Gordana Đorđević

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

BackgroundThe 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-1α (HIF-1α), 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.MethodsNinety-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-1α 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.ResultsVariable 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-1α expression (nHIF-1α) showed inverse correlation with diffuse cytoplasmic VEGF-A (p = 0.002) and VEGF-C (p = 0.053), while cytoplasmic HIF-1α expression (cHIF-1α) 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-1α and diffuse cytoplasmic VEGF-A expression. In contrary, overexpression of nHIF-1α 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).ConclusionOverexpression of VEGF-A and cHIF-1α in tumor cells highlights a more aggressive subtype of CCRCC that might have some clinical implications. The significance of nHIF-1α expression associated with better differentiated tumors should be further elucidated.

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.

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

BackgroundThe 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.MethodsThe 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.ResultsMembrane 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 patients survival, while there was no connection with pathological stage.ConclusionIn 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.

The association between the recurrence of solitary non-muscle invasive bladder cancer and tumor infiltrating lymphocytes.

Aim To evaluate whether tumor infiltrating lymphocytes (TIL) in biopsy specimens are associated with the clinical outcome of non-muscle invasive bladder cancer. Methods We retrieved tumor specimens from 115 patients with solitary papillary non-muscle invasive bladder cancer treated between 1996 and 2006 and constructed tissue microarrays. Patients were divided in two groups: those with recurrent disease (N = 69) and those without recurrent disease (N = 46) during the follow up of minimum 5 years. All patients were treated with initial transurethral resection and none received adjuvant therapy. Immunhistochemical staining was performed with anti-CD3, CD4, CD8, and Granzyme B (GrB). The CD4+:CD8+ and GrB+:CD8 ratios were determined. Results Tumor infiltrating lymphocytes were predominantly observed within cancer stroma, and only rare individual cells were observed intraepithelially. The group without recurrent disease had lower levels of CD3+ and CD8+ lymphocytes than the group with recurrent disease (P = 0.0001, P = 0.0002, respectively). The CD4+:GrB+ and GrB+:CD8+ ratios were significantly higher in patients without recurrent disease (P = 0.0002, P = 0.039, respectively). Conclusion This study revealed a possible connection between TIL number and bladder cancer recurrence. TIL subset ratio showed different patterns in recurrent and non-recurrent tumors, which is why it could become a useful a prognostic clinical index if our findings are confirmed in randomized trials.

Different Perforin Expression in Peripheral Blood and Prostate Tissue in Patients with Benign Prostatic Hyperplasia and Prostate Cancer

Perforin (P) is a prototypical cytotoxic molecule involved in cell‐mediated immunity against various pathogens, alloantigens and particularly different tumours. The purpose of this study was to determine P expression in different lymphocyte subpopulations isolated from peripheral blood and prostate tissue of patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and compare it with the P expression found in the control group. Twenty subjects were recruited in each of the groups. Prostate mononuclear cells of the BPH and PCa tissues were isolated by enzymatic digestion and gradient density centrifugation, whereas peripheral blood mononuclear cells were isolated by gradient density centrifugation alone. Cells and tissue samples were labelled using monoclonal antibodies against P and different surface antigens (CD3, CD4, CD8 and CD56) and analysed by immunofluorescence and flow cytometry. Total P expression in peripheral blood lymphocytes did not differ significantly between BPH/PCa patients and control group, although the BPH and PCa tissue showed lower P expression level. A negative correlation between prostate‐specific antigen levels and the overall percentage of P+, CD3+CD56−P+, and CD3−CD56+P+ cells in the prostate tissue was observed only in patients with PCa. Our findings indicate that the low frequency of P+ lymphocytes, including T, NKT and NK cells, in the prostate tissue of patients with BPH and, particularly, PCa could be the consequence of local tissue microenvironment and one of the mechanisms involved in the pathogenesis of prostate hyperplasia following malignant alteration.

Matrix Metalloproteinases 2 and 9 Immunoexpression in Prostate Carcinoma at the Positive Margin of Radical Prostatectomy Specimens

The aim of this study was to evaluate the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in prostate cancer in the main tumor mass and tumor cells at the positive margin as well as the influence of these biomarkers on the biochemical recurrence of the disease in prostatectomy patients. Tissue microarrays of 120 archival prostate carcinoma samples were immunohistochemically evaluated for MMP-2 and MMP-9 expression and compared with clinicopathological parameters. Tumors with positive surgical margins showed significantly higher overall expression of MMP-9 versus tumors with negative resection margins (P = 0.0121). MMP-9 expression was significantly elevated in tumors from patients who had biochemical recurrence (P = 0.0207). In the group of patients with negative margins, MMP-9 expression above the cut-off value was significantly associated with recurrence (P = 0.0065). Multivariate analysis indicated that MMP-9 is a good predictor of biochemical recurrence (odds ratio = 10.29; P = 0.0052). Expression of MMP-2 in tumor cells was significantly higher at the positive margins than in the main tumor mass (P = 0.0301). The present results highlight the potential value of MMP-2 and MMP-9 expression for predicting the behavior of prostate tumors after prostatectomy with both positive and negative surgical margins.

