5
EDUCATIONAL SERIES /3hi( .... Molecular biology of testicular germ cell tumors Eduardo Vilar, Elniliano Cairo and Josep Tahernero l)epartment of Medical Oncology. Vail d'Hel)ron University Hospital. Barcelona. Spain. Testicular gem) cell tumm~ (TCGT) con)prise a heterogeneous group of neoplasms, although all of them are originated from common precursors relat- ed to germ cell lineage. Understanding of normal development of germinal cells is essential to define new markers for diagnosis, prognostic subgroups and targeted therapies, Recent advances related to cylogenetic and molecular features have estab- lished the role of immunohistochemistry of c-kit, OCT-3/4 and determination of gain of chromosome 12 in the daily workup of premali~lant lesions and invasive tumors. This review summarizes the cur- rent knowledge in the field of molecular biology of TGCT. Ker words: testicular germ cell tumor, intratubular germ cell neoplasia unclassified, molecular biolo~,, c-kit, OCT-3/4. I t/ar E, Cah,o E, Tabernero d. Molecular biolo~o?"qf testicular germ cell llllnor,~. C/in ?)'ansi Oncol. 2006;8(12):846-50. EPIDEMIOLOGY AND HISTOLOGY An estimated 8250 new cases of testicular tumors are expected to occur among men in 2006 I. SEER statisti- cal data refer to the whole group of testicular tumors, however the vast majority are germ cell tumors (GCT) accounting for 210/0 of all malignant neoplasms in male adolescents and young adults fi'om 1975 to 2000. The incidence of testicular GCT (TGCT) as a function of age at diagnosis shows three modal peaks: *Supl)orled by an unresh'iclededt0calionalgrant bv Bristol-Myers Squibb. Correspondence:E. Cairo. l)eparlment or Medical Oncolog~,'. Vail (l'Hel)ron Universily Hospital. Ps. Vaild'Hebron, 119-129. 081)55 Barcelona.Spain. E-mail:ecah'[email protected] children under 5 years, 25- to 40-year age and 75- to 85- year age groups, being the most conamon solid tu- mors in the 15- to 29-year age group 2. An increased in the incidence in the last 40 years in most industri- alized countries has been reported. The etiology of TGCT is poorly understood and several factors have been proposed that increase tim risk for TGCT, such as eryiorebidism, high maternal hormone levels dur- ing pregnancy, familial clustering of TGCT, hernia, pre-term birth, viral orchitis and constitutional chro- mosome abnormalities (particularly Klinefelter's syn- drome). However, there is limited evidence suppml- ing the association of most of them with a higher risk to develop TGCT ~. Histologically, TGCT are divided into seminoma and non-seminoma subtypes. Two classifications devel- oped by the World Health Organization and the British Testicular Tumor Panel are summarized in table 1'4. Seminoma represents about 500/0 of TGCT with a median age of presentation in the fourth decade of life. Spermatocytic seminoma is a rare vari- ant seen in men over 45 years of age. Nonseminoma- tous TGCT (NSTGCT) are frequently presented in the third decade of life and it constitutes an entity formed by four different histologies: embryonal carcinoma, choriocarcinoma, yolk sac tumor and teratoma 5. TABLE 1. Histologio olassifications of TGCT WHO British testicular tumor panel Seminoma Typical (classical) Anaplastia Embryonal carcinoma Teratocarcinoma Mature Inmature With malignant differentiation Choriocarcinoma Yolk sac Teratoma Mixed germ cell (specify components) Seminoma Malignant teratoma undifferentiated Malignant teratoma intermediate Malignant teratoma trophoblastic Yolk sac Malignant teratoma differentiated (specify components) ~46 C lin Transl Onc ol. 2006;8(12):846-50

Molecular biology of testicular germ cell tumors

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Page 1: Molecular biology of testicular germ cell tumors

E D U C A T I O N A L S E R I E S / 3 h i ( . . . .

Molecular b io logy of test icular germ cell tumors E d u a r d o Vilar, E l n i l i a n o C a i r o a n d Josep T a h e r n e r o

l)epartment of Medical Oncology. Vail d'Hel)ron University Hospital. Barcelona. Spain.

