22C3 Antibody Research Articles

Abstract C149: Relationship between PD-L1 expression and clinicopathological characteristics in Hispanic/ Latino women with breast cancer

Abstract Background: Programmed death ligand 1 (PD-L1) expression in breast cancer serves as a prognostic marker, particularly in aggressive subtypes like triple-negative breast cancer (TNBC), where it helps in predicting response to immune checkpoint inhibitors and guides immunotherapy use. Although several studies have assessed the prevalence of PD-L1 expression in breast cancer, data from the Hispanic population, especially in Latin American countries, is lacking. This study evaluates PD-L1 expression and its correlation with pathological features in Colombian women. Methods: Women with breast cancer tested for PD-L1 were selected from the archives of the Oncologic Pathology Department at the National Cancer Institute of Colombia from September 2021 to May 2024. Information from their pathology reports was extracted, and correlation analyses were carried out using Fisher's exact test Results: A total of 60 reports were screened, comprising 56 cases of ductal invasive breast carcinoma and 1 case of lobular invasive breast carcinoma. 48 individuals were tested using the PD-L1 22C3 antibody, with 41.67% of them being positive for PD-L1 expression. Among the 48 tumors tested with 22C3, 35 cases were classified as TNBC, and 13 cases corresponded to luminal B subtype with a low positive expression (1-10%) of estrogen receptors. The percentage of positive PD-L1 cases was 37.14% for TNBC and 53.85% for the luminal B subtype. Furthermore, 5 HER2-low cases were identified with 2 of them being positive for PD-L1 expression. In the correlation analyses, PD-L1 positive expression was significantly associated with a more aggressive histologic grade compared to PD-L1 negative tumors (p = 0.007). No significant association was found between PD-L1 expression and HR, HER2, or KI67 status, nor with age, necrosis, lymphovascular involvement, perineural invasion, desmoplastic response, or immune cell infiltration. Among the 12 individuals tested for PD-L1 with the SP142 antibody, none showed positive expression, precluding correlation analyses. Conclusion: This study highlights a significant association between PD-L1 expression and aggressive histologic grade in Colombian women with breast cancer. The findings underscore the potential of PD-L1 as a prognostic marker and its relevance in guiding immunotherapy in this population. Further research is needed to explore PD-L1 expression in broader Hispanic groups to better understand its clinical implications. Citation Format: Daniel Mendivelso-González, Patricia López- Correa, Nicolás Prada-Aceros, Alfredo E Romero-Rojas, Rafael Parra-Medina. Relationship between PD-L1 expression and clinicopathological characteristics in Hispanic/ Latino women with breast cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C149.

Concordance of PD-L1 Expression in Metastatic Triple-negative Breast Cancer Between the 22C3 and E1L3N Antibodies Using Combined Positive Scoring.

For patients with metastatic triple-negative breast cancer (TNBC), treatment with pembrolizumab is dependent on the accurate determination of programmed death ligand 1 (PD-L1) expression using immunohistochemistry (IHC). This study evaluated the interobserver concordance in assessing PD-L1 expression on TNBC samples using the commercial 22C3 IHC assay and an in-house assay based on the E1L3N antibody. Concordance between the 22C3 and the E1L3N IHC assays was evaluated on TNBC samples read by a commercial laboratory and a Brigham and Women's Hospital breast pathologist (BWH reader). Each slide was given a PD-L1 combined positive score (CPS) and was considered PD-L1 positive or negative based on the CPS cutoff of 10. Interobserver concordance for the assays was also evaluated on a subset of samples between 2 and 3 independent readers. On 71 samples, 2 independent readers (1 BWH reader and commercial laboratory) using E1L3N and 22C3, respectively, reached agreement on PD-L1 status (positive/negative) on 64 samples (90.1%). Using 22C3, 2 independent readers reached agreement on PD-L1 status on 30 of 36 samples (83.3%), and 3 independent readers reached agreement on 16 of 27 samples (59.3%). Using E1L3N, 2 BWH readers reached agreement on PD-L1 status on 18 of 27 samples (66.7%). Three BWH readers reached an agreement on 2 of 12 of the most challenging samples (16.7%). In conclusion, concordance between E1L3N and 22C3 testing using CPS for PD-L1 in metastatic TNBC was >90%. However, certain cases were challenging to agree upon using current threshold criteria, highlighting the need for more standardized evidence-based methods to assess PD-L1 expression.

