Chrontosontal Abnorntalities in Cutaneous T-Cell Lyntphonta and in Its Prentalignant Conditions as Detected by G-Banding and Interphase Cytogenetic Methods

Leena Karenka, * Eija Hyytinen , t Seppa Sarna,:j: and Annamari Ranki *§ ' Department of Derm ato logy and Venerea l Diseases, Helsinki Universiry Central Hospital. Helsinki; 'I' Laboratory fo r Cancer Genetics . Ins ti tute of Medica l Techn ology, alld *Department of Dermato logy and Venereal Dise<lses, Universiry ofTalllpere, and Tampere University Hospital. Tampere; and :j:Department of Public Health, University of Helsinki . Helsinki. Finland

The etiology of cutaneous T-celllytnphotnas (CTCL) is unknown. We studied the pattern of chrotnosotnal abnormalities with G-banding and interphase in sit" hybridization tnethods in blood tnononuc1ear cells in 17 patients representing the different phases of CTCL or the prelnalignant condition, parapsoriasis en plaque, and in 10 control persons. We used biotinyl­ated centrol11ere-specific probes with fluorescent de­tection (FISH) for chrotnosotnes 1, 11, 8, and 17 and sil11ilar, enzytnatically detectable, digoxigenin-Ia­beled probes for chrotnosotnes 1, 6, 12, 17, and 18. In G-banding, all patients showed num.erical and struc­tural chrotnosotne aberrations. Numerical aberra­tions of chrol11osotnes 6, 13, 15, and 17, l11arker chrOtnOSOlnes, and structural aberrations of chrol110-sotnes 3, 9, and 13 were increased in tnycosis fun­goides (MF) cotnpared with healthy controls. In four

Mycosis fungo ides (MF), the most comm o n type of cutaneous T-cell lym ph o m as (CTCL). usu­all y deve lops slowly, during the co urse of sev­era l yea rs. T he most comm o n prem alignant skin in volveme n ts be lo ng to th e parapsorias is (PPS)

g ro up (La mbe rt and Everetc, 1981; Bunn ct al. 1980c). T he Sezary synd rome (SS) is the le ukemic form of CTCL and ca n evo lve via M F o r ca n be the first manifestation of CTCL (Abel, 1985; Bunn el

aI, 1980a). T he mechani sm and time of mali gnant transformation in CTCL arc not k nown. but the T ce ll s are tho ught to undergo malign an t transformation before they pass in to the skin (Bunn ct aI , 1980a; Whittake r et ai, 199 1). Rearrangements of thc T -ccll rcceptor gene show th 'lt the T cells at ea rly stagcs of CTCL are pol y- o r o li goclonal in o rig in (reviewcd in Wood , 19(5). H owever, a sm all percentage of cc lls in the skin lesions and even in extracu­wncous morphologi cally uninvolved tissue ma y represent a clo ne

Manuscript received Novcmber 27. 1995; rcviscdJul y 10. 1996; accepted for publication August 29. '1996 .

Il..eprill t requcsts to: Dr. Lecna Karenko. Departl1lcll t of Dermato logy and Venerca l Diseases. Helsin ki Ulliversity CClltral Hospital. Meilahdclltie 2, 00250 Helsin ki Finlalld.

Abbreviations: £015 1-1 . cll zyme-detccted ;/1 S;f/l hybridization; FISH, Auorescellcc ;/1 S;f" hybrid izatio ll ; MF. mycos is fUllgo ides ; pPS. p'Jrapsoria­sis; SS. Sczary synd r0I11c.

of five patients the detection of a chrol11osotnal clone preceded relapse or progression of the disease. In FISH of interphase cells, the cells abnonnal for chro­nl0S0tneS 8 or 11, and for all four chrotnosomes collectively, were increased in MF and in Sezary Syndrome (SS). FISH and G-banding l11ethods agreed statistically significantly for the d e tection of mono­somy. Also, digoxigenin-Iabeled probes hybridized to interphases or tnitoses detected aberrations corre­sponding to those observed with G-banding. Thus. chrotnosomally abnorl11al cells can be found in the peripheral blood of both parapsoriasis en plaque and CTCL patients. They can be detected with interphase cytogenetical methods, which obviates the need for dividing cells, often difficult to accomplish in CTCL. Key IIJoyds: mycosis fimgoides/parapsoYiasis/cill'Omosome/in situ "ybyidiz atioll. ] lIwest DeYllratol 108:22-29, 1997

tha t later beco llles dom in ant (Veelken cI aI, 1995; Wood, 1995). C lon al rearrangem ents of the T-cell recepto r genc have also been repo rted in several other T-cell diseases. such as pityriasis li chen­o idcs et va ri o liform is acuta (reviewed in W e inbe rg cl aI , 1(93), and their signifi cance in difi:c l-cntiation of malignant and bcni gn T-ce ll pro life rativ e skin disOI-ders is thus not unequivoca ll y recognized (Landa ct aI, 1993; W einbe rg 1'1 al. 1993 ; Vee lken ct aI, 1995). A lso, the diagnosis of ea rly CTCL with clinica l, histopa th o logic, and immun o hi stochem ica l m etho ds is o ften diffi cult, and the smail number of atyp ica l cells res tri cts the cho ice of di agnostic m casures.

Ea rlie r cytogenctic studies, m os tly o n blood lymph ocytes, of MF o r SS pa tien ts (N owcll cl aI, 1982; Whang-Pcng el aI, 1982; J o hnson el aI, 1985, Berger and Bernhcim, 1987; Sh apiro cl aI, 1987), have revea led a large spectrulll of chro mosom al abn o rm alities. vc r)' often nume rical. N o specifi c abn o rmality conld be detected, but cytogene ti c abno rmaLities could precede his to logic changes and we rc sugges ted to have a signifi cant d iagnostic and prognostiC va lu e (Whang-Peng ef aI, 1982). As traditional cytogen etics with G-banded chro mosomcs is tinlC consumin g and de tects o nl y spon­ta neously dividin g cells, o r cell s indu cible to divi sio n in cultivation ,_ we decided to compare if I sitff hybridi za tio n m ethods. capabl c oj

detectin g chro mosoma l aberratio ns in n o n-dividin g in te rphase cell s, with G-banding. O ur aim was to detect and chara c te ri ze eventual chro m oso m al aberratio ns in periphera l blood lymph ocyteS

0022-202XI97 IS I 0.50 • Copyright © 1997 hy T he Socicty for In vestig'ltivc Dermatology. In c.

