Secondary ocular involvement in systemic “memory” B-cell lymphocytic leukemia

Secondary Ocular Involvement in Systemic “Memory” B-cell Lymphocytic Leukemia Sarah E. Coupland, MBBS, PhD,1 Hans-Dieter Foss, MD,1 Nicholaos E. Bechrakis, MD,2 Michael Hummel, PhD,1 Harald Stein, MD1 Objective: Recently, B-cell chronic lymphocytic leukemia (B-CLL) has been subdivided into “naive” B-CLL and “memory” B-CLL on the basis of the presence of somatic mutations in the variable region of the immunoglobulin heavy chain gene (IgH). The aim of the current paper was to report the clinical, histopathologic, and molecular biologic findings of intraocular and ocular adnexal involvement in a patient with systemic B-CLL. Design: Case report. Intervention: Treatment consisted of systemic chemotherapy, conjunctival biopsies, and, ultimately, enucleation of the left eye. Methods: Histopathologic findings of a bone marrow biopsy, the conjunctival biopsies, and the enucleated eye were compared. Further, extensive immunohistochemistry, polymerase chain reaction (PCR) for the detection of heavy chain (IgH) gene rearrangement, gene scan analysis, and DNA sequencing were performed on all tissues. Results: The tumor manifestations in all specimens demonstrated similar morphologic and immunophenotypic characteristics, consistent with the diagnosis of B-CLL. Immunoglobulin-H PCR and gene scan analysis showed that the B-CLL infiltrates consisted of B cells derived from the same clone. DNA sequencing demonstrated the presence of eight somatic mutations in the variable region of IgH, consistent with “memory” B-CLL. Conclusions: Secondary ocular manifestations of B-CLL occur relatively commonly during disease progression. In the current case of memory B-CLL, ocular manifestation of the disease occurred 16 years after initial diagnosis, agreeing with clinical studies suggesting that a less aggressive course is seen in “memory” B-CLL than its counterpart, “naive” B-CLL. Somatic mutation analysis in the variable region of IgH in B-CLL should be part of routine staging investigations to aid the prediction of the individual clinical course in B-CLL patients and to determine new therapeutic strategies. Ophthalmology 2001;108:1289 –1295 © 2001 by the American Academy of Ophthalmology. Lymphomas of the eye and its adnexa are relatively uncommon neoplasms and are usually B-cell non-Hodgkin’s lymphomas. Whereas the most common lymphoma subtype occurring in the ocular adnexa is the low-grade malignant extranodal marginal zone B-cell lymphoma (EMZL),1,2 primary intraocular lymphomas are usually diffuse, large-cell B-cell lymphomas, tumors of high-grade malignancy, initially involving the retina.3 Secondary manifestations of systemic lymphoma, however, can occur both in the ocular adnexa and intraocularly; when occurring in the latter, the choroid is typically involved. Ocular involvement in leukemic disease is relatively common and may occur in as many as 80% to 90% of patients at some point in the course of the disease.4,5 Clinically, it may present with a myriad of signs and symptoms, including decrease in vision, conjunctival

Originally received: August 1, 2000. Accepted: February 27, 2001. Manuscript no. 200457. 1 Department of Pathology, University Hospital Benjamin Franklin, Freie Universita¨t, Berlin, Germany. 2 Department of Ophthalmology, University Hospital Benjamin Franklin, Freie Universita¨t, Berlin, Germany. Correspondence to Sarah E. Coupland, MBBS, PhD, Department of Pathology, Universita¨tsklinikum Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: [email protected]. © 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.

