Epithelial-Mesenchymal Transition Proteins in Neuroendocrine Neoplasms: Differential Immunohistochemical Expression in Different Sites and Correlation with Clinico-Pathological Features.

The first step leading to metastasis, or for the acquisition of local invasiveness, involves changes in the phenotype of neoplastic cells in the primary tumor. The epithelial-mesenchymal transition (EMT) is a process that determines the acquisition of a form and a transcriptional program that are characteristic of mesenchymal cells, in epithelial cells. The factors involved in this process are E-cadherin and N-cadherin adhesion proteins and some transcription factors such as Slug and Twist. EMT is a site-specific mechanism that is also active in embryogenesis-embryonic cells are affected if invested in certain points, probably due to the signals emanating from the cells or groups of surrounding cells. It is known that neuroendocrine neoplasms have a biological behavior that differs in grading, staging, and site. The aim of our study was to investigate the immunohistochemical expression of EMT factors (Twist, Slug, and E-cadherin) in the neuroendocrine neoplasms of the gastrointestinal tract, the pancreas, and lungs, in 65 cases retrieved from the archives of the Department of Pathology, of three hospitals. The immunoscores were compared in each site and correlated with the clinico-pathological parameters. Statistical evaluation revealed an association between the higher Twist immunoscore and higher grading (p value < 0.0001) and staging (p value = 0.0055). Slug was detected only in pancreatic cases where its reduced expression was associated with a higher grading (p value = 0.0033). This data could be of diagnostic utility in the case of metastases from neuroendocrine neoplasm, to define the site of the primitive tumor when the traditional immunohistochemical panel is not sufficient. In summary, our results indicated, first that the EMT is also an active process in neuroendocrine neoplasms. To the best of our knowledge, this was the first study that evaluated the expression of EMT factors in neuroendocrine neoplasms of different districts.

TCGA molecular subgroups and FIGO grade in endometrial endometrioid carcinoma.

BACKGROUND: International Federation of Gynecology and Obstetrics (FIGO) grade is a crucial factor in the current system for the risk stratification of endometrial endometrioid carcinoma (EC). The Cancer Genome Atlas (TCGA) demonstrated four molecular prognostic subgroups for EC: POLE (good prognosis), microsatellite-instable (MSI, intermediate prognosis), copy-number-high (CNH, poor prognosis), and copy-number-low (CNL, variable prognosis). OBJECTIVE: To assess how the prevalence of the TCGA molecular subgroups changes from low-grade (G1-2) to high-grade (G3) EC, to understand how it may affect the current risk-assessment system. METHODS: A systematic review and meta-analysis was carried out by searching seven electronic databases from January 2013 to September 2019 for studies assessing the TCGA classification G1-2 and G3 EC. Pooled prevalence of the TCGA subgroups was calculated in EC. The association of each subgroup with grade was assessed using odds ratio (OR), with a significant p value < 0.05. RESULTS: Nine studies with 3185 patients were included. G3 EC showed significantly higher prevalence of the POLE subgroup (12.1% vs 6.2%; OR = 2.13; p = 0.0001), of the MSI subgroup (39.7% vs 24.7%; OR = 2.15; p = 0.0003) and of the CNH subgroup (21.3% vs 4.7%; OR = 5.25; p < 0.00001), and significantly lower prevalence of the CNL subgroup (28% vs 63.5%; OR = 0.2; p < 0.00001) than G1-2 EC. CONCLUSION: The prevalence of the TCGA subgroups is not in accordance with the prognostic value of FIGO grade, indicating that the current risk stratification of EC will be heavily affected by molecular signature.

Ovarian borderline tumors, a subtype of neoplasm with controversial behavior. Role of Ki67 as a prognostic factor.

