Clinical Diagnosis

The diagnosis of retinoblastoma (Rb) is usually established by examination of the retina of the eye using indirect ophthalmoscopy. Fundus imaging, MRI, and ultrasonography are used to support the diagnosis and stage the tumor.

Retinoblastoma is:

• Unilateral if only one eye is affected by retinoblastoma. Usually, in individuals with unilateral retinoblastoma the tumor is also unifocal, i.e., only a single retinoblastoma tumor is present. However, in most persons with unilateral retinoblastoma without a family history, the tumor is large and it is not possible to determine if a single tumor is present.
• Bilateral if both eyes are affected by retinoblastoma. In individuals with bilateral retinoblastoma both eyes clearly may show multiple tumors. Some individuals have multifocal tumors in one eye (unilateral multifocal retinoblastoma). Intraocular seeding may mimic true multifocal tumor growth.
• Trilateral if bilateral (or, rarely, unilateral) retinoblastoma and a pinealoblastoma co-occur.

Testing

Histopathology. Diagnosis of retinoblastoma can be confirmed by histopathologic investigation. Careful investigation of the optic nerve is required to identify possible invasion of tumor cells.

Chromosome analysis. Cytogenetic analysis of peripheral blood lymphocytes has been used to detect deletions or rearrangements involving 13q14.1-q14.2, which are present in approximately 5% of individuals with unilateral Rb and approximately 7.5% of individuals with bilateral Rb. Cytogenetic resolution at the 600-650 band level is recommended and at least 30 metaphases should be analyzed in order to detect mosaic aberrations that are present in about 1% of individuals with Rb.

Molecular Genetic Testing

Gene. RB1 is the only gene in which mutations are known to initiate retinoblastoma.

Clinical testing

For an integrated view of the use of different testing methods, see Interpretation of Test Results and Testing Strategy.

Table 1. Summary of Molecular Genetic Testing Used in Retinoblastoma

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Gene Symbol	Test Method		Mutations Detected	Mutation Detection Frequency by Test Method 1, 2	Test Availability
RB1	Sequence analysis / mutation scanning (genomic) 3		Single-base substitutions, small intragenic deletions, insertions	70%-75%	Clinical
	Targeted mutation analysis 4		Panel of recurrent point mutations	25% 5
	Sequence analysis of RNA from blood 6		Deep intronic splice mutations, gross rearrangements	<5% 7
	Gross deletion / duplication analysis 8	Deletion / duplication analysis 8	Exonic, multiexonic, and whole-gene deletions along with large insertions, rearrangements 9	16%
		FISH 10	Submicroscopic deletions and translocations	>8%
		Heterozygosity testing	De novo submicroscopic germline deletions 11,12	8%
	Methylation analysis		Hypermethylation of the promoter region	10%-12% 13

Test Availability refers to availability in the GeneTests™ Laboratory Directory. GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests™ Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory. GeneTests does not verify laboratory-submitted information or warrant any aspect of a laboratory's licensure or performance. Clinicians must communicate directly with the laboratories to verify information.

1. The ability of the test method used to detect a mutation that is present in the indicated gene

2. Lohmann et al [2002]. In individuals with normal chromosome studies; refers to the ability to detect a germline mutation if one is present. Note: Table 2 lists the probability that a germline mutation would be present based on family history and tumor presentation.

3. Sequence analysis and mutation scanning of the entire gene can have similar mutation detection frequencies; however, mutation detection rates for mutation scanning may vary considerably between laboratories depending on the specific protocol used.

4. Particularly useful for detecting mosaic recurrent mutations in blood and can detect mutant DNA levels that are below the limit of conventional sequence analysis. Low levels of mutational mosaicism have been identified in probands with bilateral disease and in individuals with unilateral disease who have affected children who inherited the mutation; therefore, detecting mosaic recurrent mutations is clinically relevant [Rushlow et al 2009].

5. Rushlow et al [2009]. Panel of targeted mutations and detection frequency may vary.

6. May be useful when sequence analysis or mutation scanning of genomic DNA purified from peripheral blood is negative.

7. In individuals without a mutation identified by DNA-based analyses [Zhang et al 2008]

8. Testing that identifies deletions/duplications not readily detectable by sequence analysis of the coding and flanking intronic regions of genomic DNA; included in the variety of methods that may be used are: quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and chromosomal microarray (CMA) that includes this gene/chromosome segment. See CMA.

9. Mutations detected may vary with the method chosen for deletion/duplication analysis.

10. Deletions of all or parts of RB1 have been identified by FISH analysis using probes derived from sequences of RB1 (e.g., LSI 13 [RB1] 13q14 SpectrumOrange Probe).

11. The genotype of RB1 in peripheral blood DNA of the parents may suggest absence of the mutant RB1 allele of the proband, suggesting a de novo germline mutation, or low-level mosaicism for the mutant allele in the parent.

12. Testing for loss of heterozygosity in tumors. Comparative genotyping of polymorphic loci within and flanking RB1 in DNA from peripheral blood and tumor can reveal that loss of the normal allele (hemizygosity) with or without duplication (homozygosity) of the mutant allele constitutes the somatic mutation.

13. Hypermethylation of RB1 promoter (which silences gene expression) is observed in 10%-12% of tumors from individuals with sporadic, unilateral retinoblastoma [Zeschnigk et al 2004]. In these individuals, analysis of the promoter methylation status in DNA from tumor is needed to identify the two inactive RB1 alleles that triggered tumor development.

