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Detection of human papillomavirus (HPV) in cervical smears using real-time PCR and SybrGreen
330
Posters: Orthopedic Pathology / Pathology - Research and Practice 200 (2004) 330-333
Aims: Apaf-1 is one of the key regulators in hypoxia-mediated
Aims: Human papillomavirus are strongly associated with cervi-
apoptotis. Loss of Apaf-1 expression leads to apoptotic resistance in vitro. Hypoxic cervical carcinomas with a low apoptotic index have a poor prognosis. The aim of this study was to analyze the role of Apaf-1 expression in cervical cancer and to characterize its potential dependance on the intratumoural pOz-level. Methods: From 88 cervical cancer patients, tumour biopsies were taken after Eppendorf pO2-measurements. The tumour tissue was labelled immunohistochemically with polyclonal anti-Apaf-1. The tumour staining intensity and the percentage of Apaf-1 positive cells were evaluated by two independent blinded pathologists. Statistical analysis using SPSS was performed with regard to the intratumoural pOz-level, tumour classification, histopathological criteria and clinical data. Results: In our group of patients, hypoxic tumours had a significantly higher pT- and FIGO-stage than normoxic tumours (p < 0.05). 95.5% of all carcinomas showed a positive Apaf-1 staining. However, we identified a group of hypoxic tumours with an unexpected weak Apaf-1 expression. In all other carcinomas, intratumoural hypoxia and Apaf-1 expression correlated significantly (p = 0.000). Conclusions: Hypoxic tumours showed a higher pathologic and clinical stage than normoxic tumours. The majority of the cervical carcinomas expressed Apaf- 1. There is a group of hypoxic tumours with a very low expression of Apaf-1. Unlike the other carcinomas, these tumours might have lost their ability to undergo apoptosis. The lack of Apaf-1 expression in some tumours could be an explanation for the observed apoptotic resistance in hypoxic cervical cancer.
cal cancer. The polymerase chain reaction (PCR), using either consensus or type-specific primers is known as a highly sensitive and specific method for the detection of HPV. Up to now the RealTime PCR has been used for quantification of specific HPV genotypes. Aim of this study was to develop a broad spectrum screening assay for detection of HPV by Real-Time PCR. Material and methods: A total of 103 cervical smears on liquid base, interpreted cytologically either as ASCUS (n = 54), LSIL (n = 35) or HSIL (n = 14) were examined. For Real-Time PCR, SybrGreen dye was used with the CPI/CPIIG primer pair. All samples were further analysed by conventional PCR using the consensus Primers GP5+/6+ and a seminested PCR using the consensus primers MY09/GP1/GP2. HPV genotype was determined by DNA sequencing. Results: HPV DNA was detected in 67 (65%) samples by either of the methods. 22 (40.7%) of 54 ASCUS, 31 (88.6%) of 35 LSIL and 14 (100%) of 14 HSIL were HPV DNA positive. Of the 67 samples containing HPV DNA, 23 (34.3%) tested positive by Real-Time PCR, 51 (76.1%) by conventional PCR and 63 (94%) by seminested PCR. Overall 18 different HPV genotypes were identified. Conclusions: Using Real-Time PCR and SybrGreen for HPV screening would be appealing due to easy handling of samples and reduced risk of contamination. However, using a single pair of consensus primers and SybrGreen as performed in this study revealed a low sensitivity compared to the other PCR methods applied. Improvement may be achieved by using a mixture of different primers.
279 Tumorbiologic parameters of the samm cell neuroendocrine carcinoma of the uterine cervix L.-C. HORN 1, K. LINDNEW, K. BILEK 2, J. EINENKEL 2 Institut f'tir Pathologie, Gynakopathologie, Universit~it Leipzig 2Universit~its-Frauenklinik Leipzig Triersches Institut)
Aims: Small cell neuroendocrine carcinomas (SCC) of the uterine cervix are rare tumors. The knowledge of p16- and p53-expression and proliferative activity is limited. Methods: SCC were selected from our files for immunohistochemical staining (neuroendocrine markers, Ki-67, p53 and p16) and follow-up analysis. In cases with mixed tumors, the percentage of SCC-component was calculated and correlated with survival. Results: Nine out of 677 tumors (1.3%) were classified as SCC after Grimelius-staining (8/9 tumors positive) and immunoreaction against NSE and chromogranin A (7/9 positive tumors). Two SCC's represented p53-positivity. All cases showed strong p 16-immunostaining. Four patients died of the tumor after a median time of 36.7 months (range 15 to 56 months). Even a SCC-component of 17% was associated with fatal course. SCC represented significant lower proliferation (Ki-67 labeling-index) than the non-small-cell component in the same tumor (12.8 vs. 70.8%; p < 0.001). Conclusions: The results suggest that p16 is upregulated or accumulated in the SCC of the uterine cervix, probably caused by infection with high risk HPV. Even a small SCC-component in mixed carcinoma of the uterine cervix is associated with adverse outcome, despite low proliferative activity. It is necessary for the pathologist to recognize the presence of NEC-components in mixed carcinomas in order to prevent a misdiagnosis.
