Cell Signaling Technology Research Articles

PD-L1 expression in non–small cell lung cancer: evaluation of the diagnostic accuracy of a laboratory-developed test using clone E1L3N in comparison with 22C3 and SP263 assays

Different studies have evaluated the comparability of various immunohistochemical assays for PD-L1 expression evaluation, with contrasting results. Besides the important issues related to analytic performance and comparability of validated assays, not all platforms are available in all laboratories; moreover, standardized assays are very expensive, and funding for PD-L1 testing is hard to obtain, especially in the research setting. One of the most widely used and inexpensive PD-L1 clones is E1L3N (Cell Signaling Technology, Danvers, MA), which is labeled for research use only. In this work, we wanted to further study and validate in a larger cohort the analytical performance of E1L3N clone on Ventana platform (Ventana Medical Systems, Tucson, AZ) and its comparability with assays SP263 and 22C3 run onto their dedicated platforms. Serial sections of tissue microarrays built from 165 cases of resected lung cancer were stained for E1L3N onto Ventana platform following a previously reported protocol and for 22C3 and SP263 assays onto their respective platforms following manufacturer's instructions. Overall, we found very high concordance when comparing E1L3N with SP263 at both 1% and 50% cutoffs. Lower concordance was found between E1L3N and 22C3 at both cutoffs; however, 100% sensitivity was found for E1L3N compared with both SP263 and 22C3 at 50% cutoff. Given the 100% sensitivity at 50% cutoff demonstrated by E1L3N in comparison with both SP263 and 22C3 and therefore the lack of false-negative cases, we propose an algorithm for PD-L1 testing in NSCLC when considering pembrolizumab as first-line therapy.

Prognostic significance of CD73 expression in localized renal cell carcinoma (RCC).

4582 Background: CD73 or ecto-5'-nucleotidase mediates the de-phosphorylation of adenosine monophosphate to adenosine which promotes immunosuppression in the tumor microenvironment. Its prognostic significance in localized RCC has not been well characterized. Methods: We assessed CD73 protein expression using immunohistochemistry (Cell Signaling Technology, D7F9A, 1:25) on tissue microarrays (TMAs) containing tumor tissue from patients with pT1-4,N0-1, M0 RCC who underwent nephrectomy.CD73 expression was quantified using a combined score (CS: intensity x percentage of cells staining positive). CD73 positivity was defined as any CD73 expression on tumor cells (CS > 0). Patients were categorized into CD73 negative (CS = 0), low ( < median CS) and high (≥ median CS) groups. Clinical data was collected retrospectively.Baseline patient characteristics were compared between CD73 negative and positive (high + low) groups using the Cochran-Armitage trend test. Multivariable Cox regression evaluated associations of CD73 expression with disease-free and overall survival (DFS, OS) after adjusting for other baseline prognostic variables. Results: Of the 112 patients included, clear cell was the most common histology (70%) followed by chromophobe (12.5%), and papillary (12%) RCC. CD73 expression (CS > 0) was noted in 22% (n = 25) of tumors and was associated with more advanced stage (T3-4/N+: 37.5% vs. 25%; p = 0.02) and a trend towards higher nuclear grade (≥3: 48% vs. 35%; p = 0.07). Median follow-up from nephrectomy was 9.7 yrs. In multivariable analysis adjusting for nuclear grade ( < 3 vs. ≥3) and stage (T1-2, N0 vs. T3-4/N+), tumors with high CD73 expression had significantly worse DFS and OS (Table). Conclusions: CD73 expression was found in 22% of RCC patients and was associated with adverse pathologic features and poor prognosis independent of tumor stage and histologic grade. Our results provide strong rationale for further investigation of the CD73/adenosine pathway as a therapeutic target in RCC. [Table: see text]

Combined immunodeficiency in a patient with c-Rel deficiency

This study reports a homozygous mutation in REL abrogating c-Rel protein expression in a patient with combined immunodeficiency characterized by susceptibility to Mycobacterium tuberculosis, Salmonella, Cryptosporidium, and cytomegalovirus.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Calcium signals that determine vascular resistance.