Test icular gem) cell t umm~ (TCGT) con)prise a he te rogeneous group of neoplasms, a l though all of them are or ig inated from c o m m o n precursors relat- ed to germ cell l ineage. U n d e r s t a n d i n g of n o r m a l deve lopmen t of ge rmina l cells is essential to def ine new marke r s for diagnosis , prognost ic subgroups and targeted therapies, Recent advances re la ted to cylogenet ic a n d m o l e c u l a r features have estab- l ished the role of i m m u n o h i s t o c h e m i s t r y o f c-kit , OCT-3/4 and d e t e r m i n a t i o n of gain of c h r o m o s o m e 12 in the daily workup of p r e m a l i ~ l a n t lesions a nd invasive tumors . This review s u m m a r i z e s the cur- rent knowledge in the field of m o l e c u l a r biology of TGCT.

Ker w o r d s : testicular germ cell tumor, in t ra tubular germ cell neoplasia unclassified, molecular biolo~,, c-kit, OCT-3/4.

I t/ar E, Cah,o E, Tabernero d. Molecular biolo~o?" q f testicular germ cell llllnor,~. C/in ?)'ansi Oncol. 2006;8(12):846-50.

E P I D E M I O L O G Y AND HISTOLOGY

An estimated 8250 new cases of testicular tumors are expected to occur among men in 2006 I. SEER statisti- cal data refer to the whole group of testicular tumors, however the vast majority are germ cell tumors (GCT) account ing for 210/0 of all mal ignant neoplasms in male adolescents and young adults fi'om 1975 to 2000. The incidence of testicular GCT (TGCT) as a function of age at diagnosis shows three modal peaks:

*Supl)orled by an unresh'icled edt0calional grant bv Bristol-Myers Squibb.

Correspondence: E. Cairo. l)eparlment or Medical Oncolog~,'. Vail (l'Hel)ron Universily Hospital. Ps. Vail d'Hebron, 119-129. 081)55 Barcelona. Spain.

E-mail: ecah'[email protected]

children unde r 5 years, 25- to 40-year age and 75- to 85- year age groups, being the most conamon solid tu- mors in the 15- to 29-year age group 2. An increased in the incidence in the last 40 years in most industr i - alized countries has been reported. The etiology of T G C T is poorly understood and several factors have been proposed that increase tim risk for TGCT, such as eryiorebidism, high maternal hormone levels dur- ing pregnancy, familial cluster ing of TGCT, hernia, pre-term birth, viral orchitis and consti tutional chro- mosome abnormali t ies (particularly Klinefelter 's syn- drome). However, there is limited evidence suppml- ing the association of most of them with a higher risk to develop T G C T ~.

Histologically, TGCT are divided into seminoma and non-seminoma subtypes. Two classifications devel- oped by the World Health Organization and the British Testicular Tumor Panel are summar ized in table 1 '4. Seminoma represents about 500/0 of TGCT with a median age of presentat ion in the fourth decade of life. Spermatocytic seminoma is a rare vari- ant seen in men over 45 years of age. Nonseminoma- tous TGCT (NSTGCT) are frequently presented in the third decade of life and it constitutes an entity formed by four different histologies: embryonal carcinoma, choriocarcinoma, yolk sac tumor and teratoma 5.

TABLE 1. Histologio olassifications of TGCT

WHO British testicular tumor panel

Seminoma Typical (classical) Anaplastia

Embryonal carcinoma Teratocarcinoma

Mature Inmature With malignant

differentiation Choriocarcinoma Yolk sac Teratoma Mixed germ cell

(specify components)

Seminoma

Malignant teratoma undifferentiated Malignant teratoma intermediate

Malignant teratoma trophoblastic Yolk sac Malignant teratoma differentiated (specify components)

~ 4 6 C lin Tra nsl Onc ol. 2006;8(12):846-50

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VILAR E, CALVO E, TABERNERO J. MOLECULAR BIOLOGY OF TESTICULAR GERM CELL TUMORS

O R I G I N A T I N G C E L L S AND T H E ROLE O F G E N O M I C I M P R I N T I N G

It is necessary to know the embryology of the gonads in o rder to under s t and the b i o l o ~ and genesis of these tumors. This is impor tant because ge rm l ineage cells involved in this process are blocked in different stages of ma tu ra t ion becoming ma l ignan t and devel- oping a specific subtype of TGCT 4,6. The blastocyst will develop in both t ropheetoderm and inner cell mass. Proximal epiblast is the par t of the in- ner cell mass that will give rise to various types of so- matic cells including the most primitive germ cells which are embryonic stem cells (ESC). Pr imordial germ cells (PGC) are ESC so called when they start to migrate along the midl ine of the body through the hindgut to the genital ride increas ing greatly due to in- tense proliferation. Those cells isolated in z'itro fi'om the PGC population before day 12.5 of deve lopment are embryonic germ cells (EGC). PGC will develop the gonocytes when they reach the genital ride and, de- pending on gender -ch romosomal constitution, they will progress to oocytes (XX) or spermatogonia (XY) 6.

