Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

Clinical background

Retinoblastoma is the most common intraocular malignancy in children and is the prototype inherited cancer predisposition syndrome. About 60% of new patients exhibit unilateral ocular involvement with familial inheritance pattern. The remaining patients have a heritable form of retinoblastoma, which is often associated with bilateral ocular involvement, germline transmission to offspring, and second primary tumors.1 In most retinoblastoma families, the penetrance (the proportion of individuals with a germline mutation in the RB gene who develop clinical manifestations of the disease) is about 90%.2 However, about one in seven families will exhibit reduced penetrance as low as 30–60%. This chapter will focus on features that are specific to low-penetrance retinoblastoma (Box 49.1). The features of retinoblastoma in general are covered in Chapter 48.

The diseased-eye ratio (DER), calculated by dividing the number of eyes containing tumors by the number of mutation carriers in a family, has been devised as a means of more precisely defining low-penetrance families.3 A DER less than 1.5 is consistent with low-penetrance retinoblastoma. Retino­blastoma patients in low-penetrance families will often exhibit reduced expressivity (the extent to which an affected individual expresses the disease phenotype), such as unilateral ocular involvement, fewer ocular tumors, and benign retinal tumors called retinomas or retinocytomas.2,4–6 The clinical appearance of retinoblastoma in low-penetrance patients is indistinguishable from that in full-penetrance retinoblastoma patients, which is described in Chapter 48.

retinoblastoma, is thought to be mutation of the retinoblastoma (RB) tumor suppressor gene on chromosome 13q. The existence of a putative tumor suppressor gene responsible for retinoblastoma was first suggested by Knudson in 1971,7 and confirmed by the discovery of the RB gene.8–10 RB gene mutation can be demonstrated in the vast majority of retinoblastomas, as well as some other types of cancer.11,12 The mechanisms by which the RB gene is mutated, and how this leads to retinal tumors, is covered in more detail in Chapter 48.

Pathophysiology

Most retinoblastoma families demonstrate autosomaldominant inheritance with almost complete penetrance and high expressivity, due to the transmission of an inactive copy of the RB gene and subsequent loss of the remaining copy in somatic retinal cells. However, about one in seven families displays decreased penetrance and reduced expressivity. Low-penetrance retinoblastoma has been recognized for many years,2 and various mechanisms have been proposed, including immunologic factors, DNA methylation, epigenetic mechanisms, delayed mutation, host resistance factors, a second retinoblastoma locus, and “modulator genes.”2,13–16 However, recent advances in our understanding of the structure and function of the retinoblastoma protein (pRB) have shown that low-penetrance retinoblastoma results from special types of mutations at the RB gene that result in a reduced amount or activity of pRB.

Pathology

The pathologic appearance of retinoblastomas in lowpenetrance patients is indistinguishable from that in fullpenetrance retinoblastoma patients, which is described in Chapter 48.

Etiology

The rate-limiting event in the development of lowpenetrance retinoblastoma, like that of full-penetrance

Genetic mechanisms of low-penetrance retinoblastoma

In one of the first reports of low-penetrance retinoblastoma, a family was described that transmitted a constitutional chromosomal deletion involving the RB gene locus at chromosome 13q14 with unaffected carriers retaining a balanced insertional translocation.17 Although chromosomal rearrangements now appear to be a rare cause of low-penetrance retinoblastoma, this study was important in demonstrating that low-penetrance retinoblastoma could be caused by alterations at the RB locus without invoking other nongenetic mechanisms. Subsequently, it was suggested that

low-penetrance retinoblastoma may be caused by RB gene mutations that result in a “weak” copy (or allele) of the RB gene that can partially suppress tumorigenesis. In this theory, as long as one normal allele is present, an individual carrying a weak allele would be protected from retinoblastoma. In this same individual, a developing somatic retinal cell that lost the normal allele, such as by nondisjunction, and duplicated the weak allele, such as by reduplication or mitotic recombination, would have a low risk for retinoblastoma. However, if the normal allele is lost and the weak allele is not duplicated, the risk for retinoblastoma would be high (Figure 49.1). This theory appears to explain most lowpenetrance retinoblastoma. Approximately 60% of second hits that would be tumorigenic with full-penetrance mutations would have a low risk for causing tumors with a weak, or low-penetrance, mutation.

