Ovarian cancer molecular pathology.

Ovarian cancer (OVC) is the fourth leading cause of cancer mortality among women in Europe and the United States. Its early detection is difficult due to the lack of specificity of clinical symptoms. Unfortunately, late diagnosis is a major contributor to the poor survival rates for OVC, which can be attributed to the lack of specific sets of markers. Aside from patients sharing a strong family history of ovarian and breast cancer, including the BRCA1 and BRCA2 tumor suppressor genes mutations, the most used biomarker is the Cancer-antigen 125 (CA-125). CA-125 has a sensitivity of 80 % and a specificity of 97 % in epithelial cancer (stage III or IV). However, its sensitivity is 30 % in stage I cancer, as its increase is linked to several physiological phenomena and benign situations. CA-125 is particularly useful for at-risk population diagnosis and to assess response to treatment. It is clear that alone, CA-125 is inadequate as a biomarker for OVC diagnosis. There is an unmet need to identify additional biomarkers. Novel and more sensitive proteomic strategies such as MALDI mass spectrometry imaging studies are well suited to identify better markers for both diagnosis and prognosis. In the present review, we will focus on such proteomic strategies in regards to OVC signaling pathways, OVC development and escape from the immune response.

The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling.

This study reports on the C-terminal fragment of the 11S proteasome activator complex (PA28 or Reg alpha), a novel ovarian-specific biomarker of early and late stages of ovarian cancer (OVC) relapse, in patient biopsies after chemotherapy. A total of 179 tissue samples were analyzed: 8 stage I, 55 stage III-IV, 10 relapsed serous carcinomas, 25 mucinous carcinomas and 12 borderline and 68 benign ovarian tissue samples. This fragment was detected by MALDI mass spectrometry profiling in conjunction with a novel extraction method using hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propanol; HFIP) solvents for protein solubilization and by immunohistochemistry using a specific antibody directed against the C-terminal fragment of PA28. Due to its specific cellular localization, this fragment is a suitable candidate for early OVC diagnosis, patient prognosis and follow-up during therapy and discriminating borderline cancers. Statistical analyses performed for this marker at different OVC stages reflect a prevalence of 77.66 ± 8.77 % (with a correlation coefficient value p < 0.001 of 0.601 between OVC and benign tissue). This marker presents a prevalence of 88 % in the case of tumor relapse and is detected at 80.5 % in stage I and 81.25 % ± 1.06 in stage III-IV of OVC. The correlation value for the different OVC stages is p < 0.001 of 0.998. Taken together, this report constitutes the first evidence of a novel OVC-specific marker.

MALDI imaging mass spectrometry: state of the art technology in clinical proteomics.

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient's entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single "node" in the pathway.

MALDI imaging mass spectrometry in ovarian cancer for tracking, identifying, and validating biomarkers.

BACKGROUND: Among biomarkers, cancer-antigen 125 (CA-125) is the most studied. We propose an analytical tool to track ovarian carcinoma biomarkers, that is, the MALDI mass spectrometry imaging. MATERIAL/METHODS: Ovarian carcinomas and benign ovaries were directly analyzed by MALDI-TOF-MS. After automatic profiling and mass spectrometry imaging analyses, hierarchical clustering based on principal component analysis in nonsupervised mode was carried out. On the same samples, preparations were performed to investigate peptides, then proteins, followed by high mass proteins, in an automatic profiling to specific signatures for diagnosis. Using tissue bottom-up strategy on tissue digestion, and mass spectrometry imaging after by shotgun sequencing by nalano-LC-IT-MS in MS/MS mode from washing samples from on tissue digested peptides, several biomarkers were found. RESULTS: A list of specific biomarkers from the ovarian carcinoma regions was obtained and classified as proteins associated with cell proliferation, involved in immune response modulation, signaling to the cytoskeleton, and tumor progression. These specific biomarkers were then validated by immunocytochemistry using Tag-mass technology, cell biology, Western blot, and by PCR (using SKOV-3 ovarian epithelial cancer cells). A link between the immune regulation (innate immunity, tolerance) and virus cause is also discussed. CONCLUSIONS: From the biomarkers identified, proteins involved in immune response modulation and cell proliferation have been pointed out in this study. Two new markers have been identified using such a strategy, that is, fragment C-terminal of the PSME1 (Reg-Alpha) and mucin-9.

