RUBY I. THOMPSON. demonstrating the Ph1 chromosome in myeloblasts in the spinal fluid of a

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1 From by guest on November 18, 218. For personl use only. Chromosomes in the Spinl Fluid: Evidence for Metsttic Origin of Meningel Leukemi By RENATO MASTRANGELO, WOLF W. ZUELZER, PETER S. ECKLUND AND RUBY I. THOMPSON I T IS WELL KNOWN tht the blstic cells in cute leukemi often show numericl or other berrtions from the norml kryotype, which permit their chrcteriztion s stem lines.13 While these my undergo further evolution, they generlly remin identifible during consecutive relpses throughout the course of the disese,4 5 thus constituting evidence for the persistence of n utonomous cell popultion. Thus fr, chromosoml studies hve been restricted to bone mrrow nd cultured blood cells. Except for single report demonstrting the Ph1 chromosome in myeloblsts in the spinl fluid of ptient entering the blstic phse of chronic grnulocytic leukemi,#{176} the cytogenetic spects of leukemic cells in the centrl nervous system hve not been investigted. Under current therpeutic conditions, between one third nd one hlf of the children with cute leukemi show involvement of tht system t some time. The origin of the leukemic cells in the spinl fluid hs been the subject of specultion.71#{176} The demonstrtion of chromosoml constitution common to these cells nd the leukemic bone mrrow popultion would gretly strengthen the evidence for their metsttic chrcter. This report describes the cytogenetic findings in the spinl fluid of nine children with cute leukemi. CASE MATERIAL The ptients were children rnging from seven months to 1 yers in ge in whom the dignosis of cute leukemi ws mde on the bsis of blood nd bone mrrow findings, nd who hd been followed for periods of 763 months. The leukemi ws clssified s lymphoblstic ( stem cell ) in seven cses, nd s myeloblstic nd monocytic in one cse ech. Spinl fluid ws obtined during periods of symptomtic involvement of the nervous system, in severl instnces fter previous similr episodes for which intrthecl methotrexte nd rdition therpy hd been given. The shortest intervl between previous episode nd the time when the spinl fluid ws studied ws four months. The ptients From the Child Reserch Center of Michign, nd The Deprtments of Peditrics of Children s Hospitl of Michign nd Wyne Stte University School of Medicine, Dctroit, Mich. Submitted July 24, 1969; ccepted for publiction October 17, R. MASTRANCELO, M.D.: Assistnt Professor of Peditrics, Universit Cttolic del S. Cuore, Rome, Itly. W. W. ZUELZER, M.D.: Director of Lbortories, Children s hospitl of Michign; Director, Child Reserch Center of Michign; Professor of Peditric Reserch, Wyne Stte University School of Medicine, Detroit, Mich, P. S. ECKLUND, MS.: Reserch Associte, Child Reserch Center of Michign, Detroit Mich. R. I. THofPsoN, MS.: Reserch Associte, Child Reserch Center of Michign, Detroit, Mich. Supported by Ntionl Institutes of Helth USPHS Grnt CA 1175, nd Children s Leukemi Foundtion. BLooD, VOL. 35, No. 2 (FEBRUARY),

2 From by guest on November 18, 218. For personl use only. 228 MASTRANGELO ET AL. 1 C 1 Ac A AVl1 c Cl if i_ CI, 8 z (I 4 Cl 1 r fi cl CL. CI C,, C).. C ) if) CIl fifi C., C ) CI if) >< l CC, E E 5. CI CI cc_ CC L) CI if) C. I j_,>_; l C I CI fi1 CIfl #{176}4: ;; v; ZE #{231})i CC.C.C Cl C ) CC

3 From by guest on November 18, 218. For personl use only. CHROMOSOMES IN THE SPINAL FLUID 229 o i V I c44 ci% I ICC C fi fi fi fifi Cl 5 C o. CI 1 Sc, I bi o, S C,, #{149}u Ei...E,. I IL SC ohs CI C1 4: fl4: I C ) I_ CI jl,,,. II CI CI C) Cl #{163}C1 cb h _4: C <><(j< < u C? Cl C) C) tcc C ) C CC CCCCSf) If) It) if) It) 4: C) C, II 4 I ( I.E OC)i CI Cl Cl 2 C )CIO CC CC C, S C, CI,, I C.I) I) L4 ZE CI I I CI.fl C) C,, 1) CC.1 C6CC if)c6 z 4 r.v.c6. C. Cci It) CC A

