15 Maya?
`
`
`
`
`
`Ubligaiion of the International Union Against Cancer
`on de l'Union Internationale Contre le Cancer
`
`ee
`
`Miltenyi Ex. 1042 Page 1
`
`Miltenyi Ex. 1042 Page 1
`
`

`

`15 t,1ay 1977
`
`Vol. 19
`
`o. 5
`
`INTERNATIONAL
`
`J ournal
`o}Cancer
`
`JOURNAL INTERNATIONAL DU CANCER
`
`E. A. Saxen
`Editor-in-Chief:
`Assistant Editor:
`E. Saksela
`Managing Editor: F. Luder
`
`Editorial Office:
`
`International Journal of Cancer
`Third Dept. or Pathology, Uni,;crsity of Helsinki
`Helsinki 29, Finland
`'
`
`EDITORIAL BOARD
`Comile de redactiOIJ
`
`S. A. AARONSON, Bethesda, Md. (USA)
`G. I. ABEi.EV, Moscow (USSR)
`R. W. BALDWIN, Nottingham (England)
`S. BARON, Bethesda, Md. (USA)
`GEORGES BARSKI, Villejuif (France)
`M. M. BURGER, Basel (Switzerland)
`VmoRJo DEFEND1, New York, N.Y. (USA)
`LEiu DIAMOND, Philadelphia, Pa. (USA)
`s· ir RICHARD D OLL, Oxford (England)
`S. J. ECKHARDT, Budapest (Hungary)
`H. JiANAFUsA, New York, N.Y. (USA)
`JENNIFER J. HA,wEY, Harrow (England)
`H. ZUR HAUSEN, Erlangen (Germany)
`1
`· li.£t0ELB£RCeR, Madison, Wisconsin, (USA)
`AUL K.rr, Houston, Texas (USA)
`H.. G. Kw A, Amsterdam (Netherlands)
`Al.8£11.T LEVAN, Lund (Sweden)
`~ MENEGHINJ, Sao Paulo (Brazil)
`ONAIJ) MET
`E
`CALF, Melbourne (Australia)
`JU.IA Mo
`C
`lLER, Stockholm (Sweden)
`· S. MlIIR, Lyons (France)
`
`~
`
`N . P. NAPALKOV, Leningrad (USSR)
`EsKO N!KKILA, Helsinki (Finland)
`K. N1s1110KA, Tokyo (Japan)
`T. ONOE, Sapporo (Japan)
`J . PoNTtN, Uppsala (Sweden)
`R. T. PRCIIN, Philadelphia, Pa. (USA)
`V. RAMAUNGASWAMf, New Delhi (India)
`J. J. R O0ERTS, Chalfont St. Giles (England)
`E. R uOSLAIITT, Duarte, Calif. (USA)
`LEO SACHS, Rehovot (Israel)
`D. SCHMAHL, Heidelberg (Germany)
`K. SHANMUGARATNAM, Singapore
`H. O. SJOGREN, Lund (Sweden)
`J. svooooA, Prague (Czechoslovakia)
`v. THURZO, Bratislava (Czechoslovakia)
`L. T0MATIS, Lyons (France)
`J.M . VASILIEV, Moscow (USSR)
`u. VERONESI, Milan (Italy)
`o. M. WHITTLAW, Vancouver (Canada)
`A. o. Wu..LJAMS, Ibadan (Nigeria)
`
`Miltenyi Ex. 1042 Page 2
`
`

