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Published in final edited form as: Mod Pathol. 2023 Mar 15;36(7):100159. doi: 10.1016/j.modpat.2023.100159

Tissue Age Affects Antigenicity and Scoring for the 22C3 Immunohistochemistry Companion Diagnostic Test

Aileen I Fernandez 1,*, Patricia Gaule 1,*, David L Rimm 1,2
PMCID: PMC10502188  NIHMSID: NIHMS1889375  PMID: 36925070

Abstract

Programmed death-ligand 1 (PD-L1) antibody 22C3 is the approved companion diagnostic immunohistochemistry (IHC) test for treatment with pembrolizumab and cemiplimab in multiple cancer types. The 22C3 and 28–8 antibodies target the extracellular domain (ECD) of PD-L1 which is known to contain N-glycosylation sites. We hypothesize that antigenicity could be affected by degradation of the glycan part of the epitope and thus change the scoring of the assay over time. Here, we test samples over time and assess the effects of time and deglycosylation on PD-L1 signal by comparing an antibody with an extracellular domain (ECD) antigen to an antibody with an intracellular domain (ICD) antigen. Ten whole tissue sections of non-small cell lung cancer (NSCLC) from 2018 were selected for testing. Fresh cut serial sections for each case were stained on DAKO Link48 for 22C3 according to the label. In parallel, a previously described laboratory developed test using E1L3N (an ICD antibody) was performed on the Leica BondRX. Tumor proportion scores (TPS) for 22C3 and E1L3N were read by a pathologist and compared to the previous clinical diagnoses. To determine the effect using a quantitative approach, a TMA cohort with 90 NSCLC cases was similarly assessed. Finally, to determine if the possible effect of epitope glycosylation, antibodies were tested before and after enzymatic deglycosylation of specimens. We found that 6/7 archival positive samples showed significant reduction in positive staining with 22C3 compared to original diagnostic sample assessed 3 years earlier. In an older archival TMA cohort, a quantitative significant difference in signal intensity was noted when staining with 22C3 was compared to E1L3N. This loss of signal was not noted in the fresh cell line TMA consistent with a time dependent degradation of staining. Finally, quantitative assessment of fresh TMA showed significant loss of signal after a deglycosylation procedure when stained with 22C3 which was not seen when stained with E1L3N. We believe this data shows that the glycan part of the 22C3 epitope is not stable over time, and that this issue should be considered when assessing archival tissue for diagnostic or research purposes.

Introduction

Immunotherapies targeting the Programmed death-ligand 1 (PD-L1) axis revolutionized cancer treatment and resulted in the accelerated approval of immune checkpoint inhibitors (ICIs) in multiple indications.1–6. Tumor or immune cell expression of PD-L1 has been used to predict response to anti-PD-1/PD-L1 therapies but predictive value for treatment response relative to PD-L1 expression is limited.7–13. Thus, some anti-PD-1/PD-L1 therapies received Food and Drug Administration (FDA) approval without requiring a PD-L1 immunohistochemistry (IHC) test14. The cause of the variability is not understood. It could be a result of tumor-specific biological phenomena or it could be systemic issues with PD-L1 detection by IHC.15–18.

The PD-L1 IHC 22C3 pharmDx® (22C3 assay) was amongst the first to be approved by the FDA as a companion diagnostic for immunohistochemical detection of PD-L119. Other approved PD-L1 complementary diagnostic IHC tests include Ventana SP142, Ventana SP263 and Agilent PD-L1 IHC 28–8 pharmDx®. Various studies have shown the variable agreement between these diagnostic tests in measuring PD-L1 by IHC when assessed on fresh samples (excluding SP142 which showed consistently lower analytical sensitivity)19–24. The antibody clones bind unique sequences of the PD-L1 protein on the intracellular (ICD) or extracellular domain (ECD). 22C3 and 28–8 each bind separate domains of the ECD. SP142 and SP263 target identical ICD sequences, whereas E1L3N binds a close to, but not the identical ICD sequence25.

High levels of N-linked glycosylation have been characterized in PD-L1 ECD18,26. These glycosyl residues add ~17kDa, 52% of the molecular weight of PD-L1 polypeptide (33KDa)27,28. Glycan residues on the ECD of PD-L1 have also been shown to affect the binding of clinical antibodies in pre-clinical studies which may explain variation in response to anti-PD-L1 treatments29,30. While is clear that both the 22C3 and 28–8 antibodies include some portion of some glycan in their epitope, it is not clear if this affects their effectiveness over time. Indeed, mechanisms of aging artefact are poorly understood. It is generally accepted that epitopes are stable within the FFPE block but never proven.

