Antibody Dako Research Articles

PD-L1 Expression in High-Risk Non-Muscle-Invasive Bladder Cancer Is Influenced by Intravesical Bacillus Calmette-Guérin (BCG) Therapy.

In the expanding landscape of immune checkpoint inhibitors (CPI) in high-risk (HR) non-muscle-invasive bladder cancer (NMIBC), the role of programmed death ligand 1 (PD-L1) as prognostic and predictive is increasingly significant. However, data evaluating its variability and susceptibility to Bacillus Calmette-Guérin (BCG) therapy in HR NMIBC patients is scarce. This retrospective study analyzed 126 HR NMIBC tissue samples from 63 patients (38× BCG-treated, 25× BCG-naïve) at two time points to assess PD-L1 expression using the 'combined positivity score' (CPS) with the 22C3 DAKO antibody method and correlated it with clinicopathological parameters. A CPS > 10 defined PD-L1 positivity. The impact of initial PD-L1 status and its change over time on time-to-recurrence, progression-free survival, and overall survival (TTR, PFS, OS) was analyzed using Kaplan-Meier and Cox proportional hazard models. BCG treatment significantly increased PD-L1 expression (5.31 vs. 0.22, p = 0.0423), with PD-L1 positive cases rising post-treatment in the BCG group and remaining unchanged in BCG-naïve patients. Multivariate analysis including T-stage, CIS, grading, tumor size, multifocality, age, and sex revealed a significant correlation between PD-L1 status change to positivity and improved TTR (p = 0.03). Our findings demonstrate a potential modulation of the PD-L1 status by an intravesical BCG therapy. However, its prognostic value appears limited.

Mismatch-Repair Deficiency in Follicular Lymphoma Tumors Is Common and Associated with a Favorable Overall Survival

Introduction: DNA mismatch repair (MMR) is a major mechanism cells use to repair erroneous insertion or deletion of bases during DNA replication. MMR deficiency (MMR-d), including rare germline mutations (Lynch syndrome) and somatic inactivation, has been extensively studied in solid tumors, such as colon, endometrial, pancreatic, and ovarian cancers. The prevalence of MMR-d in solid tumors ranges from 1% - 20% of somatic defects, and patients with MMR-d tumors have a longer overall survival. It is also well-known that MMR is an integral part of activation-induced cytidine deaminase (AID)-mediated somatic hypermutation and immunoglobulin class switch processes in normal mature B lymphocytes, while mice deficient in the MMR genes are prone to develop T and B cell lymphomas. It is also known that follicular lymphoma (FL) carries a high degree of somatic hypermutation with some heterogeneity induced by AID. Our understanding of MMR status in human blood cancers, especially in FL remains very limited. One could hypothesize that MMR-d status could permit cells to acquire and sustain high-degree of mutation rates, better sensitivity to DNA damaging chemotherapy while hindering rapid cell proliferation, which could manifest to a better prognosis in patients. Herein, we conducted an exploratory analysis to understand the frequency of MMR-d status and its prognostic significance in FL.Methods: Following approval of the Institution Review Board, we assessed the expression of MMR proteins; MLH1, PMS2 and MSH6 on formalin fixed paraffin embedded (FFPE) lymph node tissue obtained from patients with FL by immunohistochemistry (IHC) using standard methods on Ventana Benchmark XT automated stainers (Ventana Medical Systems, Tucson, AZ). Dako (clone ES05), Cell Marque (clone EPR3947) and Biocare (clone BC/44) antibodies were used to stain MLH1, PMS2 and MSH6, respectively. Fifty cases of FL with existing FFPE blocks were selected randomly. The expression of MLH1, PMS2 and MSH6 proteins in the tumor cells was scored as 0, +1 or +2, (Figure 1A). Cases with absence of the IHC signal (score of 0) in any one of the three proteins were classified as MMR-d. Progression free survival at 12 months (PFS12) or 24 months (PFS24) was defined as being free from disease progression or death at 12 or 24 months, respectively. All time-to-event analyses were done from the time of diagnosis.Results: Fifty patients with FL were included in the study. The median age at diagnosis was 65 years (range 34-85) and 58% were females. At initial diagnosis, 36 (72%) patients had advanced stage (stage 3, n=8, stage 4, n=28) disease. Of the 50 patients, 26 (52%) required treatment; chemotherapy (n=14), radiation treatment alone (n=9), surgery alone (n=3).Out of the 50 patients, 23 (46%) were classified as MMR-d due to lack of expression of one of the three assessed proteins: MLH1 (n=1, 2%), PMS2 (n=11, 22%) and MSH6 (n=19, 38%). The patient who had MLH1 loss had concomitant PMS2 loss. Stage IV disease was observed in 52% of the MMR-d patients and 59% in the MMR proficient (MMR-p) patients, p=0.77. Median age at diagnosis was 62 (range: 34-84) years and 67 (range: 34-85) years in MMR-d and MMR-p patients, respectively, p=0.1. FLIPI score was similar between the MMR-d [low n=10 (43%), intermediate n=9 (39%), high n=4 (18%)] and MMR-p [low n=5 (18%), intermediate n=14 (52%), high n=8 (30%)] patients, p=0.16. Fourteen (61%) of MMR-d patients required initiation of therapy upon diagnosis compared to 12 (44%) of MMR-p patients, p=0.27.The median follow-up for the entire cohort was 17 years [95% confidence interval (CI): 11.7-29.4]. The median overall survival (OS) of MMR-d patients was 10 years (95%CI: 7.8-20.7) compared to 6 years (95%CI: 4.5-NR) in MMR-p patients, p=0.036, Figure 1B. In patients with MMR-d and MMR-p, 83% and 55% achieved PFS12, p=0.04 and 65% and 41% achieved PFS24, p=0.08, respectively. After adjusting for FLIPI score, MMR status remained associated with OS (MMR-d HR: 0.51, 95%CI: 0.2-1.02, p=0.05).Discussion and Conclusion: In this pilot study, MMR-d in the FL tumors was common (46%) and, as predicted, was associated with a favorable OS. Association of MMR-d status with PFS in general and patients with early progression (short PFS12 and PFS24) and relationship of MMR-d with types of therapy need further study in large FL datasets. [Display omitted] DisclosuresPaludo: Karyopharm: Research Funding. King: Celgene/BMS: Research Funding. Maurer: Celgene: Research Funding; Genentech: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Morphosys: Membership on an entity's Board of Directors or advisory committees, Research Funding; Nanostring: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nowakowski: Celgene, NanoString Technologies, MorphoSys: Research Funding; Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Witzig: Karyopharm Therapeutics, Celgene/BMS, Incyte, Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS, Acerta Pharma, Kura Oncology, Acrotech Biopharma, Karyopharm Therapeutics: Research Funding.

