Percentage Of Immunopositive Cells Research Articles

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1β treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Mast Cells in Human Cutaneous Neurofibromas: Density, Subtypes, and Association with Clinical Features in Neurofibromatosis 1

Background: Cutaneous neurofibromas (cNFs) are hallmarks of neurofibromatosis 1 (NF1) and cause the main disease burden in adults with NF1. Mast cells are a known component of cNFs. However, no comprehensive characterization of mast cells in cNFs is available, and their contributions to cNF growth and symptoms such as itch are not known. Methods: We collected 60 cNFs from ten individuals with NF1, studied their mast cell proteinase content, and compared the mast cell numbers to selected clinical features of the tumors and patients. The tumors were immunolabeled for the mast cell markers CD117, tryptase, and chymase, and the percentage of immunopositive cells was determined using computer-assisted methods. Results: The median proportions of positive cells were 5.5% (range 0.1–14.4) for CD117, 4.0% (1.2–7.0) for tryptase, and 5.0% (1.1–15.9) for chymase. The median densities of cells immunopositive for CD117, tryptase, and chymase were 280, 243, and 250 cells/mm2, respectively. Small tumors, growing tumors, and tumors from patients below the median age of 33 years displayed a high proportion of mast cells. Cells expressing both tryptase and chymase were the predominant mast cell type in cNFs, followed by cells expressing chymase only. Conclusion: The results highlight the abundance of mast cells in cNFs and that their number and subtypes clearly differ from those previously reported in unaffected skin.

English

BACKGROUND Surface epithelial ovarian tumour (SEOT) develops from the outer surface of ovary. It accounts for more than 90 % of all the ovarian tumours. Most of the SEOT are benign. Few SEOT are of low malignant potential or high malignant potential. The purpose of this study was to assess the rate of expression of proliferative marker Ki-67 and staining pattern of p53 in various histological types of SEOT. METHODS It was a randomised type of study carried out in the Department of Pathology, GSVM Medical College, Kanpur, for 2 years. It included 100 random patients with surgically resected specimens of SEOT. Ki-67 immunohistochemistry (IHC) was performed on all malignant cases and few random benign cases; and the percentage of immunopositive cells in each case was expressed as Ki-67 labelling index (Ki-67 LI). p53 expression was interpreted as positive when cells showed diffuse and intense nuclear staining in malignant cases. RESULTS Out of 100 cases, 70 were benign and 30 were malignant. Cases comprised of serous and mucinous histological subtypes. In all malignant cases, Ki-67 expression was found to be positive (Ki-67 LI > 1 %). Highest Ki-67 LI of 52 % was associated with high grade serous cystadenocarcinoma. High grade serous carcinoma (HGSC) had higher p53 positivity. 88.8 % of HGSC were p53 positive. CONCLUSIONS Ki-67 is a cost-effective proliferative marker; therefore, its assessment can be included in routine histopathological report of SEOT for better understanding of the biologic behaviour and aggressiveness of the tumour. p53 expression was more common in HGSC and can help in discriminating between HGSC and lowgrade serous carcinoma (LGSC). KEYWORDS p53, Ki-67, Ovarian Tumour, Serous, Mucinous, SEOT

Regional-specific activation of phagocytosis in the rat brain in the conditions of sepsis-associated encephalopathy

In the condition of sepsis-associated encephalopathy (SAE), the brain neuroinflammatory response is considered as one of the most critical mechanisms of tissue damage and impaired cerebral homeostasis. The main cell population of the brain responsible for the immune surveillance is microglia, and its phagocytic activity is a fundamental function providing both homeostatic and damaging properties. The aim of this study was to determine the immunohistochemical and ultrastructural specificity of the phagocytosis activation in different rat brain regions in the conditions of experimental sepsis. Materials and methods. The study was conducted in Wistar rats: 5 sham-operated animals and 20 rats with cecum ligation and puncture (CLP). The immunohistochemical study of CD68 expression in the cortex, white matter, hippocampus, thalamus, caudate/putamen was carried out in the period of 20–48 h postoperatively. The cerebral cortex was examined using transmission electron microscopy. Results. Beginning from the 20th h after CLP, there was a significant dynamic increase in the values of the relative area of CD68 expression, the number of immunopositive cells, as well as the percentage of immunopositive cells with amoeboid morphology in all animals of the CLP group, with a predominance of the indicators in the lethal group of rats. The highest levels of phagocytic activity were noted in the white matter and caudate/putamen in both the survived and non-survived animals. Ultrastructurally, the microgliocytes of the lethal group were characterized by signs of actively phagocytic cells and extensive glial-neuronal interaction; phagocytosing microglia in the survived animals showed an active involvement in the processes of necrotic debris elimination into the vasculature. Conclusions. In the conditions of SAE, there is the early and dynamic increase in phagocytosis activation with the predominant localization in the brain white matter and caudate/putamen, which could conceivably indicate a special role of these brain areas in the mechanisms of neuroinflammatory response in the conditions of systemic inflammation. In the brain of non-survived animals, the phagocytosis indices are higher than in the group of survivors, which most likely indicates a natural response of microglia to more pronounced destructive processes, but it does not preclude a concurrent neurotoxic activity of CD68-positive cells on the surrounding tissue elements.