swelling, diplopia, proptosis, retinal hemorrhages, whitegray retinal infiltrates, serous retinal detachment, as well as endophthalmitis.5,6 Conversely, the disease manifestation may be clinically undetected.5 Confirmation of clinically suspected secondary involvement of lymphomatous disease in ocular and ocular adnexal tissue is based on morphologic, immunohistochemical, and, often, molecular biologic (polymerase chain reaction [PCR]) analysis of vitrectomy or biopsy specimens, or both. In the case of lymphoma, PCR demonstrates a clonal rearrangement of the heavy (H) and light (L) chains of the immunoglobulin genes (Ig), appearing as a distinct band on the polyacrylamide gel, and as a single peak on gene scan analysis. The IgH consist of variable (VH), diversity (D), and joining (JH) gene segments, and a functional VH–D–JH complex is formed in the bone marrow during B-cell ontogeny when one of the numerous potential VH segments is combined with a D and JH gene segment.7 Maturation of the immune response is thought to occur in the germinal centers of peripheral lymphatic tissue on antigenic challenge by a process of somatic (hyper)mutation of the VH region.8 –10 It has been demonstrated that “naive” pregerminal center B cells carry nonmutated VH genes, whereas germinal center B cells and postgerminal-center (“memory”) cells express ISSN 0161-6420/01/$–see front matter PII S0161-6420(01)00594-2

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Ophthalmology Volume 108, Number 7, July 2001 mutated VH region genes.8 –11 This information is useful in the subtyping of B-cell non-Hodgkin’s lymphomas, dividing these malignancies into those descended from naive B cells and others descended from germinal-center or memory B cells. Most analyses of VH genes in B-cell chronic lymphocytic leukemia (B-CLL) have demonstrated an absence of somatic mutations,12–14 suggesting that these tumors arise from pregerminal center cells; this is supported by the immunophenotype of the B-CLL tumor cells. Recent investigations, however, show that some cases of B-CLL have some mutated VH genes.15,16 On the basis of the current understanding of the somatic mutation process, it can only be concluded that the latter B-CLL arise from memory B-cells. This discrimination between the two subtypes of B-CLL is important because clinical investigations suggest that the so-called “memory” B-CLL (with somatic mutations) behave less aggressively than their “naive” counterparts.15–18 A case of B-CLL with extensive ocular involvement and with subsequent infiltration of extraocular tissues and the meninges of the optic nerve is presented. The intraocular tumor manifestation had the same morphologic and immune profile as that of the tumor cells examined in the bone marrow and conjunctival biopsies. Further, using PCR and subsequent sequencing of the PCR products, it could be demonstrated that the tumor manifestations represented the same B-cell clone, which was derived from postgerminalcenter (memory) B-cells. Although B-CLL does not represent the most common lymphoma subtype in either the ocular adnexa2 or in intraocular tissues, a secondary manifestation of this tumor should be considered by ophthalmic pathologists in the differential diagnosis of ocular lymphoma.

poietic cell lineages. Abnormal liver function tests suggested hepatic involvement. The subsequent treatment included cyclophosphamide, vincristine, procarbazine, and prednisolone every 4 weeks. The patient was referred in August 1999 to the Ophthalmology Department because of loss of vision in the left eye. On initial examination, the visual acuity with correction was 6/6 in the right eye and light perception only in the left. The intraocular pressure measured 16 mmHg in both eyes on presentation. The additional findings on examination included conjunctival injection, a perilimbal ring-shaped salmon-colored conjunctival swelling (Fig 1), band keratopathy, partial vascularization of the cornea, a central “pseudohypopyon,” a hyphema with limited view of the iris, and only diffuse red light on funduscopy. Ultrasound examination did not reveal any abnormalities in either the vitreous cavity or posterior segment. A conjunctival biopsy was performed on September 1, 1999 on the basis of suspected B-CLL infiltration. Histologic examination confirmed this and also demonstrated purulent secretion that suggested infection. Conjunctival swab revealed colonization of Corynebacteria species. Despite broad spectrum antibiotic and antimycotic therapy, rapid corneal melting was observed 1 day later with spontaneous hemorrhage into the conjunctiva and with formation of a central corneal ulceration. Because of the risk of a purulent endophthalmitis developing in a blind eye, enucleation of the left eye without subsequent orbital implant insertion was undertaken. The postoperative course was relatively uncomplicated, and the patient was discharged on September 11, 1999 with a visual acuity of 6/6 in the right eye. A follow-up appointment was made for 6 weeks later. However, the patient died of cerebral manifestations of her disease in early October. Permission for an autopsy was not obtained.

