Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition.

About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

Retinoblastoma: From genes to patient care.

Retinoblastoma is the most common paediatric neoplasm of the retina, and one of the earliest model of cancer genetics since the identification of the master tumour suppressor gene RB1. Tumorigenesis has been shown to be driven by pathogenic variants of the RB1 locus, but also genomic and epigenomic alterations outside the locus. The increasing knowledge on this "mutational landscape" is used in current practice for precise genetic testing and counselling. Novel methods provide access to pre-therapeutic tumour DNA, by isolating cell-free DNA from aqueous humour or plasma. This is expected to facilitate assessment of the constitutional status of RB1, to provide an early risk stratification using molecular prognostic markers, to follow the response to the treatment in longitudinal studies, and to predict the response to targeted therapies. The aim of this review is to show how molecular genetics of retinoblastoma drives diagnosis, treatment, monitoring of the disease and surveillance of the patients and relatives. We first recap the current knowledge on retinoblastoma genetics and its use in every-day practice. We then focus on retinoblastoma subgrouping at the era of molecular biology, and the expected input of cell-free DNA in the field.

Targeted long-read sequencing allows for rapid identification of pathogenic disease-causing variants in retinoblastoma.

BACKGROUND: Identification of disease-causing variants of the retinoblastoma gene (RB1), the predominant cause of retinoblastoma, is challenging. Targeted long-read genome sequencing offers a novel approach to resolve the diverse range of pathogenic variants in RB1 and provides haplotype information rapidly. MATERIALS AND METHODS: Genomic DNA was isolated from a venipuncture blood draw of a retinoblastoma patient. Whole genome sequencing (WGS) was carried out using the short-read Ilumina platform. WGS and targeted sequencing of RB1 was accomplished using the long-read Oxford Nanopore Technologies (ONT) platform. Deep-learning frameworks allowed haplotagging, variant calling, and variant annotation of both short- and long-read data. RESULTS: Targeted long-read sequencing of the RB1 gene allowed for enhanced depth of read coverage for discovery of rare variants and haplotype analysis. A duplication leading to a frameshift and early termination in RB1 was identified as the most deleterious variant by all sequencing methods, with long-read technology providing additional information of methylation signal and haplotype information. More importantly, there was greater than 98% concordance of RB1 variants identified between short-read and targeted long-read sequencing modalities. CONCLUSIONS: Targeted long-read technology allows for focused sequencing effort for variant discovery. Application of this for the first time in a retinoblastoma patient allowed haplotagged variant identification and demonstrated excellent concordance with benchmark short-read sequencing. The added benefit of targeted long-read sequencing to resolve disease-causing genomic variation in RB1 rapidly from a blood draw will provide a more definitive diagnosis of heritable RB and guide management decisions for patients and their families.

Chromosome 6p amplification detected in blood cell-free DNA in advanced intraocular retinoblastoma.

BACKGROUND: In patients with retinoblastoma, gains of chromosome 6p have been associated with less differentiated tumors. In cell-free DNA from the aqueous humor (AH), 6p gain has been associated with an increased risk of enucleation. While the identification of somatic copy number alterations (SCNAs) via the AH has been well established, these alterations are not routinely identified in the blood due to low tumor fraction. MATERIALS AND METHODS: SCNAs were considered positive at 20% deflection from the baseline. Somatic RB1 pathogenic variants were identified with targeted sequencing using a panel including all RB1 exons. RESULTS: A 24-month-old patient presented with unilateral retinoblastoma (Group D/AJCC Stage cT2B) and was treated with primary enucleation. In the peripheral blood, a heterozygous mutation (c.3920T>A) in the APC gene was reported. Genomic analysis of the tumor and AH revealed two novel somatic RB1 mutations (c.1589_1590del and c.2330dupC). Both also demonstrated highly recurrent RB-related SCNAs. Chromosome 6p gain was detected in the blood with an amplitude suggesting approximately 12% tumor fraction. At a follow-up of 24 months, there has been no evidence of metastatic disease. CONCLUSIONS: To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting in some advanced eyes there may be a high enough tumor fraction to detect these alterations (>5% needed). It remains unclear whether 6p gain or increased tumor fraction in the blood is indicative of increased risk of metastatic disease or new primary cancer; studies to address this are ongoing.

