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Aims: Patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) are eligible for first‐line immune checkpoint inhibition if their tumour is positive for programmed death ligand 1 (PD‐L1) determined by the combined positive score (CPS). This nationwide study, using real‐world data, investigated the developing PD‐L1 testing landscape in the first 3 years after introduction of the test in HNSCC and examined interlaboratory variation in PD‐L1 positivity rates. Methods: Pathology reports of HNSCC patients mentioning PD‐L1 were extracted from the Dutch Pathology Registry (Palga). Tumour and PD‐L1 testing characteristics were analysed per year and interlaboratory variation in PD‐L1 positivity rates was assessed using funnel plots with 95% confidence limits around the overall mean. Results: A total of 817 PD‐L1 tests were reported in 702 patients among 19 laboratories; 85.2% of the tests on histological material were stated to be positive. The national PD‐L1 positivity rate differed significantly per year during the study period (79.7–89.9%). The use of the recommended 22C3 antibody increased from 59.9 to 74.3%. A total of 673 PD‐L1 tests on histological material from 12 laboratories were analysed to investigate interlaboratory variation. Four (33%) deviated significantly from the national mean of PD‐L1‐positive cases using CPS ≥ 1 cut‐off, while two (17%) deviated significantly for CPS ≥ 20 cut‐off. Conclusion: In the first 3 years of PD‐L1 assessment in HNSCC, the testing landscape became more uniform. However, interlaboratory variation in PD‐L1 positivity rates between Dutch laboratories was substantial. This implies that there is a need for further test standardisation to reduce this variation. [ABSTRACT FROM AUTHOR]

Aims: Immune checkpoint inhibitors targeting programmed death‐ligand 1 (PD‐L1) have shown promising clinical outcomes in urothelial carcinoma (UC). The combined positive score (CPS) quantifies PD‐L1 22C3 expression in UC, but it can vary between pathologists due to the consideration of both immune and tumour cell positivity. Methods and Results: An artificial intelligence (AI)‐powered PD‐L1 CPS analyser was developed using 1,275,907 cells and 6175.42 mm2 of tissue annotated by pathologists, extracted from 400 PD‐L1 22C3‐stained whole slide images of UC. We validated the AI model on 543 UC PD‐L1 22C3 cases collected from three institutions. There were 446 cases (82.1%) where the CPS results (CPS ≥10 or <10) were in complete agreement between three pathologists, and 486 cases (89.5%) where the AI‐powered CPS results matched the consensus of two or more pathologists. In the pathologist's assessment of the CPS, statistically significant differences were noted depending on the source hospital (P = 0.003). Three pathologists reevaluated discrepancy cases with AI‐powered CPS results. After using the AI as a guide and revising, the complete agreement increased to 93.9%. The AI model contributed to improving the concordance between pathologists across various factors including hospital, specimen type, pathologic T stage, histologic subtypes, and dominant PD‐L1‐positive cell type. In the revised results, the evaluation discordance among slides from different hospitals was mitigated. Conclusion: This study suggests that AI models can help pathologists to reduce discrepancies between pathologists in quantifying immunohistochemistry including PD‐L1 22C3 CPS, especially when evaluating data from different institutions, such as in a telepathology setting. [ABSTRACT FROM AUTHOR]

Aims: Programmed death‐ligand 1 (PD‐L1) expression is a predictive biomarker for adjuvant immunotherapy and has been linked to poor differentiation in lung adenocarcinoma. However, its prevalence and prognostic role in the context of the novel histologic grade has not been evaluated. Methods: We analysed a cohort of 1233 patients with resected lung adenocarcinoma where PD‐L1 immunohistochemistry (22C3 assay) was reflexively tested. Tumour PD‐L1 expression was correlated with the new standardized International Association for the Study of Lung Cancer (IASLC) histologic grading system (G1, G2, and G3). Clinicopathologic features including patient outcome were analysed. Results: PD‐L1 was positive (≥1%) in 7.0%, 23.5%, and 63.0% of G1, G2, and G3 tumours, respectively. PD‐L1 positivity was significantly associated with male sex, smoking, and less sublobar resection among patients with G2 tumours, but this association was less pronounced in those with G3 tumours. PD‐L1 was an independent risk factor for recurrence (adjusted hazard ratio [HR] = 3.25, 95% confidence intervals [CI] = 1.93–5.48, P < 0.001) and death (adjusted HR = 2.69, 95% CI = 1.13–6.40, P = 0.026) in the G2 group, but not in the G3 group (adjusted HR for recurrence = 0.94, 95% CI = 0.64–1.40, P = 0.778). Conclusion: PD‐L1 expression differs substantially across IASLC grades and identifies aggressive tumours within the G2 subgroup. This knowledge may be used for both prognostication and designing future studies on adjuvant immunotherapy. [ABSTRACT FROM AUTHOR]

Aims: Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody‐drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN‐4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN‐4/NECTIN‐4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. Methods and results: The TCGA BLCA (n = 405) and the CCC‐EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN‐4/NECTIN‐4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN‐4/NECTIN‐4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN‐4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H‐score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN‐4 expression was reduced in neuroendocrine‐like and/or protein‐based double‐negative tumours. TROP2‐ and NECTIN‐4‐negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD‐L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN‐4 expression. Conclusions: TACSTD2/TROP2 and NECTIN‐4/NECTIN‐4 are widely expressed in aUC, independent of FGFR3 alterations or PD‐L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(‐like) and sarcomatoid aUC. [ABSTRACT FROM AUTHOR]