Developmenta l biology has deeply studied the genom- ic and epigenetic character is t ics displayed by ESC and

subsequently derived cells such as PGC 7. Embryonic carc inoma cells (ECC) are tumora l counterpar t s of EGC and, despite of genetic changes that involve the mal ignant t ransformat ion process , they retail] certain epigenetic character is t ics that have enabled us to de- fine their cell of origin under a molecu la r point of view. Also, the identification of commonal t i e s and dif- fierences among precursors and tumora l cells have added a lot of informat ion about putative growth fac- tors, suppresso r genes and specific proteins related to molecu la r events impl icated in the genesis of TGCT.

Epigenetic changes demons t ra ted in germ cells are explained due to the fact that a f imctional difference be tween the pa te rna l and mate rna l haploid set of c h r o m o s o m e s exists in every individual . This mono- allelic express ion is based on epigenet ic modificat ion by methyla t ion and it is called genomic impr in t ing I~. Oocyte which conta ins materna l impr in t s will be complemented by the pa terna l genc~me reciprocal ly impr in ted and vice versa and this fact expla ins why two copies of each parenta l c h r o m o s o m e are needed fbr normal deve lopmen t 7. The init iation of the pro- cess for es tabl i sh ing a new set of impr in t s is confined to the germ line dur ing both spe rmatogenes i s and oo- genesis and it depends on gender -c lu 'omosomal con-

i GENOMIC IMPRINTING

GERM-CELL LINEAGE DEVELOPMENT

;;Genital ride ~nigration i ; i ; ' I

E C C I

, I Embryonic features �9

Pluripotency re

N O N S E M I N O M A

i SPERMATOZOA

S"

s

Proliferation along germ-cell lineage

S o s"

t ' s"

i ~

S .s

,s

S E M I N O M A ~

Fig. 1. Schematic representation of the generation of testicular tumors by epigenetic modifications. TGCT: tes- ticular germ cell tumors; PGC: primordial germ cells; ESC: embryonic stem cells; EGC: embryonic germ cells; ECC: embryonic carcinoma cells; ITGCNU: intratubular germ cell neoplasla unclassified.

C/in Transl Oncol. 2006;8(12).'846-50 8 4 7

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VILAR E, CALVO E, TABERNERO J. MOLECULAR BIOLOGY OF TESTICULAR GERM CELL TUMORS

stitution (uniparen ta l pat tern) , ie, the presence of the Y c h r o m o s o m e will de te rmine pa te rna l impr in ts wi- thin the germ line and the pa te rna l X chromosonae will be impr in ted in female germ lines (fig. 1). However before this process starts, it is necessa ry the e rasu re of formerly exist ing imprints . The epigenet ic modif icat ions will start in PGC when they entry into the geni tal ride showing a genome-wide demethy la - tion (e rasure of genomic impr in ts ) which is also ob- served in EGC but not in ESC which retain a bipater- nal pat tern of genomic imprin t ing . Therefore, a l though ESC, PGC and EGC have the proper ty of be- ing p lur ipotent (in fact ESC are considered omnipo- tent), they show different pa t te rns of genomic im- pr in t ing that will a l low us to dis t inguish them. Progression in germ cell deve lopment will imply loss of both p lur ipotency and b iparen ta l pat tern o f g e n o m - ic impr in t ing in a paral le l way towards a more static cell phenotype and un iparen ta l impr in ts as it is ob- served in spermatocytes and ooeytes 7.

