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A 46,XY female with mixed gonadal dysgenesis and a 48,XY,+7,+i(12p) chromosome pattern in a primary gonadal tumor
A 46,XY Female with Mixed Gonadal Dysgenesis and a 48,XY, + 7, + i(12p) Chromosome Pattern in a Primary Gonadal Tumor A. Hamers, B. de Jong, R. F. Suijkerbuijk, A. Geurts van Kessel, J. W. Oosterhuis, J. van Echten, J. Evers, and F. B o s m a n
A B S T R A C T : (;vtogenetic tmul~'sis oI u l)rinmry ,Aertn-cell , tumor ori~intaing Irom the streuk ,Uo.ud ,~! .
20-year-oht phenotypic ¢emtde with u .t6.XY karyotype und mixed gonadtH dys,4enesis rm'ctded (t 48,X Y. • 7, ÷i( 12pJ chromosonml putter.. Germ-cell tumors originating lrt,ln gonads of normal IIItlII.'S lift? tlSU(III~."tl/gtl/~,' (meUldOid. An isochrOlllOSOtlle 12 I) (~s well (Is till m ' e r r e p r c s e n h l l i o n o.t (:/irOlllOSCqltt! 7 nloteritd are tHlIOtlg lh¢? S[}et:ilit" t:h(tllgCS tilt)St co,sistenth" observed. The present case shtm.s that tumors ,,/dysgenetic gtmmts, ulbeit I)eing near-dildoid, row." exhibit sintil(n" t:hrorm)somtd chan,e, es. This o b s e r v a t i o n l e n d s addititmal SUplmrt h) the hyln~/hesis that these s Det:ific cytogenetic oi'lomtllies Ill(IV IIItIV till Jlll DOl'ltlllt ri)lf! ill the {)(lt}ltlgt'llt!SiS fit Ilttllltlll ,.4,erm-(:e I I tlllllors.
INTRODUCTION Human testicular germ-cell tumors (TGCTs) are usually highly aneuploid with a modal (:hroinosome n u m b e r in the triploid range. In addition, they are characterized
hy a very spe(:ifi(: structural (:hrolnosomal anomaly, i.e., ail isochromosome 12p, in about 80% o[ all cases [11] for review). The i(12p) chrotllOSOllle has ills() been ()hserve(t in two (lysgerlninomas, an ovarian yolk sac ttunor, and ill s(une extragona(hll mc(liastinal germ-cell tumors 12- 71. The present study deals with a 20-year-oht female who was refe,rre(t for (:ytog(;netic analysis he(:ause of prilnary amenurrhoea and c.levated follit:le-stinmlatiilg hormon(; (FSH) and luteinizing hormone (LH) values. The analysis of perit)heral hlood lymphocyle (:ullures revealed a 46,XY chrumos(imal pattern. Because phelmtypi(: females with a 46,XY karyotype art'. at risk of developiIlg a malignant:y in the gonatls, an exploratory laparottuny was performed. Floth g O l l a d s appeared to (:ontain tumors
and were removed. From the 1)epartments of t tuman Geneti(:s. University of Limburg. Maastricht (A. 11.1. University of Groningen (FI. d. J.. J. v. El. Uniw:rsity of Nijmegen (R. F. S.. A. C. ;'. K.). the l)r. l)aniel den Hoed Klmiek. Rotterdam ( J. W. O.). and the Departments ot C;yneacology ( 1. El and Paflmlogy (F. B.]. University of Limburg. Maastricht, the Netherlands.
Address re.print requests to: A. Hmners. Ph.D., l)e.partnmnt o.f (hmetics and Cell Biology. State University o] l,imburg. Postbox 616. 6200 BX Moastricht. The Netherhmds. l{ece, ived March 21. 1991: m:ceptmt April 26. 1991.
219 ,'c 1991 Elsevier Science Publishing Co., Inc. 655 Awmue of the Americas, Nev,' York, NY 10010
Cancer (.h;net Cytogenet 57:21~-~- 224 (19911 0165-4608,'91:S03.50
220
A. Hamers et al. Here we report, for the first time, the karyotype of a dysgenetic gonad tulnor. For our analyses both Giemsa-G handing and fluorescent in situ hybridization (FISH) techniques were used. Our results indicate that this type of tumor may exhibit chromosomal changes similar to those seen in germ-cell tumors of gonads of normal males, which suggests a common pathogenetic pathway for both types of neoplasms.
