Oliver Dorigo

Dual Targeting of Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Using NVP-BEZ235 as a Novel Therapeutic Approach in Human Ovarian Carcinoma

Purpose: This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy. Experimental Design: Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-rasG12D/+PtenloxP/loxP). Results: NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC50 for NVP-BEZ235. In LSL-K-rasG12D/+PtenloxP/loxP mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration. Conclusions: Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intra-abdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer. Clin Cancer Res; 17(8); 2373–84. ©2011 AACR.

Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells

UNLABELLED The exact molecular mechanisms of ovarian cancer platinum resistance are not well understood, and biomarkers to reliably predict ovarian cancer resistance to platinum and other chemotherapeutic agents are lacking. Biomechanics of cisplatin-treated ovarian cancer cells were measured quantitatively at nanoscale level using atomic force microscopy. We demonstrate that cisplatin modulates the cellular nanomechanics of ovarian cancer cells; sensitive cells show dose-dependent increase in cell stiffness, which is effected by disrupting the F-actin polymerization. In contrast, resistant cells show no significant changes in cell stiffness upon cisplatin treatment. Further, stimulated emission depletion, an emerging super-resolution microscopy, shows that at the molecular level, F-actin is indeed remodeled considerably in cisplatin-sensitive and cisplatin-resistant cells. These findings reveal a direct role of the actin remodeling mechanism in cisplatin resistance of ovarian cancer cells, suggesting potential future applications of nanomechanical profiling as a marker for cancer drug sensitivity. FROM THE CLINICAL EDITOR In this paper, nanomechanical profiling and an emerging super-resolution microscopy method was utilized to decipher the mechanisms of cisplatin resistance in ovarian cancer cells, paving the way to future studies of this and similar other problems with drug resistance in cancer biology.

Treatment outcomes in a large cohort of patients with invasive Extramammary Paget's disease

OBJECTIVE The outcome of patients with invasive Extramammary Pagets disease (EMPD) is poorly studied. The goal of the current study was to analyze the incidence, treatment approaches and outcome of patients with invasive EMPD. METHODS We searched the SEER program database for patients diagnosed with invasive EMPD between 1973 and 2007. Demographic data, outcome and therapeutic modalities were included in the analysis. Disease specific survival (DSS) was calculated from the time of original diagnosis. RESULTS 1439 patients were diagnosed with invasive EMPD. Most patients (80.4%) had localized disease, while 17.1% had locoregional spread, and 2.5% had distant disease. 1230 (86.4%) patients underwent site directed surgery, and 92 (6.4%) patients radiotherapy. 139 (9.7%) patients did not undergo any surgery or radiation therapy. The 5-year DSS was 94.9% for localized disease, 84.9% for regional disease and 52.5% for distant disease. Multivariate analysis showed a significantly shorter DSS associated with older age and advanced stage treatment modality (HR for death 1.07 and 2.5). Site directed surgery was associated with a significantly improved outcome when compared to patients who underwent no intervention (HR 0.44). Patients who received radiation, alone or in combination with site directed surgery, did not fare any better than patients who underwent surgery alone. CONCLUSIONS The DSS of patients with invasive EMPD is generally favorable. A poor outcome was associated with older age, advanced stage and treatment modality. The association between a shortened DSS and the use of radiotherapy, alone or in combination with surgery, is surprising and warrants further investigation.

Cisplatin and PI3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix‐metalloproteinase expression

Targeting of the PI3K (phosphoinositide3‐kinase)/Akt/mTOR pathway in human ovarian cancer cells is a promising novel therapeutic strategy. We investigated the effects of cisplatin and the PI3K inhibitor LY294002 on invasion, migration and the expression of essential matrix metalloproteinases (MMPs) in ovarian cancer cells. SKOV3, OVCAR5 and IGROV1 human ovarian cancer cell lines were treated with cisplatin, LY294002 and a combination of both drugs. Invasion and migration of treated cells was assessed using Matrigel and uncoated PET membrane assays. Expression levels of pro‐MMP2, MMP2, TIMP1, TIMP2 and MT1‐MMP were determined using Western Blotting. Gel zymography was used to quantitate the functional levels of active MMP2. All three cell lines showed significantly reduced invasion and migration after treatment with cisplatin, LY294002, and the combination of both drugs compared to untreated controls. In SKOV3 cells, cisplatin alone and in combination with LY294002 resulted in a 6.3 and 7.1‐fold reduction in the total amount of activated MMP2. TIMP1 expression decreased by 5.0, 6.6 and 28.4‐fold with cisplatin, LY294002 and the combination respectively (P < 0.05). In contrast, only cisplatin and the combination of both drugs resulted in a significant, 3.7 and 5.1‐fold reduction in the level of TIMP2. Expression levels of MT1‐MMP remained unchanged. These observations were corroborated in IGROV1 cell lines that showed similar changes of activated MMP2 and TIMP2 expression, but no significant decrease in TIMP1 levels. Our data suggests that inhibition of ovarian cancer cell motility is mediated via down‐regulation of activated MMP2, TIMP1 and TIMP2 expression under these treatment conditions.

Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells

ABSTRACT Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

HER-2/neu targeting for recurrent vulvar Paget's disease: A case report and literature review

BACKGROUND The treatment of Pagets disease of the vulva particularly for recurrences can be challenging. Overexpression of the HER-2/neu protein has been found in about 30% of vulvar Pagets cases therefore presenting a potential therapeutic target. CASE We report the case of a 52-year-old patient with persistent Pagets disease of the vulva despite eight surgical excisions over a 15-year period. Immunohistochemistry demonstrated overexpression of the HER-2/neu protein in the vulva resection specimen. Treatment with Trastuzumab resulted in a significant regression of her disease and resolution of symptoms. CONCLUSION Based on our case report, therapeutic targeting of HER-2/neu for patients with Pagets disease of the vulva using for example Trastuzumab is a potentially effective, alternative approach, and warrants further investigation.