Considerable interlaboratory variation in PD-L1 positivity forhead and neck squamous cell carcinoma in the Netherlands- A nationwide evaluation study.

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. 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. 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. 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 CT203: Multi-omic analysis of serial biopsies to inform biomarkers of sensitivity to olaparib and durvalumab in patients with metastatic BRCA-wildtype triple negative breast cancer (mTNBC)

Abstract Background: The phase 2 Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC) trial (NCT03801369) evaluated the efficacy of the combination of the PARP inhibitor Olaparib (Ola) and the PD-L1 inhibitor Durvalumab (Durva) in participants with BRCA-wildtype mTNBC. The combination was found to be effective with a median progression free survival (mPFS) of 5.5 months (AACR 2022). Here we report on updated biomarker analyses from paired biopsies (Bx) pre- and on-Ola therapy from 14 AMTEC participants. Methods: AMTEC participants undergo a pre-Ola Bx (Bx1), then one (28-day) cycle of Ola monotherapy with a repeat on-Ola Bx (Bx2) before adding Durva to Ola. Multi-omic profiling of DNA, RNA, and protein signals in Bx1 and Bx2 using Deep candidate gene sequencing, RNAseq, Nanostring Digital Spatial Profiling (DSP), and multiplex immunohistochemistry (mIHC) was correlated with clinical outcomes to identify predictors of Ola + Durva sensitivity, and adaptive resistance to PARPi therapy. Results: We identified over 30 biomarkers, with the optimal predictive value arising from Bx2. Key potential biomarkers were confirmed in an independent test set. Markers of good prognosis: • Basal Immune Activated (BLIA)/Basal Immune Suppressed (BLIS subtype) - A BLIA subtype on Bx2 was associated with a mPFS of 7.36 months compared to 1.71 months for BLIS/Luminal androgen receptor subtypes (p=0.001) • Multi-omic immune composite - A positive immune consensus signature (RNA, DSP, mIHC) on Bx2 was associated with a mPFS of 7.36 months compared to 2.29 months (p=0.005) • B cell activation signature - We developed a novel B-cell RNA activation signature that was highly predictive of response in Bx2, with mPFS of 8.54 and 1.04 months in signature-positive and signature-negative tumors, respectively (p=0.0004) Markers of poor prognosis: • AKT/MAPK pathway - AKT pathway gene mutations in Bx2 with concomitant protein phosphorylation of pathway members correlated with a shorter mPFS of 2.4 months compared to 7.13 months for no pathway mutation/activation (p=0.03). MAPK pathway protein phosphorylation in Bx2 was a negative prognostic factor, with mPFS of 2.17 months (p=0.004) • Angiogenesis - An upregulated Hallmark of Cancer angiogenesis RNA signature on Bx2 was associated with a shorter mPFS of 1.94 months compared to 7.95 months with no angiogenesis upregulation (p=0.0009) The following markers did not show significant association with survival outcomes on AMTEC: • PD-L1 positivity by IHC (defined as >1% Tumor Proportion Score, 22C3 antibody) • Tumor mutational burden • HRD, as measured by Rad51 foci or by mutations in HR pathway members Conclusions: Findings highlight the value of paired Bxs to identify predictive biomarkers of PARPi + immune checkpoint inhibitor sensitivity. The striking predictive value of the BLIA/BLIS signature warrants evaluation in a larger trial. Emerging resistance mechanisms justify AMTEC trial expansion to include PARPi + MEKi or PARPi + AKTi in biomarker selected patients, which is now in progress. Citation Format: Zahi I. Mitri, Allison L. Creason, Jayne M. Stommel, Daniel Bottomly, Jeong Youn Lim, SMMART Clinical Trials Program, Christopher L. Corless, Shannon McWeeney, Gordon B. Mills. Multi-omic analysis of serial biopsies to inform biomarkers of sensitivity to olaparib and durvalumab in patients with metastatic BRCA-wildtype triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT203.