Methods Specimens

Case Report In May 1983, a 59-year-old patient sought treatment from her general practitioner for postmenopausal bleeding. The patient, who appeared otherwise to be in good health, was referred to a specialist hospital in Berlin, where a curettage was performed. Although a gynecologic malignancy could be excluded, examination of the peripheral blood count revealed a lymphocytosis (77% lymphocytes with a total leukocyte count of 14.6/nl; normal range, 4 –10/nl). Consequently, the patient was referred for hematologic consultation, whereby a slightly enlarged right-sided supraclavicular lymph node was palpated. This was excised and, after fixation, examined using conventional and immunohistologic stains; the diagnosis of B-CLL was established. As part of the clinical staging investigations, a sternal marrow aspiration was performed, revealing tumor infiltration with a normal megakaryopoesis but reduced erythropoiesis and granulopoiesis (stage III, according to the Rai clinical classification; stage A, according to Binet). The initial treatment consisted of erythrocyte concentrates when required. Bilateral enlargement of inguinal lymph nodes in 1997 indicated disease progression, and the patient began combined chlorambucil and prednisolone therapy. In April 1999, anemia developed in the patient (Hb, 8.7 g/dl; normal range, 12–16 g/dl), thrombocytopenia (platelet count, 26/nl; normal range, 150 – 400/nl), and leukocytosis (leukocytes, 304/nl; normal range, 4 –10/nl). Repeat staging investigations, including bone marrow trephination, revealed extensive infiltration by the B-CLL with suppression of all hemo-

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The specimens received for pathologic examination included a bone marrow trephine, 17 mm in length and 2 mm in diameter; three conjunctival biopsies, 7 ⫻ 5 ⫻ 3 mm, 3 ⫻ 1 ⫻ 1 mm, and 3 ⫻ 2 ⫻ 1 mm; and the enucleated left eye, 25 ⫻ 24 ⫻ 23 mm, with an attached optic nerve, 7 mm in length. All specimens were fixed in 4% buffered formalin and embedded in paraffin. The bone marrow trephine required decalcification using edetic acid. Sections of all specimens were stained using hematoxylin and eosin, periodic acid–Schiff, and Giemsa. Special stains for the detection of fungi and bacteria (Gram, Grocott) were also applied to the conjunctival specimens.

Immunohistochemical Analysis Additional slides were stained for immunohistochemical studies using several monoclonal and polyclonal antibodies that are reactive in paraffin sections. An antigen retrieval method using a pressure cooker was performed before immunohistochemical staining.19 The staining consisted of a first-stage incubation with the following primary monoclonal antibodies: CD20, CD79a, CD10, CD5, CD21, MIB-1, BCL-2, cyclin D1, CD23, CD43. Polyclonal antibodies were used to test the expression of CD3 antigen and of the Ig chains ␬, ␭, ␮, ␥, ␦, and ␣. The antibodies were made visible with an indirect immunoperoxidase method for the antibodies to the heavy and light chains, whereas the alkaline phosphatase antialkaline phosphatase method was used to demonstrate the binding of the remaining antibodies.20

Coupland et al 䡠 “Memory” B-CLL with Ocular Involvement Table 1. Summary of the Morphologic, Immunohistologic, Molecular Biologic, and Clinical Features of B-cell Lymphocytic Leukemia Compared with Extranodal Marginal Zone B-cell Lymphoma B-cell Lymphocytic Leukemia Morphologic features ● “Pseudofollicular” growth pattern* ●

Prolymphocytes and paraimmunoblasts in the pseudofollicles

●

Small B-cell lymphocytic leukemia cells between the pseudofollicles

Immunohistologic features ● CD20⫹(weak), CD79a⫹, CD43⫹, CD23⫹, CD5⫹, BCL-2⫹, IgM⫹ ● CD10⫺, cyclin D1⫺ ● Usually absence of FDCs Genetics ● Clonal rearrangement of immunoglobulin H genes ● Somatic mutations in some cases Clinical features ● 20% of all NHL ● Peak age, 65 years ● Females ⬎ males ● Often BM infiltration or involvement of LN, spleen, and liver at time of diagnosis ● Not curable Cell of origin ● B-cell lymphocytic lymphoma without somatic mutations: naive B cell ● B-cell lymphocytic lymphoma with somatic mutations: memory B cell

