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Unlabelled: The angiogenic switch, a rate-limiting step in tumor progression, has already occurred by the time most human tumors are detectable. However, despite significant study of the mechanisms controlling this switch, the kinetics and reversibility of the process have not been explored. The stability of the angiogenic phenotype was examined using an established human liposarcoma xenograft model. Nonangiogenic cells inoculated into immunocompromised mice formed microscopic tumors that remained dormant for approximately 125 days (vs. <40 days for angiogenic cells) whereupon the vast majority (>95%) initiated angiogenic growth with second-order kinetics. These original, clonally derived angiogenic tumor cells were passaged through four in vivo cycles. At each cycle, a new set of single-cell clones was established from the most angiogenic clone and characterized for in vivo for tumorigenic activity. A total of 132 single-cell clones were tested in the second, third, and fourth in vivo passage. Strikingly, at each passage, a portion of the single-cell clones formed microscopic, dormant tumors. Following dormancy, like the original cell line, these revertant tumors spontaneously switched to the angiogenic phenotype. Finally, revertant clones were transcriptionally profiled and their angiogenic output determined. Collectively, these data demonstrate that the angiogenic phenotype in tumors is malleable and can spontaneously revert to the nonangiogenic phenotype in a population of human tumor cells., Implications: Leveraging the rate of reversion to the nonangiogenic phenotype and tumor dormancy may be a novel anticancer strategy., (©2014 AACR.)

The intermediate filament nestin, a neural stem-cell marker, is reported to be expressed more strongly in melanomas compared with benign melanocytic lesions, and increasingly expressed in advanced melanoma stages. However, the prognostic impact of nestin on melanoma has not been well elucidated. The aim of the present study was to evaluate the prognostic influence of nestin expression in cutaneous melanoma in comparison with standard clinico-pathologic variables. In a large series of nodular cutaneous melanoma (n=348), nestin expression was assessed by immunohistochemistry using tissue microarray (TMA) sections. For comparison, nestin staining in corresponding metastases as well as in superficial spreading melanomas and benign nevi was also examined. Nestin was expressed to varying degrees in a majority of nodular melanomas (92%), and was significantly associated with increased tumor thickness, high mitotic count, and the presence of ulceration and tumor necrosis. Also, expression was stronger in the nodular type than in superficial spreading melanomas and benign nevi, but without significant difference when compared with matched metastases from the former. Importantly, strong expression of nestin was significantly associated with reduced survival in multivariate analysis. In conclusion, increased nestin expression was associated with aggressive melanoma features, with independent prognostic impact on multivariate survival analysis when compared with clinico-pathologic factors.

In pancreatic ductal adenocarcinoma (PDAC), the benefit of current chemotherapy and radiation therapy is very limited, even in radically resected patients. New treatment strategies, for example based on the inhibition of the tumour's blood supply, need to be explored. We have investigated angiogenesis markers and their associations with relapse and survival in 52 histologically confirmed cases of PDAC. Angiogenesis in the primary tumour was evaluated by microvessel density (MVD), vascular proliferation index (VPI) and the presence of glomeruloid microvascular proliferations (GMP). These features were analysed in the context of clinicopathological variables, KRAS mutation status, relapse location and survival. MVD (median 134 microvessels/mm(2) , range 88-177) and VPI (median 3.2%, range 1.6-4.9) were associated with larger tumour size and lymph node metastasis. MVD was also related to the occurrence of liver metastases. Both variables were associated with survival in univariate and multivariate analyses. GMPs were present in 32 (62%) of the cases. Patients who exhibited MVD and VPI values above median, and GMP positivity, had a median survival of only 4.2 months after surgery. In conclusion, the angiogenesis markers MVD and VPI have a significant impact on survival. By also including GMP, a subgroup of PDAC patients with particularly short survival could be identified., (© 2013 APMIS Published by John Wiley & Sons Ltd.)

Background: The incidence of malignant melanoma in Norway is among the highest in the world and rising, with approximately 1 500 persons receiving the diagnosis annually. Correct surgical primary treatment cures 80-90%, while 10-20% experience relapses. The treatment of a metastatic malignant melanoma has changed considerably in the last 1-2 years as a result of clinical experience with new drugs. The current publication provides an updated overview of the treatment of malignant melanoma in Norway., Method: The article is based on a search in PubMed and on the authors' own research and clinical experience., Results: After several decades with almost no change in the treatment of malignant melanoma, we have seen a positive development over the past couple of years. New treatment methods for malignant melanoma with distant spreading metastases have yielded favourable results in selected patients and are currently established in cancer departments in Norway., Interpretation: Rapid and correct primary treatment is curing most patients with malignant melanoma. New drugs offer hope for selected patient groups with metastatic disease. Several new types of targeted treatment are being tested in clinical studies in Norway and elsewhere in the world.

Introduction: Tumor-associated angiogenesis is one of the essential hallmarks underlying cancer development and metastasis. Anti-angiogenic agents accordingly aim to restrain cancer progression by blocking the formation of new vessels, improving the delivery of chemotherapeutic agents to the tumor site and reducing the shedding of metastatic cells into the circulation. This review article addresses some key issues in the use of angiogenesis inhibitors in cancer., Areas Covered: The authors review the complex interactions between cell signaling pathways involved in tumor angiogenesis, and focus in particular on the molecular mechanisms that may induce resistance to angiogenesis inhibitors. They will also discuss some novel therapeutic strategies evolving within anti-angiogenic therapy such as the targeting of VEGFR-3, endothelial integrins and hepatocyte growth factor-MET signaling., Expert Opinion: Although anti-angiogenic therapy is targeted at the non-malignant part of the tumor, the intricate network of growth promoting signaling pathways and in particular the redundancy when single pathways are targeted in endothelial cells represents a major therapeutic obstacle. A key challenge will be to develop more efficient inhibitors, combined with an individualized approach based on each tumor's own endothelial signaling profile. Furthermore, reliable biomarkers which pinpoint those patients that will benefit from anti-angiogenic therapy need to be identified.

