Primary Intraocular Lymphoma

Primary Intraocular Lymphoma Improving the Diagnostic Procedure Anni Karma, MD, PhD,1 Eva O. von Willebrand, MD, PhD,2 Petri V. Tommila, MD, PhD,1 Anders E. Paetau, MD, PhD,3 Pertti S. Oskala, MD,1 Ilkka J. Immonen, MD, PhD1 Objective: To analyze the clinical features of primary intraocular lymphoma (PIOL) and to describe cytochemical and immunocytochemical findings of the vitreous specimens as well as the reasons for delayed diagnosis of PIOL. Design: Prospective noncomparative study. Participants: Eleven patients referred to the uveitis or medical retina units, Department of Ophthalmology, University of Helsinki, were diagnosed as having PIOL between 2000 and 2005. The median follow-up of the patients was 32 months. Methods: Clinical features and diagnostic workup of uveitis were described. Twelve vitrectomies were performed on 9 patients. The first 5 biopsies were fixed in an equal volume of 50% alcohol. The specimens of the next 7 vitrectomies were handled without alcohol, and tissue culture medium was added to the samples. Main Outcome Measures: Clinical features of PIOL, intervals from ocular symptoms and from first ophthalmological examination to diagnosis, and the role of a proper handling of the vitreous sample in the diagnosis of PIOL. Results: Six females (54%) and 5 males (46%) (median age, 61 years) were included. Ten patients had ocular symptoms for 1 to 30 months (median, 8) before the first contact with an ophthalmologist. Uveitis was bilateral in 9 patients. Vitreitis was seen in all patients, and it was severe in 8. Fundus lesions dominated in 3 patients. Six patients lost useful vision in one eye before the diagnosis of PIOL. Cytologic and immunohistochemical stainings prepared of the unfixed vitreous specimens showed PIOL in 6 patients. The samples fixed in alcohol were nondiagnostic in 4 patients, and in them, verification of diagnosis was based on brain biopsy (3) or cerebrospinal fluid (1) findings. Seven patients died due to primary nervous system lymphoma. Conclusions: Diagnosis of PIOL is difficult but can be improved. Severe bilateral vitreitis in an elderly patient is a characteristic finding of PIOL. Alcohol fixation may jeopardize the identification of PIOL cells in the vitreous sample. Optimal handling of the vitreous specimens and examination of the slides by an experienced cytopathologist are critical in the diagnostic workup of PIOL. Ophthalmology 2007;114:1372–1377 © 2007 by the American Academy of Ophthalmology.

Primary intraocular lymphoma (PIOL) is a subset of B-cell non-Hodgkin’s lymphoma of the nervous system, primary nervous system lymphoma (PNSL). Although it is still a rare disease, the incidence of PNSL has tripled over 3 decades.1 Systemic spread of PNSL outside the nervous system occurs rarely. However, recent studies suggest that the malignant event occurs in a lymphocyte outside the nervous system, in an extraneural germinal center, but the nervous system and the eye are the only sites of detectable disease, according to currently available diagnostic modalities.2– 4 Originally received: April 3, 2006. Accepted: November 1, 2006. Manuscript no. 2006-389. 1 Department of Ophthalmology, University of Helsinki, Helsinki, Finland. 2 Transplantation Laboratory, University of Helsinki, Helsinki, Finland. 3 Department of Pathology, University of Helsinki, Helsinki, Finland. Correspondence to Anni Karma, MD, PhD, Department of Ophthalmology, University of Helsinki, FIN-00029 Helsinki, Finland. E-mail: anni.karma@ fimnet.fi. Reprint requests to Petri V. Tommila, MD, PhD, Department of Ophthalmology, University of Helsinki, FIN-00029 Helsinki, Finland.

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© 2007 by the American Academy of Ophthalmology Published by Elsevier Inc.