INTRODUCTION: Ovarian borderline tumors (OBT) are tumors with an intermediate grade of malignancy whose diagnosis is purely based on morphological criteria. They usually occur in young women (under 40 years) and are characterized by a cellular proliferation with slight nuclear atypia and lacking stromal invasion with a destructive pattern. Aim of this study was to explore the immunohistochemical expression of Ki67 proliferative index in OBT and to correlate it with known clinicopathologic prognostic factors in patients older than 40 years. MATERIAL AND METHODS: Twenty cases of OBTdiagnosed in the period ranging from 2016 to 2018 were retrieved. Each specimen was taken from hysterectomy or adnexectomy surgery. Immunohistochemical studies were performed on the most representative sample of the tumor. Positive signal was nuclear and it was evaluated by three independent pathologists. RESULTS: Ki67 Labelling Index (L.I.) value ranged from 2% to 40%, with an average value of 14% and a median of 10%. Higher Ki67 L.I. was observed in patients older than 40 years (pvalue = 0.0194) and in those with tumors with a maximum diameter ≥ 10 cm (pvalue = 0.0547). Furthermore, a direct correlation was evident between tumor size value and Ki67 L.I. (p value<0.0001, r = 0.7745). Hitherto no known prognostic factor correlated with high Ki67 L.I. CONCLUSIONS: Overall, OBT are tumors with greater risk of evolution at a more advanced age and when they are greater in size. The assessment of Ki67 could be a valid support in the diagnosis of a more aggressive tumor. Further studies are needed to assess possible correlation with data concerning recurrences rate, that in our cases were not available.

Anti-Apoptotic and Anti-Oxidant Proteins in Glioblastomas: Immunohistochemical Expression of Beclin and DJ-1 and Its Correlation with Prognosis.

DJ-1 deglycase is a protein with anti-oxidative and anti-apoptotic properties and its role in oncogenesis is controversial. Indeed in primary breast cancer and non-small-cell lung carcinoma, its higher expression was shown in more aggressive tumors while in other neoplasms (e.g., pancreatic adenocarcinoma), higher expression was related to better prognosis. Beclin has a relevant role in autophagy and cellular death regulation, processes that are well known to be impaired in neoplastic cells. DJ-1 shows the ability to modulate signal transduction. It can modulate autophagy through many signaling pathways, a process that can mediate either cell survival or cell death depending on the circumstances. Previously, it has been suggested that the involvement of DJ-1 in autophagy regulation may play a role in tumorigenesis. The aim of our study was to investigate the link between DJ-1 and Beclin-1 in glioblastoma through the immunohistochemical expression of such proteins and to correlate the data obtained with prognosis. Protein expression was assessed by immunohistochemistry and the immunoscores were correlated with clinicopathologic parameters. Kaplan-Meier survival curves were generated. A statistically significant association between DJ-1 score and recurrence (p = 0.0189) and between the former and Isocitrate Dehydrogenase 1 (IDH1) mutation (p = 0.0072) was observed. Kaplan-Meier survival curve analysis revealed that a higher DJ-1 score was associated with longer overall survival (p = 0.0253, ĸ2 = 5.005). Furthermore, an unexpected direct correlation (p = 0.0424, r = 0.4009) between DJ-1 and Beclin score was evident. The most significant result of the present study was the evidence of high DJ-1 expression in IDH-mutant tumors and in cases with longer overall survival. This finding could aid, together with IDH1, in the identification of glioblastomas with better prognosis.

Clinicopathological factors associated with BRAF-V600E mutation in colorectal serrated adenomas.

Serrated adenomas are genetically heterogeneous, and the histological classification into sessile serrated (SSA) adenoma and traditional serrated adenoma (TSA) does not reflect the molecular landscape. The objective of this study was to assess clinical or pathological factors associated with BRAF-V600E mutation in serrated adenomas. Systematic review and meta-analysis was performed by searching electronic databases from January 2011 to January 2019 for studies assessing the association of BRAF-V600E mutation with clinical or pathological features of serrated adenomas. Odds ratio (OR) was calculated for each factor; a P-value <0.05 was considered significant. Forty studies assessing 3511 serrated adenomas (2375 SSAs and 1136 TSAs) were included. BRAF-V600E mutation was significantly associated with proximal localisation (OR = 2.71; P < 0.00001) and CIMP-H status (OR = 4.81; P < 0.0001) in both SSA and TSA, with polyp size <10 mm (OR = 0.41; P = 0.02) in TSA, and with endoscopic pit pattern II-O (OR = 13.11; P < 0.00001) and expression of MUC5A5 (OR = 4.43; P = 0.003) and MUC6 (OR = 2.28; P < 0.05) in SSA. Conversely, BRAF mutation was not associated with age <70 years (OR = 1.63; P = 0.34), age <60 years (OR = 0.86; P = 0.79), female sex (OR = 0.77; P = 0.12), flat morphology (OR = 1.52; P = 0.16), presence of any dysplasia (OR = 1.01; P = 0.59), serrated dysplasia (OR = 1.23; P = 0.72) and invasive cancer (OR = 0.67; P = 0.32), nuclear ß-catenin expression (OR = 0.73; P = 0.21) and p53 overexpression (OR = 1.24; P = 0.82). In conclusion, BRAF-V600E mutation is associated with proximal localisation and CIMP-H status in both SSA and TSA, with size <10 mm only in TSA, and with expression of MUC5A5 and MUC6 and endoscopic pit pattern II-O at least in SSA. In serrated adenomas, BRAF-V600E mutation does not seem to be associated with age and sex, with the prevalence of dysplasia and cancer and with the morphology of the dysplastic component.

