Tissue Microarray Sections Research Articles

FISHing with immunohistochemistry for housekeeping gene changes in Alzheimer's disease using an automated quantitative analysis workflow.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Androgen receptor in breast cancer: A tissue microarray-based study

Background and Aim: Breast carcinoma is the most common malignant tumor and the leading cause of cancer deaths in women. The incidence of breast cancer in India has been gradually raising accounting for 25%–33% of all cancers in women. Breast carcinomas are a heterogeneous group of tumors which show varied behavior and response to therapeutic agents. Androgen receptors (ARs) have been studied in great detail in prostate cancers, now being studied in breast cancers, but its role in breast cancer has not been elucidated completely. The aim of our study was to evaluate the expression of AR in invasive carcinoma of the breast and to evaluate its relation to estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal growth factor Receptor2 (HER2) expression on tissue microarray (TMA) sections. Materials and Methods: This was a descriptive study which included 53 cases of breast carcinoma. The specimens were fixed in 10% formalin, routinely processed. A master block for TMA was made by taking 2 mm cores from paraffin blocks containing tumor. Sections were taken and stained for ER, PR, AR, and HER2. Results: A total of 53 cases were studied. Around 63% of breast carcinomas were positive for AR. Conclusion: AR expression can be used as an additional biomarker in breast carcinomas, and a large number of cases can be studied by TMA sections.

The Correlation between Deglycosylation of PD-L1 and Gene Mutations and Their Effects on the Prognosis of Patients with Lung Adenocarcinoma

Aim: The aim of this study was to define the clinical significance of deglycosylation of PD-L1 and its correlation with EGFR and ALK mutation in lung adenocarcinoma. Methods: Lung adenocarcinoma tissue microarray sections were obtained from Outdo Biotech. We estimated the intensity of both native and deglycosylated PD-L1 signals by a 28-8 antibody. We analyzed the difference in the H-score between tumor and paratumor tissues, as well as that before and after deglycosylation. Correlations between EGFR or ALK status and PD-L1 expression were analyzed. We also evaluated the differences among survival curves. Results: The expression level of PD-L1 in lung adenocarcinoma tissues was significantly higher than that in paratumor tissues (P 0.05). Conclusion: PD-L1 deglycosylation enhances the detection of PD-L1 when utilizing a 28-8 antibody. Moreover, the response to deglycosylation of PD-L1 may predict the survival of certain patients with lung adenocarcinoma. Our research provides a more effective strategy for estimating PD-L1 expression and predicting patient outcomes in lung adenocarcinoma.

Expression and prognosis analysis of JMJD5 in human cancers.

Background: JumonjiC (JmjC) domain-containing protein 5 (JMJD5) plays an important part in cancer metabolism. However, the prognostic value of JMJD5 in most human cancers is unknown yet. We aimed to examine the expression level and prognostic value of JMJD5, immune cell infiltration in cancer patients, and simultaneously to examine the correlations among them. Materials and methods: The mRNA and protein expression of JMJD5 were analyzed through online Tumor Immune Estimation Resource (TIMER) or immunohistochemistry (IHC) of tissue microarray sections (TMAs) in cancer versus normal tissues. The Kaplan-Meier Plotter databases were used to assess the prognostic values. The connection between the expression of JMJD5 and the abundances of six infiltrating immune cells were explored by TIMER in breast cancer (BRCA), liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD) and stomach adenocarcinoma (STAD). We used the Cox proportional hazards model to investigate the correlations among clinical outcome, the abundance of immune cell infiltration and JMJD5 expression. Results: We found that the JMJD5 expression was obviously lower in BRCA, LIHC and lung cancer (LUC) but higher in STAD than in normal tissues. High expression of JMJD5 had a better prognosis only in BRCA, LIHC and LUC but a worse prognosis in STAD. The expression of JMJD5 has a significant connection with the abundance of six kind of infiltrating immune cells. The expression of JMJD5 plus the number of immune-infiltrating B cells or macrophages may jointly serve as a prognostic marker in the above four cancers. Conclusion: We provided novel evidence of JMJD5 as an essential prognostic biomarker and perspective therapeutic target in BRCA, LUAD, LIHC and STAD.