Immunotherapeutic approaches to ovarian cancer treatment

Despite advances in combinatorial chemotherapy regimens and the advent of intraperitoneal chemotherapy administration, current therapeutic options for ovarian cancer patients are inadequate. Immunotherapy offers a novel and promising therapeutic strategy for treating ovarian tumors. Following the demonstration of the immunogenicity of ovarian tumors, multiple immunotherapeutic modalities have been developed. Antibody-based therapies, immune checkpoint blockade, cancer vaccines, and chimeric antigen receptor-modified T cells have demonstrated preclinical success and entered clinical testing. In this review, we discuss these promising immunotherapeutic approaches and emphasize the importance of combinatorial treatment strategies and biomarker discovery.

Personalizing CA125 Levels for Ovarian Cancer Screening

Screening trials for the early detection of ovarian cancer in the general population and in patients at a high risk for this disease have so far failed to show a reduction of ovarian cancer–specific mortality. Current screening modalities include pelvic examinations, transvaginal ultrasounds, and cancer antigen 125 (CA125) serum marker levels, which are associated with a high false-positive rate. The last decade has witnessed significant modifications in the interpretation of serum CA125 that extend beyond a static CA125 cutoff point. The Risk of Ovarian Cancer Algorithm (ROCA) incorporates changes of CA125 levels over time and an individuals age-specific risk. Ongoing screening trials have incorporated ROCA, but it is still unclear whether the algorithm will increase the sensitivity and specificity of early ovarian cancer diagnosis. A very recent study analyzed baseline CA125 serum marker levels from high-risk patients included in ovarian cancer screening trials conducted by the Cancer Genetics Network and the Gynecologic Oncology Group. The findings show that the distribution of CA125 serum marker levels in this population is significantly affected by various demographic and clinical factors, in particular menopausal status and oral contraceptive use in premenopausal patients. The data suggest that CA125 cutoff points might have to be stratified for subgroups of patients to reduce false-positive results. These intriguing observations will need to be validated in future screening trials for ovarian cancer. Cancer Prev Res; 4(9); 1356–9. ©2011 AACR.

Comparison of gene therapy with interleukin-2 gene modified fibroblasts and tumor cells in the murine CT-26 model of colorectal carcinoma.

We compared the efficacy of gene therapy mediated by interleukin-2 (IL-2) gene-modified tumor cells to gene therapy mediated by IL-2 transduced fibroblasts in the CT-26 model of murine colorectal carcinoma. We transduced CT-26 tumor cells and BALB/c 3T3 fibroblasts with three different retroviral vectors using three different promoters for the human IL-2 gene: DC/TKIL-2 (thymidine kinase promoter), LXSN-iIL2 (long terminal repeat promoter), and LNCX-iIL2 (cytomegalovirus promoter). These transductions resulted in CT-26 and 3T3 subclones that secreted different amounts of IL-2. Immunization of animals with either CT-26/IL-2 cells or with fibroblast/IL-2 cells mixed with CT-26 induced similar levels of immunity that protected 62-82% of animals against a subsequent tumor challenge with parental CT-26. However, mice developed tumors at the site of inoculation in 46% of the animals immunized with CT-26/IL-2 cells. In a separate experiment, CT-26/IL-2 cells were exposed to 6,000 cGy of gamma irradiation to prevent tumor growth at the site of inoculation. Although the CT-26/IL-2 cells continued to secrete IL-2 after irradiation, they were no longer effective at inducing antitumor immunity. In contrast, both irradiated and nonirradiated fibroblast/IL-2 cells, mixed with irradiated CT-26, were equally effective at inducing antitumor immunity. These data suggest that in the CT-26 model, fibroblast-mediated IL-2 gene therapy has advantages for the induction of antitumor immunity and abrogation of tumorigenic potential at the site of inoculation compared with tumor cell-mediated IL-2 gene therapy.

Predictors of resolution of complex atypical hyperplasia or grade 1 endometrial adenocarcinoma in premenopausal women treated with progestin therapy

OBJECTIVE To identify clinical and pathologic predictors of response to progestin treatment in premenopausal women with complex atypical hyperplasia (CAH) and Grade 1 endometrial adenocarcinoma (Grade 1 EA). METHODS Forty premenopausal patients with Grade 1 EA or CAH who underwent progestin therapy for a minimum of 8 weeks were retrospectively identified. Patient characteristics and histopathologic features of pretreatment and first follow-up endometrial specimens were evaluated as predictors of resolution, defined as absence of hyperplasia or carcinoma. RESULTS Kaplan-Meier analysis indicated 63% resolution at 18 months of follow-up. Multivariate classification analysis showed that resolution rates were higher in individuals with a low pre-treatment qualitative abnormal architecture score and a BMI <35 (Standardized Resolution Ratio (SRR)=1.48, p=0.03). The diagnosis of benign endometrium or simple hyperplasia on the first follow-up specimen was highly predictive of resolution (SRR=2.25, p=0.002). Resolution rates were lower among subjects with a high pre-treatment qualitative abnormal architecture score (SRR=0.37, p<0.03) and lowest in subjects whose first follow-up specimen showed persistent complexity, atypia, or carcinoma with adjacent stromal decidualization (SRR=0.24, p=0.002). CONCLUSIONS Clinical and pathologic parameters can predict response to progestin therapy in premenopausal women with CAH and Grade 1 EA. A low likelihood of resolution is predicted by an unfavorable pre-treatment architectural score and lack of pathological response in the first specimen, despite adjacent stromal decidualization.