PD-L1 Expression on Cell Block Preparations of Pancreatic Adenocarcinoma

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S94-S95
Author(s):  
Victoria Costa ◽  
David Kim ◽  
Melanie Johncilla ◽  
Abha Goyal ◽  
Rema Rao
Keyword(s):  
Disease Progression ◽  
Tumor Cells ◽  
Locally Advanced ◽  
Immunohistochemical Analysis ◽  
Inflammatory Cells ◽  
Fixation Method ◽  
Lung Cancers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Combined Positive Score

Abstract Objectives Programmed death-ligand 1 (PD-L1) is an emerging molecular target in anticancer therapy, most notably non–small cell lung cancers. PD-L1 expression in pancreatic adenocarcinomas (PDAs) on surgical specimens is highly variable, with 10% to 60% of tumors showing expression. PD-L1 expression in PDA on endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs) has been rarely studied. Methods Formalin-fixed, paraffin embedded (FFPE) cell blocks (CBs) from 65 EUS-FNA samples of 55 patients, with a diagnosis of PDA, with at least 20% tumor cellularity were retrieved. The cell blocks were originally fixed in CytoRich fixative. Immunohistochemical staining (IHC) for PD-L1 was performed using the M365329-1 (22C3) clone according to the manufacturer’s approved protocol and optimized for the fixation method using appropriate controls. A combined positive score (CPS) defined per CAP guidelines as the total number of positive tumor cells and inflammatory cells as a percentage of the total number of tumor cells was assessed for each case and was grouped as <1, 1-20, or >20. Results Twenty-five samples (38%) from 21 patients showed immunoreactivity to PD-L1, with 21 (32%) having a CPS of <1 and four (6%) having a CPS of 1-20. Eight of these 25 samples had surgical correlates, of which concordant staining was noted in five (62.5%). Of the discordant three, decreased tumor cell sampling in the core biopsy was noted in one. Overall, 20 of 21 patients (95%) with PD-L1 immunoreactivity had disease progression with 17 (81%) associated with metastatic disease and three (14%) with locally advanced disease. Conclusion Immunohistochemical analysis for PD-L1 is feasible on EUS-FNA CB samples when optimized and validated for the fixation method using appropriate controls. PD-L1 expression is seen in only a minority of PDAs, albeit with a very low CPS. The potential role of PD-L1 as a prognostic marker for disease progression needs further exploration.

Characterization of inflammatory infiltrate of ulcerative dermatitis in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice

2018 ◽  
Vol 53 (5) ◽  
pp. 447-458 ◽  
Author(s):  
Davide De Biase ◽  
Francesco Esposito ◽  
Marco De Martino ◽  
Claudio Pirozzi ◽  
Antonio Luciano ◽  
...  
Keyword(s):  
Mast Cell ◽  
Immunohistochemical Analysis ◽  
Inflammatory Cells ◽  
Type I ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Student’S T ◽  
Cryopreserved Skin ◽  
Inflammatory Infiltrates ◽  
Skin Samples

Ulcerative dermatitis (UD) is an idiopathic, spontaneous and progressive disease typically affecting C57BL/6 aged mice with an unknown aetiopathogenesis. For this study, we evaluated 25 cases of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice. Formalin-fixed, paraffin-embedded skin samples were submitted to morphological investigations. Immunohistochemical analysis was performed to characterize and quantify inflammatory cells using CD3, CD45/B220, CD4, CD8 and IL-17 antibodies. Mast cell-bound IgE was investigated by immunofluorescence, whereas serum and cryopreserved skin samples were collected for molecular analysis. Student's t-test (two-tailed) was performed to assess significant differences between the two groups. Affected skin showed extensive areas of ulceration and diffuse, severe and mixed inflammatory infiltrates. No relevant changes were observed in control mice. Immunohistochemical analysis showed a predominant CD3 + CD4 + leukocyte population with fewer CD45/B220 and IL-17 immunolabelled cells and mast cell-bound IgE. Increases in TNFα, IL-1β and Il-6 mRNA expression were observed in the skin of affected animals compared to controls. Serum TNFα and IL-6 did not vary between affected and control mice. Inflammatory infiltrates and cytokine expression were consistent with both Th2/IgE and Th17 differentiation, a typical pattern of a type I hypersensitivity reaction. Overall, our data suggest an allergic-based aetiopathogenesis of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice.