Small arteries in the body control vascular resistance, and therefore, blood pressure and blood flow. Endothelial and smooth muscle cells in the arterial walls respond to various stimuli by altering the vascular resistance on a moment to moment basis. Smooth muscle cells can directly influence arterial diameter by contracting or relaxing, whereas endothelial cells that line the inner walls of the arteries modulate the contractile state of surrounding smooth muscle cells. Cytosolic calcium is a key driver of endothelial and smooth muscle cell functions. Cytosolic calcium can be increased either by calcium release from intracellular stores through IP3 or ryanodine receptors, or the influx of extracellular calcium through ion channels at the cell membrane. Depending on the cell type, spatial localization, source of a calcium signal, and the calcium-sensitive target activated, a particular calcium signal can dilate or constrict the arteries. Calcium signals in the vasculature can be classified into several types based on their source, kinetics, and spatial and temporal properties. The calcium signaling mechanisms in smooth muscle and endothelial cells have been extensively studied in the native or freshly isolated cells, therefore, this review is limited to the discussions of studies in native or freshly isolated cells. This article is categorized under: Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Mechanistic Models.

Author Correction: Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis.

In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.

Determination of ROS1 positivity by immunohistochemistry in a multicentric cohort of 426 non-small-cell lung cancer cases in India

Introduction: ROS1 is a receptor tyrosine kinase of the insulin receptor family that acts as a driver oncogene in 1%–2% of non-small cell lung carcinoma (NSCLC) patients via a gene translocation between ROS1 and other genes, the most common one being CD74. Histologic and clinical features that are associated with ROS1 translocations include never smokers, younger age, and adenocarcinoma (AC) histology. The identification of ROS1 fusion is important because it responds to targeted therapies such as crizotinib that can greatly improve the survival of the patient. The goal of this study was to determine the percentage of population harboring ROS1 mutation in India. Materials and Methods: ROS immunohistochemistry (IHC) method was optimized using Cell Signalling Technology, ROS antibody (ROS1 clone D4D6 rabbit mAb), and appropriate positive and negative controls in a fully automated procedure using Ventana automated stainer. Approximately 2000 retrospective lung cancer cases from six different Apollo hospitals were identified. The formalin-fixed paraffin-embedded blocks and corresponding diagnostic and demographic data were retrieved. A total of 426 cases with confirmed diagnosis of NSCLC were then tested by IHC for ROS1. Results: Given the retrospective nature of the cancer cases and diagnostic data being used for the study, there was high attrition in the availability of the blocks and data. Among the 2000 cases examined, there were 604 cases that were initially reported as lung cancer in the hospital medical records and for whom corresponding blocks were available, but blocks for only 485 cases had sufficient tumor content, as judged by hematoxylin and eosin staining and microscopic examination, to enable testing for ROS1 staining. Of these 485 cases, 225 were confirmed AC cases. A single case stained positive for ROS1 among the 225 AC cases. The percentage is calculated to be 0.44% based on this single positive case. No positive case was observed in squamous cell carcinomas or other cancers of the lung such as neuroendocrine tumors, germ cell tumors, and adenoid cystic tumors. Conclusion: The percentage positivity for ROS1 in the study comprising 225 Indian NSCLC AC cases appeared similar to the 0.3%–1.6% range reported for smokers and lower than 0.9%–1.7% positivity reported in mixed patient populations.

