Integrated genomic and transcriptomic analysis reveals the activation of PI3K signaling pathway in HPV-independent cervical cancers.

BACKGROUND: HPV-independent cervical cancers (HPV-ind CCs) are uncommon with worse prognosis and poorly understood. This study investigated the molecular characteristics of HPV-ind CCs, aiming to explore new strategies for HPV-ind CCs. METHODS: HPV status of 1010 cervical cancer patients were detected by RT-PCR, PCR and RNA-sequencing (RNA-seq). Whole exome sequencing (WES) and RNA-seq were performed in identified HPV-ind CCs. The efficacy of PI3Kα inhibitor BYL719 in HPV-ind CCs was evaluated in cell lines, patient-derived organoids (PDOs) and patient-derived xenografts (PDXs). RESULTS: Twenty-five CCs were identified as HPV-ind, which were more common seen in older, adenocarcinoma patients and exhibited poorer prognosis as well as higher tumor mutation burden compared to HPV-associated CCs. HPV-ind CCs were featured with highly activated PI3K/AKT signaling pathway, particularly, PIK3CA being the most predominant genomic alteration (36%). BYL719 demonstrated superior tumor suppression in vitro and in vivo. Furthermore, HPV-ind CCs were classified into two subtypes according to distinct prognosis by gene expression profiles, the metabolism subtype and immune subtype. CONCLUSIONS: This study reveals the prevalence, clinicopathology, and molecular features of HPV-ind CCs and emphasizes the importance of PIK3CA mutations and PI3K pathway activation in tumorigenesis, which suggests the potential significance of PI3Kα inhibitors in HPV-ind CC patients.

Targeting of ß-Catenin Reverses Radioresistance of Cervical Cancer with the PIK3CA-E545K Mutation.

This study aims to explore whether E545K, the most common hotspot mutation of PIK3CA in cervical cancer, confers radioresistance to cervical cancer cells, to demonstrate the underling mechanism, and to develop the effective targets. SiHa and MS751 cells with PIK3CA-WT and PIK3CA-E545K were established by lentiviral transfection. The radiosensitivity was assessed by colony formation, cell cycle, cell apoptosis, DNA damage, and repair assay. The growth and immunohistochemical assay of xenograft tumor-related toxicity were evaluated in vivo It was indicated that more cells with PIK3CA-E545K arrested in S phase. Irradiation (IR) led to more survival percentage, less apoptosis, fewer pH2A.X foci, and higher expression of Chk1/2 in SiHa and MS751 cells bearing PIK3CA-E545K. Mechanically, AKT/GSK3ß/ß-catenin pathway was highly activated, and more ß-catenin was found accumulated in nucleus in cells with PIK3CA-E545K after IR. Furthermore, targeting ß-catenin by shRNA or XAV939 enhanced IR sensitivity in cells with PIK3CA-WT and PIK3CA-E545K, whereas it was more notably in the latter. ß-Catenin shRNA and XAV939 increased IR-mediated inhibition of colony formation with highly activated p53/bcl2/bax pathway. XAV939 enhanced IR-caused apoptosis, DNA damage, overcame S-phase arrest, DNA repair and reversed ß-catenin nuclear accumulation in MS751 cells with PIK3CA-E545K. In vivo, XAV939 enhanced the radiosensitivity of cervical cancer xenografts with PIK3CA-E545K with invisible viscera toxicity. The findings demonstrate that cervical cancer cells with PIK3CA-E545K are resistant to IR by enhancing the expression and nuclear accumulation of ß-catenin. Targeting ß-catenin reverses the radioresistance, which suggests possible areas for preclinical research on ß-catenin inhibition for strengthening the radiosensitivity of cervical cancer.

The PIK3CA E542K and E545K mutations promote glycolysis and proliferation via induction of the ß-catenin/SIRT3 signaling pathway in cervical cancer.

