Impact of delayed fixation and decalcification on PD-L1 expression: a comparison of two clones

Forest, Fabien; Cote, Gaelle; Laville, David; Cruz, Vanessa Da; Col, Pierre Dal; Camy, Florian; Mobarki, Mousa; Clémenson, Alix; Yvorel, Violaine; Péoc’h, Michel

doi:10.1007/s00428-019-02613-w

Cited by 25 publications

(19 citation statements)

References 20 publications

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“…Both acid and ethylenediaminetetraacetic acid (EDTA) declacifiers reduce the proportion of PD‐L1 positive cells stained and the intensity with which they stain. Acid decalcification is more deleterious and shows an effect sooner than EDTA .…”

Section: Discussionmentioning

confidence: 99%

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

Dodson¹,

Parry²,

Lissenberg‐Witte

et al. 2019

The Journal of Pathology CR

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PD-L1 inhibitors are part of first line treatment options for patients with advanced non-small cell lung cancer. PD-L1 immunohistochemistry (IHC) assays act as either a companion or a complementary diagnostic. The purpose of this study is to describe the experience of external quality assurance (EQA) provider UK NEQAS ICC and ISH with the comparison of different PD-L1 assays used in daily practice. Three EQA rounds (pilot, run A and run B) were carried out using formalin fixed paraffin embedded samples with sample sets covering a range of epitope concentrations, including 'critical samples' near to clinical threshold cut-offs. An expert panel (n = 4) evaluated all returned slides simultaneously and independently on a multi-header microscope together with the participants own in-house control material. The tonsil sample was evaluated as 'acceptable' or 'unacceptable', and for the other samples the percentage of PD-L1 stained tumour cells were estimated in predetermined categories (<1%, 1 to <5%, 5 to <10%, 10 to <25%, 25 to <50%, 50 to <80%, 80 to 100%). In the pilot and the two subsequent runs the number of participating laboratories was 43, 69 and 76, respectively. The pass rate for the pilot run was 67%; this increased to 81% at run A and 82% at run B. For two 'critical samples', in runs A and B, 22C3 IHC had significantly higher PD-L1 expression than SP263 IHC (p < 0.001), whilst the PD-L1 scores for the other six samples were similar for all assays. In run A the laboratory developed tests (LDTs) using 22C3 scored lower than the commercial 22C3 tests (p = 0.01). After the initial testing, improvement in performance of PD-L1 IHC is shown for approved and LDT PD-L1 assays. Equivalency of approved PD-L1 22C3 and SP263 assays cannot be assumed as the scores cross the clinically relevant thresholds of 1% and 50% PD-L1 expression.

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Section: Discussionmentioning

confidence: 99%

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

Dodson¹,

Parry²,

Lissenberg‐Witte

et al. 2019

The Journal of Pathology CR

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“…22 Decalcification seems to slightly decrease the yield of staining, particularly when ethylenediaminetetraacetic acid is used in combination with 22C3 clone. 23 Furthermore, avoiding overfixation is of paramount importance because 20% and 10% of samples can be suboptimally stained with IHC using the SP142 and SP263 clones, respectively, when fixation duration is beyond 96 hours, whereas only 3% to 6% of samples may suffer suboptimal staining with 12 to 72hour fixation. 24 Similarly, cellblock processing protocols affect PD-L1 staining.…”

Section: Discussionmentioning

confidence: 99%

The International Association for the Study of Lung Cancer Global Survey on Programmed Death-Ligand 1 Testing for NSCLC

Mino‐Kenudson

Stang

Daigneault

et al. 2021

Journal of Thoracic Oncology

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“…Moreover, in another study, the type and duration of decalcification were shown to drastically reduce the PD-L1 IHC intensity and the proportion of positive cells [10,14]. When PD-L1 IHC intensities after decalcification were compared between two IHC clones (22C3 and E1L3N), there was a slight reduction in percentage of positive cells only for 22C3 clone while staining using E1L3N clone was stable [10]. Therefore, the knowledge of the effect of preanalytical variables on the performance of a predictive biomarker is essential for implementation in the clinical practice.…”

Section: Introductionmentioning

confidence: 92%

“…Recently, in a tissue model experiment on the effect of delayed fixation, reduced IHC staining of several antibodies, including PD-L1 and cytokeratins, was found [8]. Moreover, in another study, the type and duration of decalcification were shown to drastically reduce the PD-L1 IHC intensity and the proportion of positive cells [10,14]. When PD-L1 IHC intensities after decalcification were compared between two IHC clones (22C3 and E1L3N), there was a slight reduction in percentage of positive cells only for 22C3 clone while staining using E1L3N clone was stable [10].…”

Section: Introductionmentioning

confidence: 95%

“…Pre-analytical variables in daily pathology practice include steps from the sample collection until start of fixation. Potentially, most important factors for the quality of the sample and those that can be influenced by the way of sample handling are (1) warm ischemia time (relevant for the resection specimens and starts from the blood supply cut off), (2) cold ischemia time (time from removal from the body until fixation) [8], (3) temperature at which fresh sample is kept during cold ischemia time [9], (4) type of fixative and decalcification [10], but also (5) tissue to fixative ratio [11]. In general, the possible effect of variation in prenanalytical variables on predictive biomarker testing is frequently mentioned in reviews [12,13], but detailed studies on this topic are sparse.…”

Section: Introductionmentioning

confidence: 99%

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Influence of preanalytical variables on performance of delta-like protein 3 (DLL3) predictive immunohistochemistry

et al. 2020

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DLL3 might become a predictive immunohistochemical marker in small cell carcinoma of the lung (SCLC). We investigated the influence of pre-analytical handling of samples on the performance of DLL3 immunohistochemistry (IHC) using DLL3 SP347 ready to use assay (Ventana). DLL3 positive cell lines were subjected to different experimental conditions mimicking the pre-analytical variation in daily clinical practice. Formalin fixation of 24 h led to the most optimal results of DLL3 IHC. Longstanding fixation in Cytolyt, methanol-based fixative for cytology samples, but also decalcification using a mix of formic- and hydrochloracid resulted in decreased DLL3 staining. Postponed staining of blanc slides for 3 months also decreased DLL3 IHC. Postponed fixation of the SCLC cell lines did not influence the performance of DLL3 IHC, although this might be different in the tissues than in the cell lines. In conclusion, different pre-analytical variables decrease the performance of DLL3 IHC. These findings are essential for implementing novel predictive immunohistochemical biomarkers in daily pathology practice.

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Impact of delayed fixation and decalcification on PD-L1 expression: a comparison of two clones

Cited by 25 publications

References 20 publications

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

External quality assessment demonstrates that PD‐L1 22C3 and SP263 assays are systematically different

The International Association for the Study of Lung Cancer Global Survey on Programmed Death-Ligand 1 Testing for NSCLC

Influence of preanalytical variables on performance of delta-like protein 3 (DLL3) predictive immunohistochemistry

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