Laparoscopic photodynamic diagnosis of ovarian cancer peritoneal micro metastasis: an experimental study.

The goal of this study was to assess the interest of photodynamic diagnosis (PDD) for laparoscopic detection of peritoneal micro metastasis in ovarian carcinoma. Using an experimental animal model, intraperitoneal injection of aminolevulinic acid (ALA) and hexylester of aminolevulinic acid (He-ALA) were compared in order to improve laparoscopic detection of ovarian peritoneal carcinomatosis. Twenty-one 344 Fischer female rats received an intra peritoneal injection of 106 NuTu-19 cells. At day 22, carcinomatosis with micro peritoneal metastasis was obtained. Rats were randomized in three groups concerning intra peritoneal injection before laparoscopic staging: 5-ALA hydrochloride, HE-ALA and sterile water. Using D Light system, laparoscopic peritoneal exploration was performed with white light (WL) first and then with blue light (BL). The main objective was to assess feasibility and sensibility of laparoscopic PDD for nonvisible peritoneal micro metastasis of ovarian cancer. The main parameter was the confirmation of neoplasic status of fluorescent foci by histology. Concerning PDD after intraperitoneal injection of 5-ALA, mean values of lesions seen is higher than without fluorescence (32 vs 20.7; P = 0.01). Using He-ALA, mean values of detected lesions is higher than without fluorescence (42.9 vs 33.6; P < 0.001). Neoplasic status of fluorescent foci was confirmed in 92.8% of cases (39/42). Using 5-ALA, fluorescence of cancerous tissue is significantly higher than that of normal tissue in all the rats (ratio 1.17) (P = 0.01). With He-ALA, intensity of fluorescence is significantly higher in cancerous tissue compared to normal tissue, irrespective of the rat studied (ratio 1.22; P < 0.001).

Fluorescence diagnosis of cervical squamous intraepithelial lesions: A clinical feasability study.

UNLABELLED: The potential of fluorescence diagnosis (FD) is still undeveloped in gynaecology. In order to diagnose and localize squamous intraepithelial lesion (SIL) of the cervix, a new method improving the low specificity of colposcopy, would be useful. OBJECTIVE: The goal of this study was to assess the feasability and safety of colposcopic FD of SIL after topicaly application of methyl aminolevulinate (MAL). MATERIALS AND METHODS: Patients with histologic proved cervical intraepithelial neoplasia (CIN) and planned for loop electrosurgical excision procedure (LEEP) under general anesthesia, were included in a prospective study. Before general anesthesia, a thick layer of MAL (Metvix(®)-160mg/mL cream) was applied on the cervix for 35-150min. Fluorescent colposcopic inspection of the cervix was performed using a rigid 10-mm laparoscope inserted in the vaginal cavity and connected to D-light AF system (Karl Storz Endoskope, Tuttlingen Germany). In order to confirm neoplasic status, fluorescent foci underwent directed punch biopsy(ies). RESULTS: Fourteen patients were included in the study. Colposcopic fluorescence imaging revealed red fluorescent foci in 71.4% of cases (10/14) (neoplasic status of fluorescent foci was confirmed in 80%). Concerning ME-ALA, the mean of application time was 73min (35-150). Two cases presented a false-positive fluorescence and four cases of false-negative fluorescence. For all cases of false-negative fluorescence, application time of MAL was less than 60min. We observed no systemic or local toxicity of MAL application in any of the groups. CONCLUSION: Using topical application of MAL to the cervix, we demonstrated that FD of SIL is feasible. This study justifies the further development of fluorescence imaging that combines classical white light colposcopy with the use of a photosensitizer.

Management of multicentric lesions of the lower genital tract.

