Murine bilateral retinoblastoma exhibiting rapid-onset, metastatic progression and N-myc gene amplification
2007; Springer Nature; Volume: 26; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7601515
ISSN1460-2075
AutoresDavid MacPherson, Karina L. Conkrite, Mandy Tam, Shizuo Mukai, David Mu, Tyler Jacks,
Tópico(s)Polyomavirus and related diseases
ResumoArticle18 January 2007free access Murine bilateral retinoblastoma exhibiting rapid-onset, metastatic progression and N-myc gene amplification David MacPherson Corresponding Author David MacPherson Department of Embryology, Carnegie Institution, Baltimore, MD, USA Search for more papers by this author Karina Conkrite Karina Conkrite Department of Embryology, Carnegie Institution, Baltimore, MD, USA Search for more papers by this author Mandy Tam Mandy Tam Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shizuo Mukai Shizuo Mukai Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, MA, USA Search for more papers by this author David Mu David Mu Human Cancer Genome Center, Cold Spring Harbor Laboratory, Woodbury, NY, USA Search for more papers by this author Tyler Jacks Tyler Jacks Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author David MacPherson Corresponding Author David MacPherson Department of Embryology, Carnegie Institution, Baltimore, MD, USA Search for more papers by this author Karina Conkrite Karina Conkrite Department of Embryology, Carnegie Institution, Baltimore, MD, USA Search for more papers by this author Mandy Tam Mandy Tam Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shizuo Mukai Shizuo Mukai Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, MA, USA Search for more papers by this author David Mu David Mu Human Cancer Genome Center, Cold Spring Harbor Laboratory, Woodbury, NY, USA Search for more papers by this author Tyler Jacks Tyler Jacks Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Author Information David MacPherson 1, Karina Conkrite1, Mandy Tam2, Shizuo Mukai3, David Mu4 and Tyler Jacks2,5 1Department of Embryology, Carnegie Institution, Baltimore, MD, USA 2Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA 3Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, MA, USA 4Human Cancer Genome Center, Cold Spring Harbor Laboratory, Woodbury, NY, USA 5Howard Hughes Medical Institute, Chevy Chase, MD, USA *Corresponding author. Department of Embryology, Carnegie Institution, 3520 San Martin Drive, Baltimore, MD 21218, USA. Tel.: +1 410 246 3084; Fax: +1 410 243 6311; E-mail: [email protected] The EMBO Journal (2007)26:784-794https://doi.org/10.1038/sj.emboj.7601515 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Human retinoblastoma is a pediatric cancer initiated by RB gene mutations in the developing retina. We have examined the origins and progression of retinoblastoma in mouse models of the disease. Retina-specific inactivation of Rb on a p130−/− genetic background led to bilateral retinoblastoma with rapid kinetics, whereas on a p107−/− background Rb mutation caused predominantly unilateral tumors that arose with delayed kinetics and incomplete penetrance. In both models, retinoblastomas arose from cells at the extreme periphery of the murine retina. Furthermore, late retinoblastomas progressed to invade the brain and metastasized to the cervical lymph nodes. Metastatic tumors lacking Rb and p130 exhibited chromosomal changes revealed by representational oligonucleotide microarray analysis including high-level amplification of the N-myc oncogene. N-myc was found amplified in three of 16 metastatic retinoblastomas lacking Rb and p130 as well as in retinoblastomas lacking Rb and p107. N-myc amplification ranged from 6- to 400-fold and correlated with high N-myc-expression levels. These murine models closely resemble human retinoblastoma in their progression and secondary genetic changes, making them ideal tools for further dissection of steps to tumorigenesis and for testing novel therapies. Introduction In humans, inherited and somatic mutations in the RB tumor suppressor gene lead to the development of retinoblastoma, a childhood malignant tumor of the eye. In contrast, germline heterozygosity for Rb gene mutations