Association of TLR2, TLR3, TLR4 and CD14 genes polymorphisms with oral cancer risk and survival.

OBJECTIVES: The aim of this study is to investigate association between polymorphisms in TLR2, TLR3, TLR4 and CD14 genes or their haplotypes with oral squamous cell carcinomas (OSCC) risk and survival. METHODS: The study was conducted on 93 OSCC patients and 104 cancer-free controls. Polymorphisms were genotyped by real-time PCR or PCR-RFLP method. RESULTS: Significant increase in oral cancer risk was observed in individuals with mutated genotype of TLR3 rs3775291 polymorphism (OR = 1.096, P = 0.036) compared to wild-type. The heterozygous and mutated genotype of TLR3 rs5743312 polymorphism had worse survival in group of patients with stage III tumours (P = 0.043). Multivariate Cox regression analysis revealed that TLR3 rs5743312 polymorphism could be considered as prognostic marker in advanced III stage OSCC (HR = 2.456, P = 0.007), but not independently of nodal status. TLR3 rs3775291 and rs5743312 polymorphisms were in strong linkage disequilibrium. Haplotype TG was associated with worse prognosis in OSCC patients in comparison with common CG haplotype (HR = 1.717, P = 0.042). Interaction among polymorphisms in TLR2, TLR3 and CD14 genes was observed (P = 0.010). CONCLUSIONS: TLR3 rs5743312 polymorphism could be considered as potential predictor of worse overall survival in advanced oral cancer, but not independently of nodal status. Haplotypes in TLR3 gene might be associated with poor prognosis in OSCC patients.

CHEK2 1100delC and Del5395bp mutations in BRCA-negative individuals from Serbian hereditary breast and ovarian cancer families.

PURPOSE: Hereditary breast/ovarian cancer (HBOC) occurs in families with several members affected. Most HBOC is caused by mutations in high penetrance BRCA genes accounting for about 5% of all breast cancers. There is a large number of genes with moderate or low penetrance, contributing to non-BRCA aggregation of breast and ovarian cancers. In the present study, we evaluated the presence and frequency of 1100delC and Del5395 mutations in CHEK2 gene in Serbian BRCA-negative HBOC cases. METHODS: We analyzed 57 BRCA-negative subjects from high risk breast/ovarian cancer families from Serbia for CHEK2 1100delC and Del5395 mutations. We used two multiplex allele specific PCR in order to detect them. All suspected positive cases were compared with controls and confirmed by DNA sequencing. RESULTS: 1100delC was not detected in the tested group. However, we detected one Del5395 mutation in a female diagnosed with breast cancer at the age of 32 and with apparent family history of breast cancer (her mother and sister were diagnosed with breast cancer at 42 and 39 years of age, respectively). The frequency of Del5395 mutation in our tested group was 1.7% (1/57). CONCLUSIONS: 1100delC variant in CHEK2 gene was not present in the tested subjects from HBOC families in Serbia. However, the finding of Del5395 mutation does not allow us to discard a possible involvement of this gene in breast cancer susceptibility in Serbian population. It would be of great interest to assess the distribution of this large deletion in other countries from the Balkan region in order to assess its geographical distribution and possible founder effect.

Therapeutic cancer vaccines in cervical cancer: phase I study of Lovaxin-C.

Producing effective therapeutic vaccines has proved much more difficult and challenging than developing cancer preventive vaccines. Despite huge research in the area of cancer immunology, FDA/EMEA have not approved any type of cancer treatment vaccine so far. More than 99% of cervical cancers have detectable amounts of human papillomavirus (HPV) DNA. Integration of high-risk HPV into the host cell genome is followed by continual expression of HPV E6 and E7 oncoproteins, making them excellent targets for developing vaccines which could be used in high grade precancerous (CIN) lesions or invasive cancer or in the prevention of cancer recurrence. Therapeutic cervical cancer vaccines have been extensively studied. Strategies used were vaccination with HPV peptides or proteins, alone or in pulsed dendritic cells, DNA vaccines, virus-like particles or viral and bacterial vectors. Lovaxin-C is a recombinant live-attenuated Listeria monocytogenes (Lm) that secretes the antigen HPV-16 E7 fused to a non-hemolytic listeriolysin O protein. In a phase I study Lovaxin-C was administered to advanced cervical cancer patients refractory to existing therapies. The dose-limiting toxicity was hypotension and flue-like syndrome. There were no serious adverse events. Specific T-cell response was detected as well as clinical response to Lovaxin-C. Several other therapeutic HPV vaccines are in clinical development and in most of the studies specific immunological and clinical responses were seen. Efficacious therapeutic vaccine for the treatment of cervical cancer should be expected in the near future.

siRNA and miRNA for the treatment of cancer.