Extranodal Marginal Zone B-cell Lymphoma Morphologic features ● Expansive growth in the marginal zone between reactive secondary follicles ● Centrocytelike cells, monocytoid B cells, plasmacytoid cells, occasional blasts ● Possibly lymphoepithelial lesions ● Often multifocal growth Immunohistologic features ● CD20⫹, CD79a⫹, CD43⫹ (often), BCL-2⫹, IgM⫹ ● CD5⫺, CD10⫺, CD23⫺, cyclin D1⫺ ● Presence of FDCs in reactive secondary follicles Genetics ● Clonal rearrangement of immunoglobulin H genes ● Somatic mutations in almost all cases Clinical features ● 8% of all NHL ● Peak age, 65 years ● Females ⬎ males ● Rarely involvement of BM or spleen at time of diagnosis ● Possible involvement of other extranodal sites (e.g., salivary glands, stomach) ● Tendency to recur Cell of origin ● Memory B cell

BM ⫽ bone marrow; FDC ⫽ follicular dendritic cells; LN ⫽ lymph node; NHL ⫽ non-Hodgkin’s lymphoma. *In extranodal sites, the typical morphologic pseudofollicular pattern may be absent.

All antibodies were obtained from DAKO (Glostrup, Denmark), Novocastra (Newcastle-upon-Tyne, England), or Transduction Laboratories (Lexington, KY), except for MIB-1, CD79a, and CD5, which were kindly provided by Dr. J. Gerdes (Borstel, Germany), Dr. D. Mason (Oxford, England), and Dr. K. Gatter (Oxford, England), respectively. The number of cells positive for MIB-1 was determined by counting the number of cells with clear nuclear positivity for these markers for 5 ⫻ 100 tumor cells using the ⫻40 objective.

Polymerase Chain Reaction Amplification Method DNA was extracted after dewaxing from 20-␮m thick paraffin sections of all specimens using QIAEX (Qiagen, Germany), according to the manufacturer’s recommendations. A nested PCR for rearrangements of IgH was performed as described previously.2 The first round of DNA amplification was performed with six family-specific FW1 primers in conjunction with a JH consensus primer. The reamplification was carried out with an aliquot (1%) of the first PCR using six family-specific FW2 primers in conjunction with a consensus JH primer. Reactive tonsils were used as polyclonal controls, whereas B-cell line DNA served as monoclonal controls. Sufficient numbers of PCRs without DNA were included as negative controls. Products were analyzed on 6% polyacrylamide gel stained with ethidium bromide and viewed under ultraviolet light. A discrete band (230 –280 base pairs long) after electrophoresis indicated monoclonality.

Gene Scan For precise size determination, the amplificates were separated on an automated DNA-sequencer (gene scan analysis). For this purpose, the reamplification was performed as described above with the exception being the use of a fluorescence-labeled VLJH primer and separation taking place on a DNA sequencing gel. Fluorescence-labeled PCR products were detected by a laser scanner, and their size was automatically calculated according to a differently labeled size standard separated in the same lane.

DNA Sequence and Somatic Mutation Analysis The DNA sequence analysis was performed using an automated DNA sequencer (Model AB1377A, Applied Biosystems, Weiterstadt, Germany) by using the DyeDeoxy Terminator Method for use solely with this system. For direct sequencing, the amplified products were separated by Polyacrylamide gel electrophoresis (PAGE), and appropriate bands were isolated by excising them under ultraviolet light. The excised bands were placed in separate Eppendorf tubes with 20 ␮l of distilled water for 24 hours, and 5 ␮l of this were used for sequencing. The isolated products were sequenced in both directions by using the reamplification primers, FR2A and VLJH, respectively, in two separate sequencing reactions. A comparison was performed with published VH germline sequences (VBASE; German Cancer Research Center, Heidelberg, Germany).21

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Figure 1. Clinical photograph demonstrating conjunctival injection, a perilimbal ring-shaped salmon-colored conjunctival swelling, band keratopathy, as well as partial vascularization of the cornea. Figure 2. Photomicrograph demonstrating extensive and diffuse infiltration of the bone marrow by the small atypical lymphocytes, characteristic of B-cell lymphocytic leukemia (stain, hematoxylin– eosin; original magnification, ⫻40.) Figure 3. Extensive and diffuse infiltration of B-cell lymphocytic leukemia in the conjunctiva (stain, hematoxylin– eosin; original magnification, ⫻200). Figure 4. Higher magnification of the B-cell lymphocytic leukemia (B-CLL) infiltrate, demonstrating the so-called “pseudofollicular” growth pattern of B-CLL, with the pseudofollicles (arrows) consisting of paraimmunoblasts and prolymphocytes (stain, Giemsa; original magnification, ⫻400).

