Julie K. Schwarz

A miR-200 microRNA cluster as prognostic marker in advanced ovarian cancer

OBJECTIVE Ovarian cancer is one of the most deadly human cancers, resulting in over 15,000 deaths in the US each year. A reliable method that could predict disease outcome would improve care of patients with this disease. The main aim of this study is to identify novel prognostic biomarkers for advanced ovarian cancer. METHODS We hypothesized that microRNAs (miRNAs) may predict outcome and have examined the prognostic value of these small RNA molecules on disease outcome prediction. miRNAs are a newly identified family of non-coding RNA genes, and recent studies have shown that miRNAs are extensively involved in the tumor development process. We have profiled the expression of miRNAs in advanced ovarian cancer using a novel PCR-based platform and correlated miRNA expression profiles with disease outcome. RESULTS By performing miRNA expression profiling analysis of 55 advanced ovarian tumors, we have shown that three miR-200 miRNAs (miR-200a, miR-200b and miR-429) in the miR-200b-429 cluster are significantly associated with cancer recurrence and overall survival. Further target analysis indicates that these miR-200 miRNAs target multiple genes that are involved in cancer development. In addition, we have also shown that overexpression of this miR-200 cluster inhibits ovarian cancer cell migration. CONCLUSIONS miR-200b-429 may be used as a prognostic marker for ovarian cancer outcome, and low-level expression of miR-200 miRNAs in this cluster predicts poor survival. In addition, our study suggests that miR-200 miRNAs could play an important regulatory role in ovarian cancer.

A MicroRNA Expression Signature for Cervical Cancer Prognosis

Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. Furthermore, functional studies were done to characterize the effect of miRNAs in cervical cancer cells. Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.

The Role of 18F-FDG PET in Assessing Therapy Response in Cancer of the Cervix and Ovaries

For locally advanced cervical cancer, the current literature supports the use of 18F-FDG PET for assessing treatment response 3 mo after the completion of concurrent chemoradiation. 18F-FDG PET can provide reliable long-term prognostic information for these patients and, in the future, may be used to guide additional therapy. Investigational areas include the use of 18F-FDG PET for monitoring response during radiotherapy and chemotherapy in the metastatic and neoadjuvant settings. For ovarian masses, the performance of 18F-FDG PET in the detection of borderline tumors is limited, and the presence of physiologic 18F-FDG uptake in normal ovaries of premenopausal women poses another limitation. Preliminary data suggest that the performance of 18F-FDG PET and 18F-FDG PET/CT is superior to that of CT alone in initial staging, but the sensitivity of both in the detection of carcinomatosis is limited. Preliminary data also suggest that 18F-FDG PET may be promising for early prediction of response to chemotherapy and for prediction of response after the completion of chemotherapy. 18F-FDG PET and 18F-FDG PET/CT are most helpful in the evaluation of patients with suspected recurrent ovarian carcinoma, especially when CA-125 levels are rising and CT findings are normal or equivocal. PET and CT are complementary, and PET/CT should be used when available. Preliminary data suggest that the addition of 18F-FDG PET/CT to the evaluation of these patients changes management in approximately a third and reduces overall treatment costs by accurately identifying patients who will or will not benefit from surgery.

Comparison of Molecular Markers of Hypoxia and Imaging with 60 Cu-ATSM in Cancer of the Uterine Cervix