Programmed cell death ligand‐1 (PD‐L1) expression in cancer may predict clinical response to immunotherapeutic treatment with PD‐1/PD‐L1 inhibitors. Within the vulvar cancer field, PD‐L1 expression has only been assessed by a few studies. We conducted a meta‐analysis to examine the prevalence of PD‐L1 positivity in vulvar cancer. PubMed, Embase, and Cochrane were searched for articles reporting on PD‐L1 expression in vulvar cancer. Study selection and data extraction were performed independently by two authors. We extracted data on PD‐L1 prevalence in vulvar cancer according to combined positive score (CPS) and tumour proportion score (TPS). Cutoff values for positivity were ≥1 or ≥10 for CPS and ≥1% and ≥5% for TPS. Random‐effects models were used to estimate pooled PD‐L1 prevalence, with 95% confidence intervals (CIs). Tests of between‐study heterogeneity were evaluated by the I2 statistics. Sources of heterogeneity were explored by subgroup analyses and meta‐regression. In total, 19 studies were included. Pooled PD‐L1 prevalence in vulvar cancer was 83.4% (95% CI: 70.8–91.3; I2 = 80.0) and 53.9% (95% CI: 37.4–69.6; I2 = 93.0) according to CPS and TPS, respectively. Based on TPS, human papillomavirus (HPV)‐associated vulvar squamous cell carcinomas (SCC) showed a lower PD‐L1 prevalence (39.9%; 95% CI: 13.3–74.2) compared with HPV‐independent SCC (62.6%; 95% CI: 33.7–84.6), but meta‐regression showed no significant variation in PD‐L1 prevalence by HPV status. PD‐L1 prevalence was similar in advanced (44.9%; 95% CI: 29.8–61.1) and localized vulvar cancer (56.7%; 95% CI: 18.9–76.7). In conclusion, PD‐L1 expression in vulvar cancer is frequent but between‐study heterogeneity was high. Based on a subgroup of heterogenous studies, we found no strong variation in PD‐L1 prevalence according to HPV status and stage. [ABSTRACT FROM AUTHOR]

Aims: Gastric adenocarcinoma with enteroblastic differentiation (GAED) is a rare subset of alpha‐fetoprotein (AFP)‐producing carcinomas with poor prognosis. However, the molecular features associated with the malignant potential of GEAD remain partially elucidated. Methods and results: In this study, the relationship between clinicopathological parameters and aggressive biological behaviour was analysed in 37 patients with GAED. The results showed that GAED tended to infiltrate the deep layer of the gastric wall and possessed more frequent vascular invasion than conventional gastric adenocarcinoma (CGA) (P < 0.001). All distant metastases were observed in the GAED group, not the CGA group (P < 0.001). High HER2 expression was found in nearly 24.32% of the informative cases, and none showed EBV‐encoded RNA positivity or deficient mismatch repair. The most frequently mutated gene in GAED was p53. Programmed cell death‐ligand 1 (PD‐L1) immunostaining revealed 13 patients with a combined positive score (CPS) ≥ 5 (65%, 13 of 20). Thus, based on these molecular markers (immunostaining, in situ hybridisation and mutation analysis), GAED may be classified as a unique subgroup of the chromosomal instability subtype with HER2+/EBV−/MSS/TP53+/PD‐L1+. Next‐generation sequencing analyses showed that mutations in the TOPI, ELOA and NOTCH3 genes were found only in GAED, and abnormally expressed genes in GAED were significantly enriched in hepatocellular carcinoma‐, gland development‐, and gastric cancer‐related pathways. Conclusion: The HER2+/EBV−/MSS/TP53+/PD‐L1+ profile and hepatocellular carcinoma‐related pathways may be significant in the malignant potential of GAED. In addition to anti‐HER2 therapy, immune check‐point inhibitors may be an effective treatment option for patients with GAED. [ABSTRACT FROM AUTHOR]

Aims: Oesophageal small‐cell carcinoma is a rare and highly aggressive subtype of oesophageal cancer with a dismal prognosis. To explore the potential applicability of immunotherapy, we investigated the expression status of programmed death ligand 1 (PD‐L1) and human leukocyte antigen (HLA)‐class I and the degree of tumour‐infiltrating lymphocytes (TILs) in oesophageal small‐cell carcinoma. Methods and results: PD‐L1 and HLA‐class I expression levels were evaluated in 10 pure small‐cell carcinomas and five mixed neuroendocrine‐non‐neuroendocrine neoplasms (MiNENs). The combined positive score (CPS) and tumour proportion score (TPS) were used for PD‐L1 assessment. Immunohistochemistry for mismatch repair (MMR) proteins was also performed. PD‐L1 immunohistochemistry demonstrated CPS ≥1 in nine (60%), CPS ≥10 in five (33%), and TPS ≥1 in five (33%) cases. Overall survival was significantly longer in patients with CPS ≥1 than in those with CPS <1. HLA‐class I deficiency (>50% tumour cells) was noted in five cases (33%), with no significant correlation with PD‐L1 expression status. Among the five MiNENs, HLA‐class I expression was decreased in the small‐cell carcinoma component of three cases. HLA‐class I deficiency was significantly associated with higher TNM stage and reduced TIL levels. MMR deficiency was not observed in any case. Conclusion: Given that a significant subset (40%) exhibited PD‐L1 CPS ≥1 with preserved HLA‐class I expression and high levels of TIL, the PD‐1/PD‐L1 pathway is a potential therapeutic target for oesophageal small‐cell carcinoma. [ABSTRACT FROM AUTHOR]

Aims: The current criteria for surgical treatment after endoscopic resection of a pT1 colorectal carcinoma (CRC) are unsatisfactory because nodal involvement is rarely present. This study investigates the correlation between PD‐L1 expression and nodal metastasis in pT1 CRCs to enable its use for tailoring surgical treatment after endoscopic removal. Methods and results: Histopathological features of 81 surgically resected pT1 CRC, 19 metastatic and 62 non‐metastatic cases were assessed. PD‐L1 expression was evaluated by immunohistochemistry (clone 22C3) and independently assessed by two pathologists using tumour proportion score (TPS), combined positive score (CPS) and immune cell score (ICS). The correlation between PD‐L1 expression and nodal metastasis, the optimal cut‐off values, interobserver agreement and impact upon patients' surgical management were determined. PD‐L1 expression in terms of CPS and ICS independently correlated with lymph node metastasis (PD‐L1CPS: OR = –2.5, 95% CI = −4.11 to −0.97, P = 0.008 and PD‐L1ICS: OR = −1.85, 95% CI = −2.90 to −0.79, P = 0.004) and < 1.2 CPS and <1.3% ICS were identified as the optimal cut‐off values to discriminate between metastatic and non‐metastatic patients. In our cohort, the implementation of these cut‐off values would have avoided a significant rate of unnecessary surgeries in pN0 patients (PD‐L1CPS = 43.2; PD‐L1ICS = 51.9%). Ultimately, PD‐L1 evaluation showed good interpathologist concordance in absolute terms [PD‐L1CPS interclass correlation coefficient (ICC) = 0.91; PD‐L1ICS ICC = 0.793] and using the identified cut‐off values (PD‐L1CPS ICC = 0.848; PD‐L1ICS ICC = 0.756). Conclusions: Our study shows that PD‐L1 expression is an effective predictor of nodal status and could improve patient selection for surgery after endoscopic removal of pT1 CRCs. [ABSTRACT FROM AUTHOR]

