Nadim Bou Zgheib

Progression-free and overall survival of a modified outpatient regimen of primary intravenous/intraperitoneal paclitaxel and intraperitoneal cisplatin in ovarian, fallopian tube, and primary peritoneal cancer

OBJECTIVE GOG study 172 demonstrated improved progression-free (PFS) and overall (OS) survival for patients with stage III optimally debulked ovarian and peritoneal carcinoma treated with IV/IP paclitaxel and IP cisplatin compared to standard IV therapy. The inpatient administration, toxicity profile, and limited completion rate have been blamed for the lack of acceptance and widespread use of this regimen. We sought to evaluate the PFS, OS, toxicity, and completion rate of a modified outpatient IP regimen. METHODS Using a prospectively maintained database, we evaluated the outcomes of patients who underwent primary optimal cytoreduction for stage III ovarian, tubal, or peritoneal carcinoma followed by IV/IP chemotherapy from 1/05-3/09. Our modified regimen was as follows: IV paclitaxel (135 mg/m(2)) over 3h on day 1, IP cisplatin (75 mg/m(2)) on day 2, and IP paclitaxel (60 mg/m(2)) on day 8, given every 21 days for 6 cycles. RESULTS We identified 102 patients who initiated the modified IV/IP regimen and completed chemotherapy. The median follow-up was 43 months. The median age at diagnosis was 57 years (range, 23-76). Primary disease site was: ovary, 77 (75%); fallopian tube, 13 (13%); peritoneum, 12 (12%). FIGO stage was: IIIA, 8 (8%); IIIB, 4 (4%); IIIC, 90 (88%). Residual disease after cytoreduction was: none, 58 (57%); ≤ 1 cm, 44 (43%). The most frequent grade 3/4 toxicities were: neutropenia, 12 (12%); gastrointestinal, 8 (8%); neurologic, 6 (6%). Eighty-two (80%) of 102 patients completed 4 or more cycles of IV/IP therapy; 56 (55%) completed all 6 cycles. The median PFS and OS were 29 and 67 months, respectively. CONCLUSIONS By modifying the GOG 172 treatment regimen, convenience, toxicity, and tolerability appear improved, with survival outcomes similar to those of GOG 172. This modified IV/IP regimen warrants further study.

A novel c-Met inhibitor, MK8033, synergizes with carboplatin plus paclitaxel to inhibit ovarian cancer cell growth.

Elevated serum levels of hepatocyte growth factor (HGF) and high tumor expression of c-Met are both indicators of poor overall survival from ovarian cancer (OVCA). In the present study, we evaluated the role of the HGF signaling pathway in OVCA cell line chemoresistance and OVCA patient overall survival as well as the influence of HGF/c-Met signaling inhibition on the sensitivity of OVCA cells to combinational carboplatin plus paclitaxel therapy. The prevalence of the HGF receptor, c-Met, was determined by immunohistochemistry in primary OVCA samples (n=79) and OVCA cell lines (n=41). The influence of the c-Met-specific inhibitor MK8033 on OVCA cell sensitivity to combinations of carboplatin plus paclitaxel was examined in a subset of OVCA cells (n=8) by CellTiter-Blue cell viability assays. Correlation tests were used to identify genes associated with response to MK8033 and carboplatin plus paclitaxel. Identified genes were evaluated for influence on overall survival from OVCA using principal component analysis (PCA) modeling in an independent clinical OVCA dataset (n=218). Immunohistochemistry analysis indicated that 83% of OVCA cells and 92% of primary OVCA expressed the HGF receptor, c-Met. MK8033 exhibited significant anti-proliferative effects against a panel of human OVCA cell lines. Combination index values determined by the Chou-Talalay isobologram equation indicated synergistic activity in combinations of MK8033 and carboplatin plus paclitaxel. Pearsons correlation identified a 47-gene signature to be associated with MK8033-carboplatin plus paclitaxel response. PCA modeling indicated an association of this 47-gene response signature with overall survival from OVCA (P=0.013). These data indicate that HGF/c-Met pathway signaling may influence OVCA chemosensitivity and overall patient survival. Furthermore, HGF/c-Met inhibition by MK8033 represents a promising new therapeutic avenue to increase OVCA sensitivity to carboplatin plus paclitaxel.

