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V.L. Capelozzi
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MTE24 - Immunohistochemical Assessment of Biomarkers for Immune Checkpoint Inhibitors (Ticketed Session) (ID 317)
- Event: WCLC 2016
- Type: Meet the Expert Session (Ticketed Session)
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 07:30 - 08:30, Schubert 4
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MTE24.01 - Immunohistochemical Assessment of Biomarkers for Immune Checkpoint Inhibitors (ID 6582)
07:30 - 08:30 | Author(s): V.L. Capelozzi
- Abstract
- Presentation
Abstract:
Immune checkpoint inhibitors in cancer immunotherapy. Programmed death receptor-1 (PD-1) is a type 1 membrane protein of the immunoglobulin superfamily that has an important role in restrincting immune-mediated tissue danage secondary to inflammation and/or infection (1). The clinical advantage of antibodies that target either PD-1 or PD-L1 to block this ligand-receptor interface, allowing cancer killing by T cells became clear when CTLA4, an antagonist against the T-cell, such as ipilimumab, and afterward PD-1, showed an increase survival in patients with metastatic melanoma (2). Clinical investigations in lung cancer have demonstrated the benefit of PD-1 inhibitors pembrolizumab in advanced non–small cell lung cancer (NSCLC) and nivolumab in advanced squamous and nonsquamous NSCLC; both approved as second-line therapies by the US Food and Drug Administration (FDA) (3-5). Others PD-L1 inhibitors such as atezolizumab and durvalumab have demonstrated effectiveness in several tumor types (6-7) but they were not approved for clinical use until now. PD-1 inhibitors induce around of 20% of complete response frequency in patients with NSCLC, and persistent response in a subgroup of patients treated by immune checkpoint inhibitors. Garon et al (3) showed that tumors with PD-L1 expression ≥ 50% by immunohistochemistry (IHC) were significantly more expected to respond to pembrolizumab than those with less than 50% malignant cell expression. In contrast, response rates to nivolumab are significantly greater in patients with nonsquamous NSCLC, showing ≥ 1% tumor cell positivity (5). These differences are related to the combination of antibody clone and detection system as a companion diagnostic for selecting lung cancer patients for pembrolizumab therapy. Previous investigations reported response taxes in PD-L1–positive tumors of 31% to 52%, but particularly more than 16% of PD-L1–negative tumors also showed treatment response (1). This finding indicates that PD-L1 expression improves for responders but the absence of expression is not a complete indicator of advantage. PD-L1 expression did not predict differential response to nivolumab in lung squamous cell carcinoma as compared with docetaxel (4).Immunohistochemical Assessment of Immune Checkpoint Inhibitors. PD-L1 in NSCLC is expressed on the membrane of tumor cells, and/or on immune infiltrating cells dendritic cells, antigen-presenting cells and T lymphocyte. PD-1, the PDL1 receptor, is expressed on tumor infiltrating lymphocytes, mainly CD4 T cells, T and B regulatory, NK, monocytes and DC. Concerning PD-L1 binding, PD-1 inhibits kinases involved in T cell activation. Two potential mechanisms are involved in expression of immune checkpoints on tumor cells and their immune stromal component: oncogenic signaling, and response to inflammatory signals (8). Tumor cells express multiple ligands and receptors and antitumor immune response can be enhanced by multi-level blockade of immune checkpoints. PD-1/PD-L1 commitment leads to HSP-2 phosphatase activity which dephosphorylates Pi3K and thus downregulate AKT (8). The positive score on tumor cells has not been evaluated nor enhanced or standardized (3; 8). Brambilla and Ming (8) assessed a score of positivity for prognosis analysis using E1L3N Cell Signaling antibody commercially available. They found that 20% of lung tumors cell expressed PD-L1 (≥ 20% intensity 2+3+), and 29% the immune stromal cells (T, macrophages, DC ) ≥ 10% intensity 2+3+. PD-L1 positivity in both tumor and immune cells were seen in only 9% of NSCLC, 20,7% were both negative. There was no prognostic relevance of PD-L1 (tumor cells or stroma) whatever cut off by 10% increment or linear scoring was used. Only immune PD-L1 expression was correlated with a highly intense immune infiltrations. Previous published evaluations of prognostic value were discordant likely because immune checkpoints modulators play both positive and negative roles in