280 Detection of Human papillomavirus (HPV) in Cervical Smears using Real-Time PCR and SybrGreen D. KAUP, C. GERBER, M. BAUMANN, A. STALDER, R. GAUDENZ, G. CATHOMAS Kantonales Institut f'tir Pathologie Liestal, Labor ftir Infektionspathologie u. Frauenklinik Kantonsspital Liestal, Schweiz
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281
Comparative analysis of expression of cell cycle-related gene productes in localised versus diffuse pigmented villonodular synovitis H. W E C K A U F 1, B. HELMCHEN I, U. HINZ 2, C. FLECHTENMACHER 1, H. F. OTTO 1, I. BERGER 1 1Institute of Pathology, Ruprecht-Karls Univetsity, Heidelberg 2Department of Surgery, Ruprecht-Karls-University, Heidelberg
Aim: Pigmented villonodular synovitis (PVNS) is an aggressive disease with progressive joint destruction mostly considered a benign neoplasm. The study aims to examine expression patterns of proteins involved in cell cycle regulation in neoplastic synovial cells in relation to the clinical behaviour of the different forms of PVNS. Methods: Expression of p16, CDK4, cyclineD1, RB, E2F, p53, p63, p21, p27 and Ki-67 in proliferating synovial cells in primary and recurrent tumours in locafised and diffuse PVNS were examined by tissue array and analysed statistically. Results: P53 overexpression was detected in both forms of PVNS (diffuse: 34%, localised: 39.1%), there was no significant difference (p = 0.794). Nuclear staining of CDK4 was significantly more frequent in diffuse (30.4%) versus localised (8.7%) PVNS (p = 0.046). A loss of p16 was seen in 91.3% of localised versus 58.9% of diffuse PVNS (p = 0.0067). Los of nuclear staining of RB was detected in 73,9% of localised and 60,7% of diffuse PVNS (p = 0.309). All other analysed cell cycle related proteins (p21, p63, cyclineD1 and E2F) did not differ significantly in their staining pattern. Analysis of expression all investigated cell cycle related proteins did not reveal statistically significant differences between primary and recurrent tumours.
Posters: Orthopedic Pathology / Pathology - Research and Practice 200 (2004) 330-333
Aims: Apaf-1 is one of the key regulators in hypoxia-mediated
Aims: Human papillomavirus are strongly associated with cervi-
apoptotis. Loss of Apaf-1 expression leads to apoptotic resistance in vitro. Hypoxic cervical carcinomas with a low apoptotic index have a poor prognosis. The aim of this study was to analyze the role of Apaf-1 expression in cervical cancer and to characterize its potential dependance on the intratumoural pOz-level. Methods: From 88 cervical cancer patients, tumour biopsies were taken after Eppendorf pO2-measurements. The tumour tissue was labelled immunohistochemically with polyclonal anti-Apaf-1. The tumour staining intensity and the percentage of Apaf-1 positive cells were evaluated by two independent blinded pathologists. Statistical analysis using SPSS was performed with regard to the intratumoural pOz-level, tumour classification, histopathological criteria and clinical data. Results: In our group of patients, hypoxic tumours had a significantly higher pT- and FIGO-stage than normoxic tumours (p < 0.05). 95.5% of all carcinomas showed a positive Apaf-1 staining. However, we identified a group of hypoxic tumours with an unexpected weak Apaf-1 expression. In all other carcinomas, intratumoural hypoxia and Apaf-1 expression correlated significantly (p = 0.000). Conclusions: Hypoxic tumours showed a higher pathologic and clinical stage than normoxic tumours. The majority of the cervical carcinomas expressed Apaf- 1. There is a group of hypoxic tumours with a very low expression of Apaf-1. Unlike the other carcinomas, these tumours might have lost their ability to undergo apoptosis. The lack of Apaf-1 expression in some tumours could be an explanation for the observed apoptotic resistance in hypoxic cervical cancer.
