- Email: [email protected]
FXIII+ in Oral Squamous Cell Carcinoma
Poster Abstracts Oral AbstractsPoster ListOrals ListPan. Disc. & Symp. Abs.Keynote Abs.Keynote Bios.ProgramIAOOWelcomeCommittee Listings
214
Poster session III et al. / Oral Oncology Supplement 3 (2009) 201–236
Conclusion: Our results suggested that H-1 and H-1R cells may be useful for searching the candidate genes responsible for crossresistance to platinum derivatives and for further studies to understand the mechanism of platinum resistance. doi:10.1016/j.oos.2009.06.566
P3.41. Transcriptome profiling and network pathway analysis of genes associated with invasive phenotype in oral cancer A.J.C. Cheng a,*, Y.J.C. Chen a, J.T.C. Chang b, C.T.L. Liao b, H.M.W. Wang b, Z.C.Y. Yen b a b
Chang Gung University, Taiwan Chang Gung Memorial Hospital-Linko, Taiwan
The aim of this study was to clarify relevant alterations of gene expression associated with the invasive potential of oral cancer. To reduce heterogeneity and to obtain specific data on genes involved in invasive potential, we established a highly invasive ORC subline through in vitro Matrigel invasion method. Affymetrix microarrays were used for transcriptome profiling between parental and the highly invasive subline. Seventy-nine genes were differentially expressed at least 2-fold, including 38 up-regulated and 41 down-regulated. After analyzing the microarray data by MetaCoreTM algorithm, a total of 12 regulatory pathways were found to be associated with invasive phenotype (p < 0.001). Two functional pathways were most significant: the cell adhesion through extracellular matrix remodeling (p = 4.964e 06), and MHC-class-I mediated antigen presentation (p = 9.843e 05). To shed more light on the biological functions of invasiveness, two genes highly overexpressed in the invasive subline, Cyr61 and CD44 were further validated. RNAi knockdown of Cyr61 and CD44 led to significant suppression of cell growth (32% and 31%, respectively, at day 3), cell migration (45% and 96%, respectively, at 24 h), and cell invasion (83% and 87%, respectively, at day 3). These results suggested important roles of these genes in regulating invasive phenotype, and demonstrated the confidence of this study design in the search of invasive associated genes. The identified pathways associated with invasion mechanism may be novel targets for manipulation of the cancer behavior with consequences on treatment outcome. doi:10.1016/j.oos.2009.06.567
P3.42. Promoter methylation and protein expression of hMLH1 gene in oral cancer. Preliminary report ´ rez-Amador a, C. Garcı´a-Cuellar b, Y. ´ rez a,*, V. Ramı I. González-Ramı b Sánchez-Pérez , G. Anaya-Saavedra a, E. Irigoyen-Camacho a a b
Universidad Autónoma Metropolitana, Mexico Instituto Nacional de Cancerologı´a, Mexico
Background: Methylation is an epigenetic process associated with transcriptional silencing. One of the genes affected by this mechanism is hMLH1, particularly expressed in human cells undergoing rapid renewal; its reduced expression has been reported in oral cancer (OC). Objective: The aim of the present study was to analyze the relationship between protein expression and promoter methylation of the hMLH1 gene in OC of Mexican individuals. Methods: Ongoing prospective study that included individuals with newly diagnosed OC who attended an Oncology Hospital (August 2007–2008), in Mexico City. Previous informed consent, a biopsy was taken from OC lesions in all included subjects. Protein expression was evaluated by immunohistochemistry using a mono-