Defective Autophagy Process and Increased ROS Formation in Primary Myelofibrosis

Introduction: We and others have reported markedly increased ROS formation in primary myelofibrosis (MF) including post-ET or PV-MF when compared with essential thrombocytosis (ET), polycythemia vera (PV) and normal controls. A growing body of evidence illustrates the interdependency between the Keap1-Nrf2 pathway and p62-mediated selective autophagy, are playing an important role in major cellular defense mechanisms against oxidative and electrophilic stress ; dysregulation of p62-Keap1-Nrf2 axis has been implicated in tumor development. Therefore, we studied p62 mediated autophagy and Nrf2 pathways in patients with MF ,ET and PV.Materials and Methods:33 patients including 15 patients with MF, 7 patients with PV, 11 patients with ET and 10 normal controls were studied. Cellular ROS measurement: Cellular ROS was determined by a dichlorofluorescein (DCF) assay. The oxidation of H2DCFDA was measured by flow cytometry. Real-time PCR assay: Total RNA was extracted from normal control or patient mononuclear cells using RNeasy Kits from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Western blotting: Total cell lysates were obtained from normal control or patient mononuclear cells. Primary antibodies against Beclin-1, LC3 and p62 were purchased from Cell Signaling Technology (Danvers, MA).Results: Fig.1a shows significantly elevated ROS in MF (2127±261.6) when compared with PV (1090 ± 163.8) or ET (1031 ± 140.9) or controls (549.1 ± 52.50). Fig.1b. shows that Nrf2 protein levels (ratio to controls) was significantly reduced in MF (41.84 ± 29.33), PV (14.42 ± 2.96), ET (14.05 ± 3.02), than the controls ( 101.3 ± 7.64). Fig.1c shows that the RT-PCR values of p62 was significantly increased in MF (1.34 ± 0.06) and grouped PV (1.21 ± 0.07), ET (1.14 ± 0.08) and MF as MPN (1.27 ± 0.04) than controls (1.00 ± 0.05),( P<0.05); ET or PV were not significantly elevated than controls.Conclusions: Patients with MF had increased levels of ROS when compared with PV and ET while Nrf2 levels were significantly reduced in all three( ET,PV, MF) which implies defective detoxification process in MPNs. P62, a marker for defective autophagy was significantly increased in MF than controls . This implies that there is a defective autophagy process in MF, contributing further to the formation of P62 and ROS. There is also a defective detoxification mechanism which further leads to more ROS formation and further DNA damage. Further studies are in progress on other autophagy markers which will enlighten more about the pathophysiology of MF. [Display omitted] DisclosuresWang:INCYTE: Other: clinical trial.

The anticancer mechanism investigation of Tanshinone IIA by pharmacological clustering in protein network

BackgroundCancer is the second most common cause of death globally. The anticancer effects of Tanshinone IIA (Tan IIA) has been confirmed by numerous researches. However, the underlying mechanism remained to be integrated in systematic format. Systems biology embraced the complexity of cancer; therefore, a system study approach was proposed in the present study to explore the anticancer mechanism of Tan IIA based on network pharmacology.MethodAgilent Literature Search (ALS), a text-mining tool, was used to pull protein targets of Tan IIA. Then, pharmacological clustering was applied to classify obtained hits, the anticancer module was analysed further. The top ten essential nodes in the anticancer module were obtained by ClusterONE. Functional units in the anticancer module were catalogued and validated by Gene Ontology (GO) analysis. Meanwhile, KEGG and Cell Signalling Technology Pathway were employed to provide pathway data for potential anticancer pathways construction. Finally, the pathways were plotted using Cytoscape 3.5.1. Furthermore, in vitro experiments with five carcinoma cell lines were conducted.ResultsA total of 258 proteins regulated by Tan IIA were identified through ALS and were visualized by protein network. Pharmacological clustering further sorted 68 proteins that intimately involved in cancer pathogenesis based on Gene Ontology. Subsequently, pathways on anticancer effect of Tan IIA were delineated. Five functional units were clarified according to literature: including regulation on apoptosis, proliferation, sustained angiogenesis, autophagic cell death, and cell cycle. The GO analysis confirmed the classification was statistically significant. The inhibiting influence of Tan IIA on p70 S6K/mTOR pathway was revealed for the first time. The in vitro experiments displayed the selectivity of Tan IIA on HeLa, MDA-MB-231, HepG2, A549, and ACHN cell lines, the IC50 values were 0.54 μM, 4.63 μM, 1.42 μM, 17.30 μM and 204.00 μM, respectively. This result further reinforced the anticancer effect of Tan IIA treatment.ConclusionsThe current study provides a systematic methodology for discovering the coordination of the anticancer pathways regulated by Tan IIA via protein network. And it also offers a valuable guidance for systematic study on the therapeutic values of other herbs and their active compounds.

