Genetic Heterogeneity in Cellular Angiofibromas.

BACKGROUND: Cellular angiofibroma, a rare benign mesenchymal neoplasm, is classified within the 13q/RB1 family of tumors due to morphological, immunohistochemical, and genetic similarities with spindle cell lipoma. Here, genetic data reveal pathogenetic heterogeneity in cellular angiofibroma. METHODS: Three cellular angiofibromas were studied using G-banding/Karyotyping, array comparative genomic hybridization, RNA sequencing, and direct cycling sequencing. RESULTS: The first tumor carried a del(13)(q12) together with heterozygous loss and minimal expression of the RB1 gene. Tumors two and three displayed chromosome 8 abnormalities associated with chimeras of the pleomorphic adenoma gene 1 (PLAG1). In tumor 2, the cathepsin B (CTSB) fused to PLAG1 (CTSB::PLAG1) while in tumor 3, the mir-99a-let-7c cluster host gene (MIR99AHG) fused to PLAG1 (MIR99AHG::PLAG1), both leading to elevated expression of PLAG1 and insulin growth factor 2. CONCLUSION: This study uncovers two genetic pathways contributing to the pathogenetic heterogeneity within cellular angiofibromas. The first aligns with the 13q/RB1 family of tumors and the second involves PLAG1-chimeras. These findings highlight the diverse genetic landscape of cellular angiofibromas, providing insights into potential diagnostic strategies.

Clinicopathological and genetic characterization of radiotherapy-induced undifferentiated pleomorphic sarcoma following breast cancer: a case series of three tumors and comprehensive literature review.

AIMS: Compared to primary breast sarcoma (BSs), radiotherapy-induced sarcoma (RIS) is a less frequent type of secondary breast sarcoma. Undifferentiated pleomorphic sarcoma (UPS) is an even rarer occurrence within the RIS category. This study aimed to present the clinicopathologic and molecular features of breast radiotherapy-induced UPS. METHODS: A retrospective study was conducted at the Third Affiliated Hospital of Soochow University to analyze three patients with radiation-induced undifferentiated pleomorphic sarcoma (UPS) following breast cancer, spanning from 2006 to 2023. The clinical and pathological variables were extracted from the medical records, while immunohistochemistry was employed to analyze the immunophenotypes of these tumors. Genomic characteristics were assessed through DNA and RNA sequencing techniques. Another 15 cases from the literature were also reviewed to better characterize the tumor. RESULTS: The affected areas encompass the chest wall and breasts, with an incubation period ranging from 6 to 17 years. The tumor cells exhibit pleomorphism and demonstrate a high degree of pathological mitosis. Notably, two cases displayed an accelerated disease progression, characterized by recurrent tumors and metastases occurring within short intervals of 48 and 7 months respectively subsequent to the initial diagnosis. The two prevailing identified genes were TP53 (2/3, 66.7%) and RB1 (1/3, 33.3%). Through analysis of somatic copy number variation (CNV), it was discovered that two oncogenes, MCL1 (1/3, 33.3%) and MYC (1/3, 33.3%), had experienced gains in CNV. The Tumor Mutational Burden (TMB) values for case 1, case 2, and case 3 were 5.9 mut/Mb, 1.0 mut/Mb, and 3.0 mut/Mb, respectively. Moreover, the analysis of RNA-NGS (next-generation sequencing) revealed the presence of a novel gene fusion, named COL3A1-GULP1, in case 2. CONCLUSIONS: Based on our thorough analysis of research findings and previous reports, it is evident that radiotherapy-induced UPS exhibits a highly diverse and frequently severe clinical and biological behavior. Identifying tumor formation using genome sequencing can help understand its biological behavior and determine personalized treatments.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

Unraveling Copy Number Alterations in Pediatric B-Cell Acute Lymphoblastic Leukemia: Correlation with Induction Phase Remission Using MLPA.

OBJECTIVE: Acute Lymphoblastic Leukemia (ALL) is the most common malignancy occurring in children. Copy number alterations (CNA) like PAX5, CDKN2A/2B, PAR1 Region, ETV6, IKZF1, BTG1, and RB1 gene deletion are important genetic events that define and prognosticate B-cell ALL. Thus, this study aimed to evaluate associations of CNA with induction phase remission status in childhood B-cell ALL. METHODS: This study was observational with a cross-sectional design at the Dharmais Cancer Hospital, Harapan Kita Mother and Children Hospital, and Tangerang Regional Public Hospital. We evaluated 74 pediatric B-cell ALL cases with 1-18-year-olds. Genomic DNA was analyzed by Multiplex Ligation Dependent Probe Amplification Assay (MLPA). This study used the P335 ALL-IKZF1 panel kit, which contains several ALL-related genes. The patient's clinical and laboratory characteristics were collected from medical records from January to December 2019. RESULT: We observed gene copy number alteration in children with B-Cell ALL. PAX5 was the most commonly observed gene deletion, followed by CDKN21/2B, ETV6, IKZF1, BTG1, RB1, and PAR1 Region. Based on gene mutations, only the PAX5 had a significant association with the remission status of pediatric B-cell ALL (p-value <0.05; OR = 3.91). It showed that patients with PAX5 gene mutations have 3.9 times the risk of no remission and/or relapse compared to those without PAX5 gene mutations. CONCLUSION: Patients with mutations in the PAX5 gene have a higher chance of not achieving remission and/or experiencing relapse than those without such mutations. The MLPA method can be utilized for examining copy number alterations, which is valuable for achieving more precise stratification in diagnosis.. Further research is needed to expand upon this finding.