Ocular involvement is found in 20% to 25% of patients with PNSL.5,6 Primary intraocular lymphoma masquerades as different uveitis entities. In uveitis surveys, it has rarely been recognized: 2 cases of PIOL were diagnosed among 1300 consecutive uveitis patients in a tertiary survey from the northeastern United States,7 and 9 cases were found among 828 consecutive uveitis patients referred to a tertiary ophthalmologic center in The Netherlands.8 Because of the rarity of the disease, vague ocular symptoms, diverse clinical picture, and the demanding procedure for a successful vitreous specimen, the diagnosis of PIOL is a great challenge to ophthalmologists and cytopathologists. The preparation of vitreous specimens for cytological evaluation varies,9 –14 and multiple specimens may be required before an unequivocal diagnosis of PIOL can be made.11–13 More than 40% of the vitrectomy specimens may remain nondiagnostic.13,15 The disease has a poor prognosis. Patients die of cerebral tumor growth after a mean survival of 20 months from the diagnosis of the ocular disorder.15 However, as advances in the management of PNSL and PIOL have resulted in imISSN 0161-6420/07/$–see front matter doi:10.1016/j.ophtha.2006.11.009

Karma et al 䡠 Primary Intraocular Lymphoma proved survival and preservation of vision, prompt diagnosis will become increasingly critical.16 –19 We report our experience with 11 patients with PIOL diagnosed since March 2000 in the Department of Ophthalmology, Helsinki University Central Hospital. We describe the reasons for delayed diagnosis of PIOL and the process of improving the diagnosis.

Patients and Methods Between March 2000 and December 2005, the diagnosis of PIOL was established in 11 patients initially sent to the uveitis and/or medical retina units of the Department of Ophthalmology, Helsinki University Central Hospital. Nine patients were referred from either practicing ophthalmologists (6 patients) or other eye hospitals in Finland (3 patients). Two patients (nos. 1 and 11; Table 1), in whom PNSL already had been diagnosed, were referred from the Department of Oncology, Helsinki University Central Hospital. One patient (no. 4; Table 1) referred by a practicing ophthalmologist had gone through a neurological examination because of facial palsy, hemiparesis, and periodic severe headaches suspected to be due to recurrent herpetic meningitis. All patients were Caucasian, and all were immunocompetent. Human immunodeficiency virus infection was not an exclusion criterion in our study. The ophthalmological examination included slit-lamp biomicroscopy and fundus examination with indirect ophthalmoscopy and 3-mirror lens examination after pupillary dilatation. Uveitis was categorized anatomically according to the Uveitis International Study Group recommendations,20 and vitreous reaction was graded according to the scale described by Nussenblatt and Palestine.21 Laboratory and ancillary tests were taken on the basis of the differential diagnosis compiled for each case. In most patients, the tests included complete blood count, angiotensin-converting enzyme, antiborrelial and antinuclear antibodies, rheumatoid factor, liver function tests, and chest radiography. Magnetic resonance