4 From by guest on November 18, 218. For personl use only. 23 MASTRANGELO ET AL. CASE NO _I(O :4L.c. I.M. m.r.l m.r.2 mr.l nir.1 nl mr.2 flr.3 mor.4 C.S.F. 1E IJIIUI1.O? mcr.5 CASINO., 8 I IN., un.r Rmr.2 II #{149}1 c_s.,. #{149} S mor.l Dm.r.1 16 D ISII.l Fig. 1.Composite of chromosome pirs showing mrker chromosomes in cses 18. Upper rowsbone mrrow. Lower rowsspinl fluid. hd lso received vrious other ntileukemic gents. All but one ( cse eight ) were in pprent hemtologic remission. METHOD The method is modifiction of the direct hone mrrow From 23 ml. of freshly drwn spinl fluid is plced in complete tissue culture medium ( MEM ) insted of bsic slt solution. After incubtion for one hour t 37#{176}C, colcemid is dded to give concentrtion of.2g./ml. After nother hour of incubtion the specimen is centrifuged nd the superntnt fluid discrded. 5. ml. of.75 M KC1 is dded. After seven minutes t 37C two drops of Cmoy s fixtive is dded. After mixing nd centrifuging, the specimen is fixed in three chnges of Crnoy s solution. Slides re prepred by rpid flme drying nd stined with Ciems t ph 7.. Stisfctory preprtions were obtined when the spinl fluid cell count ws 2/mm.3 or higher. Chromosomes in 575 cells were counted nd suitble metphses ( s rule 12 ) were kryotyped. RESULTS In every cse in which stisfctory relpse bone mrrow preprtions were vilble for comprison, good correspondence between the cytogenetic findings in the spinl fluid nd initil or in some cses subsequent bone mrrow popultions were demonstrble. In two instnces (cses 1 nd 2, Tble 1) the initil mrrow cells nd the cells in the spinl fluid obtined 26 months nd seven months lter, respectively, hd identicl modl numbers nd mrker #{176} Chromosome Medium 1A without Phytohemgglutinin from Grnd Islnd Biologicl Compny.

5 From by guest on November 18, 218. For personl use only. CHROMOSOMES IN THE SPINAL FLUID 231 A mr.t211 )4m : UhOIS(i1,pis.s11, 6 12X bis. u.s Sg O 2122 y Bi i111 flhiui 1II11IdIii 6.12 x m **1 #{248}.* Il * v Fig. 2.Cse 3. Representtive spinl fluid kryotype 21 months fter dignosis. Fig. 2b.Representtive bone mrrow kryotype 22 months fter dignosis. Note kryotype identicl with tht shown in Fig. 2, except for dditionl supernumerry chromosome. chromosomes. In cse 1, the mrrow cells subsequently underwent further clonl evolution, s shown by the development of second mrker nd progressive increse of the modl number from 52 to 55. In cse 2 the mrker ws prticulrly striking in tht it consisted of n centric frgment which mintined fixed ssocition with D group chromosome (Fig. 1). In cse 3, similr evidence of clonl evolution first becme pprent in the spinl fluid which showed mrker (Fig. 2 A) not seen in the initil bone mrrow, but noted in single mrrow metphse during prtil relpse two months erlier. The initil modl number in the bone mrrow ws 46. The spinl fluid cells t 21 months showed mode of 49 (Fig. 3). Subsequent mrrow specimens during relpses t 22 nd 24 months showed virtully identicl kryotype with the modl number 5 nd the mrker (Fig. 2 B).

6 From by guest on November 18, 218. For personl use only. 232 MASTRANGELO ET AL. I 46 t dx mos. CSF 12 mos. r.mission 22 mos. I z U U Li 46 #{163} [ 19 mos. prtil r.lps. Js. r.lps mos. prtil w D z , mos. remission 21 24mos..i1. relpse 5 CHROMOSOME NUMBER A MARKER CHROMOSOME NUMBER Fig. 3Cse 3. Histogrm of modl chromosome numbers in bone mrrow nd spinl fluid during 24 months of observtion. Tringles denote mrker. remission remission 35mos w z 4 U U, mos. remission mos. CS, 38 mos. remission U S 53 S 28 mos. CSF mos. CSF CHROMOSOME NUMBER 53 A MARKER Fig. 4.Cse 6. Histogrm showing persistence of modl number nd mrker. In cse 4, the modl number both in the mrrow nd 25 months lter in the spinl fluid ws 46. The initil mrrow hd shown four different mrkers, ech of which ws occsionlly found in single cells during remission t 13 nd 22 months. These nd n dditionl fifth mrker were seen in vrious combintions in the spinl fluid cells.

