Detection of an EGFR mutation in cytological specimens of lung adenocarcinoma.

OBJECTIVE: Epidermal growth factor receptors (EGFR) mutation status is crucial for the prediction of a tumour response to treatment with EGFR tyrosine kinase (EGFR-TK) inhibitors. The aim of the study was to establish a protocol for the detection of EGFR-activating somatic mutations on cytological samples collected using a standard bronchoscopy procedure and to determine the frequency of EGFR mutations among pre-selected Croatian patients with non-small cell lung cancer (NSCLC) of an adenocarcinoma histological subtype. METHODS: A total of 177 cytological samples were collected from the patients diagnosed with NSCLC. DNA was isolated from the cytological material recovered from the fixed and stained slides. EGFR mutations were analysed using the polymerase chain reaction (PCR)- mediated Sanger sequencing method. RESULTS: Out of 177 collected samples, EGFR mutation analyses were successfully performed on 167 samples (94.4%); 77 (46.1%) of these were from male and 90 (53.9%) from female patients. EGFR mutations/deletions were found in 33 (19.8%) of the tested patients; exon 19 deletions in 17 (10.2%) and point mutations of exon 21 in 16 (9.6%) patients. CONCLUSION: The PCR-mediated Sanger sequencing method was found to be reproducible and reliable. Cytological samples can be used successfully to determine the EGFR mutation status in NSCLC patients providing information for targeted therapy at an early stage of the disease.

Expression of adhesion molecules and chemotactic cytokines in cultured human mesothelial cells.

The mesothelium is a flat epithelial lining of serous cavities that could gate the traffic of molecules and cells between the circulation and these body compartments. The present study was designed to elucidate the capacity of mesothelial cells to express adhesion molecules and chemoattractant cytokines, two fundamental mechanisms of regulation of leukocyte recruitment. Cultured human mesothelial cells express appreciable levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and these were increased by in vitro exposure to tumor necrosis factor (TNF), interferon gamma (IFN-gamma), or TNF and IFN-gamma. Interleukin 1 (IL-1) was a less consistent stimulus for adhesion molecule expression in vitro. Unlike endothelial cells, used as a reference cell population, resting or stimulated mesothelial cells did not express E-selectin and ICAM-2, as assessed by flow cytometry. Analysis of VCAM-1 mRNA by reverse transcriptase and polymerase chain reaction using appropriate primers revealed that mesothelial cells expressed both the seven- and the six-Ig domain transcripts, with predominance of the longer species. Monocytes bound appreciably to "resting" and, to a greater extent, to stimulated mesothelial cells. Monocytes exposed to IFN-gamma and lipopolysaccharide, used as prototypic activation signals, showed increased capacity to bind mesothelial cells. Anti-CD18 monoclonal antibody significantly inhibited binding of monocytes to mesothelial cells, and this blocking effect was amplified by anti-very late antigen 4. Mesothelial cells were able to express the chemotactic cytokines IL-8 and monocyte chemotactic protein 1 at the mRNA and protein levels. These results indicate that mesothelial cells can express a set of adhesion molecules (ICAM-1 and VCAM-1) overlapping with, but distinct from, that expressed in vascular endothelium (ICAM-1, ICAM-2, VCAM-1, E-selectin), and that these are functionally relevant for interacting with mononuclear phagocytes. The regulated expression of adhesion molecules and chemotactic cytokines by mesothelial cells is probably important in inflammatory and immune reactions that involve serous cavities, such as the long-known macrophage appearance and disappearance reactions.

Primary leiomyosarcoma of the tibia--five years without recidivism.

Primary leiomyosarcoma of the bone is a rare malignancy. Clinical follow-up suggests that primary osseous leiomyosarcoma has an aggressive biologic behaviour with poor survival time. We report a case of primary leiomyosarcoma arising from the proximal metaphysis of the right tibia of a 55-year old woman with a long follow-up period, without recidivism. Primary leiomyosarcoma has to be considered as a differential diagnostic possibility in the case of bone tumours seen on routine initial plain radiographs as lytic lesions. If the tumour has been adequately excised at the time of diagnosis, as in the present case, with adjuvant therapy protocol, the long-term prognosis of such an aggressive tumour can be exceptionally good.

