Suppressor Gene P53 Research Articles

Abstract 4483: Early life exposures and promoter methylation in breast cancer: the Western New York Exposures and Breast Cancer (WEB) Study

Abstract Abstract Background: Exposures in the peri- and post-natal period (in utero, infancy, childhood, and adolescence) are associated with breast cancer risk, particularly premenopausal breast cancer. Epigenetic alterations in response to these exposures may play a critical role in early life, and then influence breast cancer risk in adult life. Using archived tumor blocks from incident breast cancer cases in a population-based the Western New York Exposures and Breast Cancer Study (WEB Study), we examined the association between early life exposures and promoter methylation of three tumor suppressor genes (p16, E-cadherin, and RARα2). Method: DNA samples isolated from 803 paraffin embedded tumor tissue were analyzed for hypermethylation status using real time methylation-specific polymerase chain reaction (MSP). Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) for associations of early life exposure with the likelihood of promoter methylation. Results: Among premenopausal breast tumors, compared to those with normal birth weight (2.6-3.9 kg), there was increased likelihood of E - cadherin gene hypermethylation in tumors from lower birth weight (≥ 2.5 kg) (OR = 2.79, 95% CI, 1.15-6.82). Height was positively associated with the likelihood of hypermethylation of RAR-α2 gene in premenopausal tumors (OR =3.34, 95% CI, 1.19-9.39). For p16, there was significant differences in the likelihood of promoter methylation of p16 in relation to not having been breast fed in premenopausal tumors (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. The likelihood of promoter methylation of the individual genes did not differ by maternal age at delivery, maternal height, age at menarche, age of first live birth, and weight at age 20 in either strata of menopause Conclusion: We found lower birth weight, not having been breast fed, and higher height were associated with promoter methylation of E - cadherin, p16 and RAR-α2 premenopausal breast tumors, respectively, suggesting that early life exposures may affect breast carcinogenesis among younger women through aberrant DNA methylation. Further studies of more genes and larger sample size may provide additional insight. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4483. doi:1538-7445.AM2012-4483

Abstract 2342: Identification of oncogenes and tumor suppressor genes using immortalized lung bronchial epithelial cells and small airway epithelial cells

Abstract We have immortalized human normal proximal bronchial epithelial cells (HBECs) for the study of lung tumors arising in the central compartment of the lung, and now have over 30 of these strains (growing in defined KSFM media) including many isogenic derivatives with various oncogenic changes (Can Res 2004, Can Res 2006). We have extended this by immortalizing normal distal (small airway) epithelial cells that serve as models for the study of tumors arising in the peripheral compartment of the lung. Using Cdk4 and hTERT exogenous expression vectors (as used for HBECs) and serum free defined media, we have immortalized (N= 19) human small airway epithelial cells (HSAECs) from peripheral lung. We have characterized the HBECs and HSAECs and found they have the property of lung basal /stem cells. In the current study, we compared the genome-wide RNA expression profile of HBECs/ HSAECs with 108 lung tumor cell lines and identified a panel of genes that are differentially expressed in these two cell types. We found ∼ 1200 genes that are 2 fold over expressed in tumor cells lines compared with HBECs/HSAECs (p ≤ 0.005). Many known lung tumor oncogenes such as members in cancer/testis (CT) antigen families (XAGE/GAGE/MAGE), proliferation-promoting E2F2 transcription factors, and Kinesin family are over-expressed in tumor lines validating the comparison strategies. In addition, we found genes that had been shown to be involved in other types of tumors but had not been reported in lung tumors. Importantly, we found several genes that had not been implicated as oncogenes previously, thus these are potential novel oncogenes for lung tumor transformation. We also found ∼1300 genes that are 2 fold over expressed in HBECs/HSAECs compared with tumor cell lines (p≤ 0.006). Many of the genes are lung basal cell markers (Keratin and Laminin family members) or known tumor suppressor genes (p53 binding genes and metallothionein genes) as expected. Like in the case of tumor specific genes, we found several genes that had not been reported functioning in the inhibition of tumor transformation. Thus, these are candidate tumor suppressor genes. The current study showed that the immortalized HBEC and HSAECs are ideal models for the discovery of novel oncogenes and tumor suppressor genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2342. doi:1538-7445.AM2012-2342