Comparison of Dako HercepTest and Ventana PATHWAY anti-HER2 (4B5) tests and their correlation with silver in situ hybridization in lung adenocarcinoma.

BackgroundDiscordant results exist about the role of human epidermal growth factor receptor 2 (HER2) overexpression and/or HER2 amplification in lung adenocarcinoma. We aimed to compare the performance of HercepTest and PATHWAY anti-HER2 (4B5) by correlating immunohistochemistry (IHC) results with silver in situ hybridization (SISH) in adenocarcinoma lung specimens.MethodsA total of 148 surgically resected adenocarcinoma lung specimens were included.ResultsHER2 overexpression was found in 7.4% patients for HercepTest Dako and in 2.7% patients for 4B5 antibody. The overall coincidence between these two types of antibodies equals 93.9%. The incidence of HER2 amplification in lung adenocarcinoma was 17.6%, of which in 2.7% of the cases high-grade amplification was present. HER2 amplification was present in 90.9% of patients with overexpression of HER2, obtained by using HercepTest Dako and 75% patients using 4B5 antibody. A significant correlation between overexpression of HER2 receptors obtained by HercepTest Dako and 4B5 antibody and HER2 amplification was shown.ConclusionThe research of the efficiency of targeted molecular therapies with an HER2 antibody may serve as a basis for the introduction of routine HER2 status determination in lung adenocarcinoma, dictating the need for the standardized protocol for HER2 status determination in such pathology.

P–744 The features expression of some lymphocyte markers in the pelvic peritoneal adhesions’ tissue at reproductive age women

Abstract Study question To study the expression of CD4, CD8, CD20, CD 138 in the tissue of the pelvic peritoneal adhesions at women of reproductive age. Summary answer Immunohistochemical study of pelvic adhesions revealed the CD8-positive cells is directly involved in the formation of the immune response at the late stages of adhesiogenesis. What is known already One of the reason identifies the high frequency of adhesion formation is the presence of inflammation in the abdominal cavity with different severity and origin. It is known that Insufficiency of the fibrinolytic system, increased levels of a number of cytokines, including transforming growth factor- β1, and tissue hypoxia induce neoangiogenesis and fibrotization of the fibrin matrix, which leads to the formation of adhesions. Data on expression of CD4, CD8, CD20, CD138/syndecan–1 in the pelvic peritoneal adhesions in connection with their prescription, localization and origin is absent at accessible literature. Study design, size, duration Two hundred infertile women (aged 19–49 yrs) with pelvic peritoneal adhesions, who were underwent operative laparoscopy and adhesiolysis. Participants/materials, setting, methods The material for this study was the fragments of surgical material (adhesions and their parts) n = 200, taken from the women of reproductive age who suffered with infertility during operative laparoscopy.The morphological and immunohistochemical study of adhesions were carried out by standard techniques using paraffin blocks, reagents of Dako and monoclonal antibodies to CD4 (Clone 4B12 Ready-to-Use),CD8 (Clone C8/144B Ready-to-Use), CD20 (Clone L26 Ready-to-Use),CD138/syndecan–1 (Clone MI15 Ready-to-Use) of Abcam with automatic coloring Dako Cytomation. Main results and the role of chance To assess the population composition of these cell infiltrates, as well as individual diffusely located inflammatory cells, an immunohistochemical method with the main lymphocytic markers (CD4, CD8, CD20, CD138) was used. First of all, it is necessary to note the complete absence of CD20-positively colored cells in all observations, which indicates that at the final stage of the formation of adhesions, there is no element of the B-lymphocytic immune response. In an immunohistochemical study with syndecan–1 (CD138) antibodies, we identified a small number of positively colored cells that were located mainly perivascular, as part of mononuclear infiltrates. Quantitative analysis showed that the number of such cells is 0.8±0.2. When studying CD4–positive T-lymphocytes, it was found that they are usually located in the form of band-shaped infiltrates and focal perivascular clusters. The number of CD4-positive cells in the spike tissue is 5.6±0.2. CD8-positive cells were located mainly submesothelial, and in the form of perivascular clusters, the number of such cells was 9.2±0.6. Limitations, reasons for caution Age limitation, only women aged 19–49 yrs took part in this study. Exclusion criteria were the following for the groups: acute gynecological diseases, malignant diseases of female genitalia and ovarian tumors. Wider implications of the findings: The absence of B-cells in the “mature” adhesions’ tissue was found.The number of CD8-positive cells in our study was 1.5 times higher than the number of CD4-positive T-lymphocytes.CD4-positive T-lymphocytes play an important role and their number significantly prevails over the number of CD8-positive T-lymphocytes at the initial stages of adhesiogenesis. Trial registration number Case control study

PD-L1 Tumor Cell Expression in Upper Tract Urothelial Carcinomas is Associated With Higher Pathologic Stage.