the immune inhibitory pathways with some redundancy, and patients series and assays were not comparable. The two meta-analyses with different antibodies, cutoffs, patient series, ethnicities and contribution of oncogene driven cancers, initial resection sample or contemporary biopsy rendered their interpretation extremely problematic. Global result was supporting a poor prognosis of “PD-L1 positivity” on tumor cells.PD-L1 as a Predictive Biomarker for Checkpoint Inhibitors. Most of phase I trials works with four antibodies targeting PD-1 or its primary ligand PD-L1, response taxes appear higher in patients with increased tumor PD-L1 membrane expression by IHC. However, different antibody assays, absence of standardization, different score to determine PD-L1 positivity, companion test type, and a short number of specimens available for testing, accopled to the variability of the intervals between biopsy and test, has certainly disadvantaged the conclusion and prevent consensus to be reached (10). The best threshold was provided by Garon et al, with ≥ 50% of tumor cells PD-L1 positive to allow the highest response rate of 45% to pembrolizumab (3). In most trial series, biopsies or resected specimen were used and considerable difference between these samples occurs due to tumor heterogeneity. The reliability of small biopsy samples is questioned (10). Indeed lung tumor heterogeneity is characteristic and PD-L1 is typically heterogeneous in its distribution in the tumor majority as is PD-L1 positive immune cells. Multiple questions are still addressed before PD-L1 is considered as a definitive molecular predictor of effectiveness. As for prognostic evaluations, thresholds of ≥ 1%, ≥ 5%, ≥ 10%, ≥ 50% or continuous H score have been used. In addition, in a few trials, PD-L1 expression in TILs was predictive more than PD-L1 on tumor cells but the best cut off was not revealed.Conclusion. PDL1 expression predicts response to immune checkpoint inhibitors. Concordant results showing a better response if PDL1 + in several trials, using drug specific test and for Nivolumab also histology specific. We should evaluate membranous staining in tumor sample with at least 100 tumors cells and immune cells. Perspective for upgrading includes: 1) heterogeneity of the expression of PDL1 within tumor, primitive vs metastases number and size of samples; 2) surgical tissue versus biopsy and 3) archival versus new biopsy and 4) standardize the assays. Published abstracts showed high rates of concordance between primary and metastases (81%). Obtaining multiple biopsies from different areas of the tumor would enhance the validity of the results of IHC evaluation (160 patients=48% discordance).References 1. Sholl LM, Aisner DL, Allen TC, Beasley MB, Borczuk AC, Cagle PT, Capelozzi V, Dacic S, Hariri L, Kerr KM, Lantuejoul S, Mino-Kenudson M, Raparia K, Rekhtman N, Roy-Chowdhuri S, Thunnissen E, Tsao MS, Yatabe. Programmed Death Ligand-1 Immunohistochemistry--A New Challenge for Pathologists: A Perspective From Members of the Pulmonary Pathology Society. Arch Pathol Lab Med. 2016;140(4):341-4. 2.Couzin-Frankel J. Breakthrough of the year 2013: cancer immunotherapy. Science 2013;342:1432–1433. 3.Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non–small-cell lung cancer. N Engl J Med 2015;372:2018–2028. 4.Brahmer J, Reckamp KL, Baas P, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med 2015;373:123–135. 5.Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med 2015;373: 1627–1639. 6. Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 2014; 515:563–567. 7.Stewart R, Morrow M, Hammond SA, et al. Identification and characterization of MEDI4736, an antagonistic anti-PD-L1 monoclonal antibody. Cancer Immunol Res 2015;3:1052–1062. 8. Brambilla E, Le Teuff G, Marguet S, Lantuejoul S, Dunant A, Graziano S, Pirker R, Douillard JY, Le Chevalier T, Filipits M, Rosell R, Kratzke R, Popper H, Soria JC, Shepherd FA, Seymour L, Tsao MS. Prognostic Effect of Tumor Lymphocytic Infiltration in Resectable Non-Small-Cell Lung Cancer. J Clin Oncol. 2016;34:1223-30. 9. Soria JC, Marabelle A, Brahmer JR, Gettinger S. Immune checkpoint modulation for non-small cell lung cancer. Clin Cancer Res. 2015;21: 2256-62. 10. Kitazono S, Fujiwara Y, Tsuta K, Utsumi H, Kanda S, Horinouchi H, Nokihara H, Yamamoto N, Sasada S, Watanabe S, Asamura H, Tamura T, Ohe Y. Reliability of Small Biopsy Samples Compared With Resected Specimens for the Determination of Programmed Death-Ligand 1 Expression in Non--Small-Cell Lung Cancer. Clin Lung Cancer 2015;16:385-90.