in mice causes predisposition to pituitary and thyroid tumors, but these animals do not develop retinoblastoma (Clarke et al, 1992; Jacks et al, 1992). Homozygous Rb mutation results in mid-gestational embryonic lethality, which has been attributed to defects in placental and hematopoietic development (Clarke et al, 1992; Jacks et al, 1992; Lee et al, 1992; Wu et al, 2003). We and others have specifically deleted Rb in the developing mouse retina using Cre-lox technology (Chen et al, 2004; MacPherson et al, 2004; Zhang et al, 2004a). Use of Pax6 α-Cre transgenic mice to delete Rb in early retinal progenitors led to defects in proliferation, increased levels of cell death and associated inhibition of differentiation in a cell-type-specific fashion. The majority of bipolar, ganglion and many rod photoreceptor cells were selectively lost in the developing Rb-deficient retina, whereas other cell types survived (Chen et al, 2004; MacPherson et al, 2004). Although Rb deletion leads to proliferation defects in the retina, retinoblastomas did not develop. Compensation or functional overlap affecting the activity or levels of the pocket protein family members, p107 and p130, minimizes the effects of Rb loss, preventing tumorigenesis. This was first shown in a chimeric setting, where retinoblastomas did not emerge in chimeras with retinal contribution of Rb−/− cells (Maandag et al, 1994; Williams et al, 1994), but were present in chimeras composed of cells mutant for both Rb and p107 (Robanus-Maandag et al, 1998). Breedable models of retinoblastoma involving conditional Rb mutation on a p107−/− genetic background (with or without additional p53 inactivation) have now been generated (Chen et al, 2004; Zhang et al, 2004b). We recently used transgenic expression of Cre from the nestin promoter to show that inactivation of Rb in neural progenitors of p130−/− animals also results in retinoblastoma development (MacPherson et al, 2004). The apparent functional compensation among the Rb gene family may explain the fact that in many human tumors the RB pathway is disrupted not by RB mutation, but by mutations that act upstream (reviewed in (Sherr, 1996)). Examples include p16INK4a loss in glioma, melanoma and pancreatic carcinoma, CDK4 amplification in melanoma, cyclin D1 amplification in breast and esophageal cancer or translocations in B-lineage tumors. Thus, examining tumor formation in a context of mutation in multiple Rb family members in the mouse may help us understand the overlapping and unique roles of these proteins in tumor suppression. The availability of well-defined mouse models of retinoblastoma is expected to help answer longstanding questions concerning the genetic changes that contribute to retinoblastoma progression as well as the nature of the cell of origin of this tumor type. For example, Knudson's two-hit hypothesis proposed that mutations in both alleles of the RB gene represent the rate-limiting steps in retinoblastoma development (Knudson, 1971). However, comparative genomic hybridization (CGH) analysis has revealed chromosomal gains at 6p, 1q and 2p as well as losses at 16q in a significant percentage of human retinoblastomas (Mairal et al, 2000; Chen et al, 2001; Lillington et al, 2003; Zielinski et al, 2005). These data suggest that other changes also occur during tumor progression. In particular, selection for N-myc amplification has been implicated in the 2p gain, which often involves high-level gene amplifications (Lee et al, 1984). Microarray-based CGH platforms in which tumor or normal DNA is hybridized to bacterial artificial chromosomes (BAC), cDNA or oligonucleotide arrays are improving the resolution with which copy number changes can be detected (reviewed in Pinkel and Albertson, 2005). Representational oligonucleotide microarray analysis (ROMA) is a comparatively new approach for detecting copy number variation that uses PCR-based genome representations hybridized to oligonucleotide arrays, reducing genome complexity and increasing signal-to-noise ratios (Lucito et al, 2003; Lakshmi et al, 2006). Application of ROMA to mouse models of retinoblastoma, where tumor progression can be examined in a more