Epithelial Ovarian Cancer: molecular and clinical predictors for platinum resistance

Most patients with ovarian cancer are still diagnosed in advanced stage disease needing intensive therapy with debulking surgery and chemotherapy. However, 25 % of patients will progress during or will relapse within 6 months of the primary therapy. These patients are called the platinum resistant patients. Unfortunately there are not many therapeutic options for this group of patients available. This doctoral thesis was performed in an effort to identify platinum resistant patients at diagnosis by searching for molecular and clinical predictors for platinum resistance. In this way the burden of a cytotoxic therapy compromising bone marrow reserve and quality of life could be avoided and targets for more individualised therapy could be found. Proteins represent the actual functional molecules in a cell and when a mutation occurs at the DNA level, it is the protein that ultimately will be affected. Therefore the study of the proteome, meaning the entire protein content produced by a cell during its life cycle, can help us to detect changes occurring during a particular disease process. Challenges in proteomic studies in blood are the presence of high abundant proteins masking the detection of the low concentrated ones, inter- and intra-individual differences and the need for prospectively and well controlled collected samples. Therefore we took part in a European consortium, the OVCAD study, where biological samples of ovarian cancer patients were prospectively collected according to a strict protocol. In this way we were able to collect a substantial number of samples in a relative short study period. In an attempt to minimise variation in protein content and to improve the probability of finding an ovarian cancer specific marker we decided to perform proteomic analysis on tumor tissue biopsies. Moreover, as ovarian tumor tissue biopsies consist of a heterogeneous amount of cells, we preferred to work with laser microdissected ovarian cancer cells. In the molecular part of this thesis, we firstly identified current problems and obstacles in proteomic studies and subsequently defined a protocol to combine laser microdissection and SELDI-TOF MS analysis in ovarian cancer tissue to achieve robust protein profiles. We demonstrated that changes in sample handling have influences on the protein profile and that strict protocols are necessary to obtain reliable results.Using this protocol we performed a first analysis on LMD ovarian tumor tissue of platinum sensitive vs. resistant patients. Based on the obtained profiles we determined that CM10 and IMAC30 were the ProteinChip surfaces of interest to be used further SELDI-TOF MS analysis.This model was then applied on the prospectively collected OVCAD samples and a prediction model was build to classify patients according to platinum sensitivity. Furthermore, we were able to confirm differentially expressed peaks in the lower molecular mass range between platinum resistant and sensitive patients. One of the upregulated peaks in patients with platinum sensitive disease with an m/z value of 2885 Da, could be identified as Histone H2B type 1-D (H2B1D_Human). Histone modification is currently used in other cancers as target for therapy and this finding could therefore have a clinical impact and warrants further investigation. Subsequently a validation study was performed but was not able to confirm the results of the training set. We believe other techniques such as immunohistochemistry should be considered to establish the role of Histone H2B type 1-D in the predictions of platinum resistance in ovarian cancer. In the clinical part of this dissertation, we assessed the clinical data of patients included in the OVCAD study in relation to their platinum free interval to determine clinical predictors for response. After multivariate analysis we identified the presence of peritoneal carcinomatosis, high levels of CA125 at the end of therapy and the achievement of complete remission at the end of primary therapy as the most significant factors for platinum resistance. These data could guide us in the follow-up of EOC patients who are at risk for early relapse.In a next chapter we evaluated the effect of a dose dense or weekly paclitaxel carboplatinum regimen for recurrent ovarian cancer in an attempt to overcome platinum resistance. Both regimens achieved good RR of 37% in the platinum resistant group. Toxicity varied between both schemes but was acceptable. In conclusion the molecular studies in this thesis need further investigation and might have a clinical impact in the search for targeted therapy in resistant epithelial ovarian cancer. Secondly, in the clinical part we identified patients at risk for early recurrence and we suggest that even in platinum resistant patients a rechallenge with a modified paclitaxel and platinum containing regimen is a valuable treatment option.Chapter 1: General Introduction 1.1. Clinical aspects of Epithelial Ovarian Cancer 1.1.1. Epidemiology 1.1.2. Symptoms – Diagnosis 1.1.3. Treatment 1.1.3.1. Surgery 1.1.3.2. Chemotherapy 1.1.3.3. Recurrent disease 1.1.4. Prognosis 1.2. Platinum resistance 1.3. Proteomics 1.3.1. General introduction 1.3.2. Proteomics in ovarian cancer 1.3.3. Methods for proteomic analysis 1.3.3.1. MALDI-TOF MS 1.3.3.2. SELDI-TOF MS 1.4. Laser microdissection 1.5. OVCAD: OVarian CAncer: Diagnosing a silent killer Chapter 2: Objectives Chapter 3: Proteomic studies 3.1. Application of proteomics in ovarian cancer: which sample should be used? 3.2. The utility of proteomics in gynecological cancer 3.3. Methods in molecular biology: protocol for proteomic analysis of laser microdissected ovarian cancer tissue with SELDI-TOF MS 3.4. The use of laser microdissection and SELDI-TOF MS in ovarian cancer tissue to identify protein profiles 3.5. Prediction of platinum resistance by protein profiling of laser microdissected ovarian cancer cells: an OVCAD study 3.6. Protein profiles for prediction of platinum resistance in ovarian cancer fails validation, a prospective OVCAD study Chapter 4: Clinical studies 4.1. Analysis of clinical predictive factors for platinum resistance in epithelial ovarian cancer within the prospective collected dataset of the OVCAD study 4.2. The “Leuven” dose dense paclitaxel / carboplatin regimen in patients with recurrent ovarian cancer 4.3. The “Leuven” paclitaxel / carboplatin weekly regimen in patients with recurrent ovarian cancer Chapter 5: General discussion Chapter 6: Summary 6.1. Summary 6.2. Samenvatting Curriculum Vitae List of publicationsnrpages: 206status: publishe

