Characterization of immunoreactivity with whole-slide imaging and digital analysis in high-grade serous ovarian cancer.

Ovarian cancer is the most lethal of gynecological cancers with 5-year survival rate of ca. 45%. The most common histologic subtype is high-grade serous carcinoma, which typically is presented with advanced stage and development of chemoresistance. Therefore, new treatment options, including immunotherapies, are needed. Understanding the features of the immune cell populations in the tumor microenvironment is essential for developing personalized treatments and finding predictive biomarkers. Digital image analysis may enhance the accuracy and reliability of immune cell infiltration assessment in the tumor microenvironment. The aim of this study was to characterize tumor microenvironment in a retrospective cohort of high-grade serous carcinoma samples with whole-slide imaging and digital image analysis. Formalin-fixed paraffin-embedded high-grade serous carcinoma tumor tissue samples (n = 67) were analyzed for six immunohistochemical stainings: CD4, CD8, FoxP3, granzyme B, CD68, and CD163. The stained sample slides were scanned into a digital format and assessed using QuPath 0.1.2 and ImageJ software. Staining patterns were associated with clinicopathological data. The higher numbers of intraepithelial CD8+, CD163+, and granzyme B+ immune cells were associated with survival benefit when analyzed individually, while high levels of both CD8+ and granzyme B+ tumor-infiltrating lymphocytes were an independent prognostic factor in the Cox multivariate regression analysis (median progression-free survival; hazard ratio = 0.287, p = 0.002). Specimens taken after administration of neoadjuvant chemotherapy presented with lower FoxP3+ tumor-infiltrating lymphocyte density (Fisher's exact test, p = 0.013). However, none of the studied immunomarkers was associated with overall survival or clinical factors. Tumors having high amount of both intraepithelial CD8+ and granzyme B+ tumor-infiltrating lymphocytes showed better progression-free survival, possibly reflecting an activated immune state in the tumor microenvironment. The combined positivity of CD8 and granzyme B warrants further investigation with respect to predicting response to immune therapy. Neoadjuvant chemotherapy may have an effect on the tumor microenvironment and therefore on the response to immuno-oncologic or chemotherapy treatments.

Expression of Estrogen-Related Receptors in Localized Provoked Vulvodynia.

Eight percent of women suffer from vulvodynia, a chronic pain condition with unknown etiology. Inflammation and dysregulation of estrogen signaling have been suggested to play a role in the pathogenesis of localized provoked vulvodynia (LPV). Therefore, the aim of the study was to analyze protein expression levels of estrogen-related receptors ERRα, ERRß, ERRγ, estrogen receptor (ERα), and progesterone receptor (PRα) and CD3-positive T cells in the vulvar vestibulum obtained from women suffering from LPV in comparison to healthy, unaffected controls. Vulvar vestibulum tissue specimens were obtained from LPV patients (n = 12) who had undergone modified posterior vestibulectomy and from 15 healthy controls. Protein expression of ERRα, ERRß, ERRγ, ERα, and PRα and CD3-positive T cells was analyzed by immunohistochemistry (IHC). Expression of ERRß was significantly more pronounced in samples from LPV compared to healthy controls (p = 0.006). No significant difference in the expression patterns of ERRα, ERRγ, ERα, PRα, or CD3 cells was detected. To our knowledge, this is the first study reporting ERR expression in normal vestibulum and in vestibulectomy samples from LPV patients. The higher level of ERRß expression detected by IHC may reflect dysregulation of estrogen signaling in LPV.

Fluoro-Chromogenic Labelling for Detection of MCM2 to Assess Proliferation Activity in HER2-amplified Breast Carcinomas.

