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Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE 2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (s Il1ra , Nfil3 , Arg1 , Serpinb2 ) owing to upregulation of IL-10-STAT3 and PGE 2 -C/EBPβ signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Child abuse has devastating and long-lasting consequences, considerably increasing the lifetime risk of negative mental health outcomes such as depression and suicide. Yet the neurobiological processes underlying this heightened vulnerability remain poorly understood. The authors investigated the hypothesis that epigenetic, transcriptomic, and cellular adaptations may occur in the anterior cingulate cortex as a function of child abuse., Method: Postmortem brain samples from human subjects (N=78) and from a rodent model of the impact of early-life environment (N=24) were analyzed. The human samples were from depressed individuals who died by suicide, with (N=27) or without (N=25) a history of severe child abuse, as well as from psychiatrically healthy control subjects (N=26). Genome-wide DNA methylation and gene expression were investigated using reduced representation bisulfite sequencing and RNA sequencing, respectively. Cell type-specific validation of differentially methylated loci was performed after fluorescence-activated cell sorting of oligodendrocyte and neuronal nuclei. Differential gene expression was validated using NanoString technology. Finally, oligodendrocytes and myelinated axons were analyzed using stereology and coherent anti-Stokes Raman scattering microscopy., Results: A history of child abuse was associated with cell type-specific changes in DNA methylation of oligodendrocyte genes and a global impairment of the myelin-related transcriptional program. These effects were absent in the depressed suicide completers with no history of child abuse, and they were strongly correlated with myelin gene expression changes observed in the animal model. Furthermore, a selective and significant reduction in the thickness of myelin sheaths around small-diameter axons was observed in individuals with history of child abuse., Conclusions: The results suggest that child abuse, in part through epigenetic reprogramming of oligodendrocytes, may lastingly disrupt cortical myelination, a fundamental feature of cerebral connectivity.

Background: Checkpoint blockade with ipilimumab provides long-term survival to a significant proportion of patients with metastatic melanoma. New approaches to increase survival and to predict which patients will benefit from treatment are needed. This phase II trial combined ipilimumab with carboplatin/paclitaxel (CP) to assess its safety, efficacy, and to search for peripheral and tumor-based predictive biomarkers., Methods: Thirty patients with untreated unresectable/metastatic melanoma were treated with ipilimumab and CP. Adverse events (AEs) were monitored and response to treatment was evaluated. Tumor tissue and peripheral blood were collected at specified time points to characterize tumor immune markers by immunohistochemistry and systemic immune activity by multiplex assays and flow cytometry., Results: Eighty three percent of patients received all 5 cycles of CP and 93% completed ipilimumab induction. Serious AEs occurred in 13% of patients, and no treatment-related deaths were observed. Best Overall Response Rate (BORR) and Disease Control Rate (DCR) were 27 and 57%, respectively. Median overall survival was 16.2 months. Response to treatment was positively correlated with a higher tumor CD3 + infiltrate (immune score) at baseline. NRAS and BRAF mutations were less frequent in patients who experienced clinical benefit. Assessment of peripheral blood revealed that non-responders had elevated baseline levels of CXCL8 and CCL4, and a higher proportion of circulating late differentiated B cells. Pre-existing high levels of chemokines (CCL3, CCL4 and CXCL8) and advanced B cell differentiation were strongly associated with worse patient overall survival. Elevated proportions of circulating CD8 + /PD-1 + T cells during treatment were associated with worse survival., Conclusions: The combination of ipilimumab and CP was well tolerated and revealed novel characteristics associated with patients likely to benefit from treatment. A pre-existing systemic inflammatory state characterized by elevation of selected chemokines and advanced B cell differentiation, was strongly associated with poor patient outcomes, revealing potential predictive circulating biomarkers., Trial Registration: Clinicaltrials.gov , NCT01676649 , registered on August 29, 2012.

Male gender is independently and significantly associated with poor prognosis in melanoma of all clinical stages. The biological underpinnings of this sex difference remain largely unknown, but we hypothesized that gene expression from gonosomes (sex chromosomes) might play an important role. We demonstrate that loss of the inactivated X chromosome in melanomas arising in females is strongly associated with poor distant metastasis-free survival, suggesting a dosage benefit from two X chromosomes. The gonosomal protein phosphatase 2 regulatory subunit B, beta (PPP2R3B) gene is located on the pseudoautosomal region (PAR) of the X chromosome in females and the Y chromosome in males. We observed that, despite its location on the PAR that predicts equal dosage across genders, PPP2R3B expression was lower in males than in females and was independently correlated with poor clinical outcome. PPP2R3B codes for the PR70 protein, a regulatory substrate-recognizing subunit of protein phosphatase 2A. PR70 decreased melanoma growth by negatively interfering with DNA replication and cell cycle progression through its role in stabilizing the cell division cycle 6 (CDC6)-chromatin licensing and DNA replication factor 1 (CDT1) interaction, which delays the firing of origins of DNA replication. Hence, PR70 functionally behaves as an X-linked tumor suppressor gene., (Copyright © 2016, American Association for the Advancement of Science.)

The majority of endometrial carcinomas are classified as Type I endometrioid endometrial carcinomas (EECs) and have a good prognosis. Type II non-endometrioid endometrial carcinomas (NEECs) have a significant worse outcome. Yet, 20 % of the EECs are associated with an unexplained poor outcome. The aim of this study was to determine if L1CAM expression, a recently reported biomarker for aggressive tumor behavior in endometrial carcinoma, was associated with clinicopathological features of EECs. A total of 103 patients diagnosed as EEC at the Radboud University Medical Centre, based on the pathology report were selected. L1CAM status of these tumors was determined, and histologic slides were reviewed by two expert pathologists. L1CAM-positivity was observed in 17 % (18/103). Review of the diagnostic slides revealed that 11 out of these 18 L1CAM-positive tumors (61 %) contained a serous- or mixed carcinoma component that was not initially mentioned in the pathology report. L1CAM-expression was associated with advanced age, poor tumor grade, and lymphovascular space invasion. A worse five year progression free survival rate was observed for patients with L1CAM-positive tumors (55.6 % for the L1CAM-positive group, compared to 83.3 % for the L1CAM-negative group P = 0.01). L1CAM expression carries prognostic value for histologically classified EEC and supports the identification of tumors with a NEEC component., Competing Interests: Compliance with Ethical Standards Funding None of the authors have received any kind funding or disclosures to be mentioned.