CASE HISTORY A 20-year-old patient was referred for cytogeneti(: analysis because of primary amenorrhoea and ele.w~ted t"St:I and IJ't values. The analysis of peripheral blood lympho(:yt(;s revealed a 46,XY (mah;) karyotypc. For further diagnostic evaluation, the patient was referred to the Departlnent of (;ylU.'(:ology, especially to estimate the risk of developing a malignancy of the gonads. At exph)ratory laparotomy a small uterus with an extende(l cervix uteri was seen. Normal fallopian tubes were present bilaterally. At the right side a streak ovary was fnun(t, whereas at the left side a solid oval gonad was f)resent, thought to be an abnormal testis or an ovotestis. Both gotla(ls showed tumoral induration and ,.,,'ere retm)w;d together with fallopian tubes. Hish)pathologic evaluation (if the streak gonad from the right revealed a gona(loblastoma partially overgrown by (.tisgerlninc, ma. Th.e external surface was snmoth and intact an(l there was no evidence of tumor growth outside the gonad. The left gona(l also consisted of gtmad(~blastoma and (tysgerminoma trot, in ad(lition, c:(mtained mature and immature teratoma and yolk sac tumor elements. No s e m i n o m e n o u s tubules couht be identified, but fo(:i of cellular ovarian stroma vvere found in both gonads. The external (:apstde of this gonad also appeared to be inta(:l. MATERIALS AND METHODS Chromosome Analysis Chromosome analysis of pllytohenmgglutinill [P[lA)-stinullate(t perit)heral blood lyml)ho(:yles was (:arried out using stan(lar(I (:(mventiunal c.vtogelletic techniques with (;TG [)anding I11. From the left gonad a biopsy specimen was taken, inin(:ed, cultured for 10 days in RPM[-1640 m e d i u m SUl)ph,nw, nted with antibiotics, glutamin, and 15% fetal calf serum and, subsequently, harvested for cytogenetic [(;T(;) analysis. Double In Situ Hybridization and lmmunocytochemi(:al Detection l)oubh;-fluorescence in situ hybridization experiments were carried oul using a combilmtion of two different i)robes: I)~12H8 18l, which is spe.cifi(: for the perit:entrome.ri(: region of chronlost)me 12, amt DNA from lhe somatic cell hybrid M28 [.ql. which is specific for the short arm of chr(mlosome 12 [101. The hybridization t)roce(lures used we're essentially as dc's(:ribe(l I)efore [10]. Briefly, I),~12H8 an(l M28 DNAs were laheh;d with d i g o x i g e n i n - I I - d [ ITI' [Boehriuger)and biotin-11-(t1J'l'l> [Sigma) [)y standard ni(:k translation, and (liltlle(l to) a (:on(:eutralion of 5 nwttl and 200 ng."ttl, resl)ectively. After denaturatit)n [and partial premmealing of the M28 [)robe) 5/.t[ of each t)r(~l)e was adde(I onto a heat-denatured (:hromusonaal slide and. su[)sequently. mixed under a (:overslip. Hybridizations were t)erf(wnle(I at 37~:(] for 3 days. Pt~sthvbri(lization washing stel)S were (:arrie(t ()ul as (lest:riI)ed bv Kievils et al. [ I l l . Immunocvtochemical deh;cticul of the chromosomal segments, hybridizing with p,,~12118 or M28 DNA, was ac(:omplished using TRIT(] or FIT(] conjugates (l)akopalts ;m(t Vector l,aboratt)ries), respe(:tively, as descrihe(I l)v Pinke[ el al. I12]. l"inally, the
Female with Gonadal Tumor and 48,XY,+7,ti(12p)
221
chromosome slides were mounted in antifade-medium, supplemented with 1,4 diazobicyclo-(2,2,2)-octane {DABCO, Sigma) and 4,6-diamino-2-phenylind()le (DAPI, Sigma) for counterstaining, Slides were studied with a Zeiss Axiophot epifluorescence mit:roscope, equipped for the detection of DAPI, TRITC, anti FITC flunrochromes.
