Comparative genomic hybridization in pineal parenchymal tumors

Nine pineal parenchymal tumors were studied by comparative genomic hybridization. These consisted of three pineocytomas (WHO grade II), three pineal parenchymal tumors of intermediate differentiation (WHO grade III), and three pineoblastomas (WHO grade IV). An average of 0 chromosomal changes per pineocytoma, 5.3 per pineal parenchymal tumor of intermediate differentiation (3.3 gains vs. 2.0 losses), and 5.6 per pineoblastoma (2.3 gains vs. 3.3 losses) were found. The most frequent DNA copy number changes among pineal parenchymal tumors of intermediate differentiation and pineoblastomas were gains of 12q (3/6 cases), 4q, 5p, and 5q (2/6 cases each), as well as losses of 22 (4/6 cases), 9q, and 16q (2/6 cases each). Among pineal parenchymal tumors of intermediate differentiation, the most common chromosomal imbalances were +4q, +12q, and -22 (2/3 cases each), and in pineoblastomas -22 (2/3 cases). Five high level gains were identified, all of them in pineoblastomas; these were found on 1q12-qter, 5p13.2-14, 5q21-qter, 6p12-pter, and 14q21-qter. Clinically, all patients with pineocytomas and pineal parenchymal tumors of intermediate differentiation were alive after a mean observation time of 142 and 55 months, respectively, whereas all patients with pineoblastomas had died after an average of 17 months. Our findings suggest that pineal parenchymal tumors of intermediate differentiation are cytogenetically more similar to pineoblastomas and prognostically more similar to pineocytomas. Furthermore, imbalances in higher-grade pineal parenchymal tumors mainly affect gains of 12q and losses of chromosome 22.     

Neuronal and glial properties of a murine transgenic retinoblastoma model.

Antigenic properties of a murine transgenic model for hereditary retinoblastoma, induced by a chimeric gene coding for Simian virus 40 large T antigen, an oncogene that inactivates the retinoblastoma susceptibility gene product, were studied by immunohistochemistry. All transgenic mice develop bilateral intraocular retinal tumors in the inner nuclear layer with Homer Wright-like rosettes, and one quarter develop midbrain tumors resembling trilateral retinoblastoma. Cell lines TE-1 and TM-1 were established from intraocular and metastatic tumors, respectively. Intraocular tumors reacted with antibodies to neuron-specific enolase and synaptophysin, while vimentin, glial fibrillary acidic, and S-100 proteins were detected only in reactive glia derived from adjacent retina. The midbrain tumors showed weak reactivity to synaptophysin, and they blended with reactive astrocytes positive for glial markers. The tumors were negative for cytokeratins. Finally both derived cell lines expressed synaptophysin and individual neurofilament triplet proteins in immunofluorescence and Western blotting, supporting their essentially neuronal nature. The antigenic profile resembles human retinoblastoma, but differences in morphology and antigen distribution suggest a more close relationship to neurons of the inner nuclear layer than to photoreceptor cells.     

Trilateral retinoblastoma: a case report

Trilateral retinoblastoma is a rare, but well recognized syndrome. These tumors usually occur in the pineal, parasellar, or suprasellar regions several years after successful management of ocular retinoblastomas without evidence of direct extension or distant metastasis. Here we report a case of trilateral retinoblastoma presenting initially with a sellar tumor and with concurrent unilateral retinoblastoma. The patient was a 5-month-old baby girl showing poor eye contact and nystagmus for several days. She had no family history of retinoblastoma. Brain MRI revealed a midline suprasellar tumor without evidence of cerebrospinal fluid seeding or extracranial metastasis. A pathologic diagnosis of retinoblastoma was made for her brain tumor, and a small, intraocular retinoblastoma was detected in the left eye by thorough examination of the fundus. If a retinoblastoma occurs in the midline of the brain, including the pineal and sellar regions, a careful screening to detect any additional retinal tumors should be performed. Moreover, since these tumors are often hereditary and harbor a worse prognosis, the diagnosis has implications for genetic counseling. This is the first report on a case of trilateral retinoblastoma in Korea presented with a sellar mass.     

