Fig. 13.3 Same patient as in Fig. 13.1. Fluorescein angiogram demonstrated multiple areas of hyperfluorescence in both eyes. (a, b) Arteriovenous phase demonstrating multiple areas of disturbances at the level of the retinal pigment

epithelium (RPE). Deposits of older tumor cells that are either dead or sick absorb fluorescein dye, causing window defects of the RPE. A hot optic disc is seen in Fig. 13.3a (Courtesy of Raul Vianna, M.D.)

Papillitis is sometimes observed (Fig. 13.3a, b) [7]. It may be due to direct invasion of the optic disk by tumor or secondary to increased intracranial pressure related to the cerebral neoplasm [7]. Very rarely the intraocular tumor may invade the orbit [28].

Imaging

Ancillary ocular imaging modalities like fluorescein angiography (FA), optical coherence tomography (OCT), and indocyanine green angiography (ICGA) are useful adjuncts in raising the level of suspicion so that a diagnosis of PVRL is made promptly. A study from the National Eye Institute showed that despite the presence of vitritis in most patients, the FA did not show hallmarks of intraocular inflammation in most eyes [34]. CME was present in 19% and perivascular staining or leakage in 6% of eyes. Furthermore, 83% of eyes with CME had a prior history of intraocular surgery [34]. Since PVRL is often confused with a uveitic process, it is remarkable that the FA is not suggestive of an inflammatory process. The most common FA findings consist of disturbances at the level of the RPE [34]. PVRL cells are often confined to the sub-RPE space, and their viability will determine the FA findings [21, 34]. Deposits of older tumor

cells that are either dead or sick absorb fluorescein dye causing window defects of the RPE (see Fig. 13.3).

Alternatively, window defects might represent areas of tumor resolution with secondary RPE atrophic changes. In contrast, healthy tumor cells will not absorb fluorescein and will manifest as focal areas of blocked fluorescence [32, 34]. In some cases, FA detected lesions that were not seen ophthalmoscopically [30, 32, 34]. On the other hand, some eyes with vitritis will have a normal FA study [34].

Fardeau et al. [35] compared the FA, OCT, and ICGA findings among patients with PVRL, infectious uveitis, metastatic tumors, and immunemediated uveitis. Their most significant findings were clusters of small round hypofluorescent lesions 50–250 m(mu)m in diameter seen in both the early and late phases of the FA. These were identified in 45% of patients with PVRL and in only 2% of patients without PVRL. These hypofluorescent lesions corresponded to punctate white lesions seen in the fundus. Similarly hypofluorescent lesions that tended to fade in the later phase of the study were seen with ICGA in 26% of PVRL patients and 8% of non-PVRL patients. Nodular hyperreflective lesions at the level of the RPE were observed in the OCT of 42% of patients with PVRL compared to 15% of patients without PVRL. The combination of these

three imaging modalities yielded a positive predictive value of 88.9% and a negative predictive value of 85% for diagnosing PVRL [35].

Some investigators have suggested using ophthalmic echography as an adjunctive study in patients with PIOL [36]. In a series of 13 patients

Fig. 13.4 Echographic examination of a patient with primary vitreoretinal lymphoma (PVRL) demonstrating vitreous debris

with PVRL, all the patients manifested some ultrasonographic abnormality. The most common findings were vitreous debris (77%) (Fig. 13.4), choroidal scleral thickening (46%) (Fig. 13.5), and widening of the optic nerve (31%). None of these findings are specific for PVRL but in the proper clinical context might provide sufficient evidence to consider it seriously.

