R. Kevin Reynolds

Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors, but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors, compared with ALDH(-) cells, validating ALDH as a marker of ovarian CSC in cell lines. Notably, as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice, whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines, we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells, suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly, as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC, ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally, the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together, our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs.

Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3β) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-xL. Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Preliminary Experience with Robot-Assisted Laparoscopic Myomectomy

The following retrospective case series evaluated the technique and feasibility of integrating robot-assisted technology in the performance of a laparoscopic myomectomy in order to overcome the limitations of conventional laparoscopy. We attempted 35 robot-assisted laparoscopic myomectomies in a university hospital setting with a conversion rate of 8.6%. There were a total of 48 myomas removed in 31 patients with completed robot-assisted laparoscopy. The mean number of myomas removed/patient was 1.6 (range 1-5). The mean diameter of myomas removed was 7.9 +/- 3.5 cm (95% CI 6.63-9.13), with the majority greater than 5 cm. The mean myoma weight was 223.2 +/- 244.1 g (95% CI 135.8-310.6). Mean operating time was 230.8 +/- 83 minutes (95% CI 201.6-260). The average estimated blood loss was 169 +/- 198.7 mL (95% CI 99.1-238.4). One patient experienced cardiogenic shock from vasopressin, two developed postoperative infections, and one was found to have adenomatous adenomyosis instead of a leiomyoma. The median length of hospital stay was 1 day. Overall, robot-assisted laparoscopic myomectomy is a promising new technique that may overcome many of the surgical limitations of conventional laparoscopy.

Metformin targets ovarian cancer stem cells in vitro and in vivo.

PURPOSE Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC. METHODS The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts. RESULTS Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis. CONCLUSIONS Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.

Prevention of cervix cancer

Cervix carcinoma is an important health problem world-wide, being the second most common cancer among women, ranking first in many developing countries. A number of important epidemiological risk factors have been identified as contributing to the development of CIN and invasive cervix carcinoma. Of key importance is infection with human papillomavirus (HPV), which is the primary risk factor. There are evolving primary and secondary preventive strategies that could further reduce the burden from cervical carcinoma. The possible primary preventive strategies include risk reduction, diet or dietary supplements, HPV vaccines, and other chemopreventive agents. The possible advances in secondary preventive strategies include new technologies for Pap smears, HPV typing triage, and other adjuvant screening procedures. The impact of these strategies will depend upon evidence to support their use along with the characteristics of the population and environment in which they are used.

Low-hanging fruit: A clementine as a simulation model for advanced laparoscopy

Introduction Low-cost, high-fidelity models for training in advanced laparoscopic surgery techniques are not currently available. The objective of this study was to evaluate a model and assessment protocol for developing associated fine, precise laparoscopic dissection skills with accompanying surgical decision making. Methods Novice to expert laparoscopists (n = 41) were asked to remove the peel of a clementine in as few pieces as possible, separate and remove all albedo from and between all fruit segments, and return the clementine to as close to its natural state as possible with completely closed skin (sutured). Clinical decision making included deciding when unacceptable segment damage would result by removing difficult-to-extract albedo, analogous to treating lesions or metastases through other methods, rather than risking damage to vital anatomic structures. Faculty assessed deidentified video-recorded performances. Data analyses included analysis of variance with Bonferroni post hoc. Results A single-performance construct (operative ability) with 2 scoring dimensions (surgical skills and clinical judgment) was confirmed through factor analysis. There were significant performance differences between all experience levels (F2,41 = 59.175, P < 0.000). There were no statistical time differences between the groups. Conclusions Validation of this low-cost, easily facilitated model for developing advanced laparoscopic surgical skills may support the preparation of residents and fellows and provide a platform for skill acquisition, assessment, and basic critical thinking for performing laparoscopic tasks.

Identifying alemtuzumab as an anti-myeloid cell antiangiogenic therapy for the treatment of ovarian cancer

