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P203 Presence and influence of human papillomaviruses in locally advanced tonsillar squamous cell carcinomas receiving concurrent chemoradiotherapy
192 P202 Fatty acid synthase (FAS) and the isopeptidase usp2a are highly expressing in erb-b2 positive oral squamous cell carcinoma S.D. Silva1 , I.W. da Cunha2 , F.A. Soares2 , I.N. Nishimoto2 , L.P. Kowalski2 *, D.M. Carraro2 , E. Graner1 . 1 UNICAMP, Brazil, 2 Funda¸ca ˜o Antˆ onio Prudente, Brazil Background: ErbB-2 overexpression has been described in oral squamous cell carcinoma (OSCC). Fatty acid synthase (FAS), the key lipogenic enzyme responsible for the endogenous synthesis of fatty acids, is one of the downstream genes regulated by ErbB-2. Deubiquitinating enzymes or isopeptidases prevent the destruction of protein substrates through deubiquitination prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) plays a critical role in prostate cancer cell survival through FAS stabilization. This study investigates whether the expression of ErbB2, FAS and USP2a is correlated with the clinicopathological characteristics of OSCC. Methods: Were included in this study 41 frozen samples of the patients with OSCC from the Hospital do Cancer A.C. Camargo, S˜ ao Paulo, Brazil. Clinical and treatment data were obtained from the medical records and all histolopathologic diagnosis were reviewed. OSCC was microdissected using laser capture microdissection and one round of linear mRNA amplification was done based on template switch and T7-driven amplification. ErbB2, FAS and USP2a expression were analyzed by real time RT-PCR and compared to normal morphologically tissue of the same source. Results: Our study showed a strong positive correlation between ErbB2, FAS and USP2a expression in the OSCC samples. Microscopic characteristics as perineural infiltration was associated with ErbB2 (p = 0.046). The clinical stage N+ was associated with ErbB2 (p = 0.001), FAS (p = 0.002) and USP2a expression (p = 0.006). Additionally, ErbB2, FAS and USP2a were significantly associated (p = 0.001). Conclusion: Here, we have identified a molecular link between ErbB2 oncogene, FAS and USP2a. These data strongly suggest that high-level expression of ErbB2 and FAS in OSCC is linked with USP2a expression and both involved with tumor progression. Keywords: Squamous cell carcinoma, Tumor progression, Molecular Biology, Deubiquitinating enzymes
P203 Presence and influence of human papillomaviruses in locally advanced tonsillar squamous cell carcinomas receiving concurrent chemoradiotherapy J.M. Jian *, Y.L. Chung. Sun Yat-Sen Cancer Center, Tajikistan Purpose: Human papillomavirus (HPV) and Epstein-Barr virus (EBV) have both been implicated as causative agents in subsets of head and neck cancers. This study was undertaken to discern an association for HPV or EBV with tonsillar squamous cell carcinoma (TSCC), and to determine whether viral infection disturbs p53, pRB, and p21 expression and affects prognosis after concurrent chemoradiotherapy (CCRT). Experimental design: Tumor tissue array was carried out on serial sections from archival specimens from 46 continuous patients with locally advanced TSCCs (AJCC stage III-IVB). They all received CCRT at our institution (median followup, 70 months). Specimens were tested for the presence of HPV genome integration or EBV episome by use of real-time polymerase chain reaction (PCR)-based gene chip assays, DNA sequencing, southern blotting, and/or in-situ hybridization. Alterations of cell cycle-related gene expression were evaluated by immunohistochemical staining. The association of virus presence with p53, pRB, and p21 alterations was correlated to patient characteristics, tumor response and clinical outcome after CCRT.
