Clone E1L3N Research Articles

Updated efficacy results of ASKB589 combined with CAPOX and PD-1 inhibitor as first-line treatment for metastatic gastric/gastro-esophageal junction (G/GEJ) adenocarcinoma from a phase Ib/II study.

454 Background: ASKB589 is a non-fucosylated fully human IgG1 monoclonal antibody that binds CLDN18.2 with high affinity, specificity, and improved ADCC and CDC activities. Previous findings indicated that ASKB589 exhibited a well-tolerated safety profile at doses up to 20 mg/kg and demonstrated promising antitumor effects. A dose of 6 mg/kg has been selected for further evaluation. Here, we present the updated data from dose expansion part in NCT05632939 study. Methods: This phase 1b/2 study was consisted of dose-escalation part and dose-expansion part. The expansion part aimed to assess the safety and efficacy of ASKB589 combined with CAPOX plus a PD-1 inhibitor as the initial treatment for advanced G/GEJ cancer with CLDN18.2 positive, regardless of PD-L1 expression. CLDN18.2 and PD-L1 expression were evaluated through IHC analysis using clone DS-3 and clone E1L3N at a central lab. All pts were given ASKB589 intravenously at a dosage of 6 mg/kg. Responses were assessed by RECIST 1.1 every 6 weeks. Adverse events were graded using CTCAE v5.0. Results: As of July 29, 2024, 49 evaluable pts with CLDN18.2 M/H expressions (≥2+ membrane staining intensity in ≥40% of tumor cells) regardless of PD-L1 CPS score. Forty pts (81.6%) achieved a partial or complete response, with a confirmed objective response rate (cORR) of 73.5% (95%CI: 58.9, 85.1). The median duration of response was 11.14 months (6.93, NE). Three subjects underwent surgical treatment as a result of tumor reduction following treatment. All 49 pts achieved SD or better with a disease control rate of 100%. cORR was 76.2% and 74.1% in the CPS<1 and CPS<5 groups, respectively. Of all treated pts (N=50), PFS rate at 9 months was 58.1% (95% CI: 42.2, 71.0) and OS rate at 12 months was 77.1% (95% CI: 61.2, 87.2). Mature PFS and OS data will be reported at the time of the conference. The safety profile of the triplet is generally consistent with the safety data of ASB589 plus CAPOX combination in first-line G/GEJ cancer pts presented previously, which was mainly characterized by manageable on-target-off-tumor effects, including hypoalbuminemia, nausea, and vomiting. Conclusions: The combination of ASKB589 with CAPOX and a PD-1 inhibitor as the initial treatment for pts with G/GEJ cancer was well tolerated with no unexpected safety signals. It has shown a high confirmed ORR, 100% disease control, deep and durable antitumor activity, regardless of PD-L1 expression levels. A phase 3 study of the triple regimen for the 1L G/GEJ cancer is actively enrolling pts in China. Clinical trial information: NCT05632939 . cORR based on CLDN18.2 and PD-L1 expression. n/N (%) PD-L1CPS<1 PD-L1CPS 1-5 PD-L1 CPS≥5 Total CLDN18.2 M&HcORR 16/21(76.2%) 7/10 (70.0%) 13/18 (72.2%) 36/49 (73.5%) CLDN18.2 Moderate cORR 2/3(66.7%) 0/1(0.0%) 6/9(66.7%) 8/13(61.5%) CLDN18.2 High cORR 14/18(77.8%) 7/9(77.8%) 7/9(77.8%) 28/36(77.8%)

PD-L1 Expression in Ampullary Adenocarcinoma.

Ampullary adenocarcinoma is a rare neoplasm often treated by the complex Whipple's procedure. Several histological factors predict poor prognosis including pancreatobiliary morphology, presence of lymphovascular, perineural invasion and local or distant metastasis. Systemic therapy with gemcitabine, 5-fluorouracil regimens are given with variable benefits. Immunotherapy checkpoint inhibitors have shown beneficial anti-tumor effects in several carcinomas, the most remarkable being in non-small cell lung cancer. Administration of these novel drugs is based on immunohistochemical expression (which may or may not be indicative of response to therapy) along with meticulous decision making by the multidisciplinary team. Immunohistochemistry (IHC) is an effective means of immune marker demonstration and has been used in various tumor types for predictive and prognostic purposes. PD-L1 IHC (clone E1L3N) was applied in 101 cases of ampullary adenocarcinoma. Tumor infiltrating lymphocytes were also evaluated. The immunoreactivity was assessed and categorized into following staining thresholds: <1%, <5%, <10% and ≥10% for tumor cells (membranous and/or cytoplasmic staining pattern), and 5% and 10% cut-offs for immune cells. We found that at a 10% cut-off, 73.3% (74/101) patients were men (P = .006) older than 50 years of age (P < .001) presenting with a tumor measuring <3 cm (P = .001). It was significantly associated with intestinal differentiation (P = .004) and grade 1 tumors (P = .001). Twelve patients presented with recurrence as well (P = .03). In the context of ampullary adenocarcinoma, this study highlights the positivity observed with the PD-L1 IHC clone E1L3N at different thresholds, with the particularly stronger associations being evident at a 10% cut-off.