BackgroundMurine studies suggest that myeloid cells such as vascular leukocytes (VLC) and Tie2+ monocytes play a critical role in tumor angiogenesis and vasculogenesis. Myeloid cells are a primary cause of resistance to anti-VEGF therapy. The elimination of these cells from the tumor microenvironment significantly restricts tumor growth in both spontaneous and xenograft murine tumor models. Thus animal studies indicate that myeloid cells are potential therapeutic targets for solid tumor therapy. Abundant VLC and Tie2+ monocytes have been reported in human cancer. Unfortunately, the importance of VLC in human cancer growth remains untested as there are no confirmed therapeutics to target human VLC.MethodsWe used FACS to analyze VLC in ovarian and non-ovarian tumors, and characterize the relationship of VLC and Tie2-monocytes. We performed qRT-PCR and FACS on human VLC to assess the expression of the CD52 antigen, the target of the immunotherapeutic Alemtuzumab. We assessed Alemtuzumabs ability to induce complement-mediated VLC killing in vitro and in human tumor ascites. Finally we assessed the impact of anti-CD52 immuno-toxin therapy on murine ovarian tumor growth.ResultsHuman VLC are present in ovarian and non-ovarian tumors. The majority of VLC appear to be Tie2+ monocytes. VLC and Tie2+ monocytes express high levels of CD52, the target of the immunotherapeutic Alemtuzumab. Alemtuzumab potently induces complement-mediated lysis of VLC in vitro and ex-vivo in ovarian tumor ascites. Anti-CD52 immunotherapy targeting VLC restricts tumor angiogenesis and growth in murine ovarian cancer.ConclusionThese studies confirm VLC/myeloid cells as therapeutic targets in ovarian cancer. Our data provide critical pre-clinical evidence supporting the use of Alemtuzumab in clinical trials to test its efficacy as an anti-myeloid cell antiangiogenic therapeutic in ovarian cancer. The identification of an FDA approved anti-VLC agent with a history of clinical use will allow immediate proof-of-principle clinical trials in patients with ovarian cancer.

Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progesterone in normal and neoplastic endometrial cell cultures

Growth factors are polypeptides which regulate cell proliferation through binding to specific receptor proteins. Normal and neoplastic human endometrium have been shown to express epidermal growth factor (EGF) and insulin-like growth factor I (IGF-1) receptors. Endometrial cell cultures were used to test modulation of EGF and IGF-1 receptors in response to steroid hormones. Endometrial gland and stroma cells were separated by enzymatic dispersion and were incubated in medium containing estradiol (10, 100, or 1000 pg/ml) or progesterone (1, 10, or 100 ng/ml) followed by radioligand assays. Normal endometrial cultures (n = 6) treated with estradiol demonstrated 40% less EGF binding than control cultures (P less than 0.05), while IGF-1 binding was unaffected. Stromal cells treated identically decreased in only one treatment group. Progesterone treatment stimulated a significant increase in EGF and IGF-1 receptors in gland cultures. Cultures derived from adenocarcinoma (n = 2) demonstrated decreased EGF binding compared with normal endometrium (P less than 0.05). Carcinoma cells treated with progesterone resulted in a dose-dependent increase in EGF binding over control (P less than 0.05). These data illustrate effects of steroid hormones upon growth factor receptors in human endometrium, and suggest involvement of growth factors in the regulation of normal and neoplastic endometrial growth.

Characterization of epidermal growth factor receptor in normal and neoplastic human endometrium

Growth factors, including epidermal growth factor (EGF), have been implicated in the growth of several types of cancer. This study compares EGF receptors in normal and neoplastic endometrium. Membrane fractions were isolated from surgical specimens. Radioreceptor assays demonstrated the presence of receptors with a dissociation constant of 0.64 nmol/1 in normal endometrium. Affinity crosslinking revealed receptor molecular weight of 150 to 170 kiloDaltons (KD). A survey of samples (n = 37) revealed progressive decrease of EGF receptors in cancers of increasing grade: Grade 1–2 adenocarcinoma decreased 34% from control (n = 6, P < 0.01), whereas Grade 3 adenocarcinoma decreased 90% (n = 7, P <0.01) and sarcoma decreased by 72% (n = 3, P < 0.01). The dissociation constant and molecular weight of the receptor in neoplastic endometrium did not differ significantly from normal. The inverse relationship with grade suggests receptor alteration or down regulation by hormones and/or growth factors.

Vulvar melanoma: review of diagnosis, staging, and therapy.

Objectives. To update, assimilate, and bridge the contemporary literature on vulvar and cutaneous melanoma regarding diagnosis, staging, and therapy to provide a useful clinical reference for managing and counseling for affected patients. Materials and Methods. A computerized search for reports in the literature up to June 2003 was carried out using PubMed and MEDLINE databases. Multidisciplinary involvement was used in evaluating the available data and formulating conclusions. Results. More than 300 reports were reviewed. Diagnosis, staging, and therapy aspects of vulvar melanoma are summarized. Conclusions. Vulvar melanoma represents a subtype of cutaneous melanoma, with similar prognostic and staging factors. The most recent American Joint Committee on Cancer staging system for cutaneous melanoma is applicable to vulvar melanoma. Sentinel lymph node biopsy is reliable for staging the regional lymph node basin for vulvar melanoma. Standardized documentation of clinical and histopathologic parameters is needed to standardize grouping of cases for future comparison studies.