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VOL. I OH. O. I JANUA R Y 1997 COMPARISON OF G-UANDING AND FISH IN CTCL 23

Table I. Diagnosis and Treatlucnt History of Patients

Disease Activ ity" TilllC froill Sex allo Age Phototherapyl Ell i Last T herapy

Pacie n t (y) Dg allo St"ge" Time li'O IlI og At Sample Fo llow-up Time (y)' T ime" at Samplil1g

Fn S FM Sino R PUV AI2 2 M/~(I I'PS 'i }' R PUV A/ I

N o During PUV A N o 1 wk/ PUVA

3 M / 56 PI'S 12 Y A SUP No 15 mo/ UVB UVB / 2

4 F176 PI'S 5 y A SU P/ I 5 M / 32 MF, IN ,11110 R PI' PU VA / I

No 4 y/ SU I' No Sd/ PUVA

(, F/ 49 MF. IN 2 )' R PUVA / 4 7 M /~7 MF. IU" S Y R PUVA / I

No 2 y/PUV A 2 rn o 10 rno / PUV A

S M I()I MF. I13 I wk A No ~ y/E13

No 9 M / 4l:l MF. III 2 Y R PI' I' U VAI2 N o 2 wk / l'UVA

10 F/ 57 MF. II3'· 5 }' A PI' No 11 F/ 45 MF. II 11 17 111 0 A PI' PUVA I2

5 Y 2 m o / puv A' 2 )' Duril1 g PUVA

1. 0 m o / EB 12 M / 68 MF. II U" <) Y R PI' SU P/ I (, y ~ mo/ EI3

PUVA / I 13 M I7 'J CTC L. II13 ' I Y A No 14 Fl U) MF. III I 111 0 A PI' No

4 y D nrin !; EB No

15 M / 77 SS. Ill lll :2 111 0 A N o No 16 M / 66 SS. 111131 wk A No N o 17 M /58 SS. Ililli I wk A No n

.. FM . 1i.) lIi c lllar IlIl1 Cill t.>!"is: PI'S. par:lpsol' iasis; C T'CL -" Ia gillg (13 UIlII I'f 1/1. I C)SO;l) : B I. arypi c;11 cin.:III ;nill g cc ll )ri.

v R. remi ssio n ; A . active disc~l sc : PI'. b tL' r progrcssitlll {If ti ll: Jiscast..·. t" P UVA. psn ralcn + uhr:n ';o lc( A: SU P, '\c lccli vc ult r:I\'io lct the rapy ; UV B. tlltra\' in let B Ihn;, !'y . • 1 EB . dcc tro ll bC;l 1ll + r;lci i:llio ll d lc ra py . • ~ M F prc(L'dcd by pal'a psori :L'i is. , Plaqu es icli :'Ilic !" trc allllCllI .

" ClllaI1 L· tH l ~ ~<ln: o id os i ~ ill Ih l! 1 t)5Us. B-cc ll lymphoma 1 1)~ 2. Frolll 1992 ( I y hctt-ln.: sam plill g:) Ki - I + T -ce ll Iymph o lil il.

of p a tients with difJe re nt stages o f CTCL and patie nts with pre ma­

li g nant s kin les ions. We c h ose ce ntro mere-speciti c pro b es for c hro mosom es 8, '1'1, and 17. whic h had previousl y been sh own to

be among the c hro m oso m es 1lI0st o ften num eric a ll y aberrant in

CTCL (W han g - Peng 1'1 ai, 198 2). In addition. we L1 sed a pro b e

specific for c hrom oso me I. kno wn to b e involved in s tru ctura l l'earra n ge l11 e nts (Whan g -Pe n g ('I ti l. 191:12) . The h ybridized probes

were v isuali zed with Auorescen ce ill sill1 h ybridi z ation (F IS H) and

c ompa re d with the results of G -banding. A lso. e n zym e-detected ill s ill1 h ybridization (E DISH) , with digo xigenin-I abe le d probes (for

c hro mosomes I , 6. 12, 1.7. a nd 18), was e mployed .

MATERJALS AND METH O DS

Patients Altoge ther. I.'l patients with CTC L. no t undergoing radiother­ap y (exceptillg O Il C paticl1t) o r cy to static rref.ltl1lC l1ts, we re se lec ted tor th e

s tud y (Table I). T he IJati ents had been ro ll oweo lip at th e Departlllellts or Dermatology. in the Helsinki ano Talllpere Univers ity Hospil·al s. (H elsinki a nd Tampere, Finlano). since the o \\Se t o f the ir skin sYlllp tom s. The d iagnosis was based 0 11 clinical. hi sto logic. and illllllUllOltistologic findings ( rc vic 'wcd ill K uzel (' r (/1. 'I C)91), dH~ btter (\."0 in at least' two conscc llti ve biops ies. Ce ll s w ith Sczar), ce ll lII o rph o logy w e re repeatedl), sought iII the periph e ra l blood o r hOJ1l! I1lanow of MF patients . but the ), were e ithe r absent Or represented le ss th an I (l'X, of the peripheral blood lI1 o nonuciear

ce ll s. The three pa tie ll ts w ith S5 all IJresemed with erythroderma . a lld 20%-90% of their peripheral b lood lymphocy tes had Sezar), cd l morphology ( Kuzel CI nl, 199 1). III two o f the patients, n o bone m arrow o r lymph node involvclllclll" Vias fo und . howcver. :lnd they W Cfl' t re:Hed w ith sys te Jlli c predniso lo ne. T he third SS patient (case 15) later receiveo iv c1aoridine rreatment alld has rema in ed in relnissio n fo r .3 y to date (Table I) .

