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Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2 (LMP2) in nasopharyngeal carcinoma
Posters: Pathology of the Lung and Upper Respiratory Tract / Pathology - Research and Practice 200 (2004) 283-285
Aims: Array-based comparative genomic hybridization (aCGH) is a powerful new technology capable of identifying allelic imbalances at a high resolution by co-hybridizing differentially labelled test and control DNAs to a microarray of genomic clones. Here, we analysed a series of 18 Barrett's adenocarcinomas (BCA) by aCGH. Methods: For aCGH, a chip was used containing 287 genomic targets covering oncogenes, tumour suppressor genes as well as chromosome regions frequently altered in several human cancer types. For validation, selected clones found to be amplified (SNRPN, CMYC, HER2) were confirmed by Fluorescence in situ hybridization (FISH). Results: aCGH revealed DNA gains and losses in all 18 BCA. DNA sequence copy number gains (mean 41 -+ 25/BCA) were more frequent than deletions (mean 20 _+ 15/BCA). Several genes not known to be involved in BCA were identified. Highest frequency of allelic gains was seen for SNRPN (61%), GNLY (44%), NME1 (44%), DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, TNFRSF6B (39 % each), MSH2, TERC, SERPINE1, AFM137XAll, IGFR1, PTPN1 (33% each). Allelic losses were identified for PDGFB (44%), D17S125 (39%), AKT3 (28%), RASSFI, FHIT, CDKN2A (pl6) and SAS (CDK4) (28% each). Gene amplification of SNRPN, CMYC and HER2 were confirmed by FISH. Conclusions: These data show the superior sensitivity of the aCGH for the identification of allelic gains and losses in BCA. The newly identified genes may include biomarkers of increased risk in patients with Baxrett's esophagus and are therefore a starting point for studies elucidating their role in Barrett's premalignant lesions. + ....
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109 Allelic loss, MIB-1 labeling index and HPV-DNA as supportive diagnostic markers in epithelial hyperplasticlaryngeal lesions B. KLEIST 1, D. JUNGHANS I, G. LORENZ 1, M. POETSCH 2 11nstitut ftir Pathologie, Universit~it Greifswald 2Institut ftir Rechtsmedizin, Universit/it Greifswald
Aims: Various endogenous and environmental factors are involved in laryngeal carcinogenesis. Here, we tested loss of heterozygosity (LOH) and microsatellite instability (MSI) on chromosomes 2, 3, 5, 9, 10, 11, 13, 17 and 18, the p53 protein and MIB-1 expression status and presence of HPV-DNA for their potential to characterize different stages of epithelial hyperplastic laryngeal lesions (EHLL). Methods: Thirty-two laryngeal biopsies were classified into four grades of the Ljubljana classification (simple = SH, abnormal = AbH, atypical hyperplasia = AtH and carcinoma in situ) and investigated by immunohistochemical methods, PCR and direct sequencing analysis. Results: MIB-1 labeling increased continuously with histopathological progression of the lesions, whereas a remarkable difference in p53 positivity was found only in the step from SH to AbH. The frequency of LOH (in general) increased with progressive grades of the lesions, but none of the altered loci (9p, 9q, 10q, l l q , 17p) proved to be sufficient as distinctive marker between different stages of EHLL. MSI did not clearly correlate to histopathological grading. HPV-DNA was detected mostly in SH and AbH (both: 66.7%). Conclusions: MIB-1 labeling and allelic loss could be helpful in the distinction of different stages of EHLL. Presence of HPV-DNA
283
may probably characterize a group of non-risky (reactive) lesions within EHLL.
110
Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2 (LMP2) in nasopharyngeal carcinoma N. HEUSSINGER, M. BUTTNER, G. OTT 1, E. BRACHTEL 2, B .Z. PILCH 2, E. KREMMER 3, G. NIEDOBITEK Pathologisches Institut, Universit~it Erlangen-Nfimberg Pathologisches Institut, Universitat Wiirzburg 2Department of Pathology, Massachussetts General Hospital, Boston, USA 3Institut ftir Molekulare Immunologie, GSF, Mtinchen
Aims: Immunotherapeutic approaches targeting EBV-encoded antigens are being explored for the treatment of Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC). Viral proteins expressed in IIL and NPC include the nuclear antigen EBNA1 and the latent membrane proteins (LMP) 1 and 2. LMP2 is the most promising target for immunotherapy since it can elicit a cytotoxic T-cell (CTL) response. However, expression of LMP2 in NPC has been shown only at the RNA level but as yet not at the protein level. Methods: Expression of LMP1 and LMP2 was studied in a series of 28 paraffin-embedded NPC specimens (25 EBV +, 3 EBV-) using immunohistochemistry and well characterised antibodies. Results: Expression of LMP2 was detected in the tumour cells of 12 EBV + NPCs (48%), 11 cases were negative, and 2 could not be evaluated. Three EBV- cases were L M P 2 . LMP1 expression was detected in 8 cases (2 LMP2 +, 6 LMPZ). Conclusions: We show that LMP2 is expressed at the protein level in almost 50% of EBV-associated NPCs. This observation should encourage attempts to employ LMP2-targeted immunotherapy in the treatment of NPC. We also observed a tendency for LMP1 and LMP2 expression to be mutually exclusive. The significance of this observation requires further investigation.