`

`r
`
`Union lnternationale Contre le Cancer
`International U nion Against Cancer
`
`F. de ANESTI (Cbile/Chili)
`N. N. BLOK HIN (USSR/URSS)
`M. CoPEL.AND (USA/ EU)
`P. DENOIX, President (France)
`R. D o LL (UK/ RU)
`S. E CKHARDT (Hungal)/H ongrie)
`l. ELSFDAI (Eg) pt/Egypte)
`D. E. GUSTAFSSOX (S,,cden/Suede)
`E. H ECKCR (Germany/Allemagne)
`A. C. J UNQUE.IRA (Bra,il Bresil)
`D. J. J ussAWAI LA (India/Indc)
`H. l<AS"ORr ( rugu.ly)
`L. G. LAffilA (UK/RU)
`R. LEE CLARI US:\/EU)
`P. LoUSTALOT <Switzerland/Suisse)
`F. F. McEACll!m-i (Canada)
`
`COUNCfL - CONSU L
`
`D. MFTCAL F (Australia/Australic)
`A. M OJTA0AI (I ran)
`G. P. M UR PHY (USA EU)
`K. OoTA (Japan/Japan)
`I. PAOOVAN (Yugoslavia/Yougoslavie)
`E. A. SAXfN (Finland/Finlande)
`C. G. SCHMIDT (Gcrmany/AJlemagne)
`K. SHANMUOARATNAM (Singapore/Singapour)
`M. SuOIMURA (Japan/Japan)
`R. M. TAYLOR (Canada)
`N . TRAINtN (Israel/Israel)
`N. N. TRAl'EZNlKOV (USSR/ URSS)
`u. VERONESI (Italy/Italic)
`F. J. WILCOX (USA/EU)
`A . WtlLIG (USA/EU)
`
`INTERNATIONAL UNION AGAINST ·cANCER
`The International Union Against Cnncer is a non-governmcn1al, voluntary organization devoted sol~ly
`, promoting throughout the world the campaign against cancer in its research, therapeutic and prevenuve
`1x-cts. Over the years, the UICC has developed into a world-wide association with member-organizations
`1n over 79 coun1ries. It s1rivcs to achieve its aim by facilitating the exchange of information between national
`cancer organizations, holding international cancer congresses, conferences and symposia, standardizing
`nomenclatures and classifications, and by stimulating national elforts against cancer.
`The UICC publishes the International Journal of Cancer, Monographs Manuals Technical Reports,
`and 1he UICC B11/le1i11. The International Union Against Cancer ha~ recei~cd financi~I assistance for the
`publication of the lntemational Journal of Ca11cer from the U.S. National f11stitlltes of Health, 1he Cnloustt
`G11lbe11kian Fo1111datio11, the Swedish Cancer Society, the Ca11adia11 Canci>r Society 1he National Cancer
`/11stlt11te of Ca11ada, and the Cancer Society of Finland.
`'
`
`UNION INTERNATIONALE CONTRE LE CANCER
`L'Union Internationale <;on_tre le Cancer, au sein de laquelle sont groupees de<; societes me_mbrcs de
`plus de 79 pays, est uoe orgam~auon non gouvcrnementale sans but lucrntif, qui se consacn: cxclus1vcmco111
`encourager dans le mondc enuer la lullc conLre le cancer: thfrapeutique prophyla,ic ct rccherche. A cette
`fin, clle_ facili!e Jes 6<:hanges d'i~formations entrc societes na1io~ale~, organise des congrcs. conferences et
`symposiums m1crnauonaux, umfie !es nomenclatures et les classifications et stirnule I 'action mcncc conirc
`le cancer sur le pl:in mllional.
`•
`L' UIC:C pu~lie le Jou~nal_J111ematio11al du Cancer, des Monographies, Manuels, Rapporl> t.:, h01quC$i
`et le Bulle11n de I UICC. . ~ Union 1!'terna11~nale ~ontrc I~ Cancer a re<;:u pour la publication du J~umo
`Jntemarioual du Cancer I cude financ1ere des 1ns11111uons su1vantcs: the National fllstitllles of /lea/th l SA);
`Folllia1io11 Calouste Gulbe11kia11, tire S wedish Cancer Societ)', Societe Ca11adienue du Cancer !,,stillll ,\ ,1/1tl/lO
`du Ca11cer du Ca11ada, c l the Cancu Sorii:t)• of Finland.
`•
`
`-
`
`JI
`
`Miltenyi Ex. 1042 Page 3
`
`

