Figure 1. Mechanisms of Rb pathway loss and assays for their detection.

Left (DNA): Wild-type RB1 may be lost through genomic alterations (e.g., deletions, missense/nonsense mutations, indels or rearrangements) or epigenomic modifications (e.g., methylation of the RB1 gene promoter). Fluorescence in situ hybridization (FISH) is a method for detecting gene copy losses or rearrangements but not point mutations or indels; however, FISH can be performed from very sparse material and allows for in situ visualization of intratumoral heterogeneity. Next generation sequencing that includes exonic regions of the RB1 gene locus may be performed from clinical biopsy specimens or circulating tumor DNA to detect point mutations and indels; these assays also detect rearrangements when located within exonic regions and can usually identify mono- or bi-allelic deletions, though copy number losses are more challenging to detect in samples with low tumor DNA content. Whole genome sequencing would be required to detect complex genomic alterations outside exonic regions of RB1 (not depicted), and specific assays such as bisulfite sequencing would be required to detect epigenetic modifications of the RB1 gene. Middle (Protein): Biallelic loss of the RB1 gene locus leads to loss of RB1 protein expression by immunohistochemistry (IHC), but point mutations and frame shifts may result in expression of a hypofunctional or truncated polypeptide product that could potentially be recognized by RB1 antibodies for IHC. Non-genomic alterations such as epigenetic modifications, decrease in gene transcription/translation, or increased degradation could lead to decreased Rb protein levels by IHC, but the degree of decrease in levels may be indeterminate with regards to functional outcomes. Right (mRNA): Transcriptome-based assays may be performed from clinical biopsy specimens. RNA-based assays may not be able to distinguish upstream mechanisms of Rb pathway loss.