The significance of tumor necrosis in cutaneous melanoma has not been well elucidated. The purpose of this study was to explore the prognostic impact of necrosis in comparison with other known clinicopathologic factors in these tumors. Initially, 457 consecutive cases of nodular cutaneous melanoma (1981 to 2008) were included in this series. Tumor necrosis was assessed on hematoxylin and eosin-stained sections and was recorded as significant when an area of at least a quarter of a high-power field (×400; 0.07 mm) was occupied by necrotic cells and as sparse when clusters of at least 5 necrotic cells were observed. Tumor necrosis (26% of the cases) was associated with increased tumor thickness, high mitotic count, presence of tumor ulceration, and decreased survival. Stratified analyses (univariate and multivariate) with standard microscopic variables indicated the strongest prognostic influence of necrosis in tumors thicker than 4 mm. Notably, in the stratum of pT4 tumors, presence of necrosis was a stronger prognostic predictor than was ulceration. Tumor necrosis was a significant prognostic indicator providing additional information to established predictors of patient outcome in this series of nodular cutaneous melanoma, predominantly among thick tumors (>4 mm). Presence of necrosis was a stronger indicator for worse outcome compared with ulceration in pT4 tumors.

Angiogenesis is important for the growth and spread of malignant tumors, and anti-angiogenesis treatment is currently being evaluated for breast cancer and other tumors. Although microvessel density is the most commonly used tissue-based marker of tumor associated angiogenesis, it has significant limitations and has not proven effective as a predictive factor in selecting patients for treatment. We here wanted to explore the significance of vascular endothelial cell proliferation in breast carcinoma. We examined microvessel proliferation in breast cancer by dual immunohistochemical staining, using the pan-endothelial marker Factor-VIII combined with proliferation of endothelial cells by Ki-67 expression, in three independent series of breast cancer, including a total of 499 patients and 141 events during follow-up. Common statistical tests of associations as well as univariate and multivariate regression analysis of patient survival were used. By counting vessels with actively proliferating endothelium, we show that microvascular proliferation is a significant predictor of disease progression in breast cancer, especially among high-grade and ER-negative tumors. Our findings indicate that this novel marker of active tumor angiogenesis might be of value in patient management and should be further studied in the context of patient selection for anti-angiogenesis treatment.

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

Mitotic count is a known prognostic predictor in cutaneous melanoma, and is included in the current American Joint Committee on Cancer tumor-node-metastasis (TNM) staging system. The mitotic marker phosphohistone H3 (PHH3) is considered to facilitate counting of mitosis, and the purpose of this study was to evaluate the prognostic significance and strength of PHH3 in comparison with standard mitotic counting in cutaneous malignant melanoma. A total of 457 consecutive cases of nodular cutaneous melanoma were initially included in this series. The mitotic count was assessed on hematoxylin and eosin sections, and PHH3 was then examined by immunohistochemistry on standard sections of paraffin-embedded tumor tissue. Both the mitotic count and the number of PHH3-stained mitotic figures were recorded in a minimum area of 1 mm(2). Increased mitotic count and PHH3 value were both associated with unfavorable features like tumor thickness and presence of ulceration. Univariate survival analysis showed a highly significant prognostic impact of mitotic count and PHH3, whereas multivariate analysis indicated PHH3 to be a stronger prognostic indicator than mitotic count. Assessment of mitotic activity by PHH3 immunostaining might have important practical advantages, and should be further studied to consider a place in routine examination of all cutaneous melanomas.

Background: VEGF driven angiogenesis plays a key role in cancer progression. We determined the clinical efficacy of bevacizumab monotherapy in patients with metastatic melanoma., Methods and Findings: Thirty-five patients with metastatic melanoma in progression were enrolled in this phase II, single arm clinical trial. Each patient received bevacizumab monotherapy 10 mg/kg q14 d until intolerable toxicity or disease progression occurred. Clinical efficacy was evaluated as objective response, disease control (DC), and survival. We observed one complete (3%) and 5 partial (14%) responses. In addition, 5 patients experienced stable disease >6 months (14%) while 24 patients had progressive disease (PD, 69%), corresponding to a total DC at 6 months in 11 out of 35 patients (31%). Median progression free survival (PFS) was 2.14 months and median overall survival (OS) was 9 months (1.12-49). Seven of the 11 patients experiencing DC developed early hypertension (<2 months) compared to 3/24 of patients with PD (P = 0.001), and hypertension was associated with PFS (P = 0.005) and OS (P = 0.013)., Conclusion: Bevacizumab monotherapy demonstrated promising clinical efficacy in patients with metastatic melanoma with disease control in 31% of the patients. Induced early hypertension was a marker for clinical efficacy of bevacizumab., Trial Registration: ClinicalTrials.gov NCT00139360.

Background: Glomeruloid microvascular proliferation (GMP), a novel histology-based angiogenesis marker, has been associated with decreased survival in several human cancers., Methods: In this study, we evaluated the ability of GMP to predict clinical response to neoadjuvant chemotherapy in a series of locally advanced breast cancers (n=112)., Results: Presence of GMP (21% of the cases) was significantly associated with high-grade tumours and TP53 mutations in addition to the basal-like and HER2 subtypes of breast cancer as defined by gene expression data. GMP was correlated to a gene expression signature for tumour hypoxia response. The GMP pattern was also significantly associated with lack of treatment response and progressive disease (P=0.004)., Interpretation: The findings suggest that GMP might be able to predict the lack of response to neoadjuvant chemotherapy in locally advanced breast cancer. Whether GMP may be an independent predictor compared with other factors including TP53 mutation status and tumour grade needs confirmation in larger studies.

