It can also demonstrate the bcl-2 oncogene indicating the presence of a t(14;18) translocation, an immunoglobulin heavy chain gene rearrangement indicating a B-cell lymphoma, and rearrangements of the T-cell receptor gamma gene indicating the presence of a T-cell lymphoma. The presence of multiple inflammatory cells found in conjunction with the lymphoma cells renders conventional PCR techniques less specific and sensitive. A novel way of obtaining material of higher purity for PCR has been described [6]. The histological slides are prepared in the usual manner. Under direct visualization from the light microscope, the cells of interest are identified and scraped from the slide with a 30-gauge needle or with laser capture. These are then placed in a single-step extraction buffer, which happens to be the starting point for PCR. PCR is then performed as usual. Demonstration of rearrangement of the IgG heavy chain confirms monoclonality [6].

Intravitreal interleukin-10 (IL-10) levels may help in the diagnosis of PVRL [6, 7, 44]. IL-10 is a growth and differentiation factor for B lymphocytes and plays a role in the growth of lymphocytic leukemia, AIDS, lymphoma, Burkitt’s lymphoma, and other NHL. Intravitreal IL-10 levels that correlated with the severity of the vitritis were detected in 3 patients with PVRL by enzyme-linked immunosorbent assay (ELISA) [44]. In this same study, intravitreal IL-10 was not detected in any other uveitic conditions such as sarcoid, acute retinal necrosis, and endophthalmitis. Cerebrospinal fluid (CSF) levels of IL-10 were suggestive of the presence of malignant cells. Intravitreal IL-6 levels were shown to be elevated in patients with intraocular inflammation unrelated to malignancies. Others have suggested using the ratio of IL-10 to IL-6 to control for the dilution factor involved in obtaining the vitreous sample from the vitrectomy cassette. An intravitreal IL-10/IL-6 >1 provides a 75% specificity and sensitive to distinguish PVRL from other conditions. Aqueous levels of IL-10 are also significantly high in PVRL [44]. Aqueous levels of IL-10 greater than 50 pg/mL give a sensitivity of 89% and a specificity of 93% for detecting PVRL. Similarly, intravitreal levels of IL-10 greater than 400 pg/mL yield a sensitivity of 80%

and a specificity of 99%. Since T cells do not secrete IL-10, low IL-10 levels may suggest a T-cell lymphoma [6]. However, cytopathologic findings combined with immunophenotyping are considered gold standard for the diagnosis of the lymphoma and subsequent treatment.

Treatment

Effective treatment of PVRL has been difficult to determine because of its rarity. Furthermore, the small number of patients receiving each therapy makes comparisons between studies difficult. The natural history of PVRL seems to be death from progression of CNS disease [14, 25]. In patients with PVRL, the interval between diagnosis of PCNSL and death was of 12 months [25]. Therefore, the goal of treatment should be the eradication of CNS and intraocular disease. The treatment protocol will depend on whether or not the patient has isolated PVRL or has concurrent CNS disease. Treating the ocular disease prior to CNS involvement probably improves survival [29]. However, the results of an international multicenter retrospective study were not able to confirm the benefits on survival of treating ocular lymphoma [45].

In order to better understand treatment of PVRL, an understanding of the unique characteristics of PCNSL is required. Unlike other brain tumors, complete resection of PCNSL has not been shown to improve survival over supportive care and therefore has no therapeutic role [42]. Likewise, removal of an eye involved by PVRL in an otherwise healthy person does not constitute a cure either [22]. The existence of the blood-brain barrier has been a limiting factor in the penetration of systemic chemotherapeutic agents into the CNS and eye. Although PCNSL disrupts the blood-brain barrier, disruption may be limited to areas of bulky tumor, whereas areas of microscopic disease may have a relatively well-preserved blood-brain barrier. Thus, chemotherapy protocols such as CHOP (cyclophosphamide, hydroxydaunomycin, oncovin, and prednisone) that are generally effective in systemic lymphoma are ineffective in PCNSL and PVRL [46, 47].