Comparative genomic hybridization of 49 primary retinoblastoma tumors identifies chromosomal regions associated with histopathology,progression, and patient outcome

Forty-nine primary retinoblastoma (Rb) tumors were analyzed by the use of comparative genomic hybridization (CGH), and clinical/histological correlations were performed. Adverse histological factors were present in 13 patients. Chromosomal imbalance was a frequent phenomenon, seen in 96% of the tumors. Gain of 6p represented the most frequent event (69% of the tumors), whereas +1q was observed in 57%, confirming that these abnormalities are key secondary events in retinoblastoma tumor progression. Loss of 13q and 16 was significantly associated with tumors displaying adverse histo-prognostic factors, whereas -16q was significantly associated with tumors without adverse features. In three patients who developed an extra-ocular relapse, the tumors showed -13q and 2/3 had -5q, suggesting that these abnormalities may be associated with metastasis. Children >or= 36 months of age at enucleation tended to have more CGH abnormalities per tumor than children < 12 months (median numbers 11 vs. 3). In addition, +1q, +13q, -16, and -16q were more frequent in children with an older age at enucleation. Identical CGH changes were found in both tumors from one patient with bilateral tumors, suggesting a common origin. It is possible that tumors displaying loss of 13q and 5q indicate those patients who may suffer an adverse outcome and who would require alternative or more intensive therapy. CGH analysis on larger cohorts and in prospective clinical trials will be invaluable in determining whether a genetic classification of retinoblastoma represents a reliable measure of prognosis.     

Expression analysis of 6p22 genomic gain in retinoblastoma

To identify gene(s) targeted by 6p22 genomic gain, present in more than 50% retinoblastoma tumors, we used real-time RT-PCR to quantify the expression of seven genes in normal human retina and retinoblastoma. Six genes are located in the quantitative multiplex PCR-defined 0.6 Mb minimal region of gain at 6p22 (DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153), and E2F3 is 2 Mb away from the minimal region of gain on 6p22. E2F3, DEK, KIF13A, and NUP153 were most frequently overexpressed in retinoblastoma with 6p genomic gain, compared with the normal adult human retina. E2F3 and DEK mRNA levels were increased in all human tumors showing 6p22 gain, as well as in mouse retinoblastoma induced by SV40 large T antigen expression in developing retina, compared with the normal controls (adult human retina and 7-day-old mouse retina, respectively). Only DEK showed statistically significant correlation of expression and genomic copy number (P = 0.019). E2F3 and DEK, but not NUP153, showed developmental regulation. E2F3 and DEK mRNA overexpression was always associated with protein overexpression, determined by immunoblotting or immunofluorescent staining of primary tumors, relative to the adjacent normal retina. E2F3 was strongly expressed in actively proliferating cells, while DEK was overexpressed in all tumor cells. Taking into account the proliferation-promoting role of E2F3, implication of E2F3 in bladder and prostate cancer, and the translocation and overexpression of DEK in leukemia, we conclude that either DEK or E2F3 (or both) are targeted by the 6p22 gain in retinoblastoma.     

Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.     

Parietal eye-pineal morphology in lizards and its physiological implications

Pineal complexes in 85 species of lizards examined comprised seven morphological types. Members of the same family do not necessarily have the same pineal complex type. “Regressive” parietal eyes were not common except in certain arboreal lizards, primarily from the family Chameleontidae. The parietal eye is often retained in burrowing lizards, presumably because these animals are occasionally exposed to light and the parietal eye is a more suitable photoreceptor for a burrower than are lateral eyes. The pineal of certain lizards possesses a finger-like projection that extends toward the parietal eye. This extension, along with pineal wall convolutions, results in more photoreceptor cells oriented for maximal absorption of light. It is rare to find convolutions and an extension in the same pineal. Cartilage deposits and blood sinuses may modify the intensity and wavelength of light reaching the pineal. These observations suggest that the intracranial pineal of lizards is a more important photoreceptor than was previously realized, a situation that may be a factor in the occasional “failure” of parietalectomy experiments.     