Abstract 553: Assessment of CNTN4 expression and its association with PD-L1 across 18 various cancers using an artificial intelligence-powered immunohistochemistry analyzer

Abstract Background: Contactin-4 (CNTN4) has been identified as an inhibitory immune checkpoint protein and mainly expressed on tumor cells. It interacts with APP present in T cells, leading to a reduction in T cell proliferation, activation, and responsiveness to tumors. In this study, we applied artificial intelligence (AI)-powered analyzers to examine the expression of CNTN4 and its association with PD-L1 in various solid tumors. Methods: The AI-powered immunohistochemistry (IHC) analyzers were developed using pan-cancer whole slide images (WSIs) stained with 18 different IHC types. Pathologists annotated cells and tissues on the WSIs. The universal model was trained by annotations from IHC WSI, which could detect tumor cells with four grade intensities (strong, moderate, weak, and negative) and segment invasive cancer area. Finally, the AI analyzer can count the tumor cells in an invasive cancer area and calculate the ratio based on intensity. The AI analyzer was applied to the pan-cancer tissue microarray dataset (n=795) consisting of 18 cancers to validate the analyzer. The dataset was stained with rabbit monoclonal antibody (clone EPR 8735) and PD-L1 22C3 antibody, respectively. The expression levels of CNTN4 and PD-L1 were calculated with H-score (range 0 to 300) or tumor proportion score (TPS). Results: When positivity was defined as moderate-to-strong staining intensity in ≥ 30%, 50%, 75%, and 90%, 42.4% (337/795), 25.5% (203/795), 11.4% (91/795), and 5.2% (41/795) of samples were CNTN4 positive, respectively. In case of moderate-to-strong staining in ≥ 50% tumor cells, CNTN4 expression were most frequently observed in hepatocellular carcinoma (63.9%), endometrial carcinoma (50.0%), stomach cancer (43.9%), pancreatic cancer (40.0%), and prostate cancer (35.4%). Notably, 10% of cases with an H-score < 200 (75/757) showed a range of PD-L1 expression (PD-L1 TPS ≥ 1%), while all of the samples with an H-score ≥ 200 (n=38) were PD-L1 TPS <1%. Conclusions: This study describes the CNTN4 expression levels in various tumors using an AI-powered IHC analyzer. CNTN4 tends to be expressed in opposition to PD-L1, making it a viable immune therapeutic target in cancers that do not conventionally express PD-L1. Citation Format: Seunghwan Shin, Jimin Moon, Soo Ick Cho, Chan-Young Ock, Bu-Nam Jeon, Gyeongyeon Kim, Hyunkyung Yu, Miyoung Cha. Assessment of CNTN4 expression and its association with PD-L1 across 18 various cancers using an artificial intelligence-powered immunohistochemistry analyzer [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 553.

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.

PD-L1 expression and its correlation with clinicopathological and molecular characteristics in Chinese patients with non-small cell lung cancer.

Little is known about the relationship between programmed cell death-ligand 1 (PD-L1) expression and histologic and genetic features in real-world Chinese non-small cell lung cancer patients. From November 2017 to June 2019, tumor tissues were collected from 2674 non-small cell lung cancer patients. PD-L1 expression was detected with immunohistochemistry using the 22C3 and SP263 antibodies, and patients were stratified into subgroups based on a tumor proportion score of 1%, 1% to 49%, and ≥ 50%. Genetic alterations were profiled using targeted next-generation sequencing. In the total population, 50.5% had negative PD-L1 expression (tumor proportion score < 1%), 32.0% had low-positive expression (1%-49%), and 17.5% had high-positive expression (≥50%). The PD-L1 positive rate was 39.0% in squamous cell carcinomas and 53.6% in adenocarcinomas. PD-L1 expression was higher in squamous cell carcinomas (P < .001) and lower in adenocarcinomas (P < .001). Of the overall patient population, 11.2% had Kirsten rat sarcoma viral oncogene (KRAS) mutations, 44.9% had epidermal growth factor receptor (EGFR) mutations, 2.1% had BRAF V600E mutations, 0.3% had MET exon 14 skipping mutations, 5.4% had anaplastic lymphoma kinase translocations, and 0.9% had ROS proto-oncogene 1 translocations. Patients carrying ROS proto-oncogene 1 translocations (P = .006), KRAS (P < .001), and MET (P = .023) mutations had significantly elevated expression of PD-L1, while those harboring EGFR (P < .001) mutations had lower PD-L1 expression. In our study, PD-L1 expression was significantly higher in squamous cell carcinomas and lower in adenocarcinomas, and was positively associated with MET and KRAS mutations, as well as the wild-type EGFR gene state. Nonetheless, additional studies are needed to further validate those associations and determine the clinical significance for immune checkpoint inhibitors of these factors.