OBJECTIVES: To report management and outcome of multicentric lesions of the lower genital tract. To define risk factors of recurrence. STUDY DESIGN: Retrospective review of multicentric dysplasias treated in our colposcopic clinic between 1996 and 2003. Multicentric dysplasias included CIN with VAIN and/or VIN. After primary treatment, follow-up was colposcopic, cytologic and virologic. RESULTS: Forty-four patients presented multicentric lesions out of 998 patients referred for CIN (4.4%). The average age was 36.8 years. Immunologic disorders were present in 20.4%. Ninety-one percent had cervicovaginal or cervicovulvar lesions, only 9% had three sites of genital dysplasia. 53.3% of lesions were concomitant. 79.5% of CIN were high grade, 62.5% of VAIN low grade and 62.5% of VIN high grade. Therapeutic modalities were as follows: conization for CIN (70.4%), CO2 laser for VAIN (33.3%) and surgery for VIN (41.7%). Forty patients were followed and had at least one post-treatment cytologic control; 55% of them had residual disease. Out of the 23 patients with at least two negative controls after treatment, 43.5% presented recurrence. Risk of recurrence was not statistically bound to such parameters as tabagism, immunologic disorder, high grade lesions, non-surgical treatment, and persistence of HPV infection after treatment. CONCLUSION: Multicentric dysplasias are associated with high rate of residual lesion and recurrence. Management of these lesions require long term follow-up.

[Ureteric and bladder involvement of deep pelvic endometriosis. Value of multidisciplinary surgical management]. / Atteinte urétérale et vésicale de l'endométriose pelvienne profonde. Interet de la prise en charge chirurgicale multidisciplinaire.

OBJECTIVES: To evaluate the clinical features, staging by medical imaging, treatment strategy and results of surgical management of deep pelvic endometriosis with bladder and/or ureteric involvement. MATERIALS AND METHODS: Eighteen cases of ureteric and/or bladder deep pelvic endometriosis (DPE) were treated in our centre between 1996 and 2004. Preoperative data (clinical symptoms, MR imaging), intraoperative data (resection and urinary tract diversion procedures, associated procedures on the genital and gastrointestinal tracts), and postoperative data (histological results, complications, anatomical follow-up by imaging and functional assessment) were reviewed. RESULTS: Urinary symptoms were present in 55% of cases, genital symptoms were present in 83% of cases and gastrointestinal symptoms were present in 46% of cases. A combination of gynaecological and gastrointestinal lesions was demonstrated on imaging in 82% of cases. The mean postoperative follow-up was 16 months (range: 6-36 months). Six patients presented anterior vesical endometriosis. In these cases, the sensitivity and specificity of MRI were 100%. Six partial cystectomies were performed. All corresponded to endometriotic lesions on histological examination. No cases of recurrence of vesical endometriosis were observed. Posterior endometriosis with ureteric involvement was observed in 13 patients (including one with vesical endometriosis). The ureteric lesion was asymptomatic in 8 out of 13 cases (61%). The diagnostic sensitivity of MRI was 92% for posterior nodules, identifying 4 lateral parametrial nodules and 8 median retrocervical nodules. Ureterohydronephrosis was observed in 3 patients with lateral parametrial nodules and 8 patients with median retrocervical nodules, and was bilateral for 3 patients, i.e. 14 dilated renal units. Surgical management consisted of 2 ureteric resections with end-to-end anastomosis, 3 psoas bladder reimplantations, and 9 ureterolyses (8 patients). Two out of 13 patients (15%) with ureteric lesions treated by ureterolysis developed recurrence of the ureteric stricture with upper tract dilatation related to recurrence of the lateral nodule. In 14 patients, genital and/or gastrointestinal resections were associated with the urinary tract procedure. CONCLUSION: Preoperative evaluation of all DPE lesions is based on MRI with reconstruction images of the ureter in the presence of urinary tract lesions. Systematic ureteric stenting prior to surgical dissection of the pelvic wall is recommended in patients with posterior nodules and in the case of partial cystectomy for anterior nodules when the ureteric meati are adjacent to the lesion. Ureteric reimplantation onto a psoas hitch bladder must be performed when the DPE lesions are extensive and partly resected or invade the ureteric wall. The frequency of associated lesions (urinary, gynaecological gastrointestinal) justifies a multidisciplinary surgical approach.