clonal antibody against MLH1. After DNA purification and subsequent bisulfite modification, the presence of methylated CpG islands (positions 804–986) was analyzed by methylation specific polymerase chain reaction (MSP). Results: Fourteen consecutive individuals were included, median age 60 (range: 32–84) years; eight patients (57%) were females. Twelve (85%) cases were at stages III–IV. All cases were positive for MLH1 antibody, showing a positive nuclear stain. No methylated MSP bands were identified. Conclusion: This preliminary results suggest that in the sample analyzed the expression of hMLH1, seemed to be related with a non methylated pattern. Future steps would involve genomic sequencing analysis to clarify the methylation status of the gene. doi:10.1016/j.oos.2009.06.568
P3.43. Myofibroblasts and phenotypical changes in the expression of epithelial markers in lymph node metastases from patients with tongue cancer M. Vered a,b,*, D. Dayan b, A. Dobriyan a, R. Yahalom a, Y.P. Talmi a, L. Bedrin a a b
Sheba Medical Center, Tel Hashomer, Israel Tel Aviv University, Israel
Background: The pattern of distribution and frequency of myofibroblasts (MF) in metastatic cervical lymph nodes from patients with tongue carcinoma (N = 14) were assessed and compared to that of the primary tumors. Material and methods: Pattern of MF distribution was defined as ”spindle” when these cells tightly adhered to the periphery of the tumor islands/nests in one-to-three concentric layers, and as ‘‘network”, when MF were exceptionally abundant and were organized in intersecting, multilayering bundles. Frequency of MF was assessed on a five-scale scoring system: 0 – no MF; 0.5 – focal/limited MF in either in the ‘‘spindle” or ”network” pattern; 1 – predominance of the ‘‘spindle” pattern; 2 – ”network” pattern in the greater part of the section; and 3 – predominant ‘‘network” pattern. The metastatic deposits were examined with the aid of epithelial membrane antigen (EMA) to assess the degree of maintenance of the epithelial phenotype. Results: MF were found in 13 (93%) lymph nodes containing metastatic deposits; no MF were found in the lymph nodes free of tumor. ‘‘Network” pattern of distribution (score 2) was found in 8 (62%) cases, ‘‘spindle” (score 1) in 2 (15%) and focal (score 0.5) in 3 (23%) cases. The frequency of MF was similar to or higher than the corresponding primary tumor in 5 (36%) cases. EMA was identified in the metastatic carcinoma in 6 (43%) of the cases, usually in isolated cells or small clusters of cells, irrespective of the degree of differentiation of the tumor. Conclusion: The study showed that metastatic carcinoma cells create a tumor-specific microenvironment, including emergence of MF, and they undergo modifications in the epithelial phenotype. doi:10.1016/j.oos.2009.06.569
P3.44. Expression of dendritic cells HLA-DR+/FXIII+ in Oral Squamous Cell Carcinoma C.P. Pedraza-Zamora a,*, L.F. Jacinto-Alemán a, A. AlcántaraVázquez b, E. Echevarrı´a y Pérez b, M.D. Jiménez-Farfán a, J.C. Hernández-Guerrero a a b
UNAM, Mexico Hospital General de México, Mexico
Oral Squamous Cell Carcinoma (OSCC) is a malignant epithelial neoplasm, which present invasive growth behavior, metastasis to lymphatic nodes and three histological grades of differentiation (Well Differentiated – WD, Moderately Differentiated – MD and Poorly Differentiated – PD). It is well known that the immune system plays an important role in the immunosurveillance of tumors and in its prognostic. Dendritic Cells (DCs) are Antigen Presenting Cells with ability to activate a lymphocytic response throughout the Major Histocompatibility Complex (MHC). Objective: The aim of this study was to correlate the number of DCs HLA-DR+/FXII+ with the Histological Differentiation Grade of OSCC. Methods: Ten healthy oral mucosa samples from retromolar mucosa and tongue and 15 samples of OSCC were diagnosed and classified histologically by an Oral Pathologist according to their differentiation grade. The samples were conventionally paraffin embedded and serial cut for immunofluorescence to HLA-DR/FXIII. Quantification, Central Tendency, Dispersion Measures and Pearson’s Correlation test