Background: Tumor cell proliferation is a predictor of survival in cutaneous melanoma. The aim of the present study was to evaluate the prognostic impact of mitotic count, Ki-67 expression and novel proliferation markers phosphohistone H3 (PHH3), minichromosome maintenance protein 4 (MCM4) and mitosin, and to compare the results with histopathological variables., Methods: 202 consecutive cases of nodular cutaneous melanoma were initially included. Mitotic count (mitosis per mm2) was assessed on H&E sections, and Ki-67 expression was estimated by immunohistochemistry on standard sections. PHH3, MCM4 and mitosin were examined by staining of tissue microarrays (TMA) sections., Results: Increased mitotic count and elevated Ki-67 expression were strongly associated with increased tumor thickness, presence of ulceration and tumor necrosis. Furthermore, high mitotic count and elevated Ki-67 expression were also associated with Clark's level of invasion and presence of vascular invasion. High expression of PHH3 and MCM4 was correlated with high mitotic count, elevated Ki-67 expression and tumor ulceration, and increased PHH3 frequencies were associated with tumor thickness and presence of tumor necrosis. Univariate analyses showed a worse outcome in cases with elevated Ki-67 expression and high mitotic count, whereas PHH3, MCM4 and mitosin were not significant. Tumor cell proliferation by Ki-67 had significant prognostic impact by multivariate analysis., Conclusions: Ki-67 was a stronger and more robust prognostic indicator than mitotic count in this series of nodular melanoma. PHH3, MCM4 and mitosin did not predict patient survival.

Purpose: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non-small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification., Experimental Design: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation., Results: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects., Conclusion: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Tumor progression is dependent on a number of sequential steps, including initial recruitment of blood vessels (i.e., angiogenic switch). Failure of a microscopic tumor to complete one or more of these early steps may lead to delayed clinical manifestation of the cancer. In this review we summarize some of the clinical and experimental evidence suggesting that microscopic human cancers can remain in an asymptomatic, non-detectable, and occult state for the life of a person or animal. We present three clinical cases where tumors present shortly after an accidental trauma in otherwise healthy individuals. We also review current experimental human tumor dormancy models with special emphasis on the angiogenic switch which closely recapitulates clinically observed delay in tumor recurrence.

Background: Tumor necrosis and apoptotic activity are considered important in cancer progression, but these features have not been much studied in melanomas. Our hypothesis was that rapid growth in cutaneous melanomas of the vertical growth phase might lead to tissue hypoxia, alterations in apoptotic activity and tumor necrosis. We proposed that these tumor characteristics might be associated with changes in expression of cell adhesion proteins leading to increased invasive capacity and reduced patient survival., Methods: A well characterized series of nodular melanoma (originally 202 cases) and other benign and malignant melanocytic tumors (109 cases) were examined for the presence of necrosis, apoptotic activity (TUNEL assay), immunohistochemical expression of hypoxia markers (HIF-1 alpha, CAIX, TNF-alpha, Apaf-1) and cell adhesion proteins (alphavbeta3 integrin, CD44/HCAM and osteopontin). We hypothesized that tumor hypoxia and necrosis might be associated with increased invasiveness in melanoma through alterations of tumor cell adhesion proteins., Results: Necrosis was present in 29% of nodular melanomas and was associated with increased tumor thickness, tumor ulceration, vascular invasion, higher tumor proliferation and apoptotic index, increased expression of alphavbeta3 integrin and poor patient outcome by multivariate analysis. Tumor cell apoptosis did also correlate with reduced patient survival. Expression of TNF-alpha and Apaf-1 was significantly associated with tumor thickness, and osteopontin expression correlated with increased tumor cell proliferation (Ki-67)., Conclusion: Tumor necrosis and apoptotic activity are important features of melanoma progression and prognosis, at least partly through alterations in cell adhesion molecules such as increased alphavbeta3 integrin expression, revealing potentially important targets for new therapeutic approaches to be further explored.

Tumor progression is dependent on a number of sequential steps, including initial tumor-vascular interactions and recruitment of blood vessels (i.e., the angiogenic switch), as well as tumor cells interacting with the surrounding microenvironment and its different components. Failure of a microscopic tumor to complete one or more of these early stages may lead to delayed clinical manifestation of the cancer and a state of stable non-progressing disease (i.e., tumor dormancy). In this review, some of the clinical and experimental evidence is summarized, suggesting that microscopic human cancers, either primary, recurrent or metastatic, can remain in an asymptomatic, non-detectable, and occult state for a long period of time. We also review current experimental human tumor dormancy models which closely recapitulate clinically observed delay in tumor progress.