IMMUNOHISTOCHEMICAL ANALYSIS OF AUTOPHAGY-RELATED PROTEINS IN ODONTOGENIC KERATOCYSTS

Objective: To evaluate the immunoexpression of autophagy-related proteins (p62 and Atg7) in odontogenic keratocysts associated with Gorlin syndrome (SOKCs) and nonsyndromic odontogenic keratocysts (NSOKCs).Study design: The percentages of immunopositive cells (nuclear and cytoplasmic/membrane) for p62 and Atg7 in the epithelial component of 25 SOKCs and 25 NSOKCs were assessed. The results were analyzed statistically using the nonparametric Mann-Whitney U test and Spearman correlation test. Results: Expression of p62 and Atg7 was observed in all cases studied. Compared with NSOKCs, SOKCs exhibited higher nuclear expression of p62 (P< .001) and Atg7 (P< .001). No significant difference in the cytoplasmic/membrane expression of p62 was observed between groups (P = .594). On the other hand, SOKCs exhibited higher cytoplasmic/membrane expression of Atg7 compared with NSOKCs (P< .001). In NSOKCs, positive correlations were observed between cytoplasmic/membrane expressions of Atg7 and p62 (P = .004). In SOKCs, positive correlations were found between nuclear and cytoplasmic/membrane expressions of Atg7 and p62 (P< .05).Conclusion: The results suggest a potential role of autophagy in the pathogenesis of odontogenic keratocysts. The more aggressive biological behavior of SOKCs compared with NSOKCs may be related to increased nuclear translocation of p62 and higher cytoplasmic/membrane expression of Atg7. Financial support: CNPq (427931/2016-9). Objective: To evaluate the immunoexpression of autophagy-related proteins (p62 and Atg7) in odontogenic keratocysts associated with Gorlin syndrome (SOKCs) and nonsyndromic odontogenic keratocysts (NSOKCs).Study design: The percentages of immunopositive cells (nuclear and cytoplasmic/membrane) for p62 and Atg7 in the epithelial component of 25 SOKCs and 25 NSOKCs were assessed. The results were analyzed statistically using the nonparametric Mann-Whitney U test and Spearman correlation test. Results: Expression of p62 and Atg7 was observed in all cases studied. Compared with NSOKCs, SOKCs exhibited higher nuclear expression of p62 (P< .001) and Atg7 (P< .001). No significant difference in the cytoplasmic/membrane expression of p62 was observed between groups (P = .594). On the other hand, SOKCs exhibited higher cytoplasmic/membrane expression of Atg7 compared with NSOKCs (P< .001). In NSOKCs, positive correlations were observed between cytoplasmic/membrane expressions of Atg7 and p62 (P = .004). In SOKCs, positive correlations were found between nuclear and cytoplasmic/membrane expressions of Atg7 and p62 (P< .05).Conclusion: The results suggest a potential role of autophagy in the pathogenesis of odontogenic keratocysts. The more aggressive biological behavior of SOKCs compared with NSOKCs may be related to increased nuclear translocation of p62 and higher cytoplasmic/membrane expression of Atg7. Financial support: CNPq (427931/2016-9).

