• © 2008 by American Society of Clinical Oncology

Second Epstein-Barr Virus–Associated Burkitt's Lymphoma of the CNS in a Child With Progressive Renal Failure

A 14-year-old boy was admitted to the Hematology Service in November 2002, with CNS involvement. The patient had been diagnosed with Burkitt's lymphoma (BL) in March 1998, with pelvic mass presentation, severe ascitis, and bone marrow infiltration (St. Jude stage IV) associated with acute renal failure and tumor lysis syndrome. Magnetic resonance imaging (MRI) of the pelvis showed a solid mass, producing bone lesion by contiguity in L5 and parts of the sacrum, penetrating the right neural foramen in L5 with invasion of vertebral canal and neural root compression. Immediate Berlin-Frankfurt-Muenster–based treatment¹ resulted in complete clinical remission, although the patient continued to experience a neurogenic bladder and progressive deterioration of renal function. A month before his second admission, neurologic involvement was apparent, with seizures and temporary loss of consciousness. At his second admission, the patient showed 11,000/μL leukocyte, 343,000/μL platelets, 8.2 g/dL hemoglobin, 288 UI/L lactate dehydrogenase (normal range, 240 to 480 UI/L), 5.80 mg/dL uric acid, 8.34 mg/dL calcium, 5.60 μmol/L potassium, and 3.22 mg/dL serum creatinine. Viral serology was negative for human T-cell leukemia-lymphoma virus I and II, hepatitis A virus, and hepatitis C virus. Investigation of HIV infection, performed several times along the course of his second disease and afterward, yielded negative results. Brain MRI revealed a hypodense mass in the right temporal-occipital region (Fig 1A), although abdominal computed tomography scan and a bone marrow aspirate did not show tumor involvement. A stereotactic biopsy of the cerebral mass followed by histopathologic analysis showed a CD20⁺, CD10⁺, CD3⁻ and BCL2⁻–non-Hodgkin's lymphoma, compatible with BL diagnosis, which was confirmed by interphase fluorescent in situ hybridization (I-FISH). Due to severe renal failure (creatinine clearance 20 mL/min by Cockcroft-Gault formula), whole-brain radiotherapy was administered, with 36 Gy divided in 20 fractions, and a boost of 8 Gy in four fractions. Figure 1B shows brain MRI after the end of treatment. The patient achieved complete clinical remission and is at present being observed by clinicians. He has not relapsed or shown signs of neurotoxicity related to whole-brain radiotherapy. DNA was extracted from pathology specimens of the first tumor (frozen cytospins), and from the small biopsy of the second tumor together with RNA and cell imprintings. In both tumors, Epstein-Barr virus (EBV) was detected by specific polymerase chain reaction (PCR) analyses and, in the second tumor, by EBV-encoded RNA in situ hydridization (Fig 2A),² though without expressing LMP1 oncoprotein (clones CS1-4; DAKO; Glostrup, Denmark). A t(8;14)(q24;q32) translocation was detected by I-FISH with LSI IGH/MYC, CEP 8 tri-color, dual fusion translocation probes (Vysis; Downers Grove, IL). Figures 2B and 2C show I-FISH images of normal brain cells and a tumor cell, respectively. Arrows show two co-localized signals corresponding to t(8;14). Immunoglobulin (Ig) clonal gene rearrangements amplified by Framework Region 2 (FR2)–JH nested PCR³ showed clearly different clonality patterns. Figure 3 shows a monoallelic clonal rearrangement superimposed on a polyclonal background in the first tumor, and in the second tumor, two rearrangements of different size from the one observed in the first tumor (Fig 3). These results suggested that the tumors were clonally unrelated, and that the second tumor was a primary CNS lymphoma (PCNSL). The Variable Diversity–Joining rearrangements of the Ig variable region from the first tumor (FR2-JH fragments) and from the second tumor (cDNA amplified with VH1/7 family primers),⁴ after subcloning in a pCR2.1 vector (Invitrogen; Carlsbad, CA), were sequenced and compared with germline, V-BASE sequence data (http://www.mrc-cpe.cam.uk/) using MacVector 10.0 software (Accelrys, Inc, San Diego, CA). As expected from gel analysis, the first tumor showed a VH4-2/JH4b unmutated rearrangement coexisting with a large number of polyclonal rearrangements, mostly involving VH3 and VH4 families. Conversely, the second tumor showed two in-frame Variable Diversity–Joining rearrangements, V2-05/JH4b and V3-30/D3-22/JH4b, with 98.01% and 98.13% homology with germline alleles, respectively. The V3-30 allele showed intraclonal variation. In all VH genes, S (silent) and R (replacement) mutations were randomly distributed in complementarity-determining or framework regions, suggesting the absence of B-cell receptor–mediated selection in tumor cells. Activation-induced cytidin deaminase (AID) gene expression was quantified by real-time PCR in the second tumor, by a TaqMan assay (Applied Biosystems; Foster City, CA; inventoried assay), showing an approximately eight-fold lower AID expression in tumor cells with respect to quiescent, mononuclear peripheral-blood cells used for calibration.

