Distinct Gene Expression Profiles Define Anaplastic Grade in Retinoblastoma
2018; Elsevier BV; Volume: 188; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2018.06.013
ISSN1525-2191
AutoresLauren E. Hudson, Pia R. Mendoza, William Henry Hudson, Alison Ziesel, G. Baker Hubbard, Jill Fielding-Wells, Bhakti Dwivedi, Jeanne Kowalski, Sandra Seby, Viren Patel, Eldon E. Geisert, Charles A. Specht, Hans E. Grossniklaus,
Tópico(s)interferon and immune responses
ResumoMorbidity and mortality associated with retinoblastoma have decreased drastically in recent decades, in large part owing to better prediction of high-risk disease and appropriate treatment stratification. High-risk histopathologic features and severe anaplasia both predict the need for more aggressive treatment; however, not all centers are able to assess tumor samples easily for the degree of anaplasia. Instead, identification of genetic signatures that are able to distinguish among anaplastic grades and thus predict high- versus low-risk retinoblastoma would facilitate appropriate risk stratification in a wider patient population. A better understanding of genes dysregulated in anaplasia also would yield valuable insights into pathways underlying the development of more severe retinoblastoma. Here, we present the histopathologic and gene expression analysis of 28 retinoblastoma cases using microarray analysis. Tumors of differing anaplastic grade show clear differential gene expression, with significant dysregulation of unique genes and pathways in severe anaplasia. Photoreceptor and nucleoporin expression in particular are identified as highly dysregulated in severe anaplasia and suggest particular cellular processes contributing to the development of increased retinoblastoma severity. A limited set of highly differentially expressed genes also are able to predict severe anaplasia accurately in our data set. Together, these data contribute to the understanding of the development of anaplasia and facilitate the identification of genetic markers of high-risk retinoblastoma. Morbidity and mortality associated with retinoblastoma have decreased drastically in recent decades, in large part owing to better prediction of high-risk disease and appropriate treatment stratification. High-risk histopathologic features and severe anaplasia both predict the need for more aggressive treatment; however, not all centers are able to assess tumor samples easily for the degree of anaplasia. Instead, identification of genetic signatures that are able to distinguish among anaplastic grades and thus predict high- versus low-risk retinoblastoma would facilitate appropriate risk stratification in a wider patient population. A better understanding of genes dysregulated in anaplasia also would yield valuable insights into pathways underlying the development of more severe retinoblastoma. Here, we present the histopathologic and gene expression analysis of 28 retinoblastoma cases using microarray analysis. Tumors of differing anaplastic grade show clear differential gene expression, with significant dysregulation of unique genes and pathways in severe anaplasia. Photoreceptor and nucleoporin expression in particular are identified as highly dysregulated in severe anaplasia and suggest particular cellular processes contributing to the development of increased retinoblastoma severity. A limited set of highly differentially expressed genes also are able to predict severe anaplasia accurately in our data set. Together, these data contribute to the understanding of the development of anaplasia and facilitate the identification of genetic markers of high-risk retinoblastoma. Retinoblastoma is the most common intraocular cancer of childhood, accounting for 6.1% of all cancers in children younger than 5 years of age.1Broaddus E. Topham A. Singh A.D. Incidence of retinoblastoma in the USA: 1975-2004.Br J Ophthalmol. 2008; 93: 21-23Crossref PubMed Scopus (246) Google Scholar Advances in surveillance and vision-sparing techniques over the past several decades have greatly improved outcomes,2Grossniklaus H.E. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson Memorial Lecture.Am J Ophthalmol. 2014; 158: 875-881.e1Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar with 5-year survival rates now greater than 95% in developed countries.3Canturk S. Qaddoumi I. Khetan V. Ma Z. Furmanchuk A. Antoneli C.B. Sultan I. Kebudi R. Sharma T. Rodriguez-Galindo C. Abramson D.H. Chantada G.L. Survival of retinoblastoma in less-developed countries impact of socioeconomic and health-related indicators.Br J Ophthalmol. 2010; 94: 1432-1436Crossref PubMed Scopus (128) Google Scholar Depending on the level of risk and stage of retinoblastoma at presentation, eyes with retinoblastoma may be salvaged using chemoreduction, in which chemotherapy is systemically infused for five to six cycles and the primary retinoblastoma is consolidated by laser photocoagulation in the operating room.4Shields C.L. Honavar S.G. Meadows A.T. Shields J.A. Demirci H. Singh A. Friedman D.L. Naduvilath T.J. Chemoreduction plus focal therapy for retinoblastoma: factors predictive of need for treatment with external beam radiotherapy or enucleation.Am J Ophthalmol. 2002; 133: 657-664Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Alternatively, super-selective ophthalmic artery endovascular infusion of chemotherapy via intra-arterial chemotherapy is used in some centers.5Abramson D.H. Dunkel I.J. Brodie S.E. Kim J.W. Gobin Y.P. A phase I/II study of direct intraarterial (ophthalmic artery) chemotherapy with melphalan for intraocular retinoblastoma initial results.Ophthalmology. 2008; 115 (1404.e1): 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 6Shields C.L. Bianciotto C.G. Jabbour P. Ramasubramanian A. Lally S.E. Griffin G.C. Rosenwasser R. Shields J.A. Intra-arterial chemotherapy for retinoblastoma: report no. 1, control of retinal tumors, subretinal seeds, and vitreous seeds.Arch Ophthalmol. 2011; 129: 1399-1406Crossref PubMed Scopus (178) Google Scholar, 7Abramson D.H. Super selective ophthalmic artery delivery of chemotherapy for intraocular retinoblastoma: ‘chemosurgery’ the first Stallard lecture.Br J Ophthalmol. 2010; 94: 396-399Crossref PubMed Scopus (39) Google Scholar, 8Gobin Y.P. Dunkel I.J. Marr B.P. Brodie S.E. Abramson D.H. Intra-arterial chemotherapy for the management of retinoblastoma: four-year experience.Arch Ophthalmol. 2011; 129: 732-737Crossref PubMed Scopus (330) Google Scholar Both chemoreduction and intra-arterial chemotherapy have reduced enucleation rates to approximately 10% of retinoblastoma cases in developed countries. Such improvement in treatments and clinical outcomes stems from a better understanding of the risk factors associated with metastasis and mortality, which have in turn enabled better patient risk stratification and tailoring of care. To date, estimates of clinical risk in retinoblastoma have been determined by a combination of clinical and histopathologic features. Clinical features such as older age at presentation, symptom duration longer than 6 months, poor visual acuity at presentation, buphthalmos, secondary glaucoma, iris neovascularization, ectropion uveae, orbital cellulitis, and group E tumors (the most severe according to the International Classification of Intraocular Retinoblastoma9Shields C.L. Mashayekhi A. Au A.K. Czyz C. Leahey A. Meadows A.T. Shields J.A. The international classification of retinoblastoma predicts chemoreduction success.Ophthalmology. 2006; 113: 2276-2280Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) have been shown to be associated strongly with high-risk retinoblastoma.10Kaliki S. Shields C.L. Rojanaporn D. Al-Dahmash S. McLaughlin J.P. Shields J.A. Eagle R.C. High-risk retinoblastoma based on international classification of retinoblastoma: analysis of 519 enucleated eyes.Ophthalmology. 