Immunohistochemical and Histopathological Features of Persistent Gingival Enlargement in Relation to Metal Allergic Sensitisation during Orthodontic Treatment.

This study aimed to analyse the immunohistochemical profile of inflammatory infiltrates in the gingival tissue of patients undergoing orthodontic treatment in relation to patients' titanium and/or nickel allergy status. Patients with gingival enlargement received initial periodontal therapy, followed by external gingivectomy in the case of persistent gingival enlargement. The sample included 44 patients (22 had metal allergic sensitisation). Histopathological changes were assessed, and an immunohistochemical analysis was performed on formalin-fixed and paraffin-embedded gingival samples using antibodies against CD1a, CD3, CD4, CD8, CD20, CD68, and CD138. Computer-assisted image analysis was performed to evaluate the positive cell count in the gingival tissue. The gingiva of the sensitised patients was characterised by the absence of multifocal inflammatory infiltrates (p < 0.05), while pronounced exocytosis and band-like inflammatory infiltrates were more frequently observed in sensitised patients. In addition, there was an increase in Langerhans cells and T-helper lymphocytes and a decrease in naïve T-lymphocytes, cytotoxic T-lymphocytes, macrophages, and plasma cells in the sensitised subjects compared to non-sensitised. However, the differences were only statistically significant for macrophages, with a moderate effect size (82.8 vs. 133.9; p = 0.041; r = 0.308). The absence of multifocal inflammation appears to be the most characteristic histopathological feature of the gingiva of sensitised patients. Although their gingiva presented certain characteristics of late hypersensitivity immune reactions the observed changes imply dominant irritative effect e.

Cardiomyocytes calpain 2 expression: Diagnostic forensic marker for sudden cardiac death caused by early myocardial ischemia and an indicator of the duration of myocardial agonal period?

Sudden cardiac death (SCD) is an unexpected natural death of cardiac etiology and occurs within one hour of the onset of cardiac symptoms in an apparently healthy subject or within 24 h if death is not witnessed. The diagnosis of early myocardial ischemia (EMI) or acute myocardial infarction (AMI) after death is a challenge for forensic pathologists especially when death occurs in a short period of time after the onset of myocardial ischemia. Disorder of cardiomyocytes Ca2+ homeostasis caused by myocardial ischemia during SCD can lead to the activation of calcium-activated non-lysosomal cysteine protease, including calpains. They serve as a proteolytic unit for cell balance and also participate in the processes of apoptosis and necrosis. Agony is a period that precedes somatic death that differs from cellular agony which may evolve for hours after somatic death lasting differently depending on the cell type and mechanism of death. We hypothesize that the expression of calpain 2 in cardiomyocytes could be a specific and sensitive diagnostic forensic marker for SCD caused by EMI and an indicator of the duration of myocardial agonal period. We will conduct a retrospective study that will prove this hypothesis on the respondents who died of SCD by EMI and AMI, instant death by head gunshot and hanging. There is no data on such an analysis in the available literature. The standard hematoxylin-eosin staining will be used to detect cardiac tissue damage. The expression of calpain 2 in cardiomyocytes will be analyzed immunohistochemically. In SCD caused by EMI we expect lower level of calpain 2 expressionin comparison to AMI due to shorter duration of dying. Similar, we predict in the remote region lower calpain 2 expression than in the region of ischemia for both EMI and AMI. In instant death caused by perforating traumatic brain injury we expect mild or no calpain 2 expression throughout the whole myocardium because of very short (immediate) duration of dying. In death caused by hanging calpain 2 expression throughout the whole myocardium should be strong because of longer cellular agonal period. We expect that our results would indicate the immediate activation of calpain 2 in different causes of cardiomyocytes death. From the degree of expression of calpain 2 we could conclude about the duration of cardiomyocytes agony so calpain 2 could be used as a marker for the assessment the duration of somatic and cellular agony.

Osteopontin is associated with decreased apoptosis and αv integrin expression in lung adenocarcinoma.