A comprehensive pan-cancer analysis of PD-L1 expression using clone E1L3N in Chinese patients with cancer.

2656 Background: Immune checkpoint inhibitors (ICIs) benefit patients with multiple cancer types, and the expression of programmed death ligand 1 (PD-L1) protein assessed by immunohistochemistry (IHC) has been correlated with response and survival benefit from anti-PD-1/PD-L1 ICI therapies in several cancer types. IHC assay with E1L3N clone is currently approved by the NMPA for PD-L1 detection in non-small cell lung cancer (NSCLC), but its performance in other cancer types needs to be further validated. Thus, we performed this study to examine the prevalence of PD-L1 expression in a wide variety of tumor types in Chinese pan-cancer patients using the E1L3N antibody clone. Methods: From 2019 to 2022, a total of 13,647 patient across multiple tumor types were analyzed. Tumor tissue samples were subjected to IHC. PD-L1 expression was tested using the E1L3N PD-L1 IHC assay (Amoy Diagnostics, Xiamen, China), and scored with the tumor proportion score (TPS) and combined positive score (CPS). PD-L1 expression status was identified as positive and negative according to TPS or CPS. For NSCLC: negative (TPS &lt; 1%), low expression (TPS 1-49%), and high expression (TPS ≥ 50%). For gastric/gastroesophageal junction carcinoma (GC/GJC): negative (CPS &lt; 1), low expression (CPS 1-5), and high expression (CPS ≥ 5). For other cancer: negative (CPS &lt; 1), low expression (CPS 1-10), and high expression (CPS ≥ 10). Results: In total, 14 cancer types were enrolled in the present study, including 68.2% NSCLC, 6.9% colorectal carcinoma (CRC), and 3.8% GC/GJC, and the remaining 21.1% include esophageal cancer (EC), breast cancer, cervical cancer (CC), biliary tract cancer (BTC), pancreatic cancer (PC), ovarian cancer, renal cancer, small cell lung cancer, urothelial carcinoma (UC), bladder cancer, head and neck squamous cell carcinoma (HNSCC), etc. PD-L1 positivity by TPS was observed in 52.5% NSCLC patients, similar to the frequency of PD-L1 expression in Chinese NSCLC patients detected using 22C3 clone. 76.0% and 36.7% of GC/GJC patients with PD-L1-positive tumors at a CPS cutoff of ≥ 1 and ≥ 5, respectively. PD-L1 positive expression frequency was higher than that in western GC/GJC patients detected using 22C3 and 28-8 clone (as reported from previous research: PD-L1 positivity with CPS ≥ 1 was 45.5% using the 22C3 assay and 49.1% using the 28-8 assay). 73.6% and 17.5% of CRC patients with PD-L1-positive tumors at a CPS cutoff of ≥ 1 and ≥ 10, respectively. In addition, 8 tumors (EC, CC, breast cancer, PC, BTC, UC, HNSCC, and bladder cancer) had the prevalence of PD-L1 CPS ≥ 1 greater than 50%. Conclusions: E1L3N can be used as a reliable alternative antibody clone to evaluate PD-L1 expression status not only in NSCLC patients but also in other cancer types.

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 .

EGFR and ERBB2 exon 20 insertion/duplication in advanced non-small cell lung cancer: genomic profiling and clinicopathologic features.

