Population-based genetic alterations in Ewing’s tumors from Japanese and European Caucasian patients

T. Ozaki1,7,+, K.-L. Schaefer2,9, D. Wai2, R. Yokoyama3, S. Ahrens4, R. Diallo2,9, T. Hasegawa5, T. Shimoda5, S. Hirohashi6, A. Kawai7, N. Naito7, Y. Morimoto7, H. Inoue7, W. Boecker2, H. Juergens3, W. Winkelmann1, B. Dockhorn-Dworniczak8 and C. Poremba2,9,§

1 Department of Orthopaedics, 2 Gerhard-Domagk-Institute of Pathology and 4 Pediatric Hematology and Oncology, Westfaelische Wilhelms-University, Münster, Germany; 3 Department of Orthopaedic Surgery and 5 Department of Pathology, National Cancer Center Hospital, Tokyo; 6 Research Institute, National Cancer Center, Tokyo; 7 Department of Orthopaedic Surgery, Okayama University Medical School, Okayama, Japan; 8 Institute of Pathology, Kempten; 9 Institute of Pathology, Heinrich-Heine-University, Düsseldorf, Germany

Received 23 November 2001; revised 8 February 2002; accepted 7 March 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background:

The incidence of Ewing’s tumors (ETs) is lower in Asians or African-Americans than in Caucasians.

Patients and methods:

Japanese ETs were available for analysis of chromosomal aberrations by comparative genomic hybridization (n = 16) and for expression of chimeric EWS transcripts by reverse-transcriptase polymerase chain reaction (n = 11). These results in Japanese patients were compared with those of 62 ETs in European Caucasian patients registered in the European Intergroup Cooperative Ewing’s Sarcoma Study.

Results:

Japanese patients with ET had lower overall survival (P = 0.0446) and relapse-free survival (P = 0.0371) compared with European Caucasian patients. Ten of 11 Japanese ETs and 31 of 62 European Caucasian ETs had type I (EWS exon 7 to FLI1 exon 6) fusion transcripts. In Japanese ETs, the median numbers of chromosomal aberrations were 2.0 and 6.0 in 11 primary tumors and five relapsed tumors, respectively. In European Caucasian ETs, the median number of changes were 2.5 and 5.0 in 52 primary and 10 relapsed tumors, respectively. Frequent gains were 8q (38%), 8p (31%) and 12q (25%) in Japanese ETs and 8q (52%), 8p (48%) and 12q (19%) in European Caucasian ETs. Frequent losses were 19q (44%), 19p (38%) and 17p (25%) in Japanese ETs and 16q (21%), 19q (18%) and 17p (15%) in European Caucasian ETs. The incidence of losses of 19p (P = 0.0215) and 19q (P = 0.0277) were significantly higher in Japanese ETs than in European Caucasian ETs. An amplification (1p33-p34) was observed in only one Japanese ET.

Conclusions:

Japanese patients with ET in this study had a worse prognosis than European Caucasian patients. In molecular genetic analyses, Japanese ETs had a higher frequency of loss of chromosome 19 than European Caucasian ETs. Different genetic aberrations may explain the different incidences and prognoses of ET between Caucasian and Japanese patients.

Key words: Ewing’s tumor prognosis, Caucasian, c-erbB-2, comparative genomic hybridization, chromosomal aberrations, Japanese


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ewing’s tumor (ET) is the second most frequent primary malignant bone tumor in children and adolescents [1]. Ewing’s tumors are mainly composed of small round tumor cells characterized by specific chromosomal translocations; the most frequent translocation partner of EWS (22q12) is FLI1 (11q24) which occurs in 90–95% of cases [24], followed by ERG (21q22) in 5–10% of cases [5, 6]. These fusion products encode chimeric proteins which function as aberrant oncogenic transcription factors [7]. The most common fusion joins EWS exon 7 in frame with FLI1 exon 6 (type I), and was reported to be associated with a better prognosis than other fusion types [8, 9]. Other common chromosomal aberrations are trisomies 8 [10] and 12 [11] and der(1;16) [12, 13]. In recent years, chromosomal aberrations revealed by comparative genomic hybridization (CGH) have been reported; the common changes are gains of chromosome 8, 12 and 1q, and losses of 16q and chromosome 11 [14, 15].

