Janet S. Rader

Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours

Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and defined a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG). Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour lines revealed only rare inactivating mutations. Recently, de novo methylation at a CpG island associated with isoform A of RASSF1 (RASSF1A) was reported in lung tumours and tumour lines. To investigate RASSF1A as a candidate TSG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours. RASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumours and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour lines. In view of the lower frequency of RASSF1 methylation in primary breast cancers we proceeded to RASSF1 mutation analysis in 40 breast cancers. No mutations were detected, but six single nucleotide polymorphisms were identified. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methylation, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (P=0.0012), one out of five ovarian and none out of six cervical tumours with 3p21.3 loss had RASSF1A methylation. These results suggest that (a) RASSF1A inactivation by two hits (methylation and loss) is a critical step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser importance in NSCLC, breast, ovarian and cervical cancers in which other genes within LCTSGR1 are likely to be implicated.

A MicroRNA Expression Signature for Cervical Cancer Prognosis

Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. Furthermore, functional studies were done to characterize the effect of miRNAs in cervical cancer cells. Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.

Posttherapy [18F] Fluorodeoxyglucose Positron Emission Tomography in Carcinoma of the Cervix: Response and Outcome

PURPOSE The aim of this study was to evaluate response to therapy using posttherapy molecular imaging with [(18)F] fluorodeoxyglucose (FDG), and to compare the response with patient outcome. PATIENTS AND METHODS This was a retrospective medical record review of 152 patients with carcinoma of the cervix. All patients underwent a pre- and posttreatment whole-body positron emission tomography (PET) imaging scan with FDG. Patients were treated with external irradiation and intracavitary brachytherapy, and most received concurrent weekly cisplatin. Posttherapy whole-body FDG-PET was performed 1 to 12 months (mean, 3 months) after completion of treatment. RESULTS The posttherapy PET did not show any abnormal FDG uptake in 114 patients, and their 5-year cause-specific survival estimate was 80%. There was persistent (in the irradiated region) abnormal FDG uptake in the cervix or lymph nodes in 20 patients. Their 5-year cause-specific survival estimate was 32%. New anatomic sites (in unirradiated regions) of abnormal FDG uptake were present in 18 patients, and none were alive at 5 years. A Cox proportional hazards model of survival outcome indicated that any abnormal posttherapy FDG uptake (persistent or new) was the most significant prognostic factor for developing metastatic disease and death from cervical cancer when compared with pretreatment- and treatment-related prognostic factors (P <.0001). CONCLUSION Posttherapy abnormal FDG uptake (persistent or new) as detected by whole-body PET measures tumor response and might be predictive of tumor recurrence and death from cervical cancer. Prospective validation of these results is warranted.

Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers

Endometrial cancer is the most common gynecologic malignancy in the United States and the most frequent extracolonic tumor in hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC patients have inherited defects in DNA mismatch repair and the microsatellite instability (MSI) tumor phenotype. Sporadic endometrial cancers also exhibit MSI, usually associated with methylation of the MLH1 promoter. Germ-line MSH6 mutations, which are rare in HNPCC, have been reported in several families with multiple members affected with endometrial carcinoma. We reasoned that MSH6 mutation might account for loss of mismatch repair in MSI-positive endometrial cancers in which the cause of MSI is unknown. We therefore investigated MSI and MLH1 promoter methylation in 441 endometrial cancer patients unselected for age or personal and family history of cancers. MSI and MLH1 promoter methylation status were associated with age of onset and tumor histology. One hundred cases (23% of the entire series) were evaluated for MSH6 defects. Inactivating germ-line MSH6 mutations were identified in seven women with MSI-positive, MLH1 promoter unmethylated cancers. Most of the MSI in these cases was seen with mononucleotide repeat markers. The MSH6 mutation carriers were significantly younger than the rest of the population (mean age 54.8 versus 64.6, P = 0.04). Somatic mutations were seen in 17 tumors, all of which had MSI. Our data suggest that inherited defects in MSH6 in women with endometrial cancer are relatively common. The minimum estimate of the prevalence of inherited MSH6 mutation in endometrial cancer is 1.6% (7 of 441), comparable with the predicted prevalence for patients with colorectal cancer.

Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6

MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancer-derived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR-34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16(+) and HPV18(+) cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G(0)/G(1) phase cells. Ectopic expression of miR-34a in HPV18(+) HeLa cells and HPV(-) HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer.

Lymph Node Staging by Positron Emission Tomography in Cervical Cancer: Relationship to Prognosis

PURPOSE A previous retrospective study demonstrated that positron emission tomography with [(18)F]fluorodeoxyglucose (FDG-PET) was more sensitive than computed tomography for lymph node staging in patients with cervical cancer; the findings on FDG-PET were strongly associated with progression-free survival. Therefore, a prospective cohort study was initiated to evaluate FDG-PET lymph node staging in a larger patient population. PATIENTS AND METHODS The study was conducted between July 2000 and March 2009. All 560 patients with cervical cancer underwent pretreatment FDG-PET lymph node staging. Treatment included surgery alone, surgery and postoperative radiation therapy, and definitive radiation or combination radiation and chemotherapy. PET findings were correlated with the risk of disease progression and with survival. Results Overall, 47% of patients had lymph node involvement by FDG-PET at diagnosis. The frequency of lymph node metastasis increased with clinical stage and was similar to that in historical surgical series. Within a stage, patients with PET-positive lymph nodes had significantly worse disease-specific survival than those with PET-negative lymph nodes (P < .001). Disease-specific survival was stratified into distinct groups based on the most distant level of PET-detected nodal disease (none, pelvic, para-aortic, or supraclavicular; P < .001). The hazard ratios for disease recurrence increased incrementally based on the most distant level of nodal disease: pelvic 2.40 (95% CI, 1.63 to 3.52), para-aortic 5.88 (95% CI, 3.80 to 9.09), and supraclavicular 30.27 (95% CI 16.56 to 55.34). CONCLUSION Nodal involvement detected by FDG-PET in cervical cancer relates to clinical stage, is comparable to historical data, and stratifies patient recurrence and survival outcomes.