THE TOPICAL APPLICATION EFFECT OF NANO-HYDROXYAPATITE VERSUS PLATELET RICH FIBRIN ON THE EARLY STAGES OF EXTRACTION SOCKET HEALING IN RABBITS: HISTOMORPHOMETRIC, HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL STUDY

Introduction: The proper healing of extraction socket is essential for ridge preservation and better sequential prosthetic procedures. Several techniques and materials are developed for acceleration of socket healing. Two of the promising materials for this purpose are nanohydroxyapatite (NHA) and platelet rich fibrin (PRF); their mechanism of action and optimum application techniques are still not fully elucidated. Purpose: To compare between NHA and PRF topical application in the enhancement of early stages of extraction socket healing in rabbits. Materials & Methods: The mandibular first molars were extracted in twenty-four rabbits; the extraction sockets were divided into 3 groups: control group didn’t receive topical treatment. N and P groups received topical application of NHA and PRF respectively. Each group was subdivided into 2 subgroups according to the date of scarification after extraction: 1 week and 2 weeks. The extraction sockets were investigated by hematoxylin and eosin (H&E), Masson trichrome (MT) stains and proliferation cell nuclear antigen (PCNA) marker. The results were illustrated histologically and histomorphometrically. Results: Both NHA and PRF treated groups in both 1 week and 2 weeks periods showed enhanced socket healing, more regular and mature bone in both H&E and MT stains as well as increased PCNA positive cells percentage in comparison to control group. The bone area in both periods and the osteocyte count after 1 week were highest in PRF group followed by NHA group while control group revealed the least values. These differences were statistically non-significant except the percentage of immuno-positive cells. Conclusion: Both NHA and PRF improve the extraction socket healing process with slight preference of PRF particularly in the cellular proliferation in the first week after extraction.

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

To assess the utility of proliferative marker Ki-67 in surface epithelial ovarian tumor

Background: Surface epithelial ovarian tumor (SEOT) accounts for more than 90% of all the ovarian tumors and are the most lethal one. Cellular proliferation plays a significant role in the clinical behavior and aggressiveness of ovarian tumor. Proliferative activity of the tumor can be assessed using the proliferative marker Ki-67, which has both diagnostic and prognostic values. Aim and Objective: The aim and objective of this study was to assess the expression of proliferative marker Ki-67 in various SEOT and correlate its level of expression with clinicopathological parameters including the International Federation of Gynecology and Obstetrics (FIGO) staging and preoperative CA-125 level. Materials and Methods: The study was a cross-sectional and observational study carried out in the Department of Pathology, JNMC, Sawangi, Wardha, over 2 years. It included 74 patients with surgically resected specimen of SEOT. Ki-67 immunohistochemistry was performed in all the 74 cases, and the percentage of immunopositive cells was expressed as Ki-67 labeling index (Ki-67 LI). Results: Out of 74 cases, 54 were benign and 20 were malignant which comprised serous and mucinous histological subtypes. Ki-67 expression was found to be positive only in malignant tumors (Ki-67 LI >1%). High Ki-67 LI was associated with high-grade serous cystadenocarcinoma (48.5%), advanced FIGO staging (40.5%), and high CA-125 levels. However, there was no association between Ki-67 LI and histological subtype. Conclusion: Ki-67 is a cost-effective proliferative marker. Therefore, immunohistochemical assessment of Ki-67 expression can be included in routine histopathological report of SEOT for diagnosis and prognostication which will help in better understanding of the biologic behavior of the tumor and modifying treatment strategies.

Immunohistochemical Analysis of Galectins-1, -3, and -7 in Periapical Granulomas, Radicular Cysts, and Residual Radicular Cysts

IntroductionGalectins play important roles in immunoinflammatory responses, but their participation in the development of periapical lesions remains unclear. This study aimed to evaluate the expressions of galectins-1, -3, and -7 in periapical lesions, correlating them with the intensity of the inflammatory infiltrate and the pattern of the cystic epithelium. MethodsTwenty periapical granulomas (PGs), 20 radicular cysts (RCs), and 20 residual radicular cysts (RRCs) were submitted to immunohistochemistry using anti–galectin-1, -3, and -7 antibodies. The percentage of immunopositive cells in epithelial and connective tissues was determined. ResultsIn connective tissue, PGs exhibited higher cytoplasmic/membrane expression of galectins-1 and -7 than RCs and RRCs (P < .05). There was higher nuclear expression of galectin-1 in PGs compared with RCs and RRCs (P < .05). The expression of galectins-1 and -7 in connective tissue was higher in lesions with grade III inflammation (P < .05). No significant differences in galectin-3 immunoexpression were observed for any of the parameters evaluated (P > .05). In the epithelial component, a higher nuclear expression of galectin-7 was detected in RRCs (P < .05), and a higher cytoplasmic/membrane expression of this protein was found in cysts with hyperplastic epithelium (P < .05). Positive correlations were observed between the nuclear and cytoplasmic/membrane expression of galectin-1 in connective tissue (P < .05) as well as between the nuclear and cytoplasmic/membrane expression of galectin-7 in epithelial tissue of cysts (P < .05). ConclusionsGalectins-1 and -7 may play important roles in the pathogenesis of PGs, RCs, and RRCs. On the other hand, the present results suggest only a minor involvement of galectin-3 in the development of these lesions.