for positive DCs was performed using the SPSS 15 Program. Results: From the total of samples, 6 samples were taken from feminine patients and 9 from masculine. It was obtained 8 WD (53%), 4 MD (26%) and 3 PD (20%). Quantification: The average of DCs and Standard Deviation (±) according to their differentiation grade was: 4.1(±1.2) DCs for Healthy Mucosa, 3.37(±1.70) DCs for Well Differentiated; 2.03(±0.68) DCs for Moderately Differentiated and 1.53(±0.90) for Poorly Differentiated. The results were correlated getting an Statistic Significance of 0.046 (p 6 0.05) according to the DC’s number and Histological Differentiation Grade. Conclusions: The number of DCs was associated with the Differentiation Grade of OSC. Well Differentiated OSCC had a greater number of DCs than Poorly Differentiated OSCC. doi:10.1016/j.oos.2009.06.570
P3.45. The validity of topoisomerase II alpha in oral carcinomas M.E. Wali Faculty of Oral & Dental Medicine, Cairo University, Egypt The nuclear enzyme DNA topoisomerase II (topo-II) has been shown to be required for chromatin condensation and chromosomal segregation during mitosis; its isoform topo-II a is linked with active cell proliferation.
215
The aim of the present work was to examine the expression of topo-II a in oral carcinomas and to evaluate the validity of this marker as regards prognosis. For each case examined, a top-II a index was determined and compared to that obtained from a Ki-67 count. The ratio between the immunopositive cells for topo II a and Ki-67 (T/K ratio) was calculated. Significant differences were found between the immunoexpression of topo II a and Ki-67. Topo II a was highly expressed in undifferentiated neoplasms than in well-differentiated ones. On the contrary, Ki-67 did not show any difference in expression among the grades examined. In this respect, the T/K ratio demonstrated a sensitive proliferation parameter for the histologic grading of oral carcinomas. Hence, topo II a may be considered as an additional biomarker in the proliferation assessment of the malignant cell than Ki-67. doi:10.1016/j.oos.2009.06.571
P3.46. Reduced peripheral blood dendritic cells correlates with increased level of vascular endothelial growth factor in patients with oral squamous cell carcinoma Z.Y. Wang a, P.H. Shi a, Q.G. Hu a,*, X.F. Huang a, Z.C. Hua b, Y.Y. Hou c a Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School, Nanjing University, China b School of Life Science, Nanjing University, China c Department of Immunology, Medical School, Nanjing University, China
Purpose: VEGF could cause the deficient function of DCs in vitro. However, less remains understanding about peripheral blood dendritic cell (PBDC) and VEGF in patients with OSCC. Methods: PBDC levels and serum VEGF were investigated in 81 patients with OSCC, and VEGF expression in 57 available primary tumors was assessed with immunohistochemical analysis. The correlation between PBDC levels and VEGF was analyzed. The effect of VEGF on DC phenotype and function were then tested in vitro. Results: (1) Serum VEGF level was significantly increasing in OSCC patients compared with ‘no-cancer’ subjects as control (P < 0.05), but PBDC levels were significantly decreasing (P < 0.05). An elevation in the serum VEGF was significantly associated with a reduction in the PBDC levels in the OSCC patients, including the DC count (r = 0.662, P < 0.01) and the DC/PBMC percent (r = 0.652, P < 0.01), but no correlation in the control (r = 0.302, P > 0.05; r = 0.305, P > 0.05) (Figs. 1 and 2). (2) The increased serum
Fig. 1. The mononuclear cells revealed in Region P1 of (a). The Region P2 of (b) showed the mononuclear cells which had no expression of CD14 and CD16. The PBDCs which were stained representative patterns of HLA DR+, CD33+, CD14 and CD16 with FITC or PE-labeled monoclonal antibodies were gated in Region P3 of (c).
