Jeanne M. Schilder

Identification and Characterization of Ovarian Cancer-Initiating Cells from Primary Human Tumors

The objective of this study was to identify and characterize a self-renewing subpopulation of human ovarian tumor cells (ovarian cancer-initiating cells, OCICs) fully capable of serial propagation of their original tumor phenotype in animals. Ovarian serous adenocarcinomas were disaggregated and subjected to growth conditions selective for self-renewing, nonadherent spheroids previously shown to derive from tissue stem cells. To affirm the existence of OCICs, xenoengraftment of as few as 100 dissociated spheroid cells allowed full recapitulation of the original tumor (grade 2/grade 3 serous adenocarcinoma), whereas >10(5) unselected cells remained nontumorigenic. Stemness properties of OCICs (under stem cell-selective conditions) were further established by cell proliferation assays and reverse transcription-PCR, demonstrating enhanced chemoresistance to the ovarian cancer chemotherapeutics cisplatin or paclitaxel and up-regulation of stem cell markers (Bmi-1, stem cell factor, Notch-1, Nanog, nestin, ABCG2, and Oct-4) compared with parental tumor cells or OCICs under differentiating conditions. To identify an OCIC cell surface phenotype, spheroid immunostaining showed significant up-regulation of the hyaluronate receptor CD44 and stem cell factor receptor CD117 (c-kit), a tyrosine kinase oncoprotein. Similar to sphere-forming OCICs, injection of only 100 CD44(+)CD117(+) cells could also serially propagate their original tumors, whereas 10(5) CD44(-)CD117(-) cells remained nontumorigenic. Based on these findings, we assert that epithelial ovarian cancers derive from a subpopulation of CD44(+)CD117(+) cells, thus representing a possible therapeutic target for this devastating disease.

Epigenetic Resensitization to Platinum in Ovarian Cancer

Preclinical studies have shown that hypomethylating agents reverse platinum resistance in ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining study, we tested the clinical and biologic activity of low-dose decitabine administered before carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate (RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood mononuclear cells and tumors. The number of demethylated genes was greater (P < 0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244 genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included cytokine-cytokine receptor interactions, drug transporters, and mitogen-activated protein kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis (P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and HOXA11 in tumors positively correlated with PFS (P < 0.05). Together, the results of this study suggest that low-dose decitabine altered DNA methylation of genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resulting in a high RR and prolonged PFS.

Tissue transglutaminase links TGF-β, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer

Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-β, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-β-activated kinase 1-mediated activation of the nuclear factor-κB complex. TGF-β-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-β-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44+/CD117+). Furthermore, CD44+/CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-β-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype.

A phase 1 and pharmacodynamic study of decitabine in combination with carboplatin in patients with recurrent, platinum‐resistant, epithelial ovarian cancer

Aberrant DNA methylation is a hallmark of cancer, and DNA methyltransferase inhibitors have demonstrated clinical efficacy in hematologic malignancies. On the basis of preclinical studies indicating that hypomethylating agents can reverse platinum resistance in ovarian cancer cells, the authors conducted a phase 1 trial of low‐dose decitabine combined with carboplatin in patients with recurrent, platinum‐resistant ovarian cancer.

Phase II trial of combination bevacizumab and temsirolimus in the treatment of recurrent or persistent endometrial carcinoma: a Gynecologic Oncology Group study.

OBJECTIVE This two-stage phase II study was designed to assess the activity of the combination of temsirolimus and bevacizumab in patients with recurrent or persistent endometrial carcinoma (EMC). METHODS Eligible patients had persistent or recurrent EMC after receiving 1-2 prior cytotoxic regimens, measurable disease, and Gynecologic Oncology Group performance status ≤ 2. Treatment consisted of bevacizumab 10 mg/kg every other week and temsirolimus 25 mg IV weekly until disease progression or prohibitory toxicity. Primary end points were progression-free survival (PFS) at six months and overall response rate using RECIST criteria. RESULTS Fifty-three patients were enrolled. Forty-nine patients were eligible and evaluable. Median age was 63 years, and prior treatment consisted of one or two regimens in 40 (82%) and 9 (18%), respectively. Twenty (41%) received prior radiation. Adverse events were consistent with those expected with bevacizumab and temsirolimus treatment. Two gastrointestinal-vaginal fistulas, one grade 3 epistaxis, two intestinal perforations and 1 grade 4 thrombosis/embolism were seen. Three patient deaths were possibly treatment related. Twelve patients (24.5%) experienced clinical responses (one complete and 11 partial responses), and 23 patients (46.9%) survived progression free for at least six months. Median progression-free survival (PFS) and overall survival (OS) were 5.6 and 16.9 months, respectively. CONCLUSION Combination of temsirolimus and bevacizumab is deemed active based on both objective tumor response and PFS at six months in recurrent or persistent EMC. However, this treatment regimen was associated with significant toxicity in this pretreated group. Future study will be guided by strategies to decrease toxicity and increase response rates.