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.

Immunotherapeutic options for management of metastatic cervical cancer

Cervical cancer (CC) remains one of the common tumour diseases of women. It accounts for 9.8% of all neoplasia in women. The Food and Drug Administration approved pembrolizumab with PD-1 inhibitor antibodies for the treatment of RSM following the KEYNOTE-158 clinical trial. PD-L1/PD-1 inhibitors have shown promising results against objective response rate in female patients with CC. A clinical case of pembrolizumab treatment of a patient with metastatic CC with progression after radiotherapy and pelvic organ evisceration is described. The patient underwent IHC examination of the tumour material and was found to have PDL1 expression of more than 1% according to the 22C3 antibody assay. According to the instructions for Pembroria® it was decided to start systemic chemotherapy in the regimen of paclitaxel 175 mg/m2, carboplatin AUC5 and pembrolizumab at a dose of 200 mg every three weeks. After the third course, a control MRI study was performed. A partial response according to iRECIST criteria was confirmed. After the sixth course of therapy cytostatics were cancelled, the patient continued monotherapy with pembrolizumab 200 mg once every three weeks. At the control MRI after 10 months of therapy, the tumour conglomerate in the right hindbrain fossa was not detected and a complete response was confirmed. In the given observation anti-PD1 therapy and metastatic CC allowed to achieve complete remission of the disease. Thus, the clinical observation demonstrates the efficacy of pembrolizumab therapy in patients with advanced CC.

Comparative analysis of PD-L1 expression and molecular alterations in primary versus metastatic lung adenocarcinoma: a real-world study in China.

Programmed death-ligand 1 (PD-L1) is the only Food and Drug Administration-approved biomarker for monitoring response to immune checkpoint inhibitor (ICI) therapy in patients with lung adenocarcinoma. Understanding the nuances of molecular phenotypes, clinical attributes, and PD-L1 expression levels in primary and metastatic lung adenocarcinoma may help predict response to therapy and assist in the clinical management of lung adenocarcinoma. A total of 235 primary and metastatic lesion specimens from patients with non-small cell lung cancer (NSCLC) an institution in Shandong, China were analyzed. PD-L1 expression was assessed by immunohistochemistry using the 22C3 antibody, and the molecular phenotype was determined by next-generation sequencing of 450 genes. The molecular phenotypes of the primary and metastatic lesions were compared. Elevated PD-L1 expression was significantly associated with advanced and metastatic disease (P = 0.001). The distribution of PD-L1 expression varied based on the anatomical location, showing a higher frequency of elevated PD-L1 expression in distal metastases than in the primary tumor. Metastatic lesions exhibited a higher proportion of carcinogenic pathway gene alterations and a greater number of DNA damage-repair pathway gene alterations than the primary lesions. Notably, CDKN2A copy number deletions were more prevalent in metastatic lesions than in primary lesions. Clinical data stemming from research conducted at the Memorial Sloan Kettering Cancer Center revealed an association between the absence of CDKN2A expression and a poorer prognosis in stage I lung adenocarcinoma. Samples of metastatic tumors exhibited a higher proportion of elevated PD-L1 expression, a greater number of pathway alterations, and a higher occurrence of CDKN2A copy number deletions than primary samples. This highlights the importance of reinforcing the clinical management and follow-up of patients with CDKN2A deficiency, particularly within the subset of stage I lung adenocarcinoma.

Expression of PD-L1 in breast invasive lobular carcinoma.