Fundus autofluorescence (FAF) is a relatively new ophthalmic imaging modality that utilizes the fluorescent properties of lipofuscin to study the health of the RPE and photoreceptor complex. There has been recent interest in studying the FAF of several chorioretinal diseases such as central serous chorioretinopathy, non-exudative age-related macular degeneration, and Stargardt’s disease, among others [37]. Ishida and colleagues [38] have recently published their FAF findings in five eyes with PVRL. They reported that the FAF patterns were diverse but distinctive according to the individual funduscopic findings. Sub-RPE tumors were generally

Fig. 13.5 Same patient as in Figs. 13.1 and 13.3. A- and B-scan ultrasound shows an irregular internal reflectivity and no internal vascularity. No extensive serous retinal

detachment is usually seen with this type of tumor (Courtesy of Raul Vianna, M.D.)

weakly hyperfluorescent on FAF. The hyperpigmented mottling of the RPE overlying the subRPE tumors was very hyperfluorescent on FAF. In eyes with tumor infiltration into the retina causing whitening of the retina, the FAF pattern was that of hypofluorescence. Spontaneous resolution of the sub-RPE tumors usually leaves an atrophic area of RPE that appears hypofluorescent on FAF [38].

Novel ophthalmic imaging techniques including magnetic resonance spectroscopy, magnetic resonance imaging, and novel positron emission tomography agents with the ability to detect lymphoma cells between the RPE and Bruch’s membrane may be in the horizon [39]. Malignant B lymphoma cells can be differentiated from normal and activated T-cell populations from as few as 8 cells by their intrinsic autofluorescence when excited with wavelengths of 351, 458, and 488 nm.

Diagnosis and Pathology

In patients over 50 years of age with posterior or pan-uveitis unresponsive to corticosteroid treatment, the possibility of the lymphoma must be considered [6, 7, 11, 15, 16, 20, 40]. Some cases have been reported where PVRL responds briskly to steroids, thus delaying the diagnosis [6, 25, 30]. If PVRL is suspected, fluorescein angiography, OCT, and a complete medical history and physical examination are conducted. Neurological work-up including neuroimaging and lumbar puncture is then performed. If lymphoma cells are isolated from the CSF, it is not necessary to pursue further diagnostic procedures. Some have clinically defined PVRL by the presence of ocular symptoms and signs in patients with known PCNSL [25]. However, as mentioned previously, during the autopsy of a patient with PCNSL and uveitis, the vitreous was clear of neoplastic cells [19]. Conversely, Zimmerman [41] pointed out that whenever a patient with PVRL showed neurological manifestations, it was implied that PCNSL was present. However, in his experience, that was not always the case. In 18 patients with histopathologically proven

PVRL, biopsies of the brain did not always confirm the presence of PCNSL. In nine cases of PVRL with neurological symptoms, PCNSL was confirmed; however, in six cases, CNS manifestations were secondary to other causes such as nocardiosis, hemorrhage, and Behcet’s disease [41]. If PCNSL is not evident, then one should proceed with a diagnostic vitrectomy. Just like in any type of malignancy, tissue diagnosis is a prerequisite for the initiation of therapy in PVRL.

Currently, diagnosis of PVRL is based primarily on cytological evaluation, immunohistochemistry, and molecular techniques on the tumor. The CSF and the vitreous are examined and processed in the same fashion [6].

A single vitreous biopsy may not always be diagnostic of PVRL [6, 25, 30, 33]. Steroids can hinder the diagnosis by clearing some malignant cells [6, 30]. They are known to shrink CNS lymphoma lesions [42]. Some have advocated discontinuing the steroids for a period of time before the specimens are obtained [6, 30]. In certain selected cases when vitreous cytological findings are equivocal and the suspicion for PVRL remains high, retinal biopsy, chorioretinal biopsy, full thickness eye wall biopsy, and aspiration of subretinal lesions may be considered [6, 40].