Earlier studies have shown frequent mutations in the BRAF and NRAS genes in cutaneous melanoma, but these alterations have not been examined in the rare category of melanoma from black Africans. Moreover, the frequency of epidermal growth factor receptor (EGFR) mutations in melanocytic tumors is not known. We therefore examined 165 benign and malignant melanocytic lesions (including 118 invasive melanomas and 18 metastases collected as consecutive cases from various time periods and from two different pathology departments; the 51 nodular melanomas were randomly selected from a larger, consecutive, population-based series of nodular melanomas) with respect to alterations in the EGFR, BRAF and NRAS genes. Mutations in EGFR (exons 18-21) were not detected. EGFR protein expression was observed in a subgroup of melanomas, but without associations with clinicopathologic phenotype or prognosis. Cytoplasmic EGFR expression was, however, significantly increased from benign nevi to melanomas. Mutations in BRAF and NRAS were detected in superficial melanoma (25 and 29%, respectively), nodular melanoma (29 and 28%, respectively) and lentigo maligna melanoma (15 and 16%, respectively). In a series of melanomas from black Africans (n=26), only two BRAF mutations (8%) were found, both being different from the common T1799A substitution. Moreover, melanomas from black Africans exhibited mutations in NRAS exon 1 only (12%), whereas NRAS exon 2 mutations were predominant in melanomas from Caucasians. Thus, the frequencies of BRAF and NRAS mutations were particularly low in melanomas from black Africans, supporting a different pathogenesis of these tumors.

Background: Microscopic human cancers can remain dormant for life. Tumor progression depends on sequential events, including a switch to the angiogenic phenotype, i.e., initial recruitment of new vessels. We previously demonstrated that human tumors contain tumor cell populations that are heterogeneous in angiogenic activity. Here, we separated angiogenic from nonangiogenic human tumor cell populations and compared their growth., Methods: Severe combined immunodeficient (SCID) mice were inoculated with nonangiogenic human MDA-MB-436 breast adenocarcinoma, KHOS-24OS osteosarcoma, or T98G glioblastoma cells. Most of the resulting tumors remained microscopic (<1 mm diameter), but some eventually became angiogenic and enlarged and were used to isolate angiogenic tumor cells. Angiogenic and nonangiogenic tumor cells were inoculated into SCID mice, and time to the development of palpable tumors was determined. Cell proliferation was assayed in vitro by growth curves and in vivo by staining for proliferating cell nuclear antigen or Ki67. Microscopic tumors from both tumor cell populations were examined for histologic evidence of vascular development 14 days after inoculation in mice. Expression of the angiogenesis inhibitor thrombospondin-1 was examined by immunoblotting., Results: Nonangiogenic tumors of each tumor type developed palpable tumors after means of 119 days (range: 53-185 days) for breast cancer, 238 days (184-291 days) for osteosarcoma, and 226 days (150-301 days) for glioblastoma. Angiogenic cells developed palpable tumors within 20 days after inoculation. However, nonangiogenic and angiogenic cells of each tumor type had similar proliferation rates. Fourteen days after tumor cell inoculation, tumors from angiogenic cells showed evidence of functional vasculature. In contrast, nonangiogenic tumors remained microscopic in size with absent or nonfunctional vasculature. Thrombospondin-1 expression was statistically significantly lower (by five- to 23-fold, depending on tumor type) in angiogenic than nonangiogenic cells., Conclusions: This model provides a conceptual framework and a reproducible in vivo system to study unresolved central questions in cancer biology regarding the initiation, reversibility, and molecular regulation of the timing of the angiogenic switch.

Purpose: EZH2 is a member of the polycomb group of genes and important in cell cycle regulation. Increased expression of EZH2 has been associated previously with invasive growth and aggressive clinical behavior in prostate and breast cancer, but the relationship with tumor cell proliferation has not been examined in human tumors. The purpose of this study was to validate previous findings in a population-based setting, also including tumors that have not been studied previously., Patients and Methods: In our study of nearly 700 patients, we examined EZH2 expression and its association with tumor cell proliferation and other tumor markers, clinical features, and prognosis in cutaneous melanoma and cancers of the endometrium, prostate, and breast., Results: Strong EZH2 expression was associated with increased tumor cell proliferation in all four cancer types. Associations were also found between EZH2 and important clinicopathologic variables. EZH2 expression showed significant prognostic impact in melanoma, prostate, and endometrial carcinoma in univariate survival analyses, and revealed independent prognostic importance in carcinoma of the endometrium and prostate. CONCLUSION Our findings point at EZH2 as a novel and independent prognostic marker in endometrial cancer, and validate previous findings on prostate and breast cancer. Further, EZH2 expression was associated with features of aggressive cutaneous melanoma. The fact that EZH2 might identify increased tumor cell proliferation and aggressive subgroups in several cancers may be of practical interest because the polycomb group proteins have been suggested as candidates for targeted therapy. EZH2 expression should, therefore, be further examined as a possible predictive factor.

Purpose: It has been proposed that melanoma cells shift from E-cadherin to N-cadherin expression during tumor development, and recent gene profiling has shown increased expression of Wnt5a/Frizzled in aggressive melanomas possibly by interactions with beta-catenin. We therefore wanted to investigate the role of cadherin subtypes, beta-catenin, and Wnt5a/Frizzled in melanocytic tumors, with focus on prognosis in nodular melanomas., Experimental Design: The immunohistochemical expression of E-cadherin, N-cadherin, P-cadherin, beta-catenin, and Wnt5a/Frizzled was examined using tissue microarrays of 312 melanocytic tumors., Results: Cytoplasmic expression of P-cadherin was associated with increasing tumor thickness (P=0.005) and level of invasion (P=0.019), whereas membranous staining was associated with thinner (P=0.012) and more superficial (P=0.018) tumors. Increased cytoplasmic P-cadherin was associated with reduced survival (P=0.047). Lack of nuclear beta-catenin expression was related to increased tumor thickness (P=0.002) and poor patient survival in univariate (P=0.0072) and multivariate (P=0.004) analyses. Membranous expression of N-cadherin was significantly increased from primary tumors to metastatic lesions, whereas E-cadherin staining tended to be decreased. Wnt5a and its receptor Frizzled were highly coexpressed, and nuclear expression of both markers was significantly reduced from benign nevi to melanomas, with a shift from nuclear to cytoplasmic expression in malignant tumors. In addition, Wnt5a expression was significantly associated with nuclear beta-catenin expression., Conclusions: Alterations in the expression and subcellular localization of cell adhesion markers are important in the development and progression of melanocytic tumors, and strong cytoplasmic P-cadherin expression and loss of nuclear beta-catenin staining were associated with aggressive melanoma behavior and reduced patient survival.

