Tissue Selection for PD-L1 Testing in Triple Negative Breast Cancer (TNBC)
Case selection: this study falls under the banner of performance assessment/quality assurance and, as such, did not require approval by a UK Research Ethics Committee. A retrospective selection of FFPE tissue blocks from 73 TNBC cases with matched diagnostic core biopsies and excision biopsies were collected from the participating hospitals. Inclusion criteria were TNBC diagnosis on pathology records and availability of FFPE blocks from both core biopsy and excision. All cases included in this study were treated by primary surgery (no neoadjuvant chemotherapy) and therefore the time elapsed from core biopsy to excision was minimal. For each case, we stained the core biopsy and all the available tumor tissue from the excision biopsy (up to a maximum of 5 blocks) with H&E and PD-L1, using the SP142 clone on-label protocol. Immunohistochemistry: Paraffin sections were cut at 3 to 5 µm and mounted on TOMO slides (TOM-11, Matsunami Glass Ind Ltd., Japan) together with the appropriate on-slide tissue controls and stained with anti-PD-L1 (SP142) rabbit monoclonal primary antibody (Roche Ventana) on a BenchMark Ultra platform (Roche Ventana) using the validated locked-down protocol. The staining protocol for the SP142 CDx was available in the package insert. Briefly, antigen retrieval was undertaken for 48 minutes, the primary antibody was applied for 16 minutes, amplification was done for 8 minutes/8 minutes multimer, and sections were counterstained for 4 minutes with haematoxylin II and post-counterstained with bluing reagent for 4 minutes. All sections were stained within a week from being cut. All slides were mounted with glass coverslips. Slide assessment: slides were digitized using Panoramic 250 Flash-3 scanners (3D-Histech) using x20 lens. Digital slides were stored on a CaseCenter v2.9 (3D-Histech) and viewed using CaseViewer v2.2.1 (3D-Histech). Glass slides were viewed with transmitted light microscopes (Olympus BX series) fitted with planapo lenses and multi-head facility. All pathologists (FD, AB, AC, KB, M-PC, RV, ML-T, EA, MC, PT and CD) had been trained in PD-L1 assessment for TNBC according to the SP142 algorithm. Pathologists scored either glass slides, digital slides or both. Three PD-L1 scores were given as described in Figure : (A) one score for each individual slide, (B) a cumulative score of each individual excision sample by averaging the PD-L1 expression over the entire tumor area present in all slides from the excision, and (C) a cumulative score of each individual patient by averaging the PD-L1 expression over the entire tumor area present in all slides from both core biopsy and excision. All cases showing discrepancy between pathologists were re-assessed by two pathologists (P.T. and C.D.) and a consensus diagnosis reached using a multi-head microscope. A slide, case or patient status was considered positive if the PD-L1 score was ≥1%. Approach to scoring and Atlas: we realize that pathologists use different approaches for assessing PD-L1, especially in complex cases with heterogeneous staining. An example of a systematic approach to scoring is described in Figure . In the course of the work, we realized that it would be useful to have a reference atlas, demonstrating different PD-L1 staining patterns to help achieve better concordance. This atlas was prepared by taking microphotographs from digital slides all taken at the same magnification (digital ×20) from the cases used in this study. Each image was overlaid with a grid of 100 equal tiles (each tile equaling 1% of the total area). The microphotographs were then scored by 3 pathologists independently for PD-L1 IC expression (% of area occupied by PD-L1 positive IC in relation to the total area) according to the interpretation guide provided by the manufacturer and described in the original paper. Once the individual scores were collected, we realized that there was discrepancy in the assessment of some images. We therefore decided to employ the encircling technique, as described by Vennapusa et al (7, Supplemental Digital Content 3). To do this, we remeasured PD-L1 scores using a manual geometrical scoring approach: each image was reviewed and re-scored in consensus by 2 pathologists. Briefly, the positive immune cells (singly or in clusters) on the digital slides were each encircled manually with an overlaid smooth perimeter; the individual perimeters were then manually clustered together and a PD-L1 score was obtained by adding the number of tiles occupied by the clustered perimeters (see Fig. ). Figure is an extract from the atlas, the full collection is available as an online atlas (see Supplemental Digital Content 1 & 2, http://links.lww.com/AIMM/A357 & http://links.lww.com/AIMM/A358 , for images representing the full range of PD-L1 scoring). Our hope is that now this atlas can support diagnostic pathologists to find