In recent years, the molecular nature of many of these “weak” RB gene alleles has been elucidated (Box 49.2). At least 14 different RB gene mutations have now been described in low-penetrance retinoblastoma families (Tables 49.1 and 49.2).3,6,16,18–21 These mutations fall into two functional classes: (1) mutations that reduce the level of expression of normal pRB; and (2) mutations that result in a mutant pRB that is partially inactivated. There may also be mutations that both reduce protein levels and partially inactivate the protein,22 although these have not yet been convincingly proven.

Type 1 mutations that reduce the expression of normal retinoblastoma protein

Type 1 mutations that cause a reduction in the amount of functional pRB are less common than type 2 mutations and fall into two main subtypes: promoter mutations and splice site mutations. Promoter mutations presumably reduce the amount of RB mRNA produced by perturbing the interaction

between the transcriptional machinery and the promoter. For example, low-penetrance point mutations have been found in the binding sites for transcription factors, such as SP1 and ATF,18,20 both of which are known to be required for normal pRB expression.23 Another form of type 1

RB WT

First hit

Figure 49.1  Chromosomal events leading to tumorigenesis in retinoblastoma according to the Knudsen “two-hit” hypothesis.7 The first “hit” or mutation of the RB gene occurs either in the germline or in a somatic cell (i.e., retinoblast). The second hit, which is always a somatic event, disrupts the remaining RB allele by one of the indicated mechanisms (their approximate frequencies in retinoblastoma tumors are indicated).61 The predicted result of the two hits (high versus low tumor risk) is indicated when the first hit involves a full-penetrance versus a low-penetrance mutation. These predictions are based on the assumption that two copies of a low-penetrance mutation may be sufficient to suppress tumorigenesis. Approximately 60% of second hits that are tumorigenic with full-penetrance mutations may not cause tumors with low-penetrance mutations. RB, chromosome 13q bearing mutant copy of RB; WT, chromosome 13q bearing wild-type (normal) copy of RB gene.

Box 49.1  Low-penetrance retinoblastoma

•Pathologically indistinguishable from full-penetrance retinoblastoma

•Displays decreased penetrance and expressivity

•Caused by distinct types of mutations in the retinoblastoma gene

Box 49.2  Low-penetrance retinoblastoma mutations

•Type 1 mutations result in decreased production of normal retinoblastoma protein

•Type 2 mutations result in the production of partially inactivated retinoblastoma protein

Box 49.3  Structure of the retinoblastoma protein

•Pocket domain contains binding sites for chromatin remodeling proteins and E2F family transcription factors

•Carboxy terminal region contains regulatory phosphorylation sites, binding sites for oncoproteins such as Hdm2 and c-abl

•Amino terminal region is less well characterized and contains binding sites for proteins such as MCM7, EID-1, and RbK

mutation occurs when a mutation within a splice site reduces the efficiency of mRNA splicing and/or inconsistent intron excision. These mutations result in a reduced amount of correctly spliced mRNA and, consequently, a reduced amount of protein. An example of this is a G to A transition that is seen in the last base of exon 21 which causes a reduction in the match of the exon boundary acceptor site to the consensus.6 This change from the consensus results in a 90% reduction in the production of normal mRNA.

Type 2 mutations that partially inactivate the retinoblastoma protein

Type 2 mutations partially inactivate the pRB protein by affecting the coding region of the protein. These mutations are usually small in-frame deletions or missense mutations (single nucleotide changes that do not result in a premature stop codon) that have subtle effects on the overall structure and function of the protein. A better understanding of the effects of type 2 mutations can be gained by understanding the detailed structure and function of each region of the pRB protein.

Structure of the retinoblastoma protein

The retinoblastoma gene encodes a 105-kDa nuclear phosphoprotein made up of three major regions: the amino terminus, the pocket domain, and the carboxy terminus (Box 49.3; Figure 49.2). pRB is able to function as a tumor suppressor primarily through its ability to inhibit the cell cycle at the G-to-S transition (Figure 49.3). This function of pRB is due to its ability to interact with, and inhibit, the transcription of cell cycle genes through the E2F family of transcription factors.24 pRB interacts with E2Fs through both its pocket domain and its carboxy terminus, so mutations in either of these regions could affect tumor suppressor function.

E2F

HDAC

BRG1

SUV39H1

HPC2

Cell cycle control

Differentiation

MyoD

C/EBP

CBFA1

Figure 49.3  The complex role of the retinoblastoma protein (pRB) in tumor suppression. Initially, pRB was noted to inhibit cell cycle progression through binding to proliferative factors such as E2F. However, binding of pRB to chromatin remodeling proteins, such as histone deacetylases (HDAC), SWI/SNF ATPases (e.g., BRG1), histone methyltransferases (e.g., SUV39H1), and polycomb proteins (e.g., HPC2), is also critical for its ability to control the cell cycle. In addition, pRB can suppress tumorigenesis by inducing differentiation, which appears to be linked to its ability to interact with differentiation factors such as myoD (muscle differentiation), C/EBP (adipocyte differentiation), and CBFA1 (bone differentiation). Some low-penetrance mutations appear to result in the production of a mutant pRB that cannot control the cell cycle but can still induce differentiation.

