Andrzej Kulig

Polymorphisms of the promoter regions of matrix metalloproteinases genes MMP-1 and MMP-9 in breast cancer

SummaryPurposeMatrix metalloproteinases play a crucial role in the cancer invasion and metastasis, angiogenesis and tumorigenicity. A single guanine insertion – the 1G/2G polymorphism in the promoter of the matrix metalloproteinase 1 (MMP-1) gene creates a binding site for the transcription factor AP-1 and thus may affect the transcription level of MMP-1. The C→T substitution at the polymorphic site of the MMP-9 gene promoter results in a higher transcription activity of the T-allelic promoter trough the loss of binding site for a repressor protein. The aim of this work was to investigate the influence of 1G/2G and C→T polymorphisms on the MMP-1 and MMP-9 level and therefore on the occurrence and progression of breast cancer.Experimental designWe investigated the distribution of genotypes and frequency of alleles of the 1G/2G and C→T polymorphisms for 270 patients with breast cancer and 300 healthy women served as control. The genotypes were determined by RFLP-PCR. Additionally, we estimated the level of MMP-1 and MMP-9 antigens in tumor samples and normal breast tissue using ELISA.ResultsThe levels of MMP-1 in tumor samples of node positive patients ware significantly higher than in samples of node negative patients (p<0.05). Increased level of MMP-9 correlates with Bloom-Richardson grading III (p<0.05), increased tumor size (p<0.05) and absence of estrogen and progesterone receptors (p<0.01). Additionally, both MMP-1 and MMP-9 levels were higher in tumor than in the normal breast tissue. We showed the higher risk of metastasis development in lymph node for the 2G/2G genotype (OR=2.14; CI 95% 1.24;3.69) and the 2G allele carriers (OR=1.68; CI 95% 1.19;2.39). We found correlation between the T allele (OR=2.61; CI 95% 1.33;4.87), 2G (OR=2.58; CI 95% 1.35;4.91) and malignance.ConclusionThe results suggest that MMP-1 is responsible for the local invasion and MMP-9 is associated with the malignance and the growth of the tumor. We suggest that the 2G allele of the 1G/2G MMP-1 gene polymorphism may be associated with the lymph node metastasis in patients with breast cancer and therefore it can be considered as a progression marker in this disease.

Antigen levels of the urokinase-type plasminogen activator and its gene polymorphisms in colorectal cancer.

We analysed the distribution of genotypes of two polymorphisms in the urokinase-type plasminogen activator (uPA) gene: C-->T substitution in exon 6 and T-->C substitution in intron 7 in 52 subjects with colorectal cancer. Genotypes were determined in tumour tissue and distant mucosa samples by allele-specific polymerase chain reaction. The antigen levels of uPA in cancer tissue were higher than in distant mucosa as measured by enzyme-linked immunosorbent assay. The level of uPA antigens in cancer samples with the C/C genotype of C-->T polymorphism in exon 6 was higher than in samples with C/T and T/T genotypes. No differences in the level of uPA antigens between the alleles of the intron 7 T-->C polymorphism were found. As uPA can be involved in cancer invasion and metastasis, C/C genotype in exon 6 of uPA gene can be further considered as being related to colorectal cancer progression.

Production of Cytokines During Interaction of Peripheral Blood Mononuclear Cells with Autologous Ovarian Cancer Cells or Benign Ovarian Tumour Cells

Cytokines produced by tumour and immune cells may play a significant role in a modulation of immune cells response against tumour. We investigated an ability of peripheral blood mononuclear cells (PBMC) of patients with early and advanced stages of ovarian cancer and from non‐cancer patients to produce various cytokines in the presence or absence of autologous ovarian cancer (OC) cells or benign ovarian tumour (BOT) cells. Activated PBMC of patients with advanced stage of cancer produced slight amount of interferon γ (IFN‐γ) and what’s more, the production of IFN‐γ was decreased in the presence of OC cells. PBMC of patients with ovarian cancer or benign ovarian tumour generated comparable amounts of interleukin 6 and 10 (IL‐6, IL‐10), and transforming growth factor β1 (TGF‐β1). PBMC of the patients with cancer produced higher amount of tumour necrosis factor α (TNF‐α) than PBMC of non‐cancer patients. We demonstrated here that the reciprocal contact of OC cells from advanced cancer with autologous PBMC altered the direction of produced cytokines and leads to the down‐regulation of IFN‐γ and TNF‐α as well as to up‐regulation of immunosuppressive (IL‐10, TGF‐β1) and pro‐inflammatory (IL‐6) cytokines production.