The purpose of this study was to investigate the expression of PD-L1 in invasive lobular carcinoma (ILC) and to determine its implications. Tissue microarrays were constructed for 101 cases of ILC, and immunohistochemical staining for PD-L1 (using 22C3, SP142, and SP263 antibodies) was performed to examine the correlation between staining results and clinicopathologic parameters. The positive cut-off values were defined as tumor cell (TC)≥1%, immune cell (IC)>0%, and IC≥1%. The range of PD-L1 TC positivity was 0.0-2.0%, with PD-L1 SP263 TC showing the highest positivity of 2.0%. The range of PD-L1 IC positivity was 0-21.8% for IC ≥ 1%, with PD-L1 22C3 IC showing the highest positivity. When PD-L1 IC was positive (IC≥1%), the highest antibody agreement was observed between SP263 and SP142 (OA = 93.1%), while the lowest agreement was observed between 22C3 and SP263 (OA = 73.3%, κ = 0.040). PD-L1 22C3 IC positivity (≥1%) was associated with high nuclear grade (p = 0.002), HER-2 positivity (p = 0.019), and pleomorphic type (p = 0.002). PD-L1 expression in ILC shows a low TC positivity rate (0-2%) with various antibody clones and a variable IC positivity rate (0-21.8%). Pleomorphic type ILC exhibits higher PD-L1 IC positivity.

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.

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.

Equivalence of laboratory-developed test and PD-L1 IHC 22C3 pharmDx across all combined positive score indications.

We conducted an analysis across multiple PD-L1 combined positive score (CPS) indications to establish concordance of a 22C3 antibody-based laboratory-developed test (LDT) on the Ventana BenchMark XT or BenchMark ULTRA platform and the regulatory-approved PD-L1 IHC 22C3 pharmDx in cervical cancer (CC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and urothelial carcinoma (UC). Tumor specimens from each tumor type were stained with 22C3 antibody and scored using the 22C3 antibody-based LDT, and scores were compared with those using PD-L1 IHC 22C3 pharmDx. PD-L1 status was measured by the pathologist using CPS as a continuous score and using clinically relevant cutoffs (CC, ≥1 and ≥10; HNSCC, ≥1 and ≥20; ESCC, TNBC, and UC, ≥10). The agreement between the BenchMark platforms and PD-L1 IHC 22C3 pharmDx was assessed by intraclass correlation coefficient (ICC) and a contingency table for clinical interpretation. A total of 522 samples were evaluated for the pan-tumor analysis (CC, n = 77; ESCC, n = 80; HNSCC, n = 126; TNBC, n = 118, UC, n = 121). Most clinical interpretations of PD-L1 status were concordant between the BenchMark XT and PD-L1 IHC 22C3 pharmDx for all five tumor types with regard to negative percentage agreement (NPA; 83-97%), positive percentage agreement (PPA; 86-100%), and overall percentage agreement (OPA; 90-97%); the ICC by tumor type was high (≥0.88). Importantly, the pan-tumor ICC was 0.95 (95% CI 0.94-0.96). Thirty additional TNBC samples were evaluated using the BenchMark ULTRA and PD-L1 IHC 22C3 pharmDx; the NPA, PPA, and OPA were 100%. The 22C3 antibody-based LDT on Ventana BenchMark XT and BenchMark ULTRA platforms demonstrated high concordance with the regulatory-approved PD-L1 IHC 22C3 pharmDx across multiple tumor types. These findings suggest the comparability of PD-L1 IHC 22C3 pharmDx with an LDT based on the 22C3 antibody.

Comparing PD-L1 scores for predicting pembrolizumab efficacy in first-line treatment of unresectable cutaneous squamous cell carcinomas.