In patients with bilateral involvement, the eye with the worse visual acuity or the most prominent cellular infiltration in the vitritis is selected to undergo diagnostic vitrectomy. A very experienced cytopathologist is often needed to make the diagnosis since the yield from vitrectomy samples is often small [6, 30, 33]. Cytological analysis should be given precedence. An undiluted sample of vitreous (1–2 mL) is first obtained and immediately placed into 2–3 mL of cell culture medium such as RPMI (Associated Biomedic Systems, Inc., Buffalo, NY, USA) and then immediately transported to the cytology lab [6]. Rapid processing of the vitrectomy specimen is critical in maintaining the morphology and integrity of the cells [33]. The ocular pathologist then proceeds to isolate the cells by cytocentrifugation. Staining of the PVRL cells with Diff-Quick or Giemsa is superior to hematoxylin-eosin and Papanicolaou stains [6]. The supernatant is used for cytokine analysis and possibly viral polymerase

Fig. 13.6 Cytological examination of a vitrectomy specimen showing lymphoma cells with large round nuclei with indistinct cytoplasm

chain reaction (PCR) [6]. In cases where shipment of the vitrectomy specimen to different institutes is required, the material can be fixed in equal volume of 90% ethanol. Such fixed tissue is processed for cell block preparations followed by hematoxylin-eosin stain, immunophenotyping to determine clonality, and special stains for infectious agents. A core vitrectomy is completed, and the resulting diluted sample and cassette washings may be used for additional studies such as flow cytometry, immunohistochemistry, and molecular analysis [6].

Under light microscopy, tumor cells typically display large round, oval, or indented hyperchromatic nuclei with prominent eccentric eosinophilic nucleoli, mitotic figures, a coarse chromatin pattern, indistinct cytoplasm, pleomorphism, and occasionally finger-like projections from the nuclei (Fig. 13.6) [6, 13, 15, 16, 30, 33]. Reactive T lymphocytes, fibrin, cellular debris, and apoptotic and necrotic cells are often found in conjunction with the lymphoma cells, underscoring the importance of having an experienced cytopathologist [6]. Electron microscopic studies reveal that intranuclear inclusions, cytoplasmic crystalloids, pseudopodal cytoplasmic extensions, cytosomes with autophagic vacuoles, and electron-dense bundles may be seen in the intercellular space [43]. Cytological examination can

detect lymphoma cells, but it cannot differentiate between a B-cell and T-cell origin [6].

Histopathologic examination reveals that neoplastic cells are primarily seen in the vitreous, in the sub-RPE and subretinal spaces, and in the retina, optic nerve head, and rarely choroid [17, 19]. Involvement of the vitreous by neoplastic cells can produce effects similar to inflammation [7]. The vitreous can become condensed, liquefied, cloudy, and detached. A vitreous cellular infiltrate might be the only ocular manifestation of the disease. They form fluffy nondiscrete opacities. Occasionally, the anterior segment of the eye is involved. A chronic inflammatory infiltrate in the uveal tract has been described [16]. The choroid can be diffusely thickened and sometimes infiltrated with inflammatory cells primarily made up of reactive T cells, macrophages, and B lymphocytes [15, 21]. The RPE detachments from sub-RPE tumor cell deposits can evolve to RPE atrophy, photoreceptor layer atrophy, and disciform scars [32]. Tumor cells are typically arranged around blood vessels in the retina and/or brain. Extensive infiltration of the retina and optic nerve head may lead to coagulative necrosis [14, 16].

Most PVRLs are monoclonal B-cell lymphomas [3, 6–8]. A minority of cases are of T-cell origin and can simulate a reactive inflammatory process. Immunohistochemical stained sections or immunophenotyping by flow cytometry showing a monoclonal response of B-cell markers such as CD19, CD20, and CD22 with either a kappa or lambda light chain restricted expression help make the diagnosis in cases where cytopathology is equivocal [6, 30]. Flow cytometry can analyze multiple markers simultaneously. The number of cells required to perform cell typing was the limiting factor in studying immune characterization of PVRL by flow cytometry [6]. Furthermore, some B-cell lymphomas do not express surface markers, thus preventing its recognition by flow cytometry [6]. Immunohistochemistry for T-cell markers such as CD3 and/or PCR for T-cell receptor gene rearrangements can help identify PVRL of T-cell origin [6].

PCR analysis can demonstrate the monoclonal proliferation of B lymphocytes in the vitreous [6, 7].