Distribution Pattern of ER &amp; PR Immunoexpression in Endometrial Biopsies of DUB and Infertile Patients from A Tertiary Care Centre

Objective : To study endometrial estrogen and progesterone immunoexpression in different phases of menstrual cycle, in cases of dysfunctional uterine bleeding and infertility. A comparative analysis was also done for calculating ER & PR expression, between two methods: quick score and percentage of immunopositive cells. Methods : Endometrial biopsies from 107 clinically diagnosed DUB cases and 23 infertile patients were included in the study. Tissue sections were analyzed for different phases of menstrual cycle and immunoexpression of ER & PR receptors was calculated in glandular epithelium and stromal cells, using percentage of positively stained cells and Quick score method. Endometrial sections of hysterectomy specimens of uterovaginal prolapse cases were used as control sections. Result : In the present analysis, secretory endometrium was the commonest finding histopathologically. Mean total ER & PR expression in cases of DUB was statistically higher in the proliferative phase using both methods (p<0.05). Mean total ER expression difference in both the phases of infertility cases was not statistically significant by the observation of percentage of positively stained cells. However, quick score revealed significant difference between both receptor expression in infertile patients. Conclusion: The percentage of positive cells for ER and PR expression plays a more determinant role in studying ER and PR expression in cases of DUB and infertility as compared to quick score. DOI: 10.21276/APALM.1374

Bone Morphogenetic Protein Promotes Lewis X Stage-Specific Embryonic Antigen 1 Expression Thereby Interfering with Neural Precursor and Stem Cell Proliferation.

The glycoprotein Prominin-1 and the carbohydrate Lewis X stage-specific embryonic antigen 1 (LeX-SSEA1) both have been extensively used as cell surface markers to purify neural stem cells (NSCs). While Prominin-1 labels a specialized membrane region in NSCs and ependymal cells, the specificity of LeX-SSEA1 expression and its biological significance are still unknown. To address these issues, we have here monitored the expression of the carbohydrate in neonatal and adult NSCs and in their progeny. Our results show that the percentage of immunopositive cells and the levels of LeX-SSEA1 immunoreactivity both increase with postnatal age across all stages of the neural lineage. This is associated with decreased proliferation in precursors including NSCs, which accumulate the carbohydrate at the cell surface while remaining quiescent. Exposure of precursors to bone morphogenetic protein (BMP) increases LEX-SSEA1 expression, which promotes cell cycle withdrawal by a mechanism involving LeX-SSEA1-mediated interaction at the cell surface. Conversely, interference with either BMP signaling or with LeX-SSEA1 promotes proliferation to a similar degree. Thus, in the postnatal germinal niche, the expression of LeX-SSEA1 increases with age and exposure to BMP signaling, thereby downregulating the proliferation of subependymal zone precursors including NSCs. Stem Cells 2017;35:2417-2429.

English

ABSTRACT Excessive accumulation of macrophages in sciatic nerve fascicles inhibits regeneration of peripheral nerves. The aim of this study is to determine the percentage of the macrophages inside and outside of the fascicles at the proximal, at the site of injury and at the distal segment of rat model of sciatic nerve crush injury. Thirty male 3 months age Wistar rats of 200-230 g were divided into sham-operation group and crush injury group. Termination was performed on day 3, 7, and 14 after crush injury. Immunohistochemical examination was done using anti CD68 antibody. Counting of immunopositive and immunonegative cells was done on three representative fields for extrafascicular and intrafascicular area of proximal, injury and distal segments. The data was presented as percentage of immunopositive cells. The percentage of the macrophages was significantly increased in crush injury group compared to the sham-operated group in all segments of the peripheral nerves. While the percentage of macrophages outside fascicle in all segments of sciatic nerve and within the fascicle in the proximal segment reached its peak on day 3, the percentage of macrophages within the fascicles at the site of injury and distal segments reached the peak later at day 7. In conclusions, accumulation of macrophages outside the nerve fascicles occurs at the beginning of the injury, and then followed later by the accumulation of macrophages within nerve fascicles