Intrinsic and acquired resistance of metastatic melanoma to (V600E/K)BRAF and/or MEK inhibitors, which is often caused by activation of the PI3K/AKT survival pathway, represents a major clinical challenge. Given that p53 is capable of antagonising PI3K/AKT activation we hypothesised that pharmacological restoration of p53 activity may increase the sensitivity of BRAF-mutant melanoma to MAPK-targeted therapy and eventually delay and/or prevent acquisition of drug resistance. To test this possibility we exposed a panel of vemurafenib-sensitive and resistant (innate and acquired) (V600E/K)BRAF melanomas to a (V600E/K)BRAF inhibitor (vemurafenib) alone or in combination with a direct p53 activator (PRIMA-1(Met)/APR-246). Strikingly, PRIMA-1(Met) synergised with vemurafenib to induce apoptosis and suppress proliferation of (V600E/K)BRAF melanoma cells in vitro and to inhibit tumour growth in vivo. Importantly, this drug combination decreased the viability of both vemurafenib-sensitive and resistant melanoma cells irrespectively of the TP53 status. Notably, p53 reactivation was invariably accompanied by PI3K/AKT pathway inhibition, the activity of which was found as a dominant resistance mechanism to BRAF inhibition in our lines. From all various combinatorial modalities tested, targeting the MAPK and PI3K signalling pathways through p53 reactivation or not, the PRIMA-1(Met)/vemurafenib combination was the most cytotoxic. We conclude that PRIMA-1(Met) through its ability to directly reactivate p53 regardless of the mechanism causing its deactivation, and thereby dampen PI3K signalling, sensitises (V600E/K)BRAF-positive melanoma to BRAF inhibitors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Gene expression analysis in the tumor microenvironment using archived clinical samples is challenging because of formalin fixation, RNA degradation, and limiting sample volume. NanoString gene expression profiling is a RNA-DNA hybrid capture technology that does not require PCR and can accurately quantify the expression of to 800 transcripts in a single reaction. The technology requires 50-100 ng of RNA, which can be degraded (, Editor: is this correct?) to a 200 bp fragment size. In contrast to amplification technologies, nanoString counts the actual numbers of transcripts that are captured with transcript-specific and fluorescently-barcoded probes. This chapter describes protocols for RNA extraction, quantification, mRNA and miRNA profiling and data analysis.

Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of β-catenin independent of the PI3K-AKT-GSK3β axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent β-catenin transcriptional modulation in vitro, cooperates with NRAS(Q61K) to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization. The CAV1-β-catenin axis is mediated by a feedback loop in which β-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of β-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.

Background: We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3' untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA-mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs., Methods: The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155-TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3'UTR and miR-155 were analysed by RT-qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues., Results: The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the 'match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the 'match' group, whereas it positively correlated with protein but not with miR-155 in the 'mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival., Conclusion: Polymorphisms in 3'UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Cutaneous melanoma is associated with strong prognostic phenotypic features, such as gender, Breslow's thickness and ulceration, although the biological significance of these variables is largely unknown. It is likely that these features are surrogates of important biological events rather than directly promoting cutaneous melanoma progression. In this article, we address the molecular mechanisms that drive these phenotypic changes. Furthermore, we present a comprehensive overview of recurrent genetic abnormalities, both germline and somatic, in relation to cutaneous melanoma subtypes, ultraviolet exposure and anatomical localization, as well as pre-existing and targeted therapy-induced mutations that may contribute to resistance. The increasing knowledge of critically important oncogenes and tumor-suppressor genes is promoting a transition in melanoma diagnosis, in which single-gene testing will be replaced by multiplex and multidimensional analyses that combine classical histopathological characteristics with the molecular profile for the prognostication and selection of melanoma therapy., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Introduction and Hypothesis: The rs1800255, COL3A1 2209 G>A polymorphism in the alpha 1 chain of collagen type III has been associated with an increased risk of pelvic organ prolapse (POP). In one of our previous studies however, polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) misdiagnosed rs1800255, COL3A1 2209 G>A in 6 % of cases. The high-resolution melting (HRM) analysis on the contrary obtained a 100 % accordance for this specific polymorphism and was used in the present study to validate this risk factor for POP., Methods: In this case-control study, women with and without symptoms of POP were included and compared. DNA was extracted from blood samples. HRM analysis was used to assess for the presence of the homozygous rs1800255. Groups were compared using the Pearson chi-square, Mann-Whitney, and t tests. The discrepancy between HRM and PCR-RFLP results was investigated using PCR-RFLP results available from our previous study., Results: The study included 354 women: 272 patients with POP and 82 controls; 18 (7 %) cases versus 3 (4 %) controls had a homozygous rs1800255, COL3A1 2209 G>A polymorphism (odds ratio 1.9, 95 % confidence interval 0.5-6.9, compared to the wild type), and thus no association between POP and the homozygous polymorphism could be demonstrated. A discrepancy between HRM and PCR-RFLP results was found in 8 % of the samples., Conclusions: The previously found statistically significant association between the rs1800255, COL3A1 2209 G>A polymorphism as measured with PCR-RFLP and POP could no longer be demonstrated. This raises concerns regarding the results of other association studies using PCR-RFLP.

