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Despite efforts to improve chemotherapeutic efficacy in epithelial ovarian cancer, outcome for patients with advanced disease has remained unchanged since the introduction of standard carboplatin and paclitaxel. Interest has therefore shifted toward molecularly targeted therapies that interfere with important features of ovarian carcinogenesis, such as angiogenesis. Several angiogenesis inhibitors, targeting vascular endothelial growth factor (VEGF) ligands (bevacizumab, VEGF-Trap) or their receptors (VEGFR-targeted tyrosine kinase inhibitors) have been clinically evaluated. These agents demonstrated efficacy in phase II clinical trials. Results from phase III trials, in which bevacizumab was added to standard frontline chemotherapy, show a modest effect. Although the initial expectations for angiogenesis inhibitors have been tempered, further research is warranted to define their precise place in the treatment of ovarian cancer. This review summarizes the performed and ongoing studies with regard to angiogenesis inhibitors in ovarian cancer, and the available data on biomarkers for response prediction. Preclinical studies evaluating alternative angiogenesis inhibitors will also be discussed., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: Radiotherapy is associated with short-term and long-term morbidity. This study compared toxicity rates among patients with endometrial carcinoma (EC) treated with adjuvant external beam radiation therapy (EBRT) on a small pelvic field (SmPF) in comparison with a standard pelvic field (StPF) or an extended field (EF)., Methods: Patients with EC preoperatively diagnosed with high-grade histological disease (grade 3 endometrioid, papillary serous, clear cell, and mixed tumor type) or cervical involvement were treated with total abdominal hysterectomy, bilateral salpingo-oophorectomy, and lymphadenectomy in the University Medical Center Groningen between 1999 and 2008. Patients who received adjuvant EBRT were included in this study. External beam radiation therapy on SmPF (includes only the central pelvis and proximal vagina) was applied in case of negative lymph nodes after adequate lymphadenectomy (≥10 lymph nodes removed at the bilateral obturator and external iliac nodal stations). In case of positive pelvic lymph nodes or inadequate lymphadenectomy, EBRT on StPF was given. External beam radiation therapy on EF was applied in case of common iliac and/or para-aortic lymph node metastases. Retrospectively, using the Common Terminology Criteria for Adverse Events v3.0, acute toxicity was scored during radiotherapy, whereas late toxicity was scored, from 3 months onward after treatment., Results: Toxicity could be evaluated in 75 patients treated with SmPF (n = 33), StPF (n = 28), and EF EBRT (n = 14). Most patients with late adverse events had also reported toxicity during radiotherapy (71%). The most common late adverse events were gastrointestinal tract related, more frequently present in the StPF group (60.7%) compared to SmPF (33.3%; P = 0.032). In particular, nausea and anorexia were more frequent in the StPF group (32.1%) compared to the SmPF group (3.0%; P = 0.004), as well as ileus (14.3% vs 0%, P = 0.039, respectively)., Conclusions: Treatment with adjuvant EBRT on SmPF results in less gastrointestinal late adverse events compared to treatment with EBRT on StPF in patients with surgically staged EC.

The purpose of the current phase II single-arm clinical trial was to evaluate whether pretreatment with low-dose cyclophosphamide improves immunogenicity of a p53-synthetic long peptide (SLP) vaccine in patients with recurrent ovarian cancer. Patients with ovarian cancer with elevated serum levels of CA-125 after primary treatment were immunized four times with the p53-SLP vaccine. Each immunization was preceded by administration of 300 mg/m2 intravenous cyclophosphamide as a means to affect regulatory T cells (Tregs). Vaccine-induced p53-specific interferon-gamma (IFN-γ)-producing T cells evaluated by IFN-γ ELISPOT were observed in 90% (9/10) and 87.5% (7/8) of evaluable patients after two and four immunizations, respectively. Proliferative p53-specific T cells, observed in 80.0% (8/10) and 62.5% (5/8) of patients, produced both T-helper 1 and T-helper-2 cytokines. Cyclophosphamide induced neither a quantitative reduction of Tregs determined by CD4+ FoxP3+ T cell levels nor a demonstrable qualitative difference in Treg function tested in vitro. Nonetheless, the number of vaccine-induced p53-specific IFN-γ-producing T cells was higher in our study compared to a study in which a similar patient group was treated with p53-SLP monotherapy (p≤0.012). Furthermore, the strong reduction in the number of circulating p53-specific T cells observed previously after four immunizations was currently absent. Stable disease was observed in 20.0% (2/10) of patients, and the remainder of patients (80.0%) showed clinical, biochemical and/or radiographic evidence of progressive disease. The outcome of this phase II trial warrants new studies on the use of low-dose cyclophosphamide to potentiate the immunogenicity of the p53-SLP vaccine or other antitumor vaccines., (Copyright © 2011 UICC.)

Background: Concurrent platinum-based chemoradiation significantly improved survival of advanced stage cervical patients over radiotherapy alone. However, the 5-year overall survival is still only 66%. Presently, no biomarkers are available to select those cervical cancer patients that might benefit from concurrent platinum-based chemoradiation therapy. DNA methylation is a well-established contributor to the regulation of gene transcription, predominantly causing transcriptional silencing. Differences in promoter hypermethylation patterns and subsequent silencing, could contribute to the variety of responses observed in clinical practice. Several clinical trials on various malignancies reported a better response when Decitabine was administered prior to or in combination with standard therapy. This sensitization is thought to be due to re-expression of tumor suppressor genes. However, not all patients might benefit from demethylating agents, since re-expression of oncogenes could render patients more resistant., Aim: In this review, we summarized the present state of art regarding hypermethylated genes and their affected signaling pathways that are associated with outcome after cisplatin treatment, radiotherapy or chemoradiation. Since only few studies were reported in cervical cancer, other malignancies were reviewed as well., Conclusions: From the data presented in this review, we conclude that, in order to select patients that benefit most optimally from demethylating strategies, a comprehensive screening of a large panel of methylation markers, associated with both good as well as poor clinical outcome have to be investigated. Since such panels are not available at this moment, global methylation screening approaches are required to profile such methylated genes. Such methylated gene profiles might be very useful to optimize personalized treatment planning not only in cervical cancer but also in other malignancies.

In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells.