imaging of the head and examination of cerebrospinal fluid as a part of the diagnostic workup of uveitis were performed on 5 patients. Six patients (nos. 1 and 3–7; Table 1) received topical and/or systemic corticosteroids for a period of 1 to 5 months. Additionally, patients 4 and 5 received intravenous and oral acyclovir or valavir therapy, and patient 8 had received intravenous ceftriaxone followed by oral amoxicillin before referral to us. Two patients’ uveitis responded temporarily to oral corticosteroid therapy (patients 3 and 4), but the remaining patients were nonresponsive to corticosteroids and antimicrobials (Table 1). Twelve vitrectomies were performed on 9 patients. On 1 patient (no. 8; Table 1) the vitrectomy had been performed in another eye hospital, but the result was nondiagnostic. For sampling of the vitreous specimen, a 3-port pars plana vitrectomy setup was used. One milliliter of undiluted vitreous core sample was aspirated manually through a 20- or 25-gauge vitreous cutter, with the cutting mode activated at 600 to 1200 cuts per minute. The infusion line was opened after sampling. The aspirated vitreous specimens of patients 1, 3 (performed on both eyes), 4, and 5 (first vitrectomy) were fixed in an equal volume of 50% alcohol and taken immediately to the cytopathologist. The specimens were prepared using a centrifuge (Cyto-Tek, Miles Scientific, Elkhart, IL). Seven most recent vitreous specimens of 6 patients (patients 5–10; Table 1) were handled in a different way: the aspirated material was emptied into a 10-ml glass tube, in which 5 to 6 ml of Rosewell Park Memorial Institute media 1640 (Life Technologies Inc., Grand Island, NY) was added to improve cell viability. The vitreous specimen was carried immediately to the cytology laboratory for processing. In 7 cases, the diluted specimen in the vitrector cassette was delivered without delay to the cytology laboratory as well. The vitreous specimen first was centrifuged at 200 rounds per minute for 10 minutes. After that, the cells were washed and resuspended in the culture medium and counted. Then the cells were cytocentrifuged at 500 rounds per minute for 10 minutes onto glass slides. One slide was stained with May–Grünwald–Giemsa, and the rest were used for the immunostainings. Immunohistochemical staining was performed using a sensitive

Table 1. Data of Patients Evaluated for Primary Intraocular Lymphoma (PIOL) Patient Duration (mos) of No./ Ocular Symptoms Bilateral Age before Initial Eye Ocular (yrs) Examination Involvement 1/53 2/70

8 8

No Yes

3/54

7

Yes

4/54

8

Yes

5/71

12

Yes

6/68 7/61 8/51

30 6 4

Yes Yes Yes

9/56

12

10/81 11/52

1 No symptoms

Initial Diagnosis

Time (mos) from Onset of Ocular Initial CNS Symptoms to Involvement Diagnosis

Diagnosis Based On

No. and Result of Vitrectomies

Follow-up (mos) Survival

PIOL Idiopathic or sarcoidosis Idiopathic or sarcoidosis Vitreous hemorrhage ARN

Yes No

8 14

Brain biopsy 1, nondiagnostic Brain biopsy ND

47 10

Dead* Dead*

No

25

Brain biopsy 2, nondiagnostic

49

Dead*

Yes

48

CSF

1, nondiagnostic

99

Alive

No

20

32

Dead*

No No No

36 7 7

28 33 35

Alive Alive Alive

Yes

PIOL Idiopathic Disseminated chorioiditis PIOL

Brain biopsy 2, nondiagnostic and PIOL Vitrectomy 1, PIOL Vitrectomy 1, PIOL Vitrectomy 1, PIOL†

No

13

Vitrectomy

20

Dead*

Yes No

PIOL PIOL

No Yes

7 No symptoms

12 17

Dead* Dead*

2, PIOL and nondiagnostic Vitrectomy 1, PIOL Brain biopsy ND

ARN ⫽ acute retinal necrosis; CSF ⫽ cerebrospinal fluid; ND ⫽ not done. *Died due to progressive primary nervous system lymphoma of the brain. † First vitrectomy had been performed in another hospital.

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Ophthalmology Volume 114, Number 7, July 2007