Results Conventional Histologic and Immunohistologic Analysis The bone marrow trephine obtained in April 1999 was hypercellular and consisted mainly of small monomorphic atypical lymphocytes with sparse cytoplasm (Fig 2), clumped chromatin in the nuclei, and, occasionally, a small nucleolus (Table 1). The tumor cells took up approximately 90% of the total cell content in the

bone marrow. Conventional histologic examination of two of the three conjunctival specimens (Figs 3 and 4) demonstrated tumor cell infiltrates similar to those seen in the bone marrow. In addition, larger lymphoid cells (prolymphocytes and paraimmunoblasts) were present, imparting a so-called “pseudofollicular” pattern when viewed at low magnification (Fig 4) (Table 1). In the third conjunctival specimen, a fibrinous exudate with extensive neutrophil infiltration of the epithelium was seen. Macroscopic examination of the enucleated eye revealed a large central ulceration of the cornea covered by a red-yellow

Figure 5. Tumor manifestation within a massive choroidal hemorrhage (stain, hematoxylin– eosin; original magnification, ⫻200). Figure 6. Perivascular and intravascular (arrow) B-cell lymphocytic leukemia manifestation in the choroid, with drusen and detachment of the overlying retina (stain, periodic acid–Schiff; original magnification, ⫻200).

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Coupland et al 䡠 “Memory” B-CLL with Ocular Involvement

Figure 7. Perivascular B-cell lymphocytic leukemia infiltrates in the posterior extraorbital soft tissue (stain, hematoxylin– eosin; original magnification, ⫻200). Figure 8. Meningeal infiltration of the B-cell lymphocytic leukemia in the region of the optic nerve (stain, hematoxylin– eosin; original magnification, ⫻200). Figure 9. Tumor cells demonstrating positivity for CD23 (stain, alkaline phosphatase antialkaline phosphatase; original magnification, ⫻400). Figure 10. MIB-1 stain demonstrating a growth fraction of 5% of the tumor cells (alkaline phosphatase antialkaline phosphatase; original magnification, ⫻200).

gelatinous mass. Extensive hemorrhage was seen in the bulbar conjunctiva, the anterior chamber, and the posterior segment. On conventional histologic examination, fragments of anterior segment structures including the iris and the ciliary body, as well as diffuse tumor cell infiltrates, could be observed within coagulated blood. The remaining peripheral cornea showed stromal dissolution and extensive infiltration by both neutrophils as well as tumor cells. No lens material could be identified. Massive choroidal hemorrhage with accompanying retinal detachment could be observed. Tumor cell manifestation was seen within the choroidal hemorrhage, surrounding intrascleral and extraocular blood vessels and nerves as well as within the meninges around the optic nerve (Figs 5– 8). The immunophenotype of the tumor cells was identical in all three locations and is summarized in Table 1 (Fig 9). Further, the morphologic and immunohistologic characteristics of EMZL, the main differential diagnosis, are summarized in Table 1. The growth fraction, as determined using MIB-1 directed against the Ki-67 antigen, was between 5% and 10% (Fig 10).

Discussion B-cell lymphocytic leukemia represents the most common chronic lymphatic leukemia in Europe and North America. The disease, which is indolent but usually not curable, predominates in patients between the ages 58 and 70 years (mean, 65 years; Table 1).22 In most cases, there is involvement of the bone marrow and the peripheral blood. In some cases, an M-gradient (i.e., paraprotein in serum) can be demonstrated. Further, involvement of lymph nodes, the spleen, and the liver is common. Secondary involvement of

Polymerase Chain Reaction, Gene Scan, and Sequencing Analysis Polymerase chain reaction and gene scan analysis of all tissues examined revealed a clonal amplificate of the same size (230 bp; Fig 11). Subsequent direct sequencing of the PCR products and comparison with published germ line sequences revealed an IgH rearrangement involving the VH1 segment, which showed the same eight somatic mutations in all samples investigated.

