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Although several antibodies are available for immunohistochemical detection of Ki-67, even the most commonly used MIB-1 has not been validated yet. Our aim was to compare 5 commercially available antibodies for detection of Ki-67 in terms of agreement and their ability in predicting prognosis of breast cancer. Tissue microarrays were constructed from 378 breast cancer patients' representative formalin-fixed, paraffin-embedded tumor blocks. Five antibodies were used to detect Ki-67 expression: MIB-1 using chromogenic detection and immunofluorescent-labeled MIB-1, SP-6, 30-9, poly, and B56. Semiquantitative assessment was performed by 2 pathologists independently on digitized slides. To compare the 5 antibodies, intraclass correlation and concordance correlation coefficient were used. All the antibodies but immunofluorescent-labeled MIB-1 (at 20% and 30% thresholds, P=.993 and P=.342, respectively) and B56 (at 30% threshold, P=.288) separated high- and low-risk patient groups. However, there were a significant difference (P values for all comparisons≤.005) and a moderate concordance (intraclass correlation, 0.645) between their Ki-67 labeling index scores. The highest concordance was found between MIB-1 and poly (concordance correlation coefficient=0.785) antibodies. None of the antibodies except Ki-67 labeling index as detected by poly (P=.031) at 20% threshold and lymph node status (P<.001) were significantly linked to disease-free survival in multivariate analysis. At 30% threshold, this was reduced to lymph node status (P<.001) alone. Our results showed that there are considerable differences between the different Ki-67 antibodies in their capacity to detect proliferating tumor cells and to separate low- and high-risk breast cancer patient groups., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We aimed to analyze to what extent expression of four cell cycle regulation markers-minichromosome maintenance protein (MCM2), Ki-67, cyclin A, and phosphohistone-H3 (PHH3)-predict response to primary systemic therapy in terms of pathological complete remission (pCR). In search of an accurate and reproducible scoring method, we compared computer-assisted (CA) and routine visual assessment (VA) of immunoreactivity. We included 57 patients with breast cancer in the study. The cell cycle markers were detected using immunohistochemistry on pre-therapy core biopsy samples. Parallel CA (validated by manual labeling) and standard VA were performed and compared for diagnostic agreement and predictive value for pCR. CA and VA results were dichotomized based on receiver operating characteristic analysis defined optimal cut-off values. "High" was defined by staining scores above the optimal cut-off, while "low" had staining scores below the optimal cut-off. The CA method resulted in significantly lower values for Ki-67 and MCM2 compared to VA (mean difference, -3.939 and -4.323). Diagnostic agreement was highest for cyclin A and PHH3 (-0.586 and -0.666, respectively). Regardless of the method (CA/VA) used, all tested markers were predictive of pCR. Optimal cut-off-based dichotomization improved diagnostic agreement between the CA and VA methods for every marker, in particular for MCM2 (κ = 1, p < 0.000). Cyclin A displayed excellent agreement (κ = 0.925; p < 0.000), while Ki-67 and PHH3 showed good agreement (κ = 0.789, p < 0.000 and κ = 0.794, p < 0.000, respectively). We found all cell cycle markers (Ki-67, MCM2, cyclin A, and PHH3) predictive of pCR. Diagnostic agreement between CA and VA was better at lower staining scores but improved after optimal cut-off-based dichotomization.

Claudins are key tight junctional proteins between adjacent epithelial, mesothelial or endothelial cells, which are responsible for the permeability of the paracellular space. This paper describes that the endothelial cells of normal hepatic arterioles, portal venules and portal lymphatics as well as the endothelium of sinusoids from dogs show strong membranous claudin-5 cross-reactivity. In 25 liver biopsy samples taken from dogs with portal vein hypoperfusion, an increased number of arterioles was detected in the portal areas (PAs) by the use of humanised anti-claudin-5 antibody. The increased number of hyperplastic hepatic arterioles per PA was 5-6, 8-12 and 15-20 in the case of small, medium-sized and large PAs, respectively. It is suggested that the claudin-5 marker can improve the detection of hepatic arteriolar proliferation in the PAs of liver samples.