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Karma et al 䡠 Primary Intraocular Lymphoma indirect 3-layer immunoperoxidase method22 and monoclonal antibodies. The following monoclonal mouse immunoglobulin G1 antibodies were used: CD2, CD20, CD30, ␬ light chains, ␭ light chains, and, in some cases, CD68 (DakoCytomation Denmark A/S, Glostrup, Denmark). The antibodies were used at an optimal dilution in tris(hydroxymethyl)aminomethane/1% bovine serum albumin. After incubation for 30 minutes in a humid chamber at room temperature, the preparates were washed in tris(hydroxymethyl)aminomethane buffer and incubated for 30 minutes with peroxidase-labeled rabbit antimouse immunoglobulin (Dako P161, DakoCytomation Denmark), washed again, then incubated for 30 minutes with peroxidase-labeled goat antirabbit immunoglobulin (Caltag Laboratories, San Francisco, CA). The reaction was revealed by chromogen 3-amino-9-ethylcarbatzole containing hydrogen peroxide. The specimens were counterstained with Mayer’s hemalum and mounted (Aquamount, BDH Ltd., Poole, United Kingdom). In addition to the cytological and immunohistochemical analysis, fractions of the aspirated fluid from patients 1, 3 to 7, and 10 were sent to the microbiology laboratory. Gram’s stain and cultures for bacteria, fungi, and viruses were performed as well as genomic amplification by polymerase chain reaction (PCR) to test for the presence of herpes simplex virus, herpes zoster virus, herpes simplex 6 virus, cytomegalovirus, Toxoplasma gondii protozoa, Borrelia burgdorferi bacteria, and mycobacteria.

were immobile yellowish or brownish clumps and attached to the detached posterior vitreous membrane. In all patients except one, a few or multitudinous punctate white spots or clear-cut round lesions up to one quarter of a disc diameter in size were seen at the level of the outer retina and/or the pigment epithelium in the posterior pole and/or peripheral retina (Fig 1). Fundus lesions dominated the clinical picture in 1 eye in 3 patients. In 3 eyes, a poorly defined confluent yellowish retinal or subretinal mass was seen in the posterior pole (Fig 2). Because of poor visibility of the fundus in 8 patients, fluorescein angiography initially was performed on only 4 patients. It was assessed as normal in 2 cases, and in 2 patients, hypofluorescent dots intermingling with hyperfluorescent round areas were disclosed (Fig 3). The interval from the onset of ocular symptoms to diagnosis of PIOL ranged from 8 to 48 months (Table 1). The duration between the first contact in our clinic and diagnosis was considerably shorter among the 6 most recent patients than among the first 5 patients: 0 to 6 months (median, 1) versus 6 to 40 months (median, 9), respectively. The follow-up of our patients ranged from 10 to 99 months (median, 32). Seven patients died of PNSL of the brain. In 3 patients, the disease is still restricted to the eyes.

Findings of the Vitreous Biopsies

Results Patients’ demographic data, suggestions for the etiology of uveitis at the referral visit in our clinic, and the delay in the diagnostic workup as well as the procedures on which the final diagnosis was based are presented in Table 1. There were 6 (54%) females and 5 (46%) males. The median age of the patients at the first referral visit was 61 years (range, 51– 81). Ten patients had had ocular symptoms for 1 to 30 months (median, 8) before the first contact with an ophthalmologist, and 1 patient (no. 11) had no ocular symptoms. In 6 patients, the symptoms were mild and consisted of blurred vision and floaters or shadows in the visual fields (VFs). In 3 patients, the main complaint was decreased vision in one eye during previous months. Two patients complained of rapid alterations of visual acuity (VA), and additionally, 1 patient complained of deterioration of color vision. In 2 patients, VA was 20/20 in both eyes at the initial referral visit. Visual acuity of the remaining patients was 20/40 or less in one or both eyes; in 4 of them, VA was counting fingers (CF) in one eye. During the diagnostic workup period, 2 additional patients lost useful vision in one eye. Uveitis was bilateral in 9 patients (Table 1). The anterior segment was quiet in 5 patients, and 6 patients had a mild aqueous flare (⫹1). All patients had vitreitis, which was extensive (⫹3 or ⫹4) in 8 patients in either one (4 patients) or both eyes. Vitreitis consisted of sheets and opacities in addition to cells. Vitreous cells

