Elena Pereira

Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers

Background High-grade serous ovarian and endometrial cancers are the most lethal female reproductive tract malignancies worldwide. In part, failure to treat these two aggressive cancers successfully centers on the fact that while the majority of patients are diagnosed based on current surveillance strategies as having a complete clinical response to their primary therapy, nearly half will develop disease recurrence within 18 months and the majority will die from disease recurrence within 5 years. Moreover, no currently used biomarkers or imaging studies can predict outcome following initial treatment. Circulating tumor DNA (ctDNA) represents a theoretically powerful biomarker for detecting otherwise occult disease. We therefore explored the use of personalized ctDNA markers as both a surveillance and prognostic biomarker in gynecologic cancers and compared this to current FDA-approved surveillance tools. Methods and Findings Tumor and serum samples were collected at time of surgery and then throughout treatment course for 44 patients with gynecologic cancers, representing 22 ovarian cancer cases, 17 uterine cancer cases, one peritoneal, three fallopian tube, and one patient with synchronous fallopian tube and uterine cancer. Patient/tumor-specific mutations were identified using whole-exome and targeted gene sequencing and ctDNA levels quantified using droplet digital PCR. CtDNA was detected in 93.8% of patients for whom probes were designed and levels were highly correlated with CA-125 serum and computed tomography (CT) scanning results. In six patients, ctDNA detected the presence of cancer even when CT scanning was negative and, on average, had a predictive lead time of seven months over CT imaging. Most notably, undetectable levels of ctDNA at six months following initial treatment was associated with markedly improved progression free and overall survival. Conclusions Detection of residual disease in gynecologic, and indeed all cancers, represents a diagnostic dilemma and a potential critical inflection point in precision medicine. This study suggests that the use of personalized ctDNA biomarkers in gynecologic cancers can identify the presence of residual tumor while also more dynamically predicting response to treatment relative to currently used serum and imaging studies. Of particular interest, ctDNA was an independent predictor of survival in patients with ovarian and endometrial cancers. Earlier recognition of disease persistence and/or recurrence and the ability to stratify into better and worse outcome groups through ctDNA surveillance may open the window for improved survival and quality and life in these cancers.

Genomic Analysis of Uterine Lavage Fluid Detects Early Endometrial Cancers and Reveals a Prevalent Landscape of Driver Mutations in Women without Histopathologic Evidence of Cancer: A Prospective Cross-Sectional Study