The ID1 protein, an inhibitor of basic helix-loop-helix transcription factors, has been involved in multiple cellular processes including cell cycle regulation, apoptosis, and angiogenesis. To evaluate the importance of ID1 in malignant melanoma, tumour cell expression was examined by immunohistochemistry in 119 cases of nodular melanoma using tissue microarray technique, and related to multiple tumour markers including proliferation, p16 expression, angiogenesis and patient survival. Strong ID1 expression was significantly associated with increased tumour thickness, and significantly reduced survival. Also, increased ID1 was associated with loss of thrombospondin-1 (TSP-1) expression, a known inhibitor of angiogenesis, and increased intensity of ephrin-A1 and its receptor EPHA2. Presence of BRAF mutations was related to strong ID1 expression, but there was no relationship with p16 protein expression. Further, no significant correlation was found between ID1 and microvessel density. In conclusion, our study supports a significant role of the ID1 protein in melanoma progression and patient prognosis. The absence of correlation with p16 protein expression and angiogenesis suggests that other regulatory pathways and mechanisms might be influenced by ID1 in melanomas. An inverse relation between ID1 and TSP-1 expression support an important role of ID1 in the regulation of this complex multitarget protein.

Previous studies have shown frequent mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) or NRAS (neuroblastoma RAS viral [V-ras] oncogene homolog) genes in cutaneous melanoma, but the relationship between these alterations and tumor cell proliferation has not been examined in human melanoma. In our study of 51 primary nodular melanomas and 18 paired metastases, we found mutations in BRAF (codon 600, previously denoted 599) in 15 primary tumors (29%) and eight metastases (44%). The figures for NRAS mutations were 27% and 22%, respectively. Mutations in BRAF and NRAS genes were mutually exclusive in all but one case, and were maintained from primary tumors through their metastases. Mutations, however, were not associated with tumor cell proliferation by Ki-67 expression, tumor thickness, microvessel density, or vascular invasion, and there were no differences in patient survival. Although BRAF and NRAS mutations are likely to be important for the initiation and maintenance of some melanomas, other factors might be more significant for proliferation and prognosis in subgroups of aggressive melanoma.

Cell cycle regulating proteins are important in tumour development. To investigate whether alterations in Cyclin D1, p14, CDK4 and Rb are associated with tumour cell proliferation, tumour progression and patient survival in malignant melanoma, we examined 202 vertical growth phase tumours and 68 corresponding metastases for expression of Cyclin D1, p14, CDK4 and Rb, and compared the results with Ki-67 expression, p16 and p53 expression, clinico-pathological variables, and survival data. Nuclear staining of Cyclin D1 was strong in 35% of cases, and correlated with high levels of Rb (p=0.05), but not with survival or other markers tested. Strong staining of p14 was found in 63% of nodular melanomas and was associated with strong p53 expression (p=0.014), and with high levels of CDK4 (p<0.0001). Low p14 expression was associated with increased tumour thickness (p=0.008) and increasing level of invasion (p=0.020). Strong nuclear staining for CDK4 was found in 81% of cases and was associated with tumour thickness below the median value of 3.7 mm and improved survival (log-rank test, p=0.024). Further, 56% of the tumours showed strong nuclear staining for Rb, and these cases were significantly associated with absent/low levels of p16 staining (p=0.030), high levels of p14 (p=0.010), as well as high Ki-67 expression (p=0.005). Our results seem to confirm that the p16-Rb pathway plays an important role in tumour progression and prognosis in vertical growth phase melanomas, whereas alterations in the p14-p53 pathway might be less important.

Background: Tumour angiogenesis and lymphangiogenesis are important new areas of cancer research. In this paper we highlight the central aspects of these processes and describe new therapies directed at the tumour vasculature. We also present some of our own data., Material and Methods: Data from key papers in international journals on tumour angiogenesis and lymphangiogenesis are cited and discussed., Results: Research on tumour angiogenesis and lymphangiogenesis has increased dramatically over the last 30 years. Today, this knowledge is being used to predict the aggressiveness of malignant tumours and to design compounds which can halt the angiogenesis process and affect tumour growth., Interpretation: Insight into how angiogenesis and lymphangiogenesis are regulated in malignant neoplasms is important to the understanding of tumour biology. Currently, numerous clinical trials are conducted with angiogenesis inhibitors; the first completed trials indicate that tumour growth may be retarded in some cancer entities.

We tested the hypothesis that hyperbaric oxygen (HBO) alone and with chemotherapy (5-FU) attenuates tumor growth of DMBA-induced tumors in rats. Six series were performed: (1) Controls (air and vehicle 0.9% NaCl i.p.), (2) 5-FU (0.2 mg/kg i.p.), (3) HBO (2 bar for 90 min and vehicle), (4) HBO and 5-FU, (5) HBO (11 days) and air (next 12 days), (6) HBO (23 days). All treatments were applied on days 1, 4, 7, 10 (Series 1-4), as well as on days 14, 17 and 23 (Series 5-6). Tumor diameter increased by 76.7 and 41.2% in untreated controls and in the 5-FU group, respectively, after 10 days. Tumor size fell by 17-24.2% in the HBO groups and by 35.5% when combined with 5-FU (P < 0.05 compared to HBO). HBO treatment reduced the total number of blood vessels in the tumors. After completion of HBO treatment tumor size increased, but statistically insignificant, during the next 12 days., (Copyright 2004 Elsevier Ireland Ltd.)