Background and aims: In gastric cancer (GC), HER2 was the first biomarker for guided therapy registered for clinical use. Considering the recent approvals of immune check‐point blockade (ICB) in gastro‐oesophageal cancers, testing for mismatch repair deficiency (dMMR), Epstein–Barr virus (EBV) and PD‐L1 combined positive score (CPS) is becoming increasingly important. Here we describe a real‐world cohort on biomarker assessment in GC patients. Methods: Patients diagnosed with GC between 2017 and 2021 were included. Biomarker results were retrieved from electronic patient files. PD‐L1 CPS was determined retrospectively on dMMR and EBV‐positive (EBV+) tumours. Data on genomic sequencing were analysed separately. Results: Of 363 patients identified, 45% had metastatic disease. In 335 patients (92%) at least one biomarker was tested. The prevalence of HER2+, dMMR and EBV+ tumours was 10% (32 of 319), 7% (20 of 294) and 1% (three of 235), respectively. Of the dMMR and EBV+ tumours, 95% had a PD‐L1 CPS ≥ 5. Therapeutic strategy was adjusted in 31 of 55 patients and consisted of anti‐HER2 therapies as well as ICB in clinical trials. Genomic alterations were found in 44 of 60 tested patients. TP53 (73%) and PIK3CA (20%) mutations were most common, followed by KRAS mutations (11%) and amplifications (11%). Conclusions: In this real‐world cohort, testing for HER2, dMMR and EBV status affected treatment decisions in 56% of the patients. Although most dMMR and EBV+ tumours had a PD‐L1 CPS ≥ 5, not all patients with a high probability of treatment response are identified. Based on these results, a stepwise diagnostic strategy is proposed. [ABSTRACT FROM AUTHOR]

Aims: High‐grade metaplastic breast carcinoma (HG‐MBC) is a rare subtype of invasive breast carcinoma, mostly triple‐negative. Metaplastic carcinomas are less responsive to neoadjuvant chemotherapy and are associated with a worse outcome than invasive carcinomas of no special type. Methods: Clinicopathological characteristics and immunophenotype were retrospectively assessed in a series of 65 patients diagnosed with HG‐MBC between 2005 and 2017 at the Curie Institute (antibody panel: oestrogen receptor [ER], progesterone receptor [PR], androgen receptor [AR], human epidermal growth factor receptor 2 [HER2], programmed death ligand‐1 [PD‐L1], and trophoblast cell surface antigen 2 [TROP2]). Results: The median age at diagnosis was 59.5 years. Six (9%) patients had metastatic disease at diagnosis. Among the nonmetastatic patients receiving neoadjuvant therapy, 26% (5/19) achieved pathological complete response. Most tumours were pT1/pT2 (77%) and 12% were pN+. Histological subtypes (mixed, squamous, mesenchymal, and spindle cell) were 40%, 35.5%, 15.5%, and 9%, respectively. Tumour‐infiltrating lymphocytes were low or moderate except when squamous differentiation was present. Most tumours were triple‐negative (92%). AR and TROP2 were positive in 34% and 85% of the cases, respectively. PD‐L1 was positive in tumour cells in 18% (cutoff: 1% of positive tumour cells) of the cases and in tumour‐infiltrating immune cells in 40% (cutoff: 1% of tumour area) of the cases. Notably, spindle cell and mesenchymal metaplastic breast carcinomas were mostly PDL1‐negative. Lastly, 21 (32.3%) cases were HER2‐low, all being HER2 1+, with no HER2 2+. Conclusion: Metaplastic breast carcinoma could benefit from tailored therapeutic strategies adapted to the phenotypic specificities of histological subtypes. [ABSTRACT FROM AUTHOR]

Aims: A common concern among pathologists scoring PD‐L1 immunohistochemical staining is interobserver and intraobserver variability. We assessed the interobserver and intraobserver reproducibility of PD‐L1 scoring among trained pathologists using a combined positive score (CPS; tumour cell and tumour‐associated immune cell staining). Methods and results: Data were collected for 2 years (2017–2019) from 456 pathologists worldwide. Digital training encompassed unique, tumour‐specific training, and test sets. Samples were stained using PD‐L1 IHC 22C3 pharmDx and evaluated at specific CPS cutoffs for gastric cancer (GC), cervical cancer (CC), urothelial cancer (UC), oesophageal cancer (OC), and head and neck squamous cell carcinoma (HNSCC). Pathologists underwent expert‐to‐peer training and scored 20 blinded samples on day 1 and 25 blinded samples on day 2 (including 15 of the day 1 samples). Interobserver and intraobserver reproducibility were assessed. For GC (120 observers) and CC (32 observers) samples assessed at CPS ≥1, average interobserver agreement was 91.5% and 91.0%, respectively, and average intraobserver agreement was 90.2% and 96.6%, respectively. For UC (139 observers) and OC (52 observers) samples measured at CPS ≥10, average interobserver agreement was 93.4% and 93.7%, respectively, and average intraobserver agreement was 92.0% and 92.5%, respectively. For HNSCC samples (113 observers), average interobserver agreement was 94.1% at CPS ≥1 and 86.5% at CPS ≥20; intraobserver agreement was 94.7% at CPS ≥1 and 90.5% at CPS ≥20. Conclusion: The consistently high interobserver and intraobserver concordance rates support the effectiveness of face‐to‐face training of many global pathologists for scoring PD‐L1 CPS across multiple indications at several specific cutoffs. [ABSTRACT FROM AUTHOR]

