PATHOLOGY LAB III-RETINOBLASTOMADr. Santo

 CASE OUTLINE.Case Presentation: Tom S.DefinitionEpidemiologyGenetics of retinoblastomaOther genetic events in retinoblastomaPathogenesisClinical presentationGrowth pattern  Pathologic features of retinoblastomaDissemination pathwaysRisk factors for metastatic diseaseSecond nonocular primary tumorsRetinocytoma OBJECTIVES.After completing this module the student will have a clear understanding of the origins, nature, and clinical features of retinoblastoma and retinocytoma. REFERENCES.1. Poulaki V, Mukai S.  Retinoblastoma: genetics and pathology. Int Ophthalmol Clin. 2009 Winter; 49(1):155-64.2. Balmer A, Zografos L, Munier F. Diagnosis and current management of retinoblastoma. Oncogene. 2006 Aug 28; 25(38):5341-9.3. Robbins and Cotran pathologic Basis of Disease, 8th edition, 2010. CASE PRESENTATION: TOM S.This 5-month-old boy was referred to Wills Eye Institute for the treatment of left eyelid swelling and leukocoria. His left eyelid was swollen and could not be opened by himself. The conjunctiva was also swollen, and the cornea was clouded because of increased intraocular pressure. Orbital CT scan (Figure 1) demonstrated a left intraocular tumor with calcification, periocular high intensity area (anterior orbital cellulitis) and deformed eyeball. The intraocular tumor showed low signal intensity on T2-weighted image. His right eye had three small retinal tumors, and the clinical diagnosis was bilateral retinoblastoma. His left eye was enucleated, and the pathological examination revealed retinoblastoma with calcification (Figure 2), extensive necrosis, and extraocular extension. He underwent no systemic chemotherapy, but only selective ophthalmic arterial injection for the right eye. 

  DEFINITION.Retinoblastoma represents the prototypic model for inherited cancers. The Rb1 was the first tumor-suppressor gene to be identified, leading to the discovery of a whole new class of antioncogenes and greatly contributing to advances in the management of solid tumors in children. Although a rare disease, retinoblastoma is the most frequent primary eye cancer in children under 15 years old. It may also be simulated by other lesions. Retinoblastoma is almost exclusively a disease of childhood, has even been detected in the fetus but can also occur in exceptional cases, in young adults. Diagnosis is often late, in spite of clear warning signs. This malignant tumor is now considered curable. With the heritable forms, however, there remains a major risk of second nonocular primary tumors, these even more lethal than the retinoblastoma itself.

Predisposition to malignant transformation may lead to a primary intracranial neuroblastic tumor, in particular pinealoma (‘trilateral’ retinoblastoma). Early treatment of the disease is straightforward, well tolerated, effective and at little cost, saving both life and vision.  EPIDEMIOLOGY.Retinoblastoma is a malignant tumor of the immature retina, the result of a double (‘two-hit’) oncogenic mutation occurring between the start of the third month postconception and the age of 4 years, this period representing the window of apparition and final maturity of retinoblasts. The cell of origin is most probably either a precursor cone photoreceptor cell or a multipotent retinoblast. The disease may thus occur in utero and up to the age of 4 years, the average age at appearance of first signs being 13 months for bilateral cases and 24 months for unilateral cases.Retinoblastoma may be nonheritable (60%) or heritable (40%), the latter by autosomal-dominant transmission with incomplete penetrance.The incidence of retinoblastoma is given in the literature as 1 in 20,000 – 30,000 live births with no gender or race predilection and no significant environmental or socio-economic factors. Although rare, retinoblastoma represents 80% of all primary ocular cancers in children up to 15 years old, 1% of all cancer-related deaths and rates third highest of all intraocular tumors in all ages (the most common tumor is choroidal melanoma which occurs in patients at an average of 55).  GENETICS OF RETINOBLASTOMA.The pivotal genetic event in all retinoblastoma tumors is the inactivation, due to mutations or deletions, of both copies of Rb1 at the chromosomal locus 13q14. This was first predicted by Knudson (two-hit hypothesis), who first generated the concept of tumor suppressor genes. Rb1 protein is a regulator at the cell-cycle checkpoint between G1 and entry into the S-phase. 1. Hereditary Retinoblastoma. Rb1 is the prototypical tumor-suppressor gene. In hereditary cases of retinoblastoma (40% of cases), one allele has already been inactivated in the germline (usually inherited from a parent) and every cell of the body is primed to enter the carcinogenesis process as soon as the other allele is lost. If the loss occurs in a retinal cell, the carcinogenesis process will follow the direction that leads to retinoblastoma, although other cells in the body can give rise to malignancies in the absence of Rb1 function, such as osteosarcoma. These patients are prone to additional primary retinoblastoma in the same or contralateral eye. Approximately 90% of individuals with a germline Rb1 mutation will develop at least 1 retinoblastoma and most patients develop 3 to 4 tumors. Secondary nonocular malignancies are observed in survivors of germline retinoblastoma at a rate of 1%per year of life and include osteogenic sarcomas of the skull and long bones, soft tissue sarcomas, pinealomas (pinealoblastomas), cutaneous melanomas, brain tumors, Hodgkin disease, lung cancer, and breast cancer. It should be noted that exposure to radiation, including treatment for retinoblastoma with external-beam radiation therapy, contributes to the increased incidence of secondary malignancies in the irradiated area constituting a significant drawback for this treatment modality. Patients carrying germline Rb1 mutations are also more sensitive to other carcinogens, such as chemotherapy and sunlight.In cases where the first hit occurred as a new genetic event in a gamete or in the early stages of embryogenesis (leading to various degrees of mosaicism), the patient is still vulnerable to multiple primary malignancies, including bilateral retinoblastomas, but without the existence of previous family history. If such a case is unilateral, it can be mistaken to be a case of nonhereditary retinoblastoma. Interestingly, when the new Rb1 mutation is a germline mutation, it is more frequently derived from the father, suggesting that new germline Rb1 mutations arise primarily during spermatogenesis.

