwent R0 and 4 R1 resection. Pre-REP TIL expansion was successful in the majority of samples (68.2%, n=22). Twelve PDX models with a take rate of 40% have been generated. Interim analysis of tumor samples by IHC demonstrated higher median distribution of all cell types: CD3+ T cells, cytotoxic T cells CD8+, PD1+ cells, tumor associated macrophages (TAM) CD68+, TAM CD68+PD-L1+, CD20+B cells, memory T cells CD45R0, natural killer cells CD57+, regulatory FOXP3+ T cells, and cytotoxic granzyme B cells (cells/mm[2]) in the stroma as compared to the tumor compartment. Intra-tumoral regulatory FOXP3+T cells were more abundant in squamous cell carcinomas compared to adenocarcinomas (median 312 vs 51 cells/mm[2], p = 0.05). Higher concentration of intra-tumoral CD68+PD-L1+ expressing cells was observed following neoadjuvant chemotherapy (median 97 vs 60 cells/mm[2] no chemo; p= 0.077), as was the concentration of memory T cells CD45R0 (median 129 vs 30 cells/mm[2], no chemo; p = 0.077). Mass spectrometry-based immunopeptidome analysis identified several thousand peptides, of which 4 promising antigens have been chosen for further development as immunotherapeutic T-cell targets.
Conclusion:
The ICON is an ongoing, ambitious prospective project that aims to define the baseline immunologic characteristics of surgically resectable NSCLC. The rapid enrollment illustrates the enthusiasm for tumor immunoprofiling amongst patients and physicians alike. Data from this patient cohort will serve as a baseline comparison for upcoming neoadjuvant immunotherapy trials.
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P1.05 - Poster Session with Presenters Present (ID 457)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Early Stage NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.05-028 - Phenotypic and Functional Profiling of Tumor-Infiltrating Lymphocytes (TIL) in Early Stage Non-Small Cell Lung Cancer (NSCLC) (ID 6044)
14:30 - 14:30 | Author(s): B. Sepesi
- Abstract
Background:
The ICON study aims to perform a comprehensive immunogenomic characterization of early stage localized non-small cell lung cancer (NSCLC) and lay the foundation for the identification of barriers to tumor immunity that may be targeted in future trials. Earlier work has demonstrated the prognostic significance of TIL in localized NSCLC. This work evaluates the functional status of T cells infiltrating the NSCLC tumors and their capacity to expand ex-vivo and perform effector function in the first 22 patients enrolled.
Methods:
Patients enrolled on the ICON study underwent surgery. Fresh tumor samples were mechanically disaggregated and immediately stained for flow cytometry. Panels consisted of markers of T cell subsets, differentiation status, T cell function, activation and exhaustion. PD-L1 expression was assessed on malignant cells as well as CD68+ tumor-associated macrophages (TAMs) by IHC. Fragments from the tumor tissue were also placed in culture in media containing IL-2 for ex-vivo TIL expansion. TIL cultures were maintained for 3-5 weeks and subsequently underwent phenotypical and functional characterization.
Results:
Analysis of freshly disaggregated tumor tissue (n=22) from NSCLC tumor or adjacent normal tissue by flow cytometry demonstrated that effector CD8[+] T cells found in the tumor were less functional than T cells infiltrating normal tissue, revealed by a decrease in the co-expression of the cytotoxic effector molecules perforin and granzyme B (p=0.0004) together with an enhanced expression of the inhibitory receptor PD-1 (p<0.0001). Immunohistochemistry analysis showed PD-L1 expression on malignant cells and/or CD68+ TAMs on all tumor samples except one, strongly suggesting that the PD-1/PD-L1 inhibitory axis was engaged contributing to decreased T cell functionality. TIL could be expanded from the majority of samples (68.2%, n=22). The degree of infiltration predicted the ability to grow TIL ex-vivo (median CD3+ infiltrate of 15.25% of live cells in disaggregated tumor tissue for samples from which TIL could be grown versus 2.9% when TIL could not grow p = 0.015). Immunophenotyping following expansion showed an enrichment in CD8+ aβTCR+ T-cells expressing both perforin and granzyme B indicating that TILs propagated with IL-2 regained functionality (p=0.016). Lastly, NSCLC TIL were rapidly expanded using anti-CD3 antibody, feeder cells and IL-2 over two weeks (n=6) and reached clinically relevant numbers for TIL ACT (range 381-1282 fold expansion).
Conclusion:
Overall, while TILs present in NSCLC are functionally inhibited, they can be expanded ex-vivo from most tumor samples and regain a functional phenotype for potential use in adoptive T-cell therapy.
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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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P2.01-054 - Lung Cancer PD-L1 mRNA Expression Profile and Clinical Outcomes - An Analysis From The Cancer Genome Atlas and Cancer Cell Line Encyclopedia (ID 5048)
14:30 - 14:30 | Author(s): B. Sepesi
- Abstract
Background:
Programmed death ligand 1 (PD-L1) has become one of the most studied biomarkers in early, and advanced non-small cell lung cancers (NSCLC). Conflicting results have been reported in the literature on the value of PD-L1 in predicting survival in surgically resected lung cancers. Our aim was to evaluate mRNA PD-L1 expression and survival based on the data available from the Cancer Genome Atlas (TCGA), and Cancer Cell Line Encyclopedia (CCLE).