In a chance observation during some research experiments, we noted significant loss of PD-L1 positivity when assessed by the 22C3 assay, compared to the original clinical read, which was not observed when stained with the E1L3N assay. We sought to understand the mechanism by which this age dependent reduction in signal occurred. Limited research has been performed in this area, but the available data suggests that ECD targeted antibodies are subject to signal degradation by changes in humidity, oxygen concentration and direct light exposure. Other studies have confirmed loss of tumor proportion score (TPS) in aging specimens when stained with 22C331,32. These studies focus largely on expression in pre-sectioned slides rather than FFPE blocks. Here, we test the stability of 22C3 ECD assay compared to E1L3N ICD assay over time and determine the role of glycosylation in the changes seen with time.

Materials and Methods

Patient cohorts

Retrospectively collected formalin-fixed paraffin-embedded (FFPE) whole-tissue sections (WTS) from 10 NSCLC resection specimens collected at Yale School of Medicine in 2018 were obtained from Yale Pathology archives. Cases were selected based on sufficient archival FFPE tissue blocks and prior staining for PD-L1, using 22C3, as a reflex clinical order after diagnosis. All cases had archived H&E and PD-L1 slides available for review. Original PD-L1 scores were collected from clinical pathological reports.

Two TMAs (YTMA 386 and YTMA-472) were utilized for the deglycosylation assays. Tissue specimens were prepared in a TMA format as previously described33. Briefly, representative tissues areas were obtained from FFPE specimens and 0.6mm cores from each tumor block arrayed in a recipient block. YTMA386 consisted of panel of 15 isogenic cell lines expressing various amounts of PD-L1 sourced from Horizon Dx, including production and growth of cell lines, and TMA assembly as previously described34. YTMA 472 consisted of 90 patients in 2-fold redundancy from a non-serial collection of lung cancer cases ranging from 2009 to 2014. No clinicopathologic information from patients was collected from these cases. All human tissue was used in accordance with US Common Rule after approval from the Yale Human Investigation Committee protocol #9505008219.

Protein deglycosylation of TMA

Protein deglycosylation was performed using Protein Deglycosylation Mix II, (New England Biolabs, Ipswich, MA). Slides were baked at 60°C for 1 hour and incubated in xylenes twice for 20 minutes each. Slides were then rehydrated with two 1-minute, 100% ethanol washes, one 1-minute, 70% ethanol wash, and one 5-minute water rinse. Enzymatic deglycosylation was performed for one hour according to manufacturer’s instructions to simultaneously remove N-linked and O-linked glycyl groups while preserving native protein structures. Protein Deglycosylation Mix II contains both endo- and exoglycosidases including PNGase F (N-Glycosidase F), O-Glycosidase, neuraminidase (sialidase), β1–4 galactosidase, and β-N-acetylglucosaminidase (endoglycosidase H – removes the bulk of glycans). These each have a function in removing oligomannose N-linked, complex N-linked, short or long O-linked gylcoproteins, or resistant monosaccharides on O-glycans. Importantly, PNGase F (N-Glycosidase F) is an amidase that is the most effective way to remove N-linked glycoproteins35. Non-denaturing conditions were used to maintain the native protein structure. Denaturing conditions were used as a positive control to ensure the most efficient and complete level of deglycosylation (not shown). Control slides underwent the same conditions as the deglycosylated slides with the only differing substrate being the addition of the protein deglycosylation mix in the latter. Incubation time with the protein deglycosylation mix was optimized. One hour at 37C was determined to be the optimal conditions. Deglycosylation was followed by IHC staining as described below.

Chromogenic staining with PD-L1 antibodies

TMA, with and without deglycosylation, and WTS were processed for chromogenic staining as follows:

  • 22C3- Tissue sections were subject to PD-L1 22C3 (pharmDx, Santa Clara, CA, USA) staining on Agilent Link 48 using PD-L1 IHC 22C3 pharmDx© kit as per manufacturer’s instructions. Sections were de-paraffinized, dehydrated and subjected to target retrieval in PT link using Envision Flex target retrieval solution low pH. Following cycle completion slides were immersed in 1x Envision Flex Wash buffer and cooled to room temperature. Slides were transferred to Link48 and stained using pharmDx protocol on label.