controlled and less variable manner than possible using human samples, may aid in the identification of secondary changes also relevant for tumor progression. In this study, we use improved mouse models of retinoblastoma to examine tumor initiation and progression to metastatic disease. We also show that ROMA analysis can identify an important secondary oncogenic event that contributes to tumor development in these models, pinpointing a minimal region of gene amplification that includes a single known gene. Results Mutation of Rb coupled with the absence of either p107 or p130 in chimeric or in retina-specific knockout models causes retinoblastoma (Robanus-Maandag et al, 1998; Chen et al, 2004; Dannenberg et al, 2004; MacPherson et al, 2004; Zhang et al, 2004b). These results may suggest that retinoblastoma development results from an overall reduction in 'Rb family' tumor suppressor function, which can occur equivalently through loss of pRB plus either of the Rb-related proteins. However, in some experiments involving compound mutation with Rb, distinct effects of p107 and p130 mutations have been shown (Dannenberg et al, 2004; Haigis et al, 2006). Therefore, we directly compared the effects of p107 versus p130 mutation when combined with retinal-specific deletion of a conditional allele of Rb (MacPherson et al, 2003; Sage et al, 2003). For these studies, we used a transgenic strain in which Cre expression is controlled by the α-enhancer of the Pax6 promoter. In Pax6 α-enhancer Cre transgenic mice, Cre expression occurs by embryonic day (E)10.5 in mid- to far-peripheral neural retina as well as in some peripheral eye structures (Supplementary Figure 1; Marquardt et al, 2001). Here, we refer to animals with retinal-specific Rb mutation on p107−/− or p130−/− genetic backgrounds as Rb/p107 double knockout (DKO) or Rb/p130 DKO mice. Kinetics of retinoblastoma development in Rb/p107 versus Rb/p130 DKOs We aged compound mutant mice and followed tumor development by visual examination of the mouse eye over time. Mice were examined for either the presence of tumor or blood in the anterior chamber, or distortion of the eye caused by the tumor. Upon initial observation of unilateral retinoblastoma, the cohort continued to be followed for the appearance of bilateral retinoblastoma unless tumor burden either in the eye region or due to metastasis necessitated killing of the animal. Figure 1 shows the time to first observation of retinoblastoma upon examination of the eye. Rb/p130 DKOs developed visible retinoblastoma with rapid and consistent kinetics, exhibiting an average time to visible bilateral retinoblastoma of 128±18 days (mean±s.d). By contrast, mutation of Rb and p107 led to tumors that developed with delayed and variable kinetics: 27/44 (61%) developed unilateral retinoblastoma, first visible at an average time of 280±107 days. Bilateral tumors were rare in this Rb/p107 DKO model (Figure 1). Overall, the tumor kinetic data indicate that the pattern of tumorigenesis in the Rb/p107 DKO and Rb/p130 DKO models differs significantly. Figure 1.Different kinetics of retinoblastoma emergence in Rb/p107 versus Rb/p130 DKOs. Kaplan–Meier curve showing time to first observation of externally visible retinoblastoma. Inset: retinoblastoma visible in the anterior chamber of an Rb/p130 DKO mouse at 4 months of age. Download figure Download PowerPoint Rb/p130 DKO tumors emerge from the extreme periphery of retina The rapid and consistent kinetics of tumor formation in Rb/p130 DKOs provided an opportunity to examine the origins of retinoblastoma development. We examined retinas histologically at postnatal day (PND)21, a time when retinal histogenesis is normally complete. Previous work by us and others has shown that the absence of Rb alone causes hypocellularity in the retina due to loss of specific cell types, but the three nuclear layers are still detected (Chen et al, 2004; MacPherson et al, 2004). In contrast, in the Rb/p130 DKOs, the three nuclear layers could not be distinguished, except in central retina, where Cre is not widely expressed (Figure 2A). The Rb/p130 DKO retinas were very hypocellular and contained apoptotic bodies and