Exon 20 (ex20) in-frame insertions or duplications (ins/dup) in epidermal growth factor receptor (EGFR) and its analog erb-b2 receptor tyrosine kinase 2 (ERBB2) are each detected in 1.5% of non-small cell lung cancer (NSCLC). Unlike EGFR p.L858R or ex19 deletions, ex20 ins/dup is associated with de novo resistance to classic EGFR inhibitors, lack of response to immune checkpoint inhibitors, and poor prognosis. US Food and Drug Administration has approved mobocertinib and amivantamab for targeting tumors with this aberration, but the number of comprehensive studies on ex20 ins/dup NSCLC is limited. We identified 18 cases of NSCLCs with EGFR/ERBB2 ex20 ins/dup and correlated the findings with clinical and morphologic information including programed death-ligand 1 (PD-L1) expression. A total of 536 NSCLC cases tested at our institution between 2014 and 2023 were reviewed. A custom-designed 214-gene next-generation sequencing panel was used for detecting DNA variants, and the FusionPlex CTL panel (ArcherDx) was used for the detection of fusion transcripts from formalin-fixed, paraffin-embedded tissue. Immunohistochemistry (IHC)for PD-L1 was performed using 22C3 or E1L3N clones. Nine EGFR and nine ERBB2 ex20 ins/dup variants were identified from an equal number of men and women, 14 were non- or light smokers, and 15 had stage IV disease. All 18 cases were adenocarcinomas. Seven of the 11 cases with available primary tumors had acinar predominant pattern, two had lepidic predominant pattern, and the remainder had papillary (one case) and mucinous (one case) patterns. Ex20 ins/dup variants were heterogenous in-frame one to four amino acids spanning A767-V774 in EGFR and Y772-P780 in ERBB2 and were clustered in the loop following the C-helix and α C-helix. Twelve cases (67%) had co-existing TP53 variants. Copy number variation in CDK4 amplification was identified in one case. No fusion or microsatellite instability was identified in any case. PD-L1 was positive in two cases, low positive in four cases, and negative in 11 cases. NSCLCs harboring EGFR/ERBB2 ex20 ins/dup are rare and tend to be acinar predominant, negative for PD-L1, more frequent in non- or light smokers, and mutually exclusive with other driver mutations in NSCLC. The correlation of different EGFR/ERBB2 ex20 ins/dup variants and co-existing mutations with response to targeted therapy and the possibility of developing resistant mutations after mobocertinib treatment warrants further investigation.

Impact of infiltrating T cell subtypes on survival and response to FLOT chemotherapy in resectable oesophagogastric adenocarcinoma (OGA).

443 Background: The prognostic and predictive relevance of tumour infiltrating T cell subtypes and of PD-L1 expression in localised OGA treated with FLOT chemotherapy (CTx) and surgery is poorly understood. Methods: Pre-CTx biopsy tissue was retrospectively collected from 46 patients (pts) with localised OGA who received perioperative FLOT and underwent surgery at a single institution. Opal Multicolour immunofluorescence was performed with primary antibodies against CD4, CD8, FOXP3, CD45RO, PD-1 and cytokeratin. Digital image analysis was performed using inForm and HALO softwares. PD-L1 CPS was determined using the E1L3N clone and the standard CPS assessment algorithm (CPS≥5 = high; CPS &lt;5 = low). CTx responses were assessed according to Mandard Tumour Regression Grade (TRG). For each immune cell marker and T cell phenotype, high vs low groups were defined using median cell density (count/mm2) as the cut-off. Association of high vs low cell density with disease free survival (DFS) was assessed using the log-rank test and correlation with pathological response to CTx was assessed using the Chi-squared test. The paired Wilcoxon rank-sum test was used to compare differences in immune cell densities between paired pre-CTx biopsy and post-CTx resection samples in a subset of 35 pts. Results: Pts with a PD-1+ cell density above median in biopsy had significantly longer DFS compared to those with a density below median (median DFS not reached vs 1111 days; HR 0.3, 95% CI 0.1 – 0.8; p=0.028). A trend towards longer survival was also observed in the CD8+ high, FOXP3+ high, and activated CD8+ (CD8+ PD-1+) high groups. There was no association between PD-L1 CPS, CD4+, CD45RO+, memory CD8+ (CD8+ CD45RO+), regulatory CD4+ (CD4+ FOXP3+), memory CD4+ (CD4+ FOXP3- CD45RO+), and activated CD4+ (CD4+ FOXP3- PD-1+) cells and survival. Pts with higher density of PD-1+ cells achieved better responses than those with low PD-1+ cells (TRG1/2 48% vs 9%, p=0.017). Pts with PD-L1 CPS ≥10 in biopsy were numerically more likely to achieve better responses to FLOT (46% TRG1/2). PD1+ cell density decreased in resection specimens in good CTx responders but did not differ in non-responders. FOXP3+ cells significantly decreased whereas CD45RO+ cells and memory CD8+ cells significantly increased in non-responders only. Conclusions: These results indicate prognostic and predictive value of PD-1 expressing tumour infiltrating T cells in resectable OGA treated with perioperative FLOT CTx. CD8+, activated CD8+ and FOXP3+ cells were associated with a trend in longer survival. This analysis provides candidate biomarkers for further study in larger cohorts on the association between infiltrating immune cells and prognosis as well as response to standard cytotoxic CTx in Western patients.