Interaction with E2Fs directly blocks their transactivation domain, but pRB also suppresses transcription by recruiting chromatin remodeling proteins, such as histone deacetylases, SWI/SNP ATPases, DNA methylases, histone methylases and polycomb proteins, to remodel local chromatin into an inactive, closed state.24–28 The tumor suppressor function of pRB may also derive from its ability to cooperate with differentiation factors, such as myoD, C/EBP, and CBFA1, to promote terminal differentiation and cell cycle exit, which protects cells from malignant transformation (Figure 49.3).29,30 Similarly, pRB can bind and inhibit differentiation inhibitors such as Id2.31

The pocket domain

The pocket domain (amino acids 379–792) is perhaps the most well-studied region of pRB, since much of its tumor suppressor activity maps to this domain. The pocket is made up of two highly conserved regions known as the A and B boxes, which are separated by a less conserved spacer region.32 The importance of the pocket domain is due, at least in part, to its ability to bind to many different proteins, some of which contain an LxCxE motif (e.g., HDACs, BRG1, EID-1, and the viral oncoproteins simian virus-40 large T antigen, human papillomavirus E7, and adenovirus E1A).

The binding site within the pocket for the LxCxE-containing proteins is made up of at least five highly conserved amino acids that are separated within the linear polypeptide sequence.32 The crystal structure of the pocket bound to the LxCxE-containing protein E1A showed that there were as many as 20 residues within the pocket that were mediating the interaction.33 Mutations in any of these or surrounding residues could potentially reduce binding efficiency and, thus, inhibit key functions of pRB. Although the majority of the key residues for these interactions are located in the B box, mutations in the A box could also potentially disrupt binding of the B box to other proteins, since the A and B box exhibit a complex interaction with each other that is required for proper conformation and function of the B box.34

One low-penetrance mutation has been shown to involve a missense nucleotide alteration that affects Cys712 in the LxCxE-binding site.23,35 The residue directly next to this mutation site, Lys713, is one of the key residues in the LxCxE-binding site.32 Mutations in this site result in reduced binding to HDAC and E2F.26,36

The pocket domain is also responsible for binding nonLxCxE proteins, such as E2Fs, which bind to the pocket domain at a highly conserved groove that is formed at the interface between the A and B boxes.37 When key residues within this groove are mutated, the resultant pRB protein has a marked reduction in binding affinity for E2F1.37 The crystal structure of pRB bound to E2F1 showed a total of 17 pocket residues involved in their interaction,38 and some of these residues include the binding sites for LxCxE proteins.33

One mutation seen in low-penetrance families involves amino acid substitution at Arg661.3,36,39–42 This residue is in exon 20 within the B box of the pocket and is in close proximity to Lys653, which is located within the A–B groove and is critical for E2F1 binding. The Arg661 mutant protein can interact through hydrogen bonds with residues in the A box, which may help to stabilize the A–B interface, yet it can be inactivated in a temperature-dependent manner.36,40,43 This suggests that the mutant protein is not completely inactive but is subject to subtle changes in structure that affect binding efficiency.

The carboxy terminal region

In addition to the pocket domain, the carboxy terminus (amino acids 793–928) also plays an important role in the tumor suppressor activity of pRB.43 This region contains binding sites for the oncoproteins Hdm2 and c-abl,44,45 seven phosphorylation sites that regulate protein activity, and docking sites for cyclin-dependent kinases that phosphorylate pRB.46 This region also contains a nuclear localization signal that is critical for pRB to reach the nucleus, where its function is carried out.23 The carboxy terminus also contains sites that are important for binding to E2Fs.47,48

One example of a low-penetrance mutation within this region is an in-frame deletion of exon 24 and 25, which removes amino acids 830–887.16 This mutant protein lacks one of the two E2F-binding sites, as well as the binding site for HDM2 and c-abl, and the nuclear localization signal. This mutant protein is unable to bind HDM2 and has reduced binding to E2F, but still has some residual nuclear

Conclusions

localization due to sequences within the pocket that can mediate nuclear entry.49