`

`- I
`
`nt. J. Cancer: 19, 621-626 (1 977)
`
`CHARACTERIZATION OF EBY-GENOME NEGATIVE " NULL "
`AND " T " CELL LINES D ERIVED FROM CHILDR EN WITH ACUTE
`L YMPHOBLASTIC LEUKEMIA AND LEUKEMTC TRANSFORMED
`NON-HODGKIN LYMPHOMA
`
`Ulrich ScH EIDER, Hans-Ulrich S c H\>JEN K , and Georg B ORN KAMM
`Kinderkli11ik der Unh·ersitiit Erla11ge11-Niimberg, and lnstitut fiir Kli11ische Viro/ogie der Universitiit Erla11ge11-
`Niirnberg, D-8520 Erla11ge11, Loschgestr., Federal Republic of Ger111a11y
`
`Sixty-two e xplants from pe ripheral blood, bone
`marrow and ce rebral fluid of childre n with acute
`lymphoblast ic leukem ia (ALL) and le ukemic trans(cid:173)
`formed non- Hodgki n
`lympho ma (NHL) w e re
`cultivated fo r at le ast 8 w eeks. Although lymphatic
`cel ls persisted up t o 16 w eeks in tissue culture, no
`proliferation w as observed in 54 cultures. From the
`remaining cultures, eight permanently growing cell
`lines were obt ained . Five of the se we r e EBNA
`(Epstein-Bar r virus-s pecific nucle ar antigen)-posi(cid:173)
`tive. Three, however , were EBNA-nega tive and
`lacke d Epstein-Barr v irus ge nome s . Two ce ll l ines
`(KM-3 a nd S H -2) e xpresse d neithe r B nor T cell
`characteristics . One line (JM) expresse d T cell
`chara cteristi cs a nd comple m e nt re ceptors. The
`growing lympha tic cells represented leukemic cells,
`si nce the patte rn of cyto chemical s ta ining a nd that
`of m e mbr a ne r e ce ptors of lymphoblasts from the
`sa me do nor prior to cultivation we re identical .
`All leuke mic cell line s we r e de r ive d from pa t ients
`in re lapse. In contrast, no proliferation of leuke mic
`cells occurre d in expla nts from patie nts revealing
`the first manife sta tion of the disease. These results
`suggest e nhance d growth potential of lymphobla sts
`resisting antile ukem ic therapy.
`
`Acu te lymp hoblast ic leu kem ia (ALL) contributes
`up to 35 % o f a ll childhood malignancies. A ltho ugh
`the prognosis o f ALL improved after introd uctio n
`of more efficient ra dio- and c hemotherapy (Pinkel
`et al. , 1972), abo u t 50 % o f the patie nts rela psed
`within 5 years a fte r first rem issio n (Aur et al.,
`1974). Sim ila rly, a grea t number o f child ren sufferi ng
`from non-Hodgk in lympho ma (N HL) term inate in
`leukemic genera liza tio n. Therefo re, ea rly recognitio n
`of patients with h igh risk o f relapse would be
`importa nt for the prognosis a nd possib ly a lso for
`an initial intensificatio n o f the therapy in this gro up.
`A considerable nu m ber o f these patienLS express
`T cell surface markers ( Bo rella er al., 1973), a lt h o ugh
`relapses occur in T -ma rker negative ALL as well.
`In the
`la tter gro u p differen t para meters (initial
`leukocyte cou nt, age of the pa tient) may allow some
`Prediction o f prognosis. Nevertheless a better
`characterizatio n o f
`the
`lymphoblasts
`is urgently
`needed .
`Leukemic cell lines represent a n excellent tool in
`the search for tu mo r-specific characteristics. The
`almost un li mited q uantity, the lack o f co n tami-
`
`na tion by no rma l leu kocytes and their co nven ient
`availability a re helpful
`in ma ny experimen tal
`designs. As a prereq uis ite, however, the lack o f
`Epstein-Ba rr vi rus (E BY) genomes and the ident ity
`of pro perties o f cu ltured cells a nd pr imary tumo r
`cells have to be ensured. U p to now these c r iter ia
`have been fulfi lled in o n ly a few cell lines estab lished
`fro m c hildren with T-cell re:eptor-positive A LL o r
`N HL (Fo ley et al., 1965; Adams et al., 1968;
`Mi nowad a et al., 1972, 1977). T he permanent
`growth o f T and B cell receptor-negative cells in
`childhood ALL h as no t been repo r ted thus far.
`Tn this commun icat io n we describe the esta blishme nt
`'' null ·· a nd o f a T receptor(cid:173)
`o f two leukem ic
`positive line.
`
`M ATERIAL AND METHODS
`
`Origin and 111ai11te11ance of rhe cell lines used as
`controls
`The cell lines R aji (Pulvertaft, 1964), U-698 M
`(N ilsson a nd Sund strom, 1974), Ramos (Klein et al ..
`1975) a nd MO L T-4 ( M inowada er al., 1972) were
`kindly provided by Dr. H . zu r Ha usen, Erlangen.
`The cell cu lt ures were incubated at 37° C and sub(cid:173)
`c ulti va ted o nce or twice weekly with med ium
`RPM I
`I 640, supp lemented with 10 ° 0
`inactivated
`fe tal calf serum, 100 I U/m l penic illi n and 100 11g/ m l
`streptomycin.
`
`Esrablishmenr of rl,e cell lines
`Blood a nd bone-marrow sa mple were cent r ifuged
`o n a Ficoll-Hypaq ue grad ient ( Boyu m, 1968) after
`I :2 dilution in medi um RP M I I 640, supp lemented
`with feta l calf serum a nd ant ibio tics a described
`above. Ten sam ples derived from five pa tients ,,ere
`injected into Spongosta n foam (Ferro an, Malmo ,
`Sweden) and layered on a grid of stainles
`steel
`accord ing to the method of N ilsson (197 1). The
`cu ltures were incu ba ted at 37 C in a 5 % C O 2
`least 8 weeks.
`atmosphere and observed fo r at
`One-ha lf to o ne-th ird o f the med iu m was changed
`twice weekly.
`
`Rec.:ived : December 3, 1976.
`
`Miltenyi Ex. 1042 Page 4
`
`