Clinical significance of k-ras and c-erbB-2 mutations in pancreatic adenocarcinoma and chronic pancreatitis

AbstractBackground: The differentiation of chronic pancreatitis (CP) from pancreatic adenocarcinoma (PA) remains the great challenge for clinicians. The purpose of this study was to compare the prevalence of K-ras and c-erbB-2 mutations in PA and CP in order to evaluate their usefulness in differential diagnosis of those diseases. Methods: The study included 49 patients who underwent Whipple resection or distal pancreatectomy for pancreatic adenocarcinoma (26 subjects) or chronic pancreatitis (23 subjects). DNA from pancreatic tissue was analyzed for K-ras codon 12 and c-erbB-2 mutations with PCR amplifications. Results: The K-ras gene mutation has been shown in 20 (76.9%) PA cases and in 8 (34.8%) CP cases (p<0.01). Prevalence of c-erbB-2 amplification in patients with PA was 17 (65.3%), which was not different from CP, 16 (56.5%) (p=0.58). There was a significant correlation between K-ras mutation and lymph node metastases (p=0.025) as well as between K-ras mutation and G3 tumor differentiation (p=0.037). Overall median survival in patients with PA was 9.5 mo. There was no relationship between presence of K-ras (p=0.58) or c-erbB-2 (p=0.17) mutation and survival time in PA patients. Conclusion: Those results may indicate that both K-ras and c-erbB-2 play a role in pancreatic carcinogenesis, however only K-ras may provide an additional tool in differential diagnosis of CP and PC.

Analysis of APC, α-, β-catenins, and N-cadherin protein expression in aggressive fibromatosis (desmoid tumor)☆

The aims of this study were to analyze the cadherin/catenin adhesion complex in cells from abdominal and extra-abdominal aggressive fibromatosis tumors, and to estimate the correlation between the expression of the tested proteins and the clinical data of the desmoid patients. Immunohistochemistry was used to examine the expression of the cadherin/catenin adhesion complex: APC protein, alpha-, beta-catenin, and N-cadherin in archival material derived from 15 cases of extra-abdominal desmoid tumor (E-AD) and 20 cases of abdominal (AD) desmoid tumor. The tested proteins demonstrated cytoplasmic (c) staining. Furthermore, nuclear (n) or cytoplasmic and nuclear (c+n) staining was observed for beta-catenin. The mean values of the percentage of positive cells for the tested proteins between E-AD vs. AD did not demonstrate any statistically significant difference except for alpha-catenin. In the E-AD group, in both cases of recurrent tumors, no alpha-catenin expression was observed but the expression of this protein was detected in primary tumors. In the groups investigated, no statistically significant correlation was found between alpha-catenin, beta-catenin (c), (n) and (c+n) expression, and tumor size (p>0.1). The results regarding beta-catenin expression obtained in our study confirm the previous findings that nuclear accumulation of this protein plays a crucial role in the pathogenesis of aggressive fibromatosis.

The role of tumor cells in the modification of T lymphocytes activity--the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I.

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.

Different distribution of c-kit positive interstitial cells of Cajal-like in children’s urinary bladders

We describe the presence of c-kit positive interstitial cells of Cajal-like (ICCs-like) in the walls of the urinary bladders of children. An immunohistochemical study of specimens, obtained at autopsy from either the trigonum (Group A) or the corpus (Group B), was performed using antibodies against c-kit (CD 117). Histological morphometry of the immunoexpression of c-kit positive ICCs-like was performed by means of image analysis system. The c-kit positive ICCs-like were identified by their morphology and counted in the vesical muscle layer in ten adjacent high power fields, each of 0.0479 mm(2). The areas of the epithelial and subepithelial layers containing c-kit positive mast cells (rounded body with no dendritic processes) were neglected. The results were expressed as the number of ICCs-like cells per mm(2). Differences between groups were tested using unpaired Students t-test preceded by evaluation of normality and Levenes test. Results were considered statistically significant if p 〈 0.05. In Group A, the mean number of ICCs-like cells was statistically significantly higher (41.5 cells/mm(2)) than in Group B (30.4 cells/mm(2)), p 〈 0.05. ICCs-like cells were found within the smooth muscle layer of the urinary bladder. There was a different distribution of these cells in particular parts of the bladder, which was probably due to the different roles of the trigonum and the corpus in the bladders of children.