Proteomic biomarkers predicting lymph node involvement in serum of cervical cancer patients. Limitations of SELDI-TOF MS

<p>Abstract</p> <p>Background</p> <p>Lymph node status is not part of the staging system for cervical cancer, but provides important information for prognosis and treatment. We investigated whether lymph node status can be predicted with proteomic profiling.</p> <p>Material & methods</p> <p>Serum samples of 60 cervical cancer patients (FIGO I/II) were obtained before primary treatment. Samples were run through a HPLC depletion column, eliminating the 14 most abundant proteins ubiquitously present in serum. Unbound fractions were concentrated with spin filters. Fractions were spotted onto CM10 and IMAC30 surfaces and analyzed with surface-enhanced laser desorption time of flight (SELDI-TOF) mass spectrometry (MS). Unsupervised peak detection and peak clustering was performed using MASDA software. Leave-one-out (LOO) validation for weighted Least Squares Support Vector Machines (LSSVM) was used for prediction of lymph node involvement. Other outcomes were histological type, lymphvascular space involvement (LVSI) and recurrent disease.</p> <p>Results</p> <p>LSSVM models were able to determine LN status with a LOO area under the receiver operating characteristics curve (AUC) of 0.95, based on peaks with m/z values 2,698.9, 3,953.2, and 15,254.8. Furthermore, we were able to predict LVSI (AUC 0.81), to predict recurrence (AUC 0.92), and to differentiate between squamous carcinomas and adenocarcinomas (AUC 0.88), between squamous and adenosquamous carcinomas (AUC 0.85), and between adenocarcinomas and adenosquamous carcinomas (AUC 0.94).</p> <p>Conclusions</p> <p>Potential markers related with lymph node involvement were detected, and protein/peptide profiling support differentiation between various subtypes of cervical cancer. However, identification of the potential biomarkers was hampered by the technical limitations of SELDI-TOF MS.</p

The impact of enzastaurin (LY317615.HCl) on CA125 biosynthesis and shedding in ovarian cancer cells

In this study the modulatory effect of the proteinase kinase C beta (PKC beta) selective inhibitor enzastaurin on CA125 expression and shedding in ovarian cancer cells (OVCAR-3 cells) was investigated. MATERIAL ANDMETHODS: OVCAR-3 cells were cultured in vitro and treated with increasing concentrations of carboplatin (2-1000 microM), paclitaxel (0.2-100 nM) or enzastaurin (1-100 microM) single agent. Growth inhibitory effects were evaluated by MTS and luminescence assay. CA 125 was determined in supernatans and in cell lysate using an electrochemo-iluminescence immunoassay.Cell growth of OVCAR-3 cells was inhibited by single agent carboplatin, paclitaxel or enzastaurin in a dose dependent manner. Carboplatin caused a transient increase of CA125 in supernatans followed by a gradual decrease of CA125. Treatment with increasing doses of paclitaxel or enzastaurin caused an increase of CA125 shedding in culture medium but also the membrane bound fraction of CA125 was increased.These results suggest that enzastaurin, as paclitaxel, has a direct stimulatory effect on CA 125 synthesis and shedding in vitro

Robotic retroperitoneal lower para-aortic lymphadenectomy in cervical carcinoma: First report on the technique used in 5 patients