In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.

Whether a breast carcinoma is HER2-positive or -negative has a significant impact on its treatment. The most common method of evaluating the HER2 status of breast tumors is immunohistochemistry. Preliminary data suggest that the proportion of HER2-positive tumors in Hungary shows a wide range from laboratory to laboratory, and overall it just reaches the bottom levels of the incidence reported in the literature (12% 3+ and 11% 2+ cases). A 3-round quality-control test was implemented on a voluntary and anonymous basis. Participating laboratories had to immunostain and evaluate 4 tumor samples per circulation, according to their daily routine. The authors of the present article gave an expert opinion in all cases, and this was compared with the individual laboratories' evaluation and the real FISH-controlled HER2 status of the samples. On the basis of the participants' and experts' evaluation 22/218 and 21/218 HER2 3+ cases were underscored, corresponding to an underevaluation rate of 10%. As most samples were from HER2-positive tumors, overscoring was less common (1%), but reached 5% (individual laboratories' evaluation) or 7% (expert evaluation) when the 2+ scoring of negative cases was also considered. Each case was discussed interactively with the participants and technical advise was also given when deemed necessary. The evaluation of the HER2 status of breast cancers gives reliable results only if adequate quality control measures are implemented, and this study was an important step in this respect.

Aims: Invasive lobular carcinoma (ILC) has distinct morphology and association with loss of E‐cadherin function. It has special clinical and imaging features, and its proper recognition is important. Following a recent proposal, we tested the value of the routine use of E‐cadherin immunohistochemistry (IHC) in recognizing ILC. Methods and results: Five pathologists with experience in breast pathology from four Hungarian institutions histotyped 1001 breast cancers from diagnostic core biopsies or excision specimens randomly assigned to haematoxylin and eosin (HE) diagnosis first, followed by E‐cadherin IHC; or to immediate HE and E‐cadherin‐based diagnosis. Of 524 cases with HE diagnosis, 73(14%) were deemed uncertain. E‐cadherin made the initial histological type change in 14/524 cases (2.7%), including three with confident HE‐based type allocation. Use of E‐cadherin immunostaining was considered useful in 88/477 cases (18%) with immediate dual assessment, and typing uncertainty went down to 5% (25/477 cases), but was not zero. Collective assessment of 171 uncertain, difficult, nonclassical cases resulted in consensus diagnosis in most cases, but 15 cases were still doubtful as concerns their proper histological type. CDH1 gene sequencing was attempted and successful in 13; pathogenic genetic alterations were identified in seven cases. Conclusions: The routine use of E‐cadherin IHC decreases the uncertainty in typing and improves the typing accuracy at the cost of potentially redundant additional immunostains. Furthermore, this procedure does not exclude uncertainty due to E‐cadherin‐positive ILCs, which are occasionally difficult to confidently label as ILC, especially when the growth pattern is not classic. [ABSTRACT FROM AUTHOR]

