A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.


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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. We included a first series of 102 retinoblastomas studied by genomics, transcriptomics, and methylomics. The sample size represents the largest cohort of retinoblastoma analyzed by multi-omics approaches to date. We also included an independent series of 112 retinoblastoma with HRPFs (High-Risk Pathological Features), of which 19 metastasized and 93 did not. Considering the low incidence of retinoblastoma and low availability of metastatic cases, the size of the series with 19 metastatic tumors can be considered large. We studied only one subtype 2 retinoblastoma case by single-cell RNA-seq but this sample was particularly informative, as it was heterogeneous, with one cell population expressing differentiated cone markers and the other expressing neuronal/ganglion cell markers. This case is representative of 30% of type 2 retinoblastoma, as assessed by immunohistochemistry.
In our transcriptomic analysis of retinal markers in retinoblastoma, we included three fetal retinas (20, 23, 27 gestational weeks) for comparison. Our work was also largely based on a large single-cell RNA-seq study of the developing retina (Lu et al., 2020).
No data were excluded.
To confirm the identified non-synonymous somatic mutations by WES, we performed a mutation-validation experiment using Sanger sequencing, which yielded a 92% validation rate of variants with allele fractions >10% (100 variants tested). We also confirmed all the mutations identified by targeted high-throughput sequencing by Sanger sequencing. The expression of key genes was confirmed by both Microarray and NanoString. Each immunohistochemistry slide was examined by two to three specialists. tumors identified by immunohistochemistry was confirmed and further defined on a representative tumor by single cell RNA-seq. The different stages of cone differentiation in retinoblastoma observed in the transcriptomic data were confirmed by comparing retinoblastoma with retinal organoids and the developing retina.
This was a retrospective study. This study identifies two molecular subtypes and depicts their molecular and clinical landscapes. Treatment decisions were made independently of this study. Randomization is not applicable.
This was a retrospective study. This study identifies two molecular subtypes and depicts their molecular and clinical landscapes. The investigators were blinded to the clinical characteristics at the stage of identifying molecular subtypes. For the Immunohistochemistry experiments, including the study of the metastatic series, investigators were blinded to the molecular subtypes. Cell line is not authenticated but the characterization of the hiPSC-2 cell line consisted of positive alkaline phosphatase staining, immunohistochemistry, qRT-PCR analysis of pluripotency markers (NANOG, TRA1-81, OCT4, and SSEA4), capacity for embryoid body formation, differentiation towards the three main germ layers markers (endoderm, SOX17, mesoderm, BRACHYURY, SMA, and ectoderm PAX6, TUJ1), teratome formation in NSG mouse and karyotype analysis (Reichman et al., 2014).

CRX
Absence of mycoplasma contamination was verified by theMycoAlert™Mycoplasma Detection Kit (selective biochemical test of mycoplasma enzymes) used according to the manufacturer's instructions (Lonza).
No commonly misidentified cell lines used.