INTRATUBULAR G E R M C E L L NEOPLAS1A U N C L A S S I F I E D ( ITGCNU) . P A T H O G E N E S I S

The genesis of human TGCT is not yet well charac- terized. ITGCNU is the p recur so r lesion of all types of TGCT with the except ion of spermatocy t ic s e m i n o m a and it is character ized morphologica l ly by the pres- ence of a typical germ cells located above the basal membrane . Ca rc inoma in si tu was the nomenc la tu re used prev ious ly and it was substi tuted because it was related to the presence of epi thel ial differentiat ion features 8. It is seen with inc reased f requency in pa- tients with c ry torch id ism, gonadal dysgenes is or with history of b iopsies for infertility. A widely accepted theory states that s e m i n o m a s are genera l ly histologi- cally uni form and are developed fi'om t ransformed PGC into 1TGCNU cells that will prol iferate a long the germina l l ineage. Morphotgieal , u l t ras t ruetura l and compara t ive gene express ion s tudies have conf i rmed that the origin of ITGCNU are PGC. On the other side non - seminoma tous TGCT (NSTGCT) contain stem cells t e rmed ECC that resemble both ESC and EGC and also retain potential to differentiate to cells de- rived from ex t ra -embryon ic t issues, as yolk sac and t rophoblas t (plur ipotent cells). Microar rays analys is compar ing express ion pat terns of the different subgroups of T G C T and those from potent ial p recu r so r cells (ESC, ECC and PGC) have discovered genes involved in cell l ineage differentia- tion, se l f - renewal and surv iva l which are main char- acterist ics of s tem cells. POUFSF1 is one of the genes identified and it will encode OCT-3/4 which is an oc- t amer -b ind ing t ranscr ipc ion factor shown to be ex- p ressed only in the p lur ipo ten t ceils of embryo as ESC, ECC and s e m i n o m a s 9. It was observed ne i ther in normal testis, somat ic tumor samples nor in fully

differentiated ma l ignan t t issues as mature teratomas, yolk sac tumors , de rmoid cysts of the ovary and sper- matocytic semi nomas. However OCT-3/4 function is regulated by a more complex way than a b inary on- off control sys tem requir ing prec ise quanti t ies of the fhctor to t r igger different responses so, ma in ta in ing the level of diploid cells stem cells will cont inue pro- liferating, however inc reases or decreases of 50% will tr igger differentiation into e n d o - m e s o d e r m or tro- phec tode rm lineage, respect ively m. c-kit (CDIIT) is a receptor glycoprotein of the platelet derived growth factor receptor family with tyrosine ki- nase activity whose ligand is an integral membrane glyeoprotein called stem cell factor (SCF), kit l igand or steel factor 2 2. It may have a critical role in the develop- ment and migrat ion of PGC and therefore it may be a good m a r k e r for dis t inguishing s eminomas and em- bryonal carc inoma. Pre-clinical studies performed with wikl- type mice PGC have shown that c-kit is pres- ent even when these cells have reached the genital ride and when PGC are grown in vitro in the presence of SCF they have showed longer survival. Additionally, mice carD'ing mutat ions at loci encoding both c-kit and SCF are sterile. In human adul t testis, expression of c-kit has been detected on Leydig cells and sper- matogonia I~. At least two studies have showed that c- kit immunohis tochemica l s taining is positive in the majority of s eminomas and ITGCNU and negative in non-seminomas and normal testis, what is consistent with the cel lular o r i o n of each tumor cell 15,14. NKX3.1 is a prostate t umor suppresso r gene that be- longs to homeobox subfamily. NKX3.1 has demon- strated posit ive s ta ining by i m m u n o h i s t o c h e m i s t r y of germ cells in normal testis, ITGCNU and the most dif- ferentiated NSTGCT and negative in the major i ty of both s e m i n o m a s and NSTCGT. Its express ion is re- tained in the p recur so r lesions and it is ubiqui tously lost in invasive tumors , suggest ing an associat ion with progress ion in turn or igenesis 25. Therefore, in o rde r to identify ITGCNU in those cases where differential d iagnosis is difficult, prote ins relat- ed to s tem-cel l features as the a forement ioned pla- cental- l ike a lka l ine phospha tase , 0CT-5/41637 and c- kit 21~, have been tested and have shown to be s ta ined by inmunolais tochemist ry . Of interest , ploidy and cy- togenetics of ITGCNU have s imilar i t ies with the adja- cent invas ive componen t suggest ing that both compo- nents are clonally related lsJg, except for gain of" lop that has not been demons t r a t ed in compara t ive ge- nomic hybridizat ion (CGH) and f luorescent in situ hybridizat ion (FISH) studies 2~

M O L E C U L A R BIOLOGY O F TGCT. C Y T O G E N E T I C S AND C H R O M O S O N A L C O N S T I T U T I O N

TGCTs show genomic aber ra t ions that can be detect- ed by several techniques. Pre l iminary reports were

