Clone SP142 Research Articles

Assessment of PD-L1 expression and tumour infiltrating lymphocytes in early-stage non-small cell lung carcinoma with artificial intelligence algorithms

AimsTo study programmed death ligand 1 (PD-L1) expression and tumour infiltrating lymphocytes (TILs) in patients with early-stage non-small cell lung carcinoma (NSCLC) with artificial intelligence (AI) algorithms.MethodsThe study included samples...

The prognostic value of programmed death-ligand 1 (PD-L1) expression in resected colorectal cancer without neoadjuvant therapy - differences between antibody clones and cell types

BackgroundProgrammed death-ligand 1 (PD-L1) expression on tumor cells is associated with poor prognosis in several malignancies, while partly contradictory and inconclusive results have been presented for colorectal cancer (CRC). This study aimed to evaluate PD-L1 as a prognostic biomarker in CRC by comparing three different antibody clones.MethodsPatients surgically treated for CRC between January 1st, 2007, and December 31st, 2015, in Kalmar County, Sweden, were retrospectively included. Tissue microarrays from 862 primary tumors without neoadjuvant treatment were assessed for immunohistochemical expression of PD-L1 in tumor cells (TC) and immune cells (IC) using clones 73-10, SP263, and 22C3. Cox regression proportional hazard models were used to estimate hazard ratios for overall survival (OS) and disease-free interval (DFI) in univariable and multivariable analyses, with 1% and 5% set as cut-offs for positive expression in TC and IC respectively.ResultsPD-L1 expression in TC was found in 89 (10%) cases for clone 73-10, 76 (9%) for clone SP263, and 38 (4%) for clone 22C3, while the numbers for IC were 317 (37%) cases for clone 73-10, 264 (31%) for clone SP263, and 89 (10%) for clone 22C3. PD-L1 expression in IC was associated with prolonged OS and DFI in univariable analysis for all three clones. The link to prolonged DFI remained in multivariable analysis for 73-10 and SP263, but only for 73-10 regarding OS. PD-L1 expression in TC was not prognostic of OS in any analysis, while it was associated with prolonged DFI for SP263, and a trend was seen for 73-10. The link to prolonged DFI remained for SP263 and was strengthened for 73-10 in multivariable analysis.ConclusionsThe prognostic value of PD-L1 expression in both IC and TC differs between antibody clones, with 73-10 and SP263 being more reliable for prognostic information than 22C3 in resected CRC.

PD-L1 Expression and Its Modulating Factors in Anaplastic Thyroid Carcinoma: A Multi-institutional Study.

Anti-PD immunotherapy is currently under investigation in anaplastic thyroid carcinoma (ATC). Tumor cell surface PD-L1 expression is considered predictive of therapeutic response. Although papillary thyroid carcinoma has been widely studied for PD-L1 expression, there are limited data on ATC. In this retrospective multi-institutional study involving 9 centers across Asia, 179 ATCs were assessed for PD-L1 expression using the SP263 (Ventana) clone. A tumor proportion score (TPS) ≥1% was required to consider a case PD-L1-positive. PD-L1 expression was compared with the histological patterns, the type of specimen (small or large), tumor molecular profile ( BRAF V600E and TERT promoter mutation status), and patient outcome. PD-L1 expression in any co-existent differentiated thyroid carcinoma (DTC) was evaluated separately and compared with ATC. Most ATCs (73.2%) were PD-L1-positive. The median TPS among positive cases was 36% (IQR 11% to 75%; range 1% to 99%). A high expression (TPS ≥ 50%) was noted in 30.7%. PD-L1-negative cases were more likely to be small specimens ( P =0.01). A negative result on small samples, hence, may not preclude expression elsewhere. ATCs having epithelioid and pleomorphic histological patterns were more likely to be PD-L1-positive with higher TPS than sarcomatoid ( P <0.01). DTCs were more frequently negative and had lower TPS than ATC ( P <0.01). Such PD-L1 conversion from DTC-negative to ATC-positive was documented in 71% of cases with co-existent DTC. BRAF V600E, but not TERT promoter mutations, correlated significantly with PD-L1-positivity rate ( P =0.039), reinforcing the potential of combining anti-PD and anti-BRAF V600E drugs. PD-L1 expression, however, did not impact the patient outcome.