Background Endometrial cancer is the most common gynecologic malignancy, and its incidence and associated mortality are increasing. Despite the immediate need to detect these cancers at an earlier stage, there is no effective screening methodology or protocol for endometrial cancer. The comprehensive, genomics-based analysis of endometrial cancer by The Cancer Genome Atlas (TCGA) revealed many of the molecular defects that define this cancer. Based on these cancer genome results, and in a prospective study, we hypothesized that the use of ultra-deep, targeted gene sequencing could detect somatic mutations in uterine lavage fluid obtained from women undergoing hysteroscopy as a means of molecular screening and diagnosis. Methods and Findings Uterine lavage and paired blood samples were collected and analyzed from 107 consecutive patients who were undergoing hysteroscopy and curettage for diagnostic evaluation from this single-institution study. The lavage fluid was separated into cellular and acellular fractions by centrifugation. Cellular and cell-free DNA (cfDNA) were isolated from each lavage. Two targeted next-generation sequencing (NGS) gene panels, one composed of 56 genes and the other of 12 genes, were used for ultra-deep sequencing. To rule out potential NGS-based errors, orthogonal mutation validation was performed using digital PCR and Sanger sequencing. Seven patients were diagnosed with endometrial cancer based on classic histopathologic analysis. Six of these patients had stage IA cancer, and one of these cancers was only detectable as a microscopic focus within a polyp. All seven patients were found to have significant cancer-associated gene mutations in both cell pellet and cfDNA fractions. In the four patients in whom adequate tumor sample was available, all tumor mutations above a specific allele fraction were present in the uterine lavage DNA samples. Mutations originally only detected in lavage fluid fractions were later confirmed to be present in tumor but at allele fractions significantly less than 1%. Of the remaining 95 patients diagnosed with benign or non-cancer pathology, 44 had no significant cancer mutations detected. Intriguingly, 51 patients without histopathologic evidence of cancer had relatively high allele fraction (1.0%–30.4%), cancer-associated mutations. Participants with detected driver and potential driver mutations were significantly older (mean age mutated = 57.96, 95% confidence interval [CI]: 3.30–∞, mean age no mutations = 50.35; p-value = 0.002; Benjamini-Hochberg [BH] adjusted p-value = 0.015) and more likely to be post-menopausal (p-value = 0.004; BH-adjusted p-value = 0.015) than those without these mutations. No associations were detected between mutation status and race/ethnicity, body mass index, diabetes, parity, and smoking status. Long-term follow-up was not presently available in this prospective study for those women without histopathologic evidence of cancer. Conclusions Using ultra-deep NGS, we identified somatic mutations in DNA extracted both from cell pellets and a never previously reported cfDNA fraction from the uterine lavage. Using our targeted sequencing approach, endometrial driver mutations were identified in all seven women who received a cancer diagnosis based on classic histopathology of tissue curettage obtained at the time of hysteroscopy. In addition, relatively high allele fraction driver mutations were identified in the lavage fluid of approximately half of the women without a cancer diagnosis. Increasing age and post-menopausal status were associated with the presence of these cancer-associated mutations, suggesting the prevalent existence of a premalignant landscape in women without clinical evidence of cancer. Given that a uterine lavage can be easily and quickly performed even outside of the operating room and in a physician’s office-based setting, our findings suggest the future possibility of this approach for screening women for the earliest stages of endometrial cancer. However, our findings suggest that further insight into development of cancer or its interruption are needed before translation to the clinic.

The role of liver resection at the time of secondary cytoreduction in patients with recurrent ovarian cancer.

Objective The aim of this study is to determine the role of liver metastatectomy in the morbidity and survival of patients with recurrent ovarian carcinoma. Methods We retrospectively reviewed the records of all patients who had undergone hepatic resection for liver metastases from ovarian carcinoma at the time of cytoreductive surgery at our institution from 1988 to 2012. The Kaplan-Meier method was used for survival analysis. A total of 76 patients met the inclusion criteria and had undergone liver resection as part of cytoreductive surgery for ovarian carcinoma during the study period. Of these 76 patients, 27 underwent liver resection at the time of secondary cytoreduction, and these patients that are the focus of this analysis. Results Median overall survival for the study group from the time of diagnosis to the last follow-up or death was 56 months (range, 12–249 months). Twenty died of the disease with an overall median survival of 12 months from the time of the liver resection (2–190 months), and 7 patients were alive with the disease at the time of the last follow-up. Based on Kaplan-Meier survival analysis, the factors associated with the longest survival after the liver resection (2–190 months) were the interval from the primary surgery of less than 24 months versus more than 24 months (P = 0.044) and secondary cytoreduction to residual disease of less than 1 cm (P = 0.014). Conclusions Based on our analysis of a single institution’s series of ovarian cancer patients with hepatic metastasis, liver resection is feasible and safe and should be considered as an option in selected patients at the time of secondary cytoreduction.