Previous studies have shown that BRCA1-related breast cancers are often high-grade tumors that do not express estrogen receptors, HER2, p27(Kip1), or cyclin D1, but do express p53 and cyclin E. In addition, the expression of cytokeratin 5/6 (CK5/6), indicating a basal epithelial phenotype, is frequent in BRCA1-related breast cancer. Here, in a series of 247 breast cancers, we demonstrate that CK5/6 expression was associated with nearly all of the features of BRCA1-related breast cancer and was also associated with a poor prognosis. In a parsimonious multivariable proportional hazards model, protein levels of cyclin E, p27(Kip1), p53, and the presence of glomeruloid microvascular proliferation all independently predicted outcome after breast cancer. In this model, only cyclin E and p27(Kip1) levels were independent predictors in lymph node-negative cancers, whereas glomeruloid microvascular proliferation and tumor size independently predicted outcome in node-positive disease. The molecular determinants of the basal epithelial phenotype encapsulate many of the key features of breast cancers occurring in germ-line BRCA1 mutation carriers and have independent prognostic value. Basal breast cancer deserves recognition as an important subtype of breast cancer.

Glomeruloid microvascular proliferation (GMP) in breast cancer independently adversely affected survival (relative risk 1.9, 95% CI: 1.2-3.0), particularly among women who received adjuvant chemotherapy (10-year survival 27 vs 69%, P=0.0003), and was significantly associated with p53 overexpression and BRCA1 germline mutations. The presence of GMP may influence treatment decisions.

Glomeruloid microvascular proliferations (GMPs), which are focal proliferative buddings of endothelial cells resembling a renal glomerulus, can be induced experimentally by adenoviral transfer of VEGF-A(165). We recently found that GMPs were present in 13-23% of various human tumours (melanoma, breast-, endometrial- and prostate cancer), and this vascular signature was significantly associated with an impaired prognosis. In the present study, a series of 202 vertical growth phase melanomas were examined for the expression of various angiogenic factors and their receptors. Presence of GMP was associated with increased expression in tumour endothelium of the VEGF-A receptors KDR, FLT-1 and neuropilin-1, as well as VEGF-D protein. Thrombospondin-1 staining in the tumour stroma showed the same relationship. Endothelial cell expression of VEGF-A was increased in GMP endothelium when compared with other intra-tumoural vessels. In contrast, GMP expression of bFGF was decreased. Our findings suggest an important role of VEGF-A and its receptors in GMP formation in human cutaneous melanoma.

The aim of this study was to determine lymphatic vessel density (LVD) in a series of nodular melanoma and correlate the findings with the expression of several angiogenic factors, including vascular endothelial growth factor-C, basic fibroblast growth factor (bFGF), patient survival, and clinico-pathologic data. Patients with nodular melanoma and complete follow-up information were included. Lymphatic vessels were immunostained with the LYVE-1 and Podoplanin antibodies, and LVD was evaluated in both intra- and peri-tumoral (LVDpt) areas. Median LVD was 6.3 and 12.5 vessels/mm(2) in intra- and peri-tumoral areas, and coexpression of LYVE-1 and Ki-67/MIB-1 in lymphatic endothelial cells within the tumor was demonstrated, indicating active but low-grade lymphangiogenesis. Increased LVDpt was significantly associated with localization on the extremities (P = 0.005), decreased tumor thickness (P = 0.036), absence of vascular invasion (P = 0.004), brisk lymphocytic infiltration (P = 0.018), low proliferative rate by Ki-67 (P = 0.011), increased bFGF expression in tumor cells (P = 0.01) as well as in endothelial cells (P = 0.008), and decreased tumor cell expression of Ephrin-A1 (P = 0.009). Decreased LVD in intra-tumoral areas and LVDpt both predicted improved survival rates in multivariate analyses (for LVDpt, Hazard ratio: 2.1, P = 0.009). We found that decreased LVD was present in thicker and more proliferative tumors (Ki-67) and that increased LVD was significantly associated with improved patient survival in multivariate analysis. In addition, our data suggest the presence of low-grade intra-tumoral lymphangiogenesis in melanoma and a stimulating role of bFGF in lymphangiogenesis.

We evaluated the presence of glomeruloid microvascular proliferations (GMPs) in 723 patients with melanomas, breast, endometrial, or prostate cancer. Presence of GMPs was associated with markers of aggressive tumor behavior and significantly reduced survival or increased clinical recurrences in all four of the cancer types in univariate analysis. GMPs were related to increased microvessel density in prostate cancer only. In the case of melanomas, breast, and prostate cancers (but not endometrial cancers), GMPs were a significant prognostic factor in the final multivariate models (P all

Promoter hypermethylation, mutations, and loss of heterozygosity in the CDKN2A gene as well as polymorphisms at the 3'-untranslated region were determined in vertical growth phase melanomas. Methylation-specific polymerase chain reaction in soluti and in situ showed that 19% of the cases were hypermethylated at the CDKN2A promoter region, and some of these cases were heterogeneous with both methylated and unmethylated tumor cells. Methylation was associated with increased tumor cell proliferation by Ki-67 expression (P = 0.01) and decreased patient survival (P = 0.025). Point mutations in CDKN2A were found in 4% of the cases, whereas 90% had loss of heterozygosity at one or more of 4 markers studied. Furthermore, presence of the 540 C>T polymorphism at the 3'-untranslated region of CDKN2A (23%) was associated with improved survival in multivariate analysis (hazard ratio, 2.6; P = 0.02). Our results suggest that promoter methylation of the CDKN2A gene is present in a subgroup of the tumors and associated with increased tumor cell proliferation and reduced survival. Further, the 540 C>T polymorphism might define a distinct subgroup of low-grade vertical growth phase melanomas. These findings support a significant role of the CDKN2A gene in melanoma progression.