Validation of E1L3N antibody for PD-L1 detection and prediction of pembrolizumab response in non-small-cell lung cancer

BackgroundThe programmed death-ligand 1 (PD-L1) 22C3 assay is one of the approved companion diagnostic assays for receiving anti-programmed cell death ligand 1 (PD-L1) therapy. Our study evaluated the performance of E1L3N and 22C3 antibodies in estimating PD-L1 expression in non-small cell lung cancer (NSCLC).MethodsOur retrospective study included 46 patients diagnosed with unresectable EGFR/ALK/ROS1-negative NSCLC who received first-line pembrolizumab therapy between 2018 and 2021. PD-L1 immunohistochemistry of baseline tissue biopsy samples was performed using PDL1-E1L3N and PDL1-22C3 antibodies. The concordance between the PD-L1 assays and the treatment outcomes was assessed.ResultsUsing a tumor proportion score (TPS) cutoff of ≥1%, 67.4% of patients are evaluated to be positive using PDL1-E1L3N and 73.9% using PDL1-22C3. Using a TPS of ≥50% as the cutoff, 26.1% of patients are positive using PDL1-E1L3N and 30.4% using PDL1-22C3. The PDL1-22C3 and PDL1-E1L3N assays are highly concordant and reveal a correlation coefficient of 0.925 (p < 0.0001). Patients with PDL1-E1L3N TPS > 50% have a significantly higher objective response rate than patients with PDL1-E1L3N TPS < 1% (p = 0.047), with a similar trend observed for PDL1-22C3 (p = 0.051). Consistent with PDL1-22C3, patients with higher PDL1-E1L3N expression (≥50%, 1–49%) have longer progression-free survival than those with PDL1-E1L3N TPS < 1%.ConclusionOur study provides clinical evidence on the concordance of PD-L1 TPS scores between clones E1L3N and 22C3. Moreover, the treatment responses to pembrolizumab are also comparable between the PDL1-E1L3N and PDL1-22C3. These findings indicate that E1L3N is a reliable and cost-effective assay and may serve as an alternative to 22C3.

Biomarkers expression among paired serous ovarian cancer primary lesions and their peritoneal cavity metastases in treatment-naïve patients: A single-center study.

BackgroundHigh‐grade serous ovarian carcinoma (HGSOC), the most common histologic subtype of ovarian epithelial cancer, is associated with treatment resistance, enhanced recurrence rates, and poor prognosis. HGSOCs often metastasize to the peritoneal cavity, while fluid cytology examination could identify such metastases. This retrospective study aimed to identify potential biomarker discrepancies between paired HGSOC primary tissues and metastatic peritoneal fluid cytology samples, processed as cell blocks (CBs).MethodsTwenty‐four pairs of formalin‐fixed, paraffin‐embedded primary tissues and metastatic CBs from an equal number of treatment‐naïve patients were used, and immunohistochemistry (IHC) for epidermal growth factor receptor (EGFR), human epidermal growth factor receptor, programmed cell death‐1 ligand 1 (PD‐L1), and CD147 was applied.Results13/24 pairs showed discordant EGFR IHC results; in all these 13 patients, EGFR was positive (≥1+ membranous staining intensity found in at least 10% of the cancer cells) in the peritoneal, yet negative in the primary tissue samples. Notably, EGFR IHC was positive in 15/24 of the metastatic, whereas in just 2/24 of the primary HGSOC samples (p < 0.001). Although most PD‐L1 results were concordant, 5/24 and 6/24 pairs exhibited discordant results when stained with the E1L3N and 22C3 clones, respectively. Lastly, CD147 overexpression was found more often in the metastatic rather than the matched primary HGSOCs stained with CD147, though the difference was not significant.ConclusionsCytology from effusions could be considered for biomarker testing when present, even when tissue from the primary cancer is also available and adequately cellular, as it could provide additional information of potential clinical significance.

Abstract 2678: Tumoral CXCR4 and CXCL12 but not CXCR7 nor PD-L1 expression predicts disease free survival (DFS) and cancer specific survival (CSS) in resected pancreatic adenocarcinoma patients