P15 Prevalence of EML4- ALK Fusion Gene in Adenocarcinoma Lung Patients by Using Immuno Histo Chemistry

An oral ATP- competitive TKI of ALK and c- MET, Crizotinib has shown impressive clinical activity in advanced non–small cell lung cancers especially in the tumors harboring ALK rearrangements. With FISH as the mainstay for detection of ALK rearrangements, the ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, IL) has become an FDA-approved companion diagnostic for targeted therapy with the ALK inhibitor crizotinib in lung cancers. The objective of this molecular epidemiological study is to estimate the prevalence of EML4-ALK fusion gene using IHC as a cost effective alternative to FISH for Indian patients with adenocarcinoma lung. Patients with NSCLC, adenocarcinoma histology, whose tumors had been tested for EML4-ALK fusion gene using IHC were considered for this study. Permission was obtained from the Ethics committee before the start of the study. Clinical characteristics and treatment details were collected from the patient’s medical records. IHC analysis was performed using a Ventana automated immunostainer (Ventana Medical Systems, Illkirch Graffenstaden, France). Detection was performed using a multimer-technology system with the UltraView Universal DAB detection kit. A total of 204 NSCLC adenocarcinoma patients were included in the study. There were 126 (61.7%) men and 78 (38.23%) women with a median age of 57 years. Of the 204 patients, 47 (23.03%) were non-smokers and 175 (85.78%) had stage-IV disease at the time of initial diagnosis. 48 (23.52%) blocks were positive for EGFR mutations whereas 156 (76.47%) were EGFR wild type. EML4-ALK fusion gene was present in 27 (13.23%) patients whereas 177 (86.76%) tumors were EML4-ALK negative. 25 out of the 27 patients with ALK positivity received Crizotinib therapy. The incidence of EML4-ALK gene fusions (13.23%) in this Indian population is four fold high than the previous reported incidences and supports the claim of several recent studies that a relatively new ALK clone, 5A4 and D5F3 from cell signaling technology (Ventana) can accurately identify ALK rearranged lung ACA as compared to FISH. The inclusion of IHC for the detection of EML4-ALK gene fusions as a low cost alternative seems warranted.

Abstract 3037: Comparison of multiplexed imaging mass cytometry in FFPE tissue to monoplex immunohistochemistry

Abstract Introduction: Multiplexed analysis of limited tissue samples can improve our understanding of tumor biology and tumor microenvironment. Chromogenic and fluorescent multiplexed immunohistochemistry (IHC) approaches are available and offer great insights while conserving limited tissue however these approaches have their limitations. Multiplexed chromogenic IHC methods can at best accommodate up to 2-3 distinct markers. Fluorescence-based approaches can support higher degree of multiplexing however spectral overlap issues and differences in labeling efficiency/photostability complicate experimental procedures and data interpretation. Imaging mass cytometry system (IMC) has recently emerged as a novel technology for tissue imaging that enables multiplexed analysis protein expression (up to 40 markers) in a single tissue sample while circumventing the limitations of chromogenic and fluorescent IHC techniques. Metal-conjugated antibodies are used to perform qualitative and quantitative analysis of expression of multiple proteins of interest on a single formalin fixed paraffin embedded (FFPE) tissue slide. Here, we compare the performance of the IMC method with conventional, established IHC techniques using a small panel of markers. Methods: Serial sections of FFPE tonsil and non-small cell lung carcinoma tissues were assessed by monoplex IHC and multiplex IMC for CD3 (Cell Signaling Technology, D7A6E), CD8 (LS Bio, C8/144B), CD68 (Abcam, KP1), PD-L1 (Spring Bio, SP142) and Histone H3 (D1H2). Digital image analysis using Flagship's image analysis software was used to compare performance characteristics of multiplex IMC platform with standard monoplex chromogenic IHC. Results: The staining patterns of the corresponding biomarkers were similar between IMC and IHC on sequential sections. Digital image analysis demonstrated concordance in the percentage of biomarker positive cells within analyzed matched IHC and IMC areas. It was also demonstrated that cellular image segmentation can be performed on IMC images thus allowing for utilization of various software packages for high dimensional single cell analysis of IMC data. Conclusion: Comparative digital image analysis indicates that on FFPE tissues multiplexed IMC platform generates data comparable to that obtained from the monoplex chromogenic IHC platform. We believe that an IMC platform is a new tool capable of dramatically enhancing our ability to study biology of cancer using highly multiplexed analysis of limited tissue samples. Citation Format: Navi Mehra, Carsten Schnatwinkel, Elliott Ergon, Joseph Krueger, Karl Calara-Nielsen, Brad Foulk, Kirti Sharma, Denis Smirnov, Chandra Rao, Tatiana Perova, Rengasamy Boominathan. Comparison of multiplexed imaging mass cytometry in FFPE tissue to monoplex immunohistochemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3037.

