Phase II trial of Oxaliplatin and 5-Fluorouracil/Leucovorin combination in epithelial ovarian carcinoma relapsing within 2 years of platinum-based therapy

Objective To evaluate the efficacy and toxicity of Oxaliplatin and 5-Fluorouracil (5-FU)/Leucovorin (LV) combination in ovarian cancer relapsing within 2 years of prior platinum-based chemotherapy in a phase II trial. Methods Eligible patients had at least one prior platinum-based chemotherapy regimen, elevated CA-125 ≥ 60 IU/l, radiological evidence of disease progression and adequate hepatic, renal and bone marrow function. Patients with raised CA-125 levels alone as marker of disease relapse were not eligible. Oxaliplatin (85 mg/m 2) was given on day 1, and 5-Fluorouracil (370 mg/m 2) and Leucovorin (30 mg) was given on days 1 and 8 of a 14-day cycle. Results Twenty-seven patients were enrolled. The median age was 57 years (range 42-74 years). The median platinum-free interval (PFI) was 5 months (range 0-17 months) with only 30% of patients being platinum sensitive (PFI > 6 months). Six patients (22%) had two prior regimens of chemotherapy. A total of 191 cycles were administered (median 7; range 2-12). All patients were evaluable for toxicity. The following grade 3/4 toxicities were noted: anemia 4%; neutropenia 15%; thrombocytopenia 11%; neurotoxicity 8%; lethargy 4%; diarrhea 4%; hypokalemia 11%; hypomagnesemia 11%. Among 27 enrolled patients, 20 patients were evaluable for response by WHO criteria and 25 patients were evaluable by Rustin's CA-125 criteria. The overall response rate (RR) by WHO criteria was 30% (95% CI: 15- 52) [three complete responses (CRs) and three partial responses (PRs)]. The CA-125 response rate was 56% (95% CI: 37-73). Significantly, a 25% (95% CI: 9-53) radiological and a 50% (95% CI: 28-72) CA-125 response rate were noted in platinum resistant patients (PFI < 6 months). The median response duration was 4 months (range 3-12) and the median overall survival was 10 months. Conclusion Oxaliplatin and 5-Fluorouracil/ Leucovorin combination has a good safety profile and is active in platinum-pretreated advanced epithelial ovarian cancer. © 2004 Elsevier Inc. All rights reserved.

Targeting histone deacetylases for the treatment of immune, endocrine and metabolic disorders

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The proinflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment. © 2009 Bentham Science Publishers Ltd.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines. Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique. Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r=0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant. Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

Cirurgia não ginecológica em pacientes com câncer de ovário

OBJETIVO: avaliar a freqüência da utilização de procedimentos cirúrgicos não ginecológicos no tratamento do câncer de ovário, assim como descrever as suas complicações. MÉTODOS: foram incluídas retrospectivamente 82 pacientes, atendidas no período de fevereiro de 1999 a outubro de 2003, e que haviam sido submetidas a laparotomia para tratamento cirúrgico do câncer de ovário. Foram incluídas no estudo apenas pacientes com adenocarcinoma epitelial de ovário. A média de idade foi de 54,1±15,1 anos, variando de 22 a 89 anos. O estadiamento (FIGO) mostrou 5 pacientes no estádio I (6,1%), 18 pacientes no II (21,9%), 40 pacientes no III (48,8%) e 19 pacientes no IV (23,2%). As pacientes foram divididas em 2 grupos: aquelas em que foram realizados exclusivamente procedimentos ginecológicos e aquelas com realização de procedimentos não ginecológicos. As diferenças entre as médias foram analisadas pelo teste t de Student. As comparações entre grupos independentes foram feitas pelo teste do chi2. RESULTADOS: em 35 pacientes (42,7%) foram realizados procedimentos cirúrgicos não ginecológicos, incluindo: 17 colostomias, 16 enterectomias, oito peritonectomias pélvicas, sete colectomias, cinco ressecções parciais de cúpula frênica, quatro cistectomias parciais, quatro esplenectomias, duas ileostomias e uma hepatectomia parcial. Todas as pacientes submetidas a procedimentos não ginecológicos apresentavam-se nos estádios III e IV. Esse grupo apresentou maior tempo cirúrgico (5,3±1,4 versus 3,1±0,9 horas); p<0,001). Os grupos não mostraram diferenças significativas em relação à necessidade de hemotransfusão (42,2 versus 40%; p=0,512) e tempo de internação (11,5±7,2 versus 10±9,9 dias; p=0,454). A realização de procedimentos cirúrgicos não ginecológicos associou-se a maior taxa de complicações pós-operatórias 37% versus (17,1%; p=0,042), sendo que duas pacientes desse grupo (2,4%) evoluíram para o óbito. CONCLUSÕES: os procedimentos cirúrgicos não ginecológicos são freqüentemente empregados no tratamento de pacientes com câncer de ovário. A realização dessas cirurgias está associada a maior tempo cirúrgico e maior taxa de complicações pós-operatórias.

