Identification of effective substrates for the direct analysis of lipids from cell lines using desorption electrospray ionization mass spectrometry.

RATIONALE: Various disease conditions, particularly tumours, can be understood easily by studying changes in the lipid profile of cells. While lipid profiles of tissues have been recorded by desorption electrospray ionization mass spectrometric (DESI-MS) imaging, there is paucity in standardized protocols for sample preparation involving cell cultures to generate reliable results. In this study, we report a method for the direct analysis of lipids from cultured cells by incorporating them onto Whatman 42 filter paper as a substrate for reliable DESI-MS analysis. METHODS: The WERI-RB1 cell line was spotted on commonly used substrates for DESI-MS analysis, such as glass slides, Teflon coated glass slides, thin layer chromatography (TLC) plates, and Whatman 42 filter paper. A comparison of mass spectrometric images with two different lipids was made to understand the behaviour of different surfaces when the same sample was spotted on them. Relative intensities of different lipid peaks in the WERI-RB1 cell line were compared and relative lipid abundances were also compared across two different human retinoblastoma cell lines; WERI-RB1 and Y79. RESULTS: The study demonstrates that good lipid signals can be obtained by DESI-MS when the cells are spotted on Whatman 42 filter paper. Tandem mass spectrometry was performed to identify the lipids as glycerophosphocholines (PC). Better lipid images from assembly of cells were obtained with distinct boundary when they were spotted on Whatman 42 filter paper than other surfaces. CONCLUSIONS: We demonstrate the use of a simple substrate for reliable DESI-MS analysis of cultured cells. This method has the potential to understand various interactions of cells with other external agents. The current method would help in the application of DESI-MS for biology in general and medical sciences in particular.

HOW EXPRESSIONS OF CLAUDIN-1 AND MMP-2 IN RETINOBLASTOMA CORRELATE WITH HISTOLOGICAL DIFFERENTIATION AND OPTIC NERVE INVASION.

Retinoblastoma is a commonly seen and dangerous intraocular malignant tumor in infants. Studies have found that Claudin-1 and MMP-2, whose expressions may be connected, play roles in tissues of retinoblastoma. In this study we analyze and discuss changes of Claudin-1 and MMP-2 expressions, and the correlation between the expressions and retinoblastoma histological differentiation and optic nerve invasion. MaxVisionTM was applied to detect expressions of Claudin-1 and MMP-2 in 45 samples of retinoblastoma and 15 paraffin-embedded samples of normal retina. The correlation between Claudin-1 expression and MMP-2 expression was analyzed based on chi-squared test and Spearman’s correlation test. Positive expressions of Claudin-1 in retinoblastoma were fewer than those in retina; higher positive expressions were found in differentiated tissues than in undifferentiated tissues; while compared to expressions in invasive optic nerves, Claudin-1 expressed more positively in optic nerves without invasion. As for MMP-2, its expressions were higher in retinoblastoma than in normal retina; undifferentiated tissues had higher positive expressions than differentiated tissues, which were not statistically significant; higher positive expressions were detected in invasive optic nerves. Thus, it could be concluded that the correlation between Claudin-1 expression and MMP-2 expression in retinoblastoma was negative. Expressions of Claudin-1 were positively related to histological differentiation and optic nerve invasion of retinoblastoma; while MMp-2 expression had negative correlation with histological differentiation and optic nerve invasion of retinoblastoma. Claudin-1 and MMP-2 played a negative role in the optic nerve invasion and tumor development of retinoblastoma.

[Efficacy of intravitreal carboplatin plus bevacizumab in refractory retinoblastoma].

