Takuma Fujii

p16INK4a overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix

Carcinogenesis of cervical cancer has been investigated, and p16(INK4a) overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16(INK4a) overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16(INK4a) is overexpressed in small cell carcinoma, and if p16(INK4a) is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16(INK4a) overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16(INK4a) was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16(INK4a) was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma.

Abdominal radical trachelectomy as a fertility-sparing procedure in women with early-stage cervical cancer in a series of 61 women

OBJECTIVE This retrospective study was conducted as a review of a series of women who had undergone abdominal radical trachelectomy and pelvic lymphadenectomy for early-stage cervical cancer. METHODS We performed a retrospective review of patients who had undergone an abdominal radical trachelectomy. Data regarding tumor size and histology, surgical complications, recurrences, pregnancies and live births were collected and were presented as a percentage of the total number of cases. RESULTS A total of 61 women were followed up for a median of 27 months (range: one month--79 months). There were six recurrences (9.8%); none of the recurrences occurred in patients with a tumor diameter of <20 mm except in one case with adenocarcinoma. Twenty-nine women attempted to conceive; four of these women were successful. All four of these women had live births: two had preterm deliveries, and the remaining two had full-term deliveries. The cumulative pregnancy rate among the women who attempted to conceive was 13.8% (4/29). CONCLUSION Among selected women with early-stage cervical cancer, especially those with a tumor diameter of <20 mm, abdominal radical trachelectomy and pelvic lymphadenectomy is a fertility-sparing treatment option, even though the cumulative conception rate was not particularly high compared with that for women who had undergone a vaginal radical trachelectomy.

Predicting the progression of cervical precursor lesions by human papillomavirus genotyping: A prospective cohort study

Only a subset of cervical precursor lesions progress to cervical cancer and because of the lack of the predictive markers, it cannot be ascertained which lesions will progress or not. To estimate the risk of disease progression associated with human papillomavirus (HPV) genotypes, we followed 570 Japanese women with cytological LSIL (low‐grade squamous intraepithelial lesion) and histological CIN (cervical intraepithelial neoplasia) grade 1–2 lesions (479 CIN 1; 91 CIN 2) at 3 to 4 month intervals for a mean follow‐up period of 39.1 months. At entry, we detected HPV DNA in cervical samples by polymerase chain reaction‐based methodology. Over the period of follow‐up period, 46 lesions progressed to CIN 3 while 362 regressed to normal cytology. Women with multiple HPV infections were more likely to have persistent lesions (hazard ratio [HR] for regression, 0.65; 95% confidence interval [CI], 0.42–1.02; p = 0.07); however, multiple infections did not increase the risk of progression (HR for progression, 1.04; 95% CI, 0.37–2.94; p = 0.94). After adjusting for CIN grade and womens age, HRs for progression to CIN 3 (vs. women with low‐risk types or negative for HPV DNA) varied markedly by HPV genotype: type 16 (11.1, 95% CI: 1.39–88.3); 18 (14.1, 0.65–306); 31 (24.7, 2.51–243); 33 (20.3, 1.78–231); 35 (13.7, 0.75–251); 52 (11.6, 1.45–93.3); 58 (8.85, 1.01–77.6); other high‐risk types (4.04, 0.47–34.7). HPV 45 was not detected in our study subjects. The cumulative probability of CIN 3 within 5 years was 20.5% for HPV 16, 18, 31, 33, 35, 52 and 58; 6.0% for other high‐risk types; 1.7% for low‐risk types (p = 0.0001). In conclusion, type‐specific HPV testing for women with LSIL/CIN 1–2 lesions is useful for identifying populations at increased or decreased risk of disease progression.

Correlation of p16INK4A overexpression with human papillomavirus infection in cervical adenocarcinomas.