CDC7 inhibition impairs neuroendocrine transformation in lung and prostate tumors through MYC degradation.

Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.

Comprehensive identification of pathogenic variants in retinoblastoma by long- and short-read sequencing.

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. The causal variants in RB are mostly characterized by previously used short-read sequencing (SRS) analysis, which has technical limitations in identifying structural variants (SVs) and phasing information. Long-read sequencing (LRS) technology has advantages over SRS in detecting SVs, phased genetic variants, and methylation. In this study, we comprehensively characterized the genetic landscape of RB using combinatorial LRS and SRS of 16 RB tumors and 16 matched blood samples. We detected a total of 232 somatic SVs, with an average of 14.5 SVs per sample across the whole genome in our cohort. We identified 20 distinct pathogenic variants disrupting RB1 gene, including three novel small variants and five somatic SVs. We found more somatic SVs were detected from LRS than SRS (140 vs. 122) in RB samples with WGS data, particularly the insertions (18 vs. 1). Furthermore, our analysis shows that, with the exception of one sample who lacked the methylation data, all samples presented biallelic inactivation of RB1 in various forms, including two cases with the biallelic hypermethylated promoter and four cases with compound heterozygous mutations which were missing in SRS analysis. By inferring relative timing of somatic events, we reveal the genetic progression that RB1 disruption early and followed by copy number changes, including amplifications of Chr2p and deletions of Chr16q, during RB tumorigenesis. Altogether, we characterize the comprehensive genetic landscape of RB, providing novel insights into the genetic alterations and mechanisms contributing to RB initiation and development. Our work also establishes a framework to analyze genomic landscape of cancers based on LRS data.

Retinoblastoma caused by an RB1 variant with unusually low penetrance in a Danish family.

Retinoblastoma is the most common eye cancer in children. It is caused by pathogenic alterations of both alleles of the tumor suppressor gene RB1. In heritable retinoblastoma, a constitutional RB1 variant predisposes the cells to tumor formation, and loss of the other allele is a prerequisite for the development of retinoblastoma. Heritable retinoblastoma is inherited in an autosomal dominant manner; however, the majority of cases are the result of a de novo pathogenic RB1 variant. Penetrance is usually high (>90%), but with marked inter-familial variability. In some families, penetrance is incomplete and family members who develop tumors tend to remain unilaterally affected. Moreover, some families with low penetrance also show a parent-of-origin effect. We describe a patient with unilateral retinoblastoma caused by a previously unreported likely pathogenic RB1 variant (c.1199T>C) that disrupts a highly conserved amino acid residue within the A-box functional domain. Segregation analysis showed that the variant had unusually low penetrance as nine non-affected family members carried the same variant. We emphasize the use of genetic analysis on tumor DNA for classifying the RB1 variant, and underline the challenges in clinical management and counseling of families carrying the specific RB1 variant.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma.

PURPOSE: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). EXPERIMENTAL DESIGN: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. RESULTS: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. CONCLUSIONS: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

RB1: governor of the cell cycle in health and disease-a primer for the practising pathologist.

RB1 stands as the pioneering discovery in tumour-suppressor genes, marking a pivotal breakthrough in comprehending cancer development. This overview delves into the role of RB1 in both health and disease, exploring its association with the tumourigenesis of various cancers and a distinct subset of soft-tissue neoplasms. Additionally, we discuss the application of immunohistochemistry and fluorescence in situ hybridisation to detect RB1 alterations.

Genomic profiling of pleomorphic rhabdomyosarcoma reveals a genomic signature distinct from that of embryonal rhabdomyosarcoma.