Objective: Lymph node status in early-stage vulvar cancer can be accurately assessed by the sentinel-node (SN) procedure. Molecular techniques, such as DNA-methylation assay, might improve SN assessment. In this study, we selected methylation markers for vulvar cancer and determined if these methylation markers were suitable for lymph node assessment., Methods: We performed methylation specific PCR on DNA isolated from primary tumors, metastatic lymph nodes, and negative lymph nodes from twenty vulvar cancer patients using the following genes: P16INK4a, MGMT, TWIST1, CADM1, TERT, and TFPI2. For P16INK4a and MGMT immunohistochemistry was performed on primary tumors and metastatic lymph nodes in order to explore intratumor heterogeneity in gene expression patterns., Results: TERT was methylated in all vulvar cancers, P16INK4a in 13/20, TFPI2 in 12/20, CADM1 in 11/20, MGMT in 9/20, and TWIST1 in 7/20. A panel of three methylation markers (P16INK4a, TERT and TFPI2) reached a sensitivity of 67% and specificity of 100% for detection of metastatic lymph nodes. Immunohistochemistry showed intratumor heterogeneity for expression of P16INK4a and MGMT in respectively 55% and 45% of primary tumors., Conclusions: Our study shows methylation for one or more methylation markers in all vulvar cancers. Despite a specificity of 100% our panel of three methylation markers had only moderate sensitivity for metastatic lymph node detection, thereby limiting its applicability for lymph node assessment. Intratumor heterogeneity for expression of P16INK4a and MGMT may reflect intratumor heterogeneity for methylation patterns and thereby in general explain the moderate sensitivity of our marker panel for detection of metastases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing., (Copyright © 2011 UICC.)

Background and Purpose: The TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through activation of the death receptors, TRAIL-R1 and TRAIL-R2. Recombinant human (rh) TRAIL and the TRAIL-R1 directed monoclonal antibody mapatumumab are currently clinically evaluated as anticancer agents. The objective of this study was to develop radiopharmaceuticals targeting the TRAIL-R1, suitable for clinical use to help understand and predict clinical efficacy in patients., Experimental Approach: rhTRAIL was radioiodinated with (125) I, and conjugated mapatumumab was radiolabelled with (111) In. The radiopharmaceuticals were characterized, their in vitro stability and death receptor targeting capacities were determined and in vivo biodistribution was studied in nude mice bearing human tumour xenografts with different expression of TRAIL-R1., Key Results: Labelling efficiencies, radiochemical purity, stability and binding properties were optimized for the radioimmunoconjugates. In vivo biodistribution showed rapid renal clearance of [(125) I]rhTRAIL, with highest kidney activity at 15 min and almost no detectable activity after 4 h. Activity rapidly decreased in almost all organs, except for the xenografts. Radiolabelled mapatumumab showed blood clearance between 24 and 168 h and a reduced decrease in radioactivity in the high receptor expression xenograft., Conclusions and Implications: rhTRAIL and mapatumumab can be efficiently radiolabelled. The new radiopharmaceuticals can be used clinically to study pharmacokinetics, biodistribution and tumour targeting, which could support evaluation of the native targeted agents in phase I/II trials., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Objective: The aim of this study is to evaluate the current opinion of the members of the European Society of Gynecological Oncology (ESGO) on the use of neoadjuvant chemotherapy (NACT) in stage IIIC and IV ovarian cancer., Methods: A link to a 21-item questionnaire, with questions about the management of patients with stage IIIC and IV ovarian cancer, was sent 3 times to the ESGO members (N = 1177)., Results: Of the 469 (40%) responding members, 70.2% believe there is sufficient evidence to use NACT followed by interval debulking for the treatment of stage IIIC and IV ovarian cancer. On the basis of a multivariable logistic regression analysis, no relationships between the belief in evidence for NACT and practice type (P = 0.15) or level of experience (P = 0.41) were observed. Only 5.3% of respondents never use NACT, and 30% uses NACT in less than 10% of their patients. Optimal debulking, defined as "no macroscopic residual tumor," is reported in more than 60% of the patients by 20% of the respondents at primary debulking, and by 34.6% of the respondents when interval debulking is performed. Whether a patient can be optimally primarily debulked is impossible to determine preoperatively according to 51.1% of the respondents. Computed tomographic scan (79.4%) and clinical examination (72.5%) are regarded as the most important modalities to predict operability. Diagnostic laparoscopy is used by 46.3% of the respondents. The most important reasons for choosing NACT are bulky disease in the upper abdomen (64.7%) and stage IV disease (58.7%)., Conclusions: Of the responding ESGO members, 70% believe there is sufficient evidence to treat patients with stage IIIC-IV ovarian cancer with NACT, and 30% uses NACT in less than 10% of their patients.

Standard treatment of locally advanced cervical cancer currently consists of concurrent chemoradiation, leading to a 5-year disease-free survival of 66-79%, indicating that there is still ample room for improvement. Characteristic of cervical cancer is the presence of high risk (HR) human papillomavirus (HPV) DNA in more than 99% of these tumors. When the HR HPV genome integrates into the host genome, oncogenic E6 and E7 proteins become constitutively expressed. These oncogenes are also active earlier in the infection cycle and hence are available as therapeutic targets at the preneoplastic stages as well. E7 plays an important role in the early stage of carcinogenesis by stimulating proliferation. HR HPV E6-induced proteasomal degradation of p53 hampers p53 functionality in cell cycle arrest and apoptosis. As p53 plays a key role in the intrinsic apoptotic pathway, current chemoradiation cannot optimally activate this pathway. In this review, we focus on targeted anticancer drugs to eliminate the consequences of HR HPV E6 and E7 activity. Strategies for direct and indirect targeting of HR HPV E6 and E7, including RNA interference, small molecules, proteasome inhibitors, and histone deacetylase inhibitors, are described. In addition, the extrinsic apoptotic pathway as possible alternative therapeutic target for apoptosis induction is reviewed. The rational for implementing recombinant human TRAIL and death receptor agonists and the latest developments on combining these drugs with standard treatment in preclinical settings as well as clinical trials are discussed.

Background: Folate receptor alpha (FR-α) has been identified as a potential target in ovarian cancer for diagnostic and therapeutic purposes, based on its overexpression in serous epithelial ovarian carcinoma. The effect of chemotherapy on FR-α expression may be important in the applicability of FR-α directed agents in the case of residual tumor tissue. The objective of this study was to assess FR-α expression in ovarian carcinoma and to evaluate whether FR-α expression is altered by chemotherapy., Materials & Methods: FR-α expression was analyzed by semi-quantitative scoring of immunohistochemical staining on tissue microarrays (TMAs) from a database containing 361 ovarian cancer tissue samples, of which 210 serous and 116 non-serous carcinoma (35 missing). Serous carcinoma samples included 28 matched samples with tissue from both primary surgery and interval debulking surgery, and 12 matched samples with tissue from both primary surgery and surgery for recurrent disease., Results: FR-α expression was seen in 81.8% of serous ovarian cancers versus 39.9% of non-serous carcinomas (p < 0.001). In matched serous carcinoma samples, no significant change in FR-α expression in vital tumor tissue after chemotherapy was observed (p = 0.1). FR-α expression was not a prognostic marker of progression free survival (p = 0.8) or overall survival (p = 0.7)., Conclusion: FR-α was expressed in the majority of serous ovarian tumors, although >50% of cases showed only weak expression. Chemotherapy did not alter expression rates in remaining vital tumor tissue, indicating that folate-targeted agents may have a place in the treatment for ovarian cancer, before as well as after chemotherapy. Furthermore, FR-α status did not influence survival.

