Article

Antonescu CR, Nafa K, Segal NH, et al. EWS-CREB1: a recurrent variant fusion in clear cell sarcoma: association with gastrointestinal location and absence of melanocytic differentiation

Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Clinical Cancer Research (Impact Factor: 8.19). 10/2006; 12(18):5356-62. DOI: 10.1158/1078-0432.CCR-05-2811
Source: PubMed

ABSTRACT Clear cell sarcoma (CCS) usually arises in the lower extremities of young adults and is typically associated with a t(12;22) translocation resulting in the fusion of EWS (EWSR1) with ATF1, a gene encoding a member of the cyclic AMP-responsive element binding protein (CREB) family of transcription factors. CCS arising in the gastrointestinal tract is rare and its pathologic and molecular features are not well defined.
We report a novel variant fusion of EWS to CREB1, a gene at 2q32 encoding another CREB family member highly related to ATF1, detected in three women with gastrointestinal CCS. All three cases contained an identical EWS-CREB1 fusion transcript that was shown by reverse transcription-PCR. In two of the cases tested, EWS gene rearrangement was also confirmed by fluorescence in situ hybridization and the EWS-CREB1 genomic junction fragments were isolated by long-range DNA PCR.
Morphologically, all three tumors lacked melanin pigmentation. By immunohistochemistry, there was a strong and diffuse S100 protein reactivity, whereas all melanocytic markers were negative. Ultrastructurally, two of the cases lacked melanosomes. The melanocyte-specific transcript of MITF was absent in two cases, and only weakly expressed in the third case. The Affymetrix gene expression data available in one case showed lower expression of the melanocytic genes MITF, TYR, and TYRP1, compared with four EWS-ATF1-positive CCSs of non-gastrointestinal origin.
EWS-CREB1 may define a novel subset of CCS that occurs preferentially in the gastrointestinal tract and shows little or no melanocytic differentiation. Thus, evidence of melanocytic lineage or differentiation is not a necessary feature of sarcomas with gene fusions involving CREB family members.

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    • "Recently, an EWS-CREB fusion protein was also identified in a subset of CSST patients [24]. EWS-ATF1 and EWS-CREB fusion onco-proteins are constitutively active and enhance the expression of CREB target genes, independent of growth or signal stimulus. "
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    ABSTRACT: The cAMP response element-binding protein (CREB) is a nuclear transcription factor downstream of cell surface receptors and mitogens that is critical for normal and neoplastic hematopoiesis. Previous work from our laboratory demonstrated that a majority of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) overexpress CREB in the bone marrow. To understand the role of CREB in leukemogenesis, we examined the biological effect of CREB overexpression on primary leukemia cells, leukemia cell lines, and CREB overexpressing transgenic mice. Our results demonstrated that CREB overexpression leads to an increase in cellular proliferation and survival. Furthermore, CREB transgenic mice develop a myeloproliferative disorder with aberrant myelopoiesis in both the bone marrow and spleen. Additional research from other groups has shown that the expression of the cAMP early inducible repressor (ICER), a CREB repressor, is also deregulated in leukemias. And, miR-34b, a microRNA that negative regulates CREB expression, is expressed at lower levels in myeloid leukemia cell lines compared to that of healthy bone marrow. Taken together, these data suggest that CREB plays a role in cellular transformation. The data also suggest that CREB-specific signaling pathways could possibly serve as potential targets for therapeutic intervention.