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumor types and the 4th leading cause of tumor-related mortality in developed nations. PDAC is characterized by a highly fibrotic stroma that can physically exclude cytotoxic T cells from the vicinity of tumor cells. Targeting the axis CXCR4-CXCL12 improved T cell access to PDAC Tumor Microenvironment (TME) making tumors more susceptible to anti-PD-1/anti PD-L1 therapy. With the intent to evaluate the role of the entire CXCR4-CXCL12-CXCR7 axis in a context of PD-1/PD-L1 expression, 80 primary pancreatic cancers were evaluated in cancer cells and TME. Methods: 80 primary PDAC samples from patients undergone surgery collected at Ospedale P. Pederzoli Peschiera del Garda (Verona, Italy) were included in the study. Immunohistochemistry was conducted using primary antibodies: Anti-CXCR7/RDC-1 clone 11G8; Anti-CXCL12 Clone #79018; two commercial anti-CXCR4 clones #44716 and the UMB-2; two commercial anti-PD-L1, clone E1L3N and 22C3. Results: CXCR4 was expressed in 53 out of 73 tumors (72.6%). CXCL12 was predominantly identified into the membrane of cancer cells in 16 out of 75 (21.3%) tumors. CXCR7 staining was detectable in 44 out of 76 (57.8%) tumors. PD-L1 identified into the membrane of cancer cells in 35 out of 70 (50%) tumors. Patients with high CXCR4 tumor expression experienced worse outcome (DFS: 11.77 vs 22.85 months p=0.0071; CSS: 21.83 vs 35 months p&amp;gt;0.05, not significant) and predicted short DFS in R0 patients subgroup (n=48) (DFS: 11.20 months vs 33.40 months p=0.0140). CXCL12 expression predicted DFS (p=0.0002) and CSS (p=0.0046). Of note CXCL12 predicted short DFS (p=0.0015) and CSS (p=0.0213) in R0 patient's subgroup (n=48). Although stromal expression of CXCR4, CXCL12 and CXCR7 were not prognostic, CXCR4 positive inflammatory cells and tumoral CXCL12 significantly and directly correlated with the vascular invasion. Neither tumoral nor stromal expression of PD-L1 was prognostic but PD-L1 interestingly stained the perineural invasion and tumor-infiltrating inflammatory cells of body or tail tumors. In multivariate analysis, tumoral CXCR4 expression (p=0.0043), perineural invasion (p=0.0030) and AJCC lymph node status (p=0.0016) were independent prognostic factors for DFS. Tumoral CXCL12 expression (p=0.01467), AJCC Stage (p=0.0004) and perineural infiltration (p=0.03164) were independent prognostic factors for CSS. Conclusion: Tumoral CXCR4 and CXCL12 were prognostic factors at the univariate and multivariate analysis as for DFS and CSS in PDAC. Stromal PD-L1 stained perineural infiltration and stromal CXCR4 characterized inflammatory cells in the vascular invasion. Thus tumoral and stromal characterization for CXCR4 and CXCL12 in PDAC is worth to identify prognostic categories and to orient therapeutic approach. Citation Format: Crescenzo D'Alterio, Alessandro Giardino, Giosuè Scogmaliglio, Fabiana Tatangelo, Giovanni Butturini, Luigi Portella, Giuseppe Guardascione, Isabella Frigerio, Stefano Gobbo, Gerardo Botti, Stefania Scala. Tumoral CXCR4 and CXCL12 but not CXCR7 nor PD-L1 expression predicts disease free survival (DFS) and cancer specific survival (CSS) in resected pancreatic adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2678.

Fusion 3D gross sampling method to overcome heterogeneity in clear cell renal cell carcinoma (ccRCC) and grading angiogenic versus immune signatures.

e16565 Background: Grading tissue signatures in ccRCC is a potential tool to improve patients’ selection for anti-angiogenic drugs and immunotherapies. After the molecular audit to the TCGA public platforms, we aimed to grade angiogenic and immune signatures in ccRCC using a new 3D next-generation gross sampling method to overcome intratumoral heterogeneity. Methods: 100 consecutive advanced ccRCCs (≥pT3a) were sampled. Paraffin-embedded blocks were obtained after mapping the position of each sample to the whole tumor, to allow the reconstruction of the entire 3D tumor mass ( fusion 3D). Multisite tumor sampling was performed to analyze the whole tumor. TCGA platforms were assessed for angiogenic and immune molecular signatures: CD31 and CD34 to evaluate the absolute count and density of vessels, while E1L3N and sp263 clones for PD-L1 expression in tumor cells (TCs). The digital analysis was performed with image processing: comparing each tissue block to whole 3D assessment, the coefficient of variation (CV) was the statistical measure of the dispersion of data points in the data series around the mean. CV &lt; 0.2 defined the homogeneity of the assessment. Results: 656 gross photographs representative of the 3D tumoral masses were collected and 4231 paraffin blocks and tissue sections were stored. Matching gross photographs with tissue samples was performed. 6324 tissue cores were evaluated after combining standard routine sampling plus the 3D multisite sampling and tissue microarray cores. Only 18% of cores displayed homogeneous profile of angiogenesis (CV &lt; 0.2) with two distinct patterns: homogeneous high level of angiogenesis (pattern A) (10% of cases) or homogeneous low level of angiogenesis (pattern B) (8% of cases). The heterogeneous profile of angiogenesis was more frequent than the homogeneous one and was characterized by zones with high and low density of angiogenesis (82% of cases) (pattern C). On the other hand, the homogeneity of PD-L1 expression was more frequently observed both as diffuse absence ( &lt; 1% of TCs, grade 0) or high expression (≥50% of TCs, grade 2) compared to low PD-L1 expression (1-49% of TCs, grade 1) (60% plus 7% versus 33% of cases, respectively). After the comparison of grading angiogenic versus immune signatures, we observed that cases with low PD-L1 expression (grade 0/1) usually expressed high density of angiogenesis (pattern A). Conclusions: Grading angiogenic (pattern A, B and C) versus immune (grade 0, 1 and 2) signatures in ccRCCs can be performed using commonly available tissue vascular (CD31 or CD34) and immune (PD-L1) antibodies. We promoted a simple assay to perform fusion 3D gross sampling to reduce at minimum the bias of heterogeneity in RCCs analyses. Both angiogenic versus immune signature by using the grading systems may help treatment decision-making and response assessment in ccRCC patients.