Dopachrome tautomerase is a retinoblastoma-specific gene, and its proximal promoter is preferentially active in human retinoblastoma cells.

Purpose: Retinoblastoma (RB) is a malignant childhood intraocular tumor. Current treatment options for RB have undesirable side effects. A comprehensive understanding of gene expression in human RB is essential for the development of safe and effective new therapies. Methods: We reviewed published microarray and RNA sequencing studies in which gene expression profiles were compared between human RB and normal retina tissues. We investigated the expression of genes of interest using quantitative reverse transcription PCR. We examined the activities of cloned promoter DNA fragments with luciferase assay. Results: Dopachrome tautomerase (DCT) was among the most overexpressed genes in RB in published studies. We found that DCT was highly expressed in six of 13 samples microdissected from Thai RB tissues. Expression of DCT was absent or barely detected in retina tissues, various human ocular cells, and major organs. We also demonstrated that the -657 to +411 DCT promoter fragment efficiently directs RB cell-specific transcription of the luciferase reporter gene in cell lines. Conclusions: The present work highlights that DCT is one of the most RB-specific genes. The regulatory elements required for this cell-specific gene expression are likely located within its proximal promoter.

Diverse mutational spectrum in the 13q14 chromosomal region in a Brazilian cohort of retinoblastoma.

Retinoblastoma is a rare childhood tumor caused by the inactivation of both copies of the RB1 gene. Early diagnosis and identification of heritable RB1 mutation carriers can improve the disease outcome and management via genetic counseling. We used the Multiplex Ligation-dependent Probe Amplification (MLPA) method to analyze the RB1 gene and flanking regions in blood samples from 159 retinoblastoma patients previously negative for RB1 point mutations via Sanger sequencing. We detected a wide spectrum of germline chromosomal alterations, ranging from partial loss or duplication of RB1 to large deletions spanning RB1 and adjacent genes. Mutations were validated via karyotyping, fluorescent in situ hybridization (FISH), SNP-arrays (Single Nucleotide Polymorphism-arrays) and/or quantitative relative real-time PCR. Patients with leukocoria as a presenting symptom showed reduced death rate (p = 0.013) and this sign occurred more frequently among carriers of two breakpoints within RB1 (p = 0.05). All unilateral cases presented both breakpoints outside of RB1 (p = 0.0075). Patients with one breakpoint within RB1 were diagnosed at earlier ages (p = 0.017). Our findings characterize the mutational spectrum of a Brazilian cohort of retinoblastoma patients and point to a possible relationship between the mutation breakpoint location and tumor outcome, contributing to a better prospect of the genotype/phenotype correlation and adding to the wide diversity of germline mutations involving RB1 and adjacent regions in retinoblastoma.

Novel insights into RB1 mutation.

Since the discovery of the retinoblastoma susceptibility gene (RB1) decades ago, RB1 has been regarded as a prototype tumor suppressor gene providing a paradigm for tumor genetic research. Constant research has updated the understanding of RB1-related pathways and their impact on tumor and nontumor diseases. Mutation of RB1 gene has been observed in multiple types of malignant tumors including prostate cancer, lung cancer, breast cancer, and almost every familial and sporadic case of retinoblastoma. Even if well-known and long-investigated, the application potential of RB1 mutation has not been fully tapped. In this review, we focus on the mechanism underlying RB1 mutation during oncogenesis. Therapeutically, we have further discussed potential clinical strategies by targeting RB1-mutated cancers. The unsolved problems and prospects of RB1 mutation are also discussed.