Abstract IA10: Pathology of lung neuroendocrine tumors: In search of evidence for classification

Abstract Neuroendocrine (NE) tumors comprise approximately 25% of all tumors in human lung. As a group they continue to provide challenges at biological, molecular and pathological level for uniform classification. The prototype of NE tumors is the highly aggressive small cell lung cancer (SCLC) that accounts 15% of human lung cancers. For years lung cancers were clinically classified mainly into SCLCs and non-SCLCs (NSCLCs) while it has become evident that lung NE tumors actually form a complex spectrum from SCLC to carcinoid with shifting boundaries. The current WHO classification recognizes SCLC, its variant form combined SCLC, typical and atypical carcinoids as well as large cell NE carcinoma (LCNEC) and its variant form combined large cell carcinoma, the last two cancers under the main NSCLC category of large cell carcinoma. With the exception of the LCNEC, it is expected that the diagnoses will be rendered by routine hematoxylin-eosin stain according the established criteria. Benign entities include diffuse idiopathic pulmonary NE cell hyperplasia which is described in the chapter of preinvasive lesions. Over time there have been several efforts to place lung NE tumors into two to four-tier categories according to their malignant potential (high versus low grade) or degree of differentiation often leaving behind confusing terminology that is later misused. Unlike surgically resected NSCLCs, most NE tumors are frequently diagnosed from biopsies which remains a seldom recognized fact that may create its own biases. The diagnosis that experts most often agree with is SCLC, while distinctions between atypical carcinoid and LCNEC that are in the middle of the spectrum are often disputed. Many molecular studies have also “correctly” classified the endpoints of the NE spectrum while grouping in the middle of it has produced varied results. Moreover, several studies have suggested that NSCLCs with NE features (NSCLC-NEs) is a distinct carcinoma type with worse prognosis than ordinary non-NE NSCLC, while others disagree. Biologically this category is very interesting as NSCLCs and SCLCs are ultimately thought to be derived from the same cell. The regulation of NE differentiation is poorly understood. The proneural achaete-scute homolog 1 (ASCL1), a lineage-dependent oncogene may be a key factor for NE differentiation. On the other hand, it was recently shown that the deletion of the tumor suppressor genes Rb1 and p53 specifically in NE cells, a subpopulation of cells in normal airways, or in alveolar non-NE type2 pneumocytes of mice resulted in SCLCs closely mimicking human disease. These results provide experimental evidence for the close relationship of NE and non-NE epithelial cells in carcinogenesis.

English

Tp53 is an important tumor suppressor gene, which induces cell growth arrest or apoptosis when subjected to cytotoxic stimuli. Association has been reported between various cancers and p53 codon 72 polymorphism. Our objective was to investigate the possible association between p53 at codon 72 for Arg/Arg, Arg/Pro and Pro/Pro allele polymorphisms in blood samples from 187 prostate cancer patients and 185 controls in southwest Iran by nested-polymerase chain reaction (PCR) of p53 exon 4 and digestion with BstUI restriction enzyme and the DNA fragments were then resolved by electrophoresis in 2% agarose gel. The frequencies of Arg/Arg, Arg/Pro and Pro/Pro genotypes were 39.57% (74/187), 52.41% (98/187) and 8.02% (15/187), respectively, in the cases with prostate cancer, and 27.03% (50/185), 60% (111/185) and 12.97% (24/185), respectively in the healthy controls. Statistically, analyzed and combined results showed there was a significant difference in the frequency of the Arg/Arg genotype and Arg allele between prostate cancer cases and control (p>0.001). These findings suggest that p53 Arg/Arg genotype could be a risk factor for the development of prostate cancer among patients in southwest Iran. Key words: Prostate cancer, suppressor gene (p53) codon 72, polymorphism, Iran.