SALL4 expression in gestational trophoblastic tumors: a useful tool to distinguish choriocarcinoma from placental site trophoblastic tumor and epithelioid trophoblastic tumor.

SALL4 has important functions in embryonic stem cells. The aim of this study was to investigate SALL4 expression in gestational trophoblastic neoplasia. We hypothesized that it could help to distinguish choriocarcinoma, the presumed most primitive form of gestational trophoblastic neoplasia, from placental site trophoblastic tumor and epithelioid trophoblastic tumor, which would be more differentiated variants. This study included 31 gestational trophoblastic neoplasias: 19 choriocarcinomas, 9 placental site trophoblastic tumors, 1 epithelioid trophoblastic tumor, and 2 mixed tumors comprising a placental site trophoblastic tumor and an epithelioid trophoblastic tumor. Unlike usual markers of gestational trophoblastic neoplasia (p63, human chorionic gonadotrophin and human placental lactogen), SALL4 was expressed in 100% of choriocarcinomas and it was not detected in any placental site trophoblastic tumor and epithelioid trophoblastic tumor. However, the proportion of positive cells varied in a wide range, from 10% to 70%, reflecting the fact that SALL4 was specifically present in mononuclear cells consistent with neoplastic cytotrophoblast. So, SALL4 may be helpful in the differential diagnosis of gestational trophoblastic neoplasias.

In vivo Real-Time Mass Spectrometry for Guided Surgery Application.

Here we describe a new instrument (SpiderMass) designed for in vivo and real-time analysis. In this instrument ion production is performed remotely from the MS instrument and the generated ions are transported in real-time to the MS analyzer. Ion production is promoted by Resonant Infrared Laser Ablation (RIR-LA) based on the highly effective excitation of O-H bonds in water molecules naturally present in most biological samples. The retrieved molecular patterns are specific to the cell phenotypes and benign versus cancer regions of patient biopsies can be easily differentiated. We also demonstrate by analysis of human skin that SpiderMass can be used under in vivo conditions with minimal damage and pain. Furthermore SpiderMass can also be used for real-time drug metabolism and pharmacokinetic (DMPK) analysis or food safety topics. SpiderMass is thus the first MS based system designed for in vivo real-time analysis under minimally invasive conditions.

Implications of a two-step procedure in surgical management of patients with early-stage endometrioid endometrial cancer.

OBJECTIVE: Since European Society for Medical Oncology (ESMO) recommendations and French guidelines, pelvic lymphadenectomy should not be systematically performed for women with early-stage endometrioid endometrial cancer (EEC) preoperatively assessed at presumed low- or intermediate-risk. The aim of our study was to evaluate the change of our surgical practices after ESMO recommendations, and to evaluate the rate and morbidity of second surgical procedure in case of understaging after the first surgery. METHODS: This retrospective single-center study included women with EEC preoperatively assessed at presumed low- or intermediate-risk who had surgery between 2006 and 2013. Two periods were defined the times before and after ESMO recommendations. Demographics characteristics, surgical management, operative morbidity, and rate of understaging were compared. The rate of second surgical procedure required for lymph node resection during the second period and its morbidity were also studied. RESULTS: Sixty-one and sixty-two patients were operated for EEC preoperatively assessed at presumed low-or intermediate-risk before and after ESMO recommendations, respectively. Although immediate pelvic lymphadenectomy was performed more frequently during the first period than the second period (88.5% vs. 19.4%; p<0.001), the rate of postoperative risk-elevating or upstaging were comparable between the two periods (31.1% vs. 27.4%; p=0.71). Among the patients requiring second surgical procedure during the second period (21.0%), 30.8% did not undergo the second surgery due to their comorbidity or old age. For the patients who underwent second surgical procedure, mean operative time of the second procedure was 246.1±117.8 minutes. Third operation was required in 33.3% of them because of postoperative complications. CONCLUSION: Since ESMO recommendations, second surgical procedure for lymph node resection is often required for women with EEC presumed at low- or intermediate-risk. This reoperation is not always performed due to age/comorbidity of the patients, and presents a significant morbidity.

