Sari Nakao

Human papillomavirus infections among Japanese women: age-related prevalence and type-specific risk for cervical cancer

To obtain baseline data for human papillomavirus (HPV) screening and vaccination in Japan, we analyzed HPV DNA data from 2282 Japanese women (1517 normal cytology, 318 cervical intraepithelial neoplasia [CIN] grade 1, 307 CIN2–3, and 140 invasive cervical cancer [ICC]) that visited the University of Tsukuba Hospital or Ibaraki Seinan Medical Center Hospital for screening or treatment of cervical diseases between 1999 and 2007. An L1‐based PCR method was used for individual HPV genotyping. The most common HPV types in ICC were, in order of decreasing prevalence, HPV16 (40.5%), HPV18 (24.4%), HPV52 (8.4%), HPV58 (3.1%), and HPV33 (3.1%). Based on the comparison of HPV type distributions between normal cytology and CIN2–3 and ICC, estimated risk of disease progression varied considerably by genotype: HPV16, HPV18, HPV31, HPV33, HPV35, HPV52, and HPV58 (prevalence ratio, 1.92; 95% confidence interval 1.58–2.34); other oncogenic types (0.31, 95% confidence interval 0.19–0.50); and non‐oncogenic types (0.09, 95% confidence interval 0.03–0.43). HPV16 and/or HPV18, including coinfections with other types, contributed to 67.1% of ICC and 36.2% of CIN2–3 among Japanese women. More importantly, the overall prevalence of HPV16 and/or HPV18 varied greatly according to the womens age: highest in women aged 20–29 years (ICC, 90.0%; CIN2–3, 53.9%), decreasing with age thereafter, and lowest in women aged 60 years or older (ICC, 56.3%; CIN2–3, 25.0%). In conclusion, type‐specific HPV testing may help identify Japanese women at high risk of progression to CIN2–3 and cancer. In Japan, current HPV vaccines are estimated to provide approximately 70% protection against ICC and may be more useful in reducing the incidence of cervical cancer and precancer in young women of reproductive age. (Cancer Sci 2009; 100: 1312–1316)

Biased amplification of human papillomavirus DNA in specimens containing multiple human papillomavirus types by PCR with consensus primers

Genotyping human papillomavirus (HPV) in clinical specimens is important because each HPV type has different oncogenic potential. Amplification of HPV DNA by PCR with the consensus primers that are derived from the consensus sequences of the L1 gene has been used widely for the genotyping. As recent studies have shown that the cervical specimens often contain HPV of multiple types, it is necessary to confirm whether the PCR with the consensus primers amplifies multiple types of HPV DNA without bias. We amplified HPV DNA in the test samples by PCR with three commonly used consensus primer pairs (L1C1/L1C2+C2M, MY09/11, and GP5+/6+), and the resultant amplicons were identified by hybridization with type‐specific probes on a nylon membrane. L1C1/L1C2+C2M showed a higher sensitivity than the other primers, as defined by the ability to detect HPV DNA, on test samples containing serially diluted one of HPV16, 18, 51, 52, and 58 plasmids. L1C1/L1C2+C2M failed to amplify HPV16 in the mixed test samples containing HPV16, and either 18 or 51. The three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58. The data indicate that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types, and suggest that the genotyping data obtained by such a method should be carefully interpreted. (Cancer Sci 2011; 102: 1223–1227)

Interleukin-10 −1082 Gene Polymorphism and Susceptibility to Cervical Cancer Among Japanese Women

Polymorphisms in cytokine genes can influence immune responses to human papillomavirus infection, possibly modifying risks of cervical cancer. Using an amplification refractory mutation system-polymerase chain reaction method, we analyzed a single nucleotide polymorphism (A/G) at position -1082 in interleukin-10 promoter region in 440 Japanese women: 173 women with normal cytology, 163 women with cervical intraepithelial neoplasia and 104 women with invasive cervical cancer. The carrier frequency of interleukin-10 -1082 G alleles associated with higher interleukin-10 production increased with disease severity: 9.8% for normal cytology; 19.6% for cervical intraepithelial neoplasia; 29.8% for invasive cervical cancer (P for trend < 0.001). Among cytologically normal women, human papillomavirus infections were more common in those who were positive for an interleukin-10 -1082 G allele (P = 0.04). In conclusion, our data suggest that interleukin-10 -1082 gene polymorphism may serve as a marker of genetic susceptibility to cervical cancer among Japanese women.

Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2.

Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56-75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64-73 and aa58-64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56-75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs.

Loss of PTEN expression is an independent predictor of favourable survival in endometrial carcinomas.

