Nives Jonjić

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Blocking CCL2 nitration in tumors promoted CD8+ influx and reduced tumor growth and prolonged survival in mice when combined with adoptive cell therapy.

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.

Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma

Abstract. Angiogenic and anti-angiogenic factors, secreted by tumor, inflammatory, and stromal cells play an important role in regulation of neovascularization. Among the most important of these is vascular endothelial growth factor (VEGF), a specific mitogen for endothelium, which increases vascular permeability and induces proteolytic enzymes necessary for vascular remodeling. Tumor-associated macrophages (TAMs) can express complex functions related to tumor biology, including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define, using immunohistochemical analysis, the microvessel density (MVD), VEGF expression, and TAMs level in 97 human invasive ductal breast carcinomas not otherwise specified (NOS), investigate a possible relationship between them and then correlate their values with tumor grade, mitotic activity index (MAI), tumor size and lymph-node status. Statistical analysis showed a strong positive relationship between MVD and VEGF expression (P<0.001). Furthermore, both MVD and VEGF expression were significantly correlated with tumor grade and lymph-node status, and TAMs infiltration with MAI. TAM level showed a significant positive connection with VEGF expression and MVD. These in situ observations suggest that VEGF stimulates angiogenesis in human invasive ductal breast carcinoma NOS and attracts macrophages to the tumor locus, which then may be involved in angiogenesis promotion. The expression of this angiogenic molecule, and MVD and TAM level, can provide additional prognostic significance and help in the identification of patients who need postoperative adjuvant therapy.

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

Correlation of serum IL‐6, IL‐8 and IL‐10 levels with clinicopathological features and prognosis in patients with diffuse large B‐cell lymphoma

Cytokines play important roles in the pathogenesis of lymphomas. This study aimed to determine the relationship(s) between serum levels of interleukin (IL)‐6, IL‐8 and IL‐10, measured by enzyme‐immunoassay, and the clinical characteristics and outcomes in 46 untreated patients with diffuse large B‐cell lymphoma (DLBCL). Serum IL‐6, IL‐8 and IL‐10 levels were higher in DLBCL patients than in control subjects. Elevated levels of IL‐6, IL‐8 and IL‐10 correlated with more adverse disease features. Consequently, patients with elevated IL‐6, IL‐8 and IL‐10 levels prior to treatment had a lower response to therapy. Furthermore, those with elevated IL‐6 and IL‐10 levels had poor median, 3‐year and 5‐year survival, while elevated serum IL‐8 level did not correlate with overall survival. Worse survival was also confirmed in patients with combined elevated pretreatment serum levels of IL‐6, IL‐8 and IL‐10 (none, one, two or three elevated). Multivariate analysis identified elevated values of IL‐6 and IL‐10 and response to therapy as significant predictors for overall survival. Serum levels of IL‐6, IL‐8 and IL‐10 before treatment of patients with newly diagnosed DLBCL may give some insight into the possible prognosis and thus facilitate the decisions regarding therapeutic approaches for individual patients.

Distribution pattern of tenascin-C in glioblastoma: correlation with angiogenesis and tumor cell proliferation.

Tenascin-C (TN-C) is an extracellular matrix protein which participates in different processes like normal fetal development, wound healing, inflammation, keloids and rheumatoid arthritis. Furthermore, the immunostaining for TN-C is seen in the stroma of various malignant tumors as in glioblastoma multiforme (GBM), however, the significance of these findings is still not clear. In this study 62 GBM samples were analyzed immunohistochemically for distribution patterns of TN-C and correlated with angiogenesis and tumor cell proliferation. Tenascin-C in GBM localizes in two compartments, perivascular and intercellular space. Intercellular tenascin-C (TN-C ic) showed focal distribution in 66%, and diffuse one in 34% of cases. Perivascular tenascin-C (TN-C pv) showed strong correlation with microvascular density (MVD) and vascular endothelial growth factor (VEGF) expression. Moreover, it seems that TN-C pv enhanced the effect of VEGF. Intercellular TN-C did not correlate with MVD and VEGF expression, but showed strong correlation with proliferation index. Furthermore, tumors with diffuse TN-C ic expression had higher proliferation indices than tumors with focal TN-C expression. Our results indicate that TN-C plays a role in angiogenesis and tumor cell proliferation, but beside the intensity of expression, the distribution patterns are also important in these processes. This study also suggests that perivascular and intercellular TN-C compartments have probably different sources and different roles in GBM.