Spectroimmunohistochemistry: a novel form of MALDI mass spectrometry imaging coupled to immunohistochemistry for tracking antibodies.

MALDI mass spectrometry imaging (MALDI-MSI) is currently used for clinical applications, such as biomarker identification, particularly for the study of solid tumors. The ability to map specific compounds that have been determined to be biomarkers and therapeutic targets is relevant for the evaluation of the efficacy of targeted therapies. This article describes a new method called Spectro-ImmunoHistoChemistry (SIHC), which combines the use of specific antibodies against markers and mass spectrometric imaging in the MS/MS mode. SIHC is based on direct primary antibody-antigen recognition, trypsin digestion of the antibody overlaying the markers of interest in the tissue section, and MALDI-MSI of the tryptic peptides generated from the antibody. This approach has both clinical and pharmacological applications. First, it can be used as a cross-validation method to monitor the presence specifically of a marker in a tissue section. Second, SIHC could potentially be used as a novel technology for tracking specific antibodies after in vivo injection for anti-cancer treatments. Additionally, SIHC could enable novel clinical applications of MSI, such as monitoring the efficacy of cytotoxic antibody treatments.

Proteomic analyses of serous and endometrioid epithelial ovarian cancers - cases studies - molecular insights of a possible histological etiology of serous ovarian cancer.

PURPOSE: Epithelial ovarian carcinogenesis may occur de novo on the surface of ovarian mesothelial epithelial cells or from cells originating in other organs. Foreign Müllerian cell intrusion into the ovarian environment has been hypothesized to explain the latter scenario. In this study, MALDI MS profiling technology was used to provide molecular insights regarding these potentially different mechanisms. EXPERIMENTAL DESIGN: Using MALDI MS profiling, the molecular disease signatures were established in their anatomical context. MALDI MS profiling was used on serous and endometrioid cancer biopsies to investigate cases of epithelial ovarian cancer. We then applied bioinformatic methods and identification strategies on the LC-MS/MS analyses of extracts from digested formalin-fixed, paraffin-embedded tissues. Extracts from selected regions (i.e. serous ovarian adenocarcinoma, fallopian tube serous adenocarcinoma, endometrioid ovarian cancer, benign endometrium, and benign ovarian tissues) were performed, and peptide digests were subjected to LC-MS/MS analysis. RESULTS: Comparison of the proteins identified from benign endometrium or three ovarian cancer types (i.e. serous ovarian adenocarcinoma, endometrioid ovarian adenocarcinoma, and serous fallopian tube adenocarcinoma) provided new evidence of a possible correlation between the fallopian tubes and serous ovarian adenocarcinoma. Here, we propose a workflow consisting of the comparison of multiple tissues in their anatomical context in an individual patient. CONCLUSION AND CLINICAL RELEVANCE: The present study provides new insights into the molecular similarities between these two tissues and an assessment of highly specific markers for an individualized patient diagnosis and care.

Fetal microchimerism: benevolence or malevolence for the mother?

For a long time, the conventional view was that the fetus and maternal vascular system are kept separate. In fact there is a two way traffic of cells through the placenta and the transplacental passage of cells is in fact the norm. The fetal cells can persist in a wide range of woman's tissues following a pregnancy or an abortion and she becomes a chimera. Fetal cells have been found in the maternal circulation and they were shown to persist for the entire life in humans, thus demonstrating long-term engraftment and survival capabilities. Microchimerism is a subject of much interest for a number of reasons. Studies of fetal microchimerism during pregnancy may offer explanations for complications of pregnancy, such as preeclampsia, as well as insights into the pathogenesis of autoimmune diseases which usually ameliorate during pregnancy. The impact of the persistence of allogenic cells of fetal origin and of the maternal immunological response to them on the mother's health is still not clear. On the beneficial side, it has been proposed that genetically disparate fetal microchimerism provides protection against some cancers, that fetal microchimerism can afford the mother new mechanisms of protection to some diseases, that fetal microchimerism can enlarge the immunological repertoire of the mother improving her defense against aggressor. Fetal cells are often present at sites of maternal injury and may have an active role in the repair of maternal tissues.