2. Nonhereditary retinoblastoma. Truly nonhereditary retinoblastoma (60% of cases) is unilateral and unifocal and occurs when the same cell in the developing retina sequentially loses both Rb1 functional alleles. This scenario occurs in 1 in approximately 25,000 infants. As both Rb1 mutations are somatic, family members are not at risk, and the risk of second malignancy is not increased. Although radiotherapy and cytotoxic chemotherapy can still have some, albeit smaller, carcinogenic effect, this is solely from the treatment and not from a genetic predisposition.  OTHER GENETIC EVENTS IN RETINOBLASTOMA.Some patients with germline Rb1 mutations have been found to form retinocytomas. The incidence of retinocytoma is 1% of that of retinoblastoma. Retinocytomas are retinal tumors that seem to represent an intermediate step in the carcinogenesis process although it is controversial whether they are truly benign tumors or represent spontaneously regressed retinoblastomas. Although they have lost both functional Rb1 alleles, they require additional event(s) to achieve a fully malignant phenotype. Malignant transformation of retinocytomas toretinoblastomas can occur, and retinocytoma patients and families require the same clinical investigation and follow-up as retinoblastoma families. Retinocytomas have both Rb1 alleles mutated, but do not show proliferation, aneuploidy or gene expression patterns characteristic of retinoblastoma, suggesting that additional genetic events are necessary to convert a RB1– / – cell to a retinoblastoma cell. The senescence marker, p16, and the pRb family member, p130, are expressed in retinocytoma but not in retinoblastoma.Retinoblastomas are usually near diploid or hyperdiploid, but occasionally are triploid or tetraploid. Karyotypically normal tumors are rare. Multifocal tumors display quite different karyotypes, but only a single clone is evident in tumors of nonheritable unilateral patients.Chromosomal gains of 1q, 2p, 6p, 12q, and 13q and loss of 16q and 17p have been reported in retinoblastomas, suggesting that candidate oncogenes and tumor-suppressor genes lie within these regions. The overexpression of Mdm2 and Mdm4, which could lead to p53 degradation may explain how the p53 pathway is inactivated in human retinoblastoma by a genetic lesion not involving the p53 locus. Mdm2 protein functions as an ubiquitin ligase that recognizes p53 tumor suppressor protein targeting both itself and p53 for degradation by the proteasome.

PATHOGENESISMost cells comprising the tumor histologically resemble the cells of an undifferentiated retina of the embryo called retinoblasts. This resemblance prompted Verhoeff to coin the term “retinoblastoma”.In 1970, Tso established that the tumor arises from photoreceptor precursors. The most widely held concept of histogenesis of retinoblastoma is that it generally arises from a multipotential precursor cell that could develop in any type of inner or outer retinal cell. CLINICAL PRESENTATION.Leukocoria is the most common presenting sign in patients with retinoblastoma (60%). The second major presenting sign is strabismus (20%), this usually the result of macular involvement. Leukocoria is a white pupillary reflex.  The pupil appears white because of the light reflecting off of the surface of the tumor. The remaining 20% present atypical signs, often inflammatory, such as red painful eye secondary to iris invasion, and orbital cellulitis secondary to orbital invasion. These usually late signs are associated with a much poorer survival and globe salvage prognosis. 

  GROWTH PATTERN.