Interrogation of IDH1 Status in Gliomas by Fourier Transform Infrared Spectroscopy.

Simple SummaryGliomas represent the vast majority of primary brain tumours and are of significant medical importance due to the poor clinical course of affected patients. The isocitrate dehydrogenase 1 (IDH1) mutation is associated with improved prognosis, compared to patients with IDH1-wildtype lesions of the same stage. In this proof-of-concept study, Fourier transform infrared spectroscopy was used to determine the IDH1 molecular status in fixed glioma sections. Classification algorithms successfully distinguished the two IDH1 classes with high accuracies (>80%). Knowledge of the IDH1 status would be beneficial, as maximum resection may be preferred in patients with IDH1-mutant gliomas, whilst a more limited resection can be best for IDH1-wildtype gliomas. Furthermore, we examined blood serum in an attempt to identify the biomolecular alterations caused by the IDH1 mutation. Non-invasive approaches that can detect the molecular status may guide some patients to an alternative treatment prior to surgery.Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are found in a high proportion of diffuse gliomas. The presence of the IDH1 mutation is a valuable diagnostic, prognostic and predictive biomarker for the management of patients with glial tumours. Techniques involving vibrational spectroscopy, e.g., Fourier transform infrared (FTIR) spectroscopy, have previously demonstrated analytical capabilities for cancer detection, and have the potential to contribute to diagnostics. The implementation of FTIR microspectroscopy during surgical biopsy could present a fast, label-free method for molecular genetic classification. For example, the rapid determination of IDH1 status in a patient with a glioma diagnosis could inform intra-operative decision-making between alternative surgical strategies. In this study, we utilized synchrotron-based FTIR microanalysis to probe tissue microarray sections from 79 glioma patients, and distinguished the positive class (IDH1-mutated) from the IDH1-wildtype glioma, with a sensitivity and specificity of 82.4% and 83.4%, respectively. We also examined the ability of attenuated total reflection (ATR)-FTIR spectroscopy in detecting the biomolecular events and global epigenetic and metabolic changes associated with mutations in the IDH1 enzyme, in blood serum samples collected from an additional 72 brain tumour patients. Centrifugal filtration enhanced the diagnostic ability of the classification models, with balanced accuracies up to ~69%. Identification of the molecular status from blood serum prior to biopsy could further direct some patients to alternative treatment strategies.

Histological and immunohistochemical characterization of 17 gonadal tumours in koi carp (Cyprinus carpio koi).

Reports on abdominal tumours in koi carp are scarce and most are from the gonads. Their histological diagnosis is challenging due to the occurrence of mixed populations of neoplastic cells and the few availability of cross-reactive antibodies in fish tissues. The present study aims to provide a histopathological characterization of seventeen gonadal tumours, enriched by a wide antibody panel (vimentin, CD117, placental alkaline phosphatase-PLAP, AE1/AE3 cytokeratin, E-cadherin, proliferating cell nuclear antigen-PCNA, müllerian-inhibiting substance-MIS, GATA4 and Inhibin-α) applied on whole and tissue microarray (TMA) sections. Abdominal enlargement was associated with tumours filling 30%-80% of the abdominal cavity; frequently, the gonads had been completely replaced by neoplastic tissue. Twelve cases were characterized as sex cord-stromal tumours (SCSTs), three as germ cell tumours (GCTs), one as mixed germ cell sex cord-stromal tumour (MGCSCST) and one as carcinoma. By immunohistochemistry, PLAP enabled confirmation of GCTs, ovarian carcinoma and the objective identification of a further cell component in 8 out of the 12 SCSTs that were reclassified as mixed tumours. The use of an immunohistochemical panel can help in refining the histological diagnosis, but the morphological diagnosis still represents the main tool for the characterization of these tumours in koi carp.

