Diagnosis of a primary uveal extranodal marginal zone B-cell lymphoma by chorioretinal biopsy: case report

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Graefe’s Arch Clin Exp Ophthalmol (2005) 243: 482–486


DOI 10.1007/s00417-004-1050-4

Sarah E. Coupland Antonia Joussen Gerasimos Anastassiou Harald Stein

Received: 26 April 2004 Revised: 24 August 2004 Accepted: 14 September 2004 Published online: 7 December 2004 # Springer-Verlag 2004 S. E. Coupland (*) . H. Stein Department of Pathology, Charité University Hospital, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany e-mail: [email protected] Tel.: +49-30-84453077 Fax: +49-30-84454473 A. Joussen Department of Ophthalmology, University Hospital Cologne, Cologne, Germany G. Anastassiou Department of Ophthalmology, University Hospital Essen, Essen, Germany

Diagnosis of a primary uveal extranodal marginal zone B-cell lymphoma by chorioretinal biopsy: case report

Abstract Purpose: To report the clinical, histopathological and molecular biological findings of a primary extranodal marginal zone B-cell lymphoma (EMZL) of the uvea. Methods: Conventional histology, immunohistochemistry and polymerase chain reaction for immunoglobulin heavy chain gene rearrangement (IgH-PCR) and GeneScan analysis were performed on a chorioretinal biopsy. Results: Histological examination of the chorioretinal biopsy demonstrated a dense infiltrate of small centrocyte-like cells, plasmacytoid tumour cells and occasional blasts. The tumour cells were positive for CD20, showed monotypical expression for Ig-kappa and IgM, and a growth fraction of 10%. Clonality analysis using IgH-PCR disclosed a

Introduction Intraocular manifestations of lymphoma can be divided into three major types: (1) so-called primary intraocular lymphomas (PIOL), which arise in the vitreous and retina, are usually highly malignant B-cell non-Hodgkin lymphomas (NHL) and can be associated with primary central nervous system lymphomas (PCNSL); (2) the primary uveal lymphomas, which are commonly low-grade malignant B-NHL; and (3) intraocular (mainly choroidal) involvement of systemic lymphoma. The primary uveal lymphomas are the least common of the intraocular lymphoma manifestations, with only 65 cases being reported in the literature (see reviews [4, 6]). As primary uveal lymphomas are usually clinically indolent, they have been misnamed “reactive lymphoid

monoclonal B-cell population. Following localised irradiation of 35 Gy, a complete remission was achieved. Conclusion: This report describes a rare uveal EMZL diagnosed following investigation of a chorioretinal biopsy. Despite its rarity, ophthalmic pathologists should consider the diagnosis of a primary uveal EMZL when reviewing chorioretinal biopsies. Keywords Intraocular lymphoma . Extranodal marginal zone B-cell lymphoma . WHO lymphoma classification . Vitrectomy . Chorioretinal biopsy . Immunohistochemistry . Polymerase chain reaction . Treatment

hyperplasia” (RLH) and “uveal pseudotumours” in the past. The demonstration of monoclonality within the infiltrating lymphocytes using immunohistochemistry and, later, using polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) rearrangements, resulted in their redefinition as low-grade B-cell NHL. [1, 11] We recently suggested that primary uveal lymphomas are most accurately subtyped as “extranodal marginal zone B-cell lymphomas” (EMZL) of MALT type, according to the REAL lymphoma classification [8], since they demonstrate morphological, immunophenotypical and clinical features similar to EMZL in other locations [6]. Most uveal EMZL have been diagnosed in eyes enucleated due to difficulties in differentiating the uveal lymphoproliferative lesion from a malignant uveal melanoma, or to pain as a result of secondary glaucoma. We


present here a rare case of primary uveal EMZL diagnosed in a chorioretinal biopsy on the basis of immunohistochemical studies and the clonality analysis using IgH-PCR.