The incidence of Ewing’s sarcoma differs considerably according to race [16]. Ewing’s tumors are rare among African-American children [1619] and are also rare in the Chinese population [16]. Furthermore, inter-ethnic differences within the structure of EWS intron 6 have been reported [20]. The ratios of ETs to osteosarcoma were around 0.5 among Caucasians in the USA and <0.15 among non-Caucasian children in Shanghai, Beijing and the USA [16]. In Japanese patients registered with the Japanese Musculoskeletal Tumor Committee [21], the ratio of ET to osteosarcoma was 0.13. The treatment strategy and dose intensity of chemotherapy against ET are almost the same both in Japan and Germany; however, there seems to be a difference in the prognosis of patients in these two countries. As the incidence of ET differs according to race, there may be a difference of genetic alterations in ETs between both countries. A molecular genetic analysis may answer this question and finally lead to treatment strategies based on the genetic features of the tumor.

In this study, we analyzed the chromosomal aberrations in 16 Japanese ETs by CGH. The number of Japanese cases is relatively small in this study; however, the incidence in the Asian population is much lower compared to that in Caucasians [16]. In 11 of these Japanese ETs, RNA was available for evaluating the expression of chimeric EWS transcripts with reverse-transcriptase polymerase chain reaction (RT–PCR). The molecular and clinical data for Japanese ETs were statistically compared with those data from 62 European Caucasian ETs [15].


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Sixteen ET tissues from 16 Japanese patients who were diagnosed and whose treatment was initiated between 1987 and 1998 were available for this study (Table 1). Nine samples were obtained from frozen tissues, which were taken at biopsy before treatment of the lesion and preserved at –80°C. Tumor tissues were taken from viable tumor areas with at least 80% tumor cell content. Seven samples were obtained from formalin-fixed and paraffin-embedded tissue. All cases were re-evaluated by two pathologists and classified based on histology, immunostaining for the MIC2 gene product and findings of the characteristic fusion gene transcripts.


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Table 1. Clinical and histological characteristics of 16 Japanese patients with Ewing’s tumors
 
Of 16 tumors, 11 were primary ETs (10 Ewing’s sarcomas and one extraskeletal Ewing’s sarcoma), four were metastatic ETs (three Ewing’s sarcomas and one extraskeletal Ewing’s sarcoma) and one was a locally relapsed ET (atypical Ewing’s sarcoma) (Table 1).

In 11 patients with primary tumors, four patients had metastases at diagnosis. In five patients with relapsed tumors, none had metastasis at diagnosis of the primary tumor. In 11 patients with primary tumors, four tumors were centrally located (pelvis, two; ribs, one; skull, one), six were proximally located (femur) and one was distally located (ankle). In five patients with relapsed or metastatic tumors, one tumor was a local relapse of the upper arm tumor (Case 1), one was a pulmonary metastasis of a thigh tumor (Case 3), one was a sternum metastasis of a hand tumor (Case 9), one was a spinal metastasis of an iliac tumor (Case 12) and one was a pulmonary metastasis from a femoral tumor (Case 15). In 15 patients, the volume of the primary tumor could be evaluated at the time of diagnosis according to the method of Göbel et al. [22]: nine patients had tumors with large volumes (>=100 ml) and six patients had tumors with small volumes (<100 ml). In all 16 patients, detailed information on treatment and clinical course was available. In seven patients, chemotherapy was performed according to the vincristine (VCR) plus actinomycin-D (Act-D) plus cyclophosphamide (CPM) plus doxorubicin (ADR) (VACA) protocol [23]. Five patients received vincristine, actinomycin-D, IFO and doxorubicin (VAIA), one patient received etoposide plus VAIA (EVAIA) [24] and three patients received another chemotherapy protocol. The regimens of chemotherapy were similar between Japanese patients and European Caucasian patients.

Ten patients received radiotherapy: five preoperative radiation, three radiation alone and two palliative radiation. Ten patients underwent surgery and the surgical margin was classified according to the method of Enneking et al. [25]: radical, one; wide, seven; marginal, one; unknown, one. Six patients had no surgery: three (Cases 5, 8 and 16) had multiple metastases at diagnosis, one (Case 10) had spinal metastasis after treatment of primary tumor and two (Cases 11 and 12) refused surgical treatment. The follow-up period ranged from 12 to 85 months (median 29 months).