PurposeTo determine if hypoxia-related molecular markers are associated with 60Cu labeled diacetyl-bis (N4-methylthiosemicarbazone); (60Cu-ATSM) imaging of tumor hypoxia in cervical cancer.ProceduresFifteen patients were enrolled in a prospective study and underwent evaluation of tumor hypoxia with positron emission tomography (PET) using 60Cu-ATSM. 60Cu-ATSM-PET imaging was compared with the expression of tissue molecular markers, which included vascular endothelial growth factor (VEGF), cyclo-oxygenase-2 (COX-2), epidermal growth factor receptor (EGFR), carbonic anyhdrase IX (CA-9), and apoptotic index.ResultsSix patients had hypoxic tumors determined by 60Cu-ATSM, and nine had non-hypoxic tumors. The 4-year overall survival estimates were 75% for patients with non-hypoxic tumors and 33% for those with hypoxic tumors (p = 0.04). Overexpression of VEGF (p = 0.13), EGFR (p = 0.05), CA-9 (p = 0.02), COX-2 (p = 0.08), and the presence of apoptosis (p = 0.005) occurred in patients with hypoxic tumors. Cox proportional hazards modeling demonstrated hypoxia as determined by 60Cu-ATSM to be a significant independent predictor of tumor recurrence (p = 0.0287).Conclusions60Cu-ATSM hypoxia was correlated with overexpression of VEGF, EGFR, COX-2, CA-9, an increase in apoptosis, and a poor outcome.

Pathway-specific analysis of gene expression data identifies the PI3K/Akt pathway as a novel therapeutic target in cervical cancer

Purpose: Cervical tumor response on posttherapy 2[18F]fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response. Experimental Design: This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results. Results: There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines. Conclusions: Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation. Clin Cancer Res; 18(5); 1464–71. ©2012 AACR.

Metabolic Response on Post-therapy FDG-PET Predicts Patterns of Failure After Radiotherapy for Cervical Cancer

PURPOSE To determine the patterns of failure in patients with cervical cancer treated with definitive radiotherapy and evaluated for metabolic response with early posttherapy (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET). METHODS AND MATERIALS The records of 238 patients with cervical cancer were reviewed. All patients were treated with a combination of external radiotherapy and intracavitary brachytherapy. Two hundred and nineteen patients (92%) received concurrent chemotherapy. All patients underwent pretreatment FDG-PET, and posttherapy FDG-PET was performed within 8-16 weeks of the completion of radiotherapy. Posttherapy FDG-PET results were categorized as complete metabolic response (CMR), partial metabolic response (PMR), and progressive disease (PD). Failure patterns were categorized as none, isolated local failure (central pelvis ± pelvic lymph nodes), distant failure, or combined local plus distant failure. RESULTS Of the 91 patients (38%) who had a recurrence, 22 had isolated local failures, and 69 had distant failures (49 distant failures and 20 combined local plus distant failures). Of the 173 patients with a CMR, 40 (23%) experienced treatment failure. All 25 patients with PD experienced treatment failure, which was distant in 24 patients (96%). Among the 40 patients with PMR, no failure has been observed for 14 patients (35%). Of the 26 failures within the PMR group, 15 (58%) were limited to the pelvis. Differences in the patterns of failure between the three groups (CMR, PMR, PD) were statistically significant (chi-square test; p < 0.0001). CONCLUSIONS The majority of failures after definitive radiotherapy for cervical cancer include distant failures, even in the setting of concurrent chemotherapy. PMR within the cervix or lymph nodes is more commonly associated with isolated local recurrence.

18-F-FLUORODEOXYGLUCOSE-POSITRON EMISSION TOMOGRAPHY EVALUATION OF EARLY METABOLIC RESPONSE DURING RADIATION THERAPY FOR CERVICAL CANCER

