Genomic differences between retinoma and retinoblastoma

Figures & data

Table I.  Primers and probes sequences for Real-Time qPCR.

Gene symbol	Band	Primers (5′-3′)	TaqMan probe (5′-3′)	Primers & Probe location
MDM4	1q32.1	CAGCAGGAGCAGCATATGGTATA	GGAGATCTTTTGGGAGAA	Exon 3
		CTACTGGGACGTCAGAGCTTC
MYCN	2p24.1	CGTCCGCTCAAGAGTGTCATC	CTTGAGCCCCCGAAACT	Exon 2
		TTTCTGCGACGCTCACTGT
E2F3	6p22	GTACCATAAGGGAAGACTTCTTTTAACTGT	AACTCGGAATACGAACTTT	Intron 2
		GAAGCAATGTCCATTAAGGGAAGTTAAAATTAA
CDH11	16q22	AGAGAGAGCCCAGTACACGTT	ATGGCTCAGGCGGTGG	Exon 2
		ACCAATCGGCCACTGGAG

Figure 1.  Ophthalmoscopic examination of Case 1. Ophthalmoscopic examination at 2 years and 6 months showing cystic retinoma on the right fundus oculi overwhelming the optic nerve head.

Figure 2.  Histology and immunohistochemistry of Case 1. a. H&E staining and b. p75^NTR immunohistochemistry of the whole eye. Normal retina (arrow-head), retinoblastoma (asterisk) and retinoma (arrows) are shown. c. H&E staining (original magnification 100x) of retinoma (RN) and retinoblastoma (RB). d. Positive immunostaining for p75^NTR (original magnification 100x) in retinoma (RN), while retinoblastoma cells (RB) are not stained as well as inflammatory cells inside the tumor. Spikes of positivity inside the tumor represent residues of neuron bodies and their elongation and vessel walls. e. Widespread Ki-67 nuclear cell immunostaining in RB cells indicates a high proliferation index, while retinoma cells are negative (original magnification 100X). f. Higher magnification of normal retina stained with p75^NTR (original magnification 200X). p75^NTR positivity in the retina is diffuse and includes neuron bodies and elongate processes of all the retina layers; outer segments of cones and rods are not stained. g. Higher magnification of retinoma (H&E staining, original magnification 200X) showing fleurettes (see arrows). h. Higher magnification of retinoblastoma (H&E stain, original magnification 200X) displaying Homer Wright rosettes (see arrows).

Figure 3.  Histology and immunohistochemistry of Case 2. a. H&E staining and b. p75^NTR immunohistochemistry of the whole eye. Normal retina (arrow-head), retinoblastoma (asterisk) and retinoma (arrow) are shown. c. H&E staining (original magnification 100x) of retinoma (RN) and retinoblastoma (RB). d. Positive immunostaining for p75^NTR (original magnification 100x) in retinoma (RN), while retinoblastoma (RB) cells are not stained. Spikes of positivity inside the tumor represent residues of neuron bodies and their elongation and vessel walls. e. RB cells show a widespread Ki-67 nuclear cell immunostaining indicating a high proliferation index, while retinoma cells are not stained (original magnification 100X). f. Normal retina with an intraretinal RB focus stained with p75^NTR (original magnification 200X). In the normal retina p75^NTR positivity is diffuse, while in the neoplastic area positivity is restricted to vessel walls. g. Higher magnification of retinoma (H&E stain, original magnification 200X) showing fleurettes (see arrows). h. Higher magnification of retinoblastoma (H&E stain, original magnification 200X) with Homer Wright rosettes (see arrows).

Figure 4.  Genomic changes detected by Real-Time quantitative PCR. ddCT ratios and standard deviations obtained for retinoma (RN), retinoblastoma (RB) and retina (RT) of Case 1 (left) and Case 2 (right). For each graph, genomic copy number is indicated on the left (gain: 3-7 copies; amplification ≥7 copies). The asterisk shows when the difference is statistically significant (t-test, p ≥ 0.05). a. Both cases show MDM4 gain in retinoma and retinoblastoma at the same number of copies. b. Progressive gain of MYCN from retinoma to retinoblastoma respect to normal retina in both cases. c. Case 1 does not show copy number changes, while Case 2 displays gain of E2F3 in retinoma and amplification in retinoblastoma compared to retina. d. No genomic changes were detected in the two retinoma tissues. One of the two retinoblastoma samples (Case 1) presents gain of CDH11.

Figure 5.  a. Tumor multi-step model from Bowles et al., 2007. b. Retinoblastoma tumor progression model based on our results. In light grey, copy number changes found in only one of the two samples.