Figure 11. Gene scan analysis of all tissues (bone marrow, lane 9; conjunctiva, lane 10; and enucleated eye, lane 11) revealing a B-cell clonal amplificate of the same size (230 base pairs).

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Ophthalmology Volume 108, Number 7, July 2001 ocular tissues in B-CLL is frequent and may occur in 80% to 90% of patients at some point during the disease.4,5 Morphologically, the B-CLL tumor cells are small (slightly larger than a normal lymphocyte) with clumped chromatin, a round nucleus, and occasionally a small nucleolus.23 Usually clustered in so-called pseudofollicles, larger lymphoid cells, prolymphocytes, and paraimmunoblasts are also present (Table 1). In extranodal sites however, the typical morphological, “pseudofollicular” pattern of B-CLL may be absent. The characteristic immune profile of the tumor cells is summarized and compared with that of EMZL in Table 1.23 The neoplastic cells of B-CLL usually demonstrate a clonal rearrangement of IgH, confirming their malignant origin. Previous investigations demonstrated an absence of somatic mutations on sequencing of the variable region of the IgH gene in B-CLL, suggesting that the tumor cells of B-CLL were derived from so-called pregerminal center or naive B-cells.12–14 However, recent research has demonstrated that some B-CLL express mutated VH genes,15,16 indicating their derivation from postgerminal center cells (or memory B-cells). The new terminology for the two subsets of B-CLL are, consequently, naive B-CLL and memory B-CLL. These findings are not only of interest for molecular biologists and pathologists, but also for all treating clinicians, because it has been demonstrated that a less aggressive disease course occurs in memory B-CLL compared with naive B-CLL15,16; the mean survival rates are 293 and 95 months (24 and 7 years), respectively, for patients in stage A disease (according to the Binet classification).16 In the present case, eight somatic mutations were detected in the VH gene, indicating that this tumor would belong to the memory B-CLL group. The relatively indolent course of the disease in our patient over a 16-year period, with ocular (and probably cerebral) infiltration only in the latter stages, therefore reflects the molecular findings. Although reasons for the different disease courses are not clear, it has been proposed that VH analysis in B-CLL should be included in routine staging investigations to aid the prediction of the individual clinical course in B-CLL patients and to determine potential new therapeutic strategies.15,16 The presence of memory B-CLL in ocular and ocular adnexal tissues is also of concern for ophthalmic pathologists, because some B-CLLs have remarkable resemblance to the more common ocular adnexal lymphoma, EMZL, and it may require extensive immunohistochemical analysis to differentiate between the two (Table 1). Despite the two subtypes belonging to the so-called low-grade lymphomas, it is of importance to distinguish them because of their different disease progression patterns and therapies. In the case of a memory B-CLL with IgH mutations, somatic mutation analysis may not necessarily be of assistance because ocular EMZLs, arising from postgerminal center cells, also display mutated VH genes.24 Although secondary leukemic infiltration of ocular tissues is not unusual, presented here is a case of B-CLL in which it could be demonstrated using PCR and gene scan analysis that the tumor manifestations in the bone marrow, conjunctiva, and intraocular tissues arose from the same

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B-cell clone. Further, sequencing of the PCR products demonstrated somatic mutations in the variable region of the IgH gene of the tumor cells, indicating their derivation from postgerminal center cells, that is, the tumor represented a memory B-CLL, the first case to our knowledge to be reported in the ophthalmic literature. Ophthalmologists should be aware of the different disease courses and, consequently, of the different prognoses of their patients with B-CLL, particularly when determining subsequent therapies in consultation with their oncology colleagues.