111
Synchronous adenocarcinomas of the inner nose and the colon after wood dust exposure K. DONHUIJSEN, H. HANNIG, H.-G. SCHROEDER 1, I. PETERSEN 2 Institut ftir Pathologie, Klinikum Braunschweig 1 HNO-Klinik, Kfinikum Braunschweig 2 Institut fitr Pathologie, Charitr, Humboldt-Universit~itBerlin
Aims: Intestinal types of adenocarcinoma of the inner nose - typical after wood dust exposure - and colorectal adenocarcinoma present a stupendous similarity of morphological and immunohistochemical features. A very rare case with synchronous manifestations of both in a 68-year-old joiner enabled us to compare the,tumours of different localisations by molecular and other methods. Methods: After microdissection of the tumour tissue, multiplex PCR was performed for microsatellite instability with loci BAT 25, BAT 26, D5 S 346 (APC), D17 S 250 (Mfdl5CA), D2 S 123. Expression as well as mutation of p53 mad K-ras was examined by direct DNA-sequencing. Chromosomal imbalances were investigated by CGH. Results: The adenocarcinoma of the colon ascendens (pT3, pN0, cM0, G2, R0) displayed tubular structures in most cases, whereas the intestinal type adenocarcinoma of the inner nose (cT2, G2) generally exhibited a papillary pattern. Mucin production, histochemical resuits and immunohistochemical reactions were identical, even for CD X2. PCR results for the 5 loci revealed no microsatellite instabil-
Aims: Array-based comparative genomic hybridization (aCGH) is a powerful new technology capable of identifying allelic imbalances at a high resolution by co-hybridizing differentially labelled test and control DNAs to a microarray of genomic clones. Here, we analysed a series of 18 Barrett's adenocarcinomas (BCA) by aCGH. Methods: For aCGH, a chip was used containing 287 genomic targets covering oncogenes, tumour suppressor genes as well as chromosome regions frequently altered in several human cancer types. For validation, selected clones found to be amplified (SNRPN, CMYC, HER2) were confirmed by Fluorescence in situ hybridization (FISH). Results: aCGH revealed DNA gains and losses in all 18 BCA. DNA sequence copy number gains (mean 41 -+ 25/BCA) were more frequent than deletions (mean 20 _+ 15/BCA). Several genes not known to be involved in BCA were identified. Highest frequency of allelic gains was seen for SNRPN (61%), GNLY (44%), NME1 (44%), DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, TNFRSF6B (39 % each), MSH2, TERC, SERPINE1, AFM137XAll, IGFR1, PTPN1 (33% each). Allelic losses were identified for PDGFB (44%), D17S125 (39%), AKT3 (28%), RASSFI, FHIT, CDKN2A (pl6) and SAS (CDK4) (28% each). Gene amplification of SNRPN, CMYC and HER2 were confirmed by FISH. Conclusions: These data show the superior sensitivity of the aCGH for the identification of allelic gains and losses in BCA. The newly identified genes may include biomarkers of increased risk in patients with Baxrett's esophagus and are therefore a starting point for studies elucidating their role in Barrett's premalignant lesions. + ....