Evaluation of the level expression of the Russian clone of PD-L1 antibodies and comparison with validated clones from other manufacturers on stomach cancer material

The study of immunotherapeutic markers is one of the most popular and promising areas in oncology. One of the most studied targets of immunotherapy is the ligand of the programmed cell death protein 1 (PD-L1). All diagnostic kits for determining PD-L1 expression in tumors are of foreign origin. To ensure the technological sovereignty of the Russian Federation in the field of medical technologies, it is necessary to conduct research with a domestic clone of PD-L1 antibodies and compare the results with already validated analogues. The aim of the work was to evaluate the expression of a domestic clone of antibodies to PD-L1 (PBM-1A4), determine its relationship with the clinical and morphological parameters of gastric cancer and compare the expression of various PD-L1 clones in gastric cancer (SP142, SP263, 22C3 and PBM-1A4). The study was performed on samples of gastric cancer surgical material from 131 patients who had not received chemotherapy, radiation or immunotherapy before surgery. Results: PD-L1 expression detected by the PBM-1A4 antibody clone is present in 46.2% of gastric cancer samples. Among PD-L1(PBM-1A4)-positive tumors, neoplasms of the second macroscopic type according to R. Bormann and tubular adenocarcinomas significantly predominate. The SP142 clone shows the greatest consistency with PBM 1A4 expression (significant consistency, Cohen's kappa is 0.67335). Conclusion: This antibody clone undoubtedly represents an extremely promising development by Russian scientists, which makes it possible to make the molecular diagnosis of malignant neoplasms more accessible to Russian patients.

The Vanishing Clinical Value of PD-L1 Status as a Predictive Biomarker in the First-Line Treatment of Urothelial Carcinoma of the Bladder.

Our study endeavors to elucidate the clinical implications of PD-L1 positivity in individuals afflicted with advanced urothelial carcinoma of the bladder (UCB). Patients with advanced UCB were prospectively enrolled following a radical cystectomy (RC) performed within January 2017 to December 2022 at our tertiary referral center. The clinical outcome, defined as the progression-free survival (PFS) and overall survival (OS) on systemic treatment, was analyzed using an χ2-test, Mann-Whitney U-test, the Kaplan-Meier method, and a log-rank test. A total of 648 patients were included following an RC performed within January 2017 to December 2022. Their PD-L1 status was analyzed with the primary PD-L1-specific antibody (clone SP263, Ventana) and defined both by the CPS and IC-score in 282 patients (43.5%) with a high risk (pT3-pT4 and/or lymph node involvement) or metastatic UCB. While the median PFS was significantly prolonged 5-fold in PD-L1+ patients, we found no difference in OS, regardless of PD-L1 status, or treatment regimen. While PD-L1 positivity indicates prolonged PFS, the presence of PD-L1 does not influence OS rates, suggesting its limited usefulness as a prognostic biomarker in bladder cancer. However, the positive correlation between an PD-L1 status and a sustained response to ICI treatments indicates its potential role as a predictive biomarker. Further research is required to understand how the predictive value of PD-L1 positivity may extend to the use of ICIs in combination with antibody-drug conjugates.

PD-L1 Expression in Neoplastic and Immune Cells of Thymic Epithelial Tumors: Correlations with Disease Characteristics and HDAC Expression.

Programmed death-ligand 1 (PD-L1) expression in neoplastic and immune cells of the tumor microenvironment determines the efficacy of antitumor immunity, while it can be regulated at the epigenetic level by various factors, including HDACs. In this study, we aim to evaluate the expression patterns of PD-L1 in thymic epithelial tumors (TETs), while we attempt the first correlation analysis between PD-L1 and histone deacetylases (HDACs) expression. Immunohistochemistry was used to evaluate the expression of PD-L1 in tumor and immune cells of 91 TETs with SP263 and SP142 antibody clones, as well as the expressions of HDCA1, -2, -3, -4, -5, and -6. The PD-L1 tumor proportion score (TPS) was higher, while the immune cell score (IC-score) was lower in the more aggressive TET subtypes and in more advanced Masaoka-Koga stages. A positive correlation between PD-L1 and HDAC-3, -4, and -5 cytoplasmic expression was identified. Higher PD-L1 expression in neoplastic cells and lower PD-L1 expression in immune cells of TETs characterizes more aggressive and advanced neoplasms. Correlations between PD-L1 and HDAC expression unravel the impact of epigenetic regulation on the expression of immune checkpoint molecules in TETs, with possible future applications in combined therapeutic targeting.