Diagnostic value of clusterin immunostaining in hepatocellular carcinoma

BackgroundHistologic distinction between well differentiated hepatocellular carcinoma (HCC) and benign hepatocellular mass lesions is a known challenge. Existing biomarkers are of limited diagnostic value. Our previous studies observed an enhanced canalicular expression pattern of clusterin (CLU) in HCC, which was not observed in benign hepatocellular mass lesions such as hepatocellular adenoma. The aim of this study was to further investigate its diagnostic value for HCC by examining the expression pattern of CLU in a large number of non-hepatocellular tumors, and by comparing it with two other commonly used hepatocellular markers pCEA and CD10 that also show a canalicular staining pattern in HCC.MethodsEnhanced canalicular staining patterns of CLU, pCEA and CD10 were analyzed on 54 surgically resected well to moderately differentiated HCCs on whole tissue sections, of which 37 had surrounding regenerative nodules while the remaining 17 had a non-cirrhotic background. CLU immunostaining was also performed on tissue microarray sections that contained 74 HCCs (40 of which were also stained for pCEA and CD10), 55 normal liver tissue samples, and 1305 non-hepatocellular tumors from multiple organs.ResultsEnhanced CLU canalicular staining was observed in 70% (89/128) HCCs but not in regenerative nodules, normal liver tissues or any non-hepatocellular tumors. The sensitivity and specificity for enhanced canalicular staining pattern of CLU in HCCs were 0.70 and 1.00. This enhanced canalicular pattern was observed in only 26 and 23% HCCs for CD10 and pCEA, respectively. These results further demonstrate that the distinctive enhanced canalicular pattern of CLU is unique to HCC.ConclusionsCLU is superior to pCEA and CD10 as a diagnostic immunomarker in that it can help distinguish well to moderately differentiated HCC not only from non-HCC malignancies but also from benign hepatocellular mass lesions.

Detection of NTRK1/3 Rearrangements in Papillary Thyroid Carcinoma Using Immunohistochemistry, Fluorescent In Situ Hybridization, and Next-Generation Sequencing.

NTRK1/3 rearrangements have been reported in 2.3-3.4% of papillary thyroid carcinoma (PTC) and are regarded as potential therapeutic targets. Recently, the application of immunohistochemistry (IHC) to detect NTRK rearrangements has been widely discussed. The current study aimed to characterize the clinicopathological features of PTC with NTRK1/3 fusions, to examine the utility of pan-TRK IHC, and to compare IHC with fluorescent in situ hybridization (FISH) and next-generation sequencing (NGS). In a cohort of 525 consecutive PTC cases, 60 BRAFV600E-negative cases underwent complete analyses of FISH, and 12 (2.3%) cases with NTRK1/3 break-apart were found. A novel ERC1-NTRK3 fusion was identified by NGS in one case. Pathological features of non-infiltrative tumor border, clear cell change, and reduced nuclear elongation and irregularity were significantly more common in NTRK1/3-rearranged PTC when compared with 48 BRAFV600E-negative non-NTRK1/3 PTC cases. In whole tissue sections, pan-TRK IHC was positive in 3/7 (42.9%) cases with an ETV6-NTRK3 rearrangement including 2 cases with low percentage of stained tumor cells, 2/3 (66.7%) with non-ETV6 NTRK3 rearrangements, and 2/2 (100%) with NTRK1 rearrangements. All FISH-negative cases were negative for pan-TRK in tissue microarray sections. As a result, pan-TRK IHC showed a sensitivity of 58.3% and specificity of 100% for NTRK1/3 rearrangements in BRAFV600E-negative PTC. In conclusion, NTRK1/3-rearranged PTC shared some unique morphologic features. Pan-TRK IHC showed high specificity and moderate sensitivity for NTRK1/3-rearranged PTC and should be interpreted with caution due to staining heterogeneity. Based on the above findings, we propose an algorithm integrating morphology, IHC, and molecular testing to detect NTRK1/3 rearrangements in PTC.

A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment.

Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs >0.9, P-value<0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.

Cell Polarity Protein Pals1-Associated Tight Junction Expression Is a Favorable Prognostic Marker in Clear Cell Renal Cell Carcinoma.

