Immunoreactive Score Research Articles

Study of Survivin: A Biomarker in Carcinoma Colon – An Original Study

Background: Survivin, an anti-apoptotic protein has been of importance in colon carcinoma patient. Here we studied the prevalence of Survivin biomarker among cases of colon carcinoma and its relationship with other prognostic indicators.
 Design: Tissue samples from 102 patients with colon carcinoma were studied. H&E stain and immunohistochemistry were performed and evaluated. Immuno-histochemistry expression was sought according to IRS Scoring System (Immunoreactivity Scoring) for stain intensity and percentage positive cells. Survivin expression was also correlated with age at diagnosis, gender, grade, nodal status, lympho-vascular and perineural invasion, resection margins, Tumor stage (TNM) and Group Stage.
 Result and discussion: Survivin was expressed in high grade and stage tumors and showed significant association (p value= 0.002). Survivin expression was positive in lymph node positive tumors and showed significant association (p value= 0.002). Survivin was expressed in tumors with lympho-vascular and perineural invasion and showed significant association (p value= 0.02 and 0.001) respectively. Age at diagnosis showed significance of p=0.03 whereas Gender (Male/Female) and resection margins did not show any association with Survivin expression.
 Conclusion: Survivin expression increases as the tumor grade worsens in colon cancer and is maintained in approximately all high group staged tumors (TNM Staging) along with tumors showing lymphovascular and perineural invasion. Gender does not have any association.

Impact of Capicua on Pancreatic Cancer Progression.

The transcription factor capicua (CIC) regulates mammalian development and homeostasis. Growing evidence shows that CIC suppresses various human cancers by directly repressing the downstream cancer-related target genes. This study investigated the clinical and biologic significance of CIC expression in pancreatic cancer (PC). The study reviewed 132 patients with PC who underwent curative resection. The patients were divided into two groups according to CIC immunoreactivity score by immunohistochemistry, and the associations between CIC expression, clinicopathologic characteristics, and postoperative prognosis were investigated. Moreover, the influence of CIC expression on the malignant potential of PC cells was assessed with cell proliferation, motility assays, and use of quantitative real time-polymerase chain reaction and Western blot on the downstream target genes of CIC in knockdown experiments. The low-CIC expression group showed a higher proportion of lymphatic invasion (72.9% vs. 53.1%; p = 0.024), intrapancreatic neural invasion (94.1% vs. 81.3%; p = 0.021), and extrapancreatic plexus invasion (30.9% vs. 7.8%; p = 0.0006) than the high-CIC expression group as well as significantly worse overall survival (p = 0.0002) and recurrence-free survival (p = 0.0041) rates. Low CIC expression was an independent risk factor for poor prognosis (p = 0.038). Pancreatic cancer cells with knockdown CIC significantly enhanced cell motilities and cell cycle progression, promoted expression levels of ETV4 and MMP-9, and induced EMT. The study elucidated the association of low CIC expression with a poor prognosis for patients with PC and suggested that the CIC-ETV4-MMP-9 axis might control PC progression.

TROP2 is a Good Indicator for Infiltrative Nature of Carcinoma Rather than Diagnosing Malignancy in Thyroid.

TROP2 is a glycoprotein which is expressed in carcinomas rather than normal tissues and associated with poor prognosis. Immunohistohemical TROP2 staining was determined to be useful for diagnosing papillary thyroid carcinoma (PTC). In this study, we aimed to find out relationship between TROP-2 expression and the diagnosis of PTC, and clinico pathological parameters. This retrospective study was conducted on 270 cases (145 malignant and 124 benign). All histopathological parameters were re-evaluated blindly. We used 3 different scores for positivity for TROP2 expression (TES: Total estimation score, 5%, IRS:Immunoreactive score). Sensitivity was respectively according to TES, 5%and IRS; 55.5%, 54.1%, 51.4%. Specificity was 98.4% in all different scores. TROP2 positivity was correlated with older age in 5% score and TES. Absence of encapsulation, presence of infiltrating spreading, perineural invasion, extra thyroidal extension, tall cell and/or hobnail differentiation were associated with TROP2 expression in three different scores. Presence of lymphatic invasion was correlated with TROP2 positivity in 5% score and IRS. TROP2 expression was inversely proportional to follicular variant PTC and tumour size in 5% score. TROP-2 is a marker for aggressive behaviour rather than detecting malignancy, but if it stained and not malignant, it may also have potential for determining precursor lesion that demands further studies. Additionally, in 3 different scores, TES was most sensitive, but most variable that had correlation with TROP2 was seen at 5% score.

