Staining Platform Research Articles

Comparison of MIB‐1‐Specific Membrane Staining in Hyalinising Trabecular Tumor Using Mainstream Automated Immunohistochemical Staining Platforms

ABSTRACTBackgroundMIB‐1, a monoclonal antibody against Ki‐67, exhibits specific membrane staining in the immunohistochemistry of hyalinising trabecular tumor (HTT). This specific staining pattern is crucial in diagnosing HTT. Although manual immunohistochemical staining remains the established method for MIB‐1 staining, this process is complicated, inconsistent, and prone to false negatives.MethodsThis study aimed to explore whether the classical reaction pattern can be replicated by utilizing the current mainstream automated immunohistochemical staining platforms. Furthermore, we examined the effect of different conditions on staining efficiency and their value in clinical diagnosis assistance.ResultsSpecimens obtained from eight and six cases of HTT and non‐HTT, respectively, from a single center were stained using the manual staining method and the Dako Autostainer Link 48 (AS48), Dako Omnis, Ventana BenchMark ULTRA, and Leica BOND‐III automated immunohistochemical staining platforms. The Autostainer Link 48 was found to be the most stable staining platform, while the BenchMark ULTRA with primary antibody incubation at room temperature (RT) and the Omnis platform with antigen retrieval at pH 9.0 were able to reproduce membrane‐positive staining for MIB‐1 in the HTT specimens.ConclusionsOur results offer crucial reference value for clinical diagnostic assistance.

Human epidermal growth factor receptor 2 (HER2) status in breast cancer: practice points and challenges.

Human epidermal growth factor receptor 2 (HER2)-enriched breast cancer benefits significantly from anti-HER2 targeted therapies. This highlights the critical need for precise HER2 immunohistochemistry (IHC) interpretation serving as a triage tool for selecting patients for anti-HER2 regimens. Recently, the emerging eligibility of patients with HER2-low breast cancers for a novel HER2-targeted antibody-drug conjugate (T-DXd) adds challenges to HER2 IHC scoring interpretation, notably in the 0-1+ range, which shows high interobserver and interlaboratory staining platform variability. In this review, we navigate evolving challenges and suggest practical recommendations for HER2 IHC interpretation.

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 &PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast–like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 &PTEN mutated BCs and precursor lesions.

Abstract 3797: Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis

Abstract Spatial biology techniques have revolutionized our approach towards the study of the tumor microenvironment (TME) and its complex cellular interplay. On the one hand, multiplex immunofluorescence (mIF) methods have enabled a precise profiling of immune cells and other key cellular players of the TME while uncovering their spatial distribution and interactions. On the other hand, in situ hybridization (ISH) technologies have shown to provide complementary information to protein profiling such as the mapping of cytokine- and chemokine-expressing cells, essential for comprehending signaling networks and immune activation statuses. Here, we made use of a novel multiomics approach that combines these two biological inputs by integrating RNAscope™ and sequential immunofluorescence (seqIF™) protocols to achieve same-slide co-detection of RNA and protein targets. The combined workflow is automated on COMET™, an advanced tissue staining and imaging platform. Through precise control over temperature and reagent distribution, the instrument ensures maximum efficiency and reproducibility of the assays. The integrated multiomics protocol allows for up to three RNAscope™ detection cycles combined with twelve seqIF™ cycles, for a final 12-plex RNA + 24-plex protein panel. We first showed the capacity of COMET™ platform to fully automate the RNAscope™ protocol and demonstrated its sensitivity and specificity with the analysis of positive and negative control genes on HeLa cell pellets, highlighting the flexibility of sample selection. To illustrate the potential multiomics in unravelling the complexity of the TME, we designed a panel of 12-RNA-targeting probes for key biomarkers of tumor-infiltrating lymphocytes and their activation status, including multiple secreted molecules (e.g., cytokines and proteases). This was combined with a 24-antibody panel for the detection of protein biomarkers, selected to enable the single-cell profiling of different players within the TME. We applied this panel to characterize different human FFPE tumor tissues and showed that the co-detection of RNA and protein biomarkers on the same slide allows a better characterization of key cellular components involved in tumor progression and immune response. Our results highlight the potential of spatial multiomics technologies in enhancing the efficiency of investigations of immune cell behavior and generally the understanding of cellular interplay in the TME. Their full automation on a platform like COMET™ will accelerate analysis and increase robustness by minimizing user intervention, and ultimately helping in the development of prognostic and predictive biomarkers, in the refinement of cancer diagnoses, and in the selection of new personalized therapies. Citation Format: Paula Juricic, Arec Manoukian, Alice Comberlato, Pino Bordignon, Alix Failletaz, Anushka Dikshit, Emerald Doolittle, Rose Delvillar, Steve Zhou, Ching-wei Chang, Li-chong Wang, Saska Brajkovic, Maithreyan Srinivasan, Diego Dupouy. Unveiling the spatial dynamics of the tumor microenvironment: Integrated RNA and protein profiling on the same slide through automated spatial multiomics analysis [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 3797.