OBJECTIVES: To evaluate the efficacy of intravitreal carboplatin plus bevacizumab in refractory retinoblastoma. METHODS: Perspective study.Eleven patients (11 eyes) with the diagnosis of refractory retinoblastoma were enrolled in Department of Ophthalmology of Peking University People's Hospital from June 2013 to March 2014. They underwent intravitreal carboplatin plus bevacizumab every 4 weeks, an average of 4.5 times of treatment.Observe for 3 months after the last treatment. Aqueous humor was taken for cytological and VEGF detection and retinal funds were taken photos for observation.Statistical analyses between experimental group and control group and before and after intravitreal injection within experimental group were performed with independent samples t test. RESULTS: Tumor in vitreous cavity reduced significantly in seven patients, however, poor control in four cases, and three of them were recurrent after first-line treatment. Cytology detection for aqueous humor showed no tumor cells in all of them. Aqueous VEGF of patients with retinoblastoma (60.65 ± 6.20) was significantly higher than the control group (21.98 ± 6.91). The difference was statistically significant (t = 13.80, P < 0.01). And the aqueous VEGF content decreased significantly after treatment (t = 2.12, P < 0.05). CONCLUSION: Intravitreal carboplatin plus bevacizumab, is a relatively safe, effective treatment for refractory retinoblastoma, however, ineffective for recurrent tumor.

Distantly Metastatic Retinoblastoma to Soft Tissue and Bone: A Challenging Diagnosis Highlighting the Utility of CRX.

Distant metastasis of retinoblastoma to sites outside the central nervous system is rare; such cases may present years following primary treatment. Diagnosis may be difficult given the rarity of such events and considerable histologic mimics. We describe the clinicopathologic features of 6 cases of metastatic retinoblastoma to distant bone and soft tissue sites from 2 large academic centers. Patients were 3 female and 3 male children; median age was 9.5 years (range: 5 to 15 y) with a mean interval from primary disease diagnosis of 8.0 years (range: 0.75 to 14 y). Metastasis to bones of the lower extremities was most common, occurring in 4 of 6 cases. Tumors showed typical histologic features of retinoblastoma, with sheets of primitive round cells with minimal cytoplasm and indistinct nucleoli; however, characteristic Flexner-Wintersteiner rosettes were absent. A subset of cases demonstrated an alveolar growth pattern, and 2 cases showed higher grade cytology with nuclear anaplasia and prominent nucleoli. Immunohistochemistry for CRX and RB1 showed uniform positivity and loss of expression, respectively. Metastatic retinoblastoma outside the central nervous system may present following long disease-free intervals. Immunohistochemistry for CRX is helpful to confirm this challenging diagnosis.

Downregulation and aberrant promoter methylation of p16INK4A: a possible novel heritable susceptibility marker to retinoblastoma.

RB loss has long been recognized as the causative genetic alteration underlying retinoblastoma but it is increasingly evident that other alterations are required for the tumor to develop. Therefore, we set out to identify additional inheritable susceptibility markers and new potential preventive and therapeutic targets for retinoblastoma. We focused on the p16INK4A tumor suppressor gene because of its possible role in retinoblastoma pathogenesis and its involvement in predisposition to familial cancer. p16INK4A expression was analyzed in tumor samples from retinoblastoma patients by immunohistochemistry and in peripheral blood cells from both patients and their parents by real-time quantitative reverse transcription-PCR (qRT-PCR). Since promoter methylation is a common mechanism regulating p16INK4A expression, the methylation status of its promoter was also analyzed in blood samples from patients and their parents by methylation-specific PCR. A downregulation of p16INK4A was observed in 55% of retinoblastoma patients. Interestingly, in 56% of the cases showing p16INK4A downregulation at least one of the patients' parents bore the same alteration in blood cells. Analysis of p16INK4A promoter methylation showed hypermethylation in most patients with p16INK4A downregulation and in the parents with the same alteration in p16INK4A expression. The finding that p16INK4A was downregulated both in patients and their parents suggests that this alteration could be a novel inheritable susceptibility marker to retinoblastoma. The observation that p16INK4A downregulation seems to be due to its promoter hypermethylation opens the way for the development of new preventive and therapeutic strategies using demethylating agents.