Expression of VEGF-A in Intraepithelial and Invasive Cervical Neoplasia

Angiogenesis seems to be an early event that is associated with disease progression in cervical intraepithelial neoplasia (CIN). The aim of this study was to describe and analyze VEGF-A expression in CIN 3 and invasive cervical carcinoma, in tumor cells and stromal cells. This study comprised three groups: Group 1: 53 cases with CIN 3; Group 2: 24 cases with both CIN 3 and invasive carcinoma components; Group 3: 36 cases with invasive carcinoma. Protein expression was investigated in tumor and stromal cells by immunohistochemistry. Statistical analysis was carried out, considering the mean percentage of immunopositive cells. VEGF-A expression was higher in stromal cells in cases of invasive carcinoma (Group 3) than in CIN 3 cases (Group 1) and this difference was statistically significant. VEGF-A expression showed a borderline association between stromal cells in invasive carcinoma (Group 3) and invasive carcinoma associated with CIN 3 (Group 2). The mean percentage of cells expressing VEGF-A was significantly higher in tumor cells in CIN 3 than in stromal cells in CIN 3 (Group 1). The progression of CIN 3 to invasive carcinoma seems be determined by complex interactions between tumor and stromal cells. These findings reinforce the evidence of the role of stromal cells in tumor invasiveness.

An observational study on the expression of cyclooxygenase-2 in meningioma.

Background:The cyclooxygenase-2 (COX-2) enzyme is overexpressed in different types of tumors and is known to be associated with malignant behavior of tumors. We determined the association of COX-2 expression and different grades of human meningioma.Materials and Methods:This retrospective study was conducted on specimens obtained from adult patients with meningioma. Meningioma was classified according to the WHO 2007 classification protocol (I, II, and III). COX-2 expression intensity was scored based on the percentage of immunopositive cells as 0: 0-10%; +1: >10% and a part of the cell membrane; +2: >10% and complete cell membrane; and +3: >30% and complete cell membrane. Scores of +2 or +3 were considered as COX-2 positive.Results:Ninety meningioma cases (mean age = 53.0 ± 13.2 years, 71.1% female) were studied. COX-2 was positive in 25% (17/68), 68.4% (13/19), and 100% (3/3) of cases with tumor grade I, II, and III, respectively (P < 0.001). There was a significant correlation between tumor grade and COX-2 expression score (Spearman's correlation coefficient = 0.422, P < 0.001).Conclusions:There is a strong association between COX-2 expression and tumoral grade in meningioma with more aggressive tumors expressing COX-2 with more intensity. Prospective studies examining the association of COX-2 expression with tumor recurrence and interventional studies examining the role of COX-2 inhibitors anticancer therapy of meningioma are warranted.

P53, MAPK, topoisomerase II alpha and Ki67 immunohistochemical expression and KRAS/BRAF mutation in ovarian serous carcinomas