In the process of RNA interference (RNAi), small RNAs pair with complementary messenger RNAs preventing their expression. The discovery of RNAi has revolutionized our understanding of gene regulation. Since cancer is a disease of altered genes, RNAi may have tremendous potential as a therapeutic strategy by downregulating altered genes. Just one decade after its discovery, this process is already being used in clinical trials and new technical achievements in delivering small RNAs to the cells are constantly improving the efficiency of this specific cancer treatment.

Towards targeted epigenetic therapy of cancer.

Increasing number of publications in the last 10 years implicated that cancer development depends, except genetic alterations, also on inheritable gene expression patterns that are not bound to DNA sequence alterations. These epigenetic mechanisms manifest mostly through changes in chromatin packing and in localized gene promoter changes that influence the transcription of the genes involved in carcinogenesis. These changes are mitoticaly inheritable and potentially reversible, providing large possibilities of epigenetic therapy of cancer. So far this therapy lacks specificity of targeting certain genes. Instead, epigenetic therapy attempts either to reactivate or to silence genes that are important for the cancer progress. Epigenetic therapy of cancer is based mostly on the usage of inhibitors of DNA methyltransferases (DNMTs), histone deacetylase (HDAC) inhibitors and anti-micro-RNA therapy. Developments that involve integration of the latest technological advances, such as whole genome microarray expression profiling, help identify mechanisms of action of epigenetic drugs, leading to development of second generation of epi-drugs which would have greater specificity and efficacy. The obtained results are promising, leaving great possibilities for improvement of cancer therapy.

TP53 mutations in breast cancer: association with ductal histology and early relapse of disease.

PURPOSE: This study aimed to investigate the incidence of core domain TP53 mutations in Serbian breast cancer patients in view of their possible correlation with prognostic parameters, tumor characteristics and clinical disease course. METHODS: 145 breast cancer patients were included. Data on clinical disease course were available for 100 patients including 30 node-negative and 70 node-positive patients. After surgery, node-positive patients underwent adjuvant chemotherapy, mostly CMF. TP53 mutations were detected by PCR-SSCP. RESULTS: 31 mutations were found in 27/145 patients including 4/59 node-negative patients and 23/83 node-positive patients (4 double mutations). 26/31 TP53 mutations were found in patients with invasive ductal carcinoma and only 2 in patients with invasive lobular carcinoma. The presence of TP53 mutations was correlated with clinical disease course in premenopausal node-positive patients (n=70). 11/20 patients with TP53 mutations relapsed. Within the first 24 months of follow-up, significantly shorter disease-free intervals were observed in TP53-mutated patients. CONCLUSIONS: TP53 mutations correlated only with nodal status and ductal histology. The significance of the predominant distribution of TP53 mutations in tumors with a ductal histology for the aggressive behavior of these tumors has yet to be proved, since the favorable biological features of tumors with a lobular histology do not result in a better prognosis. Early relapse in mutated-TP53 carriers may support data on its predictive value with respect to adjuvant CMF.

Prognostic significance of TP53 mutations in oral squamous cell carcinoma with human papilloma virus infection.

PURPOSE: The aim of this study was to analyze the prognostic impact of mutated TP53 in patients with oral squamous cell carcinoma (OSCC) whose tumors were infected with human papillomavirus (HPV). METHODS: Thirty-two HPV-positive OSCC patients were included. Most of them were clinically classified as stage III (n=29). All patients underwent postoperative radiotherapy (follow-up from 12 to 60 months, median 32). There were 21 relapses. DNA was isolated by phenol extraction from tumor tissue. HPV DNA (type 16, 18, 31, 33) was detected in genomic DNA of the tumors by the PCR-PAGE method. TP53 mutations (exons 4-8) were detected by the PCR-SSCP method. RESULTS: A statistically significant difference in the number of relapses in HPV-infected (13/21) versus HPV-infected and TP53-mutated (8/8) patients was observed. Patients with both TP53 mutation and HPV infection had a significantly shorter disease-free interval than patients with HPV infection only (median 6 versus 31 months, respectively). CONCLUSIONS: TP53 mutations are associated with a higher risk of relapse and contribute to an even worse prognosis of patients with OSCC when the tumors are HPV infected. The shorter disease-free interval in patients with TP53 mutations indicates that the response to postoperative radiotherapy may be influenced by TP53 status. The presence of both HPV infection and TP53 mutations may define a particular group of tumors with a more aggressive phenotype in advanced OSCC.

