Summary
Immunohistochemical assays for programmed cell death ligand 1 (PD-L1) expression in non–small cell lung cancer (NSCLC) are either required or recommended to guide therapy with immune checkpoint inhibitors. Four commercially available immunohistochemical assays are currently available as either complimentary or companion diagnostic assay for their counterpart therapy. Harmonization or exchangeability of one assay for the other is a highly sought for goal. The aim of this study was to compare one assay, 22C3, with another, SP263, and examine whether they can be exchanged one for the other. Seventy samples from 70 patients with NSCLC were tested for PD-L1 using the SP263 and then the 22C3 antibody clones according to the manufacturer’s instructions in case of the SP263 assay and according to a previously described and reported method for the 22C3 assay on the Ventana’s ultra immunstainer. Results were evaluable in 51 cases, which were interpreted independently by 2 different pathologists on 2 different occasions for each case. The cases were given a percentage score based on the tumor proportion score. The Pearson correlation coefficient was calculated. A high concordance rate was found between the 2 assays. The Pearson correlation coefficient was 0.95, which indicates an almost perfect correlation (95% confidence limits, 0.92-0.97 and P<0.0001). The findings indicate that SP263 assay can be used in place of the 22C3 assay for PD-L1 assay in NSCLC, and it can be used on the Ventana platform.
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Original Article
Comparison of 22C3 PharmDx and SP263 Assays to Test PD-L1 Expression in NSCLC
Applied Immunohistochemistry & Molecular Morphology
Maher A. Sughayer, MD, Farah Alnaimy, MD, Anas M. Alsughayer, and Naim Qamhia, MD
Immunohistochemical assays for programmed cell death ligand 1 (PD-L1) expression in non–small cell lung cancer (NSCLC) are either required or recommended to guide therapy with immune checkpoint inhibitors. Four commercially available immunohistochemical assays are currently available as either complimentary or companion diagnostic assay for their counterpart therapy. Harmonization or exchangeability of one assay for the other is a highly sought for goal. The aim of this study was to compare one assay, 22C3, with another, SP263, and examine whether they can be exchanged one for the other. Seventy samples from 70 patients with NSCLC were tested for PD-L1 using the SP263 and then the 22C3 antibody clones according to the manufacturer’s instructions in case of the SP263 assay and according to a previously described and reported method for the 22C3 assay on the Ventana’s ultra immunstainer. Results were evaluable in 51 cases, which were interpreted independently by 2 different pathologists on 2 different occasions for each case. The cases were given a percentage score based on the tumor proportion score. The Pearson correlation coefficient was calculated. A high concordance rate was found between the 2 assays. The Pearson correlation coefficient was 0.95, which indicates an almost perfect correlation (95% confidence limits, 0.92-0.97 and P<0.0001). The findings indicate that SP263 assay can be used in place of the 22C3 assay for PD-L1 assay in NSCLC, and it can be used on the Ventana platform.
Immunotherapy has emerged as an important and effective modality of therapy against several types of cancer in the past few years. The immune checkpoint inhibitors anti–programmed cell death receptor 1 (anti-PD-1) and anti–programmed cell death ligand 1 (anti-PD-L1) have been variously approved for the treatment of malignant tumors such as melanoma, non–small cell lung cancer (NSCLC), urothelial malignancies, and head and neck squamous cell carcinoma among others.1
In advanced NSCLC they have been approved in second-line and then extended to first-line therapy in the case of pembrolizumab.2–11 The latter approval was for patients with high positive PD-L1 expression with >50% tumor proportion score (TPS).12
The approval was based on clinical trials that showed response in patients with NSCLC to be related to the degree of expression of the biomarker PD-L1 by immunohistochemistry (IHC).2–12 For pembrolizumab, the approval is conditioned with the use of the companion assay Dako 22C3 monoclonal antibody. The other drugs, namely nivolumab, atezolizumab, and durvalumab, have complementary rather than companion assays, which are Dako 28-8, Roche-Ventana SP142 and Roche-Ventana SP263, respectively. These complementary assays are not mandatory to prescribe the therapy, but their use is recommended to guide and optimize therapy. The PD-L1 assay is far from being perfect, and each trial has different cutoffs of what is considered as positive or negative expression.13,14 Therefore, each assay has a different scoring system. Pathology laboratories face a challenge when it comes to assaying PD-L1 to inform therapeutic options, because of the different companion or complementary assays that differ in many aspects of the testing process, ranging from the antibody clone used to the validated platform for such an assay. In some of the complementary assays (Roche-Ventatna SP142), the target cells being assayed are also different, in that both tumor cells and immune cells should be assessed, rather than only tumor cells in the other assays.
