Retinoblastoma Workup

Updated: Jul 12, 2016
  • Author: Marichelle Aventura Isidro, MD; Chief Editor: Hampton Roy, Sr, MD  more...
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Workup

Laboratory Studies

Blood counts and electrolyte determination as well as urinalysis and liver function tests are useful in excluding other conditions confused with retinoblastoma.

DNA analysis

Blood specimens should be taken not only from the patient but also from the parents and any siblings for DNA analysis, which could aid in genetic counseling.

There are direct and indirect methods in the analysis of the retinoblastoma gene. The direct method aims to find the initial mutation that precipitated the development of the tumor; then, it is determined whether that mutation is in the germline of the affected patient. Indirect methods can be used in cases where the initial mutation cannot be located or it is uncertain whether it exists.

Sources of DNA to be evaluated directly are either from tumor cells or leukocytes.

Deletions or rearrangements of the retinoblastoma gene can be detected by either karyotyping or Southern blotting techniques.

Point mutations in the retinoblastoma gene can be detected by the following techniques: ribonuclease protection, denaturing gradient gel electrophoresis, single-strand conformation polymorphism, or direct DNA sequencing amplified by the polymerase chain reaction.

Retinoblastomas also may arise by hypermethylation of the promoter region of the retinoblastoma gene, which deactivates this gene but does not alter the DNA sequence. This also can be detected by Southern blot analysis.

Indirect methods of analysis of the retinoblastoma gene rely on DNA polymorphisms within this gene.

Assays of aqueous humor enzyme levels

Assays of aqueous humor enzyme levels could offer useful information to patients with suspected retinoblastoma. Lactate dehydrogenase (LDH) is a glycolytic enzyme that uses glucose as an energy source. It is present in high concentrations within metabolically active cells. Normally, its concentration in serum and aqueous humor is low and the ratio of aqueous humor to serum LDH is less than 1.0 in patients with ocular disease other than retinoblastoma. However, aqueous humor for eyes with retinoblastoma exhibits increased LDH activity expressed as an aqueous humor/LDH ratio of greater than 1.0.

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Imaging Studies

Computed tomography

Cranial and orbital computerized tomography provides a sensitive method for diagnosis and detecting intraocular calcification and shows intraocular extent of the tumor even in the absence of calcification (examples shown below). This neuroimaging technique is also invaluable in assessing the CNS anatomy, including the optic nerve, for possible extension of retinoblastoma.

Patient with retinoblastoma, glaucomatous stage. I Patient with retinoblastoma, glaucomatous stage. Intracranial extension on CT scan.
Patient with retinoblastoma, glaucomatous stage. A Patient with retinoblastoma, glaucomatous stage. Another CT scan slice, showing the intracranial extension of the tumor.
Retinoblastoma, intraocular stage (CT scan finding Retinoblastoma, intraocular stage (CT scan findings). History: 5-month-old female with chief complaint of "cat's eye reflex." Two months prior to admission (PTA), cat's eye reflex noted with outward deviation of left eye. The patient's 29-year-old mother had bilateral retinoblastoma and underwent enucleation, left eye, at age 2 years. Examination: Regressed type stage III, left eye visual acuity (+) dazzle right eye; indirect ophthalmoscopy (+) mass nasal retina with seeding, multiple tumors in peripheral retina, left eye. E/N Retina: Right eye. Management: The patient underwent enucleation, left eye. Examination under anesthesia of right eye: E/N. Histopathology: Retinoblastoma, intraocular stage, well-differentiated left eye.

Ultrasonography

Ultrasonography is useful in distinguishing retinoblastomas from non-neoplastic conditions. It is also useful in detecting calcifications.

MRI

MRI may be beneficial in estimating the degree of differentiation of retinoblastomas but is not as specific as computerized tomography because of its lack of sensitivity in detecting calcium.

Studies show that on T1-weighted images, the tumors usually have a low intensity and are usually difficult to distinguish from surrounding vitreous, but, on T2-weighted images, retinoblastoma tumors demonstrate very low intensity compared to vitreous. Calcification is more pronounced on T2 sequences.

MRI also is useful in identifying any associated hemorrhagic or exudative retinal detachment. This is seen as a localized subretinal area of higher signal intensity compared to vitreous on both T1- and T2-weighted sequences.

Certain ADC values calculated at T3-weighted imaging are correlated with some of the accepted parameters of poor prognosis for retinoblastoma, particularly degree of differentiation of the tumor and tumor size. [14]

X-ray studies

In areas of the world where ultrasonography and computerized tomography are not available, x-ray studies may be the only means of identifying intraocular calcium in patients with opaque media.