Background:We and others previously reported the prognostic significance of PTEN mutational status on favourable survival in endometrial carcinomas. Here, we demonstrate that loss of PTEN expression in immunohistochemistry is an independent prognostic marker for favourable survival in endometrial carcinomas.Methods:We conducted immunohistochemical analyses of PTEN, PIK3CA, phosphorylated Akt (p-Akt), and p27 in primary endometrial carcinomas from 221 patients. Mutation of PTEN was analysed further.Results:Expression of PTEN was lost in 56 patients (25%), and PIK3CA was overexpressed in 159 patients (72%). Overexpression of PIK3CA was associated with p-Akt overexpression (P<0.001), which was in turn associated with loss of nuclear p27 expression (P=0.028). Loss of PTEN expression was found to be associated with endometrioid histology (P=0.03), and was inversely associated with the presence of lymphovascular space invasion (P=0.03). Univariate and multivariate survival analyses revealed that factors of PTEN loss, age <70, histological grade 1, early International Federation of Gynecology and Obstetrics (FIGO) stage, and absence of lymphovascular invasion were independent prognostic indicators for better overall survival (P=0.03, 0.04, 0.01, <0.001, and 0.03, respectively). The subset analysis showed a stronger tendency of PTEN loss towards favourable survival in advanced-stage (III and IV) disease than in early-stage (I and II) disease (P=0.05 vs 0.14). Moreover, our mutational analysis demonstrated that PTEN expression loss was associated with PTEN-truncating mutations (P=0.03).Conclusion:The current observations further support the prognostic significance of PTEN aberration on favourable outcome in endometrial carcinomas, providing useful implications for the individualised management of the disease.

Clinicopathologic implications of DNA mismatch repair status in endometrial carcinomas

OBJECTIVE Endometrial carcinoma is the most common malignancy in women with Lynch syndrome caused by mismatch repair (MMR) deficiency. We investigated the clinicopathologic significance of deficient MMR and Lynch syndrome presumed by MMR analyses in unselected endometrial carcinomas. METHODS We analyzed immunohistochemistry of MMR proteins (MLH1/MSH2/MSH6/PMS2) and MLH1 promoter methylation in primary endometrial carcinomas from 221 consecutive patients. Based on these results, tumors were categorized as sporadic or probable Lynch syndrome (PLS). Clinicopathologic variables and prognosis were compared according to MMR status and sporadic/PLS classification. RESULTS Deficient MMR showed only trends towards favorable overall survival (OS) compared with intact MMR (p=0.13), whereas PLS showed significantly better OS than sporadic (p=0.038). Sporadic was significantly associated with older age, obesity, deep myometrial invasion, and advanced stage (p=0.008, 0.01, 0.02 and 0.03), while PLS was significantly associated with early stage and Lynch syndrome-associated multiple cancer (p=0.04 and 0.001). The trend towards favorable OS of PLS was stronger in advanced stage than in early stage (hazard ratio, 0.044 [95% CI 0-25.6] vs. 0.49 [0.063-3.8]). In the subset receiving adjuvant therapies, PLS showed trends towards favorable disease-free survival compared to sporadic by contrast with patients receiving no adjuvant therapies showing no such trend (hazard ratio, 0.045 [95% CI 0-20.3] vs. 0.81 [0.095-7.0]). CONCLUSIONS The current findings suggest that analyzing MMR status and searching for Lynch syndrome may identify a subset of patients with favorable survival and high sensitivity to adjuvant therapies, providing novel and useful implications for formulating the precision medicine in endometrial carcinoma.

High Pretreatment Plasma D-dimer Levels Are Associated With Poor Prognosis in Patients With Ovarian Cancer Independently of Venous Thromboembolism and Tumor Extension

Objective Elevated plasma D-dimer (DD) is associated with decreased survival among patients with breast, lung, and colon cancers. The present study clarifies the prognostic significance of pretreatment plasma DD levels in patients with epithelial ovarian cancer (EOC). Methods We investigated pretreatment DD levels and other variables for overall survival using univariate and multivariate analyses in 134 consecutive patients with EOC stages II to IV who were initially treated between November 2004 and December 2010. Results The median follow-up period was 53 (7–106) months. Univariate analysis significantly associated elevated pretreatment DD (≥2.0 μg/mL) levels to poor 5-year overall survival rates irrespective of previously treated venous thromboembolism (72.2% vs 52.6%, P = 0.039). Cancer antigen 125 levels of 200 U/mL or higher (P = 0.011), distant metastases (P = 0.0004), residual tumors (P < 0.0001), and International Federation of Gynecology and Obstetrics stage III/IV (P = 0.0033) were also poor prognostic factors. Multivariate analysis independently associated DD levels of 2.0 μg/mL or higher (P = 0.041), distant metastases (P = 0.013), and residual tumors (P < 0.0001) with poor overall survival. Conclusions High pretreatment DD levels are associated with poor overall survival in patients with EOC independently of venous thromboembolism and tumor extension and might comprise a promising prognostic biomarker for patients with EOC.