2325 Targeting isoform-specific PI3 kinase signaling for treatment of cutaneous T cell lymphoma

OBJECTIVES/SPECIFIC AIMS: (1) Determine the anti-proliferative effect of Copanlisib (α/δ PI3 kinase inhibitor) in CTCL cell lines and synergy with other anti-tumor drugs such as HDAC inhibitors. (2) Determine the effect of Copanlisib treatment on downstream targets of the PI3K/AKT/mTOR pathway. METHODS/STUDY POPULATION: We will test the anti-proliferative effect of Copanlisib on the cell lines H9 and HH, which are well characterized for evaluating new therapies for CTCL (Netchiporouk et al., 2017). We will also test the anti-proliferative effect of Copanlisib in combination with HDAC inhibitors on CTCL cell lines. Cell Titer Glo (Promega) will be used for the proliferation assay. Briefly, Cell-Titler Glo will be added after 24, 48, and 72 hours and luminescence will be measured as a % of maximal growth. Inhibitory effect will be determined by comparison to % growth of the control cultures without Copanlisib treatment. Our next objective is to determine the effect of Copanlisib treatment on downstream targets of the PI3K/AKT/mTOR pathway using Western blot analysis. In brief, 30 μg of each lysate will be subjected to 4%–12% gradient SDS-PAGE gel eletrophoresis. All primary antibodies were purchased from Cell Signaling Technology. Membranes will be washed with TBST and incubated with 1:10,000 dilution of IRDye-conjugated secondary antibody (Licor) for 1 hour. Results will be expressed as relative intensity: the intensity of each band adjusted to that of GAPDH. Experiments will be done in triplicate and 1-way analysis of variance followed by multiple comparison test will be applied to compare the cell proliferation between different treatment groups. RESULTS/ANTICIPATED RESULTS: Previous results from the Ai lab have demonstrated the importance of the PI3 kinase signaling cascade using high-throughput proliferation assays and siRNA knockdown of individual and double PI3 kinase isoforms (unpublished data). So far I have successfully established culture of HH and H9 cells in 96 well plates (100,000 cells/200 μL). We are titrating Copanlisib at doses from 20 nm to 20 μM. Initial results suggest a promising anti-proliferative effect and currently we are optimizing the most effective pharmacologic dose. Thus we anticipate that Copanlisib will exhibit a potent anti-proliferation activity in CTCL cell lines. H9 and HH cell lysates have been collected and preserved for Western blot analysis. We anticipate that Copanlisib treatment will significantly decrease the phosphorylation of AKT and 4EB-P1, both downstream targets of PI3 kinase signaling. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will elucidate the importance of the PI3 kinase/AKT/mTOR pathway in tumor proliferation in CTCL. Identifying the importance of specific isoforms of PI3 kinase in CTCL will allow for more targeted selection of treatment. Copanlisib targets α and δ isoforms of PI3K and is newly approved by the FDA for low grade B cell lymphoma. Our results seek to quantify the anti-proliferative effect of Copanlisib and determine an on-target mechanism of action by investigating the drug’s effects on downstream signaling molecules of the PI3 kinase pathway. This project will elucidate the disease process of CTCL and provide important insight for its management.

Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma

Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

Abstract A51: Evaluation of Akt molecular targets in colorectal tumors after radiotherapy and MK2206 treatment