Sixty-two European Caucasian cases as controls
Sixty-two ET samples from 62 patients were obtained from frozen tissues preserved at –80°C. Of the 62 tumors, 52 were primary tumors [37 Ewing’s sarcomas, 11 primitive neuroectodermal tumors (PNETs) and four atypical Ewing’s sarcomas] and 10 were metastatic or locally relapsed tumors (five Ewing’s sarcomas, three atypical Ewing’s sarcomas, one PNET and one extraskeletal Ewing’s sarcoma) [15]. Among the 62 patients, the male to female ratio was 36 to 26 (1.4) and ages ranged from 4 to 33 years: median age was 15 years. Twenty of 52 patients with primary tumors and four of 10 patients with relapsed tumors had metastases at diagnosis of the primary tumors. Thirty-six tumors were centrally located, 15 were proximally located and 11 were distally located. In 57 patients, tumor volume could be evaluated according to the method of Göbel et al. [22]: 43 patients had tumors with large volumes (>=100 ml) and 14 patients had tumors with small volumes (<100 ml).

Chemotherapy was performed according to the protocol of the European Intergroup of Cooperative Ewing’s Sarcomas Study (EICESS) 92 [24]. In four cases, some modifications to the protocol were carried out. As a local treatment in 52 patients with primary tumor, 30 patients received preoperative irradiation and surgery, 10 irradiation only, four surgery and postoperative irradiation, two surgery alone, one pre- and postoperative irradiation and surgery, one no local treatment and for four patients, no information was available. Obtained surgical margins were as follows: radical, two; wide, 25; marginal, seven; intralesional, three. As an initial local therapy in 10 relapsed cases, five received preoperative irradiation and surgery, two irradiation alone, one surgery alone, one surgery and postoperative irradiation and one no local treatment. The follow-up period ranged between 24 and 72 months (median 33.5 months).

Comparative genomic hybridization (CGH)
Reference DNA from healthy blood donors and tumor DNA was labeled by the nick translation method with digoxigenin-11-dUTP (Boehringer, Mannheim, Germany) and biotin-14-dATP (Gibco-BRL, Gaithersburg, MD, USA), respectively. The hybridization was performed as described by Kallioniemi et al. [26] with some modifications [27, 28]. Ratio profiles were averaged from 10 metaphases per sample (up to 20 chromosome homologs). Gains of DNA sequences were defined as chromosomal regions with a fluorescence ratio above 1.25 and losses as regions with a ratio below 0.75. A positive control, with known aberrations, and a negative control were included in each CGH experiment as quality controls. Regional shifts of the fluorescence ratio profile exceeding the 1.5 threshold were regarded as amplifications [29]. Telomeric and heterochromatic regions were excluded from the analysis. The profiles of 1p32-pter, 16p, 17p and chromosomes 19 and 22 were interpreted with caution, as they have been known to give false-positive results [26]. Judgement was based on a consensus of at least two of three authors in all cases without reference to the patient’s clinical records.

RT–PCR for Ewing’s tumor-related gene fusion transcripts
Total RNA from frozen or paraffin-embedded material was isolated using the acid–guanidium–phenol/chloroform method [8, 9, 30, 31]. RT–PCR for EWS–FLI1 and EWS–ERG transcripts was performed using 1 µg total RNA, as published previously [8, 9, 30, 31]. For RT–PCR from paraffin-embedded tissues, the TITAN System (Roche Diagnostics, Mannheim, Germany) was used [32].

Gene dosage studies and protein immunohistochemistry for c-erb-B2/HER-2
Fluorescence in-situ hybridization (FISH)
The qualitative presence of c-erb-B2/HER-2 amplification was analyzed in two cases with chromosome 17q gains (detected by CGH) on 4 µm-thick paraffin sections by FISH using the Oncor"/Ventana" INFORM" HER-2/neu Gene Detection System (Ventana Medical Systems, Frankfurt, Germany). Assay and scoring were strictly performed according to the respective ‘Procedure and Interpretation Guide’ and as previously published [33]. Scoring of amplification was performed by counting the number of all fluorescent signals present in 20 randomly selected, non-overlapping ET cell nuclei in two distinct areas within the same section using a DAPI/Cy3 filter set and the 100x oil immersion objective of a Zeiss Axioskop 100 fluorescence microscope (Zeiss, Jena, Germany). Tumors with <=4 mean signals per nucleus were interpreted as non-amplified, whereas cases exhibiting a mean of >4 signals were classified as amplified.