Background: Serrated adenocarcinoma (SAC), a recognised WHO variant of colonic adenocarcinoma, is the purported end‐product of serrated neoplasia. However, the diagnosis of SAC is infrequently rendered, and little is known about its prognosis, immune microenvironment and molecular alterations. Materials and methods: We assessed 903 consecutive colon carcinomas and recognised tumours with ≥ 5% (n = 77) serrated and ≥ 50% serrated patterns (n = 13). We assessed precursor polyps and synchronous polyps. We recorded demographic/clinical parameters, histological features and mismatch repair (MMR) status. We performed immunohistochemistry and quantification on tissue microarray for HLA class I/II proteins, B2MG, CD8, CD163, LAG3, FoxP3, PD‐L1 and BRAF V600E. Results: We identified ≥ 5% epithelial serration prevalence in 8.5% of cases and ≥ 50% epithelial serration prevalence in 1.4% of cases. Precursor lesions were present in 21.4% of cases; these were mostly tubular adenomas with two traditional serrated adenomas identified. SAC with ≥ 5% serrations exhibited lower numbers of CD8‐positive lymphocytes (P = 0.002) and lower B2MG expression (P = 0.048), although neither value was significant at ≥ 50% serration threshold. There was no difference in HLA class I/II, or PD‐L1 expression on tumour cells and no difference in PD‐L1, LAG3, FoxP3 and CD163 expression on immune cells. There was no association with MMR status, or BRAFV600E relative to conventional adenocarcinoma. There was improved disease‐specific survival on univariate (but not multivariate) analysis between carcinomas with serrated pattern and non‐mucinous conventional colonic carcinomas at ≥ 5% epithelial serrations (P = 0.04). Conclusion: SAC category shows a limited impact on survival, and this phenotype may harbour a unique immunological milieu. [ABSTRACT FROM AUTHOR]

Aims: The aim of this multicentre study was to harmonize programmed death‐ligand 1 (PD‐L1) immunohistochemistry (IHC) and melanoma scoring. To provide a reference for PD‐L1 expression independently of the IHC protocol, PD‐L1 mRNA expression was compared with IHC. Methods and results: Standardized PD‐L1 assays (22C3, 28–8, SP142, SP263) and laboratory‐developed tests (QR1, 22C3) were evaluated on three IHC platforms with a training set (seven cases). mRNA expression was determined by RNAscope (CD274/PD‐L1 probe) and analysed with image analysis. PD‐L1 IHC findings were scored by seven blinded pathologists using the tumour proportion score (TPS), the combined positive score (CPS), and the MELscore. This method was validated by three blinded pathologists on 40 metastatic melanomas. Concordances among various antibody/platforms were high across antibodies [intraclass correlation coefficient (ICC) >0.80 for the CPS], except for SP142. Two levels of immunostaining intensity were observed: high (QR1 and SP263) and low (28–8, 22C3, and SP142). Reproducibilities across pathologists were higher for QR1 and SP263 (ICC ≥0.87 and ICC ≥0.85 for the TPS and the CPS, respectively). QR1, SP263 and 28–8 showed the highest concordance with mRNA expression. We developed a standardized method for PD‐L1 immunodetection and scoring, tested on 40 metastatic melanomas. Concordances among antibodies were excellent for all criteria, and concordances among pathologists were better for the MELscore than for other scores. Conclusion: Harmonization of PD‐L1 staining and scoring in melanomas with good concordance is achievable with the PD‐L1 IHC protocols applied to other cancers; this reproducible approach can simplify daily practice. [ABSTRACT FROM AUTHOR]

Aims: Immunohistochemical programmed death‐ligand 1 (PD‐L1) staining to predict responsiveness to immunotherapy in patients with advanced non‐small cell lung cancer (NSCLC) has several drawbacks: a robust gold standard is lacking, and there is substantial interobserver and intraobserver variance, with up to 20% discordance around cutoff points. The aim of this study was to develop a new deep learning‐based PD‐L1 tumour proportion score (TPS) algorithm, trained and validated on a routine diagnostic dataset of digitised PD‐L1 (22C3, laboratory‐developed test)‐stained samples. Methods and results: We designed a fully supervised deep learning algorithm for whole‐slide PD‐L1 assessment, consisting of four sequential convolutional neural networks (CNNs), using aiforia create software. We included 199 whole slide images (WSIs) of 'routine diagnostic' histology samples from stage IV NSCLC patients, and trained the algorithm by using a training set of 60 representative cases. We validated the algorithm by comparing the algorithm TPS with the reference score in a held‐out validation set. The algorithm had similar concordance with the reference score (79%) as the pathologists had with one another (75%). The intraclass coefficient was 0.96 and Cohen's κ coefficient was 0.69 for the algorithm. Around the 1% and 50% cutoff points, concordance was also similar between pathologists and the algorithm. Conclusions: We designed a new, deep learning‐based PD‐L1 TPS algorithm that is similarly able to assess PD‐L1 expression in daily routine diagnostic cases as pathologists. Successful validation on routine diagnostic WSIs and detailed visual feedback show that this algorithm meets the requirements for functioning as a 'scoring assistant'. [ABSTRACT FROM AUTHOR]