In add itio n. fo ur patients w ith histo logica ll y confirmed par:lpso ri as is e n p laque Or fo lli cnlar lIIucinosis les io ns (Lambert "nd Evere tt . I 9l:l 1) we re rud ied (Table I) . The periphera l hloo o ce ll s of these patiem s were

nlo rph o logicall y 11 0 rl11al. For contro l purposes . blond was drawll from six hea lthy voluntee rs (four womell and two IIl e ll ). aged 25-5 2 y (meall 39 y. SD 9 y) . III adoition. f0 1l1' pati ents w ho had rece iveo systemic o r tOIJi ca l PUVA (psora len ,md ul t ravi o let A) th erapy fo r pso riasi s \'Id ga ris or a lopecia a rcata / to tali s (ageo 34 -5 4 y. m edian 44 y). were studi ed 10 . 26. 42 oays . o r 1 1 months after the PUV A treatment.

G-Banding of Mitoses Peripheral hlood lymp hocytes we re stimulated willI I'H A. cultured 1'0 1' 3 to 4 d . and IlIetap hascs trypsil1 G-bal1dcd in the con ve n t iollal way (Venna an d Btll'lu . 1989) . Q ne hundred 111i toscs in evcry sarnplc werc exa min ed . and th e chrornoso lllal ahelT'lrio ll s were c1 nss ifi ed accordin g to ISC N 1995 (Mitclmal1 . 1995). C hromoso m ally abe rral1l cells of rhe oiltcre lll patient g ro ups were conllteo and g ro ll IJcd h )' type of ;lhc rratio Jl: 1l1l111c rical o r stru c tural, ho th in rhe S:l nl C cell. o r Other abc rr:l­tiOIlS (11Iinlltcs. pu lverization) . III additi o ll . Ilulli e ri ca l and srrll Ciural aher­rati o ns of indi vidual chrOllloso lll CS \"erc CO llllte d separatel y. As po lyclonal. Jlul11c ri ca l chro illoso ina l aberrations a rl' ("0l1 l1"1l 0 1l in CTC L. a c hro llloso ln al dOJ1e was defined as t.hn . .:c or t11 0re Illil OSCS with th e sa ine llu1l1c ri cal a l'le rration. o r two o r Ill o rc mitoses w ith th e sa lli e stru c tural al'lcfl'nti o n (Mitelmall. 19'15) pCI' 100 Il)itoses .

Probes and Probe Labeling DNA pro bes . 200-4Utl bp in si ze and spcc iti c for the pc rice ll tro ll lcric reg io ns o f the fo llowing c ilrOlltOSOIlICS.

were used. For FISH. bintin ylatco (l3 ioNick kit; G IIlC O BRL. Gaithers­burg . M D) probes fo r ch ro l\loso lll es I ( I q 121 pUC 177 D. 8 (DSZ2 IpJMI 2S I) . I I (pSP65 IpLC IIAI). :1l1d 17 (D 17Z llp1 7 hS I) Wl,re used . Probes spec ific fo r ch rolllosO l\l es I :1 no 17 (p laslllios as above) nnd chromoso ll) e 12 (pA 12 h8) "lid 18 (pI 8 R ) \\'ere also Iaheled \\'ith o igo xi­gCllin - ll-dcox ),uridinc triph os ph;ltc (Uo c hringcr Ma l111h e inl ) b y ni ck trans­latio n (ll ioNick k it). The IJro be for chro ll1 osom e () w"s a digoxigenin ­lahded cOnlmercial pro bc (On cor III C" C;a ithershllq;. M D).

III Sitll Hybridization The ill silll hybridizario n of inlerphase or milooc cdls \Va s p~ rfo rl11 cd as pre vi ous ly desc ribe d ill detai l (Hyy till l! l1 ('( 01 19<) .. q. The hybridizati o n mi x ture co l1tained the labeled prohe ( 1-5 ng) . de x tr:1I1 $lIl fate ( IO'X.. Sig lll a. Sr. Louis M ). torl1larllioc (5 S% ill stalloard sa li llc citrate). and herrill g sperl1l DN A (n. s f.lg per 1111 in Tris-EDTA hu tfe r. pH lUI:

Sigllla) . Depending on the probe (F ISH 0 1' ED ISH). the IJl'Ocedure contin ­ued in two oiltc renr wa),s. T he slides 1'0 1' FISH w ere prcblockeo wit h 1% USA (Sig l11:l ) ill -.1 sta nd;lrd s:li ine citrate . inc uhat ed with ;1\ - idil1-t~u o rt.=sceill

iso thiocyanate (5 f.lg pCI' nIl; Vec tor Labo rarories. llllrii nga l1l e. CAl. \\'n shcd \\'ith ~ X standard sal inc citrate and I'N solutio ns (0. 1 M NaH l HPO .,. 0 . 1 M Na! HI'O .,10 .1 % NP-40. pH ll.O). FlIrlhcr prcblock ing was do ne ill PN M (S'X. C al'llatio n dry lIIilk / PN) . prio r to biotin ylatco ano-avidin a lltibo oy (5 f.lJ; per 1111; Vector La bo rato ries) as desc riheo pre "io ll sly (H yytin e ll CI nl. 1 99~). Fi ll nll ),. th c slides w ere stained anew with avioill-flno r('sccin isothio­c),alla te. was heo, allo 1I10 1l1ll eO in 10 f.l l of IJro pidiul1\ iodioe ( I f.lg

24 KAREN I{O lOT At THE JOURNAL O F INVESTIGATIVE lJEltMATOLOGy

Table II. Summary of Cytogenetic Aberrations Detected with G-Banding "-

Percclltage of Cells (11 = 100) "-

Abcrnlllt

"" Numerical Structural Diagnosis n Normal 0 111 )' a nI), Both" Other" Tot'\1

"-Health), control 5 Median 92 4 2 Od 0 8'·

I~ange 88 -98 1-1 0 1-3 0-2 0 2-l ~ Pa nlpsoria sis 4 Medi~n 89 .5 7 1 OS' 0 10.S·