Minichromosome Maintenance Protein 2 (MCM2) is critical in initiating DNA replication during the cell division process. As expressed intensively in all phases of the active cell cycle, MCM2 has been proposed as a novel biomarker to determine cellular proliferation. We aimed at clarifying the prevalence and clinical significance of MCM2 in HER2-amplified breast cancer subtype. MCM2 expression was studied in 142 primary HER2-amplified breast carcinomas by applying a novel fluoro-chromogenic immunohistochemistry and tailored digital image analysis to determine labelling index (MCM2-LI). The presence of MCM2 was detected with HRP-conjugated polymer and visualized with 3, 3'-diaminobenzidine tetrahydrochloride, in cytokeratin (CK)-positive and Cy2-IgG-labelled breast cancer cells of epithelial origin. Stained slides were digitized by scanning sequentially under bright field (for MCM2) and fluorescence (for CK) illumination. Multilayer JPEG2000 images were analyzed with ImmunoRatio 2.5 (accessory in SlideVantage 1.2 software) utilizing its bright field and fluorescence image-blending mode to display MCM2-CK dual-positive cells. MCM2-LI was retrospectively compared with histopathologic characteristics and patients' clinical outcome. MCM2 protein-expressing cells (median MCM2-LI, 63.5%) were more frequent than those of Ki67 (median Ki67 labelling index, 33%). Significant correlations were found between high MCM2-LI, high Ki67 labelling index, negative hormone receptor (ER, PR) statuses, high grade of malignancy, and high cyclin E expression. MCM2-LI was not shown to be predictive of disease recurrence during the median follow-up of 5.3 years but was shown to be useful to distinguish aggressive-type HER2-amplified breast carcinomas with high malignancy grade and hormone receptor negativity. The fluoro-chromogenic double-labelling immunohistochemistry accompanied with digital image analysis provides an accurate carcinoma-specific determination of MCM2-LI on a single tumor section.

Clinicopathological and prognostic correlations of HER3 expression and its degradation regulators, NEDD4-1 and NRDP1, in primary breast cancer.

BACKGROUND: Human epidermal growth factor receptor HER3 (ErbB3), especially in association with its relative HER2 (ErbB2), is known as a key oncogene in breast tumour biology. Nonetheless, the prognostic relevance of HER3 remains controversial. NEDD4-1 and NRDP1 are signalling molecules closely related to the degradation of HER3 via ubiquitination. NEDD4-1 and NRDP1 have been reported to contribute to HER3-mediated signalling by regulating its localization and cell membrane retention. We studied correlations between HER3, NEDD4-1, and NRDP1 protein expression and their association with tumour histopathological characteristics and clinical outcomes. METHODS: The prevalence of immunohistochemically detectable expression profiles of HER3 (n = 177), NEDD4-1 (n = 145), and NRDP1 (n = 145) proteins was studied in primary breast carcinomas on archival formalin-fixed paraffin-embedded (FFPE) samples. Clinicopathological correlations were determined statistically using Pearson's Chi-Square test. The Kaplan-Meier method, log-rank test (Mantel-Cox), and Cox regression analysis were utilized for survival analysis. RESULTS: HER3 protein was expressed in breast carcinomas without association with HER2 gene amplification status. Absence or low HER3 expression correlated with clinically aggressive features, such as triple-negative breast cancer (TNBC) phenotype, basal cell origin (cytokeratin 5/14 expression combined with ER negativity), large tumour size, and positive lymph node status. Low total HER3 expression was prognostic for shorter recurrence-free survival time in HER2-amplified breast cancer (p = 0.004, p = 0.020 in univariate and multivariate analyses, respectively). The majority (82.8%) of breast cancers demonstrated NEDD4-1 protein expression - while only a minor proportion (8.3%) of carcinomas expressed NRDP1. NEDD4-1 and NRDP1 expression were not associated with clinical outcomes in HER2-amplified breast cancer, irrespective of adjuvant trastuzumab therapy. CONCLUSIONS: Low HER3 expression is suggested to be a valuable prognostic biomarker to predict recurrence in HER2-amplified breast cancer. Neither NEDD4-1 nor NRDP1 demonstrated relevance in prognostics or in the subclassification of HER2-amplified breast carcinomas.

Comparison of Antibodies for Immunohistochemistry-based Detection of HER3 in Breast Cancer.