Vaccine-induced p53-specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long-term clinical and immunological effects of the p53-synthetic long peptide (p53-SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53-SLP® vaccine between July 2006 and August 2007. Follow-up information on patients was obtained. Clinical responses to secondary chemotherapy after p53-SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease-specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN-γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53-SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease-specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53-specific proliferative responses were observed in 5/8 patients and IFN-γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53-SLP®. Thus, treatment with the p53-SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53-specific T-cells do survive chemotherapy., (Copyright © 2011 UICC.)

Objective: Insight into the expression of multiple vascular endothelial growth factor (VEGF) family members can support the implementation of anti-angiogenic therapy. This study aimed to assess VEGF family member expression in ovarian cancers and related omental metastases., Methods: Tissue microarrays encompassing 270 primary cancers and 112 paired metastases were immunostained for VEGF-A, VEGF-B, VEGF-C and VEGF-D. Staining intensities were categorized as absent, weak, moderate or strong. Expression was related to clinicopathological characteristics and survival., Results: Immunohistochemical positivity (defined as moderate or strong expression) was observed for VEGF-A in 90%, VEGF-B in 4%, VEGF-C in 41% and VEGF-D in 55% of the primary ovarian cancers. VEGF-A expression correlated with VEGF-C and VEGF-D expression (P < 0.01). Simultaneous positivity for VEGF-A and VEGF-C or VEGF-D was observed in 38% and 54% of the cancers, respectively. Metastases showed positivity for VEGF-A in 78%, VEGF-B in 5%, VEGF-C in 26% and VEGF-D in 45% of cases. VEGF family member expression showed no independent prognostic significance in multivariate survival analysis., Conclusion: VEGF-A, VEGF-C and VEGF-D are widely and often simultaneously expressed in ovarian cancer, which may contribute to bevacizumab resistance. Measuring their expression could support a rational, individualized choice of anti-angiogenic therapy and might be of predictive value. Studies are warranted to determine whether combinatorial analysis of VEGF family member expression can be used to predict anti-angiogenic drug efficacy.

Background: The cornerstone of treatment in early-stage squamous cell carcinoma (SCC) of the vulva is surgery, predominantly consisting of wide local excision with elective uni- or bi-lateral inguinofemoral lymphadenectomy. This strategy is associated with a good prognosis, but also with impressive treatment-related morbidity. The aim of this study was to determine risk factors for the short-term (wound breakdown, infection and lymphocele) and long-term (lymphoedema and cellulitis/erysipelas) complications after groin surgery as part of the treatment of vulvar SCC., Methods: Between January 1988 and June 2009, 164 consecutive patients underwent an inguinofemoral lymphadenectomy as part of their surgical treatment for vulvar SCC at the Department of Gynaecologic Oncology at the Radboud University Nijmegen Medical Centre. The clinical and histopathological data were retrospectively analysed., Results: Multivariate analysis showed that older age, diabetes, 'en bloc' surgery and higher drain production on the last day of drain in situ gave a higher risk of developing short-term complications. Younger age and lymphocele gave higher risk of developing long-term complications. Higher number of lymph nodes dissected seems to protect against developing any long-term complications., Conclusion: Our analysis shows that patient characteristics, extension of surgery and postoperative management influence short- and/or long-term complications after inguinofemoral lymphadenectomy in vulvar SCC patients. Further research of postoperative management is necessary to analyse possibilities to decrease the complication rate of inguinofemoral lymphadenectomy; although the sentinel lymph node procedure appears to be a promising technique, in 50% of the patients an inguinofemoral lymphadenectomy is still indicated.

Objective: Indoleamine 2,3-dioxygenase (IDO) suppresses the function of T-lymphocytes and is involved in immune escape of cancers. Indoleamine 2,3-dioxygenase catalyzes the initial rate-limiting step in the degradation of the essential amino acid tryptophan. In this study, we investigated cancer-induced IDO activity in sera of endometrial, ovarian, and vulvar cancer patients., Methods: Concentrations of tryptophan and kynurenine were determined in pretreatment serum samples of patients with endometrial (n = 41), ovarian (n = 28), and vulvar cancer (n = 40) and compared to 19 healthy female controls. In serum of a subgroup of endometrial (n = 22), ovarian (n = 21), and vulvar (n = 21) cancer patients, tryptophan, kynurenine, and the kynurenine-to-tryptophan ratio (kyn/trp) were determined at different time points: preoperative, at clinical remission, and at the time of diagnosis of recurrent disease. Analyses were performed by an automated online solid-phase extraction-liquid chromatographic-tandem mass spectrometric method. Indoleamine 2,3-dioxygenase activity was estimated by calculating the kyn/trp ratio., Results: Kynurenine concentrations and the kyn/trp ratio were higher in preoperative serum of endometrial, ovarian, and vulvar cancer patients compared to controls (all: P < 0.001). Preoperative serum of ovarian cancer patients contained higher kynurenine concentrations (median, 2.53 μM; interquartile range [IQR], 1.72-4.29 μM) and a higher kyn/trp ratio (median, 39.3 μmol/mmol; IQR, 26.5-61.7 μmol/mmol) compared to serum collected at clinical remission (median, 2.02 μM; IQR, 1.68-2.72 μM, P = 0.035; and median, 29.9 μmol/mmol; IQR, 23.4-38.9 μmol/mmol, P = 0.005, respectively)., Conclusions: Patients with endometrial, ovarian, and vulvar cancer have increased tryptophan degradation compared to controls resulting in higher serum kynurenine concentrations and a higher kyn/trp ratio. Our results suggest that IDO-induced immune escape may play an important role in these gynecologic cancers.

Purpose: Real-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer., Procedures: In ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system., Results: Real-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases., Conclusions: We conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity.

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Purpose: Adoptive T-cell therapy generally fails to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel strategy designed to selectively enhance the tumoricidal activity of T cells by targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface., Experimental Design: We constructed two recombinant fusion proteins, anti-CD3:TRAIL and K12:TRAIL. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed in solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model., Results: When added to T cells, K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively, leading to cell surface accretion of TRAIL. Subsequently, anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold., Conclusions: Targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy., (©2011 AACR.)