RESULTS AND DISCUSSION
Cytogenetic analysis of phytohemagglutinin (PHA)-stimulated peripheral blood lymphucytes, derivect from the phenotypic female under investigation, revealect a 46,XY chromosomal pattern (50 cells studied). Subsequent cytogenetic investigation of the left ctysgenetic streak gonact-derivect tumor cells yielded a 48.XY, ÷ 7,+i[12p) c:hromosomal pattern in all of the 24 metaphases studied [Fig. 1). Because i(12p) is a marker highly characteristic of human testicular germ-cell tumors, the identity of this putative isochronlosolne in the dysgeneti¢: gonadal tumor cells was established further using non-radioactive in situ hvbridization tet:hniques (Fig. 2) in conjunction with the (:hromusome 12-spec:ific probes M28 [12p-arm)and p,~12H8 (12 t:entromere). Visualization of the chrolnt)somal segments, hybridizing with l)~12H8 or M28, was achieved by a t)ositive red or green-yellow fluorescent staining, respet:tiw;ly. Nonhyhridizing chromosonles t)r chromosomal segments (:ould be recognized by their blue fluorescent (DAPI) counterstaining. Double-fluores(:ence in situ hybridization (using M28 and 1)¢~12tt8) on metaphase spreads from the tumor revealed the pre.sence of two chrom()somes with positively staining short arms and centromeres, representing two (normal) copies of t:hrom()sonm 12. In addition, one marker chrt)mosome with two 12p-positive staining (:hrolnosomal arllls an(I a 12-positive centromere (i.e., an isochromosnnle 12p) coul(t easily anct unambiguously be ictentified (Fig. 2). The finding of a 48,XY,- 7. ~ i(12p) (:hromosomal pattern in the tumor cells may yield some clue concerning the role of these c:ytagenetic anomalies in the development of dysgenetic gonadal germ cell tumors. In 1982 Atkin and Baker [13[ raised the possibility that i(12p) may represent a specific chromosomal change in testicular germ-cell tumors. And indee(t, their obse.rvaticms have subsequently been confirmed by many inw;stigators ([1] for review). In addition, the i[12p)(:hrom()some was fountl in two dysgernfint)mas of the ovary [2, 3], an ovarian yolk sac tumor [4], and in two mediastinal germ-cell tumors [5-7]. The ¢:onsistent occurrence of an i[12p) marker in about 80% of testicular germ cell tumors [1, 14-19] undoubtly i)oints to an important role for this aberration in the etiology of these types of net)plasias. Besides i(12p) formation, l)olyploidization followed by gain and loss of c:opies of specifi(: chromosomes, (gain resulting in overrepresentatiun of (:hromc)somes 7, 8, 12. 21, anti X. and loss resulting in underrepresentation of t:hromt)somes 11, 13, and 18} may also play an important role in the development of testi(:ular germ-c:ell tumors ([1, 15] ft)r review). Usually, the tumors retain a highly aneuploid karyotype [1]. However, Dal Cin et al. [5] described a mediastinal germ (:ell tumor with a 47.XY,4i(12p),-13,421 chromosome pattern in the primary tumor and a 47,XY,+i(12p),-13,+21/48,XY,~i(12p),~21/49,XY,+i[12p),+i(12p),+21 pattern in its residual mass following chemotherapy. Also, Chaganti et al. [6] and Ladanyi et al. [7] described a mediastinal germ (.:ell tumor with initially a 49.XY,+1, ;6,-~ i(12p) and later a 49,XY,~-l, t 21,+i{12p) and 49,XY, ÷21,+ttel(1){q32), • i(12p) chromosomal pattern. It is of interest to) note that in these near-diph)id germ-t:ell tumors, besides formation of the i(12p), losses and gains of chronmsonms were observecl similar to those specifically encmmtered in highly aneupl()id testicular germ ce.ll tumors. The fintt-
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Female w i t h Gonadal Tumor and
48,XY,-7.-i(12p)
223
Figure 2 htentitication ot twu (normal} copies of chromosome 12 [arrowsl and the small metacentric marker chromosome i[12p} (~lrrowhead} by t]uorescellt in situ hvbridizatitm uMn~ M28 (121)} and p,tl2H8 {12-(:entromere)as probes.
