Variation In Staining Intensity Research Articles

PD-L1 Immunohistochemistry in Gastric Cancer: Comparison of Combined Positive Score and Tumor Area Positivity Across 28-8, 22C3, and SP263 Assays.

The clinical application of PD-L1 immunohistochemistry (IHC) testing is complicated by the availability of multiple IHC assays, scoring algorithms, and cutoffs. This study assessed the analytical comparability of three commercially available PD-L1 assays and two scoring algorithms used to assess PD-L1 status in gastric cancer (GC) samples. Serial sections of 100 resected GC samples, with PD-L1 expression levels across the dynamic range, were stained with three in vitro diagnostic-grade PD-L1 assays (28-8, 22C3, and SP263). Three trained pathologists blindly and independently scored slides using combined positive score (CPS) and tumor area positivity (TAP) algorithms. Comprehensive statistical analyses were performed to evaluate analytical concordance. Digital image analysis (DIA) was used to objectively compare the technical performance of each assay by simulating CPS and TAP. Comparable staining patterns were observed with these three PD-L1 assays. Despite discernible variation in staining intensity, reproducible evaluations of PD-L1 positivity were observed. Inter- and intra-assay assessments of all three assays, using either CPS or TAP and the same PD-L1 cutoffs, demonstrated moderate to almost-perfect (interassay Cohen's kappa [κ] range, 0.47-0.83) and substantial to almost-perfect (intra-assay κ range, 0.77-1.00) agreement. Interpathologist assessment exhibited a significant level of concordance (intraclass correlation coefficient ≥0.92). No difference in technical performance was observed using DIA. This study highlights analytical concordance in PD-L1 testing between three major PD-L1 assays when TAP and CPS are applied. Comparability of the technical assay performance was further supported by independent DIA. These observations support cross-application flexibility of the different PD-L1 assays and scoring algorithms to characterize PD-L1 expression in GC.

Oldies, but goldies-preserved morphology and stability of antigenic determinants in decades-old cryosections of human m. vastus lateralis.

Fibre typing by immunohistochemistry on cryosections from human skeletal muscle biopsies is an essential tool in the diagnosis and research of muscular diseases, ageing, and responses to exercise training and disuse. Preserving a good quality in these frozen specimens can be challenging especially if they are stored for longer periods before histological processing, which is often the case in studies with a large number of test subjects and/or repeated sampling separated by multiple years. We demonstrate in this article that both, the morphology and reactivity of epitopes to myosin heavy chain isoforms and dystrophin are well preserved in up to 18-year-stored unfixed and unstained cryosections of human m. vastus lateralis (n = 241). Any variation in staining intensity between samples was unrelated to the age of the biopsy donor or the storage period of the unstained cryosections, and in all cases, the obtained images were appropriate for image analysis, such as the determination of the fibre type composition and the fibre cross-sectional area, and quantitative analysis of muscle capillarisation.

A Real-World Experience from a Single Center (LPCE, Nice, France) Highlights the Urgent Need to Abandon Immunohistochemistry for ROS1 Rearrangement Screening of Advanced Non-Squamous Non-Small Cell Lung Cancer.

The detection of ROS1 rearrangements in metastatic non-squamous non-small cell lung carcinoma (NS-NSCLC) permits administration of efficient targeted therapy. Detection is based on a testing algorithm associated with ROS1 immunohistochemistry (IHC) screening followed by ROS1 FISH and/or next generation sequencing (NGS) to confirm positivity. However, (i) ROS1 rearrangements are rare (1-2% of NS-NSCLC), (ii) the specificity of ROS1 IHC is not optimal, and (iii) ROS1 FISH is not widely available, making this algorithm challenging to interpret time-consuming. We evaluated RNA NGS, which was used as reflex testing for ROS1 rearrangements in NS-NSCLC with the aim of replacing ROS1 IHC as a screening method. ROS1 IHC and RNA NGS were prospectively performed in 810 NS-NSCLC. Positive results were analyzed by ROS1 FISH. ROS1 IHC was positive in 36/810 (4.4%) cases that showed variable staining intensity while NGS detected ROS1 rearrangements in 16/810 (1.9%) cases. ROS1 FISH was positive in 15/810 (1.8%) of ROS1 IHC positive cases and in all positive ROS1 NGS cases. Obtaining both ROS1 IHC and ROS1 FISH reports took an average of 6 days, while obtaining ROS1 IHC and RNA NGS reports took an average of 3 days. These results showed that systematic screening for the ROS1 status using IHC must be replaced by NGS reflex testing.