R.ange 81-95 4-16 0 - 2 0-3 0 - 3 5- 19

M)'cosis fungoidcs'· 10 Median 85 8 .5 3.5 3 .5,1 0 15" l~a l1 ge 49-95 3- 20 1-7 1-35 0-1 5-51

Sezary syndrome 2 Ml!dian 85 8 3 3 .5 0.5 15 R.angc 82- 88 3- 13 3 2- 5 0-1 12- 18

" ,I Both nume ric.1I and structural ahcrr4ltio ll s in the x;lInc c..:ll. /, Minu tes or plll\fcri z~HioTl as the (l ill y nhnormali ry ill the ce ll . I T he c!OIl ';S in M F patic llrs. Case H: a d one 47. X Y. + 12 in .1 / I Of) ~llJd ;'lCldil"iollilJl y ;n 4) I) 6 t:c lJ ... . +- 12 aho in 2}2 1 (j cell s wi rh o ther ka ryolypcS. C ase 10: i\ d one of tWO c;cll~

with ., rypi c:1f lIlarker. Cn ~c f I : a clo ll e 0 (-4 / /00 c.;c lJ. .. 5 1X. - 1. - 2, + 2 1. as COlllllJOII fC;ltUfCS, r)'pici11Ilwrkcrs. aSc 12: ;\ d Oll !! of7 / 1(10 r eUs w ith" typic,, ' marker, va riatio\\ o f chromosome Ilumhcrs. C~I SC 14 : .1 cloue of 3 'J / 1 VI) cells, ~Cc Fig 1 .

. 1 Sigllific:IIH di(fi.: re ll cc ,~ between Ih c gruups indi ca ted. p < n.02. ;md in p<lin:o cmnp;\flsons between hC.l1 lhy controls and MF gro up. p < f).OS. " Signitic'JJJ[ diffen:m ccs hct''''ccn th t.! gro ll}J !<I iJldic:ltcd, p < 0.05 . ,md in paired cu mp;\l'isons hetween healthy COlHro ls :lIld MF ~roup. p < fJ.OS.

propidiu1l1 iodide per mi . Sig1110) ill lin anti-hIde so lu tion (Vecrashield. Vector Labo ra to rie.I). 111 the EDI5J-l method . digoxigenin- l<1be led probe was detected with I11 0 U.le '11l ti-digos igen in an tibody (Bochri nger Mallll ­hci 111 ) followed by bio ri11 ylatcd anti-mouse <l ll ti bociy, a11d I1 vidi11-biotin­perox id"se-mix tllre (Vect"st;';n Eli te mOllse IgG kit: Vec tor Labor;ltori es). Dia1l1i110henzidi11e with nickel was used as Chl·ollloge n (Dialllinobenzidin e SuhSlrate Kit, Vecror Lnboratories) . T he sli des were cOLlntersta in ed with alLll11 -kenlechtrot [AI2 (SO.,).1 . l !:l H 20, I 0 g. kc rn eciltror 0 .2 g. H 2 0 200 Ill!. bo th Merck, Darmstadt, Germany] for 5 min . washed with till' water. dehydrated. ;1l1d mOl1l1ted in Ente ll an (Merck) . T he hybridiza tion results were eva luated with out knowledge of the p~ticnr's cl ini cal diagnosis and the rc,ult of G-banding. Ovcrlappin g nLl ciei were l10t ;1l1alyzed. T he sign;ds were required to be of c'] uaJ inrensity ;lnd c1c;lr ly separate . At least a hundred interphase nuclei were al1 al),zed from each sam pie. with a few exccptio llS.

Statisticia l Ana lysis The .,wtisti c:d anal),sis was performed with the UMDP package (Dickson. 1992). T h" G-banding results were analyze d \vith the Kruskal- Wa lli s no npa rallH.:tric anal ysis of varia nce cOlltinuing with the Dun11 lllu ltiple comparison rest. The FISH resul ts were evaluated with th" Mann- W hi t",, >, U-tcst. All p values < O.llS wcre considered statistica lly significalH. As the 111l1nber ofpatiencs studied "'liS 511);1 11 . P values 0 .05-0. 10 arc also reported. T he F1SH- and G-b:lIldi1Jg res"lrs were c0 1J1pared wit.h til e Kappa tcst. An "berration rate higher chan th e highes t observ ed individ"al "berratio l1 freque1Jc)' in the he:dthy comrol group in FISH was conside red ;fbnornwJ.

RESULTS

NUluerical and Structural CbrOltl0S0lne Aberrations Were Detected by G-Banding in PPS and All Stagcs ofCTCL Al l CTCL and PPS pati ents ",x;l1nined showed nu1tle rical and struc tura l chromosome abnormalities so th 'lt num erica l abe rratio n s were 1110 re cOlllmo n in eac h g roup (Table II). The median of the perce ntage o f ch rom osoma ll y abnormal mitoses was highest in the

M F g ro up (15'%), :lIld five M P patic n ts had a chrolllosoma l clone (Table II, F ig 1). In th e 5S g ro up, o nl y two G-ba nded samples we re avai lab le, and the S5 g roup was thus not included in this stati sti ca l an alys is. The dilfe rc n ce of tota l numbe r of chromosoma ll y abnormal mitoses betwccn all the other g roups was signiflcan t (p = (J.03, Kruskal-Wallis test). Also , the MF group showed sig ni ficantly hi gher valu es in paired comparison s w ith th e co ntro l g roup (Table

II) . Numerical aberration s were m ostly losses o r, to a lesse r degree.

gains of chromosomes, resulting in nea r-diploid mitoses (Fig 2) . Mitoses of othe r pl o idy lev el s were rare. (n t he PPS alld MF g rou ps, IluJ11e rica l ab ll o rm;llities of all individual chromosomes were de­tec ted. Statistica lly sign ifi can t differe nces between the g ro ups w e re detected for c hro mO.lomes 6, 13, 15. and 17 (F ig 2). An extra

chrol11osome 8 was detec ted in three PPS o r MF patients, a ll w itll active disease.

Stnl c tur,ll a berratio ns in G-banding were rare in the PPS g roUlJ (Fig 3). M 'lI'ker chrom osom es that co uld not be characterized mor~ specifica lly were found ill a ll CTCL ;lJ]d some of th e PPS pacient~.