ings ill our patient i n d i c a t e that the d e v e l o p m e n t of a dysgeneti(: g()na(lal t u m o r may find its p r i m a r y c a u s e in i(12p) formation. H o w e v e r . as it has been p r o v e d that heler()zygosity is retained in i(12p)-positive GC'I's [19], a n c u p l o i d i z a t i o n l e a d i n g to t r i s o m v 12 s h o u l d pr(;(:e(le this process. Apparently, these e v e n t s are soon f o l l o w e d by polyphfi(lization, whi(:h is suggested to take pla(:e early in the p a t h o g e n e s i s ()f testi(:ular germ-(:ell t u m o r s [15]. It is t)ossible that ill the [)resent case l ) o l y p h ) i d i z a t i o n was p r e v e n t e d by the earh. r e m o v a l of the n e o p l a s t i c gonads. N e v e r t h e l e s s , in n e a r - d i p l o i d as well as in highly aneul)loid germ-cell t u m o r s several identical s u c c e e d i n g c h r ( m l o s o m a l c h a n g e s may play an i m p o r t a n t role in the
process of ol]cogellesis. This work was supported by the Dutch (:an(:er So(:ietv (Konin~in Wilhe.hnim~ l.'onds). We thank R. |)overnlallil for ex(:ellenl h~(:hni(:al asgiMail(:e.
REFERENCES 1. de long B, Oosterhuis JW. Caste,do SMMJ, Vos AM, .Meerman (;J te (199(1): Pathogenesis of adult testicular germ cell lumors. Cancer Genet (:ytoI4enet 48:143-167. 2. Atkin NB, Baker MC (1987}: Abnormal chromosonws including small melacentrk:s in 14 ow~rian cancers. Cancer (;enet (:ytog(;net 2{i:355-3fil.
224
A. Hamers et al.
3. Jenkyn DI, Mc:Cartney A) [1987): A chronlosonle study of three ovarian tumors. Cancer Genet Cytogenet 26:327-337. 4. Speleman F, Potter C de, Dal (:in P, Mangelschots K. lngelaere H, Laureys C. Benoil Y, l,eroy J, Van Den Berghe H (1990): i(12p) in a malignant ovarian tumor. Cancer Genet Cytogenet 45:49-53. 5. Dal Cin P, Drochmans A, Moerman P, Van Den Berghe H (1989): Isochromosome 12p in mediastinal germ cell tumor. Cancer Genet Cytogenet 42:243-251. 6. Chagnati RSK, Ladanyi M, Samaniego F, Offit K, Reuter VE, Jhanwar SC, Bosl GJ (1989): I,eukemic differentiation of a mediastinal germ cell tumor. Genes Chrom Cancer 1:83-87. 7. l,adanyi M, Samaniego F, Reuter VF, Motzer RJ, Jhanwar SC, Bosl C;], Chaganti RSK (1990}: Cytogenetic and immunihistochemical evidence for the germ cell origin of a subset of acute leukemias associated with mediastinal germ cell tumors. I Natl Cancer lnst 82:221-227. 8. Looijenga LHJ, Smit VTHBM, Wessels IW, Mollewmger P. Oosterhuis JW, Cornelisse C], Devilee P (1990]: Localization and polymorphism of a chromosome 12-specific all)ha satellit(; I)NA sequence. Cytogenet Cell C;enet 53:216-218. 9. Zhang J, Marynen P, Devriendt K, Fryns JP, Van Den Berghe H, Cassiman lJ (1989): Molecular analysis of the isochromosome 12p in the Pallister-Killian syndrome. Construction of a mouse-human hybrid cell line containing an i(12p) as the sole human chromosome. Hum Genet 83:359-363. 