Dysplastic Lipoma: A Distinctive Atypical Lipomatous Neoplasm With Anisocytosis, Focal Nuclear Atypia, p53 Overexpression, and a Lack of MDM2 Gene Amplification by FISH; A Report of 66 Cases Demonstrating Occasional Multifocality and a Rare Association With Retinoblastoma.

In our routine and consultative pathology practices, we have repeatedly encountered an unusual subcutaneous fatty tumor with notable anisocytosis, single-cell fat necrosis, and patchy, often mild, adipocytic nuclear atypia. Because of the focal atypia, consultative cases have most often been received with concern for a diagnosis of atypical lipomatous tumor. Similar tumors have been described in small series under the designations "subcutaneous minimally atypical lipomatous tumors" and "anisometric cell lipoma." Sixty-six cases of this tumor type were collected and reviewed. Immunohistochemistry for p53, MDM2, CDK4, Retinoblastoma 1 (RB1) protein, CD34, S100, and CD163 was performed. Cases were tested for MDM2 gene amplification and RB1 gene deletion with fluorescence in situ hybridization (FISH) and for TP53 mutations by Sanger sequencing. Next-generation sequencing analysis using a panel of 271 cancer-related genes, including TP53, RB1, and MDM2, was also carried out. Our patient cohort included 57 male patients, 8 female patients, and 1 patient of unstated sex, who ranged in age from 22 to 87 years (mean: 51.2 y). All tumors were subcutaneous, with most examples occurring on the upper back, shoulders, or posterior neck (86.4%). Ten patients had multiple (2 to 5) lipomatous tumors, and the histology was confirmed to be similar in the different sites in 4 of them, including 1 patient who had a retinoblastoma diagnosed at age 1. The tumors were generally well circumscribed. At low magnification, there was notable adipocytic size variation with single-cell fat necrosis in the background associated with reactive histiocytes. Adipocytic nuclear atypia was typically patchy and characterized by chromatin coarsening, nuclear enlargement, and focal binucleation or multinucleation. Focal Lochkern change was frequent. In most instances, the degree of atypia was judged to be mild, but in 3 instances, it was more pronounced. Spindle cells were sparse or absent, and when present, cytologically bland. Thick ropy collagen bundles were absent. In all cases, p53 immunoexpression was noted (range: 2% to 20% of adipocytic nuclei), characteristically highlighting the most atypical cells. Twenty of 50 cases had MDM2 immunoreactivity, usually in <1% of the neoplastic cells, but in 4 cases, up to 10% of the cells were positive. Of 32 cases tested, 22 showed a near total loss of RB1 immunoexpression, and the remainder showed partial loss. Three of 13 cases showed RB1 gene deletion in >45% of the cells by FISH (our threshold value for reporting a positive result) with an additional 3 cases being very close to the required cutoff value. MDM2 gene amplification was absent in all 60 cases tested, including those with the greatest MDM2 immunoexpression and most pronounced atypia. All 5 tested cases showed no TP53 mutation with Sanger sequencing. Because of material quality issues, next-generation sequencing analysis could be performed in only 3 cases, and this did not reveal any recurrent mutations. All tumors were managed by simple local excision. Follow-up was available for 47 patients (range: 1 to 192 mo; mean: 27 mo) and revealed 2 local recurrences and no metastases. Dysplastic lipoma is a distinctive atypical fatty tumor variant that has p53 overexpression and RB1 gene abnormalities and lacks MDM2 gene amplification by FISH. These tumors have a strong male predominance and a notable tendency to involve the subcutaneous tissue of the shoulders, upper back and posterior neck. Multifocality is frequent (18.9% of patients with follow-up information), and there is a rare association with retinoblastoma. This tumor warrants separation from ordinary lipoma with fat necrosis, fat-rich spindle cell lipoma and the conventional form of atypical lipomatous tumor that features MDM2 gene amplification.

Stem cell markers: ABCG2 and MCM2 expression in retinoblastoma.