Immunohistochemistry (IHC)
As previously described [33], two different antibodies against the c-erb-B2/HER-2 protein were used: (i) a mouse monoclonal anti-erb-B2 antibody (clone CB11, Ventana Medical Systems, prediluted for automated detection) and (ii) a rabbit polyclonal antibody (anti-human erb-B2-oncoprotein, Code No. A0485, Dako). A total of 16 paraffin-embedded ETs (seven ET patients with progressive disease and nine patients in total remission after 5-year follow-up), including the two cases for which gene dosage studies by c-erb-B2/HER-2 FISH were performed (see above), were analyzed by IHC. IHC expression of c-erb-B2/HER-2 was categorized into four groups: negative (score 0), no staining at all or membrane staining <10% of the tumor cells; negative (score 1+), faint/barely perceptible partial membrane staining in >10% of the tumor cells; weakly positive (score 2+), weak to moderate staining of the entire membrane in >10% of the tumor cells; strongly positive (score 3+), strong staining of the entire membrane in >10% of the tumor cells.

Statistical analysis
Significance of differences of the ratio between or among groups was evaluated by the chi-square test with/without Fisher’s correction. The Mann–Whitney U-test evaluated differences of the mean rank between two groups. The cumulative probability of overall survival for all patients and relapse-free survival for the patients without metastasis at diagnosis were calculated by univariate analysis with the Kaplan–Meier method. Tests of the difference between survival curves were carried out using the log-rank test. The statistical software used was Stat View version 5.0 (SAS Institute Inc., Cary, NC) on the Macintosh.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prognosis of patients with ET between Japanese and European Caucasians
The overall survival of patients with ET in both groups, including 58 European Caucasian patients (48 patients with primary tumors and 10 patients with relapsed tumors) with detailed follow-up and 16 Japanese patients (11 patients with primary tumors and five patients with relapsed tumors), was analyzed. In all patients, the beginning of follow-up was the time of diagnosis of primary ET. The overall survival was different in the two patient subgroups (P = 0.0446) (Figure 1). As 24 European Caucasian patients and four Japanese patients had metastases at diagnosis, these patients were excluded and relapse-free survival was analyzed. The relapse-free survival was still significantly higher in 34 European Caucasian patients than in 12 Japanese patients (P = 0.0371) (Figure 2).



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Figure 1. Overall survival. Sixteen Japanese patients with Ewing’s tumors had significantly lower overall survival than 58 European Caucasian Ewing’s tumor patients (P = 0.0446).

 


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Figure 2. Relapse-free survival. Twelve Japanese Ewing’s tumor patients without primary metastasis had significantly lower relapse-free survival than 34 European Caucasian Ewing’s tumor patients without primary metastases (P = 0.0371).

 
Fusion gene type
In 11 Japanese patients, 10 patients expressed the type I fusion transcript (EWS exon 7 to FLI1 exon 6) and one had a non-type I fusion transcript. In 62 European Caucasian patients, 31 patients had the type I fusion transcript and 31 had non- type I fusion transcripts. The incidence of the type I fusion was more frequent in Japanese ETs than in European Caucasian ETs. As the type of fusion transcript could be analyzed in only 11 Japanese ETs, we did not perform any statistical analysis for the significance of the difference in the two populations.

Genomic imbalances in ETs
Genomic imbalances were detected in 12 of 16 (75%) Japanese ETs and 48 of 62 (79%) European Caucasian ETs. In Japanese cases, frequent chromosomal aberrations included gains of chromosome 8 or 12 or loss of 17p or chromosome 19 (Figure 3). In one locally relapsed Japanese case (Case 1), amplification of the 1p33-p34 region was detected.



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Figure 3. Chromosomal aberrations of Ewing’s tumors by comparative genomic hybridization. Continuous lines show the primary cases and dotted lines indicate the relapsed cases. Lines on the left side of the chromosome indicate loss and lines on the right side of the chromosome indicate gain.

 
In primary tumors, 8 of 11 (73%) Japanese ETs and 39 of 52 (75%) European ETs had aberrations; the median number of aberrations was 2.0 and 2.5 in 11 Japanese ETs and 52 European Caucasian ETs, respectively. In relapsed tumors, four of five (80%) Japanese ETs and nine of 10 (90%) European Caucasian ETs had aberrations; the median number of changes was 6.0 and 5.0 in five Japanese ETs and 10 European Caucasian ETs, respectively.