The clinical presentation of retinoblastoma depends on the tumor growth pattern, duration, degree of vascularization and the presence of calcifications, vitreous seeding, retinal detachment or hemorrhage. Tumors may show an endophytic growth pattern, resulting from cell division within the internal retinal layers and tumor growth towards the vitreous.

Exophytic retinoblastoma presents with retinal detachment. The tumor arises in the internal retinal layers, grows beneath the retina in the subretinal space and causes an overlying retinal detachment.

Endophytic retinoblastoma presents as one or several tumors, isolated or coalesced, round or oval-shaped, yellowish-white in color (calcifications) or pink (vascularization). Tumor localization is statistically age-dependent, the earliest tumors developing in the posterior pole, more peripheral tumors developing later. Endophytic tumors show a marked tendency to vitreous seeding.

A mixed growth represents the association of both endophytic and exophytic patterns. Diffuse infiltrating retinoblastoma is very rare (2%). This growth pattern forms a flat

plaque on the surface of, or beneath, the retina, with no obvious mass and no calcifications, progressing slowly towards the anterior segment.

Finally, retinocytoma is a benign form of retinoblastoma, characterized by greyish, homogenous, translucent masses, with calcifications and border pigmentation imitating an irradiated retinoblastoma. Importantly, this benign form of retinoblastoma is nevertheless susceptible to malignant transformation at a later stage.

 PATHOLOGIC FEATURES OF RETINOBLASTOMA.

(A) Gross appearance. Macroscopically, retinoblastomas are soft and friable tumors. On gross section, they appear as a cream-colored mass arising from the retina, containing chalky-white calcified flecks scattered within necrotic zones. The calcifications may be detectable on imaging studies.

(B) Microscopic appearance. Microscopically, undifferentiated retinoblastoma is composed of immature retinoblasts (neuroblasts): small, round or polygonal, densely packed cells with large, hyperchromatic nuclei and scant cytoplasm. Retinoblastomas are characterized by marked cell proliferation as evidenced by generally high mitosis counts. Degenerative changes are frequent in retinoblastomas as the tumor cells outgrow its blood supply, accompanied by necrosis andcalcification. Cell necrosis can be so intense that the released DNA can accumulate

extracellularly in the walls of blood vessels, and the lens capsule, giving them a bluish tinge on H&E staining. As oxygen can diffuse to a distance of approximately 100 µm from the vascular tree, viable cells are found in sleeves surrounding the vessels. The classic histopathologic description of retinoblastoma is “islands of blue cells in a sea of pink necrosis”.

Retinoblastomas exhibit various degrees of photoreceptor differentiation that lead to characteristic architecture of tumor cells. The Flexner-Wintersteiner rosettes are specific for retinoblastoma and are seen in 70% of tumors. They are clusters of cuboidal or short columnar cells arranged around a central lumen with the nuclei displaced away from the lumen. They are considered to be the result of an attempt of the tumor to form photoreceptor cells. They should not be confused with the perivascular cuff of tumor cells that are often referred as ‘‘pseudorosettes.’’ In the Flexner-Wintersteiner rosettes, the cell cytoplasm shows proliferation of long mitochondria and microtubules resembling cone inner segments. Fleurettes, flower-like groupings of tumor cells, may represent a more advanced recapitulation of retinal epithelium with photoreceptor differentiation, and are more characteristic of well-differentiated tumors. The Homer-Wright rosettes are not specific to retinoblastoma and are seen frequently in other neuroblastic tumors such as neuroblastoma and medulloblastoma.

 

 

  DISSEMINATION PATHWAYS.The poorly cohesive high-index mitotic retinoblastoma cells have a great natural tendency to infiltrate the most readily accessible spaces, these being the vitreous, subretinal fluid, anterior segment and meningeal sheaths of the optic nerve. Retinoblastoma can thus develop in three directions:

·         Anteriorly via the vitreous or subretinal space to the anterior segment, reaching the ciliary body and anterior chamber and creating a pseudohypopyon.

·         Posteriorly, step by step towards the optic nerve and cerebrospinal fluid, usual gateway to endocranial dissemination.

·         Extraocular extension to the orbit via the choroid and sclera.Past the barrier of the lamina cribrosa, infiltration may continue via the retrolaminal portion of the optic nerve to the subarachnoid space and further, transported by the cerebrospinal fluid, to the various cerebral structures.  