Upper tract urothelial carcinomas (UTUCs) are a rare and unique subset of urothelial carcinoma (UC). Patients with UTUC may qualify for treatment with immune checkpoint inhibitors if their tumor cells express programmed death ligand-1 (PD-L1). While several large studies have looked at PD-L1 expression in UC, most have not investigated UTUC as a separate group, and most have not used Food and Drug Administration approved PD-L1 stains and scoring systems. Moreover, comparison between studies of PD-L1 expression is challenging as a wide variety of different PD-L1 antibody clones, testing platforms, and cutoff values have been used in the literature. This is a retrospective study of 37 cases of resected UTUC. Representative tissue from each case was compiled into tissue microarrays and immunohistochemical stains for PD-L1 (Dako antibody clones 22C3 and 28-8) were performed. PD-L1 staining was evaluated using several established Food and Drug Administration approved scoring systems: tumor proportion score (TPS), combined positive score, and immune cell score. Associations between PD-L1 expression and clinicopathologic features were investigated. Overall expression of PD-L1 in UTUC was 29.7% when using a TPS cutoff of ≥1%. Total of, 55.6% of cases with higher pathologic stage (pT3 or pT4) were positive for PD-L1, compared with only 5.3% of cases with lower pathologic stage (pTis, pT1, or pT2; P=0.0011). When using a combined positive score cutoff of ≥10, there was no significant association between tumor stage and PD-L1 expression. There was no association between PD-L1 positivity and tumor grade, tumor location, sex, or age. There was 100% concordance between 22C3 and 28-8 in terms of positivity rate. Our study using approved testing methods shows that PD-L1 expression in UTUC is more often associated with high pathologic stage, which may reflect an immune response evasion mechanism that UC cells acquire later in disease progression. In addition we show that 29.7% of UTUCs are positive for PD-L1 TPS expression, comparable to the 20% to 30% reported in UC literature. Finally, PD-L1 22C3 and 28-8 clones show similar overall patterns of staining in this setting.

O4: A NOVEL BOWEL DISEASE MODEL: PHENOTYPIC DIVERSITY OF CONTROL VS FAP ORGANOIDS

Abstract Introduction The lifetime risk of colorectal cancer in familial adenomatous polyposis (FAP) approaches 100%. In patients who have undergone prophylactic colectomy, duodenal cancer is 100-300 times more common than in the general population, and an important cause of death. We aimed to develop in vitro models of mucosal crypt-derived organoids from patients with FAP. Method Biopsies from apparent healthy duodenal mucosa of FAP patients undergoing upper gastrointestinal endoscopy or surgery yielded crypts that were immobilised in Matrigel Basement Membrane Matrix (Corning) and cultured in IntestiCult Organoid Growth Medium (StemCell Technologies) to generate organoids for further morphologic and immunocytochemistry analyses (Dako and Abcam antibodies). Result Duodenal crypt-derived organoids from one healthy volunteer formed ring structures (days 1 -2) that progressed to expected branched structures (days 4-7). FAP-derived organoids from 9 patients all generated organoids with aberrant morphologies. These organoids expressed markers of Paneth cells (lysozyme), proliferation (Ki-67), goblet cells (Muc-2) and the single cell layer of mucosal epithelium (CK18). Conclusion This is the first report of duodenal crypt-derived organoids generated from FAP patients. Aberrant organoid morphologies were observed in all 9 patients. Immunocytochemistry confirmed markers of duodenal epithelium, suggesting a promising in vitro model to study disease aetiology in FAP. Take-home message This is a step towards a personalised model of disease for patients with familial adenomatous polyposis.

Abstract 6471: Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas

Abstract Although Epithelial-Mesenchymal Transition (EMT) in cancers remains controversial, its relevance in tumor progression is a subject of investigation. The presence of cellular changes associated with EMT in bladder tumor progression have been well investigated, unlike other human carcinomas with very little data available to date. ZEB-1 which targets E-Cadherin repression is a transcriptional regulator that has been implicated in EMT, and its nuclear expression in tumor cells has been associated with aggressive malignancies in some types of cancer. Recently, it was also reported that ZEB-1 inhibits the expression of proapoptotic factor B-cell lymphoma (Bcl)-2 interacting mediator of cell death (BIM) by binding directly to the BIM promoter and repressing its transcription. We tested this hypothesis with a human cohort of 24 Cholangiocarcinoma (CC), 18 Hepatocellular (HCC) and 31 Renal Cell (RCC) human carcinomas. Formalin fixed paraffin embedded tissue sections containing benign and tumor lesions were immunostained using the Dako Autostainers Link 48 along with ZEB-1 monoclonal OT13G6 (ABCAM), Bcl-2 monoclonal 124 (Agilent, Dako), and E-Cadherin monoclonal NCH-38 (Agilent, Dako) antibodies. Nuclear, membraneous and/or cytoplasmic immunoreactivity was scored based on the intensity and percentage of positive cells in both the benign epithelium and adjacent tumor in each case. E-Cadherin was highly expressed in both the adjacent benign and malignant components with no significant variability in all three carcinomas, with ZEB-1 and Bcl-2 evaluated for the any positivity staining in both components for all cases. Results are detailed in the table below.All positive ZEB-1 cases in HCC were in high grade cases unlike the positive ZEB-1 in RCC which were in low grade cases. But more noticeably, 8/10 (80%) positive ZEB-1 were also positive for Bcl-2 in all three carcinoma types. Despite the small number of cases analyzed in this cohort a trend in the expression of ZEB-1 and its co-expression with BCL-2 may have some prognostic implications. We are currently extending this analysis to a larger number of CC, HCC, and RCC to closely examine the significance of ZEB-1, and Bcl-2 expression across differently graded lesions to further test our hypothesis. Citation Format: Joeffrey J. Chahine, Kyungmin Ko, Bhaskar VS Kallakury, Pamela L. Tuma. Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6471.