Analysis of T-cell clonality pattern in tumor samples of breast cancer patients.

PURPOSE AND METHODS: A large body of experimental evidence has confirmed that different tumors, including breast carcinomas, can stimulate specific T-cell-mediated immune responses. In this study we have analyzed patterns of T-cell clonality in tumor samples of 54 breast cancer patients classified as lymph node negative, N0 (n=16), or lymph node positive, N+ (n=38). The clonality of T-cells was analyzed by the PCR-PAGE method. RESULTS: Monoclonal/oligoclonal (M/O) T-cell populations were found in 15 breast cancer patients, nine N+ and six N0. In all analyzed groups (N+ + N0, N+, N0) the incidence of relapse was not significantly different between patients with M/O and patients with polyclonal T-cells. Comparison of disease-free interval (DFI) between patients divided according to the presence of TCRgamma monoclonality/oligoclonality showed a marginally significant difference only in the group of N+ patients within the first 24 months of follow-up. Patients with a M/O T-cell population had a shorter DFI than patients with a polyclonal T-cell population. This difference was not observed when the complete follow-up period was considered in the same group of patients. Furthermore, there was no significant difference in overall survival (OS) between patients with M/O and patients with polyclonal T-cells. CONCLUSION: Our results imply that tumor infiltrating T-cells are usually polyclonal. The pattern of T-cell clonality does not correlate with the incidence of relapse and the duration of DFI and OS in the analyzed groups of breast cancer patients, excluding N+ patients with M/O T-cells who had a shorter DFI in the first 24 months of follow-up. This observation suggests that polyclonal T-cell populations may provide a broader spectrum of T-cell-mediated antitumor response.

Analysis of T-cell clonality pattern in tumor samples of breast cancer patients.

PURPOSE AND METHODS: A large body of experimental evidence has confirmed that different tumors, including breast carcinomas, can stimulate specific T-cell-mediated immune responses. In this study we have analyzed patterns of T-cell clonality in tumor samples of 54 breast cancer patients classified as lymph node negative, N0 (n=16), or lymph node positive, N+ (n=38). The clonality of T-cells was analyzed by the PCR-PAGE method. RESULTS: Monoclonal/oligoclonal (M/O) T-cell populations were found in 15 breast cancer patients, nine N+ and six N0. In all analyzed groups (N+ + N0, N+, N0) the incidence of relapse was not significantly different between patients with M/O and patients with polyclonal T-cells. Comparison of disease-free interval (DFI) between patients divided according to the presence of TCRg monoclonality/oligoclonality showed a marginally significant difference only in the group of N+ patients within the first 24 months of follow-up. Patients with a M/O T-cell population had a shorter DFI than patients with a polyclonal T-cell population. This difference was not observed when the complete follow-up period was considered in the same group of patients. Furthermore, there was no significant difference in overall survival (OS) between patients with M/O and patients with polyclonal T-cells. CONCLUSION: Our results imply that tumor infiltrating T-cells are usually polyclonal. The pattern of T-cell clonality does not correlate with the incidence of relapse and the duration of DFI and OS in the analyzed groups of breast cancer patients, excluding N+ patients with M/O T-cells who had a shorter DFI in the first 24 months of follow-up. This observation suggests that polyclonal T-cell populations may provide a broader spectrum of T-cell-mediated antitumor response. (Int J Biol Markers 2005; 20: 177-83).

The past and the future of cancer genetics.