Statistica ll ), significant differen ces be tween the groups were de, tect ed for chromosom es 3, 9, 13, and marke rs (Fig 3). AbeJTation~

of chro m osom c ·14 were o bse rved in six MF cases and in O Jl ~

health y control. PUV A trcatm cn t did not significa ntl y re lace to the observeti

chromosoma l abnormalities; un trcated CTCL o r PP5 patient~

sh owed a hi g her total percentage of chromosomally a be rran t ce ll~ in G-banding tha n controls treated with PUVA. Con tra stin g wirl\ the fi n din gs in patients w ith PPS or CTCL, m ost aberratio ns it( PUVA-treated controls w e re struc tural (Table III).

Chromosome Aberrations a nd Disease Activity in Patient~ with PPS or MF In fo ur sam pl es obta ined during a remiss io n, chli, tota l percentage of chro m oso mally aberrant cells (medi an 9. rang\) 5- ·1·1) wa s in the range for h ealth y person s (highest va lue 12'Y.,) bllt was ill c re'lsed (median 22 , r;lIlge 13-51) in six patients w ith activ\) sk in les io ns. Three o ther MF patients, cases 5 , 9, and 12, were ill re missio n but had an e levated level of abe rrant mitoses (14% , 14";'1, and 40'}{" respec tive ly, Tables I,ll) . T he ir disease re lapsed 6. 5, and 1 1110 la tel', respective ly. Case 12 also had ,1 chr0 1110S0I11'1.\ clone. T he PUVA treatment received by these patients was all unlikdy calise of t he e levated perce nta ges. bec:llIse four odwt pa ri e l ts (cases 1 , 2, 6, and 7) h;1d rece ived similar treatme n t but dicj not sh ow ele vated levels of aberratio ns o r subsequent rdal se ofthl) d isease.

1n fOllr (c;lses ·10, 11, 12 , and 14) of the fi ve patie n ts with a clone, the discase relapsed or prog ressed soon afte r the detection of tbtl clone (Tables I,II). T he tifth patient with a clone (case 8) a lso had active disease. The most exten sive cion c (3 1/ "100) was found in ~ patient with no previous treatment (case. 14; Fig 1) , w hose diseaS() pl·ogressed to S5 within 23 1110 despite trC;ltmCIlt. Cases 10-12 and 14 died of their dise;lse 24-29 rno after sampling. T h e reproduc, ibility of the chro m osoma l findings was demonstrated when twO o~ the pa tients (cases ·11 and 12) were inves tigated afte r an in te rva l o~ sevcra l months, a nd the sa lll e clonal mi toses were found aga in (se~ as an example Fig 4,,) .

Paticnts with CTCL Also Had More Aberrations in FISli Than Healthy Controls When the res ul ts g iven by all fOllt biotiny lated probes (chromosomes 1, 8, 11 , aod 17) ill FISH were combined , the m edian percentage of abnormal in te rpha se cells W;l;

VOL. 11)8. N O. I J ANUARY 19n

1-2 3 4

1- ,. t

6 7 8 9 10

'f\ 13 14 15 16

-, . -, 19 20 21 22 Y

1.0% (range O.O- S. O) in the healthy con tro l gro up, 3.4'X, (range 0 .0- S. 3) in the PPS gro up, 4 .S% (range 0.0 -11 .5) in the M F group. and 7.0% (ra nge 3.0-16.0) in the SS gro up. T he d ifferen ces between the healthy con tro l gro up and MF or SS gro up were sIgnifi can t (p < 0.01) . Also, the n umber of abnorma l interp hases in the SS group sign ificantl y exceeded that in th e PPS gro up o r in th e MF group (p < 0.05) . T he sign it'i cance of d ifferen ces to r ind ivid ual ch rol1loso mes is given in F ig 5. Fo r ch romosome 11, the difference b etween healthy contro ls and the SS gro up approached signi fI cance (p "" 0.06).

~ ... 2 6 8 11 1~'-'7~2,uO,lWX.J,..,-,

Chromosome

:[JI':: ... ........ .. . :: .. ~ ........ .. . · · ~ ~~~ur · · .... . .. .. _.

2 6 8 11 14 17 20 x Chromosome

b)

l:~· ·:I"l·· "··~·:· .. ~:. ~ 0.6 - .. . .. .. .

:;:

o

d) 1 ., '" ~ 1.15 ., ~ 8.' c .!!! al 0 . •

::;

2 5 B 11 , .& 17 20 X p

Chromosome

Y -5.5 ....

iig llre 2 . Numerical ab err a tio n s de tect e d b y G -banding . T he total ;,elght of each co lu mn i nd icates the m edian pe rcentage of aberran t m itoses o r the speciti c chromosom e. T he b lack • pa rt of the bar depicts the

proporti on o f ce ll s w ith extra ch rom osom es. ~nd the g ray 0 part the ~~OPOrtio l1 o f cell s w ith m iss ing chro ll1 osom es. n) H ea lth y colltro ls; (b) I S. (c) MF; (d) SS patien ts. p. po lyplo id. Signifj . alit d ifFe rences between ~e01.~thy Contro ls. the PPS gro up . and the MF group arc ind icated as *p < .. ~. and " p < 0 .02 , Kruska ll - \Vall is . In pa ired comparisons rhere waS a ~~l~ficant d iffe ren ce (p < 0.05) between hea lthy co ntro ls and pati en ts w ith

. Or chromosomes 6. 13. 15. and 17. For chromosom e 8. the n umbers of ~;tICn ts w ith noncl onal extra/miss ing chromosom es w ere : PI'S. 1/ 2 : M F. - 6; SS . 012.