Abstract 6172: Accurate PD-L1 IHC assessment in urothelial carcinoma by an AI algorithm robust across multiple sites, antibody clones, and scanners

Abstract Background: Immunotherapy against PD-L1 is used for treatment of several indications of Urothelial Carcinoma (UC). Recent results indicate that it is likely to become part of the standard first-line treatment regimen for advanced UC in the near future. Accurate readout of Immunohistochemical (IHC) PD-L1 expression is therefore essential to inform UC treatment decisions. However, manual PD-L1-scoring is prone to high inter- and intra-observer variability. Artificial intelligence (AI) may help to improve standardization, accuracy and efficiency of PD-L1 IHC scoring. Yet, previous AI-based approaches struggled to show consistency across images that are diverse with regards to the source institutions, antibody clones, and scanning hardware used. Methods: We developed an AI software for PD-L1 tumor proportion scoring (TPS/TC) on whole-slide images (WSIs) of UC without human supervision. PD-L1 analysis by the software is fully-automated, does not require any adaptation steps, and is concordant with current clinical guidelines. The performance of the software was evaluated on two cohorts of PD-L1 stained UC samples against ground-truth (GT) TPS/TC scores given by human pathologists in clinical routine. The first cohort (n = 53) consisted of samples stained with the 22C3 antibody clone (Agilent) derived from four institutions and three scanner models. It contained both tissue microarray samples and scans of UC resectates. A second validation cohort consisted of n = 83 WSIs stained with the SP263 clone (Ventana), collected from two institutions. Results: For the threshold of TPS≥1% (cut-off established for adjuvant therapy with Nivolumab), agreement rates of AI scores with human GT scores were 92.5% for cohort 1 (22C3) and 96.4% for cohort 2 (SP263). Sensitivity/specificity values were 92.3%/92.9% for cohort 1 and 100%/93.6% for cohort 2. Across both cohorts (n = 136) the agreement rate between AI and human was 94.9%. Conclusions: Across 136 UC samples stained with two PD-L1 antibody clones, and derived from a total of four institutions and three scanner models, the fully automatic AI system showed excellent agreement with human pathologist scores. These results on challenging validation data show the potential of AI applications to standardize and optimize PD-L1 scoring and demonstrate consistency and safety of suitable AI-based systems for application in clinical routine. Citation Format: Niklas Abele, Diego Calvopiña, David Mulder, Patrick Frey, Emre Karakok, Fadime G. Salman, Murat Oktay, Markus Eckstein, Sebastian Springenberg, Arndt Hartmann, Tobias Lang. Accurate PD-L1 IHC assessment in urothelial carcinoma by an AI algorithm robust across multiple sites, antibody clones, and scanners [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6172.

Assessing the relationship between tumor-infiltrating lymphocytes and PD-L1 expression in triple negative breast cancer: Identifying optimal TILs cut-off value for pathologic reporting

BackgroundTriple Negative Breast Cancer (TNBC) presents diagnostic complexities, particularly in evaluating Tumor-Infiltrating Lymphocytes (TILs) and Programmed Death-Ligand 1 (PD-L1) expression. This study aimed to identify optimal TILs percentage cut-offs predictive of PD-L1 expression and to investigate the relationship between TILs, PD-L1, and tertiary lymphoid structures (TLSs). MethodAnalyzing 141 TNBC cases, we assessed TILs, PD-L1 expression (clones 22C3 and SP142), and TLS presence. ResultsWe identified TILs cut-offs (<20 %, 20–60 %, ≥60 %) correlating with PD-L1 expression. TILs <20 % rarely express PD-L1 with either 22C3 or SP142 clones. TILs ≥60 % demonstrate PD-L1 expression across both clones. TILs within the 20–60 % range correlate with PD-L1 expression using the SP142 clone, but not 22C3. Evaluating TILs solely at the tumor edge led to inaccuracies, highlighting the need for overall assessment of TILs throughout the entire lesion. TLS presence correlated with higher TIL percentages and PD-L1 expression, particularly with SP142. Discrepancies between 22C3 and SP142 clones (15 % vs. 50 % positivity, respectively) underscored the variability in PD-L1 detection. ConclusionThis study identifies TILs cut-offs predictive of PD-L1 positivity, suggesting the need for institutions to tailor these thresholds based on the selected PD-L1 clone and treatment. Evaluating TILs solely at the tumor edge may overlook the complexity of tumor immune infiltration. While TLS presence correlates with higher PD-L1 expression, particularly with the SP142 clone, its exact predictive value for PD-L1 remains to be clarified. The SP142 clone exhibits higher positivity rates compared to 22C3.

Assessment of programmed death-ligand 1 expression in primary tumors and paired lymph node metastases of gastric adenocarcinoma.

