Case 1 -

Left Eye, Enucleation - Retinoblastoma, Moderately Differentiated, Predominantly Exophytic with 40% Necrosis - Choroidal Invasion of 1.1 Mm Maximum Width and 0.1 Mm Maximum Thickness - Prelaminar Optic Nerve Head Invasion by Tumor to a Depth of 0.34 Mm from the Inner Limiting Membrane - Focal Posterior Synechiae - Total Retinal Detachment - Calcified Fibroglial Membrane Formation Over the Rpe Focally Patricia Chevez-Barrios The Methodist Hospital Houston, TX

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Clinical History This is an 8-month-old male with history of leukocoria (white pupil) of the left eye noted by the mother 2 months prior to enucleation. Patient was seen by pediatrician and then by general ophthalmologist whom refer the patient to an ocular oncologist. The right eye was unremarkable. No treatment prior to enucleation was performed. Case 1 - Figure 1 Exophytic retinoblastoma occupying the subretinal space with chacky white calcifications Case 1 - Figure 2 Histologic section of the PO segment, geographic areas of necrosis contain multiple calcifications Case 1 - Figure 3 Sections of the two calottes in breadloaf fashion to examin more choroidal cut surfaces Case 1 - Figure 4 Retinoblastoma tumor (on top) associated with a fribroglial calcified tissue over an area of choroidal invasion Case 1 - Figure 5 Higher magnification of the invasive tumor in the choroid that replaces and spands the choroidal stroma Case 1 - Figure 6 The tumor contains Flexner-Wintersteiner (with holowed center) and Homer Wrigth rossettes (center filled by cytoplasmic proliferation) Case 1 - Figure 7 Tumor invades the prelaminar optic nerve region Case 1 - Figure 8 Freshly enucleated eye with a partial scleral window created to harvest fresh tissue Introduction: Retinoblastoma is the most frequent intraocular malignancy in children accounting for approximately 300 new cases a year in the United States of America. The incidence varies by region of the world but exact incidences are not available in all countries. The tumor may affect one or both eyes and it is the results of mutations in the Rb1 gene (chromosome 13). Retinoblastoma is usually seen in children under 4 years of age. It is sporadic in about 70% of cases and hereditary (germline mutation) in 30% of cases. The implication of providing the genetic status to patients extends to the families. If patients have the germline mutation siblings and close relatives should be examined to exclude the disease. In contrast, if the germline mutation is not found the family has less than 2% of probability of carrying the mutation. The remaining small probability is related to current technical limitations in detecting certain mutations or in patients with mosaicsism . The tumor arises from the nuclear layers of the retina, expanding the retina and invading the vitreous and subretinal space. In developed countries, the control rate of retinoblastoma is currently high should the tumor remained within the intraocular tissues, however, if the tumor invades extraocular structures or massively invades the choroid the risk of metastasis is high. Current treatment for metastatic carcinoma is available but with variable success, especially if the tumor metastatizes to the CNS the prognosis is dismal. Hence, the importance of an adequate pathologic evaluation of the enucleated eye for histopathologic high risk factors for metastasis. If high risk factors are present, most centers would implement adjuvant chemotherapy. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: Macroscopic findings: The left eye was received immediately after enucleation. Received fresh was a firm left eye measuring 21.5mm AP x 20mm horizontally x 20mm vertically. It displayed an attached optic nerve measuring 14mm in length. The cross section of the margin was obtained and submitted in cassette ON. The cornea was transparent, measuring 12mm horizontally x 11mm vertically. The anterior chamber was visible, and appeared shallow, encompassing a dark brown iris with a round 4mm-diameter pupil. The sclera was unremarkable. On transillumination, the eye showed a shadow encompassing the majority of the superonasal globe. The eye was focally opened fresh using sterile techniques and under an stereoscopic microscope. Viable tumor was harvested and saved in 4 cryotubes (about 1.5mm2 each), snap frozen using a cryobath and stored at -70 degrees for future genetic studies. The eye was then placed in 10% formalin. After adequate fixation (>24 hrs), the eye is further opened horizontally, removing the superior calotte first. The cut surface showed that the anterior chamber was shallow, without synechiae. The lens was transparent. The vitreous was replaced largely by tumor, and the retina was totally detached. The tumor appeared to be primarily exophytic. There appeared to be a 1.5mm x 0.5mm choroidal/RPE lesion in the posterior temporal area. The macula and optic nerve were covered by tumor and not visible. Photographs were obtained. Sections were submitted as follows: PO - Pupil optic nerve section IC – Inferior calotte (bread loaf sections) SC – Superior calotte (bread loaf sections) ON – Optic nerve cross section of margin (obtained before fresh opening of the eye) Microscopic findings: Sections show a tumor arising from the retina and growing mostly exophytically into the vitreous, filling the subretinal space and