  • E1L3N- Tissue sections were subject to PD-L1 E1L3N (#13684, Cell Signaling Technology, Danvers, Massachusetts, USA) staining on Leica Bond Rx using Leica Refine Polymer Detection Kit, as per manufacturer’s instructions. Briefly, sections were baked online at 60°C for 30 minutes followed by standard dewax and rehydrate program using Bond Dewax and 100% ethanol. Post-rehydration heat-induced antigen retrieval was performed using ER2 for 20 mins at 100°C and slides were cooled to ambient temperatures. Peroxide block was added for 5 mins at room temperature (RT) followed by primary PD-L1 (E1L3N- 3.33 ug/ml) antibody for 1 hour at RT. Post Primary reagent was added for 8 minutes at RT followed by Polymer for 8 mins at RT. Mixed Dab Refine was added to slides 2x at ambient temperatures and finally hematoxylin was added for 5 minutes at RT. Between each step after peroxide addition, standard Bond Rx washing protocol was applied (3 × Bond wash at ambient temperatures). This lab developed test has been described previously23.

Tumor proportion scores

Tumor proportion scores (TPS) were read as the number of PD-L1 positive tumor cells (full or partial membrane staining)/ total number of tumor cells multiplied by 100.

All sections used in this publication were freshly sectioned and used within 5 days. TPS scores were scored by a single pathologist (DLR) blinded to the staining conditions of each set.

Digital image analysis

Slides were scanned on the Aperio ScanScope XT platform (Leica Biosystems Inc., IL, USA). PD-L1 expression was quantified using open-source software QuPath (Queen’s University of Belfast, Northern Ireland). Briefly, once images obtained from Aperio ScanScope XT were loaded, cores were automatically selected through the TMA annotation module, and later classified by the module “positive cell detection”. An optimized algorithm was used for cell segmentation based on the size of the nucleus and cell expansion, and for DAB intensity quantification of PD-L1 expression for all antibodies looking at the “Cell: DAB OD mean” score in the nucleus, cytoplasm, and/or cell compartment. The settings were adjusted to avoid false positive detection. Results were shown as percentage of PD-L1+ cells or as optical density of the chromogenic staining, divided by mm2 (OD/mm2).

Statistical analysis

Mann-Whitney non-parametric t-tests were used to test the differences between antibody clones, as well as control tissue versus deglycosylated tissue. Simple linear regression was used to compare the distribution of the antibody clones and calculate the R2 value. All data analysis was performed using PRISM 9.3.1 (GraphPad, San Diego, CA).

Results

22C3 (ECD) shows reduction of TPS score over time

For proof of concept, ten NSCLC resection specimens from 2018, with sufficient archival FFPE tissue blocks, were selected. Of the 10 specimens stained with 22C3 in 2018, 2 had an original TPS score of <1, 3 had a score of 1–49, and 5 had a score of >50. Serial sections from all the samples were stained for E1L3N and 22C3. Representative images are shown in Figure 1. Of the 8 samples with original TPS >1, 7 showed a detectable decrease in their PD-L1 TPS score (4/5 and 3/3 with PD-L1 TPS score >50, or 1–49, respectively) (Table 1). Only 1 sample (Sample 1) showed no change in TPS, however, upon visual review the sample was noted to have decreased intensity relative to the original stain. 3/8 samples with TPS >1 showed a reduction in TPS score when stained by E1L3N assay. Predictably, all negative (TPS <1) remained negative by either assay. Overall, the 22C3 assay showed notably higher levels of signal loss.

Figure 1.

Figure 1.

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A selected gallery of images representative cases previously assessed for Programmed death-ligand 1 (PD-L1) (in 2018). 3 cases >50 TPS, 2 cases 1–49 TPS and 1 case <1 TPS are shown. From left to right, 22C3 clinical stain from 2018, 2021 22C3 IHC stain, and 2021 E1L3N IHC stain. Low power view paired with a 20x view.

Table 1.