Immunohistochemical features of the human retina and retinoblastoma

The immunohistochemical features of 24 retinoblastoma specimens from 22 patients, 15 with unilateral and 7 with bilateral disease, were examined by the labelled streptavidin biotin (LSAB) method and compared with those of specimens from the remaining morphologically normal retina. In the normal retina, S-100 protein, glial fibrillary acidic protein (GFAP) and vimentin were detected in astrocytes and/or Müller cells. Neurofilament protein was seen in axons of the ganglion cells, synaptophysin was present in both plexiform layers, bcl-2 oncoprotein was seen in ganglion cells and bipolar cells, and neuron-specific enolase (NSE) was detected in ganglion cells, bipolar cells and photoreceptor cells and in their cell processes. While retinoblastoma (Rb) protein expression was noted in ganglion cells, bipolar cells, and some photoreceptor cells, p53 protein was not expressed at all. In all retinoblastomas, strong NSE expression and weak bcl-2 expression was observed in almost all tumour cells and synaptophysin was localized in rosette-forming cells, while tumour cells were devoid of S-100, GFAP, vimentin and neurofilament protein. These findings support the view that retinoblastomas are composed of neuron-committed cells. In addition, no Rb protein expression was detected in retinoblastomas, whereas p53 expression was found in 18 cases (75%).     

Photoreceptor differentiation of retinoblastoma: An electron microscopic study of 29 retinoblastomas

Retinoblastomas exhibit a unique form of differentiation to produce cell elements similar to those seen in a photoreceptor cell. An ultrastructural study was performed on 29 cases of retinoblastoma to further clarify the cytologic characteristics of the tumor cells. The age of the retinoblastomas averaged 17.1 months and the tumor cells showing photo-receptor differentiation were demonstrated in 10 cases (35%). The findings were especially notable in retinoblastomas with Flexner-Wintersteiner rosette formation (seven cases, 28%). Similar photoreceptor differentiation was also evident in solid cell clusters without rosette formation (four cases, 14%). The presence of photoreceptor elements was assumed to be significantly frequent both in Flexner-Wintersteiner rosettes and in the solid cell clusters. The cell cytoplasm also showed proliferation of long mitochondria and microtubules, reflecting photoreceptor differentiation. The hereditary-type retinoblastoma showed more advanced cell differentiation than the non-hereditary type. Photoreceptor differentiated retinoblastoma showed rather indolent growth compared with the undifferentiated type, and the former can expect a curative treatment by operation. These observations provide additional findings of the biological nature of retinoblastomas.     

Genetic and cytogenetic studies in children with retinoblastoma

Genetic and cytogenetic studies were performed in 110 children with retinoblastoma (57 girls and 53 boys), with 70% of cases being unilateral and 30% bilateral. The mean age of the patients at diagnosis was 22.6 months in unilateral and 11.1 months in bilateral cases. Sporadic cases were 94.5%, and the remaining were familial. There was no difference when the paternal age of sporadic cases was compared with that of familial cases. Three patients from a family exhibiting unilateral retinoblastoma had an interstitial deletion at band 13q14. The presence of other neoplasms and the importance of the genetic and chromosomic studies, for the purpose of genetic counseling, are discussed.     