The amino terminal region

The amino terminal region of pRB (amino acids 1–378) has largely been ignored in functional studies of pRB, but a recent crystal structure of the region has helped to shed light on its potential importance. The domain is made up of tandem cyclin-like folds that form two lobes that are reminiscent of the pocket region of pRB.50 There are also binding sites for several proteins, including a replication licensing factor (MCM7),51 an E1A-like inhibitor of differentiation (EID-1),50 and a G2/M kinase (RbK).52

The amino terminus appears to interact directly with the pocket and may contribute to the overall structure and stability of the pRB protein. The crystal structure showed that the amino acids encoded by exons 4, 7, and 9 form integral parts of the core of the amino terminus, and deletion of these exons was predicted to cause misfolding and increased turnover of the protein.50 Among these three key exons that make up the core of the amino terminus, two have been shown to be mutated in low-penetrance retinoblastoma. One of these is a mutation that causes an in-frame deletion of exon 4 that removes the G2/M kinase binding site.21 The mutant protein retains some tumor suppressor functions, but it would be predicted to be unstable and to have decreased affinity for its binding partners.40,41 Another lowpenetrance mutation in the amino terminus involves residues encoded by exon 9.42,53–55 This mutation affects the polypyrimidine tract of the 5’ acceptor splice site of intron 8, causing an in-frame deletion in exon 9. As this is another of the key domains in the amino terminus, the deletion of exon 9 would be predicted to have detrimental effects on tertiary protein structure and binding affinity.

Conclusions

Low-penetrance retinoblastoma mutations have yielded important new insights into the molecular structure and cellular function of pRB. These studies have already led to improved diagnostic testing and family counseling for retinoblastoma families. We propose a classification scheme modified from Otterson et al40 that accounts for virtually all low-penetrance mutations reported to date. The common theme in all of these mutations is a reduction in the quantity or quality of cellular pRB. Insufficient quantity of normal pRB may result from mutations in the promoter or splice site sequences, whereas pRB may be partially disabled by subtle mutations that reduce the stability and binding affinity of the protein. The tumor suppressor activity of pRB derives both from its ability to arrest the cell cycle and to induce differentiation. Some low-penetrance mutations appear to compromise preferentially one or the other of these functions, suggesting that regulation of the cell cycle and differentiation may play cooperative roles in tumor suppression by pRB. Further studies are needed to understand more clearly the mechanism of low-penetrance mutations and to apply this knowledge to improved care of patients with retinoblastoma.

Acknowledgments

This work was supported by grants (to JWH) from the National Eye Institute (R01 EY13169-01), Research to

Prevent Blindness, Inc., Barnes-Jewish Hospital Foundation, the Kling Family Foundation, and a training grant (to KM) from the Cancer Biology Pathway of the Siteman Cancer Center at Barnes-Jewish Hospital and Washington University.

1.Harbour JW. Retinoblastoma: pathogenesis and diagnosis. In: Char DH (ed.) Tumors of the Eye and Orbit. Philadelphia: BC Decker, 2001:253–265.

2.Matsunaga E. Hereditary retinoblastoma: delayed mutation or host resistance? Am J Hum Genet 1978;30:406–424.

3.Lohmann DR, Brandt B, Hopping W, et al. Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma. Hum Genet 1994;94: 349–354.

7.Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA 1971;68:820–823.

17.Strong LC, Riccardi VM, Ferrell RE, et al. Familial retinoblastoma and chromosome 13 deletion transmitted via an insertional translocation. Science 1981;213:1501–1503.

24.Harbour JW, Dean DC. The Rb/E2F pathway: emerging paradigms and expanding roles. Genes Dev 2000;14: 2545–2562.

32.Lee JO, Russo AA, Pavletich NP. Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7. Nature 1998;391:859–865.

35.Harbour JW. Overview of RB gene mutations in patients with

retinoblastoma. Implications for clinical genetic screening. Ophthalmology 1998;105:1442–1447.

40.Otterson GA, Chen WD, Coxon AB, et al. Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function. Proc Natl Acad Sci USA 1997; 94:12036–12040.

41.Sellers WR, Novitch BG, Miyake S, et al. Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. Genes Dev 1998;12:95–106.

Overview and clinical context

By the mid-1980s, many uveal melanoma patients were electing treatment by vision-sparing radiation therapy, avoiding surgical removal of the affected eye (enucleation). The Collaborative Ocular Melanoma Study eventually demonstrated no significant difference in survival between patients treated by radiation therapy and enucleation.1 Although fine-needle aspiration biopsies (FNAB) were used in selected cases to help to discriminate between uveal melanomas and lesions that clinically simulated melanoma (such as metastases to the eye), such procedures were not typically used to grade the melanoma, to assign risk of metastasis to the patients.