BACKGROUND/AIM: The authors studied the expression of cancer stem cell surface marker, ABCG2, and neural stem cell marker, MCM2, in retinoblastoma and correlated clinicopathologically. METHODS: Among 39 retinoblastomas, 18 tumours were not subjected to preoperative/postoperative chemotherapy, 15 tumours underwent postoperative chemotherapy, and six tumours had preoperative chemotherapy. There were 20 tumours with no invasion and 19 tumours with invasion of choroid/optic nerve. ABCG2 and MCM2 expression was studied by immunohistochemistry. RESULTS: ABCG2 was positive in six of six and MCM2 was positive in five of six tumours that had recurred in the orbit or metastasised. ABCG2 was positive in 15/19 tumours with invasion. MCM2 was positive in 16/19 tumours with invasion. Invasive tumours showed higher expression of ABCG2 (p < 0.01) and MCM2 (p < 0.01) proteins. There was no correlation with differentiation and laterality of the tumours. Non-neoplastic retina was positive for ABCG2 and MCM2. CONCLUSION: ABCG2 and MCM2 were expressed more in invasive tumours. Further studies are needed to understand the significance of ABCG2 and MCM2 expression in retinoblastoma.

Expression of autophagy-related protein LC3B, p62, and cytoplasmic p53 in human retinoblastoma tissues.

OBJECTIVE: Dysfunction of autophagy has been implicated in development and progression of diverse human cancers. However, the exact role and mechanism of autophagy have not been fully understood in human cancers, especially in retinoblastoma (Rb). PATIENTS AND METHODS: We determined the autophagy activity in human Rb tissues by assessing the autophagy markers microtubule-associated protein light chain 3B (LC3) and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry and then associated their expression with patient clinicopathological features. We further explored the correlation between the expression of LC3B and p62 and the expression of cytoplasmic p53, a newly identified autophagy suppressor, in Rb tissues. RESULTS: Our data revealed that the expression of LC3B and p62, was significantly associated with disease progression and tumor invasion of Rb. Furthermore, we also revealed that cytoplasmic expression of p53 was inversely associated with the behavior of tumor invasion. Finally, Spearman correlation analysis demonstrated that cytoplasmic expression of p53 was significantly and inversely correlated to the expression of both LC3B and p62. CONCLUSIONS: Autophagy might play an important role in human Rb progression, and LC3B and p62 may be useful predictors of disease progression in patients with Rb.

Management of retinoblastoma: opportunities and challenges.

Nano-delivery systems have significantly evolved over the last decade for the treatment of cancer by enabling site-specific delivery and improved bioavailability. The widely investigated nanoparticle systems are biodegradable polyesters, dendrimers, liposomes, mesoporous silica and gold nanoparticles. These particles when conjugated with different targeting motifs enhance the therapeutic efficiency of the drug molecules and biocompatibility. However, the application of such systems towards the treatment of retinoblastoma (RB), a rapidly spreading childhood eye cancer, still remains in its infancy. Nanoparticle-based systems that have been investigated for RB therapy have displayed improved drug delivery to the most restricted posterior segment of the eyes and have increased intra-vitreal half-life of the chemotherapy agents highlighting its potential in treatment of this form of cancer. This review focuses on the challenges involved in the treatment of RB and highlights the attempts made to develop nano-dimensional systems for the treatment of RB.

Molecular diagnosis in patients with retinoblastoma: Report of a series of cases. / Diagnóstico molecular en pacientes con retinoblastoma: reporte de una serie de casos.