Background: Total laparoscopic hysterectomy (TLH) is safe and cost effective in early stage endometrial cancer when compared to total abdominal hysterectomy (TAH). In non-randomised data it is often hypothesised that older and obese patients benefit most from TLH. Aim of this study is to analyse whether data support this assumption to advice patients, clinicians and policy makers., Methods: Data of 283 patients enrolled in a randomised controlled trial comparing TAH versus TLH in early stage endometrial cancer were re-analysed. Randomisation by sequential number generation was done centrally, with stratification by trial centre. Using multivariate analysis, predictors of major complications and conversions to laparotomy were assessed. For the cost effectiveness analysis, subgroups of patients were constructed based on age and body mass index (BMI). For each subgroup, costs per major complication-free patient were estimated, using incremental cost effect ratios (extra costs per additional effect)., Results: Older (odds ratio (OR): 1.05; 1.01-1.09) and obese (OR: 1.05; 1.01-1.10) patients had a higher risk to develop complications, for both groups. In obese (OR: 1.17; 1.09-1.25) patients and patients with a previous laparotomy (OR: 3.45; 1.19-10.04) a higher risk of conversion to laparotomy was found. For patients>70 years of age and patients with a BMI over 35 kg/m2, incremental costs per major complication-free patients were €16 and €54 for TLH compared to TAH, respectively., Conclusion: In general, TLH should be recommended as the standard surgical procedure in early stage endometrial cancer, also in patients>70 years of age. In obese patients with a BMI>35 kg/m2 TLH is not cost effective because of the high conversion rate. A careful consideration of laparoscopic treatment is needed for this subgroup. Surgeon experience level may influence this choice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tumor-targeted fluorescence imaging for cancer diagnosis and treatment is an evolving field of research that is on the verge of clinical implementation. As each tumor has its unique biologic profile, selection of the most promising targets is essential. In this review, we focus on target finding in ovarian cancer, a disease in which fluorescence imaging may be of value in both adequate staging and in improving cytoreductive efforts, and as such may have a beneficial effect on prognosis. Thus far, tumor-targeted imaging for ovarian cancer has been applied only in animal models. For clinical implementation, the five most prominent targets were identified: folate receptor α, vascular endothelial growth factor, epidermal growth factor receptor, chemokine receptor 4, and matrix metalloproteinase. These targets were selected based on expression rates in ovarian cancer, availability of an antibody or substrate aimed at the target approved by the Food and Drug Administration, and the likelihood of translation to human use. The purpose of this review is to present requirements for intraoperative imaging and to discuss possible tumor-specific targets for ovarian cancer, prioritizing for targets with substrates ready for introduction into the clinic.

Immune response characterization at the primary tumor site enables the design of therapeutic vaccination strategies with higher efficacy in epithelial ovarian cancer (EOC). In this study, we related Wilms tumor protein 1 (WT1) overexpression, a well-established immunotherapeutic target, to clinicopathological characteristics, immunological parameters, and survival in primary EOC. WT1 overexpression was evaluated in primary EOC tissue of 270 patients by immunohistochemistry on tissue microarrays (TMAs). Clinicopathological characteristics, follow-up, and data on infiltration of CD8⁺ cytotoxic T lymphocytes (CTLs), FoxP3⁺ regulatory T lymphocytes (Tregs), major histocompatibility complex (MHC) class I, and II molecule expression, were derived from a previously published dataset. WT1 overexpression was defined as positive immunostaining for WT1. WT1 overexpression, present in 56.3% of EOC, was associated with infiltration of Tregs [odds ratio (OR), 2.7; 95% confidence interval (95% CI), 1.6-4.7; P<0.001] and up-regulation of MHC class II (OR, 2.2; 95% CI, 1.2-4.1; P=0.014). Advanced stage (OR, 4.0; 95% CI, 1.9-8.6; P<0.001) and serous histology (OR, 6.7; 95% CI, 3.2-13.6; P<0.001) were independent predictors of WT1 overexpressing EOC. High number of CTL was an independent prognostic factor for progression-free survival (hazard ratio, 0.5; 95% CI, 0.3-0.8; P=0.006) in WT1 overexpressing EOC. As WT1 overexpressing EOC is associated with CTL and Treg infiltration next to MHC class II up-regulation, future clinical trials should evaluate the combination of therapeutic WT1 vaccines with strategies depleting Tregs and/or up-regulating MHC class I, in an attempt to enhance clinical efficacy in EOC patients.

Comparing normal colorectal mucosa and adenomas focusing on deregulated pathways obtains insight into the biological processes of early colorectal carcinogenesis. Publicly available microarray expression data from 26 normal mucosa and 47 adenoma samples were analyzed. Biological pathways enriched in adenomas were identified with Gene Set Enrichment Analysis (GSEA). The analysis revealed 10, 11 and 16 gene sets distinguishing adenomas from normal mucosa according to Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Map Annotator and Pathway Profiler (GenMAPP) and Biocarta databases, respectively. Biological pathways known to be involved in colon carcinogenesis such as cell cycle (P=0.002) and Wnt signaling (P=0.007) were enriched in adenomas. In addition, we found enrichment of novel pathways such as retinoblastoma (Rb) pathway (P=0.002), Src pathway (P=0.004), folate biosynthesis (P=0.019) and Hedgehog signaling (P=0.037) in adenomas. Microarray results for Rb and Src pathway genes were validated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on mRNA isolated from an independent set of adenoma and normal colon samples. A high correlation between microarray data and qRT-PCR expression data was found. The relevance of targeting of identified pathways was shown using the Rb pathway inhibitors roscovitine and PD-0332991 and the Src pathway inhibitor dasatinib. All inhibitors used induced cell growth reduction in adenoma cells. This study shows a bioinformatical and functional approach leading to potentially new options for chemoprevention of colorectal cancer.

Background: Drug resistance is a major problem in ovarian cancer. Triggering apoptosis using death ligands such as tumour necrosis factor-related apoptosis inducing ligand (TRAIL) might overcome chemoresistance., Methods: We investigated whether acquired cisplatin resistance affects sensitivity to recombinant human (rh) TRAIL alone or in combination with cisplatin in an ovarian cancer cell line model consisting of A2780 and its cisplatin-resistant subline CP70., Results: Combining cisplatin and rhTRAIL strongly enhanced apoptosis in both cell lines. CP70 expressed less caspase 8 protein, whereas mRNA levels were similar compared with A2780. Pre-exposure of particularly CP70 to cisplatin resulted in strongly elevated caspase 8 protein and mRNA levels. Caspase 8 mRNA turnover and protein stability in the presence or absence of cisplatin did not differ between both cell lines. Cisplatin-induced caspase 8 protein levels were essential for the rhTRAIL-sensitising effect as demonstrated using caspase 8 small-interfering RNA (siRNA) and caspase-8 overexpressing constructs. Cellular FLICE-inhibitory protein (c-FLIP) and p53 siRNA experiments showed that neither an altered caspase 8/c-FLIP ratio nor a p53-dependent increase in DR5 membrane expression following cisplatin were involved in rhTRAIL sensitisation., Conclusion: Cisplatin enhances rhTRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of caspase 8 protein expression is the key factor of rhTRAIL sensitisation.