High Interobserver Variability Among Pathologists Using Combined Positive Score to Evaluate PD-L1 Expression in Gastric, Gastroesophageal Junction, and Esophageal Adenocarcinoma

Reliable, reproducible methods to interpret programmed death ligand-1 (PD-L1) expression on tumor cells (TC) and immune cells (IC) are needed for pathologists to inform decisions associated with checkpoint inhibitor therapies. Our international study compared interpathologist agreement of PD-L1 expression using the combined positive score (CPS) under standardized conditions on samples from patients with gastric/gastroesophageal junction/esophageal adenocarcinoma. Tissue sections from 100 adenocarcinoma pretreatment biopsies were stained in a single laboratory using the PD-L1 immunohistochemistry 28-8 and 22C3 (Agilent) pharmDx immunohistochemical assays. PD-L1 CPS was evaluated by 12 pathologists on scanned whole slide images of these biopsies before and after a 2-hour CPS training session by Agilent. Additionally, pathologists determined PD-L1–positive TC, IC, and total viable TC on a single tissue fragment from 35 of 100 biopsy samples. Scoring agreement among pathologists was assessed using the intraclass correlation coefficient (ICC). Interobserver variability for CPS for 100 biopsies was high, with only fair agreement among pathologists both pre- (range, 0.45-0.55) and posttraining (range, 0.56-0.57) for both assays. For the 35 single biopsy samples, poor/fair agreement was also observed for the total number of viable TC (ICC, 0.09), number of PD-L1–positive IC (ICC, 0.19), number of PD-L1–positive TC (ICC, 0.54), and calculated CPS (ICC, 0.14), whereas calculated TC score (positive TC/total TC) showed excellent agreement (ICC, 0.82). Retrospective histologic review of samples with the poorest interpathologist agreement revealed the following as possible confounding factors: (1) ambiguous identification of positively staining stromal cells, (2) faint or variable intensity of staining, (3) difficulty in distinguishing membranous from cytoplasmic tumor staining, and (4) cautery and crush artifacts. These results emphasize the need for objective techniques to standardize the interpretation of PD-L1 expression when using the CPS methodology on gastric/gastroesophageal junction cancer biopsies to accurately identify patients most likely to benefit from immune checkpoint inhibitor therapy.

Oncogenic JAK2/STAT Signaling Molecules Activation Is Associated with PD-L1 Upregulation in Acute Myeloid Leukemia