Hämangiosarkom nach brusterhaltender Therapie beim Mammakarzinom: vier Fallbeispiele mit molekulargenetischer Diagnostik und Literaturübersicht

Hemangiosarcomas of the breast represent a rare disease of the breast mainly occurring as secondary neoplasias with a latency of 5-10 years after primary treatment of breast cancer and are associated with an unfavourable prognosis. Radiation therapy, which is integrated within the concept of breast conserving therapy ranks as the main risk factor. In this report we describe the clinical course of 4 patients including their molecular genetic pattern and give a summary of the actual literature. Hemangiosarcomas occur as a secondary neoplasm with a latency of 5-10 years after primary treatment of breast cancer and have an unfavorable prognosis. A genetic predisposition is assumed, but we could not find a significant role of tumor suppressor genes BRCA1, BRCA2 or p53 in our patients. Due to limited data available for these tumors, recommendations for therapy include radical tumor resection achieving wide free margins and inconsistent regimens of chemo- and/or immunetherapy modalities. In the majority these are based on systemic therapy regimens for other cutaneous sarcomas, such as Kaposi's sarcoma. Efforts should be taken for a nation-wide systematic registration of all cases of post-irradiation hemangiosarcomas.

Inflammatory Nitrergic Dysfunction Evokes Intestinal Dysmotility

COX-2 expressing vector, and HCA-7, a COX-2 expressing human colon cancer cells were used. Stemness was estimated by ability to form spheroid known to enrich in stem cells and oct-4 expression. DNA methyltransferase activity, demethylase activity, and DNA global hypomethylation were measured. Promoter methylation of tumor suppressor genes (p16, E-cadherin, and hMLH1), and an oncogene, S100A, was determined by MSP or COBRA. Expressions of DNMT-1, -3A, and -3B and demethylase genes (AID and glycosylase) were determined by RT-PCR. [Results] RIE-COX showed significantly elevated potential for spheroid formation, oct-4 expression, DNA demethylase activity and expression, promoter hypermethylation of tumor suppressors and hypomethylation of S100A, and DNMT-3a expression and activity, and it also showed significantly downregulated global DNA methylation, compared with RIE. siRNA for COX-2 or celecoxib, a COX-2 specific inhibitor, significantly inhibited spheroid formation, global hypomethylation, hypermethylation of tumor suppressor genes, and DNA demethylase activity/expression in HCA-7. siRNA for DNMT3a or AID partly suppressed spheroid formation of HCA-7 and oct-4 expression. [Conclusion] These results indicate that COX-2 is involved in intricate epigenetic regulation; simultaneous global hypomethylation and gene-specific hypermethylation, through discrete modulation of DNMT/demethylase expression, and it is also suggested that COX-2 inhibitors have a potential to prevent epigenetic dysregulation and aberrant biology of cancer stem cell in carcinogenesis.

Novel Role of Macrophage in the Induction of TH2 Immunity

COX-2 expressing vector, and HCA-7, a COX-2 expressing human colon cancer cells were used. Stemness was estimated by ability to form spheroid known to enrich in stem cells and oct-4 expression. DNA methyltransferase activity, demethylase activity, and DNA global hypomethylation were measured. Promoter methylation of tumor suppressor genes (p16, E-cadherin, and hMLH1), and an oncogene, S100A, was determined by MSP or COBRA. Expressions of DNMT-1, -3A, and -3B and demethylase genes (AID and glycosylase) were determined by RT-PCR. [Results] RIE-COX showed significantly elevated potential for spheroid formation, oct-4 expression, DNA demethylase activity and expression, promoter hypermethylation of tumor suppressors and hypomethylation of S100A, and DNMT-3a expression and activity, and it also showed significantly downregulated global DNA methylation, compared with RIE. siRNA for COX-2 or celecoxib, a COX-2 specific inhibitor, significantly inhibited spheroid formation, global hypomethylation, hypermethylation of tumor suppressor genes, and DNA demethylase activity/expression in HCA-7. siRNA for DNMT3a or AID partly suppressed spheroid formation of HCA-7 and oct-4 expression. [Conclusion] These results indicate that COX-2 is involved in intricate epigenetic regulation; simultaneous global hypomethylation and gene-specific hypermethylation, through discrete modulation of DNMT/demethylase expression, and it is also suggested that COX-2 inhibitors have a potential to prevent epigenetic dysregulation and aberrant biology of cancer stem cell in carcinogenesis.