Inhibition of the Nuclear Export Receptor XPO1 as a Therapeutic Target for Platinum-Resistant Ovarian Cancer

Purpose: The high fatality-to-case ratio of ovarian cancer is directly related to platinum resistance. Exportin-1 (XPO1) is a nuclear exporter that mediates nuclear export of multiple tumor suppressors. We investigated possible clinicopathologic correlations of XPO1 expression levels and evaluated the efficacy of XPO1 inhibition as a therapeutic strategy in platinum-sensitive and -resistant ovarian cancer. Experimental Design: XPO1 expression levels were analyzed to define clinicopathologic correlates using both TCGA/GEO datasets and tissue microarrays (TMA). The effect of XPO1 inhibition, using the small-molecule inhibitors KPT-185 and KPT-330 (selinexor) alone or in combination with a platinum agent on cell viability, apoptosis, and the transcriptome was tested in immortalized and patient-derived ovarian cancer cell lines (PDCL) and platinum-resistant mice (PDX). Seven patients with late-stage, recurrent, and heavily pretreated ovarian cancer were treated with an oral XPO1 inhibitor. Results: XPO1 RNA overexpression and protein nuclear localization were correlated with decreased survival and platinum resistance in ovarian cancer. Targeted XPO1 inhibition decreased cell viability and synergistically restored platinum sensitivity in both immortalized ovarian cancer cells and PDCL. The XPO1 inhibitor–mediated apoptosis occurred through both p53-dependent and p53-independent signaling pathways. Selinexor treatment, alone and in combination with platinum, markedly decreased tumor growth and prolonged survival in platinum-resistant PDX and mice. In selinexor-treated patients, tumor growth was halted in 3 of 5 patients, including one with a partial response, and was safely tolerated by all. Conclusions: Taken together, these results provide evidence that XPO1 inhibition represents a new therapeutic strategy for overcoming platinum resistance in women with ovarian cancer. Clin Cancer Res; 23(6); 1552–63. ©2016 AACR.

Multicystic Benign Cystic Mesothelioma Presenting as a Pelvic Mass

Background. Benign cystic mesothelioma (BCM) is a rare tumor that arises from the abdominal peritoneum with a predilection to the pelvic peritoneum. For this reason, it can often mimic gynecologic malignancies. Case. A 47-year-old perimenopausal female presented reporting several weeks of abdominal distention associated with abdominal tenderness and constipation. Computed tomography revealed a 24 cm multiloculated pelvic mass, and tumor markers were notable for an elevated CA-125. The patient was taken to the operating room for an exploratory laparotomy, total abdominal hysterectomy, bilateral salpingoophorectomy, and removal of pelvic mass. Final pathologic evaluation revealed a benign cystic mesothelioma. Conclusion. Classically these tumors present as large multicystic masses with thin-walled septations and on preoperative evaluation BCM can mimic many different disease entities including ovarian malignancies and cystic lymphangioma. Often diagnosis can only be made at time of surgery.

Survey of Current Practice Patterns in the Treatment of Early-Stage Endometrial Cancer.

Objectives Our aim was to assess current surgical practices and use of adjuvant therapy in the treatment of FIGO (International Federation of Gynecology and Obstetrics) stage I endometrioid endometrial cancer. Methods A 19-question survey was developed and sent to all Society of Gynecologic Oncologist members by e-mail. Data were collected anonymously using Internet-based survey software. Respondents were asked questions regarding preoperative evaluation, surgical approach, lymph node dissection (LND), and adjuvant therapy. Results A total of 1399 surveys were distributed, 320 (23%) members completed the survey. Ninety-seven percent of respondents were gynecologic oncologists or fellows, and 87% treat 30 or more endometrial cancer patients yearly. Respondents were more likely to order preoperative tests such as computed tomography abdomen/pelvis and CA-125 for biopsy-proven grade 3 disease versus grade 1 (82% vs 29%). Robot-assisted laparoscopy was the preferred surgical approach (66%), followed by conventional laparoscopy (21%). Twenty-six percent of respondents perform LND in all cases. Forty-eight percent describe their LND as complete, to the level of the inferior mesenteric artery. Adjuvant therapy was recommended more often with increasing myometrial invasion, tumor grade, and lymphovascular space invasion. Vaginal brachytherapy was the most commonly recommended adjuvant therapy for stage IA. For stage IB, grade 3, positive lymphovascular space invasion disease, respondents were more likely to combine vaginal brachytherapy with external beam radiotherapy and/or chemotherapy. Older patients were more likely to have adjuvant therapy in earlier stages of disease than younger patients. Conclusions Our findings demonstrate that respondents are individualizing care based on preoperative, intraoperative, and pathologic findings. As expected, adjuvant treatment is recommended for patients with higher stage and grade disease. Robot-assisted hysterectomy and chemotherapy are now commonly used in the management of this disease. We anticipate that new trends will continue to emerge as results from additional studies become available.