Objective. Retroperitoneal para-aortic laparoscopic lymphadenectomy is a technically challenging operation. The robotic Da Vinci system might be valuable in this operation due to a steady three-dimensional visualization, instrumentation with articulating tips, and an adaptive downscaling of the surgeons movements (without tremor). To the best of our knowledge, this is the first report on robotic retroperitoneal para-aortic lymphadenectomy in patients with gynecologic cancer. Method and results. We report on the technique and operative results of the robotic retroperitoneal lower para-aortic lymphadenectomy using the Da Vinci Surgical System. Five patients with cervical carcinoma stage IIb-IIb were included. Technically the procedure was easier to perform than with the classical retroperitoneal laparoscopic approach. However using the Da Vinci Surgical System it is important to tilt the patient slightly to the left to avoid collision between the left arm of the patient and the robotic arms, and to place the endoscopic robotic arm between the 2 arms used for dissection. Finally, we experienced that using a 30 scope is advantageous for the dissection of the paracaval nodes. None of the patients had evidence of para-aortic metastases on preoperatively staging, including Positron Emission Tomography - Computed Tomography (PET-CT). One of the patients had positive para-aortic lymph nodes. Conclusion. Here we report on the surgical technique used in our first 5 patients undergoing retroperitoneal para-aortic lymphadenectomy using the robotic Da Vinci system. It is important to adapt the surgical technique using the Da Vinci Surgical System compared with the classical laparoscopic techniqu

Subjective assessment by ultrasound is superior to the risk of malignancy index (RMI) or the risk of ovarian malignancy algorithm (ROMA) in discriminating benign from malignant adnexal masses

Purpose: The combination of two tumour markers, CA125 and HE4, in the risk of ovarian malignancy assay (ROMA) has been shown to be successful in classifying patients into those who have a high or low risk of epithelial ovarian cancer. In the present study, the diagnostic accuracy of ROMA was assessed and compared to the diagnostic accuracy of the two most widely used ultrasound methods, namely the risk of malignancy index (RMI) and subjective assessment by ultrasound. Methods: From August, 2005 to March, 2009, 432 women with a pelvic mass who were scheduled to have surgery were enrolled in a single-centre prospective cohort study. A preoperative ultrasound was performed and preoperative CA125 and HE4 serum levels were measured. Once the final surgical pathology reports were obtained, the diagnostic accuracy and performance indices of ROMA, RMI and subjective assessment were calculated. Results: Of the 432 eligible patients, 374 could be analysed. Subjective assessment had the highest area under the receiver operator characteristic curve (AUC) (0.968, 95% CI: 0.945-0.984), followed by the RMI(0.931, 95% CI: 0.901-0.955). The subjective assessment and RMI both had significantly higher AUCs than the ROMA (0.893, 95% CI: 0.857-0.922; P <0.0001 and P = 0.0030, respectively). The pre- and postmenopausal populations generated similar results. Conclusion: Although new tumour markers models are promising, they do not contribute significantly to the diagnosis of ovarian cancer. Ultrasound, especially subjective assessment by ultrasound, remains superior in discriminating malignant from benign ovarian masses. (C) 2011 Elsevier Ltd. All rights reserve

Obstetric and perinatal outcome of 1655 ICSI and 3974 IVF singleton and 1102 ICSI and 2901 IVF twin births: a comparative analysis

A total of 3974 IVF and 1655 ICSI singleton births and 2901 IVF and 1102 ICSI twin births were evaluated. Pregnancies after both fresh and frozen transfers were included. IVF and ICSI singleton pregnancies were very similar for most obstetric and perinatal variables. The only significant difference was a higher risk for prematurity (< 37 weeks of amenorrhoea) in IVF pregnancies compared with ICSI pregnancies (12.4 versus 9.2%, OR = 1.39, 95% CI = 1.15-1.70). For twin pregnancies, differences were not statistically different except for a higher incidence of stillbirths in the ICSI group (2.08 versus 1.03%, OR = 2.04, 95% CI = 1.14-3.64). Intrauterine growth retardation with or without pregnancy-induced hypertension was observed more often in the ICSI group. Regression analysis of the data with correction for parity and female age showed similar results for twins. For singletons, this analysis showed similar results with the exception of low birth weight babies (< 2500 g), which were also observed more often in IVF pregnancies (9.6 versus 7.9%, OR = 0.79, CI = 0.65-0.98, P = 0.03). This large case-comparative retrospective analysis showed that the obstetric outcome and perinatal health of IVF and ICSI pregnancies is comparable.status: publishe