Macrophage level is not affected by monocyte chemotactic protein-1 in invasive ductal breast carcinoma.

PURPOSE AND METHOD: 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 that can have a complex function in tumor biology. The aim of this study was to determine the possible correlation between parenchymal MCP-1 expression and TAM level by immunohistochemical analysis of 97 invasive ductal breast carcinomas, not otherwise specified (NOS), and to investigate their relation with tumor size, histological grade, mitotic activity index (MAI) and lymph node status. Secondly, the MCP-1 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in eight samples of normal breast tissue and 27 samples of invasive breast carcinomas and compared with TAMs. RESULTS: MCP-1 immunoreactivity was present in tumor cells (17/97), but also in TAMs, fibroblasts and endothelial cells. The statistical analysis did not show a significant correlation between MCP-1 expression in tumoral epithelium and tumor size, histological grade, MAI, lymph node status or TAMs. The results of RT-PCR showed that, in all cases of breast carcinomas (27/27) and the majority of normal breast tissues (7/8), the number of detected MCP-1 cDNA copies was above the detection limit. However, carcinomas showed higher levels of MCP-1 mRNA than normal breast tissue. Nevertheless, the statistical analysis did not find a significant correlation between MCP-1 expression and macrophage infiltrations. CONCLUSION: These results indicate that MCP-1 is probably not the only and/or crucial factor involved in macrophage attraction to tumor locus in breast carcinoma.

The lectin-binding sites for peanut agglutinin in invasive breast ductal carcinomas and their role as a prognostic factor.

The present study was designed to analyze the expression of lectin-binding sites for peanut agglutinin (PNA) in paraffin sections of primary invasive ductal carcinoma not otherwise specified and to consider PNA lectin histochemistry as a further aid in the prognostic evaluation of breast cancer. The expression of lectin-binding sites was studied using the avidin-biotin complex/ immunoperoxidase technique, and analyzed in relation to the different clinical, pathological, and biological parameters of the primary disease, i.e. the presence or absence of nodal metastases, pre- or post-menopausal age, size of the tumor, mitotic activity index, morphometric prognostic index, DNA content, S-phase fraction, and steroid receptor status. The results show significant differences in PNA binding patterns among malignant epithelial breast cells. There was no expression of PNA-binding sites in 14 out of 157 tumors, while 64 showed mostly apical (membrane) staining and 124 non-apical (membrane and/or cytoplasmic) staining. Apical staining was mostly observed in patients without lymph node metastasis, with positive steroid receptor status, and those who were postmenopausal diagnosis; non-apical staining was mostly observed in lymph-node-positive premenopausal patients negative for steroid receptors and with aneuploid tumor cells. Our results indicate that, in malignant breast cells, there is an alteration of cell-surface glycoconjugates, shown by heterogeneity within a histopathologically defined group, which is related to different properties of tumor cells. The apical PNA binding pattern indicates a better differentiation of tumor cells while non-apical PNA binding suggests a higher metastatic potential. Specific PNA lectin binding patterns should be considered as a further reliable prognostic factor in breast cancer.

A new "anonymous" type of haemangioendothelioma.

BACKGROUND: Our report may contribute to a better understanding of the different possible presentations of endothelial tumors. METHODS AND RESULTS: We report a new type of haemangioendotheliomatous tumor of uncertain aggressiveness arisen in a benign haemangioma of the scalp and represented by a proliferation of small- and medium-sized arborescent vessels whose walls were totally replaced by endothelial-like, atypical cells. CONCLUSIONS: The features of our case do not fit those of the many types previously reported in the literature. As regards the name, we prefer to consider this new variety as an anonymous type in order to avoid further confusion on a topic deserving a drastic review.

Hemangiopericytoma of the nose: a case for both internal and external localization.