Pleomorphic rhabdomyosarcoma (PRMS) is a rare and highly aggressive sarcoma, occurring mostly in the deep soft tissues of middle-aged adults and showing a variable degree of skeletal muscle differentiation. The diagnosis is challenging as pathologic features overlap with embryonal rhabdomyosarcoma (ERMS), malignant Triton tumor, and other pleomorphic sarcomas. As recurrent genetic alterations underlying PRMS have not been described to date, ancillary molecular diagnostic testing is not useful in subclassification. Herein, we perform genomic profiling of a well-characterized cohort of 14 PRMS, compared to a control group of 23 ERMS and other pleomorphic sarcomas (undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma) using clinically validated DNA-targeted Next generation sequencing (NGS) panels (MSK-IMPACT). The PRMS cohort included eight males and six females, with a median age of 53 years (range 31-76 years). Despite similar tumor mutation burdens, the genomic landscape of PRMS, with a high frequency of TP53 (79%) and RB1 (43%) alterations, stood in stark contrast to ERMS, with 4% and 0%, respectively. CDKN2A deletions were more common in PRMS (43%), compared to ERMS (13%). In contrast, ERMS harbored somatic driver mutations in the RAS pathway and loss of function mutations in BCOR, which were absent in PRMS. Copy number variations in PRMS showed multiple chromosomal arm-level changes, most commonly gains of chr17p and chr22q and loss of chr6q. Notably, gain of chr8, commonly seen in ERMS (61%) was conspicuously absent in PRMS. The genomic profiles of other pleomorphic sarcomas were overall analogous to PRMS, showing shared alterations in TP53, RB1, and CDKN2A. Overall survival and progression-free survival of PRMS were significantly worse (p < 0.0005) than that of ERMS. Our findings revealed that the molecular landscape of PRMS aligns with other adult pleomorphic sarcomas and is distinct from that of ERMS. Thus, NGS assays may be applied in select challenging cases toward a refined classification. Finally, our data corroborate the inclusion of PRMS in the therapeutic bracket of pleomorphic sarcomas, given that their clinical outcomes are comparable.

Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids.

BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.

A synthetic lethal dependency on casein kinase 2 in response to replication-perturbing therapeutics in RB1-deficient cancer cells.

Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.

Regulation of the RB1 Gene through DNMT1 by SGI-1027 and its Impact on the Growth and Metastasis of Gastric Cancer Cells.

BACKGROUND: SGI-1027 is a recognized inhibitor of DNA methyltransferase 1 (DNMT1), and earlier investigations have indicated an inverse correlation between dysregulated DNMT1 expression in gastric cancer (GC) and retinoblastoma 1 (RB1) gene expression. Despite this knowledge, the precise mechanisms underlying the action of SGI-1027 in GC cells remain inadequately comprehended. The primary objective of this study is to elucidate the impact of SGI-1027 on the behavior of GC cells, encompassing aspects such as growth and metastatic potential, by intervening in DNMT1, thereby influencing the regulation of RB1 gene expression. METHOD: The acquisition of the normal gastric mucosal cell line GES-1 and the human gastric cancer cell line MKN45 was followed by employing Western blot (WB) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques to evaluate the expression levels of RB1 and DNMT1 in these two cell lines. Subsequently, the MKN45 cell line was cultured in medium containing varying concentrations of SGI-1027, and the impact of SGI-1027 on the regulation of RB1 and DNMT1 in GC cells was reassessed using WB and qRT-PCR techniques. To scrutinize the effect of SGI-1027 on GC cells, we utilized the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay to determine cell proliferation and performed Transwell experiments to assess cell migration and invasion capabilities. Throughout this process, we also employed WB to assess the levels of cell cycle-associated proteins (Cyclin D1, Cyclin E1, and Cyclin B1) and proteins related to apoptosis (BCL-2 associated protein X apoptosis regulator (BAX) and B-cell lymphoma 2 apoptosis regulator (BCL-2)). Furthermore, we injected the MKN45 cell line and MKN45 cell line cultured with the optimal concentration of SGI-1027 for 5 days and 10 days into mice subcutaneously and through the tail vein, dividing them into the Model group, Model+SGI-1027 5d group, and Model+SGI-1027 10d group. We monitored changes in tumor size and volume in mice, and tumor tissues as well as lung tissues were collected for hematoxylin and eosin (HE) staining. Finally, DNMT1 expression levels in GC tissues were detected using both WB and immunohistochemistry (IHC) techniques. Additionally, RB1 expression levels in GC tissues were assessed using WB. RESULT: In contrast to GES-1 cells, MKN45 cells displayed a distinctive profile characterized by increased DNMT1 expression and decreased RB1 expression (p < 0.05). However, upon the introduction of SGI-1027, a notable decrease in DNMT1 levels within GC cells was observed, concomitant with an elevation in RB1 gene expression, with 25 µmol/L SGI-1027 identified as the optimal concentration (p < 0.05). Functional assays demonstrated that SGI-1027-treated GC cells exhibited pronounced features of inhibited proliferation, migration, and invasion when compared to untreated MKN45 cells (p < 0.05). Moreover, in SGI-1027-treated GC cells, the levels of Cyclin D1, Cyclin E1, Cyclin B1, and BCL-2 were significantly reduced, while the expression level of BAX increased (p < 0.05). Notably, the most pronounced impact was observed at 25 µmol/L SGI-1027, further underscoring its regulatory effects on tumor cell behavior (p < 0.05). In animal experiments, the Model group exhibited a substantial increase in tumor volume, with HE staining results indicating extensive necrosis in most gastric tissues and noticeable signs of lung metastasis, accompanied by increased DNMT1 expression and decreased RB1 gene expression. In contrast, the SGI-1027 group displayed a reduction in gastric tumor volume, decreased necrosis, and reduced lung tumor metastasis (p < 0.05). Additionally, the expression of DNMT1 was significantly reduced in SGI-1027-treated GC cells, while RB1 expression increased (p < 0.05), further confirming the inhibitory effects of SGI-1027 on tumor growth and metastasis. CONCLUSIONS: SGI-1027 effectively hinders the proliferation and dissemination of GC cells by downregulating DNMT1 and promoting the expression of RB1.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Etiology including epigenetic defects of retinoblastoma.

