MMP9 potentiates pulmonary metastasis formation.

Tumor cell dissemination to distant organ sites is a complex process involving multiple cell types, soluble growth factors, adhesion receptors, and tissue remodeling. A new study in this issue of Cancer Cell shows that MMP9-expressing tumor-associated macrophages play a key role in prepping premetastatic sites for eventual malignant cell growth in a manner dependent upon vascular endothelial growth factor receptor-1 (VEGFR-1).

Concurrent endometrial intraepithelial carcinoma (EIC) and serous ovarian cancer: can EIC be seen as the precursor lesion?

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.

Pelvic organ prolapse and collagen-associated disorders.

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.

Differentiated vulvar intraepithelial neoplasia is often found in lesions, previously diagnosed as lichen sclerosus, which have progressed to vulvar squamous cell carcinoma.

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.

Specific intraepithelial localization of mast cells in differentiated vulvar intraepithelial neoplasia and its possible contribution to vulvar squamous cell carcinoma development.

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.

The prognostic value of blood and lymph vessel parameters in lichen sclerosus for vulvar squamous cell carcinoma development: an immunohistochemical and electron microscopy 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.

Type I collagen expression contributes to angiogenesis and the development of deeply invasive cutaneous melanoma.

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.

Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation.

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.

Reactive oxygen species in melanoma and its therapeutic implications.

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.

Tumours can adapt to anti-angiogenic therapy depending on the stromal context: lessons from endothelial cell biology.

It has long been recognized that interference with the blood supply of a tumour is an effective way to halt tumour progression, and even induce tumour regression. This can be accomplished by anti-angiogenic treatment which prevents the formation of a tumour neovasculature, or anti-vascular treatment, which aims at destruction of existent tumour vessels. The latter has received relatively little attention because there is a lack of specific tumour-endothelial markers. Instead, the current detailed knowledge on the factors and mechanisms, involved in angiogenesis, has enabled the development of a variety of angiogenesis inhibitors, especially those that target cellular signalling by vascular endothelial growth factor-A (VEGF-A), the most potent angiogenic factor known. These inhibitors have received lots of attention because they effectively inhibit tumour growth in pre-clinical models. However, in clinical trials these same inhibitors showed very poor anti-tumour activity. In this review we discuss this discrepancy, and we show that the tumour microenvironment is crucial to the sensitivity of tumours to anti-angiogenic therapy.

Inflammation, proteases and cancer.

Tumours are complex tissues composed of ever-evolving neoplastic cells, matrix proteins that provide structural support and sequester biologically active molecules, and a cellular stromal component. Reciprocal interactions between neoplastic cells, activated host cells and the dynamic micro-environment in which they live enables tumour growth and dissemination. It has become evident that early and persistent inflammatory responses observed in or around developing neoplasms regulates many aspects of tumour development (matrix remodelling, angiogenesis, malignant potential) by providing diverse mediators implicated in maintaining tissue homeostasis, e.g., soluble growth and survival factors, matrix remodelling enzymes, reactive oxygen species and other bioactive molecules. This review highlights recent insights into the role of chronic inflammation associated with cancer development and examines proteolytic pathways activated by infiltrating leukocytes during neoplastic programming of tissues.

The protein phosphatase 2A regulatory subunit PR70 is a gonosomal melanoma tumor suppressor gene.

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.

Stromal responses in human primary melanoma of the skin.

Tumour development and progression has long been considered as the consequence of an imbalance between apoptosis and proliferation of transformed cells. However, whereas genetic aberrations leading to the activation of oncogenes and/or loss of tumour suppressor genes are crucial for the transformation towards aberrant cell growth, progression towards a full blown malignancy requires a dynamic interaction between tumour cells and the environment in which they thrive. Over the recent years, it has become evident that the (early) inflammatory and angiogenic response, and remodelling of the extracellular proteins are key factors in creating a microenvironment that sustains tumour growth and metastasis. The host response towards cutaneous melanoma has received relatively little attention, most likely because the majority of these tumours develop without evoking a strong stromal response as can be observed in, e.g., carcinomas. This review discusses potential critical modulators of melanoma growth: turn-over of the most abundant extracellular matrix protein in skin (i.e. type I collagen), the early inflammatory response and angiogenesis.

A caveolin-dependent and PI3K/AKT-independent role of PTEN in ß-catenin transcriptional activity.