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.

p53 protein expression and cell proliferation in non-neoplastic and neoplastic proliferative skin diseases.

Aims and background The p53 protein is essential for the regulation of cell proliferation and its aberrant accumulation is usually seen in malignant tumors but also occurs in squamous epithelium of inflammatory skin diseases characterized by hyperproliferation. The aim of this study is to elucidate the role of the p53 tumor suppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and to determine the association between p53 overexpression and cell proliferation. We also investigated the influence of aging on p53 and Ki-67 protein expression. Methods One hundred and fifty skin specimens divided into 30 samples each of normal skin (NS), psoriatic skin (PS), keratoacanthomas (KA), basal cell carcinomas (BCC), and squamous cell carcinomas (SCC) were examined immunohistochemically to assess p53 and Ki-67 protein expression. Results p53 immunostaining of NS, PS, KA, BCC and SCC was detected in 39.0%, 46.7%, 66.7%, 80% and 86.7% of cases, respectively. Median values and ranges of p53 protein expression were as follows: 0.0% (range, 0.0–1.8%) in NS, 0.0% (range, 0.0–6.5%) in PS, 9.2% (range, 0.0–24.0%) in KA, 19.3% (range, 0.0–48.1%) in BCC and 30.1% (range, 0.0–68.1%) in SCC. p53- and Ki-67-positive cells were present in basal (NS) and suprabasal layers (PS), and not only in cancer nests of KA, BCC and SCC but also in dysplastic and even morphologically normal epidermis adjoining cancers. The positivity of p53 and Ki-67 proteins differed significantly among the groups, with no differences in p53 expression between NS and PS and in Ki-67 expression between PS and KA. Within all groups there was a significant correlation between p53 and Ki-67 expression. Lesion location and patient age, with the exception of location in PS and age in BCC, were significantly related to p53 and Ki-67 expression in all groups. Conclusions Our findings suggest that p53 overexpression occurs mainly in neoplastic skin lesions, although it may also occur in squamous epithelium of inflammatory skin diseases such as PS, as well as in normal skin epithelium. It is associated with cell proliferation in normal as well as altered epithelium. p53 protein overexpression is an age-related process and significantly associated with sun exposure, especially in NS and PS but also in KA and SCC. Our findings suggest that Ki-67 rate and p53 protein expression reflect the degree of malignancy in the examined cutaneous neoplasms.

Encapsulated mesenchymal stem cells for in vivo immunomodulation

Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol 2010; 28: 570–577. 11 Döhner K, Tobis K, Ulrich R, Fröhling S, Benner A, Schlenk RF et al. Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol 2002; 20: 3254–3261. 12 Schlenk RF, Döhner K, Krauter J, Fröhling S, Corbacioglu A, Bullinger L et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909–1918. 13 Haferlach C, Mecucci C, Schnittger S, Kohlmann A, Mancini M, Cuneo A et al. AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features. Blood 2009; 114: 3024–3032. 14 Grossmann V, Schnittger S, Schindela S, Klein HU, Eder C, Dugas M et al. Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology. J Mol Diagn 2011; 13: 129–136. 15 Schnittger S, Alpermann T, Eder C, Schindela S, Grossmann V, Kern W et al. The role of different genetic subtypes in CEBPA mutated AML. Blood (ASH Ann Meet) 2010; 116: 752 (Abstracts: oral presentation).

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.