  Metastatic dissemination may also occurs via the vascular pathway (orbit, optic nerve) or lymphatic pathway (conjunctiva, eyelids).The tumor may also invade the orbit directly through the sclera or by following the orbital canals of the ciliary arteries and nerves. RISK FACTORS FOR METASTATIC DISEASE.The greatest risk factors for metastatic disease are optic nerve invasion and orbital invasion. The risk of metastasis through optic nerve invasion depends on the stage of tumor penetration. The risk is insignificant if the tumor is anterior to the lamina cribrosa. The mortality rate is 50–85% if tumor cells have reached the surgical resection line. Choroidal invasion is not considered a significant risk factor. Studies have shown that isolated choroidal invasion of retinoblastoma does not statistically increase the risk for metastases. Orbital extension of retinoblastoma can lead to systemic dissemination via the blood vessels, lymphatic system or along the visual pathway to the brain. Orbital invasion is considered an important risk factor. SECOND NON-OCULAR PRIMARY TUMORS.The Rb1 mutation present in all cells of a patient with retinoblastoma predisposes them to developing other nonocular malignant tumors, albeit with a predilection for certain tissues.Second primary tumors are the greatest cause of death in these patients, rather than the retinoblastoma itself. The cumulative incidence of these additional tumors is around 1% per year, thus 50% after 50 years’ evolution. Radiotherapy greatly increases the risk, particularly if administered before the age of 1 year. The most common sites of second nonocular tumors are, in order of decreased frequency: soft head tissue i.e. in the irradiated field (24%), bone (18%), skin (15%), and brain (8%). RETINOCYTOMA.Retinocytomas are rare benign tumors that are composed entirely of benign appearing cells with numerous fleurettes and show no evidence of necrosis or mitotic activity.The median age at diagnosis is 15 years (range, 4 - 45 years).It presents as a small retinal mass. Histologically there are masses of retinal cells with low nuclear-to-cytoplasmic ratio, bland nuclear characteristics, and photoreceptor differentiation with fleurette formation. Mitoses and necrosis are characteristically absent.

Ophthalmoscopic features include the presence of a translucent retinal mass, with calcifications and associated retinal pigment epithelial alterations.From a genetic standpoint, retinocytoma is similar to retinoblastoma, with autosomal dominant inheritance involving a mutation in the Rb1 gene locus on chromosome 13q14. In familial cases, various members of a kindred can manifest either retinocytoma or retinoblastoma or even a combination of both between the 2 eyes.Some studies suggest that retinocytoma lesions can reactivate and undergo malignant transformation into retinoblastoma.  

1. A 6-month-old female child is referred to your hospital for white reflex in both eyes for 5 months. The mother first noticed white reflex on the right eye when the child was one month old and since then it has been progressively increasing to have involved the left eye within the span of 3 to 4 months. Review of systems is negative. An ophthalmologic examination, slight proptosis is noted in both eyes with eyeball and cornea appearing relatively enlarged. Yellowish-white opacity is seen on both the eyes behind the cornea. On CT scan of the orbits, a solid retinal tumor that involved the vitreous on both sides is seen. There are no signs of extraglobe extension. The patient has both eyes removed. Histopathologic examination reveals a retinal tumor composed of malignant cells with large hyperchromatic nuclei and numerous mitotic figures. Which of the following histologic structures is most characteristic of the ocular malignancy in this patient? Horn pearl formationNumerous psammoma bodiesPalissadingFlexner-Wintersteiner rosettesAccumulations of dark pigment in the cytoplasm

2. A 6-month-old female child is referred to your hospital for white reflex in both eyes for 5 months. The mother first noticed white reflex on the right eye when the child was one month old and since then it has been progressively increasing to have involved the left eye within the span of 3 to 4 months. Review of systems is negative. An ophthalmologic examination, slight proptosis is noted in both eyes with eyeball and cornea appearing relatively enlarged. Yellowish-white opacity is seen on both the eyes behind the cornea. On CT scan of the orbits, a solid retinal tumor that involved the vitreous on both sides is seen. There are no signs of extraglobe extension. The patient has both eyes removed. Histopathologic examination reveals a retinal tumor composed of malignant cells with large hyperchromatic nuclei and numerous mitotic figures. Molecular analysis of the neoplastic cells will most likely reveal abnormalities in which of the following genes?

Rbp53MycRasHer2/neu

3. A 6-month-old female child is referred to your hospital for white reflex in both eyes for 5 months. The mother first noticed white reflex on the right eye when the child was one month old and since then it has been progressively increasing to have involved the left eye within the span of 3 to 4 months. Review of systems is negative. An ophthalmologic examination, slight proptosis is noted in both eyes with eyeball and cornea appearing relatively enlarged. Yellowish-white opacity is seen on both the eyes behind the cornea. On CT scan of the orbits, a solid retinal tumor that involved the vitreous on both sides is seen. There are no signs of extraglobe extension. Which of the following is the most likely diagnosis? MeningiomaRetinoblastomaBasal cell carcinomaMelanomaSquamous cell carcinoma