The expression of several angiogenic factors and receptors was examined in a series of vertical growth phase cutaneous melanomas using high-throughput tissue microarray technology and immunohistochemistry. The results were correlated with microvessel density, clinicopathological features, and patient survival. Expression of basic fibroblast growth factor (bFGF) was significantly associated with increased microvessel density. Also, we found an independent prognostic importance of vascular phenotype by endothelial cell expression of bFGF; cases with positive vessels had the best prognosis and these tumors revealed a low frequency of vascular invasion (14%) when compared with bFGF-negative vessels (47%). This bFGF-negative phenotype was significantly increased in metastatic lesions. Strong tumor cell expression of FLT-4, ephrin-A1, and EphA2 was associated with increased melanoma thickness, and ephrin-A1 staining was related to decreased survival (P = 0.039). Expression of EphA2 in tumor cells was associated with increased tumor cell proliferation (Ki-67 positivity), indicating possible autocrine growth stimulation. Thus, our findings indicate the presence of phenotypic diversity among tumor-associated vessels, and subgroups defined by bFGF expression may be of clinical importance. bFGF was associated with microvessel density, whereas the ephrin-A1/EphA2 pathway might also be important for tumor cell proliferation and patient survival.

In this report we present the results of mutational analysis of the CDKN2B, CDKN2C, CDK4, p53 genes and 5'UTR of the CDKN2A gene in a set of 44 sporadic primary melanomas, which had been earlier analysed for mutations in the CDKN2A (p16/p14(ARF)) gene. No tumour-associated mutations were detected except in 1 melanoma where we found a CC>T* deletion-mutation in the codon 151-152 (exon 5) of the p53 gene. On the basis of our preliminary results, we did extended genotyping of the 500 C>G and 540 C>T polymorphisms in the 3'UTR of the CDKN2A gene in 229 melanoma cases and 235 controls. The T-allele frequency (for 540 C>T polymorphism) in melanomas was significantly higher than in controls (0.14 vs. 0.08; chi(2) = 5.95, p = 0.01; OR = 1.71, 95%CI = 1.11-2.66). The heterozygote frequency for this polymorphism was 0.26 (59/229) in melanomas compared to 0.13 (30/235) in healthy controls (chi(2) = 11.4; p = 0.0007; OR = 2.34, 95% CI = 1.40-3.92). The frequency of the 500 C>G polymorphism in the 3'UTR in the CDKN2A gene was not significantly higher in melanomas compared to healthy controls. The 500 C>G polymorphism, however, was in linkage disequilibrium with approximately 50 kb apart the C>A intronic polymorphism in the CDKN2B gene (determined in 44 melanomas and 90 controls; Fisher exact test, p<0.0001). Finally, the sequence analysis of genomic DNA isolated from T cell lymphocytes of healthy individuals exhibited that the codon reported as last of exon 2 of the CDKN2C gene is rather the first codon of exon 3., (Copyright 2001 Wiley-Liss, Inc.)

Microvessel density (MVD) was estimated in a series of 202 vertical growth phase (VPG) melanomas and 68 corresponding metastases, using a marker for angiogenic endothelial cells (CD105) and Factor-VIII. The expression pattern of vascular endothelial growth factor (VEGF), FLT-1, KDR and thrombospondin-1 (TSP-1) was studied by immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction. CD105 stained significantly less vessels, but gave only limited additional prognostic information compared with Factor-VIII, and MVD was an independent prognostic factor for both markers. Ninety-eight percent of all cases showed expression of VEGF, and higher expression was found significantly more frequent in thinner and less vascularized tumors. Possible autocrine loops were suggested by co-expression of VEGF and its two receptors in tumor cells, and by a significant correlation between KDR and tumor cell proliferation (Ki-67) in the subgroup of thicker tumors. Staining of VEGF receptors in endothelium was not correlated with MVD. Strong expression of TSP-1 in tumor stroma was found in 43% of the primary tumors, and was significantly correlated with increased thickness, proliferation and MVD, as well as decreased survival. These data suggest that MVD is associated with prognosis in cutaneous melanomas, and that the VEGF system and particularly TSP-1 seem to be involved in the regulation of angiogenesis and progression of these tumors.

Malignant melanomas in black Africans are predominantly located on the lower extremities. Since their biological features have not been well focused, we studied 28 such cases with special reference to proliferative activity (Ki-67 expression), p16 and p53 staining, as well as microvessel density, all known to be involved in the progression of melanomas among whites. The findings were related to clinico-pathological characteristics. The tumours had a median thickness of 6.4 mm, ulceration was present in 71%, and vascular invasion in 36%, indicating the presence of advanced and aggressive melanomas. Further, loss of p16 protein expression was found in 50%, and high proliferative activity was present (median 41%). In contrast, strong p53 staining was rare (11%), although most tumours showed low-level positivity. Angiogenesis, as estimated by microvessel density, was significantly associated with vascular invasion (p = 0.022), supporting its role in the progression of these tumours. Thus, our findings indicate that melanomas located on the lower extremities in black Africans show several features of aggressiveness; in particular, the proliferative activity was high, and p16 alterations was frequent as evidenced by loss of protein staining. Our findings also indicated that the diagnosis is delayed among black Africans.