11

17

C OM PAR.ISON or- G-BAND ING f\ NI1 FISH IN CT C L 25

5

12

1-

18 .. MA R

1

X

2

Figu re 1. An untrea t ed MF patient (case 14) h ad an extcnsiv e c hromo­so m a l clon c (3111 DO m itoses) . T he llI os t frequent ka ryotype of the done was 4S.XX. der(l )in v( l ) (p21 q32)delq4 1 q4~). de l (2)( q I 1.2q2 1 ) . - 3.ad d (3)( p 2 1). - 6. dc r(9)add (p2 1 )dcl(q34). - I O.add ( l :l) (p 1 1) . + a dd ( 14)(q».a d d( 14)( p I I ). add ( I S)(pll ) . - 17, i ( 17)( qI O).de l( 19) (pI 3.2).+ m arl . + m ar2. In som e of the llIiroses. the karyo type had changed. so that th e c hrO lll 0S0111C Iluillbe r va r ied wirh in a ncar-d ip loid range. T he n um ber of structura 1l y aberran t Ch r 0 I110S0 111 CS va r­ied . and SOllI e o ther markers cou ld be p resent. bu r de r ( l )i n v( I )(p2 1 q32)de l (q 4 1 g44) was alwa ys detec table .

D iffere n t ill S it" Hybridization Meth ods Yi e ld Comparable R esults FISH and ED ISH were com pared in fo ur samples. ED ISH detected 2.4 - 4.5% m ore interphase cell s with o nly one signal than FISH . while less than 3'Yc, differences in either direc ri on were seen in in te rph ase ce lls w ith three 0 1' m o re signals or am ong signals in mitoti c cells (data no t shown) .

III Sit" Hyb ridization Agrees w ith G-Banding T he abi lity of FISH and G-banding to detect abnormal numbers of ch ro m osomes 1, S, 11. or '17 was compared in all <l vilil able sam ples. For m o noso my of chromosom es S and 17. and for 11l0nosom y of chrom oso m es 1, S, 11, and 17 studi ed collec ri ve ly, the m ethods agreed in 76 - 93'X, of tes ts performed. (K = 0.4S - 0.S7). Combinin g

dm MAR

Y Q) X E 16 0 IfJ 14 0 13 E 0 12 ~ 9 ~

U 8 4 3 2

•• l=::=JI _________ nmmrrumllIllIl1IlIlTIllillmIlIIllIillIllli

~IIIllllIlIlIllllllllllll

~IlllIlllIID .

• IIIIIIllIl

•

0 2 4 Median percentage

O PPS .MF IlIIJ] SS

6

F ig u re 3 . Struc tural aberrations detected b y G - b andin g . T he length of each bar ind icates the m edi an pc rcclltagc o f aberrallt mi toses fo r the specifi c chro mosolll e o f each pari enr group . Median percenrage of he,I1 rh y contro ls was 0 fo r each chro llloso111 C. Signi ficant difFerences between hea lth)' control s, the PI'S g ro up, and the M F group arc indica red as *p < 0.05 and ** 1' < 0.0 '1. Krll ska ll- Wa ll i,. In pai red comparisons fo r m arker chromosom es. the difFerence be tween healthy controls and M F o r PI'S pa tients W :1S also signifi cant (p < D.OS). The SS group is no r included in the stati stics .

26 KAR.ENKO ET A L T H E JOURNAL O F INVESTIGATIVE DElt M ATOLOG\

Table Ill. Cytogenetic Findings in Patients with Parapsoriasis or CLTC and in Controls Treated with PUVA

Percentage of Cell s (11 = 100)

Aberrant

Breakage" Numerica l Structural

Treatment/ Gro up" n Normal O nl y O nly Both/' Other' C ht hag D ie. r Tota\ ---------------------------------------------------------------------------------------------------, None/pacients" 6 Median 84.5 10 1.5

Range 81-95 3- 16 0 - 3

PUVA/paticJ1ts f 5 Median 90 7

Ran ge 87- 95 3- 11 1-3

PUVA + ED/patients 3 Median 84 3 5 Ita ngo 60 - 89 3- 8 4-7

EB/ pntients 2 Mediall 7 1.5 15 4.5 I~ange 68 - 75 10-20 4-5

PUV A/ co ntro l," 4 Median 9 1 2 6.5 Ita nge 88 - 9 1 0-5 .1-7

" I'UV A. pso,.a lcn + UVA: Eli. clcG[ron beam. II NlirlI cric ~ 1 a l1 d srnlc[U r;ll abcrratio l'l !\ ill the S;1I11C cell. ( M iJlu tes or plll vcriz~' liull as the onl y " bn orll1:lli ty in the cdl.

2.5 0 0 1-35 0-3 0-2

2 0 0 0-4 0 0- '1

9 0 Il 2-2:; 0-1 0 -1

9 0 0 1-1 7 0 0

0 I 2 0-1 0-4 1-3

I 0 0-3 0

0 0 0-1 0-1

3 0 0-6 0- 1

0.5 2 0-1 2

4 0 2- 5 0

15.5 5- 5,

10 5-14

16 11- 4~

28.5 25-3~

9

9 -1 ~

d Abe rratio ns jndic,[Iting ci:lstogcni c I! xposurc scored accord\ll V; UJ lSe N 19<)5 . cht: Chr(Hn~~tid :lbc[ratio l1 s ~ rrag: fragm en t's , i ll d li rl ilJ ~ isochromaric! gaps and b re:lks. fragm ents minll tes :1I1 d pu lverization; die . r: ~l symll1ctr i c ;,hc1Tadons including d icen rric and rill ~ I.:h rOIllOSOIll CS. Th e ce ll s arc illcluded a lso in the ot ll ef cu ll1 nlll ~ . \

" Th e g ro up includes tWO p;\ticnt's w ith :J n iv c..: p arapsoriash, (C ,ISCS 3 ;lI1d 4 , 13 '% and 1 f)% ll( abl! ITa ll( ce lls. respec tive ly) . f T hc gro up includes tWo P;ltiCll ts w id, p<l r" p.~ o ti il s i s ill rcmi.~sjon (ca.H!.<; J and 2 . S'Yo ;llld H% of abcrr:lIlt· celis, rC :ipcctivc ly). g On ly co n trol.s tre ated with PUVA. or the five orhe r. hc:drh y CO li tro is. nO( trea ted witll PUVA. fo ur IHid hrC;Jkagc in t 'X, of rhe ir cel ls. sec il lso Table n.

