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Epstein-Barr Virus–Induced CD30-PositiveDiffuse Large B-Cell Lymphoma in a PatientWith Mixed-Phenotypic Leukemia Treated

With ClofarabinePavan Kumar Bhamidipati,1 Elias Jabbour,1 Sergej Konoplev,2 Zeev Estrov,1

Jorge Cortes,1 Naval Daver1

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IntroductionLymphoproliferative disorders (LPD) have been reported in sev-

eral clinical settings associated with immunosuppression, such asprimary immune deficiency, human immunodeficiency virus infec-tion, bone marrow or solid organ transplantation, methotrexate ortumor necrosis factor-therapy, and aging.1,2 A significant number ofhese cases are associated with Epstein-Barr virus (EBV). AlthoughBV� LPDs have been documented in patients with low-grade lym-

1Department of Leukemia2Department of Hematopathology

he University of Texas MD Anderson Cancer Center, Houston, TX

Submitted: Jul 10, 2012; Revised: Sep 28, 2012; Accepted: Oct 1, 2012; Epub: Dec14, 2012

Address for correspondence: Naval Daver, MD, Department of Leukemia, Unit 428,1515 Holcombe Boulevard, The University of Texas MD Anderson Cancer Center,Houston, TX 77030

Clinical Pra● Epstein-Barr virus (EBV)–positive lymphoproliferative

disorders have been reported in several clinical set-tings associated with immunosuppression, such asprimary immune deficiency, human immunodeficiencyvirus infection, bone marrow or solid organ transplan-tation, methotrexate or tumor necrosis factor therapy,and aging. We did not identify any reported cases thatdescribed EBV� lymphoproliferative disorders afterpurine analog therapy for acute myeloid leukemia ormixed-phenotype leukemia.

● Patients with acute myeloid leukemia and with persistentor progressive lymphadenopathy should undergo fineneedle aspiration to rule out extramedullary leukemia(granulocytic sarcoma) or concurrent lymphoma.

Clinical Lymphoma, Myeloma & Leukemia,Keywords: Clofarabine, Diffuse large cell lymph

E-mail contact: ndaver@mdanderson.org

Clinical Lymphoma, Myeloma & Leukemia June 2013

phomas treated with purine analogues, including cladribine3,4 orudarabine,5,6 we could not identify any reports of EBV� LPD after

purine analogue (including clofarabine) therapy for acute myeloidleukemia (AML) or mixed-phenotype acute leukemia (MPAL). Be-cause clofarabine is being used with increasing frequency in AMLtherapy,7,8 such phenomenon may be noted with increasing fre-quency in the future. In our patient, R-CHOP (rituximab, cyclo-phosphamide, doxorubicin, vincristine, prednisone) therapy9,10 ef-fectively treated the EBV� LPD, and this may help guide treatmentin future occurrences of a similar nature.

Case ReportA 50-year-old white man presented to the MD Anderson Cancer

Center’s leukemia clinic with a 30-day history of pancytopenia andfatigue. A significant medical history included hypertension and cor-onary arthrosclerosis. Results of a physical examination revealed fever

ice Points● Identifying the ideal regimen for patients with mixed-

phenotype leukemia requires large clinical trials. How-ever, our patient with mixed-phenotype leukemiaachieved complete remission with clofarabine, idaru-bicin, cytarabine, and Solu-Medrol therapy.

● Although antivirals (acyclovir, ganciclovir, and vala-cyclovir) have been proposed for prevention andtreatment of a EBV-driven lymphoproliferative dis-order, the infected latent B cells often do not ex-press thymidylate kinase, which is the target forthese antivirals, which is clearly seen in our patientbecause prophylactic valacyclovir was clearly inef-fective in preventing the occurrence of EBV-drivenlymphoma.