Background: Increased evidence suggests the PD-1/PD-L1 axis is involved in the oncogenesis of myeloid neoplasms through promoting cancer immune escape, which suggests potential benefits by blockade of this axis in these neoplasms by immune checkpoint inhibitors. However, the ongoing clinical trials on PD-1/PD-L1 blocker in MDS/AML have reported controversial results, unlike the promising therapeutic efficacy shown in solid tumors. Identifying reliable biomarkers and scoring of PD-L1 expression and targeting the subgroup of patients who might benefit the most from PD-1/PD-L1 blockade are promising directions. Janus kinase 2 (JAK2) pathway activation has been shown to enhance PD-L1 expression in malignancies by in vitro studies. Here, we investigated PD-L1 expression associated with JAK2/STAT oncogenic pathway in AML patients using the combined positive score (CPS) algorithm. Methods: Immunohistochemical staining (IHC) for PD-L1 and pSTAT3 was performed on the marrow biopsies of 31 AML patients (39 cases), including 11 patients (17 cases) with JAK2/STAT mutation and 20 patients (22 cases) with wild-type JAK2/STAT. For PD-L1 IHC, the cases were evaluated for variable staining intensity (weak, moderate, strong) and membrane staining pattern (partial, complete) in tumor and mononuclear immune cells. The PD-L1 CPS was calculated by summing the number of all PD-L1-stained cells and dividing by the total number of tumor cells, multiplied by 100. The pSTAT3 was evaluated by percentage of positive tumor cells among the total tumor cells. The status of JAK2 / STAT mutations and other molecular abnormalities was evaluated by next generation sequencing (NGS) study. Results: Among patients of JAK2/STAT mutations, nine with JAK2 V617F exon 14 mutations, one with JAK2 I540-E543delinsRG exon 12 mutation and one with STAT5B N642H mutation. Evaluation of PD-L1 expression by IHC revealed four major cell staining patterns in our study cohort: 1. Negative (Figure 1A); 2. Partial weak membrane staining (Figure 1B, CPS<10); 3. Partial-complete, weak-moderate membrane staining (Figure 1C, CPS≥10); 4. Complete, moderate-strong membrane staining (Figure 1D, CPS≥10). Patterns 3-4 were classified as positive PD-L1 staining with a CPS cut-off of 10 (CPS≥10). Comparison of the PD-L1 CPS score in the JAK2/STAT wild-type versus the JAK2/STAT mutant groups revealed significantly higher PD-L1 CPS score in the JAK2/STAT mutant group (Figure 1K, P<0.0001, CPS≥10). One patient showed wild-type JAK2 at initial AML diagnosis. At two subsequent relapses, gain of JAK2 exon 12 (I540-E543delinsRG) mutation with increased VAF from 17% to 41% was detected. Interestingly, PD-L1 expression was correspondingly increased with CPS from 25 to 75 in these subsequent biopsies (Figure 1E, F). In another case with wild-type JAK2, STAT5B N642H mutation was detected and strong PD-L1 expression with CPS of 80 was obtained (Figure 1G). To address if JAK2/STAT3 axis is involved in PD-L1 upregulation, we assessed STAT3 phosphorylation by IHC in JAK2 wild-type and mutated groups. The results revealed significantly increased p-STAT3 expression in the JAK2 mutant compared with the wild-type cases, supporting the idea that oncogenic JAK2 mutation leads to STAT3 phosphorylation (Figure 1H-J). Furthermore, p-STAT3 expression showed a significant positive correlation with the PD-L1 CPS score (Figure 1H, r=0.8663, P<0.0001). Conclusion: In the AML patients with oncogenic JAK2 or its downstream signaling molecule STAT5B mutations, there is significantly enhanced PD-L1 expression. In addition, we proposed that the PD-L1 CPS positivity cut-off score of 10 being used as a quantitative measure of PD-L1 expression in this heterogeneous myeloid neoplasm. In the patient with relapsing AML, PD-L1 expression showed a positive correlation with increased JAK2 mutant VAF. Independent of the oncogenic JAK2 mutation, PD-L1 expression upregulation was also noted with STAT5B N642H mutation. Lastly, we demonstrated enhanced p-STAT3 expression in JAK2 mutant cases and a positive correlation between p-STAT3 and PD-L1 expression. Taken together, these data support the model that JAK2/STAT pathway activation is associated with PD-L1 upregulation in AML patients (Figure 1I), together with the proposed PD-L1 CPS cut-off of 10, can serve as scanning biomarkers to select relevant subgroups of AML patients for PD-1/PD-L1 immunotherapeutic study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Expression and clinical significance of Cathepsin K and MMPs in invasive non-functioning pituitary adenomas.

BackgroundCathepsin K (CTSK) is a protease that degrades type I collagen and extracellular matrix, thereby contributing to bone resorption and tumor invasion. Some pituitary adenomas (PAs) could invade the sphenoid sinus (SS) and cavernous sinus (CS).PurposeThis retrospective cohort study aimed to study the expression of tumoral biomarkers (CTSK, MMP9, MMP2, TIMP2, and PTTG1) and evaluate their clinical significance in non-functioning pituitary adenomas (NFPAs) with different invasion patterns.MethodsWe assessed the expression levels of candidate invasion-specific protein biomarkers CTSK, MMP9, MMP2, TIMP2, and PTTG1 by immunohistochemical staining in paraffin-embedded NFPA tumor tissues. Variations in staining intensity were analyzed in cases with SS and CS invasion and non-invasive NFPAs.ResultsWe found that the levels of CTSK were higher in PA cases with SS invasion than that in PA cases with CS invasion (95.57 ± 31.57 vs. 65.29 ± 29.64, P < 0.001), and the expression of MMP9 and MMP2 was higher in CS-invasive cases than that in SS-invasive cases (145.02 ± 49.25 vs. 111.80 ± 51.37, P = 0.002, and 138.67 ± 52.06 vs. 108.30 ± 41.70, P = 0.002). Multiple Cox regression demonstrated that higher CTSK expression (P=0.011), subtotal resection (P<0.001), invasion (P=0.037), and larger tumor diameter (P=0.001) were independent risk factors for recurrence. A positive correlation was observed between CTSK expression and tumor size (r=0.671, p<0.001). There was no significant difference in TIMP2 and PTTG1 levels between CS-and SS-invasive cases (97.42± 39.80 vs. 102.10± 43.22, P = 0.58 and 13.89 ± 4.59 vs. 12.56 ± 3.96, P = 0.14).ConclusionOur data indicated that CTSK has the potential as a marker for SS invasion of PAs, whereas MMP9 and MMP2 may be markers for CS invasion. And CTSK may play an important role in tumor relapse.

