Michael T. Deavers

Dicer, Drosha, and outcomes in patients with ovarian cancer.

BACKGROUND We studied Dicer and Drosha, components of the RNA-interference machinery, in ovarian cancer. METHODS We measured messenger RNA (mRNA) levels of Dicer and Drosha in specimens of invasive epithelial ovarian cancer from 111 patients, using a quantitative reverse-transcriptase-polymerase-chain-reaction assay, and compared the results with clinical outcomes. Validation was performed with the use of published microarray data from cohorts of patients with ovarian, breast, and lung cancer. Mutational analyses of genomic DNA from the Dicer and Drosha genes were performed in a subgroup of ovarian-cancer specimens. Dicer-dependent functional assays were performed by means of in vitro transfection with small interfering RNA (siRNA) and short hairpin RNA (shRNA). RESULTS Levels of Dicer and Drosha mRNA correlated with the levels of expression of the corresponding protein and were decreased in 60% and 51% of ovarian-cancer specimens, respectively. Low Dicer expression was significantly associated with advanced tumor stage (P=0.007), and low Drosha expression with suboptimal surgical cytoreduction (P=0.02). Cancer specimens with both high Dicer expression and high Drosha expression were associated with increased median survival (>11 years, vs. 2.66 years for other subgroups; P<0.001). We found three independent predictors of reduced disease-specific survival in multivariate analyses: low Dicer expression (hazard ratio, 2.10; P=0.02), high-grade histologic features (hazard ratio, 2.46; P=0.03), and poor response to chemotherapy (hazard ratio, 3.95; P<0.001). Poor clinical outcomes among patients with low Dicer expression were validated in additional cohorts of patients. Rare missense mutations were found in the Dicer and Drosha genes, but their presence or absence did not correlate with the level of expression. Functional assays indicated that gene silencing with shRNA, but not siRNA, may be impaired in cells with low Dicer expression. CONCLUSIONS Our findings indicate that levels of Dicer and Drosha mRNA in ovarian-cancer cells have associations with outcomes in patients with ovarian cancer.

Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Grading ovarian serous carcinoma using a two-tier system.

In this study, we evaluate a two-tier system for grading ovarian serous carcinoma. This system is based primarily on the assessment of nuclear atypia with the mitotic rate used as a secondary feature. The study included 50 cases of low-grade ovarian serous carcinoma and 50 cases of high-grade ovarian serous carcinoma retrieved from the files of the Department of Pathology at the University of Texas M. D. Anderson Cancer Center from a 28-year period. Cases assigned to the low-grade category were characterized by the presence of mild to moderate nuclear atypia. As a secondary feature, they tended to show up to 12 mitoses per 10 high power fields (HPFs), whereas those in the high-grade category had marked nuclear atypia and as a secondary feature more than 12 mitoses per 10 HPFs. For comparison, the tumors were also graded using the Shimizu/Silverberg and the FIGO grading systems. Patients in the low-grade ovarian serous carcinoma group ranged in age from 19 to 75 years (mean 41.7 years) while patients in the high-grade ovarian serous carcinoma group ranged in age from 27 to 76 years (mean 55 years). All of the cases except one were advanced FIGO stage. Using the Shimizu/Silverberg system, the low-grade ovarian serous carcinoma cases were distributed as follows: grade 1, 47 cases; grade 2, 3 cases. Using the FIGO grading system, 35 cases were grade 1 and 15 cases were grade 2. Regarding the high-grade ovarian serous carcinoma group using the Shimizu/Silverberg system, 14 of the cases were grade 2 and 36 cases were grade 3. Using the FIGO grading system, 1 case was grade 1, 38 cases were grade 2, and 11 cases were grade 3. Most of the patients in both groups were treated with total abdominal hysterectomy and bilateral salpingo-oophorectomy and also received cisplatinum-based chemotherapy. On follow-up, 37 patients in the low-grade ovarian serous carcinoma group had died of disease at a median 4.2 years after diagnosis compared with 46 patients in the high-grade ovarian serous carcinoma group who died of disease at a median of 1.7 years. Eight patients in the low-grade ovarian serous carcinoma group and 4 patients in the high-grade ovarian serous carcinoma group were alive with disease at median follow-ups of 4.3 and 3.85 years, respectively. Four patients with low-grade serous carcinoma were alive without evidence of disease after a follow-up that ranged from 4.4 to 22.6 years (median 6.85 years), and one died of other causes 14 years after the diagnosis of her ovarian tumor. On multivariate analysis, residual tumor and tumor grade based on the M. D. Anderson two-tier system for grading ovarian serous carcinoma were found to be significant independent prognostic factors (P = 0.003 and 0.04, respectively). Of interest, 60% of the low-grade ovarian serous carcinomas in this study were associated with a serous neoplasm of low malignant potential, whereas this association was present in only 2% of the high-grade ovarian serous carcinomas. This finding could reflect a difference in the pathogenesis of ovarian serous carcinomas of different grades. In summary, there is usually a good correlation between the two-tier grading system herein presented and the Shimizu/Silverberg and the FIGO grading systems. Because this system is based on defined criteria that are easy to follow and because it involves only two diagnostic categories, it should provide better reproducibility in the grading of ovarian serous carcinoma. However, additional studies are required to validate these statements.