Bevacizumab combination therapy in recurrent, platinum-refractory, epithelial ovarian carcinoma: A retrospective analysis.

The study was undertaken to determine the safety and efficacy of the monoclonal, antivascular endothelial growth factor antibody bevacizumab in combination with cytotoxic chemotherapy for women with platinum‐refractory ovarian cancer.

Clinical Outcomes of Definitive Intensity-Modulated Radiation Therapy With Fluorodeoxyglucose-Positron Emission Tomography Simulation in Patients With Locally Advanced Cervical Cancer

PURPOSE This study aimed to evaluate the toxicity and clinical outcomes for cervical cancer patients treated definitively with intensity-modulated radiation therapy (IMRT) compared with non-IMRT treatment. METHODS AND MATERIALS This prospective cohort study included 452 patients with newly diagnosed cervical cancer treated with curative intent (135 IMRT and 317 non-IMRT). Treatment involved external irradiation and brachytherapy, and 85% of patients received concurrent chemotherapy. All IMRT patients underwent an F-18 fluorodeoxyglucose positron emission tomography (FDG-PET/CT) simulation. A 3-month post-therapy PET was obtained to evaluate treatment response. Toxicity was scored by the Common Terminology Criteria for Adverse Events Version 3.0. RESULTS The IMRT and non-IMRT groups had similar stage distribution and histology. For all patients, the post-therapy FDG-PET response correlated with overall recurrence risk (p < 0.0001) and cause-specific survival (p < 0.0001). Post-treatment FDG-PET findings were not significantly different between the IMRT and non-IMRT patients (p = 0.9774). The mean follow-up for all patients alive at the time of last follow-up was 52 months (72 months non-IMRT, 22 months IMRT). At last follow-up, 178 patients (39 IMRT, 139 non-IMRT) had developed a recurrence. The difference in recurrence-free survival between the two groups did not reach statistical significance (p = 0.0738), although the IMRT group showed better overall and cause-specific survivals (p < 0.0001). Of the patients, 62 patients (8 IMRT and 54 non-IMRT) developed Grade 3 or greater bowel or bladder complications, and by cumulative hazard function analysis the risk was significantly less for patients treated with IMRT (p = 0.0351). CONCLUSION Cervical cancer patients treated with FDG-PET/CT-guided IMRT have improved survival and less treatment-related toxicity compared with patients treated with non-IMRT radiotherapy.

Utility of parametrectomy for early stage cervical cancer treated with radical hysterectomy

Removal of the parametrial soft tissue is recommended for patients with cervical cancer undergoing radical hysterectomy. Parametrectomy results in significant morbidity. The objective of the study was to determine factors predictive of parametrial tumor spread and to define a subset of patients at low risk for parametrial disease.

Microsatellite Instability and Epigenetic Inactivation of MLH1 and Outcome of Patients With Endometrial Carcinomas of the Endometrioid Type

PURPOSE Most studies of microsatellite instability (MSI) and outcomes in endometrial cancer patients have included varied histologic subtypes. Nonetheless, MSI occurs almost exclusively in endometrioid tumors. The impact of MSI on outcomes in patients with endometrial cancer is controversial. We sought to determine whether MSI and MLH1 methylation are associated with clinicopathologic variables and survival outcomes in a large series of patients with endometrial carcinomas of the endometrioid type. PATIENTS AND METHODS Tumor samples, blood, and clinicopathologic data were prospectively collected and analyzed for 446 patients with endometrioid carcinomas. MSI was determined using five National Cancer Institute (NCI) consensus panel markers, and the methylation status of the MLH1 promoter was determined by combined bisulfite restriction analysis (COBRA). Associations with clinicopathologic variables and survival outcomes were evaluated. RESULTS MSI was identified in 147 cases (33%). MSI was associated with higher International Federation of Gynecology and Obstetrics (FIGO) grade (P < .0001). MSI+ tumors without MLH1 methylation were associated with younger age (P < .001). MSI was not associated with overall survival (OS; hazard ratio [HR], 1.011; 95% CI, 0.688 to 1.484; P = .96) or disease-free survival (DFS; HR 0.951; 95% CI, 0.554 to 1.635; P = .86). The combined MSI/MLH1 methylation status (treating MSI- as the reference) did not predict OS (MSI+/MLH1-U: HR, 0.62; 95% CI, 0.27 to 1.44; P = .26; MSI+/MLH1-M: HR, 0.95; 95% CI, 0.62 to 1.46; P = .82) or DFS (MSI+/MLH1-U: HR, 0.51; 95% CI, 0.22 to 1.19; P = .12; MSI+/MLH1-M: HR, 0.93; 95% CI, 0.62 to 1.40; P = .72). CONCLUSION MSI is not associated with survival in patients with endometrioid endometrial cancer.