Ksenija Lučin

EXPRESSION OF MONOCYTE CHEMOTACTIC PROTEIN-1 IN HUMAN INVASIVE DUCTAL BREAST CANCER

Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the macrophage infiltration of tumor tissue. Tumor associated macrophages (TAMs) are a population of mononuclear-phagocytic cells, which can express complex functions related to tumor biology. The present study was designed to analyse the expression of MCP-1 in parenchymal and stromal elements on frozen sections of 27 breast invasive ductal carcinomas not otherwise specified (NOS) by immunohistochemistry. The expression of MCP-1 in tumor parenchyma and the degree of tumor differentiation were assessed. MCP-1 was detected in the parenchyma in 15 of 27 ductal carcinomas. Positive immunoreactivity manifested as diffuse, homogeneous, moderate or strong, cytoplasmic staining, confined to tumor epithelium. Generally, MCP-1-negative tumors tended to be well differentiated, while chemokine-positive tumors exhibited a low level of differentiation. MCP-1 immunoreactivity was also present in TAMs (CD68 positive cells) in 23 of 27 tumors, and in endothelial cells in 11 of 27 tumors. These results indicate that parenchymal and, more variably, stromal elements of human invasive ductal carcinomas NOS can express MCP-1 in vivo. Additionally, these findings suggest that MCP-1 expression in tumor parenchyma is correlated with the histological grade of ductal invasive breast 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.

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.

Serum IL-6, IL-8, IL-10 and beta2-microglobulin in association with International Prognostic Index in diffuse large B cell lymphoma.

Aims and Background Diffuse large B-cell lymphoma displays striking heterogeneity at clinical, genetic and molecular levels. The International Prognostic Index is useful to predict the outcome of diffuse large B-cell lymphoma patients. However, patients with identical International Prognostic Index values in clinical practice exhibit marked variability in survival, suggesting the presence of significant residual heterogeneity within each category. Since cytokines such as interleukin-6, -8 and -10 play important roles in the pathogenesis of lymphomas, and plasma level of beta2-microglobulin is associated with the outcome of patients with diffuse large B-cell lymphoma, the aim of the present study was to determine whether these parameters combined with the International Prognostic Index would better stratify these patients to predict their prognosis. Patients and Methods The study included 46 untreated diffuse large B-cell lymphoma patients. Results All study parameters (International Prognostic Index, Ann Arbor stage, extra-nodal involvement, performance status, lactate dehydrogenase, beta2-microglobulin, interleukin-6 and -10, and response to therapy) except for patient age and serum interleukin-8 level were associated with overall survival. In addition, the International Prognostic Index was strongly correlated with beta2-microglobulin, interleukin-6, -8 and -10, and when combined these parameters significantly better stratified patients according to survival. On multivariate analysis, therapeutic response to the primary treatment, elevated interleukin-6 and -10 levels, and the International Prognostic Index were significant predictors of overall survival. Conclusions Our data imply that interleukins and beta2-microglobulin evaluation should be used in association with the International Prognostic Index to define prognostic subgroups in diffuse large B-cell lymphoma patients.

Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma

BackgroundEstimation of plasma cell infiltrates in bone marrow aspirates (BMA) and bone marrow biopsy (BMB) is a standard method in the diagnosis and monitoring of multiple myeloma (MM). Plasma cell fraction in the bone marrow is therefore critical for the classification and optimal clinical management of patients with plasma cell dyscrasias. The aim of the study was to compare the percentage of plasma cells obtained by both methods with the patient clinical parameters and survival.MethodsThis retrospective study included BMA and BMB of 59 MM patients. The conventional differential count was determined in BMA to estimate the percentage and cytologic grade of plasma cells. The pattern of neoplastic infiltration and percentage of plasma cells were estimated on CD138 immunostained BMB slides microscopically and by computer-assisted image analysis (CIA).ResultsSignificantly higher values of plasma cell infiltrates were observed in pathologist (47.7 ± 24.8) and CIA (44.1 ± 30.6) reports in comparison with cytologist analysis (30.6 ± 17.1; P < 0.001 and P < 0.0048, respectively). BMB assessment by pathologist counting and using CIA showed strongest correlation (r = 0.8; P < 0.0001). Correlation was also observed between the pathologist and cytologist counts (r = 0.321; P = 0.015) as well as comparing the percentage of plasma cells in BMA and CIA (r = 0.27; P = 0.05). Patients with clinical stage I/II had a significantly lower CIA plasma cell count than those with clinical stage III (P = 0.008). Overall survival was shorter in patients with more than 25% of atypical plasma cell morphology estimated in BMA (P = 0.05) and a higher percentage of tumor cell infiltrates estimated by the pathologist and CIA (P = 0.0341 and P = 0.013, respectively).ConclusionStudy results suggested the combined analyses to be useful as a routine procedure to achieve more accurate and informative diagnostic data.