solutions to their interpretation problems in daily practice. Clinical audit: the Poundbury laboratory provided centralized testing for PD-L1 in TNBC as part of the UK EAMS (Early Access Medicines Scheme) from 2018 to 2021. It was also a referral laboratory for all patients with TNBC from Ireland from 2019 to 2022. From the onset, all cases that were negative for PD-L1 and had relatively small amounts of tumor tissue in the examined blocks were offered repeat testing of additional tissue. Following retesting of the requested tissue, a final PD-L1 diagnosis was issued based on an average score for PD-L1 on the total area of tumor tissue tested (first test plus second test). The following details were recorded for each patient: the type of tissue selected for testing, the PD-L1 status, whether additional tissue was requested for testing, whether the additional tissue was sent, the PD-L1 status of the additional tissue and the final PD-L1 clinical status. This work was audited to determine the proportion of cases identified as requiring retesting and whether retesting changed the final PD-L1 diagnosis. 

PD-L1 status in core biopsies and resections: All 73 cases entered in this study were successfully assessed for PD-L1 status on both core biopsy and resection material. Of the initial core biopsies, 22 (30%) were PD-L1 positive and 51 (70%) negative. However, 16 of the negative cases converted to final positive PD-L1 status upon assessment of the matching resection tissue. This represents 22% of the total number of patients and almost a third of the PD-L1 negative cases on core biopsies. When both excision and biopsy samples were considered together, 38 patients (52%) had a positive PD-L1 status and 35 patients (48%) remained PD-L1 negative. None of the initial positive scores on core biopsies was converted to negative status upon assessment of the excision. Clinical audit: In the period audited (2019-2021), the laboratory received a total of 1458 original requests relative to 1395 patients (some patients had more than one biopsy tested at different times). The audit data is summarized in Table A. In the initial testing, 490 samples (33.6%) were positive, but after retesting on additional tissues from the patients with initial negative status, a total of 558 (38.9%) were found positive. Of the initial requests, only 22 (1.5%) were deemed insufficient for analysis. Of the 946 negative cases, 392 were considered relatively small for reliable testing and retesting was requested, however only 288 of these had sufficient tissue material available. Table B extrapolates these figures to provide a theoretical positivity rate if all negative tissue were available for retesting. 

With the approval of therapy targeting checkpoint inhibitors for the treatment of TNBC, pathologists are being asked to assess tumor tissue for PD-L1 status to guide patient selection. A number of questions have been raised in relation to the choice of tissue for testing, but very limited guidance exists. The most common questions are: is it preferable to use tissue from primary tumor or metastatic or local recurrence lesions? What tissue is the best predictor of patient response to anti-PD-L1 treatment – that taken before or after chemotherapy and/or radiotherapy? Is there a preference for using resection samples over core biopsies or vice versa? Studies of other cancer types and TNBC have highlighted differences in PD-L1 status between primary and metastasis, and there are some indications that particular metastatic sites, such as liver, may be immunologically “protected”. The clinical implications are not yet well understood. Although testing of an archival biopsy from the primary carcinoma at first clinical presentation may not be representative of the current PD-L1 status in the patient, the IMpassion130 study used any tissue types available (except for cytology samples and bone biopsies, due to lack of stroma and use of decalcification respectively). Apart from a lower incidence of PD-L1 positivity in tissue of metastatic TNBC compared to primary, no trial data suggest that particular tissue sample types may be less effective at identifying responders. At present, therefore, there is no recommendation to favor any tissue sample type over another, but there is a need to clarify this. There are also studies reporting that PD-L1 expression is likely to change after (neo)-adjuvant therapy. It remains unclear, however, if these changes are also reflected in the clinical response to anti-PD-L1 treatment. In the IMpassion130 study, patients were allowed to have received previous radiation and chemotherapy in a curative context, if treatment was completed more than 12 months before randomization. Still, the question remains to be further investigated and guidelines are needed. In immune-excluded tumors, immune cells positive for PD-L1 accumulate along the invasive margin. This has prompted the concern