Pathologist reads of 10 whole-tissue sections(WRS) resection specimens selected from 2018. Original clinical read is shown as well as 2021 reads of E1L3N and 22C3

Sample Clinical 22c3 (2018) 22C3 (2021) E1L3N LDT (2021)
1* >50 >50 >50
2* >50 1–49 >50
3 >50 1–49 >50
4* >50 <1 >50
5 >50 1–49 1–49
6* 1–49 <1 1–49
7 1–49 <1 <1
8* 1–49 <1 <1
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(*)

indicates samples with images included in Figure 1.

To confirm this hypothesis, we assessed a larger cohort, YTMA 472, which was constructed from resection samples collected from 2008 – 2014. Here we used semi-quantitative analysis to remove potential bias of pathologist reads. Optical density per area (OD/mm2) of 22C3 assay was significantly lower than E1L3N assay (Mann Whitney t-test, p<0.0001) (Figure 2A, B). Since TPS scoring by a pathologist is independent of intensity we also calculated percent positive cells by setting a threshold for each lowest visual positive cell and compared across the cores. Percent positivity was significantly lower in 22C3 assay compared to E1L3N assay (Mann Whitney t-test, p<0.0001) (Figure 2C, D).

Figure 2.

Figure 2.

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Distribution and differences in optical density per area (mm2) (A, B) and percent cells positive (C, D) between 22C3 and E1L3N Programmed death-ligand 1 antibodies in YTMA472. (Mann Whitney t-test, **** p<0.0001).

Quantitative assessment of loss of signal after deglycosylation

Due to the increased loss of signal seen with the 22C3 assay, compared to E1L3N, we hypothesized that glycosyl group degradation, and the subsequent destabilization of the three-dimensional configuration of the ECD of PD-L1, leads to sub-optimal epitope-antibody affinity. Using two TMAs, YTMA-386 (PD-L1 isogenic cell line) and YTMA 472 (NSCLC), we compared the PD-L1 optical density and percent positivity with, and without, deglycosylation. Both sets of slides were stained in a single run to reduce staining variation as a source of error. In YTMA-386 a significant decrease in OD/mm2 was observed when staining with 22C3 after deglycosylation (Figure 3A). Percent positivity also trended downwards but did not reach statistical significance (Figure 3C). No significant change was noted between conditions when staining with E1L3N (Figure 3B,D). Interestingly, there was no significant difference between control and deglycosylation in YTMA-472 for both OD/mm2 and percent positivity for either antibody (Figure S1 A–D). We believe that the glycan residues on YTMA-472 were already fully degraded, consistent with the ages of the samples (most of the samples were 8–10 years old or older).

Figure 3.

Figure 3.

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Differences in staining measured in control and deglycosylated tissue. Optical density per area (A, B) and percent cells positive (C, D) of E1L3N (A, C) and 22C3 (B, D) measured in YTMA386 (cell line array). (Mann Whitney t-test, *p<0.05).

Discussion

PD-L1 expression measured by IHC has been fraught with questions and uncertainties since their original deployment. A search in PubMed for the terms ‘PD-L1 IHC Comparison’ yields close to 100 publications in which authors have tried to evaluate and establish harmonization procedures for the discrepancy seen in these assays36. There have also been extensive technical evaluation of the assays and their limitations24,37–39. Many studies have investigated the antigen stability of PD-L1, but mostly on cut sections. Indeed, unpublished work from our own laboratory has demonstrated epitope instability beginning at 3 months room temperature storage in a dark environment on cut slides. Here, we have shown the effect of aging on the PD-L1 positivity level when assayed using 22C3 assay in NSCLC samples in FFPE blocks. Further, we show that this reduction is most likely associated with the degradation of N-terminal glycosylation sites of the PD-L1 protein. PD-L1 contains N-linked glycosylation sites at residues 35, 192, 200 and 21929,40. Others have examined the effect of deglycosylation on the PD-L1 binding ability of ECD targeted antibodies. Abrogation of N-linked glycosylation at residue 219 significantly reduced 22c3 binding ability by western blot. Interestingly, in the same experiment 28–8 binding was decreased, contrary to other studies which have stated that deglycosylation improves 28–8 binding and improves the predictive potential of 28–8 as a marker of response to Nivolumab25,28,41.

