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cDNA microarray application on cervical epithelial cells
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of DNA and mRNA E6 and E7 HPV 16 and HPV 18 was examined based on Q-PCR standard curves for p-actine by use of Perkin Elmer-kit and the application of the sequence detector ABI PRISM 7700.Taq Man. Results: The expression of both E6 and E7 proteins of HPV 16 and 18 and also overexpression of p.53 in 3 cases was found. In 1 case the overexpression of p.53 and E6 E7 HPV 18 was confirmed. The next 6 cases were p.53 negative, but in 3 patients E6 and E7 HPV 16, and in another 3 women E6 and E7 HPV 18 were found. The last 4 patients were HPV and p.53 negative. It was noticeable that in the case of high E6 expression, overexpression of ~53 was present. In recurrence of vulvar cancer, high overexpression of ~53 was confirmed. There was also found a mutation in ~53 gene in this case. Moreover in very rapidly progressing vulvar cancer in 26 year-old women coexistence of ~53 overexpression with co-infection E6, E7 HPV 16, 18 were ascertained. Conclusions: The number of analyzed patients does not allow us to make any statistical conclusions. The co-existence of high expression of E6 protein with ~53 overexpression was ascertained and indicates that both of them can play a role in the pathogenesis of vulvar cancer. On the other hand, HPV and ~53 negative cancers suggest that in the pathogenesis of vulvar cancer also other factors can be involved.
P2.13.03 P53 POLYMORPHISM AND HUMAN PAPILLOMAVIRUS INFECTION IN HONG KONG WOMEN WITH CERVICAL CANCER Y.F. Wang, T.K.H. Chug, T.H. Cheung, V.W. Wang, Dept. OB/GYN, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. Objectives: A polymorphism at codon 72 of the ~53 gene, resulting in either an arginine or a proline residue in the protein, has been reported to affect the susceptibility of ~53 to human papillomavirus (HPV) E6mediated degradation in cultured cells. This study was to investigate the involvement of ~53 polymorphism at condo 72, the infection and typing of HPV, and the correlation of ~53 polymorphism with HPV and other clinico-pathologic characteristics in Hong Kong Chinese women with cervical cancer. Study Methods: Seventy-two primary cervical cancer specimens were obtained at surgery from consenting patients at the Department of Obstetrics and Gynecology, the Chinese University of Hong Kong. We developed a Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR - RFLP) method for analysis of ~53 polymorphism at codon 72 of exon 4 in cancer tissue DNA samples. The presence and types of HPV DNA in cancer were analyzed by PCR and DNA sequencing. Results: The homozygous ~53 arginine allele (Arg/Arg) was detected in 22 (31%), homozygous ~53 proline (Pro/Pro) in 14 (19%) and heterozygous allele (Arg/Pro) in 36 (50%) cases, respectively. HPV infection was detected in 55 of 72 (76%) cases. The prevalent types were HPV 16 (55%), HPV 18 (16%) and HPV 58 (9%). The numbers of HPV-positive cases with form Arg/Arg, Pro/Pro and Arg/Pro were 17 (31%), 12 (22%) and 26 (47%), respectively. The ~53 polymorphism at codon 72 was not significantly correlated with any of HPV types (p>O.O5). No striking overrepresentation of homozygous arginine-72 ~53 was observed in HPV-associated cervical cancer. No significant correlation was found between the ~53 polymorphism and histologic type, grade of cell differentiation, or clinical stage of cancer (p>O.O5). Conclusions: The results from this study, taken together with other reports, indicate that there may be geographic and ethnic variations in the ~53 polymorphic form in cervical cancer. However, the present study did not show any ~53 polymorphic form would have prognostic significance for cervical cancer. The information on prevalent types of HPV infection in Hong Kong Chinese women with cervical cancer, is pertinent to the development of HPV diagnostic probes and vaccination strategies (to prevent HPV type specific infection) in the future with particular reference to China.