OBJECTIVES: To report the benefits of genetic diagnosis in patients with retinoblastoma. METHOD: Observational study. Patients with retinoblastoma and their families were included. Demographic and clinical data were recorded. Blood and tumour samples were obtained. Next generation sequencing was performed on the samples. When deletion 13 q syndrome was suspected, cytogenetics microarray was performed (Cytoscan® HD, Affymetrix, Santa Clara, CA, USA), with a high density chip of 1.9 million of non-polymorphic probes and 750 thousand SNP probes. RESULTS: Of the 7 cases were analysed 4 were male. The mean age at diagnosis was 21 months (range 5-36). Three cases had bilateral retinoblastoma, and 4 unilateral. None had family history. In all patients, blood was analysed, and a study was performed on the tissue from 2 unilateral enucleated tumours, in which 6 mutations were identified, all de novo. Just one was novel (c.164delC; case 1). One case of unilateral tumour revealed blood mosaicism, showing that his condition was inheritable, and that there is a high risk of developing retinoblastoma in the unaffected eye. The patient also has an increased risk of presenting with other primary tumours. CONCLUSION: Molecular diagnosis of RB1 in patients with retinoblastoma impacts on the decision process, costs, treatment, and prognosis of patients, as well as their families.

Immunoreactivity of the 14F7 Mab raised against N-Glycolyl GM3 Ganglioside in retinoblastoma tumours.

INTRODUCTION: The identification of molecules expressed selectively on the surface of retinoblastoma cells would allow applying targeted therapies. The Ganglioside, N-Glycolyl-GM3 (NeuGc-GM3), is an attractive candidate, as it has been detected in other paediatric neuroectodermic tumours, and it is not expressed in human normal tissues. The 14F7 antibody recognizes specifically the ganglioside NeuGc-GM3. PURPOSE: To characterize the expression of NeuGc-GM3 in retinoblastoma cell lines and in retinoblastoma tumours using the 14F7 monoclonal antibody. METHODS: We studied WERI-Rb1 and Y79 cell lines, 24 retinoblastoma primary tumours from unilateral and bilateral cases and two bone marrow biopsies from metastatic retinoblastoma. Tumours were classified into three groups: non-invasive (n = 13), invasive (n = 9) and metastatic (n = 2). Three eyes enucleated because of non-tumoural conditions were used as controls. Cell lines and tumour sections were studied by immunohistochemistry using the 14F7 antibody. NeuGc-GM3 expression was evaluated by analysing the percentage of positive tumoural cells and the staining intensity. These parameters were analysed comparatively among the three groups. RESULTS: Both retinoblastoma cell lines showed immunoreactivity to NeuGc-GM3 but WERI-Rb1 presented higher intensity than Y79. All the tumours studied showed strong immunoreactivity to NeuGc-GM3 with no significant differences among groups. In both bone marrow specimens, NeuGc-GM3 immunoreactivity was observed in retinoblastoma cells. In bilaterally enucleated cases, NeuGc-GM3 immunoreactivity was not altered before and after chemotherapy. Non-tumoural retinas were negative. CONCLUSIONS: NeuGc-GM3 is highly expressed in retinoblastoma cell lines, tumours and metastatic cells to the bone marrow, and it is not detectable in control eyes. There were no significant differences in the immunoreactivity to 14F7 among tumours from different disease stages. Its immunoreactivity did not change after chemotherapy.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy: An analytical technique to understand therapeutic responses at the molecular level.

Rapid monitoring of the response to treatment in cancer patients is essential to predict the outcome of the therapeutic regimen early in the course of the treatment. The conventional methods are laborious, time-consuming, subjective and lack the ability to study different biomolecules and their interactions, simultaneously. Since; mechanisms of cancer and its response to therapy is dependent on molecular interactions and not on single biomolecules, an assay capable of studying molecular interactions as a whole, is preferred. Fourier Transform Infrared (FTIR) spectroscopy has become a popular technique in the field of cancer therapy with an ability to elucidate molecular interactions. The aim of this study, was to explore the utility of the FTIR technique along with multivariate analysis to understand whether the method has the resolution to identify the differences in the mechanism of therapeutic response. Towards achieving the aim, we utilized the mouse xenograft model of retinoblastoma and nanoparticle mediated targeted therapy. The results indicate that the mechanism underlying the response differed between the treated and untreated group which can be elucidated by unique spectral signatures generated by each group. The study establishes the efficiency of non-invasive, label-free and rapid FTIR method in assessing the interactions of nanoparticles with cellular macromolecules towards monitoring the response to cancer therapeutics.