Abstract 3755: Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b

Abstract Background: Fibroblast growth factor receptor 2b (FGFR2b) is part of the FGFR transmembrane tyrosine kinase receptor family. This protein has gained more attention in recent years as a tumor progression biomarker in numerous solid tumors. The detection of FGFR2b by immunohistochemistry (IHC) has been used recently in immunotherapy clinical trials with multiple tumor types. During IHC assay development on formalin-fixed paraffin embedded tissue, a thorough study of antigen retrieval through cell conditioning or enzyme incubation, primary antibody incubation, and detection parameters is critical to evaluate staining performance. Design: A study of an investigational anti-FGFR2b (FPR2-D) assay developed on a BenchMark ULTRA automated staining platform explored the effect of antigen retrieval and detection parameters on staining performance. VENTANA System Software was used to modify steps in the IHC staining protocol resulting in 42 permutations. Ten different solid tumors were stained with these modifications to the assay and subsequently evaluated for FGFR2b expression. Result:s The results demonstrate that the staining clarity, intensity, and dynamic range within tissue cases of FGFR2b staining can vary widely between different solid tumor types. While most permutations of the initial assay parameters had minimal impact on the staining performance and readability by a pathologist, the order of the enzyme treatment and the cell conditioning steps (in addition to increased incubation time of primary antibody) had a significant impact when comparing across different solid tumors. Detection of FGFR2b seemed to have a greater susceptibility by tumor type to the antigen retrieval process than has traditionally been observed in other biomarkers. Conclusion: The overall results of this investigation show the importance of an extensive parameter screening approach when evaluating new antibody targets and indication combinations in IHC to ensure optimal staining performance. This is critical to downstream testing and the potential use of new assays in the context of in vitro diagnostics. Citation Format: Autumn Sky Watson, Catherine Le, Anne M. Wertheimer, Spencer Escobedo, Lisa So, Bhavani Bagevalu Siddegowda, John D. Palting, Susan Marion, Steven P. Stratton, Birte Aggeler. Impact of antigen retrieval and other parameters on detection of FGFR2b in various solid tumors on a BenchMark ULTRA automated staining platform: Development of a new IHC assay for FGFR2b [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 3755.

Validation and Implementation of OptiView and EnVision FLEX Detection Systems for Immunocytochemical Staining Protocols of the Ten Most Commonly Used Diagnostic Markers in Routine Cytopathological Practice.

The withdrawal of the iView detection system (iV) forced many cytopathology laboratories, including ours, to substitute immunocytochemical (ICC) staining protocols for routine practice with other detection systems. Our objective was to optimize, validate, and implement ICC protocols using OptiView (OV) and EnVision FLEX (EnV) detection systems, comparing the results with those obtained using iV. Residual cytologic samples with known diagnoses were used, testing antibodies for the ten most common markers in routine cytopathology diagnostics (calretinin, Ber-EP4, MOC-31, CKAE1/AE3, CK5/6, CD68, LCA, desmin, HBME-1, and WT1). Different staining parameters were tested using OV on BenchMark ULTRA and EnV on Dako Omnis immunostainer, respectively. Optimal staining protocols were then selected and validated on 10 positive and 10 negative cases. The staining results were compared with iV protocols through evaluation of UK NEQAS and internal scores. The optimal staining protocols with OV and EnV demonstrated similar or superior results compared to the existing iV protocols, with slightly stronger intensity regarding positive cells. We have successfully established and validated optimal ICC staining protocols for commonly used markers in routine cytopathology practice. These protocols may benefit other laboratories using similar staining platforms. However, the challenge regarding standardizing ICC protocols across different cytopathology laboratories remains unresolved.