Untreated patients with PCNSL survive an average of 1.5 months. Whole brain radiation therapy was once the treatment of choice for PCNSL [42, 48]. In one prospective study, 41 patients with PCNSL underwent whole brain radiation therapy consisting of 40 Gy with a 20-Gy tumor boost. Despite the high response rate of 90%, 68% of patients relapsed and the median survival was only 11.6 months. Similarly, the mainstay of therapy for PVRL used to be irradiation to the CNS and/or eye [46, 49, 50]. The initial response was dramatic, but recurrences were typical. Shrinkage of the retinal and/or intracranial lesions was observed following radiation therapy. The radiation dose varied between 30 and 50 Gy in fractions of 1.5–2.0 Gy. Some had proposed to irradiate the fellow eye even if not currently involved [50]. Prophylactic radiation treatment of 45 Gy to the whole brain was recommended even if no CNS involvement was documented [50]. However, Isobe et al. [49] reported that 2 of 9 patients who received prophylactic cranial irradiation developed CNS disease compared to 1 of 4 patients that did not receive prophylactic cranial irradiation. This suggests that cranial irradiation is ineffective as a prophylactic measure to prevent CNS disease. In one series despite adequate radiation therapy, 7 of 9 patients eventually died of CNS disease progression. In another study, recurrent CNS disease was the cause of death in 12 of 13 patients.

Prognosis is poor with ocular irradiation with a median survival of 20 months [46, 50]. This is slightly better than PCNSL treated with steroids and radiation where the median survival is only 10–18 months [42]. In general, radiation therapy offers satisfactory local control for patients with isolated PVRL; however, it has fallen out of favor since it does not treat or prevent PCNSL [46]. If no additional treatment is given to treat or prevent PCNSL, 90% of the patients will develop or have a relapse of PCNSL [25]. Visual acuity rarely returns to normal due to complications secondary to ocular radiation therapy such as conjunctivitis, cataract, vitreous hemorrhage, radiation retinopathy, and retinal atrophy [46, 50]. Finally, ocular radiation therapy cannot be repeated if a relapse

occurs [46]. However, Berenbom et al. [51] challenged the notion that radiotherapy should be abandoned as first-line therapy for ocular disease without CNS involvement. They claimed that PVRL is more radiosensitive than PCNSL and thus can be treated with lower radiation doses of 35–40 Gy given in 15 fractions to both globes rather than the typical 50 Gy used in the treatment of CNS disease. Furthermore, they mentioned that customized blocks made following computed tomographic simulation may minimize normal tissue toxicity [51].

B-cell neoplasms are responsive to chemotherapy [42, 47, 48]. Chemotherapy may be administered by intrathecal, intravenous, or intravitreal routes. The blood-brain barrier and the blood ocular barrier limit the penetration of systemic chemotherapeutic agents into the CNS and eye. There is not enough information to determine if one drug or drug regimen is superior over the others, but intraocular penetration as well as CNS drug penetration are essential characteristics [46]. Methotrexate (MTX) and cytosine arabinoside (ARA-C) are favored by most investigators in the field because they appear to reach therapeutic levels in the vitreous after intravenous administration [46]. Fifty to eighty percent of patients with PCNSL show a complete response to high-dose MTX [47, 48]. ARA-C also reaches cytotoxic levels in the CSF. Chemotherapy has been employed for PCNSL and PVRL at relapse causing remission but not a cure [47, 48]. Several investigators felt that chemotherapy should not be reserved for recurrent disease and recommended combined radiotherapy and chemotherapy as initial therapy for PVRL and PCNSL [42]. The response to ARA-C has not been uniform. Three patients treated with a combination of radiation therapy to the brain and orbits, intrathecal ARA-C, and intravenous MTX treatment were still alive after 36 months [50]. In another study, three patients with PVRL, two of whom also had PCNSL, were treated with intravenous MTX, intravenous high-dose ARA-C, intrathecal MTX, and radiation therapy to the brain and orbits. All patients had a complete response including the CNS and have remained disease free for at least 2 years following completion of treatment [46].

In one study, a patient with PCNSL developed bilateral vitreoretinal involvement while on highdose ARA-C [42]. In another study, 5 out of 7 patients treated with ARA-C had a relapse after treatment. They reported a 81% complete response rate and a median survival of 44.5 months [25]. Conjunctivitis, keratitis, and ocular irritation are common in patients receiving high-dose IV ARA-C. Periorbital edema, blepharitis, conjunctival hyperemia, and photophobia have been reported with intravenous MTX [46]. Despite the lack of a uniform response, chemotherapy in combination with radiation therapy improves median survival to 40 months with 25% of patients surviving 5 years or more. However, ocular relapse is common occurring in up to 50% of patients; the mean interval between diagnosis and death was almost 16 months [42, 46].