BackgroundWe investigated the immunohistochemical expression of p53, MAPK, topoisomerase II alpha (topoII alpha) and Ki67 in ovarian serous carcinomas (OSCs) along with mutational analysis for KRAS and BRAF.MethodsEighty one cases of OSCs were reviewed and examined immunohistochemically using antibodies against p53, MAPK, topoII alpha and Ki67. Staining was evaluated as a percentage of immunopositive cells with cut-off levels at 10% for p53 and topoII alpha, and 5% for MAPK. The Ki67 immunoexpression was assessed by means of Olympus Image Analysis System as a percentage of immunopositive cells in 1000 tumor cells. KRAS and BRAF mutational analysis was performed on 73 available microdissected samples.ResultsOf 81 cases of OSCs 13.6% were of low-grade and 86.4% were of high-grade morphology. In the high-grade group there was a significantly higher immunoexpression of p53 (P < 0.001) and topoII alpha (P = 0.001), with Ki67 median 56.5 vs. 19 in low-grade group (P < 0.001). The difference in immunoexpression of active MAPK between low- and high-grade group was also significant (P = 0.003). MAPK positive immunostaining was detected in 63.6% of low-grade vs. 17.1% of high-grade OSCs. The frequency of KRAS mutation was significantly higher in low-grade as compared to high-grade group (P = 0.006). None of the samples had BRAF mutation. In addition, we detected positive MAPK immunoexpression in 13/59 samples with wild-type KRAS, suggesting that activation of MAPK pathway is not ultimately related either to KRAS or BRAF mutation. Seven morphologically high-grade samples (11.7%) showed both KRAS mutation and p53 immunopositivity.ConclusionsAlthough this study is limited by its humble number of low-grade samples, our data fit the proposed dualistic pathway of ovarian carcinogenesis. Mutational analysis for KRAS and BRAF discloses some possible interactions between different tumorigenic pathways of low- and high-grade carcinomas. Immunohistochemical staining for MAPK was not sufficiently sensitive, nor specific, to precisely predict the KRAS mutation. However, it appears to be quite reliable in ruling out a KRAS mutation if the staining is negative.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9283563368804632ZusammenfassungHintergrundWir untersuchten die Immunohistochemische Expression der p53, MAPK, topoisomerase II alpha (topoII alpha) und Ki67 in Ovarialkarzinomen (OSCs) anbei mit Mutationsanalyse für KRAS und BRAF.Methode81 OSCs Fälle wurden analysiert und Immunohistochemisch untersucht mit Antikörper gegen p53, MAPK, topoII alpha und Ki67. Die Färbung war ausgewertet als der Prozent von immunopositiven Zellen mit den “cut-of” Niveau an 10% für p53 und topoII alpha und 5% für MAPK. Die Ki67 Expression war bewertet mittels Olympus Image Analysis System als der Prozent von immunopositiven Zellen in 1000 Tumorzellen. KRAS and BRAF Mutationsanalyse wurde in 73 verfügbaren microdissections Stichproben aufgeführt.ErgebnisseVon 81 OSCs Fälle 13.6% zeigte “low-grade” und 86.4% “high-grade” Morphologie. In der “high-grade” Gruppe war eine statistisch bedeutende höhere Expression von p53 (P < 0.001) und topoII alpha (P = 0.001) mit Ki67 median von 56.5 im Gegensatz zu 19 in der “low-grade” Gruppe (P < 0.001). Die Differenz in Immunoexpression von aktiver MAPK zwischen der “low-grade” und “high-grade” Gruppe war statistisch bedeutend (P = 0.003). MAPK positive Expression war in 63.6% der “low-grade” im Gegensatz von 17.1% der “high-grade” Karzinoms bemerkt. Die Häufigkeit der KRAS Mutation war bedeutend höher in “low-grade” im Verglich zu der “high-grade” Gruppe (P = 0.006). Keiner der Stichproben hate BRAF Mutation. Wir haben auch eine positive MAPK Expression in 13/59 der Stichproben mit “wild-type” KRAS bemerkt, was sugeriert das die Aktivation des MAPK Pfads ist nicht letztmalig mit KRAS oder BRAF verbunden. Sieben der “high-grade” Stichproben (11.7%) waren KRAS Mutation und p53 Expression positive.SchlussworteObwohl diese Studie mit bescheiden Nummer von “low-grade” Stichproben limitiert ist, unsere Daten passen in das dualistische Modell von Ovarial Karzinogenesis. Mutationsanalyse für KRAS und BRAF enthüllen einige mögliche Interaktionen zwischen verschieden tumorigenen Wege von “low”- and “high-grade” Karcinomen.Die Immunohistochemische Expression für MAPK war nicht empfindlich oder spezifisch genüg um den KRAS mutations Status des Tumor genau vorauszusagen.Es scheint das die MAPK Expression ziemlich verlässlich ist in ausschließen der KRAS Mutation, wenn die Expression negative ist.

Foxp3 expression in patients with non-small cell lung cancer.

e18059 Background: Lung cancer is rarely cured by the current therapeutic approaches. Although numerous studies have implicated Foxp3 positive T regulatory cells in cancer pathogenesis, the role of Foxp3 in lung cancer pathogenesis remains unknown. Methods: We determined Foxp3 expression, using immunohistochemistry in 44 NSCLC tissue specimens, 20 samples from adjacent non neoplastic lung parenchyma and 5 normal lung tissue specimens. Immunohistochemical reactivity was graded on a scale of 0–3 according to intensity of staining and percentage of immunopositive cells. Foxp3 expression in cancer cells was categorized in three groups (high vs median vs low) using as a cut-off point the 33rd and 66th percentile. The total score for each slide was the sum of the intensity and distribution (between 0 and 6). Statistical analysis was performed with SPSS 17.0. Results: Nuclear Foxp3 immunostaining was detected in al tissues assessed. Foxp3 expression levels were higher in neoplastic cells than in adjacent non neoplastic tissue and normal lung parenchyma. Moreover, Foxp3 expression in tumor cells correlated with lymphocytic Foxp3-immunopositivity and the presence of lymph node metastasis. Furthermore, Foxp3 lymphocytic expression was negatively associated with the age of the patients. Conclusions: Foxp3 is overexpressed in NSCLC cells and tumor infiltrating lymphocytes. Moreover, tumor Foxp3 expression correlates with lymph node metastasis while lymphocytic Foxp3 levels may be age-related.