Objective: To determine the cost effectiveness of total laparoscopic hysterectomy (TLH) versus total abdominal hysterectomy (TAH) in early stage endometrial cancer alongside a multicenter randomised controlled trial (RCT)., Methods: An economic analysis was conducted in 279 patients (TLH n=185; TAH n=94) with early stage endometrial cancer from a societal perspective, including all relevant costs over a three month time horizon. Health outcomes were expressed in terms of major complication-free rate and in terms of utility based on women's response to the EQ-5D. Comparisons of costs per major complication-free patient gained and costs with utility gain and costs were made, using incremental cost effectiveness ratios., Results: The mean major complication-free rate and median utility scores were comparable between TLH and TAH at three months. TLH is more costly intraoperatively (Δ$1.129) and less costly postoperatively in-hospital (Δ$-1.350) compared to TAH. Incremental costs per major complication-free patient were $-52. Higher cost ($249) were generated while no gains in utility (-0.02) were observed for TLH compared to TAH. Analysing utility at six weeks, incremental costs per additional point on the EQ-5D scale were $1.617., Conclusion: TLH is cost effective compared to TAH, based on major complication-free rate as measure of effect. Along with future cost saving strategies in laparoscopy, TLH is assumed to be cost effective for both effect measures. Therefore and due to comparable safety, TLH should be recommended as a standard-of-care surgical procedure in early endometrial cancer., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer., Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N(0)) and 19 with positive lymph nodes (N(+)), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways., Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N(0), and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N(+) group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N(0) and N(+) tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N(0) (OR: 0.20, 95% CI: 0.06-0.66) and the β-catenin pathway (p120 positivity) to N(+) (OR: 1.79, 95%CI: 1.05-3.05)., Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer., (©2011 AACR.)

Epithelial ovarian cancer, the most lethal neoplasm of the female genital tract, is usually diagnosed at an advanced stage as obvious symptoms are absent at early stages. This disease is believed to originate from malignant transformation of the ovarian surface epithelium or fallopian tube. Histologically, several subtypes are being recognized, with serous histology accounting for the majority of cases. Serous tumors include serous borderline tumors and serous carcinomas. A better understanding of the tumor biology and molecular mechanisms involved in these tumors is needed, as both patient management and prognosis differ substantially. Previous microarray analysis identified SerpinA5, a uPA inhibitor, as key regulator for indolent borderline behavior. As carcinomas are characterized by loss of SerpinA5 mRNA expression, we hypothesized that SerpinA5 protein expression is reduced or lost in carcinomas when compared with borderline tumors. We performed SerpinA5 immunohistochemical staining on 32 serous borderline tumors, 187 primary serous carcinomas and 62 serous omental metastases. Reduced or absent SerpinA5 protein staining was observed in carcinomas when compared with borderline tumors (P<0.001). SerpinA5 protein expression was significantly lowered in the omental metastases (P<0.001) when compared with the matching primary carcinoma. Interestingly, SerpinA5 protein expression was reduced in advanced-stage borderline tumors, often characterized by micropapillary growth and/or microinvasion, when compared with early-stage borderline tumors (P=0.015). In conclusion, SerpinA5 expression is significantly reduced in advanced-stage serous borderline tumors and serous carcinomas when compared with the early-stage counterparts, and reduction of expression is linked to more aggressive features of borderline tumors.

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.

Objective: Disadvantages of the combined sentinel lymph node (SLN) procedure with radiocolloid and blue dye in vulvar cancer are the preoperative injections of radioactive tracer in the vulva, posing a painful burden on the patient. Intraoperative transcutaneous imaging of a peritumorally injected fluorescent tracer may lead to a one-step procedure, while maintaining high sensitivity. Aim of this pilot study was to investigate the applicability of intraoperative fluorescence imaging for SLN detection and transcutaneous lymphatic mapping in vulvar cancer., Methods: Ten patients with early stage squamous cell carcinoma of the vulva underwent the standard SLN procedure. Additionally, a mixture of 1 mL patent blue and 1 mL indocyanin green (ICG; 0.5 mg/mL) was injected immediately prior to surgery, with the patient under anesthesia. Color and fluorescence images and videos of lymph flow were acquired using a custom-made intraoperative fluorescence camera system. The distance between skin and femoral artery was determined on preoperative CT-scan as a measure for subcutaneous adipose tissue., Results: In 10 patients, SLNs were detected in 16 groins (4 unilateral; 6 midline tumors). Transcutaneous lymphatic mapping was possible in five patients (5 of 16 groins), and was limited to lean patients, with a maximal distance between femoral artery and skin of 24 mm, as determined on CT. In total, 29 SLNs were detected by radiocolloid, of which 26 were also detected by fluorescence and 21 were blue., Conclusions: These first clinical results indicate that intraoperative transcutaneous lymphatic mapping using fluorescence is technically feasible in a subgroup of lean vulvar cancer patients., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Lichen sclerosus is considered to be the precursor lesion of vulvar squamous cell carcinoma, of which only 2-5% progress to squamous cell carcinoma. Differentiated vulvar intraepithelial neoplasia (VIN) has been proposed to be the direct precursor lesion, but this is a recently recognized, and a difficult to diagnose, entity, which may easily be mistaken for a benign dermatosis. The aim of this study was to test the hypothesis that of all lesions that have been diagnosed as lichen sclerosus in the past, a part might currently be diagnosed as differentiated VIN, and to identify histopathological differences between lichen sclerosus lesions with and without progression to vulvar squamous cell carcinoma. All lichen sclerosus slides were revised by two expert gynecopathologists and histopathological characteristics were documented. After revision of lichen sclerosus biopsies without progression (n = 61), 58 were reclassified as lichen sclerosus. Revision of lichen sclerosus biopsies with progression yielded concordant diagnoses in 18 of 60 cases (30%). Of 60 lesions, 25 (42%) were reclassified as differentiated VIN. The median time from differentiated VIN to vulvar squamous cell carcinoma was shorter (28 months) than that from lichen sclerosus to vulvar squamous cell carcinoma (84 months) (P < 0.001). Lichen sclerosus that progressed to squamous cell carcinoma, but did not meet the criteria for differentiated VIN, more often showed parakeratosis (P = 0.004), dyskeratosis (P < 0.001), hyperplasia (P = 0.048) and basal cellular atypia (P = 0.009) compared with lichen sclerosus without progression. In conclusion, differentiated VIN diagnosis has been frequently missed and is associated with rapid progression to squamous cell carcinoma. Patients with lichen sclerosus with dyskeratosis and parakeratosis, hyperplasia and/or basal cellular atypia should be kept under close surveillance as these lesions also tend to progress to squamous cell carcinoma.