Poster Abstracts Oral AbstractsPoster ListOrals ListPan. Disc. & Symp. Abs.Keynote Abs.Keynote Bios.ProgramIAOOWelcomeCommittee Listings
Poster session III / Oral Oncology Supplement 3 (2009) 201–236
214
Poster session III et al. / Oral Oncology Supplement 3 (2009) 201–236
Conclusion: Our results suggested that H-1 and H-1R cells may be useful for searching the candidate genes responsible for crossresistance to platinum derivatives and for further studies to understand the mechanism of platinum resistance. doi:10.1016/j.oos.2009.06.566
P3.41. Transcriptome profiling and network pathway analysis of genes associated with invasive phenotype in oral cancer A.J.C. Cheng a,*, Y.J.C. Chen a, J.T.C. Chang b, C.T.L. Liao b, H.M.W. Wang b, Z.C.Y. Yen b a b
Chang Gung University, Taiwan Chang Gung Memorial Hospital-Linko, Taiwan
The aim of this study was to clarify relevant alterations of gene expression associated with the invasive potential of oral cancer. To reduce heterogeneity and to obtain specific data on genes involved in invasive potential, we established a highly invasive ORC subline through in vitro Matrigel invasion method. Affymetrix microarrays were used for transcriptome profiling between parental and the highly invasive subline. Seventy-nine genes were differentially expressed at least 2-fold, including 38 up-regulated and 41 down-regulated. After analyzing the microarray data by MetaCoreTM algorithm, a total of 12 regulatory pathways were found to be associated with invasive phenotype (p < 0.001). Two functional pathways were most significant: the cell adhesion through extracellular matrix remodeling (p = 4.964e 06), and MHC-class-I mediated antigen presentation (p = 9.843e 05). To shed more light on the biological functions of invasiveness, two genes highly overexpressed in the invasive subline, Cyr61 and CD44 were further validated. RNAi knockdown of Cyr61 and CD44 led to significant suppression of cell growth (32% and 31%, respectively, at day 3), cell migration (45% and 96%, respectively, at 24 h), and cell invasion (83% and 87%, respectively, at day 3). These results suggested important roles of these genes in regulating invasive phenotype, and demonstrated the confidence of this study design in the search of invasive associated genes. The identified pathways associated with invasion mechanism may be novel targets for manipulation of the cancer behavior with consequences on treatment outcome. doi:10.1016/j.oos.2009.06.567
P3.42. Promoter methylation and protein expression of hMLH1 gene in oral cancer. Preliminary report ´ rez-Amador a, C. Garcı´a-Cuellar b, Y. ´ rez a,*, V. Ramı I. González-Ramı b Sánchez-Pérez , G. Anaya-Saavedra a, E. Irigoyen-Camacho a a b
Universidad Autónoma Metropolitana, Mexico Instituto Nacional de Cancerologı´a, Mexico
Background: Methylation is an epigenetic process associated with transcriptional silencing. One of the genes affected by this mechanism is hMLH1, particularly expressed in human cells undergoing rapid renewal; its reduced expression has been reported in oral cancer (OC). Objective: The aim of the present study was to analyze the relationship between protein expression and promoter methylation of the hMLH1 gene in OC of Mexican individuals. Methods: Ongoing prospective study that included individuals with newly diagnosed OC who attended an Oncology Hospital (August 2007–2008), in Mexico City. Previous informed consent, a biopsy was taken from OC lesions in all included subjects. Protein expression was evaluated by immunohistochemistry using a mono-
clonal antibody against MLH1. After DNA purification and subsequent bisulfite modification, the presence of methylated CpG islands (positions 804–986) was analyzed by methylation specific polymerase chain reaction (MSP). Results: Fourteen consecutive individuals were included, median age 60 (range: 32–84) years; eight patients (57%) were females. Twelve (85%) cases were at stages III–IV. All cases were positive for MLH1 antibody, showing a positive nuclear stain. No methylated MSP bands were identified. Conclusion: This preliminary results suggest that in the sample analyzed the expression of hMLH1, seemed to be related with a non methylated pattern. Future steps would involve genomic sequencing analysis to clarify the methylation status of the gene. doi:10.1016/j.oos.2009.06.568
P3.43. Myofibroblasts and phenotypical changes in the expression of epithelial markers in lymph node metastases from patients with tongue cancer M. Vered a,b,*, D. Dayan b, A. Dobriyan a, R. Yahalom a, Y.P. Talmi a, L. Bedrin a a b
