BioMed Cen al
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BMC Cance
Open Access
Resea ch a icle
Cy o oxic d ug sensi i i y o Eps ein-Ba i us ans o med
lymphoblas oid B-cells
Laszlo Ma kasz1,2, Gyö gy S ube 1, Emilie Flabe g1, Åsa Gus a sson Je nbe g3,
S a an Eksbo g4, E a Olah2, Hen ie e Sk ibek1 and Laszlo Szekely*1
Add ess: 1Depa men o Mic obiology, Tumo and Cell Biology (MTC) and Cen e o In eg a i e Recogni ion in he Immune Sys em (IRIS),
Ka olinska Ins i u e, Box 280 S-17177 S ockholm, Sweden, 2Depa men o Pedia ics Uni e si y o Deb ecen, Medical and Heal h Science Cen e ,
Deb ecen, Hunga y, 3Depa men o Pedia ics, Ka olinska Ins i u e , Ka olinska Uni e si y Hospi al, Huddinge, Sweden and 4Ka olinska
Pha macy, and Depa men o Woman and Child Heal h, Childhood Cance Resea ch Uni , Ka olinska Ins i u e , Ka olinska Uni e si y Hospi al,
S ockholm, Sweden
Email: Laszlo Ma kasz - ma kaszl@ eemail.hu; Gyö gy S ube - [email p o ec ed]; Emilie Flabe g - Emilie[email p o ec ed];
Åsa Gus a sson Je nbe g - Asa.Gus a sson.Je nbe[email p o ec ed]; S a an Eksbo g - s a an.eksbo g@ka olinska.se; E a Olah - [email p o ec ed].hu;
Hen ie e Sk ibek - [email p o ec ed]; Laszlo Szekely* - [email p o ec ed]
* Co esponding au ho
Abs ac
Backg ound: Eps ein-Ba i us (EBV) is he causa i e agen o immunosupp ession associa ed
lymphop oli e a ions such as pos - ansplan lymphop oli e a i e diso de (PTLD), AIDS ela ed
immunoblas ic lymphomas (ARL) and immunoblas ic lymphomas in X-linked lymphop oli e a i e
synd ome (XLP). The epo ed o e all mo ali y o PTLD o en exceeds 50%. Reducing he
immunosupp ession in ecipien s o solid o gan ansplan s (SOT) o using highly ac i e
an i e o i al he apy in AIDS pa ien s leads o comple e emission in 23–50% o he PTLD/ARL
cases bu will no su ice o ecipien s o bone ma ow g a s. An addi ional he apeu ic al e na i e
is he ea men wi h an i-CD20 an ibodies (Ri uximab) o EBV-speci ic cy o oxic T-cells.
Chemo he apy is used o he non- esponding cases only as he second o hi d line o ea men .
The mos equen ly used chemo he apy egimens o igina e om he non-Hodgkin lymphoma
p o ocols and he e a e no cy o oxic d ugs ha ha e been speci ically selec ed agains EBV induced
lymphop oli e a i e diso de s.
Me hods: As lymphoblas oid cell lines (LCLs) a e well es ablished in i o models o PTLD, we
ha e assessed 17 LCLs o cy o oxic d ug sensi i i y. A e h ee days o incuba ion, li e and dead
cells we e di e en ially s ained using luo escen dyes. The p ecise numbe s o li e and dead cells
we e de e mined using a cus om designed au oma ed lase con ocal luo escen mic oscope.
Resul s: Independen ly o hei o igin, LCLs showed e y simila d ug sensi i i y pa e ns agains
29 equen ly used cy os a ic d ugs. LCLs we e highly sensi i e o inc is ine, me ho exa e,
epi ubicin and pacli axel.
Conclusion: Ou da a shows ha he inclusion o epi ubicin and pacli axel in o chemo he apy
p o ocols agains PTLD may be jus i ied.
Published: 13 No embe 2006
BMC Cance 2006, 6:265 doi:10.1186/1471-2407-6-265
Recei ed: 30 Augus 2006
Accep ed: 13 No embe 2006
This a icle is a ailable om: h p://www.biomedcen al.com/1471-2407/6/265
© 2006 Ma kasz e al; licensee BioMed Cen al L d.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
BMC Cance 2006, 6:265 h p://www.biomedcen al.com/1471-2407/6/265
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Backg ound
De elopmen o malignan B-cell lymphomas a e o gan
ansplan a ion is a signi ican complica ion a ising as a
side e ec o he immunosupp ession equi ed o suc-
cess ul g a su i al. The oncogenic Eps ein-Ba i us
(EBV) is he e iologic agen in he pos ansplan lympho-
p oli e a i e diso de (PTLD) and AIDS ela ed immuno-
blas ic lymphomas (ARL) [1]. The epo ed o e all
mo ali y o PTLD o en exceeds 50% [2,3]. The p ogno-
sis o PTLDs occu ing a e bone ma ow ansplan a ion
is e en wo se [4,5]. Male pa ien s wi h he a e inhe i ed
X-linked lymphop oli e a i e synd ome, showing speci ic
immune de ec agains EBV in ec ion, also o en succumb
o EBV induced malignan lymphomas [6].