In Search of TGCT Biomarkers: A Comprehensive In Silico and Histopathological Analysis.

Testicular germ cell tumors (TGCTs) are ever more affecting the young male population. Germ cell neoplasia in situ (GCNIS) is the origin of TGCTs, namely, seminomas (SE) and a heterogeneous group of nonseminomas (NS) comprising embryonal carcinoma, teratoma, yolk sac tumor, and choriocarcinoma. Response to the treatment and prognosis, especially of NS, depend on precise diagnosis with a necessity for discovery of new biomarkers. We aimed to perform comprehensive in silico analysis at the DNA, RNA, and protein levels of six prospective (HOXA9, MGMT, CFC1, PRSS21, RASSF1A, and MAGEC2) and six known TGCT biomarkers (OCT4, SOX17, SOX2, SALL4, NANOG, and KIT) and assess its congruence with histopathological analysis in all forms of TGCTs. Cancer Hallmarks Analytics Tool, the Search Tool for the Retrieval of Interacting Genes/Proteins database, and UALCAN, an interactive web resource for analyzing cancer OMICS data, were used. In 108 TGCT and 48 tumor-free testicular samples, the immunoreactivity score (IRS) was calculated. SE showed higher frequency in DNA alteration, while DNA methylation was significantly higher for all prospective biomarkers in NS. In GCNIS, we assessed the clinical positivity of RASSF1 and PRSS21 in 52% and 62% of samples, respectively, in contrast to low or nil positivity in healthy seminiferous tubules, TGTCs as a group, SE, NS, or all NS components. Although present in approximately 80% of healthy seminiferous tubules (HT) and GCNIS, HOXA9 was diagnostically positive in 64% of TGCTs, while it was positive in 82% of NS versus 29% of SE. Results at the DNA, mRNA, and protein levels on putative and already known biomarkers were included in the suggested panels that may prove to be important for better diagnostics of various forms of TGCTs.

Overexpression of metastasis-associated in colon cancer 1 in retinoblastoma.

Metastasis-associated in colon cancer 1 (MACC1), one of the prognostic markers for colonic and other tumours was noted to be overexpressed in retinoblastoma (Rb) Y79 cancer stem cells. This prompted us to evaluate its expression in primary Rb tumour and serum samples with clinicopathologic correlation. The interacting partner, c-MET was also evaluated in primary tumour tissues to explore the activation of MACC1 signaling. This study was done following institutional review board approval from participating institutes. Semiquantitative gene expression for MACC1 was evaluated using formalin-fixed paraffin-embedded sections and unfixed tumour samples from primary Rb cases (n = 44). Immunolocalization for MACC1 was assessed in primary Rb tumours (n = 22), bone marrow aspirates with metastasis (n = 3), and c-MET expression was also assessed in Rb tumours (n = 17). Serum MACC1 levels were analysed using enzyme-linked immunosorbent assay in samples collected from Rb patients undergoing enucleation (n = 31), Rb patients with proven clinical metastasis (n = 3), and compared to appropriate controls. Clinicopathologic correlation of MACC1 expression was analysed using the medical records with specific reference to histologic risk factors (HRF) for metastasis and differentiation. High expression of MACC1 gene was noted in all the tumour samples (n = 44), more so in cases with versus without HRF (p < 0.0001). In cases with HRF, MACC1 and c-MET showed diffuse nuclear and cytoplasmic staining whereas it was predominantly cytoplasmic in cases without HRF. Mean immunoreactivity score of MACC1 and c-MET tissue immunolocalization revealed that cases with HRF showed significantly higher expression compared to cases without HRF (p < 0.05). Unlike the findings in colonic tumours, serum levels of MACC1 were lower in patients compared to normal controls. Overexpression of MACC1 and c-MET in retinoblastoma tissues, specifically those with risk factors for metastasis, suggests its role in proliferation and possibly in invasion. However, the current data do not support it to be a clinical prognostic marker in retinoblastoma tumours. The inverse serum expression is an intriguing finding, which warrants further studies especially in retinoblastoma.

Sex Specific Expression of Interleukin 7, 8 and 15 in Placentas of Women with Gestational Diabetes.