A Case of Gastric Neuroendocrine Neoplasm with Mixed Grade: a Distinct Type of "High"-grade Well-Differentiated Neuroendocrine Neoplasm.

Grade 3 neuroendocrine tumor (NET G3) is a rare new entity that has recently been introduced in the classification of neuroendocrine neoplasms of the pancreas. It is a well-differentiated form, with a high proliferative activity (Ki67 > 20%), and it represents a category whose prognosis is intermediate between NET G2 and poorly differentiated neuroendocrine carcinoma (NEC G3). In sites other than the pancreas, this new category is by far less coded. Herein is reported a case of NET G3 of the stomach, the 13th described in literature. The patient was a 71-year-old man with a mass in the gastric fundus. Histological examination revealed a neuroendocrine tumor that was mostly composed by areas with conventional well-differentiated features and intermediate proliferative rate (< 20 mitoses/10 HPFs and 7% Ki67); part of the tumor was characterized by increased nuclear atypia, a confluent growth pattern, intermediate mitotic activity, and high Ki67 value (25%). The tumor was studied immunohistochemically and molecularly, and the only alteration that came out was MEN1 gene mutation (Lau584SerfsTer5) in both the high- and low-grade components. According to the actual classification of gastric neuroendocrine neoplasms, this case is labeled as NEC G3 but it should be named NET G3, with a much better prognosis than the former. Overall, both morphological pattern and the Ki67 value should be mentioned in the histological report in cases of high-grade neuroendocrine neoplasms because of its clinical significance in defining the prognosis and for new potential therapeutic approaches.

ATM and p53 combined analysis predicts survival in glioblastoma multiforme patients: A clinicopathologic study.

Glioblastoma is one of the most malignant cancers, with a distinguishing dismal prognosis: surgery followed by chemo- and radiotherapy represents the current standard of care, and chemo- and radioresistance underlie disease recurrence and short overall survival of patients suffering from this malignancy. ATM is a kinase activated by autophosphorylation upon DNA doublestrand breaks arising from errors during replication, byproducts of metabolism, chemotherapy or ionizing radiations; TP53 is one of the most popular tumor suppressor, with a preeminent role in DNA damage response and repair. To study the effects of the immunohistochemical expression of p-ATM and p53 in glioblastoma patients, 21 cases were retrospectively examined. In normal brain tissue, p-ATM was expressed only in neurons; conversely, in tumors cells, the protein showed a variable cytoplasmic expression (score: +,++,+++), with being completely undetectable in three cases. Statistical analysis revealed that high p-ATM score (++/+++) strongly correlated to shorter survival (P = 0.022). No difference in overall survival was registered between p53 normally expressed (NE) and overexpressed (OE) glioblastoma patients (P = 0.669). Survival analysis performed on the results from combined assessment of the two proteins showed that patients with NE p53 /low pATM score had longer overall survival than the NE p53/ high pATM score counterpart. Cox-regression analysis confirmed this finding (HR = 0.025; CI 95% = 0.002-0.284; P = 0.003). Our study outlined the immunohistochemical expression of p-ATM/p53 in glioblastomas and provided data on their possible prognostic/predictive of response role. A "non-oncogene addiction" to ATM for NEp53 glioblastoma could be postulated, strengthening the rationale for development of ATM inhibiting drugs.

PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype.

