—  SYMPOSIUM #21  —

The Role of Ancillary Techniques in the Assessment of Soft Tissue Tumors
Moderators: Dr. John R. Goldblum and Dr. Cyril Fisher

Section 5 - Contribution of Molecular Biology and Markers to the Prognosis and Management of Patients with Soft Tissue Sarcoma

Louis Guillou
University Institute of Pathology
Lausanne, Switzerland


In the last 10 years significant improvements have been made in the molecular approach of soft tissue sarcomas (STS). Roughly, STS can be be separated into two two groups, those bearing specific chromosomal abnormalities (25-30% of cases) such as gene mutations (e.g. GIST) or translocations (e.g. synovial sarcoma, alveolar rhabdomyosarcoma, etc.. see Table 1), and those showing complex karyotypes (leiomyosarcoma, pleomorphic liposarcoma, MPNST, unclassified spindle cell/pleomorphic STS [1, 2]. Because of their specificity and their increasing detectibility in paraffin-embedded tissue using FISH or PCR-based techniques, these chromosomal abnormalities are more and more often used as diagnostic markers of some STS. Since diagnosis, treatment, and outcome are all interdependent variables in the management of STS, logically, improvements in sarcoma subtyping have resulted in improvements in the treatment and prognosis of STS. In addition, with the refinements of molecular techniques, it appeared that some genomic abnormalities correlate with patient outcome in, at least, a small subset of tumors. This presentation will focus on the contribution of molecular biology and markers to the prognosis and management of patients with Ewing sarcoma, alveolar rhabdomyosarcoma, synovial sarcoma, and liposarcoma. Gastrointestinal stromal tumors (GIST), which best illustrate the relationship between molecular biology, response to treatment and outcome, will not be considered here.

Ewing Sarcoma
Mostly occurring in children and adolescents, Ewing sarcoma (ES) is an aggressive neoplasm with a poor outcome. Overall, up to 25-30% of patients have clinically apparent metastatic disease at presentation [3, 4]. Despite aggressive treatment regimens, one third of patients will relapse within five years of diagnosis. It is now well established that Ewing's sarcoma (ES) and primitive peripheral neurectodermal tumors (PNET) belong to the same spectrum of neoplasms, the ES family of tumors , characterized by recurrent balanced reciprocal chromosomal translocations, involving the EWS gene on 22q12 and one of the members of the ETS family of transcription factors, mainly the FLI-1 gene on 11q24 (about 85-90% of cases), and the ERG gene on 21q22 (10-15% of cases) [3, 4]. In less than 1% of cases, other EWS fusion partners are implicated such as ETV1 (on 7p22), E1AF (on 17q22), FEV (2q33) or others (see Table 1) [3, 4]. The presence of one of these specific translocations in a round cell tumor is diagnostic of ES/PNET and is also a prerequisite for the inclusion of patients in ES therapeutic protocols (e.g. EuroEwing). Secondary chromosomal aberrations (e.g. gains of chromosome arm 1q, gains of chromosomes 8 and 12, p16INK4 mutations and deletions, and p53 mutations) have also been described, especially in patients with advanced disease [4, 5, 6, 7]. For the FLI1-EWS translocation, breakpoints tend to cluster in given regions and some fusion gene transcripts are more frequent than others, e.g. the type I EWS-FLI1 fusion in which the exon 7 of EWS fuses with exon 6 of FLI1 (65% of cases), and the type II EWS-FLI1 fusion in which the exon 7 of EWS is fused with exon 5 of FLI1 [4, 5, 6, 7]. Zoubeck et al [8] showed in univariate analyses that for patients with localized disease, type I fusion gene was associated with longer relapse-free survival compared with other type of fusion genes. In a series of 112 patients, De Alava et al. [9] observed a positive relationship between type I EWS-Fli1 fusion and overall survival suggesting that EWS-FLI1 transcript structure is an independent determinant of prognosis in Ewing's sarcoma, potentially linked to a lower transactivation potential of the EWS-FLI1 type 1 fusion oncoprotein. Similar results were more recently obtained by Avigad S et al. [10]. De Alava et al. also observed an asociation betwen EWS-FLI1 type 1 fusion and lower proliferative rate [11]. By contrast, Ginsberg et al. [12] failed to found any prognostic value of fusion gene in ES in univariate analyses. It is important to note that all these results were based on retrospective studies which are a well-known source of biases (especially regarding treatment schemes variability), a possible explanation for those discrepancies [13].