Linkage analysis. Linkage analysis using highly informative microsatellite markers within and tightly linked to RB1 can be used in two settings:

• To track the mutant allele in families with more than two affected individuals
  
  Note: Indirect testing in a two-generation family with an affected parent and an affected child may be unreliable because of the possibility of germline mosaicism in the "founder" parent [Rushlow et al 2009].
• To determine if an individual at risk in a family with only one affected individual has inherited either RB1 allele present in the affected individual. If the individual at risk does not have either RB1 allele in common with the affected relative, the individual's risk of developing retinoblastoma decreases to the risk level in the general population [Greger et al 1988, Wiggs et al 1988].

Test characteristics. Information on test sensitivity, specificity, and other test characteristics can be found at Lohmann et al [2011] (click here for full text).

Interpretation of test results. For issues to consider in interpretation of sequence analysis results, click here.

• A combination of clinical presentation, family history, and molecular genetic testing is used to determine if a proband has a germline (heritable) mutation or two somatic (non-heritable) mutations in the tumor. See Table 2.
  
  If a disease-causing RB1 mutation is found in the DNA of white blood cells of the affected individual, (s)he has a high probability of having a germline mutation.
  
  If neither disease-causing RB1 mutation identified in tumor tissue is found in the DNA of white blood cells, the affected individual has a low probability of having an RB1 germline mutation. Note: Because blood mosaicism as low as 20% can usually be detected by conventional molecular analysis such as sequencing, the failure to detect an RB1 disease-causing mutation in the DNA of white blood cells reduces but cannot eliminate the probability that the individual has an RB1 mutation in his/her germline.

Table 2. Probability of Germline Mutation Being Present in a Proband with Rb Based on Family History and Tumor Presentation

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Family History	Rb Presentation			Probability that an RB1 Germline Mutation is Present
	Unilateral		Bilateral
	Multifocal	Unifocal
Positive 1		+		100%
	+			100%
			+	100%
Negative 2			+	Close to 100% 3
	+			14%-95%
		+		~14%

1. Positive = more than one affected family member (10% of retinoblastoma)

2. Negative = only one affected individual in the family (90% of retinoblastoma)

3. RB1 mutations are identified by conventional molecular testing in 90%-95% of simplex cases with bilateral involvement; the remaining 5% may have translocations, deep intronic splice mutations, or low-level mosaic mutations which may or may not be in the germline.

Information on specific allelic variants may be available in Molecular Genetics (see Table A. Genes and Databases and/or Pathologic allelic variants).

Testing Strategy

To confirm/establish the diagnosis in a proband

• Individuals with familial or bilateral retinoblastoma. The goal is to identify the constitutional RB1 mutation that caused inactivation of one RB1 allele.
  • Molecular genetic testing including sequence analysis and some form of deletion/duplication analysis (e.g., MLPA, FISH, CMA, heterozygosity testing) is performed on peripheral blood DNA to identify the constitutional RB1 mutation. About 90%-95% of individuals have a detectable RB1 mutation in blood.
  • In some individuals with bilateral retinoblastoma and no family history of retinoblastoma, an oncogenic RB1 mutation is not detected in peripheral blood. In such cases, tumor DNA may be studied.
    • If tumor DNA demonstrates that both RB1 alleles in the tumor have an RB1 mutation or hypermethylation of the RB1 promoter region, peripheral blood DNA can be tested for the presence of the RB1 mutations identified in the tumor.
    • If neither of the two RB1 mutations identified in the tumor is detected in DNA from peripheral blood, mutational mosaicism can be assumed. If one of the RB1 mutations identified in the tumor is a large deletion, testing for mutational mosaicism in peripheral blood cells may include FISH analysis.
• Individuals with unilateral retinoblastoma and no family history of retinoblastoma (simplex cases). The goal is to identify the two RB1 mutations that caused inactivation of both RB1 alleles in the tumor, if available.
  • Molecular genetic testing including sequence analysis, some form of deletion/duplication analysis (e.g., MLPA, FISH, CMA, heterozygosity testing), and methylation analysis is first performed on tumor tissue. If tumor DNA demonstrates that each of the two tumor alleles have an RB1 mutation or hypermethylation of the RB1 promoter region, peripheral blood DNA can be tested for the presence of the RB1 mutations identified in the tumor.
  • In about 14% of individuals with unilateral retinoblastoma and no family history of retinoblastoma (see Table 2), one of the RB1 mutations identified in the tumor is also detected in peripheral blood, either as a heterozygous mutation, or in a mosaic state (indicating a mutation that occurred after conception) which may or may not be present in the germline of the proband.
  • In the future, MYCN amplification may be useful for analysis of tumors. About 1.5% of children with sporadic unilateral Rb have high level MYCN amplification but no mutational inactivation of RB1 [Rushlow et al 2013].

Predictive testing for at-risk asymptomatic family members requires prior identification of the disease-causing mutation in the family.

Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of the disease-causing mutation in the family.

Note: It is the policy of GeneReviews to include in GeneReviews™ chapters any clinical uses of testing available from laboratories listed in the GeneTests™ Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Genetically Related (Allelic) Disorders

No phenotypes other than hereditary predisposition to retinoblastoma and second cancers (see Natural History, Related tumors) are known to be associated with mutation of RB1.

Natural History

Probands with retinoblastoma (Rb) usually present in one of the following clinical settings:

• Chromosome deletion involving band 13q14. Up to 5% of all index cases with unifocal RB and 7.5% of all index cases with multifocal RB have a chromosomal deletion of 13q14. Such chromosomal abnormalities are often associated with developmental delay and birth defects [Mitter et al 2011, Castéra et al 2013].
• Normal cytogenetic study and one of the following
  • Positive family history and unilateral or bilateral Rb (~10% of index cases)
  • Negative family history and bilateral Rb (30% of index cases)
  • Negative family history and unilateral Rb (60% of index cases)

About 60% of individuals with Rb have unilateral retinoblastoma with a mean age at diagnosis of 24 months. About 40% have bilateral retinoblastoma with a mean age at diagnosis of 15 months. In individuals with a positive family history (~10%) who undergo clinical surveillance via serial retinal examinations, tumors are often identified in the first month of life.

