Hirokuni Takano

Amplification of PVT1 Contributes to the Pathophysiology of Ovarian and Breast Cancer

Purpose: This study was designed to elucidate the role of amplification at 8q24 in the pathophysiology of ovarian and breast cancer because increased copy number at this locus is one of the most frequent genomic abnormalities in these cancers. Experimental Design: To accomplish this, we assessed the association of amplification at 8q24 with outcome in ovarian cancers using fluorescence in situ hybridization to tissue microarrays and measured responses of ovarian and breast cancer cell lines to specific small interfering RNAs against the oncogene MYC and a putative noncoding RNA, PVT1, both of which map to 8q24. Results: Amplification of 8q24 was associated with significantly reduced survival duration. In addition, small interfering RNA–mediated reduction in either PVT1 or MYC expression inhibited proliferation in breast and ovarian cancer cell lines in which they were both amplified and overexpressed but not in lines in which they were not amplified/overexpressed. Inhibition of PVT1 expression also induced a strong apoptotic response in cell lines in which it was overexpressed but not in lines in which it was not amplified/overexpressed. Inhibition of MYC, on the other hand, did not induce an apoptotic response in cell lines in which MYC was amplified and overexpressed. Conclusions: These results suggest that MYC and PVT1 contribute independently to ovarian and breast pathogenesis when overexpressed because of genomic abnormalities. They also suggest that PVT1-mediated inhibition of apoptosis may explain why amplification of 8q24 is associated with reduced survival duration in patients treated with agents that act through apoptotic mechanisms.

Adjuvant chemotherapy in a pregnant woman with endodermal sinus tumor of the ovary.

BACKGROUND: The administration of chemotherapeutic drugs during pregnancy is rare. We describe a case of malignant ovarian tumor complicating pregnancy. CASE: After laparotomy at 18 weeks of gestation, the patient received three courses of cisplatin, vinblastine, and bleomycin. Elective cesarean delivery was performed at 31 weeks of gestation. A normal infant was delivered, and no evidence of tumor recurrence was observed. Metastasis to the liver was detected in the fifth month after delivery. However, this was treated successfully with three courses of cisplatin, etoposide, and bleomycin. There was no evidence of recurrence observed at 65 months after the initial treatment. CONCLUSION: Although the risk of chemotherapy to the fetus cannot be assessed based on a single case, this experience is encouraging.

CITRUS, cervical cancer screening trial by randomization of HPV testing intervention for upcoming screening: Design, methods and baseline data of 18,471 women

BACKGROUND To assess the efficacy of screening with concurrent liquid-based cytology and human papillomavirus (HPV) testing for primary cervical cancer screening, we initiated a randomized trial entitled CervIcal cancer screening Trial by Randomization of HPV testing intervention for Upcoming Screening (CITRUS). METHODS Between June 2013 and March 2015, women aged 30-64 years of age who participated in a regular cervical cancer screening program (every 2 years) were invited to enrollment of our study. After giving their informed consent, 18,402 women were randomly assigned to liquid-based cytology as the control group (n=9145) or to HPV DNA testing with liquid-based cytology as the intervention group (n=9257). We subsequently compared the incidence rate of cervical intraepithelial neoplasia (CIN), the rate of false positive tests and the rate of overdiagnosis, as well as assessing the risks and benefits of receiving screening for women in both groups. The primary outcome of our study was the incidence of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) during the study period of around 6 years. RESULTS In the control group, 97.9% of women were NILM, and 2.06% ASC-US or worse (ASC-US+). In the intervention group, 87.13% of women were NILM/HPV negative, 0.72% ASC-US/HPV negative, 10.34% NILM/HPV positive, 0.69% ASC-US/HPV positive, 0.90% worse than ASC-US/either HPV. Positive HPV testing was not linearly related to age in our study. CONCLUSIONS Insights from CITRUS will provide future prospects for cervical cancer screening focused on the use of HPV testing in Japan. CLINICAL TRIAL REGISTRATION NUMBER NCT01895517, UMIN000010843, TRIUC1312.

Comparison of Two Sampling Procedures for Diagnosing Endometrial Carcinoma and Hyperplasia: Outpatient Tissue Biopsy Versus Cytologic Examination