e21593 Background: In the CARSKIN study (NCT02883556), 1L pembrolizumab demonstrated promising activity with durable responses and manageable safety in patients (pts) with unresectable locally advanced or metastatic CSCC. Here we report best ORR and survival endpoints of the entire cohort by PD-L1 status. Methods: The imaging assessment per RECIST v1.1 was blinded with independent central review. Best ORR, PFS, DOR OS, in the overall sample and by PD-L1 status was centrally assessed by 2 blinded independent pathologists, one using the anti–PD-L1 E1L3N clone (TPSE1L3N), the other using the 22C3 antibody (TPS22C3 and CPS22C3). Among 57 pts, 6 were excluded from the analysis: 2 untreated pts, 1 early unrelated death and 3 pts tested only with 1 assay). ROC curve analysis was used to compare assays. Results: With a median follow-up of 26 mo, best ORR was 51% with 14 PR (27%) and 12 CR (24%). Relapse occurred in 3 PR and 1 PD-L1 – CR pts. Respective median PFS, DOR, and OS were 13.7, NR, and 25.3 mo; respective 1-year PFS, DOR and OS were 50% [38-66], 80% [65-97] and 73% [61-86]. With a cutoff of 1%, best ORR was higher either for TPSE1L3N + pts vs TPSE1L3N – pts (60% vs 18%, p = 0.02) and for CPS22C3 + pts (59% vs 20%, p = 0.038), but not TPS22C3 + pts (p = 0.76). The optimal cutoff of CPS22C3 for ORR using a ROC curve was estimated to be ≥ 7% with a sensitivity of 0.70 and a specificity of 0.75. For best ORR, area under the ROC curve was higher for CPS22C3 (0.72) than TPSE1L3N (0.62) but the difference was not significant (P = 0.21). Pts with TPSE1L3N ≥ 1% had a higher 1-y PFS (57% vs 27%, p = 0.004) and OS (82% vs 42%, P = 0.01). Pts with CPS22C3 ≥ 1%, had a better 1-y OS (79% vs 50%, p = 0.03), but not 1y-PFS. Conclusions: CPS22C3 ≥ 7% appeared equivalent to TPSE1L3N ≥ 1% for predicting ORR and OS in pts receiving pembrolizumab in 1L treatment of advanced or metastatic CSCCs. Replications are warranted for validation. Pts with low scores might beneficiate from combined treatments. Clinical trial information: NCT02883556 .

Phase II study evaluating the efficacy of niraparib and dostarlimab (TSR-042) in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) patients.

6046 Background: Patients with recurrent and/or metastatic (R/M) HNSCC have poor overall survival. Immune checkpoint inhibitors (ICIs), specifically programmed death-1 (PD-1) inhibitors, result in durable responses in R/M HNSCC but &lt;20% benefit. Tumor mutational burden (TMB) correlates with ICI response. DNA pathway repair mutations have been reported in 17% of sporadic HNSCC and are associated with higher rates of TMB. Poly (ADP-ribose) polymerase (PARP) inhibition has demonstrated efficacy as a single agent in cancers with a DNA repair defect. We tested the combination of PARP inhibitor (niraparib) with the PD-1 inhibitor (dostarlimab) in R/M HNSCC patients (NCT04313504) with funding support from Tesaro/GSK. Methods: Patients with R/M HNSCC who failed at least one line of prior treatment including a PD-1 inhibitor and for whom no surgical or radiation curative options existed were eligible for the study. Patients received niraparib (200 mg daily) beginning one week prior to initiation of dostarlimab (500 mg q3wks x4 followed by 1000 mg q6wks). The primary endpoint was overall response rate (ORR) including complete response (CR), partial response (PR) and stable disease (SD) using RECIST v1.1. Fourteen patients were planned for the first stage and 10 additional patients for the second stage if ≥8 patients had a confirmed response. Safety was evaluated by CTCAE v5.0. Tumor PD-L1 immunohistochemistry was performed with 22c3 antibody and reported as combined positive score (CPS). TMB was considered high if ≥10Mut/Mb. Results: Ten of 24 planned patients were enrolled. Of the first 10 patients, 8 patients were evaluable by imaging and 1 patient clinically progressed on exam. Of the 9 evaluable patients, there were 0 CR, 1 PR, 1 SD, and 7 with progressive disease (PD). The trial was ended early for futility as it was not possible for at least 8 out of 14 patients to develop response. The patient with a PR remains on treatment past 24 months, was PD-L1 positive, had low TMB and no DDR deficiency. All patients had previously failed a PD-1 inhibitor. One patient had high TMB, 2 patients had a DDR, and 6 patients were PDL1 positive but 2 patients had insufficient tissue or indeterminate results for sequencing. Grade ≥ 3 adverse events occurred in 70% of patients with most common being hypertension (20%), hyponatremia (20%), and lung infection (20%). Conclusions: PARP-1 inhibition and PD-1 inhibition combined does not appear to enhance response over PD-1 inhibitor alone in R/M HNSCC who had previously failed PD-1 inhibitor treatment. However, a more focused population including those with DNA damage response (DDR) mutations and PD-1 treatment naïve may benefit. Clinical trial information: NCT04313504 .[Table: see text]

Tumor-Infiltrating Lymphocytes and PD-L1 Expression in Pleomorphic Lobular Breast Carcinoma.