Anti-programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) immunotherapy has demonstrated promising results on gastric cancer (GC). However, PD-L1 can express differently between metastatic sites and primary tumors (PT). To compare PD-L1 status in PT and matched lymph node metastases (LNM) of GC patients and to determine the correlation between the PD-L1 status and clinicopathological characteristics. We retrospectively reviewed 284 GC patients who underwent D2-gastrectomy. PD-L1 was evaluated by immunohistochemistry (clone SP142) using the combined positive score. All PD-L1+ PT staged as pN+ were also tested for PD-L1 expression in their LNM. PD-L1(-) GC with pN+ served as the comparison group. Among 284 GC patients included, 45 had PD-L1+ PT and 24 of them had pN+. For comparison, 44 PD-L1(-) cases with pN+ were included (sample loss of 4 cases). Of the PD-L1+ PT, 54.2% (13/24 cases) were also PD-L1+ in the LNM. Regarding PD-L1(-) PT, 9.1% (4/44) had PD-L1+ in the LNM. The agreement between PT and LNM had a kappa value of 0.483. Larger tumor size and moderate/severe peritumoral inflammatory response were associated with PD-L1 positivity in both sites. There was no statistical difference in overall survival for PT and LNM according to the PD-L1 status (P = 0.166 and P = 0.837, respectively). Intra-patient heterogeneity in PD-L1 expression was observed between the PT and matched LNM. This disagreement in PD-L1 status may emphasize the importance of considering different tumor sites for analyses to select patients for immunotherapy.

Digital Pathology Applications for PD-L1 Scoring in Head and Neck Squamous Cell Carcinoma: A Challenging Series.

The assessment of programmed death-ligand 1 (PD-L1) combined positive scoring (CPS) in head and neck squamous cell carcinoma (HNSCC) is challenged by pre-analytical and inter-observer variabilities. An educational program to compare the diagnostic performances between local pathologists and a board of pathologists on 11 challenging cases from different Italian pathology centers stained with PD-L1 immunohistochemistry on a digital pathology platform is reported. A laboratory-developed test (LDT) using both 22C3 (Dako) and SP263 (Ventana) clones on Dako or Ventana platforms was compared with the companion diagnostic (CDx) Dako 22C3 pharm Dx assay. A computational approach was performed to assess possible correlations between stain features and pathologists' visual assessments. Technical discordances were noted in five cases (LDT vs. CDx, 45%), due to an abnormal nuclear/cytoplasmic diaminobenzidine (DAB) stain in LDT (n = 2, 18%) and due to variation in terms of intensity, dirty background, and DAB droplets (n = 3, 27%). Interpretative discordances were noted in six cases (LDT vs. CDx, 54%). CPS remained unchanged, increased, or decreased from LDT to CDx in three (27%) cases, two (18%) cases, and one (9%) case, respectively, around relevant cutoffs (1 and 20, k = 0.63). Differences noted in DAB intensity/distribution using computational pathology partly explained the LDT vs. CDx differences in two cases (18%). Digital pathology may help in PD-L1 scoring, serving as a second opinion consultation platform in challenging cases. Computational and artificial intelligence tools will improve clinical decision-making and patient outcomes.

PD-L1 immunohistochemical expression in bladder urothelial cancer with SP263, SP142 and 22C3 antibodies: A comparative study

Programmed death ligand 1 (PD-L1) is currently the only biomarker used for the selection of patients with bladder urothelial cancer for immunotherapy. Several platforms, antibodies and scores are currently available for the evaluation of the expression of PD-L1 in immunohistochemistry (IHC). In this study three different antibodies (SP263, SP142 and 22C3) were compared to establish their performances and concordance rates. Twenty-four consecutive cases of surgically resected urothelial cancers of the bladder were enrolled. All cases were revised, and appropriate tumor areas were selected for IHC. Three commercially available PD-L1 antibodies were tested: 22C3 pharmDx with Dako Autostainer Link 48 (Dako, Carpinteria, Ca), and SP263 and SP142 with the Ventana BenchMark (Ventana Medical Systems, Tucson, AZ) platform. All slides were evaluated by an expert pathologist and both the tumor proportion score (TPS) and the combined positive score (CPS) were determined and compared at two different cut-off levels (≥ 1 and ≥ 10). The SP263 and 22C3 clones produced more positive results with the CPS and TPS scores, respectively. The CPS score identified more positive cases than the TPS score, irrespectively of the clone or the cut-off used; the difference was statistically significant in both the SP263 and SP142 clones with the ≥1 cut-off. No statistically significant differences were found between the clones when the ≥1 cut-off was used, irrespectively of the score. At the contrary, a statistically significant difference (p = 0.024) and a trend to significance (p = 0.082) were respectively found for the TPS and CPS scores, when the SP22C3 and the SP142 clones were compared at a cut-off level of ≥10. The ICC test using CPS was 0.676 and 0.578 for the ≥1 and ≥ 10 cut-offs respectively, and 0.729 and 0.467 respectively for the same cut-offs using TPS. This suggests that the three antibodies under investigation cannot be used interchangeably, especially the 22C3 and SP142 clones which showed statistically significant difference when TPS was tested at a ≥ 10 cut-off.