Case report In January 2003, a 45-year-old systemically healthy man was referred with an intraocular tumour of uncertain nature in the left eye (OS). He complained of progressive visual loss over the previous 6 months. On examination, the visual acuity (VA) of the right eye (OD) was 20/20, the best-corrected VA OS was 32/200, the IOP of both eyes was normal and the neurological status unremarkable. Fundoscopy OS revealed a mild 360° choroidal detachment near the ora serrata and a prominent choroidal detachment in the temporal periphery, extending to the macular region with associated serous retinal detachment (Fig. 1a). The clinical diagnosis was idiopathic uveal effusion syndrome, and systemic steroid therapy was commenced. Control examinations in March 2003 demonstrated a slight improvement in the VA OS (40/200), complete regression of the peripheral choroidal detachment and shrinkage of the choroidal prominence in the temporal periphery accompanied by a flat serous retinal detachment (Fig. 1b). The steroid therapy was gradually withdrawn over a period of 4 weeks. Five months later, the patient represented with symptoms similar to the initial ones. Whereas the VA OD remained unchanged, the VA OS had decreased to 20/800. Fundoscopy OS revealed a choroidal detachment with associated serous detachment in the lower quadrants. Ultrasonography showed a tumour up to 9.6 mm in diameter (Fig. 2) without evidence of scleral infiltration. Extensively performed systemic and radiological examinations excluded a systemic malignancy or infectious disease. A chorioretinal biopsy was performed during a lenssparing three-port vitrectomy. The biopsy of the tumorous mass, which involved the macular area, was taken after 180° retinotomy. Subsequently the eye was filled with silicone oil. Histomorphological examination of the chorioretinal biopsy revealed a low-grade B-cell NHL (see Fig. 1 a Colour photograph of the left fundus taken in January 2003, showing a prominent choroidal detachment in the temporal periphery extending to the macular region. b Fundoscopy of the left eye in March 2003 after steroid therapy, showing regression of the choroidal prominence accompanied by a flat serous retinal detachment

Fig. 2 Ultrasonography of the left eye in June 2003 demonstrated a choroidal tumour up to 9.6 mm in diameter with associated serous retinal detachment

below). Subsequent staging examinations excluded the presence of a systemic lymphoma or PCNSL. Based on these findings, a primary uveal lymphoma OS was diagnosed, and local irradiation (total 35 Gy, applied in 17 fractions) was applied in October and November 2003. In January 2004, the patient’s VA was 20/20 OD and counting fingers OS. The anterior eye segment showed posterior synechiae of the iris. Fundoscopy of the left eye did not reveal any evidence of disease recurrence. In the areas of the former tumour, only chorioretinal scars were visible (Fig. 3). In March 2004, however, a endophacoemulsification was performed OS due to the development of postirradiation cataract and to the subsequent decrease in ability to view and examine the fundus. At this time, the retina was attached with scarring only at the site of the biopsy. At the final follow up in June 2004, the retina was attached with a best-corrected VA of 20/400 OS.

Materials and methods Histology and immunohistochemical analysis of the chorioretinal biopsy The formalin-fixed and paraffin-embedded choroidal biopsy was stained using conventional histochemistry and


immunohistochemistry, employing standardised methods [5, 10]. The primary monoclonal antibodies applied were CD79a, CD20, CD43, CD5, CD23, BCL-2, BCL-6, cyclin D1 and MIB-1 (Ki-67 antigen). Polyclonal antibodies investigated the expression of CD3 antigen and of the immunoglobulin (Ig) light and heavy chains. All antibodies were obtained from DAKO-Cytomation (Glostrup, Denmark). Polymerase chain reaction Fig. 3 Colour photograph of the fundus of the left eye taken in January 2004, showing chorioretinal scars in the region of the former lesion following radiotherapy Fig. 4 a Conventional cytology of the choroidal aspirate showing an infiltration by small lymphocytes with minimal cytoplasm, round to oval nuclei and condensed chromatin (May–Grunewald–Giemsa, original magnification ×40). b Immunohistology of the formalin-fixed paraffin-embedded material demonstrating positivity of the tumour cells for the B-cell antigen CD79a (APAAP, original magnification ×40). c The CD43 staining shows an aberrant expression of the B cells for this T-cell-associated antigen (APAAP, original magnification ×40). d A small number of reactive T-cell lymphocytes can be displayed using CD3 (APAAP, original magnification ×40). e A monotypical expression of the neoplastic B cells can be demonstrated for the immunoglobulin light chain kappa (PAP, original magnification ×40). f The growth fraction of the tumour cells, as determined using MIB-1 directed against the Ki-67 antigen, was low, ca. 10% (APAAP, original magnification ×40)

DNA was extracted after dewaxing from 20-μm-thick paraffin sections of the chorioretinal specimens by em-


ploying QIAEX (Qiagen, Germany), according to the manufacturers’ recommendations. For the detection of IgH rearrangements, two single-step polymerase chain reactions (PCRs) were performed employing family-specific frame work (FR) 2 and FR3 primers (six primers for each FR) in conjunction with a common JH consensus primer (JH22) [12]. The cycling conditions (50 rounds of amplification) for all PCRs have been described in detail elsewhere [12].

Molecular biological examinations: PCR and GeneScan analysis



For analysis of the PCR products, the reaction mixtures were separated on a sequencing gel and analysed in an automated DNA sequencer (ABI 377A, Applied Biosystems, Weiterstadt, Germany) by using the GeneScan software. For this purpose, the FR2 and FR3 primers were labelled with 5-carboxyfluorescein, FAM. For precise determination of the length of the amplificates, a size standard labelled with a different fluorescence dye was coseparated in each lane.