Biological characteristics of IL-24 and mechanisms of its anti-tumor action

Studies have shown that IL-24 is the only cytokine capable of inhibiting tumor growth and neovascularization as well as stimulating immune system. IL-24 exerts its inhibition effects through pathways independent of p53, Rb, p16 and other tumor suppressor genes and has no effect on normal cells. IL-24 has emerged as a hot topic in cancer therapy research. Key words: Interleukin-24; Genes, tumor suppressor; Gene expression regulation, neoplastic

A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma

AbstractAurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.

Expression analysis of Akt and MAPK signaling pathways in lung tissue of patients with idiopathic pulmonary fibrosis (IPF)

Purpose of the study: Several studies in patients with lung cancer have shown that epidermal growth factor receptor regulates various tumorigenic processes through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin and Ras/Raf/Mek/Erk (mitogen-activated protein kinase (MAPK)) signalling pathways. The aim of our study is to evaluate whether these pathways are implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and to seek indirect evidence of a common pathogenetic pathway with lung cancer. m-RNA expression of oncogenes participating in these two signaling pathways, as well as the combined m-RNA expression of the suppressor genes R-kip and p53 in lung tissue of patients with IPF were evaluated.Basic procedures: The study population was composed by two distinct groups. Patients with IPF (n = 25) and control subjects who underwent thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned oncogenes and suppressor genes was performed using real-time reverse transcription polymerase chain reaction.Main findings: We found no difference in the overall m- RNA expression between controls and IPF in both investigated pathways. However, Braf has been overexpressed in IPF samples (P = 0.01) in contrast with K-ras that has been found downregulated (P < 0.001) in comparison with controls.Principal conclusions: These findings cannot exclude the hypothesis of involvement of Akt and MAPK signalling pathways in pathogenesis of IPF. However, further investigation is needed in order to verify these data.

Study on the promoter hypermethylation of TSHR, P16, and RAS genes in human papillary thyroid carcinoma

Objective To study the expressions of the tumor suppressor gene TSH receptor( TSHR),P16, and RAS in papillary thyroid carcinoma ( PTC ) , and the correlation between the occurrence of tumor and the aberrant promoter hypermethylation of three tumor suppressor genes. Methods RT-PCR was used to detect the mRNA expression of three tumor suppressor genes in tissues of 50 cases of PTC ,20 cases of nodular goiter,and 12 cases of thyroid adenoma. The promoter methylation status of three tumor suppressor genes was examined by methylation-specific PCR technique( MSP). Gene sequencing was used to test if the hypermethylation existed in the promoter of three tumor suppressor genes. Results In 68.0% (34/50) TSHR gene, 54.0% (27/50) P16 gene, and 60.0% ( 30/50 ) RAS gene in PTCs, hypermethylation in promoter region was detected, the respective results 21.9% (7/32) , 15. 6% (5/32) ,and 31. 3% (10/32) were found in control tissues. The rates of the three genes with promoter hypermethylation in PTC were significantly higher than those in control tissues ( all P<0. 05). The mRNA expressions of TSHR,P16,and RAS were significantly lower in PTC than those in control tissues (0. 41 ± 0.11 vs 0.63±0. 08,0. 51±0. 17 vs 0. 72±0. 22,0. 56±0. 10 vs 0. 67±0. 16, all P<0. 05). The sequencing confirmed that there was CC to TC transmission in the promoters of three tumor suppressor genes. Conclusions The methylation of three tumor suppressor genes in promoter region is a common molecule event and may be involved in the genesis and development of human PTC. Key words: Papillary thyroid carcinoma; Methylation; TSHR; P16; RAS; Promoter

