EnVision FLEX Research Articles

A Prospective Study to Evaluate the Prevalence of Microsatellite Instability in Endometrial Carcinoma by using Immunohistochemistry for Mismatch Repair Proteins as a Surrogate Marker

Abstract Aim Use of immunohistochemistry for mismatch repair (MMR) proteins to identify the prevalence of microsatellite instability (MSI) in cases of endometrial carcinoma and its subsequent correlation with various histopathological parameters. Materials and Methods The expression of MMR proteins, viz PMS2, MLH1, MSH2, and MSH6, were assessed in 114 endometrial cancer cases by immunohistochemistry using Dako EnVision FLEX system, on paraffin blocks of tumor tissue fixed in 10% formalin. Results We studied 114 endometrial cases for MMR protein expression, of which the majority were of endometrioid histologic subtype (n = 93, 81.6%), whereas the remainder comprised serous carcinoma (n = 12, 10.5%), clear cell carcinoma (n = 1, 0.9%), carcinosarcoma (n = 5, 4.4%), and dedifferentiated uterine carcinoma (n = 3, 2.6%). Twenty-one (18%) of these cases were found to be deficient for MMR proteins, of which 20 were of endometrioid histologic subtype and only 1 was dedifferentiated uterine carcinoma. Loss of MMR protein expression occurred in pairs of either PMS2 and MLH1 or MSH2 and MSH6. Conclusion MSI is one of the major molecular pathways contributing to tumorigenesis in endometrial carcinomas. Immunohistochemistry for MMR proteins is a highly sensitive and cost-effective alternative for molecular testing for MSI. It is also a great tool for screening patients for Lynch syndrome. Immunohistochemical testing for MMR should be offered to all patients of endometrial cancers.

Abstract 2535: Development of Claudin 18.2 IHC 14G11 pharmDx as a clinical trial assay for the clinical development of anti-claudin 18.2 monoclonal antibody osemitamab (TST001) in gastric and gastroesophageal junction adenocarcinoma

Abstract Clone 14G11 is a mouse anti-Claudin 18.2 monoclonal antibody developed by Transcenta. The antibody was generated against a linear epitope located on the extracellular domain of loop1 and has a binding site that overlaps with the binding site of therapeutic antibody Osemitamab. It was selected to further develop for the detection of Claudin 18.2 expression in human cancer. Agilent Technologies, Inc. is developing Claudin 18.2 IHC 14G11 pharmDx as an immunohistochemical (IHC) assay for the detection of Claudin 18.2 protein in gastric and gastroesophageal junction (GEJ) adenocarcinoma in support of clinical studies conducted by Transcenta Therapeutics. Analytical verification for Claudin 18.2 IHC 14G11 pharmDx included sensitivity, specificity, and precision assessment. Testing was performed on human formalin-fixed, paraffin-embedded (FFPE) gastric and GEJ tissues using Autostainer Link 48 with EnVision FLEX visualization system. Biomarker expression was evaluated using Tumor Proportion Score (TPS). TPS is defined as the percentage of viable invasive tumor cells that exhibit convincing partial or complete membrane staining at any intensity. Sensitivity testing of 102 commercially procured FFPE gastric/GEJ specimens demonstrated a broad dynamic range of Claudin 18.2 positivity that covered TPS from 0% to 100%. Specificity tests confirmed specific targeting of the Claudin 18.2 protein in a variety of normal and abnormal tissues. For laboratory precision testing, which evaluated the assay variability in relation to run, instrument, operator, and reagent lot, ≥ 95% of TPS values were within expected variability limits for each parameter. For observer precision testing, two-sided 95% confidence intervals were ≥ 85% for negative, positive, and overall agreements when assessing two clinical cutoffs. These results demonstrated the Claudin 18.2 IHC 14G11 pharmDx assay can provide reliable and reproducible results in day-to-day testing. Analytical verification testing indicates Claudin 18.2 IHC 14G11 pharmDx is a sensitive, specific, and precise assay for detecting Claudin 18.2 in gastric and GEJ adenocarcinoma. Claudin 18.2 IHC 14G11 pharmDx for Investigational Use Only/for Performance Evaluation Only will be used for patient selection in the upcoming phase III trial of gastric/GEJ adenocarcinoma where applicable ethics committee and regulatory approvals have been granted. Citation Format: Hayley Alderson, Katie Lefley, Lesly Fregoso, Achintya Bharadwaj, Dan Castellanos-McKenney, Siena Tabuena-Frolli, Donna Kell, Xinlai Yao, Xueming Qian, Li Xu, Caroline Germa, Emin Oroudjev. Development of Claudin 18.2 IHC 14G11 pharmDx as a clinical trial assay for the clinical development of anti-claudin 18.2 monoclonal antibody osemitamab (TST001) in gastric and gastroesophageal junction adenocarcinoma [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 2535.