2013; 120: 997-1003Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar High-risk histopathologic features (HRPFs) known to be associated with worse prognosis include tumor invasion of the optic nerve, choroid, or anterior chamber and indicate the need for adjuvant chemotherapy after enucleation.11Shields C.L. Shields J.A. Baez K. Cater J.R. De Potter P. Optic nerve invasion of retinoblastoma. Metastatic potential and clinical risk factors.Cancer. 1994; 73: 692-698Crossref PubMed Scopus (212) Google Scholar, 12Shields C.L. Shields J.A. Baez K.A. Cater J. De Potter P.V. Choroidal invasion of retinoblastoma: metastatic potential and clinical risk factors.Br J Ophthalmol. 1993; 77: 544-548Crossref PubMed Scopus (181) Google Scholar, 13Haik B.G. Dunleavy S.A. Cooke C. Ellsworth R.M. Abramson D.H. Smith M.E. Karcioglu Z.A. Retinoblastoma with anterior chamber extension.Ophthalmology. 1987; 94: 367-370Abstract Full Text PDF PubMed Scopus (48) Google Scholar However, a small percentage of retinoblastoma cases that progress to metastasis or death are not captured by these well-validated clinical and histopathologic indicators, prompting a search for additional histologic and genetic markers capable of more accurately predicting high-risk retinoblastoma and the need for chemotherapy.14Mabtum E.D. Bonanomi M.T. Lima P.P. Almeida M.T. Orbital retinoblastoma: case report.Arq Bras Oftalmol. 2013; 76: 247-249Crossref PubMed Scopus (4) Google Scholar Cellular anaplasia recently was identified as an additional histopathologic indicator complementary to HRPFs in detecting high-risk retinoblastoma.15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Anaplasia, an entity similar to but distinct from cellular differentiation, is defined by specific and measurable cytologic features such as pleomorphism, hyperchromatic nuclei, and a high nucleus-to-cytoplasm ratio.15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Anaplasia is commonly used in the classification of pediatric medulloblastoma, another embryonal central nervous system tumor, which is known to be associated with aggressive clinical behavior.16Eberhart C.G. Kepner J.L. Goldthwaite P.T. Kun L.E. Duffner P.K. Friedman H.S. Strother D.R. Burger P.C. Histopathologic grading of medulloblastomas.Cancer. 2002; 94: 552-560Crossref PubMed Scopus (274) Google Scholar We recently determined that cellular anaplasia similarly can indicate high-risk retinoblastoma and serve as a predictor of metastases even in the absence of HRPFs.15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar However, there are a limited number of ophthalmic pathology laboratories worldwide that are capable of properly examining enucleated eyes for anaplasia and HRPFs, preventing many clinical centers from taking advantage of this predictive tool. The goal of the present study was to determine the gene expression profiles of different grades of anaplasia. Identifying differentially expressed genes may lend insight into the pathways contributing to increased anaplastic severity and also serve as the first step in defining genetic markers of anaplastic grade. Determining the gene expression profiles that distinguish anaplastic grades may lead to a gene-based clinical test to facilitate risk stratification and treatment management of retinoblastoma patients. Here, we present the gene expression profiles of 28 retinoblastoma samples of mild, moderate, and severe grade anaplasia in addition to normal retina and retinocytoma, a benign precursor of retinoblastoma. We furthermore identified genes that are differentially expressed in severe versus mild and moderate anaplasia and found that although mild and moderate anaplasia are highly similar in terms of gene expression, severe anaplasia showed significant changes in the expression of nonoverlapping genes and pathways. We describe these genetic pathways that appear to distinguish severe versus mild and moderate anaplasia and discuss how they may underlie increased severity in retinoblastoma. Finally, we determined particular genes that are able to predict severe anaplasia among our retinoblastoma samples. These data comprise the first genetic characterization of anaplasia in retinoblastoma and advance our understanding of this important predictor of severity in this childhood cancer. Enucleation and exenteration specimens with a diagnosis of retinoblastoma were identified from the LF Montgomery Laboratory at the Emory Eye Center from January 1, 1940, to August 31, 2013. Exclusion criteria included any treatment before enucleation, fewer than two low-power (20×) fields of tumor, or retinoblastoma with diffuse growth patterns.17Jijelava K.P. Grossniklaus H.E. Diffuse anterior retinoblastoma: a review.Saudi J Ophthalmol. 