Background: We compared the sensitivity of 2 diagnostic procedures—tissue biopsy and cytologic examination— for detecting endometrial carcinoma and hyperplasia in outpatients. The patients’ degree of acceptance of these methods was also evaluated. Methods: The study included 124 women who had been diagnosed with carcinoma and hyperplasia by histological examination in private clinics or were suspected to have endometrial carcinoma and hyperplasia—for example, those presenting with uterine bleeding and/or abnormal endometrial morphology on cytologic examination—at Jikei University Hospital, University of Yamanashi Hospital and National Hospital Organization Kure Medical Center from January 28, 1999, to August 28, 2006. Both cytologic examination (using Endocyte ® ) and tissue biopsy (using Suresample ™ ) of the endometrium were performed before complete curettage and/or hysterectomy. The diagnosis made using these two outpatient procedures was compared to the final diagnosis made using curettage and/or hysterectomy. McNemar’s chi-square test was used to evaluate the statistical significance. Results: The sensitivity of tissue biopsy for detecting endometrial carcinoma and hyperplasia was 84% and 91%, respectively, and that of cytologic examination was 78% and 55%, respectively. There was a significant difference in the sensitivity of the 2 methods for detecting hyperplasia (p =0.045). No patients complained of severe pain, and no other complication occurred during both methods. Both methods were well tolerated by the patients. Conclusion: Our data indicate a certain diagnostic superiority of tissue biopsy over cytologic examination.

PIK3CA and KRAS mutations in cell free circulating DNA are useful markers for monitoring ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) exhibits distinct phenotypes, such as resistance to chemotherapy, poor prognosis and an association with endometriosis. Biomarkers and imaging techniques currently in use are not sufficient for reliable diagnosis of this tumor or prediction of therapeutic response. It has recently been reported that analysis of somatic mutations in cell-free circulating DNA (cfDNA) released from tumor tissues can be useful for tumor diagnosis. In the present study, we attempted to detect mutations in PIK3CA and KRAS in cfDNA from OCCC patients using droplet digital PCR (ddPCR). Here we show that we were able to specifically detect PIK3CA-H1047R and KRAS-G12D in cfDNA from OCCC patients and monitor their response to therapy. Furthermore, we found that by cleaving wild-type PIK3CA using the CRISPR/Cas9 system, we were able to improve the sensitivity of the ddPCR method and detect cfDNA harboring PIK3CA-H1047R. Our results suggest that detection of mutations in cfDNA by ddPCR would be useful for the diagnosis of OCCC, and for predicting its recurrence.

Clinical and genetic analysis of recurrent adult-type granulosa cell tumor of the ovary: Persistent preservation of heterozygous c.402C>G FOXL2 mutation

Background Adult-type granulosa cell tumors of the ovary (aGCTs) are rare tumors that represent 2–5% of ovarian malignancies. The prognosis of this tumor is favorable, and it is characterized by slow progression. 10–30% of these tumors recur after 4–7 years of the primary surgery and the 5-year survival rate from the first recurrence is 55%, for the incompletely resected patients. At this time, complete resection is the only prognostic factor for better outcome, and establishing a novel strategy for identification and/or treatment of recurrent tumors is crucial. After the discovery of heterozygous c.402C>G FOXL2 mutations in 97% of cases of aGCT, much effort has been made to find the role of the mutation on the pathogenesis of aGCT, however, little is known about the role of the mutation in disease progression. Methods We analyzed the clinical data of 56 aGCT patients to find a marker of recurrence. In particular, we compared the FOXL2 status in 5 matched primary and recurrent samples by immunohistochemistry, and TaqMan allelic discrimination assay to address the role of FOXL2 in potential mechanisms of recurrence. Results The clinical data analysis was consistent with complete resection as an indicator of disease eradication, though the sample size was limited. The genetic analysis showed all the samples, including recurrent tumor samples up to 14 years after the primary surgery, expressed heterozygous c.402C>G FOXL2 mutation and the FOXL2 protein expression. Conclusion This report describes the preservation of heterozygous c.402C>G FOXL2 mutation in recurrent aGCTs. This finding adds further credence to the concept that the c.402C>G FOXL2 mutation is oncogenic and integral to this disease.

Prognostic impact of interleukin-6 expression in stage I ovarian clear cell carcinoma

OBJECTIVE Ovarian clear cell carcinoma (OCCC) frequently presents at an early stage. In stage I OCCC, the prognosis differs according to substage. In particular, predictive biomarkers and new treatment strategies are needed for stage IC2/IC3 disease. We investigated tumor biology and prognostic factors for stage I OCCC from a clinicopathological perspective, including the expression of ARID1A and IL-6, which are considered critical for OCCC carcinogenesis. METHODS A retrospective cohort study of 192 patients with stage I OCCC treated at a single institution was performed. We calculated overall survival (OS) with respect to 12 clinicopathological parameters that included the unique and diverse histological features of OCCC. RESULTS The estimated 5-year OS rate in patients with all stage I OCCC was 88.9% during a median of 91months of follow-up. The multivariate analysis indicated that substage classification and IL-6 expression status were associated with poor OS (p=0.010 and p=0.027, respectively). Loss of ARID1A expression had no impact on survival; however, it was associated with substage (p=0.001), capsule rupture status (p=0.011), and ascites cytology (p=0.016). No clear association was found between ARID1A and IL-6 expressions. Histological findings, including the presence of endometriosis, adenofibroma, architectural pattern, and tumor cell type, showed no prognostic effects. CONCLUSIONS Both substage classification and IL-6 expression status may be independent prognostic factors in stage I OCCC. Therefore, IL-6 molecular stratification may be crucial in optimizing therapeutic strategies for early stage OCCC to improve survival.