Several studies showed that circulating tumor cells could be detected by molecular techniques in peripheral blood and/or bone marrow samples in patients with ES. The persistence of these cells after treatment or their reappearing after a period of latency is synonymous with shortened relapse-free intervals as compared with tumor-free samples [10, 14, 15, 16, 17, 18, 19].

Secondary molecular alterations in genes regulating cellular growth and differentiation occur frequently in ES, especially in those cases associated with EWS-FLI1 transcripts other than type 1 [8, 9]. Among them, alterations (deletions, mutations) of the p16INK4 , p14ARF, p27KIP1 and p53 tumor suppressors genes are of great importance, correlating frequently with aggressive behavior and poor chemoresponse [20]. hTERT upregulation by EWS-ETS fusion proteins also contribute to the developement of ES [21] and, indeed, telomerase activity was found to be a strong negative prognosticator in ES [22]. Dysregulation of many other pathways including that of cyclin D1, PDGF-C, c-Myc, TGF- b receptor II, insulin-like growth factor-1 receptors, FHIT, and VEGF have also been associated with the development of ES [23, 24]. Gene expression profiling technology has provided us with new insights into the molecular biology of ES, leading to the identification of high-risk and low-risk patient groups [24], and has brought some arguments in favor of targeted therapeutic strategies such as antiangiogenic agents or antityrosine kinase receptors (anti-IGF-1 receptor). The recent discovery that ES could originate from bone marrow derived mesenchymal progenitor cells is another major and promising step in the understanding of ES genesis [25].

Alveolar Rhabdomyosarcoma
Recent prognostic classifications of rhabdomyosarcoma (RMS) of infancy and childhood showed that spindle cell and botryoid RMS subtypes have the best prognosis, alveolar RMS (20-25% of RMS cases), undifferentiated RMS and RMS with extensive/diffuse anaplasia the worst, and that the prognosis of conventional embryonal RMS is intermediate between the two former categories [26]. Because of the propensity of alveolar RMS for local recurrence, early metastatic dissemination and resistance to treatment, patient with this neoplasm are usually enrolled in intensive (chemo)therapeutic protocols. Thus, identifying an alveolar RMS component is of paramount importance as it directly influences treatment and outcome. As opposed to embryonal RMS which is characterized by a loss of heterozygosity on the short arm of chromosome 11 (11p15.5), most alveolar rhabdomyosarcomas bear either the t(2;13) (q35;q14) (70-80% of cases) or the t(1;13) (p36;q14) (10%) translocation (see table1). From a diagnostic point of view, detection of PAX3-FKHR and PAX7-FKHR fusion transcripts (resulting from the t(2;13) and t(1;13) translocations, respectively), in a small round cell tumor displaying adequate histopathologic and immunohistochemical features, is synonymous with alveolar RMS. Molecular testing is particulartly important either for the recognition of the solid variant of alveolar RMS or for reliably identifying an alveolar component in an otherwise embryonal-looking tumor. RT-PCR-based techniques are highly sensitive for the detection of tumor-specific fusion transcripts allowing for the detection of a few tumor cells admixed with many nontumor cells. This method is, thus, particularly suitable for assessing the presence of few residual tumor cells in bone marrow or peripheral blood samples before and after chemotherapeutic protocols [27, 28]. The PAX3-FKHR fusion is often associated with the classical alveolar growth pattern and the presence of multinucleate giant cells, whereas tumors with PAX7-FKHR fusion tend to show lower apoptotic/mitotic activity [9]. From a prognostic point of view, tumors with PAX7-FKHR fusion tend to behave less aggressively than PAX3-FKHR. In a series of 34 cases, Kelly et al. [30] first noticed better outcomes among the PAX7-FKHR group by univariate analysis. A subsequent retrospective anaylsis [31] confirmed these results, showing that, in the context of metastatic disease, patients with t(1;13)-positive alveolar RMS do significantly better in terms of survival than those with the t(2;13) translocation (4-year survival: 75% versus 8%). Bone marrow involvement was also significantly more frequent in PAX3-FKHR-positive patients [31]. Since the number of cases (n=19) examined was very small, these results need to be confirmed in well-controlled prospective studies incorporating a larger number of patients.