Sheba Medical Center, Tel Hashomer, Israel Tel Aviv University, Israel
Background: The pattern of distribution and frequency of myofibroblasts (MF) in metastatic cervical lymph nodes from patients with tongue carcinoma (N = 14) were assessed and compared to that of the primary tumors. Material and methods: Pattern of MF distribution was defined as ”spindle” when these cells tightly adhered to the periphery of the tumor islands/nests in one-to-three concentric layers, and as ‘‘network”, when MF were exceptionally abundant and were organized in intersecting, multilayering bundles. Frequency of MF was assessed on a five-scale scoring system: 0 – no MF; 0.5 – focal/limited MF in either in the ‘‘spindle” or ”network” pattern; 1 – predominance of the ‘‘spindle” pattern; 2 – ”network” pattern in the greater part of the section; and 3 – predominant ‘‘network” pattern. The metastatic deposits were examined with the aid of epithelial membrane antigen (EMA) to assess the degree of maintenance of the epithelial phenotype. Results: MF were found in 13 (93%) lymph nodes containing metastatic deposits; no MF were found in the lymph nodes free of tumor. ‘‘Network” pattern of distribution (score 2) was found in 8 (62%) cases, ‘‘spindle” (score 1) in 2 (15%) and focal (score 0.5) in 3 (23%) cases. The frequency of MF was similar to or higher than the corresponding primary tumor in 5 (36%) cases. EMA was identified in the metastatic carcinoma in 6 (43%) of the cases, usually in isolated cells or small clusters of cells, irrespective of the degree of differentiation of the tumor. Conclusion: The study showed that metastatic carcinoma cells create a tumor-specific microenvironment, including emergence of MF, and they undergo modifications in the epithelial phenotype. doi:10.1016/j.oos.2009.06.569
P3.44. Expression of dendritic cells HLA-DR+/FXIII+ in Oral Squamous Cell Carcinoma C.P. Pedraza-Zamora a,*, L.F. Jacinto-Alemán a, A. AlcántaraVázquez b, E. Echevarrı´a y Pérez b, M.D. Jiménez-Farfán a, J.C. Hernández-Guerrero a a b
UNAM, Mexico Hospital General de México, Mexico
Oral Squamous Cell Carcinoma (OSCC) is a malignant epithelial neoplasm, which present invasive growth behavior, metastasis to lymphatic nodes and three histological grades of differentiation (Well Differentiated – WD, Moderately Differentiated – MD and Poorly Differentiated – PD). It is well known that the immune system plays an important role in the immunosurveillance of tumors and in its prognostic. Dendritic Cells (DCs) are Antigen Presenting Cells with ability to activate a lymphocytic response throughout the Major Histocompatibility Complex (MHC). Objective: The aim of this study was to correlate the number of DCs HLA-DR+/FXII+ with the Histological Differentiation Grade of OSCC. Methods: Ten healthy oral mucosa samples from retromolar mucosa and tongue and 15 samples of OSCC were diagnosed and classified histologically by an Oral Pathologist according to their differentiation grade. The samples were conventionally paraffin embedded and serial cut for immunofluorescence to HLA-DR/FXIII. Quantification, Central Tendency, Dispersion Measures and Pearson’s Correlation test for positive DCs was performed using the SPSS 15 Program. Results: From the total of samples, 6 samples were taken from feminine patients and 9 from masculine. It was obtained 8 WD (53%), 4 MD (26%) and 3 PD (20%). Quantification: The average of DCs and Standard Deviation (±) according to their differentiation grade was: 4.1(±1.2) DCs for Healthy Mucosa, 3.37(±1.70) DCs for Well Differentiated; 2.03(±0.68) DCs for Moderately Differentiated and 1.53(±0.90) for Poorly Differentiated. The results were correlated getting an Statistic Significance of 0.046 (p 6 0.05) according to the DC’s number and Histological Differentiation Grade. Conclusions: The number of DCs was associated with the Differentiation Grade of OSC. Well Differentiated OSCC had a greater number of DCs than Poorly Differentiated OSCC. doi:10.1016/j.oos.2009.06.570
P3.45. The validity of topoisomerase II alpha in oral carcinomas M.E. Wali Faculty of Oral & Dental Medicine, Cairo University, Egypt The nuclear enzyme DNA topoisomerase II (topo-II) has been shown to be required for chromatin condensation and chromosomal segregation during mitosis; its isoform topo-II a is linked with active cell proliferation.