8 4 8 c/i,, Transl OncoL 2006:8(12):846-50

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VILAR E, CALVO E, TABERNERO J. MOLECULAR BIOLOGY OF TESTICULAR GERM CELL TUMORS

based on convent iona l citogenetic techniques , as karyotype and flow cytometry, a l though the most re- cent s tudies have conf i rmed and expanded these re- sults with molecu la r eytogenet ie methods such as FISH, SKY and CGH arrays 2t. Three different subgroups can be defined as a fimclion of age at diagnosis and these correlate well with specif- ic clinical, histological and molecular characteristics. In tile first subgroup patients are in the pediatric age and tile most common histologies are tera tomas and yolk sac tumors. The second includes seminomas and NST- GCT and they occur typically dur ing the postpuberal period. Finally, spermatocytie seminoma affects older" patients in tile range of 75 to 85 years of age s. Both s e m i n o m a s and NSGCT have c h r o m o s o m e number s in tile triploid range, a l though the fi)rmer are hyper t r ip loid and the later" are hypotr ip lo id 22. Adjacent p re -mal ignan t lesions have been analyzed by CGH to s tudy if these a l tera t ions are an early event in tumor igenes i s or, on tile contrary , they are in- volved in tumor progress ion. It has been observed that invas ive tumors have the same c h o r o m o s o m a l aber ra t ion pat tern than pre -mat ignan t lesions 23. The only consis tent c h r o m o s o m a l abnorma l i t y presented in both adult subtypes of TGCT is the over represen ta - tion of ch romosome 12 by two different forms mutu- ally exclusive. The presence of one or more copies of i(12)p is observed in 800/0 of the cases but also, in those cases lacking i(12)p, t andem amplif icat ion of the entire 12p has been repor ted represent ing 10% of the total of adul t TGCT 2425. Moreover a more specific subregional ampli f icat ion at 12ptl .2-p12.1 has been identif ied in 8% of TGCT 26. This res t r ic ted amplif ica- tion has been corre la ted with cl inical and biological character is t ics in s e m i n o m a s such as younger age at presentation, absence of apoptosis and enhaced in r i tro

surviva l 27. Express ion profile of 12p have identified highly expressed known genes as LDHB, KRAS2, CC- ND22s and two novel with unknown function called GCTI and 2 located at 12p l l . 2 -p l2 . t amplic(m 29. Several works have been focused in CCND2 gene which encodes cyclin D2 showing that overexpres- sion of CCND2 was specif ical ly observed in ITGCNU and it was inverse ly corre la ted with differentiat ion of cell l ines 5~ Therefore it may be a possible early event in TGCT tumor igenes i s 5'5~ Among pediat r ic TGCT, differentiated te ra toma (DT) and yolk sac tumors (YST) display ch romosoma l ab- normal i t ies that are different to those detected in adult tumors : diploid karyotype in DT 5t and aneu- ploid in YST 52. Tile major i ty of the studies conf i rmed that DT and YST do not show the p resence o f i soch ro - mosome 12p ( i l2p) , however gains in cl l ronaosome 12 have been often reported. Delection of the sub- te lomer ic region of the shor t a rm of chronaosome 1 (1p56.5) is f requent in this subgroup and it may have value as prognost ic m a r k e r 55.

A complex ch romosoma l t rans locat ion involving 6p21, 6p22 and 6q25, and l lq15 has been descr ibed in severa I ex t ragonada l var iants of im matu re tera toma. This charac ter i s t ic al terat ion is associa ted with a new entity with pa r t i cu la r c l inical features as mid l ine lo- cation and blood spread metas tas is 54"55. Tile most common genetic abnormal i ty presented in spermatocyt ic s e m i n o m a is gains in c h r o m o s o m e 9, a l though there is a lack of s tudies in this rare entity s. Genomic losses observed by different molecu la r ap- p roaches have suggested the role of severa l t u m o r suppresso r genes such as RB1, APC, MGMT, DNMT2, CCND2 and TP55 in the patogenes is of TGCT, al- though this is open to discuss ion cur ren t ly 56.

T R E A T M E N T SENSITIVITY AND R E S I S T A N C E O F T G C T

TGCT are more sensi t ive to c l l emothe rapy and i r ra- diation than o ther types of cancer cells, w he rea s NSTCGT are apparen t ly able to repair radia t ion- in- duced damage. Addit ionally, approx ima te ly 10-500/0 of pat ients d iagnosed with metastat ic d isease will de- velop incomple te response or relapses. It has been debated the role of p53 levels a l though nei ther the presence of TP55 muta t ions nor the overexpress ion of the wi ld- type form co r r e sponds necessar i ly to drug resis tance and sensit ivity, respect ively J'. Defects in DNA mismatch repai r genes, apoptosis media tors as caspases , BAX, BCL2 and pro te ins re la ted with drug influx and efflux as mul t id rug resis tance related pro- teins have been suggested to be related with t reat- ment res is tance 6'57. However more s tudies are need- ed in o rde r to clarify the mechanis t i c basis of re lapse to t reatment .