many cells exhibiting large and/or irregular-shaped nuclei (Figure 2A, inset; data not shown). Strikingly, at the extreme periphery in 9/12 eyes examined, we observed early dysplastic lesions with histological similarities to retinoblastomas (Figure 2A, right). Such lesions contained Homer–Wright rosettes, which consist of a radial arrangement of cells surrounding a central tangle of neuronal processes and are found in a subset of human retinoblastomas (Yuge et al, 1995) as well as murine retinoblastomas (Robanus-Maandag et al, 1998; Dannenberg et al, 2004; MacPherson et al, 2004). The detection of early tumors specifically at the extreme periphery of the retina points to a possible niche for the cell of origin of retinoblastoma in this model. Importantly, these results do not simply reflect the expression of Cre in the extreme distal retina as Pax6 α-Cre is expressed much more broadly (Supplementary Figure 1). Figure 2.Rb/p130 DKO tumors emerge from the extreme periphery of retina. Early retinoblastomas in Rb/p130 DKOs. (A–F) Histology and immunostaining of PND21 retinas from wild-type (left panel) and Rb/p130 DKO (middle, right panels) animals. The upper inset in the middle panel shows details of Rb/p130 DKO retina adjacent to tumor. The panel in the right shows details from Rb/p130 DKO early tumor. Scale bars in the left and middle low-power panels=200 μm, high-magnification insets and right panels, 40 μm. (A) Hematoxylin and eosin (H+E) stain with normal eye structures labeled. Distinct regional phenotypes and early retinoblastoma in the Rb/p130 DKO are noted. (B) BrdU labeling of proliferating cells. (C) Syntaxin immunostaining. Note the depletion of amacrine cells adjacent to tumor (inset, middle panel) and the positive staining of early retinoblastoma (right). (D) Calretinin immunostaining labeling an amacrine and ganglion subset. (E) Calbindin immunostaining. Arrows indicate calbindin-positive horizontal cells. (F) GLAST immunostaining labeling Müller glia. (G) Fundus photograph of a p130−/− control mouse at 6 weeks of age. The ciliary body, peripheral to the neural retina, is indicated (white arrow). (H) Fundus photograph of Rb/p130 DKO mouse at 6 weeks of age. Peripheral retina is shown and black arrows point to early retinoblastoma at the extreme periphery. Retinal pigment epithelial changes due to retinal degeneration are present. The ciliary body, adjacent to the neural retina, is indicated (white arrow). Download figure Download PowerPoint Loss of Rb leads to increased proliferation beyond the normal period of retinogenesis (Chen et al, 2004; MacPherson et al, 2004). At PND12, we found extensive BrdU incorporation in Rblox/lox α-Cre retinas, and this phenotype was exacerbated in Rb/p130 DKO retinas (Figure 3A and C). Inappropriate proliferation was accompanied by increased apoptosis, which was also found at higher levels in Rb/p130 DKO over single Rb mutant retinas (Figure 3B and C). By PND21, proliferation could not be detected in Rb mutants (Chen et al, 2004; data not shown). In contrast, BrdU-positive cells were found in Rb/p130 DKOs throughout the PND21 distal retina, and were particularly concentrated at the extreme periphery, where early tumors were detected (Figure 2B). BrdU-positive cells were also found away from the tumor regions (Figure 2B, middle panel inset) where the retina was hypocellular and were also found in PND21 Rb/p130 DKO retinas that did not yet exhibit histological evidence of early retinoblastoma formation (data not shown). These data suggest that the combined loss of Rb and p130 function in the retina causes broad defects in cell-cycle control, accompanied by cell death. Figure 3.Proliferation and apoptosis in Rb/p130 DKOs at PND12. (A) BrdU incorporation in retinas from wild-type, Rblox/lox αCre (Rb KO) and Rb/p130 DKO animals. Ganglion (g), inner nuclear (i) and outer nuclear (o) layers are indicated. Scale bar=200 μm (low magnification) and 40 μm (high magnification). (B) Caspase3 immunostaining in retinas from wild-type, Rblox/loxαCre and Rb/p130 DKO animals. Scale bar=40 μm. (C) Quantification of Brdu and Caspase3 staining. Quantification was performed on horizontal sections at the