MicroRNAs (miRNAs) in circulation have received an increasing amount of interest as potential minimal invasive diagnostic tools in oncology. Several diagnostic, prognostic and predictive signatures have been proposed for a variety of cancers at different stages of disease, but these have not been subjected to a critical review regarding their validity: reproducible identification in comparable studies and/or with different platforms of miRNA detection. In this review, we will critically address the results of circulating miRNA research in oncology that have been published between January 2008 and June 2013 (5.5 years), and discuss pre-analytical challenges, technological pitfalls and limitations that may contribute to the non-reproducibility of circulating miRNA research., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Objective: Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive., Design: Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays., Results: In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib., Conclusions: Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

Background: Despite the excellent prognosis of Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage I, type I endometrial cancers, a substantial number of patients experience recurrence and die from this disease. We analyzed the value of immunohistochemical L1CAM determination to predict clinical outcome., Methods: We conducted a retrospective multicenter cohort study to determine expression of L1CAM by immunohistochemistry in 1021 endometrial cancer specimens. The Kaplan-Meier method and Cox proportional hazard model were applied for survival and multivariable analyses. A machine-learning approach was used to validate variables for predicting recurrence and death., Results: Of 1021 included cancers, 17.7% were rated L1CAM-positive. Of these L1CAM-positive cancers, 51.4% recurred during follow-up compared with 2.9% L1CAM-negative cancers. Patients bearing L1CAM-positive cancers had poorer disease-free and overall survival (two-sided Log-rank P < .001). Multivariable analyses revealed an increase in the likelihood of recurrence (hazard ratio [HR] = 16.33; 95% confidence interval [CI] = 10.55 to 25.28) and death (HR = 15.01; 95% CI = 9.28 to 24.26). In the L1CAM-negative cancers FIGO stage I subdivision, grading and risk assessment were irrelevant for predicting disease-free and overall survival. The prognostic relevance of these parameters was related strictly to L1CAM positivity. A classification and regression decision tree (CRT)identified L1CAM as the best variable for predicting recurrence (sensitivity = 0.74; specificity = 0.91) and death (sensitivity = 0.77; specificity = 0.89)., Conclusions: To our knowledge, L1CAM has been shown to be the best-ever published prognostic factor in FIGO stage I, type I endometrial cancers and shows clear superiority over the standardly used multifactor risk score. L1CAM expression in type I cancers indicates the need for adjuvant treatment. This adhesion molecule might serve as a treatment target for the fully humanized anti-L1CAM antibody currently under development for clinical use.

Purpose: We identified microRNA driven mechanisms in clear cell renal cell carcinoma associated with the tumor response to the multitargeted receptor tyrosine kinase inhibitor sunitinib., Materials and Methods: We performed screening genome-wide microRNA real-time quantitative polymerase chain reaction on 20 freshly frozen clear cell renal cell carcinoma tissue samples of patients who received sunitinib as first line targeted therapy. Nine patients with progressive disease within 6 months after initiating therapy were considered poor responders and 11 with at least 1-year progression-free survival were considered good responders. We studied microRNA-141 function in vitro by stable up-regulation of microRNA-141, quantification of target gene expression and cell viability in normoxic and hypoxic conditions. Relative expression in clinical and cell line samples was determined by real-time quantitative polymerase chain reaction. Localization of microRNA-141 and its targets was assessed by microRNA in situ hybridization and immunohistochemistry. Hypoxia induced cytotoxicity was assessed by a luminescence adenosine triphosphate detection assay., Results: Compared to good responders, microRNA-141 was significantly down-regulated in tumors of poor responders to sunitinib. This seemed spatially linked to epithelial-to-mesenchymal transition in vivo. Reintroduction of microRNA-141 in vitro reversed epithelial-to-mesenchymal transition and decreased cell viability in hypoxic conditions., Conclusions: In our study microRNA-141 down-regulation driven epithelial-to-mesenchymal transition in clear cell renal cell carcinoma was linked to an unfavorable response to sunitinib therapy. Reintroduction of microRNA-141 in vitro led to epithelial-to-mesenchymal transition reversal and increased sensibility to a hypoxic environment. Future experiments should be done in vivo to see whether microRNA-141 driven reversal of epithelial-to-mesenchymal transition could affect the efficacy of sunitinib treatment., (Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression. Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients., Methods: TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load. TYRP1/S100B ratios were calculated and median was used as cutoff value. TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion., Results: A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries. Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration. Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups., Conclusion: High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.

Introduction and Hypothesis: There is growing evidence that pelvic organ prolapse (POP) is at least partly caused by underlying hereditary risk factors. The aim of our study was to provide a systematic literature review and meta-analysis of clinical studies on family history of POP as a risk factor for POP in individual women., Methods: The databases PubMed and Embase were searched. Clinical studies reporting on family history of POP in relation to POP in individual women were included., Results: Sixteen studies were included, of which eight enabled us to calculate a pooled odds ratio (OR). The pooled OR of POP in case of a positive family history of POP was 2.58 (95 % confidence interval 2.12-3.15)., Conclusions: Women with POP are substantially more likely to have family members with the same condition compared to women without POP. This strengthens the hypothesis that genetic predisposition plays an important role in the development of POP.