TUESDAY, SEPTEMBER 5 P2.13.04 cDNA MICROARRAY APPLICATION ON CERVICAL EPITHELIAL CELLS W.B. Battistutti (l), M. Manavi (2), K. Pischinger (l), E. Kubista (2), K. Czerwenka (l), University Hospital, Vienna, Austria. (1) Dept. GYN/PATH (2) Dept. SPEC/GYN Objectives: The biology and structural behavior of a cell is determined by the gene expression within that cell. Today, there are over l,OOO,OOO human tag, sequences are available on public database (httu://www.ncbi.nlm.-nih.eovh. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. Study Methods: Gene expression monitoring using microarrays was first described using radioactive targets hybridized into filter-immobilized cDNA clones by Drmanac et al. (1996). The “Atlas array” method makes use of nylon membranes with specially prepared cDNA spots. This enables a direct comparison of the expression profiles of mRNA populations. In the first step, 2 ml of each Poly A+RNA population are catalyzed with the reverse transcriptase enzyme and with (a-32 P) dATP. All complex radioactively-labeled cDNA probes are then individually hybridized on the “Atlas array”, analyzed using autoradiography and semiquantified. The level of cDNA expression of two different mRNA matrices can be estimated by comparing signal intensities. Results: In normal and cancerous cervical cells, the transcribed gene patterns demonstrate up/down -regulation of functional groups, similar to transcription e.g.: 1) oncogenes, 2) tumor suppressor genes, 3) cell-cycle regulator proteins, 4) transcription factors. Conclusions: Neoplastic cells have numerous acquired genetic abnormalities and these changes in signal transduction in cellular neoplasias give rise to different levels of gene expression. The variations in the gene expression patterns are observable and are characteristic in the cancerogenesis of malignant tumors.
P2.13.05 LOSS OF HETEROZYGOSITY AT 6~21.3 AND THE PRESENCE OF THE HPV-16 E6 350G VARIANT IN CERVICAL CANCER. lz3H. ‘V.V.V.S. Murty,3N. Martinez,3C.M. Wheeler, 4N. Beleiio, 5G. Martinez, 6H. Posse. ‘Genetics and Molecular Biology, 4Pathology, 5Gynecology, 6Epidemiology, Instituto National de Cancerologia, Santa Fe de Bogot6, Colombia. ‘Patology, Columbia University, New York. 3Health Sciences Center, Epidemiology and Cancer control, U. of New Mexico, Albuquerque. Objectives: The aim of the study was to analyze loss of heterozygosity (LOH) at 6~21.3 and the presence of human papillomavirus (HPV) type 16 E6 variants in invasive cervical cancer (ICC). Study Methods: 61 ICC were analyzed for the presence of LOH, and HPV 16 E6 variants were detected by lineage-specific hybridization. Results: LOH was observed in at least one locus in 37 of 61 (61%) tumors. The highest frequency of LOH was for D6S265 (54%) followed by MOGCAl(50%), D6S276 (48%), D6S273 (47%), D6S291(43%), and TAP1 (41%). HPV 16 was found in 48 of 61(79’?‘)D samples. A significant difference in the prevalence of HPV 16 positive tumors was detected between those presenting with LOH and those in which no LOH was observed. In this tumors, the most prevalent HPV 16 variant was the 350G (E-350G, 52%) followed by the Prototype (E-P, 31%) and the Asian-American (AA, 17%) variant. This difference in variant distribution is significant (p=O.O19). Further, in the presence of LOH, the 350G variant frequency was significantly different when compared to the AA variant (p=O.OOS), but not when compared to the prototype (p=O.ll). Conclusions: LOH is a frequent event in the 6~21.3 region, which contains the genes of the major histocompatibility complex (MHC). This observation could in part explain the derregulation of the MHC molecules, an event commonly observed in ICC. In addition, the presence of the 350G variant can influence the appearance of genetic instability as measured by LOH and thus may contribute to the malignant potential of HPV 16 infected epithelia.