At this time, the only parameter of risk that could be assessed from tumor cells extracted by FNAB was cell type, assigned by the Callender classification.2,3 The Callender classification of uveal melanomas had been known to be strongly associated with outcome, but the assignment of risk based on the morphological assessment of cells was challenging for two reasons: (1) Callender classification had been shown to be poorly reproducible between pathologists; and (2) epithelioid cells – those cells most strongly associated with adverse outcome – were distributed heterogeneously throughout these tumors. Discrepancies between FNAB-based cytological classifications and the assignment of cell type on the subsequent enucleation were reported, and in one case, the FNAB needle track was traced through the tumor and was found to have missed a pocket of epithelioid cells, leading to an erroneous classification on the FNAB sample.3,4 Discrepancies were also reported between the more objective assignments of risk based on the morphometric measurements of nucleolar size between FNAB samples and matched enucleation specimens.

Some experts began to question why it was necessary to assign patients into risk categories if no effective treatments were available to administer to patients with metastatic uveal melanoma. Sadly, even to this day, there is no effective regimen to treat patients with metastatic uveal melanoma. The development of new classification schemes to assign risk was justified then, and is justifiable today, on the basis of

Vasculogenic mimicry

Robert Folberg and Andrew J Maniotis

two observations: (1) many patients simply want to know the risk to their life expectancy so that they can either make plans to put their affairs in order, or to make longer-term financial and personal commitments5; and (2) there is hope that with intensive research into the molecular basis of metastasis in uveal melanoma, new treatment strategies will emerge and, when this happens, it will be helpful to be able to stratify patients into risk categories to design meaningful clinical trials. Vasculogenic mimicry was therefore discovered in the context of a search to develop a noninvasive substitute for biopsy of uveal melanomas.

In 1992 a pilot study6 reported the association between death from metastatic uveal melanoma and the presence of histological patterns that stained positive with the periodic acid–Schiff (PAS) reagent. This was followed by a description of nine patterns, including the incorporation of pre-existing choroidal vessels, incomplete circles (arcs) around packets of tumor cells, arcs that bifurcated (arcs with branching), circles around packets of tumor cells (loops), back-to-back loops (three back-to-back loops were designated as networks), parallel linear patterns, and parallel patterns that cross-linked. A subsequent study of the association of these patterns and outcome disclosed that the detection of loops and networks – and the parallel with cross-linking pattern

–were associated independently with death from metastatic melanoma in multivariate statistical models.7 Most patients whose tumors lacked these patterns were long-term survivors of uveal melanoma after enucleation, and fewer than half of the patients whose tumors contained these patterns survived disease-free after enucleation. These patterns, when detected histologically, were interpreted as markers of the risk for metastasis. The association between the detection of these patterns and metastasis was confirmed by multiple independent laboratories8–10 and the patterns were subsequently identified in cutaneous melanoma11 and other cancers.12

Investigators eventually discovered a number of important associations between the detection of these patterns and other markers of metastatic behavior, including the presence of epithelioid cells, location in the ciliary body,13 monosomy 3,14 and gene expression profiles.15 Because many markers

–cytological, molecular, and cytogenetic – are distributed heterogeneously throughout tumors, a healthy skepticism

began to surface about the use of FNAB to extract material from which tumors could be graded on the basis of these markers. Thus, attention was directed to the detection of PAS-positive patterns by noninvasive means as a surro­ gate marker for more sensitive molecular and cytogenetic markers.

Two approaches were taken to image PAS-positive patterns clinically. A strong association was demonstrated between the analyses of raw radiofrequency ultrasound data and the histological detection of PAS-positive patterns,16 but the equipment to image and analyze these patterns was never made available commercially. Using indocyanine green angiography and laser scanning confocal ophthalmoscopy, perfusion channels, corresponding to the histologically detected PAS-positive patterns could be imaged clinically for tumors in the posterior pole,17 and the detection of these patterns was found to be predictive of growth of indeterminate-sized lesions in a prospective trial.18 Nevertheless, the practice of imaging tumors to detect these patterns clinically by noninvasive means never became part of the clinical ophthalmic oncology workup. However, the detection of fluid within these patterns through angiography strengthened the suspicion that PAS-positive patterns were part of the microcirculation.

Indeed, in histological sections, PAS-positive patterns were associated with blood vessels and red blood cells were detected within the patterns. It was assumed that these patterns were remodeled blood vessels, and the patterns were initially designated as “microcirculatory patterns.”19 No one had any reason to challenge this assumption until the histogenesis of these patterns was studied in vitro.