The most common presenting sign of Rb is a white pupillary reflex (leukocoria). Strabismus is the second most common presenting sign and may accompany or precede leukocoria [Abramson et al 2003]. Unusual presenting symptoms include glaucoma, orbital cellulitis, uveitis, hyphema, or vitreous hemorrhage. Most affected children are diagnosed before age five years. Atypical manifestations are more frequent in older children.

In most children with bilateral tumors, both eyes are affected at the time of initial diagnosis. Some children who are initially diagnosed with unilateral retinoblastoma later develop a tumor in the contralateral unaffected eye.

Retinoma and associated eye lesions. These lesions range from retinal scars to calcified phthisical eyes resulting from spontaneous regression of retinoblastoma, and include benign retinal tumors (called retinoma) that have undergone spontaneous growth arrest [Dimaras et al 2008].

Related tumors. Individuals with germline RB1 mutations are at increased risk of developing tumors outside the eye.

Pinealoblastomas occur in "retina-like" tissue in the pineal gland of the brain. Co-occurrence of pinealoblastomas or primitive neuroectodermal tumors and retinoblastoma is referred to as trilateral retinoblastoma. Pinealoblastoma is rare and usually fatal, unlike retinoblastoma of the eye which is generally curable [Kivelä 1999].

The risk for other specific extraocular primary neoplasms (collectively called second primary tumors) is increased in individuals with heritable Rb and in heterozygous carriers of a cancer-predisposing RB1 mutation. Most of the second primary cancers are osteosarcomas, soft tissue sarcomas (mostly leiomyosarcomas and rhabdomyosarcomas), or melanomas [Kleinerman et al 2007, Marees et al 2008, Kleinerman et al 2012]. These tumors usually manifest in adolescence or adulthood. The incidence of second primary tumors is increased to more than 50% in individuals with retinoblastoma who have received external beam radiation therapy [Wong et al 1997]. Survivors of hereditary retinoblastoma who are not exposed to high-dose radiotherapy have a high lifetime risk of developing a late-onset cancer [Fletcher et al 2004, Kleinerman et al 2012].

Genotype-Phenotype Correlations

In the majority of families with retinoblastoma, all members who have inherited a germline mutation develop multiple tumors in both eyes. It is not unusual to find, however, that the founder (i.e., the first person in the family to have retinoblastoma) has only unilateral retinoblastoma. Most of such families segregate RB1 null alleles that are altered by frameshift or nonsense mutations. With few specific exceptions, RB1 null alleles show nearly complete penetrance (<99%) [Lohmann et al 1996, Sippel et al 1998].

Fewer than 10% of families show a "low penetrance" phenotype with reduced expressivity (i.e., increased prevalence of unilateral retinoblastoma) and incomplete penetrance (i.e., 25% or lower). This low penetrance phenotype is usually associated with mutant RB1 alleles showing in-frame or missense changes, distinct splice mutations, or mutations in the promoter region.

A third category of families shows reduced penetrance but no reduced expressivity in family members with retinoblastoma [Klutz et al 2002].

Cytogenetically visible deletions involving 13q14 that also result in deletions of additional genes in the same chromosomal region as RB1 may cause developmental delay [Castéra et al 2013] and mild-to-moderate facial dysmorphism. As sizeable deletions of 13q14 show reduced expressivity, a considerable proportion of individuals with such deletions show unilateral retinoblastoma only; some of these children develop no tumors at all [Mitter et al 2011].

Penetrance

See Genotype-Phenotype Correlations.

Anticipation

Milder phenotypic expression in founders has been associated with mutational mosaicism. No multigenerational anticipation has been observed to date.

Nomenclature

Glioma retinae is another name for retinoblastoma.

Prevalence

The incidence of retinoblastoma is estimated at between 1:15,000 and 1:20,000 live births [Moll et al 1997, Seregard et al 2004].

Evaluations Following Initial Diagnosis

To establish the extent of disease in an individual diagnosed with retinoblastoma, the following evaluations are recommended:

• Prior to the planning of therapy, the extent of the tumor within and outside the eye should be determined. Each affected eye is assigned a classification, depending on the extent of disease and the risk that the cancer has spread outside the eye. In the absence of family history, most commonly the affected eye(s) contain large tumors, directly visible through the pupil as a white pupillary reflex. Extent of tumor is estimated by clinical examination under anesthetic and imaging techniques such as ultrasound and MRI, particularly focusing on the tumor-optic nerve relationship. CT scan is contraindicated because of the risk of radiation in children with germline RB1 mutations. Head MRI is also useful to evaluate for a pinealoblastoma, indicating trilateral retinoblastoma.
• For very large tumors with risk factors for extraocular disease, bone marrow aspiration and examination of cerebrospinal fluid (CSF) may also be performed at diagnosis, or performed when pathologic examination of the enucleated eye reveals optic nerve invasion or significant choroidal invasion.
• If retinoblastoma has spread outside the eye, the stage of cancer will need to be evaluated to set out the most appropriate care of the child.
• In those individuals with a family history of retinoblastoma (Rb), and in uncommon circumstances in which the child presents with strabismus or poor vision, the retinal tumors may be small and detected on clinical examination under anesthetic.
• Medical genetics consultation at the time of diagnosis is recommended to clarify for the family the heritable aspects of retinoblastoma, especially as pertains to their child. Molecular diagnosis now has a sensitivity of 96% in identifying the precise RB1 mutant allele in each child and is an important component of care of the affected individual and the family.

