Towards new therapeutic strategies in chondrosarcoma Schrage, Y.M.

Schrage, Y.M.

Citation

Schrage, Y. M. (2009, November 5). Towards new therapeutic strategies in chondrosarcoma. Retrieved from https://hdl.handle.net/1887/14327

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/14327

Note: To cite this publication please use the final published version (if applicable).

localisation, despite normal Exostosin, in central chondrosarcoma

Yvonne M. Schrage, Liesbeth Hameetman, Karoly Szuhai, Anne-Marie Cleton-Jansen, Antonie H.M. Taminiau, Pancras C.W. Hogendoorn, Judith V.M.G. Bovée.

Am J Pathol 2009;174:979-88



Abstract

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central chondrosarcomas is unknown.

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EXT1 and EXT2 were evaluated in central chondrosarcoma at the DNA PXWDWLRQDOVFUHHQLQJDUUD\&*+DQGP51$OHYHO,PPXQRKLVWRFKHPLVWU\

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VWXGLHGE\T3&5DQGin vitro.

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chondrosarcomas.

Introduction

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The protein products of EXT1 and EXT2([RVWRVLQDQG([RVWRVLQDUH

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formation of the morphogens hedgehog, decapentaplegic and wingless^. The human homologues for these morphogens are Indian and Sonic hedgehog, 7*)%%03DQG:17UHVSHFWLYHO\^,QGLDQ+HGJHKRJ,++RUFKHVWUDWHV

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While it is evident that inactivation of the EXT genes is the driving force for WKHGHYHORSPHQWRIEHQLJQSHULSKHUDOFDUWLODJLQRXVWXPRXUV^, in the far PRUHFRPPRQFHQWUDOFKRQGURVDUFRPDVWKHUROHRI(;7DQGLWVGRZQVWUHDP

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Material and methods Patient material

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Enchondroma Chondrosarcoma

))3( fresh frozen ))3( fresh frozen

Total number of

tumours 33 7* 62 27

Grade I - - 27 

Grade II - - 25 7

Grade III - -  9

0DOH)HPDOH   32:30 

Enchondromatosis  5  7

Median age at diagnosis

years (range) 

 

 50,0

 



Median follow up

months (range)  

Table 3.1 Clinicopathological data of the 110 patients. ))3( IRUPDOLQ À[HG SDUDIÀQ

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Quantitative real time reverse transcriptase PCR (q-RT-PCR)

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conditions not related to cartilaginous tumours, were used as controls.

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Sequence (5’ - 3’) T_m Product

Size SDC2-ex1F TGTAAAACGACGGCCAGTGTACTCTGCTCCGGATTCGT 

SDC2-ex1R AACAGCTATGACCATGAGGGCTCCTCTCGTAGCTTCA  

SDC2-ex2F TGTAAAACGACGGCCAGTCTCAACATCCTGACTCCCTTG 

SDC2-ex2R AACAGCTATGACCATGAATCCTGCCTGTCTCCTTGAA  

SDC2-ex3F TGTAAAACGACGGCCAGTTCATGATTGCCATGCTCAGT 

SDC2-ex3R AACAGCTATGACCATGAGATAATGCAATGCAATGGAAA  

SDC2-ex4F TGTAAAACGACGGCCAGTTTTCTTCTTTCCAACACATTTCC 

SDC2-ex4R AACAGCTATGACCATGAGTAGGCACCCTCCCACCT 59.92 270 SDC2-ex5F TGTAAAACGACGGCCAGTTGTCTGCAACCCTTGAATCTC 

SDC2-ex5R AACAGCTATGACCATGATGCAAAAGCTTTATTTTGAAAAGTT  

Table 3.2 Primer sequences SDC2 mutation analysis

52

Array comparative genomic hybridisation (CGH)

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Immunohistochemistry

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Statistical analysis

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ZHUH FDOFXODWHG E\ &KLVTXDUH ;² WHVW 'LIIHUHQFHV LQ FHOO YLDELOLW\ in vitro ZHUH FDOFXODWHG E\ 6WXGHQWV WWHVW 3YDOXHV  ZHUH FRQVLGHUHG

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Results

Normal EXT1 and EXT2 in central chondrosarcoma

7KHH[SUHVVLRQRIEXT1 and EXT2P51$LQFHQWUDOFDUWLODJLQRXVWXPRXUV

is not decreased as compared to growth plate samples, which is in contrast to the low EXT1H[SUHVVLRQOHYHOVSUHYLRXVO\IRXQGLQSHULSKHUDOWXPRXUV