C O N C L U S I O N S

Oncology research in the field of TGCT is one of the best examples of how classical cytogenet ics , converl- tional pathological techniques and h igh- t l l roughput molecu la r technologies can be in tegrated in o rde r to study in depth tumora l biology. Advances in develop- menta l biology have been successful ly appl ied to pa thology diagnosis , or ig ina t ing pract ica l tools like i n l n m n o h i s t o c h e m i c a l markers . Factors related with init iat ion and ITGCNU progress ion towards invas ive les ions rnay help to es tabl ish ear ly detect ion and t rea tment of p r e m a l i g n a n t p r ecu r so r s in a young populat ion. Better definit ion of prognos t ic groups us- ing mo lecu l a r cytogenet ics or gene express ion pro- files is needed for pat ients with low risk of re lapse in order to avoid unneces sa ry treat inent . Final ly and despite of tile fact that tile percen tage of pat ients with TGCT that r e lapse is low, new ra t ional ly des igned target -based drugs are still needed for these young patients.

Clhl Transl Oncol. 2006;8(12):846-50 849

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VILAR E, CALVO E, TABERNERO J. MOLECULAR BIQLOGY OF TESTICULAR GERM CELL TUMORS

R e f e r e n c e s

I. Society AC, editor. +lancer Facts i i l ld Pigu- l'eS 200('L A tlall la : A lllerJc;l II ( ] ance r SlicJe- ly: 2008.

2. Fal lon R, ( ) l shan A, Saxn ian S, edi tors . [Male geni tal t ract c ance r . Bethesda , MD: National Cancer Institute: 2008.

5. Bel'nstein I., Smith MA, [,ill I,, D e a p e n I), l~u 1)1,, editors. ( ]ern l ('ell, Iro- ph!>blastic and other ~ n a d a l neophlsms ICCC X. Belhesda, M[) : Nat i .na l Cancer hlstitute, SEER Program. NIH 1999.

4. Horvvich A, Shipley .I, Huddart R. Tesl icu- lar gernl-cel l cancer. I,ancel. 2006;567 (9512):754-65.

5. Bosl G J, Bajorin I)F, Scheinfekl .I, Mutze r R J, Chagan l i RS, edi tors . C a n c e r o r the testis. 71h ed. Phih ide lphia , PA.: Lippin- coil, Ui' i l ] ianls and VVilkins: 2(X)5.

6. Oos t e rhu i s ,IW. Lo - i i enga LH. T e s t i c u l a r gernl-ce]] IunloIIrS il l ii l)l'o;Ider pel'spec- live. Nat Re,, Cancer. 2005;5(5):210-22.

7. Surani MA. Rep r -~ ' an lm i l l g o f genorne funct ion Ihrough epigenetic inheri tance. Na tu re. 2(X) I ;414((;859) :122-8.

8. Retl ler Vt~:. Or ig ins and nlolec t l la r biolog3' of tes t icu la r g e r m cell t t lnlors. Mod Pa- thol. 2005:18 Suppl 2:$51-60.

9. Spe rge r JM, C h e n X, D r a p e r ,IS, et al. Ge- lie exl)ressiol l pat te rns Jn I I I I l l la l l en lb ry- ChiC stein cells and ht ln lan i)hn' ipolent gel'Ill cell tlllllOI'S. Proc Nat] Acad Sci [J S A. 2005:100(25):15550-5.

I0. N iwa H, Miyazaki .I, Snli th AG. Quant i t a - li ' ,e express ion of ()cl-5/4 def ines differ- enl ia t ion, dedi f ferent ia t ion m' sell '-renevv- al o f ES cells. Nat Genel . 2000;24(4):572-6.

11. I)e Miguel MP, C h e n g I,, Hol land EC, Federspie l M J, Donowln PJ. I ) i ssec t ion of Ihe c-Kil sigllal ing p a t h w a y ill m o u s e i)ri- n lordia l g e r m cel ls by r e t rov i r a l -n l ed ia t ed g e n e h-ansfer. Proc Nail A c a d Sci U S A. 2002 ;99( l (;): 10458-(; 5.