PD-L1 expression with QR1 and E1L3N antibodies according to histological ovarian cancer subtype: A series of 232 cases.

Therapeutic strategies for epithelial ovarian cancers are evolving with the advent of immunotherapy, such as PD-L1 inhibitors, with encouraging results. However, little data are available on PDL-1 expression in ovarian cancers. Thus, we set out to determine the PD-L1 expression according to histological subtype. We evaluated the expression of two PD-L1 clones – QR1 and E1L3N – with two scores, one based on the percentage of labeled tumor cells (tumor proportion score, TPS) and the other on labeled immune cells (combined proportion score, CPS) in a consecutive retrospective series of 232 ovarian cancers. PD-L1 expression was more frequent in high grade serous carcinoma (27.5% with E1L3N clone and 41.5% with QR1 clone), grade 3 endometrioid carcinoma (25% with E1L3N clone and 50% with QR1 clone), and clear-cell carcinomas (27.3% with E1L3N clone and 29.6% with QR1 clone) than other histological subtypes with CPS score. Using the CPS score, 17% of cases were labeled with E1L3N vs 28% with QR1. Using the TPS score, 14% of cases were positive to E1L3N vs 17% for QR1. For TPS and CPS, respectively, 77% and 78% of the QR1 cases were concordant with E1L3N for the thresholds of 1%. Overall and progression-free survival between PD-L1 positive and PD-L1 negative patients were not different across all histological types, and each subtype in particular for serous carcinomas expressing PD-L1. Expression of PD-L1 is relatively uncommon in epithelium ovarian tumors. When positive, usually <10% of tumor cells are labeled. QR1 clone and CPS appear the best tools to evaluate PD-L1 expression.

PD-L1 Expression after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancers Is Associated with Aggressive Residual Disease, Suggesting a Potential for Immunotherapy

Simple SummaryImmune checkpoint inhibitors (ICI) are now part of the therapeutical arsenal for cancers at several sites and in several settings. PD-L1 expression is assessed to predict treatment response. We used immunohistochemistry (E1L3N clone) to assess PD-L1 expression on tumor and immune cells from a cohort of 89 surgical specimens of T1-T3NxM0 triple-negative breast cancers (TNBC) from patients treated with neoadjuvant chemotherapy (NAC) with residual disease. PD-L1 expression levels were low in tumor and immune cells from post-NAC surgical specimens. PD-L1 positivity in tumor cells was significantly associated with aggressive post-NAC tumor characteristics. A small subset of TNBC patients displaying PD-L1 expression in the context of a more extensive post-NAC tumor burden could benefit from ICI treatment after NAC.The consequences of neoadjuvant chemotherapy (NAC) for PD-L1 activity in triple-negative breast cancers (TNBC) are not well-understood. This is an important issue as PD-LI might act as a biomarker for immune checkpoint inhibitors’ (ICI) efficacy, at a time where ICI are undergoing rapid development and could be beneficial in patients who do not achieve a pathological complete response. We used immunohistochemistry to assess PD-L1 expression in surgical specimens (E1L3N clone, cutoff for positivity: ≥1%) on both tumor (PD-L1-TC) and immune cells (PD-L1-IC) from a cohort of T1-T3NxM0 TNBCs treated with NAC. PD-L1-TC was detected in 17 cases (19.1%) and PD-L1-IC in 14 cases (15.7%). None of the baseline characteristics of the tumor or the patient were associated with PD-L1 positivity, except for pre-NAC stromal TIL levels, which were higher in post-NAC PD-L1-TC-positive than in negative tumors. PD-L1-TC were significantly associated with a higher residual cancer burden (p = 0.035) and aggressive post-NAC tumor characteristics, whereas PD-L1-IC were not. PD-L1 expression was not associated with relapse-free survival (RFS) (PD-L1-TC, p = 0.25, and PD-L1-IC, p = 0.95) or overall survival (OS) (PD-L1-TC, p = 0.48, and PD-L1-IC, p = 0.58), but high Ki67 levels after NAC were strongly associated with a poor prognosis (RFS, p = 0.0014, and OS, p = 0.001). A small subset of TNBC patients displaying PD-L1 expression in the context of an extensive post-NAC tumor burden could benefit from ICI treatment after standard NAC.