Activity of 2 methoxyestradiol (Panzem® NCD) in advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis: A Hoosier Oncology Group trial

BACKGROUND 2-Methoxyestradiol (Panzem, 2ME2) is an endogenous metabolite of estradiol that destabilizes microtubules and exerts anti-angiogenic properties. This study was conducted to determine the activity and safety of 2ME2 administered as a NanoCrystal dispersion (NCD) formulation in patients with recurrent, platinum-resistant epithelial ovarian cancer (EOC). METHODS Eligible patients had relapsed, platinum-resistant or refractory EOC with measurable or detectable disease. There was no limit on the number of prior treatment regimens. 2ME2 NCD 1000 mg orally four times daily (q.i.d.) was administered continuously during 4 week cycles. The primary endpoint was objective response rate (ORR). Secondary endpoints were assessment of toxicity, rate of clinical benefit defined as the number of patients experiencing an objective response, a CA125 response or stable disease (SD) >3 months, mean change in CA-125, progression-free survival (PFS), and pharmacokinetic analyses of 2ME2. RESULTS Eighteen patients were enrolled. Median age was 65.5 (range 40-73). Patients had received a median of five prior treatments. The most common adverse events were fatigue (78%), nausea (78%), diarrhea (39%), neuropathy (50%), edema (39%), and dyspnea (44%), the majority being grade 1-2. There were no objective responses, but seven patients had SD as best response. Of those, two patients had SD for greater than 12 months. The rate of clinical benefit was 31.3%. Fairly stable plasma levels of 2ME2 ranging within the predicted therapeutic window were observed. CONCLUSIONS The NCD formulation of 2ME2 is well tolerated in patients with heavily pretreated EOC. Few of these heavily pretreated patients had sustained stable disease.

Imatinib mesylate in combination with docetaxel for the treatment of patients with advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis : a Hoosier Oncology Group trial.

Ovarian tumors frequently express c‐Kit and/or platelet‐derived growth factor receptors (PDGFRs). Imatinib mesylate blocks the growth of ovarian cancer cells in vitro and may enhance the activity of chemotherapy. This study was conducted to determine the activity of imatinib in combination with docetaxel in patients with recurrent, platinum‐resistant epithelial ovarian cancer (EOC).

Sorafenib in combination with weekly topotecan in recurrent ovarian cancer, a phase I/II study of the Hoosier Oncology Group

OBJECTIVE This trial determined the efficacy and tolerability of sorafenib and weekly topotecan in patients with platinum-resistant ovarian cancer (OC) or primary peritoneal carcinomatosis (PPC). METHODS Primary endpoints were maximum tolerated dose of sorafenib with weekly topotecan (phase I) and response rate (phase II). Secondary endpoints were progression free survival (PFS), overall survival (OS), toxicity, and rate of clinical benefit. Eligibility included recurrent platinum-resistant OC or PPC, <3 prior regimens, normal end-organ function. 3+3 dose escalation was used for phase I, sorafenib being tested at 400mg and 800 mg orally daily. Topotecan dose was reduced from 4 mg/m(2) to 3.5mg/m(2) IV weekly. The phase II regimen was sorafenib 400mg daily and topotecan 3.5mg/m(2) weekly on days 1, 8, 15 of a 28 days cycle. RESULTS 16 patients were enrolled in phase I and 14 patients in phase II. Median age was 52.5 years (range 35-79), 27 patients had OC, and 3 PPC. Median number of cycles administered was 2.5 (0-15). There were 5 partial responses (PR) (16.7%), and 14 patients (46.7%) with stable disease (SD). Four PRs were recorded during phase I and 1 during phase II. One of those PRs occurred in a patient with platinum-sensitive disease. Grade 3/4 toxicities included leukopenia/neutropenia (23%), thrombocytopenia (17%), anemia (10%), fatigue, nausea, vomiting (7% each). One case of grade 3 hand-foot syndrome was recorded. CONCLUSIONS The combination of sorafenib and topotecan causes significant toxicity, precluding administration of full doses and resulting in modest clinical efficacy in platinum resistant OC or PPC.