Treatment of Manifestations

Goals of treatment are first preservation of life, and then of sight. As optimum treatment may be complex, specialists skilled in the treatment of retinoblastoma from various fields including ophthalmology, pediatric oncology, pathology and radiation oncology should collaborate in care.

In addition to eye classification and tumor stage, choice of treatment depends on many factors, including the number of tumor foci (unifocal, unilateral multifocal, or bilateral), localization and size of the tumor(s) within the eye(s), presence of vitreous seeding, the potential for useful vision, the extent and kind of extraocular extension, and the resources available.

Treatment options for the eye include enucleation; cryotherapy; laser, systemic, or local ocular chemotherapy combined with or followed by laser or cryotherapy; radiation therapy using episcleral plaques; and, as a last resort, external beam radiotherapy.

Prevention of Secondary Complications

If possible, any radiation (including x-ray, CT scan, and external beam radiation) should be avoided to minimize the lifetime risk of developing late-onset second cancers. If such tests are absolutely necessary in essential health care, then they should be used.

Surveillance

Further information regarding medical surveillance for those who have had or are at risk of developing Rb is available in the guidelines for retinoblastoma care (see ).

Detection of subsequent Rb after initial diagnosis. Following successful treatment, children require frequent follow-up examination for early detection of newly arising intraocular tumors.

• It is recommended that children known to have an RB1 germline mutation have an eye examination under anesthesia every three to four weeks until age six months, then less frequently until age three years. Clinic examinations with cooperative children are performed every three to six months until age seven years, then annually and eventually biannually for life.
• Individuals who have unilateral retinoblastoma are at risk of developing tumors in their normal eye [Temming et al 2013].
  • If the two RB1 mutant alleles are identified in the tumor and if the individual is shown to have one of those two mutations in leukocyte DNA (~14% of individuals), the children are followed as described above. Mosaicism involving more than 15% of blood cells is molecularly detectable by conventional methods.
  • If the RB1 mutant alleles identified in the tumor are not detected in leukocyte DNA, there is still risk that the individual has low-level mosaicism (involving <15% of blood cells) for the mutant allele and will develop a tumor in the other eye [Rushlow et al 2009]. This risk is small enough that examination under anesthesia may be replaced with regular clinical examination of the eyes, including clinical ultrasound (a simple, non-invasive procedure).

Detection of second non-ocular tumors in individuals with retinoblastoma. Because of the high risk for second cancers, including sarcomas, melanoma, and specific other cancers, prompt investigation of any signs or symptoms is indicated. Where available, total body MRI at regular intervals may be indicated. No specific screening protocols have been published.

Individuals at risk for retinoblastoma who warrant surveillance for early manifestations of Rb include the following:

• Individuals with retinomas (premalignant retinal lesions associated with Rb)
• Children who have inherited an RB1 cancer-predisposing mutation OR children at risk for Rb who have not undergone molecular genetic testing:
  • Eye examinations by an ophthalmologist experienced in the treatment of retinoblastoma starting directly after birth as described above in Detection of subsequent Rb after initial diagnosis. Young or uncooperative children may require examination under anesthesia.
• At-risk children who have not inherited the cancer-predisposing mutation known to be present in the family as determined by RB1 mutation analysis:
  • Examination by an ophthalmologist familiar with retinoblastoma shortly after birth. Subsequent eye examinations should be performed as needed for routine pediatric care.

Agents/Circumstances to Avoid

It has been suggested by Fletcher et al [2004] that cancer risks in hereditary retinoblastoma survivors may be reduced by limiting exposure to DNA-damaging agents (radiotherapy, tobacco, and UV light).

Evaluation of Relatives at Risk

Asymptomatic at-risk children. Use of molecular genetic testing for early identification of at-risk family members improves diagnostic certainty and reduces the need for costly screening procedures in those at-risk family members who have not inherited the disease-causing mutation [Noorani et al 1996, Richter et al 2003]. The American Society of Clinical Oncologists identifies Rb as a Group 1 disorder, i.e., a hereditary syndrome for which genetic testing is considered part of the standard management for at-risk family members [American Society of Clinical Oncology 2003].

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Pregnancy Management

When there is a family history of Rb, various options are available to optimize management of an at-risk pregnancy [Canadian Retinoblastoma Society 2009]

• When the RB1 mutant allele of the proband is known:
  • Most commonly, the fetus can be tested for that specific RB1 mutant allele at any time, including amniocentesis at 32 weeks’ gestation.
  • If the fetus does not carry the mutation, the risk for Rb is the same as that for any individual in the general population.
  • If the fetus carries the RB1 mutant allele present in the family, it is recommended that early delivery at 36 weeks’ gestation be performed, which is within the normal range for spontaneous birth. This will allow the earliest detection of visually threatening tumors at a time that they can still be treated with minimally invasive therapies to achieve potential good vision. Rb tumors grow rapidly around the time of birth, and every day can make a difference in the ultimate outcome.
• If the RB1 mutant allele of the proband is NOT known:
  • Obstetric ultrasound or MRI may reveal a large Rb in the eye of a fetus; however, these tests are not sensitive to small Rb tumors.
  • Depending on the relationship of the infant to the proband, children should have examination of both retinas with the pupils widely dilated by an ophthalmologist knowledgeable about Rb within a few days of birth, or in the first few weeks after birth.
  • Clinic examinations by an ophthalmologist knowledgeable about Rb can then follow as described above; if a tumor is seen, the child needs an examination under anesthesia to fully evaluate the stage and extent of disease to help direct appropriate treatment (see Evaluations Following Initial Diagnosis).