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P1.07 - Poster Session with Presenters Present (ID 459)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: SCLC/Neuroendocrine Tumors
- Presentations: 2
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.07-027 - 13-Gene Signature of EMT Reveals Impact on Invasion and Metastasis of Neuroendocrine Carcinomas of the Lung: A Preliminary Study (ID 5466)
14:30 - 14:30 | Author(s): V.L. Capelozzi
- Abstract
Background:
Metastasis are responsible for the death of 90% of patients with lung cancer, indicating the need to know the multiple signaling pathways involved. Neuroendocrine lung carcinomas (NELC) encompass a wide spectrum of tumors, from the low-grade typical carcinoid (TC) and atypical carcinoid (AC), to the high-grade large cell neuroendocrine carcinoma (LCNEC) and the small cell lung carcinoma (SCLC). Low-grade NELC are indolent, while high-grade NELC invade and metastasize rapidly. Biomarkers of NELC aggressiveness remain to be determined. Epithelial to mesenchymal transition (EMT) genes profile emerge promise as indicator of invasion and metastasis. Our aim was to evaluate: (1) EMT gene expression in NELC; (2) its relationship with the histologic subtypes and (3) its impact on behavior of the tumors.
Methods:
Patients with SCLC (n = 10), LCNEC (n=5), AC (n=2) and TC (n=7) were included, EMT gene expression was quantified with a quantitative real-time (RT)- PCR carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems), with RT2 Profiler PCR Array System for EMT (Qiagen, Dusseldorf, Germany). Associations of the gene signature and clinicopathological features, as well as prognostic factors were evaluated.
Results:
A 13-gene signature (AHNAK, COL3A1, DSP, IL1RN, MSN, PDGFRB, SNAI1, SNAI3, TCF3, TGFβ1, TGFβ2, TGFβ3 and VIM) that was related to EMT was up-regulated in tumor-tissue from all NELC patients, mainly in those with high-grade NELC. An increased expression of DSP, TCF3 and TGFβ3 was found in SCLC compared to AC, TC and LCNEC, and associated with lymph nodes metastasis with statistical significance respectively for DSP (p=0.03 and 0.02), TCF3 (p=0.02) and marginal significance for TCF3 and TGFβ3 (p=0.08 and p=0.08). TCF3 was also associated with tobacco history (p=0.04). A significant correlation was found between enolase and IL1RN (p=0.03), chromogranin and TGFβ2 (p=0.04), synaptophysin and TGFβ1 and TGFβ2 respectively (p=0.04 and p=0.02).
Conclusion:
The EMT analysis identified genes involved in cell proliferation, motility, invasion and metastasis of NELC. We further inferred DSP, TCF3 and TGFβ3 as target against lung cancer metastasis and invasion, thus arising as promising therapeutic agent.
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P1.07-030 - Gene Signature of EMT in Neuroendocrine Lung Carcinoma: A Comparative Analysis with Adenocarcinoma and Squamous Cell Carcinoma (ID 5366)
14:30 - 14:30 | Author(s): V.L. Capelozzi
- Abstract
Background:
Recurrence and metastasis are responsible for 90% of the death of patients with lung cancer. Adenocarcinomas (ADc) primarily invade blood vessels with distant metastasis, whereas squamous cell carcinoma (SqCC) involves the mediastinal lymph nodes. Neuroendocrine carcinomas of low-grade (typical and atypical carcinoid) are indolent, while high-grade NE carcinoma (large cell NE and small cell carcinomas) metastasize rapidly. Biomarkers of invasiveness in lung carcinomas still cannot be definitely determined. Epithelial to mesenchymal transition (EMT) genes profile emerge promise as indicator of invasion and metastasis. Our aim was to compare EMT gene expression in NELC, ADc and SqCC and its impact on behavior of these tumors.
Methods:
EMT gene expression was quantified with a quantitative real-time (RT)- PCR carried out on StepOnePlus™ Real-Time PCR System (Applied Biosystems) with RT2 Profiler PCR Array System for EMT (Qiagen, Dusseldorf, Germany).