A Standardized and Reproducible Workflow for Membrane Glass Slides in Routine Histology and Spatial Proteomics

Defining the molecular phenotype of single cells in situ is key for understanding tissue architecture in health and disease. Advanced imaging platforms have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. Furthermore, high-precision laser microdissection (LMD) of tissue on membrane glass slides is a powerful method for spatial omics technologies and single-cell type spatial proteomics in particular. However, current histology protocols have not been compatible with glass membrane slides and LMD for automated staining platforms and routine histology procedures. This has prevented the combination of advanced staining procedures with LMD. In this study, we describe a novel method for handling glass membrane slides that enables automated eight-color multiplexed immunofluorescence staining and high-quality imaging followed by precise laser-guided extraction of single cells. The key advance is the glycerol-based modification of heat-induced epitope retrieval protocols, termed “G-HIER.” We find that this altered antigen-retrieval solution prevents membrane distortion. Importantly, G-HIER is fully compatible with current antigen retrieval workflows and mass spectrometry–based proteomics and does not affect proteome depth or quality. To demonstrate the versatility of G-HIER for spatial proteomics, we apply the recently introduced deep visual proteomics technology to perform single-cell type analysis of adjacent suprabasal and basal keratinocytes of human skin. G-HIER overcomes previous incompatibility of standard and advanced staining protocols with membrane glass slides and enables robust integration with routine histology procedures, high-throughput multiplexed imaging, and sophisticated downstream spatial omics technologies.

An Optimized Melanin Depigmentation Method for Histopathological and Immunohistochemical Staining of Formalin-Fixed Paraffin-Embedded Tissues.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3'-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues.

Abstract 4336: Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system

Abstract The PD-1/PD-L1 signaling pathway is essential for immune control and maintaining immune system balance. PD-1 and PD-L1 proteins exert most functions in cells and tissues by undergoing modifications and forming dynamic complexes – effects that cannot be explored by genomics, transcriptomics, or conventional immunostaining methods. Numerous cancer therapies are being developed that affect PD-1/PD-L1 signaling, and tools to study the PD-1/PD-L1 axis are, therefore, essential. One prerequisite for successfully establishing diagnostic assays is reproducibility, standardization, and, ideally, high sample throughput via automation. Here, we aimed to introduce a new technological solution to allow for the simultaneous detection of PD-L1 and PD-1 interaction and the adjacent immune cell context in tissue samples on the Lunaphore’s automated microfluidic staining platform. The assay for detecting PD-1-PD-L1 interaction was developed and technically validated by Navinci Diagnostics. Although the detection of interaction alone provided important information on the immune status in the tumor microenvironment, we believed that PD-1/PD-L1 readout would be most powerful in the context of its cellular ecosystem. Therefore, the PD-1/PD-L1 assay was combined with the analysis of immune and cancer cells to determine their involvement in this signaling. The feasibility of combining multiplex immunofluorescence with proximity ligation assay was conducted and confirmed on human tonsils. The spatial profiling panel included immune markers for all T-cells (CD3), helper T-cell (CD4), cytotoxic T-cells (CD8), B-cells (CD19/20) and activation/exhaustion markers granzyme B (activated cytotoxic T-cell), Ki67 (proliferating cells), and LAG3 (malignant B cells, CD8 T cells, CD4 Tregs). We evaluated, implemented, and optimized the assays on the Lunaphore staining instrument to reduce labor time and cost for the end users. In the next phase, the potential of our assay to be benchmarked as a diagnostic tool in clinical praxis will be thoroughly tested and validated on diagnostic tissue samples from NSCLC patient cohorts. We believe this approach will enable spatial and functional studies of the interface between the tumor and the immune system and provide necessary information about signaling pathway activation in situ, the latest representing a novel state-of-the-art in tissue diagnostics. Citation Format: Hampus Elofsson, Agata Zieba Wicher, Carolina Oses Sepulveda, Tony Ullman, Maria-Giuseppina Procopio, Alix Faillétaz, Diego G. Dupouy, Charlotte Stadler. Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system. [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 4336.