`

`Int. J. Cancer: 19, 621-626 (1977)
`
`CHARACTERI ZAT ION OF EBY-GENOME NEGATIVE "NULL "
`AND "T " CELL LIN ES D ERIV ED FROM CHILDREN WITH ACUTE
`LYMPHOBLASTIC LEUKEMIA AND LEUKEMIC TRANSFORMED
`NON-HODGKIN LYMPHOMA
`
`Ulrich S C HNEIDER, H a n -Ulrich S C H\VEN K , a nd Georg B ORN KAMM
`Kinderklinik der Unil'ersitiit Erlange11- N iirnberg, and lnstitut fur Klinische Virologie der Universiriit Erlangen(cid:173)
`Niirnberg, D-8520 Erla11ge11, Loschgestr. , Federal Republic of Germany
`
`Sixty-two explants from peripheral blood, bone
`marrow and cerebral fluid of children with acute
`lymphoblastic leukemia (ALL) and leukemic trans(cid:173)
`formed non-Hodgkin
`lymphoma (NHL) were
`cultivated for at least 8 weeks. Although lymphatic
`cells persisted up to 16 weeks in tissue culture, no
`proliferation was observed in 54 cultures. From the
`r_emaining cultures, eight permanently growing cell
`lines we re obtained. Five of these were EBNA
`(Epstein-Barr virus-specific nuclear antigen)-posi(cid:173)
`t ive. Three, however, were ESNA-negative and
`lacked Epstein-Barr virus genomes . Two cell lines
`(KM-3 and SH-2) expressed neither B nor T cell
`characte ristics. One line (JM) expressed T cell
`characteristics and complement receptors. The
`g_rowi ng lymphatic cells represented leukemic cells,
`since the pattern of cytochemical staining and that
`of membrane receptors of lymphoblasts from the
`same donor prior to cultivation were identical.
`~II le uke mic cell lines were derived from patients
`1n relapse. In contrast, no proliferation of leukemic
`cells occurred in explants from patients revealing
`the first manifestation of the disease. These results
`suggest enhanced growth potential of lymphoblasts
`resisting anti leukemic therapy.
`
`Acute lymphoblas tic le u kemia (ALL) contributes
`up to 35 % o f a ll c hildhood malignancies. A ltho ugh
`the Prognosis o f ALL improved a fte r introduc tion
`of mo re efficie nt radio - a nd c hemo therapy ( Pinke l
`et_ al., 1972), about SO % o f the patients re lapsed
`WHhin S years a fter fir t remission (Aur er al.,
`1974). Similarly, a great numbe r o f child re n suffering
`frorn non-H odgkin lympho ma (N H L) terminate in
`leukemic gene ra liza tio n . Therefo re. early recogn ition
`?f patients w ith high ris k of relapse wo uld be
`tmpo rtant for the progno is a nd possibly a lso fo r
`an iniiial intensificatio n of the the rapy in this group.
`A considerable number of these patienlS express
`1 cell s urface ma rkers (Borella er al. , 1973), a ltho ugh
`relapses occur in T -marker negative ALL as _ ,~~II.
`lo the la tte r group differe nt paramete rs (1ni11a l
`leukocyte count, age o f the patient) may a llow some
`Prediction of progno is. Ne ertheless_ a better
`characterization o f
`the
`tymphoblasts 1s urgentl y
`needed .
`II 1·1nes represe nt a n excellent tool i.n
`·
`
`Le k u em1c ce
`.
`.
`the earch fo r LUmo r-specific charactens11cs. Th_e
`alrno t unlimite.d qua ntity, the lack o f contam1-
`
`nation by normal leu kocytes a nd their co nve n ient
`a vailability are he lpful
`in m a ny experimen ta l
`designs. A s a prerequisite, however, the lack o f
`Epste in-Barr virus (EBV) genomes and t he identity
`of properties o f cultured cells and primary tumor
`cells have to be ensured . Up to now these criteria
`have been fulfilled in o nly a few cell lines es tablis hed
`from children w ith T-cell re:::epto r-positive ALL or
`NHL (Foley et al. , 1965 ; Adams er al., 1968;
`Minowada et al. , 1972, 1977). The perma ne nt
`growth o f T and B cell rece ptor-negative cells in
`childhood ALL has n ot been re po rted th us far.
`In this communicatio n we describe the establis hment
`o f two leukemic " null ·· a nd o f a T receptor(cid:173)
`positive line.
`
`MATERI AL AN D METH ODS
`
`Origin and maintenance of the cell lines used as
`controls
`The cell lines R aji (Pulve rtaft, 1964), U-698 M
`( Nilsson and Su nds tro m, 1974), R amos (Klein er al ..
`1975) a nd MOLT-4 (M inowada er al., 1972) were
`kindl y provided by Dr. H. zur Ha usen, Erlange n.
`The cell cultures were incuba ted a t 37° C a nd u b(cid:173)
`cultivated once or
`twice weekly with med ium
`RPMI 1640, s upplemented with 10 °~ inactivated
`fetal calf serum. I 00 I U/ml penicillin a nd I 00 11g/ m l
`s treptomycin.
`
`E stablishme/11 of the cell lines
`Blood and bone-ma rro w sample were centrifuged
`o n a F icoll-Hypaq ue grad ient ( Boyum , 1968) a fter
`1 :2 dilution in medium RPM I I 640, supplemented
`w it h fe ta l calf serum and antibiotics as descr ibed
`abo, e. T en ~am ple deri ed from fi ve patients were
`injected into Spo ngo~tan foam (Ferrosa n, Malmo,
`Sweden) and layered on a grid o f s ta inle s s teel
`accord ing to
`the method o f Nils o n ( 197 1 ). The
`cul tures were inc ubated a t 37 C in a 5 % CO 2
`lea t 8 \\-eek .
`a tmosphere a nd o b erved fo r a t
`O ne-half to one-third o f the medium , as c ha nged
`twice weekly.
`
`Received: December 3, 1976.
`
`Miltenyi Ex. 1042 Page 5
`
`