SUN-133 Intratumoral Expression of Steroid Receptors, CD68+ Macrophages and Mdm2 in Different Molecular-Biological Types of Endometrial Cancer

Background and aims: In Risk Classifier for Endometrial Cancer/EC (ProMisE) 4 molecular biological types of this tumor are described recently [1,2,3] and need an additional research, including evaluation of hormone-associated characteristics of both tumor tissue and patients [4]. Aim of the work was a study of estrogen (ER) and progesterone (PR) receptors in comparison with CD68+ macrophage infiltration (which promotes the invasion of tumor cells into the myometrium [5]) and expression of MDM2 protein, a negative regulator of p53 [6], in EC types presented in the ProMisE classification. Materials and methods: The tumor tissue of 218 EC patients included in the study (mean age 60.6 years) was assigned according to the data of genetic and immunohistochemical analysis to the types of carcinomas with gene POLE mutations, deficiency of mismatch repair proteins (MMR-D), expression (positive or diffuse) of p53 oncoprotein and to the type without characteristic molecular profile, WCMP. Immunohistochemistry was used also for evaluation of ER (Ventana antibodies, clone SP1) and PR (Ventana antibodies, clone 1E2) according to Allred, MDM2 (antibodies ABCAM, dilution 1:200) and macrophage marker CD68 (DAKO antibodies, clone CD8/144B). Results: According to the averaged data, the highest expression of ER and PR was found in EC types MMR-D and WCMP, and the lowest, respectively, in types POLE and p53+. Most often, positive expression of MDM2 (in 93.2% and 96.9% of studied cases) was detected respectively in MMR-D and p53+ type of EC, indicating, therefore, a positive relationship between MDM2 and the presence of steroid receptors in the first of these types (MMR-D) and negative - in the second of them (p53+). Expression of CD68+ macrophages demonstrated (contrary to the EC types POLE and p53+) a tendency to the lower values in types MMR-D and WCMP (128.0 ± 8.1 and 113.5 ± 6.3 cond.un.), i.e. in tumors with potential sensitivity to estrogen. Conclusions: The results indicate the importance of taking into account of both - the molecular biological type of the EC as well as the role of microenvironment of tumor cells, including the colonization of the neoplasm’ tissue by macrophages (and possibly lymphocytes) and the features of hormonal signal transmission in it. For further analysis, it is desirable to consider also the EC histotype, as one of the factors underlying various prognostic groups of this tumor [7].

Structural heterogeneity of epileptic foci in local drug-resistant epilepsy

To study etiopathogenesis is one of the most important tasks of modern neurology. Various types of structural changes occur in drug-resistant epilepsy (DRE); however, they are described as distinct phenomena. To provide a comprehensive characterization of structural changes in the cortex and adjacent white matter in the electrophysiological activity zone (in the epileptic focus) in patients undergoing surgery for DRE. Biopsy material of fragments of the temporal lobe and hippocampus from 16 patients aged 21 to 54 years (mean age, 25 years) with DRE were intraoperatively obtained at the Prof. A.L. Polenov Russian Research Institute of Neurosurgery. The investigators studied histological sections stained with H&E, toluidine blue according to the Nissl method and the Spielmeyer method, as well as the results of immunohistochemical reactions with glial fibrillary acidic protein (GFAP), vimentin, and neurofilaments (NF) (Dako antibodies, Denmark). Histological examination revealed a set of heterogeneous changes, reflecting the complex pathogenetic interactions that developed during the formation of an epileptic focus. Structural brain damage involved both gray and white matter. Focal cortical dysplasia was diagnosed in 14 (87.5%) cases; white matter neuronal heterotopia in 100%; neuronal reactive and destructive changes in 100%; epileptic leukoencephalopathy (vascular demyelination, microcysts, sclerosis and dystonia, gliosis) in 100%, cortical atrophy in 12.5%, and hippocampal sclerosis in 20% (in 2 out of the 10 examinees). The morphopathological heterogeneity in the structure of epileptic foci reflects the complexity of etiopathogenetic interactions, the polymorphism of epileptic manifestations, and the individual nature of formation of the epileptic system, which requires an integral approach to understanding the pathogenesis and morphogenesis of formation of the epileptic system and provides a direction for a personalized approach to epilepsy treatment.