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.

Immunohistochemical Identification and Assessment of the Location of Immunoproteasome Subunits LMP2 and LMP7 in Acquired Cholesteatoma.

Cholesteatoma, accompanied by chronic inflammatory response, is characterized by invasive growth and osteolytic activity. As specific proteasome isoforms, the immunoproteasomes serve as an important modulator of inflammatory responses. The aim of the present study was to determine the biological activity of cholesteatoma through the analysis of the expression and localization of immunoproteasome subunits of low molecule weight protein (LMP) 2 and LMP7. Cholesteatoma specimens were obtained from 15 adults who underwent ear surgery due to acquired attic cholesteatoma. Normal skin specimens were taken from retro-auricular skin incisions from the same patients. The specimens were stained with anti-LMP7 antibody, using immunohistochemistry techniques based on the binding of biotinylated secondary antibody with the enzyme-labeled streptavidin and the Envision FLEX system. In all specimens of cholesteatoma, the immunohistochemical reaction with the antibody against the LMP2 was positive, in both the cytoplasm of the cholesteatoma matrix and the perimatrix. A negative reaction with anti-LMP2 was observed in the cytoplasm and nuclei of control skin cells. A positive nuclear and cytoplasmic immunohistochemical reaction with anti-LMP7 has been demonstrated in numerous cells, in both the matrix and perimatrix of cholesteatoma. We present evidence of the presence of expressions of LMP2 and LMP7 within cholesteatoma tissue. Our results might bring new information concerning immunoproteasome-dependent pathophysiologic mechanisms in cholesteatoma.

Low-Grade Mammary Gland Tumors in Dogs Have Greater VEGF-A and BMP2 Immunostaining and Higher CD31 Blood Vessel Density

Tumor angiogenesis is an important process in tumor growth, and different molecules are involved in its regulation including VEGF-A, BMP2, and CD31, which can be considered possible prognostic markers. The aim of this study was to verify whether the VEGF-A and BMP2 immunostaining area, and microvascular density (MVD) might be associated with the degree of malignancy in malignant mammary neoplasms of dogs. For this purpose, samples of mammary malignancies from female dogs embedded in wax were used and separated into 4 main histomorphological types: tubulopapillary carcinomas, solid, complex, and carcinosarcoma, which were separated based on high and low degrees of malignancy. Immunohistochemical analysis was performed on tissue microarray blocks using anti-CD31 antibodies for evaluation of MVD and vascular lumen area, and with anti-VEGF-A and anti-BMP2 to determine the immunostaining area using the DAKO EnVision FLEX+ kit. MVD and vascular lumen area were higher in tubulopapillary carcinomas as were the areas stained by VEGF-A and BMP2. Immunostaining for CD31 was higher in low-grade carcinomas as well as in areas immunostained by VEGF-A and BMP2. There was a positive correlation between VEGF and BMP2 in high (r = 0.556, P < .0001) and low-grade (r = 0.287, P < .0001) carcinomas and between MVD and VEGF-A in low-grade carcinomas (r = 0.267, P = .0064). Thus, the markers evaluated showed greater immunostaining in canine mammary tumors with a lower degree of malignancy.

Programmed Death Ligand 1 (PD-L1) Expression and its Association with Driver Mutations among Patients with Non-Small Cell Lung Cancer in a Private Tertiary Care Setting

Objective. The advent of immunotherapy has significantly changed the treatment and management of patients with advanced non-small cell lung cancer (NSCLC). Prior to initiation of immunotherapy,evaluation of programmed death ligand 1 (PD-L1) expression is required. One factor that affects PD-L1 expression in NSCLC is the presence of oncogenic driver mutations; however, little data on itsassociation is available, especially in the Philippine setting. The study aims to determine the prevalence of PD-L1 expression and its association with driver mutations among patients with non-small cell lung cancer in a private tertiary care hospital in the Philippines.Methodology. The study was undertaken for a period of two years from July 2017-July 2019 at St. Luke’s Medical Center and included 446 NSCLC samples. PD-L1 was evaluated by immunohistochemistry using 22C3 anti-PD-L1 antibody clone, EnVision FLEX visualization system on Autostainer Link 48. Patient demographics and data on driver mutation testing were recorded. Statistical analysis was performed using logistic regression.Results. PD-L1 expression was observed in 273 (61.20%) of 446 cases, 119 (61.20%) of whichdemonstrated high PD-L1 expression while 154 (34.50%) had low PD-L1 expression. There was nosignificant association between PD-L1 expression and EGFR mutation, ALK mutation, age, and gender.For histologic type, high PD-L1 expression was significantly associated with adenocarcinoma and non-small cell carcinoma, NOS.Conclusion. The overall prevalence of PD-L1 expression in non-small cell lung carcinoma is 61.20%based on the cases included. Although we did not find an association between PD-L1 expression andEGFR and ALK mutation, our study observed that ALK-mutated NSCLCs have 4.7 odds of having highPD-L1 expression, however, a higher sample size is warranted to truly determine significant association. The outcome of this study may provide help in the stratification of patients and predict those who will benefit from immunotherapy.