Identification of novel genes by targeted exome sequencing in Retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is initiated by mutation in both alleles of RB1 gene. However, few cases may occur even in the absence of RB1 mutation suggesting the role of genes other than RB1. METHODOLOGY: The current study was planned to utilize targeted exome sequencing in Indian RB patients affected with unilateral non-familial RB. 75 unilateral RB patients below 5 years of age were enrolled. Genomic DNA was extracted from blood and tumor tissue. From peripheral blood DNA, all coding and exon/intron regions were amplified using PCR and direct sequencing. Cases which did not harbor pathogenic variants in peripheral blood DNA were further screened for mutations in their tumor tissue DNA using targeted exome sequencing. Three pathogenicity prediction tools (Mutation Taster, SIFT, and PolyPhen-2) were used to determine the pathogenicity of non-synonymous variations. An in-house bioinformatics pipeline was devised for the mutation screening by targeted exome sequencing. Protein modeling studies were also done to predict the effect of the mutations on the protein structure and function. RESULTS: Using the mentioned approach, we found two novel variants (g.69673_69674insT and g.48373314C>A) in RB1 gene in peripheral blood DNA. We also found novel variants in eight genes (RB1, ACAD11, GPR151, KCNA1, OTOR, SOX30, ARL11, and MYCT1) that may be associated with RB pathogenesis. CONCLUSION: The present study expands our current knowledge regarding the genomic landscape of RB and also highlights the importance of NGS technologies to detect genes and novel variants that may play an important role in cancer initiation, progression, and prognosis.

Complex translocation leading to13q interstitial deletion in a Moroccan child with retinoblastoma and intellectual disability.

BACKGROUND: Retinoblastoma (RB) is the most common malignant intraocular tumor in children; it affects their eyes often even prenatally. RB may be sporadic or familial, due to germinal mutation in RB1 gene or by abnormal chromosomal abnormalities involving RB1 gene, located in 13q14. Monosomy of subband 13q14 as a partial deletion can also be responsible for RB with additional symptoms. The latter may be RB associated with psychomotor retardation, macrocephaly, broad forehead, thick earlobes, and bulbous nose. MATERIALS AND METHODS: We present here the case of a boy from a consanguineous marriage with bilateral retinoblastoma, intellectual disability and facial dysmorphic features. Classical and molecular cytogenetics were used to recognize genotype-phenotype association. RESULTS: The karyotype showed a three way translocation involving chromosomes 5, 12 and 13. Further molecular cytogenetics analysis revealed a deletion of 13q14 involving the tumor suppressor gene RB1. CONCLUSION: This case highlights the impact of classical and molecular cytogenetics in diagnosis of rare genetic syndromes and for the genetic counselling of patients and their families. Pure molecular karyotyping analyses would miss the underlying chromosomal mechanism leading to the rearrangement.

Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC­derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a­regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Characterization of the Portuguese population diagnosed with retinoblastoma.

The purpose of this study is to characterize demographically and genetically the Portuguese population with retinoblastoma; to report the clinical stage at presentation and its impact on survival and ocular preservation rate and, finally, to assess the incidence of retinoblastoma in Portugal. Retrospective observational study including children consecutively diagnosed with retinoblastoma at the Portuguese National Referral Center of Intraocular Tumors, between October 2015 and October 2020. Twenty-eight children were diagnosed with retinoblastoma at our center, 15 hereditary from which 12 presented with bilateral retinoblastoma and 3 were unilateral. The overall mean age at diagnosis was 13.6 ± 11.1 months with hereditary retinoblastomas diagnosed slightly earlier at 9.6 ± 6.3 months. A familial history of retinoblastoma was found in only 4 (14.3%) of the cases. A pathogenic mutation in the RB1 gene was found in 13 (46.4%) of the children. The most frequent sign at referral was leukocoria in 71.4% of patients. Considering the ICRB classification of the tumors, 84.6% of non-hereditable hereditary retinoblastomas were referred to our center in advanced stages. In the group of hereditable retinoblastomas 86.7% presented with one of the eyes with advanced intraocular retinoblastoma. Fourteen children had one eye enucleated due to retinoblastoma. No deaths were registered during the study period. Considering the incidence analysis, we registered a year-of-birth controlled incidence analysis of 4.04 per 100.000 living births (IC 95% 1.59-6.49). This is the first characterization of the Portuguese Population diagnosed with Retinoblastoma in the National Reference Center.

