Belgard, Christopher

Dirnhofer, Stephan

Mihatsch, Michael J.

Moch, Holger

Raggi, Anna

Schraml, Peter

Singer, Gad

Went, Philip

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CORRESPONDENCERe: Discrete Alterations inthe BZLF1 Promoter in Tumorand Non-Tumor-AssociatedEpstein–Barr VirusIn their recent article, Gutie´rrez et al.(1) identified three Epstein–Barr virus(EBV) variants in the BZLF1 promoterZp (Zp-P [considered the prototypicalsequence], Zp-V3, and Zp-V4) and pos-tulated that these might result in differ-ent lytic potentials and be differentiallyexpressed among individuals with non-malignant and malignant diseases. HongKong and Southern China are consid-ered to be endemic regions, with theEBV variants that are different fromother localities.We analyzed EBV type and sequencevariations in EBV Zp domains by directDNA sequencing in a total of 95 EBV-associated lesions (96 isolates) from in-dividuals from Hong Kong and South-ern China (Table 1). Eighty-seven of 95(91.6%) lesions contained type A EBV.Eight (8.4%) lesions contained type BEBV, all of which carried Zp-V3. Of the88 type A EBV isolates, 31 (35.2%)contained Zp-P and 53 (60.2%) con-tained Zp-V3. From 36 nonmalignantsamples with type A EBV, 19 (52.8%)contained Zp-P, and 16 (44%) containedZp-V3. From 51 malignant samples, weobtained 52 EBV isolates with type AEBV, of which 12 (23.1%) containedZp-P and 37 (71.2%) contained Zp-V3.In addition, from one nonmalignant andthree malignant samples with type AEBV, we identified four novel Zp-Pvariants. Three variants differed fromZp-P at one position only: Zp-V1_104(–104C-A), isolated from a healthy car-rier; Zp-V1_105 (–105C-T), isolatedfrom a natural killer (NK)/T-cell lym-phoma; and Zp-V1_119 (–119G-A), iso-lated from a nasopharyngeal carcinoma(NPC). All these substitutions occurredwithin the ZIIIB domain of Zp, whichcan strongly bind ZEBRA (Z, EB repli-cation activator) and contribute to thetransactivation of Zp (2). A fourth vari-ant (Zp-V3+118), isolated from an NPC,contained four nucleotide substitutionsrelative to Zp-P, three of which wereidentical to those found in Zp-V3 (1),and the fourth a C to T substitution atposition –118. This position is alsowithin the functional domain of ZIIIB. Itis noteworthy that this variant with fournucleotide substitutions was differentfrom the Zp-V4 variant described byGutie´rrez et al. (1). Taken together,these results suggest that the ZIIIB do-main may be a mutational hotspot.It was suggested that only Zp-P andZp-V4, but not Zp-V3, were found inthe nonmalignant samples (1). However,in the 40 nonmalignant samples fromour locality, Zp-P and Zp-V3 occurredwith similar frequency (19 of 40 and 20of 40, respectively). None of our 95samples contained Zp-V4, suggestingthat the Zp-V4 identified by Gutierrez´et al. (1) may be restricted to people in aparticular geographic region. In our ma-lignant NPC samples, 80.5% (33/41)contained Zp-V3 and 14.6% (6/41) con-tained Zp-P, whereas in the nonmalig-nant samples, 50% (20/40) containedZp-V3 and 47.5% (19/40) containedZp-P. Thus, Zp-V3 was more prevalentin NPC samples than in nonmalignantsamples (P  .001, chi-square test). Ifwe considered only type A EBV iso-lates, then the association between NPCand Zp-V3 was still statistically signifi-cant (P  .001).In our locality, the distribution ofEBV variants with respect to Zp invarious EBV-associated conditions isdistinct from other geographic regions.Although Zp-V3 is detectable in nonma-lignant samples, it is statistically signifi-cantly associated with NPC. Whetherthese variants have a functional con-sequence remains to be determined.However, the possible link(s) betweendifferent variants and lytic potential orlatency-to-lytic