Osteopontin (OPN) is a glycoprotein involved in invasion, progression and metastasis of many carcinomas. It contains several functional domains including binding sites for αv integrins, cell surface molecules playing a major role in mediating cell migration and adhesion. The aim of the study was to evaluate the expression of osteopontin in human non-small cell lung cancer (NSCLC) and to determine its possible prognostic significance as well as relation to apoptosis and αv integrin expression. We analyzed 111 surgically resected NSCLC for immunohistochemical expression of OPN and αv integrin. OPN expression was compared to apoptotic rate and clinicopathological parameters such as tumor size, histological grade, lymph node status, pT, and TNM stage. Apoptotic rate was measured by TUNEL staining method. OPN expression in NSCLC was significantly higher in lung adenocarcinomas (AC) then in squamous cell carcinomas (p<0.001). There was no correlation between OPN expression and clinicopathological parameters. The level of OPN expression in AC was associated with decreased apoptotic activity of tumor cells (p=0.006), and correlated with αv integrin expression (p=0.048), particularly in low stage tumors (p=0.013). Prolonged tumor cell survival in lung AC due to OPN and αv integrin overexpression may have an impact on tumor progression and resistance to therapy.

Osteopontin expression is an independent factor for poor survival in oral squamous cell carcinoma: a computer-assisted analysis on TMA sections.

INTRODUCTION: Osteopontin (OPN) is non-collagenous extracellular matrix protein involved in various physiological and pathological events, including tumor progression. The aim of this study was to analyze the expression of OPN in normal oral mucosa and oral squamous cell carcinoma (OSCC) and to assess its prognostic significance. METHODS: The expression of OPN was immunohistochemicaly analyzed in 86 OSCC and compared with clinicopathological variable such as tumor size, nodal stage, WHO clinical stage, Ki-67 proliferation index, and patients' outcome. OPN mRNA was analyzed using quantitative real-time PCR and compared with protein OPN expression and clinical outcome in 18 OSCC samples. RESULTS: The expression of OPN protein was found in OSCC tumor cells (t-OPN) and various stromal cells (s-OPN). High level of t-OPN expression was associated with higher nodal stage (P = 0.045), higher WHO clinical stage (P = 0.033), and poor clinical outcome (P = 0.022). In multivariate analysis, t-OPN emerged as an adverse independent factor for survival (P = 0.049). Although correlated with t-OPN (P = 0.005), s-OPN was not significantly associated with clinical parameters, including patients' outcome. Also, there was no association between OPN and clinical parameters at the mRNA level. CONCLUSION: OPN is upregulated in tumor and stromal OSCC cells. Tumor cell-derived OPN is involved in tumor progression and can independently predict the clinical outcome. Stromal-derived OPN probably has a different function compared with OPN secreted from tumor cells.

JAK2-v617F mutation is associated with clinical and laboratory features of myeloproliferative neoplasms.

The aim of this study is to investigate the differences of clinical and laboratory parameters between patients with JAK2-V617F positive myeloproliferative neoplasms (MPNs) and JAK2 wild type MPNs. DNA was isolated from peripheral blood granulocytes of 106 patients treated at Rijeka University Hospital Center: 41 with polycythemia vera (PV), 43 with essential thrombocythemia (ET), 9 with primary myelofibrosis (PMF) and 13 with myeloproliferative neoplasm--unclassifiable (MPN-u). The JAK2-V617F mutation was detected using allele specific PCR. Laboratory and clinical parameters were obtained from patient's medical records. The JAK2-V617F mutation was detected in 69% (73/106) patients with MPNs. The results revealed significantly different prevalence of JAK2-V617F mutation, between MPNs entities: 88% in PV 58% in ET, 56% in PMF and 54% in MPNs-unclassified disorders. The JAK2-V617F mutation significantly correlated with higher leukocyte count and alkaline phosphatase co re in ET group and with higher platelets count, leukocyte alkaline phosphatase score and serum lactate dehydrogenase in PV group. Vascular events were associated with elevated platelets count in whole MPNs group, with higher platelets and leukocyte count in ET and with splenomegaly in PVpatients. Clinical and laboratory data revealed significant contribution ofJAK2-V617F mutation to the development of clinical phenotype in patients with distinct subgroups of MPNs.

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma.

BACKGROUND: The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival. METHODS: The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining. RESULTS: Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage. CONCLUSION: In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma.

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells. Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples. Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p=0.015) and protein (p<0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p=0.006 and p=0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.

Prognostic values of morphological and clinical parameters in pT2-pT3 prostate cancer in elderly people.