PURPOSE To document changes in cervical tumor (18)-F-fluorodeoxyglocose (FDG) uptake during radiation therapy and to correlate those changes with post-treatment tumor response and survival outcome. METHODS AND MATERIALS A total of 36 patients with Stage Ib1 to IIIb cervical cancer were enrolled in an institutional protocol examining the use of fluorodeoxyglucose-positron emission tomography (FDG-PET) for brachytherapy treatment planning. As part of this study, FDG-PET or PET/computed tomograpy (CT) images were obtained before, during, and after the completion of radiation therapy. Tumor metabolic responses were assessed qualitatively and semi-quantitatively by measurement of the maximal standardized uptake value (SUV(max)). RESULTS Post-treatment FDG-PET images were obtained for 36 patients in this study. Of the patients, 29 patients had a complete metabolic response on the post-treatment PET, 4 had a partial metabolic response, and 3 had new sites of FDG uptake. Six patients had a complete metabolic response observed during radiation therapy, 26 had a partial metabolic response and 4 had stable or increased tumor metabolic activity. For patients with complete metabolic response during radiation therapy, median time to complete response was 29.5 days (range, 18-43 days). The mean cervical tumor SUV(max) decreased from 11.2 (SD, 6.3; range, 2.1-38.0) pretreatment to 2.4 (SD, 2.7; range, 0-8.8) mid treatment, and 0.5 (SD, 1.7; range, 0-8.3) post-treatment. CONCLUSIONS During radiation therapy for cervical cancer, FDG-PET can be used to monitor treatment response. Complete metabolic response during radiation therapy was observed for a subset of patients. Recommendations regarding the optimal timing of FDG-PET during treatment for cervical cancer will require further systematic study.

AKT inhibitors promote cell death in cervical cancer through disruption of mTOR signaling and glucose uptake

Background PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability. Experimental Design Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with 18F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay. Results Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment. Conclusions The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.

Tumor volume and subvolume concordance between FDG-PET/CT and diffusion-weighted MRI for squamous cell carcinoma of the cervix.

To compare [18F]fluorodeoxyglucose (FDG) / positron emission tomography (PET) / computed tomography (CT) and magnetic resonance imaging (MRI) for evaluating patients with cervical cancer. We compared tumor characteristics on FDG‐PET and apparent diffusion coefficient (ADC) maps on diffusion‐weighted MRI (DWI) to evaluate concordance of two functional imaging techniques.

Cervical Gross Tumor Volume Dose Predicts Local Control Using Magnetic Resonance Imaging/Diffusion-Weighted Imaging—Guided High-Dose-Rate and Positron Emission Tomography/Computed Tomography—Guided Intensity Modulated Radiation Therapy

PURPOSE Magnetic resonance imaging/diffusion weighted-imaging (MRI/DWI)-guided high-dose-rate (HDR) brachytherapy and (18)F-fluorodeoxyglucose (FDG) - positron emission tomography/computed tomography (PET/CT)-guided intensity modulated radiation therapy (IMRT) for the definitive treatment of cervical cancer is a novel treatment technique. The purpose of this study was to report our analysis of dose-volume parameters predicting gross tumor volume (GTV) control. METHODS AND MATERIALS We analyzed the records of 134 patients with International Federation of Gynecology and Obstetrics stages IB1-IVB cervical cancer treated with combined MRI-guided HDR and IMRT from July 2009 to July 2011. IMRT was targeted to the metabolic tumor volume and lymph nodes by use of FDG-PET/CT simulation. The GTV for each HDR fraction was delineated by use of T2-weighted or apparent diffusion coefficient maps from diffusion-weighted sequences. The D100, D90, and Dmean delivered to the GTV from HDR and IMRT were summed to EQD2. RESULTS One hundred twenty-five patients received all irradiation treatment as planned, and 9 did not complete treatment. All 134 patients are included in this analysis. Treatment failure in the cervix occurred in 24 patients (18.0%). Patients with cervix failures had a lower D100, D90, and Dmean than those who did not experience failure in the cervix. The respective doses to the GTV were 41, 58, and 136 Gy for failures compared with 67, 99, and 236 Gy for those who did not experience failure (P<.001). Probit analysis estimated the minimum D100, D90, and Dmean doses required for ≥90% local control to be 69, 98, and 260 Gy (P<.001). CONCLUSIONS Total dose delivered to the GTV from combined MRI-guided HDR and PET/CT-guided IMRT is highly correlated with local tumor control. The findings can be directly applied in the clinic for dose adaptation to maximize local control.