Gestational diabetes mellitus (GDM) is known to increase the risk for feto-maternal complications during pregnancy. A state of low-grade inflammation, with elevated levels of proinflammatory molecules, similar to patients with obesity or diabetes mellitus type 2 has also been partly described in GDM. The placenta, as unique interface between mother and fetus, is not only passively affected by changes in one of these organisms, but also acts as a modulator by expressing hormones and cytokines. This study aimed to investigate the expression of the proinflammatory cytokines Interleukin (IL) 7, 8 and 15 in GDM in placental tissue. A total number of 80 placentas were included (40 GDM/40 control group). The expression of IL-7, 8 and 15 was investigated in extravillous trophoblast (EVT) and syncytiotrophoblast (SCT) by immunohistochemistry and immunofluorescence double staining. The immunohistochemical staining was evaluated with the semiquanitfied immunoreactive score (IRS). While the expression IL-15 was significantly upregulated in EVTs of women with GDM. The expression of IL-8 was significantly decreased in EVT of the GDM group. Furthermore, significant fetal sex specific differences were detectable in all three cytokines. Our findings suggest an involvement of the investigated cytokines in the maintenance of a state of chronic low-grade inflammation on placental level in patients suffering from GDM.

Nuclear Enolase-1/ MBP-1 expression and its association with the Wnt signaling in epithelial ovarian cancer

Background:Enolase-1, primarily known for its role in glucose metabolism, is overexpressed in various cancer entities. In contrast its alternative spliced nuclear isoform MBP-1 acts as a tumor suppressor. The aim of this study is to analyze the prognostic impact of Enolase-1/ MBP-1 and its functional significance in epithelial ovarian cancer (EOC). Methods:By immunohistochemistry, Enolase-1 staining was examined in 156 EOC samples. Evaluation of Enolase-1 staining was conducted in the nucleus and the cytoplasm using the semi-quantitative immunoreactive score. Expression levels were correlated with clinical and pathological parameters as well as with overall survival to assess for prognostic impact. Results:Cytoplasmic and nuclear Enolase-1 expression did not show a significant difference between the histological subtypes (p = 0.1). High nuclear Enolase-1/ MBP-1 staining negativly correlated with the tumor grading (p<0.001; Cc= −0.318). Cytoplasmic Enolase-1 did not correlate with clinicopathological data. Higher nuclear Enolase-1/ MBP-1 staining was detected in low-grade serous cancer cases compared to high-grade ones (median IRS 3 (range 0–8) vs. median IRS 2 (range 0–4), p<0.001). Nuclear Enolase-1/ MBP-1 expression correlated with the Wnt signaling markers membranous beta-catenin (p = 0.007; Cc=0.235), serine residue 9-phosphorylated glycogen synthase kinase 3 beta (p<0.001; Cc=0.341) and snail/slug (p = 0.004; Cc= −0.257). High nuclear Enolase-1/ MBP-1 expression was associated with improved overall survival (88.6 vs. 33.1 months, median; p = 0.013). Conclusion:Additional knowledge of Enolase-1/ MBP-1 as a biomarker and its interactions within the Wnt signaling pathway and epithelial–mesenchymal transition potentially improve the prognosis of therapeutic approaches in EOC.

Anti-fibrotic effect of intravenous umbilical cord-derived mesenchymal stem cells (UC-MSCs) injection in experimental rats induced liver fibrosis.

Aim To investigate the effect of umbilical cord-derived mesenchymal stem cells (UC-MSCs) administration among liver fibrosis experimental rat model via the regulation of angiotensin II type 1 receptor (AT1R) and platelet-derived growth factor-β (PDGF-β) due to their therapeutic potential to replace liver transplantation for advanced liver fibrosis. Yet the mechanism of action has been questionably associated with UC-MSCs fibrosis regression properties. Methods Sprague-Dawley (SD) rats (n=18) were separated into three groups (control, untreated liver fibrosis, and UC-MSCs treated group). Serum PDGF-β level was determined by enzymelinked immunosorbent assay (ELISA) following 14 days of UCMSCs injection. Meanwhile, AT1R expression was interpreted based on immunoreactive score (IRS) stained using polyclonal antibody and liver fibrosis stained with hematoxylin & eosin was graded using the METAVIR score. Results UC-MSCs were isolated successfully from rat umbilical cord. Liver fibrosis was observed following 14 weeks of CCl4 injection concurrent with higher serum level of PDGF-β, but the UC-MSCs-treated group had lower level (980.08 ±289.41 and 606.42±109.85 for untreated liver fibrosis and UC-MSCs treated group, respectively; p=0.004). There was also a high expression of AT1R among untreated liver fibrosis group, as well as highgrade liver fibrosis versus localized fibrosis and low level of AT1R expression among UC-MSCs treated-group (p=0.001). Conclusion UC-MSCs administration could ameliorate liver fibrosis by reducing the AT1R expression and PDGF-β serum levels, and intervention through this signaling pathway could be alternative evidence for the causative of positive outcome.