Abstract Introduction: Colorectal cancer is the third most frequent cause of cancer-related mortality in the world. The available treatment comprises surgical resection and pre- or postoperative chemoradiotherapy. Neoadjuvant radiochemotherapy (RQTn) has been widely used in the treatment of some types of cancers, including rectum cancer, which leads to complete remission of the tumor in some cases. However, there is a lack of response to this treatment in a significant number of patients (approximately 60%). Previous data from our group suggest that activation of the Akt pathway plays an important role in the response to chemoradiotherapy treatment as observed by differential gene expression followed by pathway enrichment analysis and immunohistochemistry between rectal tumors that are responsive or not to RQTn. As Akt is involved in many biologic functions, we evaluated Akt signaling in order to understand molecular effectors that could promote radioresistance in rectal tumors. Materials and Methods: Colorectal cancer cell lines, sensitive (SW48) and resistant (SW480) to RQTn, respectively, were compared regarding basal Akt signaling status. We also assessed Akt signaling after treatment of the resistant cell line SW480 with radiation and 5-FU combined with the Akt inhibitor MK2206. To evaluate the phosphorylation of some proteins in the Akt pathway, we performed the immunoarray assay using the PathScan® Akt Signaling Antibody Array Kit Chemiluminescent Readout (Cell Signaling), following the protocol provided by the manufacturer. The images were acquired by revelation in photographic film, and the subsequent analysis of densitometry was performed by the software Image J. To validate the phosphoarray data we carried out immunoblots using both antibodies to phosphorylated AMPKα (Thr172) and total AMPK (Cell Signaling Technology, Beverly, Massachusetts, USA) with both lineages. Results and Conclusions: Using the phosphoarray immunoassay for Akt pathway, it was observed that some proteins such as AMPKα, GSK3α, PTEN, PDK1, and PRAS40 showed different levels of basal phosphorylation in SW48 and SW480 cells. Interestingly, according to our data there is a higher basal phosphorylation of AMPKα in SW48 cells than in SW480.The treatment with MK2206 in SW480 cells demonstrated efficiency in Akt inhibition, culminating in different levels of phosphorylation of some substrates of this protein, especially AMPKα. It is possible to suggest a prosurvival role of Akt since when inhibited, phosphorylation of AMPKα, which acts as a tumor suppressor, increases, and PRAS40, an inhibitor of apoptosis through mTOR regulation, decreases. It is emphasized that the study of the components of the PI3K/Akt pathway are promising targets for therapeutic intervention, since this pathway has the ability to inhibit several types of tumor suppressors. Citation Format: Jennifer Marx Fernandes, Anamaria A. Camargo, Fernanda C. Koyama. Evaluation of Akt molecular targets in colorectal tumors after radiotherapy and MK2206 treatment [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A51.

RARE-29. PROGRAMMED DEATH LIGAND 1 EXPRESSION AND TUMOR INFILTRATING LYMPHOCYTES IN TUMORS ASSOCIATED WITH NEUROFIBROMATOSIS TYPE 1 AND 2

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or its ligand (PD-L1) have been shown to be effective in treating patients with a variety of cancers. Positive associations between clinical response rates and PD-L1 expression on tumor and immune cells, as well as the presence of tumor infiltrating lymphocytes (TILs) have been found in multiple studies. It is currently unknown whether tumors associated with neurofibromatosis types 1 and 2 (NF1 and NF2) express PD-L1. We hypothesized that NF-associated tumors express PD-L1 and therefore might be amenable to immunotherapy with immune checkpoint inhibitors. We performed immunohistochemistry for PD-L1 (Spring Bioscience, clone SP142 and Cell Signaling Technology, clone E1L3N), CD3, CD20, CD8, and CD68 in 17 NF1-related tumors (11 dermal neurofibromas and 6 plexiform neurofibromas) and 20 NF2-related tumors (10 meningiomas and 10 schwannomas) using archival formalin-fixed paraffin-embedded tissues. Expression of PD-L1 was considered positive in cases with >5% membranous staining of tumor cells, in accordance with previously published biomarker studies. PD-L1 positive tumor cells with >5% membranous staining using clone SP142 or E1L3N were observed in 100% of plexiform neurofibromas, 82% and 58% of dermal neurofibromas, 70% and 100% of schwannomas, and 40% and 20% of meningiomas, respectively. Sparse to moderate presence of CD68, CD3, or CD8 positive tumor-infiltrating lymphocytes (TILs) was found in 37/37 (100%) of tumor specimens. However, expression of CD20 positive lymphocytes was limited to rare cells detectable in 4/37 (11%) of tumors. Our findings indicate that adaptive resistance to cell-mediated immunity plays a major role in the tumor immune environment of NF1 and NF2-associated tumors. Expression of PD-L1 on tumor cells and presence of TILs suggests that these tumors may be responsive to immune therapy with immune checkpoint inhibitors, which should be explored in clinical trials for NF patients.

