Vascularization of cutaneous melanoma involves vessel co-option and has clinical significance

This study was undertaken to determine the role and the fate of the peritumoural vascular plexus during the vascularization of human malignant melanoma (hMM) and in an appropriate murine melanoma model system. The prognostic significance of the vascularity of different tumour areas was also evaluated. Despite morphometry revealing several-fold higher microvessel densities (MVDs) in the peritumoural tissue than at the centre of the tumour, the development of visceral metastases of hMM was exclusively correlated with the MVD of the tumour centre. Furthermore, the 5-year survival of the patient group with low tumour centre MVD (<30/mm(2), n=29) was 100%, compared to 1/16 patients alive with high tumour centre MVD (>30/mm(2), n=16). Morphometric analysis and three-dimensional reconstruction of vessel networks of both human and murine melanomas showed clearly that the peritumoural vascular plexus present at the melanoma base is continuously being incorporated into the growing tumour mass. Once vessels become incorporated, sprouting ceases and the proliferating endothelial cells (EC) take part only in vessel dilatation. Moreover, the immunohistochemical and ultrastructural characterization of microvessels demonstrated that the pericyte coverage of endothelial tubes was complete in all of the investigated areas, in both human and murine melanomas.     

Microvessel density for melanoma prognosis

AIMS: Two-hundred and four accessible cases of malignant melanoma from the Grampian region of Scotland, collected over a period of 4 years, were studied for standard prognostic indicators for comparison with microvessel density. METHODS AND RESULTS: The range of tumour thickness varied from in-situ melanoma to 14.8 mm. Microvessel density was assessed using the Chalkley technique on sections immunostained with CD31 antibody to identify endothelium. Vessel counts were performed in the peritumoral host tissue of all specimens. Strong correlation was observed between microvessel density at the tumour edge and tumour thickness (P < 0.001). Multifactorial regression analysis confirms Chalkley estimation as a reliable predictor of the risk of recurrence of melanoma (P < 0.005). The predictive value was found to be superior to the Breslow score, for tumours more than 2 mm thick. CONCLUSIONS: Microvessel assessment of primary melanoma using the Chalkley score technique provides reliable prognostic information on the risk of recurrence of the tumour, particularly for melanomas deeper than 2 mm. It remains to be seen whether this methodology can predict response to novel anti-angiogenic therapies currently entering trials.     

Cutaneous malignant melanoma: correlation between neovascularization and peritumor accumulation of mast cells overexpressing vascular endothelial growth factor

To investigate the possible role of mast cells (MC) in the angiogenic process in cutaneous melanoma, we examined tissue samples from 35 adult patients with primary malignant melanoma and compared with 20 intradermal benign nevi. MC were identified by anti-tryptase, microvessels by anti-CD34, and vascular endothelial growth factor (VEGF) expression by standard immunohistochemical methods. Tryptase-positive MC expressing VEGF were identified by double immunostaining. The numbers of MC and microvessels around the tumor were determined by the point counting method. MC density was significantly greater in melanoma compared with benign nevi (197.6 +/- 19.4 v 95.7 +/- 5.0/mm2, P < .001). Vascular density was also significantly higher in melanoma than in benign lesions (3.6-fold, P < .001). Double immunostaining showed the presence of VEGF in the cytoplasm of tryptase-positive peritumoral MC. The percentage of this MC-subtype was significantly higher in melanoma than in nevus tissues (71.9 +/- 2.4% v 30.6 +/- 2.5%, P < .001). A strong significant correlation was shown between the number of VEGF+ MC and microvessel density (r = .811, P < .001). MC count and VEGF+ MC count, as well as microvessel density were significantly higher in aggressive (metastasizing) melanomas (P < .001). Our results suggest that peritumoral accumulation of MC and the subsequent release of potent angiogenic factor such as VEGF may thus represent a tumor-host interaction that may favor progression of this tumor.     

