Patterns of neural differentiation in melanomas

Background Melanomas, highly malignant tumors arise from the melanocytes which originate as multipotent neural crest cells during neural tube genesis. The purpose of this study is to assess the pattern of neural differentiation in relation to angiogenesis in VGP melanomas using the tumor as a three dimensional system. Methods Tumor-vascular complexes [TVC] are formed at the tumor-stroma interphase, by tumor cells ensheathing angiogenic vessels to proliferate into a mantle of 5 to 6 layers [L1 to L5] forming a perivascular mantle zone [PMZ]. The pattern of neural differentiation is assessed by immunopositivity for HMB45, GFAP, NFP and synaptophysin has been compared in: [a] the general tumor [b] tumor-vascular complexes and [c] perimantle zone [PC] on serial frozen and paraffin sections. Statistical Analysis: ANOVA: Kruskal-Wallis One Way Analysis of Variance; All Pairwise Multiple Comparison Procedures [Tukey Test]. Results The cells abutting on the basement membrane acquire GFAP positivity and extend processes. New layers of tumor cells show a transition between L2 to L3 followed by NFP and Syn positivity in L4&L5. The level of GFAP+vity in L1&L2 directly proportionate to the percentage of NFP/Syn+vity in L4&L5, on comparing pigmented PMZ with poorly pigmented PMZ. Tumor cells in the perimantle zone show high NFP [65%] and Syn [35.4%] positivity with very low GFAP [6.9%] correlating with the positivity in the outer layers. Discussion From this study it is seen that melanoma cells revert to the embryonic pattern of differentiation, with radial glial like cells [GFAP+ve] which further differentiate into neuronal positive cells [NFP&Syn+ve] during angiogenic tumor-vascular interaction, as seen during neurogenesis, to populate the tumor substance.


Background
Mammalian melanocytes originate as multipotent neural crest cells that detach from the neural tube to arrive at the dorsolateral surface by day 8 [1,2]. Melanomas are highly malignant tumors arising from the melanocytes, which are present primarily in the basal layer of the epidermis, but are found in various other locations such as uveal tract of the eyes, inner ear, mucous membrane, genital organs, anus and leptomeninges [3]. Cutaneous melanoma is a tumor derived from activated or genetically altered epidermal melanocytes, the result of complex interactions between genetic, constitutional, and environmental factors [4]. Malignant melanoma may arise from melanocytes in normal appearing skin, activated melanocytes of solar lentigo, or less frequently from atypical or relatively benign nevomelanocytic lesions. The incidence and mortality of cutaneous malignant melanoma has substantially increased among all Caucasian populations in the last few decades. Susceptibility to melanomas are influenced by various factors such as familial incidence, race, background, skin types and gender; constitutional factors such as age, number, size and type of pigmented nevi; accumulative and lifetime exposure to solar light [5].
The ability of melanoma cells to undergo proliferation in three dimensions is clinically known as the vertical growth phase (VGP). VGP melanoma is a highly angiogenic and proliferative lesion. Further genetic changes convert melanoma into an invasive tumor capable of three dimensional growth, increased angiogenesis, and metastasis [6,7]. The purpose of this study is to assess the pattern of neural differentiation within the tumor substance of a series of melanomas in vertical growth phase [VGP], using the tumor as a three dimensional system.

Materials and methods
A random sample of 27 nodular melanomas in the vertical growth phase [VGP], were received from the Cancer Surgery Unit fixed in 10% formol glutaraldehyde.
The formaldehyde-glutardehyde cold fixation can be used both in frozen, paraffin sections as well as electron microscopy. 10 nodules were taken from each tumor in the in the ratio of pigmented to poorly pigmented areas in the entire tumor. As the specimen were received and sampled the blocks were arranged in a grid, according to the pigment level which varied between 7% to 95% [ Figure 1].
Serial sections 5 μm thick    [9][10][11]. As negative control all slides included a serial section stained with no mAb. The same mAb were used simultaneously against known positive sections from human skin as positive controls.
Presence of pigment; a positive DOPA reaction; and HMB-45 positivity are criteria for diagnosis. In the absence of pigment a positive dopa reaction, HMB45 positivity and the presence of premelanosomes on electron microscopy is diagnostic of poorly pigmented melanomas. These criteria form the basis of diagnosing each tumor included in this study.

Immunohistochemistry
Neural marker positivity has been examined and compared in: [a] the general tumor; [b] perimantle zone [PMZ] of tumor-vascular complexes [TVC] formed during angiogenesis; [c] perimantle cells [PC] Marginal zone between the tumor and stroma were selected to study the tumor/vascular interaction during angiogenesis. 51 blocks are from pigmented and 52 from poorly pigmented nodules [ Figure 1].