scholarly journals Detection of circulating sarcoma tumor cells using a microfluidic chip-type cell sorter

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nobuhiko Hasegawa ◽  
Ikuko Takeda Nakamura ◽  
Toshihide Ueno ◽  
Shinya Kojima ◽  
Masahito Kawazu ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Microfluidic Chip ◽  
Locally Advanced ◽  
Normal Fibroblast ◽  
Nonsynonymous Mutation ◽  
Cell Sorter ◽  
Chip Type ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
On Chip

AbstractAnalyses of circulating tumor cells have been shown to be effective for the detection of cancer relapse and prognosis prediction. However, research regarding its utility in sarcoma remains scarce. In this study, the microfluidic chip-type cell sorter On-chip Sort was used to construct a system for detecting circulating sarcoma cells (CSCs). A pilot study using normal fibroblast or sarcoma cell lines was designed to establish a reliable protocol to separate CSCs by On-chip Sort. A single CSC was separated and recovered from 10 ml of whole blood from a patient with locally advanced myxofibrosarcoma. The nonsynonymous mutation for KMT2B p.Ile2602Val identified in the formalin-fixed paraffin-embedded tumor sample was also confirmed in the CSC. Use of the developed protocol may allow CSCs to become an early predictor for metastasis and recurrence of sarcoma. Further, it may aid in optimizing post-operative therapies for patients without metastasis.

scholarly journals Semaphorin Signaling in Cancer-Associated Inflammation

2019 ◽  
Vol 20 (2) ◽  
pp. 377 ◽  
Author(s):  
Giulia Franzolin ◽  
Luca Tamagnone
Keyword(s):  
Immune Response ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Disease Progression ◽  
Tumor Cells ◽  
Cross Talk ◽  
Immune Cells ◽  
Major Element ◽  
Inflammatory Cells ◽  
Anti Cancer

The inflammatory and immune response elicited by the growth of cancer cells is a major element conditioning the tumor microenvironment, impinging on disease progression and patients’ prognosis. Semaphorin receptors are widely expressed in inflammatory cells, and their ligands are provided by tumor cells, featuring an intense signaling cross-talk at local and systemic levels. Moreover, diverse semaphorins control both cells of the innate and the antigen-specific immunity. Notably, semaphorin signals acting as inhibitors of anti-cancer immune response are often dysregulated in human tumors, and may represent potential therapeutic targets. In this mini-review, we provide a survey of the best known semaphorin regulators of inflammatory and immune cells, and discuss their functional impact in the tumor microenvironment.

PD-L1 expression in primary clear cell renal cell carcinomas (ccRCCs) and their metastases.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 467-467 ◽  
Author(s):  
Marcella Callea ◽  
Elizabeth M Genega ◽  
Mamta Gupta ◽  
André P Fay ◽  
Jiaxi Song ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Primary Tumor ◽  
Clear Cell ◽  
Predictive Biomarkers ◽  
Primary Tumors ◽  
Metastatic Lesions ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Membranous Staining ◽  
Formalin Fixed

467 Background: Clinical trials evaluating anti-PD-1 and anti-PD-L1 antibodies (Abs) in ccRCC have shown promising efficacy in a subset of patients. Preliminary studies have demonstrated that tumor PD-L1 expression increases the likelihood of benefit with anti-PD-1 Ab, but fails to identify all responders. One potential explanation for these results is that predictive biomarkers are usually evaluated in the primary tumors, which are more readily available; however, biomarker expression in nephrectomy samples may not accurately reflect expression in the metastases that are being targeted by therapy. In this study, we compared PD-L1 expression in a series of ccRCCs and their metastases. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue blocks from 34 primary ccRCCs and corresponding metastases were retrieved. Multiple areas of the primary tumors, including areas of predominant and highest Fuhrman nuclear grade (FNG), were selected for analysis. Slides were immunostained with a mouse monoclonal anti-PD-L1 antibody (405.9A11). The assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry. The presence of tumor cells with membranous staining was assessed. A case was considered positive when any tumor cell positivity was detected. Results: Positive membranous PD-L1 expression in tumor cells was observed in 10/34 (29%) primary ccRCCs. In 3 of these 10 cases (30%), the metastases were negative. In 2 cases the primary tumor was negative but the metastases were positive. In twenty-two cases, both the primary tumor and the corresponding metastasis were negative. The pattern of PD-L1 staining was highly heterogeneous in the primary tumors and was restricted to areas of highest FNG. The staining was more homogeneous in the metastases. PD-L1 expression by the tumor infiltrating immune cells is currently being evaluated. Conclusions: Discordant expression of PD-L1 between the primary tumor and the corresponding metastases was detected in 5/34 (15%) cases, suggesting that accurate assessment of predictive biomarkers for PD-1 blockade in ccRCC might require analysis of metastatic lesions. Analysis of a larger patient cohort is ongoing to confirm these findings.