Loss of E-cadherin expression in melanoma correlates with increased tumor thickness and reduced disease-free survival. The molecular mechanisms underpinning its differential expression in melanoma tissue remain elusive. MicroRNAs (miRNAs) have been implicated in tumor progression and regulation of E-cadherin expression. Here, we demonstrate a significant correlation between tumor thickness and loss of expression of miR-200a, miR-200c, and miR-203 in a series of 23 frozen primary melanomas, where it was confirmed in two subsequent validation series (series 1: six nevi, 15 primary melanomas, and 16 metastases; series 2: 11 matched pairs of primary melanomas and metastases). Decreased levels of miR-200a, miR-200c, and miR-203 correlated with increasing thickness in the combined validation series (P = 0.024, 0.033, and 0.031, respectively). In addition, progressive loss of miR-200a expression with disease progression was observed in series 1 (P < 0.001) and in series 2 (P = 0.029). MiR-200 in situ hybridization and E-cadherin immunohistochemistry demonstrated reduced expression of both at the deep invasive margin of the tumor. Furthermore, a functional validation study using an anti-miR200 strategy demonstrated that loss of miR-200 expression in melanoma cell lines reduced E-cadherin expression. Collectively, our data point towards an important role for miR-200 and miR203 expression in regulating E-cadherin during melanoma progression.

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a progression marker of a variety of cancers, including melanoma, and is a marker for mesenchymal stem cells. ALCAM expression triggers matrix metalloproteinase activity and correlates with the transition between superficial melanoma growth and deep dermal invasion in vivo. We previously showed that manipulating ALCAM functionality could both decrease and increase melanoma invasion, depending on the manner by which ALCAM function was altered. How ALCAM exerts these opposing invasive phenotypes remained elusive. In the present study, we analyzed differences in melanoma cell gene expression in two- and three-dimensional cultures as function of ALCAM-mediated adhesion. We identified a cluster of genes highly responsive to ALCAM functionality and relevant for melanoma invasion. This cluster is characterized by known invasion-related genes similar to L1 neuronal cell adhesion molecule and showed a remarkable induction of several innate immune genes. Unexpectedly, we identified major variations in the expression of genes related to an immunological response when modulating ALCAM function, including complement factors C1r and C1s. The expression and function of these proteinases were confirmed in protein assays and in vivo. Together, our results demonstrate a link between ALCAM functionality and the immune transcriptome, and support the assumption that ALCAM-ALCAM interactions could function as a cell signaling complex to promote melanoma tumor invasion.

Introduction and Hypothesis: Pelvic organ prolapse (POP) and other disorders, such as varicose veins and joint hypermobility, have been associated with changes in collagen strength and metabolism. We hypothesized that these various disorders were more prevalent in both POP patients and their family members., Methods: In this study, the prevalence of various collagen-associated disorders, including POP, was compared between POP patients (n = 110) and control patients (n = 100) and their first and second degree family members., Results: POP patients reported a higher prevalence of varicose veins, joint hypermobility and rectal prolapse and were more likely to have family members with POP as compared to the control group (p < 0.01). In contrast, the family members of the POP group did not report a higher prevalence of collagen-associated disorders compared to the family members of the control group (p = 0.82)., Conclusions: POP and other collagen-associated disorders may have a common aetiology, originating at the molecular level of the collagens.

Objective: The pathogenesis of serous ovarian carcinoma (SOC) is still unknown. Recently, endometrial intraepithelial carcinoma (EIC) was proposed to be the precursor lesion of SOC. This study examines the model of EIC as precursor for SOC., Methods: Cases of SOC with a noninvasive or superficially invasive serous lesion, a hyperplastic lesion with/without atypia, or EIC in the endometrium were selected for inclusion in this study. Tissue sections from both ovaries, the fallopian tubes, and the uterus were extensively reviewed by an expert gynecopathologist. For both EIC and SOC, immunostaining for p53, Ki-67, estrogen receptor, and progesterone receptor; TP53 mutation analysis; and in situ ploidy analysis were performed., Results: Nine cases of SOC with concurrent EIC in the endometrium were identified. Immunostaining for p53, Ki-67, estrogen receptor, and progesterone receptor revealed almost identical expression patterns and similar intensities in each pair of EIC and coincident SOC. Identical TP53 mutations were found in SOC and coinciding EIC in 33% of the cases, suggesting a clonal origin. DNA ploidy analysis, as a marker for neoplastic progression, demonstrated an increased number of aneuploid nuclei in SOC compared to their corresponding EIC (P = 0.039). In addition, the mean amount of DNA per nucleus in SOC was higher (ie, more aneuploid) compared to EIC (P = 0.039)., Conclusion: This study provides a first indication of EIC as possible precursor lesion for SOC. This finding could have major clinical implications for future ovarian cancer management and underscores EIC as a possible target for early SOC detection and prevention.

Objective: Taking a biopsy is a standard procedure to make the correct diagnosis in patients with suspicious premalignant vulvar lesions. The use of a less invasive diagnostic tool as triage instrument to determine whether biopsy is necessary may improve patient comfort especially in patients with chronic vulvar disorders that may warrant consecutive biopsies. This study was conducted to investigate whether vulvar brush cytology is feasible and may be used to detect (pre)malignant vulvar lesions., Methods: A pilot study was performed with patients having clinically normal vulvar skin, lichen sclerosus (LS), usual or differentiated vulvar intraepithelial neoplasia or squamous cell carcinoma. A total of 65 smears were taken with the use of a vulvar brush and biopsies were performed for histopathological analysis., Results: Out of 65 smears, 17 (26%) were discarded because of poor cellularity. A total of 28 of 29 (97%) smears with a histological proven (pre)malignancy had a smear classified as 'suspicious' or 'uncertain'. Cytology classified 11 smears as 'non-suspicious', of which 10 (91%) were indeed normal skin or LS. The accuracy, based on the presence of a lesion, for (pre)malignant lesions with the use of the brush showed a sensitivity of 97% and a negative predictive value of 88%., Conclusion: Vulvar brush cytology is feasible and may be a first step in the development of a triage instrument to determine whether subsequent biopsy of a clinically (pre)malignant lesion is necessary.