Introduction: The Pals1-associated tight junction (PATJ) is a Crumbs (CRB) complex component that regulates epithelial cell apico-basal polarity and directional migration. This study assessed PATJ expression in clear cell renal cell carcinoma (ccRCC) vs. normal tissues and associated with ccRCC progression and prognosis. Methods: The effects of PATJ knockdown were investigated on regulation of normal kidney epithelial cell viability and protein expression in vitro. The PATJ mRNA data in ccRCC were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and analyzed with UALCAN, LinkedOmics, Kaplan-Meier Plotter, GEPIA, and SurvExpress tools. Immunohistochemistry was performed for PATJ in tissue microarray sections (n = 150 ccRCC and 30 normal renal specimens). Normal human kidney tubular epithelial cell (HKC) cells were transfected with PATJ and negative control siRNA for cell viability CCK-8 assay, flow cytometry, and western blots. Results: The data showed that PATJ mRNA and protein were downregulated in ccRCC tissues and cell lines. Downregulation of PATJ mRNA was associated with male patients, advanced tumor stages, grades, and ccB subtypes as well as poorer overall and disease-free survival of patients. Furthermore, PATJ protein was also significantly downregulated in ccRCC tissues and associated with advanced tumor pathologic, TNM stages and poorer overall. In vitro, knockdown of PATJ expression promoted HKC proliferation and the activation of mitogen-activated protein kinases (MAPK) pathway proteins. Conclusions: This study revealed that a decrease of PATJ in ccRCC, which was associated with male patients, advanced tumor, and poorer survival, suggesting that PATJ may be a useful prognostic biomarker and therapeutic target for ccRCC.

Clinicopathological significance of CD47 expression in hepatocellular carcinoma

AimsCD47 is upregulated on the surface of various tumour cells, and it is known to inhibit the phagocytosis of tumour cells by macrophages. Immunotherapy that targets CD47 has demonstrated success...

Retinoblastoma protein expression and its predictors in triple-negative breast cancer

Retinoblastoma protein (Rb) is a product of the RB tumor suppressor gene. Its expression is highly prevalent in luminal breast cancers and is critical to the success of cyclin-dependent kinase (CDK) 4/6 inhibitor therapy. Expression of Rb in triple-negative breast cancer (TNBC), tumors generally associated with basal biology, is not well known. However, heterogeneity among TNBC and presence of subtypes with luminal features are well described. The purpose of this study was to determine prevalence and predictors of Rb protein expression in BRCA1-associated and sporadic TNBCs. We studied 180 TNBC patients (70 BRCA1-associated and 110 sporadic). The clinical and pathologic features of these cases were previously assessed and reported. For this study, immunohistochemical stains for Rb were performed on tissue microarray sections. Details of treatment and outcome were abstracted from medical records. Fifty-one percent of TNBC were Rb positive (≥10% nuclei staining), and 85% of these cases had ≥50% nuclei staining. Rb expression was significantly associated with sporadic TNBC (71.4% vs 49.4%; p < 0.001), androgen receptor (AR) expression (16.5% vs 3.4%; p = 0.007), histologic grade 1 or 2 (9.9% vs 2.2%; p = 0.04), and first recurrence in bone (8.8% vs 1.1%; p = 0.03). Expression of p53 was not associated with Rb expression. Expression of Rb in TNBC was significantly associated with sporadic TNBC, AR expression, lower histologic grade, and metastasis to bone. These observations characterize a TNBC subtype with features suggestive of luminal-like biology and the potential to benefit from CDK 4/6 inhibition.

A Machine-learning Approach for the Assessment of the Proliferative Compartment of Solid Tumors on Hematoxylin-Eosin-Stained Sections.