Association of high copy number instability (CNI) score with prognosis in patients with gastric cancer after surgical resection.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e15552-e15552
Author(s):  
Yanling Zhang ◽  
Dandan Ren ◽  
Beibei Mao ◽  
Xue Song ◽  
Wanning Yang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Adjuvant Chemotherapy ◽  
Surgical Resection ◽  
Copy Number ◽  
Locally Advanced ◽  
Clinical Staging ◽  
General Measure ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Formalin Fixed Paraffin Embedded

e15552 Background: As the fifth most common malignancies and the third leading cause of cancer death in the world, gastric cancer (GC) is often diagnosed in locally advanced or metastatic stage and, therefore, has a poor prognosis. Copy number instability (CNI) score, interpreted as the general measure of genomic instability, was reported as a prognosis predictor of some types of cancer, but its role in prognosis of GC patients remains unknown. The aim of the present study was to investigate its prognostic value in patients with GC after surgical resection and adjuvant chemotherapy. Methods: The present study included 120 patients who had received gastrectomy and adjuvant chemotherapy with stage II-IV GC. Follow-up was available for them. DNA was extracted from primary formalin-fixed paraffin-embedded (FFPE) tumor specimens and matched blood samples. Genomic profiles were analyzed by using a designed panel (1408 genes) based on next generation sequencing (NGS). Results: The most frequently mutated genes were TP53 (49%), PIK3CA (8%), RNF43 (6%) ERBB3 (6%) and APC (6%), and the most frequently amplified genes were TRPS1 (41%), COL1A2 (31%), CSMD3 (29%), ZFHX4 (29%), NAV3 (23%). CNI score was negatively correlated with OS, the CNI-high group had markedly shorter OS than CNI-low group ( p= 0.0093). In addition, there were statistically significant differences in OS between different clinical staging ( p= 0.0468). Moreover, the Cox proportional hazard model showed that CNI score was an independent prognosis factor in GC. Conclusions: The current study indicates that CNI score in primary tumor tissue is an independent predictive prognostic biomarker for GC.

PD-L1 expression, tumor mutational burden, and microsatellite instability status in 746 pancreas ductal adenocarcinomas.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 757-757
Author(s):  
Amanda Hemmerich ◽  
Claire I. Edgerly ◽  
Daniel Duncan ◽  
Richard Huang ◽  
Natalie Danziger ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Mutational Burden ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Tumor Mutational Burden ◽  
Positive Score ◽  
Combined Positive Score ◽  
High Level ◽  
Positive Groups

757 Background: Pancreas ductal adenocarcinomas (PDA) has a 5-year survival rate of 6% with a need for new therapeutic options. The approval of pembrolizumab for some gastrointestinal cancers shows the potential of immunotherapy (IMT) in PDA. We evaluated the IMT-associated biomarkers of PD-L1 expression, tumor mutational burden (TMB), microsatellite instability (MSI) and PD-L1 amplification in PDAs. Methods: 746 formalin-fixed paraffin embedded samples were evaluated for PD-L1 IHC using the Dako 22C3 pharmDx assay and scored using tumor proportion score (TPS). The cases had comprehensive genomic profiling (CGP) via DNA sequencing, using a hybrid-capture next-generation sequencing assay (FoundationOne and FoundationOneCDx) for genomic alterations (GAs), TMB, and MSI. Results: PD-L1 was positive (TPS ≥ 1%) in 29% (214/746) and negative in 71% (532/746). 43/214 (20%) of positive cases were high positive (TPS ≥ 50%). TMB (590 cases) had a mean of 3.20, 3.46, and 3.61 mutations/Mb for PD-L1 negative, positive, and high positive groups. 3 hypermutated (TMB ≥ 20) were negative for PD-L1 expression. 3/581 cases were MSI-high with a high TMB score (average 23.53 mutations/Mb). 2 MSI-high cases were negative for PD-L1 and 1 was high positive. PD-L1 amplification was not detected (0/746). Only BCOR was significantly different between PD-L1 high positive and PD-L1 negative tumors (Table). Conclusions: Of 729 PDA cases, 29% were positive (TPS ≥ 1%) for PD-L1 expression while only 6% of all cases showed a high level of PD-L1 expression on tumor cells. TMB high (3/729) and MSI-High (3/729) cases were rare. Only 2 of the TMB high cases were also MSI-high. PD-L1 amplification was not detected. Comparing GAs in PD-L1 high positive vs negative cases was only significantly different for BCOR. Further investigation is needed to see if a combined positive score of PD-L1 expression may identify a subset of patients with PDA who are more likely to respond to IMT. [Table: see text]