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Other Tests

Immunohistopathologic staining

The aim of immunohistochemical studies is to decide whether retinoblastomas come from a common progenitor cell capable of differentiation into either glial or neuronal cells or from neuron-committed cells.

Numerous variables alter the results in these studies. These variables include tissue fixation, staining procedures, specific areas taken into consideration, tumor cell differentiation, antigen expressivity, and age of tumor.

Caution is required when interpreting most immunohistochemical results because of the related controversies associated with these tests. An experienced immunopathologist is required to provide worthwhile results.

Immunohistochemical and biochemical studies show an S-antigen detected in well-differentiated retinoblastomas using immunoperoxidase staining of paraffin sections. Felberg and Donoso have performed several related studies. [15]

Bridges and colleagues performed biochemical assays and showed interphotoreceptor retinoid-binding protein (IRBP) in retinoblastoma. These findings suggested an embryonic origin of the cells.

Numerous contradictory studies providing evidence for a neuronal nature and differentiation exist.

Transmission electron microscopy

Ultrastructural investigations have paved the way for more definitive descriptions of retinoblastoma. Research using this technology provided evidence of the presence of photoreceptor cell elements in retinoblastoma, and a strong evidence of retinoblastoma to human fetal retina has been demonstrated.

The ultrastructural findings of retinoblastoma investigations have been described previously.

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Procedures

Patients noted to have presenting signs of retinoblastoma should undergo prompt office examination.

Complete eye examination should be performed including an estimation of the patient's visual acuity for both eyes.

A dilated fundus examination with indirect ophthalmoscopy should be completed since ancillary diagnostic studies play only a secondary role when the fundus can be visualized clearly.

Bone marrow aspiration and biopsy

A bone marrow aspiration and biopsy could be performed as well as lumbar puncture with cytocentrifuge examination for tumor cells. These may prove useful in the early diagnosis of distant spread since the primary mode of spread of retinoblastoma is hematogenous to the bone marrow and back through the optic nerve into the cerebrospinal fluid (CSF).

Results of a study by Moscinski et al recommends performing bone marrow and CSF evaluations only in patients with clinical, histologic, or radiologic evidence of local or systemic extension or in patients presenting with 1 R-E group V eye with retrolaminar or extrascleral extension of their tumor. They also recommend limiting follow-up bone marrow and CSF determinations to those patients who develop objective signs and symptoms of metastasis or recurrence. [16]

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Histologic Findings

The classic histologic findings of retinoblastoma are Flexner-Wintersteiner rosettes (shown in the image below) and less commonly fleurettes.

Flexner-Wintersteiner rosettes in retinoblastoma Flexner-Wintersteiner rosettes in retinoblastoma

A Homer-Wright rosette can be encountered, but they are also seen in other neuroblastic tumors.

Considerable variability exists in the histologic features. Some neoplasms display marked necrosis and prominent foci of calcification. Few show areas of glial differentiation.

Note: In an enucleated eye that is being prepared for gross examination and fixation for histopathologic examination, it is essential that adequate fixation is attained (fixation is usually complete within 48 h). Thorough fixation is especially important for eyes removed for retinoblastoma because the tumor is friable and may be spilled into the uvea or outside of the eye when the eye is sectioned, thereby confusing the assessment of the confinement of tumor to the interior of the eye (a feature that is important for the assessment of survival).

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Staging

The International Classification for Intraocular Retinoblastoma is the newer retinoblastoma staging system. In this staging system, intraocular retinoblastoma is differentiated into 5 groups, from A to E. A indicates a better prognosis, and E indicates a poorer prognosis using existing treatment modalities. Staging is as follows:

  • Group A: Small tumors (≤3 mm in diameter) that are only in the retina and are not near important structures such as the optic disc (where the optic nerve enters the retina) or the foveola (the center of vision)
  • Group B: All other tumors (≥3 mm in diameter or small but close to the optic disc or foveola) that are still only in the retina
  • Group C: Well-defined tumors with small amounts of spread under the retina (subretinal seeding) or into the jellylike material that fills the eye (vitreous seeding)
  • Group D: Large or poorly defined tumors with widespread vitreous or subretinal seeding; the retina may have become detached from the back of the eye
  • Group E: The tumor is very large, extends near the front of the eye, is bleeding or causing glaucoma (high pressure inside the eye), or has other features indicating almost no likelihood that the eye can be salvaged

Prior to the International Classification for Intraocular Retinoblastoma, the Reese-Ellsworth classification system (see image below) was the most useful system when external beam radiation therapy (EBRT) was the standard of treatment for eye salvage. However, now that chemotherapy has supplanted radiation, this classification system is not as predictive of outcome and survival.

Reese-Ellsworth classification of retinoblastoma Reese-Ellsworth classification of retinoblastoma
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