The FoxP3 (forkhead box P3) gene is an X-linked gene that is submitted to inactivation. It is an essential transcription factor in CD4(+)CD25(+)FoxP3 regulatory T cells, which are therapeutic targets in disseminated cutaneous melanoma. Moreover, FoxP3 is an important tumor suppressor gene in carcinomas and has putative cancer suppressor gene function in cutaneous melanoma as well. Therefore understanding the structure and function of the FoxP3 gene is crucial to gaining insight into the biology of melanoma to better develop immunotherapeutics and future therapeutic strategies., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. 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.

Intratumoral hypoxia has been associated with poor prognosis in several solid tumors. The aim of this study was to determine whether the hypoxia-associated markers glucose transporter (GLUT)-1 and carbonic anhydrase (CA)-IX expression and preoperative hemoglobin (Hb) levels correlate with presence of inguinofemoral or distant metastases, and disease-free survival (DSS) in vulvar squamous cell carcinoma (SCC) patients. Vulvar SCC (n=103) were reviewed for histopathological characteristics by an expert gynecopathologist and stained for GLUT-1 and CA-IX. Clinical data and preoperative Hb levels were obtained from medical records. No significant correlations were observed between GLUT-1 or CA-IX expression patterns and preoperative Hb levels, presence of inguinofemoral or distant metastases and DSS. However, anemic patients (Hb<11.2 g/dL) had significantly more inguinofemoral metastases and lower Hb level was an independent prognostic factor for a worse DSS (p<0.001). The number of comorbidic conditions was inversely correlated with preoperative Hb level. Preoperative Hb levels are associated with poor DSS for vulvar SCC patients, whereas tumor hypoxia reflected by GLUT-1 and CA-IX expression does not have a predictive value. Because preoperative Hb levels inversely correlated with the number of comorbidic conditions and not with GLUT-1 or CA-IX expression, it is most likely that preoperative Hb levels represent overall physical condition.

Retained placenta is caused by abnormal adherence of the placenta to the uterine wall, leading to delayed expulsion of the placenta and causing postpartum haemorrhage. The mildest form of retained placenta is the placenta adhesiva (PA), of which the cause is unknown. The aim of our study was to explore possible differences in immune response in the basal decidua between PA and control placentas (CP). We performed a descriptive analysis of immunohistochemical differences in 17 PA and 10 CP. Our results show that in PA the amount of uterine natural killer (uNK) cells is significantly reduced (0.2 uNK cell/standardised area) as compared to CP (9.8 uNK cell/standardised area, p < 0.001) whereas the number of trophoblast cells and the expression of HLA-G by trophoblast are similar in the decidua of PA and CP. We speculate that adequate numbers of uNK cells in the basal decidua are needed for normal expulsion of the placenta., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Melanoma progression is a multistep progression from a common melanocytic nevus through the radial growth phase, the invasive vertical growth phase finally leading to metastatic spread into distant organs. Migration and invasion of tumor cells requires secretion of proteases to facilitate remodeling of the extracellular matrix including basement membranes. Here we used a reconstructed skin model to investigate melanoma growth and invasion in vitro. Using this model we show that the dermoepidermal basement membrane prevents the invasion of metastatic melanoma BLM and MV3 cells in the absence of a stratified epidermis. In the reconstructed skin model, matrix metalloproteinase-9, a protease activated early in melanoma development, is secreted by the keratinocytes and subsequently activated by an unknown soluble factor secreted by the melanoma cells. The dynamic interplay between keratinocytes and melanoma cells is further shown by an altered growth pattern of melanoma cells and the finding that a reconstructed epidermis induces invasion. Overall, our findings show that the invasive behavior of melanoma cells is determined by the melanoma cells themselves, but that the interplay between surrounding keratinocytes and the melanoma cells plays an important role in melanoma invasion.

The molecular pathogenesis of human papilloma virus-unrelated vulvar squamous cell carcinoma is not well known. Whether malignant progression of lichen sclerosus and differentiated vulvar intraepithelial neoplasia to vulvar squamous cell carcinoma could be accompanied by altered DNA content has not been studied extensively. DNA content in isolated nuclei of microdissected normal vulvar epithelium (n = 2), lichen sclerosus (n = 9), differentiated vulvar intraepithelial neoplasia (n = 13), and squamous cell carcinoma (n = 17) from 22 patients was measured via DNA image cytometry. For additional analysis, 6 differentiated vulvar intraepithelial neoplasia lesions were selected, bringing the number of patients to 28. p53 expression was determined by immunohistochemistry on consecutive tissue sections. Thirty-eight percent (5/13) of differentiated vulvar intraepithelial neoplasia lesions and 65% (11/17) of squamous cell carcinomas were DNA aneuploid or tetraploid. In lesions that contained differentiated vulvar intraepithelial neoplasia and adjacent squamous cell carcinoma, the ploidy status of differentiated vulvar intraepithelial neoplasia did not exceed that of squamous cell carcinoma. We observed a strong correlation between high p53 expression and DNA aneuploidy. This relation was also present at the level of a single nucleus, measured by sequential image cytometry of p53 immunohistochemistry followed by DNA image cytometry on formalin-fixed tissue sections. Similarly, we found p53-positive nonproliferating cells with increased DNA content in the superficial compartment of 6 additional solitary differentiated vulvar intraepithelial neoplasia lesions that were not associated with squamous cell carcinoma, indicating ascending aneuploid cells from the basal compartment. DNA ploidy measurements suggest that differentiated vulvar intraepithelial neoplasia has a higher malignant potential than lichen sclerosus and thus is a more likely precursor of squamous cell carcinoma. Furthermore, high p53 expression correlates with increased DNA content and aneuploidy; but it requires further research to unveil a possible causal relation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aims: The aetiology of vulvar squamous cell carcinomas (SCC) that are not causally associated with high-risk human papillomavirus remains largely elusive. The aim of this study was to analyse the inflammatory response in its presumed precursor lesions, lichen sclerosus (LS) and differentiated vulvar intraepithelial neoplasia (dVIN), and provide evidence that dVIN is a likely precursor of vulvar SCC., Methods and Results: Immunohistochemical analyses for CD4+, CD8+, CD20+, CD68+, S100+ and tryptase-positive immune cells were performed and quantified in LS (n = 7), dVIN (n = 19), SCC (n = 11), and normal vulvar tissue (n = 8). The subepithelial inflammatory response in dVIN and SCC was comparable, but absent in LS. Abundant intraepithelial mast cells were observed in dVIN only, and confirmed by electron microscopy, toluidine blue staining and cKIT expression. Adjacent keratinocytes displayed increased proliferation as determined by MIB-1 positivity. Electron microscopy revealed intraepithelial mast cell degranulation. Intraepithelial mast cells were not or infrequently observed in vulvar hyperplasia (n = 13), condylomata acuminata (n = 5), keratinocytic intraepidermal neoplasia of sun-exposed skin (n = 15), epidermal hyperplasia of head and neck (n = 12), and psoriasis (n = 3)., Conclusions: These data indicate that dVIN can be recognized by intraepithelial mast cells and that they might promote the progression of dVIN to SCC., (© 2010 Blackwell Publishing Limited.)