Abstract 4535: MDM2 inhibitor MI-319 in combination with cisplatin/oxaliplatin is an effective treatment for pancreatic cancer independent of p53 function

Abstract Pancreatic cancer (PC) is considered to be among the most intractable malignancies as it is refractory to chemotherapy and radiation. Among the various genetic alterations in PC, mutation in the tumor suppressor p53 (mut-p53) gene has been reported to be about 50%. In the remaining 50%, the p53 is normal, but its function is inhibited by MDM2 protein which plays a key role in promoting ubiquitination and proteasomal-dependent degradation of p53. An attractive approach to p53 activation is the use of MDM2 inhibitors that disrupt the MDM2-p53 interaction and restore the p53 function. However, these small molecule inhibitors (SMIs) are not effective against tumors with mut-p53. Our group has designed a new class of SMI of MDM2 that is different from Nutlin-3. Here we are reporting, for the first time, that MI-319 can induce cell growth inhibition and apoptosis in PC irrespective of p53 mutational status when combined with cisplatin/oxaliplatin. MI-319-cisplatin or oxaliplatin combination synergistically suppressed cell growth (MTT CI&amp;lt;1), colony formation (clonogenic assay) and induced apoptosis in both mut-p53 Colo-357, BxPC-3 and Panc-28 or wt-p53 Capan-2 cells. Western blot analysis and siRNA silencing studies in mutant as well as p53 null cells highlighted a mechanism involving p73 which is under the regulation of MDM2, and unlike p53, it is rarely mutated in PC. In order to substantiate its in vivo relevance we tested this potent combination in mice xenograft models carrying Capan-2 and BxPC-3 tumors. 4 groups, each containing 8 animals were treated either with; Control (Vehicle only), MI-319 treated 200mg/Kg orally twice a day for three weeks, Cisplatin treated 4 mg/kg (i.v.) twice a week for two weeks and combination treated (MI-319 200 mg/Kg orally + Cisplatin 4 mg/kg). The combination effectively reduced tumor growth in both wt-p53 and mut-p53 tumor xenograft models (50% Capan-2 animals were tumor free). Consistent with our in vitro results, remnant tumor tissue analysis showed up-regulation of p73 and the cell cycle regulator p21. In conclusion, this study highlights a new role of MDM2 inhibitors in combination with cisplatin or oxaliplatin, and thus warrants further clinical investigation in human pancreatic tumors containing both wt-p53 and mut-p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4535.

Occurrence and expression of p53 suppressor gene and c-Myc oncogene in dog eyelid tumors

To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c-Myc in eyelid tumors of dogs using the PCR, RT-PCR, PCR-ELISA and RT-PCR-ELISA techniques. These genes have not been described in dog eyelid tumors before. Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin-eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR-ELISA and RT-PCR-ELISA were used to detect p53 and c-Myc genes. The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c-Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Eyelid and third eyelid tumors of dogs express both the p53 and the c-Myc genes as shown by PCR and RT-PCR. However, PCR ELISA and RT-PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis.

Expression of oncogenes and tumor suppressor genes in lungs of rats exposed to sulfur dioxide and benzo(a)pyrene

Concurrent exposure to SO2 and benzo(a)pyrene (B(a)P) resulted in an increased incidence of lung tumors in rodents compared to exposure to B(a)P alone. A synergistic effect on the expression of c-fos and c-jun between SO2 and B(a)P was observed in lungs after SO2 and B(a)P exposure. However, tumorigenesis occurs by multiple events that may involve the activation of more than one oncogene, as well as the functional loss of the tumor suppressor gene. In order to further investigate the interactions between SO2 and B(a)P, male Wistar rats were exposed via intratracheal instillation of B(a)P (3 mg) or SO2 (56 mg/m3) inhalation, alone or together. The mRNA and protein levels of oncogenes (c-myc and H-ras) and tumor suppressor genes (p53, p16, and Rb) were analyzed in lungs by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that all treatments increased mRNA and protein expression levels of c-myc, H-ras, and p53, and reduced expression levels of p16 and Rb. In general, the combination of SO2 and B(a)P was more effective in influencing these expression levels than either agent alone, except for H-ras expression. These findings indicate that multiple cell cycle regulatory proteins play key roles in the toxicity of SO2 and B(a)P. It might involve the activation of c-fos, c-jun, c-myc, and p53. And the p16-Rb pathway might also participate in the progress. Elucidating the expression patterns of those factors after SO2 and B(a)P exposure may be critical to understanding the mechanisms of SO2 cocarcinogenesis and helpful for therapeutic intervention.