Glycoconjugates in retinoblastoma

Summary The binding of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities to tumour tissue and remaining morphologically normal retina was studied in ten formalin-fixed and paraffin-embedded human eyes with retinoblastoma. In undetached retinas, outer and inner segments of photoreceptors bound concanavalin A (ConA) as well asLens culinaris (LCA), wheat germ (WGA)Ricinus communis (RCAI) and peanut (PNA) agglutinins. Both nuclear and plexiform layers bound ConA, LCA and, in some specimens, WGA and RCAI. These results agree with those obtained with normal adult human retina, the main difference being that PNA labelled some rods in addition to cones in the retinoblastoma eyes. Flexner-Wintersteiner rosettes reacted with ConA and LCA, and often with WGA, PNA and RCAI. Undifferentiated retinoblastoma cells always bound ConA and LCA, and in some tumours WGA, PNA and RCAI. Pretreatment with neuraminidase increased the number of cells that bound PNA and RCAI, but diminished binding of WGA. Pokeweed mitogen andBandeiraea simplicifolia I, Dolichos biflorus, soybean,Ulex europaeus I andLotus tetragonolobus agglutinins labelled only vascular endothelial cells. Retinoblastoma cells most closely resembled photoreceptor cells in their lectin-binding patterns.This study was supported by grants from the Emil Aaltonen Foundation and Rasmussens Stiftelse     

Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization

Mutation of both alleles of the retinoblastoma gene (RB1) initiate oncogenesis in developing human retina, but other common genomic alterations are present in the tumors. In order to sublocalize the altered genomic regions, 50 retinoblastoma tumors were examined by comparative genomic hybridization (CGH). The minimal regions most frequent gained were 1q31 (52%), 6p22 (44%), 2p24-p25 (30%) and 13q32-q34 (12%). The minimal region most frequently lost was 16q22 (14%). The overall total number of gains or losses evident on CGH was significantly greater in those tumors with either or both 6p or 1q gain, than in tumors with neither 6p nor 1q gain suggesting that chromosomal instability may be associated with acquisition of these changes. Genes mapping to 6p22 and 1q31 may be important in tumor development in retina subsequent to the loss of RB1 alleles.     

Photoreceptor cell differentiation in retinoblastoma demonstrated by a new immunohistochemical marker mucin-like glycoprotein associated with photoreceptor cells (MLGAPC)

AIMS: For further understanding of specific differentiation in retinoblastoma, we studied the expression of newly detected mucin-like glycoprotein associated with photoreceptor cells (MLGAPC), which is specific for photoreceptor cells of retina and analogous to interphotoreceptor matrix proteoglycan-1 (IMPG1). METHODS AND RESULTS: Surgically enucleated retinoblastomas (n=21; undifferentiated type, n=15, differentiated type, n=6) were immunohistochemically studied with a polyclonal antibody against MLGAPC, and 17/21 cases (81%) showed positive staining of tumour cells. We classified various staining patterns and structures into four groups: type 1 showing a granular intracellular scattered staining pattern with round small cells; type 2 showing a reticular staining pattern between spindle-shaped tumour cells; type 3 showing radiating staining from the centre of Homer-Wright rosettes; type 4 showing ring-shaped, radiating and granular staining associated with Flexner-Wintersteiner rosettes. Eleven of 15 undifferentiated retinoblastomas (73%) showed type 1 or 2, and all the six differentiated cases showed type 3 or 4. Image analysis of immunostaining revealed an increase in MLGAPC-positive area from 0.48% in undifferentiated cases to 1.60% in differentiated cases, and a negative correlation was shown between mitotic frequency and MLGAPC-positive area. CONCLUSIONS: This study proved MLGAPC as a valuable marker of retinoblastoma, and that photoreceptor differentiation takes place even in 'undifferentiated' retinoblastoma.     