Background: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling., Methods: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided., Results: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors., Conclusion: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

Objective: The objective of the study was to quantify vessel type and density in lichen sclerosus (LS) to find a marker for its malignant potential., Study Design: Quantitative analysis was performed on paraffin-embedded tissue samples of 28 patients with LS (7 adjacent to vulvar squamous cell carcinoma, 21 solitary) and immunohistochemical staining for CD34 (vascular and lymphangiogenic lymph endothelial cells), D2-40 (lymphatic-specific marker), and alpha-SMA (pericyte marker). Electron microscopy was performed on fresh tissue., Results: No significant differences in vessel density or other vessel parameters could be demonstrated between the 2 groups. In hyalinized lesions, vessel diameter, and alpha-SMA positivity was reduced compared with nonhyalinized lesions. Electron microscopy revealed detachment of pericytes from vascular endothelial cells and increased thickening of basement membrane, whereas endothelial cell function did not appear strongly impaired., Conclusion: Malignant potential of LS cannot be predicted by vessel characteristics. Hyalinization in LS is associated with pericyte detachment from the basal lamina of vascular endothelial cells., (Copyright (c) 2010 Mosby, Inc. All rights reserved.)

Cutaneous melanoma still represents a paradox among all solid tumors. It is the cancer for which the best prognostic markers ever identified in solid tumors are available, yet there is very little understanding of their biological significance. This review focuses on recent biological data that shed light on the clinical-biological correlations underlining the 2010 American Joint Committee on Cancer (AJCC) melanoma staging system. A major challenge is to replace outcome clustering based on artificial biomarker breakpoints by a continuous multidimensional prognostic model. Major improvement will come from shared computerized tools that allow the generation of continuous likelihood scores for diagnosis, prognosis, and response prediction. This will lead to the development of platforms which can be used by scientists from different fields to integrate and share high-quality data in the pre-competitive setting and generate new probabilistic causal models.

Background: Reliable population-based incidence and survival data on extracutaneous melanoma (ECM) are sparse., Methods: Incidence data (1989-2006) from the Netherlands Cancer Registry were combined with vital status on January 1, 2008. Age-adjusted annual incidence rates were calculated by direct standardization, and the estimated annual percentage change was estimated to detect changing trends in incidence. Additionally, we carried out cohort-based relative survival analysis., Results: Ocular melanomas were the most common ECM subsite with European standardized incidence rates (ESR) of 10.7 and 8.2 per 1,000,000 person-years for males and females, respectively. In comparison, for cutaneous melanoma (CM), the ESRs for men and women were 122 and 155 per million person-years, respectively. No statistically significant trends in the incidence of ECM were detected, whereas an annual increase of 4.4% for men and 3.6% for women was detected in the incidence of CM. Relative survival for ECM was poor, but differed largely between anatomic subtypes ranging from a 5-year relative survival of 74% for ocular melanomas to 15% for certain subsites of mucosal melanomas., Conclusions: Of all ECM subsites, ocular melanomas had the highest incidence and the best survival. Mucosal melanomas were the second most frequent subsite of ECM. Five-year relative survival for all ECM subtypes was worse if compared with CM. No statistically significant trends in the incidence of (subsites of) ECM were determined., Impact: This study gives insight into the relative sizes of the different subgroups of ECM as well as an estimate of 5-year survival, which varies substantially by subsite., (Copyright 2010 AACR.)

Adhesion molecules endow tumor cells with the necessary cell-cell contacts and cell-matrix interactions. As such, adhesion molecules are involved in cell signalling, proliferation and tumor growth. Rearrangements in the adhesion repertoire allow tumor cells to migrate, invade and form metastases. Besides these membrane-bound adhesion molecules several soluble adhesion molecules are detected in the supernatant of tumor cell lines and patient body fluids. Truncated soluble adhesion molecules can be generated by several conventional mechanisms, including alternative splicing of mRNA transcripts, chromosomal translocation, and extracellular proteolytic ectodomain shedding. Secretion of vesicles (ectosomes and exosomes) is an alternative mechanism mediating the release of full-length adhesion molecules. Soluble adhesion molecules function as modulators of cell adhesion, induce proteolytic activity and facilitate cell signalling. Additionally, adhesion molecules present on secreted vesicles might be involved in the vesicle-target cell interaction. Based on currently available data, released soluble adhesion molecules contribute to cancer progression and therefore should not be regarded as unrelated and non-functional side products of tumor progression., (2010 Elsevier GmbH. All rights reserved.)

