Agnes Bankfalvi

Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast.

Aims:

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN‐related alterations in HNSCC, the role of tumor‐infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN‐related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil‐to‐lymphocyte ratio and serum concentrations of CXCL8 (interleukin‐8), CCL4 (MIP‐1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC‐conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor‐derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host‐mediated changes in the tumor microenvironment.

Gains and losses of adhesion molecules (CD44, E‐cadherin, and β‐catenin) during oral carcinogenesis and tumour progression

The aim of this study was to define whether or not the impaired expression of CD44, E‐cadherin (E‐cad), and β‐catenin (β‐cat) correlates with the clinical evolution and prognosis of oral cancer. Ninety‐three primary oral squamous cell carcinomas (OSCCs) with tumour‐adjacent normal and/or dysplastic mucosa, 30 associated metastases, and 12 recurrences were immunostained for CD44s, ‐v3, ‐v4, ‐v5, ‐v6, ‐v7, ‐v9, E‐cad, and β‐cat. In non‐neoplastic epithelium, all molecules investigated were constitutively expressed in the basal layers. In the majority of dysplasias, immunoreactivity for all adhesion molecules was increased, but there was restricted loss for CD44s, E‐cad, and β‐cat in a few cases. In carcinomas, a striking accumulation of CD44s, v3, v4, v9 and a loss of E‐cad/β‐cat were observed at the invasive tumour front. In metastases and recurrences, besides a loss of CD44s, v4, v7, and E‐cad, a significant increase of v9 was recorded, whereas CD44v5 and v6 remained unchanged. Clinically, reduced expression of CD44v3, E‐cad, and changes of CD44v9 phenotype within the primary tumours correlated significantly with poor prognosis; decreased β‐cat expression was a predictive marker for nodal metastases. These findings indicate that there is some perturbed expression of adhesion molecules during the stepwise course of oral carcinogenesis and tumour progression. Distinct phenotypic alterations project poor prognosis, while others predict metastasis. Some of these restricted molecular changes may serve as potential targets for future antibody‐based tumour therapy. Copyright

Telomerase as a prognostic marker in breast cancer: high‐throughput tissue microarray analysis of hTERT and hTR

Telomerase activity (TA) has been shown to correlate with poor clinical outcome in various tumour entities, indicating that tumours expressing this enzyme may be more aggressive and that TA may be a useful prognostic marker. For breast cancer, however, TA is a controversial prognostic marker; whereas some studies suggest an association between TA and disease outcome, others do not find this association. This study used tissue microarrays (breast carcinoma prognosis arrays) containing 611 samples (each 0.6 mm in diameter) from the tumour centre of paraffin‐embedded breast carcinomas to analyse the catalytic subunit of telomerase, human telomerase reverse‐transcriptase (hTERT), and the internal RNA component (hTR), which are the core components of the telomerase holoenzyme complex. hTERT protein expression was obtained by immunohistochemistry (human anti‐telomerase antibody Ab‐2, Calbiochem), and hTR RNA was measured by radioactive in situ hybridization. hTERT and hTR expression were determined semi‐quantitatively and graded (scores 1–4). Clinical data, such as histological subtype, pT stage, tumour diameter, pN stage, BRE grade, tumour‐specific survival (in months), patients age and others, were available for statistical analysis. A statistically significant correlation was found between tumour‐specific survival (overall survival) and hTERT expression (p < 0.0001) or hTR expression (p = 0.00110). Tumours with higher scores (scores 3, 4) for hTR and/or hTERT were associated with a worse prognosis. In multivariate analysis, hTERT expression was an independent prognostic factor. Previous studies, focusing on analysis of TA in smaller numbers of fresh‐frozen breast carcinomas by the TRAP assay, gave controversial results with respect to TA as a prognostic marker. Using tissue microarrays from 611 breast carcinomas, this study has demonstrated that increased expression levels of the telomerase core components, hTERT and hTR, are associated with lower overall survival. These findings suggest that TA should be included in future validation studies as a prognostic marker in breast cancer. Copyright

Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor‐derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)‐dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C‐C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor‐derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor‐derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high‐grade osteosarcomas (17.0) was significantly higher than in low‐ or intermediate‐grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21‐p31 and 1q21‐q24, and the most frequent losses were 10q, 5q and 13q. High‐level gains were observed on 8q23‐q24, 17p13 and 1q21‐q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high‐level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event‐free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21‐22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma.