HERG1 Potassium Channel Expression in Colorectal Adenomas: Comparison with Other Preneoplastic Lesions of the Gastrointestinal Tract.

Preneoplastic lesions represent a useful target for early diagnosis and follow-up of gastrointestinal malignancies. hERG1 channel expression was tested by immunohistochemistry (IHC) in a cohort of colorectal adenoma samples belonging to Italian subjects. Overall, hERG1 was expressed in 56.5% of cases with both high staining intensity and a high percentage of positive cells. Moreover, hERG1 was expressed in a higher percentage of dysplastic adenomas with respect to hyperplastic lesions, and the proportion of positive samples further increased in patients with high-grade dysplasia. Comparing hERG1 expression in other preneoplastic lesions of the GI tract (gastric dysplasia and Barrett’s esophagus), it emerged that in all the conditions, hERG1 was expressed with a diffused pattern, throughout the cell, with variable staining intensity within the samples. The highest expression was detected in gastric dysplasia samples and the lowest in Barrett’s esophagus at similar levels observed in colorectal adenomas. Our results show that hERG1 is aberrantly expressed in human preneoplastic lesions of the gastrointestinal tract and has a different pattern of expression and role in the different sites. Overall, the detection of hERG1 expression in preneoplastic lesions could represent a novel diagnostic or prognostic marker of progression in the gastrointestinal tract.

Development of dynamic image analysis methods to measure vascularisation and syncytial nuclear aggregates in human placenta.

Histological examination of the placenta significantly contributes to diagnosis in adverse birth outcomes. One challenge in image analysis is variation in staining intensity caused by batch variation. We investigated if dynamic threshold image analysis methods may increase accuracy. Placenta samples were stained for endothelial cells and syncytial nuclear aggregates and analysed in Qupath software. Dynamically setting the threshold resulted in data more similar to manual method data. The method is simple and effective at modelling the dynamic interpretation of variation in staining intensity achieved by manual methods. We anticipate dynamic methods could be used to enhance placental diagnosis.

P53 Immunohistochemistry Patterns Are Surrogate Biomarkers for TP53 Mutations in Gastrointestinal Neuroendocrine Neoplasms.

Aims The aim of this study was to establish p53 immunohistochemistry (IHC) patterns to predict TP53 mutations in gastrointestinal neuroendocrine neoplasms (GI-NENs) and to determine whether p53 IHC patterns could be used for the differential diagnosis of neuroendocrine neoplasms. Methods TP53 gene sequencing and p53 IHC were performed on formalin-fixed paraffin-embedded (FFPE) tissue samples from 92 patients diagnosed with GI-NENs from five medical centers. Results The cohort included 35 well-differentiated neuroendocrine tumors and 57 poorly differentiated neuroendocrine carcinomas. Gene sequencing revealed 38 wild-type TP53 and 54 TP53 mutations. p53 expression was interpreted as follows: pattern A, p53 was absent from all tumor cells; pattern B, scattered and weak p53 expression in 1-20% of tumor cells; and pattern C was subclassified as pattern C1: variable p53 staining intensity in 21-60% of tumor cells and tumor cell nests with focal strong positive p53 staining and pattern C2: strong p53 staining in more than 60% of tumor cells. p53 IHC patterns were evaluated as a binary classifier where pattern B predicted wild-type TP53, and patterns A and C predicted TP53 mutations. The sensitivity, specificity, and overall accuracy of this binary classification to predict TP53 status were 0.963, 0.868, and 0.924, respectively. p53 IHC patterns were also correlated with TP53 mutation types. Most cases with pattern A harboured loss-of-function (LOF) mutations, whereas patterns B and C tended to indicate wild-type TP53 and gain-of-function (GOF) mutations, respectively. Furthermore, most of the well-differentiated NETs showed pattern B, whereas pattern C2 was more common in poorly differentiated NECs. Finally, staining interpretation between different observers also yielded high reproducibility. Conclusions p53 IHC patterns may be used as predictors of TP53 gene mutations and therefore could be potential surrogate markers for TP53 mutations in GI-NENs and could distinguish between well-differentiated NETs and poorly differentiated NECs.