We introduce a machine learning-based analysis to predict the immunohistochemical (IHC) labeling index for the cell proliferation marker Ki67/MIB1 on cancer tissues based on morphometrical features extracted from hematoxylin and eosin (H&E)-stained formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples. We provided a proof-of-concept prediction of the Ki67/MIB1 IHC positivity of cancer cells through the definition and quantitation of single nuclear features. In the first instance, we set our digital framework on Ki67/MIB1-stained OSCC (oral squamous cell carcinoma) tissue sample whole slide images, using QuPath as a working platform and its integrated algorithms, and we built a classifier in order to distinguish tumor and stroma classes and, within them, Ki67-positive and Ki67-negative cells; then, we sorted the morphometric features of tumor cells related to their Ki67 IHC status. Among the evaluated features, nuclear hematoxylin mean optical density (NHMOD) presented as the best one to distinguish Ki67/MIB1 positive from negative cells. We confirmed our findings in a single-cell level analysis of H&E staining on Ki67-immunostained/H&E-decolored tissue samples. Finally, we tested our digital framework on a case series of oral squamous cell carcinomas (OSCC), arranged in tissue microarrays; we selected two consecutive sections of each OSCC FFPE TMA (tissue microarray) block, respectively stained with H&E and immuno-stained for Ki67/MIB1. We automatically detected tumor cells in H&E slides and generated a “false color map” (FCM) based on NHMOD through the QuPath measurements map tool. FCM nearly coincided with the actual immunohistochemical result, allowing the prediction of Ki67/MIB1 positive cells in a direct visual fashion. Our proposed approach provides the pathologist with a fast method of identifying the proliferating compartment of the tumor through a quantitative assessment of the nuclear features on H&E slides, readily appreciable by visual inspection. Although this technique needs to be fine-tuned and tested on larger series of tumors, the digital analysis approach appears to be a promising tool to quickly forecast the tumor’s proliferation fraction directly on routinely H&E-stained digital sections.

Immunohistochemical biomarker expression in gastro-entero-pancreatic neuroendocrine tumors at ENETS Centre of Excellence, University Hospital Zurich, Switzerland.

e15673 Background: Gastro-entero-pancreatic neuroendocrine tumors (GEP-NET) are a heterogeneous tumor entity with respect to biological behaviour and prognosis. Definition of new predictive and prognostic biomarkers as well as new therapy targets are of upmost clinical interest. Methods: At the ENETS CoE Zurich tissue microarray (TMA) blocks were constructed with 147 tissue samples of tumors diagnosed from 2000 to 2017, including primary tumors and metastasis. Tissue microarray sections were immunostained for SOX-9, SOX-10, SSTR-2 +, PDL-1, thyroid transcription factor 1 (TTF1), estrogen receptor-α (ER-α) and -β (ER-β), progesterone receptor (PR), androgen receptor (AR), BRAF and HER2. Results: In total 270 patients were screened between 2000 - 2017 and 92 patient’s material were sufficient for TMA analysis. Subgroup analysis showed 32 pancreatic, 37 ileum, 10 duodenum, 7 appendix, 3 colorectal, 3 gastric and 1 NET of gallbladder. 50% were male, the median age 63.5 years (range 19-88y). AR, ER-β, TTF1, PDL-1 and SOX10 was only present in a small number of NETs. HER2 and BRAF were negative in all samples. We found ER-α expression in 27.2%, 30% were PR positive and 67.4% showed SOX9 expression. Conclusions: We identified a frequent expression of new markers as SOX9, progesterone receptor and estrogen receptor-α in a broad amount of samples and a potential correlation with tumor grade. This finding might implicate a prognostic or predictive value of these markers, as well as it reveals new potential therapy targets for GEP-NETs. Therefore, further analyses are planned.

Cancer genomics predicts disease relapse and therapeutic response to neoadjuvant chemotherapy of hormone sensitive breast cancers

Several studies provide insight into the landscape of breast cancer genomics with the genomic characterization of tumors offering exceptional opportunities in defining therapies tailored to the patient’s specific need. However, translating genomic data into personalized treatment regimens has been hampered partly due to uncertainties in deviating from guideline based clinical protocols. Here we report a genomic approach to predict favorable outcome to treatment responses thus enabling personalized medicine in the selection of specific treatment regimens. The genomic data were divided into a training set of N = 835 cases and a validation set consisting of 1315 hormone sensitive, 634 triple negative breast cancer (TNBC) and 1365 breast cancer patients with information on neoadjuvant chemotherapy responses. Patients were selected by the following criteria: estrogen receptor (ER) status, lymph node invasion, recurrence free survival. The k-means classification algorithm delineated clusters with low- and high- expression of genes related to recurrence of disease; a multivariate Cox’s proportional hazard model defined recurrence risk for disease. Classifier genes were validated by Immunohistochemistry (IHC) using tissue microarray sections containing both normal and cancerous tissues and by evaluating findings deposited in the human protein atlas repository. Based on the leave-on-out cross validation procedure of 4 independent data sets we identified 51-genes associated with disease relapse and selected 10, i.e. TOP2A, AURKA, CKS2, CCNB2, CDK1 SLC19A1, E2F8, E2F1, PRC1, KIF11 for in depth validation. Expression of the mechanistically linked disease regulated genes significantly correlated with recurrence free survival among ER-positive and triple negative breast cancer patients and was independent of age, tumor size, histological grade and node status. Importantly, the classifier genes predicted pathological complete responses to neoadjuvant chemotherapy (P < 0.001) with high expression of these genes being associated with an improved therapeutic response toward two different anthracycline-taxane regimens; thus, highlighting the prospective for precision medicine. Our study demonstrates the potential of classifier genes to predict risk for disease relapse and treatment response to chemotherapies. The classifier genes enable rational selection of patients who benefit best from a given chemotherapy thus providing the best possible care. The findings encourage independent clinical validation.

