Mitochondrial Function of CKS2 Oncoprotein Links Oxidative Phosphorylation with Cell Division in Chemoradioresistant Cervical Cancer.

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer.

Reference MicroRNAs for RT-qPCR Assays in Cervical Cancer Patients and Their Application to Studies of HPV16 and Hypoxia Biomarkers.

MicroRNA (miRNA) expressions in tumor biopsies have shown potential as biomarkers in cervical cancer, but suitable reference RNAs for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays in patient cohorts with different clinicopathological characteristics are not available. We aimed to identify the optimal reference miRNAs and apply these to investigate the potential of miR-9-5p as human papilloma virus (HPV) 16 biomarker and miR-210-3p as hypoxia biomarker in cervical cancer. Candidate reference miRNAs were preselected in sequencing data of 90 patients and ranked in a stability analysis by RefFinder. A selection of the most stable miRNAs was evaluated by geNorm and NormFinder analyses of RT-qPCR data of 29 patients. U6 small nuclear RNA (RNU6) was also included in the evaluation. MiR-9-5p and miR-210-3p expression was assessed by RT-qPCR in 45 and 65 patients, respectively. Nine candidates were preselected in the sequencing data after excluding those associated with clinical markers, HPV type, hypoxia status, suboptimal expression levels, and low stability. In RT-qPCR assays, the combination of miR-151-5p, miR-152-3p, and miR-423-3p was identified as the most stable normalization factor across clinical markers, HPV type, and hypoxia status. RNU6 showed poor stability. By applying the optimal reference miRNAs, higher miR-9-5p expression in HPV16- than HPV18-positive tumors and higher miR-210-3p expression in more hypoxic than less hypoxic tumors were found in accordance with the sequencing data. MiR-210-3p was associated with poor outcome by both sequencing and RT-qPCR assays. In conclusion, miR-151-5p, miR-152-3p, and miR-423-3p are suitable reference miRNAs in cervical cancer. MiR-9-5p and miR-210-3p are promising HPV16 and hypoxia biomarkers, respectively.

Association Between Proportion of Nuclei With High Chromatin Entropy and Prognosis in Gynecological Cancers.

Background: Nuclear texture analysis measuring differences in chromatin structure has provided prognostic biomarkers in several cancers. There is a need for improved cell-by-cell chromatin analysis to detect nuclei with highly disorganized chromatin. The purpose of this study was to develop a method for detecting nuclei with high chromatin entropy and to evaluate the association between the presence of such deviating nuclei and prognosis. Methods: A new texture-based biomarker that characterizes each cancer based on the proportion of high-chromatin entropy nuclei (<25% vs ≥25%) was developed on a discovery set of 175 uterine sarcomas. The prognostic impact of this biomarker was evaluated on a validation set of 179 uterine sarcomas, as well as on independent validation sets of 246 early-stage ovarian carcinomas and 791 endometrial carcinomas. More than 1 million images of nuclei stained for DNA were included in the study. All statistical tests were two-sided. Results: An increased proportion of high-chromatin entropy nuclei was associated with poor clinical outcome. The biomarker predicted five-year overall survival for uterine sarcoma patients with a hazard ratio (HR) of 2.02 (95% confidence interval [CI] = 1.43 to 2.84), time to recurrence for ovarian cancer patients (HR = 2.91, 95% CI = 1.74 to 4.88), and cancer-specific survival for endometrial cancer patients (HR = 3.74, 95% CI = 2.24 to 6.24). Chromatin entropy was an independent prognostic marker in multivariable analyses with clinicopathological parameters (HR = 1.81, 95% CI = 1.21 to 2.70, for sarcoma; HR = 1.71, 95% CI = 1.01 to 2.90, for ovarian cancer; and HR = 2.03, 95% CI = 1.19 to 3.45, for endometrial cancer). Conclusions: A novel method detected high-chromatin entropy nuclei, and an increased proportion of such nuclei was associated with poor prognosis. Chromatin entropy supplemented existing prognostic markers in multivariable analyses of three gynecological cancer cohorts.

Vulvar carcinoma in Norway: A 50-year perspective on trends in incidence, treatment and survival.

OBJECTIVE: To explore trends in vulvar squamous cell carcinoma (SCC) incidence, age and stage at diagnosis, treatment and survival in Norway from 1961 to 2010. METHODS: From 1961 to 2010, 2233 cases of vulvar SCC were extracted from the Cancer Registry of Norway. Data on age at diagnosis, tumor morphology, stage of the disease and treatment were analyzed. Age-standardized incidence rates, adjusted to the Norwegian standard population, were computed. Relative survival was calculated as a ratio of the observed survival in the study population over the expected survival in the background population. Multivariate Cox model was fitted to estimate hazard ratios. RESULTS: The overall incidence of vulvar SCC increased >2.5 fold (from 1.70 to 4.66 per 100,000 women/year; P<0.01). Age-specific incidence rates increased among women aged ≤60years (by 150% in age group 0-39years, 175% in age group 40-49years and 68% in age group 50-59years). From 1971 to 2010, the percentage of patients receiving surgery as only treatment decreased from 81% to 61%, whereas the use of radiation and combination therapy (surgery and radiation) increased from 3% to 11% and 6% to 20%, respectively. 5-year relative survival increased significantly among women ≤80years (from 72% to 83% among women aged ≤60years and from 60% to 65% among women aged 61-80years). CONCLUSIONS: The incidence of vulvar SCC has increased since the sixties, particularly among women younger than 60years. Despite less aggressive surgical treatment, survival has improved.