The histogenesis of PAS-positive patterning: vasculogenic mimicry

Because PAS-positive patterns were thought to represent remodeled blood vessels, the histogenesis of these patterns was first explored in vitro in co-cultures of uveal melanoma cells, endothelial cells, and fibroblasts. However, looping patterns, identical to those seen in tissue sections, were generated by highly invasive uveal melanoma cells, in the absence of endothelial cells and fibroblasts in threedimensional culture conditions. Interestingly, poorly invasive uveal melanoma cells did not generate these patterns under any culture condition, thus establishing a functional relationship between the in vitro observations and the association between the identification of these patterns in tissue sections and death from metastatic melanoma. Moreover, these patterns – formed in vitro exclusively by highly invasive uveal melanoma cells – conducted fluid after direct injection and after iontophoresis, strengthening the hypothesis that these patterns conducted fluid in vivo. The histogenesis of these patterns was described as vasculogenic mimicry20

– vasculogenic because, although these pathways do not form from pre-existing vessels, they distribute plasma and may contain red blood cells; and mimicry because the pathways are not blood vessels and merely mimic vascular function by functioning as a “fluid-conducting meshwork.”21

384

Questions asked about vasculogenic mimicry

Are vasculogenic mimicry patterns   blood vessels?

Looping PAS-positive patterns are not blood vessels. The patterns do not stain with endothelial cell markers22 and they are composed ultrastructurally of sheets of electrondense material in which tumor cells are embedded.23 The patterns are composed of laminin, fibronectin, collagens IV and VI, and possibly heparan sulfate proteoglycan.24 Threedimensional reconstructions have shown these patterns to represent sleeves of extracellular matrix material wrapped around branching cylindrical projections of melanoma cells.25 Plasma and some red blood cells are conducted by the patterned extracellular matrix which connects focally to blood vessels.23 Thus, vasculogenic mimicry patterns are not blood vessels by composition, ultrastructure, or topology, although they do conduct fluid.

Investigators have described the formation of tubes by melanoma cells and have identified tubular structures in tissue sections of melanomas and other tumors that are lined by tumor cells and not endothelial cells. One group has advanced the hypothesis that highly invasive and genetically dysregulated melanoma cells undergo transdifferentiation into an endothelial cell genotype (because of the upregulation of genes such as VE-cadherin).26 There are several challenges to this approach. First, vascular spaces in tissue may be formed by tumor cells replacing endothelial cells – a “complete” manifestation of mosaic tumor vessels. Second, even if transdifferentiation does provide a mechanism for the generation of tubes of tumor cells mimicking the appearance of blood vessels, the transdifferentiation is incomplete because tumor cells do not form cobblestone monolayers in vitro as do endothelial cells and because angiogenesis inhibitors do not block the formation of tumor cell-generated cords.23

The generation of the highly patterned fluid-conducting meshwork has been designated as vasculogenic mimicry of the patterned matrix type, and the formation of tubes by tumor cells has been called vasculogenic mimicry of the tubular type.12 The material that follows in this chapter refers only to vasculogenic mimicry of the patterned matrix type.

Are vasculogenic mimicry patterns a stromal response to the tumor (i.e., are these patterns fibrovascular septa)?

Before the histogenesis of PAS-positive patterns was described, some investigators assumed that these patterns represented fibrovascular septa.9 Even after it had been shown that tumor cells generated these patterns, some investigators persisted in describing these patterns as stromal response to the tumor.27 Although fibrovascular septa have been identified in uveal melanomas, their prevalence is low and fibrovascular septa do not have any prognostic associations with outcome.24 Perhaps the most convincing evidence in support of the tumor cell generating these patterns comes from a set of experiments in which human uveal melanoma

cells were injected into the livers of immunosuppressed mice. Polyclonal antibodies to laminin, not species-specific, labeled vasculogenic mimicry patterns generated by the tumor cells as well as mouse liver structures. However, a monoclonal antibody that was species-specific for human laminin labeled only vasculogenic mimicry patterns within the tumor and not the mouse liver. Thus, the laminin within the tumor was not co-opted from the mouse stroma and vasculogenic mimicry patterns are therefore not a stromal response by host tissue to the tumor.28

Does fluid flow through vasculogenic   mimicry patterns?