13, No. 3, 342-6 Published by Elsevier Inc.Epstein-Barr Virus, Mixed phenotype leukemia

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and splenomegaly. A complete blood cell count showed a white cell

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count of 1 � 109/L (reference range, 4-11 � 109/L), hemoglobinevel of 72 g/L (reference range, 140-180 g/L), platelet count of 31 �

09/L (reference range, 140-440 � 109/L), and absolute neutrophilount of 0.06 � 109/L (reference range, 1.7-7.3 � 109/L). Bone

marrow biopsy revealed cellular marrow with 65% blasts. Flow cy-tometry immunophenotype analysis of bone marrow aspirate mate-rial detected a distinct population of immature cells with mixedmyeloid–T-lymphocyte phenotype. The neoplastic cells were posi-tive for CD7 (partial), CD34, CD117, HLA-DR (partial), myelo-peroxidase (small subset), CD13, CD64, CD33 (partial), CD71,CD123 (partial), TdT, CD10, CD38, and CD33 (partial), and forcytoplasmic CD3. The neoplastic cells were negative for CD1a,CD2, surface CD3, CD4, CD5, CD8, CD14, CD19, CD41, TCRalpha/beta, and TCR gamma/delta. The immunophenotype of theblasts fulfilled diagnostic criteria for MPAL, T/myeloid as defined bythe European Group for the Immunologic Characterization of Leu-kemias11 and by the 2008 World Health Organization classifica-tion.12 Conventional cytogenetic studies revealed a complex karyo-type, including -3, add (5) (q35), -7, -11, -15, -16, -18, and -20chromosomal aberrations. The patient was seen by the stem celltransplant service at initial diagnosis of biphenotypic leukemia withcomplex karyotype and was thought to be a good candidate for con-solidation with allogeneic stem cell transplantation. He received in-duction therapy with clofarabine (15 mg/m2 for 3 days), cytarabine0.75 g/m2 for 3 days), idarubicin (8 mg/m2 for 3 days), and meth-

ylprednisolone (40 mg intravenous for 3 days). Day-21 bone marrowaspirate showed complete morphologic and cytogenetic remission.

After chemotherapy, the patient became pancytopenic. Twenty-eight days after completion of induction therapy, he presented to theemergency department with a sore throat and progressive swelling inthe neck. Computerized tomography (CT) of the neck showed ne-crotic lymphadenopathy in bilateral submandibular regions and en-hancement of the palatine tonsils. A throat swab and blood culturesgrew vancomycin-resistant enterococci. The initial antibiotics in-cluded meropenem and linezolid. Subsequently, fine needle aspira-tion of a submandibular lymph node revealed inflammatory infiltratewith no neoplastic cells and no microbial growth on cultures.

The patient went on to receive 2 cycles of consolidation therapy at4-weekly intervals per protocol with clofarabine, cytarabine, and ida-rubicin. Four weeks after completion of the second consolidationcycle, he presented with worsening neck swelling. A repeated CT ofthe neck showed resolving submandibular lymphadenitis. A CT ofthe chest showed multiple lung nodules. Biopsy of a lung noduleshowed diffuse infiltration by neoplastic cells with perivascular infil-tration and necrosis. The neoplastic cells were large, with irregularshaped nuclei and inconspicuous nucleoli (Figure 1C). They werepositive for CD20 (Figure 1D), CD30 (Figure 1E), and PAX-5, andwere negative for CD3, CD33, CD34, and TdT. In situ hybridiza-tion studies for EBV small encoded RNA (EBER) demonstrated thatthe neoplastic cells were strongly positive for EBER (Figure 1F).Peripheral blood EBV quantitative polymerase chain reaction (PCR)identified a viral load of 16,626 EBV copies/mL of plasma (normallyundetectable). These results led to a diagnosis of EBV� diffuse largeB-cell lymphoma (DLBCL). We retrospectively performed EBER onan initial bone marrow specimen to see if EBV was present at the time

of initial diagnosis and may have caused the MPAL. EBER was neg- b

ative on the initial bone marrow. We were unable to perform fluo-rescence in situ hybridization for c-myc or BCL2/IGH because ex-tensive necrosis in the tissue sample precluded fluorescence in situhybridization analysis.

Whole-body positron emission tomography–CT showed in-creased fluorine-18 fluorodeoxyglucose uptake in bilateral pulmo-nary nodules, bilateral cervical lymph nodes, stomach, spleen, andliver (Figure 1A). Bone marrow biopsy showed 0% abnormal blastsand no morphologic evidence of DLBCL or morphologic evidence ofMPAL. The patient was staged as IVE, S DLBCL (Cotswold modi-fication of the Ann Arbor staging system). He continued to be fol-low-up with the stem cell transplant service, and a 10/10 siblingdonor was identified. Unfortunately, he developed the DLBCL be-fore proceeding to allogeneic transplant for the biphenotypicleukemia.

