Assessment of early-stage optic nerve invasion in retinoblastoma using high-resolution 1.5 Tesla MRI with surface coils: a multicentre, prospective accuracy study with histopathological correlation.

OBJECTIVES: To assess the accuracy of high-resolution (HR) magnetic resonance imaging (MRI) in diagnosing early-stage optic nerve (ON) invasion in a retinoblastoma cohort. METHODS: This IRB-approved, prospective multicenter study included 95 patients (55 boys, 40 girls; mean age, 29 months). 1.5-T MRI was performed using surface coils before enucleation, including spin-echo unenhanced and contrast-enhanced (CE) T1-weighted sequences (slice thickness, 2 mm; pixel size <0.3 × 0.3 mm(2)). Images were read by five neuroradiologists blinded to histopathologic findings. ROC curves were constructed with AUC assessment using a bootstrap method. RESULTS: Histopathology identified 41 eyes without ON invasion and 25 with prelaminar, 18 with intralaminar and 12 with postlaminar invasion. All but one were postoperatively classified as stage I by the International Retinoblastoma Staging System. The accuracy of CE-T1 sequences in identifying ON invasion was limited (AUC = 0.64; 95 % CI, 0.55 - 0.72) and not confirmed for postlaminar invasion diagnosis (AUC = 0.64; 95 % CI, 0.47 - 0.82); high specificities (range, 0.64 - 1) and negative predictive values (range, 0.81 - 0.97) were confirmed. CONCLUSION: HR-MRI with surface coils is recommended to appropriately select retinoblastoma patients eligible for primary enucleation without the risk of IRSS stage II but cannot substitute for pathology in differentiating the first degrees of ON invasion. KEY POINTS: • HR-MRI excludes advanced optic nerve invasion with high negative predictive value. • HR-MRI accurately selects patients eligible for primary enucleation. • Diagnosis of early stages of optic nerve invasion still relies on pathology. • Several physiological MR patterns may mimic optic nerve invasion.

Effects of matrine on proliferation and apoptosis of cultured retinoblastoma cells.

BACKGROUND: Extracted from the traditional Chinese medicine of Kushen, matrine is an alkaloid with potential anti-neoplastic and anti-inflammatory effects. Here, we examined the effect of matrine on proliferation and apoptosis of cultured retinoblastoma cells. METHODS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 were treated with matrine in increasing concentrations from 0.2-1.1 mg/ml for 24 hours, and the cell proliferation rate was measured. The cells were exposed to matrine at 50% inhibition concentration (IC50) for 12, 24 and 48 hours. Cell cycle was analyzed by flow cytometry, concentration of proteins regulating cell cycle and apoptosis was determined by Western blot, apoptosis rate was measured by TUNEL staining, and cell morphology was assessed by electron transmission microscopy. RESULTS: The retinoblastoma cell lines Y79, WERI-RB1 and SO-RB50 showed an increased inhibition of cell proliferation with increasing matrine concentrations. Applying the IC50 concentration of matrine, the alteration of the cell cycle, including a reduced percentage of the S phase, was significantly (P < 0.01) associated with a longer treatment time by matrine. Correspondingly, the cell-cycle-associated proteins P21 and P27 were up-regulated and the protein cyclinD1 was down-regulated. The apoptosis-associated protein Bcl-2 was down-regulated, and Bax was up-regulated. In a similar manner, the apoptosis rate was significantly increased with longer treatment time. CONCLUSIONS: Matrine added to cultures of immortalized retinoblastoma cells led to a reduced tumor cell proliferation, decreased rate of mitosis and an increased tumor cell apoptosis, paralleled by corresponding changes in the proteins regulating the cell cycle or apoptosis.

Sonoporation enhances chemotherapeutic efficacy in retinoblastoma cells in vitro.