The aim of this study was to systematically review the prognostic and predictive significance of cell biological markers in cervical cancer patients primarily treated with (chemo)radiation. A PubMed, Embase, and Cochrane literature search was performed. Studies describing a relation between a cell biological marker and survival in ≥50 cervical cancer patients primarily treated with (chemo)radiation were selected. Study quality was assessed, and studies with a quality score of 4 or lower were excluded. Cell biological markers were clustered on biological function, and the prognostic and predictive significance of these markers was described. In total, 42 studies concerning 82 cell biological markers were included in this systematic review. In addition to cyclooxygenase-2 (COX-2) and serum squamous cell carcinoma antigen (SCC-ag) levels, markers associated with poor prognosis were involved in epidermal growth factor receptor (EGFR) signaling (EGFR and C-erbB-2) and in angiogenesis and hypoxia (carbonic anhydrase 9 and hypoxia-inducible factor-1α). Epidermal growth factor receptor and C-erbB-2 were also associated with poor response to (chemo)radiation. In conclusion, EGFR signaling is associated with poor prognosis and response to therapy in cervical cancer patients primarily treated with (chemo)radiation, whereas markers involved in angiogenesis and hypoxia, COX-2, and serum SCC-ag levels are associated with a poor prognosis. Therefore, targeting these pathways in combination with chemoradiation may improve survival in advanced-stage cervical cancer patients., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages., Methods: Lavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohen's Kappa (k)., Results: In lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001)., Conclusions: DNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The objective of this study is to correlate the expression of epidermal growth factor receptor (EGFR) components with clinical behavior of early-stage cervical cancer. Tissue samples of 336 consecutive Federation of International Gynecologists and Obstetricians stage IB-IIA cervical cancer patients all treated primarily by radical surgery were collected. Clinicopathologic and follow-up data were prospectively obtained during standard treatment and follow-up. As representatives for the EGFR pathway, expression of EGFR, pEGFR, PTEN, pAKT, and pERK was assessed by immunohistochemistry on tissue microarrays. Positive immunostaining was observed for EGFR in 32.1%, for pEGFR in 21.0%, for PTEN in 38.3%, for pAKT in 5.3%, and for pERK in 4.3% of tumor samples. Positive EGFR immunostaining was associated with squamous cell carcinoma of the cervix (odds ratio [OR], 7.41; 95% confidence interval [CI], 3.38-16.23, P < .001), negative pEGFR immunostaining with poor differentiation (OR, 0.39; 95% CI, 0.20-0.73, P = .004), and negative PTEN immunostaining with metastatic pelvic lymph nodes (OR, 0.51; 95% CI, 0.30-0.90, P = .019). In multivariate analysis, only pelvic lymph node metastasis (hazard ratio, 6.11; 95% CI, 3.46-10.77, P < .001) and poor differentiation (hazard ratio, 1.91; 95% CI, 1.12-3.26, P = .018) were related to disease-specific survival. In early-stage cervical cancer, loss of PTEN expression is associated with pelvic lymph node metastasis, suggesting PTEN to be one of the tumor suppressor genes affecting pelvic lymph node metastasis. However, expression of EGFR pathway components does not appear to have prognostic impact in surgically treated early-stage cervical cancer., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the risk of cardiovascular events (CVE) in patients with cervical cancer treated with radiotherapy or chemoradiation., Methods and Materials: The incidence of CVE in patients treated between 1989 and 2002 by radiotherapy or chemoradiation was compared with a Dutch reference population. Standardized incidence ratios (SIRs) were calculated for myocardial infarction (MI), angina pectoris (AP), congestive heart failure (CHF), cerebrovascular accident (CVA) separately and for any cardiac event combined (MI, AP, and CHF)., Results: In 277 patients with a median follow-up of 4.5 years (range, 0.1-17 years) and a median survival of 9.2 years, 27 cardiac events occurred. The 5-, 10-, and 15-year actuarial incidence of any cardiac event were 9, 14, and 16%, respectively. For the whole population, the SIR for MI was elevated (2.05, 95% CI: 1.12-3.43). The radiotherapy group (n = 132) was older and had more cardiovascular risk factors than the chemoradiation group (n = 145). The SIR for MI in the radiotherapy group was 2.88 (95% CI: 1.44-5.15) and in the chemoradiation group 1.00 (95% CI: 0.21-7.47). In multivariate analyses, there was no relation between treatment modality and the risk for MI., Conclusions: In this cohort of cervical cancer patients, an increased risk for developing a MI was observed. This increased risk of MI, in combination with the high prevalence of cardiovascular risk factors in cervical cancer patients, urges the need to explore strategies to reduce their risk for cardiovascular morbidity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases. Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema. As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor. In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix. The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion. It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used. The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Objective: Unexplained menorrhagia can be caused by underlying bleeding disorders. The aim of this study was to investigate the current work-up of menorrhagia in routine gynaecological practice, with a special interest in haemostatic evaluation. Secondly, we investigated the outcome of individualized treatment in our centre., Study Design: Retrospective medical chart review of 112 consecutive patients referred with menorrhagia to a general gynaecology clinic of a university teaching hospital in the Netherlands between January 2006 and January 2007. In April 2008 we performed a structured telephone interview evaluating the effectiveness of their therapy., Results: We included 112 patients, whose median age was 42 years. Twenty-nine percent were anaemic (hemoglobin <12.0g/dL). Seventy-one (63%) had unexplained menorrhagia. Only two patients had haemostatic evaluation and neither had von Willebrand's disease. Forty percent (29/71) needed two or more different therapies, 17% (12/71) needed three different therapies and two patients needed a total of seven different therapies. Eight patients underwent hysterectomy, six of them after endometrial ablation. Most patients (80%) were successfully treated medically or surgically and were satisfied with their therapy during follow-up. Eleven patients declined therapy and accepted their heavy periods., Conclusion: Haemostatic evaluation in women with unexplained menorrhagia is uncommon in gynaecological practice in our centre. Although most of the patients were satisfied with their treatment, a significant number required hysterectomy and another important proportion had to accept their menorrhagia. We hypothesize that the identification of haemostatic disorders might improve care for these women., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Quality of surgery is one of the most important determinants of the outcome in ovarian cancer patients. Surgery by a gynaecological oncologist in a specialised, high-volume environment and removal of all visible tumours are associated with a higher likelihood of favourable outcome for patients with advanced-stage ovarian cancer. Population-based studies in Europe however show that a substantial number of patients do not receive optimal surgical care. Less than half of the patients suffering from advanced-stage ovarian cancer are operated by a gynaecological oncologists. Also the proportion of patients operated in a high-volume or specialised hospital is lower than 50%. In a substantial number of patients, minimum standard procedures are not performed and optimal tumor debulking is not achieved. To improve the quality of care, efforts are needed to develop and implement robust evidence-based European guidelines, provide surgical training for gynaecological oncologists and establish comprehensive cancer networks with sufficient resources., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Purpose of Review: The purpose of this review is to outline current controversies in management of early-stage vulvar cancer. The main focus will be on the procedures for assessing the sentinel node and the treatment of those with evidence of metastatic involvement., Recent Findings: Assessment of the sentinel node has recently been introduced into the standard treatment of early-stage squamous cell vulvar cancer. The combination of a radioactive tracer and blue dye is the most accurate technique for sentinel node detection. Preoperative imaging is recommended to rule out gross nodal involvement and ultrasound with fine needle aspiration cytology by an experienced radiologist appears to have the highest sensitivity/specificity for detecting metastases, although large comparative studies are not available. All patients with sentinel node metastases require additional treatment to the groin, independent of the size of metastasis in the sentinel node and currently this involves inguinofemoral lymphadenectomy. Further research is ongoing to investigate the role of radiotherapy instead of lymphadenectomy. The little experience there is of sentinel node biopsy in vulvar melanoma suggests that the procedure is feasible and inclusion criteria should follow those of cutaneous melanoma., Summary: Sentinel node biopsy is safe in treatment of early-stage vulvar cancer. Ongoing studies are investigating the optimal additional treatment for patients with a positive sentinel node in terms of efficacy and morbidity.