PIWI-Like 1 and PIWI-Like 2 Expression in Breast Cancer.

Simple SummaryA family of proteins, the PIWI proteins, play a crucial role in the regulation of the development of germ cells and self-preservation of so-called stem cells. Former studies have shown that these proteins can be over- or underrepresented (over-/underexpressed) in some cancers and, in the case of abnormal expression, may be correlated with worse outcomes of tumor patients. In our study, we investigated the influence of the two PIWI proteins, PIWI-like 1 and PIWI-like 2, on the survival of breast cancer patients and their correlation with certain breast cancer subtypes. If a breast cancer showed a higher expression of PIWI-like 1 protein but less PIWI-like 2 protein than in non-tumorous tissue, the patient suffered from a more aggressive breast cancer subtype and had shorter survival. By analyzing these two proteins in breast cancer, we were able to predict tumor aggressiveness and prognosis.PIWI-like 1 and PIWI-like 2 play a role in stem cell self-renewal, and enhanced expression has been reported for several tumor entities. However, few studies have investigated PIWI-like 1 and PIWI-like 2 expressions in breast cancer subtypes regarding prognosis. Therefore, we examined protein expression in a large consecutive cohort of breast cancer patients and correlated it to breast cancer subtypes and survival outcome. PIWI-like 1 and PIWI-like 2 expressions were evaluated using immunohistochemistry in a cohort of 894 breast cancer patients, of whom 363 were eligible for further analysis. Percentage and intensity of stained tumor cells were analyzed and an immunoreactive score (IRS) was calculated. The interaction of PIWI-like 1 and PIWI-like 2 showed a prognostic effect on survival. For the combination of high PIWI-like 1 and low PIWI-like 2 expressions, adjusted hazard ratios (HRs) were significantly higher with regard to overall survival (OS) (HR 2.92; 95% confidence interval (CI) 1.24, 6.90), disease-free survival (DFS) (HR 3.27; 95% CI 1.48, 7.20), and distant disease-free survival (DDFS) (HR 7.64; 95% CI 2.35, 24.82). Both proteins were significantly associated with molecular-like and PAM50 subgroups. Combining high PIWI-like 1 and low PIWI-like 2 expressions predicted poorer prognosis and both markers were associated with aggressive molecular subtypes.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients.

Simple SummaryThe expression of the androgen receptor (AR) and its splice variant AR-V7 is crucial for prostate cancer (PCa) biology. An immunohistochemical staining was performed on a tissue microarray with specimens from 410 PCa patients. AR staining, neither in the nucleus nor in the cytoplasm was associated with prognosis. AR-V7 staining of the general cytoplasm was associated with a shorter relapse free survival (RFS), whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. Further subgroup stratification for AR-V7 granular staining revealed it as an independent prognostic factor in younger patients (age ≤ 65), patients with negative CK20 staining and patients with perineural invasion. Altogether, AR-V7 protein detected in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

ERK phosphorylation as a marker of RAS activity and its prognostic value in non-small cell lung cancer

BackgroundDeregulated signal transduction pathways play a key role in development, progression and therapeutic resistance of non-small cell lung cancers (NSCLC). The purpose of this study is to assess the downstream markers of two well-characterized pathways and to correlate them with clinical outcome. Design670 patients with metastatic NSCLC were prospectively enrolled in a comprehensive biomarker profiling program at a single center from 2012 to 2016. Phosphorylation of extracellular signal-regulated kinase (p-ERK), and protein kinase B (p-AKT) was assessed by standardized immunohistochemistry. Product of scores for quantity and quality of staining were calculated (immunoreactive score, 0–9). Somatic mutations of Kirsten rat sarcoma viral oncogene homolog [KRAS], epithelial growth factor receptor [EGFR], v-Raf murine sarcoma viral oncogene homolog B [BRAF] and phosphatidylinositol 3-kinase [PIK3CA]) were detected by Sanger (2012-03/2015) and amplicon NGS (04/2015-02/2016). Patients enrolled during the first year (2012) were used as discovery cohort. Patients enrolled from 2013 to 02/2016 were used as validation cohort. Clinical data were retrieved from the electronic medical records and were analyzed retrospectively. ResultsUsing a discovery cohort, we identified an immunoreactive score of p-ERK ≥3 to be prognostically relevant. The validation cohort confirmed that higher levels of p-ERK correlated with worse overall survival (OS) and higher proportion of RAS mutations. Multivariate analysis including established risk factors such EGFR, ALK or ROS mutations and metastatic disease showed a trend of a detrimental effect of high p-ERK on OS (HR 1.23, CI 0.94–1.59, p = 0.131 for p-ERK immunoreactive score ≥3) and time to treatment failure after first-line therapy in the validation cohort. Phosphorylated AKT did not correlate with clinical outcome. ConclusionWhile serving as a prognosticator in univariate analysis, highly phosphorylated ERK does not convey a significant prognostic effect for OS in the presence of other prognostic factors. Phosphorylated ERK indicates a higher activity of RAS in advanced NSCLC.