As patients survive longer with combination therapy, late neurological sequelae of radiation therapy that affect quality of life such as neurocognitive disorders and irreversible visual loss have been observed, especially in patients older than 50 years of age [42, 46–48]. Protocols utilizing chemotherapy alone without radiation therapy as primary therapy have been investigated [47, 48]. In one trial, the median survival was 30.5 months, while in another, the median survival had not been attained after a mean follow-up of 3.3 years. High doses of intravenous MTX (8 g/m2) achieve therapeutic levels in both the aqueous and vitreous [46]. Nine patients with either PCNSL and PVRL or isolated PVRL were treated with only intravenous MTX at high doses. This protocol consisted of an induction phase of 8 g/m [2] MTX every 14 days until a complete response, a consolidation phase of 8 g/m [2] MTX twice a month for a month, and a maintenance phase of 8 g/m [2] MTX every 28 days for 11 doses. Four of the nine patients had a sustained response to the intravenous MTX. The remaining five patients required ocular radiation therapy. The lack of a sustained response of these 5 patients is probably a reflection of the fact that intravitreal levels of MTX are much lower than the aqueous levels. In a retrospective study of eight patients with PVRL that were treated solely with systemic chemotherapy, 100% relapsed. The chemotherapeutic

regimen consisted of either ARA-C or MTX in all patients. Some patients also received procarbazine and vincristine. The relapse occurred in the eyes in 75% and in the CNS in 25% [46]. Others have favored hyperosmolar blood-brain barrier disruption (HBBBD) chemotherapy to treat PCNSL [52]. Intra-arterial mannitol is used to disrupt the blood-brain barrier. The chemotherapeutic agents used included cyclophosphamide, MTX, leucovorin, procarbazine, and dexamethasone. Patients treated with this protocol experienced a greater mean survival from diagnosis in addition to preservation of cognitive function as compared to patients treated with conventional radiotherapy [52]. Fifty-four to sixty-five percent of patients treated with HBBBD may develop a pigmentary maculopathy [46]. This maculopathy is bilateral but asymmetric. It is characterized by fine clumps of RPE hyperpigmentation in the foveal region associated with a variable loss of the RPE. Permanent and progressive visual loss may result from this maculopathy.

The alkylating cytostatic agent, trofosfamide, that has been used as maintenance therapy in hematologic malignancies, and its main metabolite ifosfamide, which can reach cytostatic levels in the CNS, have both been investigated in PVRL [53]. Trofosfamide has an excellent bioavailability of almost 100%. It has been used successfully in the treatment of PCNSL. Its side effects include a dose-related hematotoxicity, nausea, and vomiting. The response rate was high but so was the relapse rate. Thus, the authors concluded that both ifosfamide and trofosfamide were promising candidates for combination therapy [53]. Clearly systemic chemotherapy without local therapy does not appear to be sufficient in the treatment of PVRL.

The addition of high-dose MTX to the treatment protocol of PVRL and PCNSL has greatly improved the prognosis for these patients [47, 48]. Nevertheless, anywhere from 35% to 60% of patients do not respond to treatment. Furthermore, up to 60% of patients that initially have a complete clinical remission develop a relapse. If no treatment is given, the overall survival of recurrent PCNSL is only 5 months. Less than 50% of

these patients have a second complete remission. Salvage radiotherapy, which can only be given to those patients who did not receive it, previously increases the overall survival to 11 months. Intensive chemotherapy followed by hematopoietic stem cell rescue (IC + HCR) appears to be promising [47]. In a small prospective trial of 11 patients with PVRL, 11 patients were prospectively treated with ESHAP (cisplatin, VP-16, ARA-C, methylprednisolone) plus intrathecal steroids, MTX, and ARA-C, and whole brain and ocular radiation therapy in some cases; or alternating ESHAP and high-dose MTX; or just highdose MTX as primary treatment. All the patients experienced either treatment failure or relapse. Second-line therapy was given to nine patients and included ocular and whole brain radiotherapy, high-dose MTX, or a combination of holoxan, natulan, and thiotepa. Disease progression was documented in all the nine patients that underwent second-line treatment. Of these nine patients, five were treated with doses of busulfan, thiotepa, and cyclophosphamide, followed by autologous bone marrow transplant. They achieved complete remission. None of the patients experienced CNS progression. Of these patients, two relapsed after 6 months but the other three were disease free after 15 months. A more recent study from the same group expands on their initial experience. Selection criteria for IC + HCR included patients with refractory or recurrent PCNSL that responded to two salvage cycles of ARA-C and etoposide (VP-16) or patients with PVRL that failed treatment with high-dose MTX and ARA-C. Nine out of 12 patients achieved clinical remission with a median survival greater than 53 months. However, 5 of 7 patients older than 60 years old died during therapy. A larger multicenter trial of 43 patients validated the value of IC + HCR as salvage treatment for PCNSL and PVRL. After a median follow-up of 36 months, the median overall survival and the median progression-free survival were 18.3 and 11.6 months in the whole cohort compared to 58.6 and 41.1 months in patients that completed IC + HCR, respectively. Patients who responded to the salvage cycles of ARA-C and VP-16 and went on to IC + HCR had the best prognosis. In