Abstract P6-04-07: Primary Diagnosis of Breast Biopsies supported by AI versus Microscope: Multi-Site Clinical Reader Study

Abstract Objective This study aimed to clinically validate the use of an AI-based solution by pathologists for the primary diagnosis of breast core needle biopsies as compared with the gold standard practice (review on the microscope). Methods A two-arm prospective reader study comparing the performance of pathologists using an AI-based solution with pathologists using a microscope was performed at two sites (different staining and digital scanners). Both arms were compared to ground truth (GT) established by the consensus of two breast pathologists. Rates of major discrepancies between each arm and GT, as determined by an adjudicating pathologist, were compared. Results Eight pathologists participated in the study and reported on 385 cases (442 HES and 330 H&E slides), each case being reported twice, once in each study arm. Pathologists first reviewed only H&E/HES slides, if requested and available, they were provided with IHCs, while the AI results were on H&E/HES only. The major discrepancy rates of the microscope arm and of the AI arm against GT were 4.42% and 3.12%, respectively, demonstrating a 29.4% reduction in major discrepancies. Pathologists with AI demonstrated very high accuracy for the detection of invasive carcinoma with sensitivity and specificity of 100% for both, as well as for DCIS/ADH with sensitivity of 92.4% and specificity of 97.8%. Conclusions This multi-site reader study reports diagnostic accuracy improvements by pathologists performing diagnosis and reporting with the support of a first read AI solution for breast biopsies. The AI solution performed accurately and generalized well for different staining platforms and different scanners. Thus, AI solutions could be used as significant aiding tools for pathologists in clinical decision-making in routine pathology practice, enhancing the quality and reproducibility of diagnosis. Citation Format: Anne Salomon, Alona Nudelman, Joanna Cyrta, Marina Maklakovski, Anat Albrecht Shach, Geraldine Sebag, Giuseppe Mallel, Ira Krasnitsky, Tali Feinberg, Manuela Vecsler, Judith Sandbank. Primary Diagnosis of Breast Biopsies supported by AI versus Microscope: Multi-Site Clinical Reader Study [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 P6-04-07.

Immunohistochemical detection of double-stranded RNA in formalin-fixed paraffin-embedded tissue.

Double-stranded RNA (dsRNA) is produced during most viral infections, and immunohistochemical detection of dsRNA has been proposed as a potential screening marker for viral replication. The anti-dsRNA monoclonal antibody clone 9D5 is more sensitive than the established clone J2 but has not been validated in formalin-fixed paraffin-embedded (FFPE) tissue. This study aimed to test and compare the performance of the anti-dsRNA monoclonal antibodies, 9D5 and J2, in FFPE tissue using an automated staining platform. Archived clinical tissue samples with viral infections (n= 34) and uninfected controls (n= 30) were examined. Immunohistochemical staining for dsRNA (9D5 and J2) and virus-specific epitopes was performed. 9D5 provided a similar staining pattern but a higher signal-to-noise ratio than J2. The following proportions of virus-infected tissue samples were dsRNA-positive: SARS-CoV-2 (5/5), HPV (6/6), MCV (5/5), CMV (5/6), HSV (4/6), and EBV (0/6). Also, 18 of 30 uninfected samples were dsRNA positive, and an association between fixation time and intensity was observed. However, signals in all samples were markedly reduced by pretreatment with dsRNA-specific RNAse-III, indicating a specific reaction. In conclusion, dsRNA can be demonstrated in most viral infections with immunohistochemistry in FFPE tissue but with low clinical specificity. The antibody clone 9D5 performs better than clone J2.

Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone.

An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics. OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA). Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako. New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics.

Indirect Clinical Validation of a Programmed Death-Ligand 1 Laboratory-Developed Test for Gastric/Gastroesophageal Junction Adenocarcinoma with 22C3 Antibody Concentrate.

The PD-L1 IHC 22C3 pharmDx used on the Dako Autostainer Link 48 (ASL48) staining platform is an established method for assessing programmed death-ligand 1 (PD-L1) expression in tumor tissue and determining patient eligibility for pembrolizumab treatment; however, the availability of this platform is limited in Europe and Asia. The aims of this study were to develop and optimize protocols for the PD-L1 22C3 antibody concentrate with multiple immunohistochemistry staining platforms and to validate these protocols using PD-L1 combined positive score (CPS) with a cut-off of ≥1 in gastric or gastroesophageal junction adenocarcinoma. The 22C3 antibody concentrate was tested and optimized protocols were developed for use with three staining platforms: Dako ASL48, Ventana BenchMark ULTRA, and Leica BOND-MAX. Tumor specimens (N = 120) from patients with gastric or gastroesophageal junction adenocarcinoma were used for the validation study; these specimens were evaluated independently by three pathologists for PD-L1 CPS as a continuous variable and using a cut-off of ≥1. PD-L1 IHC 22C3 pharmDx used on the Dako ASL48 platform served as the reference or gold standard. The intraclass correlation coefficient of CPS as a continuous variable between the gold standard and each staining platform assessed was 0.910-0.989. When CPS was dichotomized based on a cut-off of ≥1, depending on the pathologist and the platform used, positive percentage agreement was 81-99% and negative percentage agreement was 90-100%. Interobserver agreement using the gold standard showed substantial agreement (κ=0.779). The PD-L1 22C3 antibody concentrate can potentially be used with the laboratory-developed test on three commercially available immunohistochemistry staining platforms to determine PD-L1 expression in tumor samples from patients with gastric or gastroesophageal junction adenocarcinoma.