BACKGROUND: Nasal and paranasal cavities are sites of predilection for hemangiopericytoma (HPC), a rare vascular tumor described for the first time in 1942. Because of their characteristics, i.e. clinical presentation, age distribution and biological behavior, nasal HPCs are frequently reported as "hemangioperycitoma-like intranasal tumors" although such a distinction from other HPCs is debated. PATIENTS, METHODS AND RESULTS: out of nearly 64,000 autopsies and 336,000 surgical and endoscopic biopsies performed at the University of Trieste over 30 years, only three cases of HPC have been found, two of them affecting the external nose; for both there was a good agreement between histology--supported by immunohistochemistry and flow cytometry--and biological behavior. CONCLUSIONS: On the basis of the sparse literature on skin HPC and of our two cases, we suggest that not only the internal, but also the external nose should be considered a predilection site for HPC.

Gastric epithelium proliferation in early Hp+ and Hp- gastritis: a flow cytometry study.

BACKGROUND: Helicobacter pylori increases cellular turn-over causing hyperproliferation and possible assumption of neoplastic characteristics by the gastric epithelial cells. To verify whether patients at risk of cancer can be identified at the very first stages of gastric disease, we studied a sample of patients affected by early Hp+ and Hp- gastritis by flow cytometry and assessed the methods commonly adopted to study gastric cell proliferation. METHODS: 48 fresh biopsies taken from the gastric antrum and body of 24 patients who had undergone endoscopy for dyspepsia, and 48 paraffin-embedded antrum and body biopsies taken from the files of our Department were studied by flow-cytometry. The following markers and parameters were considered: S-phase, proliferation index, PCNA and ploidy. RESULTS: No correlation was found between Hp+ or Hp- gastritis and gastric cell proliferation and no cases of aneuploidy were observed. Gastric proliferation was found to vary depending on the methods, markers and type of biopsy employed. Furthermore, proliferation expressed by PCNA was significantly different in antrum and body. CONCLUSIONS: The commonly studied proliferation markers do not allow the early detection of patients at risk of gastric cancer by flow cytometry. Proliferation differences between body and antrum must be taken into account in the investigation of gastric diseases.

Epidermal growth factor-receptor expression correlates with tumor cell proliferation and prognosis in gastric cancer.

Currently, the most accurate prognostic indicator in gastric cancer is stage. Studies on proliferating cell nuclear antigen (PCNA) in gastric cancer have demonstrated that the PCNA labeling index correlates with depth of invasion, organ metastasis, vascular invasion, and tumor stage, suggesting that this marker may be a valuable prognostic factor. Epidermal growth factor (EGF) promotes the growth of cells of both ectodermal and mesodermal origin, and plays an important role in cellular proliferation and differentiation. Furthermore, there has been increasing evidence that growth factors and their receptors are involved in carcinogenesis. The aim of this study was to investigate the correlation between expression of the EGF-receptor (EGF-r) and proliferative activity (PCNA labeling index) in gastric cancer by immunohistochemical analysis. Our preliminary results on 56 gastric cancers indicate that the PCNA labeling index correlates with EGF-r immunoreactivity. Furthermore, survival was significantly lower in patients with EGF-r positive tumors and a high PCNA labeling index. These in situ observations suggest that EGF-r may play an important role in the growth regulation of human gastric carcinomas.

Comparison of microvessel density with tumor associated macrophages in invasive breast carcinoma.

Neovascularization, the growth and formation of capillary blood vessels, is an essential component of solid tumor growth and a critical step in metastasis. Tumor associated macrophages (TAMs) have several functions related to tumor biology including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define the TAM and microvessel density (MD) in human invasive breast carcinoma NOS and to correlate their values with lymph node status, tumor size, tumor grade and mitotic activity index (MAI), and, finally, to determine whether MD is connected with TAMs. A total number of 57 invasive breast carcinomas NOS were processed for immunohistochemical analysis using mAb to F-VIII to visualize endothelial cells and mAb to CD68 antigens for macrophages. Statistical analysis showed only a positive correlation between TAMs and MAI (p = 0.004). These results support the notion that intensity of tumor angiogenesis does not provide additional prognostic significance, while TAMs may play a positive role in breast cancer micro system since they regulate tumor proliferation.

Prognostic significance of T-cell infiltrates, expression of beta 2-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.

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.

The lectin-binding sites for peanut agglutinin in invasive breast ductal carcinomas and their metastasis.