Lymphatic metastasis of oral squamous cell carcinoma (SCC) is important for prognosis and clinical decision making concerning the treatment of the neck but may be difficult to detect. Activated leukocyte cell adhesion molecule (ALCAM), has been shown to correlate with prognosis or tumor grade in different tumor types and may be a predictor of lymphatic metastasis. ALCAM expression at the invasive front in fresh frozen tissue samples of oral SCC's (n=41) was studied immunohistochemically, using a polyclonal antibody directed against ALCAM's extracellular domain. Membranous expression of ALCAM at the invasive front was significantly related to lymph node metastasis (p=0.001, sensitivity 69%, specificity 84%) and tumor grade (p=0.035). There was no significant relationship with tumor thickness (p=0.394). Lymph node status (p=0.030), correlated with 5-year overall survival. A significant relation between ALCAM and prognosis could not be established, due to an insufficient sample size. However, ALCAM expression does appears to increase risk of early death. Membranous ALCAM expression at the invasive front serves as a molecular marker for lymphatic metastasis and may facilitate better treatment choices concerning the neck in patients with oral SCC., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Introduction and Hypothesis: A familial tendency has been demonstrated in the etiology of pelvic organ prolapse (POP), but the specific genetic defects have not been identified. Type III collagen is an important factor in the repair of connective tissue, and gene polymorphisms may impair the tensile strength. We hypothesized that polymorphisms in the alpha I chain of the type III collagen protein-encoding gene (COL3A1) pose women at risk for POP., Methods: In this case-control study, the prevalence of type III collagen polymorphisms was compared in women with and without signs and symptoms of POP., Results: Two hundred and two POP patients and 102 normal parous controls were included. A homozygous single-nucleotide substitution in the coding region of type III collagen (COL3A1 2209G>A, rs1800255) was identified in 27 (13%) POP patients and three (3%) controls (odds ratio, 5.0; 95% confidence interval, 1.4-17.1)., Conclusions: The probability of POP was higher in women with COL3A1 2209G>A. This polymorphism showed to be a relevant risk factor for POP.

Purpose: High-risk human papilloma virus (HPV) plays a role in the development of a subset of vulvar squamous cell carcinomas. Uncertainty exists about the true impact of HPV in this tumor type because conflicting reports have been published with diverging prevalence rates. This study was done to fine tune the role of high-risk HPV infection in vulvar squamous cell carcinoma development in relation to clinical prognosis., Experimental Design: 130 vulvar squamous cell carcinomas of patients with known survival data were analyzed for histology of the adjacent lesion (differentiated or HPV-associated usual vulvar intraepithelial neoplasia), in relation to p16(INK4A) expression as marker of HPV activity, and presence and integration of high-risk HPV DNA., Results: Usual vulvar intraepithelial neoplasia was present adjacent to vulvar squamous cell carcinoma in 25 of 130 cases. Usual vulvar intraepithelial neoplasia-associated squamous cell carcinomas had high p16(INK4A) expression, and 24 of 25 squamous cell carcinomas contained integrated high-risk HPV DNA. Differentiated vulvar intraepithelial neoplasia was found adjacent to 105 of 130 vulvar squamous cell carcinomas. High-risk HPV was detected in 11 (10.5%) differentiated vulvar intraepithelial neoplasia-associated vulvar squamous cell carcinoma but correlated with high p16(INK4A) expression in only one case. Integration of viral DNA was never observed in differentiated vulvar intraepithelial neoplasia-associated squamous cell carcinomas, which suggests that a causal relationship of high-risk HPV in differentiated vulvar intraepithelial neoplasia-associated tumors is highly unlikely. The disease-specific survival of the differentiated vulvar intraepithelial neoplasia-associated vulvar squamous cell carcinoma patients was significantly worse compared with patients with a usual vulvar intraepithelial neoplasia-associated tumor., Conclusions: High-risk HPV is causally associated with the development of usual vulvar intraepithelial neoplasia associated squamous cell carcinomas, which comprise 19% of all vulvar squamous cell carcinomas, but not with differentiated vulvar intraepithelial neoplasia-associated vulvar squamous cell carcinomas. Differentiated vulvar intraepithelial neoplasia-associated vulvar squamous cell carcinomas have a significantly worse prognosis.

Objective: The purpose of the present study is to investigate the trends in incidence of both usual (u) and differentiated (d) vulvar intraepithelial neoplasia (VIN) separately, their malignant potential and the relation with other HPV related anogenital lesions in the Netherlands during a 14-year-period., Methods: The incidences of both types of VIN and vulvar SCC were retrieved from the Nationwide Netherlands Database of Histo- and Cytopathology. Population data were retrieved from the Database of Statistics Netherlands., Results: In the study period, the incidence of uVIN and dVIN increased, while the incidence of vulvar SCC remained stable. The overall percentage of uVIN patients that were later diagnosed with vulvar SCC was 5.7%, which was significantly lower than the percentage for dVIN patients (32.8%). In addition to this 5.6-fold increased conversion rate, the time of progression from dVIN to SCC development was significantly shorter than that of uVIN (p=0.005). Percentage of uVIN patients that were later diagnosed with SCC significantly increased with age (p=0.005), whereas the time to SCC significantly shortened with age (p=0.05). Forty-one percent of uVIN patients had a past, concomitant or future HPV-associated lesion of the lower genital tract, which is in contrast to the 3% for dVIN patients., Conclusions: An increase in diagnoses of both uVIN and dVIN has not led to an increase in vulvar SCC incidence. The malignant potential of dVIN is higher than that for uVIN. For uVIN the malignant potential increases with age.

The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin-12 (rhIL-12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL-12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B-blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL-12 treated patients, increased IFN-gamma mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL-12 treatment. In conclusion, these data indicate that after i.t. IL-12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients., ((c) 2008 Wiley-Liss, Inc.)