Expression of Cyclin D1 in Cutaneous Xanthogranuloma and Solitary Reticulohistiocytoma.

Expression of Cyclin D1 in Cutaneous Xanthogranuloma and Solitary Reticulohistiocytoma.

P982: MYELOID-DERIVED SUPPRESSOR CELLS: CHARACTERIZATION IN PERIPHERAL BLOOD AND SPLEEN TISSUE OF PATIENTS WITH MYELOFIBROSIS

Background: Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that accumulate in patients with malignancies, sepsis or chronic inflammation. Features of MDSCs are: myeloid origin, immature state, strong ability to reduce cytotoxic functions of T/NK cells, potential to differentiate into endothelial cells favoring neoangiogenesis. MDSCs are conventionally divided into polymorphonuclear (PMN)- and monocytic (M)-MDSCs. PMN-MDSCs are characterized by the production of reactive oxygen species and arginase-1 (Arg-1), that directly inhibits T-cell proliferation and activation, while M-MDSCs predominantly express inducible nitric oxide synthase and produce nitric oxide. Myelofibrosis (MF) is characterized by clonal expansion of a hematopoietic stem cell, extramedullary hematopoiesis, bone marrow (BM) fibrosis, splenomegaly, BM and splenic neoangiogenesis and an acquired mutation of JAK2, CALR or MPL gene. Inflammation plays a relevant role in MF pathogenesis, as proven by high levels of inflammatory cytokines with prognostic significance and by a state of chronic oxidative stress. Aims: To characterize circulating MDSCs and assess their localization and quantification in spleen samples from MF patients. Methods: Peripheral blood mononuclear cells (PBMCs) from MF patients (n=15) and healthy subjects (HDs; n=10) were stained with surface antibodies (CD11b, CD14, HLA-DR, CD15, CD33) and intracellular Arg-1 and analysed by flow cytometry. Formalin-fixed paraffin-embedded samples of spleen specimens (MF n=41; HDs n=20) were immunostained with anti-Arg-1 antibody using the automated platform Dako Omnis Envision Flex with the EnVision FLEX, High pH, HRP Rabbit/Mouse High pH [GV800] revelation kit. Results: In PBMCs, the percentage of PMN-MDSCs (identified as CD11b+HLA-DRlow/-CD33dimCD14-CD15+) was higher (p=0) in patients (median 9.2, range 1-59) than HDs (median 1.7, range 0.1-4.5) and directly correlated with the allelic burden (R=0.86, p=0.006) in JAK2-mutated patients. Patients in pre-fibrotic phase (n=7) had levels of PMN-MDSCs (median 9.2, range 1-31.4) comparable to HDs; on the contrary, the percentage of PMN-MDSCs in patients with BM fibrosis (n=8; median 13.3, range 3.5-59) was higher (p=0) than in HDs. All PMN-MDSCs were Arg-1+, both in MF and in HDs. Gene expression studies are ongoing. The expression of the CXCR4 receptor on PMN-MDSCs was lower, although not statistically significant, in MF (median 1.1, range 0-9.3) than in HDs (median 2.5, range 0-11.9). PMN-MDSCs in MF inversely correlated (R=-0.89 p=0.007) with CXCR4 expressed on CD34+ cells. The percentage of M-MDSCs (identified as CD11b+HLA-DRlow/-CD33hiCD14+CD15-) was higher (p=0.015) in HDs (median 0.49, range 0.25-1.5) than in MF (median 0.29, range 0.02-0.76). Immunohistochemical staining of spleen sections showed a higher (p=0) percentage of Arg-1+ cells (calculated on total spleen cells) in MF (median 20, range 5-60) than in HDs (median 7, range 2-20); Arg-1+ cells were present in all patients and predominantly located in the red pulp in MF patients, while in HDs were detected in the white pulp. Summary/Conclusion: The identification of MDSCs and their relation with the mutational status suggest a new mechanism of MF pathogenesis, either related to the chronic inflammatory status of patients or as players of the neoangiogenesis that characterizes the disease. Moreover, the low expression of CXCR4 on PMN-MDSCs could be, on one hand, related to the low expression of this receptor on CD34+ cells or suggest a MF-specific recruitment mechanism, other than that observed in solid tumors.