The Influence of Ketogenic Diets on Psoriasiform-Like Skin Inflammation

The Influence of Ketogenic Diets on Psoriasiform-Like Skin Inflammation

Abstract 5246: A paradox: Expression of the myelin and lymphocyte protein 2 (MAL2) is down-regulated in human hepatocholangio and renal carcinomas in contrast to its up-regulation in other cancers

Abstract Recent work has found that Myelin and Lymphocyte Protein 2 (MAL2), a protein that functions in polarized protein sorting, is frequently overexpressed in many epithelial-derived human cancers, and has been linked to poor prognosis in patients with colorectal and pancreatic cancers. Yet, its role in tumorigenesis is unknown. Our recent overexpression/knockdown studies in polarized hepatic WIF-B cells, Hep3B hepatocellular carcinoma cells and hepatoma-derived Clone9 cells all indicate that MAL2 is a tumor suppressor. How can MAL2’s function as a tumor suppressor be reconciled with its upregulated expression and link to bad prognosis in human cancers? MAL2 resides on chromosome region 8q24, a region frequently amplified in multiple epithelial-derived human cancers along with c-Myc, a well-known oncogenic driver whose overexpression dysregulates gene expression leading to downstream decreased expression of MAL2 via inactivation of the Miz1 transcription factor. Our prediction is that lower grade lesions from human epithelial-derived cancers display high MAL2 expression that will diminish as cancers progress into higher grade lesions and metastases with a concomitant increase in c-Myc expression. We tested this hypothesis with a cohort, of 23 cholangiocarcinoma (CC), 18 hepatocellular (HCC) and 20 renal cell (RCC) human carcinoma cases whose formalin fixed paraffin embedded tissue sections containing benign and later-stage tumor lesions were immunostained using the Dako Autostainers Link 48 along with Mal2 rabbit polyclonal (ABCAM), Miz-1 monoclonal ZBTB17(NOVUS), c-Myc monoclonal Y69 (ABCAM) and Ki-67 monoclonal Mib-1(Dako) antibodies. Nuclear and/or cytoplasmic immunoreactivity was scored based on the intensity and percentage of positive cells in both the benign epithelium and adjacent tumor component in each case. In all three carcinoma types, both MAL2 and Miz-1 expression was high in the benign tissue. In the tumor component MAL2 and Miz-1 expression was respectively lost in 15/23(65%) and 22/23(96%) CC, 13/18(72%) and 17/18(94%) HCC, 13/20(65%) and 16/20(80%) RCC cases. In contrast only 2/20 (10%) and 1/20(5%) RCC cases showed an upregulated expression of MAL2 and Miz-1 respectively compared to the benign component, with all three remaining carcinoma cases showing no up-or-down regulation in protein expression. However, no enhanced c-Myc expression was detected in the tumor lesions despite the higher proliferative index as indicated by Ki-67 labeling. These results indicate a concordant down-regulation of both MAL2 and Miz-1 in CC, HCC, and RCC supporting a tumor suppressor role in these cancers. We are currently extending these studies to other epithelial-derived cancer tissue types and also are more closely examining MAL2, Miz1 and c-Myc expression across graded lesions to better test our hypothesis. Note: This abstract was not presented at the meeting. Citation Format: Alfonso Lopez-Coral, Joeffrey J. Chahine, Bhaskar V. Kallakury, Pamela L. Tuma. A paradox: Expression of the myelin and lymphocyte protein 2 (MAL2) is down-regulated in human hepatocholangio and renal carcinomas in contrast to its up-regulation in other cancers [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 5246.

273-OR: Intrapancreatic Adipose Tissue of Nondiabetic and Type 2 Diabetic Subjects and Relationships with Islet Morphometry

Little information is available on adipose tissue features in the human pancreas (AdTP). In the present study we performed morphologic and morphometric studies on AdTP of 9 overweight/obese nondiabetic (ND, age: 65±5 years; 4M/5F; BMI: 31.3±0.4 Kg/m2, mean±SEM) and 13 matched type 2 diabetic (T2D, age: 70±2 years, 8M/5F; BMI: 30.0±0.8 kg/m2) organ donors. Adipocytes were identified and quantified on a fluorescence DM5500 Leica microscope, using the MetaMorph v 1.8.0 software with a tresholded mask applied in exclusive mode. Macrophages were identified by immunohistochemistry using anti-CD68 (Dako) antibody. Assessment of insulin (Abcam Ab) and glucagon (Sigma Ab) positive cells was also performed. The number of adipocytes trended higher (+32.8%) in T2D (8.9±1.4/mm2) compared to ND (6.7±2.1/mm2) and their size was greater in T2D (9,319±1,028 μm2) than ND (5,639±639 μm2, p=0.01). AdTP area in relation to acinar tissue was 90.4% higher (p=0.07) in T2D (7.6±1.3%) than ND (3.9±1.5%). The amount of adipocytes with adjacent ≥ 3 CD68+ cells was higher (p=0.048) in T2D (31/281, 11%) than ND (7/147, 5%). Insulin positive area (T2D: 0.49±0.06%; ND: 0.55±0.09%) as well as glucagon positive area (T2D: 0.24±0.03%; ND: 0.32±0.07%) did not differ significantly between the two groups. Adipocyte size was negatively associated with islet number in ND+T2D (p=0.06) and positively with insulin area in ND (p=0.058); there was a significant, positive relationship between the amount of adipocytes and the number of islets in T2D (p=0.017). In conclusion, we found significant differences in the AdTP of T2D as compared to ND, including increased adypocite size and presence of adipocyte adjacent CD68+ cells; some of these parameters correlated with islet morphometric indices. Disclosure M. Suleiman: None. C. De Luca: None. P. De Simone: None. U. Boggi: None. F. Grano: None. M. Bugliani: None. P. Marchetti: None. L. Marselli: None. Funding Innovative Medicines Initiative 2 Joint Undertaking (115881); European Union; European Federation of Pharmaceutical Industries and Associations; Swiss State Secretariat for Education, Research and Innovation (16.0097-2)

Observations of foetal heart veins draining directly into the left and right atria.