Progesterone Receptor Expression Is an Independent Prognosticator in FIGO Stage I Uterine Leiomyosarcoma.

OBJECTIVES: To analyze the clinical role of hormone receptors in a large uterine sarcomas series with long-term follow-up. METHODS: Protein expression of estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemistry was studied in tissue microarrays from 294 patients diagnosed with uterine sarcoma in Norway from 1970 to 2000 and analyzed for an association with clinicopathologic parameters and outcome. RESULTS: ER and PR were detected in 136 of 291 and 184 of 291 tumors (three noninformative cases each), respectively. Expression was unrelated to histology, patient age, tumor diameter, the degree of atypia, the presence of necrosis or vascular invasion, or mitotic counts. ER and PR expression was unrelated to survival in the analysis of the entire cohort. When survival analysis was confined to stage I leiomyosarcoma (n = 147), higher PR score was significantly related to longer overall survival (OS) (P = .042). Clinicopathologic prognosticators in this group were age (P = .041), tumor diameter (P = .001), and mitotic count (P = .007), with a trend for atypia (P = .087). In Cox multivariate analysis, PR score (P = .019), tumor diameter (P = .013), and mitotic count (P = .002) were independent prognosticators of OS. CONCLUSIONS: Hormone receptor expression is not informative of outcome in the analysis of uterine sarcomas of all stages and histologic types. PR expression identifies patients with longer survival in stage I leiomyosarcoma.

DNA ploidy may be a prognostic marker in stage I and II serous adenocarcinoma of the endometrium.

In patients with serous adenocarcinoma (SAC) of the endometrium, we evaluated the prognostic importance of clinicopathological parameters, DNA ploidy, and immunoexpression of p53, estrogen receptor (ER), progesterone receptor (PR), and Ki-67. In a series of 73 stage I and II SAC, DNA ploidy analysis was performed on hysterectomy specimens using DNA image cytometry. Immunohistochemical analysis of p53, ER, PR, and Ki-67 expression was additionally performed. In the review of the histological slides by three gynecologic pathologists, the presence of a serous component was not agreed upon in 17 (23 %) cases. The remaining 56 cases, consisting of pure SAC or SAC mixed with endometrioid adenocarcinoma, were further analyzed. Tumor recurrence was observed in 14 patients, and 28 patients died during the follow-up period. Patients with diploid (n = 19), aneuploid (n = 29), and tetraploid (n = 8) tumor had 5-year recurrence rates of 10, 38, and 53 %, respectively (p = 0.09). A DNA ploidy parameter, 5c exceeding rate, was found to be a prognostic marker for recurrence (p = 0.03), progression-free survival (p < 0.01), and overall survival (p = 0.02). Immunoexpression of p53, ER, PR, and Ki-67 did not have prognostic value, and the same was true for FIGO stage, lymphovascular invasion, the extent of myometrial invasion, and lymphadenectomy. The histological diagnosis of SAC may be difficult in some cases. Established clinicopathological parameters do not seem to be strong prognosticators in stage I and II disease. A DNA ploidy parameter, 5c exceeding rate, may be a prognostic marker in this patient group and should be further validated in larger series.

A study of chronic pelvic pain after radiotherapy in survivors of locally advanced cervical cancer.

INTRODUCTION: Chronic pelvic pain (persisting pain in hips, groins or lower back) is poorly described in studies of cervical cancer survivors (CCSs). The aims of this study were to describe chronic pelvic pain and associated variables in CCSs surveyed >5 years post-radiotherapy, and to compare the presence of pain in hips and lower back in CCSs with findings in the general female population. METHODS: Ninety-one CCSs treated with radiotherapy between 1994 and 1999 were in 2005 included in a cross-sectional questionnaire-based study. They were asked about demographic variables, clinical symptoms, mental distress, and quality of life (QOL). Normative data (NORM) were collected from a population-study of Norwegian females. RESULTS: Pain in lower back and hips was significantly more prevalent (p < .001) in CCSs compared to NORMs. 35/92 (38%) of the CCSs had chronic pelvic pain. These women had significantly lower QOL, higher levels of anxiety and depression and more bladder and intestinal problems than those without chronic pelvic pain. In a multivariable regression model, use of analgesics and intestinal and bladder problems were significantly associated with chronic pelvic pain in the CCSs. CONCLUSIONS: CCSs have a higher prevalence of pain in lower back and hips than women in the general population, which might be due to late effects of radiation. 35/92 (38%) of the CCSs suffer from chronic pelvic pain, shown to be associated with high overall mental and somatic morbidity. IMPLICATIONS FOR CANCER SURVIVORS: Evaluation and management of pelvic pain is important in follow-up of CCSs treated with radiotherapy.