BACKGROUND: Growth factor receptor HER3 (ErbB3) lacks standardized immunohistochemistry (IHC)-based methods for formalin-fixed paraffin-embedded (FFPE) tissue samples. We compared 4 different anti-HER3 antibodies to explain the differences found in the staining results reported in the literature. MATERIALS AND METHODS: Four commercial HER3 antibodies were tested on FFPE samples including mouse monoclonal antibody clones, DAK-H3-IC and RTJ1, rabbit monoclonal antibody clone SP71, and rabbit polyclonal antibody (SAB4500793). Membranous and cytoplasmic staining patterns were analyzed and scored as 0, 1+, or 2+ according to the intensity of the staining and completeness of membranous and cytoplasmic staining. A large collection of HER2-amplified breast cancers (n=177) was stained with the best performing HER3 antibody. The breast cancer cell line, MDA-453, and human prostate tissue were used as positive controls. IHC results were confirmed by analysis of flow cytometry performed on breast cancer cell lines. Staining results of FFPE samples were compared with samples fixed with an epitope-sensitive fixative (PAXgene). RESULTS: Clear circumferential cell membrane staining was found only with the HER3 antibody clone DAK-H3-IC. Other antibodies (RTJ1, SP71, and polyclonal) yielded uncertain and nonreproducible staining results. In addition to cell membrane staining, DAK-H3-IC was also localized to the cytoplasm, but no nuclear staining was observed. In HER2-amplified breast cancers, 80% of samples were classified as 1+ or 2+ according to the HER3 staining on the cell membrane. The results from FFPE cell line samples were comparable to those obtained from unfixed cells in flow cytometry. IHC conducted on FFPE samples and on PAXgene-fixed samples showed equivalent results. CONCLUSIONS: We conclude that IHC with the monoclonal antibody, DAK-H3-IC, on FFPE samples is a reliable staining method for use in translational research. Assessment of membranous HER3 expression may be clinically relevant in selecting patients who may most benefit from pertuzumab or other novel anti-HER3 therapies.

Cyclin E amplification, over-expression, and relapse-free survival in HER-2-positive primary breast cancer.

Cyclin E is a well-characterized cell cycle regulator and an amplified oncogene in breast cancer. Over-expression of cyclin E has generally been associated with poor survival. Recent studies have shown an interaction between HER-2 (ERBB2) and cyclin E, but the exact mechanism is unknown. Interestingly, cyclin E over-expression has been associated with trastuzumab resistance. We studied cyclin E over-expression, CCNE1 amplification, and relapse-free survival in HER-2-positive primary breast cancers treated with and without trastuzumab therapy. Formalin-fixed paraffin-embedded tissue samples from 202 HER-2-positive breast carcinomas were studied. Expression levels of cyclin E and proliferation marker Ki-67 were determined using immunohistochemistry. Chromogenic in situ hybridization (CISH) with a gene-specific bacterial artificial chromosome (BAC) probe was used to analyze presence of CCNE1 amplification. Majority of HER-2-positive breast carcinomas exhibited nuclear staining for cyclin E protein. Cyclin E was highly expressed (≥50 % cells) in 37 % of cases. Incidence of CCNE1 amplification (≥6 gene copies/cell or clusters) was 8 %. Cyclin E amplification and over-expression were strongly associated with each other, grade, hormone receptors, and Ki-67. Neither high cyclin E expression nor CCNE1 amplification was associated with relapse-free survival (RFS) irrespective of short-term (9-week regimen) adjuvant trastuzumab therapy. These results confirm cyclin E and HER-2 gene co-amplification in a fraction of HER-2-positive breast cancers. Cyclin E is frequently over-expressed but appears to have limited value as a prognostic or predictive factor in HER-2-positive breast cancer regardless of trastuzumab therapy.

Effect of fermented milk product containing lactotripeptides and plant sterol esters on haemodynamics in subjects with the metabolic syndrome--a randomised, double-blind, placebo-controlled study.