Fig. 13.7 Same patient as in Figs. 13.1, 13.3, and 13.5. Slit lamp examination of a patient with intraocular lymphoma. The right eye demonstrates the presence of keratic precipitates more prominent in the right eye (shown) and 1+ cells in the anterior chamber (Courtesy of Raul Vianna, M.D.)

this subgroup, the median overall survival and median progression-free survival had not been reached at 36 months. Of concern was the development of late neurotoxicity in 5 (11%) patients, including 3 with severe cognitive impairment, which proved to be fatal in one patient. Since neuropsychometric testing was not performed at baseline, it remains unclear if IC + HCR was responsible for this adverse event as many patients had received radiation therapy as part of their primary or secondary treatment [47].

Intravitreal chemotherapy for PVRL has been advocated given the disadvantages of ocular radiation therapy and the poor ocular penetration of systemic chemotherapy [46]. Several chemotherapeutic agents, including MTX, have been tested intravitreally in rabbits. No evidence of retinal toxicity was found for MTX using electroretinography and light microscopy. A single intravitreal injection of 400 mg of MTX can lead to a prolonged intravitreal tumoricidal concentration (>0.5 mM for 48–72 h in the rabbit eye) lasting longer than that achieved by systemic administration. Several investigators have reported that serial injections of 400 mg of intravitreal MTX clear the eye of lymphoma cells (Figs. 13.7, 13.8, and 13.9a, b). Another group reported their success with intravitreal MTX in achieving remission in a patient with an aggressive recurrent

PVRL. Based on their good experience of 10 years, Frenkel et al. [54] proposed intravitreal MTX as first-line treatment for PVRL. Their protocol consisted of an induction phase of two injections of 400 mg of intravitreal MTX per week for a month, followed by a consolidation phase consisting of a weekly injection for 2 months, and finally a maintenance phase of a monthly injection for a year. In their series of 44 eyes that were followed for a median of 21 months (range, 3–120 months), clinical remission was obtained after a mean of 6.4 injections (range, 2–16 injections). Fourteen patients died from their CNS or systemic lymphoma after a median

Fig. 13.8 Same patient as in Figs. 13.1, 13.3, 13.5, and 13.7. Slit lamp examination of the patient with intraocular lymphoma (right eye) in Fig. 13.1 after 2 months of intravitreal methotrexate (Courtesy of Raul Vianna, M.D.)

of 17 months (range, 3–84 months). None of the patients had an ocular relapse. Interestingly, in those patients with bilateral disease, there was no difference in the response to intravitreal MTX between the eye that underwent the diagnostic vitrectomy and the fellow eye. The visual acuity in most patients remained stable or improved [54].

Local recurrences and MTX resistance following intravitreal MTX monotherapy have been reported by others [39]. A patient with PVRL and PCNSL that had a complete resolution of her disease with systemic chemotherapy developed a relapse in her right eye and was treated with multiple injections of intravitreal MTX. Complete resolution of her disease was documented, but recurrences were noted over the next 18 months. It was noted that the PVRL became less and less responsive to intravitreal MTX. Examination of the intraocular lymphoma cells revealed that there was an aberrant expression of multidrug resistance protein (MRP) and decreased expression of both reduced folate carrier (RFC) and folate binding protein (FBP). This suggests that this patient’s PVRL cells acquired the ability to reduce the intracellular accumulation and metabolism of MTX [55]. To decrease the chances of resistance, cases of recurrent PVRL have been treated with a combination of MTX and dexamethasone in one case and MTX and thiotepa in the other case. Another patient with recurrent bilateral PVRL was treated with several treatment