Genetic analysis in retinoblastoma and peripheral blood correlation.

OBJECTIVE: To determine the importance of intratumoral genetic analysis in the diagnosis of germ-line mutations in patients with retinoblastoma. To underline the importance of performing these genetic tests in every case of retinoblastoma. METHOD: Intratumoral genetic analysis of RB1 mutation was performed on 17 enucleated eyes that were non-responsive to conservative treatment. Patients had no family history of retinoblastoma, and lesions were always single. The identified mutations were then also studied in peripheral blood analysis. RESULTS: There were 12 (70.6%) cases with positive results in intratumoral analysis. In 8 cases (47.1%) mutation of both RB1 alelli were detected, and in 4 (23.5%) cases only one allele was found mutated. In 5 patients (29.4%) no mutation was identified. In the first hit, mutations comprised 7 frameshift or nonsense and 2 splice, whereas in the second hit, one splice mutation, 2 nonsense and 8 loss of heterozygosity were identified. Among 6 patients where intratumoral analysis detected a single mutation associated with a loss of heterozygosity, the peripheral blood analysis was able to detect the same mutation in 3 cases (50%). CONCLUSIONS: Intratumoral genetic analysis of sporadic retinoblastoma can detect germ-line mutations. These patients are at higher risk of bilateralization and development of second tumors or trilateral retinoblastoma. Genetic screening is recommended in every patient diagnosed with retinoblastoma.

Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients: Phenotypic and molecular epidemiological implications.

Mutation analysis of retinoblastoma is considered important for genetic counseling purposes, as well as for understanding the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of an analysis of 43 hereditary retinoblastoma Spanish patients and kindred, using direct PCR sequencing, we have observed 29 mutations; most of them (62%) have not been reported previously. Of the mutations, 69% correspond to nonsense mutations (mainly CpG transitions) and frameshifts, with the expected outcome of a truncated Rb protein that lacks the functional pocket domains and tail. The remainder corresponds to splicing mutations, most of them (62%) targeted to invariant nucleotides, with the predicted consequence of out of frame exon skipping. Two of the splicing mutations in our study were found associated to families with a low-penetrance phenotype. Additionally, most of the mutations affecting splice junctions corresponded to retinoblastoma cases of either sporadic or hereditary nature with delayed onset (32 months on average). In contrast, most of the nonsense and frameshift mutations are associated with an early age at diagnosis (8.7 months on average). These differences are discussed in the context of the relationships between genotype and low expressivity phenotype. The differences in the spectrum of RB1 mutations found in this and other European surveys are also discussed in the context of alternate DNA methylation and mismatch repair phenotypes.

Increased chromokinesin immunoreactivity in retinoblastoma cells.

chromokinesin is a developmentally down-regulated gene with specific expression in proliferating cells during embryonic chick development. It encodes a DNA-binding motor protein localized along the chromosome arm during mitosis, suggesting that the protein may be a component of the long-observed, yet poorly understood 'ejection force' hypothesized to be involved in controlling the direction and speed of chromosome movement. We have isolated human chromokinesin; with affinity-purified antibodies we demonstrated immunocytochemically that Chromokinesin was present at a much higher level in cultured retinoblastoma cells than in primary cultures of human dermal fibroblasts. The increase in immunoreactivity was particularly prominent in interphase cells, whereas in primary cultures of fibroblasts immunopositive cells were predominantly M-phase cells. These observations imply a deregulation of chromokinesin in retinoblastoma cells. Data presented here may be useful in designing strategies to modulate chromosome movement and cell proliferation with either antisense oligonucleotides or specific antibodies, and hence may set the stage for further investigations of the involvement of chromosome motor molecules in mitosis under normal and pathological conditions.

Second primary rhabdomyosarcomas in patients with bilateral retinoblastoma: a clinicopathologic and immunohistochemical study.