)LJXUH$DQG%²². No correlation of EXT1 or EXT2H[SUHVVLRQZLWK

KLVWRORJLFDO JUDGH ZDV IRXQG QRW VKRZQ 'LUHFW PXWDWLRQ VFUHHQLQJ DQG

0/3$IRUERWKEXT1 and EXT2GLGQRWUHYHDODQ\JHQHWLFDEHUUDWLRQVQRW

VKRZQ&RPSDUDWLYHJHQRPLFK\EULGLVDWLRQRQDT%$&WLOLQJDUUD\RIWKH

WZRFHQWUDOFKRQGURVDUFRPDVZLWKWKHORZHVWH[SUHVVLRQRIEXT1 did not UHYHDODQ\DOWHUDWLRQVRQTWKH

locus for EXT 5HVXOWV IRU /

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DUH UHSUHVHQWDWLYH IRU / QRW

VKRZQ1RRWKHUJDLQVRUORVVHVLQT

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were found in the EXT1 and EXT2 genes in the 7 chondrosarcomas K\EULGLVHG DW WKH ROLJRQXFOHRWLGH

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Figure 3.1$(;7P51$H[SUHVVLRQOHYHOV

RIFHQWUDOFKRQGURVDUFRPDDUHFRPSDUDEOHWR

growth plate levels while peripheral tumours SXEOLVKHG SUHYLRXVO\^ VKRZ D WKUHH IROG

GHFUHDVH%(;7P51$OHYHOVDUHVOLJKWO\

KLJKHULQFHQWUDODQGVOLJKWO\ORZHULQSHULSK- eral chondrosarcoma, compared to growth SODWH ([SUHVVLRQ OHYHOV RI WKH WXPRXUV DUH

log2 transformed and relative to the growth SODWHOHYHOV&TWLOLQJDUUD\&*+SDWWHUQ

RI FHQWUDO FKRQGURVDUFRPD / LV VKRZQ

*HQRPLFDOWHUDWLRQVDUHDEVHQWGHVSLWHORZ

(;7 H[SUHVVLRQ DW T3&5 / VKRZHG

VLPLODUUHVXOWVDV/GDWDQRWVKRZQ)RU

comparison an osteochondroma with homo- ]\JRXV ORVV RI PXOWLSOH FORQHV FRYHULQJ WKH

(;7 JHQH DQG KHPL]\JRXV ORVV RI D ODUJHU

SDUWRITLVVKRZQLQ'ÀJXUHFRPSRVHG

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Aberrant location of HSPGs SDC2 and CD44v3 in central chondrosarcoma

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VHFWLRQV RI WRQVLO VKRZLQJ PHPEUDQRXV H[SUHVVLRQ SDWWHUQ RI &'Y LQ

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FHOO PHPEUDQH DQG LQ WKH H[WUDFHOOXODU PDWUL[ VXUURXQGLQJ K\SHUWURSKLF

FKRQGURF\WHVQRWVKRZQ1XFOHDUORFDOLVDWLRQRI6'&DVIRXQGE\,+&ZDV

YHULÀHGLQWKHQXFOHDUFRPSRQHQWRIWKHFHOOIUDFWLRQDWLRQE\LPPXQREORWWLQJ

)LJXUH'

No aberrations in other genes important for HSPG formation

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DEHUUDWLRQVLQRWKHUUHJXODWRUVRI+63*IRUPDWLRQ0XWDWLRQVFUHHQLQJRI

WKHÀYHFRGLQJH[RQVRIWKHSDC2JHQHGLGQRWUHYHDODQ\PXWDWLRQVLQWKH

VHYHQWXPRXUVQRWVKRZQ8VLQJWKHROLJRQXFOHRWLGHWLOLQJDUUD\FRYHULQJ

(;7(;7OLNHDQGRWKHUJHQHVLQYROYHGLQ+63*ELRV\QWKHVLVZHGLGQRW

ÀQGDQ\VSHFLÀFJHQRPLFORVVHVRUJDLQVLQWKHVHVHYHQWXPRXUV

Decreased WNT signalling and increased TGFB signalling in high grade chondrosarcomas

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HQFKRQGURPDV LQ   RI JUDGH , DQG LQ   RI JUDGH ,,

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of canonical WNT signalling was increased in grade I chondrosarcomas FRPSDUHGWRHQFKRQGURPDV+RZHYHUXSRQIXUWKHULQFUHDVHLQKLVWRORJLFDO

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HH signalling in central chondrosarcoma in vivo and in vitro