7 From by guest on November 18, 218. For personl use only. CHIOMOSOMES IN THE SPINAL FLUID 233 A X.** i..i Y :znu *IIIfl$ii 612X ill z_il I.I.*. 13_.i iii eusrl 21. y Fig. 5.Cse 7. Kryotype of spinl fluid t seven months showing mrker chromosome (mr2), lso present in mrrow t this time. Fig. 5b.Bone mrrow metphse obtined t sme time showing sme mrker plus nother (mrl) present in originl bone mrrow. Cse 5 exhibited Bq chromosome in the initil bone mrrow. The sme chromosome ws frequently found in single mrrow cells during remission over period of lmost two yers. This chromosome ws present in spinl fluid cells 23 months nd 27 months fter onset of the disese. Both the initil mrrow nd the spinl fluid cells showed mode of 46 with rnge from 4653 nd 46Si, respectively. The spinl fluids lso showed mrker in the hyperdiploid cells. In cse 6, no initil mrrow specimen ws vilble. Spinl fluid cells 28 months fter dignosis hd modl number 53 nd mrker ( Fig. 1 ) which persisted in two subsequent episodes of meningel leukemi, s did the modl number (Fig. 4). As yet, no mrrow relpse hs been observed in this ptient, but interestingly enough, single 53 cell ws found during remission in bone mrrow obtined t 35 months. In cse 7, some spinl fluid cells crried mrker one seen in the mrrow t

8 From by guest on November 18, 218. For personl use only. 234 MASTRANGELO ET AL. dignosis. In this specimen, obtined seven months fter dignosis, mrker two ws lso seen ( Fig. 5 A ) nd this mrker simultneously ppered in the bone mrrow cells ( Fig. 5 B). In cse 8, the initil mrrow popultion ws bimodl 45/46 without mrkers. The spinl fluid t 15 months showed only 46 cells. Approximtely 4 per cent of these crried Bq chromosome. Cse 9 lcked relpse bone mrrow preprtion for comprison. The spinl fluid cells 63 months fter dignosis showed modl number 55. Figure 1 shows composite of the mrkers seen in hone mrrow nd spinl fluid in the group s whole. DIscussIoN Whtever the cuse of the mlignnt trnsformtion, the nture of cute leukemi s n utonomous neoplsm is no longer in serious doubt. The leukemic cell popultion in the bone mrrow often exhibits the cytogenetic chrcteristics of single stem line which my show further clonl evolution in the course of time. Such findings re consistent with clonl origin of the leukemic cells from mlignnt mutnt. If this concept is vlid, it is priori likely tht leukemic involvement of the centrl nervous system is metsttic, rther thn the result of n utochthonous new growth, possibility suggested by the demonstrtion tht the choroid plexus nd the leptorneningel mesenchyme of the humn embryo hs the potentil for hemopoiesis. The identity or similrity of the chromosoml constitution of the leukemic popultions in the spinl fluid with those in the bone mrrow constitutes direct evidence for common origin nd thus for the metsttic nture of meningel leukemi. In view of the nonrndom effects of certin drugs nd viruses on the chromosoml pprtus observed in vitro,13 14 the possibility cnnot be completely excluded tht the unknown leukemogen might produce identicl kryotypic bnormlities in l)one mrrow nd spinl fluid cells nd tht the ltter might thus be of seprte, locl origin. It would be difficult, however, to explin on this bsis the finding t both sites of certin lrge mrkers, which by their size indicte their formtion s the result of trnsloctions, for while brekge of chromosomes nd formtion of mrkers due to deletions might be nonrndom, the recombintion of the frgments should be lrgely mtter of chnce. An even stronger rgument derives from the observtion of identicl clonl evolution in spinl fluid nd bone mrrow elements in the course of the disese, for such prllelism of secondry chnges would be difficult to explin if two independent cell popultions were involved. The findings re therefore interpreted s evidence for the metsttic origin of the leukemic cells in the spinl fluid nd, by extension, in other, less ccessible extrmedullry sites such s kidney nd gonds. By the sme token these observtions militte ginst multicentric origin of humn cute leukemi. SUMMARY The cytogenetic findings in the spinl fluid of nine children with cute leukemi re presented. Identity or similrity of the chromosoml constitution