Interferon regulatory factor 1 is an independent predictor of platinum resistance and survival in high-grade serous ovarian carcinoma

OBJECTIVE High-grade serous ovarian cancer (HGSOC) that is resistant to platinum-based chemotherapy has a particularly poor prognosis. Response to platinum has both prognostic survival value and dictates secondary treatment strategies. Using transcriptome analysis, we sought to identify differentially expressed genes/pathways based on a tumors platinum response for discovering novel predictive biomarkers. METHODS Seven primary HGSOC tumor samples, representing two extremes of platinum sensitivity/timing of disease recurrence, were analyzed by RNA-Seq, Ingenuity Pathways Analysis (IPA) and Upstream Regulator Analysis (URA), and used to explore differentially expressed genes and prevalent molecular and cellular processes. Progression-free and overall survival (PFS, OS) was estimated using the Kaplan-Meier method in two different sample sets including GEO and TCGA data sets. RESULTS IPA and URA highlighted an IRF1-driven transcriptional program (P=0.0017; z-score of 3.091) in the platinum sensitive improved PFS group. QRT-PCR analysis of 31 HGSOC samples demonstrated a significant difference in PFS between low and high IRF1 expression groups (P=0.048) and between groups that were platinum sensitive versus not (P=0.016). In a larger validation data set, increased levels of IRF1 were associated with both increased PFS (P=0.043) and OS (P=0.019) and the effect on OS was independent of debulking status (optimal debulking, P=0.025; suboptimal, P=0.041). CONCLUSION Transcriptome analysis identifies IRF1, a transcription factor that functions both in immune regulation and as a tumor suppressor, as being associated with platinum sensitivity and an independent predictor of both PFS and OS in HGSOC.