Роль ангиотензинпревращающего фермента и рецепторов ангиотензина ii второго типа в патогенезе пролиферативных заболеваний предстательной железы

Angiotensin converting enzyme, angiotensin II, angiotensin II receptors of the first and second types represent the "classical" axis of regulation of the renin-angiotensin system. OBJECTIVE: to analyze the role of the components of the renin-angiotensin system in the pathogenesis of proliferative lesions of the prostate glan MATERIALS AND METHODS: The study included 63 patients who underwent transrectal prostate biopsy. The first group consisted of 19 patients with benign prostatic hyperplasia, the second group consisted of 19 men whose prostate cancer was detected during repeated biopsy, the third group consisted of 25 men with prostate cancer detected during primary prostate biopsy. The expression of angiotensin II type II (AT2-R) receptors in prostate tissue was evaluated using primary polyclonal antibodies Angiotensin II Type 2 Receptor and the EnVision FLEX imaging system (Dako, Denmark) according to a standard technique. The activity of angiotensin converting enzyme (ACE) was determined in the secret of the prostate gland, RESULTS: It was found that the activity of ACE in the secret of the prostate gland in proliferative diseases is significantly higher than in the "healthy" prostate. The highest activity of ACE was noted for benign prostatic hyperplasia, and the minimum - for prostate cancer. The expression of AT2-R in prostate tissues in proliferative diseases of the prostate gland has its own characteristics. The expression of AT2-R in the prostate stroma turned out to be the same, in the nuclei of epithelial cells, the level of expression of AT2-R decreased in the range of BPH-PIN-CP. Thus, an increase in the activity of ACE, the accumulation of angiotensin II in prostate secretions in proliferative prostate diseases against the background of a deficiency of AT2-R is the metabolic basis of malignant transformation of the prostate gland. CONCLUSION: The levels of ACE activity in prostate secretion and the expression of AT2-R in prostate tissue during primary prostate biopsy can be considered as promising prognostic tools for early detection of prostate malignancy.

Content and location of lymphocyte subpopulations with markers CD4+, CD8+ and CD20+ in the esophageal tonsil of chickens and the Meckel diverticulum of ducks

Immune formations of birds' digestive organs, including the esophageal tonsil and Meckel’s diverticulum, protect the body from foreign antigens that enter the body with food and water and play an important role in maintaining the genetic constancy of its internal environment. This unique property of the immune system is formed during ontogenesis and is associated with maintaining the selection of lymphocyte clones that are able to respond to foreign antigens and carry out a specific immune response of two types: humoral and cellular. This article presents the results of a study of T- and B-lymphocyte subpopulations of the esophageal tonsil of Shever 579 cross chickens at the age of 25, 180 and 300 days, the Meckel diverticulum of the Blagovarsky cross ducks at the age of 30, 150 and 180 days. Immunohistochemical and statistical research methods were used to determine the localization and quantitative parameters of cell populations of lymphocytes (CD4+, CD8+, CD20+) using monoclonal antibodies and the DAKO EnVision FLEX+ imaging system (Dako Cytomation, Denmark). Separate subpopulations of T-lymphocytes (CD4+ - helpers, CD8+ -cytotoxic / T-suppressors) and mature B-lymphocytes (CD20+) were found in the esophageal tonsil and Meckel diverticulum of birds. Their presence confirms that antigen-independent proliferation and differentiation of lymphocytes into effector cells occur in the immune formations of the digestive system. The lymphoid tissue of these formations is represented mainly by a well-defined diffuse form and nodules with light centers (secondary). In the esophageal tonsil of chickens, these structures are located in the tunica mucosa and tela submucosa, and in the Meckel diverticulum of ducks – also in the tunica muscularis. The content of lymphocytes with these markers predominates in diffuse lymphoid tissue compared to that in secondary lymphoid nodules. In the diffuse lymphoid tissue of the esophageal tonsil, lymphocytes are located mainly near the adenomeres and excretory ducts of the esophageal glands, blood vessels, and under the surface epithelium, and in Meckel’s diverticulum – around the crypts, in their epithelium and in the epithelium of the villi. They are also found in the light centers of lymphoid nodules and on their periphery. The indices of the content of lymphocytes with the indicated markers in the esophageal tonsil and Meckel diverticulum which we determined were associated with age characteristics of the poultry in the postnatal period of ontogenesis. According to our observations, the content of CD20+ lymphocytes was the highest, while the populations of CD4+ and CD8+ lymphocytes were much smaller. This indicates an increase in the activity and predominance of the humoral immunity over the cellular one. The content of CD20+ lymphocytes was highest in birds at the age of 180 days, that is, during their sexual maturity. The data presented in the work can be used by morphologists researching the organs of the immune system, immunologists, poultry specialists involved in breeding, using and raising poultry and in educational work.