As human papillomavirus (HPV) infection is the main risk factor for squamous cell carcinoma of the cervix and overexpression of p16INK4a occurs when retinoblastoma protein is inactivated by high-risk HPV, the authors studied the association of HPV infection and expression of p16INK4a in cervical adenocarcinomas. Specimens of cervical glandular neoplasias were immunostained with a p16INK4a-specific monoclonal antibody (clone E6H4). Approximately 80% of glandular neoplasms showed overexpression of p16INK4a. Exfoliated cells from 14 adenocarcinomas were further examined by p16INK4a-specific immunocytochemistry, and 12 cases showed overexpression of p16INK4a, suggesting that immunostaining for p16INK4a may be a useful diagnostic tool for cervical adenocarcinomas. The authors further examined HPV DNA in cervical adenocarcinomas with the polymerase chain reaction method. Overexpression of p16INK4a was positive in 94% of cases in which HPV16 or 18DNA was positive, a finding suggesting that HPV16 or 18 may play an important role in cervical adenocarcinomas. Overexpression of p16INK4a may be an indicator of pathogenic activity of high-risk HPVs.

Age-Related Decrease of Meiotic Cohesins in Human Oocytes

Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.

Reduction of NOTCH1 expression pertains to maturation abnormalities of keratinocytes in squamous neoplasms

Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.

Lymph node metastasis in grossly apparent stages I and II epithelial ovarian cancer

Objectives: Incidence of lymph node metastasis is relatively high even in early-stage epithelial ovarian cancers (EOC). Lymphadenectomy is important in the surgical treatment of EOC; however, the exact role of lymphadenectomy in the management of EOC remains unclear. In this study, we evaluated lymph node metastasis in stages I and II EOC patients. Patients and Methods: Seventy-nine patients with stage I/II EOC underwent initial surgery, and 68 patients received adjuvant platinum and taxane chemotherapy after surgery at Keio University Hospital. The patients were evaluated with respect to age at diagnosis, clinical stage, histology, histological grade, and tumor laterality. Results: Of the 79 patients, 10 (12.7%) had lymph node metastasis. Of these, 4 (5.1%) had lymph node metastasis in paraaortic lymph node (PAN) only, 1 (1.3%) in pelvic lymph node (PLN) only, and 5 (6.3%) in both PAN and PLN. The incidence of serous-type lymph node metastasis in PAN, PAN + PLN, and total was higher than nonserous type (25% vs 1.5%, P < 0.0001; 25% vs 3.0%, P = 0.001; 50% vs 5.9%, P < 0.0001). However, there was no significant difference between lymph node status and T factor or histological grade. In 78% of patients (7/9), metastases in contralateral lymph nodes were present (contralateral, 2; bilateral, 5). There was no significant difference in progression-free survival between node-positive and node-negative groups (P = 0.47). Conclusions: Based on diagnostic value, the result suggests that the role of lymphadenectomy might differ by histological type, as its therapeutic effect might be unclear. A multicenter analysis is essential for confirmation.

Tobacco smoking and regression of low-grade cervical abnormalities

The role of tobacco smoking in the multistage carcinogenesis at the cervix is not fully understood because of a paucity of prospective data. To assess the relationship between smoking and spontaneous regression of cervical precursor lesions, a total of 516 women with low‐grade squamous intraepithelial lesion (LSIL) were monitored by cytology and colposcopy every 4 months. Probability of LSIL regression within 2 years was analyzed in relation to smoking behaviors, with regression defined as at least two consecutive negative Pap smears and normal colposcopy. Women’s age, initial biopsy results, and human papillomavirus (HPV) genotypes were included in the multivariate models for adjustments. Our study subjects included 258 never‐smokers and 258 smokers (179 current and 79 former smokers). During a mean follow‐up time of 39.8 months, 320 lesions regressed to normal cytology. Probability of regression within 2 years was significantly lower in smokers than in never‐smokers (55.0%vs 68.8%, P = 0.004). The risk of LSIL persistence increased with smoking intensity and duration and with younger age at starting smoking (P = 0.003, P < 0.001, and P = 0.03, respectively). Smokers had twice as high a risk of persistent HPV infection compared to never‐smokers (odds ratio, 2.50; 95% confidence interval, 1.30–4.81; P = 0.006). In young women, passive smoking since childhood reduced probability of regression within 2 years (56.7%vs 85.9%, P < 0.001). Further adjustments for a wide range of cervical cancer risk factors did not change the findings. In conclusion, tobacco smoking may interfere with regression of cervical precursor lesions. Childhood exposure to second‐hand smoke may increase a risk of persistent cervical abnormalities among young women. (Cancer Sci 2010)

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor

Background:Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated.Methods:The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ.Results:In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells.Conclusions:NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.