Relevance of PD-L1 Non-Coding Polymorphisms on the Prognosis of a Genetically Admixed NSCLC Cohort.

PurposeAlthough non-small cell lung cancer (NSCLC) remains a deadly disease, new predictive biomarkers have emerged to assist in managing the disease, of which one of the most promising is the programmed death‐ligand 1 (PD-L1). Each, PD-L1 variant seem to modulate the function of immune checkpoints differently and affect response to adjuvant treatment and outcome in NSCLC patients. We thus investigated the influence of these PD-L1 genetic variations in genetically admixed NSCLC tissue samples, and correlated these values with clinicopathological characteristics, including prognosis.Materials and MethodsWe evaluated PD-L1 non-coding genetic variants and protein expression in lung adenocarcinomas (ADC), squamous cell carcinomas (SqCC), and large cell carcinomas (LCC) in silico. Microarray paraffin blocks from 70 samples of ADC (N=33), SqCC (N=24), and LCC (N=13) were used to create PD-L1 multiplex immunofluorescence assays with a Cell Signaling E1L3N clone. Fifteen polymorphisms of the PD-L1 gene were investigated by targeted sequencing and evaluated in silico using dedicated tools.ResultsAlthough PD-L1 polymorphisms seemed not to interfere with protein expression, PD-L1 expression varied among different histological subtypes, as did clinical outcomes, with the rs4742098A>G, rs4143815G>C, and rs7041009G>A variants being associated with relapse (P=0.01; P=0.05; P=0.02, respectively). The rs7041009 GG genotype showed a significant correlation with younger and alive patients compared to carriers of the A allele (P=0.02 and P<0.01, respectively). The Cox regression model showed that the rs7041009 GG genotype may influence OS (P<0.01) as a co-dependent factor associated with radiotherapy and recurrence in NSCLC patients. Furthermore, the Kaplan–Meier survival curves showed that rs7041009 and rs4742098 might impact PPS in relapsed patients. In silico approaches identified the variants as benign.ConclusionPD-L1 non-coding variants play an important role in modulating immune checkpoint function and may be explored as immunotherapy biomarkers. We highlight the rs7041009 variant, which impacts OS and PPS in NSCLC patients.

High concordance of programmed death-ligand 1 expression with immunohistochemistry detection between antibody clones 22C3 and E1L3N in non-small cell lung cancer biopsy samples.

BackgroundThe detection of programmed death-ligand 1 (PD-L1) expression can enrich for patients who respond to anti-programmed cell death 1 (PD-1)/PD-L1 therapies. Though, for most laboratories, the cost of PD-L1 22C3 pharmDx is prohibitive for widespread use, whereas the laboratory-developed test (LDT) PD-L1 E1L3N antibody clone is widely available and inexpensive. This study aims to explore the analytical performance of E1L3N on the Dako Autostainer Link-48 platform and further evaluate the concordance of E1L3N and 22C3 expression in non-small cell lung cancer (NSCLC) biopsy samples.MethodsA total of 171 NSCLC biopsy samples were utilized in this study. Cases with less than 100 tumor cells were excluded. Serial sections of representative blocks were used for immunohistochemistry (IHC) staining. The staining protocol was performed according to the standard PD-L1 IHC 22C3 pharmDx package. PD-L1 staining on the tumor cell membrane was detected by immunofluorescence.ResultsAt a 1% cutoff value, PD-L1 was positive in 46.2% of patients using clone 22C3 and 42.1% of patients using E1L3N assays. At a 50% cutoff value, PD-L1 was positive in 16.4% of patients using clone 22C3 and 15.2% of the patients using E1L3N assays. Cohen’s kappa was used to evaluate the concordance of the PD-L1 expression between clone 22C3 and E1L3N. The kappa values were 0.893 [95% confidence interval (CI): 0.826–1] at the 1% cutoff and 0.868 (95% CI: 0.764–1) at the 50% cutoff. An evaluation of the intraclass correlation coefficients (ICCs) between the antibodies was used to quantify the interassay variability for PD-L1 expression in tumor cells. ICCs showed high concordance between the two antibodies (0.955, 95% CI: 0.939–0.967). Cohen’s kappa was also used to assess the consistency of the PD-L1 evaluation between two pathologists. The kappa values were 0.941 and 0.912 at the 1% cutoff, and 0.904 and 0.909 at the 50% cutoff for clone 22C3 and E1L3N expression, respectively.ConclusionsThe results indicated that the clone E1L3N assay has a high concordance with 22C3. The PD-L1 clone E1L3N assay is reliable and cost-effective, and could be used as a primary screening agent for PD-L1 IHC staining in pathological laboratories, especially in a research setting.

