Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

84.Myatt N, Aristodemou P, Neale MH, Foss AJE, Hungerford JL, Bhattacharya S, Cree IA. Abnormalities of the transforming growth factor-beta pathway in ocular melanoma. J Pathol 192:511–518, 2000.

85.Esser P, Grisanti S, Bartz-Schmidt KU. TGF-b in uveal melanoma. Microsc Res Tech 52:396–400, 2001.

86.Manenti S, Malecaze F, Chap H, Darbon JM. Overexpression of the myristoylated alanine-rich C kinase substrate in human choroidal melanoma cells affects cell proliferation. Cancer Res 58:1429–1434, 1998.

87.Heine B, Coupland SE, Kneiff S, Demel G, Bornfeld N, Hummel M, Stein H. Telomerase expression in uveal melanoma. Br J Ophthalmol 84:217–223, 2000.

88.Harris CC. p53 tumor suppressor gene: From the basic research laboratory to the clinic—an abridged historical perspective. Carcinogenesis 17:1187–1198, 1996.

89.Freedman DA, Wu L, Levine AJ. Functions of the MDM2 oncoprotein. Cell Mol Life Sci 55:96–107, 1999.

90.Kishore K, Ghazvini S, Char DH, Kroll S, Selle J. p53 gene and cell cycling in uveal melanoma. Am J Ophthalmol 121:561–567, 1996.

91.Janben K, Kuntze J, Busse H, Schmid KW. p53 oncoprotein overexpression in choroidal melanoma. Mod Pathol 9:267–272, 1996.

92.Tobal K, Warren W, Cooper AS, McCartney A, Hungerford J, Lightman S. Increased expression and mutation of p53 in choroidal melanoma. Br J Cancer 66:900–904, 1992.

93.Jay V, Yi Q, Hunter WS, Zielenska M. Expression of bcl-2 in uveal malignant melanoma. Arch Pathol Lab Med 120:497–498, 1996.

94.Vito P, Lacana E, D’Adamio L. Interfering with apoptosis: Calcium-binding protein ALG-2 and Alzheimer’s disease gene ALG-3. Science 271:521–525, 1996.

95.Kvanta A, Steen B, Seregard S. Expression of vascular endothelial growth factor (VEGF) in retinoblastoma but not in posterior uveal melanoma. Exp Eye Res 63:511– 518, 1996.

96.Pe’er J, Folberg R, Itin A. Vascular endothelial growth factor upregulation in human central vein occlusion. Ophthalmology 105:412–416, 1998.

97.Stitt AW, Simpson DAC, Boocock C, Gardiner TA, Murphy GM, Archer DB. Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours. J Pathol 186:306–312, 1998.

98.Vinores SA, Ku¨chle M, Mahlow J, Chiu C, Green WR, Campochiaro PA. Blood-ocular barrier breakdown in eyes with ocular melanoma—A potential role for vascular endothelial growth factor vascular permeability factor. Am J Pathol 147:1289–1297, 1995.

99.Sheidow TG, Hooper PL, Crukley C, Young J, Heathcote JG. Expression of vascular endothelial growth factor in uveal melanoma and its correlation with metastasis. Br J Ophthalmol 84:750–756, 2000.

100.IJland SA, Jager MJ, Heijdra BM, Westphal JR, Peek R. Expression of angiogenic and immunosuppressive factors by uveal melanoma cell lines. Melanoma Res 9:445–450, 1999.

101.Maniotis AJ, Folberg R, Hess A, Seftor EA, Gardner LMG, Pe’er J, Trent JM, Meltzer PS, Hendrix MJC. Vascular channel formation by human melanoma cells in vivo and in vitro: Vasculogenic mimicry. Am J Pathol 155:739–752, 1999.

102.Hess AR, Seftor EA, Gardner LMG, Carles-Kinch K, Schneider GB, Seftor REB, Kinch MS, Hendrix MJC. Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: Role of epithelial cell kinase (Eck/EphA2). Cancer Res 61:3250–3255, 2001.

103.Pasquale EB. The Eph family of receptors. Curr Opin Cell Biol 9:608–615, 1997.

