Critical Appraisal of Programmed Death Ligand 1 Reflex Diagnostic Testing: Current Standards and Future Opportunities

Abstract

IntroductionPatient suitability to anti–programmed death ligand 1 (PD-L1) immune checkpoint inhibition is key to the treatment of NSCLC. We present, applied to PD-L1 testing: a comprehensive cross-validation of two immunohistochemistry (IHC) clones; our descriptive experience in diagnostic reflex testing; the concordance of IHC to in situ RNA (RNA-ISH); and application of digital pathology.MethodsEight hundred thirteen NSCLC tumor samples collected from 564 diagnostic samples were analyzed prospectively, and 249 diagnostic samples analyzed retrospectively in tissue microarray format. Validated methods for IHC and RNA-ISH were tested in tissue microarrays and full sections and the QuPath system were used for digital pathology analysis.ResultsAntibody concordance of clones SP263 and 22C3 validation was 97% to 98% in squamous cell carcinoma and adenocarcinomas, respectively. Clinical NSCLC cases were reported as PD-L1–negative (48%), 1% to 49% (23%), and more than 50% (29%), with differences associated to tissue-type and EGFR status. Comparison of IHC and RNA-ISH was highly concordant in both subgroups. Comparison of digital assessment versus manual assessment was highly concordant. Discrepancies were mostly around the 1% clinical threshold. Challenging IHC interpretation included 1) calculating the total tumor cell denominator and the nature of PD-L1 expressing cell aggregates in cytology samples; 2) peritumoral expression of positive immune cells; 3) calculation of positive tumor percentages around clinical thresholds; and 4) relevance of the 100 malignant cell rule.ConclusionsSample type and EGFR status dictate differences in the expected percentage of PD-L1 expression. Analysis of PD-L1 is challenging, and interpretative guidelines are discussed. PD-L1 evaluations by RNA-ISH and digital pathology appear reliable, particularly in adenocarcinomas.

Cytology cell blocks from malignant pleural effusion are good candidates for PD-L1 detection in advanced NSCLC compared with matched histology samples

“…To better contrast our work with that in previous studies, we used three TPS cut-offs (< 1, 1-49% and ≥ 50%), which were used in the 22C3 assay, to evaluate PD-L1 expression. This decision was carefully considered and was based on the high concordance between the 22C3 and SP263 assays [14,24,27,28]. In our study, the expression rate of PD-L1 with TPS ≥50% as a cut-off for either histology (29.7%) or cytology (37.7%) was EBRT external beam radiotherapy; a different from previous treatments; Specimen 1 obtained earlier than specimen 2 similar to that noted in two previous studies of consecutive sample analyses [23,25].…”

“…As mentioned in many studies, PD-L1 evaluation is indeed a challenge for pathologists because it is sometimes difficult to distinguish tumor cells from immune cells, and this challenge is even more pronounced in cytology samples [23,24]. To our knowledge, it has not yet been reported whether IHC double staining for nuclear proteins such as thyroid transcription factor-1 (TTF-1) and PD-L1 can resolve this issue.…”

PD‐L1 expression in cell‐blocks of non‐small cell lung cancer: The impact of prolonged fixation

“…Developing a standardized procedure for cytological samples is undoubtedly challenging, since specimens can be fixed by a variety of methods and processed by different workflows. Moreover, although CBs can be prepared with distinct techniques, 18,30‐32 most studies applying PD‐L1 on cytology (Table 3) provide only few details on laboratory processing and fixation time 6‐9,11‐18 …”

“…Interestingly, 22 CBs have been included in the Blueprint (BP) PD‐L1 Immunohistochemistry Comparability Phase 2 Project, which aims to harmonize in real life practice the clinical use of different commercial PD‐L1 IHC assays on 81 routine lung cancer samples 5 . Noteworthy, several studies carried out on matched cytological and histological samples from the same patients have reported comparable results between CBs, surgical resections and small biopsy specimens in terms of adequacy rate, level of PD‐L1 expression, and clinical outcomes 6‐18 . Moreover, CBs also represent a valuable source of diagnostic material even in the early stages of the disease.…”

Improving the Diagnostic Accuracy of the PD-L1 Test with Image Analysis and Multiplex Hybridization

“…However, the accelerated adoption of these diagnostic tests has highlighted several difficulties in the pathological assessment of PD-L1. We recently reported on the routine challenges faced clinically in assessment of PD-L1 [5]. This is in addition to the myriad of companion diagnostic assays available for PD-L1 and the variation in assessment criteria across tumor types [6][7][8].…”