Localization of 4-hydroxy 2-nonenal immunoreactivity in aging human retinal Müller cells

Müller cells play a pivotal role in maintaining retinal homeostasis of the extracellular fluid environment. Information on whether human retinal Müller cells suffer from oxidative stress with normal aging is lacking. We examined post mortem human retinas for the localization of a biomarker of lipid peroxidation (4-hydroxy 2-nonenal, 4-HNE) by immunohistochemistry. We procured human eyes from donors (N=11; age: 45-91 years; post mortem delay: 1-3h), who had no history of ocular diseases. They were fixed in 4% paraformaldehyde and the retinas cryosectioned and labeled against anti-4-HNE employing the immunoperoxidase method. Compared to the lower age group (45-56 years), in the advanced age group (67-91 years), immunoreactivity (IR) to 4-HNE was prominent in peripheral Müller cell end-feet, select cells in the inner nuclear layer and in outer fibers located in the macular fiber layer of Henle. Colocalization with glutamine synthetase revealed that the 4-HNE positive profiles in the inner nuclear layer were Müller cells. Quantitative analysis revealed that the percentage of immunopositive cells in the inner nuclear layer as well as the grey levels of the immunoreaction products in the parafoveal and peripheral retinal regions significantly increased in the advanced age group. The findings indicate that Müller cells of human retina suffer from lipid peroxidation and are susceptible to damage in the course of normal, advanced aging.

Oncocytic carcinoma of the breast: frequency, morphology and follow-up

Oncocytic breast carcinomas are tumors composed of no fewer than 70% of oncocytic cells (World Health Organization). The purpose of this study was to determine the frequency, morphologic, immunohistochemical, and clinical features of invasive oncocytic carcinoma in a large series. Twenty-eight cases of putative oncocytic breast carcinoma (selected cases group) and 76 consecutive cases of invasive breast carcinoma (consecutive cases group) were analyzed. Immunohistochemistry for mitochondria, gross cystic disease fluid protein 15, chromogranin, estrogen receptor, progesterone receptor, androgen receptor, HER2/Neu, cytokeratin 7, cytokeratin 14, epithelial membrane antigen, and differentiation cluster 68 was performed. Score for mitochondria was based on intensity and percentage of immunopositive cells. Classes were as follows: (1) oncocytic carcinoma: at least 70%, 3+; (2) mitochondrion-rich carcinoma: 50% to 70%, 3+, or more than 50%, 2+; and (3) all the other cases were referred to as invasive breast carcinoma. Ultrastructural examination was available for 6 cases of oncocytic carcinoma. Morphologic and immunohistochemical features of the 3 groups were compared using Fisher exact test (P < .05). For overall survival analysis, Kaplan-Maier curves were compared using log-rank and Wilcoxon tests (P < .05). Our results suggest that oncocytic breast carcinoma is a morphologic entity with distinctive histologic and ultrastructural features. Mitochondrion-rich carcinomas are histologically similar to oncocytic carcinomas and constitute 19.7% of all invasive carcinomas, indicating that cytoplasmic eosinophilia in breast cancer cells is often due to accumulation of mitochondria. Oncocytic carcinomas and mitochondrion-rich carcinomas are more often grade III tumors and show human epidermal growth factor receptor 2 overexpression. Clinical features and overall survival of oncocytic carcinomas are not distinctive because they are similar to those of the other cases when matched for grade and stage.

Study on breast carcinoma Her2/neu and hormonal receptors status assessed by automated images analysis systems: ACIS III (Dako) and ScanScope (Aperio).