PURPOSE: To study the ability of ultrasound (US) and microbubbles (MB) to enhance chemotherapeutic efficacy against retinoblastoma Y79 cells in vitro. METHODS: The experiment was performed in three stages. The authors first compared cell viability of Y79 cells exposed to doxorubicin versus cells exposed to doxorubicin combined with low-intensity, low-frequency US + MB. They then evaluated enhanced cell permeability by studying the intensity of intracellular fluorescence in cells exposed to doxorubicin versus those exposed to doxorubicin with US + MB. Lastly they evaluated the morphologic characteristics of the cells by scanning electron microscopy (SEM) to identify the presence of pores. RESULTS: The Y79 cells exposed to doxorubicin with US + MB showed a significant decrease in cell viability at 72 hours compared with those exposed to doxorubicin alone (P = 0.02). Cells also showed immediate increased permeability to doxorubicin with the addition of US + MB compared with doxorubicin alone, which continued to increase over 60 minutes. SEM did not demonstrate physical pores at the lowest US + MB intensity shown to enhance intracellular doxorubicin fluorescence. CONCLUSIONS: US + MB facilitates the uptake of chemotherapy in retinoblastoma Y79 cells in vitro. This occurs in the absence of visible pores, suggesting a possible secondary mechanism for increased drug delivery. This experiment is the first step toward enhancing chemotherapy with sonoporation in the treatment of intraocular tumors. This technique may lead to more effective chemotherapy treatments with less collateral damage to ocular tissues and may allow reduced systemic dosage and systemic side effects.

Treating retinoblastoma in tissue culture and in a rat model with a novel isoquinoline derivative.

PURPOSE. To investigate the effectiveness of a novel isoquinoline derivative, EDL-155, in killing retinoblastoma in vitro and in vivo. METHODS. Dose-response curves were generated in which Y79 retinoblastoma cells tagged with luciferase (Y79-Luc) were treated with serial concentrations of EDL-155. Electron microscopy was used to evaluate the ultrastructural morphology of EDL-155-treated Y79 cells. To determine whether autophagy was induced in EDL-155-treated Y79-Luc cells, staining with acridine orange and LC-3 immunoblot analysis was performed. To evaluate the efficacy of EDL-155 in vivo, Y79-Luc retinoblastoma cells were injected into the vitreous cavity of newborn rats, followed by periocular injections of EDL-155 (20 mg/kg/day) or an equivalent dosage of saline. RESULTS. EDL-155 appeared to destroy the retinoblastoma cells in vitro with an EC(50) of 9.1 micriM. EDL-155-treated retinoblastoma cells displayed a lack of viable mitochondria and the presence of autophagosomes wrapped in the characteristic double membranes. Acridine orange staining of EDL-155-treated retinoblastoma cells demonstrated the accumulation of vacuoles, and the immunoblots displayed a shift in molecular weight of LC-3, indicative of incorporation into autophagosome vesicles. In the retinoblastoma animal model, four doses of EDL-155 were delivered over 4 days, which was sufficient to see a significant decrease (P = 0.01) in viable intraocular tumors. Seven of the 25 rats treated with EDL-155 had no detectable living tumor. No significant decrease in viable tumor was observed in control animals. CONCLUSIONS. EDL-155 appears to eliminate retinoblastoma cells by disrupting mitochondria and inducing autophagy. Local delivery of EDL-155 may be an effective therapy for some types of ocular cancers.

Retinal ependymoma: an immunohistologic and ultrastructural study.

Glial tumors of the retina are rare. Most are syndrome associated and include pilocytic astrocytoma in neurofibromatosis type 1 and subependymal giant cell astrocytoma in tuberous sclerosis complex. Acquired, more conventional, diffuse astrocytomas are less frequent. Ependymoma is exquisitely rare. Herein, we report the clinicopathologic features of the second case of retinal ependymoma. The tumor was sporadic in occurrence and unilateral, low grade, and of cellular type. Its chronic course and large size prompted an initial pathologic diagnosis of "massive retinal gliosis." The literature regarding retinal glial neoplasia including ependymoma as well as the so-called massive retinal gliosis is discussed.

Primary glial tumor of the retina with features of myxopapillary ependymoma.