After discussion with lymphoma specialists, the R-CHOP regi-men was initiated. After 1 course, he had significant shrinkage ofpalpable lymph nodes and significantly diminished fluorine-18 fluo-rodeoxyglucose uptake on the positron emission tomography (Figure1A, B). A repeated EBV serology performed 1 week later showedundetectable EBV levels in serum. He received a total of 2 cycles ofR-CHOP therapy for the DLBCL. The stem cell transplant servicecontinued to follow up the patient with the hope to proceed toallogeneic transplantation as a potential therapy for both the biphe-notypic leukemia and DLBCL. Unfortunately, before he could pro-ceed to allogeneic stem cell transplantation, the patient developed aprogressive fungal pneumonia with sepsis and expired in the inten-sive care unit.

DiscussionWe report a case of EBV� DLBCL in a patient treated with clo-

farabine for MPAL. EBV� LPD has been reported in several clinicalsettings associated with primary or acquired immunosuppres-sion.1,2,11,12 Although cases of EBV� LPD have been documentedn patients with low-grade lymphomas and hairy cell leukemiareated with fludarabine and cladribine,3-6 we did not find any re-orted cases of EBV� LPD in patients with MPAL or AML treatedith purine analogues.Our patient initially presented with MPAL, which accounts for

ewer than 4% of all acute leukemias.13,14 Results of several studiesave suggested that patients with MPAL have a worse clinical out-ome than AML and/or acute lymphoblastic leukemia (ALL).15,16

There is no consensus as to whether induction therapy should bewith lymphoid drugs, myeloid drugs, or combinations.17 A retro-spective study reported that induction with combined AML andALL drugs led to a high rate of early death in patients with adult andpediatric MPAL, although most of these patients had t(9,22), which,on its own, portends a poor prognosis.16 In pediatric MPAL, induc-ion with ALL-type therapy or combined therapy with agents that arective against lymphoid and myeloid leukemias has shown to yieldigher complete remission rates than AML-type induction ther-py.18,19 It is unknown whether similar strategies are applicable todults.

The mechanisms by which EBV stimulates lymphoid prolifera-ion and malignant transformation are poorly understood. EBV

inds to receptors on B cells and pharyngeal surface molecule

Clinical Lymphoma, Myeloma & Leukemia June 2013 343

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CD21.20 The initial EBV infection is often subclinical. It then per-ists in the memory cells as a latent infection through integration ofiral DNA into B-cell DNA. It is proposed that the EBV infectionhen induces upregulation of BCL2 and MYC genes associated withnti-apoptosis and cell proliferation, respectively.21 This transformshe B cell into a constantly proliferating lymphoblastoid cell. NF-�B

and mammalian target of rapamycin (mTOR) pathways may alsoplay a role in this transformation. EBV has been associated with

Figure 1 (A) A Positron Emission Tomography–Computed Tomocyclophosphamide, doxorubicin, vincristine, prednisoSections of Lung Biopsy Demonstrate Numerous LargNucleoli (H&E, original magnification �500. (D) The n(Immunohistochemistry (IHC), original magnification �(IHC, original magnification �500. (F) In situ hybridizDemonstrated That the Neoplastic Cells Were Strongl

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multiple B- and T-cell lymphoproliferative disorders, including a

Clinical Lymphoma, Myeloma & Leukemia June 2013

DLBCL, Hodgkin lymphoma, posttransplant lymphoproliferativediseases, AIDS-associated lymphomas, Burkitt lymphoma, primarycentral nervous system lymphoma, mucosa-associated lymphoidtissue, angioimmunoblastic lymphoma and peripheral T-cell lym-phoma.2,22,23

Clofarabine is a new-generation purine nucleoside (deoxyadenos-ine) analogue and is closely related to other members of this class,such as cladribine and fludarabine.24 It has more cytotoxic properties

hy (PET-CT) Before Therapy With R-CHOP (rituximab,B) PET-CT After 1 Cycle of R-CHOP. (C) Routinely Stainedoplastic Cells With Irregular-shaped Nuclei and Inconspicuousastic cells are B cells as evident by CD20 Expression). (E) The Neoplastin Cells are Also Strongly Positive for CD30studies for Epstein-Barr Virus Small Encoded RNA (EBER)itive for EBER (original magnification �500)