The CDKN2A (p16INK4alpha) cell cycle-inhibitory gene has been associated with development of familial melanoma. Additionally, recent studies indicate that p16 alterations occur frequently in sporadic melanomas. To investigate whether differences in p16 expression are associated with tumor cell proliferation, tumor progression, and patient survival, we examined the immunohistochemical staining of p16 protein in a consecutive series of 202 vertical growth phase melanomas and 68 corresponding metastases and compared the results with Ki-67 expression, p53 expression, clinicopathological variables, and survival data. Forty-five percent of the primary tumors showed absent or minimal nuclear staining for p16 protein. These cases were significantly associated with high Ki-67 expression (P < 0.0001), ulceration (P = 0.001), and vascular invasion (P = 0.03). Further loss of p16 expression was observed in metastatic lesions (77% were negative; P < 0.0001). Absent/minimal nuclear p16 staining significantly predicted poor patient survival (log-rank test, P = 0.0003), with 37% and 67% estimated 10-year survival rates for cases with absent or present p16 expression, respectively. In multivariate analysis, p16 staining was an independent prognostic factor (hazard ratio, 2.5; 95% confidence interval, 1.5-4.2; P = 0.0008), along with p53 expression, Ki-67 expression, anatomical site, Clark's level of invasion, and vascular invasion. Our findings indicate that loss of nuclear p16 protein expression in vertical growth phase melanomas is associated with increased tumor cell proliferation (Ki-67) and independently predicts decreased patient survival. Cases without p53 expression had improved survival.

The association between microvessel density (MVD) and patient survival has recently been established for various types of human cancers. Immunohistochemical staining (factor-VIII related antigen) was performed on a series of 102 vertical growth phase melanomas and vessels were counted in areas. Increased MVD was associated with large histologic tumor diameter (p=0.006) and tumor ulceration (p=0.04). Further, high vascular density was significantly related to decreased patient survival (p=0.04), with borderline significance (p=0.10) in the final multivariate model. The results of this study suggest that microvessel density is of prognostic importance in aggressive vertical growth phase melanomas.

The frequency and significance of p53 alterations in cutaneous melanoma have not been completely clarified. In the present study, 31 primary melanomas of the nodular type and 15 metastases occurring between 1981 and 1983 were studied with respect to mutations in exons 7 and 8, as well as to p53 protein immunostaining using different antibodies. Altogether 13% of the primary tumors showed strong p53 staining using the DO-7 antibody. Different results were obtained with other antibodies. Seven mutations were found in primary and metastatic tumors; all of these were single base changes, most of which occurred in the core domain of the p53 protein responsible for sequence-specific DNA binding (residues 102-293). The mutations were not significantly associated with p53 staining results, and p53 alterations (mutations or marked immunopositivity) had no prognostic value. Our results indicate that point mutations in exons 7 and 8 are more frequent than previously reported in primary melanomas, and such changes may be important for the development of certain melanoma subgroups.

The role of p16 and p53 alterations in cutaneous melanoma has been recently discussed, but it remains to be clarified. In the present immunohistochemical study, the expression of p16 and p53 proteins and their possible prognostic relevance have been examined in 102 melanomas of the aggressive nodular type. Twelve percent showed a strong expression of p53 protein, and these cases were significantly more frequent in the head/neck area compared with other sites (32% vs. 6%). Expression of p16 protein was negative or weak in 9% of the cases, and this tended to be less frequent in head/neck tumors compared with the others (0% vs. 12%). Whereas p53 staining was not prognostically important, loss of p16 staining was significantly associated with markedly reduced recurrence free and patient survival in univariate analysis (product-limit method). In multivariate analysis, lack of p16 staining was significantly associated with recurrent disease (p = 0.013). Our findings indicate an important role of altered p16 protein expression in a subgroup of melanoma patients.

Background: The role of vascular invasion as an independent predictor of outcome in cutaneous melanoma is controversial, mostly because of a close relationship with tumor thickness. Therefore, the prevalence of vascular involvement and its prognostic impact has been studied in a series of patients with aggressive cutaneous melanoma., Methods: One hundred and two patients with nodular malignant melanoma diagnosed between 1981 and 1989 were studied retrospectively. The vessels were stained with factor VIII antibody, and vascular invasion as well as other features of the primary tumor and essential clinical variables were related to time to recurrence and death due to melanoma using univariate survival analysis (product-limit method) and the multivariate Cox regression method., Results: Definite vascular invasion was present in 15 cases (15%), and it was significantly associated with tumor thickness, histologic diameter, ulceration, and presence of lymph node metastases and distant spread at the time of diagnosis. In univariate analysis, vascular invasion was a significant predictor of patient survival (P = 0.006), and this variable also showed an independent prognostic impact in multivariate analysis (P = 0.02). Furthermore, anatomic site (P = 0.03), histologic diameter (P = 0.004), and Clark's level of invasion (P = 0.006) were independently associated with decreased patient survival. In the multivariate analysis of time to recurrence, anatomic site (P = 0.003), tumor thickness (P = 0.002), and histologic diameter (P = 0.003) were independent risk factors., Conclusions: Although vascular invasion was significantly associated with tumor thickness and histologic diameter of the primary tumor, it provided an independent prognostic information by multivariate analysis of patient survival, in addition to anatomic site and indicators of tumor size (histologic diameter and level of invasion). Our findings further indicate that the prognostic importance of vascular invasion is associated with both lymphatic and hematogeneous spread, although the latter is probably more important.