Carcinogenesis represents a multistep process associated with accumulation of somatic mutations in the classes of genes that regulate cell proliferation, apoptosis as well as DNA repair. Oncogenes, positive regulators of cell proliferation are activated during carcinogenesis. On the contrary, tumor suppressor genes, negative regulators of cell proliferation have to be inactivated. Mutations in genes that function in the maintenance of genomic stability are manifested by increase in the mutation rate in cancer cells that drive tumor progression. In general, on the basis of malignant transformation lies the abrogation of the balance between cell proliferation and cell apoptosis. The genetic mechanisms included in the transformation of normally acting genes comprise a wide spectrum of events, such as gene mutation, gene and chromosome rearrangement and gene amplification. Besides the role of somatic gene alteration in the development of sporadic cancer, germline mutations are the basis of a substantial number of inherited cancer syndromes. The future decades will be marked with the expansion of data exploiting cancer genetics, epigenetics and genomics into clinical practice. Consequently, translational cancer research should provide the generating of new targeted therapies, since individual molecular profiling of a patient?s tumor should increase efficacy of conventional anticancer therapies such as chemotherapy and radiotherapy.

Progesterone receptor status of breast cancer metastases.

PURPOSE: The usefulness of steroid receptor content in breast cancer metastases for metastatic disease therapy planning was examined in this study. METHODS: Steroid receptors in primary tumors and corresponding metastases in the same breast cancer patients ( n=23) were determined by five-point DCC assay. We carried out an analysis of the therapeutic response and comparison of the progression-free interval of patients treated with endocrine/chemo-endocrine therapy for metastatic disease according to the positive/negative progesterone receptor status of primary tumors, or of breast cancer metastases. RESULTS AND CONCLUSIONS: It seems that the lack of positive progesterone receptors in metastasis (0/8) and conversion from PR+ primary to PR- metastasis (5/8) may be important in describing the non-responder phenotype. We obtained a similar progression-free interval in patients with progesterone receptor-positive/negative primary tumors, but a longer progression-free interval in the patients with progesterone receptor-positive metastases ( n=9) than with negative ones ( n=14), indicating the possibility of using steroid receptor content from metastases for metastatic disease therapy planning.

PS2 protein in breast carcinomas: cut-off value of estrogen-regulated expression.

This study includes 152 patients with histologically confirmed breast carcinoma. Steroid hormone receptors (SR), estrogen (ER) and progesteron (PR) receptors, and pS2 protein were assayed on the same cytosolic extract in accordance with the recommendation of EORTC. Our results showed menopausal- and histologic grade-related expression of pS2 protein. Unfavorable carcinoma subgroups, in relation to expression of pS2 protein were defined: postmenopausal carcinomas with histologic grade II, and pre-, as well as postmenopausal carcinomas with histologic grade III. There were overlappings of individual pS2 protein values between favorable and unfavorable carcinoma subgroups in relation to the expression of pS2 protein. Otherwise, no overlapping of pS2 protein values was obtained between ER-positive and ER-negative carcinomas within defined unfavorable menopausal - and histologic grade-related expression of pS2 protein subgroups. The highest pS2 protein level observed in ER-negative unfavorable subgroups (15 ng/mg) was considered as the cut-off value which defined estrogen-regulated expression of pS2 protein.

Content of epidermal growth factor receptor in metastatic breast cancer: its role in endocrine sensitivity prediction.

Epidermal growth factor receptor (EGF-R) is known as an indicator of endocrine independence of breast cancer. However, a small proportion of EGF-R expressing tumors was found to respond to endocrine treatments. On the other side, a cut-off point of EGF-R positivity is not yet defined. In the aim to find out whether there exists a cut-off value that sharply discriminate the endocrine sensitive and endocrine insensitive breast cancers, the quantitative EGF-R content was analyzed in a group of 42 female patients with metastatic disease, being routinely treated with chemo-, chemo-endocrine, or endocrine therapy alone. Steroid receptors (SR) and EGF-R were determined by biochemical methods in tissue samples of an unselected group of patients. Patients with metastatic disease, either at diagnosis, or developed after the treatment of operable or locally advanced breast cancer, were included in the present analysis. According to the treatments used, and their therapeutic response, all patients were divided in endocrine sensitive or resistant, and chemo-sensitive or resistant. The SR and EGF-R status and content was analyzed in relation to the sensitivity to both systemic treatments. The EGF-R content was significantly lower in responders to endocrine treatments, compared to non-responders, while there was no difference in EGF-R level, in relation to the sensitivity to chemotherapy. In addition, the EGF-R content was significantly higher in chemo-sensitive tumors, than in endocrine sensitive. On the contrary, ER content was significantly higher in endocrine sensitive, than in endocrine resistant, and in chemo sensitive patients, as well. Similar differences were found in PR content, but they were less pronounced. While the individual ER contents in endocrine sensitive and endocrine resistant tumors overlapped, the EGF-R ranges were different: no one endocrine sensitive tumor exceeded the EGF-R content of 26 fmol/mg, thus suggesting the EGF-R cut-off point of endocrine sensitivity. The clinical use of EGF-R, with the cut-off point of 26 fmol/mg, in addition to clinical criteria of endocrine sensitivity and SRs, would significantly improve the correct endocrine sensitivity prediction (from 52 to 78%). In conclusion, in a group of metastatic breast cancer patients, treated routinely by systemic therapies it was found, that the use of higher cut-off point for EGF-R positivity can improve the prediction of endocrine sensitivity. The prognostic relevance of this cut-off value remains to be analyzed.