Early detection of RMS cells in bone marrow or peripheral blood samples has been shown to be prognostically relevant. In 1997, Kelly et al. [27] showed that, using RT-PCR, tumor cells from alveolar rhabdomyosarcoma were detectable in bone marrow (but not in peripheral blood samples) from patients for whom there was no histologic evidence of disease by conventional light microscopic examination. This capacity of RT-PCR to detect ocult metastatic disease was subsequently confirmed by others [32], which supports a positive role for RT-PCR in staging and follow-up procedures. As for Ewing sarcoma, the presence of alveolar rhabdomyosarcoma tumor cells in bone marrow, as detected by RT-PCR alone, seems to be predictive of shortened disease-free survival and/or overall survival [27, 32], suggesting that these patients should be treated with more intensive therapy. RT-PCR can also be used as a method to assess the efficacy (chemosensitivity) of chemotherapeutic protocols, based on the monitoring of tumor cell clearance in bone marrow and/or peripheral blood samples after drug administration [28].

Beside the potential prognostic value of fusion transcripts in terms of survival, molecular studies also confirmed that myogenin is a reliable immunohistochemical marker of alveolar RMS and that this marker could be used in routine practice in making the distinction between alveolar and embryonal RMS [33]. In the series of Hostein et al. [33], 72% of those neoplasms containing >50% myogenin-positive cells were PAX fusion positive, including 89% of alveolar rhabdomyosarcomas. Interestingly, 11% of these tumors had been classified as embryonal rhabdomyosarcomas prior to molecular examination, underlining the fact that any tumor displaying >50% myogenin-positive cells should be tested for PAX fusion transcripts before being (mis)diagnosed as an embryonal RMS. By contrast, all tumors containing less than 50% myogenin-positive cells were PAX fusion negative. Recently, gene expression profiling analyses identified a list of genes that might be of value in discriminating between RMS of favorable and unfavorable outcome (e.g. essentially between ARMS and non ARMS), including AP2 b , P-cadherin, EGFR, and fibrillin-2 [34]. AP2 b and P-cadherin were expressed essentially in alveolar RMS (specificity: 98%; sensitivity: 64%) whereas EGFR and fibrillin-2 were detected in embryonal rhabdomyosarcoma with a specidicity of 90% and a sensitivity of 60% [35].

Amplification and/or overexpression of N-MYC was associated with adverse outcome in alveolar rhabdomyosarcoma [36]. The development of molecular-based targeted therapies (e.g. epidermal growth factor tyrosine kinase inhibitors, rapamycin analogues, a vaccine directed against small peptide fragments spanning the PAX3-FKHR fusion, etc..) in the treatment of rhabdomyosarcoma is just at the beginning [reviewed in ref. 37].

Synovial Sarcoma
Synovial sarcoma (SS) accounts for 10 to 15% of STS and can be confused with numerous other STS types including MPNST, Ewing sarcoma, and fibrosarcoma. As opposed to many other sarcoma subtypes, SS is (relatively) chemosensitive and, thus, accurate identification is of relevance for patients. SS bears the t(X;18) translocation which involves the SYT gene on chromosome 18 (18q11) and either the SSX1 or the SSX2 genes on chromosome X (p11.23), only rarely SSX4 [38]. SSX1 and SSX2 are involved in 95% of SS, regardless of morphology. The t(X;18) translocation is specific for SS and is not observed outside this tumor type. Besides the diagnostic usefulness of the detection of the t(X;18) translocation, it has been suggested that fusion type could be an important prognostic factor in SS patients [39, 40, 41]. In 1998, a preliminary study [39] suggested that tumors harboring the SYT-SSX1 fusion were more aggressive and had a higher propensity for metastatic dissemination than SYT-SSX2 neoplasms. These results were confirmed by subsequent studies [40] , including a large multi-institutional study of 243 patients [41]. In the latter study, fusion type emerged as the most important prognostic factor for overall survival, by multivariate analysis, in patients with localized disease at diagnosis. A similar work performed by the French Sarcoma Group failed to confirm these results [42], showing that histologic grade, not fusion type, was the best predictor of outcome. Even worse, In the study of the French sarcoma Group, SSX2 tumors had a worse outcome compared to SSX1 tumors, a finding also observed by Nakagawa et al. [43] in a smaller series. In both the Ladanyi et al., and the FNCLCC studies, biphasic SS were rarely associated with SYT-SSX2 transcripts, suggesting a close relationship between morphology and genetics. The prognostic value of chromosomal instabilities in SS has also been examined. A recent study showed that tumors harbouring an increased number of genetic aberrations (≥3) had a worse clinical course, and that gains of SAS and loss of CCND1 genes were negative prognostic indicators [43]. Studies of gene expression in SS showed that many SS overexpress EGFR, C-Erb2 (especially in the epithelial component of biphasic SS), IGF2, insulin-like growth factor receptor-1 and insulin-like growth factor binding proteins [44, 45]. As these proteins seem to play a significant role in SS growth and maintenance, inhibition of these signalling pathways may represent a promising therapeutic approach. High Ki-67 proliferative index and p53 overexpression also correlated with an increased risk of tumor recurrence [38, 46].