BACKGROUND: The study aims to present the effect of PIK3CA E542K and E545K mutations on glucose metabolism and proliferation and identify their underlying mechanisms in cervical cancer. METHODS: The maximum standard uptake value (SUVmax) of tumors was detected by18F-FDG PET/CT scan. In vitro, glycolysis analysis, extracellular acidification rate analysis, and ATP production were used to evaluate the impact of PIK3CA E542K and E545K mutations on glucose metabolism. The expression level of key glycolytic enzymes was evaluated by western blotting and immunohistochemical staining in cervical cancer cells and tumor tissues, respectively. Immunofluorescence analysis was used to observe the nuclear translocation of ß-catenin. The target gene of ß-catenin was analyzed by using luciferase reporter system. The glucose metabolic ability of the xenograft models was assessed by SUVmax from microPET/CT scanning. RESULTS: Cervical cancer patients with mutant PIK3CA (E542K and E545K) exhibited a higher SUVmax value than those with wild-type PIK3CA (P = 0.037), which was confirmed in xenograft models. In vitro, enhanced glucose metabolism and proliferation was observed in SiHa and MS751 cells with mutant PIK3CA. The mRNA and protein expression of key glycolytic enzymes was increased. AKT/GSK3ß/ß-catenin signaling was highly activated in SiHa and MS751 cells with mutant PIK3CA. Knocking down ß-catenin expression decreased glucose uptake and lactate production. In addition, the nuclear accumulation of ß-catenin was found in SiHa cells and tumors with mutant PIK3CA. Furthermore, ß-catenin downregulated the expression of SIRT3 via suppressing the activity of the SIRT3 promotor, and the reduced glucose uptake and lactate production due to the downregulation of ß-catenin can be reversed by the transfection of SIRT3 siRNA in SiHa cells with mutant PIK3CA. The negative correlation between ß-catenin and SIRT3 was further confirmed in cervical cancer tissues. CONCLUSIONS: These findings provide evidence that the PI3K E542K and E545K/ß-catenin/SIRT3 signaling axis regulates glucose metabolism and proliferation in cervical cancers with PIK3CA mutations, suggesting therapeutic targets in the treatment of cervical cancers. TRIAL REGISTRATION: FUSCC 050432-4-1212B. Registered 24 December 2012 (retrospectively registered).

Establishment and molecular characterization of a human ovarian clear cell carcinoma cell line (FDOV1).

BACKGROUND: Ovarian clear cell carcinoma is a distinct histologic subtype with grave survival. The underlying molecular mechanism is not fully elucidated. However, we don't have many cell lines, which are useful experimental tools for research. We describe the establishment and characterization of a new ovarian clear cell carcinoma cell line from a Chinese patient. RESULTS: FDOV1 has been subcultured for more than 80 generations. Monolayer cultured cells are polygonal in shape, showing a transparent cytoplasm full of vacuoles. The number of chromosomes ranges from 45 to 90. FDOV1 cells produces CA-125, but not CA-199. The cells could be transplanted and produced tumors mimicking the donor tumor morphologically and immunohistochemically. Whole exome sequence showed both FDOV1 and tissue block harbored PIK3CA H1047R mutation and ARID1A frameshift mutations (p.L2106 fs, p.N201 fs). More interestingly, we observed SPOP mutation (p.D82H) and ZNF217 (chromosome 20q13) amplification in FDOV1, which are quite novel. CONCLUSIONS: Only a few patient-derived ovarian clear cell carcinoma cell lines have been reported in the literature. FDOV1 is the very first one, to the best of our knowledge, from a Mainland Chinese patient. It showed infinite multiplication until now and tumorigenicity in vivo. FDOV1 has co-existing PIK3CA and ARID1A mutations. It also harbored SPOP mutation and ZNF217 amplification, which would probably be a good model for exploring the molecular mechanism of ovarian clear cell carcinoma.

Mutational analysis of KRAS and its clinical implications in cervical cancer patients.