Expression of epidermal growth factor receptor in breast cancer, from early stages to advanced disease.

Epidermal growth factor receptor was determined in 106 newly diagnosed breast cancer patients, using the biochemical method. The group consisted of 58 patients in stage I-II, and 48 patients in stage III-IV. Although a significant inverse correlation was found between EGF-R status, and ER or PR status, quantitative content of EGF-R did not correlate either with quantitative ER, or PR levels. The ER/PR content was similar in all clinical stages, suggesting their stability during the clinical course of the disease. EGF-R content was significantly higher in stage IV, compared to stage I, while intermediate clinical stages and all substages did not differ according to the EGF-R content. EGF-R was confirmed as a weak prognostic factor within clinical stages. However, in a whole group, the overall survival was significantly better in patients whose tumors EGF-R content was lower than 26 fmol/mg, compared to those with higher ERF-R content. EGF-R content was highly predictive for the response to systemic endocrine treatment, in metastatic breast cancer patients. In locally advanced breast cancer a trend towards higher levels of EGF-R was found in inflammatory breast cancers, compared to non-inflammatory ones. Slightly higher levels were found in responders to local non-endocrine primary treatments (radiotherapy with or without chemotherapy), compared to non-responders, suggesting the possible predictive role of EGF-R for the response to such treatments. Our results emphasized the usefulness of quantitative receptor determination suggesting the relative stability of EGF-R content during the clinical course of breast cancer, its independence from ER, its significant predictive and weak prognostic values, and a possible correlation with the aggressiveness of the disease, and response to non-endocrine treatments.

Cathepsin D: association between TN-stage and steroid receptor status of breast carcinoma.

PURPOSE: To assess whether the expression of estrogen-induced protease, cathepsin D, might facilitate biological subgrouping of patients with breast carcinomas, and accordingly, its potential applicability in clinical oncology. PATIENTS AND METHODS: This study includes 70 patients with histologically confirmed breast carcinoma. Pathological findings were classified according to tumor size (T) and the presence or absence of metastases in regional lymph nodes (N). Steroid hormone receptor (SR) density as well as cathepsin D concentrations were assayed in the cytosol of breast carcinomas in accordance with the recommendation of the EORTC. RESULTS AND DISCUSSION: Statistically significant direct correlations were observed between cathepsin D expression and axillary node status as well as SR status. However, it is important to point out that in spite of these statistically significant findings, there were no biologically significant associations due to a wide range of individual cathepsin D values. Baseline levels of cathepsin D expression were found in patients with SR-negative status and node-negative tumors as well as in patients with SR-negative status and tumors of

pS2 in breast carcinoma: association with steroid hormone receptor status.