Glioblastoma (GBM), the most malignant of the brain tumors, has been classified on the basis of molecular signature into four subtypes: classical, mesenchymal, proneural and neural, among which the mesenchymal and proneural subtypes have the shortest and longest survival, respectively. Here we show that the transcription factor PATZ1 gene is upregulated in gliomas compared to normal brain and, among GBMs, is particularly enriched in the proneural subtype and co-localize with stemness markers. Accordingly, in GBM-derived glioma-initiating stem cells (GSCs) PATZ1 is overexpressed compared to differentiated tumor cells and its expression significantly correlates with the characteristic stem cell capacity to grow as neurospheres in vitro. Interestingly, survival analysis demonstrated that PATZ1 lower levels informed poor prognosis in GBM and, specifically, in the proneural subgroup, suggesting it may serve a role as diagnostic and prognostic biomarker for intra-subtype heterogeneity of proneural GBM. We also show that PATZ1 suppresses the expression of the mesenchyme-inducer CXCR4, and that PATZ1 and CXCR4 are inversely correlated in GSC and proneural GBM. Overall these findings support a central role of PATZ1 in regulating malignancy of GBM.

Can recurrences be predicted in craniopharyngiomas? ß-catenin coexisting with stem cells markers and p-ATM in a clinicopathologic study of 45cases.

BACKGROUND: Recurrence is a common feature of craniopharyngiomas, benign tumors that origin from squamous epithelial remnants of Rathke's pouch- arising at any segment of its whole course. There are two histotypes, showing different morphology and clinical behavior: adamantinomatous(adaCP) and papillary (papCP). An univocal strategy of management has not yet been defined, being considered the combination of surgery and radiotherapy the most effective, especially in case of incomplete resection. Therefore, the identification of factors influencing the biological and clinical behaviour is of paramount importance. ß-catenin is a cell-cell adhesion protein, whose nuclear localization has been linked to the pathogenesis of adaCP: its nuclear accumulation is associated to the presence of a tumor stem cell subpopulation. The latter is made of cells capable of self-renewal, hence believed to be responsible of recurrence, metastases and resistance to therapy in all tumors. ATM is a kinase activated by autophosphorylation (p-ATM) upon DNA double-strand breaks. It is involved not only in DNA repair, but also in tumor migration and invasiveness. Its expression may have prognostic implications in many neoplastic diseases. METHODS: In this study, we measured the immunohistochemical expression of ß-catenin, stem cell markers (CD133, CD166), Ki67 and pATMin 45 craniopharyngiomas and correlated it with clinicopathologic features. RESULTS: Statistical analysis revealed strong correlation of ß-catenin with recurrence (p = 0.0039), Ki67 (p = 0.0011, r = 0.4903) and CD166 (p = 0.0002, r = 0.6218). A slight tendency to a higher expression of ß-catenin was recorded for adaCP rather than papCP (p = 0.0895).Fisher's exact test showed that CD166 was significantlyrelated with recurrence (p = 0.0040). Furthermore, cytoplasmic pATM was more expressed in adaCPs (p = 0.0470), compared to papCPs that displayed a more evident nuclear signal (p = 0.0313) instead. CONCLUSIONS: Backing upon these data, we could weigh in on the need of identifying ß-catenin and CD166 as prognostic markersthat could be useful in predicting thebiologicalbehavior, as recurrence risk incraniopharyngiomas. The final goal is to drew up a prognostic algorithm to be of aid in the planning of an appropriate treatment strategy. Furthermore, our findings demonstrate that pATM could be used as additional distinction-marker between the two histotypes.

Immunohistochemical expression of stem cell markers CD44 and nestin in glioblastomas: Evaluation of their prognostic significance.

Glioblastoma (GB) is a highly aggressive brain tumor with dismal prognosis and its standard of care consists of surgery followed by radiotherapy plus adjuvant chemotherapy with temozolomide (Stupp protocol). The response to therapy is variable and may be affected by the presence, within the tumor, of a proportion of cancer stem cells, implicated in chemo- and radioresistance. The expression of the stem cell markers nestin and CD44 in GBs has been extensively investigated. The aim of our study was to compare the immunohistochemical expression of nestin and CD44 in 25 cases of GBs and evaluate their possible prognostic value in relation to overall survival time. We found that their expression was strongly correlated (p=0.0090e r=0.5320) and this finding was confirmed by observation with double staining techniques. For the first time, we made a separate immunohistochemical examination of membrane and cytoplasmic nestin staining, finding that although the two signals correlated (p=0.0184, r=0.4678), they had different impact on survival (respectively p=0.0255 and p=0.6376). Enhanced CD44 expression was associated with poor survival rates even if this finding was not statistically significant (p=0.4481, ĸ(2)=0.5755). Overall, membrane nestin signal was found to be a valid prognostic factor. Furthermore, the detection of CD44 in GBs could be of value also as predictive marker; hyaluronan-based nanoparticles have already shown an active targeting of this antigen in other tumors.