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    ABSTRACT: Background: The Ewing sarcoma family of tumors (ESFT) are rare but highly malignant neoplasms that occur mainly in bone or but also in soft tissue. ESFT affects patients typically in their second decade of life, whereby children and adolescents bear the heaviest incidence burden. Despite recent advances in the clinical management of ESFT patients, their prognosis and survival are still disappointingly poor, especially in cases with metastasis. No targeted therapy for ESFT patients is currently available. Moreover, based merely on current clinical and biological characteristics, accurate classification of ESFT patients often fails at the time of diagnosis. Therefore, there is a constant need for novel molecular biomarkers to be applied in tandem with conventional parameters to further intensify ESFT risk-stratification and treatment selection, and ultimately to develop novel targeted therapies. In this context, a greater understanding of the genetics and immune characteristics of ESFT is needed. Aims: This study sought to open novel insights into gene copy number changes and gene expression in ESFT and, further, to enlighten the role of inflammation in ESFT. For this purpose, microarrays were used to provide gene-level information on a genomewide scale. In addition, this study focused on screening of 9p21.3 deletion sizes and frequencies in ESFT and, in another pediatric cancer, acute lymphocytic leukemia (ALL), in order to define more exact criteria for highrisk patient selection and to provide data for developing a more reliable diagnostic method to detect CDKN2A deletions. Results: In study I, 20 novel ESFT-associated suppressor genes and oncogenes were pinpointed using combined array CGH and expression analysis. In addition, interesting chromosomal rearrangements were identified: (1) Duplication of derivative chromosome der(22)(11;22) was detected in three ESFT patients. This duplication included the EWSR1-FLI1 fusion gene leading to increase in its copy number; (2) Cryptic amplifications on chromosomes 20 and 22 were detected, suggesting a novel translocation between chromosomes 20 and 22, which most probably produces a fusion between EWSR1 and NFATC2. In study II, bioinformatic analysis of ESFT expression profiles showed that inflammatory gene activation is detectable in ESFT patient samples and that the activation is characterized by macrophage gene expression. Most interestingly, ESFT patient samples were shown to express certain inflammatory genes that were prognostically significant. High local expression of C5 and JAK1 at the tumor site was shown to associate with favorable clinical outcome, whereas high local expression of IL8 was shown to be detrimental. Studies III and IV showed that the smallest overlapping region of deletion in 9p21.3 includes CDKN2A in all cases and that the length of this region is 12.2 kb in both Ewing sarcoma and ALL. Furthermore, our results showed that the most widely used commercial CDKN2A FISH probe creates false negative results in the narrowest microdeletion cases (<190 kb). Therefore, more accurate methods should be developed for the detection of deletions in the CDKN2A locus. Conclusions: This study provides novel insights into the genetic changes involved in the biology of ESFT, in the interaction between ESFT cells and immune system, and in the inactivation of CDKN2A. Novel ESFT biomarker genes identified in this study serve as a useful resource for future studies and in developing novel therapeutic strategies to improve the survival of patients with ESFT. Ewingin sarkooma on pahanlaatuinen luukasvain, jota esiintyy sen harvinaisuudesta huolimatta etenkin lapsilla ja murrosikäisillä. Ewingin sarkoomalle ominainen piirre on kromosomien 11 ja 22 välinen uudelleenjärjestäytyminen eli translokaatio, joka johtaa kimeerisen EWSR1-FLI1 -fuusiogeenin