Aims: The introduction of immunotherapy for patients with head and neck squamous cell carcinoma (HNSCC) raises the need for harmonisation between different types of antibody and immunohistochemistry platform for evaluating the expression of PD-L1 by use of the combined positive score (CPS) in this tumour. The aim of this study was to compare the expression of PD-L1 as determined with the CPS and two widely used assays (the 22C3 PharmDx assay and the SP263 assay) in a cohort of HNSCCs. Methods and results: We analysed 43 whole sections of HNSCC with two different anti-PD-L1 antibodies, 22C3 and SP263. The results, expressed as the CPS, were evaluated by 10 trained pathologists and statistical analyses were performed. We found a very similar results for PD-L1 expression between the 22C3 PharmDx assay and the SP263 assay in our cohort, and a strong and significant correlation between the two assays for all specimens (P < 0.0001). The interobserver reliability among pathologists for the continuous scores of CPS with the intraclass correlation coefficient and the correlation between the two assays were both good. Moreover, the rate of agreement between assays was high at all cut-offs and was best for the most relevant cut-off of CPS = 1, and the kappa values were always in the range of almost perfect. Conclusions: Two different assays (the 22C3 PharmDx assay and SP263 assay) for PD-L1 in HNSCC showed high agreement. These data suggest that these two assays are interchangeable in the selection of patients with HNSCC for immunotherapy. [ABSTRACT FROM AUTHOR]

Aims: In the 2020 World Health Organization classification of female genital tumours, endocervical adenocarcinomas (ECAs) are subclassified into human papillomavirus (HPV)-associated (HPVA) and HPVindependent (HPVI) groups on the basis of their distinct aetiologies and clinical behaviours. The aim of this study was to investigate programmed death-ligand 1 (PD-L1) expression and its prognostic value in HPVI ECA and HPVA ECA, and compare these between the two entities. Methods and results: A total of 93 ECAs accessioned between 2013 and 2020 were selected for further analysis, including 48 usual-type HPVA ECAs and 45 HPVI ECAs. Then, we evaluated PD-L1 expression in whole tissue sections of these cases by using the tumour proportion score (TPS) and the combined positive score (CPS). Heterogeneous PD-L1 expression was observed in both HPVI ECAs and usual-type HPVA ECAs. However, no significant difference in PD-L1 expression was seen among different histological types of ECA when either the CPS or the TPS was used. Gastric-type ECA (GEA) was associated with higher clinical stage (P = 0.001), worse progression-free survival (PFS) (P = 0.008) and worse overall survival (OS) (P = 0.02) than usual-type HPVA ECA and non-GEA HPVI ECA. When the TPS was used, PD-L1-positive GEA was associated with significantly worse PFS (P = 0.03) and OS (P = 0.015) than PD-L1-negative GEA. Conclusions: Our data show frequent PD-L1 expression in HPVI ECAs, supporting the potential role of the programmed cell death protein 1/PD-L1 pathway as a therapeutic target for these tumours. Our data also support PD-L1 as a negative prognostic marker associated with a potentially unfavourable outcome for GEAs. [ABSTRACT FROM AUTHOR]

Aims: The introduction of immunotherapy for patients with head and neck squamous cell carcinoma (HNSCC) raises the need for harmonisation between different types of antibody and immunohistochemistry platform for evaluating the expression of PD‐L1 by use of the combined positive score (CPS) in this tumour. The aim of this study was to compare the expression of PD‐L1 as determined with the CPS and two widely used assays (the 22C3 PharmDx assay and the SP263 assay) in a cohort of HNSCCs. Methods and results: We analysed 43 whole sections of HNSCC with two different anti‐PD‐L1 antibodies, 22C3 and SP263. The results, expressed as the CPS, were evaluated by 10 trained pathologists and statistical analyses were performed. We found a very similar results for PD‐L1 expression between the 22C3 PharmDx assay and the SP263 assay in our cohort, and a strong and significant correlation between the two assays for all specimens (P < 0.0001). The interobserver reliability among pathologists for the continuous scores of CPS with the intraclass correlation coefficient and the correlation between the two assays were both good. Moreover, the rate of agreement between assays was high at all cut‐offs and was best for the most relevant cut‐off of CPS ≥ 1, and the kappa values were always in the range of almost perfect. Conclusions: Two different assays (the 22C3 PharmDx assay and SP263 assay) for PD‐L1 in HNSCC showed high agreement. These data suggest that these two assays are interchangeable in the selection of patients with HNSCC for immunotherapy. [ABSTRACT FROM AUTHOR]

Aims: In the 2020 World Health Organization classification of female genital tumours, endocervical adenocarcinomas (ECAs) are subclassified into human papillomavirus (HPV)‐associated (HPVA) and HPV‐independent (HPVI) groups on the basis of their distinct aetiologies and clinical behaviours. The aim of this study was to investigate programmed death‐ligand 1 (PD‐L1) expression and its prognostic value in HPVI ECA and HPVA ECA, and compare these between the two entities. Methods and results: A total of 93 ECAs accessioned between 2013 and 2020 were selected for further analysis, including 48 usual‐type HPVA ECAs and 45 HPVI ECAs. Then, we evaluated PD‐L1 expression in whole tissue sections of these cases by using the tumour proportion score (TPS) and the combined positive score (CPS). Heterogeneous PD‐L1 expression was observed in both HPVI ECAs and usual‐type HPVA ECAs. However, no significant difference in PD‐L1 expression was seen among different histological types of ECA when either the CPS or the TPS was used. Gastric‐type ECA (GEA) was associated with higher clinical stage (P = 0.001), worse progression‐free survival (PFS) (P = 0.008) and worse overall survival (OS) (P = 0.02) than usual‐type HPVA ECA and non‐GEA HPVI ECA. When the TPS was used, PD‐L1‐positive GEA was associated with significantly worse PFS (P = 0.03) and OS (P = 0.015) than PD‐L1‐negative GEA. Conclusions: Our data show frequent PD‐L1 expression in HPVI ECAs, supporting the potential role of the programmed cell death protein 1/PD‐L1 pathway as a therapeutic target for these tumours. Our data also support PD‐L1 as a negative prognostic marker associated with a potentially unfavourable outcome for GEAs. [ABSTRACT FROM AUTHOR]