We report a primary retinal tumor with features of myxopapillary ependymoma. The lesion occurred in a 33-year-old man with a long history of phthisis bulbi and a more recent history of pain to the right eye. Enucleated ocular globe revealed a lesion occupying most of the retinal surface. Histologically, the retina was replaced by a tumor composed of spindle cells with fibrillary cytoplasm and round to ovoid nuclei forming fascicles, perivascular pseudorosettes, microcysts, and deposition of extracellular mucins. Calcifications, metaplastic bone, and lymphoplasmacytic inflammatory infiltrate were also seen. Tumor cells expressed GFAP and S-100 and to lesser extent carbonic anhydrase II. The immunoreaction for EMA showed diffuse granular positivity, decorated a few extracellular lumina, and highlighted intracytoplasmic lumina in a few cells. Ultrastructurally, there was accumulation of extracellular material between cells and around capillaries, long interdigitating cytoplasmic processes, extracellular lumina packed with microvilli, a few junctions evident around lumina, and some ciliary basal bodies and ciliary basal rootlets. As control cases, we also investigated expression of EMA and carbonic anhydrase II in an ocular globe with retinal gliosis and three cases of myxopapillary ependymoma of the cauda equina. The lesion described here represents the first example of retinal tumor with features of myxopapillary ependymoma. Pathologic features and particularly expression of carbonic anhydrase II suggest a derivation from intrinsic glial cells of retina otherwise known as Muller cells.

Pathological, ultrastructural and immunohistochemical observations of adenoma of retinal pigment epithelium.

PURPOSE: To Study the clinical, pathological, ultrastructural and immunohistchemical characters of adenoma of the retinal pigment epithelium in order to offer evidence to diagnose this tumor. METHODS: Routine paraffin slices HE stain, histochemistry PAS and VG stain, transmission electron microscopy, and immunohistochemistry for S-100 and vimentin with LSAB method were used. RESULTS: The tumor cells were oval and cuboidal in shape. Part of the tumor had a tubular arrangement. Around the sheets of tumors cells there was a large amount of uniform red stick-like substances. The above matter represented positive in PAS stain. Most of the above matter was yellow, while less of the matter showed red in VG stain. Transmission electron microscopy showed that there were tight junctions between tumor cells. Immunohistochemistry showed positive for S-100, negative for vimentin. CONCLUSIONS: The ultrastructural and immunohistochemical characters of the adenoma of retinal pigment epithelium are consistent with the retinal pigment epithelium.

Cell death in retinoblastoma: electron microscopic, immunohistochemical, and DNA fragmentation studies.

Apparent cell loss by apoptosis occurs in carcinomatous tissue. To investigate cell death in retinoblastoma (Rb), ultrastructural examination, ApopTag staining, electrophoresis to detect apoptotic DNA fragmentation, and flow cytometric studies were performed. Immunostaining for the oncogenic products bcl-2 and p53 was also carried out. Relationships between the proliferation fraction (PF), apoptotic index (AI), and the distribution of bcl-2 and p53 were investigated according to the degree of histologic differentiation of Rb. Ultrastructurally, two patterns of cell death were seen. Necrotic cells exhibited vacuolation of cytoplasmic organelles with a marked lytic change in the karyoplasm and cytoplasm. In contrast, apoptotic cells were characterized by crescentic margination of chromatin, condensation of karyoplasm and cytoplasm, and fragmentation of the nucleus. Differentiated Rb had a low AI value (< 1%), whereas undifferentiated Rb had a high AI value (> 8%). The PF of undifferentiated RB (31%) was significantly higher than that of differentiated RB (14%). Analysis of DNA fragmentation using 3'-end labeling with terminal transferase indicated that undifferentiated Rb has increased DNA cleavage. The distribution of apoptotic bodies within Rb was inversely correlated with the expression of bcl-2. A majority of tumor cells of differentiated Rb were negative for p53, whereas 20-40% of tumor cells of undifferentiated Rb showed a positive reaction for p53. These findings suggest that the degree of susceptibility to apoptosis is closely related to PF, is inversely related to the degree of differentiation of Rb, and is protected by oncogene bcl-2.

Glutamate and antimitotic agents induce differentiation, p53 activation, and apoptosis in rodent neostriatal cell lines immortalized with the tsA58 allele of SV40 large T antigen.

The tsA58 allele of SV40 large T antigen has the ability to immortalize cells, which is thought to be due, in part, to binding of p53 protein by T antigen at 33 degrees C. At the nonpermissive temperature (39.5 degrees C), it is thought that p53 is released, inducing growth arrest, vulnerability to apoptosis, and loss of the immortal phenotype. In cell lines derived from the rat neostriatum immortalized with tsA58, the toxic agents Adriamycin, cytosine arabinoside, and glutamate induced apoptosis and increased p53 activity and differentiation. The apoptosis and p53-inducing effects of the drugs were not greater at 39.5 degrees C compared to 33 degrees C, suggesting that p53 is not effectively blocked even at 33 degrees C. Growth arrest was not induced under most treatment conditions despite p53 induction. On the other hand, process extension was enhanced at 39.5 degrees C compared to 33 degrees C. Therefore, these cell lines are temperature sensitive with respect to differentiation, but not growth regulation or apoptosis.