104.Easty DJ, Hill SP, Hsu M, Fallowfield ME, Florenes VA, Herlyn M, Bennet DC. Upregulation of Ephrin-A1 during melanoma progression. Int J Cancer 84:494–501, 1999.

105.El-Shabrawi Y, Langmann G, Hutter H, Kenner L, Hoefler G. Comparison of current methods and PCR for the diagnosis of metastatic disease in uveal malignant melanoma. Ophthalmologica 212:80, 1998.

106.Baker JKL, Elshaw SR, Mathewman GEL, Nichols CE, Murray AK, Parsons MA, Rennie IG, Sisley K. Expression of integrins, degradative enzymes and their inhibitors in uveal melanoma: Differences between in vitro and in vivo expression. Melanoma Res 11:265–273, 2002.

107.Va¨isa¨nen A, Kallioinen M, von Dickhoff K, Laatikainen L, Ho¨yhtya¨M, TurpeenniemiHujanen T. Matrix metalloproteinase-2 (MMP-2) immunoreactive protein—A new prognostic marker in uveal melanoma? J Pathol 188:56–62, 1999.

108.Elshaw SR, Sisley K, Cross N, Murray AK, MacNeil SM, Wagner M, Nichols CE, Rennie IG. A comparison of ocular melanocyte and uveal melanoma cell invasion and the implications of [alpha]1[beta]1, [alpha]4[beta]1 and [alpha]6[beta]1 integrins. Br J Ophthalmol 85:732, 2001.

109.El-Shabrawi Y, Ardjomand N, Radner H, Ardjomand N. MMP-9 is predominantly expressed in epithelioid and not spindle cell uveal melanoma. J Pathol 194:201–206, 2001.

110.Seftor REB, Seftor EA, Koshikawa N, Meltzer PS, Gardner LMG, Bilban M, StetlerStevenson WG, Quaranta V, Hendrix MJC. Cooperative interactions of laminin 5 g2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma. Cancer Res 61:6322–6327, 2001.

111.Hewitt RE, Brown KE, Corcoran M, Stetler-Stevenson WG. Increased expression of tissue inhibitor of metalloproteinase type 1 (TIMP-1) in more tumourigenic colon cancer cell line. J Pathol 192:455–459, 2000.

112.Hernandez-Barrantes S, Toth M, Bernardo MM, Yurkova M, Gervasi DC, Raz Y, Sang QA, Fridman R. Binding of active (57kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation. J Biol Chem 275:12080– 12089, 2002.

113.de Vries TJ, Mooy CM, van Balken MR, Luyten GPM, Quax PHA, Verspaget HW, Weidle UH, Ruiter DJ, Van Muijen GNP. Components of the plasminogen activation system in uveal melanoma—A clinico-pathological study. J Pathol 175:59–67, 1995.

114.Ma D, Gerard RD, Li XY, Alizadeh H, Niederkorn JY. Inhibition of metastasis of intraocular melanomas by adenovirus-mediated gene transfer of plasminogen activator inhibitor type I (PAI-1) in an athymic mouse model. Blood 90:2738–2746, 1997.

115.Marshall JF, Rutherford DC, Happerfield L, Hanby A, McCartney ACE, NewtonBishop J, Hart IR. Comparative analysis of integrins in vitro and in vivo in uveal and cutaneous melanomas. Br J Cancer 77:522–529, 1998.

116.Ten Berge PJM, Danen EHJ, Van Muijen GNP, Jager MJ, Ruiter DJ. Integrin expression in uveal melanoma differs from cutaneous melanoma. Invest Ophthalmol Vis Sci 34:3635–3640, 1993.

117.Larouche N, Larouche K, Be´liveau A, Leclerc S, Salesse C, Pelletier G, Gue´rin SL. Transcriptional regulation of the a4 integrin subunit gene in the metastatic spread of uveal melanoma. Anticancer Res 18:3539–3548, 1998.

118.Natali PG, Bigotti A, Nicotra MR, Nardi RM, Delovu A, Segatto O, Ferrone S. Analysis of the antigenic profile of uveal melanoma lesions with anti-cutaneous melanoma-associated antigen and anti-HLA monoclonal antibodies. Cancer Res 49:1269–1274, 1989.