Analysis of Tumor Depth Invasion With Anti-Smoothelin Antibody in Equivocal Transurethral Resection of Urinary Bladder Tumor Surgical Specimens.

To examine the expression and value of the smoothelin marker in control cases, to standardize the working method, and to analyze its application in pathologic staging process of problematic transurethral resection of bladder tumor (TURBT) cases. Immunohistochemical (IHC) staining was performed on tumor-free bladder wall sections, tumor-free large bowel sections, TURBTs with unequivocal tumor stage, and TURBTs with equivocal stage. The IHC staining of muscularis mucosa (MM), muscularis propria (MP), and blood vessels was evaluated semiquantitatively. Smoothelin IHC staining pattern ranged from negative (30% to 67% cases) to 2+ (0% to 15% cases) in MM and from 1+ (10% to 50% cases) to 3+ (9% to 48% cases) in MP. When compared on the same slide, the smoothelin expression of MP showed a stronger staining intensity than the one of the MM in all the analyzed cases. Blood vessel muscle cells stained in a constant intensity as the MM (r = 0.9808; r = 0.9604). Smoothelin determined restaging of 33% of the problematic TURBT cases. Smoothelin is an IHC marker that shows differential staining between coexistent MM and MP; however, variations in staining intensity and pattern may occur, aspects that can be influenced by different technique variables. We recommend using this marker as a diagnostic tool in problematic TURBT cases only when there is sufficient experience in control cases with this antibody.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

RESTORE: Robust intEnSiTy nORmalization mEthod for multiplexed imaging

Recent advances in multiplexed imaging technologies promise to improve the understanding of the functional states of individual cells and the interactions between the cells in tissues. This often requires compilation of results from multiple samples. However, quantitative integration of information between samples is complicated by variations in staining intensity and background fluorescence that obscure biological variations. Failure to remove these unwanted artifacts will complicate downstream analysis and diminish the value of multiplexed imaging for clinical applications. Here, to compensate for unwanted variations, we automatically identify negative control cells for each marker within the same tissue and use their expression levels to infer background signal level. The intensity profile is normalized by the inferred level of the negative control cells to remove between-sample variation. Using a tissue microarray data and a pair of longitudinal biopsy samples, we demonstrated that the proposed approach can remove unwanted variations effectively and shows robust performance.

NTRK3 overexpression in undifferentiated sarcomas with YWHAE and BCOR genetic alterations.

The BCOR family of tumors includes a number of undifferentiated sarcomas, occurring in various age groups and anatomic sites, characterized by a spindle and round cell phenotype and diffuse immunoreactivity for BCOR. Prior RNA sequencing data revealed that NTRK3 was a top upregulated gene in BCOR-CCNB3 sarcomas. In this study we investigate a large cohort of tumors harboring BCOR/YWHAE genetic alterations for NTRK3 upregulation at both the mRNA and protein levels, compared to other sarcoma types. Pan-Trk immunohistochemistry was assessed for intensity and extent. A correlation between NTRK3 expression and the type of BCOR alteration and BCOR immunoreactivity was also performed. Most soft tissue undifferentiated round cell sarcomas with YWHAE or BCOR rearrangements or BCOR internal tandem duplications (ITD) showed NTRK3, but not NTRK1 or NTRK2, up-regulation by RNA sequencing data analysis. Cytoplasmic pan-Trk immunoreactivity was also observed in most soft tissue round cell sarcomas with YWHAE rearrangements (100%), BCOR ITD (80%), and BCOR-CCNB3 fusions (67%), as well as clear cell sarcomas of kidney (75%), another BCOR family tumor, and ossifying fibromyxoid tumors with ZC3H7B-BCOR fusion (100%), with variable staining intensity and extent. Pan-Trk staining was also seen in solitary fibrous tumors (100%) and less frequently in synovial sarcoma and Ewing sarcoma, but rarely in other sarcomas tested. Tumors harboring rare fusion variants of BCOR, such as BCOR-CHD9, a novel fusion identified by targeted RNA sequencing, and KMT2D-BCOR, were also positive for pan-Trk staining and NTRK3 overexpression. In conclusion, NTRK3 upregulation resulting in pan-Trk overexpression is common in the BCOR family of tumors as well as in subsets of BCOR expressing sarcomas through alternative mechanisms. The therapeutic implication of this finding awaits further investigation.

MUC4 Expression in Angiomatoid Fibrous Histiocytoma.