12. I , ove land KL, Schlatt S. Stenl ('ell factor and ('-kit in the i l l annl la l ian teslis: les- sons or ig ina lh lg I'ronl Mother Nature's gelle knockotlts. J 14:n(Iocl'inol. 1997;155 (5):557-44.

15. Rajper l - I )e Meyts E, S k a k k e b a e k NE. Exl)ressiort o1" the c-kit p ro teh l I)roducl in ca rc inon la - in - s ih i and i n v a s i v e tes t icu la r g e r m cell lUnlours, hll J Androl . 1994;17 (2):85-92.

14. I ,e roy X, Augus to D, l , e t eu r t r e E, ( ]osse l in B. (.]I)50 and CI) I 1 7 (c-kiD used i l l conlbi- nal ion are IlSe[lll till" dJslJll~%lJshJng ell l- I)ryonal Cal'cinonl~l D'oln Selllinll111~l, J H ist ochenl Cy t . c h e l n . 2002:50(2) :285-5.

15. 8ko the im RI, K o r k l n a z ItS, Klokk TI, el al. NKXS.I exp res s ion is lost in lesticuhu" g e r m (:ell tllnIol's. Anl J Palhol . 2005;185 (fi):2149 -54.

16. I,ooijenga I,H,I, Sloop H, tie I , e e u w HP.IC, el al. POLI5FI (OCT3/4 ) Ident i f ies Cells wi th P lur ipo ten t Potential ill H u n l a n G e r m Cell T u n l o r s . C a n c e r Res. 2005;65 (9):2244-50.

17. Jones TI ) , Ulbr ighl TM, Eble JN, Cl leng 1,. OGT4: A sens i t ive a n d specific b i o m a r k e r fro" inh 'a tu l )u la r g e r m cell n e . p l a s i a or the leslJs. Cl in Cancer Res. 2004;10(24):8544-7".

18. de GraalT WE, Ooslerhuis Jl1.', tie Jong if, el al. Phfidy o[ tesl ieular carcinonla il l situ. Lalb hlvesL 1992;00(2):166-8.

19. van Echten J, van ( ;urp R J, Sloepker M, l ,ooi jenga LH, tie Jong J, Ooslerhuis %'. Cylogenelic evidence that carcinonla il l siIu is the prevursor lesion Ibr invasi~e lest icular gel'nl cell tun}ors. Cancer (]enel (, 'ytogelleI. 1995;85(2):155-7.

20. R o s e n b e r g C, ~,an Gurp R J, Gee len E, t ) o s l e rhu i s JW, Lnoi jenga I,H. O v e r r e p r e - senta t ion of the sho r t a r m o f c h r o n l o s o n l e 12 is rehl ted to i n v a s i v e ~'ovvtll of h u n l a n testicular selninol l laS alld nOllSelUlnO- inas. O n c o g e n e . 2000;I 9(51 ):5858-fi2.

21. I ,ooi jenga I,H, t )o s t e rhu i s J~,V. Pa thogene - sis of tes t icu la r ge rn l cell tun lours . Rev Repl ' .d . 1999:4(2):90-100.

22. van Eeh ten J, Oos t e rhu i s .I~X, I ,ooi jenga I,H, el al. No l 'eClll'rellI Stl ' l lOtlll 'al a I l l l l i l ' - reali t ies apa r t f rom i(12p) in p r i m a r y gernl cell t u m o r s or tile ,'ltllllt testis. G e n e s Chron lo so lne s Cance r . 19,(15:14(2):135-44.

25. l ,,,oijenga I,H, R o s e n b e r g C, v a n G u r p R J, el al. Conl l lar ; l l ive genolnic hybrkl iz ; i t ion (if illiCl'odissecled sail lples l'ronl dirt'erenl stages il l tile devehip lnenl o fa senl inonla alld a nol l-senl i l lOlna. J Palhol. 2t)00:191 (2):187-92.

24. Su i jkerbu l jk RF, Silike R J, IMelonl AM, el al. O v e r r e p r e s e n t a t i o n Of c h l ' o n l o S O n l e 12p s e q u e n c e s and karyo typ ic evolu t ion in i (12p) -nega t ive tes t icu la r g e r m - c e l l lll- n lors r e v e a l e d by I l u o r e s c e n c e ill silu hy- I)ridiz, ation. Cance r (}enel Cyhlgenet . 1995; 70(2):85-95.