Validation of an immunohistochemical assay, CEACAM5 IHC 769, under development for use with the antibody-drug conjugate tusamitamab ravtansine (SAR408701).

e21030 Background: Despite recent advances in non-small cell lung cancer (NSCLC) treatment, a need remains for treatment options at disease progression. Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate of a humanized CEACAM5-specific monoclonal antibody linked to the maytansinoid DM4, a potent anti-tubulin agent. In a Phase 1/2 study, tusamitamab ravtansine showed promising antitumor activity in pretreated advanced non-squamous NSCLC (NSQ NSCLC) patients with high CEACAM5 expression, defined as ≥ 50% of the tumor cells per specimen with CEACAM5 positive staining at ≥ 2+ intensity (Gazzah A et al. J Clin Oncol 2020;38[15]: 9505). Sanofi and Agilent have partnered to develop an immunohistochemical (IHC) assay, CEACAM5 IHC 769, for detection of CEACAM5 protein in formalin-fixed paraffin-embedded (FFPE) NSQ NSCLC tissue using the EnVision FLEX visualization system on Dako Omnis. This assay is in development for the assessment of patients for whom tusamitamab ravtansine is being considered. Methods: CEACAM5 IHC 769 is a qualitative IHC assay using mouse monoclonal anti-CEACAM5 clone 769. Murine anti-CEACAM5 clone 769 is based on tusamitamab ravtansine, with the same specificity as the drug for CEACAM5. The clone was developed by Sanofi and used to evaluate CEACAM5 expression in a Phase 1/2 trial. The CEACAM5 IHC 769 scoring algorithm defines positive staining as any partial or complete tumor cell plasma membrane staining at ≥ 2+ intensity. Membrane staining may be linear or punctate. Staining may appear as complete or partial staining involving the basal, lateral, or basolateral aspects of the membrane, or as apical staining of the luminal aspect of the cell. CEACAM5 IHC 769 is being validated by Agilent Technologies for NSQ NSCLC at the cutoff of ≥ 50% at ≥ 2+ intensity. Internal studies included sensitivity, specificity, intra-block heterogeneity, observer precision, laboratory repeatability/precision, and robustness. Results: Sensitivity studies demonstrated detection across a dynamic range of CEACAM5 expression. Specificity studies indicated that the antibody is specific for CEACAM5. An intra-block heterogeneity study showed good overall diagnostic agreement for sections from the same tumor (point estimate ≥ 85%). Inter- and intra-observer precision, laboratory repeatability/precision, and robustness validation studies met acceptance criteria; the lower bound of the two-sided 95% confidence interval was ≥ 85% for positive, negative, and overall percent agreement. Conclusions: Internal validation studies demonstrated that CEACAM5 IHC 769 is sensitive, specific, precise, reproducible and robust in evaluating CEACAM5 expression in NSQ NSCLC tissue at the cutoff of ≥ 50% at ≥ 2+ intensity. CEACAM5 IHC 769 is being used for patient selection/stratification in ongoing Phase 2 and 3 clinical trials of tusamitamab ravtansine.

A simple digital image analysis system for automated Ki67 assessment in primary breast cancer.

Ki67 is a well-established immunohistochemical marker associated with cell proliferation that has prognostic and predictive value in breast cancer. Quantitative evaluation of Ki67 is traditionally performed by assessing stained tissue slides with light microscopy. Automated image analysis systems have become available and, if validated, could provide greater standardisation and improved precision of Ki67 scoring. Here, we aimed to evaluate the use of the Cognition Master Professional Suite (CogM) image analysis software, which is a simple system for scoring Ki67 in primary breast cancer samples. Sections from 94 core-cut biopsies, 20 excision specimens and 29 pairs of core-cut biopsies and excision specimens were stained for Ki67 with MIB1 antibody and the Dako EnVision FLEX Detection System. Stained slides were scanned to convert them to digital data. Computer-based Ki67 scoring was performed with CogM. Manual Ki67 scoring assessment was conducted on previously stained sections from the same biopsies with a clinically validated system that had been calibrated against the risk of recurrence. A high correlation between manual and digital scores was observed [rCores =0.92, 95% confidence interval (CI) 0.87-0.94, P<0.0001; rExcisions =0.95, 95% CI 0.86-0.98, P<0.0001] and there was no significant bias between them (P=0.45). There was also a high correlation of Ki67 scores between paired core-cut biopsies and excision specimens when CogM was used (r=0.78, 95% CI 0.59-0.89, P<0.0001). CogM image analysis allows for standardised automated Ki67 scoring that accurately replicates previously clinically validated and calibrated manual scores.