Poster abstracts, Saturday 19 May Results: HPV DNA integration was found in 23 (50%) of the 46 patients. The HPV types detected were: HPV-16 (20 patients), and HPV-18, -26, and -33 (one patient each). In contrast, only 1 (2%) of the 46 patients was positive to EBV. The incidence between HPV and EBV infection in TSCC is significantly different (p < 0.0001). HPV status was not related to age, gender, and use of alcohol and/or tobacco, but the cells of HPV-positive TSCCs was more poorly differentiated than the cells of HPV-negative TSCCs (61% vs. 9%, p = 0.0002). Because the nuclear immunoreactivities of p53, pRB and p21 showed no detectable or only low levels in normal tonsillar tissues, they were considered altered or mutated when at least 10% of tumor cells demonstrated strong nuclear activity. Thirteen (58%) of the HPV-negative patients and 7 (30%) of the HPVpositive patients had either p53 or pRB alteration (p = 0.07). As compared with the HPV-negative TSCCs, the HPV-positive TSCCs were less likely to have >50% cells with p53 or pRB overexpression (9% vs 35%, p = 0.0024), which is consistent with the degradation of p53 and pRB by the HPV oncoproteins. The incremental numbers of altered cell cycle markers of p53, pRB, and p21 overexpression were independently associated with not only HPV-negative TSCCs (HPV-negative vs. HPV-positive: 35% vs. 17%, p = 0.02) but also decreased clinical outcomes after CCRT. Patients with HPV-positive TSCCs had better overall survival rates (91% vs. 43% at 3-year, and 86% vs. 35% at 5-year, p = 0.0003), local control rates (87% vs. 46% at 3-year, and 87% vs. 41% at 5-year, p = 0.0006), and free of distant metastasis (96% vs. 83% at 3-year, and 96% vs. 72% at 5-year, p = 0.077) when compared with HPV-negative TSCC patients. Conclusions: The results suggest that HPV may be involved in the genesis of a subset of head and neck cancers, such as TSCC. These data extend molecular and epidemiologic studies and strongly suggest that HPV-positive TSCC comprises a distinct molecular, clinical, and pathologic disease entity that has a markedly improved prognosis after the treatment of CCRT. Keywords: Human papillomavirus, Tonsillar cancer, Cocurrent chemoradiotherapy
P204 Effects of heparin-binding epidermal growth factor on the invasion activity of a oral cancer cell line M. Furukawa *, Y. Ohnisi, K. Kakudo. Osaka Dental University, Japan Aims: There has been reported that Oral squamous cell carcinoma (SCC) display overexpression of EGF receptors, so EGF and EGF reseptors are one of the regulatory factors for the production of matrix metalloproteinase (MMP) and modulates growth and differentiation of various cancer cells. In the present study, we analyzed the effects of heparin-binding(HB)EGF, one of the EGFR family of growth factor, on the invasion activity of a cultured oral cancer cell line, HSC3, using the short interfering RNA (siRNA)method. Methods: Cells were grown in the presence or absence of HB-EGF. An oral squamous carcinoma cell line, HSC3, was transfected with short interfering RNA (siRNA) of HB-EGF (HSC3-siRNA). Expressions of HB-EGF and MMP-9 were analyzed by RT-PCR. The invasive ability of the transfected cells was determined using a matrigel invasion assay and MMP-9 production was measured using gelatin zymography. Results: The expressions of HB-EGF and MMP-9 were dramatically reduced in HSC3-siRNA. Matrigel invasion assay demonstrated that the HSC3 had a higher invasive ability, but HSC3-siRNA was lowere. Gelatin zymography demonstrated that in HSC3-siRNA, MMP-9 production was decreased. Conclusions: These findings suggest that HB-EGF siRNA is reduse the invasion activity of oral cancer, particularly by regulation of MMP-9. Keywords: HB-EGF, SCC, invasion, MMP9
P203 Presence and influence of human papillomaviruses in locally advanced tonsillar squamous cell carcinomas receiving concurrent chemoradiotherapy J.M. Jian *, Y.L. Chung. Sun Yat-Sen Cancer Center, Tajikistan Purpose: Human papillomavirus (HPV) and Epstein-Barr virus (EBV) have both been implicated as causative agents in subsets of head and neck cancers. This study was undertaken to discern an association for HPV or EBV with tonsillar squamous cell carcinoma (TSCC), and to determine whether viral infection disturbs p53, pRB, and p21 expression and affects prognosis after concurrent chemoradiotherapy (CCRT). Experimental design: Tumor tissue array was carried out on serial sections from archival specimens from 46 continuous patients with locally advanced TSCCs (AJCC stage III-IVB). They all received CCRT at our institution (median followup, 70 months). Specimens were tested for the presence of HPV genome integration or EBV episome by use of real-time polymerase chain reaction (PCR)-based gene chip assays, DNA sequencing, southern blotting, and/or in-situ hybridization. Alterations of cell cycle-related gene expression were evaluated by immunohistochemical staining. The association of virus presence with p53, pRB, and p21 alterations was correlated to patient characteristics, tumor response and clinical outcome after CCRT.