Aims: Human papilloma virus (HPV)‐independent cervical adenocarcinoma (CA) is usually diagnosed at an advanced stage, while the therapeutic options are limited. Therefore, effective treatment options are required. The programmed cell death 1 (PD‐1) inhibitor pembrolizumab has been approved for the treatment of patients with recurrent or metastatic cervical squamous cell carcinoma expressing PD‐ligand 1 (PD‐L1). However, no data regarding PD‐L1 expression in HPV‐independent CA are available. Thus, we evaluated the association between PD‐L1 expression and the clinicopathological characteristics and survival of patients with HPV‐independent CA. Methods: We evaluated PD‐L1, mismatch repair (MMR) protein expression and the immune stromal features of 44 patients with HPV‐independent CA. PD‐L1 expression was defined as a combined positive score (CPS) ≥1 and a tumour proportion score (TPS) ≥1%. Results: PD‐L1 expression was observed in 14 cases (31.8%) with CPS ≥1 and 12 cases (27.3%) with TPS ≥1%. PD‐L1 expression, based on either the CPS or the TPS, was associated with a high tumour‐infiltrating lymphocyte percentage (CPS = P < 0.001; TPS = P < 0.001). Patients with a PD‐L1 CPS ≥1 showed worse progression‐free survival and overall survival than PD‐L1‐negative patients (P = 0.004 and P = 0.023, respectively). Forty‐two cases demonstrated intact MMR expression and two cases demonstrated loss of MSH2/MSH6. Conclusions: Our data demonstrated that PD‐L1 was expressed in HPV‐independent CA, especially in clear cell carcinoma, and that PD‐L1 expression is a negative prognostic marker. Our data support the role of PD‐L1 in HPV‐independent CA and its potential as an immunotherapeutic target. [ABSTRACT FROM AUTHOR]

Aims: Programmed death‐ligand 1 (PD‐L1) immunostaining is used to predict which non‐small‐cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD‐L1 inhibitors. PD‐L1 immunostaining is sometimes performed on alcohol‐fixed cytological specimens instead of on formalin‐fixed paraffin‐embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) results in diminished PD‐L1 immunostaining as compared with formalin fixation. Methods and results: FFPE cell blocks from EBUS‐TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD‐L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory‐developed test (LDT). PD‐L1 positivity was determined with two cut‐offs (1% and 50%). Concordance of PD‐L1 positivity between the formalin‐fixed (gold standard) and ethanol‐prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol‐prefixed specimens showed false‐negative results at the 1% and 50% cut‐offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol‐prefixed specimens showed false‐negative results at the 1% cut‐off (kappa 0.67). At the 50% cut‐off, concordance was higher (kappa 0.91), with 12% of the ethanol‐prefixed specimens showing false‐negative results. Conclusion: Ethanol fixation of EBUS‐TBNA specimens prior to formalin fixation can result in a considerable number of false‐negative PD‐L1 immunostaining results when a 1% cut‐off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut‐off when immunostaining is performed with the 22C3 LDT. [ABSTRACT FROM AUTHOR]

Aims: Immunotherapies represent a new alternative therapeutic approach for hepatocellular carcinomas (HCCs), and have shown promising results when used in combination therapy. The aim of this study was to evaluate the potential of transarterial chemoembolisation (TACE) to modulate programmed death‐1 (PD‐1) and programmed death‐ligand 1 (PD‐L1) expression profiles in a cohort of surgically treated HCCs. Methods and results: A total of 82 surgically treated HCCs from patients who had undergone (n = 32) or not undergone (n = 50) preoperative TACE were included in the study. Immunohistochemical expression of PD‐1 and of PD‐L1 were analysed and compared according to TACE treatment. Pretreatment biopsies, which were available for 30 cases (20 with TACE and 10 without), were similarly analysed. Follow‐up data were retrieved from patients' charts. PD‐1 expression (≥1%) in intratumoral inflammatory cells (ICs) was observed in 46% of HCCs, and PD‐L1 expression (≥1%) in ICs and PD‐L1 expression in tumour cells (TCs) were observed in 46% and 16% of HCCs, respectively. A low level of PD‐1 expression (<1%) was associated with strong and diffuse glutamine synthetase overexpression (8% versus 27%, P = 0.024). HCCs from patients with TACE pretreatment showed significantly higher PD‐L1 expression in TCs than those from patients without TACE pretreatment (2% versus 0.4%, P = 0.027). PD‐1 expression in ICs and PD‐L1 expression in both ICs and TCs were higher in TACE‐resected tumours than in corresponding pre‐TACE biopsies (respectively: 1.8% versus 8.1%, P = 0.034; 0.8% versus 7.1%, P = 0.032; and 0% versus 2.4%, P = 0.043). Conclusion: Our results, showing increases in PD‐1 expression and PD‐L1 expression in HCCs following TACE, support the use of TACE in combination with immunotherapy in selected cases to optimise tumour response. [ABSTRACT FROM AUTHOR]

Aims: Studies in various cancer types have demonstrated discordance between results from different programmed death‐ligand 1 (PD‐L1) assays. Here, we compare the reproducibility and analytical concordance of four clinically developed assays for assessing PD‐L1‐positivity in tumour‐infiltrating immune cells in the tumour area (PD‐L1‐IC‐positivity) in triple‐negative breast cancer (TNBC). Methods and results: Primary TNBC resection specimens (n = 30) were selected based on their PD‐L1‐IC‐positivity per VENTANA SP142 (<1%: 15 cases; 1–5%: seven cases; >5%: eight cases). Serial histological sections were stained for PD‐L1 using VENTANA SP142, VENTANA SP263, DAKO 22C3 and DAKO 28‐8. PD‐L1‐IC‐positivity and tumour cell expression (≥1 versus <1%) were scored by trained readers from seven sites using online virtual microscopy. The adjusted mean of PD‐L1‐IC‐positivity for SP263 (7.8%) was significantly higher than those for the other three assays (3.7–4.9%). Differences in adjusted means were statistically significant between SP263 and the other three assays (P < 0.0001) but not between the three remaining assays when excluding SP263 (P = 0.0961–0.6522). Intra‐class correlation coefficients revealed moderate‐to‐strong inter‐reader agreement for each assay (0.460–0.805) and poor‐to‐strong inter‐assay agreement for each reader (0.298–0.678) on PD‐L1‐IC‐positivity. Conclusions: In this first multicentre study of different PD‐L1 assays in TNBC, we show that PD‐L1‐IC‐positivity for SP142, 22C3 and 28‐8 was reproducible and analytically concordant, indicating that these three assays may be analytically interchangeable. The relevance of the higher PD‐L1‐IC‐positivity for SP263 should be further investigated. [ABSTRACT FROM AUTHOR]