The BCL2 antagonist of cell death pathway influences endometrial cancer cell sensitivity to cisplatin

OBJECTIVE To identify pathways that influence endometrial cancer (EC) cell sensitivity to cisplatin and to characterize the BCL2 antagonist of cell death (BAD) pathway as a therapeutic target to increase cisplatin sensitivity. METHODS Eight EC cell lines (Ishikawa, MFE296, RL 95-2, AN3CA, KLE, MFE280, MFE319, HEC-1-A) were subjected to Affymetrix Human U133A GeneChip expression analysis of approximately 22,000 probe sets. In parallel, endometrial cell line sensitivity to cisplatin was quantified by MTS assay, and IC(50) values were calculated. Pearsons correlation test was used to identify genes associated with response to cisplatin. Genes associated with cisplatin responsiveness were subjected to pathway analysis. The BAD pathway was identified and subjected to targeted modulation, and the effect on cisplatin sensitivity was evaluated. RESULTS Pearsons correlation analysis identified 1443 genes associated with cisplatin resistance (P<0.05), which included representation of the BAD-apoptosis pathway. Small interfering RNA (siRNA) knockdown of BAD pathway protein phosphatase PP2C expression was associated with increased phosphorylated BAD (serine-155) levels and a parallel increase in cisplatin resistance in Ishikawa (P=0.004) and HEC-1-A (P=0.02) cell lines. In contrast, siRNA knockdown of protein kinase A expression increased cisplatin sensitivity in the Ishikawa (P=0.02) cell line. CONCLUSION The BAD pathway influences EC cell sensitivity to cisplatin, likely via modulation of the phosphorylation status of the BAD protein. The BAD pathway represents an appealing therapeutic target to increase EC cell sensitivity to cisplatin.

The O-glycan pathway is associated with in vitro sensitivity to gemcitabine and overall survival from ovarian cancer.

Ovarian cancer (OVCA) is the most lethal gynecological malignancy. The high mortality rate associated with this disease is due in large part to the development of resistance to chemotherapy; however, the biological basis of this remains unclear. Gemcitabine is frequently used for the treatment of patients with platinum-resistant OVCA. We report molecular signaling pathways associated with OVCA response to gemcitabine. Forty-one OVCA cell lines were subjected to gene expression analysis; in parallel, IC50 values for gemcitabine were quantified using CellTiter-Blue viability assays. Pearsons correlation coefficients were calculated for gene expression and gemcitabine IC50 values. The genes associated with gemcitabine sensitivity were subjected to pathway analysis. For the identified pathways, principal component analysis was used to derive pathway signatures and corresponding scores, which represent overall measures of pathway expression. Expression levels of the identified pathways were then evaluated in a series of clinico-genomic datasets from 142 patients with stage III/IV serous OVCA. We found that in vitro gemcitabine sensitivity was associated with expression of 131 genes (p<0.001). These genes include significant representation of three molecular signaling pathways (p<0.02): O-glycan biosynthesis, Role of Nek in cell cycle regulation and Antiviral actions of Interferons. In an external clinico-genomic OVCA dataset (n=142), expression of the O-glycan pathway was associated with overall survival, independent of surgical cytoreductive status, grade and age (p<0.001). Expression levels of Role of Nek in cell cycle regulation and Antiviral actions of Interferons were not associated with survival (p=0.31 and p=0.54, respectively). Collectively, expression of the O-glycan biosynthesis pathway, which modifies protein function via post-translational carbohydrate binding, is independently associated with overall survival from OVCA. Our findings shed light on the molecular basis of OVCA responsiveness to gemcitabine and also identify a signaling pathway that may influence patient survival.

BAD-mediated apoptotic pathway is associated with human cancer development

The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, p<0.001), breast (n=185, p<0.0008; n=61, p=0.04), colon (n=22, p<0.001) and endometrial (n=33, p<0.001) cancers, as well as with ovarian endometriosis (n=20, p<0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, p<0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

Micro-RNAs associated with the evolution of ovarian cancer cisplatin resistance

OBJECTIVES Ovarian cancer (OVCA) is the leading cause of mortality among women with gynecologic malignancy, in part due to the development of chemoresistance. We sought to identify micro-RNAs (miRNAs) associated with in vitro development of OVCA chemoresistance that may also represent potential targets for therapy. METHODS In this study, four OVCA cell lines (A2780CP, A2780S, IGROV1, and OVCAR5) were serially treated with cisplatin in parallel with measurements of miRNA expression changes. RESULTS Nine miRNAs were found to be associated with increasing cisplatin resistance (IC50) (p<0.01); however, only 5 of these miRNAs have publically available information. Pathway analysis identified 15 molecular signaling pathways that were represented by genes predicted to be targets of the 5 miRNAs (false discovery rate<0.05), 11 of which are associated with the epithelial-mesenchymal transition (EMT). Further analysis identified 2 of those pathways as being associated with overall survival in 218 patients with OVCA. CONCLUSIONS Collectively, this panel of miRNAs associated with in vitro evolution of OVCA cisplatin resistance and the pathways identified to be associated with EMT and overall patient survival provide a framework for further investigations into EMT as a therapeutic target in patients with OVCA.