In Japanese ETs, the frequent gains were chromosomes 8q (38%), 8p (31%), 12q (25%), 14q (19%) and 17q (19%) and frequent losses were chromosomes 19q (44%), 19p (38%) and 17p (25%). In European Caucasian ETs, frequent gains involved chromosomes 8q (52%), 8p (48%), 12q (19%), 1q (19%), 20p (18%) and 20q (18%) and frequent losses involved chromosomes 16q (21%), 19q (18%), 17p (15%) and 10 (13%). Between Japanese and European Caucasian ETs, six aberrations of interest were statistically analyzed (Figure 4). Of these aberrations, the incidence of gain of 17q (P = 0.0577), loss of 19p (P = 0.0215) and loss of 19q (P = 0.0277) were higher in 16 Japanese ETs (11 primary and five relapsed) than 62 European Caucasian ETs (52 primary and 10 relapsed). In primary ETs alone, the incidence of 19p loss was still significantly higher in 11 Japanese ETs than in 52 European Caucasian ETs (P = 0.0174).



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Figure 4. Rate of chromosomal aberrations in 16 Japanese Ewing’s tumors and 62 European Caucasian Ewing’s tumors. Including relapsed cases, loss of 19p (P = 0.0215) and loss of 19q (P = 0.0277) were significantly more frequent in the Japanese cases than in European Caucasian cases (marked with *).

 
Gene dosage studies and protein immunohistochemistry for c-erb-B2/HER-2
As gains of chromosome 17q were found in 19% of the Japanese and only 3.2% of the European Caucasian ETs by CGH, we sought to determine if c-erb-B2/HER-2, which is located at chromosome 17q12, was amplified or overexpressed in ETs. To our knowledge, there is no published study to date evaluating the amplification or overexpression of c-erb-B2/HER-2 in ETs. First, two tumors with 17q gains by CGH were analyzed by c-erb-B2/HER-2 FISH. One case exhibited 1.875 signals per nucleus as a mean (range 1–3; SD 0.4043) and the other case 1.775 signals per nucleus as a mean (range 1–3; SD 0.5305), indicating non-amplification of c-erb-B2/HER-2. Second, a total of 16 ETs (both with and without 17q gains) were analyzed for c-erb-B2/HER-2 oncoprotein expression by immunohistochemistry, including the two cases for which gene dosage studies by c-erb-B2/HER-2 FISH were performed. All 16 Ewing’s tumors were completely negative (score 0) for the c-erb-B2/HER-2 oncoprotein.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of ET varies according to race. Ewing’s tumors occur more frequently in Caucasians than in African-Americans or Chinese [16]. A report on inter-ethnic polymorphism in EWS intron 6, which includes a very high density (65%) of Alu elements, describes the size of the intron 6 region decreasing by over 50% as the result of homologous recombination between two Alu sequences; this rare allele has only been observed in individuals of African origin [20]. At the present time, there are no further reports which prove that ethnic variations in the EWS gene and fusion-transcript subtypes are relevant to prognostic differences. Therefore, it was of great interest to study the molecular genetic differences in ET patients from different ethnic backgrounds.

In our study, the regimens of chemotherapy were similar between Japanese patients and European Caucasian patients, as the treatment regimens used in Japan were adapted from the European protocols CESS86 and -92. Nevertheless, the prognosis of Japanese patients with ET is worse than that of European Caucasian patients. To further elucidate our understanding of these findings, we analyzed ETs by CGH and RT–PCR in a total of 78 ET cases from both Japan and Europe. However, we realize that the number of Japanese patients in our study is relatively low. We were limited by the low incidence of ET in the Japanese population and the fact that we wanted to only investigate patients who were subjected to similar treatment strategies comparable to the European cases included in our study.

Incidence of the type I fusion was higher in Japanese ETs than in European ETs. While we observed EWS–FLI1 type I (EWS exon 7 to FLI1 exon 6) fusions in nine of 10 Japanese cases (90%), Ida et al. [34] had reported a rate of only 50% (five of 10). Genetic variation within the Japanese population may account for the observed differences in frequency of type I fusions.

In previous studies, it was determined that fusion gene type was important in the prognosis of patients with ET: localized disease with type I fusion was reported to be associated with a better clinical outcome than other fusion types [8, 9]. This was explained by the fact that the type I fusion encodes a significantly weaker transactivator than the product of other fusion types [35], and tumors with the type I fusion had a lower immunoreactivity for Ki-67 and IGF-1R than those with other fusion types [36]. Unexpectedly, we observed that Japanese patients with a high number of type I fusion transcripts had a worse prognosis than European Caucasian patients. This finding may challenge the hypothesis that type I fusion transcripts are an indicator for a more favorable outcome in ETs. In a recent follow-up study, reported by Frohlich et al. at the EMSOS (European Musculoskeletal Tumour Society) meeting 2000, held in London, a significant difference in outcome between EWS–FLI1 fusion type I compared with other transcripts was no longer observed: 4-year EFS was 0.5 in both ETs with type I fusion transcripts and those with other types (P = 0.6771) (B. Frohlich, personal communication).

