Retinoblastoma

Retinoblastoma (RB) is an intraocular malignancy of the developing retina associated with germline and/or somatic mutations of the RB1 tumor suppressor gene. 

PrintPrint

Retinoblastoma (RB) is an intraocular malignancy of the developing retina associated with germline and/or somatic mutations of the RB1 tumor suppressor gene. 

Quick Links to Relevant Resources

Approximately 60% of RB is unilateral (affecting a single eye), while the other 40% of cases are bilateral (affecting both eyes).3 RB occurs in hereditary and non-hereditary forms, with all bilateral RB and some unilateral RB cases being hereditary. Children with hereditary RB harbor a germline RB1 mutation due to parental transmission or, more commonly, a de novo event. Hereditary RB is an autosomal dominant, highly penetrant cancer predisposition syndrome characterized by an increased risk of RB and a spectrum of secondary, non-ocular malignancies, most commonly osteosarcoma, pinealoma and melanoma.4,5 In addition, germline RB1 mutations have been shown to confer susceptibility to radiation-induced malignancies.4,6

The Ambry Test: Retinoblastoma detects germline mutations in the RB1 gene by full gene sequence analysis of all coding domains and splice junctions, and by gross deletion/duplication analysis. This Ambry test identifies >99% of described mutations (analytic sensitivity). Approximately 90-92% of patients with bilateral retinoblastoma have a detectable germline mutation in the RB1 gene sequence, whereas approximately 13-14% of patients with unilateral retinoblastoma have a mutation in the gene (clinical sensitivity).7,8

Disease Name 
Retinoblastoma
Disease Information 

Retinoblastoma (RB) is an intraocular malignancy of the developing retina associated with germline and/or somatic mutations of the RB1 tumor suppressor gene. RB is a childhood cancer; typically presenting in the first 5 years of life and occurring at a frequency of approximately 1 in 15,000-20,000 live births.1,3 The most common sign of RB is a visible whiteness in the pupil called leukocoria or "cat's eye reflex".2 Other signs and symptoms of retinoblastoma include strabismus, irritation, redness, persistent eye pain and vision impairment in the affected eye. Approximately 60% of RB is unilateral (affecting a single eye), while the other 40% of cases are bilateral (affecting both eyes).3 RB occurs in hereditary and non-hereditary forms, with all bilateral RB and some unilateral RB cases being hereditary.

Children with hereditary RB harbor a germline RB1 mutation due to parental transmission or, more commonly, a de novo event. Hereditary RB is an autosomal dominant, highly penetrant cancer predisposition syndrome characterized by an increased risk of RB and a spectrum of secondary, non-ocular malignancies, most commonly osteosarcoma, pinealoma and melanoma.4,5 In addition, germline RB1 mutations have been shown to confer susceptibility to radiation-induced malignancies.4,6

The RB1 gene (NM_000321.2) comprises 27 exons, spanning approximately 178kb of genomic DNA, and maps to chromosome 13q14.2.  It encodes a 928 amino acid nuclear phosphoprotein pRB, which functions as a negative regulator of the cell cycle and cell proliferation. To date, 673 RB1 mutations have been reported in the Human Gene Mutation Database (HGMD), the majority of which are small deletions, missense/nonsense and splicing.

Testing Benefits & Indication 

Identification of a germline RB1 mutation can aid diagnosis, provide accurate recurrence risks, guide treatment and may dramatically improve outcome, If detected and treated at an early stage, the prognosis for retinoblastoma (RB) is excellent, with a cure rate of 95% in the US.1  RB1 molecular testing is indicated in patients who are clinically suspected to have RB. RB1 testing may also be considered for differential diagnosis, carrier testing for individuals with a family history, and for at risk pregnancies. 

Test Description 

RB1 coding exons 1-27 and well into the 5’ and 3’ ends of all the introns and untranslated regions are analyzed by sequencing. Gross deletion/duplication analysis determines gene copy number for coding exons 1-27. Clinically significant intronic findings beyond 5 base pairs are always reported. Intronic variants of unknown or unlikely clinical significance are not reported beyond 5 base pairs from the splice junction. Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using standardized methodology and quantified. Sequence enrichment of the targeted coding exons and adjacent intronic nucleotides is carried out by a bait-capture methodology, using long biotinylated oligonucleotide probes followed by polymerase chain reaction (PCR) and next generation sequencing (NGS). Sanger sequencing is performed for any regions missing or with insufficient read depth coverage for reliable heterozygous variant detection. Reportable small insertions and deletions, potentially homozygous variants, variants in regions complicated by pseudogene interference, and single nucleotide variant calls not satisfying 100x depth of coverage and 40% het ratio thresholds are verified by Sanger sequencing.9  Gross deletion/duplication analysis of RB1 using multiplex ligation-dependent probe amplification (MLPA) and/or targeted chromosomal microarray is also performed.

Mutation Detection Rate 

90-92% of patients with bilateral retinoblastoma have a detectable germline mutation in the RB1 gene, whereas 13-14% of patients with unilateral retinoblastoma have a mutation in the gene (clinical sensitivity).7,8 Ambry's RB1 analysis can detect >99.9% of described mutations in the gene, when present (analytic sensitivity).

Turnaround Time 
TEST CODE TECHNIQUE CALENDAR DAYS
5426 RB1 Gene Sequence and Deletion/Duplication Analyses  14 - 21 days
5422 RB1 Specific Site Analysis  7-14 days

 

Specialty 
Genes 
RB1
References 
  1. Aerts I, et al. Retinoblastoma. Orphanet J Rare Diseases. 2006;1:31. [PMID: 16934146]
  2. Balmer A, et al. Diagnosis and current management of retinoblastoma. Oncogene. 2006;25:5341-5349. [PMID: 16936756]
  3. Draper GJ, et al. Patterns of risk of hereditary retinoblastoma and applications to genetic counselling. Br. J. Cancer. 1992;66:211-219. [PMID: 1637670]
  4. Woo KI, et al. Review of 676 second primary tumors in patients with retinoblastoma: association between age at onset and tumor type. Arch Ophthalmol. 2010;128(7):865-870. [PMID: 20625047]
  5. Kivela T, et al. Trilateral retinoblastoma: a meta-analysis of hereditary retinoblastoma associated with primary ectopic intracranial retinoblastoma.  J. Clin. Oncol. 1999;17:1829-1837. [PMID: 10561222]
  6. Chauveinc L, et al. Osteosarcoma following retinoblastoma: age at onset and latency period. Ophthalmic Genet. 2001;22:77-88. [PMID: 11449317]
  7. Rushlow D, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009:30(5):842-51. [PMID: 19280657]
  8. Nichols KE, et al. Recent advances in retinoblastoma genetic research. Curr Opin Ophthalmol. 2009;20(5):351-5. [PMID: 19587599]
  9. Mu W, et al. Sanger confirmation is required to achieve optimal sensitivity and specificity in next-generation sequencing panel testing. J Mol Diagn. 2016. 18(6):923-932.