2013; 27: 135-139Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 18Shields C.L. Ghassemi F. Tuncer S. Thangappan A. Shields J.A. Clinical spectrum of diffuse infiltrating retinoblastoma in 34 consecutive eyes.Ophthalmology. 2008; 115: 2253-2258Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Patient medical records were reviewed for RB1 mutational status (heritable or sporadic) and demographic parameters, including age at presentation, age at enucleation, sex, and race. Clinical findings including laterality, symptoms, ophthalmologic findings, Reese-Ellsworth Classification,19Reese A. Tumors of the Eye.ed 3. Harper and Row, Hagerstown, MD1976Google Scholar International Classification of Retinoblastoma,20Murphree A.L. Intraocular retinoblastoma: the case for a new group classification.Ophthalmol Clin North Am. 2005; 18: 41-53Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar length of follow-up evaluation, and treatments also were reviewed. Clinical outcomes of interest as determined from chart review were local recurrence and the presence of distant metastasis and secondary tumors. Study approval was obtained from the Emory University Institutional Review Board. Histopathologic evaluation of enucleation specimens was conducted as previously described to determine tumor size, growth pattern, level of differentiation, degree of apoptosis, grade of anaplasia, tumor seeding, extent of tissue invasion, and presence of retinocytoma.15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Two ophthalmic pathologists (C.S. and H.E.G.) blinded to patient data reviewed standard pupil–optic nerve sections and transverse optic nerve sections at the surgical margin of each specimen according to American Joint Committee on Cancer pathologic classifications.21Sastre X. Chantada G.L. Doz F. Wilson M.W. De Davila M.T.G. Rodríguez-Galindo C. Chintagumpala M. Chévez-Barrios P. Proceedings of the Consensus Meetings from the International Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma.Arch Pathol Lab Med. 2009; 133: 1199-1202PubMed Google Scholar, 22Edge S.B. Compton C.C. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM.Ann Surg Oncol. 2010; 17: 1471-1474Crossref PubMed Scopus (6247) Google Scholar Calottes were obtained and examined for all cases collected subsequent to protocol standardization [retinoblastoma (RB) 7, RB21-24, RB30, RB32, RB33].21Sastre X. Chantada G.L. Doz F. Wilson M.W. De Davila M.T.G. Rodríguez-Galindo C. Chintagumpala M. Chévez-Barrios P. Proceedings of the Consensus Meetings from the International Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma.Arch Pathol Lab Med. 2009; 133: 1199-1202PubMed Google Scholar Calotte examination provides more thorough and conclusive determination of HRPFs; however, all cases lacking calottes were examined according to standard contemporary practices as described earlier in this paragraph. High-risk features were determined as defined by the International Retinoblastoma Staging Working Group and included any invasion of the postlaminar optic nerve, massive choroidal invasion, anterior segment invasion, or prelaminar or laminar optic nerve invasion in conjunction with nonmassive choroidal invasion.21Sastre X. Chantada G.L. Doz F. Wilson M.W. De Davila M.T.G. Rodríguez-Galindo C. Chintagumpala M. Chévez-Barrios P. Proceedings of the Consensus Meetings from the International Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma.Arch Pathol Lab Med. 2009; 133: 1199-1202PubMed Google Scholar, 23Eagle