FISH and ED ISH for chro m osom e 17 , the analysis was also concordan t for extra chrom osom e 17. and fo r al\ ce lls aberran t for ch romosome 17 (agreem en t 84')/'" and 72'1." /( '= 0.56, I( = 0.47, respect ively; pairs evenl y aro und the medi an, /( = (1 .45 , and 0.43 , respectively) . [n other cases, the observed agreeme nt r<lte was usually larger than that expected by chance , but no t significa n t (data not shown).

T he num e ri cal chrom osom e abe rrations observed with G-band­in g we re also detected with ED ISH (Table IV). T he tota l number of aberrant EDISH sign'lls in th e interphase cells paraJl ell ed the number of aberrant c hromosOllles in G-ba nding in all but one case (Table IV, case 4, chromosom e I).

W hen three clones observed in G-banding were examined with EDISH (Table IV), the 1l1 0n osomy of chrom osom es 1 or 6, (cases 1 '1 and 14, respectivel y), were detected at almost tbe sa m e fre­q uency as by G-banding (Tables II and IV). T h e absence o f the o ther cbrol11 osom e 17 centro me re resultin g fi'om isochro m osom e formatio n (case 14) was also observa ble w ith E D ISH, thou gh with a lesser frequ en cy (Table IV). T he c lo ne 47,XY, + 12 of case 8 could be observed botb in interphases (4.5%,) and in mi toses (2%,) (Tables II, IV, Fig 4).

D ISCUSSION

T his stud y shows that chro mosom all y abno rma l lymphocytes, found in the b lood of CTCL-patients , call also be found in the pe riphe ra l blood of patients with pa rapsoriasis en plaq ue or follic­Uhll' mLl cin osis. PPS pati ents also have marker chromoso mes, a phenomeno n considered typical to CTCL (rev iewed in Whan g­Peng d (II, 1982). W e also show that the abnormal cell s are detecta bl e by interphase cyto gene tics (FISH and EDlSH) , w ith

12

~10 C'G -; 8 ~ (I) 6 a. t:

4 CU

"0 (I)

2 :E

0

p<:O.01

p<O.05

_____ p<O.05

Contr PP5 MF 55 n=5 "=4 "=10 "=3

Patient 9 rou P IIilm chr 1 .chr 8 D chr 11 . chr 17

Figure 5. Median percent.ages of a bnormal interphase cells in different patient groups and healthy controls as d etected by FISH. Signifi cnnt differences between individual chro mo somes arC indicated in th~ figure. (For chrom osome 11 in PPS grou p n = 1.)

agreement rates up to 93'Vo w ith con ventional G-banding. T his obviates th e need for dividing cell s in cytogenetic analysis.

In o ur FISH stu dy, abnormal ce lls w e re most frequent in the SS group , but with G-bandill g ill the MF gro up . Som e of these observed dilfercnces m ay reRect a real difference be tween nondi,

Figure 4 , Hybridization l'eslIlts with centrol11ere-specific probes (in p arenthesis) detected imlt1l1noilistocilemically (EDISH) or with FISH, In so me cases, for an unknown n .!aSO IJ, the mitotic ch rOlJJosomes visuali ze as painted. (n) Detail o f a mitosis in the clo ll e of case 14. Fo llow-up sample 2 )' after the first samp le (sec Fig 1) with a simi lar clone. T he derivative chromosome 1 is visible w ith a full-l ength chro moso me I (cho ll1osome I /ED ISH); (b) Normal mitosis and inte rphases o f case 8; one in te rphase shows th ree signals (ch rol11oso ttl c12/ED ISH); (c) A tl;so l11i c interphase of case 8, (chromosome 12/ EDISH) ; (d) A tri somic mitosis of case 8 (chro mo so m e 12 EDlSH) ; (e) A normal mi tos is and interphascs ofa health y persoll (chromosom c II / F ISH); (1) Normal mitose and monosomic in terphase of case 9 (chromosome 1 IFISH ); (~) An interphase ce ll w ith four signa ls of case 12 (ch.romosome l / F1SH ); (h) In te rphase cell s with o ne to three signals ofensc '13 (chrom osom e 17/FISH). Scale !. (f rs, 10 (.kill.

VOL. 108. NO.1 J ANUA R Y 1997 COMPA RI SON OF G-llAND ING AND FISH IN CTCL 27

•

a - b -•

28 I<ARENI<O /iT AI. Ti l E JOURN AL 01' INVESTIGATIVE DEI\.MATOLOCY

Table IV. Comparision of Chromosome Aberratiol1s as Detected with G-Banding and EDISH in Selected Patients and Comparison of EDISH Results with COl1trols Treated with PUV A

----------------------------------

Patient

F 4

11

F 11 13 I"

F 4 8

12

F 4

l(j 14

F 12

I)i;'gllose

Healthy Parapsoriasis Mycosis fUllgoides PUVA"

HC<l ltl' l' Mycosis fU ll goides Mycosis fungoides Mycosis fungo idcs PUVA

Healthy P~\rapsol-ia si s

Mycosis fungoidcs Mycos is fungoidcs PUVA

HC:1lthy Pnraspo riasis Mycosis fungo ides Mycosis fungo ides PUVA

Heil lthy Mycosis fungoid l!s PUVA

Ml!rli;ln Range

Median I~a nge

Media n Range

M edian R;lI1gc

Median Range

Chromosollle

(,

6 6 (j

(,

12 12 12 12 12

17 17 17 17 17

18 I I:! I II

Tota l Nllmber

2112 3 14 202 250

21)0-300

208 22(, 405 119 165

% - 200

209 409 221 513 101

92- 103

400 230 100 222 207

124 - 28')

SOO 101 203

200 -40S

EI ISH" t~, Ahe rrant Ill te rph ascs G-U:1I1r1ill p;