Epithelial-mesenchymal transition in ovarian cancer.

Ovarian cancer is a highly metastatic disease and the leading cause of death from gynecologic malignancy. Hence, and understanding of the molecular changes associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions. The conversion of an epithelial cell to a mesenchymal cell plays a key role both in the embryonic development and cancer invasion and metastasis. Cells undergoing epithelial-mesenchymal transition (EMT) lose their epithelial morphology, reorganize their cytoskeleton and acquire a motile phenotype through the up- and down-regulation of several molecules including tight and adherent junctions proteins and mesenchymal markers. EMT is believed to be governed by signals from the neoplastic microenvironment including a variety of cytokines and growth factors. In ovarian cancer EMT is induced by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and endothelin-1 (ET-1). Alterations in these cellular pathways candidate them as useful target for ovarian cancer treatment.

Breast cancer during pregnancy.

Breast cancer in pregnancy is an uncommon situation but poses dilemmas for patients and their physicians. There is a paucity of prospective studies regarding diagnosis and treatment of breast cancer during pregnancy. Women diagnosed with breast cancer during pregnancy have similar disease characteristics to age-matched controls. Current evidence suggests that diagnosis may be carried out with limitations regarding staging. Surgical treatment may be performed as for non-pregnant women. Radiotherapy and endocrine or antibody treatment should be postponed until after delivery. Chemotherapy is allowed after the first trimester. Physicians should be aggressive in the workup of breast symptoms in the pregnant population to expedite diagnosis and allow multidisciplinary treatment without delay.

Specific MALDI imaging and profiling for biomarker hunting and validation: fragment of the 11S proteasome activator complex, Reg alpha fragment, is a new potential ovary cancer biomarker.

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.

In vivo expression and antitumor activity of p53 gene transfer with naked plasmid DNA in an ovarian cancer xenograft model in nude mice.

INTRODUCTION: Abnormalities in the p53 and p16 tumor suppressor genes are one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 or p16 functions is seen as a particularly promising approach for cancer gene therapy. In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and ovarian tumor regression. AIM: To evaluate the efficiency of intratumoral injection of naked DNA in tumor growth inhibition in an ovarian xenograft model. For that purpose, plasmid vectors encoding wild-type p53 (wt-p53) or p16 alone or in combination were used. METHODS: Nude mice were injected subcutaneously with the human ovarian adenocarcinoma cell line SKOV3. Three weeks after xenograft, tumor-bearing mice were injected twice a week with plasmid vectors carrying WT-p53 and/or WT-p16 cDNA. Empty plasmids and saline buffer were used as control. Tumor growth was monitored to evaluate the inhibition potential with p53 and/or p16 restoration. RESULTS: When compared to the control, intratumoral repeated injections of naked plasmid DNA encoding wt-p53 were inhibiting tumor growth. This inhibition was not observed with p16 and no synergy could be obtained between p53 and p16. p53 expression was restored in 84% of mice injected with plasmid encoding wt-p53. p16 expression was restored in 63% of mice injected with plasmid encoding p16. CONCLUSIONS: In this report we demonstrated that: (i) naked DNA represents an efficient gene transfer in the SKOV3 xenograft model; (ii) restoration of wt-p53 gene allows tumor growth inhibition; and (iii) this inhibition could be correlated with p53 expression as seen in 84% of treated mice after repeated naked DNA injections. These results allow us to envisage naked DNA as a therapeutic adjuvant in ovarian cancer treatment, concomitantly with tumor resection and chemotherapy.