As for the total number of chromosomal aberrations per tumor, there was no significant difference between Japanese and European Caucasian patients. However, significant differences were detected in the incidences of specific aberrations including gain of chromosome 17q and loss of chromosome 19.

In Japanese ETs, loss of 17p was more frequent than in European ETs (not significant). The loss of 17p may affect the tumor suppressor gene TP53 on 17p13.1. The p53 protein functions as transcription factor and apoptosis regulator [37]. It is frequently inactivated in various types of malignant tumors [3840]. The relationship between overexpression of TP53 and markedly poor prognosis of patients with ET was recently reported by de Alava et al. [37]. On the long arm of chromosome 17, the HER2/neu (17q12-q21) oncogene and the BRCA1 (17q21) and NF1 (17q11.2) tumor-suppressor genes are located [41, 42]. HER2/neu functions as growth factor and overexpression of HER2/neu is associated with tumorigenesis and drug resistance [41]. Three different ET cell lines (TC-71, RD-ES and A4573) were reported to express high levels of the HER2/neu protein [41]. However, we did not find any amplification or overexpression of c-erb-B2/HER-2, either in tumors with or without chromosome 17q gains. Therefore, we conclude that c-erb-B2/HER-2 status cannot serve as a prognostic marker in Ewing’s sarcoma and does not explain the different outcomes in Japanese versus European Caucasian ET patients. Furthermore, clinical trials with antibodies that target the erb-B2/HER-2 receptor protein, such as the anti-HER-2 monoclonal antibody rhuMAB HER-2 (Herceptin"; Genentech, San Francisco, CA, USA), are probably not warranted for the treatment of Ewing’s sarcoma. The protein encoded by BRCA1 (17q21) functions as a component of the RNA polymerase II holoenzyme [42]. This gene is related to transcription factors involved in DNA repair, and mutations or deletions in the BRCA1 gene are associated with ovarian and breast cancer, in some cases as an inherited predisposition [43]. In musculoskeletal tumors, loss of heterozygosity of BRCA1 was reported in 21% of osteosarcomas [44]. The protein encoded by NF1 (17q11.2), fibromin, acts as a negative regulator of p21/ras. Abnormalities of this gene are associated with neurofibromatosis type 1, which in turn may predispose to malignant peripheral nerve sheath tumor or neurofibrosarcoma [45]. However, NF1 mutations do not inevitably give rise to cancer, since the majority of neurofibromas are benign. Therefore, the potential role of these genes and their derived proteins needs to be further elucidated in ETs.

STK11/LKB1 is located on chromosome 19p [46]. The STK11/LKB1 gene encodes a serine-threonine kinase which is involved in tumorigenesis in patients with Peutz–Jehgers syndrome [46]. A high frequency of alterations on chromosome 19p has been reported in lung carcinomas [47]. In the current study, loss of either 19p or 19q was significantly more frequent in Japanese tumors than in European Caucasian tumors. Our observations may indicate the existence of an important tumor suppressor gene located on chromosome 19p. Information on aberrations of chromosome 19q in human tumors is limited; however, loss of 19q has been commonly found in gliomas [4850].

We also observed amplification of 1p33-34 in one Japanese ET. Up to now, there have been no reports of amplification of this locus in ETs. The amplified region contains several candidate oncogenes, such as MYCL1 which is involved in the development of small cell lung cancer [51]. This patient (Case 1) had a locally relapsed tumor and has since had a rash clinical course. At present, we have no proof that MYCL1 is actually over expressed in this case. Furthermore, since we observed 1p33-34 amplification in only one sample, its relevance in ET is questionable.

In conclusion, the prognosis of ET is different in patients diagnosed in Japan and Germany. In Japanese ETs, the type I fusion is more frequent and loss of 19p is more common than in European ETs. Genomic instability of chromosome 19 may be related to a poorer prognosis for Japanese patients with ET.


    Acknowledgements
 
This work was supported by grants from the Alexander von Humboldt Foundation and the German Research Foundation DFG (Grant number PO 529/5-1).


    Footnotes
 
+ The first three authors contributed equally to this study. Back

§ Correspondence to: Prof. C. Poremba, Institute of Pathology, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany. Tel: +49-211-81-18339; Fax: +49-211-81-18353; E-mail: poremba{at}med.uni-duesseldorf.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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