119.van der Pol JP, Jager MJ, De Wolff-Rouendaal D, Ringens PJ, Vennegoor C, Ruiter DJ. Heterogeneous expression of melanoma-associated antigens in uveal melanomas. Curr Eye Res 6:757–765, 1987.

120.Carrel S, Schreyer M, Gross N, Zografos L. Surface antigenic profile of uveal melanoma lesions analyzed with a panel of monoclonal antibodies directed against cutaneous melanoma. Anticancer Res 10:81–90, 1990.

121.Mooy CM, Luyten GPM, De Jong PTVM, Jensen OA, Luider TM, Van der Ham F, Bosman FT. Neural cell adhesion molecule distribution in primary and metastatic uveal melanoma. Hum Pathol 26:1185–1190, 1995.

122.Ma D, Niederkorn JY. Role of epidermal growth factor receptor in the metastasis of intraocular melanomas. Invest Ophthalmol Vis Sci 39:1067–1075, 1998.

123.Radinsky R. Molecular mechanisms for organ-specific colon carcinoma metastasis. Eur J Cancer 31:1091–1095, 1995.

124.Hurks HMH, Metzelaar-Blok JAW, Barthen ER, Zwinderman AH, De WolffRouendaal D, Keunen JEE, Jager MJ. Expression of epidermal growth factor receptor: risk factor in uveal melanoma. Invest Ophthalmol Vis Sci 41:2023–2027, 2000.

125.Scholes AGM, Hagan S, Hiscott P, Damato BE, Grierson I. Overexpression of epidermal growth factor receptor restricted to macrophages in uveal melanoma. Arch Ophthalmol 119:373–377, 2001.

126.Hendrix MJC, Seftor EA, Seftor REB, Gardner LM, Boldt HC, Meyer M, Pe’er J, Folberg R. Biologic determinants of uveal melanoma metastatic phenotype: Role of intermediate filaments as predictive markers. Lab Invest 78:153–163, 1998.

127.Hendrix MJC, Seftor EA, Seftor REB, Kirschmann DA, Gardner LM, Boldt HC, Meyer M, Pe’er J, Folberg R. Regulation of uveal melanoma interconverted phenotype by hepatocyte growth factor scatter factor (HGF/SF). Am J Pathol 152:855–863, 1998.

128.Ma¨kitie T, Summanen P, Tarkkanen A, Kivela¨ T. Tumor-infiltrating macrophages (CD68þ cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci 42:1414–1421, 2001.

129.Vaherie A, Carpen O, Heiska L. The ezrin protein family: Membrane-cytoskeleton interactions and disease associations. Curr Opin Cell Biol 9:659–666, 2002.

130.Crepaldi T, Gautreau A, Comoglio PM, Louvard D, Arpin M. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol 138:423–434, 1997.

131.Lamb RF, Ozanne BW, Roy C. Essential functions of ezrin in maintenance of cell shape and lamellipodial extensions in normal and transformed fibroblasts. Curr Biol 7:682– 688, 1997.

132.Hiscox S, Jiang WG. Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/b-catenin. J Cell Sci 112:3081–3090, 1999.

133.Ma¨kitie T, Carpe´n O, Vaherie A, Kivela¨ T. Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma. Invest Ophthalmol Vis Sci 42:2442–2449, 2001.

134.Yu H, Rohan T. Role of the insulin-like growth factor family in cancer development and progression. J Natl Cancer Inst 92:1472–1489, 2000.

135.All-Ericsson C, Girnita L, Seregard S, Bartolazzi A, Jager MJ, Larsson O. Insulin like growth factor-1 receptor in uveal melanoma: a predictor for metastatic disease and a potential therapeutic target. Invest Ophthalmol Vis Sci 43:1–8, 2002.

136.van Ginkel PR, Gee RL, Subramanian L, Walker TM, Albert DM, Meisner LF, Varnum BC, Polans AS. Over-expression of the receptor tyrosine kinase axl promotes ocular melanoma cell survival. submitted 2002.

137.Ma D, Luyten GP, Luider TM, Jager MJ, Niederkorn JY. Association between nm23H1 gene expression and metastasis of human uveal melanoma in an animal model. Invest Ophthalmol Vis Sci 37:2293–2301, 1996.