Association Between PD-L1 status Detected by SP142 and SP263 Antibody Clones and Clinical and Morphological Features of Gastric Cancer Factors

The aim is to study the relationship of PD-L1 status of SP142 and SP263 clones in gastric cancer with its clinical and morphological parameters. Material and methods. Surgical material obtained from 131 patients with a confirmed diagnosis of stomach cancer. Antibodies to PD-L1 SP263 were stained 127 cases, antibodies to PD-L1 SP142 – 126 cases. Reactions with PD-L1 were performed using the Ventana BenchMark Ultra device (Roche Ventana, USA) according to the protocols recommended by the manufacturer of primary antibodies. The results of immunohistochemical staining were compared with the clinical and morphological characteristics of gastric cancer using statistical analysis. Results. The positive PD-L1 status detected by clones SP263 and SP142 is significantly associated with type 2 macroscopic form according to the R. Bormann classification (p=0,003/p=0,003), tubular morphological type according to the WHO 5th edition, 2019 classification (p=0,001/p=0,018), intermediate morphological type according to the classification of P. Lauren (p=0,027/p=0,005) and the absence of signet ring cells (p=0,001/p=0,010). Staining with both clones used was not associated with the gender and age of patients, the size and localization of the tumor, the degree of its differentiation, the presence or absence of emboli in the lumen of blood and lymph vessels, the presence of affected lymph nodes and distant metastases, as well as the clinical stage (p&gt;0,05). The largest proportion of PD-L1 positive cases in the study of SP263/SP142 clones was found in tubular type tumors (75,6%/84,6%) according to the WHO 5th edition, 2019 classification, intermediate type (60,0%/68,0%) according to P. Lauren, type 2 (65,4%/68,0%) according to R. Bormann and in the absence of signet ring cells (70,7%/76,9%). Conclusion. Statistically significant clinical and morphological parameters of gastric cancer, where a reliable relationship between them and the expression of PD-L1 clones SP263 and SP142 was revealed, are: type 2 macroscopic form according to R. Bormann (p=0,003/p=0,003), tubular morphological type according to the classification of WHO 5th edition, 2019) (p=0,001/p=0,018), intermediate type according to P. Lauren (p=0,027/p=0,005) and the absence of signet ring cells (p=0,001/p=0,010).

PD-L1 immunohistochemical expression considering HPV status in oropharyngeal squamous cell carcinoma.

This systematic review aims to determine whether the presence of human papillomavirus (HPV) influences the immunohistochemical expression of programmed cell death-1 ligand (PD-L1) in oropharyngeal squamous cell carcinoma (OPSCC). PD-L1 immunohistochemical expression varies in OPSCC, and the presence of HPV is a plausible explanation for this variability. Comprehending these findings is crucial, as high PD-L1 expression in the tumor microenvironment of OPSCC can help identify patient subgroups that could be suitable for immunotherapy. Therefore, a systematic review was conducted following PRISMA guidelines (CRD42023437800). An electronic literature search was performed without time or language restrictions. The search included PubMed/MEDLINE, Embase, Scopus, Web of Science, https://clinictrials.gov, and relevant journals. A meta-analysis was performed using RStudio. Fourteen studies involving 1,629 participants were included. The sample consisted predominantly of males (81.26%) with a mean age of 58.3 years. Concerning clinical and pathological characteristics, the most frequently described anatomical location was the tonsils (68.54%), and most participants were either current or former smokers (78%) and alcohol users (79%). Advanced TNM IV was the most common stage. Regarding histopathological characteristics, HPV 16 was the only type mentioned, and half of the cases were detected through immunohistochemistry. The SP142 clone (35.7%) and the pattern of membrane immunostaining in tumor cells (71%) were the most commonly employed methods. The most prevalent findings were positive expression of PD-L1 (64.28%) and negative HPV status (57.14%). The association between PD-L1 positivity and HPV positivity (78.57%) was confirmed by meta-analysis. The conclusion was that HPV-positive status has an impact on immunohistochemical expression of PD-L1 in OPSCC.