Jr., R.C. High-risk features and tumor differentiation in retinoblastoma: a retrospective histopathologic study.Arch Pathol Lab Med. 2009; 133: 1203-1209Crossref PubMed Google Scholar Anaplastic grade was determined by two independent ophthalmic pathologists (C.S. and H.E.G.) blinded to patient data and included assessment of cell shape, cell wrapping, mitosis, and nuclear size, contour, and chromaticity.15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Specifically, retinocytoma was defined as cells showing unenlarged nuclei, abundant eosinophilic cytoplasm and evenly dispersed chromatin, proper differentiation as reflected by fleurettes, and the absence of mitotic figures and pleomorphism. Mild anaplasia was defined by unenlarged nuclei, differentiation (Flexner-Wintersteiner and Homer Wright rosettes), and the presence of rare mitotic figures and mild pleomorphism. Moderate anaplasia was defined by enlarged nuclei, moderate to poor differentiation, frequent mitotic figures, and moderate pleomorphism. Severe anaplasia was defined by very large hyperchromatic nuclei, poor differentiation, numerous mitotic figures, extreme pleomorphism (angular, rhomboid, or fusiform), and cell wrapping. Grade was assigned to a sample according to the highest grade identified in any focal area. Tissue cores were obtained from formalin-fixed, paraffin-embedded tissue blocks for RNA preparation. Samples with less than 70% tumor content determined by histology were excluded from analysis. Total RNA was extracted using the AllPrep DNA/RNA formalin-fixed, paraffin-embedded tissue kit (Qiagen, Valencia, CA) and Mag-Bind XP formalin-fixed, paraffin-embedded tissue RNA kit (Omega Bio-tek, Norcross, GA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). First- and second-strand cDNA synthesis, labeling, and hybridization to Affymetrix (Santa Clara, CA) Human Gene 2.0ST arrays were performed according to standard Affymetrix protocols by the Emory University Integrated Genomics Core. Data were deposited to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo; accession number GSE110811). Samples that failed amplification (three retinoblastoma and two normal retina samples) were eliminated from the study. Microarray expression data were processed, normalized by robust multiarray average, and log2 transformed using the Oligo Bioconductor R package version 1.36.1.24Carvalho B.S. Irizarry R.A. A framework for oligonucleotide microarray preprocessing.Bioinformatics. 2010; 26: 2363-2367Crossref PubMed Scopus (984) Google Scholar, 25Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. Hornik K. Hothorn T. Huber W. Iacus S. Irizarry R. Leisch F. Li C. Maechler M. Rossini A.J. Sawitzki G. Smith C. Smyth G. Tierney L. Yang J.Y.H. Zhang J. Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5:R80Google Scholar Differentially expressed genes were identified using t-tests for each gene between groups of interest (retinoblastoma versus normal retina, severe anaplasia versus mild and moderate anaplasia, and mild and moderate anaplasia versus normal retina), and P values were corrected for multiple comparisons using the Benjamini–Hochberg false discovery rate procedure. Principal component analysis (PCA) was performed on normalized expression data using the FactoMineR version 1.3926Lê S. Josse J. Husson F. FactoMineR: an R package for multivariate analysis.J Stat Softw. 2008; 25: 1-18Crossref Scopus (5108) Google Scholar package in R. For hierarchal clustering, affinity propagation clustering was performed with the APCluster package version 1.4.4 in R for all differentially expressed genes (P < 0.05, |log2 fold change| > 0.6).27Bodenhofer U. Kothmeier A. Hochreiter S. APCluster: an R package for affinity propagation clustering.Bioinformatics. 2011; 27: 2463-2464Crossref PubMed Scopus (278) Google Scholar, 28Frey B.J. Dueck D. Clustering by passing messages between data points.Science. 