cycle switch requiresfurther investigation.JOANNA H. M. TONGKWOK WAI LOFLORENCE W. L. AUDOLLY P. HUANGKA-FAI TOREFERENCES(1) Gutierrez MI, Ibrahim MM, Dale JK, GreinerTC, Straus SE, Bhatia K. Discrete alterationsin the BZLF1 promoter in tumor and non-tumor-associated Epstein-Barr virus. J NatlCancer Inst 2002;94:1757–63.Table 1. Distribution of BZLF1 promoter variants in type A and type B Epstein–Barr virus (EBV)isolates from healthy carriers and various EBV-associated conditions*Total Zp-P Zp-V3 Zp-V3+118 Zp-V1Type A EBVNon-malignantHC 21 11 9 0 1IM 15 8 7 0 0Total 36 19 16 0 1MalignantNPC† 37 6 29 1 1NK/T 12 6 5 0 1‡PTLD 3 0 3 0 0Total 52 12 37 1 2Type B EBVNon-malignantHC 3 3IM 1 1Total 4 4MalignantNPC 4 4Total 4 4*A total of 96 EBV isolates from 40 non-malignant samples and 55 malignant samples were analyzedby direct sequencing. Briefly, amplification of position −221 to +12 (with reference to the transcriptionstart site) of the BZLF1 promoter region was performed by using the polymerase chain reaction (PCR) asdescribed by Gutie´rrez et al. (1). Purified PCR products were sequenced using ABI PRISM Big Dyeterminator cycle sequencing Kit (Applied Biosystems, Foster City, CA), and the sequencing products wererun on an ABI 377 DNA auto-sequencer (Applied Biosystems). The non-malignant samples included 24gargle specimens collected by rinsing the mouth with phosphate-buffered saline from healthy donors whowere EBV carriers (HC) and 16 peripheral blood samples from subjects with infectious mononucleosis(IM). The malignant samples included biopsies of 39 nasopharyngeal carcinomas (NPC), 11 natural killer(NK)/T-cell lymphomas (NK/T), three EBV-positive post-transplant lymphoproliferative disease (PTLD)samples, and two NPC xenografts (Xeno-666 and Xeno-2117).†Including two NPC xenografts, both carried type A EBV and contained Zp-V3.‡This sample was co-infected with a variant containing Zp-P.1008 CORRESPONDENCE Journal of the National Cancer Institute, Vol. 95, No. 13, July 2, 2003(2) Flemington E, Speck SH. Autoregulation ofEpstein-Barr virus putative lytic switch geneBZLF1. J Virol 1990;64:1227–32.NOTESSupported by Research Grants Committee Ear-marked Grant CU4301/99M, Hong Kong SpecialAdministrative Region of the People’s Republic ofChina.Affiliation of authors: Department of Anatomi-cal and Cellular Pathology, Prince of Wales Hos-pital, The Chinese University of Hong Kong,Hong Kong.Correspondence to: Ka-Fai To, F.R.C.P.A., De-partment of Anatomical and Cellular Pathology,Prince of Wales Hospital, The Chinese Universityof Hong Kong, Shatin, NT Hong Kong (e-mail:kfto@cuhk.edu.hk).RESPONSETong et al. have extended the studyrelated to BZLF1 polymorphic variantsof Epstein–Barr virus (EBV) to samplesfrom Hong Kong and Southern China.This study is of special interest becauseit incorporates malignant and nonmalig-nant samples from regions that are en-demic for nasopharyngeal carcinoma(NPC). Their observations both confirmand contradict several conclusions fromour study (1). First, they provide inde-pendent confirmation of the existence ofthe Zp-P and Zp-V3 variants in anothergeographic region. Our earlier observa-tions suggested that Zp variants segre-gated strongly by EBV type, with type Bvirus exclusively associating with Zp-V3 and type A virus often associatingwith Zp-P. Exceptions were noted insamples from Far East Asia, which con-tained type A virus with a Zp-V3 se-quence, suggesting that type A Zp-V3may be prevalent in this geographic re-gion. Tong et al. confirm these findingsand validate the existence and the preva-lence of the type A Zp-V3 variant in theSoutheastern Asian population.Type B virus is generally consideredto be more readily inducible than