Retrospective analysis of sites of recurrence in stage I epithelial ovarian cancer

Objective The aim of the study is to investigate recurrence of stage I epithelial ovarian cancer. Methods Six hundred two patients diagnosed with stage I epithelial ovarian cancer at 4 hospitals between 2000 and 2013 were retrospectively analyzed. Age, surgical procedure, substage, histologic type, adjuvant chemotherapy, recurrence, initial recurrence site (peritoneal dissemination [P], hematogenous recurrence [H], lymphogenous recurrence [L], and others [O]), and frequency of recurrence at each site were investigated retrospectively. Results Median age was 54 years and median follow-up was 60 months. The stage was IA in 180 cases (30%), IB in 8 (1%), IC1 in 247 (41%), IC2 in 63 (10%), and IC3 in 104 (17%). Systematic lymph node dissection including both pelvic and para-aortic lymph nodes was performed in 224 patients (37%), and 412 patients (68%) received adjuvant chemotherapy. Recurrence occurred in 70 patients (11.6%). The median time to recurrence was 18 months, and the stage was IA in 13 (19%), IB in 1 (1%), IC1 in 24 (34%), IC2 in 9 (13%), and IC3 in 23 (33%) cases. The numbers of recurrence at the P, H, L, and O sites, including overlapping cases, were 49 (70%), 18 (26%), 9 (13%), and 6 (9%), respectively, and recurrence by peritoneal dissemination in the pelvis occurred in 43 cases (61%). Conclusion Recurrence of stage I epithelial ovarian cancer by peritoneal dissemination was frequent, especially in the pelvis. There is a need to elucidate the pathogenesis of peritoneal recurrence and to prepare a treatment strategy to prevent pelvic peritoneal recurrence.

Clinical associations of Trousseau's syndrome associated with cerebral infarction and ovarian cancer

Objective Since there have been few large series studies to date, we investigated the relationship between Trousseaus syndrome associated with cerebral infarction and its clinical associations with ovarian cancer. Methods In this study, we investigated the association between cerebral infarction onset and ovarian cancer. Eight-hundred twenty-seven consecutive ovarian cancer patients from 4 affiliated academic institutions were included in the study over a 12 years period. All patients were histopathologically diagnosed as epithelial ovarian cancer and were analyzed retrospectively. Results The 27 patients (3.2%) presented with cerebral infarction during the study period, 14 patients onset prior to treatment (1.7%), and 13 patients onset after start of initial treatment (1.5%). Univariate analysis and multivariate analysis was performed for onset of Trousseaus syndrome and various clinical and pathological parameters. There was no statistical significance between the occurrence of Trousseaus syndrome with age or International Federation of Gynecology and Obstetrics (FIGO) stage; however, univariate analysis and multivariate analysis demonstrated a statistically significant association between clear cell carcinoma (CCC) and non-CCC histology. Conclusion Thus, our results demonstrate that Trousseaus syndrome with cerebral infarction occurred with greater incidence among CCC cases compared to non-CCC cases.

Liquid-based cytology versus conventional cytology for detection of uterine cervical lesions: a prospective observational study

Objective Liquid-based cytology (LBC) and conventional cytology (CS) are routine diagnostic techniques in cervical cytology, but few studies have compared their diagnostic performances with each other and with histologic diagnosis. This study aimed to compare the diagnostic performances of these techniques in subjects with abnormal cervical cytology of atypical cells of undetermined significance (ASC-US) or worse. Methods A total of 312 patients diagnosed with ASC-US or worse were enrolled in this prospective study in Japan from 2013 to 2014. LBC and CS samples were prepared by a split-sampling technique and evaluated blindly. The results were classified using the Bethesda System 2001. Colposcopy and biopsy were conducted simultaneously or within 4 weeks of cytology-specimen collection in all cases. Diagnostic performance was calculated in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of CIN2 or worse, with a cut-off ASC-US or worse. Results There was one unsatisfactory CS sample and the remaining 311 cases were evaluated. The sensitivities of LBC and CS were 100.0% and 98.8%, specificities were 17.2% and 23.8%, PPVs were 56.1% and 57.9% and NPVs were 100.0% and 94.7%, respectively. LBC had slightly higher sensitivity and NPV for detection of CIN2, but there was no significant difference between the two methods. Conclusions There was no significant difference in the diagnostic performances of LBC and CS in patients with ASC-US or worse. LBC may therefore be an alternative approach to CS for cervical cancer screening.