%\ T3&5 DFWLYH ++ VLJQDOOLQJ ZDV VKRZQ LQ FHQWUDO FKRQGURVDUFRPD

irrespective of histological grade, since PTCH, GLI1 and GLI2ZHUHH[SUHVVHG

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Figure 3.2$&HQWUDOFKRQGURVDUFRPDVKRZLQJF\WRSODVPLFGRWOLNHDFFXPXODWLRQRI&'Y

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three genes in chondrosarcomas were in the same range of growth plate samples and large variations within the groups ZHUHREVHUYHG1RUHODWLRQWRKLVWRORJLFDO

JUDGH ZDV IRXQG ([SUHVVLRQ OHYHOV DUH

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Figure 3.5$In vitroWKH*/,P51$H[SUHVVLRQ

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whereas the other four chondrosarcoma cell cul- WXUHVGLGQRWVKRZDQHIIHFWRIF\FORSDPLQHWUHDW- PHQW7KHEODFNEDUVUHSUHVHQWWKHLQDFWLYHFRP- SRXQG WRPDWLGLQH 70 ZKLFK GHPRQVWUDWH WKH

selective effect of CP. Data represent 3 individual H[SHULPHQWVSHUIRUPHGLQTXDGUXSOLFDWH

LQGLFDWHV6WXGHQWVWWHVWIRUSDLUHGGDWDS

treatment resulted in a profound decrease of GLI1 P51$ H[SUHVVLRQ

FRPELQHG ZLWK GHFUHDVHG FHOO YLDELOLW\ LQ RQO\ RQH FHOO FXOWXUH &+

ZKHUHDVDOORWKHUFXOWXUHVGLGQRWUHVSRQGE\GHFUHDVHG*/,OHYHOVRUFHOO

YLDELOLW\)LJXUH,Q/DQLQFUHDVHRI*/,ZDVREVHUYHGZKLOH/

VKRZHGLQFUHDVHGFHOOYLDELOLW\,QDOOFKRQGURVDUFRPDFHOOFXOWXUHVPTCH OHYHOVZHUHXQFKDQJHGRUHYHQLQFUHDVHGDIWHUF\FORSDPLQHWUHDWPHQW)LJXUH

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compound.

Discussion

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WDUJHWVLQFHQWUDOFKRQGURVDUFRPD:HFRQÀUPWKDWSHULSKHUDODQGFHQWUDO

chondrosarcomas are clear distinct genetical entities also with respect to their EXTH[SUHVVLRQ:KHUHDVLQDFWLYDWLRQRIEXT is the driving force for WKHGHYHORSPHQWRIEHQLJQSHULSKHUDOFDUWLODJHWXPRXUV²², we demonstrate in central cartilage tumours the EXTJHQHVWREHQRUPDOERWKDWWKH'1$

DVZHOODVDWWKHP51$H[SUHVVLRQOHYHO7KHDEVHQFHRIODUJHUGHOHWLRQVLQ

the EXT1UHJLRQLVLQFRQFRUGDQFHZLWKWKHSUHYLRXVO\UHSRUWHGDEVHQFHRI

/2+^DQGNDU\RW\SLFDEEHUDWLRQV³⁹DWTLQFHQWUDOFKRQGURVDUFRPDV

7KH (;7 SURWHLQV DUH LQYROYHG LQ WKH ELRV\QWKHVLV RI KHSDUDQ VXOSKDWH

'HVSLWHQRUPDOH[SUHVVLRQRIWKHEXT genes in central tumours, heparan VXOSKDWHSURWHRJO\FDQV&'YDQG6'&XQH[SHFWHGO\DFFXPXODWHGLQ

WKHF\WRSODVP7KLVLVVLPLODUWRZKDWZHSUHYLRXVO\GHVFULEHGIRUSHULSKHUDO

WXPRXUV FDUU\LQJ EXT mutations²²,ZKLOH LQ RWKHU WXPRXUV PHPEUDQRXV

H[SUHVVLRQRI&'YDQG6'&LVGHVFULEHG^.,QDGGLWLRQRIFHQWUDO

FKRQGURVDUFRPDVDOVRGHPRQVWUDWHGQXFOHDU6'&H[SUHVVLRQZKLFKZDV

QRWREVHUYHGLQSHULSKHUDOFKRQGURVDUFRPDVQRULQVRIWWLVVXHVDUFRPDV

,Q DGGLWLRQ WR WKH JHQHUDO IXQFWLRQ RI 6'& LQ PRGXODWLQJ H[WUDFHOOXODU

ligands, intracellular actions are implicated for SDC2. In osteosarcoma 6'&KDVEHHQVKRZQWRLQGXFHDSRSWRVLV^.:HH[FOXGHGPXWDWLRQVLQ