2007; 315: 972-976Crossref PubMed Scopus (5144) Google Scholar Gene set enrichment analysis (GSEA) was performed with the GSEA preranked algorithm with rank determined by −log10(padj) * sign (fold change).29Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci U S A. 2005; 102: 15545-15550Crossref PubMed Scopus (26767) Google Scholar Gene set collections searched included the hallmark, curated (C2), computational (C4), GO (C5), and oncogenic (C6) gene sets from the molecular signatures database,30Liberzon A. Subramanian A. Pinchback R. Thorvaldsdottir H. Tamayo P. Mesirov J.P. Molecular signatures database (MSigDB) 3.0.Bioinformatics. 2011; 27: 1739-1740Crossref PubMed Scopus (2736) Google Scholar as well as gene sets generated from differentially expressed genes described by Kapatai et al.31Kapatai G. Brundler M.A. Jenkinson H. Kearns P. Parulekar M. Peet A.C. McConville C.M. Gene expression profiling identifies different sub-types of retinoblastoma.Br J Cancer. 2013; 109: 512-525Crossref PubMed Scopus (57) Google Scholar Linear discriminant analysis and leave-one-out cross-validation were used to identify and test genes for the prediction of anaplastic grade.28Frey B.J. Dueck D. Clustering by passing messages between data points.Science. 2007; 315: 972-976Crossref PubMed Scopus (5144) Google Scholar Data were visualized using the Plotly32Plotly Technologies Inc.Collaborative Data Science. Plotly Technologies Inc., Montreal, QC2015Google Scholar and ggplot2 packages in R.33Wickham H. ggplot2: Elegant Graphics for Data Analysis.ed 2. Springer-Verlag, New York, NY2016Crossref Google Scholar There were 28 retinoblastoma samples (3 with matched normal retina and 3 with retinocytoma components) that met criteria for the present study, for a total of 34 samples. The majority of patients were male (64.2%), with age at diagnosis ranging from 1 to 54 months (Tables 1 and 2). Nineteen retinoblastoma cases (67.9%) presented with unilateral disease, and 6 cases developed secondary tumors (7.1%) or distant metastases (14.2%) (Tables 1 and 2). RB1 mutational status was determined in 17 (60.7%) retinoblastoma cases. Of these, six cases were found to have germline mutations. One sample had neither RB1 mutations nor MYCN amplification, which drives a unique subset of unilateral retinoblastoma cases,34Rushlow D.E. Mol B.M. Kennett J.Y. Yee S. Pajovic S. Thériault B.L. Prigoda-Lee N.L. Spencer C. Dimaras H. Corson T.W. Pang R. Massey C. Godbout R. Jiang Z. Zacksenhaus E. Paton K. Moll A.C. Houdayer C. Raizis A. Halliday W. Lam W.L. Boutros P.C. Lohmann D. Dorsman J.C. Gallie B.L. Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies.Lancet Oncol. 2013; 14: 327-334Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar but instead showed 100% hypermethylation of the RB1 promoter (sample RB31).35Ohtani-Fujita N. Fujita T. Aoike A. Osifchin N.E. Robbins P.D. Sakai T. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene.Oncogene. 1993; 8: 1063-1067PubMed Google Scholar, 36Stirzaker C. Millar D.S. Paul C.L. Warnecke P.M. Harrison J. Vincent P.C. Frommer M. Clark S.J. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors.Cancer Res. 1997; 57: 2229-2237PubMed Google Scholar All patients with high-risk histology received adjuvant chemotherapy.Table 1Patient DemographicsPatient characteristics (N = 28)N (%)Sex Male18 (64.2) Female10 (35.8)Laterality Unilateral19 (67.9) Bilateral9 (32.1)Age at diagnosis (months) Mean17.7 Range1–54RB1 mutational status Not known or test not performed11 (39.2) Known17 (60.7) Germline6/17 (35.3) Nongermline10/17 (58.8) No RB1 mutation identified1/17 (5.9)Secondary tumors2 (7.1)Distant metastasis4 (14.2) Open table in a new tab Table 2Retinoblastoma CharacteristicsAnaplastic gradeSampleHRPFDistant metastasisSecondary tumorsRB1 mutationLateralityAge at diagnosis (months)Follow-up period (months)MildRB 3NoNoNoNDBilateral3120RB 5Postlaminar ONNoPinealomaGBilateral156RB 6NoNoNoNDBilateral269RB 7Postlaminar ON, massive choroidal invasionNoNoGBilateral110RB 19NoNoNoNDUnilateral1181RB 23NoNoNoNGUnilateral1742ModerateRB 1NoNoRCCNDBilateral3212RB 2NoNoNoNDBilateral10179RB 4NoNoNoNDBilateral4186RB 