It is clear from animal model studies that intravenous tracers co-localize to vasculogenic mimicry patterns. In a recent clinical study, patients with posterior uveal melanomas were injected with indocyanine green in the antecubital vein of one arm, and shortly after injection, blood was phlebotomized from the contralateral arm while a confocal angiogram was being taken. The blood removed after injection continued to fluoresce weeks after the injection, while fluorescence within intratumoral vasculogenic mimicry patterns was extinguished within 15 minutes after injection, thus demonstrating indirectly that fluid flows through vasculogenic mimicry patterns.29 It is possible that leaky intratumoral vessels permit blood to enter into the tumor cell-generated extracellular matrix, but that once in the matrix, plasma and red blood cells circulate throughout the patterns.

What is the relationship between vasculogenic mimicry and angiogenesis?

In the early 1990s, an association was demonstrated between increased microvascular density in breast cancer and adverse outcome,30 and a large series of papers then followed demonstrating similar associations in other cancers, including uveal melanoma.31 Although it was intuitive that increased risk of metastasis should accompany angiogenesis, these associations were somewhat paradoxical because highly invasive cancers are typically destructive of the host microenvironment: by what mechanisms would new blood vessels be able to penetrate into the cellular compartment of highly malignant tumors when these tumors were simultaneously elaborating a variety of substances leading to the degradation of stroma? Indeed, a careful examination of angiogenesis in breast cancer revealed that angiogenic blood vessels were situated in the fibrous connective tissue surrounding tumor cells and were not in direct contact with the tumor cell compartment, consistent with the notion of the tumor stroma representing a form of scar tissue.32 Furthermore, when endothelial cells were co-cultured with highly invasive uveal melanoma cells, the uveal melanoma cells destroyed the endothelial cells on contact.23

It was known that highly invasive uveal melanoma cells are genetically dysregulated and express markers that are inappropriate for cells of neural crest lineage (like fetal cytokeratins33 and endothelial cells such as VE-cadherin34). Therefore, the relationship of microvascular density to

Vasculogenic mimicry as a tumor biofilm: therapeutic implications

adverse outcome in uveal melanoma was studied by doublelabeling histological sections with CD34 (a nonspecific endothelial cell marker found by one group to provide for the highest microvascular density measurements), and with S100 protein (a nonspecific marker of cells of neural crest lineage such as melanocytes that do not label vascular endothelial cells). A high level of co-expression of CD34 and S100 protein was identified, and as “microvascular density”

– as measured by CD34 labeling – increased, so did coexpression of this protein by melanoma cells. Therefore, the association between high microvascular density and metastasis in uveal melanoma may be explained on the basis of a population of highly invasive and genetically dysregulated tumor cells rather than angiogenesis.35 Aberrant expression of CD34 in cutaneous melanoma was discovered subsequently.36

The microcirculation of uveal melanoma is therefore complex, including normal choroidal vessels that are incorporated into tumors, angiogenic vessels (especially next to zones of necrosis or in tumors previously treated by radiation therapy), mosaic vessels (lined by tumor cells and endothelial cells), and vasculogenic mimicry patterns.12 Vasculogenic mimicry patterns provide at least 11-fold increased perfusion surface area in comparison to incorporated tubular vessels or angiogenic vessels.37

One might speculate that vasculogenic mimicry patterns facilitate metastasis, functioning like lymphatic channels in a location devoid of lymphatic vessels. However, there is no direct evidence implicating vasculogenic mimicry in the dissemination of tumor cells. Because vasculogenic mimicry patterns are formed by highly aggressive melanoma cells, it is possible that the detection of these patterns is merely a marker for the presence of an aggressive tumor phenotype and therefore has little or nothing to do with the actual spread of tumor.

Similarly, one might speculate that plasma and red blood cells circulating through vasculogenic mimicry patterns provide sufficient oxygen and nutrients to prevent necrosis. Most uveal melanomas lack zones of necrosis: the architecture of most uveal melanomas is far different from highly angiogenic retinoblastomas which feature zones of necrosis beyond a narrow cuff of tumor cells surrounding blood vessels. However, plasma flowing through vasculogenic mimicry patterns is not likely to be well oxygenated, and only scattered red blood cells – most often in a rouleaux formation – are identified histologically within these patterns. Is it possible, therefore, that uveal melanomas may not require a high degree of oxygenation? Might vasculogenic mimicry patterning serve a function other than perfusion or the facilitation of metastasis? Indeed, there is evidence that vasculogenic mimicry patterning regulates the behavior of tumor cells.