OBJECTIVE: The predictive and prognostic role of KRAS mutations in cervical cancer remains inconclusive. The aim of this study was to explore the clinicopathological and prognostic relevance of KRAS mutations in invasive cervical cancers (ICC). METHODS: Reverse transcription polymerase chain reaction (PCR) and Sanger sequencing were employed to detect KRAS mutations in 876 ICC patients. Quantitative real-time PCR was used to detect human papillomavirus (HPV) 16 and HPV 18. RESULTS: Non-synonymous mutations of KRAS were identified in 30 (3.4%) patients. These mutations were more common in non-squamous cell carcinoma than in squamous cell carcinoma (SCC) (8.2% vs. 2.2%, respectively, p<0.001) and were associated with HPV 18 infection (p=0.003). The prevalence of mutations was highest (18.2%) in the uncommon histological subtypes followed by adenocarcinoma (AC, 7.3%) and adenosquamous carcinoma (ASC, 5.8%). During the median follow-up of 55 months, compared to patients with wild-type KRAS, a greater percentage of patients with mutant KRAS relapsed (20.0% vs. 42.9%, respectively, p=0.007). The 3-year relapse-free survival was poorer in patients with mutant KRAS than in patients without KRAS mutations (57.1% vs. 81.9%, respectively, p=0.001). Furthermore, the multivariate analysis showed that the presence of a KRAS mutation was an independent predictor for disease recurrence (hazard ratio [HR]=2.064; 95% confidence interval [CI]=1.125-3.787; p=0.019). CONCLUSION: KRAS mutations were predominant in non-SCCs of the cervix and were associated with HPV 18 infection. A combination of KRAS mutation detection and HPV genotyping would be useful in identifying patient with poor prognosis for further interventions.

ERBB2 mutation: A promising target in non-squamous cervical cancer.

OBJECTIVE: ERBB2 mutations have been found in a subset of invasive cervical cancer (ICC). Nevertheless, the prevalence, mutation spectrum, clinicopathological relevance, human papillomavirus (HPV)-genotype association and prognostic significance of ERBB2-mutated ICCs have not been well established. METHODS: In this study, ICC samples (N=1015) were assessed for mutations in ERBB2, KRAS, and PIK3CA by cDNA-based Sanger sequencing. RESULTS: Somatic ERBB2 mutations were detected in 3.15% patients. The ERBB2 mutation rate was significantly higher in adenocarcinoma (4.52%, 7/155), adenosquamous carcinoma (7.59%, 6/79) and neuroendocrine carcinoma (10.34%, 3/29) than that in squamous carcinoma (2.14%, 16/749) (P=0.004, Fisher exact test). In addition, 18.75% of the patients carrying ERBB2 mutations concomitantly harbored PIK3CA or KRAS mutations. Patients with ERBB2-mutated ICCs tended to have a worse prognosis than those with wild-type or PIK3CA-mutated ICCs but a better prognosis than those with KRAS-mutated ICCs. CONCLUSIONS: This study provided a promising rationale for the clinical investigation of tyrosine kinase inhibitors for the treatment of cervical cancer with ERBB2 mutations. Patients with non-squamous cell carcinomas have priority as candidates for ERBB2-targeted therapy. Concurrent PIK3CA/RAS mutations should be considered in the design of clinical trials.

Cycles of cisplatin and etoposide affect treatment outcomes in patients with FIGO stage I-II small cell neuroendocrine carcinoma of the cervix.

OBJECTIVE: This study sought to explore the outcomes and prognostic factors of patients with small cell neuroendocrine carcinoma of the cervix (SCNEC) and to determine the effects of adjuvant treatment on survival in patients with FIGO stage I-II SCNEC after radical surgery. METHODS: A single-institution retrospective analysis was performed in 92 patients who underwent radical surgery for SCNEC. All clinicopathological variables and treatment strategies were reviewed. Kaplan-Meier and Cox regression methods were used for survival analyses. RESULTS: During a median follow-up period of 38months (23.6-52.4), 43 (46.7%) patients experienced disease recurrence, and distant metastases were documented in 35 (81.4%) patients. The 3-year recurrence-free survival (RFS) for the entire group was 50.1%. The median RFS was 39months. The multivariate analysis confirmed that lymph node metastasis, positive parametrial extension and cycles of etoposide plus platinum (EP) were independent prognostic factors for disease recurrence. Adjuvant chemotherapy for at least 5cycles of EP (EP 5+, n=39) was associated with improved 5-year RFS compared with other treatments (n=46) (67.6% vs. 20.9%, p<0.001). Additional radiotherapy or concurrent chemoradiation failed to validate further improved RFS in patients with EP 5+, and this finding was consistent in the subset of patients with high-risk factors (positive lymph nodes or positive parametrium). CONCLUSIONS: Half of stage I-II SCNEC patients experienced disease failure within 3years, and distant metastasis was an outstanding issue. EP regimen for at least 5cycles improved long-term RFS after radical surgery. Additional radiation might be unnecessary, even in patients with high-risk factors.