Vascular counts: [Figure 2]
Vascular channels are counted at the tumor margins in each of the 103 blocks to a depth of two high power fields

Perimantle cells
The percentage immunopositive cells around the mantle zone were counted to a depth of one HPF.

Statistical Analysis
Anova Analysis: Kruskal-Wallis One Way Analysis of Variance; and Tukey Test: All Pairwise Multiple Comparison Procedures.

Pattern of Neural differentiation
The expression of neural markers [GFAP, NFP and Syn], by melanocytes in association with pigmentation and the tumor morphology has been examined in this section. It is observed that the general tumor areas differ from areas of angiogenesis where there is a patterned neural expression and melanocyte morphology.

General Tumor
There is a marked anisocytosis and anisonucleosis. Pleomorphism, increased nuclear-cytoplasmic ratio, hyperchromatin, enlarged nucleoli, abnormal mitoses and giant cells are seen. Mononucleate and multinucleate giant cells with 10-12 nuclei are also present. There is no definite pattern of neural differentiation in areas unrelated to angiogenesis.  Figure 4].

Pattern of neural differentiation in relation to Angiogenesis
The pattern of neural differentiation and cell morphology is regimented and well defined at the tumor/stroma interphase where the tumor cells interact with the neovascular angiogenic vessels. This pattern is lost within the general tumor away from the margins.

Angiogenesis: [Figure 2]
The adjacent stromal blood vessels proliferate, to extend endothelial buds which grow towards the tumor margin. These cannelise and acquire a basement membrane at the tumor margins. The blood vessels branch extensively within the tumor substance. Angiogenesis is significantly higher at the margins as quantified by counting the blood vessels [bv] at the margins and well within the tumor growth. On an average 8.18 bv/HPF are observed near the invasive margins and an average of 1.9 bv/HPF in the tumor. At the margins a maximum of 19 bv/HPF and a minimum of 5 bv/HPF are observed. In the areas of main tumor growth a maximum of 4 bv/HPF and a minimum of 0 bv/HPF are observed [ Figure 2] Thus as there is a significant difference between angiogenic vessels at the invasive margins and within the tumor, in a rapidly growing tumor the central portions recede from the margins and are deprived of vascularisation. The tumor cells interact with the angiogenic vessels at the margins to form a mantle of 5 to 6 cell layers giving a lobular or spheroid appearance.

Tumor vascular interaction: [Figure 3a-d]
A single layer of tumor cells surround the endothelial tubes and grow out into 5 to 6 concentric layers to form a compact spheroidal structure clearly demarcated from the surrounding tumor.
Pattern of neural differentiation related to neovasculature: [ Figure 3&4] The pattern of differentiation in the tumor cell layers around the angiogenic vessel, is examined for neuronal markers GFAP, NFP and Syn. Quantitation and comparison has been given below.