focally into the small amount of vitreous. There is roughly 40% necrosis and multiple areas of calcification. The tumor is moderately differentiated, showing both Flexner-Wintersteiner and Homer-Wright rosette formation. Photoreceptor differentiation is not seen. The retina is totally detached. The tumor occupies most of the optic nerve head, extending 0.34 mm from the inner limiting membrane into the prelaminar optic nerve. The remainder of the optic nerve, including the surgical margin and meninges, is negative for tumor cells. There is focal choroidal invasion by the tumor (noticed in the IC sections) of 1.1mm maximum width and 0.1mm maximum thickness. There is also focal fibroglial membrane formation (IC sections) over the RPE posteriorly. The anterior chamber is unremarkable, and there is no neovascularization of the iris or cataractous changes in the lens. However, there is focal posterior synechiae formation between the iris and the anterior lens. The sclera is unremarkable. Final Diagnosis: Left eye, enucleation - Retinoblastoma, moderately differentiated, predominantly exophytic with 40% necrosis - Choroidal invasion of 1.1 mm maximum width and 0.1 mm maximum thickness - Prelaminar optic nerve head invasion by tumor to a depth of 0.34 mm from the inner limiting membrane - Focal posterior synechiae - Total retinal detachment - Calcified fibroglial membrane formation over the RPE focally Note: Results of genetic studies showed no germline mutation Differential Diagnoses: Clinical and histopathologic differential diagnoses include: Coat's disease (vascular abnormality of the retinal vessels accompanied by exudative retinal detachment simulating a mass) Toxocariasis (parasitic larva invades the uveal and retinal tissues with associated inflammatory (eosinophils) infiltrate, vitreous condensation that mimics a mass and retinal traction) Persistent Hyperplastic Primary Vitreous (PHPV)(vitreous vascularization with fibroglia membrane mimicking a mass, tractioning the retina and the ciliary processes) Medulloepithelioma (frequently a ciliary body tumor formed by neuroepithelial structures surrounded by hyaluronic acid and basement membrane material. Occasionally this tumor may contain undifferentiated malignant component sometimes indistinguishable from retinoblastoma) Final Diagnosis: Left eye, enucleation - Retinoblastoma, moderately differentiated, predominantly exophytic with 40% necrosis - Choroidal invasion of 1.1 mm maximum width and 0.1 mm maximum thickness - Prelaminar optic nerve head invasion by tumor to a depth of 0.34 mm from the inner limiting membrane - Focal posterior synechiae - Total retinal detachment - Calcified fibroglial membrane formation over the RPE focally Case Discussion: This case demostrates an exophytic retinoblastoma (Fig 1). Exophytic retinoblastoma grows from the retina into the subretinal space and often causes serous detachments of the retina. These tumors may invade the choroid through Bruch's membrane. This case shows focal choroidal invasion (< 3mm) (Fig 4,5)and prelaminar optic nerve invasion (Fig 7). The tumor is moderatelly differentiated as it contains moderate amount of rossettes (Fig 6). It contains 40% of tumor necrosis associated with dystrophic calcification (Fig 2). Microscopic examination of the eye with retinoblastoma reveals a tumor with large areas of necrosis and multifocal calcifications. The majority of the tumor is formed by small hyperchromatic cells with a high nuclear to cytoplasmic ratio, mitotically and apoptotically active.The viable cells surround blood vessels in a range of 90-110 micra forming a collarette (pseudorosettes) . Viability of the tumor cells depends on the intrinsic tumor blood supply. Histopathology shows many areas of coagulative necrosis contain multiple foci of dystrophic calcification which are a very helpful feature for making the clinical diagnosis of retinoblastoma. Ultrasonography, magnetic resonance imaging (MRI), and computed tomography (CT) of the orbit are the imaging studies most frequently used to confirm the diagnosis of retinoblastoma. CT is better for detecting intraocular calcification that could provide confirmation of retinoblastoma. However, in recent years, CT of young children has fallen into disfavor due to the long-term risks for radiation-induced second malignancies. Combination of ultrasound and MRI may help confirm the diagnosis in very young children. Some retinoblastomas show large areas of undifferentiated or poorly differentiated tumor while other tumors show a certain degree of differentiation represented by formation of rosettes. Flexner-Wintersteiner rosettes are highly characteristic of retinoblastoma although they are also seen in pinealoblastomas and medulloepitheliomas. Flexner- Wintersteiner rosettes are lined by tall cuboidal cells that circumscribe an apical lumen. The apical ends attach to each other by terminal bars and the cells may have apical cytoplasmic projections into the lumen of the rosette. Homer Wright rosettes are less common than Flexner- Wintersteiner rosettes and they are found in a variety of neuroblastic tumors in addition to retinoblastoma. These rosettes do not surround a lumen but rather extend cytoplasmic processes that fill the center of the rosette. About 5 -10% of tumors show benign photoreceptor differentiation into groups of cells with short cytoplasmic processes, abundant