Prostate Cancer and New Insights in Angiogenesis

Angiogenesis is considered one of the hallmarks of tumor initiation and progression therefore it is also the widely accepted therapeutic target. It has been shown that angiogenesis blockers prolong patient’s survival for several months (1). In order to progress, cancer cells acquire several distinct morphological alterations increasing their capacity to proliferate and independently sustain growth, and at the same time, inhibit homeostatic, physiologic signals from the microenvironment they are in, and under the control of which they should have remained. This is the main principle behind a tumor gaining the ability to metastasize (2). Till now, several agents have been approved by the Food and Drugs Administration (FDA) but their efficacy is still limited and this observation is repeatedly proven by recurrence of more aggressive tumor after application of anti-angiogenic therapy (3). So far stated is highly suggestive to back up and justify the existence of hyperplasia in the field of research for novel therapies, which would induce suppression of angiogenesis in carcinogenesis cascade and limit tumor progression. The complexity of angiogenesis becomes one of the greatest challenges in understanding various processes of inflammation, reparation, tumorigenesis, and metastases. In following paragraphs, we will try to express opinion where in near future we should probably search for the next generation of promising therapeutics for management of prostate cancer. It has been established that new blood vessel formation is required for tumor growth (4). The mutations, which predispose tissue to neoangiogenesis and induce tissue invasion, are reactivated genetic programs normally turned off in adult human especially those activated during reparation processes in the surrounding microenvironment, where pro-angiogenic factors play a key role in establishing a capillary network from the surrounding host tissues (5). The surrounding stromal cells, tumor-associated macrophages, and other components of the extracellular also actively constitute matrix chemical milieu, and not just the cancer cells alone as previously implied. To be more precise, angiogenic switch by its definition implies impaired angiogenesis and importance of high vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) levels holding responsible for PCA progression (6). The level of oxygenation in prostate is important for estimation of its hypoxic state. Namely, hypoxic state is informative as relative tissue oxygenation status, which correlates with overall VEGF expression in prostate (7–10). Progressive induction of hypoxia influences metabolic changes at cellular level in prostate epithelial cells and their energetic demands are being modified accordingly. Impaired systems of cellular repair are not capable to restore in full sublethal cellular damage induced by hypoxia therefore pro-inflammatory cytokines are being increasingly secreted by prostate gland epithelia. This leads toward further promotion of immunologic response inducing chronic inflammation. From other tumor models it has been known that long lasting chronic inflammation stimulates cellular proliferation and favors fasten growing, which is often in hypoxic circumstances deranged at certain level. Oxygen delivery and consumption are regulated with hypoxia-inducible factors (HIFs) among other molecules, which also induce transcription of VEGF stimulating, in turn, tumor angiogenesis due to increased oxygen demand. Hypoxic cancer cells are stimulated to acquire invasive and metastatic properties and they are promptly developing resistance to chemotherapy too (11). Although significance of inflammation in cancer progression has been discussed and researched for a long time, till recently, research has extrapolated pathways directed to connection between inflammation and cancer evolution. Different studies strongly point out that chronic inflammation is involved in progression of chronic prostatic disease, such as benign prostatic hyperplasia (BPH) and consequent development of prostate cancer (12). The coincidence of chronic inflammation and tumorigenesis in the peripheral zone identified as so-called proliferative inflammatory atrophy is proposed as possible precursor of prostatic intraepithelial neoplasm. This can be explained since inflammatory microenvironment releases growth factors and cytokines that may influence the activation of the vascular endothelial cells and signal transduction in these cells. Continuous inflammatory stimuli as it have been previously shown result in endothelial sprouting. This links process of inflammation with angiogenesis, which predisposes preexisting endothelial cells to continuous activation and usually has its gene activation for consequence. Neovascularization includes also presence of dysfunctional endothelial cells, which is particularly present during chronic inflammation and among others related with sustained p38 MAP kinase pathway activity and increased levels of migration of cytokines. When observed, described gene changes reactive oxygen species (ROS) levels are elevated (13). The novel factors could possibly influence angiogenic switch consequently leading to progression from low-grade prostatic intraepithelial neoplasia (PIN) to high-grade PIN and beyond to prostate cancer or even more aggressive, poorly differentiated, and androgen-independent histological subtypes. From morphological point of view, it is important to recognize the presence of even small amount of microscopic patterns, which may be responsible for prompt tumor progression, advancement, and transition from low-grade lesion to the high grade one. Today, we are aware of their importance and connection with different molecular pathways, which prevail in interplay among tumor cells and environment interaction. Recently, it has been shown that the p38-MAPK pathway can be activated under continuous extensive anti-androgen exposure in prostatic cancer cells, and that the p38-MAPK pathway has a critical role in the induction of resistance, as well as in the acquisition of a more aggressive and invasive phenotype (13). This observation is at least interesting if we would not exaggerate since it offers potentially new therapeutic escape window. Namely, proposed role of p38 in controlling the phosphorylation and activity of transcription factors that has been established would regulate tumor and stromal cells proliferation and the expression and release of multiple tumor-derived cytokines. Cytokines may in turn promote tumor angiogenesis and lymphangiogenesis, thereby favoring the establishment of a permissive tumor microenvironment. The prostate microenvironment appears relatively simple in comparison with some other milieus (i.e., bone marrow for instance) since it consists of the components of supporting stromal elements’ extracellular matrices, and predominantly, carcinoma-associated fibroblasts. Activation and increased expression of several adhesion molecules correlate with a progression of malignant tumors. CD44 has been recognized to recruit and accumulate matrix metalloproteinase on the cell surface, enabling indirectly tumor cell angiogenesis and invasion (14). CD44 is also recognized as a potential target for cells gained pre-metastatic phenotype and entering process of epithelial–mesenchimal transition (15). Understanding a different expression of CD44s marks and metalloproteinase in the field of invasion may help in recognizing patients with more aggressive forms of prostate tumors, who should be treated surgically and also additionally. Fibroblast actively participates in extracellular matrix remodeling thereby promoting actively carcinogenesis and stimulating prostate cancer cell proliferation and angiogenesis (15, 16). From this concept, two options of therapeutic actions are deriving one targeting reactive stroma and other is aiming inflammatory, stromal and circulating cells (17). There is a constant need of new anticancer agents, in general oncologic therapy. Though exact molecular events enabling prostate cell malignant transformation and the ability to gain aggressive metastatic phenotype remain elusive, it is important not to overlook the importance that new therapeutic agents should be able to provide a therapeutic solution for both local and metastatic prostate cancer. As previously mentioned, as agents capable of disrupting MAPK signaling pathway and inducing irreversible inhibition of tumor proliferation, angiogenesis and metastatic potential, and as moderators of expression and function of adhesion molecules, molecule CD44 would probably be the next generation of promising therapeutics for management of prostate cancer.

