Development of a diagnostic PD-L1 immunohistochemical assay for nivolumab therapy in urothelial carcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e14585-e14585 ◽  
Author(s):  
Rose Ann De Los Santos ◽  
Josette William ◽  
Debra Ann Hanks ◽  
Adam Northrup ◽  
Dipeshkumar Jaiswal ◽  
...  
Keyword(s):  
Urothelial Carcinoma ◽  
Tumor Cells ◽  
Staining Intensity ◽  
Observer Agreement ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Staining Protocol ◽  
Visualization Technology ◽  
Slide Type

e14585 Background: PD-L1 IHC 28-8 pharmDx is a qualitative assay developed by Agilent Technologies for the Autostainer Link 48 platform and is based on EnVision FLEX visualization technology and monoclonal rabbit anti-PD-L1, clone 28-8 antibody. The assay has been co-developed with the immunotherapeutic agent nivolumab; initially as an aid in assessing PD-L1 expression in non-squamous non-small cell lung cancer (NSCLC) and melanoma patients. Here we describe the efforts to validate this assay for urothelial carcinoma (UC). Methods: IHC staining was performed on Autostainer Link 48 platform using an automated staining protocol stated per the assay’s instruction for use (IFU). Specimens were coverslipped and interpreted for % PD-L1 positive tumor cells. The assay was analytically validated on commercially acquired formalin-fixed, paraffin-embedded (FFPE) human UC invasive tumor specimens at ≥1% and ≥5% PD-L1 positive tumor cells expression levels. Results: A wide range of % PD-L1 positive tumor cells at all staining intensity levels have been detected. Assay in-house precision was validated for inter-operator, inter-instrument, inter-day, inter-run and intra-run as well as inter-observer and intra-observer agreement. Robustness studies evaluated the assay under multiple conditions for target retrieval pH, temperature and incubation time, slide type as well as tissue section thickness. Assay reproducibility was evaluated at three external sites by testing samples for intra-site/inter-day and inter-site agreement measures. Specimens were also evaluated by an observer at each site, with three reads for each observer to assess intra-observer and inter-observer agreement. All validation studies demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 84%. Conclusions: Results of all conducted studies show high robustness and reproducibility of the assay on UC.

scholarly journals Impact of Preanalytical Factors During Histology Processing on Section Suitability for Digital Image Analysis

2020 ◽  
pp. 019262332097053
Author(s):  
Elizabeth A. Chlipala ◽  
Mark Butters ◽  
Miles Brous ◽  
Jessica S. Fortin ◽  
Roni Archuletta ◽  
...  
Keyword(s):  
Image Analysis ◽  
Digital Image ◽  
Digital Image Analysis ◽  
Digital Pathology ◽  
Staining Intensity ◽  
Protocol Design ◽  
Formalin Fixed Paraffin ◽  
Mouse Tissues ◽  
Formalin Fixed Paraffin Embedded ◽  
Staining Protocol

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables—staining protocol design, reagent quality, section attributes, and instrumentation—on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

scholarly journals PD-L1 in Cytological Samples: A Review and a Practical Approach

2021 ◽  
Vol 8 ◽  
Author(s):  
Eva Tejerina ◽  
Laura García Tobar ◽  
José I. Echeveste ◽  
Carlos E. de Andrea ◽  
Elena Vigliar ◽  
...  
Keyword(s):  
Predictive Biomarkers ◽  
Cost Effective ◽  
Standard Of Care ◽  
Observer Agreement ◽  
Cancer Treatments ◽  
Formalin Fixed Paraffin ◽  
Programmed Death ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Programmed Death 1

With a growing number of predictive biomarkers needed to manage patients with non-small cell lung cancer (NSCLC), there has been a paradigm shift in care and handling of diagnostic samples. Among the various testing methods, immunohistochemistry (IHC) is the most cost- effective and widely available. Furthermore, over the past decade immunotherapy has emerged as one of the most promising cancer treatments. In this scenario IHC is the most used testing method available for PDL-1/PD1 immunotherapy. Several monoclonal antibodies targeting programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) pathways have been integrated into standard-of-care treatments of a wide range of cancer types, once provided evidence of PD-L1 expression in tumor cells by immunohistochemistry (IHC). Since currently available PD-L1 assays have been developed on formalin-fixed paraffin embedded (FFPE) histological specimens, a growing body of research is being dedicated to confirm the feasibility of applying PDL-1 assays also to cytological samples. Albeit promising results have been reported, several important issues still need to be addressed. Among these are the type of cytological samples, pre-analytical issues, cyto-histological correlation, and inter-observer agreement. This review briefly summarizes the knowledge of the role of cytopathology in the analysis of PD-L1 by immunocytochemistry (ICC) and future directions of cytopathology in the immunotherapy setting.