cal cancer. The polymerase chain reaction (PCR), using either consensus or type-specific primers is known as a highly sensitive and specific method for the detection of HPV. Up to now the RealTime PCR has been used for quantification of specific HPV genotypes. Aim of this study was to develop a broad spectrum screening assay for detection of HPV by Real-Time PCR. Material and methods: A total of 103 cervical smears on liquid base, interpreted cytologically either as ASCUS (n = 54), LSIL (n = 35) or HSIL (n = 14) were examined. For Real-Time PCR, SybrGreen dye was used with the CPI/CPIIG primer pair. All samples were further analysed by conventional PCR using the consensus Primers GP5+/6+ and a seminested PCR using the consensus primers MY09/GP1/GP2. HPV genotype was determined by DNA sequencing. Results: HPV DNA was detected in 67 (65%) samples by either of the methods. 22 (40.7%) of 54 ASCUS, 31 (88.6%) of 35 LSIL and 14 (100%) of 14 HSIL were HPV DNA positive. Of the 67 samples containing HPV DNA, 23 (34.3%) tested positive by Real-Time PCR, 51 (76.1%) by conventional PCR and 63 (94%) by seminested PCR. Overall 18 different HPV genotypes were identified. Conclusions: Using Real-Time PCR and SybrGreen for HPV screening would be appealing due to easy handling of samples and reduced risk of contamination. However, using a single pair of consensus primers and SybrGreen as performed in this study revealed a low sensitivity compared to the other PCR methods applied. Improvement may be achieved by using a mixture of different primers.
279 Tumorbiologic parameters of the samm cell neuroendocrine carcinoma of the uterine cervix L.-C. HORN 1, K. LINDNEW, K. BILEK 2, J. EINENKEL 2 Institut f'tir Pathologie, Gynakopathologie, Universit~it Leipzig 2Universit~its-Frauenklinik Leipzig Triersches Institut)
Aims: Small cell neuroendocrine carcinomas (SCC) of the uterine cervix are rare tumors. The knowledge of p16- and p53-expression and proliferative activity is limited. Methods: SCC were selected from our files for immunohistochemical staining (neuroendocrine markers, Ki-67, p53 and p16) and follow-up analysis. In cases with mixed tumors, the percentage of SCC-component was calculated and correlated with survival. Results: Nine out of 677 tumors (1.3%) were classified as SCC after Grimelius-staining (8/9 tumors positive) and immunoreaction against NSE and chromogranin A (7/9 positive tumors). Two SCC's represented p53-positivity. All cases showed strong p 16-immunostaining. Four patients died of the tumor after a median time of 36.7 months (range 15 to 56 months). Even a SCC-component of 17% was associated with fatal course. SCC represented significant lower proliferation (Ki-67 labeling-index) than the non-small-cell component in the same tumor (12.8 vs. 70.8%; p < 0.001). Conclusions: The results suggest that p16 is upregulated or accumulated in the SCC of the uterine cervix, probably caused by infection with high risk HPV. Even a small SCC-component in mixed carcinoma of the uterine cervix is associated with adverse outcome, despite low proliferative activity. It is necessary for the pathologist to recognize the presence of NEC-components in mixed carcinomas in order to prevent a misdiagnosis.
280 Detection of Human papillomavirus (HPV) in Cervical Smears using Real-Time PCR and SybrGreen D. KAUP, C. GERBER, M. BAUMANN, A. STALDER, R. GAUDENZ, G. CATHOMAS Kantonales Institut f'tir Pathologie Liestal, Labor ftir Infektionspathologie u. Frauenklinik Kantonsspital Liestal, Schweiz
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281
Comparative analysis of expression of cell cycle-related gene productes in localised versus diffuse pigmented villonodular synovitis H. W E C K A U F 1, B. HELMCHEN I, U. HINZ 2, C. FLECHTENMACHER 1, H. F. OTTO 1, I. BERGER 1 1Institute of Pathology, Ruprecht-Karls Univetsity, Heidelberg 2Department of Surgery, Ruprecht-Karls-University, Heidelberg
Aim: Pigmented villonodular synovitis (PVNS) is an aggressive disease with progressive joint destruction mostly considered a benign neoplasm. The study aims to examine expression patterns of proteins involved in cell cycle regulation in neoplastic synovial cells in relation to the clinical behaviour of the different forms of PVNS. Methods: Expression of p16, CDK4, cyclineD1, RB, E2F, p53, p63, p21, p27 and Ki-67 in proliferating synovial cells in primary and recurrent tumours in locafised and diffuse PVNS were examined by tissue array and analysed statistically. Results: P53 overexpression was detected in both forms of PVNS (diffuse: 34%, localised: 39.1%), there was no significant difference (p = 0.794). Nuclear staining of CDK4 was significantly more frequent in diffuse (30.4%) versus localised (8.7%) PVNS (p = 0.046). A loss of p16 was seen in 91.3% of localised versus 58.9% of diffuse PVNS (p = 0.0067). Los of nuclear staining of RB was detected in 73,9% of localised and 60,7% of diffuse PVNS (p = 0.309). All other analysed cell cycle related proteins (p21, p63, cyclineD1 and E2F) did not differ significantly in their staining pattern. Analysis of expression all investigated cell cycle related proteins did not reveal statistically significant differences between primary and recurrent tumours.