Vasculogenic mimicry as a tumor biofilm: therapeutic implications

When vasculogenic mimicry patterns form in vitro, some highly invasive and genetically dysregulated melanoma cells become entrapped within the extracellular matrix that they generate. Typically, spindle A melanoma cells are entrapped

within vasculogenic mimicry patterns while the patterns themselves surround packets of epithelioid melanoma cells.38 The mechanisms underlying this observation were explored in a series of in vitro studies.

In general, the chromatin of cancer cells is more sequestered than chromatin of noncancerous cells. However, laminin in the extracellular matrix microenvironment can induce chromatin sequestration of benign cells, and can increase the sequestration of chromatin in malignant cells through a novel mechanical signaling pathway mediated by the cytoskeleton (mechanogenomic signaling).39 Dramatic shifts in gene expression have been shown to accompany the sequestration of uveal melanoma cells in vitro, especially during the formation of vasculogenic mimicry patterns by the highly invasive melanoma cells that generate these patterns. Paradoxically, when vasculogenic mimicry patterns form, genes that are typically associated with invasive behavior (such as CD44) are downregulated while genes associated with differentiation and suppression of the cell cycle (such as p21) are upregulated. Indeed, after highly invasive uveal melanoma cells form vasculogenic mimicry patterns, their ability to proliferate and migrate is significantly compromised. When highly invasive epithelioid uveal melanoma cells are seeded over discrete zones of laminin that have been stenciled on to plastic surfaces, the cells retain their epithelioid morphology on plastic, but after contact with the edge of the laminin stencil, these cells elongate, the nucleolus diminishes in size, and a nuclear fold appears – morphologic evidence of a dramatic change in morphology from epithelioid to spindle A morphology. That these in vitro observations are clinically relevant is reflected in tissue microarray studies of human uveal melanoma samples in which the proliferation index (measured by Ki67 labeling) was significantly decreased in melanoma cells adjacent to vasculogenic mimicry patterns compared with melanoma cells located where vasculogenic mimicry patterning is absent.38

Therefore, in addition to any role that vasculogenic mimicry patterns may play in tumor perfusion or the dissemination of tumor cells, these patterns regulate higherorder chromatin structure, gene expression, phenotypic behavior, and morphology. The generation of extracellular matrix proteins, especially laminin, by highly invasive melanoma cells provides a microenvironment in which these cells generate vasculogenic mimicry patterns. In the formation of these patterns, tumor cells become entrapped within the matrix and revert to an indolent (spindle A) phenotype, and the proliferation rate of melanoma cells in the vicinity of these patterns decreases. Thus, paradoxically, although vasculogenic mimicry patterns are histological features associated with metastatic behavior because the generation of these patterns requires the presence of highly invasive tumor cells, the generation of these patterns dampens malignant behavior.

Thus, vasculogenic mimicry itself mimics another biological phenomenon: the formation of microbial biofilms.40 The generation of an extracellular matrix by certain microbial organisms entraps these potential pathogens and renders them phenotypically quiescent as long as they are within the biofilm. Disruption of the biofilm can disperse the organisms and increase their pathogenicity. Interestingly, the organisms in a microbial biofilm may be highly drug-resistant.

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Vasculogenic mimicry may therefore provide at least one explanation for the well-documented latency between the detection of the primary tumor and the development of clinically detectable metastases in the liver. Ophthalmic oncologists seldom encounter patients who have evidence of metastatic uveal melanoma when the primary tumor is detected, and it is well known that metastases may emerge many years after treatment of the primary tumor. The formation of vasculogenic mimicry in hepatic metastases may render these subclinical metastases biologically quiescent and it is possible that clinically relevant metastatic disease emerges when vasculogenic mimicry patterning in hepatic micrometastases is disrupted, allowing for the phenotypically indolent melanoma cells entrapped within the vasculogenic mimicry patterning to emerge from “hibernation.” There is some experimental evidence supporting this hypothesis. When primary uveal melanoma cells are placed directly into the liver of severe combined immunodeficient (SCID) mice, the earliest lesions – only a few cells in diameter – are typically encased in looping matrices rich in laminin, suggesting that vasculogenic mimicry may be an early response to the colonization of the liver by tumor cells. Over a period of time in these experiments, large masses of melanoma cells develop in the mouse liver, and in highly invasive tumors; vasculogenic mimicry patterning may not be easily identified in these large experimental tumors. However, in animals with large intrahepatic masses, secondary micrometastases to the lungs are identified, and almost without exception, these micrometastases feature looping patterns rich in laminin, characteristic of vasculogenic mimicry.28 These observations are consistent with the study of vasculogenic

Box 50.1  Vasculogenic mimicry: key points