Corrigendum] Farnesoid X receptor deletion improves cardiac function, structure and remodeling following myocardial infarction in mice.

Following the publication of this article, an interested reader drew to our attention that we had incorrectly reported (in the Materials and methods section, 'Western blot analysis', on p.674) that the anti-farnesoidX receptor (FXR) antibody of Cell Signalling Technology, Inc., cat.no.#12295, had been used in this study to probe for FXR. In fact, the antibodies used in the above-mentioned study were a gift from the group of DrXin-Liang Ma at Thomas Jefferson University (Philadelphia, PA, USA), as referenced in the following article: [PulJ, YuanA, ShanP, GaoE, WangX, Wang Y, Lau WB, KochW, Xin-MaXL and HeB: Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury. Eur Heart J34: 1834-1845, 2013]. The antibody that was used for the western blotting analysis was raised against the C-terminus of FXR (C-20; cat.no.sc-1204, Santa Cruz Biotechnology, San Diego, CA, USA). We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. The error made in our incorrect referencing of the antibody did not affect the conclusions reported in this study. Furthermore, we regret the inconvenience that this mistake caused. [the original article was published in the Molecular Medicine Reports 16: 673-679, 2017; DOI: 10.3892/mmr.2017.6643].

Abstract 5350: A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens

Abstract This poster describes a novel fully integrated method for the multiplexed DNA, RNA and Protein analysis of formalin fixed paraffin embedded tumor specimens. Cancer progression is typically a result of aberrations in the molecular pathways that regulate cell growth. Identifying and understanding these changes is necessary for the detection of drug targets as well as the identification of novel biomarkers that can predict response to such drugs. Deep molecular profiling of clinical tumor samples is challenged by limited material and the compromised quality resulting from variable handling and the fixation processes that are commonly employed in the clinical laboratories. While multiplexed gene expression and mutational analysis of such samples has become well established, we describe a new method that enables multiplexed DNA, RNA and protein analysis from a single formalin fixed paraffin embedded (FFPE) tumor specimen on the NanoString nCounter system. We have adapted the sample preparation methods to enable the multi-analyte analysis of FFPE samples and have demonstrated concordance with the clinical gold standard immunohistochemistry (IHC) methodology. As a proof-of-concept, five FFPE non-small cell lung cancer (NSCLC) specimens were simultaneously stained with a panel of antibodies specific to a variety of cancer-relevant proteins, five of which were validated on serial sections by IHC using antibodies from Cell Signaling Technology. In addition to the protein analysis, from the same specimen, expression of 770 cancer-related genes and &amp;gt;100 single nucleotide variants (SNVs) were also analyzed on the nCounter platform. By integrating the analysis of DNA SNV’s, as well as RNA and protein expression from single FFPE specimens, it is now possible to reveal the molecular mechanisms of tumor progression, uncover novel drug targets and identify biomarkers from precious specimens. Citation Format: Douglas Hinerfield, Jennifer Mellen, Gary Geiss, Philippa Webster, Chris Merritt, Kristi Barker, Gokhan Demirkan, Brian Filanoski, Roberto Polakiewicz, Katherine Crosby. A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5350. doi:10.1158/1538-7445.AM2017-5350

Identification of CD146 as a novel molecular actor involved in systemic sclerosis

We highlight for the first time that CD146/sCD146 is involved in fibrotic process during SSc. sCD146 could thus constitute a new biomarker to assess disease activity, and potentially a new target for therapeutic applications.

Immunohistochemical staining for programmed cell-death ligand 1 (PD-L1) in malignant thymoma and thymic carcinoma.