Morphological and Immunocytochemical Characterization of Tumor Spheroids in Ascites from High-Grade Serous Carcinoma.

Tumor spheroids in the ascites of high-grade serous carcinoma (HGSC) are poorly described. Our objective was to describe their morphological features, cellular composition, PD-1 and PD-L1 expression, and survival correlation of these parameters. The density and size of spheroids were assessed in Giemsa-stained smears; the cell composition of spheroids, including tumor cells, immune cells, capillaries, and myofibroblasts, as well as PD-1 and PD-L1 expression on tumor and immune cells was assessed in immunocytochemically stained cell block sections. Forty-seven patients with primary HGSC and malignant ascites were included. A cut-off value for a spheroid density of 10% was established, which significantly predicted overall survival. However, spheroid size did not correlate with survival outcomes. Spheroids were primarily composed of tumor cells, but the presence of lymphocytes and macrophages was also confirmed. Moreover, capillaries were present in the spheroids of three patients, but the presence of myofibroblasts was not confirmed. PD-1 was expressed on lymphocytes but not on tumor cells. PD-L1 expression was seen on both tumor and immune cells, assessed by 22C3 and SP263 antibody clones but not by the SP142 clone. Our results highlight the potential of routine cytopathological techniques to analyze spheroids in HGSC ascites as a valuable tool to investigate their potential as prognostic markers.

Clear cell carcinoma of the uterine cervix; an unusual HPV-independent tumor: Clinicopathological features, PD-L1 expression, and mismatch repair protein deficiency status of 16 cases.

Endocervical clear cell carcinoma (c-CCC) is a rare and HPV-independent adenocarcinoma type of cervix. Being usually resistant to conventional chemotherapy. Immunotherapy has recently been added as a preferred regimen as a second-line treatment option for programed cell death-ligand 1 (PD-L1)-positive or mismatch repair (MMR) deficient cervical carcinomas. In this study, clinicopathological features, PD-L1 expression, and MMR deficiency status of c-CCCs were investigated. Sixteen c-CCC diagnosed cases were included in this study. PD-L1 expression was evaluated using two different PD-L1 clones (22C3 and SP263). MMR deficiency status of the cases was evaluated using four MMR proteins (MLH1, PMS2, MSH2, and MSH6). Most of the c-CCC cases were presented as FIGO Stage I (68.75%). PD-L1 expression in either tumoral or tumor-infiltrating immune cells (TILs) was present in 62.5% (10/16) and 69% (11/16) of the 22C3 and SP263 clones, respectively. Most of the cases with high TIL density were also positive for PD-L1. The PD-L1 expression rate was less than 50% in most of the cases and 12.5% of the cases shared extensive PD-L1 staining. Overall, MMR deficiency was observed in 31.25% of the cases. Most of the MMR-deficient cases (80%) were PD-L1 positive. Although our study cohort is limited, we have shown that PD-L1 expression and MMR deficiency can be found in c-CCCs in variable degrees. These findings suggest that accompanying TIL density and MMR deficiency could be used as candidates for predicting PD-L1 positivity for c-CCCs. However, to indicate the clinical importance of these findings, objective treatment outcomes of cases treated with immunotherapy should be seen.

Programmed death-ligand 1 (PD-L1) expression in primary gastric adenocarcinoma and matched metastases

BackgroundCombination of immunotherapy and chemotherapy is recommended for first line treatment of gastric adenocarcinoma (GC) patients with locally advanced unresectable disease or metastatic disease. However, data regarding the concordance rate between PD-L1 combined positive score (CPS) in primary GC and matched regional lymph node metastasis (LNmet) or matched distant metastasis (Dmet) is limited.MethodsTissue microarray sections from primary resected GC, LNmet and Dmet were immunohistochemically stained with anti-PD-L1 (clone SP263). PD-L1 expression was scored separately in tumour cells and immune cells and compared between matched primary GC, LNmet and/or Dmet. CPS was calculated and results for CPS cut-offs 1 and 5 were compared between matched samples.Results275 PD-L1 stained GC were analysed. 189 primary GC had matched LNmet. CPS cut-off 1 concordance rate between primary GC and LNmet was 77%. 23 primary GC had matched Dmet but no matched LNmet, CPS cut-off 1 concordance rate was 70%. 63 primary GC had both matched LNmet and matched Dmet, CPS cut-off 1 concordance rate of 67%. CPS cut-off 5 results were similar. The proportion of PD-L1 positive tumour cells increased from primary GC (26%) to LNmet (42%) and was highest in Dmet (75%).ConclusionOur study showed up to 33% discordance of PD-L1 CPS between primary GC and LNmet and/or Dmet suggesting that multiple biopsies of primary GC and metastatic sites might need to be tested before considering treatment options. Moreover, this is the first study that seems to suggest that tumour cells acquire PD-L1 expression during disease progression.