Aims: Gastric neuroendocrine carcinoma (NEC) is a rare and aggressive subtype with a poor prognosis. We aim to investigate expression profiles of HER2 and programmed death ligand 1 (PD‐L1) in gastric NEC to test the potential applicability of drugs targeting these molecules. Methods and results: Expression levels of HER2 and PD‐L1 were evaluated in 25 gastric NECs, including 10 pure NECs and 15 mixed adenocarcinoma–NECs, and a combined positive score (CPS) was used to evaluate PD‐L1 expression. The correlations of expression levels with both clinicopathological features and the expression of p53, retinoblastoma protein (Rb) and mismatch repair proteins were also analysed. Eighteen of the 25 (72%) cases showed a PD‐L1 CPS of ≥ 1, which was previously shown to be associated with response to pembrolizumab. Positive nodal metastasis and low tumour‐infiltrating lymphocyte (TIL) levels at the invasive margin were significantly associated with a PD‐L1 CPS of < 1. The NEC component was HER2‐negative in all cases, whereas HER2 positivity was observed in the adenocarcinoma component of six of 15 (40%) mixed adenocarcinoma–NECs. Mismatch repair deficiency, a mutant pattern of p53 expression and loss of Rb expression were observed in four (16%), 17 (68%) and nine (36%) cases, respectively, although these alterations were not associated with the PD‐L1 CPS or other clinicopathological characteristics. Conclusions: HER2 is unlikely to be an effective target in gastric NEC owing to the lack of HER2 expression, whereas the PD‐1/PD‐L1 pathway is a potential therapeutic target for gastric NEC because of the relatively high prevalence of a PD‐L1 CPS of ≥ 1 in this subtype. [ABSTRACT FROM AUTHOR]

Aims: This study was performed to elucidate the clinicopathological characteristics, genetic alterations and therapeutic targets of primary malignant melanoma of the oesophagus (PMME). Methods and results: The clinicopathology and molecular pathology of 13 PMME cases and 10 skin malignant melanoma (SKMM) cases were analysed with next-generation sequencing (NGS) and immunohistochemistry. The 3-year overall survival rate and the median survival time for PMME patients were 23.1% and 11.9 months, respectively. Three (23.1%) and eight (61.5%) PMME cases showed a papillary structure and lymph node metastasis, respectively. DNA and RNA hybridization capture-based NGS analysis revealed that NF1 was the most frequently mutated gene (30%) in 10 of the PMME cases. Other mutations detected in PMME included SF3B1 (20%), KRAS (10%), BRCA2 (10%), KIT (10%) and TP53 (10%) mutations. Commonly detected BRAF mutations in SKMM were not detected in PMME. Immunohistochemistry and mutation status were concordant between p53/c-Kit and TP53/KIT, respectively. Focal expression of programmed death-ligand 1 was observed in one PMME sample. The tumour mutation burden in PMME was significantly lower than that in SKMM (P = 0.030). No PMME case showed high microsatellite instability. RNA sequencing revealed a distinctive pattern with respect to RNA expression. Tcell co-stimulation differed between PMME and SKMM. Conclusions: The RAS-mitogen-activated protein kinase pathway is one of the main pathways involved in PMME. The genetic profile of PMME was similar to that of mucosal/acral melanoma, but differed from the SKMM profile. A subset of PMMEs may contain actionable mutations. Immunotherapy seemed to be less effective for most PMMEs in this series. [ABSTRACT FROM AUTHOR]

Aims: This study was performed to elucidate the clinicopathological characteristics, genetic alterations and therapeutic targets of primary malignant melanoma of the oesophagus (PMME). Methods and Results: The clinicopathology and molecular pathology of 13 PMME cases and 10 skin malignant melanoma (SKMM) cases were analysed with next‐generation sequencing (NGS) and immunohistochemistry. The 3‐year overall survival rate and the median survival time for PMME patients were 23.1% and 11.9 months, respectively. Three (23.1%) and eight (61.5%) PMME cases showed a papillary structure and lymph node metastasis, respectively. DNA and RNA hybridization capture‐based NGS analysis revealed that NF1 was the most frequently mutated gene (30%) in 10 of the PMME cases. Other mutations detected in PMME included SF3B1 (20%), KRAS (10%), BRCA2 (10%), KIT (10%) and TP53 (10%) mutations. Commonly detected BRAF mutations in SKMM were not detected in PMME. Immunohistochemistry and mutation status were concordant between p53/c‐Kit and TP53/KIT, respectively. Focal expression of programmed death‐ligand 1 was observed in one PMME sample. The tumour mutation burden in PMME was significantly lower than that in SKMM (P = 0.030). No PMME case showed high microsatellite instability. RNA sequencing revealed a distinctive pattern with respect to RNA expression. T‐cell co‐stimulation differed between PMME and SKMM. Conclusions: The RAS–mitogen‐activated protein kinase pathway is one of the main pathways involved in PMME. The genetic profile of PMME was similar to that of mucosal/acral melanoma, but differed from the SKMM profile. A subset of PMMEs may contain actionable mutations. Immunotherapy seemed to be less effective for most PMMEs in this series. [ABSTRACT FROM AUTHOR]