Evaluation of semiserial sections of 14 normal hearts from human foetuses of gestational age 25-33 weeks showed that all of these hearts contained thin veins draining directly into the atria (maximum, 10 veins per heart). Of the 75 veins in these 14 hearts, 55 emptied into the right atrium and 20 into the left atrium. These veins were not accompanied by nerves, in contrast to tributaries of the great cardiac vein, and were negative for both smooth muscle actin (SMA) and CD34. However, the epithelium and venous wall of the anterior cardiac vein, the thickest of the direct draining veins, were strongly positive for SMA and CD34, respectively. In general, developing fibres in the vascular wall were positive for CD34, while the endothelium of the arteries and veins was strongly positive for the present DAKO antibody of SMA. The small cardiac vein, a thin but permanent tributary of the terminal portion of the great cardiac vein, was also positive for SMA and CD34. A few S100 protein-positive nerves were observed along both the anterior and small cardiac veins, but no nerves accompanied the direct dra- inage veins. These findings suggested that the latter did not develop from the early epicardiac vascular plexus but from a gulfing of the intratrabecular space or sinus of the atria. However, the immunoreactivity of the anterior cardiac vein suggests that it originated from the vascular plexus, similar to tributaries of the great cardiac vein.

MON-309 Intratumoral Proliferation and Proinflammatory Macrophages in Obese and Non-Obese Patients with Different Molecular-Biological Types of Endometrial Cancer

Background and aims: Endometrial cancer (EC) is characterized in times of modern obesity epidemic by the rapid incidence increase and the recent update of the molecular-biological classification, which now distinguishes not two, as before, but four types of this tumor (Kandoth et al., 2013; Talhouk et al., 2017; Berstein et al., 2017) that need to be comprehensively studied. The objective of this work was to compare the proliferative potential of a tumor tissue (Ki67 index) and compare it with the expression of proinflammatory CD68-macrophages in obese and non-obese patients with different types of EC according to the PROMISE classification. Materials and methods: In all predominantly postmenopausal 216 treatment naive EC patients included in the study, the body mass index (BMI) was estimated due to which the females were divided into subgroups with BMI ≥30.0 and <30.0. According to genetic (high-resolution melting and sequencing) and immunohistochemical (DACO antibodies) analysis, endometrial carcinomas were classified in accordance with recommendations (Talhouk et al, 2017) into types with POLE gene mutations, defect of mismatch repair proteins (MMR-D), expression (positive or diffuse) of p53 oncoprotein and variant without characteristic molecular profile, WCMP. Immunohistochemistry was also used to evaluate the expression of Ki67 (DBS antibodies, clone SP6) and CD68 (DAKO antibodies, clone CD8/144B). Results: Obesity was more common in patients with a WCMP tumor type and, especially, in MMR-D group (BMI ratio ≥30.0/<30.0, respectively, 1.62 and 2.32). The highest Ki67 index values were detected with positive (59.3 ± 3.8%) and a combination of positive and diffuse (50.5 ± 3.7%) p53 expression, and the lowest (27.6 ± 1.7%) in the WCMP group. In all EC types (except for cases with p53+), Ki67 expression was lower at BMI ≥30.0, which was confirmed within this subgroup by negative Spearman correlation of Ki67 level with BMI value. CD68 expression showed a downward trend in the MMR-D and WCMP types (124.4 ± 7.8 and 115.0 ± 5.9 cond.un.) and a tendency to positive correlation with Ki67 in obese women belonging to the POLE and p53 groups. Conclusions: The findings suggest that the level of such important prognostic tumor markers as Ki67 and CD68 (Soeda et al., 2008) is characterized by a relationship with the presence/absence of obesity as well as with EC molecular-biological type, that expands target range for preventive and therapeutic interventions. Acknowledgement to grant 18-015-00026

CD57 (Leu-7, HNK-1) immunoreactivity seen in thin arteries in the human fetal lung

CD57 (synonyms: Leu-7, HNK-1) is a well-known marker of nerve elements including the conductive system of the heart, as well as natural killer cells. In lung specimens from 12 human fetuses at 10–34 weeks of gestation, we have found incidentally that segmental, subsegmental, and more peripheral arteries strongly expressed CD57. Capillaries near developing alveoli were often or sometimes positive. The CD57-positive tissue elements within intrapulmonary arteries seemed to be the endothelium, internal elastic lamina, and smooth muscle layer, which corresponded to tissue positive for a DAKO antibody reactive with smooth muscle actin we used. However, the lobar artery and pulmonary arterial trunk as well as bronchial arteries were negative. Likewise, arteries in and along any abdominal viscera, as well as the heart, thymus, and thyroid, did not express CD57. Thus, the lung-specific CD57 reactivity was not connected with either of an endodermal- or a branchial arch-origin. CD57 antigen is a sugar chain characterized by a sulfated glucuronic acid residue that is likely to exist in some glycosphingolipids. Therefore, a chemical affinity or an interaction might exist between CD57-positive arterioles and glycosphingolipids originating from alveoli, resulting in acceleration of capillary budding to make contact with the alveolar wall. CD57 might therefore be a functional marker of the developing air-blood interface that characterizes the fetal lung at the canalicular stage.