215
The aim of the present work was to examine the expression of topo-II a in oral carcinomas and to evaluate the validity of this marker as regards prognosis. For each case examined, a top-II a index was determined and compared to that obtained from a Ki-67 count. The ratio between the immunopositive cells for topo II a and Ki-67 (T/K ratio) was calculated. Significant differences were found between the immunoexpression of topo II a and Ki-67. Topo II a was highly expressed in undifferentiated neoplasms than in well-differentiated ones. On the contrary, Ki-67 did not show any difference in expression among the grades examined. In this respect, the T/K ratio demonstrated a sensitive proliferation parameter for the histologic grading of oral carcinomas. Hence, topo II a may be considered as an additional biomarker in the proliferation assessment of the malignant cell than Ki-67. doi:10.1016/j.oos.2009.06.571
P3.46. Reduced peripheral blood dendritic cells correlates with increased level of vascular endothelial growth factor in patients with oral squamous cell carcinoma Z.Y. Wang a, P.H. Shi a, Q.G. Hu a,*, X.F. Huang a, Z.C. Hua b, Y.Y. Hou c a Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School, Nanjing University, China b School of Life Science, Nanjing University, China c Department of Immunology, Medical School, Nanjing University, China
Purpose: VEGF could cause the deficient function of DCs in vitro. However, less remains understanding about peripheral blood dendritic cell (PBDC) and VEGF in patients with OSCC. Methods: PBDC levels and serum VEGF were investigated in 81 patients with OSCC, and VEGF expression in 57 available primary tumors was assessed with immunohistochemical analysis. The correlation between PBDC levels and VEGF was analyzed. The effect of VEGF on DC phenotype and function were then tested in vitro. Results: (1) Serum VEGF level was significantly increasing in OSCC patients compared with ‘no-cancer’ subjects as control (P < 0.05), but PBDC levels were significantly decreasing (P < 0.05). An elevation in the serum VEGF was significantly associated with a reduction in the PBDC levels in the OSCC patients, including the DC count (r = 0.662, P < 0.01) and the DC/PBMC percent (r = 0.652, P < 0.01), but no correlation in the control (r = 0.302, P > 0.05; r = 0.305, P > 0.05) (Figs. 1 and 2). (2) The increased serum
Fig. 1. The mononuclear cells revealed in Region P1 of (a). The Region P2 of (b) showed the mononuclear cells which had no expression of CD14 and CD16. The PBDCs which were stained representative patterns of HLA DR+, CD33+, CD14 and CD16 with FITC or PE-labeled monoclonal antibodies were gated in Region P3 of (c).
Poster Abstracts Oral AbstractsPoster ListOrals ListPan. Disc. & Symp. Abs.Keynote Abs.Keynote Bios.ProgramIAOOWelcomeCommittee Listings
Poster session III / Oral Oncology Supplement 3 (2009) 201–236