Programmed death-ligand 1 (PD-L1) expression in cervical intraepithelial neoplasia and cervical squamous cell carcinoma of HIV-infected and non-infected patients

Programmed death-ligand 1 (PD-L1) is overexpressed in cervical carcinoma, hindering tumor destruction. The aim of this study was to assess PD-L1 expression by immunohistochemistry in cervical squamous cell carcinoma (SCC) and squamous intraepithelial lesions (SILs) from human immunodeficiency virus–positive (HIV+) and human immunodeficiency virus-negative (HIV-) patients. A total of 166 SCC and SIL samples of HIV+ and HIV- patients were included and analyzed for PD-L1 expression through tumor proportion score (TPS), and results were stratified in five TPS groups using SP263 antibody and, combined positive score (CPS) using 22C3 antibody. In cohort 1 (SP263 clone), all HIV+ patients were negative for intraepithelial lesion or malignancy (NILM), and low-grade squamous intraepithelial lesions (LSILs) scored < 1; and 87.5% of high-grade squamous intraepithelial lesions (HSILs) adjacent to SCC, 19% of HSILs non-adjacent to SCC, and 69% of SCCs scored ≥ 1 (15.4% scored 5). In HIV- patients, all NILM, LSILs, HSILs adjacent to SCC, and two HSILs non-adjacent to SCC scored < 1. SCC: 88.2% scored ≥ 1 and 5.9% scored 5. In cohort 2 (SP263 and 22C3 clones), 16.7% of HIV+ patients with SCC were positive with both clones, CPS ≥ 1 (22C3) or score 5 (≥ 50%) (SP263), showing no significant differences in positivity between both clones. These results indicate that a relatively low percentage of SCCs (16.7%; both in HIV+ and in HIV- patients) express PD-L1 (TPS ≥ 50% and CPS > 1), which may be due to some samples being archival material, sample characteristics, or use of different methodologies, highlighting the need for standardization of PD-L1 assessment in SCC of the cervix. The fact that PD-L1 is overexpressed in SILs of HIV+ patients suggests potential additional applications for immunotherapy in this disease.

Abstract 1018: Multiplex immunofluorescence and image analysis to investigate the role of the immune contexture and fibroblast activation for tumor cell budding in colorectal cancer

Abstract The advent of immune oncology had a significant impact on the stratification of cancer patients in the past few years. Furthermore, immune cell phenotyping of the tumor microenvironment is becoming a tool not only for the identification of novel predictive biomarkers for cancer immunotherapy, but also for prognostic markers that may help to understand several mechanisms like invasion and epithelial-mesenchymal transition (EMT). We demonstrate how multiplexed immunofluorescence (mIF) assays and digital image analysis helped to investigate tumor budding in colorectal cancer by evaluating the tumor microenvironment and activated fibroblasts. Akoya Bioscience’s Opal detection system and fluorophores were optimized on LEICA Bond RX for use with human normal tonsil control tissues and FFPE colorectal cancer specimens. The 6-plex mIF panel included CD4 (clone SP35), CD8 (clone C8/144B), CD68 (clone PG-M1), FoxP3 (clone SP97), PD-L1 (clone SP263) and pan-Cytokeratin (clones AE1/AE3). In addition, FAP-alpha single-plex IHC was used to investigate reactivity of the stroma surrounding tumor cell buds. Each single marker of the mIF panel was independently validated for specificity regarding stripping efficacy and epitope stability, and for accuracy compared to single plex bright field immunohistochemistry. Automated image analysis and cellular phenotyping followed a workflow of custom Visiopharm apps. The tissue was segmented into regions of interest, such as areas with and without tumor cell budding, and bud microenvironment. Additional parameters like MSI (Microsatellite Instability) status and TNM classification were also taken into consideration for data analysis. The applied mIF immune cell panel and automated image analysis workflow identified a broad number of different immune cell phenotypes, including rare double- or triple-positive cell subtypes, as well as their spatial relation to tumor cell buds within the activated stroma region. This revealed new insights into the complexity of the tumor microenvironment in colorectal cancer around tumor cell buds, suggesting a potential effect of both the immune system and the stromal cells on tumor cell invasion into the surrounding tissue. Citation Format: Dirk Zielinski, Rebekka Vogtmann, Anne Siemon, Christina Koppel, Christina Schipper, Anastasiia Tereshchenko, Suso Platero, Hans-Ulrich Schildhaus. Multiplex immunofluorescence and image analysis to investigate the role of the immune contexture and fibroblast activation for tumor cell budding in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1018.