Angiomatoid fibrous histiocytoma (AFH) is a rarely metastasizing neoplasm that typically occurs in the deep dermis and subcutis of the extremities of young patients, characterized by a t(2;22) translocation involving EWSR1 and CREB1. Because of its distinctive histologic features, the diagnosis of AFH is generally straightforward, although the immunohistochemistry (IHC) findings are relatively nonspecific. We recently encountered a case of primary cranial AFH that showed strong MUC4 IHC expression, which has not yet been reported previously. Prompted by this surprising finding, we investigated MUC4 expression in a series of AFH to evaluate this potential diagnostic pitfall. The expression of ALK by IHC, recently discovered in AFH, was also assessed in this study. We also analyzed EWSR1 rearrangement by fluorescence in situ hybridization using a dual color break-apart probe to confirm the diagnosis. The results showed MUC4 expression in 22.2% of AFH cases (4/18 cases), demonstrating a variable intensity of cytoplasmic staining. Most notably, one of the positive cases showed strong and diffuse expression. ALK IHC expression was observed in 17 of 18 cases (94.4%), usually in a diffuse and strong cytoplasmic pattern. EWSR1 rearrangement was demonstrated by fluorescence in situ hybridization in 81.2% of cases (13 of 16), including all the MUC4-positive cases. Our results indicate that although the significance of MUC4 expression in AFH is unknown, it is important to be aware that a subset of AFH can express the protein by IHC, expanding a variety of MUC4-positive mesenchymal tumors.

Abstract LB-318: Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow

Abstract Background: Emerging data suggests that predictive biomarkers based on the spatial arrangement of multiple cell types in FFPE tissue sections will be an important component of precision medicine in immune-oncology. Multiplexed immunofluorescence (mIF) facilitates such assessments. If mIF is to play a translational role in research and ultimately clinical practice, it is vital to refine, standardize, and validate an end-to-end workflow that supports large scale multi-site trials and clinical laboratory processes. To this end, six institutions collaborated to develop an automated 6-plex assay focused on the PD-1/PD-L1 axis and assessed its inter- and intra-site reproducibility. Specific attention was paid to assessment of %PD-L1 expression by immune cells (ICs), as pathologists have poor concordance for this parameter as noted in the Blueprint 2 and multi-institutional NCCN studies on PD-L1 IHC. Methods: A 7-color mIF panel (PD-L1, PD-1, CD8, CD68, FoxP3, Cytokeratin, and DAPI) was optimized on a Leica Bond Rx autostainer. Serial sections of tonsil and a lung cancer tissue-microarray (TMA), antibodies and TSA-Opal detection reagents (Akoya Biosciences) were distributed. Cell pellet arrays were also distributed and used to normalize batch variation in intensity measurements. Tonsil and TMA sections were stained at each site and imaged at 20x using a Vectra Polaris. Cells were segmented and phenotyped using image analysis algorithms. In tonsil sections, the average intensity of the top quartile of cells positive for each marker was assessed to identify potential variation in staining intensity. In lung TMAs, cell densities and %PD-L1 expression in immune cells (CD68+ and CD8+ cells) was determined. Results: The average staining intensity coefficients of variation (CV) for all markers within sites was 10% in tonsil samples. Inter-site concordance for tumor cell and immune cell subset densities in TMAs, had an average R2 value of 0.86 and slope of 0.96. Inter-site concordance for %PD-L1+ ICs had an average R2 value of 0.81, and slope of 0.82, in contrast to ICC values of &amp;lt;0.3 in the NCCN and Blueprint 2 studies. Conclusions: We demonstrate a reproducible end-to-end process for mIF characterization of the PD-1/PD-L1 axis including automated staining, multispectral imaging, and machine-learning-trained image analysis algorithms. This approach improved reproducibility of %PD-L1 IC assessment and brought it in line with %PD-L1 tumor cell assessment by pathologists. Sources of variation were identified and will be discussed. The described approach may serve as a template for assessing reproducibility of emerging mIF panels for other investigative teams, with an eye toward translating such approaches into clinical trials and ultimately into the clinic. Citation Format: Clifford Hoyt, Kristin Roman, Liz Engle, Chichung Wang, Carmen Ballesteros-Merino, Shawn M. Jensen, John McGuire, Yi Zheng, Carla Coltharp, Mei Jiang, Justin Lucas, Edwin Parra, Ignacio Wistuba, Darren Locke, Bernard A. Fox, David L. Rimm, Janis Marie Taube. Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-318.