Expression of thymosin beta10 and its role in non-small cell lung cancer

The exact role of thymosin beta10 in lung cancer progression remains unclear. We investigated by immunohistochemistry the expression of thymosin beta10 protein in tumors and tumor-adjacent tissues from 69 patients with non-small cell lung cancer. The relationship of thymosin beta10 expression with vascular endothelial growth factor, vascular endothelial growth factor-C, microvessel density, and lymphatic vessel density was determined; clinicopathologic factors and surgical treatment outcome were also studied. The results showed that thymosin beta10 was mainly expressed in the cytoplasm of lung cancer cells, and the overexpression of thymosin beta10 was correlated with advanced clinical stage (P = .026), distant metastases (P = .016), lymph node metastases (P = .007), poor degree of differentiation (P = .03), and poor postoperative survival (P = .004). Furthermore, thymosin beta10 overexpression was associated with vascular endothelial growth factor (P = .004), vascular endothelial growth factor-C (P = .017), microvessel density (P = .000), and lymphatic vessel density (P = .002). The lowest survival rate was observed in the patients with high thymosin beta10, positive vascular endothelial growth factor, and high microvessel density (P = .007) or in the patients with high thymosin beta10, positive vascular endothelial growth factor-C, and high lymphatic vessel density (P = .005). These results suggest that thymosin beta10 might induce microvascular and lymphatic vessel formation by up-regulating vascular endothelial growth factor and vascular endothelial growth factor-C in lung cancer tissues, thus promoting the distant and lymph node metastases and being implicated in the progression of non-small cell lung cancer.     

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8-30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.     

Importance of vascular phenotype by basic fibroblast growth factor, and influence of the angiogenic factors basic fibroblast growth factor/fibroblast growth factor receptor-1 and ephrin-A1/EphA2 on 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.     

Expression and prognostic role of MMP2, MMP9, MMP13, and MMP14 matrix metalloproteinases in sinonasal and oral malignant melanomas

Sinonasal and oral malignant melanomas are rare malignancies accounting for less than 2% of all melanomas. Matrix metalloproteinases (MMPs) are proteolytic enzymes required for extracellular matrix degradation in a variety of physiological and pathologic processes including wound healing, embryogenesis, tumor invasion, and metastases. We studied the correlation between expression of MMPs, nucleolar diameter of melanoma cells, different clinical and histologic parameters, and patient's outcome. Seventeen cases of sinonasal and oral malignant melanoma were studied. The expression of MMP2, MMP9, MMP13, and MMP14 was assessed immunohistochemically on paraffinized sections and measured by computer morphometry as well as silver-stained nucleolar diameter. A significant correlation was found between MMP2 and MMP14 expression and patient's outcome. Greater overall survival was seen in patients with average MMP2 expression less than 8000 microm(2)/x20 high-power field (P = .016). In patients with negative MMP14 staining, survival rate by the end of the follow-up was 38% compared with patients with positive MMP14 staining where survival rate was 0 (P = .03). A correlation with age at onset was also found; patients younger than 66 years had better overall survival rates than patients aged 66 years or older (P = .03). The maximal nucleolar diameter (MaxND) was another parameter that significantly correlated with clinical outcome. Patients with MaxND of 8 microm or larger showed a significant worse prognosis compared with the group with MaxND less than 8 microm (P = .0009). Our pilot study demonstrates that MMP2, MMP14, MMP9, and MaxND might be used as prognostic markers in patients with sinonasal and oral malignant melanoma.     

Neovascularization of poly(ether ester) block-copolymer scaffolds in vivo: long-term investigations using intravital fluorescent microscopy

Poly(ether ester) block-copolymer scaffolds of different pore size were implanted into the dorsal skinfold chamber of balb/c mice. Using intravital fluorescent microscopy, the temporal course of neovascularization into these scaffolds was quantitatively analyzed. Three scaffold groups (diameter, 5 mm; 220-260 thickness, microm; n = 30) were implanted. Different pore sizes were evaluated: small (20-75 microm), medium (75-212 microm) and large pores (250-300 microm). Measurements were performed on days 8, 12, 16, and 20 in the surrounding normal tissue, in the border zone, and in the center of the scaffold. Standard microcirculatory parameters were assessed (plasma leakage, vessel diameter, red blood cell velocity, and functional vessel density). The large-pored scaffolds showed significantly higher functional vessel density in the border zone and in the center (days 8 and 12) compared with the scaffold with the small and medium-sized pores. These data correlated with a larger vessel diameter and a higher red blood cell velocity in the large-pored scaffold group. Interestingly, during the evaluation period the microcirculatory parameters on the edge of the scaffolds returned to values similar to those found in the surrounding tissue. In the center of the scaffold, however, neovascularization was still active 20 days after implantation. Plasma leakage and vessel diameter were higher in the center of the scaffold. Red blood cell velocity and functional vessel density were 50% lower than in the surrounding tissue. In conclusion, the dorsal skinfold chamber model in mice allows long-term study of blood vessel growth and remodeling in porous biomedical materials. The rate of vessel ingrowth into poly(ether ester) block-copolymer scaffolds is influenced by pore size and was highest in the scaffold with the largest pores. The data generated with this model contribute to knowledge about the development of functional vessels and tissue ingrowth into biomaterials.     

Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression.

Angiogenesis and lymphangiogenesis are essential for breast cancer progression and are regulated by vascular endothelial growth factors (VEGF). To determine clinical and molecular correlates of these processes, we measured blood and lymphatic vascular microvessel density in 29 invasive carcinomas (22 ductal, six lobular, one papillary), using the vascular marker CD31 and the novel lymphatic marker D2-40. Microvessel density was assessed microscopically and by image cytometry, and was compared with tumor histology, grade, stage, lymph node metastasis, hormone receptors, HER2/neu status, and expression of VEGF, VEGF-C and VEGF-D by immunohistochemistry or quantitative RT-PCR. Strong correlation was observed between visual and image cytometric microvessel density using D2-40 but not CD31 (P=0.016 and 0.1521, respectively). Image cytometric CD31 microvessel density correlated with tumor size, grade, stage and lymph node metastasis (P=0.0001, 0.0107, 0.0035 and 0.0395, respectively). D2-40 microvessel density correlated with tumor stage (P=0.0123 by image cytometry) and lymph node metastasis (P=0.0558 by microscopy). Immunohistochemical VEGF signal in peritumoral blood vessels correlated with image cytometric CD31 and D2-40 microvessel density (P=0.022 and 0.0012, respectively), consistent with the role of VEGF in blood and lymphatic vascular growth. Intratumoral VEGF-C and VEGF-D expression by quantitative RT-PCR correlated with D2-40 (P=0.0291 by image cytometry) but not with CD31 microvessel density, which could suggest a selective role of VEGF-C and VEGF-D in lymphangiogenesis. CD31 and D2-40 microvessel density correlated significantly with several prognostic factors, including lymph node metastasis. Thus, measurements of angiogenesis and lymphangiogenesis may have utility for breast cancer pathology, particularly for estimation of metastatic risk.     

Morphometric microvascular characteristics predict prognosis in superficial and invasive bladder cancer

Recent research has shown that neovascularization, quantitated by microvessel density (MVD), constitutes a strong prognostic indicator in patients with invasive urothelial carcinomas. These studies, however, have focused only on MVD as the only factor reflecting angiogenesis in transitional-cell carcinomas (TCCs). The objective of this report was to evaluate multiple morphometric microvascular characteristics besides MVD in superficial and muscle-invasive TCCs separately, to provide a better approach to the relationship between angiogenesis, clinicopathological parameters, and prognosis. Histologic sections from 115 TCCs [35 superficial (T1) and 80 muscle-invasive] were immunostained for CD31 and evaluated using image analysis for the quantitation of MVD, area, total vascular area, major axis length, minor axis length, perimeter, compactness, shape factor, and Feret diameter. Patients were followed-up until death (n=31) or for an average of 42.2 months (median 38.5 months). MVD increased with progressing T category (P=0.049) but area (P=0.033), major axis length (P=0.022), perimeter (P=0.043), and Feret diameter (P=0.042) were highest in T2 tumors. Area was the single independent predictor of adverse significance in T1 TCCs, whereas for muscle-invasive tumors, survival was independently predicted by MVD. Regarding disease-free survival in superficial tumors, the single significant independent parameter was compactness, whereas area was an independent favorable indicator of disease-free survival for patients with invasive TCCs. It is concluded that the prognostic significance of neovascularization is better assessed by area and shape-related morphometric characteristics, whereas MVD becomes influential only with regard to overall survival of patients with invasive tumors.     

Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival

Malignant melanomas of the skin are distinguished by their propensity for early metastatic spread via lymphatic vessels to regional lymph nodes, and lymph node metastasis is a major determinant for the staging and clinical management of melanoma. However, the importance of tumor-induced lymphangiogenesis for lymphatic melanoma spread has remained unclear. We investigated whether tumor lymphangiogenesis occurs in human malignant melanomas of the skin and whether the extent of tumor lymphangiogenesis may be related to the risk for lymph node metastasis and to patient survival, using double immunostains for the novel lymphatic endothelial marker LYVE-1 and for the panvascular marker CD31. Tumor samples were obtained from clinically and histologically closely matched cases of primary melanomas with early lymph node metastasis (n = 18) and from nonmetastatic melanomas (n = 19). Hot spots of proliferating intratumoral and peritumoral lymphatic vessels were detected in a large number of melanomas. The incidence of intratumoral lymphatics was significantly higher in metastatic melanomas and correlated with poor disease-free survival. Metastatic melanomas had significantly more and larger tumor-associated lymphatic vessels, and a relative lymphatic vessel area of >1.5% was significantly associated with poor disease-free and overall survival. In contrast, no differences in the density of tumor-associated blood vessels were found. Vascular endothelial growth factor and vascular endothelial growth factor-C expression was equally detected in a minority of cases in both groups. Our results reveal tumor lymphangiogenesis as a novel prognostic indicator for the risk of lymph node metastasis in cutaneous melanoma.     

Primary acquired melanosis of the conjunctiva

Forty-one cases of conjunctival primary acquired melanosis (PAM) were studied to determine the frequency of progression to malignant melanoma and to establish prognostic parameters for progression to melanoma. Two subdivisions were identified: lesions with cytologically atypical melanocytes (PAM with atypia, 28 lesions, 68.3 per cent) and those lacking cytologically atypical melanocytes (PAM without atypia, 13 lesions, 31.7 per cent). None of the lesions of PAM without atypia progressed to melanoma. Thirteen of the 28 lesions of PAM with atypia (46.4 per cent) progressed to melanoma. Progression to melanoma was more frequent in the lesions of PAM with atypia if basilar hyperplasia was not the dominant histologic pattern (90 per cent progression, P = 0.02) or if any epithelioid cells were present (75 per cent progression, P = 0.02). It was not possible to determine which lesions were atypical on the basis of clinical appearance. Lesions at risk for the development of melanoma should be totally extirpated.     

Radiogenic lymphangiogenesis in the skin

The time course of microvascular changes in the environment of irradiated tumors was studied in a standardized human protocol. Eighty skin biopsies from 40 patients with previously treated primary breast cancer were taken from irradiated skin and corresponding contralateral unirradiated control areas 2 to 8 weeks, 11 to 14 months, or 17+ months after radiotherapy (skin equivalent dose 30 to 40 Gy). Twenty-two biopsies of 11 melanoma patients who had undergone lymph node dissection were used for unirradiated control. We found an increase of total podoplanin(+) lymphatic microvessel density resulting mainly from a duplication of the density of smallest lymphatic vessels (diameter <10 microm) in the samples taken 1 year after radiation. Our findings implicate radiogenic lymphangiogenesis during the 1st year after therapy. The numbers of CD68(+) and vascular endothelial growth factor-C(+) cells were highly elevated in irradiated skin in the samples taken 2 to 8 weeks after radiotherapy. Thus, our results indicate that vascular endothelial growth factor-C expression by invading macrophages could be a pathogenetic route of induction of radiogenic lymphangiogenesis.     

Expression and possible role of hPTTG1/securin in cutaneous malignant melanoma.

Human pituitary tumour-transforming gene 1 or hPTTG1 is a proto-oncogene that codes for securin, a protein involved in sister chromatid separation. Based on previous microarray data, we studied the expression of hPTTG1/securin in melanocytic lesions. In contrast to nevi and radial growth phase melanomas, securin was expressed by scattered cells in the vertical growth phase, suggesting a role in tumour progression. In a series of 29 nodular and 29 superficial spreading melanomas, matched for all histological prognostic parameters, securin expression was significantly correlated with the nodular subtype (P=0.018) and not related to thickness. In other cancers, hPTTG1 is involved in various oncogenic pathways, including induction of neovascularisation and aneuploidy, and inhibition of p53 activity. We found coexpression of securin with wild-type p53 in the same neoplastic cells in a minority of melanomas. Expression of securin was significantly correlated with the extent of aneuploidy but not with basic fibroblast growth factor immunoreactivity or microvessel density. DNA cytometry revealed that nuclei-overexpressing securin frequently showed tetraploidy or aneuploidy. Our data show that hPTTG1 is frequently overexpressed in nodular melanoma, and suggest that hPTTG1 may act as an oncogene in the vertical growth phase, either by inhibiting anaphase, thereby causing aneuploidy and genomic instability, or by modulating the function of p53, thereby impairing apoptosis.     

Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes.

Cutaneous melanoma is a common melanocytic neoplasm that can quickly metastasize to regional lymph nodes. Currently, prognosis is determined by measuring tumor thickness but more reliable markers for metastatic spread are urgently needed. We investigated whether the extent of tumor lymphangiogenesis can predict melanoma metastasis to sentinel lymph nodes. We quantified the extent of tumor lymphangiogenesis, as well as other factors, in excised primary tumors and in sentinel lymph node biopsy samples from 45 patients with primary cutaneous melanoma. The results were correlated with histological and clinical outcome. Primary melanomas from patients whose tumors had metastasized to the sentinel lymph nodes contained prominent 'hot spots' of increased lymphatic vessel density, compared to nonmetastatic tumors. Multivariate risk analysis revealed that the lymphatic vascular area of primary melanomas, an index of tumor lymphangiogenesis, was the most sensitive prognostic marker for sentinel lymph node metastasis, and was even able to more accurately predict which tumors were metastatic to sentinel lymph nodes than the currently used method of measuring tumor thickness. Highly lymphangiogenic melanomas maintained their lymphangiogenic activity after metastasis to the sentinel lymph node. The extent of tumor lymphangiogenesis is a highly sensitive (83%) and specific (89%) prognostic marker of lymph node metastasis. Assessment of lymphangiogenesis in primary melanomas may be a more effective approach than the currently used technique of measuring tumor thickness in selecting patients with early metastatic disease for aggressive therapy.     

Microvessel morphology and vascular endothelial growth factor expression in human colonic carcinoma with or without metastasis

We quantified microvessel morphology and vascular endothelial growth factor (VEGF) expression in human colonic carcinoma with or without metastasis. The cancerous growth and the noncancerous section of surgical specimens from 36 patients with colorectal carcinoma (14 without metastasis and 22 with metastasis) were studied. Tissue slices immunostained with CD34 were processed for microvessel counts (per mm(2)), the mean diameter of microvessels (microm), and the mean spatial direction of microvessels (degree), defined by the angle between the longitudinal axis of microvessels and the direction perpendicular to the surface of the mucosa. Tissue slices immunostained with anti-VEGF antibody were processed for total epithelial cell counts (per mm(2)), VEGF-positive cell counts (per mm(2)), and VEGF-positive ratio (%). Carcinoma without metastasis had significantly larger microvessel counts (213 +/- 77, p < 0.01), larger microvessel diameter (7.99 +/- 1.77, p < 0.05), and larger spatial direction (47.2 +/- 8.3, p < 0.01) than normal tissue (144 +/- 49 for microvessel counts; 7.03 +/- 0.90 for microvessel diameter; 39.5 +/- 6.6 for spatial direction). Compared with carcinoma without metastasis, carcinoma with metastasis had a significantly larger microvessel diameter (9.75 +/- 2.65, p < 0.03) and lower microvessel counts (180 +/- 92, p = 0.51). Carcinoma without metastasis had a significantly larger VEGF-positive cell count (1276 +/- 805, p < 0.05) and larger VEGF-positive ratio (53.6 +/- 39.3, p < 0.05) than normal tissue (571 +/- 553 for VEGF-positive cell counts; 24.6 +/- 23.2 for VEGF-positive ratio). Carcinoma with metastasis had a significantly lower total cell count (1443 +/- 237, p < 0.001) and lower VEGF-positive cell count (716 +/- 463, p < 0.05) than carcinoma without metastasis. With tumor progression, microvessel diameter significantly increased and microvessel counts decreased, which can be in part explained by VEGF expression. The microvessel diameter seems to be the dominant parameter responsible for cancer cell intravasation as the first step of metastasis.     