Dinucleotide repeat polymorphisms of RAD51, BRCA1, BRCA2 gene regions in breast cancer.

Recent studies suggest that genetic polymorphisms of the DNA repair genes have been implicated in breast cancer risk. BRCA1 and BRCA2, two breast cancer susceptibility genes, are essential to maintain chromosomal integrity. This is mediated via regulation of RAD51 during homologous recombination. Dinucleotide polymorphism repeats in the 15q14–21, 17q21 and 13q12–13 regions, where the RAD51, BRCA1 and BRCA2 genes are located, respectively, have been evaluated. The polymorphism was determined using the following microsatellite markers: D15S118, D15S214, D15S1006, D17S855, D17S1323, D13S260 and D13S290. Genotypes containing the (CA)17 or (CA)19 alleles in the RAD51 region were found to be associated with a decreased breast cancer risk. Genotype containing the (CA)17 allele in the 13q12–13 region was found to be associated with an increased breast cancer risk. The results indicate that dinucleotide CA repeat polymorphism at RAD51 and BRCA2 gene regions might be associated with genetic susceptibility to breast cancer.

Interdisciplinary treatment of the patient with adenoid cystic carcinoma of the Bartholin's gland resulting in 15 years' survival: a case report and review of literature

Carcinoma of the Bartholins gland is very rare, comprises below 2% of Bartholins gland lesions and adenoid cystic carcinoma (ADC) is one of the most uncommon variants and accounts for 10-15% of Bartholins gland malignancies. There is no consensus on treatment of ADC of the Bartholins gland: reported cases were treated with local excision or vulvectomy with or without lymphadenectomy followed or not by radiotherapy. The survival of patients varies significantly, so we present a case of interdisciplinary treatment of ADC resulting in 15 years’ survival. The patient was initially treated with local excision, but the margins were not clear. Then vulvectomy, inguinal lymphadenectomy and adjuvant brachytherapy were performed resulting in 7 years free of the disease. Relapses were excised by abdominoperineal amputation of the rectum and distal part of the vagina with sigmoideostomy, excisions of local recurrences in vagina and metastasectomy of isolated lung metastases. The patient died manifesting multiple lung metastases 15 years after the initial diagnosis. Based on our experience and world literature, in cases of adenoid cystic carcinoma of the Bartholins gland, vulvectomy with or without lymphadenectomy should be considered as a treatment of choice and in patients with positive margin, surgery should be extended by adjuvant radiotherapy.

Genetic instability in the RAD51 and BRCA1 regions in breast cancer

Breast cancer is the most prevalent cancer type in women. Accumulating evidence indicates that the fidelity of double-strand break repair in response to DNA damage is an important step in mammary neoplasias. The RAD51 and BRCA1 proteins are involved in the repair of double-strand DNA breaks by homologous recombination. In this study, we evaluated loss of heterozygosity (LOH) in the RAD51 and BRCA1 regions, and their association with breast cancer. The polymorphic markers D15S118, D15S214 and D15S1006 were the focus for RAD51, and D17S855 and D17S1323 for BRCA1. Genomic deletion detected by allelic loss varied according to the regions tested, and ranged from 29 to 46% of informative cases for the RAD51 region and from 38 to 42% of informative cases for the BRCA1 region. 25% of breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 31% of breast cancer cases manifested LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA1 regions. LOH in the RAD51 region, similarly as in the BRCA1 region, appeared to correlate with steroid receptor status. The obtained results indicate that alteration in the RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and BRCA1 and thus enhance the risk of breast cancer development.