Background: Breast cancer has been categorized into molecular subtypes using immunohistochemical staining (IHC) and fluorescence in situ hybridization (FISH) since the early 2000s. However, recent research suggests that gene expression testing, specifically Prosigna® Prediction Analysis of Microarray 50 (PAM50), provides more accurate classification methods. In this retrospective study, we compared the results of IHC/FISH and PAM50 testing. We also examined the impact of various PAM50 parameters on overall survival (OS) and progression-free survival (PFS). Results: We analyzed 42 unilateral breast cancer samples, with 18 classified as luminal A, 10 as luminal B, 8 as Human epidermal growth factor receptor 2 (HER2)-positive, and 6 as basal-like using PAM50. Interestingly, 17 out of the 42 samples (40.47%) showed discordant results between histopathological assessment and the PAM50 classifier. While routine IHC/FISH resulted in classification differences for a quarter to a third of samples within each subtype, all basal-like tumors were misclassified. Hormone receptor-positive tumors (hazard rate: 8.7803; p = 0.0085) and patients who had higher 10-year recurrence risk scores (hazard rate: 1.0539; p = 0.0201) had shorter OS and PFS. Conclusions: Our study supports the existing understanding of molecular subtypes in breast cancer and emphasizes the overlap between clinical characteristics and molecular subtyping. These findings underscore the value of gene expression profiling, such as PAM50, in improving treatment decisions for breast cancer patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The addition of chromosome 17 polysomy/HER2 amplification status to the updated EAU and AUA scores improves their accuracy, allowing molecular reclassification of EAU high-risk NMIBCs and G2 tumors. Based on this, we propose to reclassify the non-HER2 amplified, non-polysomic EAU 3–4 (high- and very high-risk) cases to the EAU 2 (intermediate) risk group to prevent unnecessarily strict follow-up and treatment for these patients. Furthermore, to classify Chr17 polysomic and/or HER2 amplified G2 tumors as high-grade (HG) and non-HER2 amplified, non-polysomic G2 tumors as low-grade (LG) NMIBCs (G1 and G3 tumors remain graded as low- and high-grade, respectively). Thus, the implementation of Chr17 polysomy/HER2 amplification testing would provide an immediate and simple solution to further refine the prognostic risk assessment of NMIBCs in the uro-oncology practice. Progression of non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive disease (MIBC) significantly worsens life expectancy. Its risk can be assessed by clinicopathological factors according to international guidelines. However, additional molecular markers are needed to refine and improve the prediction. Therefore, in the present study, we aimed to predict the progression of NMIBCs to MIBC by assessing p53 expression, polysomy of chromosome 17 (Chr17) and HER2 status in the tissue specimens of the tumors of 90 NMIBC patients. Median follow-up was 77 months (range 2–158). Patients with Chr17 polysomy or HER2 gene amplification had a higher rate of disease progression (hazard ratio: 7.44; p < 0.001 and 4.04; p = 0.033, respectively; univariate Cox regression). Multivariable Cox regression models demonstrated that the addition of either Chr17 polysomy or HER2 gene amplification status to the European Association of Urology (EAU) progression risk score increases the c-index (from 0.741/EAU/ to 0.793 and 0.755, respectively), indicating that Chr17 polysomy/HER2 amplification status information improves the accuracy of the EAU risk table in predicting disease progression. HER2/Chr17 in situ hybridization can be used to select non-progressive cases not requiring strict follow-up, by reclassifying non-HER2-amplified, non-polysomic NMIBCs from the high- and very high-risk groups of EAU to the intermediate-risk group. [ABSTRACT FROM AUTHOR]

Breast cancer can occur in either gender; however, it is rare in men, accounting for <1% of diagnosed cases. In a previous transcriptomic screen of male breast cancer (MBC) and female breast cancer (FBC) occurrences, we observed that Stanniocalcin 2 (STC2) was overexpressed in the former. The aim of this study was to confirm the expression of STC2 in MBC and to investigate whether this had an impact on patient prognosis. Following an earlier transcriptomic screen, STC2 gene expression was confirmed by RT‐qPCR in matched MBC and FBC samples as well as in tumour‐associated fibroblasts derived from each gender. Subsequently, STC2 protein expression was examined immunohistochemically in tissue microarrays containing 477 MBC cases. Cumulative survival probabilities were calculated using the Kaplan–Meier method and multivariate survival analysis was performed using the Cox hazard model. Gender‐specific STC2 gene expression showed a 5.6‐fold upregulation of STC2 transcripts in MBC, also supported by data deposited in Oncomine™. STC2 protein expression was a positive prognostic factor for disease‐free survival (DFS; Log‐rank; total p = 0.035, HR = 0.49; tumour cells p = 0.017, HR = 0.44; stroma p = 0.030, HR = 0.48) but had no significant impact on overall survival (Log‐rank; total p = 0.23, HR = 0.71; tumour cells p = 0.069, HR = 0.59; stroma p = 0.650, HR = 0.87). Importantly, multivariate analysis adjusted for patient age at diagnosis, node staging, tumour size, ER, and PR status revealed that total STC2 expression as well as expression in tumour cells was an independent prognostic factor for DFS (Cox regression; p = 0.018, HR = 0.983; p = 0.015, HR = 0.984, respectively). In conclusion, STC2 expression is abundant in MBC where it is an independent prognostic factor for DFS. [ABSTRACT FROM AUTHOR]