Expression of Phosphoinositide 3-Kinase p110α and p110β Subunits and PIK3CA Mutation in Patients With Advanced Gastric Carcinoma.

Activation of phosphoinositide 3-kinase (PI3K) is pivotal for the activity of the oncogenic PI3K/AKT signaling pathway. This study assessed the expression of 2 PI3K isoform proteins, p110α and p110β, and PIK3CA mutational status in advanced gastric carcinoma (AGC) and their correlation with clinicopathologic factors. Tissue microarray blocks were generated from 99 AGCs and immunohistochemically stained for p110α and p110β. Analysis of mutations in the PIK3CA gene, which encodes p110α, was performed using the PNAClamp PIK3CA Mutation Detection kit. Of the 99 tumors, positivity was seen in 62 (62.6%) for p110α and 97 (98.0%) for p110β with variable intensity and extent of staining. The median H-scores were 40 (range: 0 to 300) for p110α and 180 (range: 0 to 300) for p110β. Isoform p110α was more highly expressed in tumors with a lower pathologic T stage (P=0.035) and TNM stage (P=0.165), while p110β was not significantly associated with clinicopathologic factors. Samples with high p110α expression had a trend toward longer overall survival (OS) although it was not statistically significant (P=0.271), whereas high p110β expression correlated with shorter OS (P=0.016). In addition, p110β was an independent factor for poor prognosis in multivariate analysis for OS. Eight (8.1%) samples had PIK3CA mutations in exon 9. Mutational status at this locus was not significantly correlated with clinicopathologic factors. These results imply that p110β could have a more important role in the progression and aggressiveness of AGC than p110α and has potential as a prognostic biomarker in patients with AGC.

Investigating Various Thresholds as Immunohistochemistry Cutoffs for Observer Agreement.

Clinical translation of immunohistochemistry (IHC) biomarkers requires reliable and reproducible cutoffs or thresholds for interpretation of immunostaining. Most IHC biomarker research focuses on the clinical relevance (diagnostic, prognostic, or predictive utility) of cutoffs, with less emphasis on observer agreement using these cutoffs. From the literature, we identified 3 commonly used cutoffs of 10% positive epithelial cells, 20% positive epithelial cells, and moderate to strong staining intensity (+2/+3 hereafter) to use for investigating observer agreement. A series of 36 images of microarray cores stained for 4 different IHC biomarkers, with variable staining intensity and percentage of positive cells, was used for investigating interobserver and intraobserver agreement. Seven pathologists scored the immunostaining in each image using the 3 cutoffs for positive and negative staining. Kappa (κ) statistic was used to assess the strength of agreement for each cutoff. The interobserver agreement between all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.64, 0.59, and 0.62, respectively, for 10%, 20%, and +2/+3 cutoffs. A good agreement was observed for experienced pathologists using the 10% cutoff, and their agreement was statistically higher than for junior pathologists (P=0.02). In addition, the mean intraobserver agreement for all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.71, 0.60, and 0.73, respectively, for 10%, 20%, and +2/+3 cutoffs. For all 3 cutoffs, a positive correlation was observed with perceived ease of interpretation (P<0.003). Finally, cytoplasmic-only staining achieved higher agreement using all 3 cutoffs than mixed staining patterns. All 3 cutoffs investigated achieve reasonable strength of agreement, modestly decreasing interobserver and intraobserver variability in IHC interpretation. These cutoffs have previously been used in cancer pathology, and this study provides evidence that these cutoffs can be reproducible between practicing pathologists.

Distribution of 5-HT1F Receptors in Monkey Vestibular and Trigeminal Ganglion Cells.