Prostate cancer is a disease of elderly men, the incidence of which increases in an age dependent manner. This study presents the correlation of clinical and morphological parameters in locally confined (pT2) and locally advanced (pT3) prostate cancer. We analyzed a group of elderly men treated with radical prostatectomy in the period 1999-2008 in the University Hospital Rijeka. We found no statistical association between pT stage and age categories, preoperative prostate-specific antigen, digitorectal examination and biopsy Gleason score. There was a significant correlation of higher Gleason score in prostate specimens after radical prostatectomy and a higher frequency of a positive surgical margin in tumors with pT3 than in pT2 stage (p = 0.003; p = 0.011 respectively). Recurrence-free survival was shorter in patients with tumors with positive surgical margins as well as in patients with pT3 stage (p = 0.030; p = 0.001 respectively). We conclude that higher tumor grade and positive surgical margins are indicators of a worse prognosis in our patients.

Predicting the likelihood of additional nodal metastases in breast carcinoma patients with positive sentinel node biopsy.

Axillary lymph node dissection (ALND) is an important procedure in the staging of breast cancer patients. However, it is associated with a significant morbidity rate. In addition, using early diagnosis a high number of cases with negative lymph nodes can be identified. A lymph node defined as sentinel lymph node (SLN) would be the first to receive tumoral drainage. A less morbid but accurate staining procedure using mapping and SLN biopsy has been introduced. The aim of this study was to estimate the likelihood of additional disease in the axilla after SLN analysis. A total of 259 breast carcinomas and SLN biopsies followed by ALND were examined. The patient median age was 59 years, approximately 75% of them postmenopausal. Tumor size was 1.4 +/- 0.8 cm (almost 80% in pT1). SLNs were positive in 59 of 259 (22.8%) carcinomas, 30 (11.6%) with micrometastases (<2.0 mm) and 29 (11.2%) with metastases. Tumor size ( P = .004) and presence of lymphovascular invasion (LVI; P = .034) were found to be significant predictors of pathologically positive SLN. Following ALND, positive non-SLNs were present mostly in patients with metastasis >2 mm in SLN (P = .003), in carcinoma with higher nuclear grade ( P = .044), decreased estrogen receptor (ER; P = .042), and progesterone receptor (PR; P = .042). Finally, lymph node status (pN) following SLN and ALND was found to be significantly associated with tumor size ( P = .006), LVI (P = .037), PR (P = .023), and Her-2 status (P < .001). These results point to detailed analysis of primary tumor and SLN that may increase the precision of patient selection for further axillary surgery or radiotherapy.

Hypoxia inducible factor-1alpha correlates with vascular endothelial growth factor A and C indicating worse prognosis in clear cell renal cell carcinoma.

BACKGROUND: The role of angiogenesis in the pathogenesis of renal cell carcinoma is well recognized, however, the influence of tumor cells in this activity has not yet been fully clarified. The aim of this study was to analyze the expression of hypoxia inducible factor-1alpha (HIF-1alpha), a regulatory factor of angiogenic switch, in comparison to vascular endothelial growth factor A and C (VEGF-A and VEGF-C), recognized to be involved in blood and lymph vessel neoangiogenesis, with potential association in the prognosis of patients with renal cell carcinoma. METHODS: Ninety-four patients with diagnosis of clear cell renal cell carcinomas (CCRCC), all clinicopathological characteristics and overall survival were unrolled in this study. Immunohistochemicaly VEGF-A, VEGF-C, HIF-1alpha and Ki67 were detected on tumor cells and the staining was performed on tissue microarrays (TMA). The staining was evaluated as a percentage of cytoplasmic or nuclear positive tumor cells. RESULTS: Variable expression of all three proteins was confirmed. Both angiogenic factors demonstrated perimembranous or diffuse cytoplasmic staining, with diffuse pattern positively associated (p < 0.001). Nuclear HIF-1alpha expression (nHIF-1alpha) showed inverse correlation with diffuse cytoplasmic VEGF-A (p = 0.002) and VEGF-C (p = 0.053), while cytoplasmic HIF-1alpha expression (cHIF-1alpha) showed positive correlation with diffuse staining of both angiogenic factors (p < 0.001; p < 0.001, respectively). In comparison to clinicopathological characteristics, a higher nuclear grade (p = 0.006; p < 0.001, respectively), larger tumor size (p = 0.009; p = 0.015, respectively), higher stage (p = 0.023; p = 0.027, respectively) and shorter survival (p = 0.018; p = 0.024, respectively) were associated with overexpression of cHIF-1alpha and diffuse cytoplasmic VEGF-A expression. In contrary, overexpression of nHIF-1alpha was associated with better diagnostic parameters i.e. lower nuclear grade (p = 0.006), smaller tumor size (p = 0.057), and longer survival (p = 0.005). CONCLUSION: Overexpression of VEGF-A and cHIF-1alpha in tumor cells highlights a more aggressive subtype of CCRCC that might have some clinical implications. The significance of nHIF-1alpha expression associated with better differentiated tumors should be further elucidated.