thetype A virus (2). We have speculatedthat the association of a virus with ma-lignant and nonmalignant lesions is in-fluenced by the lytic potential of the vi-ral types, which in turn may be dictatedby the variation in Zp. Tong et al. founda statistically significant association be-tween type A Zp-V3 and NPC. One pos-sible interpretation of these observationswould be that a type A Zp-P variant ismore responsive to lytic induction than atype A Zp-V3 variant. However, addi-tional regulatory elements beyond Zpalso modulate BZLF1, and the possibil-ity that variations in these regions occurbetween type A Zp-P and type A Zp-V3needs to be considered.Several important differences alsoarise between our observations (1) andthose of Tong et al. For example, there isthe complete absence of Zp-V4 in theirsamples. We identified 53 Zp-V4 vari-ants among our nonmalignant samples.It is highly unlikely that these differ-ences are merely technical. An impor-tant element in these two studies per-tains to the origin of the samples. Oursamples from South and North America,Saudi Arabia, and Japan—all regionsnon-endemic for NPC (3)—containedEBV with a Zp-V4 variant. If indeed theprevalence of Zp-V4 is geographicallyrestricted, it raises important epidemio-logic considerations that may affect thepathogenesis of EBV-associated malig-nancies. If the absence of Zp-V4 in tu-mors reflects lower oncogenic potential,the diminished prevalence of this strainin a population endemic for EBV-asso-ciated malignancies is of notable in-terest. Additional studies from otherendemic and non-endemic areas are re-quired to establish these associations.The apparent mutual exclusivity ofZp-V4 and Zp-V3 EBV in non-endemicand endemic populations, respectively,is intriguing. Given the low levels ofEBV DNA in nonmalignant samples,the possibility that Zp-V3 and Zp-V4variants are present at undetectable lev-els cannot be ruled out. Furthermore, theidentification of other variants—albeitat low frequency—raises the possibilitythat ongoing mutational events may in-deed play a role in the genesis of suchvariations (4).MARINA I. GUTIE´RREZSTEPHEN E. STRAUSMUNA M. IBRAHIMJANET K. DALEKISHOR BHATIAREFERENCES(1) Gutie´rrez MI, Ibrahim MM, Dale JK, GreinerTC, Straus SE, Bhatia K. Discrete alterationsin the BZLF1 promoter in tumor and non-tumor-associated Epstein-Barr virus. J NatlCancer Inst 2002;94:1757–63.(2) Buck M, Cross S, Krauer K, Kienzle N, Scul-ley TB. A-type and B-type Epstein-Barr virusdiffer in their ability to spontaneously enterthe lytic cycle. J Gen Virol 1999;80:441–5.(3) Waterhouse JAH, Muri C, ShanmugaratnamK. Cancer incidence in five continents. Vol. V.Lyon (France): International Agency for Re-search on Cancer (IARC) Sci Publ 1987(88).p. 42.(4) Gutie´rrez MI, Spangler G, Kingma D, RaffeldM, Guerrero I, Misad O, et al. EBV in nasallymphomas contains multiple ongoing muta-tions in the EBNA-1 gene. Blood 1998;92:600–6.NOTESAffiliations of authors: M. I. Gutie´rrez, M. M.Ibrahim, K. Bhatia, Research Centre, King FahadNational Children’s Cancer Centre and Research,Riyadh, Saudi Arabia; S. E. Straus, J. K. Dale,Laboratory of Clinical Investigation, Division ofIntramural Research, National Institute of Allergyand Infectious Diseases, National Institutes ofHealth, Bethesda, MD.Correspondence to: Kishor Bhatia, Ph.D., KingFaisal Specialist Hospital, P.O. Box 3354, MBC98-16, Riyadh 11211 Saudi Arabia (e-mail: kishor_bhatia@kfshrc.edu.sa).Journal of the National Cancer Institute, Vol. 95, No. 13, July 2, 2003 CORRESPONDENCE 1009

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KIT in Ovarian Carcinoma: Disillusion About a Potential Therapeutic Target

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