Therapies Under Investigation

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.

Other

Genetics clinics, staffed by genetics professionals, provide information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

Mode of Inheritance

Hereditary retinoblastoma (Rb), caused by germline RB1 mutations, is inherited in an autosomal dominant manner.

Risk to Family Members

Parents of a proband. Some individuals diagnosed with retinoblastoma have an affected parent or a parent who has an RB1 mutation but is not affected; the majority of individuals with retinoblastoma have the disorder as the result of a de novo gene mutation.

The recommendations for determining the genetic status of the parent of a proband with retinoblastoma or the conclusions about the genetic status of the parent depend on the following findings in the proband:

• Cytogenetically detectable chromosome 13 deletion or rearrangement
  • Recommendation. Parental cytogenetic studies to determine if either parent carries a balanced chromosome translocation or rearrangement
• Positive family history (i.e., the parent had retinoblastoma or a close relative of one parent had retinoblastoma)
  • Conclusion. The parent has an RB1 cancer-predisposing germline mutation.
• Negative family history
  • Recommendation. Examination of apparently unaffected parents by an ophthalmologist knowledgeable about retinoblastoma, retinoma, and retinoblastoma-associated eye lesions
  • Conclusion. If such a lesion is detected, the parent is likely to have an RB1 cancer-predisposing germline mutation.
• Presence of a germline RB1 cancer-predisposing mutation in the proband
  • Recommendation. Molecular genetic testing of both parents
  • Conclusions
    • If a heterozygous mutation is identified in either parent, the parent is at risk of developing non-ocular second primary tumors and is at 50% risk of transmitting the cancer-predisposing RB1 mutation to other offspring. In approximately 10% of simplex cases that have a heterozygous RB1 mutation detected in blood, one of the proband’s unaffected parents also has the mutation. In most cases this is a heterozygous “reduced penetrance” mutation, such as a missense mutation [Rushlow et al 2009].
    • For approximately 1% of simplex cases that have a heterozygous RB1 mutation detected in blood, one of the proband’s unaffected parents has mutational mosaicism, possibly at levels too low to be detectable by sequencing of DNA extracted from leukocytes, but detectable by more sensitive methods such as allele-specific PCR [Rushlow et al 2009]. Sibs of the proband are at risk of inheriting the mutation.
    • A parent may have germline mosaicism even in the absence of a detectable mutation in leukocyte DNA. Therefore, even if the RB1 mutation cannot be detected in leukocyte DNA from either parent, sibs of the proband should still be tested for the presence of the proband’s mutation.
• Presence of a mosaic RB1 cancer-predisposing mutation in the proband [Carlson & Desnick 1979, Rushlow et al 2009]
  • Conclusion. The mutation occurred de novo as a post-zygotic event in the proband.
  • Recommendation. Molecular genetic testing of the parents is not necessary.

Sibs of a proband. The risk to sibs of a proband depends on the genetic status of the parents.

• If a parent is determined to have a germline RB1 cancer-predisposing mutation either by positive family history, by an eye examination that reveals a retinoblastoma-associated eye lesion (retinoma), or by molecular genetic testing that reveals the presence of a cancer-predisposing RB1 mutation, the risk to each sib of the proband is 50% (or lower if the parent with the RB1 mutation has mosaicism) of inheriting the cancer-predisposing RB1 mutation. Given the approximately 99% penetrance of most RB1 cancer-predisposing mutations, the actual risk for retinoblastoma in these individuals is about 50% (or lower if the parent with the RB1 mutation has mosaicism). (In rare families with "familial low-penetrance retinoblastoma," the risk for tumor development in an individual with the mutation is also reduced.)
• If neither parent shows the cancer-predisposing RB1 germline mutation that was identified in the proband, germline mosaicism in one parent is still possible. Thus, it is recommended that each sib be tested for the RB1 mutation identified in the proband.
• If the proband clearly shows mosaicism for an RB1 cancer-predisposing mutation in non-cancer cells such as leukocyte DNA, it is assumed that the mutation arose as a post-zygotic event and that neither parent has an RB1 germline mutation. The risk to the sibs is not increased and thus the testing of sibs for the RB1 mutation identified in the proband is not warranted.
• If molecular genetic testing is not available or is uninformative, empiric risks based on tumor presentation (i.e., unifocal or multifocal) and family history can be used (Table 3). The low, but not negligible, risk to sibs of a proband with a negative family history presumably reflects the presence of either a germline RB1 mutation with reduced penetrance in one parent or somatic mosaicism (that includes the germline) for an RB1 mutation in one parent.
• If a parent has a cytogenetically detectable balanced chromosome 13 translocation or rearrangement, the sibs are at increased risk of inheriting an unbalanced chromosome rearrangement.

Offspring of a proband. The risk to the offspring of a proband depends on the following:

• If the proband has bilateral RB and no family history of RB, the presence of a germline RB1 cancer-predisposing mutation is assumed and the risk to each offspring of inheriting the mutation is 50%. Predictive DNA testing in offspring is possible if the cancer-predisposing RB1 mutation has been identified in the proband.
• If the proband has had unilateral multifocal RB and no family history of RB, recurrence risk to offspring is lower [Sippel et al 1998, Rushlow et al 2009].
• The risk to offspring of a proband with unilateral unifocal disease and a negative family history is 6%, reflecting the possibility that the proband has mosaicism for a germline mutation or a germline RB1 mutation associated with milder phenotypic expression. In families with "familial low-penetrance retinoblastoma," the risk for tumor development in persons with the low-penetrance RB1 allele is lower than the 95% observed with highly penetrant RB1 “null” alleles.