Peanut agglutinin (PNA) lectin-binding site patterns in primary invasive breast ductal not otherwise specified (NOS) carcinomas are related to aggressiveness of the tumor. The present study was designed to compare the expression of PNA-binding sites in the primary tumor and in local lymph node metastases. The expression of lectin-binding sites was studied using the avidin-biotin complex/immunoperoxidase technique and analyzed in relation to age of the patient and size of the breast cancer. Breast cancers and their metastases showed negativity or positivity, the latter being divided into "apical" and "non-apical" (i.e. membrane and/or cytoplasmic) depending on the main localization of staining in tumor cells. No correlation was found between primary tumors and metastases as regards PNA-binding patterns, which confirms the opinion that advanced primary tumors are polyclonal and that selected subclones of malignant cells give rise to metastases. Furthermore, the fact that primary tumors with PNA non-apical expression, a feature related to aggressiveness and poor differentiation, may have lymph node metastases with apical expression, suggests that this pattern, although no longer evident in the primary tumor, is involved in the process of cell metastasis.

Influence of tumor thickness and vascularity on survival in cutaneous melanoma.

Eighty-eight cases of cutaneous melanoma (CM) were analyzed in order to elucidate the relationship between thickness, angiogenesis, and prognosis. The thickness of the tumor was measured according to the Breslow method, and the microvessels were identified by an immunohistochemical study using anti-factor VIII monoclonal antibody on specimens from 40 patients with superficial spreading melanoma (SSM), and 48 with nodular type (NM). Microvessels were counted in the area of highest density. The overall survival and disease-free period were analyzed retrospectively. The proportion of patients with thicker CMs (> 1.5 mm) increased with age in both sexes. Mean vascular count was statistically significant different only between thinner and thicker tumors in the SSM group (P < 0.05). Prognosis was correlated with the thickness of CM (P = 0.0002), mean vascular count alone (P = 0.004), mean vascular count in association with CM thickness less than 1.5 mm (P = 0.0005), and with mean vascular count in NM (P = 0.02). These findings suggest that increasing microvessel density indicates a worsening prognosis.

CD30-positive cutaneous anaplastic large cell lymphoma with ichthyosis acquisita.

Anaplastic large cell lymphoma (ALCL) is a high-grade non-Hodgkin's lymphoma recognized by the expression of the CD30 marker and by its morphology. We report an unusual case of ALCL in a 42-year-old woman. For ten years the patient only had pruritic erythematous skin lesions, then a plaque with nodules and ulcers on the right thigh and leg developed, followed by ichthyosis acquisita. The development of ALCL in women and the association of ichthyosis acquisita with this type of lymphoma are uncommon.

EGFR expression is linked to osteopontin and Nf-κB signaling in clear cell renal cell carcinoma.

AIM AND BACKGROUND: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in many important aspects of cell biology that are related to tumorigenesis. There are opposite evidences of the role of EGFR in renal cancer and the outcome of EGFR-targeted therapies, suggesting the complexity of EGFR signaling pathways. In vitro, osteopontin (OPN) and nuclear factor kappa B (NF-κB) are thought to be involved in specific ligand-independent EGFR activation that could have a role in resistance to EGFR mAb therapy. Aim of this study was to analyze the relationship between EGFR and OPN at the protein and mRNA level, as well as their relation to NF-κB in clear cell renal cell carcinoma (CCRCC). MATERIALS AND METHODS: Expression of EGFR, OPN, and p65 NF-κB protein was analyzed using immunohistochemistry and compared mutually in 88 CCRCC samples. Expression of EGFR and OPN mRNAs was analyzed using quantitative Real-time PCR in 22 CCRCC samples and compared mutually and with NF-κB protein expression. RESULTS: Epidermal growth factor receptor mRNA level was higher in CCRCC samples in comparison with normal renal tissue (p = 0.012) and was associated with high OPN mRNA level, and with NF-κB activation (p < 0.001 and p = 0.045, respectively). Immunohistochemical staining showed the inverse association; high EGFR protein expression was related with low OPN and NF-κB protein expression (p < 0.001 and p = 0.047, respectively). CONCLUSION: Epidermal growth factor receptor gene is upregulated in CRCC and associated with OPN gene expression and NF-kB signaling. The inverse relation between OPN and EGFR at the protein level could probably reflect dynamic changes that EGFR undergoes following activation.