Poster abstracts, Saturday 19 May Results: HPV DNA integration was found in 23 (50%) of the 46 patients. The HPV types detected were: HPV-16 (20 patients), and HPV-18, -26, and -33 (one patient each). In contrast, only 1 (2%) of the 46 patients was positive to EBV. The incidence between HPV and EBV infection in TSCC is significantly different (p < 0.0001). HPV status was not related to age, gender, and use of alcohol and/or tobacco, but the cells of HPV-positive TSCCs was more poorly differentiated than the cells of HPV-negative TSCCs (61% vs. 9%, p = 0.0002). Because the nuclear immunoreactivities of p53, pRB and p21 showed no detectable or only low levels in normal tonsillar tissues, they were considered altered or mutated when at least 10% of tumor cells demonstrated strong nuclear activity. Thirteen (58%) of the HPV-negative patients and 7 (30%) of the HPVpositive patients had either p53 or pRB alteration (p = 0.07). As compared with the HPV-negative TSCCs, the HPV-positive TSCCs were less likely to have >50% cells with p53 or pRB overexpression (9% vs 35%, p = 0.0024), which is consistent with the degradation of p53 and pRB by the HPV oncoproteins. The incremental numbers of altered cell cycle markers of p53, pRB, and p21 overexpression were independently associated with not only HPV-negative TSCCs (HPV-negative vs. HPV-positive: 35% vs. 17%, p = 0.02) but also decreased clinical outcomes after CCRT. Patients with HPV-positive TSCCs had better overall survival rates (91% vs. 43% at 3-year, and 86% vs. 35% at 5-year, p = 0.0003), local control rates (87% vs. 46% at 3-year, and 87% vs. 41% at 5-year, p = 0.0006), and free of distant metastasis (96% vs. 83% at 3-year, and 96% vs. 72% at 5-year, p = 0.077) when compared with HPV-negative TSCC patients. Conclusions: The results suggest that HPV may be involved in the genesis of a subset of head and neck cancers, such as TSCC. These data extend molecular and epidemiologic studies and strongly suggest that HPV-positive TSCC comprises a distinct molecular, clinical, and pathologic disease entity that has a markedly improved prognosis after the treatment of CCRT. Keywords: Human papillomavirus, Tonsillar cancer, Cocurrent chemoradiotherapy
P204 Effects of heparin-binding epidermal growth factor on the invasion activity of a oral cancer cell line M. Furukawa *, Y. Ohnisi, K. Kakudo. Osaka Dental University, Japan Aims: There has been reported that Oral squamous cell carcinoma (SCC) display overexpression of EGF receptors, so EGF and EGF reseptors are one of the regulatory factors for the production of matrix metalloproteinase (MMP) and modulates growth and differentiation of various cancer cells. In the present study, we analyzed the effects of heparin-binding(HB)EGF, one of the EGFR family of growth factor, on the invasion activity of a cultured oral cancer cell line, HSC3, using the short interfering RNA (siRNA)method. Methods: Cells were grown in the presence or absence of HB-EGF. An oral squamous carcinoma cell line, HSC3, was transfected with short interfering RNA (siRNA) of HB-EGF (HSC3-siRNA). Expressions of HB-EGF and MMP-9 were analyzed by RT-PCR. The invasive ability of the transfected cells was determined using a matrigel invasion assay and MMP-9 production was measured using gelatin zymography. Results: The expressions of HB-EGF and MMP-9 were dramatically reduced in HSC3-siRNA. Matrigel invasion assay demonstrated that the HSC3 had a higher invasive ability, but HSC3-siRNA was lowere. Gelatin zymography demonstrated that in HSC3-siRNA, MMP-9 production was decreased. Conclusions: These findings suggest that HB-EGF siRNA is reduse the invasion activity of oral cancer, particularly by regulation of MMP-9. Keywords: HB-EGF, SCC, invasion, MMP9