EBV is a ubiqui ous human he pes i us ha pe sis s o
li e. P ima y EBV in ec ion can lead o mononucleosis
(IM) in adolescence and in adul s, mani es ed by a mas-
si e expansion o B cells. EBV-encoded ans o ma ion-
associa ed p o eins d i e he p oli e a ion o B lymphob-
las s in IM, in PTLDs and in immunode iciency syn-
d ome-associa ed immunoblas ic lymphomas. The EBV
ans o med cells exp ess nine la ency-associa ed i al
p o eins: EBNA1-6, LMP-1, -2A and -2B. This la ency p o-
g am is e e ed o as he ype III la ency. The same la ency
p og am is p esen in he in i o p oli e a ing lymphob-
las oid cell lines (LCLs), gene a ed by in ec ion o no mal
human B cells wi h EBV. The ac ion o B cells ha is sus-
cep ible o in i o ans o ma ion can be any hing
be ween 10% and 100% [7].
EBV d i es he p oli e a ion o human B cells in i o and
du ing p ima y in ec ion in i o. S ong T cell-media ed
immune esponses ha e been documen ed agains EBV
encoded la en p o eins and a wide ange o HLA class I
molecules wi h EBV o igina ed pep ide epi opes ha e
been iden i ied [8-10]. EBV associa ed lymphop oli e a-
i e disease can de elop only in he absence o a compe-
en cy o oxic T cell immune su eillance. EBV associa ed
lymphop oli e a i e disease may disappea upon ea -
men es o ing he immune esponse agains EBV-in ec ed
B cells.
His o ically LCLs we e o en ega ded as non- umo igenic
in immunosup essed mice upon subcu aneous inocula-
ion, especially in compa ison wi h highly umo igenic
Bu ki 's lymphomas. Howe e in ape i oneal inocula-
ion egula ly leads o de elopmen o gene alized lym-
phomas wi h mul io gan in ol emen . SCID mice
inocula ed in ape i oneally wi h pe iphe al blood lym-
phocy es (PBL) om EBV-se oposi i e dono s o wi h
human LCLs, de elop EBV-induced human lymphop oli -
e a ions wi hin a ew weeks. These lymphomas a e classi-
ied as immunoblas ic lymphomas, o en wi h
plasmacy oid ea u es [11]. His ologically he PBL de i ed
human-SCID umo s e y much esemble he EBV posi-
i e la ge cell lymphomas o immunosupp essed pa ien s
[12]. The umo s o he immunocomp omised pa ien s o
he expe imen al umo s g owing in immunode ec i e
mice as well as he in i o g owing LCLs show e y simila
pheno ypes. All h ee exp ess he same spec um o cell
su ace ma ke s, B cell ac i a ion an igens and adhesion
molecules. All h ee ha e no mal ka yo ype and show
iden ical i al gene exp ession pa e ns.
The isk o PTLD has been ound o depend upon he ype
o he ansplan ed o gan, he immunosupp essi e egi-
men, he age, he unde lying illness and he EBV s a us o
he ecipien a he ime o ansplan a ion. The es ima ed
incidence o PTLD anges om 1–4% a e enal ans-
plan a ion o 19% a e in es inal ansplan a ion. In
bone ma ow allog a ecipien s PTLD is ela i ely
uncommon (1%) [13,14] excep o when ce ain high
isk egimes, such as in i o T-cell deple ion (TCD) a e
used, when he isk may ise o 30%[4,15] PTLD ollow-
ing allogenic s em cell ansplan a ion usually de i es
om dono lymphocy es. The isk o PTLD is g ea e i he
hos is EBV-se onega i e a he ime o ansplan a ion
and/o i he e is a misma ch be ween he dono and
ecipien HLA ypes [1].
No con olled s udies ha e been pe o med in he man-
agemen o PTLD and mos o he ecommenda ions o
he apy come om small coho s a single ins i u ions [1].