Molecular genetic testing of tumor DNA from probands who are simplex cases (i.e., a single occurrence in a family) with a unilateral tumor

• If both mutant RB1 alleles are identified and if one of the mutant alleles is heterozygous in DNA from leukocytes of the proband, the risk to offspring of a proband of inheriting the mutant alleles is 50%.
• If neither mutant allele is detected in DNA from leukocytes of the proband, there is an estimated 1.2% chance that the proband has germline mosaicism for the mutant allele, giving the offspring a 0.6% risk of inheriting a germline mutation [Richter et al 2003]. Molecular genetic testing in offspring must check for the two mutant alleles identified in the tumor of the proband.
• If one of the mutations identified in the tumor is mosaic in DNA from leukocytes of the proband, the level of germline involvement is uncertain. Quantitative mutation analysis of sperm may be helpful [Rushlow et al 2009]. All offspring should be checked for the mutation identified in leukocyte DNA.

Other family members of a proband. The risk to other family members depends on the status of the proband's parents. If a parent has a disease-causing mutation, his or her family members are at risk.

Related Genetic Counseling Issues

See Management, Evaluation of Relatives at Risk for information on evaluating at-risk relatives for the purpose of early diagnosis and treatment.

Family planning

• The optimal time for determination of genetic risk and discussion of the availability of prenatal testing is before pregnancy.
• It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk [Dommering et al 2012].

Genetic cancer risk assessment and counseling. For a comprehensive description of the medical, psychosocial, and ethical ramifications of identifying at-risk individuals through cancer risk assessment with or without molecular genetic testing, see Elements of Cancer Genetics Risk Assessment and Counseling (part of PDQ^®, National Cancer Institute).

DNA banking is the storage of DNA (typically extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. See for a list of laboratories offering DNA banking.

Prenatal Testing

Molecular genetic testing. Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at about 15 to 18 weeks' gestation or chorionic villus sampling (CVS) at about ten to 12 weeks' gestation. The disease-causing allele of an affected family member must be identified or linkage phase established in the family before prenatal testing can be performed.

Cytogenetic testing. Prenatal diagnosis for pregnancies at increased risk of having an unbalanced chromosome rearrangement is possible by chromosome analysis of fetal cells obtained by amniocentesis usually performed at about 15 to 18 weeks' gestation or chorionic villus sampling (CVS) at about ten to 12 weeks' gestation.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Ultrasound examination. If a disease-causing RB1 mutation is identified in the fetus, ultrasound examination may be used to identify intraocular tumors. If tumors are present, preterm delivery to enable early treatment may be considered [Sahgal et al 2006]. Even if no tumors are visible on obstetric ultrasound, delivery of the fetus at 36 weeks’ gestation may be recommended, as 30% of babies with an RB1 mutation will have a tiny vision-threatening tumor [unpublished data].

Requests for prenatal testing for conditions (such as retinoblastoma) that do not affect intellect and have treatment available are not common. Differences in perspective may exist among medical professionals and within families regarding the use of prenatal testing, particularly if the testing is being considered for the purpose of pregnancy termination rather than early diagnosis. Decisions about prenatal testing are the choice of the parents, who can benefit by full discussion of the available options.

Preimplantation genetic diagnosis (PGD) may be available for families in which the disease-causing mutations have been identified. For laboratories offering PGD, see .

Note: It is the policy of GeneReviews to include in GeneReviews™ chapters any clinical uses of testing available from laboratories listed in the GeneTests™ Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Molecular Genetic Pathogenesis

With very rare exceptions, tumor development starts from cells that do not have a normal RB1 allele [Rushlow et al 2013]. See Figure 1.

Figure

Figure 1. Schematic of the molecular genetic mechanisms that result in non-hereditary and hereditary retinoblastoma (Rb). The development of Rb is initiated if both alleles of RB1 are mutated (rb rb).

In non-hereditary Rb, both mutations (more...)

Normal allelic variants. Twenty-seven exons are transcribed and spliced into a 4.7-kb mRNA. There is no indication of functional alternative splicing. No frequent polymorphic sites within the 2.7-kb open reading frame are known, but there are intronic variants and two highly polymorphic microsatellites (Rb1.20, Rbi2) and one minisatellite (RBD).

Pathologic allelic variants. More than 2500 distinct point mutations have been observed in white blood-cell DNA of individuals with retinoblastoma or in tumors, 1400 are archived (see Table A, Locus Specific). The majority of RB1 mutations result in a premature termination codon, usually through single base substitutions, frameshift mutations, or splice mutations. Mutations have been found scattered throughout exon 1 to exon 25 of RB1 and its promoter region. In a single family, a possible disease-causing variant in exon 27 was identified [Mitter et al 2009]. Recurrent mutations are observed at 14 methylated CpG dinucleotides. Other important types of pathogenic allelic variants are gross rearrangements and deletions [Albrecht et al 2005, Rushlow et al 2009, Castéra et al 2013].

Normal gene product. RB1 encodes a ubiquitously expressed nuclear protein that is involved in cell cycle regulation (G1 to S transition). The RB protein is phosphorylated by members of the cyclin-dependent kinase (cdk) system prior to the entry into S-phase. Upon phosphorylation, the binding activity of the pocket domain is lost, resulting in the release of cellular proteins. For a review see Goodrich [2006].

Abnormal gene product. The majority of mutant alleles, if expressed at all, code for proteins that have lost cell cycle-regulating functions. Retention of partial activities has been observed in proteins resulting from mutant alleles that are associated with low-penetrance retinoblastoma [Lohmann et al 1994, Bremner et al 1997, Otterson et al 1997].