We investigated the effects of fermented milk product containing isoleucine-proline-proline, valine-proline-proline and plant sterol esters (Pse) on plasma lipids, blood pressure (BP) and its determinants systemic vascular resistance and cardiac output. In a randomised, double-blind, placebo-controlled study, 104 subjects with the metabolic syndrome (MetS) were allocated to three groups in order to receive fermented milk product containing (1) 5 mg/d lactotripeptides (LTP) and 2 g/d plant sterols; (2) 25 mg/d LTP and 2 g/d plant sterols; (3) placebo for 12 weeks. Plasma lipids and home BP were monitored. Haemodynamics were examined in a laboratory using radial pulse wave analysis and whole-body impedance cardiography in the supine position and during orthostatic challenge. There were no differences between the effects of the two treatments and placebo on the measurements of BP at home or on BP, systemic vascular resistance index and cardiac index in the laboratory, neither in the supine nor in the upright position. The changes in plasma LDL-cholesterol concentration were - 0.1 (95% CI - 0.3, 0.1 and - 0.3, 0.0) mmol/l in the 5 and 25 mg/d LTP groups, respectively, and +0.1 (95% CI - 0.1, 0.3) mmol/l during placebo (P= 0.024). Both at baseline and at week 12, the increase in systemic vascular resistance during head-up tilt was lower in the 25 mg/d LTP group than in the 5 mg/d LTP group (P< 0.01), showing persistent differences in cardiovascular regulation between these groups. In subjects with the MetS, intake of LTP and Pse in fermented milk product showed a lipid-lowering effect of borderline significance, while no antihypertensive effect was observed at home or in the laboratory.

Activities of angiotensin-converting enzymes ACE1 and ACE2 and inhibition by bioactive peptides in porcine ocular tissues.

PURPOSE: An active local renin-angiotensin system (RAS) has recently been found in the human eye. The aim of the present study was to compare the activities of central RAS enzymes (ACE1 and 2) in porcine ocular tissues, morphologically and physiologically close to the human eye. In addition, the effects of three ACE-inhibitory tripeptides on these enzymes were evaluated. METHODS: Enucleated fresh porcine eyes were used. Activities of ACE1 and ACE2 and their inhibition by bioactive tripeptides (Ile-Pro-Pro, Val-Pro-Pro, Leu-Pro-Pro) as well as by a standard ACE-inhibitor captopril were assayed in the vitreous body, the retina and the ciliary body using fluorometric detection methods. RESULTS: Activity of ACE1 as well as ACE2 was found in all tissues evaluated. ACE1 activity was markedly higher in the ciliary body (3.7 +/- 0.7 mU/mg protein) than in retina (0.2 +/- 0.02 mU/mg), whereas ACE2 activities in the ciliary body (0.2 +/- 0.02 mU/mg) and retina (0.2 +/- 0.01 mU/mg) were at the same level. In the vitreous body ACE1 activity (8.2 +/- 0.31 nmol/min/mL) was manifold compared to that of ACE2 (0.1 +/- 0.02 nmol/min/mL). The tripeptides inhibited ACE1 at one-thousandth of the concentration needed to inhibit ACE2. All peptides studied evinced about equal inhibitory activities. CONCLUSION: To our knowledge the present findings constitute the first evidence of ACE2 activity in the ciliary and vitreous bodies, in addition to previously described activity in the retina. The known favorable effects of ACE2 products vs. those of ACE1 suggest a counterbalancing interaction of these two enzyme homologues in physiological regulation of ocular circulation and pressure and possible protective role in certain ophthalmic disorders such as glaucoma and diabetic retinopathy.

Does the renin-angiotensin system also regulate intra-ocular pressure?

The renin-angiotensin-aldosterone system is known to play an essential role in controlling sodium balance and body fluid volumes, and thus blood pressure. In addition to the circulating system which regulates urgent cardiovascular responses, a tissue-localized renin-angiotensin system (RAS) regulates long-term changes in various organs. Many recognized RAS components have also been identified in the human eye. The highly vasoconstrictive angiotensin II (Ang II) is considered the key peptide in the circulatory RAS. However, the ultimate effect of RAS activation at tissue level is more complex, being based not only on the biological activity of Ang II but also on the activities of other products of angiotensinogen metabolism, often exerting opposite effects to Ang II action. In recent studies, orally administered angiotensin II type 1 receptor blockers and angiotensin-converting enzyme inhibitors lower intra-ocular pressure (IOP), likewise topical application of these compounds, the effect being more prominent in ocular hypertensive eyes. Based on previous findings and our own experimental data, it can strongly be suggested that the RAS not only regulates blood pressure but is also involved in the regulation of IOP.