Liposarcoma


Myxoid Liposarcoma
Most myxoid liposarcomas bear the translocation t(12;16) (>95% of cases), involving the FUS/TLS gene on 16p11 and the CHOP gene on 12q13, or the t(12;22) translocation (see Table 1) [47]. As in many translocation-positive sarcomas, the genomic breakpoints of the t(12;16) are widely dispersed in specific introns of the TLS and CHOP genes and vary from one tumor to another. In a recent study, Antonescu et al. [48] showed that the molecular variability of chimeric fusion transcripts in myxoid LPS had no significant impact on disease-free survival and histologic grade. Overexpression of P53 and reduced expression of p14ARF and p16INK4, as assessed by immunohistochemistry are observed more frequently in the round cell component and correlate with poor prognosis [48, 49]. Myxoid liposarcoma can easily be confused with a well-differentiated LPS showing extensive myxoid changes, especially in the retroperitoneum. In this situation, the detection of fusion transcripts characteristic of the t(12;16) or t(12;22) translocations is of paramount importance. Along the same lines, it has been shown that multifocal myxoid liposarcomas do not exist but, rather, represent metastatic deposits from a single, monoclonal lesion [50].

Well-Differentiated and Dedifferentiated Liposarcoma
In the last 10 years, significant achievements have been made in the diagnostic approach and molecular biology of the well-differentiated/dedifferentiated liposarcoma category. Well-differentiated liposarcomas can easily be confused with benign lesions (lipoma with myxoid changes, spindle cell/pleomorphic lipoma, lipoblastoma, angiomyolipoma, etc.) whereas dedifferentiated liposarcomas are often confused with spindle cell/pleomorphic sarcomas such as so-called MFH, fibrosarcoma, MPNST, leiomyosarcoma, myxofibrosarcoma and even myxoid liposarcoma, if myxoid changes predominate. The distinction between dedifferentiated liposarcoma and other sarcoma subtypes is of import, since the clinical course of the former is much more indolent, with a high risk of local recurrence but low risk for metastatic dissemination (15-20%). Karyotypically, well-diff/dediff LPS harbour characteristically supernumerary ring and/or giant chromosomes composed of 12q13-15 amplicons Several genes situated in this region can be amplified including HMG-A2 (HMGI-C), MDM2 (murine double minute-2), CDK4 (cyclin dependent kinase 4), SAS (sarcoma amplified sequence), GLI, CHOP, etc... [reviewed in ref 47 and 51]. In contrast to well-differentiated LPS, dedifferentiated LPS show additional chromosomal abnormalities especially in the 1p32, 12q24 et 6q23 regions which might correlate with loss of lipogenic differentiation and increased aggressiveness [52]. Mdm2 and cdk4 amplifications/overexpression, which can be detected using FISH, quantitative PCR or immunohistochemistry are seldom observed in other sarcoma types [reviewed in ref 51], and, thus, can be used as a diagnostic tool to identify the well-diff/dediff liposarcoma category. As a matter of fact, it appeared that many spindle cell and pleomorphic sarcomas of the retroperitoneum and of the inguinal/paratesticular region called "MFH" were actually unrecognized dedifferentiated liposarcomas [53, 54]. Areas of heterologous differentiation in dedifferentiated liposarcomas are consistently positive for mdm2 and/or cdk4. Crucially, whereas pure myogenic sarcomas (leiomyosarcoma, rhabdomyosarcoma) display aggressive clinical behaviour, dedifferentiated liposarcomas showing myogenic (i.e. smooth muscle or skeletal muscle) heterologous differentiation have the same indolent clinical course as conventional dedifferentiated liposarcomas.

Sarcomas with Myogenic Differentiation
In a recent re-evaluation of 100 so-called MFH, Fletcher et al. [55] observed that pleomorphic sarcomas which showed myogenic differentiation (i.e leiomyosarcoma, rhabdomyosarcoma and myogenic sarcoma NOS) were associated with a more aggressive behavior, higher metastatic rate and shorter time to metastasis than those lacking myogenic differentiation. Although the minimum quantity of cells positive for myogenic markers and the intensity of staining required for a tumor to be classified as "myogenic" were not clearly stated in their paper, it was the first time that myogenic differentiation, as a whole, was shown to be an adverse prognostic factor. This was confirmed subsequently by other studies [56, 57]. Deyrup et al. [56] showed that this negative effect was maintained even after adjusting for tumor grade, tumor size, tumor extent, and patient age, and that increasing myoid differentiatioin correlated with worse survival (additive effect of myoid differentiation). Thus, patients with pleomorphic sarcomas that express myoid antigens might benefit from the development of better adjuvant therapies.