Paraneoplastic Thrombocytosis in Ovarian Cancer

BACKGROUND The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Clinical behavior of stage II-IV low-grade serous carcinoma of the ovary

OBJECTIVE: To analyze the clinical behavior of patients with stage II-IV low-grade serous carcinoma of the ovary seen at our institution who underwent primary surgery followed by platinum-based chemotherapy. METHODS: Patients with stage II-IV low-grade serous carcinoma of the ovary from 1978 to 2003 were identified using existing databases. Clinicopathologic information was obtained from medical records. Progression-free survival and overall survival were estimated by the method of Kaplan and Meier. The log-rank test was used to compare differences between survival curves. Univariable and multivariable analyses were performed using Cox proportional hazards regression. RESULTS: We identified 112 eligible patients. Median age was 43 years.; 90% had stage III disease. Preoperative serum CA 125 was elevated in 86% of patients. The most common sites of extraovarian disease were omentum, fallopian tubes, pelvic peritoneum, and uterus. Response rate to platinum-based chemotherapy in 10 evaluable patients (15% of patients with gross residual disease) was 80%, and 42 patients underwent second-look surgery: microscopically negative findings, 2 (5%); microscopically positive disease, 13 (33%); macroscopically positive disease, 24 (62%); and insufficient information, 3 (7%). Median progression-free survival and overall survival times were 19.5 and 81.8 months. Persistent disease after primary chemotherapy was the only factor associated with shorter overall survival time (hazard ratio 3.46, 95% confidence interval 2.00–5.97, P<.001). CONCLUSION: Metastatic low-grade serous carcinoma of the ovary is characterized by young age at diagnosis and prolonged overall survival. Segregating women with this diagnosis in future clinical trials is warranted. LEVEL OF EVIDENCE: II-3

EphA2 expression is associated with aggressive features in ovarian carcinoma.

Purpose: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study. Experimental Design: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables. Results: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P = 0.02) and advanced stage of disease (P = 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P = 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival. Conclusions: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.

Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis

Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.

In vivo expression of the novel CXC chemokine BRAK in normal and cancerous human tissue.

Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.

Response and survival in patients with progressive or recurrent serous ovarian tumors of low malignant potential

OBJECTIVE To evaluate the response to therapy and survival of patients with progressive or recurrent serous ovarian tumors of low malignant potential. METHODS Fifty‐three patients with progressive or recurrent serous ovarian tumors of low malignant potential were identified. Response was assessed and progression‐free and overall survival were analyzed. The influence of clinicopathologic factors on survival was determined. RESULTS In 49 patients with known histology of progression or recurrence, 36 (73%) had low‐grade serous carcinoma, and 13 (27%) had serous ovarian tumors of low malignant potential. Forty‐five patients received nonsurgical therapy and had an evaluable response. There were six (13%) patients with a complete response and six (13%) patients with a partial response. The median time to first progression or recurrence was 5.6 years. Median survival from diagnosis of first recurrence was 7.7 years. Median survival from initial diagnosis was 21 years. Nineteen (36%) patients are dead of tumor. Patients who recurred with low‐grade serous carcinoma were more likely to die of tumor than those with serous ovarian tumors of low malignant potential (47% versus 0%, P = .045). Optimal cytoreduction was associated with improved survival (P = .007). CONCLUSION Patients with progressive or recurrent serous ovarian tumors of low malignant potential have a long interval from diagnosis to progression and from progression to death, resulting in extended overall survival. Recurrence as low‐grade serous carcinoma and failure to achieve optimal secondary cytoreduction were adverse prognostic factors. There were few responses to nonsurgical therapy.

Interobserver and intraobserver variability of a two-tier system for grading ovarian serous carcinoma.

Although grading has been demonstrated to be an important prognostic factor in ovarian serous carcinoma, there is no system universally used to perform this task. A few years ago, we proposed a two-tier system for grading ovarian serous carcinoma that is based primarily on the assessment of nuclear atypia (uniformity vs. pleomorphism) in the worst area of the tumor. Tumor grade in this two-tier system is correlated with survival. After being used by numerous pathologists and trainees at The University of Texas M.D. Anderson Cancer Center (MDACC) for 15 years, we have observed that this system is user-friendly and reproducible. We undertook this study to evaluate the interobserver and intraobserver variability among a group of 7 gynecologic pathologists and 2 general surgical pathologists using this grading system. A total of 80 cases of ovarian serous carcinoma, 40 low-grade and 40 high-grade, were circulated twice among these pathologists. Slides with examples of low-grade and high-grade serous carcinoma were sent with the unknowns. A website was used to provide diagnostic criteria, images of examples of ovarian low-grade and high-grade carcinoma, and a log form to facilitate data entry. Statistical analysis demonstrated an overall κ statistic among the different observers of 0.909. The intergrader κs ranged from 0.717 to 1.000 in the first round of the review and from 0.701 to 1.000 in the second round. Eight of the participants had an intragrader κ ranging from 0.775 to 1.000 (excellent agreement), whereas a single participant had an intragrader κ of 0.725 (good agreement). This study demonstrates that the two-tier grading system (the MDACC grading system) for ovarian serous carcinoma on the basis of the assessment of nuclear atypia is easy to learn and is highly reproducible. These findings would support its universal use, which would be beneficial for the standardization of clinical trials and protocols, thus facilitating the understanding of this disease and investigation into the treatment of patients affected by these tumors.