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.
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. Intracranial extension on CT scan.
Patient with retinoblastoma, glaucomatous stage. Another CT scan slice, showing the intracranial extension of the tumor.
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.
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.
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]
Histologic Findings
The classic histologic findings of retinoblastoma are Flexner-Wintersteiner rosettes (shown in the image below) and less commonly fleurettes.
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).
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:
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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)
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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
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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)
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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
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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.
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Classic histologic finding of retinoblastoma (Flexner-Wintersteiner rosettes)
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Retinoblastoma, intraocular stage (leukocoria). History: NB, 1-year-old male from Quezon Province, Philippines, with chief complaint of opacity, left eye. Born full-term spontaneous vaginal delivery (FTSVD) to a 27-year-old gravida 3, para 2 (2002) at home. Four months prior to admission (PTA), opacity was noted in the left eye (no consultation/medications). Five days PTA, consultation with an ophthalmologist. Examination: (+) leukocoria with visual acuity of central, steady, and maintained fixation on right eye, (-) dazzle on left eye; (+) Marcus Gunn (MG) reflex. Diagnostics: Ocular ultrasound was performed, revealing intraocular retinoblastoma. Management: Patient underwent enucleation of left eye. Examination under anesthesia of right eye: E/N retina. Histopathology: Retinoblastoma, intraocular stage left eye.
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Retinoblastoma, glaucomatous stage. History: AB, 2-year-old female from Marikina City, Philippines, with chief complaint of proptosis, right eye. The patient is an adopted child. Prior to admission (PTA), with child aged 6 months (time of adoption), surrogate mother noted an opacity in the right eye. No medical consultation. One year PTA, physician consultation; told AB had an "eye mass" and needed to see an ophthalmologist. No compliance. One month PTA, proptosis was noted in the right eye. Examination: Visual acuity (VA) of right eye is no light perception; VA of left eye is central, steady, and maintained fixation. Sensorium: Awake but irritable. Diagnostics: Intracranial extension on CT scan. Skeletal survey: E/N. Management: The patient underwent exenteration (right side).
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Patient with retinoblastoma, glaucomatous stage. Intracranial extension on CT scan.
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Patient with retinoblastoma, glaucomatous stage. Another CT scan slice, showing the intracranial extension of the tumor.
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Retinoblastoma, extraocular stage (neglected with necrosis). History: RC, 2-year-old male with chief complaint of left orbital mass. Born full-term spontaneous vaginal delivery (FTSVD) to a gravida 3, para 2 (2001) at home. Three months prior to admission (PTA), an inward deviation of the left eye was noted. No consultation. Six months PTA, opacity in the left eye was noted. Five months PTA, proptosis of the left eye with pain and bleeding was noted. Family/Social History: Indigent family. Youngest of 3 siblings; eldest sibling had no retinoblastoma; second sibling had retinoblastoma and underwent enucleation, dying after 2 sessions of chemotherapy. A cousin passed away with retinoblastoma. Examination: Indirect ophthalmoscopy of right eye revealed a large intraocular mass occupying the inferior half of the retina. Mass on left side. Management: The patient was scheduled for exenteration, left side. The mother and child went home against medical advice; what happened to the patient is not known.
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Status post (S/P) enucleation for retinoblastoma, right eye retinoblastoma, recurrence, right eye. History: IJ, 3-year-old male with chief complaint of right orbital mass. At age 2 months, opacity in right eye is noted. Five months prior to admission (PTA), consultation with an ophthalmologist for proptosis, right eye. Four months PTA, the patient underwent enucleation, right eye, with no alleged tumor involvement of the tumor resection margins on histopathology. One month PTA, gradually enlarging orbital mass, right side, was noted. Examination: Visual acuity right eye, not applicable (S/P enucleation); visual acuity left eye, at least 6/12 (20/40). No masses are seen in left eye on indirect ophthalmoscopy. Diagnostics: Skeletal survey showed lytic lesions on the humerus, femur, and pubic bones.
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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.
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Flexner-Wintersteiner rosettes in retinoblastoma
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Presenting signs or symptoms in retinoblastoma. (This table is modified from Abramson DH, Frank CM, Susman M, et al. Presenting signs of retinoblastoma. J Pediatr 1998 Mar; 132(3 Pt 1): 505-8.)
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Reese-Ellsworth classification of retinoblastoma
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Classic regression patterns of retinoblastoma
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Genetic counseling for retinoblastoma. (This table is modified from Vogel F. Genetics of retinoblastoma. Hum Genet 1979; 52:1.)
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Vitreous seeding (intraocular retinoblastoma). Courtesy of Manolette Roque, MD, Ophthalmic Consultants Philippines Co, EYE REPUBLIC Ophthalmology Clinic.
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Reese-Ellsworth Stage V: vitreous seeding. Courtesy of Manolette Roque, MD, Ophthalmic Consultants Philippines Co, EYE REPUBLIC Ophthalmology Clinic.