Metastatic and nonmetastatic models of retinoblastoma

To generate animal models of retinoblastoma that closely resemble metastatic and nonmetastatic human disease for the purposes of examining tumor biology and developing alternate treatments, human retinoblastoma cell lines were injected into the vitreal cavities of immunodeficient mice. Two reproducible animal models with contrasting biological behaviors analogous to human retinoblastoma have been developed. The Y79 retinoblastoma model demonstrated specific tumor evolution similar to that seen in human invasive and metastatic disease. Y79 retinoblastoma cells formed intraocular tumors that were initially confined to the vitreal cavity. Tumors progressively invaded the retina, subretinal space, choroid, optic nerve head, and anterior chamber of the eye. Tumors progressed into the subarachnoid space and focally invaded the brain. Metastases were detected in the contralateral optic nerve. Large tumors developed extraocular extensions. The histology of the tumors showed a poorly differentiated pattern with high mitotic rate, foci of necrosis, and calcification. The WERI-Rb model more closely resembled nonmetastatic human retinoblastoma. WERI- Rb tumors were localized in the eye with only anterior choroidal invasion at late stages. To examine potential biological differences in vitro, the retinoblastoma cell lines were cocultured with adherent choroid cells or adherent glioma cells which represent the targets of invasive retinoblastoma in vivo. Consistent with the in vivo observations, Y79 cells but not WERI-Rb cells adhere specifically to both the choroidal and the glioma cell lines.     

Primitive neuroectodermal tumors of the central nervous system

Primitive neuroectodermal tumors are morphologically similar malignant tumors arising in intracranial and peripheral sites of the nervous system, showing varying degrees of cellular differentiation with a tendency to disseminate along cerebrospinal fluid pathways. They occur primarily in children and young adults. Under the designation primitive neuroectodermal tumors are included medulloblastomas and tumors that may differentiate in other directions, such as medulloepithelioma, neuroblastoma, polar spongioblastoma, pineoblastoma, ependymoblastoma, retinoblastoma, and olfactory neuroblastoma. From a practical, histologic point of view, these tumors are often indistinguishable from one another and are best thought of as primitive neuroectodermal tumors with or without differentiating features.     

The pineal gland in wild-type and two zebrafish mutants with retinal defects

Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.     

Aspiration cytology of retinoblastoma: light and electron microscopic correlations

A series of 16 cases of retinoblastoma diagnosed by fine-needle aspiration biopsy (FNAB) and confirmed by histologic examination is reviewed, and the salient cytomorphologic features are described. Two types of cells were encountered in the aspiration smears; type I cells were undifferentiated while type II cells showed more differentiation and frequently revealed cytoplasmic processes that are probably indicative of early photoreceptor differentiation. Flexner-Wintersteiner rosettes characteristic of retinoblastoma were found in 10 of 16 cases. These findings were further correlated with ultrastructural examination of the tumors in nine cases. It is concluded that the presence of rosettes and type II cells with cytoplasmic processes are the two features that are most helpful in the FNAB diagnosis of retinoblastoma.     

Loss of retinoblastoma gene and amplification of N-myc gene in retinoblastoma.

We have analyzed paired samples of genomic DNA from peripheral leukocyte and primary tumor tissue from nine patients with retinoblastoma (RB) and from two RB cell lines, WERI-Rb-1 and Y79, to detect the molecular alterations of the retinoblastoma susceptibility gene (RB-1) and N-myc gene. In Southern analysis, RB-1 deletions in tumor tissues were detected in five patients (56%), one of these revealed a total loss of RB-1. N-myc amplification was found only in one (11.1%) out of nine patients. We also observed a total loss of RB-1 in WERI-Rb-1, and a more than 100-fold amplification of N-myc in Y79. The analysis of the relationship between molecular events and clinical characteristics such as age, sex, tumor laterality did not reveal any specific correlation. These results suggest that genetic backgrounds of RB in Korean patients are quite similar to those of reported cases elsewhere. The high sensitivity of our method in detecting the RB-1 loss indicates that this method can be a useful tool for initially screening a large number of tumors.     

Diagnosis of retinoblastoma by fine-needle aspiration and aqueous cytology

This article reports the cytodiagnosis of three cases of retinoblastoma in children aged 1.5, 2.5, and 5 yr. Two of them were diagnosed by fine-needle aspiration cytology of the primary tumor and one by aqueous cytology. The tumor cells were usually round to oval, small and uniform, with scanty cytoplasm; they generally occurred in closely packed clusters of variable sizes. We discuss the differential diagnosis of retinoblastoma with other round-cell tumors of childhood involving the orbit.