e20003 Background: Recent development of anti-PD-1/L1 antibodies has demonstrated activity in various neoplasms. Thymic malignancies (TMS) are rare and treatment in advanced disease is limited. To evaluate the potential impact of anti-PD-1/L1 therapy in TMS, we examined the expression of PD-L1 in previously resected thymoma (TM) and thymic carcinoma (TC). Methods: We examined resected specimens from patients at Hartford Hospital with TM and TC between 2000 and 2014. Expression of PD-L1 was evaluated on formalin-fixed paraffin-embedded tissue. Immunohistochemical testing was done using four different clones of PD-L1 antibodies on the Leica Bond Max automated platform. The four clones include: E1L3N (Cell Signaling Technology), 28-8 (Epitomics) and SP142 (Spring Bioscience), and CAL10 (BioCare). PD-L1 expression was evaluated based on the percentage of tumor cells positive and their intensity graded as negative, weak (1+), moderate (2+), and strong (+3). The scoring was performed by three pathologists and was blinded for clinicopathologic data and antibody clones. Results: We evaluated a total of 29 patients, including 26 patients with TM and 3 with TC. Among the 29 available specimens, 12 had completed PD-L1 expression assessment at the time of submission. PD-L1 expression is present in 75-100% of the evaluated patients. All had positive PD-L1 staining by SP142 and CAL10. Three patients showed strong intensity by CAL10, and one by SP142. E1L3N and 28-8 had positive PD-L1 expression in 9 and 8 patients respectively with weak/moderate intensity. SP142 and CLA10 demonstrated the strongest concordance (R2 = 0.91) but there was significant variation between antibodies (R2 = 0.31-0.91). No correlation was detected between tumor grade and PD-L1 expression. There were focal areas that lacked expression in all of the evaluated specimens. Conclusions: There is increased expression of PD-L1 in TMS. The level of PD-L1 expression varies between the four PD-L1 antibodies. Increased PD-L1 expression provides evidence for the use of PD-L1 inhibitors in TMS. The variable staining highlights the heterogeneity of TMS and challenges in developing predictive biomarker in this cancer.

Outcomes of anti-PD-1 therapy in mesothelioma and correlation with PD-L1 expression.

8514 Background: Early phase trials of anti-programmed death 1 (PD-1) antibodies have demonstrated important responses in malignant mesothelioma (MM). Expression of the ligand, PD-L1, is a potential biomarker for PD-1 directed therapy use and is expressed in a significant proportion of MM. We present results for a cohort treated with PD-1 inhibitory antibodies and assessed for PD-L1 expression. Methods: Patients (pts) with unresectable pleural or peritoneal mm treated with anti-PD-1 antibodies were included. Data was collected retrospectively. Radiological response was assessed using RECIST 1.1. Overall survival (OS) and progression-free survival (PFS) were evaluated. PD-L1 expression was assessed with IHC clone E1L3N (Cell Signaling Technology). PD-L1 positivity was defined as membranous expression on tumour cells: &gt; 5% for PD-L1+ and &gt; 50% for PD-L1hi. Results: Forty-six pts were treated between July 2015 and January 2017. Median age was 66.5 years, ECOG PS was 0/1 in 3/46 (7%) and 35/46 (76%) respectively. Most were male (83%), and 43/46 (93%) had ≥1 prior therapy, with a median of 2 (range 0 - 5). The predominant histology was epithelioid (n = 32/46; 70%). Pembrolizumab was used in 45/46 and BGB-A317 (a PD-L1 antibody) in 1 pt. Of the 46 pts, the overall response rate (ORR) was 15% (7 PR) with 15/46 (33%) achieving stable disease, giving a DCR of 44%. Progression was seen in 24/46 (52%). Median OS for the entire cohort was 8.0 months (95% CI: 2.3 – 11.9). Median duration of response was not yet reached (range 1.5 -19.8). PD-L1 testing was performed in 14 samples, with PD-L1+ in 5 (36%) and PD-L1hi in 4 (29%). The ORR was 40% (2 PR, 3 SD) with PD-L1+, 50% (2 PR, 2 SD) with PD-L1hi and 22% (2/9 patients) with negative expression. PFS and OS were greater with both PD-L1+ (PFS HR: 0.26, 95% CI 0.05 – 1.32, p = 0.10) and PD-L1hi(PFS HR: 0.17, 95% CI 0.02 – 1.47, p = 0.11). PD-L1+ positivity remained a borderline predictor of improved survival on multivariate analysis (p = 0.06). Complete PD-L1 analysis will be presented at the meeting. Conclusions: PD-1 targeted therapy demonstrated a clinically significant response rate in this cohort of mm patients. Initial analysis suggests PD-L1 expression is correlated with improved response and survival.