Abstract 2163: Increased overall survival in platinum-resistant ovarian cancer: paradigmatic use of novel SINE (selective inhibitor of nuclear export), which restores p53 nuclear localization and activation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer (OvCa) is the most lethal female reproductive tract malignancy worldwide with >190,000 new cases diagnosed yearly. Overall, ∼60% of patients will relapse after primary therapy and for the majority of women with advanced disease, their platinum-resistant cancers are currently incurable. During OvCa progression, multiple tumor suppressor proteins (TSPs), most notably p53 (>95% of late-stage cases), are inactivated. Chromosomal region maintenance 1 (CRM1) is solely responsible for the nuclear export of the major TSPs, and increased CRM1 expression has been linked to advanced OvCa stage and poor overall survival. To address the therapeutic role of CRM1 inhibition in OvCa, we tested two novel SINEs in vitro and in vivo. For in vitro studies, we used KPT-185 as a single agent and in combination with cisplatinum in the isogenic cell lines A2780 and CP70. KPT-185 cytotoxicity was highly specific for tumor cell lines. The IC50 for the normal ovarian surface epithelial cell line IOSE527 (4000nM) was 40-90 times higher than A2780 (46.5nM) and CP70 (111.7nM) lines. Combination treatment demonstrated an additive effect on cell death, overcoming cisplatinum resistance. A2780 displayed G1/G2 arrest whereas CP70, only G2 arrest. p53 nuclear accumulation, and downstream activation of targets, was demonstrated following SINEs treatment. Two complementary in vivo models were used. First, the orally bioavailable SINE KPT-330, currently in Phase 1 clinical studies, was given as a single agent in a Rag1 KO mouse model with 4 unrelated patient-derived chemonaive cell lines. Oral KPT-330 inhibited subcutaneous tumor growth and markedly increased survival: all chronically treated mice continued to survive longer (>78d) than controls (all died at 23d). Cross-over of control mice to KPT-330 resulted in tumor growth arrest. In the second model, highly aggressive, luciferase positive, cisplatin resistant CP70 cells were injected i.p into nu/nu mice (n=34) and tumor growth followed in real-time using WBLI. SINE treatment led to marked reductions in tumor burden as well as survival improvements: control mice were all dead by 25 days whereas cisplatin (2.5mg/kg, 2x wk; 31d), KPT-330 (15mg/kg, twice weekly; 41d) and KPT-330+cisplatin (same as single treatments, respectively; 48d) treated mice have significantly improved survival outcomes (p<0.0005 for each treatment versus control). Together, these studies demonstrate the first successful use of highly selective, potent and irreversible CRM1 inhibitors in overcoming cisplatin resistance and prolonging survival in OvCa models. Restoration of p53 nuclear localization and functional activity and RNASeq identified pathways at serially increasing doses will be discussed. First-in-man trials have begun and trials in ovarian cancer are planned for 2013. Citation Format: Ying Chen, Eva Kalir, Catalina Camacho-Vanegas, Fei Huang, Elena Pereira, Yosef Landesman, Dilara McCauley, Mansoor Raza Mirza, Michael Kauffman, Sharon Shacham, Peter R. Dottino, John A. Martignetti. Increased overall survival in platinum-resistant ovarian cancer: paradigmatic use of novel SINE (selective inhibitor of nuclear export), which restores p53 nuclear localization and activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2013-2163

Concurrent chemoradiation versus radiotherapy alone for the treatment of locally advanced cervical cancer in a low-resource setting

Our goal was to determine the clinical treatment response following radiation administered with or without chemotherapy for locally advanced cervical cancers in Honduras. This is a retrospective study of patients treated with either concurrent chemoradiation (CCRT) or external beam radiation therapy (EBRT) alone at a hospital in Tegucigalpa, Honduras. 70 Gy of EBRT to the pelvis was given in all cases. Brachytherapy was not available. Chemotherapy was given when available. Extrafascial hysterectomy was performed 6 weeks after completion of treatment in patients with a complete clinical response (cCR). Records for 165 women with locally advanced cervical cancer were reviewed; 25 (15.2%) stage IB2, 15 (9.1%) stage IIA, 90 (54.5%) stage IIB, and 35 (21.2%) stage IIIB. Ninety (54.5%) patients received EBRT alone; 75 (45.5%) received CCRT. Twenty-three (33.3%) of CCRT patients received weekly cisplatin, the remainder receiving other agents. Seventy (77.8%) of the 90 patients who received EBRT had a cCR; 25 out of 75 (33.3%) patients in the CCRT group achieved a cCR. The CCRT group treated with weekly cisplatin achieved an 80% cCR; while the CCRT group given alternative agents had only a 31% cCR. Patients unable to receive platinum-based CCRT had the worst outcome, and their responses were inferior to patients who received EBRT. The challenges of treating women with locally advanced cervical cancer in a low-resource setting are multifactorial and include treatment delays, the lack of brachytherapy and the unpredictable availability of chemotherapy.

Ovarian and Adnexal Masses

Incidental finding of an adnexal mass during abdominal surgery may present as a challenge to surgeons. This chapter will provide a detailed review of the various types of adnexal masses commonly encountered. Management of adnexal masses depends on the age of the patient and the pathology of the mass. Specific surgical approaches are addressed based on the age of the patient. A conservative surgical approach is emphasized for younger patients of childbearing age, in which the goal is to remove the abnormal adnexal mass while preserving the contralateral adnexa. However, in older patients and in the case of advanced ovarian malignancies, full surgical staging and removal of all visible disease is paramount to the management of this disease.