Ana Čarić

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.

The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary.

The influence of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins on the cell death (caspase-3, TUNEL) of different ovarian cell lineages was immunohistochemically analyzed in six fetal and five adult human ovaries in order to disclose possible mechanisms of cell number control. Mild to moderate expression of Bcl-2 characterized ovarian surface epithelium, follicular cells and oocytes of 15 and 22 week human ovaries, while expression of Bax and caspase-3 gradually increased in all ovarian cell populations, except caspase-3 in the ovarian surface epithelium. Different levels of Bax and Bcl-2 proteins co-expression characterized fetal ovarian cells, while TUNEL and caspase-3 co-expression was found only in some of them. In adult ovaries, Bcl-2 was moderately and Bax strongly expressed in the surface ovarian epithelium and stroma. Bcl-2 and Bax expression in granulosa and theca interna cells varied depending on the stage of follicular atresia. Caspase-3 apoptotic cells characterized granulosa cells of adult atretic follicles. Our results indicate that intracellular levels of Bcl-2 and Bax protein might regulate the final destiny of developing germ cells. Caspase-3 dependent apoptosis seems to be the most important, but not the only cell death pathway in ovaries. In adult ovaries, caspase-dependent cell death characterized granulosa cells, but not the germ cells.

Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serous carcinoma.

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologically analyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immunohistochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the 15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cells in week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expression characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildly to moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle where IGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the tertiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausal human ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were strongly positive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausal ovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indicate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovarian cell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSC as well.

The influence of exercise on morphological and neurochemical properties of neurons in rat nodose ganglia

Physical exercise can induce immunohistochemical changes and cell proliferation in the hippocampus. One of the main effects of prolonged exercise is resting bradycardia, most probably caused by enhanced vagal activity. To investigate whether physical exercise can cause neurochemical and morphological changes in vagal afferent neurons, we performed immunohistochemical studies of nodose neurons using isolectin B4 (IB4), 200-kDa neurofilament protein (N52) and calretinin in adult female rats. To distinguish subpopulations of neurons projecting to the left ventricle, we applied a Fast Blue patch to the epicardial surface of the left ventricle. Treadmill running for 8 weeks significantly increased the size of N52-positive cardiac projecting neurons. Furthermore, the proportion of IB4-positive neurons among all nodose ganglia neurons was significantly higher in trained animals. These data indicate that exercise leads to plastic changes in nodose ganglia neurons that may initiate changes of vagal activity caused by prolonged exercise.

Apoptotic pathways in ovarian surface epithelium of human embryos during embryogenesis and carcinogenesis: close relationship of developmental plasticity and neoplasm.

Cell differentiation and different pathways of cell death were immunohistochemically analyzed in ovaries of six human embryos, 20 serous borderline tumors (SBT) and ovarian serous carcinomas (OSC) using markers for apoptosis (caspase-3, AIF, TUNEL) and stemness (Oct-4). In the 5-8-week ovaries, caspase-3 was absent in the ovarian surface epithelium (ose) and mildly positive in the ovarian stroma (os), AIF was expressed moderately, while Oct-4 expression gradually decreased during that period. Some ovarian cells expressed only caspase-3 or AIF together with TUNEL, while both caspase-3 and AIF were co-expressed in other ovarian cells. Mild expression of Oct-4 and caspase-3 characterized some cells of SBT, while their expression varied from mild to strong in OSC. AIF displayed mild to strong expression in ose of SBT and moderate to strong expression in OSC, while no expression of AIF was observed in os of both tumors. In the ose of both SBT and OSC, caspase-3 and AIF were co-expressed only occasionally, while AIF and Oct-4 were co-expressed strongly. Our study showed the presence of stemness cells and different pathways of cell death (caspase-3 and AIF-mediated) in the ovarian tissue during development and carcinogenesis, indicating the correlation between developmental plasticity in human embryonic ovaries and OSC.