The ela i e impo ance o T cell impai men , EBV and
clonal p oli e a ion has led o he ollowing s a egies:
educ ion o immunosupp ession o p ophylac ic es o a-
ion o T-cell immuni y [16], an i i al he apy and chem-
o he apy. Reducing he immunosupp ession leads o
comple e and du able emission o PTLD [17] o 23–
50% o pa ien s a e o gan ansplan a ion (SOD) bu
will no be e icien in he BMT se ing. Reduc ion o
immunosupp ession is equen ly he i s he apeu ic
s ep, and pa ien s who ha e had o gan ejec ion ha e a
much poo e p ognosis.
Fo seconda y EBV lymphomas pos BMT none o he
abo e egimens will su ice, excep when speci ically
es o ing EBV speci ic immuni y [16]. An i-B-cell mono-
clonal an ibodies a e an e ec i e he apy o PTLD. A
combina ion o an i CD21 and an i-CD24 an ibodies
we e used. Howe e hese an ibodies a e no comme -
cially a ailable and in e es has he e o e ecen ly u ned
owa ds Ri uximab, a humanized monoclonal an ibody
agains he pan-B cell ma ke CD20. The esponse a e o
Ri uximab ea men is 65% wi h a elapse a e o 18%
[18,19].
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In cases ha a e no esponding o he abo e men ioned
he apy, chemo he apy is used as he second o hi d line
o op ion.
The mos equen ly used chemo he apy p o ocols a e he
an h acycline based CHOP (cyclophosphamide, doxo u-
bicin, inc is ine and p ednisolone) and VAPEC-B (doxo-
ubicin, e oposide, cyclophosphamide, me ho exa e,
bleomycin, and inc is ine) [20]. In he s udy o Mu i e
al. 17 o 40 PTLD pa ien s we e ea ed wi h a combina-
ion o educed immunosupp ession and chemo he apy
(CHOP, VACOP-B – cyclophosphamide, inc is ine, ble-
omycin, p ednisolone, wi h inc easing doses o doxo u-
bicin and e oposide- o DHAP -cispla inum, cy osine-
a abinoside, dexame hasone) [21]. Remission a e o
an h acycline-based combina ion he apy is 69%.
PTLD pa ien s a e exposed o a high mo ali y isk due o
chemo he apy ea men ela ed oxici y [1].
The side e ec s o a d ug a e he limi ing ac o in de e -
mining he dose. Inc easing he dose o a d ug leads o a
mo e equen appea ance o side e ec s. Chemo he a-
peu ic agen s can also comp omise he su i al o he
g a . D ugs ha a e e ec i e e en a low doses could
he e o e, no only gi e ewe side e ec s, bu also p o ide
a be e g a su i al.
The aim o his s udy was o ind he mos e ec i e cy o-
oxic d ugs agains EBV-induced lymphop oli e a ions.
We ca ied ou o e 6500 cell su i al and p oli e a ion
assays, on single cell le el, in o de o es ablish he pa e n
o in i o e icacy o he mos commonly used cy o oxic
d ugs agains EBV- ans o med lymphoblas oid cells.
Me hods
Cell lines and cul u e condi ions
17 lymphop oli e a i e cell lines (LCLs) we e used in he
p esen s udy. 980215, 031016, 040113, 051018, JAK, LP,
MIN, AF, GK, FUR, HA, VMB we e es ablished by in i o
in ec ion o no mal B lymphocy es, om di e en heal hy
dono s, wi h he B95.8 s ain o EBV. These cell lines we e
expanded in IMDM (Sigma) medium supplemen ed wi h
10% e al cal se um (FCS). LSspon an is a spon aneous
LCL es ablished om a heal hy dono . IARC171 was
es ablished using B95.8 i us in ec ion o no mal lym-
phocy es om a pa ien wi h Bu ki 's lymphoma. TR was
es ablished om B cells o an XLP pa ien , wi h dele ion
in he SAP gene. The cell line IB4 con ains an in eg a ed
EBV genome.
Du ing he ime o expe imen he LCLs we e cul u ed in
IMDM (Sigma) supplemen ed wi h 20% FCS (Sigma),
100 mmol L-glu amine (Sigma) and 80 µg/ml gen amicin
(Sigma). Cell suspensions we e g own in a humidi ied
incuba o a 37°C in an a mosphe e con aining 5% CO2.
Cell coun s we e adjus ed o an op imal concen a ion o
1 × 106 cells/ml and he cells we e ed wice a week. The
absence o mycoplasma con amina ion was assu ed by
egula moni o ing wi h Hoechs 33258 s aining.