Perifosine, an AKT inhibitor, modulates ovarian cancer cell line sensitivity to cisplatin-induced growth arrest

OBJECTIVES AKT, a key regulator of diverse tumor signaling, is associated with progression of many cancers. Here, we investigated 1) the influence of AKT on survival from ovarian cancer (OVCA), 2) the activity of the AKT inhibitor perifosine ± cisplatin, and 3) the molecular determinants of perifosine-response. Phospho-AKT expression values and Affymetrix U133a expression data were downloaded from The Cancer Genome Atlas. METHODS Pearson correlation was used to determine associations between overall survival from OVCA and therapy response. Genes and represented signaling pathways associated with perifosine-response were explored in OVCA cells (n=10) and the NCI60 cancer cell panel. Pathway expressions, modeled by PCA, were evaluated for influences on survival using publically available clinico-genomic datasets. RESULTS Phospho-AKT (serine473) expression correlated with survival from OVCA (P<0.05) and platinum-response (P=0.004). In vitro, perifosine showed anti-proliferative effects against OVCA cells and potentiated cisplatin-induced growth arrest. Perifosine-response was associated with the expression (FDR<0.05) of 7 signaling pathways in OVCA cells and 64 signaling pathways in the NCI60 cell panel. Three pathways were found in common: 1) Cytoskeleton remodeling/cytoskeleton remodeling (cyto), 2) cell adhesion/chemokines and adhesion (chemokines), and 3) cytoskeleton remodeling/TGF-WNT (TGF-WNT). The TGF-WNT was associated with survival from OVCA (P=0.0055). CONCLUSIONS AKT signaling is an important determinant of OVCA response to chemotherapy and overall patient survival. Our data provide insight into the molecular basis to perifosine activity and identifies pathways associated with perifosine sensitivity and patient clinical outcome.

Factors associated with improved toxicity and tolerability of intraperitoneal chemotherapy in advanced-stage epithelial ovarian cancers.

OBJECTIVE We sought to evaluate the toxicity and tolerability of the intraperitoneal/intravenous regimen by comparing the modified regimen that is used at the Moffitt Cancer Center vs the published findings of the Gynecologic Oncology Group Study 172. STUDY DESIGN Using the Moffitt database, we evaluated the outcomes of patients who underwent primary optimal cytoreduction for stage IIC-IV epithelial ovarian, tubal, and peritoneal carcinoma followed by the intent-to-treat with intraperitoneal/intravenous chemotherapy. National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0) was used to grade adverse events. RESULTS We analyzed patient data from 2006-2011 and identified 69 patients who met our inclusion criteria. The most frequent grade 3/4 toxicities were neutropenia (48%), gastrointestinal (9%), metabolic (9%), and infection (5%). Remaining toxicities occurred in <5% of patients. Patients received a greater number of cycles compared with the Gynecologic Oncology Group Study 172 (4.28 vs 3.66, respectively; P = .0088). CONCLUSION With the use of supportive care and the preemptive management of established side-effects, the associated toxicities and tolerability of intraperitoneal chemotherapy can be improved.

Prediction of Optimal Cytoreductive Surgery of Serous Ovarian Cancer With Gene Expression Data.

Objectives Cytoreductive surgery is the cornerstone of ovarian cancer (OVCA) treatment. Detractors of initial maximal surgical effort argue that aggressive tumor biology will dictate survival, not the surgical effort. We investigated the role of biology in achieving optimal cytoreduction in serous OVCA using microarray gene expression analysis. Methods For the initial model, we used a gene expression signature from a microarray expression analysis of 124 women with serous OVCA, defining optimal cytoreduction as removal of all disease greater than 1 cm (with 64 women having optimal and 60 suboptimal cytoreduction). We then applied this model to 2 independent data sets: the Australian Ovarian Cancer Study (AOCS; 190 samples) and The Cancer Genome Atlas (TCGA; 468 samples). We performed a second analysis, defining optimal cytoreduction as removal of all disease to microscopic residual, using data from AOCS to create the gene signature and validating results in TCGA data set. Results Of the 12,718 genes included in the initial analysis, 58 predicted accuracy of cytoreductive surgery 69% of the time (P = 0.005). The performance of this classifier, measured by the area under the receiver operating characteristic curve, was 73%. When applied to TCGA and AOCS, accuracy was 56% (P = 0.16) and 62% (P = 0.01), respectively, with performance at 57% and 65%, respectively. In the second analysis, 220 genes predicted accuracy of cytoreductive surgery in the AOCS set 74% of the time, with performance of 73%. When these results were validated in TCGA set, accuracy was 57% (P = 0.31) and performance was at 62%. Conclusion Gene expression data, used as a proxy of tumor biology, do not predict accurately nor consistently the ability to perform optimal cytoreductive surgery. Other factors, including surgical effort, may also explain part of the model. Additional studies integrating more biological and clinical data may improve the prediction model.