Incidence of venous thromboembolism before treatment in cervical cancer and the impact of management on venous thromboembolism after commencement of treatment

INTRODUCTION Silent venous thromboembolism (VTE) often occurs before treatment in ovarian or endometrial cancer and management can decrease VTE after treatment. However, the incidence of VTE before treatment and the impact of management are still unclear in cervical cancer. MATERIALS AND METHODS We investigated the incidence of VTE before treatment in 272 consecutive patients with cervical cancer, and the impact of management on prevention of VTE during and after treatment. D-dimer levels before treatment were examined in all patients. Venous ultrasonography of the lower extremities was performed in patients with D-dimer ≥1.5μg/ml. Deep vein thrombosis (DVT) in the pelvis or abdomen was diagnosed by enhanced computed tomography. RESULTS Thirteen patients (4.8%; 3 preoperatively, 10 before radiotherapy or concurrent chemoradiotherapy) were diagnosed with DVT, although DVT was symptomatic in only 1 patient. None of the 13 patients showed pulmonary embolism on pulmonary scanning. Although 4 of 128 patients (3.1%) developed VTE after radical hysterectomy, none of the 124 patients who underwent radiotherapy or concurrent chemoradiotherapy developed VTE during or after treatment. CONCLUSIONS These data suggest that VTE before treatment occurs less frequently with cervical cancer than with ovarian or endometrial cancer. However, management may decrease VTE during and after treatment, especially radiotherapy.

Separate analysis of human papillomavirus E6 and E7 messenger RNAs to predict cervical neoplasia progression

A few studies previously suggested that human papillomavirus (HPV) E6 messenger RNA (mRNA) may exist uniformly in all grades of cervical intraepithelial neoplasia (CIN), whereas the detection rate of E7 mRNA may increase with disease progression from low-grade CIN to invasive carcinoma. The aim of this study was to clarify the different roles of E6 and E7 mRNAs in cervical carcinogenesis. The presence of each E6 and E7 mRNA was analyzed in 171 patients with pathologically-diagnosed CIN or cervical carcinoma. We utilized a RT-PCR assay based on consensus primers which could detect E6 mRNA (full-length E6/E7 transcript) and E7 mRNAs (spliced E6*/E7 transcripts) separately for various HPV types. E7 mRNAs were detected in 6% of CIN1, 12% of CIN2, 24% of CIN3, and 54% of cervical carcinoma. The presence of E7 mRNAs was significantly associated with progression from low-grade CIN to invasive carcinoma in contrast with E6 mRNA or high-risk HPV (HR-HPV) DNA (p = 0.00011, 0.80 and 0.54). The presence of both E6 and E7 mRNAs was significantly associated with HPV16/18 DNA but not with HR-HPV DNA (p = 0.0079 and 0.21), while the presence of E6 mRNA was significantly associated with HR-HPV DNA but not with HPV16/18 DNA (p = 0.036 and 0.089). The presence of both E6 and E7 mRNAs showed high specificity and low sensitivity (100% and 19%) for detecting CIN2+ by contrast with the positivity for HR-HPV DNA showing low specificity and high sensitivity (19% and 89%). The positive predictive value for detecting CIN2+ was even higher by the presence of both E6 and E7 mRNAs than by the positivity for HR-HPV DNA (100% vs. 91%). In 31 patients followed up for CIN1-2, the presence of both E6 and E7 mRNAs showed significant association with the occurrence of upgraded abnormal cytology in contrast with E6 mRNA, HR-HPV DNA, or HPV16/18 DNA (p = 0.034, 0.73, 0.53, and 0.72). Our findings support previous studies according to which E7 mRNA is more closely involved in cervical carcinogenesis than E6 mRNA. Moreover, the separate analysis of E6 and E7 mRNAs may be more useful than HR-HPV DNA test for detecting CIN2+ precisely and predicting disease progression. Further accumulation of evidence is warranted to validate our findings.

Expression of Tissue Factor in Epithelial Ovarian Carcinoma Is Involved in the Development of Venous Thromboembolism

Objectives Our 2007 study of 32 patients with ovarian cancer reported the possible involvement of tissue factor (TF) in the development of venous thromboembolism (VTE) before treatment, especially in clear cell carcinoma (CCC). This follow-up study further investigated this possibility in a larger cohort. Methods We investigated the intensity of TF expression (ITFE) and other variables for associations with VTE using univariate and multivariate analyses in 128 patients with epithelial ovarian cancer initially treated between November 2004 and December 2010, none of whom had received neoadjuvant chemotherapy. Before starting treatment, all patients were ultrasonographically screened for VTE. The ITFE was graded based on immunostaining of surgical specimens. Results Histological types were serous carcinoma (n = 42), CCC (n = 12), endometrioid carcinoma (n = 15), mucinous carcinoma (n = 53), and undifferentiated carcinoma (n = 6). The prevalence of VTE was significantly higher in CCC (34%) than in non-CCC (17%, P = 0.03). As ITFE increased, the frequencies of CCC and VTE increased significantly (P < 0.001 and P = 0.014, respectively). Multivariate analysis identified TF expression and pretreatment dimerized plasmin fragment D level as significant independent risk factors for VTE development. These factors showed particularly strong impacts on advanced-stage disease (P = 0.021). Conclusions The 2007 cohort was small, preventing multivariate analysis. This study of a larger cohort yielded stronger evidence that the development of VTE in epithelial ovarian cancer may involve TF expression in cancer tissues.