Results:
Younger patients expressed higher amount of AHNAK, IL1RN, MSN, TCF3 and VIM than older (p<0.05), whereas SNAI3 and TGFβ2 were more expressed in smokers (p<0.05). ADc and SqCC presented significant higher expression of COL3A1, DSP and MSN in tumor compared to normal tissue (p<0.05). 13-gene signature (AHNAK, COL3A1, DSP, IL1RN, MSN, PDGFRB, SNAI1, SNAI3, TCF3, TGFβ1, TGFβ2, TGFβ3 and VIM) was up-regulated in tumor-tissue from all NELC patients. In ADc and NELC, AHNAK, IL1RN, TCF3 and VIM was significantly different (p<0.05). ADc and SqCC compared with high-grade NELC also presented differences in COL3A1 (p<0.01). Interestingly, only NELC expressed PDGFRB, SNAI1, SNAI3, TCF3, TGFβ1, TGFβ2, TGFβ3. Advanced tumors, usually with metastasis, showed higher expression of AHNAK, DSP, IL1RN, MSN and VIM (p<0.05), as well as association with poor outcome (p <0.01).
Conclusion:
Different expression of EMT gene signature in endocrine and non-endocrine lung carcinomas, its relationship with histologic types, advanced stage, lymph node metastasis and death suggest a possible role of these markers in this malignancy, but more importantly provide a potential biomolecular marker to predict outcome. The correlation between NELC, ADc, SqCC and specific EMT genes involved in cell proliferation and motility provides a possible role of these genes on the development and aggressiveness in these tumors. Moreover, the specific genes expressed only in NELC emerges as promise biomarker of behavior. Further studies are needed to validate EMT gene expression to predict prognosis and tumoral aggressiveness.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- 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): V.L. Capelozzi
- 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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P2.03b - 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): V.L. Capelozzi
- 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.
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P3.02c - Poster Session with Presenters Present (ID 472)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02c-009 - Anti-VEGF and Anti-EGFR Reduce Malignant Pleural Effusion and Morbidity in an Experimental Adenocarcinoma Model (ID 5501)
14:30 - 14:30 | Author(s): V.L. Capelozzi
- Abstract
Background:
Lung cancer is a main cause of death by cancer worldwide and adenocarcinoma the most common cell type. Most of patients present pleural effusion at an advanced stage of the disease with high morbidity and mortality, however its pathogenesis is still poorly understood and therapeutic options are limited. OBJECTIVE: Evaluate the effects of intrapleural anti-VEGF and anti-EGFR in malignant pleural effusion induced in an experimental model.
Methods:
One hundred and twenty C57BL/6 mice received intrapleural injection of 0.5x10[5] of LLC cells and were divided into four groups that received, after 3, 7, 10 and 14 days, anti-VEGF, anti-EGFR, anti-VEGF+anti-EGFR or PBS (control) intrapleurally. Ten animals for each group were followed until death to evaluate the survival curve. Eighty animals were euthanized after 7, 10, 14 or 21 days after LLC injection and had weight (g), mobility (score 0-3), pleural fluid volume evaluated. Presence of tumor in pleura and pericardium, inflammatory cells in lung parenchyma, histological changes in kidney, liver and spleen and tumor apoptosis (TUNEL) and proliferation (PCNA) were evaluated by score (0-4). Statistical analysis: One Way ANOVA, Kaplan–Meier curve, p<0.05.
Results:
In the survival analysis, pleural carcinomatosis was lethal showed maximum survival of 25 days without statistical differences among groups (p=0.739). Reduction of body weight mice was observed in all groups after 21 days (p<0.05). However, the animals mobility was better in the groups that received anti-EGFR (p=0.026). The fluid volume was higher in all control groups no matter the study time (p=0.010). Tumor implants in the pleura were more evident in control groups compared to treated groups after 14 days (p=0.001). Neoplastic infiltration of lung parenchyma was observed only in a few animals. However, lung parenchymal inflammation was minimal in all groups. Histological evaluation of pericardium and heart muscle showed tumor implants mainly in the 21-day in the control group. In liver and kidney steatosis were observed after 14 days in control group (p<0.001). Hyperplasia of the white pulp of the spleen was observed at all evaluation time points with greater evidence at 21-day in the control group (p<0.001). High scores of apoptosis and lower scores of tumor proliferation were observed in the groups that received treatment with anti-EGFR and anti-VEGF+antiEGFR.
Conclusion:
In this experimental model, the target therapies reduced significantly the pleural fluid volume, morbidity and histological parameters mainly in the therapies with EGFR, although its action did not increased the survival of the animals.