Expression of Hedgehog signalling molecules inmicrocystic adnexal carcinoma.

Microcystic adnexal carcinoma (MAC) is a rare skin neoplasm that has not been characterized on a molecular basis. To assess expression profiles of Hedgehog (HH) signalling molecules in MAC and control tumours. Immunohistochemistry was performed for Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched 1 (PTCH1) and Smoothened (SMO) on patient MAC tissue (n=26) and control tumour tissue, including syringoma (SyG; n=11), trichoepithelioma (TE; n=11) and basal cell carcinoma (BCC; n=12) tissues. Patched 1 and SMO immunoreactivity was significantly higher in BCC than in SyG, TE or MAC (P<0.001 and P<0.03, respectively). The highest IHH expression was observed in BCC and TE compared with SyG and MAC (P<0.04). Notably, the highest SHH protein expression was observed in SyG compared with MAC, TE and even BCC (P<0.001). In patients with MAC, SMO immunoreactivity significantly (r=0.51; P<0.01) correlated with PTCH1 expression. Further correlation studies did not show significant associations between the HH expression markers assessed (P>0.05). Our results indicate that alterations of the HH signalling are unlikely to play a major role in the pathogenesis of MAC, which is in contrast to the morphologically similar BCC and TE. Our observation provides additional information to the limited molecular pathology knowledge on this rare tumour.

Immunohistochemical Analysis of Mismatch Repair Deficiency in Colorectal Cancer Patients in Kuantan, Pahang

INTRODUCTION: The deficient mismatch repair (dMMR) status of colorectal cancer (CRC) is the hallmark of a defective DNA mismatch repair (MMR) system. Immunohistochemistry (IHC) indirectly detects the affected gene by the loss of its protein product. This study aimed to assess the frequency of different types of dMMR status and associate them with the clinicopathological characteristics of CRC patients diagnosed at Hospital Tengku Ampuan Afzan (HTAA) and the Sultan Ahmad Shah Medical Centre (SASMEC) using an immunohistochemical method. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue blocks of 123 CRC cases were retrieved from the Pathology Department of HTAA and SASMEC for patients diagnosed between 1 January 2017 and 31 December 2018. IHC was performed manually using an Envision Flex Polymer detection kit (Dako) along with four primary anti-mouse antibodies (MLH1, PMS2, MSH2, and MSH6) for the MMR proteins to assess dMMR status. RESULTS: Out of 123 cases, 21 (17.07%) showed the loss of one or more MMR protein expression and were dMMR. There was no statistically significant association between pMMR (proficient Mismatch repair) and dMMR cases with regards to clinicopathological factors (age, sex, race, site of the tumour, TNM (Tumour Node Metastasis) staging, bowel wall invasion, lymph node metastasis, lymphovascular invasion, histological type, and tumour differentiation). CONCLUSION: IHC is the preferred method and most reproducible test for assessing dMMR status in CRC patients in the histopathology diagnostic laboratory.

Effects of immunohistochemical conditions on the results of PD-L1 (22C3) staining

Objective: To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection. Methods: Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A. Results: In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A's classification (positive vs. negative) and would not change the choice of clinical treatments. Conclusions: The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.

Development of a clinical trial immunohistochemistry (IHC) assay using a novel antibody to CD38.

7561 Background: CD38 is a type II transmembrane glycoprotein expressed on normal lymphoid and myeloid cells and can be highly expressed in hematologic malignancies. An IHC prototype assay was developed to detect CD38 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens from three relapsed or refractory non-Hodgkin’s lymphoma (NHL) subtypes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL) for selection of patients for treatment with daratumumab in a Phase 2 clinical trial (NCT02413489). Methods: This assay is based on EnVision FLEX IHC technology using CD38, clone DAK-CD38, primary antibody that has been developed and manufactured by Agilent Technologies. The assay staining protocol was developed for Dako PT Link and Autostainer Link 48. Specificity of DAK-CD38 staining was demonstrated by Western blot analysis of cancer cell lysates, as well as IHC on both normal and cancer tissue. Assay precision and robustness were evaluated using commercially procured FFPE DLBCL, FL and MCL specimens. Results: CD38 IHC DAK-CD38 detected a broad range of CD38 expression in DLBCL, FL, and MCL specimens. FFPE specimens derived from cancer cell lines exhibited a range of IHC staining intensity and confirmed the reported expression of CD38 in the scientific literature. All precision and robustness results met acceptance criteria for both IHC intensity and percent positive cells staining. Conclusions: Our studies demonstrate that the Dako CD38 IHC DAK-CD38 assay is sensitive, specific, precise, and robust for the detection of CD38 expression in DLBCL, FL and MCL. This assay may also have the potential to be used for the detection of CD38 in additional tumor types.