'% l\1 itoscs (II = 1(0) Sigll:1ls/Ccll with. C hrO tl H1SO tll C

----- --> .1 Tol'a l Miss itlg Ex tra Tot,,1

~.5 0 4.5 n \l 0 6.4 1. 9 IU 0 fJ 0 5.9 0 5.9 5 0 5' 2.3 1.3 2.8

1.7-2.5 0-5.5 2.5-3.7

3.9 () 3.9 0 () 0 '1.3 0.4 9.7 2 () 2 6.9 1.7 8 .6 1 2

28.6 1.7 30.3 29 0 29" n.9 4.25 5.40

0-2.5 3-·1. 6 4-6 .2

1.0 U 1.0 0 0 0 10.3 ! .7 12.0 2 () 2 1.4 4.5 5.9 II 4 4" 7.0 2.5 9.6 7 0 7 1.5 2. 1 4.4

0-3.9 1-5. 11 3.3-5.0

3 0.3 3 .3 0 () I) 7.t! 3.n 111.9 1 2 3 6 5 I 1.0 2 2 4

17. 1 1.4 18.5 3() (l 3(1'" 3.4 1. 5 5.0

3.0-4 0.5- 1 .7 3 .5-5.7

2. 6 0.2 2.8 (J () () 5.11 3.0 7.9 4 2 6 3 11 .9 4

2.4 -4.9 0-1.5 2.9-5 .7

,r Ahcrr:ltion r;ltc~ exceeding that of the highest v;lhH~ of the h C:l lth y control wcn:: cn ll ~idCTCd ahnnrlll::l l. /, Four cOJnroJ path:J1I 'i LTc,Hed w ith P UVA ( In G-bandin g a d01l e of 4/1 UO cells and onc llonc\ol1;) \ cell with one chro mosome I missing. sec Table n . " I'll G-b.mdin g- 27 of the 3 'f I r (iO dOlle ce lls and two IInncional cel l!; wen! IIIUIIOSQ llIic lo r chromoso me fi , scc Fig. 3. " In G-b:11lding a clone of 27 ,X Y. + 12 in 3/ I 00 cel ls. :md 1/ 100 ce lls wi th + 12 in i\ more complicated kiH·yotypc.. seC Table 11 . 2/94 trisomic mi toses detected w il-h [D ISH.

sec Fig. 4. J ill G-bOlnd ing the ;lbcrr:Hions of 2(, nflh · 3 1/ 100 cione cells illcluded - 17, i(l7q); three done celts and nBC l1onclnu;d cdl wCl'e Ilullisom ic l'Or chromosom e 17. sec Fig. 3 ,

viding and mi totic cell populations, as CTCL-cells arc difficu lt to cuJture (Nowell et aI, 1982; I-lindkj re r el aI, 1993; Abrams cl (II, '1993). Sim ilarl y. fl ow cyto metry of inte rpha se ce lls ha s shown 111 ore ane uploidy than G-b3ndillg (D unn el (/1. 1980b). T hus, o ur FISH results in the late r stages of CTCL are probably a better re fle ction of the true situation than th e G-banding findings. Additionally, ;n situ hybridi zation w ith centromeric probes detects marker chromo­somes, fi'equcnt in C TCL.

)n G-ba nding or FISH we found statis tically increased aberrat i OJ~ fj'equ encies for chromosom es 3,6,8,9, 11 . 13. and J 7. al l of which car ry oncogenes or tulllor suppressor ge nes o r region s (Genetic Maps, 1993; Karlsen el (II. 1994; Nobo ri cl (/1, .1994). C hromosom e 9 abe 'Tations and deletions of tumor-suppressive regions in 6q associate w ith subsets of non-Hodgkin's Iymphonn (OfEt ('I III, '1 993a. 1993b). De letio ns of 6q or tota l loss of chromosom e 6 have been observed previous ly in CTCL (Whallg-Peng et aI, 1982; Schlegel berger cJ (/1, 1994). Most abe rrations of chromoso m e 6, as well ;IS aberrations of chromosome 17. in o ur stud y in vo lved total losses of the chromosome. In the largest eione of our study, the loss of chromosome 6 was associ.ated w ith the presence of an isochr'o­l11osome 17q, a p henomenon observed in J11any lymphatic di seases (reviewed in Mertens eI aI, 1. 994). We did not observe isochromo­some i(18q), previously reported in culti vated cell s of o ne Scza ry patient (Ka ltoft el aI, 19\.12, 1994).

We found chro m o.lama l clones il1 five patients, all w ith an acrive

or relapsing M F. Four of the patients had complex ch romosomal aberrations. and one of the m deve loped SS. All four died w ithin 29 mo of the detection o f th e clone, whi ch is in agreement with prev ious repo rts 011 the associati on of chromo_ somal clones with poor prog nosis (Whang-Pcng el (/1, 1982; Shapiro cl ai, 1987).

Thl.! majority of the chro mosomal aberr,ltiOlls observed in the patients w ith PI'S o r CTCL seemed not to be cau sed by treatment. Diccntric o r ring chromosomes previously observed after photon therapy (15 Me V) (Step han el (II, 1990) were not fi'equ ent in our patients, and chromatid breaks detected after PUV A ;11 11;1,." (Youssefi c l al. 1. 994) were ra re . Al so . our untreated PPS and CTCL patients showed higher tota l numbe rs of aberrated cells than psoriasis or alopecia patien ts treated with PUVA.

C hromoso mal instability and karyotype variation is typica l of CTCL (Whang-Peng eJ aI, 1982; Berger and Bernheim, 1987). Based on studi es of CTCL-deri ved ce ll lines, a hypothesis of polyc lo nal. "genotraumatic," geneticall y uns ta ble ce lls has been presented (Kit I toft eJ fll, J 992, 1994; Thcstrup-Pedersen el (/1, .1994). T hus, o ur app roa ch o f assess ing geneti c instability not on ly by qua li tative chromosoma l anal ysis, but nlso usin g quantitari ve, ro u'­tinc )y app licab le ill silll hybridiza tio n m ethods, sho uld aid diagnosis and prognosi s in CTCL. A certain degrce of instability apparently also exist·s in som e cases o f parapsoriasis en p laque.

VOL. I IiH. NO. J ANUAIt Y 1')')7

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COMPAIU S() N OF C - IlAND ING ANI) FIS II IN eTC!. 29

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