Aims: Immune checkpoint inhibitors (ICIs) have revolutionised the treatment of advanced malignancies by boosting immune‐mediated destruction of neoplastic cells, but are associated with side effects stemming from generalised immune system activation against normal tissues. Checkpoint ligand expression in non‐tumoral cells of tissues affected by immune‐related adverse effects has been described in ICI‐associated hypophysitis, myocarditis, and acute interstitial nephritis. We aimed to investigate the tissue expression of the immune checkpoint receptor programmed cell death‐1 (PD‐1) and its ligand, programmed death‐ligand 1 (PD‐L1), in PD‐1 inhibitor‐associated colitis (PD1i colitis). Methods and results: PD‐1 and PD‐L1 immunohistochemical expression levels were analysed in 15 cases of PD1i colitis and potential mimics—infectious colitis and inflammatory bowel disease (IBD). Increased epithelial expression of PD‐L1 was observed in PD1i colitis as compared with normal colon and infectious colitis, but the expression level was lower than that in IBD. Conversely, PD‐1 expression in inflammatory cells was higher in infectious colitis, intermediate in IBD, and minimal or absent in normal colon and in patients receiving PD‐1 inhibitors. Conclusions: Although our results do not justify the use of PD‐L1 as a discriminatory marker of PD1i colitis against other entities within the differential diagnosis, they support the concept that PD1i colitis and IBD have similar pathogenetic mechanisms. They also highlight the fact that PD‐L1 epithelial overexpression is a commonly used mechanism of the gastrointestinal tract mucosa to protect itself from inflammatory‐mediated damage resulting from different aetiologies, which probably underpins the high incidence of gastrointestinal immune‐related adverse effects in patients receiving ICI therapies, in whom this mechanism is disrupted. [ABSTRACT FROM AUTHOR]

Aims: The importance of programmed cell death ligand 1 (PD‐L1) expression has emerged in clinical trials of PD‐L1 target therapy in renal cell carcinoma (RCC). This study compares PD‐L1 assays in RCC. Methods and results: Two US Food and Drug Administration‐approved PD‐L1 assays (22C3 and SP142) and one research‐use only antibody (E1L3N) were used in a retrospective cohort of 591 patients with RCC. PD‐L1 positivity on tumour cells (TCs) and immune cells (ICs) and combined positive score (CPS) were evaluated. With the 22C3, SP142 and E1L3N assays, positive PD‐L1 expression on TCs ≥1% was observed in 24 (4.1%), 12 (2.0%) and 16 (2.7%) cases and on ICs ≥1% was observed in 132 (22.3%), 120 (20.3%) and 65 (11.0%) cases, respectively. PD‐L1 expression scores among the three assays showed moderate–high positive correlation (ρ = 0.599–0.835, P < 0.001). Assays appeared similar, although staining in ICs was comparatively less frequent with E1L3N. 22C3 showed frequent positivity in TCs. PD‐L1 expression on TCs was associated with papillary type 2 RCC (P < 0.001). IC infiltration and PD‐L1 expression on ICs were predominantly found in clear cell and papillary type 1 RCC (P < 0.05). Conclusions: Programmed death 1 (PD‐1)/PD‐L1 target therapy may be beneficial for patients with papillary type 2 RCC, even if they are categorised as a heterogeneous group. PD‐L1 assays should be carefully selected, and accurate histological subtyping of RCC is needed prior to decisions on PD‐L1 testing, because of the different PD‐L1 expression observed among varying RCC subtypes. [ABSTRACT FROM AUTHOR]

Aims: Accurate assessment of programmed death‐ligand 1 (PD‐L1) levels in non‐small cell lung carcinoma (NSCLC) samples is complicated by intratumoral heterogeneity. We aimed to: (i) establish whether intratumoral PD‐L1 variation is associated with differences in local histotype; (ii) identify histotypes associated with a tendency for there to be higher or lower PD‐L1 scores; and (iii) estimate the frequency of clinically significant discordance in PD‐L1 levels between intratumoral histotype areas. Methods and results: We reviewed 166 NSCLC resection specimens clinically tested for PD‐L1 with the 22C3 pharmDx assay. Multiple histotypes were present in 55% (68/123) of non‐mucinous adenocarcinoma samples. Solid histotypes had significantly higher PD‐L1 levels than other histotypes, both when samples were grouped by predominant histotype, and when histotype areas within a tumour were compared (P < 0.02). Lepidic areas had significantly lower PD‐L1 levels than other histotype areas within the same tumour (P < 0.02). Discordance between intratumoral histotype areas at a clinically relevant threshold (PD‐L1 tumour proportion score of 1% or 50%) was present in 32% (22/68) of non‐mucinous adenocarcinoma specimens with multiple histotype areas. The lepidic histotype was most frequently involved in discordance. Conclusions: Intratumoral heterogeneity in PD‐L1 is associated with variation in histotype. Over‐representation of solid areas may increase the PD‐L1 score assigned to a tumour, whereas over‐representation of lepidic areas may decrease the PD‐L1 score. Evaluation of how histotype representation impacts on the predictive value of PD‐L1 testing is warranted. [ABSTRACT FROM AUTHOR]

Aims: As immunomodulatory therapy is being integrated into treatment regimens for non‐small‐cell lung carcinoma, we aimed to prospectively collect data on the immunohistochemical profile of tumours assessed in our institution and to correlate this with morphological tumour features. Methods and results: Immunohistochemistry for programmed death‐ligand 1 (PD‐L1) was considered to be adequate when >100 tumour cells were seen microscopically. When adequate, PD‐L1 staining was scored as <1%, ≥1–49% or ≥50% positive membrane staining within tumour cells only. There were 197 assessable cases, of which 87% of those with pleomorphic features (n = 39) showed ≥50% positivity for PD‐L1 expression, as compared with only 33% of cases without pleomorphic features (P < 0.05) (90% versus 25% in resected cases). Further correlation of PD‐L1 expression with architectural patterns within the tumours was performed in 74 adenocarcinoma resections. All invasive mucinous adenocarcinomas scored <1%. All lepidic components in non‐mucinous adenocarcinoma resections scored <1%. Thirty‐five per cent of the acinar/papillary components and 53% of the solid/micropapillary components were positive for PD‐L1 expression. Conclusions: There are significant differences in PD‐L1 expression in relation to histological patterns, with particularly high levels in those with pleomorphic features and low/undetectable levels in invasive mucinous adenocarcinomas and the lepidic components of non‐mucinous adenocarcinomas. Assessment of PD‐L1 expression in a resected adenocarcinoma with a lepidic component may therefore not be reliable when immumodulatory therapy for recurrent disease is being considered, and either re‐biopsy or limiting assessment to the invasive component may be more appropriate. [ABSTRACT FROM AUTHOR]