Background: The role of nipple discharge cytology (NDc) in the surgical management of breast cancer patients is unclear. We aimed: (i) to evaluate the effect of malignant NDc on the surgical approach to the nipple-areola complex, and (ii) to verify the association between malignant NDc and nipple malignancy. Methods: We retrospectively analyzed a case series of 139 patients with NDc who underwent breast surgery. The clinical and histological findings, types of surgery with emphasis on nipple-areola complex amputation, immunohistochemical phenotypes of the carcinomas and measurements of the tumor-nipple distance were recorded. Additionally, in patients who showed HER2-positive lesions on definitive surgery, we evaluated the HER2 immunocytochemistry of the NDc smears. Results: Thirty-two malignant and 107 benign/borderline NDc diagnoses were identified. All 32 malignant-NDc cases were histologically confirmed as malignant. Thirty borderline/benign-NDc cases were histologically diagnosed as malignant (sensitivity 58%). The majority of the patients with malignant NDc were treated with nipple-areola complex amputations in both the mastectomy and conservative surgery groups (P<0.001, χ251.77). Nipple involvement was strongly associated with HER2-positive ductal carcinoma in-situ (P<0.001, χ211.98). HER2 immunocytochemistry on the NDc revealed a 100% correlation with the immunocytochemistry performed on the surgical tissues. Conclusions: Malignant NDc influenced surgical management. The association of malignant NDc with nipple involvement is highly related to ductal carcinoma in-situ with HER2 overexpression. In case of HER2 positive NDc, nipple-areola complex involvement is more likely than in HER2 negative cases. [ABSTRACT FROM AUTHOR]

Background: Phosphatase 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. Methods: Retrospectively 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. Results: The 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. Conclusions: While 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. [ABSTRACT FROM AUTHOR]

Parallel studies of primary breast carcinomas and corresponding distant metastases samples reveal considerable differences. Our aim was to highlight this issue from another perspective and provide further data based on 98 patient samples: 69 primary breast carcinoma and 85 distant metastases from bone, central nervous system (CNS) and lung (56 paired). Two independent series of immunohistochemical reactions with different antibodies for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (Her2), along with HER2 fluroscence in situ hybridization (FISH) were performed on tissue microarrays to classify breast carcinoma and distant metastases samples into Luminal A, Luminal B-proliferating, Luminal B-HER2+, HER2+ and triple negative (TNBC) surrogate breast cancer groups. Correlation and agreement between the two assessments of ER and PgR were fair-to-moderate, and almost perfect for HER2 and Ki67. There was 40% discordance concerning immunophenotype between breast carcinomas and distant metastases. Most common metastatic site of ER+ breast carcinoma was the skeletal system (59.2%), whereas that of TNBCs was the CNS (58.8%) and lungs (23.5%). Distant metastases in bones were mostly luminal (54.3%), in the CNS, Luminal B (53.2%), and in the lung, TNBC (37.5%). The change of drugable properties of primary breast cancers in the respective bone and CNS metastases suggests that characterization of the metastasis is necessary for appropriate treatment planning. [ABSTRACT FROM AUTHOR]

Aims To assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and aurora-kinase A (aurkA); and to compare the expression of these markers in early MBC (EMBC) and late MBC separated into groups according to median time point on metastatic event occurred (28 months). Methods The expression of the above mentioned markers was analysed in a total of 47 primary MBCs and 59 DMs (out of which 37 were pairs) by immunohistochemistry. Fourteen breast carcinomas with no relapse over a 10-year follow-up period were utilised as control cases (CBC). Results Among the MBCs, 22 metastasised to the bone, 4 to the lung and 21 to the central nervous system (CNS). Geminin (p<0.001) and Ki67 (p=0.001) were increased in the MBCs while aurkA and cyclin A showed no difference when compared with CBCs. There were no differences between aurkA, cyclin A and geminin expression in MBCs and DMs in general. Expression of Ki67 was, however, elevated (p=0.027) in DMs. In CNS metastases all markers showed elevated expression as compared to MBCs. In bone metastases, geminin was lower (p<0.001) compared with primary MBCs. In the metastases of the lung, the evaluated markers did not show different expression. According to the median follow-up until the metastatic event, Ki67 was found to be significantly elevated in EMBC (p=0.018). Conclusions Ki67 index and geminin distinguish a fraction of MBC with worse prognosis, showing increased levels in the latter in comparison to CBC being tumour-free over a 10-year follow-up period. Ki67 could possibly identify a group of MBCs that develop early DMs. [ABSTRACT FROM AUTHOR]