Treatment of RB-deficient retinoblastoma with Aurora-A kinase inhibitor.

Retinoblastoma, also known as ocular cancer, usually affects children under the age of five. The standard of care for managing early-stage retinoblastoma is a combination of vincristine, carboplatin, and etoposide. However, this combination-based modality has limited applications owing to its side and late effects. Moreover, in advanced tumor stages, nearly 50% of patients would suffer a partial or full loss of vision. Therefore, therapies that preserve vision and reduce side effects are urgently required. Here, we focused mainly on the common loss-of-function (LOF) mutation of retinoblastoma gene 1 (RB1) in advanced retinoblastoma and investigated the synthetic lethality between RB1-LOF and Aurora kinase inhibition. We showed that Aurora kinase A inhibition could lead to cell mitotic abnormality and apoptosis, and demonstrated in vivo efficacy in a mouse model xenografted with RB1-deficient retinoblastoma. Our findings provide a promising druggable molecular target and potential clinical strategy for tackling retinoblastoma disease.

Identification of novel RB1 genetic variants in Retinoblastoma patients and their impact on clinical outcome.

BACKGROUND: Retinoblastoma (RB) is an intraocular childhood cancer develops due to inactivation of RB1 gene. Identification of RB1 genetic variants, correlating and confirming genetic test results with clinical outcomes are crucial for effective RB management. METHODS: Retrospective study of 62 RB patients and 14 family members who underwent genetic testing either by next generation sequencing (NGS) or multiplex ligation-dependent probe amplification (MLPA) or by both for screening RB1 germline mutations present in peripheral blood. Mutational outcomes were correlated with clinical outcomes evaluated over a follow-up period of 12 months. RESULTS: Of the 62 patients, 35 (56%) had bilateral RB and 27 (44%) had unilateral RB. Out of 24 (52%) variants detected by NGS, 9 (37.5%) were novel and 15 (62.5%) were known in 46 probands. Six (18%) gross deletions were detected by MLPA in 34 probands. The mutation detection rate by NGS and MLPA in unilateral cases was 15% (n = 4) and 74% (n = 26) in bilateral cases. In patients with RB1 genetic mutations versus those without, the rate of primary enucleation (7 (12%) vs 18 (44%) eyes; p = .0008) was inversely proportional to tumor recurrence (25 (45%) vs 6 (15%) eyes; p = .002). There was no difference in the rate of globe salvage and metastasis, over a mean follow-up period of 12 months. CONCLUSION: The mutations screening is important for risk assessment in future siblings and offspring of RB patients and most important in unilateral RB for determining if hereditary or not hereditary RB. Its role in predicting clinical outcomes is yet to be determined.

Stepwise correlation of TP53 mutations from pancreaticobiliary maljunction to gallbladder carcinoma: a retrospective study.

BACKGROUND: The genetic changes underlying carcinogenesis in patients with risk factors of gallbladder carcinoma (GBC) remains controversial, especially in patients with pancreaticobiliary maljunction (PBM). This study aimed to clarify the association between risk factors of GBC and genetic changes using next-generation sequencing (NGS). METHODS: We retrospectively analyzed resected tissues of 64 patients who were diagnosed with GBC (n = 26), PBM [with GBC (n = 8), without GBC (n = 20)], and chronic cholecystitis, used as a control group (n = 10). DNA was extracted from tumors and their surrounding tissues, which were precisely separated by laser-capture microdissection. Gene alterations of 50 cancer-related genes were detected by NGS and compared with clinical information, including PBM status. RESULTS: The most frequent gene alterations in GBC tissues occurred in TP53 (50%), followed by EGFR (20.6%), RB1 (17.6%), and ERBB2 (17.6%). Gene alterations that were targetable by molecular targeted drugs were detected in 20 cases (58.8%). Statistical analysis of gene alterations and risk factors revealed that TP53 alteration rate was higher in GBC patients with PBM than those without PBM (p = 0.038), and the TP53 mutation rates in the epithelium of control patients, epithelium of PBM patients without GBC, peritumoral mucosa of GBC patients with PBM, and tumor tissue of GBC patients with PBM were 10, 10, 38, and 75%, respectively (p <  0.01). CONCLUSIONS: TP53 alteration more than KRAS mutation was revealed to underlie carcinogenesis in patients with PBM.