optic nerve level in a region from the peripheral tip of the retina extending 1000 μm toward central retina. WT N=3; Rb KO N=4, Rb/p130 DKO N=8. Error bars represent standard deviation. P-values (Student's t-test) are shown. Download figure Download PowerPoint To characterize the early lesions further, we stained histological sections of Rb/p130 DKO retinas at PND21 for cell-type markers. Consistent with previous descriptions of murine retinoblastomas lacking Rb and p107 or p130, we found that early tumors expressed syntaxin, which stains amacrine cells and a subset of progenitor cells (Figure 2C) (Alexiades and Cepko, 1997). Away from the tumors, the amacrine layer was significantly reduced (Figure 2C, inset), suggesting that many amacrine cells do not survive in the absence of Rb and p130 function. The early tumors also stained for calretinin, which labels a subset of amacrine and ganglion cells (see control retina; Figure 2D). Calretinin in the tumors was expressed in a more focal and heterogeneous fashion that was variable from animal to animal. In normal retina, calbindin labels horizontal cells strongly (arrowheads) and a subset of amacrine cells weakly (Figure 2E). We found calbindin-positive cells in early tumors, including some intensely stained cells that were reminiscent of horizontal cells. Interestingly, adjacent to the early tumors, and in contrast to the overall hypocellularity in this area, Rb/p130 DKO retinas exhibited a clear increase in horizontal cells (Figure 2E, middle panel inset). The glial glutamate/aspartate transporter (GLAST) labels Müller cells, which survived both Rb and p130 mutation and were present in the early tumor (Figure 2F). At this stage, some of the cells present in the early tumors may be non-neoplastic cells derived from normal retina. A cone subset (stained for M-opsin; Zhu et al, 2003), rod bipolar cells (stained for PKCα) and Tuj1-positive retinal ganglion cells were either very rare or completely absent from the Rb/p130 DKO retina and early tumors (data not shown). Retinoblastoma progression in Rb/p130 DKOs Beyond PND21, cells in the periphery of Rb/p130 DKO retinas continued to proliferate. Larger tumors were found at PND31 (four animals) and PND60 (three animals), and all mice at PND31 and PND60 had retinoblastomas in each eye. Upon ophthalmoscopic examination, early tumors could be visualized by 6–8 weeks, and these were adjacent to the ciliary body at the extreme periphery of the neural retina (Figure 2H). By 4 months of age, retinoblastoma seeding the vitreous and anterior chamber of the eye was detected upon visual examination of the mouse (Figure 1). To investigate late-stage tumor progression, mice continued to be monitored beyond the initial observation of tumor until the mouse was moribund due to retinal tumor burden or retinoblastoma presence at sites outside of the eye. Rb/p130 DKOs were killed at an average age of 183±39 days of age. By this advanced stage, retinoblastomas had grossly distended the eye, filled the anterior chamber and had often invaded local tissue outside of the eye (Figure 4A and B). Tumor cells could also be found infiltrating the optic nerve (Figure 4B). We found that 11/29 (38%) of these Rb/p130 DKO mice exhibited enlarged cervical lymph nodes. Histological analysis revealed that these were retinoblastoma metastases (Figure 4C). Although the kinetics of tumor development in the Rb/p130 DKO model was consistent at early time points, the properties and size of the metastatic tumors varied significantly. Figure 4.Tumor progression in murine Rb/p130 DKO retinoblastoma. Histology and immunostaining of late retinoblastoma and metastases in Rb/p130 DKOs from 5 to 7 months of age. Scale bar=200 μm for low power and 60 μm for high power (inset) images. (A) Bilateral retinoblastoma in a 5-month-old mouse (left). Fundus photograph of late-stage retinoblastoma that fills the vitreous at 5 months (right). (B) H+E stain of late retinoblastoma, with blood and tumor in the anterior chamber (AC) and invasion of the optic nerve (inset). (C) H+E stain of lymph node metastasis, adjacent to the salivary gland. Tumor rosettes are apparent on the high-magnification