D ugs
Fo he in i o d ug sensi i i y es 29 d ugs we e used
(summa ized in Table 1). All he d ugs we e dissol ed in
50% dime hyl sul oxide (DMSO) and p in ed on 384 well
pla es, using a high densi y me al pin a ay (wi h 50 nl
eplica olumes) in Biomek 2000 luid dispense obo
(Beckman). The same obo was used o gene a e he d ug
mas e pla es con aining he iplica es o ou di e en
d ug dilu ions using a single ip au oma ic dispense
head.
The highes d ug concen a ion was selec ed as he physi-
cochemical maximally achie able d ug concen a ion a
600 imes dilu ion (50 nl d ug in 30 µl suspension assay
olume)
The Ra io o Maximum Achie ed Plasma Concen a ion
(RMAPC) was de e mined o each d ug concen a ion in
o de o compa e he in i o es ed concen a ions o he
in i o maximally achie ed plasma concen a ion (Cmax)
le els.
RMAPC was calcula ed acco ding o he ollowing equa-
ion:
RMAPC = in i o es ed concen a ion/Cmax
In i o d ug sensi i i y assay
In i o d ug sensi i i y o LCLs was assessed using a 3-day
cell cul u e on mic o i e pla es. 28 d ugs we e es ed, each
a 4 di e en concen a ions in iplica es on 384-well
pla es (G eine ). Each well was loaded wi h 30 µl cell sus-
pension con aining 3000 cells. A e h ee days o incuba-
ion he li e and dead cells we e di e en ially s ained
using luo escen Vi alDye (Bioma ke , Hunga y). The
exac numbe o li e and dead cells was de e mined using
a cus om designed au oma ed lase con ocal luo escen
mic oscope (a modi ied Pe kin-Elme Ul aView LCI sys-
em) a he Ka olinska Ins i u e isualiza ion co e acili y
(KIVIF). The images we e cap u ed using he compu e
p og am Quan Cap u e 4.0 and he li e and dead cells
we e iden i ied and indi idually coun ed using he p o-
g am Quan Coun 3.0. Bo h p og ams we e de eloped a
he KIVIF using OpenLab Au oma o p og amming en i-
onmen (Imp o ision). 15 con ol wells, ha we e used
o de e mine he con ol cell su i al (CCS), con ained
cells wi h only cul u e medium and 50 nl DMSO wi hou
d ugs, 5 wells con ained cells wi h cul u e medium alone.
Compa ing he wo ypes o con ol wells no oxic e ec
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Table 1: Used d ugs, Cmax, AUC le els
D ug g oups D ug name B and name, company In i o used
concen a ions
(µg/ml)
Cmax Value
(µg/ml)
In i o dose
(Cmax)
AUC
(µg × h/ml)
In i o dose
(AUC)
Re e ence
an h acyclines epi ubicin Epi ubicin Meda, Meda 0.026–1.66 9 120 mg/m22.412 90 mg/m2(Fogli e al. 2002) [23]
dauno ubicin Ce ubidin®, A en is Pha ma 0.065–4.166 0.49 0.95 mg/kg in 45 min 1.2786 1.5 mg/kg (Ande sson e al. 1979) [6]
doxo ubicin Doxo ubicin Te a, Te a 0.026–1.66 0.93 50 mg/m20.82464 50 mg/m2(To oli e al. 2004) [24]
epipodophyllo oxin e oposide Sigma 0.325–20.833 2.5 53 mg/m2/day 5.06 100 mg/m2/day (Minami e al. 1995) [25] (G ube e al.1995)
[26]
camp o hecins opo ecan Hycam in®, GlaxoSmi hKline 0.052–3.33 0.0084 2.3 mg/m2/day 0.0196 1.2 mg/m2/day (Ge i s e al. 1999) [27]
axanes pacli axel Taxol, O i a m 0.078–5 3.38 175 mg/m2 by 3 h 13.49 175 mg/m2(Fogli e al. 2002) [23]
doce axel Taxo e e®, A en is Pha ma 0.52–33.33 2 60 mg/m23.326 85 mg/m2(Rischin e al. 2002) [28]
inca alkaloids inc is ine Vinc is ine Mayne, Mayne Pha ma 0.0065–0.416 0.37 1.4 mg/m20.182 1.32 mg/m2(Te ashi e al. 2000) [29] (Desai e al. 1982)
[30]
inblas ine Velbe®, STADApha m 0.013–0.833 0.005 1.5 mg/m2 by 24 h 0.218 1.7 mg/m2(S ewa e al. 1983) [31] (Ba es e al.) [32]
ino elbine Na elbine®, Pie e Fab e 0.13–8.33 0.83 200 mg/week 0.899 80 mg/m2/week (Zhou e al. 1991) [33] (F eye e al. 2003)
[34]
pla inum analogs ca bopla in Ca bopla in Mayne, Mayne Pha ma 0.13–8.33 0.046 360 mg/m2348000 360 mg/m2(Ghazal-Aswad e al. 1996) [35]
oxalipla in Eloxa in, Sano i-Syn helabo 0.065–4.166 3.2 130 mg/m271.5 130 mg/m2(Gamelin e al. 1997) [36] (G aham e al.