138.Diatchenko L, Lau Y-FC, Campbell AP, Chenckik A, Mooadam F, Huang B, et al. Suppression subtractive hybridization: a method for generating differentially regulated

or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030, 1996.

139.Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 100:57–70, 2000.

140.Novatchkova M, Eisenhaber F. Can molecular mechanisms of biological processes be extracted from expression profiles? Case study: Endothelial contribution to tumorinduced angiogenesis. Bioessays 23:1159–1175, 2002.

141.Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix M, Radmacher M, Simon R, Yakhini Z, Ben-Dor A, Sampas N, Dougherty E, Wang E, Marincola F, Gooden C, Lueders J, Glatfelter A, Pollock P, Carpten J, Gillanders E, Leja D, Dietrich K, Beaudry C, Berens M, Alberts D, Sondak V, Hayward N, Trent J. Molecular classification of cutaneous melanoma by gene expression profiling. Nature 406:536–540, 2000.

Figure 1 Theoretical events driving progression from normal multipotent retinoblasts to retinoma, retinoblastoma, dissemination by vitreous and subretinal seeding, and ultimately multidrug-resistant metastasis. The initiating loss of pRB by mutation of both alleles of RB1 and the promotion of drug resistance by MDR1 are well established. The hypothetical roles of p75NTFR, RBKIN, CDH11, and MRP are not yet established.

understood, they may constitute good targets for retinoblastoma treatment and prevention strategies to cure tumors, decreasing the risk of blindness and loss of life in predisposed children.

I.FEATURES OF THE RETINAL CELL UNIQUELY SENSITIVE TO LOSS OF RB1 ALLELES

A.Developmental Window

1.The Retina: Between Precursor and Mature Cells

Only cells that normally require the intact RB1 gene for the critical role played by its protein product, pRB, can give rise to retinoblastoma. All retinal cells arise from a common precursor of neural origin. In the fully mature retina, cells are organized into three nuclear layers: the ganglion cell layer (GCL); the inner nuclear layer (INL), containing horizontal, bipolar, amacrine, and Mu¨ller glia cells; and the outer

nuclear layer (ONL), containing rod and cone photoreceptor cells. There is a welldefined temporal pattern of progression in which proliferating pluripotent precursor cells undergo terminal mitosis (‘‘birth’’), followed by terminal differentiation. In rodents, the ganglion, amacrine, horizontal, and cone photoreceptor cells are ‘‘born prenatally,’’ whereas the bipolar and Mu¨ller glia cells are mainly ‘‘born postnatally.’’ Rod photoreceptor cells, by far the predominant retinal cell type, are ‘‘born both preand postnatally,’’ with the peak terminal mitosis occurring around birth [reviewed in Ref. 6]. Ganglion cells are unique in that their birth and terminal differentiation occur over a very short, early period of time. In contrast, acquisition of features of terminal differentiation in other retinal cell types is delayed for several days after terminal mitosis [7]. Although postmitotic, multipotent progenitors give rise to mature retinal cells in a predictable fashion, their specific fates may be altered by growth factors [e.g., rod photoreceptors may be respecified by the ciliary neurotrophic factor (CNTF)] [8,9].

Our preliminary data suggests that only a few retinal cell types express detectable RB1: the postmitotic cone photoreceptor, Mu¨ller glia, ganglion, and a rare horizontal cell type. Both murine models of retinoblastoma and human retinoblastoma frequently arise from the inner nuclear layer (Fig. 2), making the developing Mu¨ller glia or horizontal cells prime candidates to be the retinoblastoma precursor (Fig. 1). In humans, the loss of both alleles of the RB1 gene in the precursor cells predisposes to development of retinoblastoma. In mice, loss of RB1 from the earliest stages of retinal development does not cause retinoblastoma, but loss of both RB1 and p107 does [10]. Jacks et al. now suggest that acute loss of RB1 alone within the window of ongoing retinal development, rather than before retinal differentiation starts, results in retinoblastoma [11].