Co-expression and prognosis analyses of GLUT1-4 and RB1 in breast cancer.

BACKGROUND: Current treatment methods for patients with triple-negative breast cancer (TNBC) are very limited, and the prognosis of TNBC is relatively poor. It has been reported that glucose transporter 1 (GLUT1) is overexpressed in breast cancer cells; however, its association with the prognosis is mostly unclear. Moreover, retinoblastoma gene 1 (RB1) might be used as a biomarker for the sensitivity of breast cancer cells to GLUT1 inhibitors, which brought us to the hypothesis that there might be a close correlation between the expression of GLUT1-4 and the expression of RB1. METHODS: In this study, we systematically analyzed the co-expression of GLUT1-4 and the influence of GLUT1-4 gene expression on the prognosis of breast cancer using data mining methods. We also explored possible relationships between GLUT1-4 and RB1 expression in breast cancer tissues. We used public databases such as ONCOMINE, GEPIA, LinkedOmics, and COEXPEDIA. RESULTS: According to the results, the mRNA expression of SLC2A1 was significantly higher in breast cancer, while the expression levels of SLC2A2-4 were downregulated. The results also indicate that GLUT1 expression does not have significant influence on the overall survival of patients with breast cancer. The mRNA expression of SLC2A1 and RB1 is significantly correlated, which means that tissues with high RB1 mRNA expression might have relatively higher mRNA expression of SLC2A1; however, further study analyzing their roles in the expression regulation pathways with human samples is needed to verify the hypothesis. CONCLUSIONS: The mRNA expression of SLC2A1 was significantly higher in breast cancer. The overall survival of breast cancer patients wasn't significantly correlated with GLUT1-4 expression. The mRNA expression of SLC2A1 and RB1 is significantly correlated according to the analysis conducted in LinkedOmics. It provides reference for future possible individualized treatment of TNBC using GLUT1 inhibitors, especially in patients with higher mRNA expression of RB1. Further study analyzing the roles of these two genes in the regulation pathways is needed.

Correlation between Family RB1 Gene Pathogenic Variant with Clinical Features and Prognosis of Retinoblastoma under 5 Years Old.

Retinoblastoma (RB) is the most common primary intraocular malignant tumor in infants and the prototype of human hereditary tumors. Its occurrence and development are closely related to the pathogenic variant of tumor suppressor RB1 gene. We aim to analyze the characteristics of RB1 gene pathogenic variant and clinical phenotype in retinoblastoma patients and their relatives. Children with RB were recruited from August 2007 to November 2017. QT-PCR, probing, and gene sequencing were used to analyze the sequence of RB1 gene in RB children, their parents, or grandparents with a clear history of illness. The SPSS20.0 software was used to analyze the correlation between polymorphisms of RB1 gene and the incidence and prognosis of the enrolled children and relatives. 40 RB children (20 males and 20 females) were recruited, unilateral RB accounted for 52.5% (21/40), bilateral RB accounted for 42.5% (17/40), and trilateral RB accounted for 5.0% (2/40). 6 patients had a clear family history (15.0%, 6/40). It had been verified that 19 probands (47.5%) have RB1 gene pathogenic variants (11 frameshift and 8 missense pathogenic variants), of which germline inheritance accounted for 47.4% (9/19) and nongermline heredity accounted for 52.6% (10/19). Pathogenic variants of 10 nucleic acid sites without reported were found, among which c.2455C>G (p.L819V) was confirmed to have heterozygous pathogenic variants in both a bilateral RB patient and his mother with unilateral RB. Family genetic high-risk factors, bilateral/trilateral RB, >12-month-onset RB have a higher proportion of RB1 gene pathogenic variant than children with no family history, unilateral RB, and ≤12-month (P = 0.021, 0.001,0.034). The proportion of pedigree inheritance of infantile retinoblastoma with bilateral disease is high. There was a certain proportion of RB1 gene pathogenic variant in 3-5-year-old children with bilateral RB, even if they had no family genetic history. Therefore, the detection of RB1 gene pathogenic variant should not only focus on infants but also on the phenotype of RB1 gene pathogenic variant in children over 3 years old with bilateral eye disease.