of DNA and mRNA E6 and E7 HPV 16 and HPV 18 was examined based on Q-PCR standard curves for p-actine by use of Perkin Elmer-kit and the application of the sequence detector ABI PRISM 7700.Taq Man. Results: The expression of both E6 and E7 proteins of HPV 16 and 18 and also overexpression of p.53 in 3 cases was found. In 1 case the overexpression of p.53 and E6 E7 HPV 18 was confirmed. The next 6 cases were p.53 negative, but in 3 patients E6 and E7 HPV 16, and in another 3 women E6 and E7 HPV 18 were found. The last 4 patients were HPV and p.53 negative. It was noticeable that in the case of high E6 expression, overexpression of ~53 was present. In recurrence of vulvar cancer, high overexpression of ~53 was confirmed. There was also found a mutation in ~53 gene in this case. Moreover in very rapidly progressing vulvar cancer in 26 year-old women coexistence of ~53 overexpression with co-infection E6, E7 HPV 16, 18 were ascertained. Conclusions: The number of analyzed patients does not allow us to make any statistical conclusions. The co-existence of high expression of E6 protein with ~53 overexpression was ascertained and indicates that both of them can play a role in the pathogenesis of vulvar cancer. On the other hand, HPV and ~53 negative cancers suggest that in the pathogenesis of vulvar cancer also other factors can be involved.
P2.13.03 P53 POLYMORPHISM AND HUMAN PAPILLOMAVIRUS INFECTION IN HONG KONG WOMEN WITH CERVICAL CANCER Y.F. Wang, T.K.H. Chug, T.H. Cheung, V.W. Wang, Dept. OB/GYN, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. Objectives: A polymorphism at codon 72 of the ~53 gene, resulting in either an arginine or a proline residue in the protein, has been reported to affect the susceptibility of ~53 to human papillomavirus (HPV) E6mediated degradation in cultured cells. This study was to investigate the involvement of ~53 polymorphism at condo 72, the infection and typing of HPV, and the correlation of ~53 polymorphism with HPV and other clinico-pathologic characteristics in Hong Kong Chinese women with cervical cancer. Study Methods: Seventy-two primary cervical cancer specimens were obtained at surgery from consenting patients at the Department of Obstetrics and Gynecology, the Chinese University of Hong Kong. We developed a Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR - RFLP) method for analysis of ~53 polymorphism at codon 72 of exon 4 in cancer tissue DNA samples. The presence and types of HPV DNA in cancer were analyzed by PCR and DNA sequencing. Results: The homozygous ~53 arginine allele (Arg/Arg) was detected in 22 (31%), homozygous ~53 proline (Pro/Pro) in 14 (19%) and heterozygous allele (Arg/Pro) in 36 (50%) cases, respectively. HPV infection was detected in 55 of 72 (76%) cases. The prevalent types were HPV 16 (55%), HPV 18 (16%) and HPV 58 (9%). The numbers of HPV-positive cases with form Arg/Arg, Pro/Pro and Arg/Pro were 17 (31%), 12 (22%) and 26 (47%), respectively. The ~53 polymorphism at codon 72 was not significantly correlated with any of HPV types (p>O.O5). No striking overrepresentation of homozygous arginine-72 ~53 was observed in HPV-associated cervical cancer. No significant correlation was found between the ~53 polymorphism and histologic type, grade of cell differentiation, or clinical stage of cancer (p>O.O5). Conclusions: The results from this study, taken together with other reports, indicate that there may be geographic and ethnic variations in the ~53 polymorphic form in cervical cancer. However, the present study did not show any ~53 polymorphic form would have prognostic significance for cervical cancer. The information on prevalent types of HPV infection in Hong Kong Chinese women with cervical cancer, is pertinent to the development of HPV diagnostic probes and vaccination strategies (to prevent HPV type specific infection) in the future with particular reference to China.