image (inset). (D) H+E stain of brain with retinoblastoma invading into brain parenchyma (inset). (E) Syntaxin immunohistochemistry of lymph node metastasis. (F) Calretinin immunohistochemistry of lymph node metastasis. (G) Calbindin immunohistochemistry of lymph node metastasis. (H) Glial fibrillary acidic protein (GFAP) immunohistochemistry of lymph node metastasis. Download figure Download PowerPoint Human retinoblastoma is known to invade the brain via the optic nerve (Shields et al, 1994). Given the presence of retinoblastoma cells in the optic nerve in the mouse model, we examined 27 Rb/p130 DKO animals for brain involvement. Seven animals were found to contain retinoblastoma lesions in the brain (26%, average age 199±41 days) (Figure 4D). To determine whether tumors spread to other tissues as well, we performed full necropsies on eight Rb/p130 DKO animals. Tumors were found only locally near the eye and surrounding tissues, in the brain and in cervical lymph nodes but not in other distant sites. Tumor progression in Rb/p107 DKOs To further assess functional differences between p130 and p107 mutation, we also carefully examined tumor progression in the Rb/p107 DKO animals. Although the Rb/p107 DKOs exhibit slower tumor kinetics and incomplete retinoblastoma penetrance (Figure 1), the end-stage tumors were histologically similar to the Rb/p130 DKO tumors (Figure 4B and C, and Supplementary Figure 2A). Because monitoring externally visible retinoblastomas may have led to an underestimation of the incidence of bilateral tumors in this model, we performed histological examination of the second eye in 11 Rb/p107 DKO animals killed due to unilateral retinoblastoma tumor burden. Retinas from all 11 eyes exhibited disorganization and degeneration, and 3/11 retinas contained an early retinoblastoma. These early lesions had abundant mitotic figures, high levels of apoptosis and Homer–Wright rosettes (data not shown). To determine whether the extreme peripheral localization of early tumors seen in the Rb/p130 DKO model was also applicable to the Rb/p107 DKO model, we examined Rb/p107 DKOs at PND31 and PND60 by histological analysis. At PND60, obvious retinoblastomas were present in four of 14 eyes examined at the level of the optic nerve head, and, in each case, the tumor was present at the extreme periphery of the retina (Supplementary Figure 3A). In 6/24 eyes examined at PND31, dysplastic lesions containing Homer–Wright rosettes were seen in this location, suggestive of early retinoblastoma. Moreover, BrdU-labeling studies performed at PND31 demonstrated proliferation concentrated at the extreme retina periphery in Rb/p107 DKOs that did not yet exhibit histological evidence of retinoblastoma (Supplementary Figure 3B and C). These data point to peripheral, late-proliferating cells as candidates for the cell of origin in the Rb/p107 DKO model as well. Consistent with overall tumor progression in this model, metastatic spread was also delayed in the Rb/p107 DKO model. In 4/14 Rb/p107 DKO mice examined histologically, retinoblastoma was observed in the CNS (average age of 296±68 days). We also observed metastasis to the cervical lymph nodes in Rb/p107 DKO mice. Late-stage retinoblastomas are heterogeneous To further characterize the cell-type composition of metastatic tumors, we studied the expression of other retinal cell-type markers in metastases from Rb/p107 DKO and Rb/p130 DKO mice. We focused on lymph node metastases, which are less likely than primary tumors to have infiltration of non-tumor cells from normal retina. Rb/p107 DKO and Rb/p130 DKO metastases stained positively for syntaxin (Figure 4E and Supplementary Figure 2B), whereas calretinin stained Rb/p130 and Rb/p107 DKO metastatic tumors in a patchy pattern, variable from animal to animal (Figure 4F and Supplementary Figure 2B). Calbindin was found to strongly label tumor cells in late lesions in the Rb/p130 DKOs, suggestive of a horizontal cell component to the tumors. This staining was also variable, ranging from only scattered positive cells in some tumors to others in which the majority of cells stained strongly (Figure 4G). In Rb/p107 DKOs, calbindin staining was