Fig. 13.9 Same patient as in Figs. 13.1, 13.3, 13.5, 13.7, and 13.8. (a) Fundus examination of a patient with intraocular lymphoma (right eye) in Figs. 13.2 and 13.3 after 2 months of intravitreal methotrexate. (b) Arteriovenous phase fluorescein angiogram demonstrat-

ing multiple areas of disturbances at the level of the retinal pigment epithelium (RPE). Window defects might represent areas of tumor resolution with secondary RPE atrophic changes (Courtesy of Raul Vianna, M.D.)

cycles of 200 mg of intravitreal MTX (given on days 1, 5, and 8) and 7.5 mg of periocular dexamethasone phosphate (injected on day 9) over a course of 5 months. Complete regression of the PVRL was sustained over a period of 24 months [46]. Based on pharmacokinetic data obtained in the rabbit eye, Velez and associates [56] devised an intravitreal chemotherapy treatment cycle consisting of 400 mg of intravitreal MTX (given on days 1, 4, and 6), fluorouracil 500 mg (injected on day 2), and 500 mg of dexamethasone sodium phosphate on day 7. They reasoned that using this protocol would provide intravitreal antitumor activity for 8 days. This would allow most cells that are active in the cell cycle to pass through the S phase and be exposed to the drug. MTX exhibits a maximal antineoplastic cytotoxic synergistic response with fluorouracil when fluorouracil is given 24 h after MTX exposure. Since steroids are cell cycle nonspecific and are cytotoxic to lymphoma cells at all stages of the cell cycle, dexamethasone is given at the end of the cycle to treat those cells that do not progress through the S phase. In addition, steroids can treat a potential inflammatory reaction that may result from the necrosis of tumor cells [56]. Adverse reactions to intravitreal MTX have been reported and include filamentary keratitis that resolved with topical folinic acid, corticosteroid responsive sterile endophthalmitis, and vitreous hemorrhage [46, 54]. Other complications that could not be directly attributed to the intravitreal MTX included irreversible loss of visual acuity, neovascular glaucoma, optic atrophy, maculopathy, and progression of cataract. In order to avoid the repetitive intravitreal injections of MTX and its potential complications, researchers have studied alternative drug delivery methods in rabbits such as subconjunctival injections and trans-scleral hydrogel iontophoresis. Unfortunately, therapeutic levels of MTX were achieved in the aqueous but not in the vitreous with both methods [46].

Most PVRL and PCNSL are B-cell neoplasms that express the cell surface molecule CD20 [45, 46, 57]. Rituximab (Rituxan, Genentech, Inc., South San Francisco, CA, USA) is a humanized mouse monoclonal antibody directed against

the CD20 B-cell antigen [46, 57]. It has been approved for systemic use in patients with B-cell lymphomas. Its efficacy in PCNSL has been limited by its poor penetration of the bloodbrain barrier when given as a systemic infusion. To enhance CSF levels, intraventricular injection of rituximab through an Ommaya reservoir has been reported. In an animal model, intravitreal and intracerebral rituximab eradicated lymphoma in more than 50% of animals and significantly inhibited tumor progression in the remainder. Light microscopic histopathologic studies in rabbits have shown a lack of retinal toxicity following a single intravitreal injection of 1 mg/0.1 mL of rituximab. Rituximab has been shown to be able to penetrate full thickness retina in rabbit eyes, which is an important property given that subretinal infiltrates are often found in PVRL. Clinical studies in rabbits showed mild vitritisinallfoureyesexaminedwithinflammatory cells infiltrating the optic nerve and ciliary body. Serial intravitreal injections of rituximab appear to be well tolerated in human eyes. Pharmacokinetic studies in rabbits demonstrate a halflife of 4.7 days, implying that injections should be given every 2 weeks. Preliminary results regarding the use of intravitreal rituximab in the treatment of PVRL are encouraging and promising. However, further research is needed in particular to determine the most appropriate dose, dosing sequence, and length of treatment. Validation of its efficacy against PVRL also is needed [46, 57].

Several novel experimental immunotherapies are currently under investigation [39, 46]. Targetspecific killing using recombinant immunotoxin HA22 therapy has been successfully tested in a mouse model of PVRL. Ocular immune privilege promotes tumor growth by interfering with the development of innate and adaptive immunity. The membrane only form of Fas ligand (FasL) terminates immune privilege inducing systemic protective immunity. Co-inoculation of the anterior chamber of several mice with lymphoma cells and microvesicles expressing either membrane FasL or no FasL showed that membrane FasL eliminated the lymphoma cells and prevented metastatic spread in most of the treated mice [46].