Storage Conditions and Immunoreactivity of Breast Cancer Subtyping Markers in Tissue Microarray Sections.

Loss of immunoreactivity in tissue sections has been shown to occur when slide sections are stored at room temperature for prolonged periods of time. We conducted a systematic investigation to determine the extent of staining loss in various storage conditions to determine an optimal storage method. We investigated 6 antibodies that are commonly used for breast cancer subtyping in research studies with immunohistochemistry (ER, PR, HER2, CK5/6, EGFR, and Ki67) in formalin-fixed paraffin-embedded breast tissue microarrays consisting of 148 patients. Tissue microarrays were sectioned at various time points: fresh, 1 week, 1 month, 6 months, and 12 months before staining. Slides sectioned at each time point were stored in 5 storage conditions: desiccator, paraffin dipped, 4°C, -20°C, and -80°C. Immunohistochemistry scores were assessed over time with McNemar Test and Bowker Test of Symmetry. Desiccator storage was the only storage condition that did not show any loss in immunoreactivity for any antibody or time point in our study. Paraffin coated slides were the most difficult storage method operationally and also showed the most loss in immunoreactivity. Storing sections in a desiccator was the most effective method for minimizing immunoreactivity loss. Cold storage at 4°C is an intermediate option that is not as protective as a desiccator, but offers the advantage of being accessible to virtually all research labs.

Expression of the hippo signalling pathway effector YAP during canine epidermal tumourigenesis

Cutaneous papilloma (CP) and cutaneous squamous cell carcinoma (CSCC) are frequent epidermal tumours in dogs. In this regard, the study of the deregulated activity of signalling molecules during the epidermal tumourigenesis process could be the basis for the development of novel molecular mechanism-based antitumour treatments for CP and CSCC canine patients. Recent evidence suggests that the development and progression of CP and CSCC involve the dysregulated activation of the Hippo signalling pathway effector YAP. Thus, in the present study, we evaluated the immunohistochemical expression pattern of YAP in sections of tissue microarrays constructed from canine samples of normal epidermis, CP, preneoplastic epidermis, and CSCC. In samples of CP, preneoplastic epidermis, and CSCC, YAP expression was significantly increased relative to normal epidermis. This emerging evidence suggests that the dysregulated activity of the Hippo signalling pathway effector YAP represents a frequent event during canine epidermal tumourigenesis, pointing to this protein as a potential therapeutic target for the treatment of CP and CSCC in dogs.