2000) [37]
an i ola e me ho exa e Me ho exa e Pha macia, P ize 0.325–20.833 363 8–12 g/m213200000 12 g/m2(Bacci e al. 2006) [38] (C ews e al. 2004)
[39]
pu in an agonis s 6-me cap opu ine Sigma 1.085–69.44 2 2.5 mg/kg o al dose 0.2587 85 mg/m2(Chan e al. 1989) [26]
clad ibine Leus a in, Janssen-Cilag 0.013–0.833 0.0356 5 mg/m20.1541 5 mg/m2(Albe ioni e al. 1998) [40]
py imidine an agonis s luo ou acil Fluo ou acil Mayne, Mayne Pha ma 0.65–41.66 55.4 400 mg/m211.59 400 mg/m2(Casale e al. 2004) [41]
cy a abine Cy a abine P ize , P ize 1.302–83.33 10.8 3 g/m2523.4 1 g/m2(DeAngelis e al. 1992) [42] (G ube e al.
1995) [26]
gemci abine Gemza , O i a m 0.781–50 22.3 1000 mg/m29.3 1000 mg/m2(Fogli e al. 2002) [23]
an i umo an ibio ics bleomycin Bleomycin Bax e , Bax e 0.39–25* 0.19* 30 IU/day 0.089 8 IU/kg/day (B ough on e al. 1977) [43] (Peng e al.1980)
[44]
dac inomycin Cosmegen®, MSD 0.006–0.416 0.1 1.5 mg/m2300 1.5 mg/m2(Veal e al. 2005) [45]
p o ease inhibi o s bo ezomib Velcade®, Janssen-Cilag 0.013–0.833 0.08 1.45 mg/m20.0438 1.45 mg/m2(Papand eou e al. 2004) [46]
MG132 Sigma 0.13–8.33 #
alkyla ing agen s cyclophosphamide Sendoxan, Bax e 0.52–33.33 37.2 50 mg/kg 367 50 mg/kg (Xie e al. 2006) [47]
i osphamide Holoxan®, Bax e 0.52–33.33 56.63 16 g/m2 in 96 h 1827.7 3 g/m2/day (W igh e al. 1995) [48] (Boddy e al. 1996)
[45]
chlo ambucil Sigma 1.302–83.33 0.49 0.2 mg/m20.883 0.2 mg/m2(GlaxoSmi hKline Resea ch T iangle Pa k)
[49]
s ep ozo ocin Sigma 0.65–41.66 ¤
miscellaneous aspa aginase Aspa aginase Medac, Medac 0.065–4.166* 4.48* 10000 IU/m20.943 30000 IU/m2(Ho e al. 1981) [50] (Ylikangas e al. 2002)
[51]
hyd oxyu ea Sigma 0.65–41.66 21 18 mg/kg 82.49 15 mg/kg (Gwil e al. 2003) [52] (Yan e al. 2005) [46]
p ednisolone Di-Ad eson F Aquosum, O ganon 1.56–100 0.325 1 mg/kg/day 1.33 1 mg/kg/day (Ros in e al. 1990) [53]
Cmax: maximum plasma le el a e adminis a ion o he clinical dose o he d ug
*In case o bleomycin and aspa aginase concen a ions a e in IU/ml
AUC: a ea unde cu e
#: no used in he clinical p ac ice
¤: no da a is a ailable
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o DMSO could be seen. Mean cell su i al (MCS) was
de e mined om he a e age o cell su i al o all 17
LCLs.
Resul s
Compa ison o he d ug sensi i i y pa e n o he di e en
LCLs
The 17 di e en LCLs ha we e es ed in he p esen s udy
ep esen a la ge a ie y o cells wi h di e en o igins and
in i o his o y. The in es iga ion included cell lines wi h
se e al yea s o con inuous in i o cul u ing along wi h
eshly es ablished ans o med B-cell cul u es o only 3
weeks o age. Many o he lines we e ans o med by he
B95-8 s ain o EBV and some o hem we e spon aneous
ou g ow hs d i en by he dono s own i us. Among he
lines, we had LCLs om heal hy dono s, om XLP
pa ien s o lines es ablished om he no mal B-cells o
Bu ki 's lymphoma pa ien s. We es ed lines wi h bo h
episomal and in eg a ed EBV genome.