Methods:
To determine the expression profile and clinical correlations of PD-L1 in lung cancers we used publicly available lung adenocarcinoma and squamous cell carcinoma (SCC) data from TCGA, and small cell and NSCLC line data from the CCLE. We performed Kaplan-Meier and correlation analyses to show how PD-L1 expression correlates with overall survival and other clinical variables. Lung cancer and normal tissue expression comparisons were also performed using normal tissue expressions from the Genotype-Tissue Expression (GTEx) project.
Results:
Results: PD-L1 mRNA expression from RNA sequencing was available for 517 lung adenocarcinoma and 501 lung SCC samples in the TCGA. The CCLE database contained PD-L1 expression for 12 small cell and 75 NSCLC cell lines. Lung cancers demonstrated a higher PD-L1 expression than most other cancers and normal tissues, and we found that PD-L1 expression was significantly higher in SCC than in adenocarcinoma (p<0.001). Furthermore, PD-L1 showed a significantly higher expression level in pathologic stage II SCC compared to stages I, III, and IV (p<0.05, p<0.05, p<0.001, respectively). Interestingly, stage IV was associated with a lower PD-L1 expression compared to stages I, II, and III (p<0.001). We did not identify similar expression associations with pathologic stage in adenocarcinoma. However, we found that current and also reformed smokers for less than 15 years had a higher PD-L1 expression in adenocarcinoma (p<0.05), but not in SCC. PD-L1 expression was not significantly associated with a survival difference in any stage or histology subgroups. Using the CCLE data we found that NSCLC cell lines show various expression of PD-L1 that is significantly higher compared to lung small cell carcinoma (p<0.001).
Conclusion:
The value of PD-L1 expression based on mRNA sequencing in predicting survival in anti PD-1 naïve patients appears to be limited. However, the levels of PD-L1 expression in various disease stages and subgroups of lung cancer patients provide rational for neoadjuvant or window-of-opportunity immunotherapy trials, which would enable us to sort out the mechanisms and to identify patients best suited for immunotherapy.
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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): B. Sepesi
- 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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P2.02 - Poster Session with Presenters Present (ID 462)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Locally Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.02-014 - Perioperative Outcomes and Downstaging Following Neoadjuvant Therapy For Lung Cancer – Analysis of the National Cancer Database (ID 4929)
14:30 - 14:30 | Author(s): B. Sepesi
- Abstract
Background:
Administration of chemotherapy prior to surgical resection is one of the strategies for the treatment of locally advanced non-small cell lung cancer (NSCLC). Potential benefits of this approach include improved treatment tolerance, tumor downstaging, and the evaluation of tumor response. Utilizing the National Cancer Database (NCDB), we sought to compare short-term perioperative outcomes and treatment response of neoadjuvant chemotherapy followed by surgery with surgery alone.
Methods:
We queried the NCDB Participant User File (PUF) for patients with clinical stage IB-IIIA NSCLC who underwent definitive surgical resection for NSCLC between 2006-2013. We identified 83,274 patients with complete datasets who met the inclusion criteria. Patients were grouped by stage and perioperative outcomes were assessed, comparing those who underwent neoadjuvant therapy to surgery alone. Neoadjuvant therapy response was assessed by downstaging on final pathology in both unmatched and matched cohorts.
Results:
Neoadjuvant chemotherapy was administered to 11.9% (9,961/83,274) of potentially eligible patients. The incidence of neoadjuvant therapy increased with clinical stage; rates of 2.7% (995/37,453) for IB, 5.4% (724/13,435) for IIA, 15% (2,048/13,619) for IIB, and 33% (6,194/18,767) for IIIA. All cause 30-, and 90-day mortality was 3.1% and 6.3% vs. 3.1% and 6.0% for neoadjuvant vs. surgery alone across all stages, (p=0.159, p<0.001). The unplanned 30-day re-admission rates were 3.8% vs. 4.3% for neoadjuvant vs. surgery alone (p<0.001). Median length of hospital stay was similar between the groups, 7.6 vs. 7.2 days for neoadjuvant vs. surgery alone (p=0.015); stage specific analysis revealed similar results. Overall downstaging was seen in 29.5% in the neoadjuvant group compared to 17% in the surgery group (p<0.001). Primary tumor downstaging occurred in 31.5% vs 9.5% (p<0.001) and nodal downstaging in 23% vs 14.4% (p<0.001) for neoadjuvant and surgery groups respectively. Additionally, significantly improved R0 resection rate was achieved for stages IIIA and IIB in the neoadjuvant group 88.1% and 86.1% vs. 82.0% and 84.5% in the surgery alone group respectively (p<0.001 for IIIA and IIB).
Conclusion:
In this largest review of perioperative outcomes and downstaging effect of neoadjuvant chemotherapy prior to definitive surgical resection for NSCLC, we demonstrate that the treatment strategy of neoadjuvant chemotherapy followed by surgery is safe and effective. Tumor downstaging and increased R0 resection rate in locally advanced lung cancer stages support the utilization of this treatment paradigm.