10. Suijkerbuijk RF, Veen AY van de, Ec:hten J van, Buys CHCM. de Jong B, Oosterhuis JW, Warburton DA, Cassiman Jl. Schonk D, Geurts van Kessel A (1991): I)emonstration of the genuine iso(12p) character of the standard marker chromosome of testicular germ cell tumors and identification of further chromosome 12 aberrations by competitive in situ hybridization. Am J Hum (;(;net 48:269-273. 11. Kievits T, Devilee P. Wagenaar MC, Cornelisse CJ, Ommen GIB win, Pearson PI, (1990): Direct nonradioac:tive in situ hybridization of somatic cell hybrid I)NA to human lympho(:yte chromosomes. Cytometry 11:105-109. 12. Pinkel D, Straume T. Gray ]W (1986): Cytogeneti(: analysis using quantitative, high-sensitivity, fluoresc:ence hybridization. PNAS 84:2934-2938. 13. Atkin NB, Baker MC [1982): Specific t.:hromosome changl!, i(12p), in tl~sticular tumours? [,am:el i i: 1348-1349. 14. (]astedo SMMJ, de ]ong B. Oosterhuis IW, Seruca R, ldenburg VJ. Buist l, Sleijfer DT {1988): i( 12p)-negative testicular germ cell tumors. Cancer Gellet Cytogenet 35:171 - 178. 15. Oosterhuis IW, Casted() SMMJ, de ]crag B, Cornelisse CJ, Dam A. Sleijfer DTh, Schraffordt Koops H (1989): Ploidy of subtypes primary germ cell tumors of the testis. Pathogenetic and clinical relevance. Lab Investigation 60:14-21. 16. Castedo SMMJ, de long B. Oosterbuis IW. Seruca R, Meerman GJ te, Dam A, Schraffordt Koops H (1989): Cytogeneti(: analysis of ten human seminomas. Cancer Res 49:439-443. 17. Castedo SMMJ, de long B, Oosterhuis JW, Seruca R, Idenburg VJS, l)am A, Meerman (,J te, Dam A, Schraffordt Koops tl, Sleijfer DT (1989): Chromosomal changes in h u m a n primary testicular nonseminomatous germ (:ell tumors. Cancer Res 49:5696-5701. 18. (]astedo SMM], de Jong B, Oosterhuis JW, Idenburg JW. ldenburg VIS. Seru(:a R, Buist ], Meerman GJ re, Dam A, Schraffordt Koops tt, Sleijfer DT (1989]: Chromosomal changes in mature residual teratomas following poly('hemotherapy. Cancer Res 49:672-676. 19. Geurts van Kessel A, Drunen E van. de Jong B, Oosterhuis JW, Langeveld A, Mulder MP [1989) Chr(,mosome 12( I heterozygosity is retaim;d in i{12p)-positive testit:ular germ t:e]l ttllllOr [:ells. ( :an(:er Genet ( :ytogenet 40:129 - 1 :,14.
A B S T R A C T : (;vtogenetic tmul~'sis oI u l)rinmry ,Aertn-cell , tumor ori~intaing Irom the streuk ,Uo.ud ,~! .
20-year-oht phenotypic ¢emtde with u .t6.XY karyotype und mixed gonadtH dys,4enesis rm'ctded (t 48,X Y. • 7, ÷i( 12pJ chromosonml putter.. Germ-cell tumors originating lrt,ln gonads of normal IIItlII.'S lift? tlSU(III~."tl/gtl/~,' (meUldOid. An isochrOlllOSOtlle 12 I) (~s well (Is till m ' e r r e p r c s e n h l l i o n o.t (:/irOlllOSCqltt! 7 nloteritd are tHlIOtlg lh¢? S[}et:ilit" t:h(tllgCS tilt)St co,sistenth" observed. The present case shtm.s that tumors ,,/dysgenetic gtmmts, ulbeit I)eing near-dildoid, row." exhibit sintil(n" t:hrorm)somtd chan,e, es. This o b s e r v a t i o n l e n d s addititmal SUplmrt h) the hyln~/hesis that these s Det:ific cytogenetic oi'lomtllies Ill(IV IIItIV till Jlll DOl'ltlllt ri)lf! ill the {)(lt}ltlgt'llt!SiS fit Ilttllltlll ,.4,erm-(:e I I tlllllors.