Abstract: The evaluation of the role of claudins (CLDNs) in breast carcinogenesis has recently begun. We investigated the expression of CLDNs 1, 2, 3, 4, and 7 in pT1pN0 and pT1pN1 invasive ductal breast carcinomas. Tissue arrays of 30–30 pT1pN0 and pT1pN1 invasive ductal breast carcinomas of different grades were constructed, and the expression of CLDN 1, 2, 3, 4, and 7 proteins was analyzed using standard and immunofluorescent immunohistochemistry. The results were evaluated by light and confocal microscopy. Regarding CLDN 1, 4, and 7 expressions, differences were noted between normal and tumor cells and also between tumors of different grades, while no remarkable differences were noted between pT1pN0 and pT1pN1 tumors. CLDNs 1 and 7 were found to be downregulated in tumor cells compared to the normal epithelium, while CLDN 4 expression was decreased in grade 1 tumors. CLDN 7 protein was abundant in normal epithelia, and the staining decreased in grade 3 tumors. There were no differences between normal and neoplastic cells regarding CLDN 2 and 3 expressions. As a preliminary result, our observations suggest that the analyzed CLDNs do not promote tumor metastasis. On the basis of our findings, it seems that CLDN 1, CLDN 4, and CLDN 7 may rather have an important role in tumorigenesis or in cell-to-cell adhesion. [Copyright &y& Elsevier]

Simple Summary: Metabolic rewiring, as an important hallmark of cancer supports the bioenergetic needs of growing tumours at the primary site or in metastases. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to metabolic plasticity and progression in cancers. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Using a set of immunoreactions, we observed that the characterisation of metabolic pathways has a prognostic potential in human cases. High in situ metabolic plasticity was considered with high mTOR activity and high expression of at least two other studied metabolic enzymes (lactate dehydrogenase A, glutaminase, fatty acid synthase or carnitine palmitoyltransferase). According to our metabolic analyses and immunohistochemistry results, breast cancer is characterised by considerable metabolic diversity in individual cases. Consequently, we suggest selecting patients who may benefit from more accurate follow-up and specific therapies (including the combination of anti-metabolic treatments and recent therapies). In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases—accumulating genomic alterations and further heterogeneity—are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies. [ABSTRACT FROM AUTHOR]

The present study evaluated the expression of claudin-7 in 10 intact perianal gland and 67 hyperplastic and neoplastic lesions of the canine perianal (hepatoid) gland. The results have shown intense typical membrane expression of claudin-7 in intact perianal glands, hyperplasia, adenoma, differentiated and anaplastic carcinoma of this gland. In intact glands, hyperplasias, adenomas of the hepatoid gland, normal, hyperplastic and neoplastic basal cells never expressed claudin-7 molecule. Epitheliomas of the hepatoid gland were negative for claudin-7 molecule. Intense membrane-bound claudin-7 immunoreactivity was found in well-differentiated carcinomas, in addition claudin-7 overexpression in poorly differentiated carcinomas of the canine hepatoid gland. Claudin-7 seems to be one of the integral constituents of tight junction structures of intact perianal gland. In addition, claudin-7 seems to be helpful in distinguishing well-differentiated carcinomas and poorly differentiated carcinomas from epitheliomas of the gland; and in distinguishing well-differentiated carcinomas from adenoma of the perianal gland. [ABSTRACT FROM AUTHOR]