Metformin and silymarin afford protection in cyclosporine A induced hepatorenal toxicity in rat by modulating redox status and inflammation.

The use of cyclosporine A (CsA) as an immunosuppressive agent is often limited owing to its hepatotoxic and nephrotoxic properties. The present study was designed to evaluate the protective effect of metformin and silymarin in a rat model of CsA induced hepatorenal toxicity. The study included seven groups of Wistar albino rats (n = 6 per group): normal control, experimental control (CsA alone, 25 mg/kg), CsA + metformin (50 and 500 mg/kg), CsA + silymarin (50 and 200 mg/kg) and CsA + vitamin E (100 mg/kg). All the drugs were given daily for a period of 21 days by oral gavage and their effect was evaluated on serum levels of organ function markers (serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase, bilirubin, urea/blood urea nitrogen, creatinine), markers of oxidative stress (thiobarbituric acid reactive substances, glutathione, superoxide dismutase), inflammation (nitrite, myeloperoxidase, tumour necrosis factor-alpha, prostaglandin E2 ), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling positivity) in addition to tissue histology, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) immunoreactivity. Administration of metformin and silymarin along with CsA ameliorated functional damage to liver and kidneys in a dose-dependent manner. Significant and comparable improvement in the tissue levels of oxidative stress, inflammation, apoptotic markers was also observed following treatment with both the test drugs. Normalization of histology scores, as well as COX-2 and iNOS immunoreactivity scores, further strengthened these findings. The hepatoprotective and nephroprotective effects of metformin and silymarin were comparable and matched with that of reference drug, vitamin E. The findings of the present study suggest that both metformin and silymarin have a potential for clinical use in patients receiving long-term CsA treatment to maintain their liver and kidney functions.

EXPRESSION OF TERATOCARCINOMA-DERIVED GROWTH FACTOR-1 (CRIPTO-1) IN EPITHELIAL ODONTOGENIC LESIONS

Objective: To evaluate the immunohistochemical expression of CRIPTO-1 in odontogenic cysts and tumors. Study Design: Thirty ameloblastomas, 30 keratocysts, 30 dentigerous cysts, and 2 ameloblastic carcinomas were evaluated using the biotin-free immunoperoxidase technique. The immunohistochemical expression was analyzed from the immunoreactive score (IRS). Kruskal Wallis and Mann-Whitney U tests were performed for statistical analysis (P ≤ .05). Results: Ages ranged from 9 to 75 years, with female gender being the most prevalent (n = 49; 53.3%). The mandible was the most affected anatomic site (n = 69; 75.0%). Immunoexpression of CRIPTO-1 was observed in all cases of ameloblastoma, keratocyst, and ameloblastic carcinoma; however, 9 cases (30%) of dentigerous cysts were negative. Expression scores were higher in cases of ameloblastomas, keratocysts, and ameloblastic carcinomas (median, 8-11) compared with cases of dentigerous cysts (median, 2) being statistically significant (P

Identification of G2 and S Phase-Expressed-1 as a Potential Biomarker in Patients with Prostate Cancer.

BackgroundThis study aimed to predict and explore the possible clinical value and mechanism of genetic markers in prostate cancer (PCa) using a bioinformatics analysis method.Materials and MethodsThe RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) database to identify the differentially expressed genes (DEGs). The hub genes were screened by building protein–protein interaction (PPI) subnetworks with four topological analysis methods. The overall survival analysis of hub genes was conducted using Kaplan–Meier curves. Furthermore, the bioinformatics results were confirmed in 102 PCa samples collected in our hospital. Gene Set Enrichment Analysis (GSEA) was performed to provide information about the molecular mechanisms underlying PCa.ResultsAmong 13 hub genes, the high expression of GTSE1 or KIF18B was associated with worse overall survival according to the TCGA samples. Immunoreactive scores for GTSE1 staining were significantly higher in PCa tissues than in paracancerous tissues (P<0.01). The overall survival time of patients with high GTSE1 expression was shorter than that of patients with low GTSE1 expression (P=0.015). GSEA demonstrated that high GTSE1 expression was mainly enriched in the cell cycle (P<0.001), DNA replication (P<0.001), mismatch repair (P<0.001), and p53 signaling pathway (P<0.001).ConclusionGTSE1 expression was significantly high in PCa and associated with poor prognosis. GTSE1 may serve as a potential biomarker and therapeutic target in PCa patients.