Primary uveal lymphomas are the rarest of the three forms of intraocular lymphomatous manifestations. These lymphomas are considered to be primary tumours arising in the uvea, due to the absence of systemic lymphoma at the time of diagnosis and to their unilaterality in most patients [11]. Typical presenting symptoms in patients, usually men in the fifth decade, include recurrent episodes of blurred vision, painless loss of vision and metamorphopsia due to secondary serous detachment of the macula [4, 6, 11]. There may be an initial response, as in the current case, to steroid therapy. Ultimately, a diffuse thickening of the uveal tract and, in advanced cases, subconjunctival or episcleral extension can occur. In most cases of primary uveal EMZL reported, the eyes were ultimately enucleated either due to difficulties in determining the nature of the uveal mass clinically or because of pain as a result of secondary glaucoma. Some authors performed biopsies of the episcleral tumour nodules to establish the diagnosis of either malignant lymphoma [9] or RLH [2]. Chorioretinal biopsy was used to diagnose RLH of the uvea by Cheung et al. [3]. The present case illustrates one of the few cases of primary uveal EMZL diagnosed on the basis of chorioretinal biopsy. The usefulness of chorioretinal biopsies—e.g. in differentiating neoplastic and inflammatory diseases of the uvea, in allowing exact subtyping of the intraocular lymphoma when present, and in formulating a rational treatment plan—has been demonstrated by a number of centres. Fine-needle aspiration biopsy is an alternative diagnostic tool successfully used in some centres in the diagnosis of uncertain intraocular tumours [7]. Although this method may be associated with less trauma than chorioretinal biopsy, one should bear in mind that mechanical artefacts, insufficient material and sampling errors can limit their interpretation of the specimens obtained. The morphological, immunohistochemical and molecular biological characteristics of primary uveal lymphomas are similar to those of EMZL of other locations [6]. Briefly, the primary uveal lymphomas consist morphologically of a dense infiltration of small tumour cells with occasional blasts in the marginal zone surrounding reactive follicles. Features such as “follicular colonisation” or “lymphoepithelial lesions” may be present and repre-

Results Histopathology and immunophenotypic studies The chorioretinal biopsy was infiltrated by small atypical lymphocytes, consisting of centrocyte-like cells, monocytoid cells, plasmacytoid cells, some with obvious Dutcher and Russell bodies, as well as occasional blasts (Fig. 4a). The neoplastic lymphocytes expressed CD79a (Fig. 4b) and CD20, the proto-oncogene BCL-2 and, aberrantly, the T-cell antigen CD43 (Fig. 4c). They were negative for CD3 (Fig. 4d), CD5, CD23 and cyclin D1. A monotypical expression for Ig-kappa and IgM was demonstrated (Fig. 4e). The tumour cell growth fraction was small (ca. 10%) (Fig. 4f).

Fig. 5 GeneScan analysis of the DNA products obtained following IgH-PCR using the primers FR2 and FR3, demonstrating a monoclonal B-cell peak of 245 base pairs. The red signals represent the internal size standard

Both primer sets (FR2 and FR3) led to the detection of dominant amplification products which were identically reproducible in repeated, independently performed PCR assays. GeneScan analysis of the dominant amplificates confirmed the presence of a clonal B-cell population (Fig. 5).


sent supporting evidence of the neoplastic nature of these lesions [8]. Immunohistochemical studies show a B-cell dominance of the infiltrating lymphocytes, with (often) an aberrant expression of the T-cell antigen CD43 [6] as well as monotypical expression of an Ig light and/or heavy chain. Although not a major feature in the present case, the degree of plasmacellular differentiation of the tumour cells can be extensive in some uveal EMZL [4, 6, 11]. These neoplastic plasmacytoid cells usually demonstrate loss of plasma-cell related antigens compared with reactive plasma cells [6]. Finally, the neoplastic nature of the B-cells in EMZL can be further supported through clonality analysis using IgH-PCR. In the present case, a monoclonal B-cell population could be shown using IgH-PCR and GeneScan analysis of the formalin-fixed biopsy material.

Although most PIOL arising in the retina are associated with a poor prognosis, those primary intraocular lymphomas originating in the uvea are less aggressive in their clinical course, with the overall survival being very good [4, 6]. Although resolution of uveal lymphomas has been achieved with moderately heavy doses of systemic corticosteroids, low-dose irradiation is considered the treatment of choice. In summary, presented here is a rare case of a primary uveal EMZL. Ophthalmic pathologists should be aware of the diagnosis when reviewing chorioretinal biopsies in patients with choroidal tumours of unclear aetiology. An exact subtyping of these intraocular lymphomas is indicated where possible, in order to obtain a better understanding of their differing pathogenesis, and in order to establish appropriate treatment regimens.

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