Matrix metalloproteinase-7 as a marker of metastasis and predictor of poor survival in bladder cancer

Matrix metalloproteinases (MMPs) play an important role in tumor progression and metastasis. Here, we investigated the prognostic relevance of MMP‐7 in urinary bladder cancer. MMP‐7 gene expression was measured in tissue samples of 101 patients using quantitative real‐time PCR. Circulating MMP‐7 serum levels of 98 individuals (79 patients and 19 controls) were analyzed by enzyme‐linked immunosorbent assay. The results were compared with the clinical follow‐up data, performing Kaplan–Meier log‐rank test as well as univariate and multivariate Cox analysis. In representative cases, immunohistochemical analysis for MMP‐7 was performed. We detected significantly elevated MMP‐7 levels both in tissue and serum samples of patients with metastatic disease (P = 0.001 and P = 0.002). Multivariate analysis revealed that high MMP‐7 tissue expression and serum concentration are stage‐ and grade‐independent predictors of both metastasis‐free (hazard ratio [HR] = 3.80, 95% confidence interval [CI], 1.29–11.23, P = 0.016, and HR = 2.53, 95% CI, 1.01–6.37, P = 0.048) and disease‐specific survival (HR = 1.89, 95% CI, 1.00–3.55, P = 0.050 and HR = 1.95, 95% CI, 1.03–3.71, P = 0.041). Based on these findings, we conclude that MMP‐7 is a promising marker to detect present and to predict future metastasis. Serum MMP‐7 analysis provides information about the risk of metastasis before surgery which could help to optimize therapeutic procedures. Furthermore, high MMP‐7 tissue and/or serum levels could identify patients most likely to benefit from early adjuvant chemotherapy.

Gains and losses of CD44 expression during breast carcinogenesis and tumour progression

This study was performed to investigate whether the CD44 immunophenotype of breast lesions correlates with the clinical evolution and prognosis of breast cancer.

Secretory carcinoma of the breast: a distinct variant of invasive ductal carcinoma assessed by comparative genomic hybridization and immunohistochemistry

Secretory carcinomas (SCA) are distinguished from infiltrating ductal carcinomas (IDC) of the breast by their characteristic histomorphology and more favorable prognosis and by the expression of a chimeric tyrosine kinase that is encoded by the ETV6-NTRK3 fusion gene. On this basis, we evaluated 13 SCAs (12 of them with ETV6-NTRK3 gene fusion) by molecular and immunohistochemical (IHC) methods. DNA was obtained from 8 of 13 microdissected SCAs and was analyzed for genetic alterations (GA) by comparative genomic hybridization (CGH). IHC staining was performed for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67 (MIB1) in all 13 cases. Molecular and immunohistochemical results in SCAs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. An average of 2.0 GAs (range: 0 to 6) were detected, including recurrent gains of chromosome 8q (37.5%) and 1q (25%) and losses of 22q (25%). Four of 13 (31%) SCAs were positive for ER, and 2 were positive for PR. The mean MIB1-labeling index was 11.4% (range: <1 to 34%). Her-2/neu protein overexpression was detected in 2 cases, including 1 with strong (score 3+) and 1 with weak HER2/neu expression (score 2+). Fluorescence in situ hybridization analysis of the latter case showed no evidence of HER-2/neu-gene amplification. Compared with previous findings in IDCs, SCAs are characterized by a relatively low number of GAs, a low proliferative rate, infrequent HER2/neu protein overexpression, decreased steroid hormone receptor expression, and expression of ETV6-NTRK3 fusion gene. These results support the hypothesis that SCAs have immunohistochemical and genetic features that distinguish them from IDCs of the usual type.

Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer?

To determine whether it is possible to stratify patients with superficial bladder cancer into low‐ and high‐risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow‐up testing.