`

`622
`
`SCIINI:IOf R ET AL.
`
`Tra11s111issioll electron microscopy
`in
`pproximatcl) 10~ cells
`,,ere cen trifuged
`ni1rocellulo~c lube a1 400 g for 10 min. The pellet
`,, a re us pended in a few drop of agarose (3 ° 0 w/ v
`in dis1illcd wa1cr) and cooled in an ice bath. The
`bottom of 1he tube wa cut off a nd the pellet divided
`in10 mall piece . After prefixing in 2.5 % phosphate(cid:173)
`buffered glutara ldehyde a nd postfixing
`in
`I %
`o mium 1e1rox ide, furt her processing was performed
`according to a procedure pu blished elsewhere (Luft,
`196 I). The sections were examined in a Zeiss EM 10
`microscope.
`
`Cytoche111istry
`Staining w ith PAS (Mc Ma nus, 1946) and detectio n
`o f acid phospha tase (Leder, 1965), non-specific
`e 1era e ( Lo ftlcr, I 96 1) a nd peroxidase (Schaefer
`and Fischer, I 968) were performed identica lly wit h
`primary and es ta blished ly mphoblasts.
`
`EB V serology and detectio11 of EBV-specific 1111clear
`a111ige11
`The liters o f anti-viral-capsid (ant i- VCA) and
`a nti-ea rly-antigen (anti-EA) antibodies were deter(cid:173)
`mined by indirect immunofl uorescence (Henle and
`Henle, 1966 ; Henle et al. , I 970). Presence of EBV-
`pecific nuclea r antigen (EBNA) (Reedman and
`Klein, 1973) was 1es1ed by sequentia l incubation of
`meth ano l-fixed cell smears with EBNA-positive
`erum (di lu tio n
`I : I 0,
`inactiva ted), human com(cid:173)
`plement (dilutio n I : I 6, corresponding to 2 hemolytic
`uni! ) a nd FITC-conjugated an ti-h uman-P1C/ f] 1A(cid:173)
`glo bulin (Traveno l Internationa l, Munich, dilution
`I :20). Cell o f !he lines Raji and MOL T-4 served
`as posi ti ve o r negative contro ls, respectively.
`
`Detection of £ 8 V-D A i11 the establi'.rhed tell lines
`Cellula r D N A was tes ted fo r the presence o f
`E BV-D A equences by reas ocialio n kinetic a
`de cribed pre vio u ly ( Bo rn ka mm et al., 1976) with
`fragmented by
`slight m odificatio n . D A was
`boiling in 0.25 N
`aOH fo r 10 min, chilled in an
`ice balh a nd neutralized by add ing 2 HC I. D A
`was incuba ted fo r hybridizatio n at 67 C fo r 12 h
`aCI, 0. 1 M T ris-HCI (pH 8) and 0.05 ° 0
`in 0.5 M
`sa rkosyl. AJiq uol o f 50 td ,, ere removed every
`hour, d il uted in10 0.5 ml 0.14 M sod ium phospha te
`and kepi at 0 C until chromatogra phing o n hydroxyl(cid:173)
`apa lite columns. The method was ensiti, e eno ugh
`to detect less 1han one E BY-genome equiva lent
`per cell.
`
`Cell 111e111bra11e receptors
`T est'> ,, i1h permanently gro11 ing ce lls ,,ere per(cid:173)
`formed during
`their
`logarithmic gro11 1h phase.
`T he viability ,,as confirmed by trypa n b lue exclusion.
`In each experiment cell lines wi th B cell cha racier-
`
`-698 '\1, Ramos), a T cell line (MO L T-4)
`i~lics (
`and blood lymphocytes from norma l donors served
`as controls . .Each experiment was ca rried out at
`least three t ime . At least 200 cells were micro(cid:173)
`scopically examined for a ll receptor .
`Sheep red blood cell ( SR BC ) -receptors and Fc(cid:173)
`receptors. T-rose ttes 11ere detected with untreated
`SRBC (Jondal et al., 1972) a fter incu ba tio n at
`37° C o r 4 C. Fe-rosetting was performed by
`incu bation wit h ox red blood cells, coa ted with a n
`inactivated ra bbit a ntiseru m, o b tained a fter four
`IV injections o f SRBC (Ha llberg et al. , 1973).
`Co111ple111ent receptors. SRBC were trypsinized for
`h a t 37° C (trypsi n supplied by Serva, Heidelberg,
`4 mg try psin/ ml in PBS). The following incubatio n
`fo r 30 m i,n. ( I)
`s teps were performed a t 37 C
`An equal vol ume of a 5 % s u pension of trypsinized
`SRBC was incubated with antiserum diluted I :5 12
`(inact iva ted rabbit erum obtained 5 days after a
`single injection of 2 ml o f a 50 % su pension o f
`SRBC). (2) E rythrocyte-anti erum-complement-com(cid:173)
`plexes (EAC) were prepa red by incu bation with
`fresh mo use complement at a dilution of 1 : IO in
`0.9 % sa line. (3) One volume of s uspended ly mpho(cid:173)
`blas1s (0.25 ml, Jo• cells) was m ixed with an eq ua l
`volume of a 1 % EAC s uspensio n an d agai n in(cid:173)
`cubated.
`In111111110/l11orescence staining with Fl TC-conjugated
`antisera. Abo ut I 0° cells were suspended in ice-cold
`PBS-sodium-azide a nd incuba ted fo r 30 m in per
`incubation step in the presence o f a ntiserum. The
`cells were spread on slides a nd exam ined under a
`Leitz U V-microscope, equipped with a Ploem
`vertical illumina to r.
`Cell-111e111brane-bo11nd i1111111111oglob11lin. The cells
`were sta ined according to the direc t tech niq ue of
`Raff ( 1970) with po lyvalent ra bbit a nti-human lg
`or monospecific sera direc ted aga in t hea vy chain
`o f human Ig_G , fg M o r lgA. Indi rect sta ining wa
`perfo rmed wtt h polyva lent rabbi t a nti-huma n serum
`and a swine a nti-rabbit conjuga te. All sera \\ere
`upplied by Medac, Ham burg.
`Cell-111e111bra11e-bo1111d fl !-111icrogfob11/in and Co11-A
`receptors. The cells were
`incubated wit h rabbit
`fl,-microglo bulin a nd a swine ant i(cid:173)
`a nt i-huma n
`ra bbit lg conj uga te. T he di lut ions ,~ ere I :20 and
`1 :40, re pec1ively. Bot h sera were su pplied by Medac,
`Hamburg. F IT C-conj ugated Co n-A ( Mile . Fra nl,,.(cid:173)
`fur1) was u ed a t a d ilutio n o f I : JO.
`
`R[SU LTS
`Cultil'm'.on °1 primary lp11phoblas1s and es1ablifl1111ent
`of cel/ l111es
`
`is sum·
`T l_,e de_r ivaiion of a ll cultivated explant
`man ~cd 111 Table I. In bone-marro11 cultures and
`a lso in some prepa ra tions of cult ivated blood cells,
`
`C
`
`l
`
`Miltenyi Ex. 1042 Page 6
`
`