9 From by guest on November 18, 218. For personl use only. CHROMOSOMES IN THE SPINAL FLUID 235 of the leukemic popultions in the spinl fluid nd in the bone mrrow ws observed. The findings re presented s direct evidence for the metsttic nture of meningel leukemi. ACKNOWLEDGMENTS The uthors wish to cknowledge the vluble technicl ssistnce of Mrs. Lur Brown nd Mrs. Gerldine Dbney. REFERENCES 1. Reismn, L. E., Mitni, M., n(l Zuclzer, \V. W. : Chromosome studies in leukemi. I. Evidence for the origin of leukemic stem lines from neuploid mutnts. New Eng. J. Med. 27:591, Kiossoglou, K. A., Mitus, W. J., nd Dmeshek, W. : Chromosoml berrtions in cute leukemi. Blood 26:61, Sndberg, A. A., Ishihr, T., Kikuchi, 1., nd Crosswhite, L. 11. : Chromosoml differences mong the cute leukemis. Ann. N. Y. Acd. Sci. 113:633, Reismn, L. E., Zuelzer, W. W., nd Thompson, H. I. : Further observtion on the role of neuploidy in cute leukemi. Cncer lies. 24:1448, Sndbcrg, A. A., Tkgi, N., Sofuni, T., nd Crossvhite, L. II. : Chromosomes rk1 custion of humn cncer nd leukemi. V. Kryotypic spects of cute leukemi. Cncer 22:1268, Kwn, H. C., Pierre, R. V., nd Long, D. L.: Meningel involvement s first mnifesttion of cute myeloblstic trnsformtion in chronic grnulocytic leukemi. Blood 33:348, Nieri, R. L., Burgert, E.., nd Groover, R. V.: Centrlnervous system complictions of leukemi: A review. Myo Clin. Proc. 43:7, Leidler, F., nd Russell, \V..: The brin in leukemi. A clinicopthologic study of twenty cses with review of the literture. Arch. Pthol. 4: 14, Sullivn, M. P. : Intrcrnil complictions of leukemi in children. Peditrics 2: 757, Moore, E. W., Thoms, L. B., Show, R. K., nd Freireich, E. J. : The centrl nervous system in cute leukemi. A postmortem study of 117 consecutive cses, with prticulr reference to hemorrhges, leukemic infiltrtions, nd the syndrome of meningel leukemi. Arch. Intern. Med. 15: 141, Tijo, J. II., nd Whng, J.: Direct chromosome preprtions of bone mrrow cells. In J. J. Yunis ( Ed. ) : humn Chromosome Methodology. New York, Acdemic Press, 1965, pp. 51fl. 12. Kppers, J. A. : Structurl nd functionl chnges in the telencephlic choroid plexus during humn ontogenesis. ln Wolstenholme, G. E. W., nd O Conner, M.: CIBA Foundtion Symposium on the Cerebrospinl Fluid: Production, Circultion nd Absorption. Boston, Little, Brown, Stich, II. F., Ilsu, T. C., nd Rpp, F.: Viruses nd mmmlin chromosomes. I. Locliztion of chromosome berrtions fter infection with herpes simplex virus. Virology 22:439, Cohen, M. M., nd Shw, M. W.: Specific effects of viruses nd ntimetbolites on mmmlin chromosomes. In Vitro 1:5, 1965.

10 From by guest on November 18, 218. For personl use only : Chromosomes in the Spinl Fluid: Evidence for Metsttic Origin of Meningel Leukemi RENATO MASTRANGELO, WOLF W. ZUELZER, PETER S. ECKLUND nd RUBY I. THOMPSON Updted informtion nd services cn be found t: Articles on similr topics cn be found in the following Blood collections Informtion bout reproducing this rticle in prts or in its entirety my be found online t: Informtion bout ordering reprints my be found online t: Informtion bout subscriptions nd ASH membership my be found online t: Blood (print ISSN 64971, online ISSN 15282), is published weekly by the Americn Society of Hemtology, 221 L St, NW, Suite 9, Wshington DC 236. Copyright 211 by The Americn Society of Hemtology; ll rights reserved.