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD), a marker of various cancers and mesenchymal stem cells, is involved in melanoma metastasis. We have exploited a secreted NH(2)-terminal fragment, sALCAM, to test the hypothesis that ALCAM coordinates tissue growth and cell migration. Overexpression of sALCAM in metastatic melanoma cells disturbed clustering of endogenous ALCAM and inhibited activation of matrix metalloproteinase-2 (MMP-2). Exposure of HT1080 fibrosarcoma cells to sALCAM similarly inhibited MMP-2, suggesting a broader effect on ALCAM-positive tumor cells. In contrast to the previously reported, promotive effects of an NH(2)-terminally truncated, transmembrane variant (DeltaN-ALCAM), sALCAM impaired the migratory capacity of transfected cells in vitro, reduced basement membrane penetration in reconstituted human skin equivalents, and diminished metastatic capacity in nude mice. Remarkably, L1 neuronal cell adhesion molecule (L1CAM/CD171), another progression marker of several cancers including melanoma, was suppressed upon sALCAM overexpression but was up-regulated by DeltaN-ALCAM. The partially overlapping and opposite effects induced by alternative strategies targeting ALCAM functions collectively attribute an integrative role to ALCAM in orchestrating cell adhesion, growth, invasion, and proteolysis in the tumor tissue microenvironment and disclose a therapeutic potential for sALCAM.

Tumors are complex tissues composed of neoplastic cells, soluble and insoluble matrix components and stromal cells. Here we report that in melanoma, turn-over of type I collagen (Col(I)), the predominant matrix protein in dermal stroma affects melanoma progression. Fibroblasts juxtaposed to melanoma cell nests within the papillary dermis display high levels of Col(I) mRNA expression. These nests are enveloped by collagen fibers. In contrast, melanoma-associated fibroblasts within the reticular dermis express Col(I) mRNA at a level that is comparable to its expression in uninvolved dermis and reduced amount of collagen protein can be observed. To determine the significance of Col(I) expression in melanoma, we pharmacologically inhibited its transcription in a porcine cutaneous melanoma model by oral administration of halofuginone. When administered before melanoma development, it reduced melanoma incidence and diminished the transition from microinvasive toward deeply invasive growth by limiting the development of a tumor vasculature. Whereas invasive melanoma growth has been correlated with increased blood vessel density previously, our data for the first time demonstrate that the proangiogenic effect of Col(I) expression by fibroblasts and vascular cells precedes the development of invasive melanomas in a de novo tumor model., ((c) 2007 Wiley-Liss, Inc.)

Oxidative phosphorylation in the mitochondria is an important energy-producing process for eukaryotic cells, but this process can also result in producing potentially cell-damaging side products. Oxygen is the final proton acceptor in this cascade of electron/proton transfer and results in harmless water. The electron transfer, however, is not completely efficient and results in the production of reactive oxygen species (ROS). Low amounts of these ROS are important for cellular-signalling pathways. Excessive ROS, however, can induce cell damage that can culminate in cell death. Therefore, the cell has developed an antioxidant network to scavenge excessively produced ROS. In general, the balance between the production and scavenging of ROS leads to homeostasis. Disturbance of this equilibrium can alter normal cellular processes; it often occurs in tumour cells. In this review, the role of ROS in cutaneous melanoma development and progression is described. Cutaneous melanoma arises from epidermal melanocytes in skin, which is a relatively hypoxic tissue. ROS are generated as a result of increased metabolism of transformed cells, immune reaction against the developing tumour, ultraviolet radiation, melanin production and an altered antioxidant system. Knowledge of the role of ROS in melanoma development and the mechanisms that alleviate oxidative stress can aid in the development of better antimelanoma therapies.

Approximately 10% of all colorectal carcinomas are mucinous carcinomas, characterized by extracellular mucin. Occasionally, mucin accumulates intracellularly in these tumours, causing signet ring cell differentiation. We hypothesized that signet ring cells arise from a separate genetic pathway. In this study the molecular background of signet ring cell differentiation was investigated by analysing genetic changes, changes in the expression of adhesion molecules, and mucin content. Furthermore, its clinical relevance was addressed. Cell lines of colorectal tumours with non-mucinous (AC), mucinous (MC), and signet ring cell phenotype (MCSRC) were used for Multiplex Ligation-dependent Probe Amplification to detect deletions and amplifications in specific oncogenes and tumour suppressor genes. Furthermore, the expression of E-cadherin, beta-catenin, ITF (intestinal trefoil factor), and MUC2 in signet ring cells was studied by immunohistochemistry, immunofluorescence, and mRNA in situ hybridization. Results were validated using a large cohort of rectal carcinomas from which clinicopathological data were available. Specific amplifications and deletions in cell lines of AC, MC, and MCSRC were detected. Bcl-2 was amplified in MCSRC and MC cell lines, but not in AC cell lines. Bcl-2 FISH analysis confirmed this in patient material. Signet ring cells had decreased expression of adhesion molecules (E-cadherin, beta-catenin) and were strongly positive for ITF and MUC2, two peptides which are normally only produced by goblet cells. RNA in situ hybridization confirmed the production of ITF. Mucinous carcinomas with signet ring cell differentiation presented at a higher T stage than adenocarcinomas and mucinous carcinomas (16% pT4 versus 3-5%, p<0.001) and were more frequently node positive (77% vs 39-44%; p<0.001). Prognosis was significantly worse. In conclusion, the presence of signet ring cells in carcinomas with mucinous differentiation correlates with increased T-stage and poor prognosis. These cells, characterized by ITF and MUC2 production, show disruption of the E-cadherin/beta-catenin complex, as well as amplification of Bcl-2., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)