Clinical Significance of P53 Protein Expression in Papillary Thyroid Carcinoma

Although mutations in the p53 suppressor gene in thyroid carcinoma have usually been detected in anaplastic carcinoma, P53 protein expression has been detected immunohistochemically in papillary thyroid carcinoma (PTC). In the present study, we examined the immunohistochemical expression of P53 protein in PTC to investigate the relations between its expression and the clinicopathologic features. The study was performed on 68 patients in whom thyroidectomy with lymph node dissection had been performed to treat PTC at Teikyo University Hospital. Expression of P53 protein was evaluated immunohistochemically in sections of paraffin-embedded tissue in 68 primary tumors and 196 lymph node metastases. Overexpression of P53 protein in the primary tumor was observed in 29 cases (43%). Statistical analysis revealed significant correlation between P53 protein expression in the primary tumor and large tumor size (unpaired t-test: p < 0.01), the presence of lymph node metastasis (unpaired t-test: p < 0.05), and the mean number of lymph node metastases (unpaired t-test: p < 0.05). Although 29 (43%) of the primary tumors overexpressed P53 protein, 143 (73%) of the metastatic lymph nodes overexpressed P53 protein irrespective of whether there was P53 overexpression by the primary tumor. The results of this study suggest that immunohistochemistry for P53 in the primary tumor could be useful in the clinical evaluation of patients with PTC. Moreover, P53 protein overexpression in lymph node metastasis may be useful as a treatment guide or target for lymph node recurrences.

Familial Adenomatous Polyposis: Analysis of Genetic Instability of Microsatellites Loci and Genetic Alternations of Tumor Suppressor Genes

Familial adenomatous polyposis (FAP) is an autosomal dominant illness with the highest risk for appearance of colorectal cancer's disease. In our study, we have used Bethesda criteria that define colorectal cancers which can be tested on microsatellite instability. The aim of our study is make an analysis of microsatellite instability (MSI), appearance of RER+ phenotype, genetic alteration of tumor suppressor genes as like as one of responsible factor for genesis of adenomatous polyposis. The base for this study were shown families with clinical diagnosed FAP. In this study two families with clinical diagnosed adenomatous polyposis were involved. Our study of both families showed that three tumor tissues belonged to RER negative phenotype, but only one belonged to RER positive phenotype. Microsatellite analysis showed instability of mononucleotide marker Bat 40 at 4 samples and Bat 26 at 2 samples, but Bat 25 and in 1 sample. Dinucleotide marker TP 53 did no show any microsatellite alterations. Genetic alteration of tumor suppressor gene APC appeared at 4 samples, p53 at 3 samples, RB1 at 2 samples and NM23 only at 1 sample, but tumor suppressor genes DCC1 and DCC2 were homozygote. Our results are agree with results of earlier studies and also the got results confirm the fact that loss of heterozygosity of tumor suppressor gene APC and p53 are responsible for genesis of adenomatous polypose and it also represents the characteristic of genetic changes FAP's patients in our region.

Evidence of Pre-Diagnostic Monoclonality in Blood Obtained up to 6.4 Years Preceding Diagnosis of Chronic Lymphocytic Leukemia (CLL).