There are a number of limitations to this work. Since the original observation was fortuitous, we used only 10 WTS that were randomly selected based on their original clinical stain but did not expand the WTS beyond this set due to the challenge of obtaining these specimens. It is another limitation that we used TMA for some aspects of this work. However, we felt this was valid since TMAs were not read, but rather quantitative scored for intensity. We also did not evaluate the effect of this deglycosylation on other FDA-approved complementary diagnostics and compared only to an E1L3N LDT. In fact, there are also other potential explanations for the change in expression beyond deglycosylation. While our data are consistent with epitope deglycosylation being a critical issue, this work does not definitively prove this fact. Further, the E1L3N assay has proven to be equivalent with other assays when freshly stained. A recent publication examined the LOD of the common PD-L1 antibody clones36. The E1L3N antibody has the lowest LOD compared to any of common PD-L1 antibody clones which supports our results noted on YTMA472 and YTMA386 demonstrating higher baseline OD/mm2. Though the deglycosylation protocol was carried out with both positive and negative controls, the use mass spectrometry or liquid chromatography would be ideal for measuring the extent of glycosylation. However, since this was carried out on slides and not in a test tube to preserve spatial information, these methods were not viable. Other groups have also used antibody measurement by IHC or immunofluorescence, after deglycosylation, as a direct read out of the impact of deglycosylation28,42, however it important to note they used a less robust set of enzymes and different antibodies. Future studies can include using these methods, as well as stepwise deglycolsylation, to measure the rate of occupancy and determine the fine structure of the glycans, however this is beyond the scope of this work. Finally, this study is limited to a single histologic indication of primary NSCLC. We did not examine additional tumor types and/or metastatic lesions to analyze consistency of degradation across histologies. Since our hypothesis is protein based and no evidence exists suggesting PD-L1 structural variance between histologies, we believe this single histology approach does not compromise the conclusion. Despite these limitations this study provides insight into the hazard of using aged specimens for patient qualification when using 22C3 assay. The PD-L1 IHC 22C3 pharmDx kit is projected to hold over 50% of the PD-L1 biomarker market share by 2032 and is still the only approved companion diagnostic43 for some drugs. The assay label lists a requirement of slides freshly sectioned within the previous 6 months but provides no specific guidelines as to block age. Pembrolizumab is currently approved in 16 cancer types across 26 specific conditions. Eight conditions require PD-L1 expression by an FDA approved test for treatment qualification. Of these 8, 7 are prescribed in recurrent and/or after progressive disease43. Standard of care does not necessarily support re-biopsy of patient lesions unless medically necessary and often the archival diagnostic specimen (if not previously tested) will be used for qualification. In this case a false negative result may be misleading to clinicians. The situation may also disproportionally affect late stage/metastatic patients whom upon exhaustion of all standard of care options, participate in exploratory clinical trials. Many trials are underway using combination treatments with an anti-PD-1 inhibitors combined with other agents. PD-L1 expression with a combined positive score >1 is often used as a qualifying metric in these combination trials in previous diagnostic specimens where a fresh biopsy is not mandated or medically unsafe44. Overall, our results suggest that all assays on blocks >1 year old who present with a negative result by 22C3 assay may want to consider repeating the assay using another PD-L1 assay against the ICD to confirm true negativity.

Supplementary Material

1

Acknowledgments

Thank you to Lori Charette and the Yale Pathology Tissue Services core facility.

Conflicts of Interest

David L. Rimm has served as an advisor for Astra Zeneca, Agendia, Amgen, BMS, Cell Signaling Technology, Cepheid, Danaher, Daiichi Sankyo, Genoptix/Novartis, GSK, Konica Minolta, Merck, NanoString, PAIGE.AI, Roche, and Sanofi. Amgen, Cepheid, NavigateBP, NextCure, and Konica Minolta have recently funded research in David L. Rimm’s lab. Patricia Gaule is now an employee of Bristol Meyers Squibb. Other authors have no relevant conflicts of interest.

Funding

This work is supported by a sponsored research agreement from Cepheid and by a grant from the Breast Cancer Research Foundation and by the Yale SPORE in Lung Cancer and the Yale Cancer Center CCSG. Dr. Aileen Fernandez is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Program Award PDEP1022351.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ethics Approval and Consent to Participate

Written informed consent or waiver of consent was provided by all the patients. This study was approved by Yale Human Investigation Committee protocol ID 9505008219. This study was performed in accordance with the Declaration of Helsinki.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1889375-supplement-1.pdf (104.2KB, pdf)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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