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 ß-catenin independent of the PI3K-AKT-GSK3ß axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent ß-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-ß-catenin axis is mediated by a feedback loop in which ß-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 ß-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.

The tumor microenvironment: a critical determinant of neoplastic evolution.

Evolution of neoplastic cells has generally been regarded as a cumulative intrinsic process resulting in altered cell characteristics enabling enhanced growth properties, evasion of apoptotic signals, unlimited replicative potential and gain of properties enabling the ability to thrive in ectopic tissues and in some cases, ability to metastasize. Recently however, the role of the neoplastic microenvironment has become appreciated largely due to the realization that tumors are not merely masses of neoplastic cells, but instead, are complex tissues composed of both a non-cellular (matrix proteins) and a cellular 'diploid' component (tumor-associated fibroblasts, capillary-associated cells and inflammatory cells), in addition to the ever-evolving neoplastic cells. With these realizations, it has become evident that early and persistent inflammatory responses observed in or around many solid tumors, play important roles in establishing an environment suitable for neoplastic progression by providing diverse factors that alter tissue homeostasis. Using cutaneous melanoma and squamous cell carcinoma as tumor models, we review the current literature focussing on inflammatory and tumor-associated fibroblast responses as critical mediators of neoplastic progression for these malignancies.

Truncation of activated leukocyte cell adhesion molecule: a gateway to melanoma metastasis.

Progression of human cutaneous primary melanoma is, among others, accompanied by de novo expression of activated leukocyte cell adhesion molecule (ALCAM/CD166) and enhanced activity of proteolytic cascades in the invasive, vertical growth phase (VGP) of lesions. The homophilic cell adhesion function of wild-type ALCAM mediates homotypic clustering of melanoma cells and would, thus, antagonize cell release from the primary tumor, an early prerequisite for metastasis. Stable transfection of a transmembrane, amino-terminally truncated ALCAM (DeltaN-ALCAM) into metastatic cells diminished cell clustering mediated by wild-type ALCAM. We have addressed the biological effects of DeltaN-ALCAM on tumorigenicity and found that the relief of cell clustering constraints promoted motility in vitro and the transition from expansive tumor growth to tissue invasion in reconstructed skin in culture. In a transplant tumor model, the changes were reflected in reduced subcutaneous tumor growth and in accelerated, spontaneous lung metastasis. These data indicate that the intact cell adhesion function of ALCAM may both favor primary tumor growth and represent a rate-limiting step for tissue invasion from VGP melanoma. ALCAM induction could, thus, provide an attractive target for proteolysis as a part of a more complex cellular program that couples growth and migration and facilitates dissemination.

Modulation of activated leukocyte cell adhesion molecule-mediated invasion triggers an innate immune gene response in melanoma.

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.

Identification of six loci associated with pelvic organ prolapse using genome-wide association analysis.

OBJECTIVE: There is evidence that both environmental and genetic factors contribute to pelvic organ prolapse. We conducted a genome-wide association study to investigate whether common genetic variants modify the risk of pelvic organ prolapse. METHODS: We recruited women who had been evaluated and treated for pelvic organ prolapse at the University of Utah from 1996 to 2008 and their affected female relatives. Those in the case group were genotyped on the Illumina 550K platform. We genetically matched 2,976 white control participants available from Illumina as the control group. Association tests were adjusted for related participants using two different software programs: EMMAX and Genie. Confirmation of findings was performed in a cohort of Dutch women (n=76) with recurrent pelvic organ prolapse and family history of pelvic organ prolapse. RESULTS: The Utah study sample included 115 case group participants treated for pelvic organ prolapse, in most case group participants with surgery (n=78) or repeat surgery (n=35). Results from association analyses using EMMAX software identified five single-nucleotide polymorphisms (SNPs) significantly associated with pelvic organ prolapse (P<1×10). Independent association analysis with Genie software identified three of the same SNPs and one additional SNP. The six SNPs were located at 4q21 (rs1455311), 8q24 (rs1036819), 9q22 (rs430794), 15q11 (rs8027714), 20p13 (rs1810636), and 21q22 (rs2236479). Nominally significant findings (P<.05) or findings trending toward significance (P<.1) were observed for five of the six SNPs in the Dutch cohort. CONCLUSION: Six SNPs have been identified that are significantly associated with pelvic organ prolapse in high-risk familial case group participants and that provide evidence for a genetic contribution to pelvic organ prolapse. LEVEL OF EVIDENCE: II.