Rema kably, he LCLs o di e en o igins showed a e y
simila sensi i i y pa e n agains he di e en d ugs. This
phenomenon is illus a ed in Figu e 1, which summa izes
he su i al cu es o all 17 LCLs agains he ou di e en
concen a ions o epi ubicin. Each indi idual hin cu e
ep esen s he a e age o h ee independen measu e-
men s. The hick line is he mean o all cu es, he Mean
Cell Su i al (MCS). The g ey shaded a ea ma ks he ±
s anda d de ia ion (SD). Figu e 2 summa izes he Mean
Cell Su i al cu e along wi h he s anda d de ia ions o
28 d ugs again emphasizing he e y simila esponse o
he di e en cell lines. The alues o MG132 a e ep e-
sen ed only in Table 2.
Iden i ica ion o highly e ec i e and non-e ec i e d ugs
The s a ing concen a ions o he dilu ion se ies (1×, 4×,
16×, 64×) o he indi idual d ugs we e ini ially de e -
mined based on he solubili y o he di e en agen s. To
make he cell su i al da a compa able wi h each o he ,
we i s compiled he known Maximum Achie ed Plasma
Concen a ion da a (Cmax) o all 28 d ugs s a ed om
he li e a u e (Table 1-column 5). Di iding he ac ual
d ug concen a ions in he dilu ion se ies wi h he Cmax
o he pa icula d ug yielded he Ra io o Maximum
Achie ed Plasma Concen a ion (RMAPC). The calcula ed
alues o RMAPC o all he d ugs a e summa ized in
Table 2 along wi h MCS and ± SD alues. Plo ing he cell
su i al agains he RMAPC alues allowed a di ec com-
pa ison o he e ec i eness o di e en d ugs. Figu e 3
shows he compa ison o he e ec i eness o he membe s
o h ee di e en d ug amilies: he an h acyclines, inca
alcaloids and axans. Figu e 4 summa izes he mean cell
su i al cu es o all he d ugs along a common RMAPC
axis. Based on hei e ec i eness on LCLs, we ha e di ided
he d ugs in o h ee di e en g oups:
G oup 1: The d ugs we e conside ed o be highly e ec i e
i he mean cell su i al (MCS) was below 30% a RMAPC
0.3 o below ha .
G oup 2: The d ug was pa ially e ec i e i MCS was unde
60% and RMAPC was be ween 0.3 and 1.
G oup 3: The d ug was ine ec i e, e.g. he LCLs we e
esis an o he d ug, i MCS was abo e 60% o RMAPC
was abo e 1.
We ound ha he ou mos e ec i e d ugs agains LCLs
we e: inc is ine, pacli axel, me ho exa e and epi u-
bicin (G oup 1).
Gemci abin, cy osine-a abinoside, doxo ubicin, luo ou -
acil, dac inomycin, doce axel, dauno ubicin, e oposide,
ino elbine was a ed as pa ially e ec i e (G oup 2.)
Mos LCLs we e esis an o cyclophosphamide, aspa agi-
nase, opo ecan, oxalipla in, bleomycin, 6-me cap opu-
in, hyd oxyu ea, clad ibine, chlo ambucil, ca bopla in,
bo ezomib, cy a abine, p ednisolone and inblas ine
(G oup 3). Almos no d ug esponse could be seen wi h
oxalipla in, bleomycin, cyclophosphamide, aspa aginase,
hyd oxyu ea and i osphamide whe eas inblas ine, chlo-
ambucil, p ednisolone and opo ecan we e e ec i e, bu
only a e y high concen a ions, well abo e he maxi-
mum achie able plasma concen a ions.
Al hough he G oup 3 d ugs we e no e ec i e agains
LCLs, hese d ugs show concen a ion dependen g ow h
D ug sensi i i y pa e n o 17 lymphoblas oid cell lines o epi ubicinFigu e 1
D ug sensi i i y pa e n o 17 lymphoblas oid cell
lines o epi ubicin. The mean alue o su i al o LCLs is
ep esen ed by he black cu e, ± SD alue is ma ked wi h
g ey. 1× : highes concen a ion, 16×: 16 imes dilu ion o he
highes concen a ion e c.

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Mean alues and s anda d de ia ions o d ug sensi i i y o 17 lymphoblas oid cell lines o 28 di e en cy os a ic d ugsFigu e 2
Mean alues and s anda d de ia ions o d ug sensi i i y o 17 lymphoblas oid cell lines o 28 di e en cy o-
s a ic d ugs. y axis: ac ion o su i ing cells 0–100% x axis: d ug dilu ions (1× ep esen s he highes concen a ion).