A phase II evaluation of nintedanib (BIBF-1120) in the treatment of recurrent or persistent endometrial cancer: an NRG Oncology/Gynecologic Oncology Group Study.

INTRODUCTION Patients presenting with advanced, recurrent, or metastatic endometrial cancer have limited treatment options. On behalf of the Gynecologic Oncology Group, we conducted this phase II trial of nintedanib (BIBF 1120), a potent small molecule triple receptor tyrosine kinase inhibitor of PDGFR α and β, FGFR 1/3, and VEGFR 1-3, in this population. OBJECTIVES The primary objectives were to estimate event-free survival (EFS) at 6 months and the proportion of patients who have an objective tumor response. In addition, we sought to determine the nature and degree of toxicity. Secondary objectives were to estimate progression-free and overall survival. METHODS This was a two-stage, single-arm phase II study. Eligible patients were treated with single-agent nintedanib at a dose of 200mg twice daily. RESULTS Of 37 patients enrolled, 32 were eligible. There were zero complete and three partial responses for an overall response rate of 9.4% (90% 2-sided CI=2.6-22.5%). Seven patients (21.9%; 90% 2-sided CI=10.7-37.2%) were EFS at 6 months, with one patient continuing on study at the time of this writing. Serious toxicity included the following grade 3 events: gastrointestinal toxicity (5), neutropenia (1), edema (1), hypertension (1), and liver function abnormalities (5). CONCLUSIONS Nintedanib lacked sufficient activity as a single agent to warrant enrollment to second stage. However, preclinical data indicate it may be synergistic with paclitaxel in a population of patients enriched for specific p53 mutations that result in loss of function. Subsequent studies may evaluate this agent in combination with paclitaxel.

Low-dose abdominal radiation as a docetaxel chemosensitizer for recurrent epithelial ovarian cancer: a phase I study of the Gynecologic Oncology Group.

OBJECTIVES The aim of this study was to determine the maximum tolerated dose and dose-limiting toxicity (DLT) of whole abdomen radiation as a chemosensitizer of weekly docetaxel for women with recurrent epithelial ovarian fallopian tube, or peritoneal cancers. PATIENTS AND METHODS Women were enrolled on one of three dose levels of docetaxel (20, 25, or 30 mg/m(2)) administered weekly with concurrent low-dose whole abdominal radiation given as 60 cGy bid 2 days weekly for a total of 6 weeks. RESULTS Thirteen women were enrolled and received 70 weekly treatments of docetaxel in combination with radiation therapy. At the first dose level, docetaxel 25mg/m(2), grade 3 fatigue and thrombocytopenia were observed. At the next dose level, docetaxel 30 mg/m(2), grade 3 febrile neutropenia, grade 4 thrombocytopenia with epistaxis, and grade 3 diarrhea were observed. Given these dose-limiting toxicities, a lower dose of docetaxel 20mg/m(2) was administered and found to be tolerable. No objective responses were observed among the 10 patients with measurable disease; however, the median progression-free survival (PFS) in all patients was 3.3 months, and 3 of the patients with measurable disease were free of tumor progression after 6 months (30%; 90% confidence interval 8.7-61%). CONCLUSIONS Twice weekly low-dose whole abdomen radiation during weekly docetaxel 20 mg/m(2) was well-tolerated. Given the PFS demonstrated in these women with resistant ovarian cancer, further study of whole abdominal radiation and concurrent chemotherapy may be warranted.