Published Guidelines/Consensus Statements

1. American Society of Clinical Oncology. Statement on genetic testing for cancer susceptibility. Available online. 2003. Accessed 3-21-13.
2. Canadian Retinoblastoma Society. National Retinoblastoma Strategy Canadian Guidelines for Care: Stratégie thérapeutique du rétinoblastome guide clinique canadien. Available online. 2009. Accessed 3-26-13. [PubMed: 20237571]

Literature Cited

1. Abramson DH, Beaverson K, Sangani P, Vora RA, Lee TC, Hochberg HM, Kirszrot J, Ranjithan M. Screening for retinoblastoma: presenting signs as prognosticators of patient and ocular survival. Pediatrics. 2003;112(6 Pt 1):1248–55. [PubMed: 14654593]
2. Albrecht P, Ansperger-Rescher B, Schüler A, Zeschnigk M, Gallie B, Lohmann DR. Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2005;26:437–45. [PubMed: 16127685]
3. American Society of Clinical Oncology. Statement on genetic testing for cancer susceptibility. Available online. 2003. Accessed 3-21-13.
4. Bremner R, Du DC, Connolly-Wilson MJ, Bridge P, Ahmad KF, Mostachfi H, Rushlow D, Dunn JM, Gallie BL. Deletion of RB exons 24 and 25 causes low-penetrance retinoblastoma. Am J Hum Genet. 1997;61:556–70. [PMC free article: PMC1715941] [PubMed: 9326321]
5. Canadian Retinoblastoma Society; National Retinoblastoma Strategy Canadian Guidelines for Care: Stratégie thérapeutique du rétinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44 Suppl 2:S1–88. [PubMed: 20237571]
6. Carlson EA, Desnick RJ. Mutational mosaicism and genetic counseling in retinoblastoma. Am J Med Genet. 1979;4:365–81. [PubMed: 120116]
7. Castéra L, Dehainault C, Michaux D, Lumbroso-Le Rouic L, Aerts I, Doz F, Pelet A, Couturier J, Stoppa-Lyonnet D, Gauthier-Villars M, Houdayer C. Fine mapping of whole RB1 gene deletions in retinoblastoma patients confirms PCDH8 as a candidate gene for psychomotor delay. Eur J Hum Genet. 2013;21:460–4. [PMC free article: PMC3598316] [PubMed: 22909775]
8. Dimaras H, Khetan V, Halliday W, Orlic M, Prigoda NL, Piovesan B, Marrano P, Corson TW, Eagle RC, Squire JA, Gallie BL. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Hum Mol Genet. 2008;17:1363–72. [PubMed: 18211953]
9. Dommering CJ, Garvelink MM, Moll AC, van Dijk J, Imhof SM, Meijers-Heijboer H, Henneman L. Reproductive behavior of individuals with increased risk of having a child with retinoblastoma. Clin Genet. 2012;81:216–23. [PubMed: 21954974]
10. Draper GJ, Sanders BM, Brownbill PA, Hawkins MM. Patterns of risk of hereditary retinoblastoma and applications to genetic counselling. Br J Cancer. 1992;66:211–9. [PMC free article: PMC1977909] [PubMed: 1637670]
11. Fletcher O, Easton D, Anderson K, Gilham C, Jay M, Peto J. Lifetime risks of common cancers among retinoblastoma survivors. J Natl Cancer Inst. 2004;96:357–63. [PubMed: 14996857]
12. Goodrich DW. The retinoblastoma tumor-suppressor gene, the exception that proves the rule. Oncogene. 2006;25:5233–43. [PMC free article: PMC2799241] [PubMed: 16936742]
13. Greger V, Kerst S, Messmer E, Höpping W, Passarge E, Horsthemke B. Application of linkage analysis to genetic counselling in families with hereditary retinoblastoma. J Med Genet. 1988;25:217–21. [PMC free article: PMC1015499] [PubMed: 3163379]
14. Kivelä T. Trilateral retinoblastoma: a meta-analysis of hereditary retinoblastoma associated with primary ectopic intracranial retinoblastoma. J Clin Oncol. 1999;17:1829–37. [PubMed: 10561222]
15. Kleinerman RA, Tucker MA, Abramson DH, Seddon JM, Tarone RE, Fraumeni JF. Risk of soft tissue sarcomas by individual subtype in survivors of hereditary retinoblastoma. J Natl Cancer Inst. 2007;99:24–31. [PubMed: 17202110]
16. Kleinerman RA, Yu CL, Little MP, Li Y, Abramson D, Seddon J, Tucker MA. Variation of second cancer risk by family history of retinoblastoma among long-term survivors. J Clin Oncol. 2012;30:950–7. [PMC free article: PMC3341108] [PubMed: 22355046]
17. Klutz M, Brockmann D, Lohmann DR. A parent-of-origin effect in two families with retinoblastoma is associated with a distinct splice mutation in the RB1 gene. Am J Hum Genet. 2002;71:174–9. [PMC free article: PMC384976] [PubMed: 12016586]
18. Lohmann D, Gallie B, Dommering C, Gauthier-Villars M. Clinical utility gene card for: retinoblastoma. Eur J Hum Genet. 2011;19(3) [PMC free article: PMC3061998] [PubMed: 21150892]
19. Lohmann D, Scheffer H, Gaille B. Best Practice Guidelines for Molecular Analysis of Retinoblastoma. European Molecular Genetics Quality Network. Available online. 2002. Accessed 3-21-13.
20. Lohmann DR, Brandt B, Höpping W, Passarge E, Horsthemke B. Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma. Hum Genet. 1994;94:349–54. [PubMed: 7927327]