Relationship between vascular endothelial growth factor and nuclear factor-kappaB in renal cell tumors.

AIM: To assess the relationship between protein and messenger RNA (mRNA) levels of vascular endothelial growth factor (VEGF) and subcellular localization of nuclear factor-kappa B (NF-kappaB), proliferation rate of tumor cells, and clinicopathological characteristics of renal cell tumors. METHODS: We analyzed 31 one renal cell tumors - 22 clear cell renal cell carcinomas (CCRCC) and 9 other histologic types (non-CCRCC). VEGF expression and subcellular localization of p65 member of NF-kappaB and Ki67 were immunohistochemically evaluated for the proliferation rate of tumor cells. Expression of VEGF mRNA was assessed using quantitative real-time polymerase chain reaction after total RNA extraction from snap-frozen tumor tissue samples. RESULTS: Cytoplasmic localization of VEGF protein in renal cell tumors showed a perimembranous and diffuse pattern, the former being more evident in CCRCC (27.1 -/+ 18.9 vs 3.3 -/+ 10 % tumors, P<0.001) and the latter in non-CCRCC type (71.7 -/+ 23.2 vs 31.1 -/+ 22.1 % tumors, P<0.001). Heterogeneity in VEGF gene expression was more pronounced in CCRCC type than in non-CCRCC type (P=0.004). In addition, perimembranous VEGF pattern was associated with higher VEGF mRNA levels (P=0.006) and diffuse VEGF pattern with lower VEGF mRNA levels (P<0.001). Nuclear and cytoplasmic staining of NF-kappaB/p65 was observed in the majority of tumor cells. A significant association was recorded between cytoplasmic NK-kappaB/65 staining and VEGF staining of diffuse pattern (P=0.026). Association between NF-kappaB/65 and proliferation rate of tumor cells was significant for cytoplasmic staining (P=0.039) but not for nuclear NFkB/p65 staining (P=0.099). CONCLUSION: Higher but inhomogeneous expression of VEGF in tumor cells, especially in CCRCCs, is associated with NF-kappaB/65 activity. This indicates that both VEGF and NF-kappaB/65 may be important in renal carcinogenesis, representing a possible molecular target in the treatment of renal cell carcinoma.

Osteopontin expression correlates with angiogenesis and survival in malignant astrocytoma.

Osteopontin (OPN) is a phosphorylated glycoprotein with diverse functions including angiogenesis, cancer development, invasion and metastasis. The aim of the study was to analyze the expression of OPN in human astrocytomas and to correlate it with angiogenesis and patients' outcome. Seventy-six human astrocytomas including eight pilocytic astrocytomas (grade I), 10 diffuse astrocytomas (grade II), 8 anaplastic astrocytomas (grade III) and 50 glioblastomas (grade IV) were immunohistochemically stained for OPN protein. The distribution of OPN staining (cytoplasmic and/or interstitial) was assessed and compared to microvessel number and patients' survival. In normal brain tissue some glial and neuronal cells showed weak cytoplasmic staining, while interstitium was negative. Astrocytomas were heterogeneous regarding the OPN expression. High cytoplasmic OPN expression in glioblastomas was associated with poor patients' survival (p = 0.012). Also, we found the association of interstitial OPN expression and angiogenesis (p = 0.033), i.e. the number of newly formed blood vessels was higher in tumors showing high interstitial OPN expression. Our results indicate the overexpression of OPN protein in astrocytoma cells and suggest the role of OPN in astrocytoma progression and angiogenesis.