TUESDAY, SEPTEMBER 5 P2.13.04 cDNA MICROARRAY APPLICATION ON CERVICAL EPITHELIAL CELLS W.B. Battistutti (l), M. Manavi (2), K. Pischinger (l), E. Kubista (2), K. Czerwenka (l), University Hospital, Vienna, Austria. (1) Dept. GYN/PATH (2) Dept. SPEC/GYN Objectives: The biology and structural behavior of a cell is determined by the gene expression within that cell. Today, there are over l,OOO,OOO human tag, sequences are available on public database (httu://www.ncbi.nlm.-nih.eovh. The cDNA microarray technique is a recently developed tool that exploits this wealth of information for the analysis of gene expression. Study Methods: Gene expression monitoring using microarrays was first described using radioactive targets hybridized into filter-immobilized cDNA clones by Drmanac et al. (1996). The “Atlas array” method makes use of nylon membranes with specially prepared cDNA spots. This enables a direct comparison of the expression profiles of mRNA populations. In the first step, 2 ml of each Poly A+RNA population are catalyzed with the reverse transcriptase enzyme and with (a-32 P) dATP. All complex radioactively-labeled cDNA probes are then individually hybridized on the “Atlas array”, analyzed using autoradiography and semiquantified. The level of cDNA expression of two different mRNA matrices can be estimated by comparing signal intensities. Results: In normal and cancerous cervical cells, the transcribed gene patterns demonstrate up/down -regulation of functional groups, similar to transcription e.g.: 1) oncogenes, 2) tumor suppressor genes, 3) cell-cycle regulator proteins, 4) transcription factors. Conclusions: Neoplastic cells have numerous acquired genetic abnormalities and these changes in signal transduction in cellular neoplasias give rise to different levels of gene expression. The variations in the gene expression patterns are observable and are characteristic in the cancerogenesis of malignant tumors.
P2.13.05 LOSS OF HETEROZYGOSITY AT 6~21.3 AND THE PRESENCE OF THE HPV-16 E6 350G VARIANT IN CERVICAL CANCER. lz3H. ‘V.V.V.S. Murty,3N. Martinez,3C.M. Wheeler, 4N. Beleiio, 5G. Martinez, 6H. Posse. ‘Genetics and Molecular Biology, 4Pathology, 5Gynecology, 6Epidemiology, Instituto National de Cancerologia, Santa Fe de Bogot6, Colombia. ‘Patology, Columbia University, New York. 3Health Sciences Center, Epidemiology and Cancer control, U. of New Mexico, Albuquerque. Objectives: The aim of the study was to analyze loss of heterozygosity (LOH) at 6~21.3 and the presence of human papillomavirus (HPV) type 16 E6 variants in invasive cervical cancer (ICC). Study Methods: 61 ICC were analyzed for the presence of LOH, and HPV 16 E6 variants were detected by lineage-specific hybridization. Results: LOH was observed in at least one locus in 37 of 61 (61%) tumors. The highest frequency of LOH was for D6S265 (54%) followed by MOGCAl(50%), D6S276 (48%), D6S273 (47%), D6S291(43%), and TAP1 (41%). HPV 16 was found in 48 of 61(79’?‘)D samples. A significant difference in the prevalence of HPV 16 positive tumors was detected between those presenting with LOH and those in which no LOH was observed. In this tumors, the most prevalent HPV 16 variant was the 350G (E-350G, 52%) followed by the Prototype (E-P, 31%) and the Asian-American (AA, 17%) variant. This difference in variant distribution is significant (p=O.O19). Further, in the presence of LOH, the 350G variant frequency was significantly different when compared to the AA variant (p=O.OOS), but not when compared to the prototype (p=O.ll). Conclusions: LOH is a frequent event in the 6~21.3 region, which contains the genes of the major histocompatibility complex (MHC). This observation could in part explain the derregulation of the MHC molecules, an event commonly observed in ICC. In addition, the presence of the 350G variant can influence the appearance of genetic instability as measured by LOH and thus may contribute to the malignant potential of HPV 16 infected epithelia.