It would therefore be reasonable to harmonize the various assays available, so that they can be exchangeable. Each laboratory would then have only 1 assay or 1 platform rather than having multiple assays with their respective platforms. Several studies have addressed the issue of harmonization and comparison of the various assays.15–18 It would be even more reasonable and feasible if an assay can be validated on a platform different from the one on which it was originally validated. Such cross-validation has been performed19,20 and shown that, indeed, one may run an assay on a different platform, as in the case of Dako’s 22C3 on Ventana’s platforms, Benchmark XT and Ultra.
In this study, we aimed to compare 2 different assays for the total positive percentages in NSCL to see whether these assays are interchangeable in that regard. The emphasis is put on the comparability of the assays at the 50% TPS cutoff used in the US Food and Drug Administration (FDA)-approved test for first-line therapy in NSCLC. The 2 assays are Ventana SP263 and Dako 22C3, run on Ventana’s Ultra platform. Both of these, as well as the other 2, are FDA approved for testing before second-line therapy in NSCLC, but only Dako 22C3 is of greater interest, being the only companion test for an approved first-line therapy in NSCLC.
Materials and Methods
A total of 70 samples from 70 patients with stages III-IV NSCLC were submitted for PD-L1 assay. The samples were retrieved from the archives of the Department of Pathology and Laboratory Medicine, King Hussein Cancer Center, Amman, Jordan. In total, 63 of the specimens were from biopsy samples, with only 7 samples from surgical resection specimens. They were accessioned consecutively during 2016 and 2017. However, the final number of samples with evaluable results was 51.
The histologic subtypes of the cases were as follows: 35 adenocarcinomas, 9 squamous cell carcinomas, 4 adenosquamous carcinomas, and 4 NSCLC not otherwise specified.
Immunostaining was performed on all the specimens using the Ventana SP263 antibody clone (Ventana Medical Systems Inc., Tucson, AZ) as per the manufacturer’s instructions. The same specimens were then stained using the Dako 22C3 antibody clone (Dako, Carpinteria, CA) according to the protocol described by Ilie et al20 with 1 modification: the antibody used was the prediluted antibody extracted from the PD-L1 IHC 22C3 pharmDx kit rather than using dilutions of an antibody concentrate.
The interpretations were independently performed for each stain by 2 of the authors (M.A.S. and F.A.), wherein the pathologist was blinded to the results of the other stain, and with at least 1 week washout interval between the readings. Each case was given a score on the basis of the proportion of tumor cells (TPS) that showed partial or complete membranous staining of any intensity as a percentage of the total number of viable tumor cells, provided that each specimen had at least 100 viable cells.
Pearson correlation coefficient was calculated for the paired TPS results of the samples.
The cutoffs for each antibody clone, as described by the corresponding trials, were compared using descriptive statistics.
Results
The quality of the staining was excellent and comparable between the 2 clones (Fig. 1).
A high concordance rate was obtained between the 22C3 and the SP263 clones when the TPS readings were compared. The Pearson correlation coefficient was 0.95 (95% confidence limits, 0.92-0.97 and P<0.0001 (Fig. 2).