Diagnosis of PIOL by vitreous biopsy was reached in 6 patients (54%). The vitreous specimens of patients 1, 3 (both eyes), 4, and 5 (first biopsy) were nondiagnostic. From these samples, only 1 to 2 Cyto-Tek preparations could be made. They showed a few inflammatory cells and debris or were totally acellular. The repeated vitreous biopsy from the fellow eye of patient 5 and 5 biopsies thereafter of patients 6 to 10 were diagnostic (Table 2). The number of the cells ranged from 0.02⫻106 to 0.2⫻106, enabling 10 to 21 slides to be prepared. In 2 cases, the specimens prepared from the cassette fluid were as representative as that from the core vitreous. Despite that, the material was sufficient for assessment of clonality in only 4 cases. The PCR technique to detect B- and T-cell gene rearrangements was tried in 2 cases and flow cytometry in 1, but the cell count was too scanty for that. The cytologic picture of the successful specimens of patients 5 to 10 was as follows: 5% to 80% of the cells were large basophilic pleomorphic blasts that intermingled with lymphocytes (10%– 63%) and monocytes (4%–32%), apoptotic cells, and debris (Fig 4). The blasts had basophilic cytoplasm and large round, oval, bean-shaped, or cloverleaf nuclei with coarse and prominent nucleoli. In all specimens, 40% to 80% of the blasts showed B-cell markers in immunohistochemical staining (Table 2, Fig 5). In 2 specimens, the blasts expressed ␬ light chain clonality (Table 2, Fig 6). Microbiological analysis of the vitreous fluid showed no positive results in any patients.

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Figure 1. Case 1. Clear-cut round lesions at the level of the outer retina and the pigment epithelium. Figure 2. Case 5. Ill-defined confluent yellowish deep retinal and subretinal lesions in the posterior pole of the right eye. Figure 3. Case 4. Arteriovenous phase of a fluorescein angiogram of the left eye. There are areas of granularity at the level of the pigment epithelium; hypofluorescent dots intermingling with hyperfluorescent dots; and a larger area of blockage of staining temporal to the macula, suggestive of an aggregate of tumor cells under the retinal pigment epithelium (arrows). Figure 4. Cytologic staining of the cytocentrifuged vitreous specimen. Large basophilic pleomorphic blast cells with scanty cytoplasm are seen. Some cells have cloverleaf nuclei. Nucleoli are prominent (stain, May–Grünwald–Giemsa; original magnification, ⫻400). Figure 5. Immunocytologic staining of the specimen with a B-cell marker (CD20). Nearly all blast cells expressed CD20 positivity (original magnification, ⫻400). Figure 6. ␬ light clonality in the B cells (original magnification, ⫻400).

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Ophthalmology Volume 114, Number 7, July 2007 Table 2. Results of Cytologic and Immunohistochemical Staining of Vitreous Cellular Infiltrates Results of Cytologic Staining

Results of Immunohistochemical Staining

Patient Cell Count ⫻ B Lymphocytes T Lymphocytes No. 106 Lymphoblasts Lymphocytes Monocytes (CD20) (CD2) 5 6 7 8 9 (a) 9 (b) 10

0.2 0.04 0.04 0.12 0.04 0.02 0.05

60% 47% 51% 80% 50% ⫹ 5%

20% 42% 38% 10% 40% ⫹ 63%

10% 9% 11% 10% 10% ⫹ 32%

80% 50% 50% 80% 50% ND 40%

15% 40% 40% 5% 40% ND 5%

␬

␭

CD30 Cells

PIOL

Negative 45% ND 60% ND ND Negative

Negative ⬍1% ND ⬍1% ND ND Negative

Negative Negative ND Negative ND ND ND

Yes Yes Yes Yes Yes Insufficient specimen Yes

ND ⫽ not done; PIOL ⫽ primary intraocular lymphoma.