Bladder perforation during transurethral resection of bladder tumour is not a result of a deficient structure of the bladder wall

BackgroundTransurethral resection of the bladder tumour (TUR) is associated with a risk of bladder perforation. The underlying mechanisms and risk factors are not fully understood. The aim of this study was to determine if the bladder wall structure affects the risk of bladder perforation during TUR.MethodsFifteen patients who underwent TUR complicated by a bladder perforation (group 1) and fifteen matched controls who underwent uncomplicated TUR (group 2) were retrospectively enrolled in this morphological analysis. Surgical specimens were collected from all participating patients to describe the quality and architecture of urothelium and bladder submucosa. Immunohistochemical studies were performed with primary mouse anti-human E-cadherin, beta-catenin, type IV collagen, cytokeratin 20 and epithelial membrane antigen antibodies. The intensity of the immunohistochemical reaction was assessed using an immunoreactive score (IRS). Ultrastructural examinations were performed by transmission electron microscopy. The microscopic assessment was focused on the intensity of fibrosis in the bladder submucosa and the presence of degenerative changes in the urothelium.ResultsPatients’ age, sex distribution, tumour diameters, surgeon experience or cancer stage did not differ between study groups. The immunohistochemical analysis did not reveal statistically significant differences between group 1 and group 2. From a clinical point of view, ultrastructural analysis by electron microscopy showed a higher rate of severe fibrosis in group 1 (63.6% vs. 38.5%), with no differences in the rate and degree of urothelial changes. However, these differences were not statistically significant (p = 0.32).ConclusionsBladder perforation during TUR is not a result of a deficient structure of the bladder wall. Based on available evidence, the surgical technique seems to play the most important role in its prevention.

Antimicrobial peptides in human synovial membrane as (low-grade) periprosthetic joint infection biomarkers

BackgroundSafe diagnosis of periprosthetic joint infection (PJI) is of utmost importance for successful exchange arthroplasty. However, current diagnostic tools show insufficient accuracy in the clinically common and challenging chronic low-grade infections. To close this diagnostic gap, reliable (bio)markers display the most promising candidates. Antimicrobial peptides (AMPs) are part of the innate immune response towards microbial growth. Recently we could show significant intraarticular levels of human cathelicidin LL-37 and β-defensin-3 (HBD-3) with high diagnostic accuracy in PJI synovial fluid. Consequently, these promising biomarkers were evaluated in PJI synovial membrane and synoviocytes, which may significantly facilitate histological diagnosis of PJI to improve outcome of septic joint replacement.MethodsIn this prospective single-center controlled clinical study (diagnostic level II), consecutive patients with total hip (THR) and knee (TKR) replacements were included undergoing primary arthroplasty (n = 8), surgical revision due to aseptic loosening (n = 9) and septic arthroplasty with coagulase-negative staphylococci (n = 8) according to the criteria of the Musculoskeletal Infection Society (MSIS). Semiquantitative immunohistochemical (IHC) analysis of LL-37, HBD-3 and HBD-2 in synovial membrane and isolated synoviocytes based on Total Allred Score (TS) and Immunoreactive Remmele and Stegner score (IRS) was performed. For statistical analysis, SPSS 26.0/R3.6.3 (p < 0.05) was used.ResultsThe AMPs LL-37 and HBD-3 were significantly elevated (up to 20×) in synovial membranes from PJI compared to aseptic loosening or primary arthroplasty. The area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 1.0 for both scores revealing excellent diagnostic accuracy. Isolated synoviocytes as cellular AMP source showed comparable results with a significant LL-37/HBD-3-increase up to 3 × in PJI. In contrast, local HBD-2 levels were negligible (p > 0.23) upon PJI with a lower diagnostic accuracy (AUC = 0.65) in analogy to our previous findings with synovial fluid.ConclusionsOur results implicate AMPs as promising and specific biomarkers for the histological diagnosis of PJI.