scholarly journals Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1601 ◽  
Author(s):  
Gretchen H. Delcambre ◽  
Junjie Liu ◽  
Jenna M. Herrington ◽  
Kelsey Vallario ◽  
Maureen T. Long
Keyword(s):  
Polyclonal Antibodies ◽  
Inflammatory Cells ◽  
Specific Protein ◽  
Proteinase K ◽  
Antigen Retrieval ◽  
Phenotypic Characterization ◽  
Buffer Solution ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Species Specific

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+(pAb A0452, Dako) T-lymphocytes, CD79αcy+B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

scholarly journals ­Immunohistochemistry for the detection of neural and inflammatory cells in equine brain tissue

2016 ◽  
Author(s):  
Gretchen H Delcambre ◽  
Junjie Liu ◽  
Jenna M Herrington ◽  
Kelsey Vallario ◽  
Maureen T Long
Keyword(s):  
Polyclonal Antibodies ◽  
Inflammatory Cells ◽  
Specific Protein ◽  
Proteinase K ◽  
Antigen Retrieval ◽  
Phenotypic Characterization ◽  
Buffer Solution ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Species Specific

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

scholarly journals Pathology Quality Control for Multiplex Immunofluorescence and Image Analysis Assessment in Longitudinal Studies

2021 ◽  
Vol 8 ◽  
Author(s):  
Rossana Lazcano ◽  
Frank Rojas ◽  
Caddie Laberiano ◽  
Sharia Hernandez ◽  
Edwin Roger Parra
Keyword(s):  
Quality Control ◽  
Image Analysis ◽  
Inflammatory Cells ◽  
Analysis Data ◽  
Malignant Cells ◽  
Core Needle ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Organ Site ◽  
Immune Profiling

Immune profiling of formalin-fixed, paraffin-embedded tissues using multiplex immunofluorescence (mIF) staining and image analysis methodology allows for the study of several biomarkers on a single slide. The pathology quality control (PQC) for tumor tissue immune profiling using digital image analysis of core needle biopsies is an important step in any laboratory to avoid wasting time and materials. Although there are currently no established inclusion and exclusion criteria for samples used in this type of assay, a PQC is necessary to achieve accurate and reproducible data. We retrospectively reviewed PQC data from hematoxylin and eosin (H&amp;E) slides and from mIF image analysis samples obtained during 2019. We reviewed a total of 931 reports from core needle biopsy samples; 123 (13.21%) were excluded during the mIF PQC. The most common causes of exclusion were the absence of malignant cells or fewer than 100 malignant cells in the entire section (n = 42, 34.15%), tissue size smaller than 4 × 1 mm (n = 16, 13.01%), fibrotic tissue without inflammatory cells (n = 12, 9.76%), and necrotic tissue (n = 11, 8.94%). Baseline excluded samples had more fibrosis (90 vs 10%) and less necrosis (5 vs 90%) compared with post-treatment excluded samples. The most common excluded organ site of the biopsy was the liver (n = 19, 15.45%), followed by soft tissue (n = 17, 13.82%) and the abdominal region (n = 15, 12.20%). We showed that the PQC is an important step for image analysis and that the absence of malignant cells is the most limiting sample characteristic for mIF image analysis. We also discuss other challenges that pathologists need to consider to report reliable and reproducible image analysis data.

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