Clinical significance of cadherin-6 expression in primary and recurrent epithelial ovarian cancer and its association with outcomes: A potential therapeutic target for epithelial ovarian cancer (206)

<b>Objectives:</b> Cadherin-6 (CDH6) is a membrane glycoprotein, and aberrant expression of CDH6 has been reported in several human carcinomas, suggesting a possible role in metastasis and invasion. Recently, antibody-drug conjugates (ADCs) targeting CDH6 have drawn attention to new cancer therapies and demonstrated their antitumor activity in preclinical models of epithelial ovarian cancer (EOC). However, limited data are available regarding the frequency of CDH6 expression and its relationship to clinical factors in EOC. Our goal was to understand the clinical significance of CDH6 expression in EOC. <b>Methods:</b> We performed immunohistochemical (IHC) staining on CDH6 expression in a total of 232 EOC surgical samples (including 181 primary tumors and 51 recurrent tumors). CDH6 IHC was performed using the Leica BOND III staining platform with an anti-CDH6 antibody (HPA007456, ATLAS ANTIBODIES). The CDH6 IHC scoring standard refers to the HER2 detection guidelines for gastric cancer (0, 1+, 2+, 3+). We define a score greater than or equal to 1 as positive for the expression of CDH6. <b>Results:</b> We investigated 181 patients with primary EOC for CDH6 expression through IHC. Total 117 (64.6%) patients tested positive. In which, 18 (15.4%), 84 (71.8%) and 15 (12.8%) cases were scored as 1+, 2+ and 3+, respectively. We then investigated the relationship between CDH6 expression and clinical-pathologic features in EOC. CDH6 expression was observed more frequently in high-grade serous carcinomas (89.0%, p < 0.0001), stage III/IV tumors (84.8%, p < 0.0001) and gross residual tumors in primary surgery (84.7%, p < 0.0001). CDH6-positive patients showed shorter progression-free survival (PFS) and overall survival (OS) (p < 0.0001 and p=0.0006) than that of CDH6-negative patients in all EOC patients. In addition, patients with CDH6 3+ had shorter PFS and OS than that of patients who had 0, 1+ and 2+ (p =0.028 and p=0.015) in the high-grade serous subgroup. We also investigated the expression of CDH6 in 51 recurrent EOC; 38/51 (74.5%) recurrent tumors tested positive for CDH6 expression, and nine (23.7%), 17 (44.7%) and 12 (31.6%) received scores of 1+, 2+ and 3+, respectively. CDH6 expression from recurrent tumors was observed in 18/24 (75.0%) high-grade serous, 11/16 (68.8%) clear cell, 4/5 (80.0%) endometrioid and 5/6 (83.3%) other histotypes. <b>Conclusions:</b> CDH6 expression is frequently observed in primary and recurrent EOC of all histotypes, especially in high-grade serous carcinoma. The frequency of CDH6 expression is consistent between primary and recurrent tumors. CDH6 is a promising therapeutic target for the treatment of EOC.

Digital Image Analysis and Quantitative Bead Standards in Root Cause Analysis of Immunohistochemical Staining Variability: A Real-world Example.

Assessment of automated immunohistochemical staining platform performance is largely limited to the visual evaluation of individual slides by trained personnel. Quantitative assessment of stain intensity is not typically performed. Here we describe our experience with 2 quantitative strategies that were instrumental in root cause investigations performed to identify the sources of suboptimal staining quality (decreased stain intensity and increased variability). In addition, these tools were utilized as adjuncts in validation of a new immunohistochemical staining instrument. The novel methods utilized in the investigation include quantitative assessment of whole slide images (WSI) and commercially available quantitative calibrators. Over the course of ~13 months, these methods helped to identify and verify correction of 2 sources of suboptimal staining. One root cause of suboptimal staining was insufficient/variable power delivery from our building’s electrical circuit. This led us to use uninterruptible power managers for all automated immunostainer instruments, which restored expected stain intensity and consistency. Later, we encountered one instrument that, despite passing all vendor quality control checks and not showing error alerts was suspected of yielding suboptimal stain quality. WSI analysis and quantitative calibrators provided a clear evidence that proved critical in confirming the pathologists’ visual impressions. This led to the replacement of the instrument, which was then validated using a combination of standard validation metrics supplemented by WSI analysis and quantitative calibrators. These root cause analyses document 2 variables that are critical in producing optimal immunohistochemical stain results and also provide real-world examples of how the application of quantitative tools to measure automated immunohistochemical stain output can provide a greater objectivity when assessing immunohistochemical stain quality.