Discussion
Tumor growth and proliferation is not totally chaotic and uncontrolled as often misconstrued. This study provides an interesting aspect of the methods within the madness of malignant growth in melanomas. Melanomas provide a mass of cells as one sees in a 3D matrix. Analysis of the growth patterns would be of benefit for the study of embryonic growth patterns as well as for the study of stem cells. The patterns of neuronal differentiation have been detailed in this work including the localisation of neural markers [GFAP, NFP and Syn] by tumor cells in relation to pigmentation. There is a distinct difference between the general tumor matrix and areas of angiogenesis where there is a patterned neural expression and melanocyte morphology.
GFAP positivity identifies the radial glial multipotent astrocytic stem cells [MASC] during embryogenesis as described in several studies [12][13][14][15][16]. GFAP, a 50 kDa intracytoplasmic protein, constitutes the major cytoskeletal protein in astrocytes and is traditionally referred to as a specific marker for astrocytes of the CNS [13] GFAP positivity and glial differentiation is related to pigmentation and is inversely proportional to astrocytic anaplasia as is well brought out in this study [17].
Reciprocal paracrine interactions between astrocytes, endothelial cells and ependymal cells have been demonstrated in recent studies. Vascular endothelial growth factor (VEGF) is released from both astrocytes and neurons eliciting a burst of mitotic angiogenesis, which is followed by the production of brain-derived neurotrophic factor (BDNF) by the stimulated microvascular cells [25][26][27]. In foci of concurrent angiogenesis and neurogenesis, neuronal progenitor cells are spatially associated with mitotic endothelial cells, [28][29][30][31].
From this study it is observed that the melanoma cells express characteristics of radial glia, on interaction with the endothelial tubes and further proliferate and differentiate into cells positive for neuronal markers and thus resemble MASC which give rise to neuronal differentiation in neurospheres in cultures [14,.
At the tumor/stroma interphase the sprouted endothelial tubes cannelise and acquire a reticulin positive basement membrane. Initially, a single layer of tumor cells surround the vessels on the outer surface of the basement membrane. The cells abutting on the basement membrane acquire GFAP positivity and extend processes. Concentric layers of tumor cells grow out from this layer, supported by GFAP positive processes which extend outward through the layers of tumor cells towards the periphery [ Figure 3a]. Where GFAP positivity is absent there is no further proliferation. As the new layers of tumor cells grow out there is a zone where all three markers are co-localized between L2 to L3 followed by NFP and Syn positivity in L4&L5.
Neurofilaments are neuron-specific intermediate filaments which can be localized by NFP positivity for neuronal differentiation [12]. They form the dynamic axonal cytoskeleton together with other axonal components such as microtubules to maintain and regulate neuronal cytoskeletal plasticity [reviewed by Kesavapany et al, 2003] [46]. During development neuroepithelial cells in the neuronal lineage lose nestin and vimentin [47] to express NF-H when the maturing cells are forming synapses [48,49]. NFP positivity is seen in differentiated ganglion cells, neoplasms of neuronal or mixed cell origin as well as neuroendocrine tumor cells. Ramirez et al [50] found rabbit choroidal melanocytes, perivascular and intervascular fibers positive for NFP.
Synaptophysin is a vesicular integral membrane protein specifically expressed in neural tissues [51]. Synaptophysin labels small synaptic like microvesicles (SLMV) present in neuroendocrine cells such as the pituitary and adrenal medulla. Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. These vesicles characterized by synaptophysin contain considerable amounts of the biogenic amines [51,52]. Earlier studies have identified the presence of biogenic amines in melanocytes. These include catechol amines as well as indole amines [53][54][55][56][57][58].
The percentage of GFAP+vity in L1&L2 correlates with the percentage of NFP/Syn+vity in L4&L5. In the poorly pigmented PMZ the very low GFAP+vity is associated with a low NFP/Syn +vity. NFP does not increase beyond L3. This is in contrast to the pigmented PMZ where high GFAP+vity in L1/L2 is associated with a similar spike in NFP/Syn+vity in L4/L5 suggesting that the neuronal positivity results from the GFAP+vity after passing through a transitional phase. Thus in those areas where the level of differentiation is low as seen by the absence of pigment, the differentiation of the tumor cells into glial cells on interaction with the neovascular channel is low. This in turn results in low neuronal differentiation.
Immunopositivity in the immediate proximity of the PMZ in the perimantle zone reflects that of the peripheral layers of the mantle there being a very low GFAP +vity [6.9%], and high Syn [35.4%] and NFP [65%] positivity. This suggests that most GFAP +ve cells proliferate into NFP and Syn +ve cells which then populate the tumor [ Figure 5].
The sequence of progression from radial glial to neuronal positive cells in the [PMZ] simulates the differentiating patterns in invitro neurospheres and early embryogenesis of the neural tube. The astrocyte-like stem cells have the ability to generate neurons [36][37][38][39][40], while newly-generated neurons can assume or revert to an astrocytic phenotype. In differentiating primary floating neurospheres neurons can shift into cells with astrocyte characteristics by transitioning through an "asteron" (neuron/astrocyte hybrid) morphotype which coexpress a variety of neuron and astrocyte proteins and genes [42].
From this data it is seen that in melanomas which are known for pleomorphism and highly variant morphology, there is an organized pattern of differentiation as the tumor spreads and vascularises. Interaction with the neovascular angiogenic channels functions as during neurogenesis. As the single interacting cell layer proliferates into a layered mantle a wave of step wise differentiation from tumor cells to glial followed by neuronal cells positive for NFP and Syn occurs.
These cells then merge with the expanding tumor cells to populate it with GFAP, NFP and Syn +ve cells which acquire the haphazard pattern seen in the general tumor substance. This mode of patterned growth is prominent in the pigmented nodules and is low in the poorly pigmented nodules and rare in the amelanotic melanomas. Thus the more differentiated the tumor the more regimented the growth pattern.
These results show that the melanoma cells have the potential for differentiating into glial as well as neuronal cells. The formation of structured PMZ during tumor cell-vascular interaction recapitulates embryogenic neurogenesis. Melanoma cells could potentially serve as neuronal stem cells, when grown as cocultures with angiogenic/endothelial cells, since in the tumor system the regimentation is confined to the PMZ, beyond which the neoplastic cells revert to a chaotic growth pattern. Although dendritic, Syn positive cells, resembling early neurons are seen in the outer layers of the PMZ [ Figure 3d], in vitro studies are required to confirm this potential. In addition the metabolic activity of melanoma derived stem cells have to be carefully monitored.