We reviewed six cases of rhabdomyosarcoma as a rare second primary malignancy in children with bilateral retinoblastoma after irradiation treatment. The patients comprised four females and two males (age range 1 year 4 months-7 years 11 months). Second tumors arose in the temporal muscle inside or close to the previously irradiated fields. All the children were alive and well 24-72 months after diagnosis. Microscopic examination showed proliferation of closely packed, small round cells with scanty cytoplasm, coarse nuclear chromatin, and increased mitotic activity without a myxoid background nor obvious alveolar architecture. The most characteristic feature was the presence of rosette-like structures in four tumors. Immunoreactivity for many skeletal muscle markers was evident, including desmin (six of six), muscle-specific actin (HHF35) (six of six), sarcomeric actin (six of six), myogenin (six of six), vimentin (six of six), and myoglobin (three of six). On reverse transcriptase-polymerase chain reaction examination, three second tumors lacked specific chimeric transcripts for alveolar rhabdomyosarcoma and Ewing's sarcoma. Unexpectedly, variable reactivity for neurofilament (150 kd) was identified in six of six second tumors as well as 15 of 20 sporadic primary rhabdomyosarcomas (75%) examined as controls, the result being confirmed by Western blot analysis. In addition, staining for retinoblastoma-susceptibility gene protein was negative in all second tumors, in contrast to positivity in 14 of 17 sporadic primary tumors (82%). This finding suggests that retinoblastoma-susceptibility gene abnormalities could be associated with the development of second primary rhabdomyosarcoma. We consider that knowledge of the occurrence of rhabdomyosarcoma and appropriate immunohistochemical study are helpful for avoiding a misdiagnosis of recurrent retinoblastoma or Ewing's sarcoma when encountering patients with a history of bilateral retinoblastoma who developed second small round cell neoplasms.

Modulation of retinoblastoma cell characteristics by hexamethylene bis-acetamide and other differentiating agents in culture.

The undifferentiated Y-79 retinoblastoma cell line can be induced by specific agents to express characteristics of mature retinal cells. In the present study, attached Y-79 cell cultures were treated with hexamethylene bis-acetamide (HMBA) and other differentiating agents and examined for "neuronal" and other properties. Immunocytochemical staining was performed with antibodies against neuron- and retina-specific antigens, [synaptophysin, interphotoreceptor retinoid-binding protein (IRBP), neural cell adhesion molecule (N-CAM), and rod- and cone-specific transducin (TR alpha and TC alpha)] and microtubule-associated protein (MAP-1) and tubulin. Enhanced expression of tubulin was observed with cAMP treatment in FBS media. Expression of N-CAM was observed in all groups. Morphological differentiation was pronounced with HMBA and butyrate treatment, with HMBA inducing increased tubulin expression after 2 weeks of treatment. Expression of TR alpha was minimal under all culture conditions, whereas TC alpha was ubiquitously expressed. This supports the concept that Y-79 retinoblastoma is predominantly of cone neuronal origin and that, surprisingly, immunocytochemical differentiation is not correlated with the marked morphological changes induced by the major differentiating agents used.

Cytokine-induced modulation of cellular proteins in retinoblastoma. Analysis by flow cytometry.

Cytokines are a group of specialized, hormone-like proteins that can exert profound influences on cellular development and on a variety of cellular functions. Retinoblastoma cells are an important model for exploring human malignancy and differentiation. These multipotent embryonic cells are capable of differentiating into neuronal, glial-like and retinal pigment epithelium (RPE)-like elements. This report shows that flow cytometric analysis can be used to measure the expression of both cytoplasmic and cell surface proteins in retinoblastoma cells. The authors used this technique to monitor changes in the expression of selected cellular proteins after exposure to specific cytokines and found that MHC class I molecules were augmented by interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma), but not by tumor necrosis factor (TNF). However, the MHC class II molecules were augmented by IFN-gamma but not by IFN-alpha or TNF. The neuronal markers, IRBP and PR-6, the glial-like marker, GFAP, and the RPE cell markers, RPE-9 and RPE-15, were not altered by any of the cytokines tested. Furthermore, IFN-gamma induced a striking enhancement of the expression of the photoreceptor cell protein, S-antigen. In contrast, IFN-alpha and TNF did not affect the expression of S-antigen. These studies show that the cytokine, IFN-gamma, can enhance a distinct cellular protein associated with cells committed to a specific cell lineage.