Background: Monoclonal B-cell lymphocytosis (MBL) is reported in about 3% of the general adult population 65 yrs or older. Risk of progression from MBL to CLL requiring treatment has been estimated to about 1% per yr. For the first time we established the occurrence of preceding MBL using prospectively collected pre-diagnostic samples from CLL pts. Also, we tested the hypothesis that indolent (IgVH mutated) CLL occurs after MBL while aggressive (IgVH unmutated) CLL has an alternative route that develops rapidly or bypasses MBL.Methods: Using the population-based nationwide PLCO (Prostate, Lung, Colorectal and Ovarian) cancer screening trial (n>144,500 cancer-free adults), we identified 46 CLL pts with available baseline cryopreserved whole blood obtained 3 to 77 months prior to CLL diagnosis. Clinical information was retrieved from screening questionnaires and medical records. 10 healthy adult controls were included. 6-color flow cytometry (CD45/CD19/CD5/CD10/kappa/lambda) and reverse transcription/polymerase chain reaction (RT/PCR) for IgH gene were used to determine evidence of expression of monoclonal IgH gene family and to establish the VH gene mutation status. Direct sequencing without subcloning (for clonal cases) and with subcloning (for non-clonal cases) was used to determine mutation status. We sequenced exons 5, 6, 7, 8, and 9 of the p53 suppressor gene for mutations.Results: After exclusion of one subject with absent viable cells, we analyzed pre-diagnostic blood specimen from 45 CLL pts. CLL diagnosis was based on clinical criteria and confirmed by review of clinical records. Immunophenotyping data were available for 39 (87%) pts. The Rai CLL stage distribution was: 0 (n=26), 1 (n=9), 2 (n=2), 3 (n=1), missing/unclear (n=7). The mean time between pre-diagnostic blood draw and subsequent CLL diagnoses was 3 yrs. Using flow cytometry, sequencing, and PCR, we demonstrated evidence of monoclonality in 43/45 (96%) pre-diagnostic samples from CLL pts. Among individuals whose blood was obtained <3 yrs vs. >3 yrs prior CLL diagnosis, the proportion of pre-diagnostic monoclonality was 28/28 (100%) vs. 15/17 (88%), respectively; the proportion of IgVH mutated pre-diagnostic clones was the same in both groups: 23/28 (82%) vs. 14/17 (82%). The most frequently expressed VH gene family was VH3 (40%) followed by VH4 (29%), VH1 (9%), VH5 (9%), VH2 (7%), and VH6 (2%), with no expression of VH7 gene families. We found 2 pts with mutations in the p53 suppressor gene; one in exon7/M237K, and one in exon 6, P22 that is silent and has been reported as in some tumors and a polymorphism. One sample with unmutated IgVH appeared to have a deletion at p53 in both alleles. Immunophenotyping analyses for pre-diagnostic clones showed kappa n=27, lambda n=8, n=4 biclonal, and missing/indeterminate n=6 (kappa:lambda ratio >3:1). Comparisons of pre-diagnostic and diagnostic kappa/lambda status in the same persons showed 100% concordance. All negative controls lacked evidence of monoclonality.Conclusions: In this first prospective study examining pre-diagnostic blood obtained 3 to 77 months prior to CLL diagnosis, we found a monoclonal B-cell population in virtually all the persons; 82% were IgVH mutated. Our findings suggest that CLL is commonly preceded by the precursor condition MBL.

P141 Do the “new drugs” antagonize the impact of unfavourable cytogenetic markers in multiple myeloma?