Development of a diagnostic PD-L1 immunohistochemical assay for nivolumab therapy in urothelial carcinoma.

e14585 Background: PD-L1 IHC 28-8 pharmDx is a qualitative assay developed by Agilent Technologies for the Autostainer Link 48 platform and is based on EnVision FLEX visualization technology and monoclonal rabbit anti-PD-L1, clone 28-8 antibody. The assay has been co-developed with the immunotherapeutic agent nivolumab; initially as an aid in assessing PD-L1 expression in non-squamous non-small cell lung cancer (NSCLC) and melanoma patients. Here we describe the efforts to validate this assay for urothelial carcinoma (UC). Methods: IHC staining was performed on Autostainer Link 48 platform using an automated staining protocol stated per the assay’s instruction for use (IFU). Specimens were coverslipped and interpreted for % PD-L1 positive tumor cells. The assay was analytically validated on commercially acquired formalin-fixed, paraffin-embedded (FFPE) human UC invasive tumor specimens at ≥1% and ≥5% PD-L1 positive tumor cells expression levels. Results: A wide range of % PD-L1 positive tumor cells at all staining intensity levels have been detected. Assay in-house precision was validated for inter-operator, inter-instrument, inter-day, inter-run and intra-run as well as inter-observer and intra-observer agreement. Robustness studies evaluated the assay under multiple conditions for target retrieval pH, temperature and incubation time, slide type as well as tissue section thickness. Assay reproducibility was evaluated at three external sites by testing samples for intra-site/inter-day and inter-site agreement measures. Specimens were also evaluated by an observer at each site, with three reads for each observer to assess intra-observer and inter-observer agreement. All validation studies demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 84%. Conclusions: Results of all conducted studies show high robustness and reproducibility of the assay on UC.

Assay performance of the PD-L1 IHC 28-8 pharmDx assay in squamous cell carcinoma of the head and neck (SCCHN).

e14588 Background: The PD-L1 IHC 28-8 pharmDx assay has been developed for use in detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-squamous NSCLC and melanoma tissues for associated treatment effect with nivolumab. Validation of the assay for measuring PD-L1 expression level in human squamous cell carcinoma of the head and neck (SCCHN) FFPE sections has been conducted and results are described. Methods: Visualization of the PD-L1 IHC 28-8 pharmDx assay is based on the EnVision FLEX IHC technology. As described in the Instructions For Use, antigen retrieval was performed using the Dako PT Link, and the automated staining was conducted with Autostainer Link 48. The assay performance was validated on commercially procured FFPE SCCHN specimens. Results: Assessment of assay sensitivity to PD-L1 expression was measured on 236 unique specimens originating from squamous cell carcinoma of the tongue, tonsil, nasopharynx, oropharynx, hypopharynx, and larynx. The study demonstrated PD-L1 positive staining across a range of 0-95% positive tumor cells and 0 to 3+ staining intensity. Among these tested specimens, 44% of specimens had PD-L1 expression levels ≥1%. Assay robustness was validated by assessing critical parameters such as target retrieval solution pH, target retrieval solution temperature, target retrieval time, and cut section thickness. Assay reproducibility was evaluated in a study at three external sites. Reproducibility of intra-site, inter-day, and inter-site agreement was assessed in addition to inter- and intra-observer agreement. Assay robustness and reproducibility demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 85%. Conclusions: These studies demonstrate that the PD-L1 IHC 28-8 pharmDx assay is reproducible and robust when used for automated detection of PD-L1 protein in FFPE human SCCHN specimens using Autostainer Link 48.

The complex morphologic diagnostic of marginal zone lymphoma.