The number of cases that were above or equal to the 50% high positive cutoff for the 22C3 assay was 15 of 51 (29.4%), whereas the number of cases above the same cutoff by the SP263 clone was 18 (35.3%). However, when the discordant cases were reviewed, it turned out that the readings were very close and around the cutoff. Four cases had a TPS of 50% by the SP263 assay while the TPSs by the 22C3 assay were 40% for 2 cases and 30% for the other 2. In contrast, 1 case with a TPS of 50% by the 22C3 assay had a TPS of 30% by the SP263 assay. The discordant cases were all from small biopsy samples, and, upon review for the total number of evaluable tumor cells, each was found to contain at least 100 tumor cells, although the sections were noticed to be smaller on the 22C3-stained consecutive sections, as these were carried out later.
When considering the intended 25% cutoff for the SP263 assay in NSCLC, 27 of the 51 (52.9%) cases were at or above this cutoff by this assay, and 26 (51%) cases were above or at this cutoff by the 22C3 assay. There were 3 discordant cases, 1 scored at 30% by the 22C3 and 20% by the SP263 assay, and 2 cases were exactly the opposite. Again, they were all around the cutoff. They also were from small biopsy specimens and contained at least 100 evaluable tumor cells.
When considering the low positive cutoff of 1% in the 22C3 assay, the concordance rate was 100% between the 2 assays.
Discussion
Treatment of advanced NSCLC has witnessed several breakthroughs recently. The most important of those is the use of immunotherapy in the form of checkpoint blockade in second-line and just recently in the first-line treatments with impressive improvements in patients’ outcome over traditional therapies.1–12
The use of checkpoint inhibitors to treat patients with advanced NSCLC has been approved in association with either companion or complementary diagnostic assays. Pembrolizumab in particular requires the use of the FDA-approved companion diagnostic assay Dako’s PD-L1 IHC 22C3 pharmDx kits on Dako’s platform Autostainer Link 48. However, this may not be practical in many pathology laboratories for various reasons among which is the unavailability of the specified platform and the availability instead of other platforms. For this reason the usability of the 22C3 clone on other platforms including the Roche-Ventana system has been previously evaluated and validated.19,20
In contrast, 2 studies examined the comparability of the various commercially available PD-L1 antibody clones. The blueprint phase 1 study, which compared 4 of the antibody clones, concluded that 3 of them, namely 22C3, SP263, and 28-8, were comparable in estimating the percentage of tumor cell positivity and the fourth, SP142, showed lower estimates.15 Phase 2 study which has just been presented at the 2017 IASLC conference confirmed the previous findings.21 In addition, a study carried out by Astra-Zeneca, which only compared 3 clones, had similar conclusions.18 On the basis of the latter study, Roche obtained the CE mark for in vitro diagnostic use of SP263 assay in testing PD-L1 for the purpose of prescribing pembrolizumab in second and then first-line treatments of patients with advanced NSCL.22
In this study, we compared 2 commercially available antibody clones to see whether we can use one in exchange for the other. Our aim was to use SP263 assay in place of 22C3 assay to guide pembrolizumab therapy in NSCLC. We found a high concordance rate in the TPS percentage, which confirms the findings of the 2 previously mentioned studies.
The concordance rate at the cutoff of 1% was 100%. The few discordant cases at the other cutoff points are easily explained by the subjectivity of the methods of estimating the TPS. Especially so if we know that the estimated values were very close and around the cutoff point. This is a well-known artifact that is common to values that are estimated by eye balling in pathology, such as the percentage of Ki-67 in a tumor. It has been recommended to use a more objective method in these cases, such as image analysis. It is clear that a subjective estimate of 40% positivity can easily be estimated to be 50% by a different person or even the same person.
The limitations of this study include the fact that the original FDA-approved assay, Dako 22C3, was not used in the comparison, but rather a laboratory-developed test using the same antibody clone but on a different platform. In addition, we do not have outcome data yet to validate the results of these assays in our patients.
Nevertheless, on the basis of this study and the previous studies, we conclude that SP263 assay can be used in place of the 22C3 assay for PD-L1 testing for the purposes of guiding therapy with anti-PD-1/PD-L1 in NSCLC.
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