RB1 germline mutation spectrum and clinical features in patients with unilateral retinoblastomas.

Background: Retinoblastoma is the most common intraocular cancer in children in which above 90% of bilateral cases and 10-25% of unilateral cases have germline RB1 mutations. We summarized the spectrum of RB1 germline mutations and the clinical manifestations of unilateral retinoblastomas to guide clinical treatments.Methods: Two hundred and sixty-three unrelated patients with unilateral retinoblastoma and their parents were included between February 2014 and August 2020. Next-generation sequencing and Sanger sequencing analysis of the core promoter region and exons 1-27 including flanking intronic regions of the RB1 gene were performed. If a germline mutation was identified in a retinoblastoma patient, the parental blood sample was requested to test for the identified mutation.Results: RB1 germline mutations were identified in 39/263 (14.8%) unilateral retinoblastoma patients and 11 (28.2%) had a missense mutation, 10 (25.6%) had nonsense mutations, 2 (5.1%) had frameshifts, 1 (2.6%) had synonymous mutation, and 7 (17.9%) had a large deletion, 2 (5.1%) had splice site mutations, 6 (15.4%) had variant of uncertain significance. Moreover, 27 (69.2%) of 39 patients identified RB1 mutations were predicted to have pathogenic mutation. The median age at diagnosis of patients with identified RB1 pathogenic mutations was 16.9 months and the patients with the wild-type allele was 21.1 months (P = .323).Conclusion: The rate of germline RB1 mutations is 14.8% in our cohort of unilateral retinoblastomas. The high incidence of germline mutations indicates that genetic testing and counseling for families of unilateral retinoblastoma patients would be beneficial.

Establishment and characterization of a novel treatment-related neuroendocrine prostate cancer cell line KUCaP13.

The prevalence of neuroendocrine prostate cancer (NEPC) arising from adenocarcinoma (AC) upon potent androgen receptor (AR) pathway inhibition is increasing. Deeper understanding of NEPC biology and development of novel therapeutic agents are needed. However, research is hindered by the paucity of research models, especially cell lines developed from NEPC patients. We established a novel NEPC cell line, KUCaP13, from tissue of a patient initially diagnosed with AC which later recurred as NEPC. The cell line has been maintained permanently in vitro under regular cell culture conditions and is amenable to gene engineering with lentivirus. KUCaP13 cells lack the expression of AR and overexpress NEPC-associated genes, including SOX2, EZH2, AURKA, PEG10, POU3F2, ENO2, and FOXA2. Importantly, the cell line maintains the homozygous deletion of CHD1, which was confirmed in the primary AC of the index patient. Loss of heterozygosity of TP53 and PTEN, and an allelic loss of RB1 with a transcriptomic signature compatible with Rb pathway aberration were revealed. Knockdown of PEG10 using shRNA significantly suppressed growth in vivo. Introduction of luciferase allowed serial monitoring of cells implanted orthotopically or in the renal subcapsule. Although H3K27me was reduced by EZH2 inhibition, reversion to AC was not observed. KUCaP13 is the first patient-derived, treatment-related NEPC cell line with triple loss of tumor suppressors critical for NEPC development through lineage plasticity. It could be valuable in research to deepen the understanding of NEPC.