Vasculogenic mimicry has no prognostic significance in pT3 and pT4 cutaneous melanoma

The concept of vasculogenic mimicry has been introduced to define periodic acid-Schiff (PAS)-positive channels and loops lined by tumor cells, instead of endothelium, able to contribute to microcirculation in uveal melanomas. Previous studies have shown that the PAS-positive patterns are associated with a poor prognosis in uveal melanoma. The aim of the current study was to investigate whether vasculogenic mimicry has a prognostic impact in pT3 and pT4 cutaneous melanoma. Fifteen patients with pT3 and pT4 cutaneous melanoma who did not experience progression after 10 years of follow-up and 30 matched controls who underwent progression were selected. Tumor sections were stained with PAS reaction, omitting the nuclear counterstaining. For immunohistochemistry, sections were stained with CD31, CD105 (endoglin), and laminin. Differences in the distribution of the PAS-positive patterns and a series of clinicopathological variables were evaluated by the Pearson chi(2) and Mann-Whitney U tests. We observed PAS-positive linear sheets, arcs, elliptical loops, and networks encircling roundish to oval aggregates of melanoma cells. The overall distribution of the PAS-positive patterns did not match with the blood microvessels' architecture as detected by immunohistochemical analysis. No statistically significant differences in the distribution of PAS-positive patterns were found between cases and controls. The presence of a parallel pattern correlated significantly with thickness (P = 0.04), whereas an inverse correlation was found with vessel area (P = 0.05). In conclusion, our results suggest that there is a mismatch between vasculogenic mimicry and tumor angiogenesis and do not support any prognostic role of vasculogenic mimicry in thick cutaneous melanoma.     

DEK expression in melanocytic lesions

The diagnosis of malignant melanoma presents a clinical challenge and relies principally on histopathological evaluation. Previous studies have indicated that increased expression of the DEK oncogene, a chromatin-bound factor, could contribute to the development of melanoma and may be a frequent event in melanoma progression. Here, we investigated DEK expression by immunohistochemistry in a total of 147 melanocytic lesions, including ordinary nevi, dysplastic nevi, Spitz nevi, melanoma in situ, primary invasive melanomas, and metastatic melanomas. Most benign nevi (ordinary, dysplastic, and Spitz nevi) were negative or exhibited weak staining for DEK, with only 4 of 49 cases showing strong staining. Similar to benign nevi, melanoma in situ also demonstrated low levels of DEK expression. In contrast, the expression of DEK in primary invasive melanomas was significantly higher than benign nevi (P < .0001). Moreover, DEK expression was significantly increased in deep melanomas (Breslow depth >1 mm) and metastatic melanomas as compared with superficial melanomas (Breslow depth ≤1 mm) (P < .05). Our findings indicate that DEK overexpression may be a frequent event in invasive melanomas, and further augmentation of DEK expression may be associated with the acquisition of ominous features such as deep dermal invasion and metastasis. These data suggest a role of DEK in melanoma progression.     

Contribution of the microvessel network to the clonal and kinetic profiles of adrenal cortical proliferative lesions.

Monoclonal adrenocortical lesions have been characterized by an inverse correlation between proliferation and apoptosis, and polyclonal lesions show a direct correlation. Their relationship with the vascular pattern remains unknown in adrenocortical nodular hyperplasias (ACNHs), adenomas (ACAs), and carcinomas (ACCs). We studied 20 ACNHs, 25 ACAs, and 10 ACCs (World Health Organization classification criteria) from 55 women. The analysis included X-chromosome inactivation assay (on microdissected samples), slide and flow cytometry, and in situ end labeling. Endothelial cells were stained with anti-CD31, and the blood vessel area and density were quantified by image analysis in the same areas. Appropriate tissue controls were run in every case. Regression analyses between kinetic and vascular features were performed in both polyclonal and monoclonal lesions. Polyclonal patterns were observed in 14 of 18 informative ACNHs and 3 of 22 informative ACAs, and monoclonal patterns were seen in 4 of 18 ACNHs, 19 of 22 ACAs, and 9 of 9 ACCs. A progressive increase in microvessel area was observed in the ACNH-ACA-ACC transition but was statistically significant between benign and malignant lesions only (191.36 +/- 168.32 v 958.07 +/- 1279.86 microm(2); P < .0001). In addition, case stratification by clonal pattern showed significant differences between polyclonal and monoclonal benign lesions; 6% of polyclonal and 57% of monoclonal lesions had microvessel area >186 microm(2) (P = .0000008). Monoclonal lesions showed parallel trends (but with opposite signs) for microvessel area and density in comparison with proliferation and apoptosis, whereas polyclonal lesions showed inverse trends. In conclusion, the kinetic advantage of monoclonal adrenal cortical lesions (increased proliferation, decreased apoptosis) is maintained by parallel increases in microvessel area and density.     

Increased expression of stem cell markers in malignant melanoma.

The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P=0.005), CD133 (P=0.003), and nestin (P=0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P=0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P=0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.