Mitsuo Yoshinouchi

Vascular endothelial growth factor-C expression and its relationship to pelvic lymph node status in invasive cervical cancer.

Vascular endothelial growth factor-C (VEGF-C) has been implicated in lymphangiogenesis, the process of new lymphatics formation. The present study investigated VEGF-C mRNA expression in invasive cervical cancer tissue. Additionally, the association of VEGF-C mRNA with clinicopathological features was examined. VEGF-C mRNA expression was assessed by reverse transcription-polymerase chain reaction using β-action as an internal control. 75 patients presenting with invasive cervical cancer were included in the trial. VEGF-C mRNA expression was markedly higher in tumours in which pelvic lymph node metastasis was diagnosed by magnetic resonance (MR) imaging (P = 0.002). 53 patients displaying stage Ib–IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. VEGF-C expression was significantly higher in tumours exhibiting deep stromal invasion, pelvic lymph node metastasis and lymph-vascular space involvement (P = 0.016, P = 0.006 and P = 0.036, respectively). Multivariate analysis revealed VEGF-C mRNA expression to be the sole independent factor influencing pelvic lymph node metastasis. Subjects demonstrating VEGF-C mRNA expression displayed significantly poorer prognoses than those lacking VEGF-C mRNA expression (P = 0.049). These findings provide evidence supporting the involvement of VEGF-C expression in the promotion of lymph node metastasis in cervical cancer. Furthermore, examination of VEGF-C expression in biopsy specimens may be beneficial in the prediction of pelvic lymph node metastasis.

The expression of vascular endothelial growth factor and transforming growth factor-β associates with angiogenesis in epithelial ovarian cancer

The object of this study was to clarify the association between microvessel density and clinicopathologic factors, as well as the association of angiogenic factors (vascular endothelial growth factor [VEGF] and transforming growth factor-β [TGF-β]) with tumor angiogenesis and patient survival in epithelial ovarian cancer. The expression of VEGF and TGF-β was evaluated by immunohistochemical staining in 60 patients with epithelial ovarian cancer. The microvessel density, assessed by immunostaining for factor VIII-related antigen in the most neovascularized areas, varied depending on histological types but not on International Federation of Gynecology and Obstetrics stage. Patients with stage III carcinomas and positive TGF-β had more extensive peritoneal dissemination and a worse outcome. The microvessel density of VEGF-rich and TGF-β positive tumors was significantly higher than that of VEGF-poor and TGF-β negative tumors. Angiogenesis appears to be an early event in epithelial ovarian cancer and may be induced differently in tumors of different histological types. The expression of VEGF and TGF-β associates with the promotion of angiogenesis, and the expression of TGF-β is a prognostic indicator in epithelial ovarian cancers.

Rapid and sensitive detection of physical status of human papillomavirus type 16 DNA by quantitative real-time PCR

ABSTRACT A rapid quantitative real-time PCR method was employed to quantify the copy number of E2 and E6 genes for analysis of the physical status of human papillomavirus type 16 (HPV-16) DNA. Significant differences with respect to both copy numbers were found when more than 40% of HPV-16 DNA was integrated with disruption of the E2 gene in an experimental model. The physical status of HPV-16 DNA in 50 clinical samples was exclusively episomal in 21 cases (42%), concomitant in 14 cases (28%), and integrated in 15 cases (30%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. Four of 11 cervical intraepithelial neoplasia involved integrated forms of HPV-16 DNA partially or exclusively. This rapid, sensitive technique is useful in the analysis of the physical status of HPV DNA.

Serum C-reactive protein as a prognostic factor in patients with epithelial ovarian cancer

OBJECTIVE It is well known that the serum level of Interleukin-6 (IL-6) correlates with the level of C-reactive protein (CRP). The purpose of this study is to determine the significance of CRP as a prognostic factor in epithelial ovarian cancer. STUDY DESIGN The present study is comprised of 120 patients with epithelial ovarian cancer from 1985 to 1992. In this study, CRP levels above 50 mg/l were considered high CRP. Univariate and multivariate analyses were performed to identify clinicopathological variables associated with poor survival. RESULTS The serum CRP value was significantly associated with the volume of ascites (P = 0.000004). Univariate analysis showed that the FIGO stage, primary tumour diameter, size of residual tumour, histologic grade, volume of ascites and high serum level of CRP were significant prognostic factors. Coxs multivariate proportional hazard model showed that histologic grade was the most important prognostic factor (P = 0.0026). FIGO stage and volume of ascites were also independent factors for 5-year survival (P = 0.0310 and P = 0.0216, respectively). However, the serum CRP value was not an independent prognostic factor. CONCLUSION CRP is an adverse prognostic factor in univariate analysis, but not in multivariate analysis.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16−cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21WAF1/CIP1, as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

Vascular endothelial growth factor is implicated in early invasion in cervical cancer.