The prognostic and predictive role of stromal tumor-infiltrating lymphocytes (sTILs) is undetermined in pleomorphic invasive lobular cancer (pILC). The same applies for the expression of PD-1/PD-L1 in this rare breast cancer subtype. Here, we aimed to investigate the expression of sTILs and analyze the PD-L1 expression levels in pILC. Archival tissues from sixty-six patients with pILC were collected. The sTIL density was scored as a percentage of tumor area using the following cut-offs: 0%; <5%; 5-9%; and 10-50%. The PD-L1 expression was analyzed using IHC on formalin-fixed, paraffin-embedded tissue sections using SP142 and 22C3 antibodies. A total of 82% of the sixty-six patients were hormone receptor positive and 8% of cases were triple negative (TN), while 10% showed human epidermal growth factor receptor 2 (HER2) amplification. sTILs (≥1%) were present in 64% of the study population. Using the SP142 antibody, 36% of tumors demonstrated a positive PD-L1 score of ≥1%, and using the 22C3 antibody, 28% had a positive PD-L1 score of ≥1. There was no correlation between sTILs or PD-L1 expression and tumor size, tumor grade, nodal status, expression of estrogen receptor (ER), or amplification of HER2. Our data did not show any difference in survival between the three molecular subtypes of pILC with respect to sTILs and PD-L1 expression. This study shows that pILCs show some degree of sTILs and PD-L1 expression; however, this was not associated with a survival improvement. Additional large trials are needed to understand immune infiltration in lobular cancer, especially in the pleomorphic subtype.

Variable Landscape of PD-L1 Expression in Breast Carcinoma as Detected by the DAKO 22C3 Immunohistochemistry Assay.

In 2020, pembrolizumab was approved as a therapy for triple-negative breast cancer (TNBC) with the companion diagnostic DAKO 22C3 programmed death ligand-1 (PD-L1) immunohistochemistry assay. The study aimed to determine the landscape of PD-L1 expression as detected by the DAKO 22C3 PD-L1 assay in breast cancer subtypes and compare the clinicopathologic and genomic characteristics of PD-L1 positive and negative TNBC. PD-L1 expression using the DAKO 22C3 antibody was scored using a combined positive score (CPS) and positive status was defined as CPS ≥10. Comprehensive genomic profiling was performed using the FoundationOne CDx assay. Of the 396 BC patients stained with DAKO 22C3, the majority were HR+/HER2- and TNBC (42% and 36%, respectively). Median PD-L1 expression and frequency of CPS ≥10 was highest in TNBC cases (median: 7.5, 50% CPS ≥10) and lowest in the HR+/HER2- group (median: 1.0, 15.5% CPS ≥10) (P < .0001). A comparison of PD-L1 positive and PD-L1 negative TNBC demonstrated no significant differences in clinicopathologic or genomic characteristics. TNBC tissue samples from the breast did have an observed enrichment for PD-L1 positivity compared to TNBC tissue samples from a metastatic site (57% vs. 44%), but this was not statistically significant (P = .1766). In the HR+/HER2- group, genomic alterations in TP53, CREBBP, and CCNE1 were more prevalent and genomic loss of heterozygosity was higher in the PD-L1(+) group compared to the PD-L1(-) group. The subtypes of breast cancer have distinct patterns of PD-L1 expression, supporting that further research of immunotherapies may include specific evaluation of optimum cutoffs for non-TNBC patients. In TNBC, PD-L1 positivity is not associated with other clinicopathologic or genomic features and should be integrated into future studies of immunotherapy efficacy.

Abstract P6-01-23: Role of tumor infiltrating lymphocytes and PD-L1 expression in the response to eribulin and pembrolizumab in metastatic triple negative breast cancer (mTNBC) on the ENHANCE1 trial