Background In our study, we have concentrated on four aberrations in multiple myeloma (MM) malignant plasma cells: deletions of tumor suppressor genes RB (locus 13q14) and p53 (17q13), translocation involving IGH gene t(4;14), and actually focused amplification of CKS1B gene in 1q21 locus. All of these aberrations are known as negative prognostic factors in patients (pts) treated by conventional therapy or stem cell transplantation. Aims The aim of this study is to show if these selected aberrations act as negative prognostic factors also in patients treated by Velcade or by thalidomide, new drugs used in relapsed patients. Methods We have an increasing group of (recently 42) MM patients. Overall clinical characteristics of the patients at the start of treatment: Average age 63.7 years (SD = 9.4), 57 % (24) of men. 12 % (5/42 pts) were in stage IA, 26 % (11/42 pts) were in stage IIA, 60 % (25/42 pts) were in stage IIIA and 2 % (1 patient) was in stage IIIB. 69 % (29/42 pts) were in first relapse, 24 % (10/42 pts) were in second relapse and the other 7 % (3/42 pts) were in third relapse. 85 % (36/42) pts received at least four cycles of therapy. 64 % (27/42) pts reached overall response (OR) during the follow up (average follow up was 11.0 months, SD = 5.3 months). For identification of malignant plasma cells in bone marrow samples, we use cytoplasmic immunoglobulin (cIg) labeling methodology (Ahmann et al. 1998). This method allows us to identify simultaneously monotypic plasma cells by monoclonal antibody fluorescence (anti-kappa or anti-lambda) and to detect chromosomal abnormalities by FISH (cIg-FISH). Results Cytogenetic findings: Deletion of RB gene was found in 57 % (21/37) of successfully examined patients, deletion of p53 gene was in 53 % (17/32) pts, translocation t(4;14) was in 61 % (22/36) pts and amplification of 1q21 was in 65 % (22/34) pts. Statistical comparison of clinical response in patients with or without each aberration: We have found no significant difference when compared overall survival (OS), time to progression (TTP), progression free survival (PFS), duration of response (DOR) and overall response in subgroups of patients with and without of any selected aberration. Summary/Conclusions In our group of patients, no one of monitored aberrations seems to have any impact, neither positive nor negative, on efficiency of used treatment. It is possible that new drugs, Velcade and thalidomide, antagonize the impact of cytogenetic negative prognostic factors in relapsed patients with MM. However, we will continue in this research to reach larger data set to confirm or disconfirm our results. If this data will be confirmed on larger group of patients, it looks necessary to find other chromosomal abnormalities, relevant for prediction of the prognosis in these patients.

Study of a functional polymorphism in thep53 gene in systemic lupus erythematosus: lack of replication in a Spanish population

The aim of this study was to assess the possible association between the p53 suppressor gene codon 72 polymorphism and systemic lupus erythematosus (SLE). Our study population consisted of 513 SLE patients and 567 healthy controls. All the individuals were of Spanish Caucasian origin. Genotyping of the p53 codon 72 polymorphism was performed by allele-specific PCR. No statistically significant differences were observed between SLE patients and healthy controls when p53 codon 72 genotype and allele frequencies were compared. In addition, no evidence for association with clinical subfeatures of SLE was found. In conclusion, the p53 codon 72 polymorphism associated with SLE in a Korean population does not appear to play a major role in the susceptibility or severity of SLE in the Spanish population.

Do Molecular Markers Predict the Electromagnetic Field Treatment of Cancer Through p53 Suppressor Gene

In recent years there have been enormous studies made toward understanding diagnosis and treatment of cancer. Although there has been a great deal learned about cancer, the treatments available for it have not progressed nearly as much. Attempted removal of the tumor followed by chemotherapy and radiotherapy still prevail as the most effective treatments used. The present study used the electromagnetic fields (4.5 Hz) to treat tumor implanted in mice. The Polymerase Chain Reaction/Restriction Fragment Length Polymorphisms (PCR/RFLPs) technique was selected as a biomarker to evaluate the effect of exposure to electromagnetic fields in implanted Ehrlich tumor of female BALB/C mice. Eighty mice used and divided into four groups (20 each); control, radiated (control exposed to 4.5 Hz), infected (control infected by Ehrlich tumor) and infected exposed (infected exposed to 4.5 Hz). The duration of exposure was for two hours every two days. Electromagnetic field exposure includes group 2 and group 4. DNA genome was extracted and p53 suppressor gene detected (~2130 bp). AatI, BanII, EaeI restriction endonucleases did not differentiate between the PCR products (p53 genes) of the four groups (control, radiated, infected and infected exposed mice groups). BanI, DraI, DraIII, HaeII and PstI differentiated between the four groups. The results approved that the electromagnetic fields could treat the tumor and PCR\RFLPs could be useful diagnostic technique.