INTRODUCTION Human testicular germ-cell tumors (TGCTs) are usually highly aneuploid with a modal (:hroinosome n u m b e r in the triploid range. In addition, they are characterized
hy a very spe(:ifi(: structural (:hrolnosomal anomaly, i.e., ail isochromosome 12p, in about 80% o[ all cases [11] for review). The i(12p) chrotllOSOllle has ills() been ()hserve(t in two (lysgerlninomas, an ovarian yolk sac ttunor, and ill s(une extragona(hll mc(liastinal germ-cell tumors 12- 71. The present study deals with a 20-year-oht female who was refe,rre(t for (:ytog(;netic analysis he(:ause of prilnary amenurrhoea and c.levated follit:le-stinmlatiilg hormon(; (FSH) and luteinizing hormone (LH) values. The analysis of perit)heral hlood lymphocyle (:ullures revealed a 46,XY chrumos(imal pattern. Because phelmtypi(: females with a 46,XY karyotype art'. at risk of developiIlg a malignant:y in the gonatls, an exploratory laparottuny was performed. Floth g O l l a d s appeared to (:ontain tumors
and were removed. From the 1)epartments of t tuman Geneti(:s. University of Limburg. Maastricht (A. 11.1. University of Groningen (FI. d. J.. J. v. El. Uniw:rsity of Nijmegen (R. F. S.. A. C. ;'. K.). the l)r. l)aniel den Hoed Klmiek. Rotterdam ( J. W. O.). and the Departments ot C;yneacology ( 1. El and Paflmlogy (F. B.]. University of Limburg. Maastricht, the Netherlands.
Address re.print requests to: A. Hmners. Ph.D., l)e.partnmnt o.f (hmetics and Cell Biology. State University o] l,imburg. Postbox 616. 6200 BX Moastricht. The Netherhmds. l{ece, ived March 21. 1991: m:ceptmt April 26. 1991.
219 ,'c 1991 Elsevier Science Publishing Co., Inc. 655 Awmue of the Americas, Nev,' York, NY 10010
Cancer (.h;net Cytogenet 57:21~-~- 224 (19911 0165-4608,'91:S03.50
220
A. Hamers et al. Here we report, for the first time, the karyotype of a dysgenetic gonad tulnor. For our analyses both Giemsa-G handing and fluorescent in situ hybridization (FISH) techniques were used. Our results indicate that this type of tumor may exhibit chromosomal changes similar to those seen in germ-cell tumors of gonads of normal males, which suggests a common pathogenetic pathway for both types of neoplasms.
CASE HISTORY A 20-year-old patient was referred for cytogeneti(: analysis because of primary amenorrhoea and ele.w~ted t"St:I and IJ't values. The analysis of peripheral blood lympho(:yt(;s revealed a 46,XY (mah;) karyotypc. For further diagnostic evaluation, the patient was referred to the Departlnent of (;ylU.'(:ology, especially to estimate the risk of developing a malignancy of the gonads. At exph)ratory laparotomy a small uterus with an extende(l cervix uteri was seen. Normal fallopian tubes were present bilaterally. At the right side a streak ovary was fnun(t, whereas at the left side a solid oval gonad was f)resent, thought to be an abnormal testis or an ovotestis. Both gotla(ls showed tumoral induration and ,.,,'ere retm)w;d together with fallopian tubes. Hish)pathologic evaluation (if the streak gonad from the right revealed a gona(loblastoma partially overgrown by (.tisgerlninc, ma. Th.e external surface was snmoth and intact an(l there was no evidence of tumor growth outside the gonad. The left gona(l also consisted of gtmad(~blastoma and (tysgerminoma trot, in ad(lition, c:(mtained mature and immature teratoma and yolk sac tumor elements. No s e m i n o m e n o u s tubules couht be identified, but fo(:i of cellular ovarian stroma vvere found in both gonads. The external (:apstde of this gonad also appeared to be inta(:l. MATERIALS AND METHODS