Program death ligand-1 immunocytochemistry in lung cancer cytological samples: A systematic review.

In this era of personalized medicine, targeted immunotherapies like immune checkpoint inhibitors (ICI) blocking the programmed death‐1 (PD‐1)/program death ligand‐1 (PD‐L1) axis have become an integral part of treating advanced stage non‐small cell lung carcinoma (NSCLC) and many other cancer types. Multiple monoclonal antibodies are available commercially to detect PD‐L1 expression in tumor cells by immunohistochemistry (IHC). As most clinical trials initially required tumor biopsy for PD‐L1 detection by IHC, many of the currently available PD‐1/PD‐L1 assays have been developed and validated on formalin fixed tissue specimens. The majority (>50%) of lung cancer cases do not have a surgical biopsy or resection specimen available for ancillary testing and instead must rely primarily on fine needle aspiration biopsy specimens for diagnosis, staging and ancillary tests. Review of the literature shows multiple studies exploring the feasibility of PD‐L1 IHC on cytological samples. In addition, there are studies addressing various aspects of IHC validation on cytology preparations including pre‐analytical (e.g., different fixatives), analytical (e.g., antibody clone, staining platforms, inter and intra‐observer agreement, cytology‐histology concordance) and post‐analytical (e.g., clinical outcome) issues. Although promising results in this field have emerged utilizing cytology samples, many important questions still need to be addressed. This review summarizes the literature of PD‐L1 IHC in lung cytology specimens and provides practical tips for optimizing analysis.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

The Immunohistochemical Expression of MCM-3, -5, and -7 Proteins in the Uterine Fibroids

Uterine fibroids are the most common mesenchymal uterine neoplasms; their prevalence is estimated in 40%–60% of women under 35 and in 70%–80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs—i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. Materials and methods: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. Results: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. Conclusions: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.

Abstract LB238: Multiplex immunohistochemistry profiling with UltraPlex IHC on FFPE lung cancer provides a fast and robust staining platform compatible with clinical laboratory workflows

Abstract A wide range of technologies are now available to perform multiplex immunofluorescence(mxIF) to examine the tumor immune microenvironment in FFPE tissue sections, offering the potentialto reveal signatures of anti-tumor immune response. However, mxIF assay performance is often limitedby autofluorescence and other artifacts, particularly in lung and prostate cancer samples. These artifactscan frustrate detection even using dedicated instrumentation to obtain images and advanced softwareto identify and measure the different biomarkers. Compared to immunofluorescence,immunohistochemistry (IHC) displays far less background and uses tools readily available in anypathology lab, but IHC has been traditionally difficult to multiplex especially regarding co-localization oncells. Recently, Cell IDx has overcome this barrier by developing UltraPlex™ multiplex IHC (mxIHC),where we leverage Cell IDx's antibody hapten-tagging technology to enable independent detection of 3-4 primary antibodies from any species on a single tissue section, using different chromogen colors todistinguish each biomarker. In this study of various NSCLC tumors, we display how mxIHC can define theTME while maintaining staining integrity. Using UltraPlex™ mxIHC, we demonstrate a useful clinicalchromogenic triple stain of CD8, PD-L1, and panCK with the ability to produce accurate quantitation forphenotype subsets. Our result show that UltraPlex™ mxIHC on FFPE tissue sections can provideincreased confidence for multiplexing on challenging tissues while requiring only conventionallaboratory methods and apparatus. Citation Format: Helen Tighe-Snyder, Matt Levin, Zohreh Akhananaghdam, Yanfei Jiang, David Schwartz. Multiplex immunohistochemistry profiling with UltraPlex IHC on FFPE lung cancer provides a fast and robust staining platform compatible with clinical laboratory workflows [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 LB238.