8NoNoNoGBilateral4141RB 9NoNoNoGUnilateral1196RB 11Postlaminar ONNoNoNDUnilateral471RB 14Postlaminar ON, massive choroidal invasionBrainNoNDUnilateral2822RB 16Massive choroidal invasionBoneNoNDUnilateral24140RB 17NoNoNoGUnilateral2176RB 18NoNoNoNGUnilateral291RB 22NoNoNoNGUnilateral1116RB 26NoNoNoNDBilateral26138RB 31NoNoNoNoneUnilateral2364SevereRB 12Postlaminar ONNoNoNDUnilateral1845RB 13Postlaminar ON, massive choroidal invasionNoNoGUnilateral2778RB 21Postlaminar ON, massive choroidal invasionNoNoNGUnilateral3435RB 24NoBone, LiverNoNGUnilateral1919RB 25NoNoNoNGUnilateral2840RB 28Laminar ON, nonmassive choroidal invasionNoNoNGUnilateral4658RB 30NoLiver, Bone marrowNoNGUnilateral279RB 32NoNoNoNGUnilateral5443RB 33Massive choroidal invasionNoNoNGUnilateral3712G, germline; ND, not determined; NG, nongermline; ON, optic nerve invasion; RB, retinoblastoma; RCC, renal cell carcinoma. Open table in a new tab G, germline; ND, not determined; NG, nongermline; ON, optic nerve invasion; RB, retinoblastoma; RCC, renal cell carcinoma. Anaplastic grade and the presence of HRPFs were determined and compared with demographic and genetic data (Table 2). Ten samples were found to have HRPFs. Nine samples (32.1%) had severe anaplasia, 13 samples had moderate anaplasia (46.4%), and 6 samples had mild anaplasia (21.4%). Interestingly, HRPFs were identified in samples of each anaplastic grade from mild to severe, as described by Mendoza et al15Mendoza P.R. Specht C.S. Hubbard G.B. Wells J.R. Lynn M.J. Zhang Q. Kong J. Grossniklaus H.E. Histopathologic grading of anaplasia in retinoblastoma.Am J Ophthalmol. 2015; 159: 764-776Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar (Figure 1). To determine whether increasing degree of anaplasia in retinoblastoma is associated with different patterns of gene expression, microarray analysis was performed on the 28 retinoblastoma specimens in addition to the 3 retinocytoma and 3 normal retina specimens (Figure 2A, Supplemental Table S1). A total of 783 genes were differentially expressed in retinoblastoma compared with normal retina (583 down-regulated, 200 up-regulated) (|log2 fold change| > 0.6; P < 0.05). In retinocytoma versus normal retina, 90 genes were differentially expressed (76 down-regulated, 14 up-regulated) (P < 0.05). Further analysis showed clear separation of samples into retinoblastoma, retinocytoma, and normal retina groups using both PCA (Figure 2B) and hierarchical clustering (Figure 2C). One normal retina sample (Supplemental Figure S1) did not cluster with the other normal retina samples, possibly because of inclusion of some tumor cells. GSEA showed that genes up-regulated in retinoblastoma versus normal retina were strongly enriched for those identified previously by Kapatai et al31Kapatai G. Brundler M.A. Jenkinson H. Kearns P. Parulekar M. Peet A.C. McConville C.M. Gene expression profiling identifies different sub-types of retinoblastoma.Br J Cancer. 2013; 109: 512-525Crossref PubMed Scopus (57) Google Scholar as retinoblastoma-associated genes, whereas down-regulated genes were enriched for normal retina-associated genes (Supplemental Figure S2). GSEA of retinoblastoma versus normal retina using the molecular signatures database showed enrichment of gene sets related to cell-cycle checkpoint, DNA replication, and nuclear chromatin among genes up-regulated in retinoblastoma versus normal retina, whereas gene sets such as the rhodopsin pathway were enriched among down-regulated genes (Figure 2, D and E, Supplemental Tables S2 and S3). This suggests that the samples not only separated into distinct groups, but also showed expected patterns of gene expression. It was next determined whether retinoblastoma with different histopathologic patterns could be distinguished by gene expression. Of interest, PCA of the 28 retinoblastoma samples did not show a clear separation of tumors with versus without HRPFs (Figure 3A). In contrast, PCA of retinoblastoma samples showed grouping of tumors with
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