Alterations of cell cycle regulators affecting the RB pathway in nonfamilial retinoblastoma.

We undertook the present study to examine alterations affecting the RB pathway in the G1 checkpoint and to determine their potential clinical significance in children affected with nonfamilial retinoblastoma. Using immunohistochemistry, patterns of expression of pRB, p16/INK4A, and E2F1 were analyzed in tissue from a cohort of 86 well-characterized patients with nonfamilial retinoblastoma diagnosed at the "Instituto Nacional de Pediatria" in Mexico City. The relationship of these phenotypes to proliferative index was assessed by analysis of Ki67 antigen expression. pRB expression was found in 11 (13%) cases. Using a hypophosphorylated specific pRB antibody, we observed low levels of underphosphorylated pRB expression in only 1 of 9 evaluable positive cases. These data suggest that the detected pRB products were hyperphosphorylated and thus had decreased functional activity. Increased p16 nuclear expression was found in only 6 tumors. No tumors showed deletions or mobility shifts of the INK4A gene. Undetectable pRB levels were significantly associated with undetectable p16 expression (odds ratio, 10.8; 95% confidence interval, 1.4-81.3; P =.03). All tumors showed nuclear immunoreactivities for E2F1 and Ki67. Increased Ki67 proliferative index was associated with increased staining for E2F1 (r =.44; P =.008) and increasing clinical stage (P =.03). Among children with unilateral disease, the mean Ki67 proliferative index was significantly higher in children with advanced clinical disease (stages 3 and 4) (mean 81.25; SD 6.78) than in those with earlier stage disease (mean 69.50; SD 9.45) (P = 0.001). Among children with bilateral disease, however, the mean proliferative index was not significantly higher for children with advanced clinical stage. When examining all cases together, there was a significant trend toward increasing proliferative index with increasing clinical stage (P =.03). In unilateral tumors, we also found that presence of detectable pRB was associated with a lower percentage of cells expressing E2F1 (46.7% v 70.8%) (P = 0.05), whereas there was no association between presence of pRB and E2F1 among bilateral tumors. We have found that expression of some of the cell cycle markers examined varies according to laterality, suggesting underlying differences in the capacity for cell cycle regulation between these 2 forms of the disease. Differences in capacities for cell cycle regulation may account for some differences in clinical behavior. Thus, the inclusion of molecular markers may become useful adjuncts to clinicopathological staging and subsequent determination of therapy.

Immunocytochemical features of retinoblastoma in an adult.

OBJECTIVE: To describe the clinical course and immunocytochemical characteristics of an unusual intraocular tumor. METHODS: Immunocytochemical analysis of the enucleated eye with an intraocular mass that markedly waxed and waned in size during 1 year of close observation of a 29-year-old woman. RESULTS: Most of the tumor was composed of either dying or rapidly proliferating cells. One area located near the retina consisted mostly of well-differentiated cells in uniform sheets (bacillettes) with lacelike glial processes between the tumor cells. Almost all of the differentiated tumor cells were positive for S antigen. In particular, the dominant cell type stained positively for both antibodies known to be specific for those isoforms of S antigen found only in blue cones and rods but not in red or green cones. Only a few of these cells labeled positively with an anti-rhodopsin antibody. CONCLUSIONS: This is the first case of adult retinoblastoma to be confirmed immunocytochemically. The tumor was unusual because the differentiated regions contained bacillettes composed mostly of blue cones. It is possible that this and other adult retinoblastomas may arise from previously existing retinocytomas.