B.Learning from Differences and Similarities in Mice and Humans

1.Impact of Loss of RB1 in the Mouse Embryo

Homozygous deletion of the RB1 gene in mice results in embryonic lethality at day E13.5–15.5 gestation [12–14], with death from abnormal hematopoiesis and neurogenesis. The embryos show a significant increase in immature erythrocytes, and massive apoptosis and ectopic mitoses in the central and peripheral nervous system. There are also significant defects in lens development and myogenesis, showing the important role of pRB in proliferation and differentiation in many tissues [15,16].

2.Different Impact of Loss of RB1 in Mouse Embryos and in Humans

Surprisingly, the neural retina of RB1 / mice is normal up to day E15.5 gestation, compared to wild type RB1þ/þ mice [12–14]. Beyond day E16, however, loss of pRB in individual cells of RB1 / ;RB1þ/ chimeric mice in which embryonic lethality has been prevented by the presence of RB1þ/ still resulted in abnormal development of the lens, with ectopic mitoses and cellular degeneration of the retina [17,18]. RB1þ/ mice and chimeric RB1 / ;RB1þ/ mice tend to develop pituitary adenoma in which the second RB1 allele has been lost, as in human retinoblastoma [14,17,19]. However, RB1þ/ humans do not develop pituitary adenoma, whereas RB1þ/ and

Figure 2 Murine and human retinoblastomas commonly arise in the inner nuclear layer. A. SV40 TAg–induced murine retinoblastoma arises from the inner nuclear layer of developing retina. B. Human retinoblastoma arises from the inner nuclear layer of the retina. G, ganglion cell layer; I, inner nuclear layer; O, outer nuclear layer; arrows point to Flexner–Wintersteiner rosettes.

chimeric RB1 / ;RB1þ/ mice do not develop retinoblastoma. This suggests that loss of pRB prior to the development of mouse retina results in cell death rather than increased retinal cell proliferation, in contrast to loss of pRB in developing human retina, which results in cell cycle progression.

3.Other pRB Family Members Compensate for Loss of RB1 in the Murine Embryo

In mice, the other pRB family members may partially compensate for loss of pRB by suppressing the development of retinoblastoma. For instance, both p107 / and p130 / mice are viable without retinal dysplasia [20,21], whereas loss of p107 as well as one copy of the RB1 gene in RB1þ/ ;p107 / mice causes retinal dysplasia [20]. Loss of p107 as well as both copies of the RB1 gene in RB1 / ;p107 / mice causes death at E11.5 so the impact on the developing retina cannot be determined [20], whereas only simultaneous loss of both p107 and pRB proteins in the retinoblasts

can cause retinoblastoma in RB1 / ;p107 / chimeric mice [22]. This retinoblastoma tumor originates in cells in the inner nuclear layer (Fig. 2) and expresses amacrine cell markers.

4.A Murine Model Comparable to Human Retinoblastoma

In an attempt to make a mouse model for pituitary adenoma, the simian virus-40 large T antigen (SV40 TAg) was linked to the promoter of the human luteinizing hormone beta subunit, LHb, so as to restrict expression to gonadotrophic cells [23]. Surprisingly, one founder lineage developed focal bilateral retinal tumors as a result of the high expression of TAg also in the retina, the first transgenic mouse model of retinoblastoma. These focal tumors are histologically, ultrastructurally, and immunohistochemically identical to human retinoblastoma. The pRB interacts with the high expression of TAg in the murine retinoblastoma cells. The retinaspecific promoter, interphotoreceptor retinoid-binding protein (IRBP), which differentiates the photoreceptors and is expressed early in retinal cell differentiation, is used to produce TAg transgenic mice [24–26]. However, the opsin promoter resulted in no retinal tumors, presumably because opsins are expressed later in photoreceptor differentiation when the retinal cells no longer have the potential for mitosis. In the murine retinoblastoma model, the entire photoreceptor layer was malignant, probably reflecting the early timing of inactivation of all the pRB family proteins by TAg rather than specific binding to pRB.