Background: Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer., Methods: For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated., Results: The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment., Conclusion: Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.

Background: The standard surgery for early-stage endometrial cancer is total abdominal hysterectomy (TAH) and bilateral salpingo-oophorectomy, which is associated with substantial morbidity. Total laparoscopic hysterectomy (TLH) and bilateral salpingo-oophorectomy is less invasive and is assumed to be associated with lower morbidity, particularly in obese women. This study investigated the complication rate of TLH versus TAH in women with early-stage endometrial cancer., Methods: This randomised trial was done in 21 hospitals in The Netherlands, and 26 gynaecologists with proven sufficient skills in TLH participated. 283 patients with stage I endometrioid adenocarcinoma or complex atypical hyperplasia were randomly allocated (2:1) to the intervention group (TLH, n=187) or control group (TAH, n=96). Randomisation by sequential number generation was done centrally in alternate blocks of six and three participants, with stratification by trial centre. After assignment, the study coordinators, patients, gynaecologists, and members of the panel were not masked to intervention. The primary outcome was major complication rate, assessed by an independent panel. Data were analysed by a modified intention-to-treat analysis, since two patients in both groups were excluded from the main analysis. This trial is registered with the Dutch trial registry, number NTR821., Findings: The proportion of major complications was 14.6% (27 of 185) in the TLH group versus 14.9% (14 of 94) in the TAH group, with a difference of -0.3% (95% CI -9.1 to 8.5; p=0.95). The proportion of patients with an intraoperative major complication (nine of 279 [3.2%]) was lower than the proportion with a postoperative major complication (32 of 279 [11.5%]) and did not differ between TLH (five of 185 [2.7%]) and TAH (four of 94 [4.3%]; p=0.49). The proportion of patients with a minor complication was 13.0% (24 of 185) in the TLH group and 11.7% (11 of 94) in the TAH group (p=0.76). Conversion to laparotomy occurred in 10.8% (20 of 185) of the laparoscopic procedures. TLH was associated with significantly less blood loss (p<0.0001), less use of pain medication (p<0.0001), a shorter hospital stay (p<0.0001), and a faster recovery (p=0.002), but the procedure took longer than TAH (p<0.0001)., Interpretation: Our results showed no evidence of a benefit for TLH over TAH in terms of major complications, but TLH (done by skilled surgeons) was beneficial in terms of a shorter hospital stay, less pain, and quicker resumption of daily activities., Funding: The Dutch Organization for Health Research and Development (ZonMw), programme efficacy., (2010 Elsevier Ltd. All rights reserved.)

Background: Currently, all patients with vulvar cancer with a positive sentinel node undergo inguinofemoral lymphadenectomy, irrespective of the size of sentinel-node metastases. Our study aimed to assess the association between size of sentinel-node metastasis and risk of metastases in non-sentinel nodes, and risk of disease-specific survival in early stage vulvar cancer., Methods: In the GROningen INternational Study on Sentinel nodes in Vulvar cancer (GROINSS-V), sentinel-node detection was done in patients with T1-T2 (<4 cm) squamous-cell vulvar cancer, followed by inguinofemoral lymphadenectomy if metastatic disease was identified in the sentinel node, either by routine examination or pathological ultrastaging. For the present study, sentinel nodes were independently reviewed by two pathologists., Findings: Metastatic disease was identified in one or more sentinel nodes in 135 (33%) of 403 patients, and 115 (85%) of these patients had inguinofemoral lymphadenectomy. The risk of non-sentinel-node metastases was higher when the sentinel node was found to be positive with routine pathology than with ultrastaging (23 of 85 groins vs three of 56 groins, p=0.001). For this study, 723 sentinel nodes in 260 patients (2.8 sentinel nodes per patient) were reviewed. The proportion of patients with non-sentinel-node metastases increased with size of sentinel-node metastasis: one of 24 patients with individual tumour cells had a non-sentinel-node metastasis; two of 19 with metastases 2 mm or smaller; two of 15 with metastases larger than 2 mm to 5 mm; and ten of 21 with metastases larger than 5 mm. Disease-specific survival for patients with sentinel-node metastases larger than 2 mm was lower than for those with sentinel-node metastases 2 mm or smaller (69.5%vs 94.4%, p=0.001)., Interpretation: Our data show that the risk of non-sentinel-node metastases increases with size of sentinel-node metastasis. No size cutoff seems to exist below which chances of non-sentinel-node metastases are close to zero. Therefore, all patients with sentinel-node metastases should have additional groin treatment. The prognosis for patients with sentinel-node metastasis larger than 2 mm is poor, and novel treatment regimens should be explored for these patients., (2010 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate classical MHC class I and nonclassical MHC (human leukocyte antigen-G [HLA-G]) expression in a large cohort of patients with endometrial cancer, to determine the prognostic value of these cell surface markers and their relation with clinicopathological variables. Tissue microarrays containing epithelial endometrial carcinoma tissue from 554 patients were stained for classical and nonclassical MHC class I using the following monoclonal antibodies: 4H84 (anti-HLA-G), beta2-m (anti-beta-2-microglobulin) and HC-10 (MHC class I antigen heavy chain). Expression data were linked to known clinicopathological characteristics and survival. HLA-G upregulation and MHC class I downregulation in neoplastic cells was observed in 40% and 48%, respectively. Nonendometrioid tumor type, advanced stage disease (FIGO stage > or = II) and poorly or undifferentiated tumors were associated with MHC class I downregulation. Absence of HLA-G expression was independently associated with MHC class I downregulation. In univariate analysis, MHC class I downregulation was a predictor of worse disease-specific survival. Prognostic unfavorable tumor characteristics were correlated with downregulation of MHC class I expression in endometrial cancer cells. Furthermore, downregulated MHC class I has a negative impact on disease-specific survival, observed in a large cohort of patients with endometrial cancer. As there seems to be a relation between classical and nonclassical MHC class I molecules (HLA-G), further research is warranted to unravel this regulatory mechanism.