Correlation of tumor-associated macrophages and NK cells with bladder cancer size and T stage in patients with solitary low-grade urothelial carcinoma

SummaryObjectiveThe aim of the present study was to correlate the level of tumor-infiltrating immune cells with bladder cancer size and T category in patients with solitary low-grade non-muscle invasive bladder cancer (NMIBC).Patients and methodsBetween 1996 and 2006, 115 patients with solitary low-grade NMIBC after transurethral resection of the bladder without adjuvant therapy were retrospectively identified from the institutional database. Tumor specimens were retrieved and tissue microarrays were constructed. Immunhistochemical staining for tumor-infiltrating immune cells with anti-CD3, CD4, CD8, CD20, CD56, CD68, and granzyme B (Gr B) was performed.ResultsImmune cells were predominantly observed within the cancer stroma. Statistically significant higher levels of CD56 cells in small tumors and CD68 cells in T1 tumors (p = 0.0310, 0.0151, respectively) were established.ConclusionThe current study propose a possible correlation of CD56+ and CD68+ cells with bladder cancer size and stage in patients with solitary low-grade NMIBC.

Deskriptivna i funkcionalna definicija jezgrice – put prema terapiji raka

Modern tumour therapy should be target specific and comprehensive. Recent clinical trials test small molecular inhibitors of transcription of ribosomal genes and activators of apoptosis. We treatise on models of activation of the suppressors of tumour growth acting in the nucleolar domain. Tumour cells demonstrate a disordered protein regulation and mutation of P53 gene which codes for a suppressor of growth. Therapy is guided by pathogenesis. Modern disease definition is based on etiopathogenesis of cell stress, wherein participate numerous organelles, including the nucleolus. Nucleus and nucleolus are forms seen on light microscopy. This presentation focuses on the description of the visible correlate of the notion of “nucleolus”. Further to this, acid non-histone proteins which gather around the transcriptional machinery of RNA polymerase I are described, and the significance of their visualisation towards the alteration of the definition of nucleolus is pointed out. Besides being a particle within the nucleoplasm and a subnuclear organelle, the nucleolus is visualized through its functional structures – the AgNORs. The AgNOR proteins have a role in nucleolar stress. The aim of this article is to discuss the nucleolar stress.

Pregled nove SZO-ove histološke klasifikacije epitelnih tumora bubrega

Progress in the diagnostics based on morphology and immunohistochemistry, genetic and molecular methods has led to the identification of new entities in renal tumor pathology. Therefore the International Society of Urological Pathology (ISUP) recommended modifications of the WHO 2004 renal tumor classification and called it ISUP Vancouver Modification of WHO (2004) Histologic Classification of Kidney Tumors which is officially published in new“ blue book“, The 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs. The aim of this article was to briefly introduce a new classification of renal cell carcinoma and new tumor entities identified by their histological and genetic characteristics. We also wanted to focus attention on the importance of those characteristics that determine the tumor grade and the stage of the disease, which have prognostic significance. The new classification will allow pathologists to harmonize their reports, and other disciplines to understand the changes in the findings of the pathologist.