5.Loss of pRB and Default Pathway of Retinal Differentiation

Our preliminary data indicate that pRB is not expressed in adult rod photoreceptors or bipolar cells of human or mouse, and only in a very narrow window of differentiation, if at all, in murine development. However, loss of pRB may result in the transformed cells assuming a default pathway of differentiation that does not require pRB. Indeed, retinal progenitor cells go through a series of changes in intrinsic properties that control their competence to make different cell types [27]. If a pRB-dependent lineage is disrupted, a cell may follow another lineage pathway, depending on extrinsic cues from the neighboring cells. Therefore, identification of retinal specific pathways that are regulated by pRB will provide clues to the identity of the cell of origin in retinoblastoma. This will not only provide a system to test experimental therapies for prevention of human retinoblastoma but also help define the fundamental properties of the stem cells that are capable of forming cancer. This has relevance beyond the rare disease of retinoblastoma.

C.The Retinoma Way Station

1.Attributes of the Benign Human Retinoma

Nonprogressing retinal lesions were originally considered to represent ‘‘spontaneous regression’’ of retinoblastoma. Then Gallie et al. proposed designating nonmalig- nant-looking retinal lesions associated with retinoblastoma as retinoma since there was no clear evidence that they were ‘‘regressed retinoblastomas’’ [28,29]. A retinoma is typically a translucent-gray retinal mass frequently associated with calcification and hyperplasia of the retinal pigment epithelium [28,30–32], unlike the

actively proliferating retinoblastoma, which shows calcification but appears opaquewhite [30]. Electron microscopy of six retinomas showed a haphazard arrangement of neuronal cells with photoreceptor differentiation, a few axons, Mu¨ller glia, and astrocytes [29]. Alio´ and coworkers found, in intraocular fine-needle aspiration biopsy of a retinoma, mainly degenerated cells with lipid cytoplasmic vacuoles and disintegrating mitochrondria, but no Flexner-Wintersteiner rosettes characteristic of retinoblastoma [33].

2.Retinoma Predisposed by RB1 Mutations

Although retinomas are ‘‘benign,’’ they may rarely undergo malignant progression to retinoblastoma. RB1 germline mutations predispose to both retinoma and retinoblastoma. Therefore individuals with retinoma should be followed regularly [30]. Of 32 individuals with retinoma studied, two-thirds had a family history of retinoblastoma or had retinoblastoma in their other eye, and 23 of 37 (62%) of their offspring developed retinoblastoma [30]. Another study showed 11 individuals with retinoma in 103 retinoblastoma families, 7 of which had a family history of retinoblastoma, and 12 of their 16 offspring developed retinoblastoma [34,35]. Keith and Webb reported a chromosome 13q-deleted patient with active retinoblastoma in one eye and a retinoma in the other eye, suggesting that retinoma and retinoblastoma were induced by similar genetic changes [36]. Although no molecular analysis of retinoma has been reported, we have identified the germline RB1 mutations in several patients with bilateral or unilateral multifocal retinomas as their only clinical manifestation.

3.Malignant Transformation of Retinoma to Retinoblastoma

Retinoma has been reported to undergo malignant transformation to retinoblastoma [32,37–39]. Histopathology of a rapidly growing, newly elevated area of a retinoma that had remained stable for 3 years showed undifferentiated retinoblastoma, while the retinoma base was characteristic of benign retinoma [32]. This suggests that additional mutations, presumably in genes involved in cell cycle regulation or apoptosis, are necessary to convert a benign retinoma cell into a malignant retinoblastoma cell [40].

II.INITIATION OF RETINOBLASTOMA: M1 AND M2 EVENTS

Since the discovery of the retinoblastoma gene, RB1, in 1986 [41], molecular identification of RB1 mutations has been used to determine the risk of retinoblastoma in other family members [42–44]. Bilaterally affected patients all have one germline RB1 mutant allele, which results in autosomal dominant transmission of the predisposition to retinoblastoma. Clinical testing requires study of blood DNA to identify the precise mutant allele in each affected family. Unilaterally affected patients have only a 15% chance that they carry a germline RB1 mutant allele. Since 85% of unilateral sporadic patients have no germline mutations, testing of blood would be prohibitively expensive and inconclusive if no mutation is found. Therefore the standard approach for unilateral patients is testing the retinoblastoma tumor DNA to identify the two mutant RB1 alleles, followed by