Abstract P5-04-26: Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers

Abstract Though CTLA4 expression was reported in various tumor types, expression of CTLA4 is not well known in breast cancers, especially HER2-positive breast cancers. TILs level has significant role in treatment response and prognosis in HER2-positive breast cancers. Total 167 patients with HER2-positive breast cancers were included. All patients were pre-operatively chemotherapy and radiotherapy naïve. All received adjuvant standard treatment. Histologic TILs evaluation was performed with full face hematoxylin and eosin sections. Immunohistochemistry for CTLA4 was performed with tissue microarray sections. CTLA4 expression was evaluated separately for tumor cells and TILs. Cytoplasmic and/or membranous stains were assessed and human appendix was used as positive control. The intensity and percentage of positivity were evaluated collectively. Immunostaining score was calculated by multiplying percentage of positive cells by staining intensity. Total immunostaining score was obtained by adding tumor cells score and TILs score. Expression level was divided into two categories (low and high) according to mean value. Only 11 patients (6.6%), 47 patients (28.1), and 10 patients (6.0) showed total negative staining for CTLA4 on tumor cells, TILs and both on tumor cells and TILs, respectively. According to mean score on tumor cells, 97 (58.1%) patients showed low level of CTLA4 expression and 70 (41.9%) showed high level of CTLA4 expression. One hundred and two (61.1%) patients had low expression and 65 (38.9%) patients had high expression of CTLA4 on TILs based on the mean value of score of TILs. According to mean value of total score both on tumor cells and TILs, 70 patients (41.9%) were categorized as high expression and 97 patients (58.1%) were categorized as low CTLA4 expression. TILs level, CTLA4 immunostaining score on tumor cells, CTLA4 immunostaining score on TILs, and total CTLA4 immunostaining score on tumor cells and TILs were significantly correlated each other. High expression level of CTLA4 on tumor cells was significantly associated with negative expression of hormone receptor and high TILs level. Also, high CTLA4 expression on TILs significantly correlated with high histologic grade, negative expression of hormone receptor and high TILs level. High level of total CTLA4 expression both on tumor cells and TILs was significantly correlated with negative expression of hormone receptor and high TILs level. However, expression level of CTLA4 was not associated with overall survival and disease-free survival. In conclusions, CTLA4 expression was common in HER2-positive breast cancers and the level of CTLA4 expression was associated with hormone receptor negativity and TILs level in HER2-positive breast cancers. Table 1. Correlation of TILs level, CTLA4 immunostaining score on tumor cells, TILs, and total immunostaining scoreimmunostaining score on tumor cellsimmunostaining score on TILsTotal immunostaining score on tumor cells and TILsTILs level0.254 (&amp;lt;0.001)0.394 (&amp;lt;0.001)0.312 (&amp;lt;0.001)Immunostaining score of CTLA-4 in tumor cells0.565 (&amp;lt;0.001)0.815 (&amp;lt;0.001)Immunostaining score of CTLA-4 in TILs0.783 (&amp;lt;0.001) Table 2. Correlation between CTLA-4 expression and clinicopathologic factorsLowHighLowHighLowHighHistologic grade1.0000.0390.754Low (grade 1, 2)51 (52.6)36 (51.4)60 (58.8)27 (41.5)52 (53.6)35 (50.0)High (grade 3)46 (47.4)34 (48.6)42 (41.2)38 (58.5)45 (46.4)35 (50.0)Necrosis0.3251.0000.325Absent30 (30.9)27 (38.6)35 (34.3)22 (33.8)30 (30.9)27 (38.6)Present67 (69.1)43 (61.4)67 (65.7)43 (66.2)67 (69.1)43 (61.4)Lymphovascular invasion0.7540.2650.754Absent51 (52.6)39 (55.7)51 (50.0)39 (60.0)51 (52.6)39 (55.7)Present46 (47.4)31 (44.3)51 (50.0)26 (40.0)46 (47.4)31 (44.3)Hormone receptor expression0.0120.0400.005Negative37 (38.1)41 (58.6)41 (40.2)37 (56.9)36 (37.1)42 (60.0)Positive60 (61.9)29 (41.4)61 (59.8)28 (43.1)61 (62.9)28 (40.0)TILs level0.001&amp;lt;0.001&amp;lt;0.001≤10%42 (43.3)19 (27.1)50 (49.0)11 (16.9)44 (45.4)17 (24.3)20-60%45 (46.4)29 (41.4)43 (42.2)31 (47.7)43 (44.3)31 (44.3)&amp;gt;60%10 (10.3)22 (31.4)9 (8.8)23 (35.4)10 (10.3)22 (31.4) Citation Format: Ahrong Kim, Jee Yeon Kim, So Jeong Lee. Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-26.