WKHQXFOHDUORFDOLVDWLRQVLJQDO1/6ORFDWHGDWWKHÀUVWH[RQRIWKH6'&

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,Q DGGLWLRQ LW LV WHPSWLQJ WR VSHFXODWH WKDW IRU H[DPSOH other JO\FRV\OWUDQVIHUDVHV WKDQ (;7 RU WKH VXOSKRWUDQVIHUDVHV RU HSLPHUDVHV

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are affected in central chondrosarcoma %\ XVLQJ D FXVWRP GHVLJQHG

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In Drosophila+63*VDUHHVVHQWLDOIRUJUDGLHQWIRUPDWLRQRIWKHPRUSKRJHQV

hedgehog, decapentaplegic and wingless^.,Q KXPDQ WKH ,++ SDWKZD\

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as well^.,Q RVWHRFKRQGURPDV ,++ VLJQDOOLQJ LV DFWLYH ZKLOH DFWLYLW\

decreases in peripheral chondrosarcoma with increasing histological grade²³.7LHW HW DO UHSRUWHG DFWLYH ,++ VLJQDOOLQJ LQ FKRQGURVDUFRPD EXW

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compared to the growth plate ^,QWKHSUHVHQWODUJHUVHULHV,++VLJQDOOLQJ

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chondrosarcoma and is vital for cellular proliferation and therefore a putative WKHUDSHXWLFWDUJHWLQDVPDOOVXEVHWRIWKHP6LQFH+63*VDUHWKRXJKWWREH

LPSRUWDQWIRUWKHGLIIXVLRQRI++WRLWVUHFHSWRURQWDUJHWFHOOVLWPLJKWEH

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for osteochondromas^.

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WR WKH DFWLYLW\ LQ SHULSKHUDO FKRQGURVDUFRPD²³. Our results suggest that DFWLYHFDQRQLFDO:17VLJQDOOLQJPLJKWEHLPSRUWDQWIRUWKHWUDQVLWLRQIURP

enchondroma towards low-grade central chondrosarcoma, however it is QRWFUXFLDOIRUSURJUHVVLRQWRZDUGVKLJKHUJUDGH)LJXUH,QFRQWUDVW

TGFB signalling was shown to increase with increasing histological grade E\ ERWK WKH SUHVHQFH RI 3$, DQG WKH ÀQGLQJ RI QXFOHDU ORFDOLVDWLRQ RI

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the main characteristics of high grade chondrosarcoma⁵⁰.

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6'&ORFDOLVDWLRQWKDWZDVVSHFLÀFIRUFHQWUDOFKRQGURVDUFRPDV,QDGGLWLRQZKLOH,++VLJQDO- ling decreased with grade in peripheral chondrosarcomas, it is still active in high grade central chondrosarcomas.

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the EXT genes are normal in central chondrosarcoma, with nevertheless DEHUUDQW ORFDOLVDWLRQ RI +63*V 'HVSLWH WKLV ,++ VLJQDOOLQJ LV DFWLYH LQ

central chondrosarcoma and is important for proliferation and therefore a SRWHQWLDOWKHUDSHXWLFWDUJHWLQDVPDOOVXEVHWRIFHQWUDOFKRQGURVDUFRPDV

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which is similar to peripheral chondrosarcoma and the nuclear SDC2 ORFDOLVDWLRQWKDWLVH[FOXVLYHO\VHHQLQFHQWUDOFKRQGURVDUFRPDLVGLIÀFXOW

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Acknowledgements

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References

 $KQ-/XGHFNH+-/LQGRZ6+RUWRQ:$/HH%:DJQHU0-HWDO&ORQLQJRI

WKHSXWDWLYHWXPRXUVXSSUHVVRUJHQHIRUKHUHGLWDU\PXOWLSOHH[RVWRVHV(;7

Nature Genet

  :X\WV:9DQ+XO::DXWHUV-1HPWVRYD05H\QLHUV(9DQ+XO(HWDO

3RVLWLRQDOFORQLQJRIDJHQHLQYROYHGLQKHUHGLWDU\PXOWLSOHH[RVWRVHVHum Mol Genet

  6WLFNHQV ' &OLQHV * %XUEHH ' 5DPRV 3 7KRPDV 6 +RJXH ' HW DO 7KH

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genes. Nature Genet

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