References 1. White WL, Ferry JA, Harris NL, Grove AS Jr. Ocular adnexal lymphoma. A clinicopathologic study with identification of lymphomas of mucosa-associated lymphoid tissue type. Ophthalmology 1995;102:1994 –2006. 2. Coupland SE, Krause L, Delecluse HJ, et al. Lymphoproliferative lesions of the ocular adnexa. Analysis of 112 cases. Ophthalmology 1998;105:1430 – 41. 3. Whitcup SM, de Smet MD, Rubin BI, et al. Intraocular lymphoma. Clinical and histopathologic diagnosis. Ophthalmology 1993;100:1399 – 406. 4. Duke-Elder S, editor-in-chief. System of Ophthalmology, Diseases of the Retina. Vol. X. St. Louis: Mosby, 1966;387–93. 5. Kincaid MC, Green WR. Ocular and orbital involvement in leukemia. Surv Ophthalmol 1983;27:211–32. 6. Rosenthal AR. Ocular manifestations of leukemia. A review. Ophthalmology 1983;90:899 –905. 7. Tonegawa S. Somatic generation of antibody diversity [review]. Nature 1983;302:575– 81. 8. Berek C, Berger A, Apel M, Maturation of the immune response in germinal centers. Cell 1991;67:1121–9. 9. Kelsoe G. The germinal center: a crucible for lymphocyte selection. Semin Immunol 1996;8:179 – 84. 10. Liu YJ, Joshua DE, Williams GT, et al. Mechanism of antigen-driven selection in germinal centres [letter]. Nature 1989; 342:929 –31. 11. Ku¨ppers R, Zhao M, Hansmann ML, Rajewsky K. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J 1993;12:4955– 67. 12. Aoki H, Takishita M, Kosaka M, Saito S. Frequent somatic mutations in D and/or JH segments of Ig gene in Waldenstro¨m’s macroglobulinemia and chronic lymphocytic leukemia (CLL) with Richter’s syndrome but not in common CLL. Blood 1995;85:1913–9. 13. Hummel M, Tamaru J, Kalvelage B, Stein H. Mantle cell (previously centrocytic) lymphomas express VH genes with no or very little somatic mutations like the physiologic cells of the follicle mantle. Blood 1994;84:403–7. 14. Meeker TC, Grimaldi JC, O’Rourke R, et al. Lack of detectable somatic hypermutation in the V region of the Ig H chain gene of a human chronic B lymphocytic leukemia. J Immunol 1988;141:3994 – 8. 15. Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840 –7. 16. Hamblin TJ, Davis Z, Gardiner A, et al. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848 –54. 17. Fais F, Ghiotto F, Hashimoto S, et al. Chronic lymphocytic

Coupland et al 䡠 “Memory” B-CLL with Ocular Involvement leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest 1998;102:1515–25. 18. Capello D, Fais F, Vivenza D, et al. Identification of three subgroups of B cell chronic lymphocytic leukemia based upon mutations of BCL-6 and IgV genes. Leukemia 2000; 14:811–5. 19. Norton AJ, Jordan S, Yeomans P. Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 1994;173:371–9. 20. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219 –29.

21. Tomlinson IM, Cook GP, Walter G, et al. A complete map of the human immunoglobulin VH locus [review]. Ann N Y Acad Sci 1995;764:43– 6. 22. A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin’s lymphoma. The Non-Hodgkin’s Lymphoma Classification Project. Blood 1997;89:3909 –18. 23. Harris NL, Jaffe ES, Stein H, et al. A revised EuropeanAmerican classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group [review]. Blood 1994;84:1361–92. 24. Coupland SE, Foss HD, Anagnostopoulos I, et al. Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa. Invest Ophthalmol Vis Sci 1999;40:555– 62.

OPHTHALMIC PATHOLOGY FELLOWSHIP Research to Prevent Blindness and the American Ophthalmological Society–Knapp Fund is offering a new two-year postgraduate fellowship for training in ophthalmic pathology with an annual stipend of $52,500. The first year of the proposed fellowship program will be spent in the study of diagnostic pathology and in the initiation of experimental eye pathology laboratory research. The second year of fellowship training will include experimental pathology research combined with exclusive time in diagnostic pathology or time in a relevant clinical subspecialty. Applicants must be graduates of a medical school accredited by the American Medical Association, citizens of the United States, and have plans for an academic career. Deadline for submission of applications: January 15, 2002 for fellowship starting in July, 2002. Please direct all inquires and requests for application materials to: Froncie A. Gutman, M.D. AOS-Knapp Fund Cleveland Clinic Foundation 9500 Euclid Avenue, Desk i-32 Cleveland, OH 44195 216-445-8145

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