Tumor Microenvironment in Male Breast Carcinoma with Emphasis on Tumor Infiltrating Lymphocytes and PD-L1 Expression.

Male breast cancer (MBC) is rare and usually presents as a locally advanced disease. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with a better response to neoadjuvant chemotherapy and improved prognosis in all molecular subtypes of female breast cancer, but their role in MBC is less clear. We studied sTILs and the expression of programmed cell death ligand 1 (PD-L1) and pan-TRK in MBC. We retrospectively studied 113 cases of MBC surgically treated between 1988 and 2015. The tumors were evaluated for histological type and grade, stage, intrinsic subtype and sTILs. We performed immunohistochemistry for PD-L1 (clone SP142) and pan-TRK (clone EPR17341) on tissue microarrays. Pan-TRK positive cases were further analyzed by next-generation sequencing. The median age was 69 years (range 60-77). Invasive carcinoma of no special type was found in 94.7% of cases, of which 53.1% were grade 2. Estrogen receptor was positive in 92% of the tumors, progesterone receptor in 85.8%, androgen receptor in 70.8%; 4.4% were human epidermal growth factor receptor 2 (HER2)-positive, and 55.8% HER2-low. 40.7% of tumors were luminal A and 51.3% luminal B, 4.4% HER2-enriched and 3.5% triple negative carcinoma. sTILs density was &lt;50% in 96.4% of the tumors, &gt;50% in 3.6% of the tumors. PD-L1 immune cell score &gt;1% was found in 7.1% of the tumors (all of luminal subtype). A weak focal cytoplasmic pan-TRK staining was present in 8.8% but without NTRK fusion. Neither sTILs nor PD-L1 had statistically significant outcomes. Our findings suggest that a subset of MBC patients harbors an immunological environment characterized by increased sTILs with PD-L1 expression. These patients may potentially benefit from immune checkpoint inhibitor therapy. Frequent HER2-low may offer novel anti-HER2 treatment options.

Enhanced PD-L1 expression on tumor cells in primary CD30-positive cutaneous large T-cell lymphoma: a report of lymph node lesions of four cases

Scarce data are available regarding neoplastic PD-L1 (nPD-L1, clone SP142) expression in cutaneous T-cell lymphoma. We recently documented a possible association of increased nPD-L1 expression with tumor progression to secondary nodal involvement in two cases of CD30-positive primary cutaneous large T-cell lymphoma (PC-LTCL) (Pathol Int 2020;70:804). Notably, the nodal sites exhibited classic Hodgkin lymphoma (CHL) mimicry related to both morphology and tumor microenvironment (TME), i.e., abundant PD-L1-positive tumor-associated macrophages and low-level PD-1 expression on T-cells. Immunohistochemistry highlighted distinctly different nPD-L1 positivity between the cutaneous and nodal lesions. In the present study, we aimed to validate this unique phenomenon in a larger series of four cases with FISH and targeted-capture sequencing (targeted-seq) analysis. We retrospectively identified two more cases of CD30-positive PC-LTCL with secondary nodal involvement among all patients consecutively diagnosed between 2001-2021. All cases immunohistochemically exhibited elevated nPD-L1 expression on ≥50% of lymphoma cells in nodal tumors, clearly contrasting with the scarce nPD-L1 positivity (≤1%) in cutaneous tumors. Moreover, all nodal lesions exhibited CHL-like TME, with abundant PD-L1-positive tumor-associated macrophages and low-level PD-1 expression on T cells, although the CHL-like morphology was limited in the two original cases. None showed CD274/PD-L1 copy number alteration by FISH analysis, or structural variations of PD-L1 3'-UTR by targeted-seq analysis. These findings indicated that nPD-L1 expression is linked with tumor progression and CHL-like TME in nodal involvement of PC-LTCL. Interestingly, one autopsied case exhibited heterogeneity of nPD-L1 expression at different disease sites.