Prognostic significance of T-cell infiltrates, expression of β2-microglobulin and HLA-DR antigens in breast carcinoma

In this study immunohistological staining was used to assess the presence of T-cell infiltrates and the expression of beta 2-microglobulin (beta 2-m) and HLA-DR antigens on tumor cells of 75 ductal invasive carcinomas. The results were compared with the morphometric prognostic index (MPI) that seems to be the most accurate prognostic predictor. The extent of T-cell infiltrates differed widely between tumors, but statistically significant correlation was found only with the lymph node status, namely, tumors with a high degree of infiltration had predominantly negative lymph nodes and vice versa (p < 0.05). Only 19 (25.3%) out of 75 carcinomas were beta 2-m+, 34 cases (45.3%) showed heterogeneous staining pattern and 22 tumors (29.3%) were completely negative. We could not find any significant correlation between beta 2-m expression and MPI or T-cell content. While normal breast epithelium was always HLA-DR negative, tumor cells displayed positivity in 25 cases (33.3%), 5 tumors (6.7%) were completely positive and 20 tumors (26.7%) displayed only focal expression of class II antigens. This expression did not correlate with any single prognostic parameter, nor with MPI. The results suggest that T-cell infiltrates and the expression of histocompatibility antigens can not be accepted as prognostic indicators in breast carcinoma.

Expression of Beta-1 Integrins on Tumor Cells of Invasive Ductal Breast Carcinoma

The integrins are transmembrane alfabeta heterodimers mediating cell-cell as well as cell-extracellular matrix interactions. The present study was designed to analyse the expression of beta-1 integrins on cryostat sections of invasive ductal carcinomas not otherwise specified by avidin-biotin complex immunoperoxidase technique, and to compare it with the morphometric prognostic index (MPI). The results show that the expression of beta-1 integrins is heterogeneous in the tumors. This heterogeneity was observed in quantitative and qualitative staining pattern. There was an absent expression of beta-1 integrins in 22 out of 55 tumors while 33 showed staining, weak on 23 cases and strong on 10 infiltrative ductal carcinomas. Statistical analysis pointed to some correlation of beta-1 integrins with some morphometric parameters. Low or absent expression of beta-1 integrins correlated significantly with tumors exceeding 2 cm (p < 0.0245). Moreover, a larger proportion of tumors with positive lymph nodes showed absence of beta-1 expression compared with negative lymph node, and this was also statistically significant (p < 0.0076). Correlation between mitotic activity index and staining intensity for beta-1 integrins was not found (p < 0.372). When tumors with different beta-1 expression were subdivided according to MPI values into two groups, one group with a low-risk, < 0.6, and second with a high risk, > 0.6, concordance in prognostic value was shown between MPI and beta-1 expression (p < 0.0193). These results support the idea that loss of beta-1 integrins correlates with the invasive and metastatic potential of tumor cells.

Assessment of Bone Marrow Fibrosis and Angiogenesis in Monitoring Patients With Multiple Myeloma

The aim of our study was to emphasize the importance of accurate and standardized techniques for detailed monitoring of the microenvironment in multiple myeloma (MM). Bone marrow fibrosis, angiogenesis, and plasma cell infiltrates in bone marrow biopsy (BMB) samples at the time of diagnosis and on completion of therapy were analyzed for 42 patients with newly diagnosed MM. Computerized image analysis was used for all slides stained with anti-CD138 and anti-CD34. The patients with fibrosis in pretreatment BMB samples had significantly higher microvessel density (MVD) and plasma cell infiltrates. In posttreatment BMB samples, nonresponders had a significantly higher frequency and grade of fibrosis and higher values of MVD, total vascular area, and plasma cell percentage. The overall survival of nonresponders and patients with increased marrow fibrosis in posttreatment BMB samples was significantly shorter. The obtained results confirm that complex morphologic examination of the bone marrow microenvironment during the monitoring of MM can provide better prognostic significance.

Expression of osteopontin and CD44 molecule in papillary renal cell tumors.

The aim of the study was to analyze the expression of CD44 adhesion molecule and its ligand osteopontin in papillary renal cell tumors, and to assess the possible prognostic significance of CD44 and osteopontin expression in papillary renal cell carcinomas. The expression of the standard and v6 exon containing isoforms of CD44 molecule, as well as of its ligand osteopontin, was immunohistochemically evaluated in 43 papillary renal cell tumors, which included 5 adenomas and 38 carcinomas. In order to assess their prognostic significance, the results obtained in papillary renal cell carcinomas were compared to usual clinicopathological parameters such as tumor size, histological grade, pathological stage, and Ki-67 proliferation index. Normal renal tissue was negative for CD44s and v6 isoforms, while the expression of osteopontin was found in distal tubular epithelial cells in the form of cytoplasmic granular positivity. CD44s and v6 isoforms were upregulated in 22 (58%) and 12 (32%) out of 38 carcinomas, respectively. Among all clinicopathological parameters examined, we only found significant association of CD44s-positive carcinomas with lower pathological stage (p=0.026). Papillary renal cell adenomas were generally negative for CD44s, except for focal positivity found in one sample. The osteopontin protein was detected in all adenomas and all papillary renal cell carcinomas, except one. Our results show constitutive expression of osteopontin in papillary renal tumors, including papillary renal cell adenomas. The upregulation of CD44s and v6 isoforms, although found in a considerable number of papillary renal cell carcinomas, does not appear to have any prognostic value in this type of renal cancer.

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.