Objective: Death ligand FasL, its agonistic receptor Fas, tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its agonistic death receptors DR4 and DR5 are implied in carcinogenesis, tumor immune surveillance and response to chemotherapy. TRAIL receptor agonists are evaluated as anti-cancer agents. This study aimed to relate expression of death ligands/receptors and downstream initiator caspase 8 and its anti-apoptotic homologue FLICE like inhibitory protein (c-FLIP) in ovarian cancers to chemotherapy response and survival., Methods: Fas, FasL, TRAIL, DR4, DR5, caspase 8 and c-FLIP were determined immunohistochemically on a tissue microarray containing 382 ovarian cancers. Protein expression profiles were correlated with clinicopathologic variables, chemotherapy response and survival., Results: Most tumors expressed DR4, DR5, caspase 8 and c-FLIP. High c-FLIP expression was associated with expression of caspase 8 and both TRAIL receptors. TRAIL and Fas were associated with low tumor grade and better progression-free survival (HR 0.63, p=.018 and HR 0.54, p=.012), respectively, and Fas with disease-specific survival (HR 0.49, p=0.009) in univariate analysis., Conclusions: Fas and TRAIL loss is associated with dedifferentiation and worse prognosis. Expression of DR4, DR5, caspase 8 and c-FLIP by most ovarian cancers does not correlate with survival. High c-FLIP expression should be taken into account for death receptor targeted therapies.

Currently, standard treatment for early-stage vulvar cancer typically includes wide local excision of the primary tumor and inguinofemoral lymphadenectomy. The morbidity of this treatment is high. The sentinel lymph node (SLN) procedure provides us with a technique for determining the status of the regional lymph nodes with less treatment-related morbidity. Recently, a large multicenter observational study provided level 3 evidence indicating that it appears safe to omit inguinofemoral lymphadenectomy in case of a negative SLN. This review focuses on the different aspects of the SLN procedure in vulvar cancer.

The prognosis of epithelial ovarian cancer (EOC), the primary cause of death from gynaecological malignancies, has only modestly improved over the last decades. Immunotherapeutic treatment using a cocktail of antigens has been proposed as a "universal" vaccine strategy. We determined the expression of tumor antigens in the context of MHC class I expression in 270 primary tumor samples using tissue microarray. Expression of tumor antigens p53, SP17, survivin, WT1, and NY-ESO-1 was observed in 120 (48.0%), 173 (68.9%), 208 (90.0%), 129 (56.3%), and 27 (11.0%) of 270 tumor specimens, respectively. In 93.2% of EOC, at least one of the investigated tumor antigens was (over)expressed. Expression of MHC class I was observed in 78.1% of EOC. In 3 out 4 primary tumors, (over)expression of a tumor antigen combined with MHC class I was observed. These results indicate that a multiepitope vaccine, comprising these antigens, could serve as a universal therapeutic vaccine for the vast majority of ovarian cancer patients.

Objectives: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL)., Methods: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings., Results: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-beta(2) at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05)., Conclusion: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.

Purpose: Activation of the epidermal growth factor receptor (EGFR) signaling pathway has been reported to induce resistance to (chemo)radiation in cancers, such as head and neck cancer, whereas EGFR-targeted agents in combination with (chemo)radiation seem to improve treatment efficacy. The aim of this study was to determine the relation between proteins involved in the EGFR pathway and response to (chemo)radiation and survival in a large, well-documented series of cervical cancer patients., Experimental Design: Pretreatment tissue samples of 375 consecutive International Federation of Gynecologists and Obstetricians stage Ib to IVa cervical cancer patients treated with (chemo)radiation between January 1980 and December 2006 were collected. Clinicopathologic and follow-up data were prospectively obtained during standard treatment and follow-up. Protein expression of EGFR, phosphorylated EGFR (pEGFR), PTEN, phosphorylated AKT, and phosphorylated extracellular signal-regulated kinase (pERK) was assessed by immunohistochemistry on tissue microarrays., Results: EGFR staining was present in 35.3%, pEGFR in 19.7%, PTEN in 34.1%, phosphorylated AKT in 4.1%, and pERK in 29.2% of tumors. pEGFR staining was related to PTEN (P = 0.001) and pERK staining (P = 0.004). EGFR staining was inversely related to PTEN (P = 0.011). In multivariate analysis, membranous staining of EGFR [hazard ratio (HR), 1.84; 95% confidence interval (95% CI), 1.20-2.82; P = 0.005] and cytoplasmic staining of pEGFR (HR, 1.71; 95% CI, 1.11-2.66; P = 0.016) were independent predictors of poor response to (chemo)radiation. Membranous EGFR staining also was an independent prognostic factor for poor disease-specific survival (HR, 1.54; 95% CI, 1.09-2.17; P = 0.014)., Conclusions: EGFR and pEGFR immunostainings are frequently observed and independently associated with poor response to therapy and disease-specific survival in cervical cancer patients primarily treated by (chemo)radiation. Our data present the EGFR pathway as a promising therapeutic target in already ongoing clinical trials.

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53-SLP) vaccine, twenty patients with recurrent elevation of CA-125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53-SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no > or = grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN-gamma producing p53-specific T-cell responses were induced in all patients who received all 4 immunizations as measured by IFN-gamma ELISPOT. An IFN-gamma secretion assay showed that vaccine-induced p53-specific T-cells were CD4(+), produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53-specific response. P53-specific T-cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53-specific T-cells. As best clinical response, stable disease evaluated by CA-125 levels and CT-scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine-induced immunity. This study shows that the p53-SLP vaccine is safe, well tolerated and induces p53-specific T-cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper-1 polarization and clinical efficacy., ((c) 2009 UICC.)