Retinoblastoma (RB), originating from the developing retina, is an aggressive intraocular malignant neoplasm in childhood. Biallelic loss of RB1 is conventionally considered a prerequisite for initiating RB development in most RB cases. Additional genetic mutations arising from genome instability following RB1 mutations are proposed to be required to promote RB development. Recent advancements in high throughput sequencing technologies allow a deeper and more comprehensive understanding of the etiology of RB that additional genetic alterations following RB1 biallelic loss are rare, yet epigenetic changes driven by RB1 loss emerge as a critical contributor promoting RB tumorigenesis. Multiple epigenetic regulators have been found to be dysregulated and to contribute to RB development, including noncoding RNAs, DNA methylations, RNA modifications, chromatin conformations, and histone modifications. A full understanding of the roles of genetic and epigenetic alterations in RB formation is crucial in facilitating the translation of these findings into effective treatment strategies for RB. In this review, we summarize current knowledge concerning genetic defects and epigenetic dysregulations in RB, aiming to help understand their links and roles in RB tumorigenesis.

Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. CONCLUSION: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Development and Independent Validation of a Prognostic Gene Expression Signature Based on RB1, PTEN, and TP53 in Metastatic Hormone-sensitive Prostate Cancer Patients.

BACKGROUND: Androgen deprivation therapy (ADT) with docetaxel (D) and/or antiandrogen receptor therapies (ARTs) are the standard therapies in metastatic hormone-sensitive prostate cancer (mHSPC). Alterations in the tumor suppressor genes (TSGs) RB1, PTEN, and TP53 are associated with an aggressive evolution and treatment resistance in castration-resistant prostate cancer (CRPC). OBJECTIVE: To study the clinical implications of TSG mRNA expression in mHSPC patients. DESIGN, SETTING, AND PARTICIPANTS: This is a multicenter retrospective biomarker study in mHSPC patients. TSGlow status was defined when two or more out of the three TSGs presented low RNA expression by nCounter in formalin-fixed paraffin-embedded samples and TSGwt for the remaining cases. The microarray data from the CHAARTED trial were analyzed as an independent validation cohort. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Molecular data were correlated with CRPC-free survival (CRPC-FS) and overall survival (OS) by the Kaplan-Meier method and multivariate Cox analysis. RESULTS AND LIMITATIONS: A total of 226 patients were included, of whom 218 were eligible: 93 were treated with ADT and 125 with ADT + D; 75.7% presented de novo stage IV and 67.9% high-volume disease. TSGlow (19.2%) was independently correlated with shorter CRPC-FS (hazard ratio [HR] 1.8, p = 0.002) and OS (HR 2, p = 0.002). In the CHAARTED trial, TSGlow was independently correlated with lower CRPC-FS (HR 2.2, p = 0.02); no differences in clinical outcomes according to treatment were observed in TSGlow patients, while a significant benefit was observed for ADT + D in the TSGwt group for CRPC-FS (HR 0.4, p < 0.001) and OS (HR 0.4, p = 0.001). However, no interaction was observed between TSG signature and treatment in either series. Study limitations are the retrospective design, small sample size, and lack of inclusion of patients treated with ADT + ART. CONCLUSIONS: TSGlow expression correlates with adverse outcomes in patients with mHSPC. The investigation of new therapeutic strategies in these patients is warranted. PATIENT SUMMARY: The low RNA expression of tumor suppressor genes in the tumors is correlated with adverse outcomes in patients with metastatic hormone-sensitive prostate cancer.