Abstract 4327: Progranulin (GP88) is an independent prognostic factor for prostate cancer patients

Abstract Prostate cancer, is still a major cause of morbidity and mortality among men worldwide. The expression of the survival and proliferation factor progranulin (GP88) has not yet been comprehensively studied in PCa tumors. The aim of this study was to characterize GP88 protein expression in PCa by immunohistochemistry and to correlate the findings to the clinico-pathological data and prognosis. Immunohistochemical staining for GP88 was performed by TMA with samples from 442 PCa patients using an immunoreactive score (IRS). Altogether, 233 cases (52.7%) with negative GP88 staining (IRS&amp;lt;2) and 209 cases (47.3%) with positive GP88 staining (IRS≥2) were analyzed. A significant positive correlation was found for the GP88 IRS with the PSA value at prostatectomy and the cytoplasmic cytokeratin 20 IRS, whereas it was negatively associated with follow-up times. The association of GP88 staining with prognosis was further studied by survival analysis (Kaplan-Meier, univariate and multivariate Cox's regression analysis). Increased GP88 protein expression appeared as independent prognostic factor for overall, disease-specific and relapse-free survival in all PCa patients. Interestingly, in the subgroup of younger PCa patients (≤65 years), GP88 positivity was associated with a 3.8-fold (P=0.004), a 6.0-fold (P=0.008) and a 3.7-fold (P=0.003) increased risk for death, disease-specific death and occurrence of a relapse, respectively. This finding gains impact against the background of increasing numbers of PCa cases among younger men below 65 years of age. Summarizing, GP88 protein positivity appears to be an independent prognostic factor for PCa patients. Citation Format: Helge W. Taubert, Amer Abdulrahman, Markus Eckstein, Rudolf Jung, Juan Guzman, Katrin Weigelt, Ginette Serrero, Binbin Yue, Carol Geppert, Robert Stöhr, Arndt Hartmann, Bernd Wullich, Sven Wach. Progranulin (GP88) is an independent prognostic factor for prostate cancer patients [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 4327.

Abstract 4380: Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer

Abstract Background: The estrogen receptor beta (ERβ) is the predominant estrogen receptor in the normal colon mucosa, and its expression is downregulated in colorectal cancer (CRC). On the other hand, ER alpha (ERα) is very little expressed in the normal colon tissue. An increased ERα/β ratio has been shown in colon carcinomas. However, the role of ERα in CRC development and progression needs further evaluation. Aim: To investigate the role of ERα expression in CRC prognosis. Methods: A tumor tissue microarray of primary CRCs from 351 patients was stained for the expression of ERα. The nuclear staining intensity was evaluated using the Immuno-Reactive Score (IRS). Cox regression analysis and Kaplan-Meier curves were used to evaluate overall (OS) and disease-free survival (DFS). Colon cancer (CC) cells lines, SW-480, HCT-116 and HT-29 were used for in-vitro investigations. Two CC mouse models were used to investigate the expression of ERα. The zebrafish xenograft model was used to study CC cell metastasis. Untreated and ERα specific agonist (PPT, 40 nM) treated colon cancer cells were injected into the zebrafish perivitelline space. After 48 h the tale was checked for the presence of the metastasized cells. Results: ERα nuclear expression was negative in 232 and positive in 119 CRC patients, with a significantly higher expression in cancer tissue compared to normal tissue. Patients with positive ERα expression had higher TNM stage and more lymph node and distant metastasis. Positive ERα expression was independently associated with worse OS (HR=0.42; 95% CI: 0.26-0.51) and DFS (HR=0.32; 95% CI: 0.18-0.56), after adjustment for age and TNM stage. Moreover, patients with positive ERα expression had higher frequency of activated K-RAS mutation and higher expression levels of cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear β-catenin, both with potent pro-tumorigenic properties. In the colon tissue from the colitis-associated colon cancer (CAC) mouse model with the Cysltr1 gene disruption, ERα expression was significantly decreased compared to wild-type (WT) mice, while in the colon of ApcMin/+ mice the ERα expression was significantly increased. In-vitro, PPT treatment significantly increased cell migration and wound closure compared to untreated cells. The zebrafish xenograft model showed that fish injected with PPT stimulated CC cells had significantly more tail metastasis compared with the control group (80% versus 67% respectively). Conclusion: Positive ERα expression correlates with poor prognosis and metastasis in colorectal cancer. Our results suggest that ERα-selective antagonists might be beneficial to combine therapeutic opportunities for improving the prognosis of CRC patients. Citation Format: Geriolda Topi, Shakti R Satapathy, Roy Roy Ehrnström, Marie-Louise Lydrup, Anita Sjölander. Positive expression of estrogen receptor alpha correlates with poor prognosis and metastasis in colorectal cancer [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 4380.