: ::
: ~ P~t~:o[~::: i :: ~ ::: :
: ::::::: :: (
++
+ :
g ~d:::::
..:::::: ......:::....:..............: . ::: ~:
109 Allelic loss, MIB-1 labeling index and HPV-DNA as supportive diagnostic markers in epithelial hyperplasticlaryngeal lesions B. KLEIST 1, D. JUNGHANS I, G. LORENZ 1, M. POETSCH 2 11nstitut ftir Pathologie, Universit~it Greifswald 2Institut ftir Rechtsmedizin, Universit/it Greifswald
Aims: Various endogenous and environmental factors are involved in laryngeal carcinogenesis. Here, we tested loss of heterozygosity (LOH) and microsatellite instability (MSI) on chromosomes 2, 3, 5, 9, 10, 11, 13, 17 and 18, the p53 protein and MIB-1 expression status and presence of HPV-DNA for their potential to characterize different stages of epithelial hyperplastic laryngeal lesions (EHLL). Methods: Thirty-two laryngeal biopsies were classified into four grades of the Ljubljana classification (simple = SH, abnormal = AbH, atypical hyperplasia = AtH and carcinoma in situ) and investigated by immunohistochemical methods, PCR and direct sequencing analysis. Results: MIB-1 labeling increased continuously with histopathological progression of the lesions, whereas a remarkable difference in p53 positivity was found only in the step from SH to AbH. The frequency of LOH (in general) increased with progressive grades of the lesions, but none of the altered loci (9p, 9q, 10q, l l q , 17p) proved to be sufficient as distinctive marker between different stages of EHLL. MSI did not clearly correlate to histopathological grading. HPV-DNA was detected mostly in SH and AbH (both: 66.7%). Conclusions: MIB-1 labeling and allelic loss could be helpful in the distinction of different stages of EHLL. Presence of HPV-DNA
283
may probably characterize a group of non-risky (reactive) lesions within EHLL.
110
Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2 (LMP2) in nasopharyngeal carcinoma N. HEUSSINGER, M. BUTTNER, G. OTT 1, E. BRACHTEL 2, B .Z. PILCH 2, E. KREMMER 3, G. NIEDOBITEK Pathologisches Institut, Universit~it Erlangen-Nfimberg Pathologisches Institut, Universitat Wiirzburg 2Department of Pathology, Massachussetts General Hospital, Boston, USA 3Institut ftir Molekulare Immunologie, GSF, Mtinchen
Aims: Immunotherapeutic approaches targeting EBV-encoded antigens are being explored for the treatment of Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC). Viral proteins expressed in IIL and NPC include the nuclear antigen EBNA1 and the latent membrane proteins (LMP) 1 and 2. LMP2 is the most promising target for immunotherapy since it can elicit a cytotoxic T-cell (CTL) response. However, expression of LMP2 in NPC has been shown only at the RNA level but as yet not at the protein level. Methods: Expression of LMP1 and LMP2 was studied in a series of 28 paraffin-embedded NPC specimens (25 EBV +, 3 EBV-) using immunohistochemistry and well characterised antibodies. Results: Expression of LMP2 was detected in the tumour cells of 12 EBV + NPCs (48%), 11 cases were negative, and 2 could not be evaluated. Three EBV- cases were L M P 2 . LMP1 expression was detected in 8 cases (2 LMP2 +, 6 LMPZ). Conclusions: We show that LMP2 is expressed at the protein level in almost 50% of EBV-associated NPCs. This observation should encourage attempts to employ LMP2-targeted immunotherapy in the treatment of NPC. We also observed a tendency for LMP1 and LMP2 expression to be mutually exclusive. The significance of this observation requires further investigation.
111
Synchronous adenocarcinomas of the inner nose and the colon after wood dust exposure K. DONHUIJSEN, H. HANNIG, H.-G. SCHROEDER 1, I. PETERSEN 2 Institut ftir Pathologie, Klinikum Braunschweig 1 HNO-Klinik, Kfinikum Braunschweig 2 Institut fitr Pathologie, Charitr, Humboldt-Universit~itBerlin
Aims: Intestinal types of adenocarcinoma of the inner nose - typical after wood dust exposure - and colorectal adenocarcinoma present a stupendous similarity of morphological and immunohistochemical features. A very rare case with synchronous manifestations of both in a 68-year-old joiner enabled us to compare the,tumours of different localisations by molecular and other methods. Methods: After microdissection of the tumour tissue, multiplex PCR was performed for microsatellite instability with loci BAT 25, BAT 26, D5 S 346 (APC), D17 S 250 (Mfdl5CA), D2 S 123. Expression as well as mutation of p53 mad K-ras was examined by direct DNA-sequencing. Chromosomal imbalances were investigated by CGH. Results: The adenocarcinoma of the colon ascendens (pT3, pN0, cM0, G2, R0) displayed tubular structures in most cases, whereas the intestinal type adenocarcinoma of the inner nose (cT2, G2) generally exhibited a papillary pattern. Mucin production, histochemical resuits and immunohistochemical reactions were identical, even for CD X2. PCR results for the 5 loci revealed no microsatellite instabil-