cytoplasm and small round nuclei similar to photoreceptors. These groups of cells which resemble a bouquet of flowers are called "fleurettes". Neither significant mitotic activity nor necrosis is observed within the fleurettes. The tumor in this case is moderately differentiated showing bothe types of rossettes (Fig 6). The tumor in this case is focally invading the optic nerve in the prelaminar region only (Fig 7). The most common route of spread is by invasion through the optic nerve. However, the size of the tumor is not always the defining factor for invasion as there are small tumors that invade the optic nerve. The tumor may invade the optic nerve head only and this is considered intraocular invasion and carries almost the same prognosis than no optic nerve invasion . Once in the nerve, the tumor tends to spread directly along the nerve fiber bundles towards the optic chiasm passing through the lamina cribrosa and into the retrolaminar area. This is now considered extraocular extension as the lamina cribrosa is the equivalent of the sclera. Patients with retrolaminar invasion carry worst prognosis for metastasis, and the prognosis is worse as the tumor infiltrates farther towards the chiasm and nearer the surgical margin. The tumor may also infiltrate through the pia into the subarachnoid space and from there into the brain and the spine. The tumor is invading focally (<3mm) into the choroid. The second major route of spread is through massive involvement of the choroid into the orbit via either scleral canals or by direct extension through the sclera. Extraocular extension generally occurs within six months if intraocular tumors are left untreated. The definition of focal versus massive choroidal invasion has been controversial and subjective. Recently, the Children's Oncology Group, and the International Retinoblastoma Staging Working Group have proposed an objective classification based on specific anatomic location and size of the tumor. Focal choroidal invasion is defined as any focus that is less than 3 mm in maximum diameter and does not reach the sclera. Massive choroidal invasion is any focus of tumor measuring 3 mm or more in maximum diameter and reaches the sclera. However, if the tumor is more than 3mm but does not reaches the sclera it is considered massive invasion. There are ongoing clinical prospective trials testing this definitions. Extraocular extension dramatically increases the chances of hematogenous and lymphatic spread. Metastatic disease is still associated with a poor prognosis. Most clinical findings are not useful in predicting the occurrence of metastasis in children with retinoblastoma. However, histopathologic features may provide a good evaluation Multivariate statistical analysis has suggested the correlation of certain histopathologic findings and prognostic risk factors. The most important prognostic indicators for the development of metastasis are the presence of tumor in the optic nerve posterior to the lamina cribrosa at the site of surgical transection and extrascleral extension of tumor into the orbit. The extent of tumor invasion in the optic nerve correlates with prognosis. Superficial invasion of the optic disc is associated with a mortality rate of 10%, a rate similar to that seen when the optic nerve is not involved. The presence of tumor up to the lamina cribrosa is associated with a mortality rate of 29%. Invasion of tumor posterior to the lamina cribrosa is associated with a mortality rate of 42%, while the presence of tumor at the transected surgical margin is associated with a mortality of 80%. Massive (>3mm), but not focal, invasion of the choroid by tumor increases the possibility for hematogenous spread, either through vascular permeation of choroidal vessels or more frequently by extension through the sclera into the orbital tissues. Review of the Literature/Treatment Options (if applicable): The current treatment options are still under analysis as there is controversy for the adjuvant treatment versus treatment of metastasis/recurrence if presented. However, the majority of centers would give adjuvant chemotherapy for patients with post-laminar optic nerve invasion with or without tumor at margin. If tumor is at margin or extraocular radiation therapy may also be implemented. Massive choroidal invasion may or maynot be adjuvantly treated. The Children's oncology group in its prospective trial treated patients with the concomitant optic nerve invasion (at any level including prelaminar) and any degree of choroidal invasion. Thus, this patient would fit this criteria and needed adjuvant therapy (3 agents, 6 cycles). Proposed guidelines to gross eyes with retinoblastoma: Harvesting fresh tumor tissue from unfixed globes for molecular studies Fresh tumor tissue for molecular genetic studies should be harvested from fresh unfixed globes immediately after enucleation. NOTE: A transverse segment of optic nerve containing the true surgical margin should be removed from the unfixed specimen and submitted separately prior to tumor harvesting to avoid contaminating the surgical margin with tumor. The margin should be marked with ink or colored pencil before sectioning the nerve. The fresh tumor is harvested through a window that is carefully made in the sclera and choroid (Fig 8). The scleral window is made with a sharp blade or scalpel under a stereoscopic microscope. The