PD-L1 expression is an unfavourable prognostic indicator in Asian renal cell carcinomas

Background/aimsThe programmed cell death receptor 1 (PD-1) checkpoint inhibitor, nivolumab, has been approved for the treatment of metastatic renal cell carcinoma (RCC). However, the understanding of the expression and distribution...

Clinicopathological associations and prognostic values of IDH1 gene mutation, MGMT gene promoter methylation, and PD-L1 expressions in high-grade glioma treated with standard treatment

Introductionthe objective was to evaluate the impact of IDH1 R132H mutation, MGMT methylation and PD-L1 expression in high grade glioma that received standard therapy (surgery, radiation and chemotherapy) to overall survival (OS).Methodsthis is a retrospective study of 35 high grade glioma cases. Genotyping of IDH1 gene alteration on the mutation hotspot R132 (Sanger sequencing method with Applied Biosystems 3500 Genetic Analyzer), EZ DNA Methylation-Gold kit (Zymo Research) is used to study the methylation, Cell line BT549 (ATCC HTB-122) and HCT-116 (ATCC CCL-247) were used as unmethylated control and partially methylated control respectively. Anti-human PD-L1 antibody clone E1L3N®from Cell Signalling Technology (USA) and Rabbit XP®were used to see PDL-1 expression.Resultsanaplastic astrocytoma cases had more MGMT promoter methylation (50%) than glioblastoma multiforme (GBM) (20%), more IDH1 R132H mutation (42%) than GBM (4.3%). Immunohistochemistry tumor proportion score method (TPS) identified 17% and 8.7% were PD-L1 positive in AA and GBM groups, respectively. Cases with IDH1 R132H mutation and MGMT methylation still showed better OS although with high PD-L1 expression.ConclusionIDH1 R132H mutation and MGMT methylation were good prognostic markers. High expression of PD-L1 apparently might not indicate poor overall survival in the presence of IDH1 R132 mutation and MGMT methylation.

From This Month's Histopathology

From This Month's Histopathology

Tumour immune microenvironment in primary and metastatic papillary renal cell carcinoma.

Among renal cell carcinoma (RCC) the tumour immune microenvironment has been best characterised in clear cell RCC. In this study we investigated the expression of several immune markers, including PD-L1, FoxP3 and CD8 in primary and metastatic papillary RCC. Three tissue microarrays were constructed from 78 cases with primary papillary RCC and paired metastatic tumour (24 cases) from 78 patients treated between 1982 and 2014. Immunohistochemistry analysis was performed using commercially available antibodies for PD-L1 (clone E1L3N), FoxP3, CD8 and Ki-67. Markers expression level in tumour and/or associated immune cells was analysed by tissue type (non-tumour versus primary tumour versus metastatic tumour) and correlated to clinicopathological features and outcome. We found PD-L1 expression in up to one-quarter of primary and metastatic papillary RCC. On univariate analysis, CD8/FoxP3 ratio >1 was associated with favourable outcome, whereas papillary RCCs with high numbers of dual CD8/Ki-67-positive lymphocytes showed an increased likelihood for tumour progression and overall and cancer-related mortality. The association of CD8/FoxP3 ratio >1 and high count of CD8/Ki-67 with outcome remained significant on multivariate analysis when adjusting for stage, grade and patient's age.

P2.09-31 High Concordance of PD-L1 Antibody Between Clone 22C3 and Clone E1L3N in Non-Small Cell Lung Cancer Biopsy Samples

P2.09-31 High Concordance of PD-L1 Antibody Between Clone 22C3 and Clone E1L3N in Non-Small Cell Lung Cancer Biopsy Samples

Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies

Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.