syntymisen ja muuttuneen geenien ilmentymisen kautta solujen pahanlaatuistumiseen. Koska kasvain lähettää aggressiivisesti etäpesäkkeitä, jää potilaiden eloonjääntiluku alhaiseksi uusista yhdistelmähoidoista huolimatta. Kun edelleenkään ei tunneta tarkkoja syitä näiden luukasvainten kehittymiselle ja etenemiselle, on potilaiden hoito haastavaa eikä tarpeeksi tehokasta ja spesifistä hoitoa ole annettavissa. Tämän takia tarve uusien kliinisesti merkittävien kohdegeenien löytymiselle Ewingin sarkoomassa on suuri, jotta taudin diagnosointi ja ennusteen määrittäminen helpottuisi sekä uusien kohdennettujen hoitomuotojen kehittäminen mahdollistuisi. Tutkimuksessa käytettiin mikrosirupohjaisia menetelmiä geenien kopiomäärän ja ilmentymisen muutosten kartoittamiseen koko perimän laajuisesti kliinisesti merkittävien kohdegeenien ja geneettisten muutosten löytämiseksi Ewingin sarkoomassa. Lisäksi mikrosirumenetelmää hyödynnettiin kasvurajoitegeeni-CDKN2A:n häviämien tutkimisessa, jotta saataisiin tietoa geenin häviämien koosta ja yleisyydestä Ewingin sarkoomassa sekä toisessa lasten syövässä, akuutissa lymfaattisessa leukemiassa (ALL). Ensimmäisessä osatyössä saimme tarkempaa tietoa mahdollisista Ewingin sarkooma -kasvaimen kehitykseen johtavista geneettisistä muutoksista yhdistämällä geenien kopiomäärä- ja ilmentymisprofiilit potilasnäytteissä. Tutkimuksemme osoitti 20 uutta potentiaalista onkogeeniä ja kasvurajoitegeeniä, jotka voivat osallistua kasvaimen kehittymiseen ja etenemiseen. Geenien kopiomäärätutkimuksessa havaitsimme kolmessa potilasnäytteessä myös derivatiivikromosomin der(22)(11;22) kahdentuman seurauksena EWSR1-FLI1 -fuusiogeenin kopiomäärän lisääntymisen. Lisäksi yhdessä potilasnäytteessä havaittiin korkea-asteisia kromosomien 20 ja 22 monistumia, jotka viittaavat translokaatioon EWSR1- ja NFATC2-geenien välillä. Toisessa osatyössä tutkimme Ewingin sarkoomakasvainten ja niitä ympäröivien kudosten välistä vuorovaikutusta analysoimalla tulehdukseen liittyvien geenien ilmentymistä. Tutkimuksessamme havaittiin tilastollisesti merkittävä tulehduksellisten geenien ilmentymisen lisääntyminen Ewingin sarkoomapotilasnäytteissä. Näytteistä voitiin todeta myös merkittävä makrofaagigeenien aktivoituminen. Mielenkiintoisimpana tuloksena tutkimuksemme osoitti komplementtitekijä C5:n paikallisen ilmentymisen vaikuttavan Ewingin sarkoomapotilaiden eloonjääntiin annosvasteesta riippuen. Tietääksemme tutkimuksemme on ensimmäinen, joka yhdistää komplementtitekijä C5:n ilmentymisen potilaiden eloonjääntiin missään syöpätyypissä. Komplementti on elimistön yleiseen immuunivasteeseen kuuluva puolustusjärjestelmä, jonka tarkoituksena on tuhota elimistölle vieraita tai muuntuneita soluja ja tehostaa adaptiivisen immuunivasteen toimintaa. Kolmannessa ja neljännessä osatyössä kartoitimme kromosomiraidan 9p21.3 häviämien koon ja yleisyyden sekä Ewingin sarkooma- että ALL-näytteissä. Totesimme, että kaikkien havaittujen 9p21.3-häviäminen pienin yhteinen alue sisältää CDKN2A-geenin ja että se on noin 12.2 kiloemästä pitkä sekä Ewingin sarkoomassa että ALL:ssa. Tutkimuksemme perusteella voidaankin todeta, että yleisimmin käytetyt kaupalliset FISH-koettimet CDKN2A-poikkeamien analysoinnissa tuottavat vääriä negatiivisia tuloksia pienimpien CDKN2A-häviämien osalta. Uusien kohdegeenien ja ennusteellisten tekijöiden tunnistamisen myötä tutkimuksemme mahdollistaa entistä tarkemman diagnoosin ja tehokkaampien hoitomuotojen kehittämisen Ewingin sarkoomaan. Lisäksi tutkimuksemme tuotti arvokasta tietoa CDKN2A-geenin häviämien koosta sekä yleisyydestä Ewingin sarkoomassa ja ALL:ssa, minkä perusteella voitaisiin kehittää entistä tarkempi menetelmä CDKN2A-häviämien havainnointiin potilasnäytteistä.
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    Cells Tissues Organs 01/2002; 172(2):133-44. DOI:10.1159/000065614 · 2.14 Impact Factor
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