`

`E$TI\ULl!il l ~IE ' I' 0 1 I CUKEM IC CELL LI NES
`
`623
`
`TA BLE I
`
`D CRI VATION OF FXPLA NTS
`
`Diagnosis
`
`Clinic,1 l ) ldgc
`
`No. of
`p:1t.ienls
`
`'lo. of
`bone.marrow
`culture-.
`
`'lo . of
`pcri 1,hcr J I blood
`euhurcs
`
`o. o f
`cultures from
`ccrcbrnl flu,d
`
`ALL
`A LL
`ALL
`LS
`
`First manifestation
`First relapse
`Second relapse
`Leukemic transfor111ation
`
`l9
`3
`2
`8
`
`19
`3
`2
`8
`
`15
`3
`2
`4
`
`.j
`
`J
`
`macrophages and occasionally fibroblasts began to
`spread as a feeder layer a few days after explan(cid:173)
`tatio n. Fibroblasts usually overgrew the proliferating
`macrop hages. In the presence of a feeder
`layer
`lymphatic cells persisted up to 16 weeks with no
`~ign of proliferation, then their viability decreased
`s lowly. Cultured lymphoblasts deri ved from the
`cerebral fluid, in contrast, s urv ived only for a few
`days. Permanently growing cell lines were obtained
`from eight cultures. Five of these harbored EBV,
`s ince the cell reactions were ESNA-positive, and
`revealed surface-bound immunoglobulin.
`
`Pa,ients and establisl1111e111 of cell lines
`Leukemic cell lines were established from samples
`of the following patients.
`Patient K.M. The line KM-3 was established after
`30 days' c ulti vation of a blood sample from a
`l 2-year-old boy during his second relapse of A L L.
`The patient initially showed hepa tomegaly and a
`lotal white blood cell count of 20,000 cells/111111 3
`•
`After chemotherapy aDd s ubsequent irradiation of
`the central nervous sys1em, two relapses occurred
`a fter 27 a nd 32 months, followed by death after a
`furlher 4 months.
`Parient S.H. The line Sl-1-2 was derived from cells
`o r a bone-marrow sample obtained from a 14-year(cid:173)
`old boy with N HL after le ukemic trans fo rmatio n.
`Proliferation was observed after 60 days of culti(cid:173)
`vation. At the beginning of the disease the patient
`had an appendectomy
`fo llowed by
`inte tina l
`o bs truction 4 weeks later. A renewed laparotomy
`establis hed a N H L which obs truc te d the ileocecal
`valve. After e ra dication o f the tumor by hemicolec(cid:173)
`tonw and combined ileotransversostomy, chemo(cid:173)
`therapy and radiatio n therapy were slarled. Three
`rnonths later leukemk cells appeared in bone marro ,,
`and cerebral nuid. D eath occurred after 2 further
`OlOnths. The line was lo s t for un k no w n reaso ns afler
`3
`rnonths of cultivation during the 12th passage.
`Pa1ie111 J.M. The line JM was deri ved from the
`Peripheral blood of a 14-ycar-old boy wilh A LL.
`No n1edias tinal mass o r lymphoma was detected.
`l3eforc therapy
`the bone marrow consis led o f
`9~•/ I
`JI
`l
`0 Ym phoblas ts, and the w hile blood ce coun
`
`.......
`
`amounted to 300,000 cclls/ mm3
`• After s uccessful
`chemotherapy and preventive irradiation of the
`s kull, four relapses occurred within 7 months. The
`patient 's condition detcrio raled due to meningeal
`leukemia during the fourth re lapse a nd he died.
`The line was established from cells o bta ined during
`the ftrst relapse. Proliferation of J M lymphoblasts
`s tarted as early as 7 days after explantation; the
`cells were passaged for the first time a fter o nly
`l4 days.
`
`l.e11ke111ic cell lines
`The three lines grew as s ingle-cell s uspensio ns
`with no attachment to the surface of the culture
`vessel. Neither cytoplasmic projections n or colo ny
`forma tion were observed . KM-3 and SH-2 were
`s ubcultivatcd weekly by changing half of the medium.
`J M
`require d
`s ubcuhivation
`t\\ ice weekly and
`achieved a saturation dens ity as high as 6 10"
`cells/ ml.
`Ul1rc1stru,·1111·e. A rather s moothly co n to ured cell
`membrane, sparse cytoplasm, dcn tnlc nuckus,
`marginal distribulion o f the chromal,n and pro(cid:173)
`minent nucleolus were common to all three cell lines.
`fn contras t to the cndopla ma tic retic ulum, 1he
`Golg i apparatus was \\ell dc\'clopcd an<l s11 o llcn
`mitochondria \\ ere frequently delectcd.
`o vir u~
`particles 11cre detected.
`c •toche111icol staining. Prima ry and c~ta bli<;he<l
`le u:emic cells of palicnt K I stained PAS-r ositilc :
`cdls from pa tient SH 11e re negali1c _with all : ta inings.
`Acid pho phatasc II as dc lcc ted m the . f rc\ h ~ 1
`ly mphobla!>lS a~ 11cll a
`in the cs tabll~hcd hne
`(Table I I). The ~mining 11 as confined lO _ th.: para-
`I , r ar"a After 8 pJ~, agc~ the C)tabh\ hetl cell\ nuc c<1
`
`, ~ •
`.
`•
`had an a~·id-phos pha la c-negatl\C reaction.
`/)t'll'Ction of EB \ ',I and r.B V- D1\ ..i in the /e11/..e111ic
`II I .
`,-81
`o EBNA
`.11 rologJ (lj the donors.
`IIIC'.I, ' ·
`Cl'
`· ·
`o r non-~~.:ific background nu orc~ccncc
`~u11ning
`1· -
`d IC •t •rJ The num ber of El:lV ~eno mc:1. deter-
`were e •;;;; ·
`.
`.
`.
`. ~
`• d bv tile rc: 1,,oc1a 11o n k111et1c, a m o u nted to
`.
`'
`.
`mme
`t ha n o ne geno me equl\ ,ile nt per cell (Table Ill ,
`I
`f
`h
`I
`.
`c-~s
`Fig . I). In 1,, 0 o f lhe scrn r~m t ~ B1V1re~ patient\
`t•
`tilers \\ere
`i hom the lines 11 crc dcrl\ ed c
`rom \
`de rccled (Tab le Ill).
`
`◄
`
`Miltenyi Ex. 1042 Page 7
`
`