Abstract 1271: UV irradiation and epigenetic change in melanocyte 5-hydroxymethylcytosine

Abstract Background Melanoma is the main cause of death from skin cancer. Excessive sun exposure with resultant UV irradiation, particularly during childhood, is established as its major environmental risk factor. Epigenetics are becoming increasingly recognized as potential reversible regulators of genomic anomalies responsible for cancerous growth. Loss of DNA hydroxymethylation marks 5-hydroxymethylcytosine (5-hmC) a functionally significant epigenetic event in melanomagenesis, but whether decreased 5-hmC is driven by UV irradiation remains unknown. The aim of this study is to characterize 5-hmC levels in potential melanoma precursor lesions and cultured samples by UV irradiation in vitro. Methods 70 human skin excision cases of chronic UV exposed skin and matched controls were evaluated. In addition, discarded human foreskins were exposed to biologically relevant doses of UVB (302 nm) and UVA (365 nm) irradiation. Tissue samples were then incubated in DMEM medium and after 24 hours fixed in 10% formalin overnight and stored in 70% ethanol prior to sectioning and staining. A dual anti-5-hmC and anti-MART-1 immunohistochemical (IHC) staining protocol was performed, and resultant semiquantitative immunoreactivity scores were analyzed statistically by one-way ANOVA analysis and Bonferroni's Multiple comparisons test or unpaired T-test using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA). All p-values were two-tailed, with a p&amp;lt;0.05 considered statistically significant. Results When comparing sun-protected versus chronically sun-exposed sites of the excision specimens, lower levels of 5-hmC immunoreactivity were noted in melanocytes of sun-protected specimens than sun-exposed samples (p = 0.017). Moreover, a more significant difference (p = 0.0053) was detected between 5-hmC staining in normal skin melanocytes versus 5-hmC loss detected in so-called “atypical melanocytic hyperplasia of sun-damaged skin” that surrounds melanomas. Finally, we sought to determine whether we could induce in vitro a UV effect on 5-hmC similar to that seen with chronic sun-damage. Significant decreased immunoreactivity for 5-hmC was observed in foreskin melanocytes after UV irradiation- a finding that replicated what we observed in patient samples containing chronically UV-altered melanocytes. Conclusions Our results suggest that UVB irradiation's effect on the epigenome may play a crucial role in the development of melanoma. Implementation of the dual staining for anti-5-hmC and anti-MART-1 may be helpful in defining melanocytes altered in the direction of melanomagenesis, and thus may serve as a practical assay in determining potential melanoma precursor fields amenable to surveillance and prophylactic therapies. Citation Format: Kevin Y. Wang, Tanja Zdravkovic, Christine Lian. UV irradiation and epigenetic change in melanocyte 5-hydroxymethylcytosine [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 1271.

18F]FEPPA PET imaging for monitoring CD68-positive microglia/macrophage neuroinflammation in nonhuman primates

PurposeThe aim of this study was to examine whether the translocator protein 18-kDa (TSPO) PET ligand [18F]FEPPA has the sensitivity for detecting changes in CD68-positive microglial/macrophage activation in hemiparkinsonian rhesus macaques treated with allogeneic grafts of induced pluripotent stem cell-derived midbrain dopaminergic neurons (iPSC-mDA).MethodsIn vivo positron emission tomography (PET) imaging with [18F]FEPPA was used in conjunction with postmortem CD68 immunostaining to evaluate neuroinflammation in the brains of hemiparkinsonian rhesus macaques (n = 6) that received allogeneic iPSC-mDA grafts in the putamen ipsilateral to MPTP administration.ResultsBased on assessment of radiotracer uptake and confirmed by visual inspection of the imaging data, nonhuman primates with allogeneic grafts showed increased [18F]FEPPA binding at the graft sites relative to the contralateral putamen. From PET asymmetry analysis of the images, the mean asymmetry index of the monkeys was AI = − 0.085 ± 0.018. Evaluation and scoring of CD68 immunoreactivity by an investigator blind to the treatment identified significantly more neuroinflammation in the grafted areas of the putamen compared to the contralateral putamen (p = 0.0004). [18F]FEPPA PET AI showed a positive correlation with CD68 immunoreactivity AI ratings in the monkeys (Spearman’s ρ = 0.94; p = 0.005).ConclusionThese findings reveal that [18F]FEPPA PET is an effective marker for detecting increased CD68-positive microglial/macrophage activation and demonstrates sufficient sensitivity to detect changes in neuroinflammation in vivo following allogeneic cell engraftment.