Abstract P3-08-12: An open source, automated tumor infiltrating lymphocyte algorithm for prognosis in triple-negative breast cancer

Abstract Background: The presence of high TILs (tumor infiltrating lymphocytes) have been shown to be predictive of response to chemotherapy and is also a prognostic factor associated with better outcome in breast cancer, especially in early stage triple-negative (TNBC) and HER2-positive breast cancers. TIL assessment, while now more standardized due to the efforts of Salgado and the International TIL Working Group (https://www.tilsinbreastcancer.org/), are still a subjective test with variability in evaluation that has prevented broad adoption. Given the advances in application of artificial intelligence to pathology images, we believe the next step for TILs is to make them automated and objective and to identify a standardized and meaning TIL cut-point. The aim of this study is to build an open source, H&amp;E image-based automated TIL assessment algorithm for breast cancer that allows global standardization of TILs for prognostic value. Materials and methods: Using QuPath open source software, we first built a neural network classifier for image-based, automated assessment of TILs. It distinguishes tumor cells, lymphocytes, stromal cells and other cells on hematoxylin-eosin (H&amp;E) stained sections. We then defined “eTILs%” calculated as follows for the percentage of machine defined TILs: (TILs/TILs+Tumor cells) *100. A retrospective collection of 63 TNBC cases was used as the training set (Set A) and then tested for cell classifier accuracy and the optimal “eTILs%” cut point. The validation sets were a retrospective collection of 354 TNBC patients comprised of three independent validation subsets (Set B; N=87, Set C; N=183, and Set D; N=83) in both tissue microarray (TMA) and whole tissue section (WTS) format. Results: Using an optimal cut point (30%) derived from TNBC cohort training set A, patients with high eTILs% displayed an overall survival benefit (HR 0.4, p=0.0150). This algorithm was then applied in other three TNBC validation sets (Set B: HR=0.42, p=0.0033; Set C: HR=0.42, p=0.0127; Set D in TMA format: HR=0.39, p=0.0089). For Set D, we also tested WTS format which showed HR=0.23, (p=0.0155). The validation sets were combined to assess independence from clinical status in a multi-variable analysis where eTILs% was independently associated with improved overall survival (HR=0.35, p&amp;lt;0.0001). Conclusion: We have constructed a single institutional algorithm built from open source QuPath software that is a robust and independent prognostic factor. With further validation in tissues from other institutions and larger cohorts, this algorithm could be potentially useful as a broadly applicable, standardized and globally available method for assessment of TILs in triple negative breast cancer patients. Citation Format: Yalai Bai, Balazs Acs, Jon Zugazagoitia, Sandra Martinez-Morilla, Fahad Shabbir Ahmed, David L. Rimm. An open source, automated tumor infiltrating lymphocyte algorithm for prognosis in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-12.

Association between low estrogen receptor positive breast cancer and staining performance

Estrogen receptor (ER) expression in breast carcinomas, determined by immunohistochemistry, indicates statistically significant benefit to endocrine therapy in patients with tumors expressing ER in ≥1% of tumor cells. Rare cases with low ER expression (1–10%) lead to the dilemma of treating these tumors as ER positive or negative. We hypothesize that low ER positive result from poor staining performance and that we may detect this artefact by assessing the average dynamic range of normal ducts adjacent to low ER positive tumors. Using quantitative tools, we compare the dynamic range of normal background ER expression in patients with low (1–10%) ER tumors to dynamic range of ER expression in normal epithelium from control patient populations, to determine if low ER cases are accompanied by decreased dynamic range. Low ER cases were infrequent (1% of invasive breast carcinomas). Twenty-one cases with low ER staining and two control cohorts, including a tissue microarray (TMA) of 10 benign breast sections and a group of 34 control breast carcinomas (reported as ER negative or >10% ER positive) with normal background epithelium, were digitally scanned. QuPath was utilized to quantify ER staining for each cell as the mean optical density of nuclear DAB staining. The dynamic range of ER expression in normal epithelium surrounding low ER tumors was significantly lower (range 2–240, median 16.5) than that of the benign epithelium in the control tumors (range 3–475, median 30.8; p < 0.001) and benign TMA sections (range 38–212, median 114; p < 0.001) suggesting inconsistent stainer performance.