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farabine was initially approved, in 2007, by the US Food and DrugAdministration for treatment of pediatric ALL.25 The therapeuticadvantage of clofarabine-based therapies in the treatment of refrac-tory AML and high-risk myelodysplastic syndrome in the elderly iswell known.7,8

Clofarabine primarily inhibits DNA polymerases, which results inimpaired DNA repair and apoptosis. Treatment with purine ana-logues can induce prolonged suppression of CD4� and CD8� cells,sometimes exceeding 24 months.20,26 The resultant immunosup-pression may allow EBV reactivation and the occurrence of LPDs.Such phenomena have been well documented in early rather thanlate posttransplant LPDs, which are 100% EBV driven.20,23 A de-rease in EBV-specific cytotoxic T lymphocytes and an increase inhe EBV viral load have been found to be strongly associated with theevelopment of posttransplant lymphoproliferative disease.27 Thessociation of EBV with AML is unknown at this time. Clofarabines being used with increasing frequency in the therapy of AML. Tour knowledge, this is the first documented report of a LPD thatccurred after purine analogue therapy for AML. With such limitedxperience, it would be premature to recommend routine evaluationith EBV PCR for all patients exposed to purine analogue therapy.owever, with close clinical follow-up and assessment with EBV

CR when clinically indicated, we may identify similar cases in theear future.Corticosteroids at high doses can induce significant immunosup-

ression that may potentiate the occurrence of EBV LPDs. However,ur patient received Solu-Medrol at a dose of 40 mg/d for 5 days. Athis dose, we believed it unlikely that steroids alone caused sufficientmmunosuppression to be implicated as the primary cause of the

LBCL. Clofarabine is known to induce a more prolonged immu-osuppression than cytarabine. Viral infections were noted withreater frequency with single-agent clofarabine at a dose of 30 mg/m2

compared with clofarabine 30 mg/m2 in combination with cytara-bine 20 mg/m2.7 In a phase III trial that compared clofarabine incombination with cytarabine with cytarabine alone, severe infectionsand deaths related to adverse events were significantly more in theclofarabine-alone arm.28 Results of these studies suggest that clofara-ine produces a more potent and prolonged suppression of lympho-yte-mediated immunity than cytarabine. Hence, we thought it to behe primary culprit.

EBV� LPDs represent a wide spectrum of entities that range fromeactive follicular or paracortical hyperplasia to full-blown DLBCLr Hodgkin lymphoma. The prognosis depends on the morphologicntity, being excellent for reactive conditions and dismal for DLBCLnd Hodgkin lymphoma.2,22 Currently, most EBER � DLBCL are

treated with standard DLBCL regimens by using a combination ofanthracycline, steroids, alkylating agents, and vincristine (CHOP).29

The addition of rituximab has improved 3- and 5-year overall sur-vival in comparison with CHOP alone.9 Feugier et al10 documentedimproved outcomes, specifically in patients with CD20�, EBER �

DLBCL treated with rituximab in combination with standardCHOP therapy. Newer therapeutic approaches, including treatmentof posttransplant lymphoproliferative diseases with autologous EBV-specific cytotoxic T lymphocytes have been shown to reduce viralloads and to shrink tumor size.30 Current clinical trials are exploring

he efficacy of administering such EBV cytotoxic T lymphocytes

2

xpressing CD30 chimeric receptors in CD30� non-Hodgkin lym-homa (http://ClinicalTrials.gov Identifier: NCT01192464) andthers (NCT00058617, NCT00779337).

Although antivirals (acyclovir, ganciclovir, and valacyclovir) haveeen proposed for the prevention and treatment of EBV-drivenPDs, latent B cells often do not express thymidylate kinase, which ishe target for these antivirals. Thus, their role in prevention may beimited, which may have been the case in our patient, who was onalacyclovir prophylaxis and yet developed EBV infection. A recenthase I/II trial has shown a promising response with simultaneousse of arginine butyrate (a thymidylate kinase inhibitor) and ganci-lovir (an antiviral) in the treatment of patients with EBV-associatedymphoid disorders.31

DisclosureThe authors have stated that they have no conflicts of interest.

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