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Table 2: The a e age Mean Cell Su i al (MCS) o he 17 LCLs a di e en d ug concen a ions, exp essed as he Ra io o Maximum
Achie ed Plasma Concen a ion (RMAPC)
64× dilu ion 16× dilu ion 4× dilu ion 1× no dilu ion 64× dilu ion 16× dilu ion 4× dilu ion 1× no dilu ion
G oup 1 Vinc is ine Epi ubicin
RMAPC 0.018 0.071 0.282 1.128 RMAPC 0.003 0.012 0.046 0.184
MCS 81.498 46.541 20.776 18.995 MCS 85.206 65.736 29.435 16.306
SD 27.995 25.737 15.258 12.209 SD 15.548 23.394 13.593 11.314
Pacli axel Me ho exa e
RMAPC 0.020 0.081 0.326 1.303 RMAPC 0.001 0.004 0.014 0.057
MCS 48.119 29.999 24.653 23.148 MCS 76.618 44.824 32.924 29.314
SD 28.435 17.418 12.869 13.364 SD 22.796 25.843 15.590 16.455
G oup 2 Gemci abin Dac inomycin
RMAPC 0.035 0.140 0.561 2.242 RMAPC 0.066 0.262 1.050 4.200
MCS 49.354 38.411 27.808 23.784 MCS 86.206 54.700 20.520 16.465
SD 34.718 30.034 16.945 12.153 SD 16.701 22.386 13.642 8.370
5-Flu ou acil Vino elbine
RMAPC 0.012 0.047 0.188 0.752 RMAPC 0.156 0.625 2.501 10.005
MCS 74.861 58.001 42.337 30.505 MCS 82.833 42.823 25.176 24.683
SD 23.686 22.987 21.370 20.645 SD 18.131 23.930 16.506 14.634
Doxo ubicin Dauno ubicin
RMAPC 0.028 0.112 0.447 1.789 RMAPC 0.133 0.531 2.126 8.502
MCS 82.503 52.477 28.292 11.499 MCS 83.171 61.159 25.940 20.439
SD 21.753 24.867 22.314 10.301 SD 21.886 25.402 23.933 17.961
E oposide Doce axel
RMAPC 0.130 0.521 2.083 8.333 RMAPC 0.260 1.042 4.166 16.665
MCS 56.281 35.375 25.315 16.778 MCS 32.990 23.036 23.866 26.331
SD 21.045 19.983 15.497 11.166 SD 22.690 17.587 16.810 19.810
G oup 3 Cy a abine 6-Me cap opu ine
RMAPC 0.121 0.482 1.929 7.716 RMAPC 0.543 2.170 8.680 34.720
MCS 87.321 79.450 48.255 40.124 MCS 84.450 78.834 62.325 55.419
SD 17.396 22.850 26.423 26.315 SD 22.147 16.514 29.820 32.858
Oxalipla in Clad ibine
RMAPC 0.020 0.081 0.325 1.301 RMAPC 0.366 1.462 5.850 23.399
MCS 87.951 84.477 83.331 73.511 MCS 102.832 101.241 72.497 52.237
SD 21.010 26.545 21.839 30.744 SD 8.130 11.160 26.804 29.545
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Aspa aginase Chlo ambucil
RMAPC 0.015 0.058 0.232 0.930 RMAPC 2.646 10.586 42.342 169.370
MCS 92.021 91.570 90.371 82.978 MCS 89.903 71.879 13.225 11.099
SD 13.718 13.222 18.153 15.919 SD 17.578 26.506 10.994 7.527
Cyclophosphamide Topo ecan
RMAPC 0.014 0.056 0.224 0.896 RMAPC 6.194 24.777 99.107 396.429
MCS 86.670 93.412 89.498 84.041 MCS 93.897 72.802 23.250 24.784
SD 21.910 18.833 17.832 17.920 SD 19.406 21.275 16.659 21.380
I osphamide Ca bopla in
RMAPC 0.009 0.037 0.147 0.588 RMAPC 2.814 11.257 45.027 180.108
MCS 92.729 95.906 90.101 88.518 MCS 91.805 79.175 77.962 52.322
SD 16.694 14.965 15.299 16.677 SD 16.670 30.927 22.065 31.295
Hyd oxyu ea Bleomycin
RMAPC 0.031 0.124 0.496 1.984 RMAPC 2.078 8.311 33.245 132.979
MCS 95.118 96.489 86.340 68.134 MCS 90.007 88.553 82.168 69.803
SD 19.595 13.933 19.759 24.782 SD 19.294 17.678 19.720 16.686
Vinblas ine S ep ozo ocin
RMAPC 2.603 10.413 41.650 166.600 RMAPC no Cmax da a was ound
MCS 81.507 53.331 28.885 21.323 MCS 95.430 92.335 89.919 87.805
SD 25.921 25.678 17.941 15.889 SD 15.828 18.710 14.094 22.637
Bo ezomib MG-132
RMAPC 0.162 0.648 2.590 10.361 RMAPC no used in clinical p ac ice