PD-L1 in breast cancer: comparative analysis of 3 different antibodies

The interaction of programmed cell death-1 and its ligand-1 (PD-L1) serves as a regulatory check against excessive immune response to antigen and autoimmunity. We compared the performance of 3 different PD-L1 antibodies (Ventana SP263, Dako 22C3, and BioCare RbMCAL10 antibodies) in 136 invasive ductal carcinoma specimens including 43 primary, 48 locally metastatic, and 46 distantly metastatic diseases. PD-L1 expression was correlated with clinicopathologic parameters including tumor size, grade, lymphovascular invasion, estrogen receptor, progesterone receptor, HER2, Ki67, molecular type, and triple-negative status. There was excellent agreement between the 3 antibodies, with highly significant κ values (P≤.001). PD-L1 expression was more likely to be associated with higher tumor grade and estrogen receptor-negative, progesterone receptor-negative, triple-negative, and highly proliferative tumors (P<.001). When we studied PD-L1 expression at 0, 1%-9%, 10%-49%, and ≥50% cutoff points by the 3 antibodies, there were 20 discordant cases between the antibodies. Sixteen were of inconsequential impact as far as low and high PD-L1 expression. The 4 differences between antibodies did exhibit an interesting pattern of expression, where there was a general agreement between the BioCare and Ventana antibodies with consistently higher PD-L1 expression compared with the Dako antibody. Given the high concordance, it is not surprising that all 3 antibodies demonstrated the same associations with all pathologic and clinical parameters studied. Standardization studies to identify reliable biomarkers that help in patient selection for immune therapy to improve the risk-benefit ratio for these drugs are still needed.

In depth evaluation of the prognostic and predictive utility of PTEN immunohistochemistry in colorectal carcinomas: performance of three antibodies with emphasis on intracellular and intratumoral heterogeneity.

BackgroundPhosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications.MethodsRetrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization.ResultsThe Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody.ConclusionsWhile PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0508-0) contains supplementary material, which is available to authorized users.

Abstract OT2-01-03: A phase 1/2 study of once-daily oral VT-464 in patients with advanced androgen receptor (AR) positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC)

Abstract VT-464, an oral dual lyase-selective CYP17 inhibitor and AR antagonist (wild-type and mutated forms [e.g., F876L and T877A]), is in multiple Phase (Ph) 2 studies as treatment for men with castration-resistant prostate cancer (CRPC). VT-464 inhibits the growth of multiple BC cell lines in vitro including MCF7 (ER(+)/AR low), tamoxifen-resistant MCF7, and MDA-MB-453 (ER(-)/AR(+)) in a dose-dependent manner and with greater potency/efficacy than enzalutamide (submitted, Ellison et al., 2015). A subset of TNBC and most ER(+) BC express AR, making them potential targets for VT-464 since it directly inhibits both androgen/estrogen synthesis and AR transcriptional activity. Objectives: The primary objective of Ph 1, now enrolling, is to establish the once-daily dose of VT-464 in women. Secondary objectives include safety, PK and efficacy endpoints, including determination of clinical benefit rate (CBR) which is the primary objective of Ph 2. Exploratory objectives include the determination of the extent of AR expression and signaling in breast tissue and to evaluate the relationship of expression with VT-464 effects on circulating tumor biomarkers, circulating hormones and clinical outcomes. Study Design: This study is an open-label, single arm, Ph 1/2 study of VT-464 in women with AR(+) TNBC or ER(+)/HER2 normal unresectable locally advanced or metastatic BC. Ph 1 will follow a modified 3+3 Fibonacci design with cohort expansion to 6 patients following a single DLT in the first 28-days of treatment. Approximately 2-3 dose-levels will be explored in Ph 1. Ph 1 start dose will be the MTD for men with CRPC. AR(+) TNBC and ER(+)/HER2 BC cohorts will be expanded in Ph 2 using the MTD from Ph 1. Ph 2 will follow a Simon's two-stage design with pre-determined futility parameters. Eligible patients will have ER≥1% BC or AR≥1% (as determined by central IHC testing using the Dako antibody) TNBC. ER(+) patients must be postmenopausal and must have received at least 1 prior line of endocrine therapy. Additional eligibility criteria include: ≥ 18 years of age, ECOG PS ≤ 1, unresectable locally advanced or metastatic BC, available representative tumor specimen to enable correlative science. Treatment Plan: Eligible patients will receive VT-464 once-nightly with dinner in a continuous dosing schedule. Adverse events and concomitant medications will be collected from the time of signing of informed consent until 30 days after end of study visit (EOS). Safety labs will be monitored monthly through EOS. Dense PK will be collected after the first dose of study drug in Ph 1 and single morning samples collected approximately every two cycles thereafter in Ph 1 and Ph 2 until EOS. Blood samples for steroids, circulating tumor DNA and circulating tumor cells will be collected through Cycle 2 and then at EOS. Tumor biopsy will be collected at baseline and at disease progression. Radiographic response will be assessed every 8 weeks and EOS. Patient Accrual: Accrual is ongoing with 12-18 patients expected to be enrolled in Ph 1. Citation Format: Gucalp A, Hudis C, Norton L, Patil S, Kurman MR, Eisner JR, Moore WR, Traina TA. A phase 1/2 study of once-daily oral VT-464 in patients with advanced androgen receptor (AR) positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-01-03.

Hypoxia Inducible Factor (HIF) Adaptation After Mechanical Circulatory Unloading of the Heart. A Gene Expression Analysis Study of Paired Myocardial Samples