`

`624
`
`SCHNUDER ET AL.
`
`Cell 111e111bra11e receptors. KM-3 and SH-2 failed
`to reveal B cell or T cell characteristics (Table l V).
`J M ex pre ed T cell characteris tics as detected by
`rosette fo rmatio n w it h S RBC. Differences between
`fresh p reparations and establis hed leu kemic cells
`(Table IV) a re expla ined by the presence of no rma l B
`a nd T cells in peripheral b lood a nd bone marrow.
`SR BC-rosettes wit h prima ry and established leu-
`
`TABLE II
`
`C YTOC H E M ICAL STAIN I NG
`
`Stain
`
`Reference
`
`Cell lines
`
`JM
`
`K M -3
`
`SH-2
`
`PAS
`Acid
`phosphatase
`Non-specific
`esterase
`Peroxidase
`
`McManus (1946)
`Leder ( I 965)
`
`- ( - )'
`I- '( + )
`
`1-( + )
`- (- )
`
`Loffier ( 1961)
`
`- (- )
`
`- (
`
`)
`
`Schaefer and
`Fischer ( 1968)
`
`- (- )
`
`- (- )
`
`• DaL~ fro m tcstS with primary lympho blas ts in pa rentheses. -
`• Acid phosphatase reaction nega1ive after eight passages.
`
`TAB LE Ill
`
`E BY-SEROLOG Y AND PRESENCE O F E DNA
`A N D EBV-DNA
`
`Delcclion o f
`
`Cell lines
`
`JM
`
`KM-3
`
`SH-2
`
`EBNA
`EBY-DNA
`
`Anti-Y A
`An ti-EA
`
`Presence of EBNA and EBY-DNA
`in the cell li nes
`
`Presence o f EBY-antibodies in
`the autologou~ sera
`I :20
`.,-- I :5
`I :20
`< I :5
`
`I :10
`• I :5
`
`1 Less than one genome equivnlcnl per cell.
`
`5
`
`6
`
`8 hours
`
`F IGURE I
`the
`Reassociation kinetics of 5 µg EBY-DNA in
`presence of I mg DNA each, from JM (• ), KM -3 ( ),
`SH-2 (■ ) and BJAB ( .J) cells a nd 100 pg Raji-DNA,
`• · supplemented wit h 900 /lg salmon perm D A.
`BJAB and Raji DNA served as negative and positive
`con trols. respectively.
`
`kem ic cells fro m patient J M were s table at 37 C a
`to norma l periphe ra l T
`opposed
`lymphocyte .
`A certa in percentage of es tablished J M cells bound
`EAC; tryps inizcd SRBC were n ot bound under
`iden tical cond itions. Con-A recep to rs a nd /J2-m icro(cid:173)
`globulin were detected in J M and KM-3: the line
`S H-2 was not te led .
`
`D ISCUSSIO
`
`EBY-ha rboring ly mphocyte may pers ist fo r many
`weeks in I issue cul tu re befo re permanent gro\\ th
`occ urs to a high deg ree (N il son, 19 7 1). As a con(cid:173)
`sequence, tumo r cell are ra pidly overgrown and
`
`TA0LI:. I V
`
`D ETECl ION OF- C CLL M E MBRA
`
`[ R l:CLPTOR S
`
`•
`
`Receptor
`
`Tcdin,que appltcd
`for dctecuo n
`
`SRO
`
`' -receptor
`
`Rosette forma tion
`
`Fe-receptor
`Complement-recep tor
`Membrane-bound lg
`P.-m1croglobulin
`Con-A
`
`Ro eu e fo rma11on
`Ro elle forma tion
`Direct and indirect I I-
`Indirect IF
`Direct rr
`
`4
`37
`
`JM
`
`Cell Imes
`
`K/11 -J
`
`56.0° 0 (76.0 ° ) t
`59.0° 0
`°
`2.0 ° 0
`18.0 ° 0
`o.o• 0 (2 o· .>
`
`0.0 " 0 (2.0 ° ol
`0.0 ° 0 (0.0 ° 0 )
`4.o •. O.0 °0 >
`2.5 ° 0 (9.5 •• >
`0.5° 0 ( 100 ° 0 )
`
`H-2
`
`o o·.
`T'
`T
`0.0 °.
`NT
`T
`
`1 !>beep red blood cell ( RBC)-rcccpto r
`
`' Data from primary lym phobl:uu •n parcnthcscs
`
`0 1 tcsml.
`
`Miltenyi Ex. 1042 Page 8
`
`