21. Lohmann DR, Brandt B, Höpping W, Passarge E, Horsthemke B. The spectrum of RB1 germ-line mutations in hereditary retinoblastoma. Am J Hum Genet. 1996;58:940–9. [PMC free article: PMC1914612] [PubMed: 8651278]
22. Marees T, Moll AC, Imhof SM, de Boer MR, Ringens PJ, van Leeuwen FE. Risk of second malignancies in survivors of retinoblastoma: more than 40 years of follow-up. J Natl Cancer Inst. 2008;100:1771–9. [PubMed: 19066271]
23. Mitter D, Rushlow D, Nowak I, Ansperger-Rescher B, Gallie BL, Lohmann DR. Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma. Fam Cancer. 2009;8:55–8. [PubMed: 18509746]
24. Mitter D, Ullmann R, Muradyan A, Klein-Hitpass L, Kanber D, Ounap K, Kaulisch M, Lohmann D. Genotype-phenotype correlations in patients with retinoblastoma and interstitial 13q deletions. Eur J Hum Genet. 2011;19:947–58. [PMC free article: PMC3179359] [PubMed: 21505449]
25. Moll AC, Kuik DJ, Bouter LM, Den Otter W, Bezemer PD, Koten JW, Imhof SM, Kuyt BP, Tan KE. Incidence and survival of retinoblastoma in The Netherlands: a register based study 1862-1995. Br J Ophthalmol. 1997;81:559–62. [PMC free article: PMC1722238] [PubMed: 9290369]
26. Noorani HZ, Khan HN, Gallie BL, Detsky AS. Cost comparison of molecular versus conventional screening of relatives at risk for retinoblastoma. Am J Hum Genet. 1996;59:301–7. [PMC free article: PMC1914748] [PubMed: 8755916]
27. Otterson GA, Chen WD, Coxon AB, Khleif SN, Kaye FJ. Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function. Proc Natl Acad Sci U S A. 1997;94:12036–40. [PMC free article: PMC23695] [PubMed: 9342358]
28. Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, Anderson J, Han L, Panton R, Branco P, Gallie B. Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72:253–69. [PMC free article: PMC379221] [PubMed: 12541220]
29. Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, Gallie BL. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009;30:842–51. [PubMed: 19280657]
30. Rushlow DE, Mol BM, Yee S, Pajovic S, Thériault BL, Prigoda-Lee NL, Spencer C, Dimaras H, Corson TW, Pang R, Massey C, Godbout R, Jiang Z, Zacksenhaus E, Paton K, Moll AC, Houdayer C, Raizis A, Halliday W, Lam WL, Boutros PC, Lohmann D, Dorsman JC, Gallie BL. Characterisation of retinoblastoma tumours without RB1 mutations: genomic, gene expression and clinical studies. Lancet Oncol. 2013 [PubMed: 23498719]
31. Sahgal A, Millar BA, Michaels H, Jaywant S, Chan HS, Heon E, Gallie B, Laperriere N. Focal stereotactic external beam radiotherapy as a vision-sparing method for the treatment of peripapillary and perimacular retinoblastoma: preliminary results. Clin Oncol (R Coll Radiol). 2006;18:628–34. [PubMed: 17051954]
32. Seregard S, Lundell G, Svedberg H, Kivelä T. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology. 2004;111:1228–32. [PubMed: 15177976]
33. Sippel KC, Fraioli RE, Smith GD, Schalkoff ME, Sutherland J, Gallie BL, Dryja TP. Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling. Am J Hum Genet. 1998;62:610–9. [PMC free article: PMC1376960] [PubMed: 9497263]
34. Temming P, Viehmann A, Biewald E, Lohmann DR. Sporadic unilateral retinoblastoma or first sign of bilateral disease? Br J Ophthalmol. 2013;97:475–80. [PubMed: 23355526]
35. Wiggs J, Nordenskjöld M, Yandell D, Rapaport J, Grondin V, Janson M, Werelius B, Petersen R, Craft A, Riedel K. et al. Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the retinoblastoma gene. N Engl J Med. 1988;318:151–7. [PubMed: 2892131]
36. Wong FL, Boice JD, Abramson DH, Tarone RE, Kleinerman RA, Stovall M, Goldman MB, Seddon JM, Tarbell N, Fraumeni JF, Li FP. Cancer incidence after retinoblastoma. Radiation dose and sarcoma risk. JAMA. 1997;278:1262–7. [PubMed: 9333268]
37. Zeschnigk M, Böhringer S, Price EA, Onadim Z, Masshöfer L, Lohmann DR. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res. 2004;32:e125. [PMC free article: PMC519124] [PubMed: 15353561]
38. Zhang K, Nowak I, Rushlow D, Gallie BL, Lohmann DR. Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2008;29:475–84. [PubMed: 18181215]

Author History

Norbert Bornfeld, MD; University of Essen (2000-2004)
Brenda L Gallie, MD (2004-present)
Bernhard Horsthemke, PhD; University of Essen (2000-2004)
Dietmar R Lohmann, MD (2000-present)
Eberhard Passarge, MD; University of Essen (2000-2004)

Revision History

• 28 March 2013 (me) Comprehensive update posted live
• 10 June 2010 (me) Comprehensive update posted live
• 7 May 2007 (me) Comprehensive update posted to live Web site
• 21 January 2005 (dl) Revision: Risk to offspring of a proband
• 28 December 2004 (me) Comprehensive update posted to live Web site
• 21 January 2003 (me) Comprehensive update posted to live Web site
• 18 July 2000 (me) Review posted to live Web site
• 21 January 1999 (dl) Original submission