Adenocarcinoma in females detected in serous effusions: Cytomorphologic aspects and immunocytochemical reactivity to cytokeratins 7 and 20

OBJECTIVE: To investigate the usefulness of assessing the immunoreactivity of cytokeratins 7 (CK7) and 20 (CK20) as well as several cytomorphologic parameters in effusions with metastatic adenocarcinomas in the search for the primary site of the tumor. STUDY DESIGN: From the files of the Pathology Department, A. C. Camargo Hospital, we studied cytologic smears from 73 metastatic adenocarcinomas originally from the breast, 63 from the ovary, 40 from the lung and 32 from the stomach, looking for morphologic parameters that could have discriminant potential in suggesting the primary site in a routine situation, including intranuclear inclusions, prominent nucleoli, mitosis, signet-ring cells, psammoma bodies, nuclear crease, binucleation and multinucleation, papillary features, acinar profile (including ball cells) and single cells. Immunoreactions were performed with monoclonal antibodies to CK7 (OV-TL 12/30 and CK20 (Ks 20.8) and included morphologic analysis. Both analyses were studied in a blind fashion regarding the primary site of the tumors. RESULTS: Positivity ratios for breast, ovary, stomach and lung cases were 67.6%, 63.5%, 29.7% and 45.5%, respectively, for CK7 and 17.2%, 15.8%, 13.5% and 32.2%, respectively, for CK20. Discriminant analysis of morphologic and immunocytochemical parameters had an error rate of 42.9% in recognizing the primary site and a Wilk's lambda of .7290. CONCLUSION: The more efficient parameter with discriminant function was the papillary appearance showed by CK7, which should be used in further studies with a similar scope. The set of parameters used in this study were insufficient to discriminate the primary site of female adenocarcinomas in effusions with significant accuracy.

Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

Correlation of the expression of human kallikrein-related peptidases 4 and 7 with the prognosis in oral squamous cell carcinoma

Background Very few articles have been written about the expression of kallikreins (KLK4 and KLK7) in oral cancers. Therefore, the purpose of this study was to examine and report on their prognostic potential. Methods Eighty archival blocks of primary oral cancers were sectioned and stained for KLK4 and KLK7 by immunohistochemistry. The percentage and the intensity of malignant keratinocyte staining were correlated with patient survival using Cox regression analysis. Results Both kallikreins were expressed strongly in the majority of tumor cells in 68 of 80 cases: these were mostly moderately or poorly differentiated neoplasms. Staining was particularly intense at the infiltrating front. Patients with intense staining had significantly shorter overall survival (p < .05). Conclusion This is the first observation on the patient survival influenced by kallikrein expression in oral carcinoma. The findings are consistent with those for carcinomas at other sites, in particular the prostate and ovary. KLK4 and/or KLK7 immunohistochemistry seems to have diagnostic and prognostic potential in this disease.

Melatonin attenuates Her-2, p38 MAPK, p-AKT, and mTOR levels in ovarian carcinoma of ethanol-preferring rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of interleukin-6 in the angiogenesis of ovarian carcinoma

The purpose of this study was to characterize the effects of IL-6 on endothelial cells and to investigate the role of IL-6 in the angiogenesis of ovarian carcinomas. We evaluated human ovarian carcinoma clinical specimens and determined that high expression of IL-6 was associated with increased tumor vascularization. Additionally, endothelial cells derived from the ovary and mesentery expressed the IL-6 receptor (IL-6R), and their stimulation with the exogenous ligand activated downstream signaling molecules and enhanced cell migration. Dual immunohistochemical staining for CD-31 and IL-6R revealed IL-6R expression on human endothelial cells within normal ovary and ovarian carcinomas. To further investigate the possible proangiogenic function of IL-6, Gelfoam sponges containing IL-6 or bFGF were implanted into the subcutis of BALB/c mice. IL-6 containing sponges were vascularized to the same extent as bFGF containing sponges. ^ Chronic stress can adversely affect disease progression. Stimulation of ovarian carcinoma cell lines with concentrations of catecholamines achieved in individuals experiencing chronic stress resulted in a substantial increase in IL-6 production. It was determined that stress mediators regulate IL-6 expression through the β-adrenergic receptor and Src. These data illustrate one mechanism by which chronic stress may influence tumor progression. ^ To investigate whether IL-6 contributes to the angiogenesis of ovarian carcinomas, we isolated low IL-6 expressing clones from the SKOV3.ip1 cell line and transfected them with a plasmid encoding the IL-6 gene. We observed no difference in tumor weight between high and low IL-6 expressing cells. However, while low IL-6 expressing tumors were highly vascularized, high IL-6 expressing tumors appeared hypervascularized. Immunohistochemical analysis revealed that all tumors exhibited robust expression of additional proangiogenic molecules. ^ Collectively, these studies indicate that IL-6 secreted by ovarian cancer cells is a highly proangiogenic cytokine. However, IL-6 is but one of several proangiogenic molecules produced by ovarian cancer, and its inhibition may not be sufficient to inhibit angiogenesis of ovarian carcinoma. The findings presented in this dissertation provide insight into the function of IL-6 as a regulator of angiogenesis. Understanding of the role of proangiogenic molecules such as IL-6 in ovarian carcinoma may have important implications for therapy directed at the vascular component of this disease. ^