25. Rodr iguez E, H o u k l s w o r t h J, Reuter VE, el at. Mo lecu l a r cy togene t ic ana ly s i s o r i (12p)- l legative h u l n a n nlale c-~e.l'lll cell ttl- nlOl'S. (JelleS C h r o l n o s o n l e s Calleel ' . 1995; 8(4) :25o -8.

26. Mosterl MC, Ve rke rk A J, van de Pol IM, el al. Ident i f icat ion o f the cr i t ical region of 121) o v e r - r e p r e s e n t a lion ill testicula r ge rm cell Iulllors ( i f adolescents and +nduils. Oncogene. 1998:16(20):2617-27.

27. I%elol's H, Mosler l MC, Polnpe K, el al. Rest r ic ted 12p ampl i f i ca t ion and RAS m u - tat ion in hulnan gei'nl cell tunlors of tile adul l leslis. Anl J Pathol+ 2000;157(4): I 155-66.

28. Rod r iguez S, J a f e r O, G o k e r H, et al. Ex- p ress inn pr,,file o f genes fi't)ln I 2p i l l les- lielllar ~el'lll ('ell IIlnlors f)f adolescents and adults associaled with i(12p) and anl- pIificalhm at 12pII.2-pI2.1. Oncogene. 2005122(12):1880-91.

29. Bourdon V, Naef F, Rao PH. et al. t ; eno - nl ic and expres s ion ana ly s i s of the I 2pl I- i)12 anlp l icon us ing EST a r r a y s ident i f ies two nove l aml/ l i f ied and o v e r e x p r e s s e d genes . C a n c e r Res. 2(X)2:82(21 ):6218-25.

50. H o u k l s w o r t h .1, Reuter V, Bosl G J, Cha- ganli RS. A b e r r a n t express ion of cyclin D2 is an early e v e n t ill l l u m a n m a l e gel'in ('ell tunlorigenesis. Cell Grow lh l ) i f fer . 199 7;8(5) :295 -9.

51. Hoffner I,, l )eka R. C h a k r a v a r l i A. Surti LJ. Gytogene t i c s and or ig ins of ped ia t r i c gel'Ill cell t l lnlors. C a n c e r (}el lel Cyloge- net . 1994:74( I ) :54-8.

52. Oos t e rhu i s Jl%, Cas ledo SM. de J o n g B, el al. Ploidy o f p r i m a r y g e r m cell t u m o r s of tile testis. Pathog 'enet ic a nd clinical rele- vance . I ,ab h lves l . 1989;80(I):14-21.

55. Stock C, A m b r o s IM, IAon T , el al. l )e lec- tioll o[ ntlmeric+ll and all'uClUl'al (hi 'ohiO- s o m e abnorn l a l i t i e s ill pedia t r ic g e r m cell IIIIIIOI'S by l l leans ofJnterpl lase cytogenel - ics. G e n e s Chronl(~sonles Cancer . I 994:1 I ( I ) :40-50.

54. Vail Echten .1, de . long [a% 8inke R J , Xle- ghu i s 1)O, Sleijfer DT, Oos t e rhu i s JW. I)e- finilion ,)f a new entity t)f nlal igqlanl ex- I r agonada I gerl l l (yell I I.i IllO I'S. G e n e s (3h romoson l e s Cancer . I ̀ ()95:12(I ):8-15.

35. Strike R.I, W e g h u i s Il l) . Suiikel 'buijk RF, el al+ Mtdecu l a r cha rac t e r i za t i on of a re- CIIl'l'illg COllll)lex clll'Onl|ls/inl;ll II'+lllSloc;I- tiOll in tWO htllllan exl r f lgonadaI gel'Ill cell hlnlors, t.~ancer (}enel CylogeneL 1994: 75(1):1 I-6.

5( ; .Skothein l RI, l ,othe RA. T h e tes t i cu la r gern l cell t lmlol i r g e n o m e . Aprnis. 2005: III (1):156-50; discuss ion 50-1.

57. Zur i la A J, I) iestra JE, (3ondonl E, el al. I , ung r e s i s t ance - re l a t ed pro te in a s a pre- dict ,)r of clinical ou lcon le ill a d v a n c e d tes t i cu la r g e r m - c e l l lun lours . Br J Ca ncer . 2005:88(6):879-86.

~0 CIm Transl OncoL 2006:8(12):846-50