Diaphragmatic Surgery and Related Complications In Primary Cytoreduction for Advanced Ovarian, Tubal, and Peritoneal Carcinoma.

BACKGROUND: To evaluate the procedures and complications of diaphragm peritonectomy (DP) and diaphragm full-thickness resection (DFTR) during primary cytoreduction for advanced stage epithelial ovarian cancer. METHODS: All the patients with epithelial ovarian carcinoma who underwent diaphragm procedures at our institution between January 2009 and August 2015 were identified. Clinicopathological data were retrospectively collected from the patients' medical records. Postoperative morbidities were assessed according to the Memorial Sloan-Kettering Cancer Center (MSKCC) grading system. RESULTS: A total of 150 patients were included in the study. The majority of the patients had ovarian cancer (96%), stage IIIC disease (76%) and serous histology (89.3%). DP and DFTR were performed in 124 (82.7%) and 26 (17.3%) patients, respectively. A total of 142 upper abdominal procedures in addition to the diaphragmatic surgery were performed in 77 (51.3%) patients. No macroscopic residual disease was observed in 35.3% of the patients, while 84% of the total patient cohort had residual disease ≤1 cm. The overall incidence of at least one major morbidity (MSKCC grades 3-5) was 18.0%, whereas pleural effusions (33.3%), pneumonia (15.3%) and pneumothorax (7.3%) were the most commonly reported morbidities. The rate of postoperative pleural drainage was 14.6% in total, while half the patients in the DFTR group received drainage intraoperatively (11.5%) and postoperatively (38.5%). The incidence of postoperative pleural effusion was associated with stage IV disease (hazard ratio [HR], 17.2; 95% confidence interval [CI]: 4.5-66.7; P < 0.001), DFTR (HR, 4.9; 95% CI: 1.2-19.9; P = 0.028) and a long surgery time (HR, 15.4; 95% CI: 4.3-55.5; P < 0.001). CONCLUSIONS: Execution of DP and DFTR as part of an extensive upper abdominal procedure resulted in an acceptable morbidity rate. Pleural effusion, pneumonia and pneumothorax were the most common pulmonary morbidities. The pleural drainage rate was not high enough to justify prophylactic chest tube placement for all the patients. However, patients who underwent DFTR merited special consideration for intraoperative prophylactic drainage.

Distal pancreatectomy with splenectomy for the management of splenic hilum metastasis in cytoreductive surgery of epithelial ovarian cancer.

OBJECTIVE: Distal pancreatectomy with splenectomy may be required for optimal cytoreductive surgery in patients with epithelial ovarian cancer (EOC) metastasized to splenic hilum. This study evaluates the morbidity and treatment outcomes of the uncommon procedure in the management of advanced or recurrent EOC. METHODS: This study recruited 18 patients who underwent distal pancreatectomy with splenectomy during cytoreductive surgery of EOC. Their clinicopathological characteristics and follow-up data were retrospectively analyzed. RESULTS: All tumors were confirmed as high-grade serous carcinomas. The median diameter of metastatic tumors located in splenic hilum was 3.5 cm (range, 1 to 10 cm). Optimal cytoreduction was achieved in all patients. Eight patients (44.4%) suffered from postoperative complications. The morbidity associated with distal pancreatectomy and splenectomy included pancreatic leakage (22.2%), encapsulated effusion in the left upper quadrant (11.1%), intra-abdominal infection (11.1%), pleural effusion with or without pulmonary atelectasis (11.1%), intestinal obstruction (5.6%), pneumonia (5.6%), postoperative hemorrhage (5.6%), and pancreatic pseudocyst (5.6%). There was no perioperative mortality. The majority of complications were treated successfully with conservative management. During the median follow-up duration of 25 months, nine patients experienced recurrence, and three patients died of the disease. The 2-year progression-free survival and overall survival were 40.2% and 84.8%, respectively. CONCLUSION: The inclusion of distal pancreatectomy with splenectomy as part of cytoreduction for the management of ovarian cancer was associated with high morbidity; however, the majority of complications could be managed with conservative therapy.