EGFR expression by immunohistochemistry (IHC) and response to chemotherapy and cetuximab in squamous cell carcinoma of the head and neck (SCCHN)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 6024-6024 ◽  
Author(s):  
M. S. Kies ◽  
M. S. Ghebremichael ◽  
T. L. Katz ◽  
R. S. Herbst ◽  
H. Youssoufian ◽  
...  
Keyword(s):  
Quantitative Methods ◽  
Staining Technique ◽  
Staining Intensity ◽  
Ordinal Scale ◽  
Tumor Resistance ◽  
Egfr Expression ◽  
Formalin Fixed Paraffin ◽  
Response To Chemotherapy ◽  
Initial Cohort ◽  
Formalin Fixed Paraffin Embedded

6024 Background: There is modest activity for cetuximab in recurrent SCCHN. Molecular markers for patient selection are desirable. Burtness et al (JCO 23:8646, 2005) reported that high EGFR expression predicted for tumor resistance to chemotherapy (CT) with or without cetuximab. We have studied an independent cohort of patients from the IMC-9816 study (Herbst et al, JCO 23:5578, 2005) using the same EGFR immunoreactivity staining technique. Methods: Slides were prepared from formalin fixed paraffin embedded tissue with the DAKO kit, with staining intensity graded on an ordinal scale 0–3 and staining density assessed according to the percentage of cells stained. High expression was defined as staining intensity 3 + on = 80% of cells. Tumor samples were available in 77 pts from the initial cohort of 132 patients with recurrent SCCHN who received cisplatin-based CT. Of the latter, 76 patients were assessed to have stable (SD 51) or progressive disease (PD 25) and then treated with cetuximab and cisplatin. An amendment permitted further enrollment of patients with PD after off study cisplatin therapy (N 54). Of a total of 130 pts who received cetuximab and cisplatin, tumor samples were available from 86. Results: There was no association of response with EGFR expression. Responses to CT are tabulated. Of the 86 SD and PD pts receiving CT and cetuximab, responses were observed in 3 of 44 and 7 of 42 pts with low and high EGFR expression, respectively (p=0.191). In a survival analysis of patients treated with cetuximab, the hazard ratio for subjects with high EGFR was 0.835, p=0.40. Conclusions: Conventional IHC, even when supplemented by staining intensity and staining density, does not predict for responsiveness of SCCHN to CT, with or without cetuximab. Future studies may require more quantitative methods, possibly subcellular localization, and measurement of other EGFR pathway targets, ligand content and probably “down-stream” effectors. No significant financial relationships to disclose. [Table: see text]

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.

Immunohistochemical Detection of Canine Adenovirus in Paraffin Sections of Liver

1986 ◽  
Vol 23 (4) ◽  
pp. 478-484 ◽  
Author(s):  
P. M. Rakich ◽  
K. W. Prasse ◽  
P. D. Lukert ◽  
L. M. Cornelius
Keyword(s):  
Immunohistochemical Detection ◽  
Hepatic Inflammation ◽  
Paraffin Sections ◽  
Inflammatory Lesions ◽  
Long Term Stability ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus-positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis.

scholarly journals NeuN: a useful neuronal marker for diagnostic histopathology.

1996 ◽  
Vol 44 (10) ◽  
pp. 1167-1171 ◽  
Author(s):  
H K Wolf ◽  
R Buslei ◽  
R Schmidt-Kastner ◽  
P K Schmidt-Kastner ◽  
T Pietsch ◽  
...  
Keyword(s):  
Neuronal Cell ◽  
Cell Types ◽  
Specific Protein ◽  
Antigen Retrieval ◽  
Neuronal Marker ◽  
Formalin Fixed Paraffin ◽  
Wide Range ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Diagnostic Histopathology

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

A Standardized Staining Protocol for L1CAM on Formalin-Fixed, Paraffin-Embedded Tissues Using Automated Platforms

2014 ◽  
Vol 29 (2) ◽  
pp. 180-183 ◽  
Author(s):  
Mina Fogel ◽  
Ayelet Harari ◽  
Elisabeth Müller-Holzner ◽  
Alain G. Zeimet ◽  
Gerhard Moldenhauer ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Staining Procedure ◽  
Type I ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Staining Protocol ◽  
Automated Platform ◽  
Endometrial Carcinomas ◽  
L1 Cell Adhesion Molecule ◽  
Formalin Fixed

The L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers and can serve as a biomarker for prognosis in most of these cancers (including type I endometrial carcinomas). Here we provide an optimized immunohistochemical staining procedure for a widely used automated platform (VENTANA™), which has recourse to commercially available primary antibody and detection reagents. In parallel, we optimized the staining on a semi-automated BioGenix (i6000) immunostainer. These protocols yield good stainings and should represent the basis for a reliable and standardized immunohistochemical detection of L1CAM in a variety of malignancies in different laboratories.

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