Abstract Background Treatment with combination chemo-immunotherapy has become the front-line standard for eligible patients with PD-L1 positive mTNBC. PD-L1 is the only approved biomarker for pembrolizumab in metastatic breast cancer for response to combination chemo-immunotherapy, however given that it is not predictive of response in all cases additional biomarkers are needed. Tumor infiltrating lymphocytes (TILs) have been shown to be both predictive and prognostic in operable TNBC, but there are fewer data regarding the role of TILs in mTNBC. In this study we report the associations between TILs and outcomes in patients treated prospectively on the ENHANCE-1 study with eribulin and pembrolizumab. Methods ENHANCE-1 was a single arm phase Ib/II trial which evaluated the efficacy and safety of eribulin and pembrolizumab in 167 patients (pts) with mTNBC who had received 0-2 prior lines of therapy (66 pts in the first line setting [stratum 1] and 101 pts with 1-2 prior lines of therapy [stratum 2]). Objective response rate (ORR) was defined as percentage of pts with either complete response (CR) or partial response (PR) by RECIST 1.1. The ORR was 25.8% in stratum 1 and 21.8% in stratum 2. Stromal TILs (sTIL) and intratumoral TILs were evaluated on whole slide H&amp;E sections from biopsy specimens used for enrollment on ENHANCE-1 by breast pathology according to the International TILs Working Group guidelines. PD-L1 positivity was determined via immunohistochemistry using the Agilent 22C3 antibody. We also assessed TIL density digitally using machine learning classifiers to identify tumor/stromal tissue areas and individual lymphocytes. Results We found that there was statistically significant increase in sTIL counts in responders compared to non-responders in stratum 1 (p=0.002) but not in stratum 2 (p=0.99). We did not find any associations between intratumoral TILs and response. Quantitative PD-L1 scoring via combined proportion score (CPS) was also positively associated with response in stratum 1 (p=0.01) but not in stratum 2 (p=0.34). We also find that sTIL counts are most correlated to CPS scores (continuous) for non-responders within stratum 1 (R2=+0.55, p&amp;lt; 0.01). Conclusion In this population of patients with mTNBC treated prospectively with eribulin and pembrolizumab, sTILs and PDL1 CPS were each individually associated with a positive response in patients treated with front-line combination chemo-immunotherapy. Neither was predictive for patient response in stratum 2. One important caveat is the biopsies were not required immediately prior to enrollment, possibly confounding the tumor microenvironment (TME) at the time of analysis. Our data contribute to emerging data that sTILs can act as a biomarker for response to immune checkpoint inhibition when utilized in the front-line setting for mTNBC. Further characterization of the TME via quantitative immunofluorescence is ongoing. This study was funded by Eisai IIS-E7389 Citation Format: Mathew Kearney, Hua Guo, Rami Vanguri, Qi Wang, Michelle Garlin, Courtney Connelly, Kevin Kalinsky, Eileen P. Connolly. Role of tumor infiltrating lymphocytes and PD-L1 expression in the response to eribulin and pembrolizumab in metastatic triple negative breast cancer (mTNBC) on the ENHANCE1 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-23.

21TiP Molecular alterations predictive of outcome in early staged cervical cancer: A translational investigation in the international validation study of sentinel node biopsy in early cervical cancer SENTICOL III

Cervical cancer remains the second cause of cancer death in women worldwide. Despite of good histological and clinical features, some patients relapse after the treatment of early stage cervical cancer. Recent studies identified correlation between some genetic alterations and poor prognosis in advanced-staged cervical cancer. Yet, the literature remains scarce about molecular alterations and outcome correlation in early stage cervical cancer. Our translational study investigate recurrent molecular alterations predictive of outcome in early stage cervical cancer. We included the first 100 patients randomized in the Senticol III trial. This large multicentric, prospective, randomized, and international “validation study” tries to validate sentinel node biopsy as nodal staging of early cervical cancers (stage Ia – IIa1). Cervical tumor slides with contributive FFPE sample are analyzed and stratified based on well-established histological criteria (SEDLIS criteria). The immune microenvironment characteristics including TIL’s infiltration and PDL1 expression. PDL1 expression is assessed by IHC using the 22C3 antibody and quantified using CPS score. We made HPV detection and typing by PCR of the tumor samples. We performed DNA and RNA extractions from the FFPE tumor specimens. Using the dedicated gene panel developed by our team, we analyzed 571 genes commonly altered in cancer. We performed high throughput RNA sequencing to establish the gene expression profile of each tumor and its associated stroma. The genomic and transcriptomic analysis assessed the tumor mutational load, the most frequently altered genes and their expression. The biostatistical analyses will correlate molecular alterations, histopathological and clinical classical features, with patient outcome. The different parameters will be first analyzed independently (univariate analysis) and then in a multiparametric manner (logistic regression). NCT03386734. Fabrice Lecuru, MD PhD.