Her-2/neu is overexpressed in 20-30% of breast cancer patients and is associated with a more aggressive disease. Identification of Her-2/c-erbB-2-neu overexpression is based on immunohistochemical [ihc] detection of protein and/or gene amplification in fluorescence in situ hybridization test (FISH). Also Estrogen receptors [ER] and Progesterone receptors [PR] are the prognostic and predictive biomarkers, recently analysed by ihc methods. Subjective, manual scoring of the ihc Her-2/neu expression and expression of the ER/PR reported as the percentage of immunopositive cells are the most common mode of interpretation among pathologists. Automated microscopy and computerised processing have provided increased accuracy in quantification and standardisation. to evaluate the scoring reproducibility of Her-2 /neu ihc expression tested by two automated systems: ACIS (Dako) and ScanScope (Aperio); to estimate the ER/PR expression in ihc staining methods with different anti-ER/anti-PR antibodies (the monoclonal and the ER/PR pharmDx TM Kit) by the ACIS system. Her-2/neu ihc expression was measured in 114 primary invasive breast carcinomas by the manual and the automated scoring (ACIS and Aperio system). 106 slides stained ihc with two types of anti-ER/anti-PR antibodies entered the quantisation. The results of our investigations showed very high reproducibility of Her-2/neu scores in intra- and interobserver analysis by ACIS evaluation. The major concordance was present in strong 3+ ihc cases; very small discordance was shown by cases with low expression of Her-2/neu. The accuracy of scoring by the Aperio was little lower in comparison to ACIS but it might result from the smaller and variable series of samples analysed by Aperio. The concordance in scoring of two automated systems was 86.5% (p<0.0001; gamma=0.887); the discordance was referred only to the lower expression of Her-2/neu. The concordance in manual scoring performed by the single observer and the panel was 84.2% (p<0.0001, gamma = 0.99); the discordance comprised a few cases with strong expression (2+ vs 3+). Very high intra- and interobserver reproducibility of the ER/PR ihc measurements was present in the readers results (referred to the percentage of immunoreactive carcinomatous cell population in the breast carcinomas acc. to the ACIS algorithm). No differences were disclosed in the percentage of ER-immunoreactive and PR-immunoreactive carcinomatous cell populations when used 2 different type of antibodies, in the ACIS automated method.

Determination of Immunohistochemical Markers for Oral Squamous Cell Carcinoma Invasions

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells. The cellular differentiation of the invasion site as well as the pattern of invasion is crucial for the tumor behavior. Both resection margins and the pattern of invasions are important predictors of local recurrence and survival in surgically treated patients. Therefore, surgical margins should be free from carcinoma or dysplasia after surgical intervention and the accepted definition of a close margin is tumor within 5 mm of the inked resection margin, but has not so clear yet. Others use less than 2 mm or 3 mm within one high power field or a variable definition related to the pattern of invasion at the tumor host interface. The aim of this study was to determine the immunohistochemical markers for identification of invasion pattern of oral squamous cell carcinoma (OSCC) and detection of epithelial cells of surgical margins. The study analyzed the immunohistochemical characteristics of 32 cases : 8 cases each of carcinoma in situ (CIS), OSCC border, OSCC early invasion and OSCC front using Ki-67, p53, CK 17, CK 13, laminin -5γ2 and type IV collagen. The correlation between the morphological pattern on invasive sites and immunohistochemical staining were studied. The results were obtained as follows :1)The average number of Ki-67 positive basal cells was 22.7% in CIS, 57.5% in OSCC border, 62.0% in OSCC early invasion and 60.2% in OSCC front, respectively. The percentage of immuno-positive cells in OSCC border, OSCC early invasion and OSCC front are significantly higher than those of CIS (p<0.05). p53 appeared in almost all tumor cells and highest in OSCC front.2)CK 17 was positive and strong in superficial cells of OSCC border, parabasal cells of OSCC early invasion and pearl nests of OSCC front, although the degree of positivity was variable. It was negative to discrete positive in CIS.3)CK 13 was positive in superficial and parabasal cells of CIS and pearl nests of OSCC front, negative in basal cells of CIS and OSCC early invasion, and weakly present in variable distribution in OSCC border.4)The cytoplasmic accumulation of laminin -5γ2 expression was intense in OSCC border, moderate in OSCC early invasion and OSCC front, and discrete in CIS.5)Type IV collagen was strong in both OSCC border and OSCC early invasion and weak in CIS and OSCC front.These results suggested that the combined expression of Ki-67 and p53 confirmed the proliferative and mutational activities of these OSCC invasions in contrast to CIS. CK 17 and CK 13 biomarkers detected their histological differentiation and variation in epithelial cells. The migration-promoting activity and role in cancer invasion were regulated by laminin -5γ2 and type IV collagen, and was clearly observed in OSCC border. The present study also revealed high proliferative activity and high invasiveness of epithelial cells in OSCC border similar to cancer and the combined used of the markers for safety margins should be considered important by surgeon.