Evidence of serotonergic involvement in vestibular pathway contributions to migraine and balance disorders is compelling. Serotonergic 5-HT1B and 5-HT1D receptors are expressed extensively in inner ear ganglia of monkeys and rats. The serotonergic 5-HT1F receptor is also a target of triptans. This study describes its distribution in vestibular and trigeminal ganglia of monkeys. Using primary polyclonal antibodies raised against oligopeptides specific for the human 5-HT1F receptor, neuronal somatic area and intensity of immunoreactive vestibular and trigeminal ganglia were quantified. Virtually all vestibular and considerable trigeminal ganglia showed positive 5-HT1F receptor immunoreactivity. Inferior and superior vestibular ganglia staining appeared confined to distinct cell regions, varying considerably among cells of different sizes: more intense in small, punctate in some medium and regionally polarized in some large cells. Analyses of average somatic vestibular neuronal immunoreactive intensity identified mainly medium sized cells with high standard deviation of intensity corresponding to punctately stained cells. Less variability occurred in somatic intensity staining and cellular distribution among 5-HT1F receptor immunopositive trigeminal ganglia. Most exhibited similar punctate staining patterns, higher mean somatic immunoreactive intensity and larger neuronal somatic size proportions per size distribution subpopulation compared to vestibular ganglia size distribution populations. Centrally directed vestibular ganglion neuronal processes, cochlear inner hair cells, vestibular hair cells and blood vessels in vestibular maculae and cristae were immunoreactive. The 5-HT1F receptor expression in vestibular ganglia shows complex variable staining intensity patterns associated with cell size of immunopositive neurons, not seen in immunopositive trigeminal ganglia and not previously evident with 5-HT1B and 5-HT1D receptor subtype immunoreactivity in vestibular ganglia. These data motivate exploration of 5-HT1 receptor oligomerization and ligand functional selectivity in differential serotonergic involvement in co-morbidity of migraine and balance disorders. Similar findings in cochlear inner hair cell afferents are applicable to migraine-related tinnitus or hypercusis (phonophobia).

Spermatogonial stem cell organization in felid testis as revealed by Dolichos biflorus lectin.

Spermatogonial stem cells are being exploited in many species as a tool to recover fertility, but may also be used to manipulate the genetic pool. Whatever the purpose, these cells must be fully characterized and easily identifiable, and our goal was to improve this procedure in the domestic cat, used as an animal model for endangered felid species and for some human diseases/physiological processes. We have therefore screened several markers that might be used to distinguish and study the undifferentiated spermatogonia population insitu and invitro via immunohistochemistry applied to tissue sections and whole mounts of the domestic cat seminiferous tubules. Our results show that, although they label the cytoplasm and nucleus of gonocytes and spermatogonia in pre-pubertal animals, PGP9.5 and FoxO1 cannot be considered markers of undifferentiated spermatogonia in adult animals, as almost all spermatogonia, namely type A and B, express these proteins. Nonetheless, the Dolichos biflorus agglutinin (DBA lectin) was able to label the cell surface and cytoplasm of a small type A spermatogonial population in the adult animals. Analysis of the number and distribution of the DBA-labeled cells showed they were present in low number, which did not vary with epithelium seminiferous stage. Morphometric analysis revealed that DBA-labeled cells present tropism to a peculiar area of the seminiferous tubules, namely the area in direct contact with Leydig cells. Whole mounts of DBA-stained seminiferous tubules revealed the arrangement of DBA-stained cells in small clones up to eight cells. Noteworthy, the clonal cells presented variable staining intensity suggesting the existence of asymmetric distribution of O-glycosylated proteins within each clone. Our results strongly suggest that the DBA lectin is a marker of undifferentiated spermatogonia in domestic cat, and illustrate the peculiar characteristics of spermatogonial stem cell development and organization in this species.

Fuchs Endothelial Corneal Dystrophy: A Systematic Immunofluorescence Study of Collagen Type VIII Suggests Heterogeneous Pathophysiology.

The purpose of this study was to investigate the pathophysiologic heterogeneity of Fuchs endothelial corneal dystrophy (FECD). We conducted a systematic immunofluorescence study on 39 Descemet membrane samples from FECD patients and compared these with 10 Descemet membrane samples from patients with pseudophakic bullous keratopathy (PBK) and 7 normal corneas. Samples were analyzed with immunofluorescence using antibodies to the α1-chain [collagen VIII α1-chain (COL8A1)] and α2-chain (COL8A2). Intensity of staining was assessed using a subjective grading scale from 0 to 3. The presence of specific staining patterns was noted. The overall distribution of COL8A1 staining intensity between groups was significantly different (P = 0.002). There was marked/intense staining in 85% (33/39) of the FECD samples, 40% (4/10) of the PBK samples (P = 0.034), and 29% (2/7) of normal samples (P = 0.004). The overall distribution of COL8A2 staining intensity was not significantly different between groups (P = 0.39). There was marked/intense staining in 33% (13/39) of the FECD samples, 10% (1/10) of PBK samples, and 14% (1/7) of the normal samples. There was substantial variation in staining intensity in the FECD group, a phenomenon that was especially pronounced for the COL8A2 antibody. We found increased staining for COL8A1, but not COL8A2 in FECD samples. Further, there was striking variation of staining intensity in FECD patients, indicating pathophysiological heterogeneity.