scleral window technique minimizes tissue distortion and preserves anatomic relationships, permitting good quality histopathologic sections. The incision should be made at a safe distance from the optic nerve. Manipulation and sectioning should be performed gently to avoid collapsing the eye and inadvertently expulsing the tumor tissue and other intraocular contents and creating artifacts that can interfere with histopathologic interpretation. Tumor tissue should be harvested by excising small samples using small scissors to avoid pulling the tumor. Traction of the tumor and other intraocular structures should be avoided to evade tissue distortion and loss of intraocular tissues. Viable tumor should be harvested and saved in 4 cryotubes (about 1.5 - 2mm2 each), snap frozen using a cryobath and stored at -70 degrees for future genetic studies. The specimen should be fixed immediately after tumor harvesting to maximize tissue preservation and avoid distortion of tissue architecture. The eye should be gently immersed in a container of 50-100 mL of 10% Formalin. The specimen should be carefully rotated to expel air bubbles that can cause floatation to the surface of the fixative resulting in inadequate fixation. At least 48 hours of fixation are recommended. Grossing the fixed eye Eyes should be carefully sectioned using a standard double- edge razor blade or half a blade. The two ends of the blade are held between the apposed surfaces of the thumb and forefinger. Removing the First Calotte During the initial cut (removal of the first calotte), the eye is placed corneal side-down on the cutting block and steadied with the non-dominant hand. The first incision is made under the opened calotte used for harvesting tumor, in the same plane of section but away from the opening and rather close to the optic nerve. Guidelines drawn on the eye with colored pencil facilitate sectioning and help to assure that the eye is oriented properly prior to sectioning. The incision is started posteriorly and extended anteriorly. At the start of the incision, the blade is positioned close to the optic nerve, but external to its dural sheath. A gentle sawing motion is used. Try not to bend the blade during sectioning. In addition, care must be taken to avoid compressing the eye too forcefully, as this can collapse the eye and express the tumor and intraocular contents. The cut should enter the periphery of the anterior chamber anteriorly. The first calotte should include about one-fifth to one-fourth of the peripheral anterior chamber . Stereomicroscopic Examination After the first calotte is removed, the eye should be examined with a dissecting microscope. If uveal invasion by tumor is noted or suspected grossly it should be documented. Removing the Second Calotte The second calotte is then removed. This is most easily done by placing the eye cut-surface down on the cutting block, and making a second parallel cut. The main pupil optic nerve (PO) segment is submitted in one cassette. Submission of Additional Tissue Segments from the Calottes to Rule-Out Uveal Invasion. It is extremely important to examine the residual tissue in the calottes to exclude uveal invasion that may not be evident grossly. Both calottes should be sectioned in a "breadloaf" fashion anterior-posteriorly and submitted entirely . One calotte per cassette (Fig 3). Conclusion(s): Histopathologic features for making the diagnosis of retinoblastoma include: high N:C ratio neuroblastic highly mitotically active tumor cells replacing retina and vitreous, geographic areas of necrosis, dystrophic calcifications, and Flexner-Wintersteiner and Homer Wright rossettes. Quantitative and qualitative characteristics of tumor invasion into choroid and optic nerve are important to evaluate high risk factors for metastasis High risk histopathologic features for metastasis include retrolaminar involvement of optic nerve, massive choroidal invasion (>3mm),tumor at cut margin of optic nerve and extraocular invasion Adequate handling (grossing) of the enucleated eye with retinoblastoma is essential for evaluation of prognostic factors and molecular/genomic studies Histopathologic examination of levels from 4 paraffin blocks is necessary to evaluate the entire eye (central portion (P.O.), 2 calottes, and optic nerve margin) References: Hurwitz RL, Chévez-Barrios P, Chintagumpala M, Shields C, Shields J (2002) Retinoblastoma. In: Pizzo PA, Poplack D (eds) Principles and practice of pediatric oncology, 4th edn. Lippincott-Raven, Philadelphia, pp 825–846 (4th 2002 and 5th ed 2006) Arrigg PG, Hedges TR III, Char DH. Computed tomography in the diagnosis of retinoblastoma. Br J Ophthalmol 1983;67:588–591. Mills DM, Tsai S, Meyer DR, et al. Pediatric ophthalmic computed tomographic scanning and associated cancer risk. Am J Ophthalmol 2006;142:1046–1053. Kopelman JE, McLean IW, Rosenberg SH. Multivariate analysis of risk factors for metastasis in retinoblastoma treated by enucleation. Ophthalmology 1987;94:371–377. Sastre, X; Chantada, GL; Doz, F; Wilson, MW; de Davila, MTG; Rodrıguez-Galindo,C; Chintagumpala, M; Chévez-Barrios P.; for the International Retinoblastoma Staging Working Group: Consensus Pathology Processing Guidelines For the Examination of Enucleated Eyes with Retinoblastoma. A Report from the International Retinoblastoma Staging Working Group. Arch Pathol Lab Med. 2009 Aug;133(8):1199- 202.