that tissue samples that do not contain the tumor margin may result in underestimation of PD-L1. In addition, we have observed that in a significant proportion of all clinical samples the distribution of the immune infiltrate is heterogeneous. Thus, sampling and size of biopsy might introduce bias in PD-L1 assessment. In this study we aimed to address whether the size of the tissue tested had an influence on PD-L1 status. To do so, we compared core biopsies with excision samples. We did not attempt to measure the tumor area in each sample, but assumed that core biopsies in general are smaller than excision samples. Indirectly, our aim was also to look for indications of the impact of non-controlled preanalytical differences between biopsies and surgical specimens in daily practice. We already know that the PD-L1 SP142 CDx is sensitive to variations in preanalytics, including cold ischemia time, prolonged or insufficient fixation and suboptimal processing. There is more abundant tissue from excision samples (either mastectomy or wide local excision) and, in general, they include the invasive margin, but they are more prone to preanalytical variations, while core biopsy tissue is usually small but has better preanalytics. Rather than choosing random tissue samples from the excision, we elected to investigate a set of cases with matched core biopsy and excision, as these would resemble more closely the clinical settings, particularly for what is affected by bias related to anatomic access during the core biopsy procedure. Our data do not support the idea that core biopsies should be preferred due to preanalytical reasons. However, it might be necessary to opt for biopsies for reasons of patient management workflow. We could not find any negative resection sample that had a positive antecedent biopsy in our study. The Impassion130 positivity rate was 40.9% based on a cohort of 902 patients, with a mix of available FFPE tumor tissue (primary and metastatic, biopsy and resection). In our series, only 30% of the cohort had a positive PD-L1 score when core biopsies alone were evaluated. This is in contrast to 52% when resection samples were assessed. We do not know if the 22% of patients who “converted” from negative to positive PD-L1 status would have clinical benefit from anti-PD-L1 treatment. The published trial data did not provide any precise information regarding tissue selection and indicated that any available tissue can be used for testing. The failure to consider tissue selection is common in clinical trials but it is a limitation, and a better understanding of the impact of tissue selection is crucial for effective implementation into clinical routine. Our results indicate that tissue selection should be considered as an important parameter to be assessed in clinical trials, especially for heterogeneous biomarkers, as has been indicated in other studies. The primary objective of the pathologist, however, is to identify all patients eligible for treatment, so with the information we have at hand, we propose the implementation of a workflow which follows up patients with a negative PD-L1 status on core biopsies with a repeat test on the subsequent resection. Our data (including our theoretical extrapolation from the audit data in Table B) indicate that this workflow will result in the capture of more patients eligible for treatment, and more closely represent the numbers seen in the initial clinical study. To date, we have no reason to believe that this approach would include patients unlikely to benefit from the treatment, but future studies are required to narrow down the best algorithm for identifying the right patients and ruling out the nonresponders. Ever since the introduction of PD-L1 scoring in TNBC, pathologists have had a need for a visual guide to aid their daily practice. A limited number of examples are provided as a part of the interpretation guide, but they are without precise numerical scores. Our work resulted in a large number of digital cases, from which it was easy to produce an atlas providing examples of different amounts of IC staining with a precise numerical annotation. We decided to use the manual encircling technique to validate the pathologist consensus score since we would like our atlas to provide a precise, definitive and practical aid to support PD-L1 scoring in TNBC. In conclusion, our study demonstrates that small tissue samples with PD-L1 status below the 1% threshold should be re-assessed using additional tissue (resection samples or additional biopsy tissue). Although we are still uncertain of the real-world prevalence of PD-L1-positive TNBC, and await larger trials, we have obtained positivity rates closer to the published rates by repeat testing in our clinical practice, and we have developed an atlas to aid other pathologists. 

SUPPLEMENTARY MATERIAL Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.appliedimmunohist.com . 