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Fig 1.

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Fig 2.

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Fig 3.

PCNSL are aggressive non-Hodgkin's lymphomas the incidence of which has been increasing in immunocompetent patients in the last few years.⁵ More than 90% of PCNSL patients have diffuse large B-cell lymphomas (DLCBLs); the remaining 10% comprise a suite of rare and poorly-characterized lymphomas, including BL. Conversely to HIV-associated patients, PCNSLs in immunocompetent individuals are mostly non-EBV associated and usually occur in patients aged older than 50 years.^5,6 Cell origin and differentiation stage of PCNSL are controversial. The high somatic hypermutation (SHM) frequency of Ig VH genes (mean, 13% v 5% to 6% of normal germinal center [GC] cells) with ongoing mutation in some patients^7-9 initially suggested a GC origin, but recent studies, based on the expression of GC and activation of B-cell markers,^10,11 rather suggest a late GC or early post-GC origin. We studied a Burkitt's PCNSL in a patient with progressive renal failure. This case was especially unique in several respects because: (1) it was a second BL, clonally unrelated to the first tumor; (2) it consisted of an EBV-associated CNS lymphoma in an HIV-negative child; and (3) it showed a very different Ig SHM pattern from BL and from previously reported PCNSLs. Detection of t(8;14) in tumor imprintings was especially relevant for confirming diagnosis in view of the complexity and rarity of this case and the difficulties in obtaining tumor material. The disparate clonality patterns of tumor samples clearly indicate that the second tumor did not originate from residual clones of the first. This is most unusual because BL rarely arises as a secondary malignancy. In this case, an abnormal CD4+/CD8+ T-cell ratio (0.48), consistently detected after the onset of the second tumor, suggested an underlying subclinical immunodeficiency. Patients with end-stage renal disease show clinical signs of immune deficiency characterized by increased susceptibility to infections and low response to B-cell vaccination.^12,13 The mechanisms accounting for this deficiency remain unknown, although changes in T-cell function may substantially contribute to this condition, given that end-stage renal disease is associated with B and T lymphopenia.^12,13 In addition, reduced CD4+/CD8+ T-cell ratios and low numbers of CD4+ and B memory cells are also found in patients with chronic renal failure.^14-16 In the case herein described, the reduced CD4+/CD8+ ratio and other immune defects resulting from progressive, chronic renal failure might have impaired the effective EBV immune control and contributed to the development of a second EBV-associated BL. The remarkably low SHM frequency found in this case, when compared with BL (4% to 8%) and PCNS-DLCBL,^7-10 suggested a different cell origin with respect to previously described patients. In our patient, the finding of ongoing mutations was unexpected in view of the absolute requirement of AID for SHM, although in one study of PCNS-DLCBLs, the majority of the tumors did not express AID despite high SHM frequencies and presence of ongoing mutation.¹⁰ This suggests that SHM and intraclonal variation might have occurred at an earlier phase of clonal expansion when tumor cells expressed AID. Thus, AID-silencing in PCNSLs might not be a developmentally regulated process but rather a postoncogenic event likely mediated by the CNS microenvironment. Nevertheless, in this case, it is difficult to envisage the proper stimulus for maintaining intraclonal variation in the tumor, in the absence of antigenic selection. Investigating the factors involved in the maintenance of clonal diversity in PCNSLs might be relevant for therapeutic development in these conditions.