Background: Endothelial to Mesenchymal Transition (EndMT) and the reverse phenomenon of mesenchymal to endothelial transitioning (MET) has been shown to contribute to fibrosis in rodent models of heart failure. There are no studies documenting the same in human cardiac tissue. Methods: We performed immunofluorescence dual staining on end stage heart failure myocardial samples obtained from our bio repository. Staining for endothelial and mesenchymal markers were performed with Anti-VE-Cadherin (Santa Cruz Biotechnologies) and anti-Fibroblast specific protein 1 (Dako Antibodies) respectively using standard immunofluoresence techniques. Presence of dual staining on fluorescence microscopy was considered as positive for the presence of EndMT. Vascular density was assessed using a DAB (3-3’ Diaminobenzidine) technique with Anti-CD31 (PECAM, Abcam). Results: Of the end stage heart failure samples, 18 were from left ventricular core during LVAD implant and 5 during a direct transplant as status 1A (axillary intraaortic balloon pump). 10 hearts of patients bridged to transplant with a continuous flow LVAD (Days under support 3436228) were also studied. Mean age was 55y with 82% males and 74% white. 70% of all samples had evidence of EndMT. The number of cells ranged from 1 to 5 per high power field. There was no significant difference between patient characteristics between those with evidence of EndMT versus not, except a higher prevalence of diabetes in the latter group. (Fig 1B) 74% of the end-stage heart failure samples and 60% of the post-VAD samples showed the presence of EndMT cells. Vascular Density was higher in the samples exhibiting EndMT phenomenon. (Figure 1A). Conclusion: We have documented for the first time, the existence of endothelial-mesenchymal transition in human end stage myocardial samples corroborating earlier pioneering studies in rodents. Interestingly the existence of such dual staining cells in end stage hearts (which is highly fibrotic) suggests a possible role of ongoing fibrosis with endothelial to mesenchymal transitioning or potential antifibrotic mechanism though mesenchymal to endothelial transitioning. Further studies of the later process will be warranted to explore potential anti-fibrotic therapeutic implications.

The role of sodium hydrogen exchanger regulatory factor 1 in osteoarthritis

Purpose: In a pilot study, selected gene expression in tissue samples from patients with OA and osteoporosis (OP) were compared. The gene, SLC9A3R1 which encodes for a member of the sodium/hydrogen regulatory factor protein family (NHERF1) was found to be differentially expressed in osteoblasts. NHERF1 plays a role in tumour suppression, via PTEN, and intracellular pH (pHi) regulation by acting on the sodium/hydrogen exchanger (NHE). We are investigating whether a decrease in NHERF1 causes a change in the pHi of chondrocytes and leads to chondrocyte proliferation. The aim of the project is to test the hypothesis that dysregulated expression of NHERF1 is a key part of the abnormal behaviour of articular chondrocytes. Methods: Full depth cartilage explants or shavings were removed from osteoarthritic femoral heads or knee joints, and from healthy controls (amputees or osteoporotic femoral heads). Explants from 17 patients (4 OA and 4 non-OA femoral heads, 4 OA knees and 5 amputees) were decalcified and fixed in formalin for paraffin embedding and sectioning, whilst shavings were placed in Dulbecco’s Modified Eagles Medium (DMEM) and washed prior to enzymatic chondrocyte isolation. Sections were deparaffinised and either stained with Fast-Green-FCF and Safranin ‘O’, or treated with Target Retrieval Solution and hyaluronidase prior to incubation with glycine, blocking with non-immune serum and overnight incubation with either rabbit anti-human NHE3 (pAb, C-20), mouse anti-human NHERF1 (mAb, IgG2b), or goat anti-human PTEN (pAb, N19) diluted in Dako Antibody Diluent (1:50: PTEN 1:100). Bound NHE3, NHERF1 or PTEN antibodies were detected with goat anti-rabbit (488 nm), goat anti-mouse (555 nm) or donkey anti-goat (488 nm) secondary antibodies (1:100, PTEN 1:200) respectively. Sections were mounted with Prolong Gold anti-fade mounting media containing DAPI and visualized with a Zeiss Imager M2 fluorescent or LSM 700 Zeiss Imager M2 confocal laser scanning microscope (CLSM). Cell counts and co-localisation were analysed using Image J software. Isolated articular chondrocytes (n=3) were incubated with BCECF-AM (5 μM, 15 mins, 37 oC), washed in DMEM without phenol red, aliquoted ( 14 x 104) and placed in a sterile Ibidi μ-Slide I (0.4) Flow Chamber (Thistle Scientific, UK) for 20-30 min (37 oC) to attach. They were washed into the appropriate saline,After saline washing, and BCECF was then alternately excited at 488 nm and 458 nm, and emission collected at >505 nm using a CLSM (Zeiss LSM 710) from individual cells. The background from each wavelength was subtracted and the 488:458 nm ratio (R) converted to pHi using a calibration curve constructed by permeabilizing the chondrocytes to H+ in high-potassium (K+) solutions of different pH containing nigericin (2 μM). Results: NHE3, NHERF1 and PTEN were expressed in all zones of OA and non-OA articular cartilage. Image J analysis showed that NHERF1 co-localised with both proteins in the cytoplasm and perinuclear region, but little was found near the cell membrane. It was also expressed in the nucleus in 50% of chondrocytes in OA and non-OA samples. The median diameter of the nuclei of OA chondrocytes was also found to be significantly larger (7.47 μm) than in non-OA chondrocytes (6.78 μm, P=0.001???). The pHi of normal, healthy chondrocytes was 6.9 + 0.2 (n=3) but OA chondrocytes appeared to be more acidic. The pHi of these chondrocytes was too low to be recorded successfully and reliably using BCECF (pKa 6.98) which is used for measuring near neutral pH, so this necessitated exploring the use of another fluorescein derivative, DCFDA (pKa 4.8) for pH measurements from pH 4-5 Conclusions: This is the first study to identify NHE3, NHERF1 and PTEN expression and co-localisation in human articular cartilage, and to measure pHi in single isolated chondrocytes using confocal microscopy and the fluorescent dyes, BCECF-AM and DCFDA. NHERF1 was expected at the cell membrane but, surprisingly, location was predominantly cytoplasmic. Detection of NHERF1 expression and function in articular cartilage may open new approaches for OA therapy in the future.