AIMS AND BACKGROUND: Knowledge of the steroid hormone receptors has proved to be of significant value in breast cancer. In the present study the possible importance of estrogen-regulated pS2 protein was investigated. Our direct purpose was to answer the question whether the expression of pS2 may be a marker of functional heterogeneity with respect to the steroid hormone receptor status. METHODS AND STUDY DESIGN: The study included 152 patients with primary, operable, histologically confirmed breast carcinomas. Histology specimens were reviewed and classified according to type, nodal status, tumor size and grade. Steroid hormone receptors were assayed by biochemical methods according to the procedures recommended by the EORTC. pS2 protein measurement was performed in breast carcinoma cytosols using an immunoradiometric assay. The results were analyzed by non-parametric statistical methods. RESULTS: A statistically significant inverse correlation between pS2 protein expression and histological tumor grade was found. The expression of pS2 protein was confirmed to be correlated with steroid hormone receptor status. However, it is important to point out that in spite of these statistically significant findings there were no significant biological associations due to overlapping individual pS2 protein values. The baseline level of expression of pS2 protein was obtained in histological grade III carcinomas with a negative steroid hormone receptor status. It was shown that the distribution of carcinomas according to the baseline level of pS2 protein expression was heterogeneous among estrogen receptor-positive carcinomas, and strikingly homogeneous among estrogen and progesterone-negative carcinoma. CONCLUSION: Our study suggested that PR and pS2 protein may identify distinct subsets of estrogen receptor-positive breast carcinomas.

Evaluation of the nutritional status and tumor characteristics in premenopausal and postmenopausal breast cancer patients.

Evaluation of the nutritional status, fat tissue distribution, and tumor characteristics was carried out in patients with primary breast cancer. The patients were classified into two groups according to their menopause: premenopausal and postmenopausal. Breast cancer prevalence was considerably higher in postmenopausal patients (61%). The patients' nutritional status was shown through the body mass index. Based on this indicator, the patients were characterized as nonobese and obese. In the premenopausal group, there was no significant difference between these categories, whereas the number of obese patients was significantly higher (80%) in the postmenopausal group. The analysis of tumor parameters as related to menopause and body size did not yield any significant differences. However, the estrogen receptor content was significantly higher in postmenopausal patients (p < 0.0001). Distribution of fat tissue of the android type was higher in obese postmenopausal women than in premenopausal ones (77%). The investigation showed that the breast cancer incidence odds are 3.5 times higher in obese postmenopausal than in premenopausal patients.

Variations in steroid hormone receptor content throughout age and menopausal periods, and menstrual cycle in breast cancer patients.

Variations in steroid hormone receptor contents throughout age and menopausal periods define three breast carcinoma groups: younger premenopausal carcinomas (aged up to 45), middle-aged carcinomas (pre-, peri- and postmenopausal aged 45-59) and older postmenopausal carcinomas (aged over 59). Age-related steroid hormone receptor contents within premenopausal and postmenopausal carcinoma groups are characterized by the important increase of both receptor contents, while menopausal-related steroid hormone receptor contents within middle-aged carcinoma group (aged 45-59) are characterized by the important decrease of progesterone receptor content and estrogen receptor functionality. No variations in steroid hormone receptor contents throughout menstrual cycle within the follicular and the luteal phases were obtained. The important decrease of estrogen receptor content in the mid-cycle phase versus the perimenstrual phase was found. Variations in steroid hormone receptor contents throughout age and menopausal periods, as well as throughout menstrual cycle could not be associated with variations in the blood steroid hormone concentrations. However, important association between steroid hormone receptor contents and the blood steroid hormone concentrations was found within the luteal phase carcinoma group and within older postmenopausal carcinoma group. It is interesting that within carcinoma group with the highest concentration of progesterone, progesterone receptor content increases with an increase of the ratio of estradiol and progesterone blood concentrations, while within carcinoma group with the lowest steroid hormone concentration and the highest content of estrogen receptor content, estrogen receptor content decreases with an increase of either the blood estradiol concentration or the ratio of the blood estradiol and progesterone blood concentrations.

Steroid receptors in pleural effusions of advanced breast cancer patients.

The steroid receptor content in breast carcinoma correlates with the responsiveness of malignant cells to endocrine manipulation. Although the steroid receptor status of the primary tumor is mostly used to select systemic therapy, it was suggested that steroid receptor content should be evaluated in metastatic lesions whenever possible. In this study the estrogen and progesterone receptor content was determined biochemically in 38 pleural effusions from advanced breast cancer patients. In 17/38 patients the steroid receptor status was assessed twice during the course of the disease - at diagnosis in the primary tumor/lymph nodes, and subsequently in metastatic pleural effusion fluid. A trend towards lower receptor values in pleural fluids was evident. There was no correlation between pleural steroid receptor content and pleural response to endocrine or chemo/endocrine therapy, indicating that the usefulness of effusional steroid receptors for therapy planning of advanced breast cancer could not be confirmed in this study.