The purpose of study: To determine possibilities of complex morphological diagnostic (routine cytology and histology, immunocytochemistry and immunohistochemistry, molecular genetics) of lymphoma of marginal zone. The study included 10 patients with diagnosis of lymphoma of marginal zone cells, established on the basis of application of morphological (cytological and histological), immunomorphologic (immunocytochemical and immunohistochemical) and molecular genetic techniques. The immunofenotyping was implemented using immunocytochemical technique (EnVision FLEX) applying monoclonal antibodies by DAKO manufacturer. The immunofenotyping was implemented by flow cytofluorometry technique (flow cytofluorometer FACS Calibur by Becton Dikinson, USA) using antibodies by DACO manufacturer labeled by fluorescent marks (FITC or RPE). The antibody panel included: common leukocytic antigen, common cytokeratins CD19, CD20, CD79a,CD10, Bcl2, Bcl6, CD23, CD34, TdT, CD3, CD4, CD5, CD8, CyclinD1, Ki67, κ, λ. The FISH technique was applied using probes Bcl2 FISH DNA Probe, Split Signal и MALT1 FISH DNA Probe Split Signal by DAKO manufacturer. In one patient a gene Bcl2 change was detected with purpose of differentiating diagnostic with follicular lymphoma. In three patients, diagnosing lymphoma of marginal zone was implemented by FISH technique using detection of translocation (11;18)(q21;q21), involving gene of inhibitor of apoptosis API2 and gene MALT1. The routine cytological analysis of ten cases permitted to establish an exact diagnosis of lymphoma only in five cases and with no indication that it is lymphoma of marginal zone. In two cases under routine cytological analysis only a suspicion about lymphoma was suggested. In all ten patients a positive expression of pan-B-cellular markers CD19, CD20, CD79a was observed both under immunocytochemistry and immunohistochemistry and flow cytofluorometry. In 6 patients (60%) a positive expression of gene Bcl2 was observed both under immunocytochemistry and immunohistochemistry and flow cytofluorometry. The comparison of immunocytochemistry and immunohistochemistry and flow cytofluorometry established absence of expression of CD5, CD3, CD10, CD34, CD23, Bcl6, TdT, cyclin D1. Under immunocytochemistry and immunohistochemistry in ten cases proliferative activity protein Ki-67made up no more than 30%. Under flow cytofluorometry clonality on light chains of immunoglobulins κ or λ (Igλ/Igκ) were established. Overall, correlation coefficient (r, p < 0,05) between immunocytochemistry and immunohistochemistry, flow cytofluorometry made up to 1. The four patients were applied FISH-reaction for adjustment of diagnosis. In patient with nodal lymphoma of marginal zone gene Bcl2 change was absent. In three patients with MALT-lymphoma a gene MALT1 change was established. Thereby, accuracy of routine cytological analysis at diagnosing lymphoma without indication of its type in case of lymphoma of marginal zone made up to 50%, sensitivity - 50%, specificity - 100%. The accuracy of immunofenotyping permitting diagnosing lymphoma of marginal zone made up to 100%, sensitivity - 100%, specificity - 100%. The correlation coefficient (r, p < 0,05) between data of immunocytochemistry and immunohistochemistry, flow cytofluorometry made up to 1. The accuracy, sensitivity and specificity of complex analysis (cytology, immunocytochemistry and FISH-technique) in diagnostic of lymphoma of marginal zone made up to 100%.

The Presence of Endometrial Cells in Peritoneal Fluid of Women With and Without Endometriosis

To reinforce Sampson's theory of retrograde menstruation in the pathogenesis of endometriosis, proof should be provided that during menstruation endometrial cells are present in peritoneal fluid (PF). We hypothesize that the prevalence of PF samples containing endometrial cells is higher in patients with endometriosis than in controls without endometriosis during menstruation. We selected from our biobank PF samples of 17 reproductive-age women with (n = 9) or without (n = 8) endometriosis who had received a diagnostic laparoscopy for investigation of pain/infertility. Peritoneal fluid had been collected during laparoscopy in the menstrual phase of the cycle, centrifuged, and the resulting pellet was stored at -80°C. About 5-(j,m sections of frozen PF pellets were stained using the Dako Envision Flex system with primary antibodies against epithelial cell adhesion molecule (Ep-CAM; endometrial epithelial cells), CD 10 (endometrial stromal cells), prekeratin (epithelial/mesothelial cells), vimentin (endometrial/mesothelial/immune cells), calretinin (mesothelial cells), and CD68 (macrophages). The PF cells positive for Ep-CAM were detected in 5 of 9 patients with endometriosis and 6 of 8 controls (P =.62). CD10 stained positively in 6 of the 9 patients with endometriosis and 3 of the 8 controls (P =.35). Calretinin and prekeratin staining showed the presence of mesothelial cells in all pellets. Vimentin stained approximately 100% of the PF cells. CD68+ macrophages represented >50% of cells in all pellets. The prevalence of PF samples containing endometrial epithelial and stromal cells was not higher in patients with endometriosis than in controls without endometriosis during menstruation. Our findings question the relevance of endometrial cells in PF for the pathogenesis of endometriosis and support the importance of other mechanisms such as immune dysfunction and/or endometrial stem cells.