typically found in a more scattered pattern (Supplementary Figure 2B) and many tumors were completely negative, indicative of some difference in the composition of Rb/p107 versus Rb/p130 DKO tumors. Some Rb/p130 and Rb/p107 DKO tumors exhibited glial fibrilary acidic protein (GFAP) and GLAST positivity (Figure 4H and Supplementary Figure 2B; data not shown). GFAP staining in retinoblastomas has been observed in murine and human retinoblastoma, but reactive gliosis from nearby Müller cells has often been implicated. GFAP and GLAST staining in the metastatic tumors in cervical lymph nodes is significant as it suggests that the Müller glial cells may indeed derive from tumor cells. This population was, however, a minor component and not present in all tumors. Overall, these data show that, although they progress at different rates, the retinoblastomas arising in Rb/p107 DKO and Rb/p130 DKO animals share many characteristics, including site of origin, overall histological appearance and routes to invasion and metastasis. ROMA analyses of murine retinoblastomas Although combined mutation in Rb and either p107 or p130 produced retinoblastomas at high frequency, the tumors were focal and, presumably, clonal. Thus, it is likely that additional genetic events contribute to tumor progression. To begin to catalog these changes, we utilized ROMA, a form of array CGH that uses PCR-generated genome representation to measure genomic DNA copy number alterations (Lucito et al, 2003). To maximize the chances of finding clonal genomic changes, we focused on metastatic tumors isolated from Rb/p130 DKO mice; six tumors were tested initially. As summarized in Table I, recurrent changes were identified in this tumor collection. For example, whole chromosome gains were frequently observed for chromosomes 1 and 12. These chromosomes may harbor one or multiple genes that contribute to tumorigenesis when expression is increased. More informatively, we also found focal regions of high-level amplification in a subset of tumors (Table I). Table 1. ROMA analysis of chromosomal changes in metastatic Rb/p130DKO retinoblastomas Tumor ID Gain Amplification Loss 9806 1, 12qA1.1qter 12qA1.1, 12qF2 12qa1.1, 11qA1 7217 12 4834 10qA4qter 2, 12, 18, 9qA5.3qter, 4qB3qter 4726 1 4848 1, 12, 19 4827 12qF2, 12qC1 4836a,b, a,b 1 12qA1.1, 3qf3, 12qF1-2 3qa3, 17qe2, 17qe1.1 drb13a 12 12qA1.1 Amplicons at 12qA1.1 harboring N-myc gene are in bold. a Samples selected for ROMA analysis based on the presence of N-myc amplification detected by Southern blot. b Tail DNA used for ROMA hybridization was not from the tumor-containing mouse; thus, polymorphisms could contribute to focal changes. N-myc amplification in a subset of murine retinoblastomas In our initial sampling of six tumors, we observed some focal amplifications, including two amplicons in tumor 9806 at 12qA1.1 and 12qF2 (Table I, and Figure 5A). Interestingly, the N-myc oncogene resides on 12qA1.1, and this gene has been reported to be amplified in approximately 10% of human retinoblastomas (Lee et al, 1984; Squire et al, 1986; Mairal et al, 2000; Lillington et al, 2002). To confirm the results from ROMA as well as to examine additional tumors, we performed Southern blot analyses of tumor DNA using N-myc as a probe. As shown in Figure 5, among metastatic tumors in the Rb/p130 DKO model, N-myc was found amplified in 3/16 samples, ranging from 6- to 17-fold (Figure 5B). N-myc amplifications may be more frequent in metastases in this model, as Southern blot analyses of 17 primary Rb/p130 DKO tumors did not reveal amplification of the gene (data not shown). Of note, we did not have the matched primary tumors for those metastases that did exhibit N-myc amplification, and, therefore, we could not assess whether the amplification was specific to the metastases. Interestingly, from a series of 21 primary retinoblastomas from the Rb/p107 DKO model, two tumors exhibited N-myc amplifcation (Figure 5C). A metastasis from one of these tumors (tumor 4459; Figure 5C) also exhibited amplification. N-myc amplification was not detected in seven other metastatic Rb/p107 DKO tumors
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