Establishment and characterization of a second primary osteosarcoma cell line (OSrb/N-M) from a patient cured of bilateral retinoblastoma.

A cell line, designated OSrb/N-M, was established from the second primary osteosarcoma that developed in a 17-year-old Japanese female patient who had suffered from bilateral retinoblastoma at infancy. The OSrb/N-M cells grew as an adherent monolayer and retained some osteogenic biochemical phenotypes. In cytogenetic analyses, this cell line revealed many structural and numerical abnormalities, however, the bands q14 of both chromosomes 13 appeared to be normal, whereas the constitutional cells displayed normal female karyotypes. Immunoblot studies using monoclonal antibodies specific to RB protein demonstrated that the tumor cells did not express RB protein, suggesting that the OSrb/N-M cells might suffer from a loss-of-function mutation at this gene locus. Thus, this cell line is useful to study the molecular mechanism for the tumorigenesis of osteosarcoma with regard to an association with retinoblastoma.

Diffuse anterior retinoblastoma.

PURPOSE: The authors describe the clinicopathologic features of an anterior variant of diffuse retinoblastoma. METHODS: The clinical history of a child with an anterior chamber infiltrate and cells in the anterior vitreous was reviewed. An anterior chamber aspirate was processed by a Millipore filter technique. The eye was enucleated and routinely processed for light and transmission electron microscopic examination. RESULTS: An 8-year-old girl was treated for anterior uveitis in her right eye that failed to resolve. Examination of an anterior chamber aspirate showed cells suggestive of retinoblastoma. The eye was enucleated, and the enucleation specimen showed retinoblastoma confined to the iris, ciliary body, and anterior vitreous with one microscopic focus of tumor in the peripheral retina. CONCLUSION: This variant of diffuse retinoblastoma involved only anterior ocular structures with minimal involvement of the retina.

[Ultramicroscopic studies in retinoblastoma]. / Cercetari ultramicroscopice în retinoblastom.

Were examined three cases with undifferentiated retinoblastoma. Were effected cross sections on the samples, remarking tumoral cells with normal aspect interspersed with tumoral necrosis cells. Tumoral cells of the undifferentiated retinoblastoma has low size and a poor cytoplasmic with a big nucleus that occupies nearly the whole cell; the ratio nucleus/cytoplasmic is increased. The cells are uni- or binucleus and the nucleus is monstrous deep invagination of the cover nucleus. Cytoplasmic has poor organelles and mitochondria are swelling following a high degree of hypoxia. Also tumoral cells on remark nontumoral cells (melanocyte).

Observation of surface structure of retinoblastoma cell line.

OBJECTIVE: We study the adhesion molecules on the surface of SO-Rb50 retinoblastoma cell line. METHODS: The distribution of proteoglycans in the retinoblastoma SO-Rb50 cell line was analyzed by histochem-electron microscopy, using Colloidal Iron in combination with a series of enzyme digestions. In addition, immunohistochemistry was performed using a panel of specific antibodies including neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), S-100 protein, fibronectin, laminin, and collagen IV. RESULTS: Immunohistochemical stains showed the most marked cytoplasmic reactivity of SO-Rb50 cells with anti-NSE and anti-S100. The cells member and surface was postive with anti-NSE. No reactivity was noted with antibodies against laminin, GFAP, and collagen IV. After incubated with colloidal iron solution, three types of colloidal iron-positive stained material could be distinguished based on differences in shape, size, electron density: (1) electron dense particles, (2) the larger colloidal iron-positive filaments, (3) the smaller colloidal iron-positive filaments, which were observed present on the tumor cells surface and in the extracellular matrix. CONCLUSION: We think that the cell surface proteoglycans is the main component of adhesion molecules of retinoblastoma, which may play a central role in mediating tumor cell adhesion to extracellular matrix and adjacent host cell.