scholarly journals Detection of N-Glycolyl GM3 Ganglioside in Neuroectodermal Tumors by Immunohistochemistry: An Attractive Vaccine Target for Aggressive Pediatric Cancer

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Alejandra M. Scursoni ◽  
Laura Galluzzo ◽  
Sandra Camarero ◽  
Jessica Lopez ◽  
Fabiana Lubieniecki ◽  
...  
Keyword(s):  
Pediatric Cancer ◽  
Nonsmall Cell Lung Cancer ◽  
Potential Utility ◽  
Formalin Fixed Paraffin ◽  
Neuroectodermal Tumors ◽  
Formalin Fixed Paraffin Embedded ◽  
Normal Human ◽  
Secondary Antibodies ◽  
Membranous Staining ◽  
Formalin Fixed

The N-glycolylated ganglioside NeuGc-GM3 has been described in solid tumors such as breast carcinoma, nonsmall cell lung cancer, and melanoma, but is usually not detected in normal human cells. Our aim was to evaluate the presence of NeuGc-GM3 in pediatric neuroectodermal tumors by immunohistochemistry. Twenty-seven archival cases of neuroblastoma and Ewing sarcoma family of tumors (ESFT) were analyzed. Formalin-fixed, paraffin-embedded tumor samples were cut into 5 μm sections. The monoclonal antibody 14F7, a mouse IgG1 that specifically recognizes NeuGc-GM3, and a peroxidase-labeled polymer conjugated to secondary antibodies were used. Presence of NeuGc-GM3 was evident in 23 of 27 cases (85%), with an average of about 70% of positive tumors cells. Immunoreactivity was moderate to intense in most tumors, showing a diffuse cytoplasmic and membranous staining, although cases of ESFT demonstrated a fine granular cytoplasmic pattern. No significant differences were observed between neuroblastoma with and without NMYC oncogene amplification, suggesting that expression of NeuGc-GM3 is preserved in more aggressive cancers. Until now, the expression of N-glycolylated gangliosides in pediatric neuroectodermal tumors has not been investigated. The present study evidenced the expression of NeuGc-GM3 in a high proportion of neuroectodermal tumors, suggesting its potential utility as a specific target of immunotherapy.

scholarly journals A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue

2015 ◽  
Vol 59 (4) ◽  
Author(s):  
I.M.S. Paulsen ◽  
H. Dimke ◽  
S. Frische
Keyword(s):  
Staining Pattern ◽  
Simple Procedure ◽  
Semiquantitative Analysis ◽  
Single Procedure ◽  
Formalin Fixed Paraffin ◽  
Labelling Intensity ◽  
Formalin Fixed Paraffin Embedded ◽  
Intracellular Vesicles ◽  
Secondary Antibodies ◽  
Formalin Fixed

<p>Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue.</p>

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.

scholarly journals Analysis ofKRASMutations of Exon 2 Codons 12 and 13 by SNaPshot Analysis in Comparison to Common DNA Sequencing

2010 ◽  
Vol 2010 ◽  
pp. 1-5 ◽  
Author(s):  
Rica Zinsky ◽  
Servet Bölükbas ◽  
Holger Bartsch ◽  
Joachim Schirren ◽  
Annette Fisseler-Eckhoff
Keyword(s):  
Dna Sequencing ◽  
Mutation Detection ◽  
Nonsmall Cell Lung Cancer ◽  
Tissue Samples ◽  
Exon 2 ◽  
Mutation Status ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Status Analysis ◽  
Formalin Fixed

Due to the call for fastKRASmutation status analysis for treatment of patients with monoclonal antibodies for metastatic colorectal cancer, sensitive, economic, and easily feasible methods are required. Under this aspect, the sensitivity and specificity of the SNaPshot analysis in comparison to the commonly used DNA sequencing was checked. We examinedKRASmutations in exon 2 codons 12 and 13 with DNA sequencing and SNaPshot analysis in 100 formalin-fixed paraffin-embedded tumor tissue samples of pancreatic carcinoma, colorectal carcinoma, and nonsmall cell lung cancer specimens of the primary tumor or metastases. 40% of these samples demonstrated mutatedKRASgenes using sequencing and SNaPshot-analysis; additional five samples (45/100) were identified only with the SNaPshot.KRASmutation detection is feasible with the reliable SNaPshot analysis method. The more frequent mutation detection by the SNaPshot analysis shows that this method has a high probability of accuracy in the detection ofKRASmutations compared to sequencing.

scholarly journals Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

2021 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Anna Von Schoenfeld ◽  
Peter Bronsert ◽  
Michael Poc ◽  
Andrew Fuller ◽  
Andrew Filby ◽  
...  
Keyword(s):  
Gas Phase ◽  
Signal Intensity ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Secondary Antibodies ◽  
Formalin Fixed

We describe a sequential multistaining protocol for immunohistochemistry, immunofluorescence and CyTOF imaging for formalin-fixed, paraffin-embedded specimens (FFPE) in the formalin gas-phase (FOLGAS), enabling sequential multistaining, independent from the primary and secondary antibodies and retrieval. Histomorphologic details are preserved, and crossreactivity and loss of signal intensity are not detectable. Combined with a DAB-based hydrophobic masking of metal-labeled primary antibodies, FOLGAS allows the extended use of CyTOF imaging in FFPE sections.

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