MCS 93.119 81.671 75.498 28.700 MCS 87.345 96.374 88.097 27.045
SD 13.779 21.572 19.617 26.948 SD 13.980 17.493 16.974 24.310
P ednisolone
RMAPC 4.8 19.23077 76.92308 307.6923
MCS 75.55877 76.25595 62.46537 37.23379
SD 4.647172 11.09091 13.97021 11.65414
G oup 1. Highly e ec i e d ugs: RMAPC < 0.3; MCS < 30%
G oup 2. Pa ially e ec i e d ugs: RMAPC be ween 0.3 and 1, MCS < 60%
G oup 3. Ine ec i e d ugs: RMAPC > 1 o MCS > 60%
SD: s anda d de ia ion in mean cell su i al
Table 2: The a e age Mean Cell Su i al (MCS) o he 17 LCLs a di e en d ug concen a ions, exp essed as he Ra io o Maximum
Achie ed Plasma Concen a ion (RMAPC) (Con inued)
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inhibi o y e ec s on o he cell lines o p ima y umo s
(da a no shown).
No RMAPC could be de e mined o s ep ozocin because
no pha macokine ical ials wi h es ablished Cmax
plasma le els we e ound. RMAPC was also no a ailable
o MG132 because i has no been used clinically. S ep-
ozocin had no e ec on su i al a he concen a ions
used in he s udy. Al hough a he highes concen a ions
he p o easome inhibi o MG132 e ec i ely dec eased he
su i al o he cells, i s clinically licensed unc ional
homologue, bo ezomib, was no e ec i e.
An al e na i e way o calcula e ela ionship be ween he in
i o d ug concen a ions and he in i o used alues is o
use he a ea unde cu e (AUC) alues o he indi idual
d ugs. To compa e AUC alues wi h he RMAPC da a he
ollowing o mula was used:
in i o used concen a ion (µg/ml) × 72 h/AUC (µgxh/
ml) in i o.
In i o AUC le els om he li e a u e a e summa ized in
Table 1. Using he o mula o ep esen ing he mean cell
su i al cu es o all he d ugs along a common " a io o
AUC" axis, ca bopla in, me ho exa e and dac inomycin
showed highe e icacy in compa ison o RMAPC igu es
whe eas he sensi i i y o de o he o he d ugs o o he
g oup classi ica ions did no change.
Discussion
The p esen ed da a sugges ha many EBV ans o med B-
cell lines sha e a common cy o oxic d ug sensi i i y p o-
ile independen o hei o igin. This p o ile does no
change e en a e many yea s o in i o cul u ing. EBV
appea s o be he necessa y and su icien e iological agen
behind he malignan immunoblas ic B-cell lymphop o-
li e a ions in immunosupp essed pa ien s. In all hese
cases he EBV encoded, la ency associa ed i al p o eins
d i e he cell p oli e a ion wi hou any ob ious need o
addi ional gene ic changes. The pheno ype o hese
umo s closely esembles he in i o g owing LCLs and he
expe imen ally induced umo s ha appea ed upon in a-
pe i oneal implan a ion o LCLs in o SCID mice [11].
Conside ing he pheno ypic and ka yo ypic s abili y o
EBV ans o med B-cells, ou da a aises he hope ha
PTLDs, AIDS associa ed CNS lymphomas and XLP associ-
a ed lymphop oli e a ions may show simila pa e ns o
d ug sensi i i y as he ones ha we ha e es ablished in he
p esen s udy on a coho o di e se LCLs.
Sugimo o e al. sugges ed ha EBV media ed ans o ma-
ion is a wo s ep p ocess whe e, a e p olonged passage,
he cells may become aneuploid, accumula e p53 mu a-
ions, down egula e he p16/ e inoblas oma p o ein
D ug sensi i i y o LCLs o an h acyclines, inca alkaloids and axansFigu e 3
D ug sensi i i y o LCLs o an h acyclines, inca alkaloids
and axans.