Discussion Primary intraocular lymphoma is a subset of non-Hodgkin’s B-cell nervous system lymphoma. In Finland, with a population of 5.2 million, 118 new cases of PNSL were registered by the Finnish Cancer Registry during 1999 to 2003, 3 times more than during a 5-year period 2 decades earlier (unpublished data). The same tendency has been described in the U.S. as well.1 Eleven patients with intraocular–primary nervous system lymphoma were diagnosed at the Helsinki University Central Hospital during a 6-year period. Nine patients presented with ocular disease. In 4 of them, only the development of the nervous system malignancy led to the right diagnosis. To our knowledge, they are the first patients with PIOL reported in Finland. There are many reasons for the delay of the proper diagnosis. Minor ocular symptoms do not urge the patient with PIOL to seek ophthalmologic examination, the biomicroscopical features of PIOL mimic many uveitis entities, and the vitreous biopsy procedure may be nondiagnostic. Vague and diffuse ocular complaints in PIOL patients also have been pointed out by others.6,9,11,23 Visual acuity was normal in both eyes in only 2 patients, and in as many as 4 patients, VA was CF in one eye. Despite that, it was the blurring of vision and shadows in the VF most patients complained of. We agree with others that PIOL mimics other uveitis entities.8,11,23–25 In the diagnostic workup of uveitis, patient age, the anatomic localization of uveitis, bilaterality, biomicroscopic appearance, onset and duration of uveitis, and response to therapy play an important role.21 Typically, PIOL is a bilateral uveitis in elderly patients. Although it may be seen rarely in young patients,26 the median age is usually 50 to 60 years.6,9,11,15 The median age of our patients was 61 years. The yield of the first 5 vitreous biopsies was nondiagnostic. The main reason for the unsuccessful result was probably the 50% alcohol added to the fresh samples. Further, corticosteroids may be cytolytic in PNSL and may contribute to the difficulty in obtaining viable cells from a vitrectomy specimen.11,25,27 Although alcohol as a fixative has been used with success by others,9,12 it did not work in our laboratory. After we improved the process and began to handle the samples unfixed, represen-

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tative specimens were received in the remaining 6 patients (6/7 specimens). Freeman et al15 and Coupland et al13 used a cytospin preparation of the unfixed vitreous specimens and obtained successful biopsy results in 56% and 58% of their PIOL patients, respectively. Cytologic assessment has been the basic method in the diagnosis of PIOL since the 1970s.9 –11,28 More sophisticated techniques in the diagnosis of PIOL have been developed as well: flow cytometric analysis,29 gene rearrangements by PCR technique,30,31 and measurement of the interleukin 10/interleukin 6 ratio of the vitreous sample.32,33 However, PCR methodology is found to work best in tissue biopsy specimens,30 and on the basis of an increased interleukin 10/interleukin 6 ratio, the diagnosis of PIOL cannot be made.32,33 Because the number of viable cells in the vitreous sample is often limited, newer techniques should be used only if a sufficient number of slides for cytologic and immunohistochemical stainings has been guaranteed. In one patient with recently diagnosed PNSL, the intraocular findings were compatible with PIOL. She had no ocular symptoms, a fact reported by others as well.6,11 Because of vague ocular symptoms or even their total absence, patients with PNSL should be examined for ocular disease. On the other hand, magnetic resonance imaging of the brain should be repeated in patients with PIOL to plan the management properly. In conclusion, our study indicates that diagnosis of PIOL is difficult and that it can be improved. A high index of clinical suspicion is most important to diagnostic success. Severe bilateral vitreitis in an elderly patient should be recognized as a characteristic finding of PIOL. In such a patient, vitreous biopsy should not be postponed. Optimal handling of the specimen and evaluation of the slides by an experienced cytopathologist are critical in the diagnostic workup of PIOL. To achieve the best possible result, direct communication between an ophthalmic surgeon and cytopathologist is essential. The patients should be observed oncologically with respect to a high risk of developing cerebral PNSL disease subsequently. Acknowledgment. The authors acknowledge Risto Sankila, MD, PhD, of the Finnish Cancer Registry for provision of the registry data.

Karma et al 䡠 Primary Intraocular Lymphoma

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