`

`ESTADLISHME T OF L EU KEMIC CELL LINES
`
`625
`
`ly a few EBY-negative tumo r cell lines derived
`on
`"
`. 1
`.
`from donor wich de11n1te y proven a nt1-YCA titers
`ha,e been esta bli hed so far ( Meneze et al., 1975 ;
`Epstein et al., 1976). The lines S H-2 and KM-3 were
`obtained from patients with e ndogeno us E BY titers,
`since
`the transmissio n of a ntibodies by blood
`transfusion o r a ppl ication of human gammaglobulin
`was excluded. The lack o f E BY in the line KM-3,
`SH-2 and JM was shown by the lack of E BNA, by
`the failure to detect EBY genomes by reassocia tion
`with radioactively labelled E BY-DNA, a nd by the
`Jack of typical c:1aracteristics of EBY-transformed
`B lymphocytes (membrane-bound immunoglobulin,
`formation o f colonies and cytoplasmatic project ions
`in tissue culture). The leukemic origin of the estab(cid:173)
`lished cells was confirmed by the identical pattern
`of cytochemical sta ining and cell membrane recep(cid:173)
`tors as compared to fresh
`leukemic cells. Acid
`phosphatase staining in the line JM was negative
`after eight passages. Therefore a change in other
`properties of cells kept in tissue culture has to be
`considered. All three lines were derived fro m male
`donors and revealed diploid male karyotypes. In
`addition each of them was characterized by distinct
`chromosomal markers (Schwanitz, 1976, unpub(cid:173)
`lished).
`KM-3 and SH-2 revealed only P~-microglobul in
`and Con-A recepto rs, but neither B nor T charac(cid:173)
`teristics. T he establishment of leukemic
`·• null ,.
`lines from patients with a blast crisis of a chronic
`rnyeloblastic leukemia was a lready reported (Mino(cid:173)
`Wada er al., 1977). The patients KM and SH,
`however, had a n ALL or leukemic-tra nsformed
`NHL, respectively, a nd no Philadelphia chromo(cid:173)
`somes were detected in the es tablished leukemic cells
`(Schwanitz, 1976, unpublished).
`A few cell lines with T-cell c :1aracteristics have
`already been established (Foley er al., 1965; Ada ms
`~ al., 1968; M in owada et al., 1972, 1977). The
`. cell character o f established J M cells was pre(cid:173)
`I
`\'10
`(S us Y demonstrated by SR BC-rosette formatio n
`r chwenk and Schneider, 1975). The stability o f the
`ose11cs at 37 C
`.
`. .
`.
`.
`suggests a thym1c o rigin, s ince
`: rip~leral T lymphocytes bind no S RBC under
`entical conditions. T he detectio n of complement
`
`recepto rs (after inhi bition of T-rosette fo rmation by
`try ps inizat ion o f SRBC) o n the mem brane o f JM
`cells correspo nds
`to
`the findings obtained with
`another T cell line (Theofilopoulos et al., 1974) a nd
`a lso p rimary T lympho ma cells (Ste in e l al., 1976).
`Fetal thymocytes a lso reveal complement receptors
`and this was regarded as a n ind ication for the
`o rigin of T lym phoblasts (Stein el al., 1976).
`The presence of autologo us macrophages o r
`fibroblasts obvio usly prolonged
`the survival o f
`prima ry cultured lymphoblasts. Viable lymphatic
`cells persisted for periods as long as 16 weeks (as
`opposed to the short surviva l of normal unstimu(cid:173)
`lated lymphocytes). Similarly, the presence o f fibro(cid:173)
`blasts or macro phages enhances the esta blishment
`of EBY-transformed B lymphocytes (Schneider and
`zur Ha usen, 1975).
`An important aspect is that all leukemic cell lines
`were derived from explants obtained during a
`relap e or leu kemic transformation. Most o f the
`T cell lines (Adams et al., 1968 ; M inowada el al.,
`1972, 1977) and a leukemic·· null'. line (NALM-1,
`M inowada et al .. 1977) were also obtained during
`a rela pse. In contrast, no ne of the 34 ex plants
`derived from patients with a primary manifestatio n
`resulted in a permanent growth. T aken together,
`these results suggest a n enhanced growth potential
`of
`those
`lymphoblasts which
`resisted chemo(cid:173)
`therapy, although the efficiency o f es tablishment _is
`very low. None of the IO grid culture' resulted in
`permanent growth a nd the tissue culture techniques
`ha ve to