Chromosome Analysis Chromosome analysis of pllytohenmgglutinill [P[lA)-stinullate(t perit)heral blood lyml)ho(:yles was (:arried out using stan(lar(I (:(mventiunal c.vtogelletic techniques with (;TG [)anding I11. From the left gonad a biopsy specimen was taken, inin(:ed, cultured for 10 days in RPM[-1640 m e d i u m SUl)ph,nw, nted with antibiotics, glutamin, and 15% fetal calf serum and, subsequently, harvested for cytogenetic [(;T(;) analysis. Double In Situ Hybridization and lmmunocytochemi(:al Detection l)oubh;-fluorescence in situ hybridization experiments were carried oul using a combilmtion of two different i)robes: I)~12H8 18l, which is spe.cifi(: for the perit:entrome.ri(: region of chronlost)me 12, amt DNA from lhe somatic cell hybrid M28 [.ql. which is specific for the short arm of chr(mlosome 12 [101. The hybridization t)roce(lures used we're essentially as dc's(:ribe(l I)efore [10]. Briefly, I),~12H8 an(l M28 DNAs were laheh;d with d i g o x i g e n i n - I I - d [ ITI' [Boehriuger)and biotin-11-(t1J'l'l> [Sigma) [)y standard ni(:k translation, and (liltlle(l to) a (:on(:eutralion of 5 nwttl and 200 ng."ttl, resl)ectively. After denaturatit)n [and partial premmealing of the M28 [)robe) 5/.t[ of each t)r(~l)e was adde(I onto a heat-denatured (:hromusonaal slide and. su[)sequently. mixed under a (:overslip. Hybridizations were t)erf(wnle(I at 37~:(] for 3 days. Pt~sthvbri(lization washing stel)S were (:arrie(t ()ul as (lest:riI)ed bv Kievils et al. [ I l l . Immunocvtochemical deh;cticul of the chromosomal segments, hybridizing with p,,~12118 or M28 DNA, was ac(:omplished using TRIT(] or FIT(] conjugates (l)akopalts ;m(t Vector l,aboratt)ries), respe(:tively, as descrihe(I l)v Pinke[ el al. I12]. l"inally, the
Female with Gonadal Tumor and 48,XY,+7,ti(12p)
221
chromosome slides were mounted in antifade-medium, supplemented with 1,4 diazobicyclo-(2,2,2)-octane {DABCO, Sigma) and 4,6-diamino-2-phenylind()le (DAPI, Sigma) for counterstaining, Slides were studied with a Zeiss Axiophot epifluorescence mit:roscope, equipped for the detection of DAPI, TRITC, anti FITC flunrochromes.
RESULTS AND DISCUSSION
Cytogenetic analysis of phytohemagglutinin (PHA)-stimulated peripheral blood lymphucytes, derivect from the phenotypic female under investigation, revealect a 46,XY chromosomal pattern (50 cells studied). Subsequent cytogenetic investigation of the left ctysgenetic streak gonact-derivect tumor cells yielded a 48.XY, ÷ 7,+i[12p) c:hromosomal pattern in all of the 24 metaphases studied [Fig. 1). Because i(12p) is a marker highly characteristic of human testicular germ-cell tumors, the identity of this putative isochronlosolne in the dysgeneti¢: gonadal tumor cells was established further using non-radioactive in situ hvbridization tet:hniques (Fig. 2) in conjunction with the (:hromusome 12-spec:ific probes M28 [12p-arm)and p,~12H8 (12 t:entromere). Visualization of the chrolnt)somal segments, hybridizing with l)~12H8 or M28, was achieved by a t)ositive red or green-yellow fluorescent staining, respet:tiw;ly. Nonhyhridizing chromosonles t)r chromosomal segments (:ould be recognized by their blue fluorescent (DAPI) counterstaining. Double-fluores(:ence in situ hybridization (using M28 and 1)¢~12tt8) on metaphase spreads from the tumor revealed the pre.sence of two chrom()somes with positively staining short arms and centromeres, representing two (normal) copies of t:hrom()sonm 12. In addition, one marker chrt)mosome with two 12p-positive staining (:hrolnosomal arllls an(I a 12-positive centromere (i.e., an isochromosnnle 12p) coul(t easily anct unambiguously be ictentified (Fig. 2). The finding of a 48,XY,- 7. ~ i(12p) (:hromosomal pattern in the tumor cells may yield some clue concerning the role of these c:ytagenetic anomalies in the development of dysgenetic gonadal germ cell tumors. In 1982 Atkin and Baker [13[ raised the possibility that i(12p) may represent a specific chromosomal change in testicular germ-cell tumors. And indee(t, their obse.rvaticms have subsequently been confirmed by many inw;stigators ([1] for review). In addition, the i[12p)(:hrom()some was fountl in two dysgernfint)mas of the ovary [2, 3], an ovarian yolk sac tumor [4], and in two mediastinal germ-cell tumors [5-7]. The ¢:onsistent occurrence of an i[12p) marker in about 80% of testicular germ cell tumors [1, 14-19] undoubtly i)oints to an important role for this aberration in the etiology of these types of net)plasias. Besides i(12p) formation, l)olyploidization followed by gain and loss of c:opies of specifi(: chromosomes, (gain resulting in overrepresentatiun of (:hromc)somes 7, 8, 12. 21, anti X. and loss resulting in underrepresentation of t:hromt)somes 11, 13, and 18} may also play an important role in the development of testi(:ular germ-c:ell tumors ([1, 15] ft)r review). Usually, the tumors retain a highly aneuploid karyotype [1]. However, Dal Cin et al. [5] described a mediastinal germ (:ell tumor with a 47.XY,4i(12p),-13,421 chromosome pattern in the primary tumor and a 47,XY,+i(12p),-13,+21/48,XY,~i(12p),~21/49,XY,+i[12p),+i(12p),+21 pattern in its residual mass following chemotherapy. Also, Chaganti et al. [6] and Ladanyi et al. [7] described a mediastinal germ (.:ell tumor with initially a 49.XY,+1, ;6,-~ i(12p) and later a 49,XY,~-l, t 21,+i{12p) and 49,XY, ÷21,+ttel(1){q32), • i(12p) chromosomal pattern. It is of interest to) note that in these near-diph)id germ-t:ell tumors, besides formation of the i(12p), losses and gains of chronmsonms were observecl similar to those specifically encmmtered in highly aneupl()id testicular germ ce.ll tumors. The fintt-
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223
Figure 2 htentitication ot twu (normal} copies of chromosome 12 [arrowsl and the small metacentric marker chromosome i[12p} (~lrrowhead} by t]uorescellt in situ hvbridizatitm uMn~ M28 (121)} and p,tl2H8 {12-(:entromere)as probes.
ings ill our patient i n d i c a t e that the d e v e l o p m e n t of a dysgeneti(: g()na(lal t u m o r may find its p r i m a r y c a u s e in i(12p) formation. H o w e v e r . as it has been p r o v e d that heler()zygosity is retained in i(12p)-positive GC'I's [19], a n c u p l o i d i z a t i o n l e a d i n g to t r i s o m v 12 s h o u l d pr(;(:e(le this process. Apparently, these e v e n t s are soon f o l l o w e d by polyphfi(lization, whi(:h is suggested to take pla(:e early in the p a t h o g e n e s i s ()f testi(:ular germ-(:ell t u m o r s [15]. It is t)ossible that ill the [)resent case l ) o l y p h ) i d i z a t i o n was p r e v e n t e d by the earh. r e m o v a l of the n e o p l a s t i c gonads. N e v e r t h e l e s s , in n e a r - d i p l o i d as well as in highly aneul)loid germ-cell t u m o r s several identical s u c c e e d i n g c h r ( m l o s o m a l c h a n g e s may play an i m p o r t a n t role in the
process of ol]cogellesis. This work was supported by the Dutch (:an(:er So(:ietv (Konin~in Wilhe.hnim~ l.'onds). We thank R. |)overnlallil for ex(:ellenl h~(:hni(:al asgiMail(:e.
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