Conclusion
Many sarcomas bear histotype-specific chromosomal abnormalities, including reciprocal translocations. These abnormalities are currently used as diagnostic markers but can also be of value in assessing the prognosis of a given neoplasm, in detecting residual disease after treatment, or for early detection of infraclinical relapse. In terms of prognosis, Ewing sarcoma, alveolar rhabdomyosarcoma, and GIST have benefited most from molecular advances, whereas the prognostic value of fusion transcripts in synovial sarcoma is still under discussion. New techniques such as gene expression profiling will provide us with new insights in the pathogenesis, maintenance, and outcome of soft tissue sarcomas, allowing the development of more specific therapies.

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Table 1 : Main chromosomal abnormalities that can be used for the diagnosis and/or prognosis of soft tissue sarcomas

Sarcoma type Chromosomal Abboldities Genes Involved Prevalence
Ewing sarcoma/PNET t(11;22) (q24;q12)
t(21;22) (q22;q12)
t(7;22) (p22;q12)
t(17;22) (q12;q12)
t(2;22) (q33;q12)
t(1;22) (p36;q12)
t(16;21) (p11;q22) 		EWS-FLI1
EWS-ERG
EWS-ETV1
EWS-ETV4 (E1AF)
EWS-FEV
EWS-ZSG
FUS-ERG 		85-95%
5-10%
rare(<1%)
rare
rare
rare
rare
Synovial sarcoma t(X;18) (p11;q11) SYT(SS18)-SSX1
SYT(SS18)-SXX2
SYT(SS18)-SSX4 		65%
35%
rare
Myxoid liposarcoma t(12;16) (q13;p11)
t(12;22) (q13;q12) 		FUS(TLS)-CHOP(DDIT3)
EWS-CHOP(DDIT3) 		95%
rare
Alveolar rhabdomyosarcoma t(2;13) (q35;q14)
t(1;13) (p36;q14)
t(X;2) (q13;q35)
t(2;2) (q35;p23) 		PAX3-FKHR(FOXO1A)
PAX7-FKHR(FOXO1A)
PAX3-AFX
PAX3;NCOA1 		60-80%
10-20%
rare
rare
Clear cell sarcoma t(12;22) (q13;q12)
t(2;22) (q32;q12) 		ATF1-EWS
EWS-CREB1 		>90%
rare
Extraskeletal myxoid chondrosarcoma t(9;22) (q22;q12)
t(9 ;17) (q22;q11)
t(9 ;15) (q22;q21) 		EWS -TEC(NR4A3/CHN/TEC)
TAF2N(RBP56)-TEC/CHN
TCF12-TEC(CHN) 		75%
25%
rare
Desmoplastic small round cell tumor t(11;22) (p13;q12) WT1-EWS >90%
Low-grade fibromyxoid sarcoma t(7;16) (q32-34;p11)
t(11;16) (p11;p11) 		FUS-CREB3L2
FUS-CREB3L1 		90%
10%
Dermatofibrosarcoma protuberans /giant cell fibroblastoma t(17 ;22) (q22;q13)
ring 17q, ring 22q, der(22) 		COL1A1-PDGFB
COL1A1-PDGFB 		>90%
Alveolar soft part sarcoma t(X ;17) (p11.2;q25) ASPL-TFE3 >90%
Infantile fibrosarcoma (cell. mesoblastic nephroma) t(12 ;15) (p13;q25) ETV6(TEL)-NTRK3(TRKC) 80-90%
Well-differentiated / dediff. liposarcoma giant chromosomes / ring chromosomes (12q14-15) MDM2, CDK4 amplified 80%
Inflammatory myofibroblastic tumor t(2;19) (p23;p13.1)
t(1;2) (q22-23;p23)
t(2;17) (p23;q23)
t(2;11) (p23;p15.5)
t(2;2) (p23;q13)
other 2p23 rearrangements 		TPM4- ALK
TPM3-ALK
CLTC-ALK
CARS-ALK
RANBP2-ALK
ALK-other partners 		nbsp;
Rhabdoid tumor -22q11.2 INI1 (hSNF5/SMARCB1) loss 70%