The association between the expression of vascular endothelial growth factor (VEGF) and clinicopathological factors has scarcely been examined in cervical cancer. This study examines the level of VEGF messenger RNA (mRNA) expression in invasive cervical cancer and its association with clinicopathological features including microvessel density. The level of VEGF mRNA was assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta-actin as an internal control in 66 patients with stages Ia-IVb invasive cervical cancer. In 42 patients who underwent surgery, the microvessel count was also assessed by immunostaining for factor VIII-related antigen in the most neovascularised area of the specimen. The highest level of VEGF mRNA expression was observed in early invasive cervical cancers. Except for stage IVb, the stage of the disease inversely correlated with the level of VEGF mRNA (P < 0.05). There was no significant difference in the level of VEGF mRNA with respect to histological cell types. 38 patients with stages Ib-IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. There was no significant difference in the level of VEGF mRNA with respect to lymph node metastasis, depth of stromal invasion, tumour size, parametrial involvement or vaginal involvement among these patients. A significant relationship was found between the microvessel density and the level of VEGF mRNA (P < 0.01). These findings provide evidence that the expression of VEGF is involved in the promotion of angiogenesis in cervical cancer and plays an important role in early invasion.

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

Vascular endothelial growth factor and platelet-derived endothelial cell growth factor expression are implicated in the angiogenesis of endometrial cancer.

Although many angiogenic factors have been described, it is not well defined which factors are expressed in endometrial cancer. The object of this study was to examine mRNA levels of the two angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in endometrial cancer tissues and their association with clinicopathological features including microvessel density. The level of VEGF and PD-ECGF mRNAs was assessed by semi-quantitative reverse transcription-polymerase chain reaction using beta-actin as an internal standard in 38 patients with endometrial cancer. Microvessel counts were also assessed by immunostaining for factor VIII-related antigen in the most vascularised area of the specimen. VEGF/beta-actin ratios of non-endometrioid tumours were significantly higher than those of endometrioid tumours (P = 0.013). VEGF/beta-actin ratios of cases with lymph-vascular space involvement were significantly higher than those of cases without lymph-vascular space involvement (P = 0.021). Although it was not statistically significant, PD-ECGF/beta-actin ratios in grade 3 tumours were higher than those in grade 1 and 2 tumours (P = 0.066). The microvessel density was significantly correlated with the level of VEGF and PD-ECGF mRNA expression (P = 0.041 and P < 0.0001, respectively). Our findings provide evidence that the expression of both VEGF and PD-ECGF is involved in the promotion of angiogenesis in endometrial cancer. In addition, VEGF and PD-ECGF might contribute to the aggressive potential of high grade tumours or certain histological subtypes with unfavourable prognosis through the induction of angiogenesis.

CD44 exon v6 is not implicated in the progression and metastasis of endometrial cancer

We examined the presence of mRNA for CD44v6 and assessed the association with clinicopathological features in 42 patients with endometrial cancer by RT-PCR and subsequent Southern blot hybridization with oligonucleotide probe specific for v6. The standard form of CD44 was expressed in all specimens and 20 out of 42 endometrial cancers expressed an isoform containing exon v6 in combination with other variant exons. However, there was no correlation between the expression of CD44v6 and any clinicopathological factors. These findings suggested that the expression of CD44v6 is not implicated in the progression and metastasis of endometrial cancer.

Optimal surgery and diagnostic approach of stage IA2 squamous cell carcinoma of the cervix

BACKGROUND Most patients with International Federation of Gynecology and Obstetrics (FIGO) stage IA2 squamous cell carcinoma of the cervix, opt for radical surgery at present. OBJECTIVE To review surgical and diagnostic approaches in such patients. STUDY DESIGN Our patient population consisted of 394 patients with a diagnosis of stage I squamous cell cervical carcinoma (with depth of stromal invasion 10mm or less) according to the 1995 FIGO definition. Biopsy and surgical specimen slides were reassessed retrospectively in all cases. The findings of T2-weighted MR imaging were available from the individual medical charts. RESULTS None of the patients with stromal invasion of 5mm depth or less showed pelvic lymph node metastasis. However, metastasis to the parametrial connective tissue was found in one case with stage IA1 exhibiting marked lymph-vascular space involvement. There were no deaths due to disease in cases with stromal invasion of 5mm depth or less. The lesions were detected in all 20 cases exhibiting stromal invasion of greater than 5mm in depth. In contrast, the lesions were not detected with T2 imaging in four of six cases (67%) with stage IA2. CONCLUSION Simple or modified radical hysterectomy with pelvic lymph node dissection may be sufficient for cases of stage IA2 cervical squamous cell carcinoma where lymph-vascular space involvement is absent. T2-weighted MR imaging with no detectable tumor would prove beneficial in the selection of these patients.