The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal

ARTICLE IN PRESS Current Diagnostic Pathology (2005) 11, 361–370

www.elsevier.com/locate/cdip

MINI SYMPOSIUM: BONE AND SOFT TISSUE PATHOLOGY

The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal Hejin P. Hahna,b, Christopher D.M. Fletchera,b, a

Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA Harvard Medical School, Boston, MA, USA

b

KEYWORDS Sarcoma; Soft tissue; Cytogenetics; Genetics; Molecular; Diagnosis

Summary Molecular genetic and cytogenetic analysis has revealed that many soft tissue tumours, both benign and malignant, carry simple, reproducible karyotypic aberrations that are tumour-specific. Many of these mutations are chromosomal translocations and the resulting fusion gene products have been cloned. Classification, diagnosis and prognostication of soft tissue tumours has already been influenced by these genetic findings. Furthermore, examination of fusion gene products has helped increase our understanding of the molecular pathogenesis of soft tissue tumours and will hopefully aid in the development of new therapeutic agents. However, in current ‘routine’ practice, it remains to be defined when molecular genetic and cytogenetic techniques should be used, which technique should be used and how testing should be performed. Herein we briefly review the impact that cytogenetic and molecular genetic analysis has had on soft tissue tumour pathology and discuss both the benefits and limitations of these techniques in the current practice of diagnostic pathology. & 2005 Elsevier Ltd. All rights reserved.

Introduction Understanding of soft tissue tumour biology has been advanced significantly by molecular genetic and cytogenetic studies. Similar to many haematological malignancies and in contrast to most epithelial neoplasms, a substantial subset of soft tissue tumours contain simple, specific and reproCorresponding author. Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel.: +1 617 732 8588; fax: +1 617 566 3897. E-mail address: [email protected] (C.D.M. Fletcher).

ducible karyotypic aberrations. This knowledge has been beneficial not only in providing insight into soft tissue tumour pathogenesis, but also in assisting soft tissue tumour diagnosis, classification, prognostication and therapeutic development. However, this area of research is a work in progress. There are numerous limitations still to be overcome, ranging from the rarity of soft tissue tumours in general to the technological and financial constraints preventing widespread availability of molecular genetic testing. How this knowledge should be utilized in current ‘routine’ practice remains to be defined.

0968-6053/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cdip.2005.08.001

ARTICLE IN PRESS 362

H.P. Hahn, C.D.M. Fletcher

Genetic abnormalities in soft tissue tumours Molecular genetic analysis has revealed that soft tissue sarcomas appear to fall into two main biological groups: (1) tumours with simple and specific chromosomal alterations and (2) tumours with complex unbalanced karyotypes that are not tumour-specific.1 Some of the soft tissue tumours containing simple and specific karyotypic alterations are listed in Table 1. Akin to leukaemias, the majority of these tumours contain reciprocal chromosomal translocations. The simplicity and specificity of these chromosomal translocations has allowed researchers to clone most of the genes involved and identify their fusion gene products (Table 1).2,3 The epidemiology of this subset of sarcomas is also distinct, since they tend to occur mainly in children or younger adults. In contrast, the second group are sarcomas with complex unbalanced karyotypes that do not appear to be tumour-specific (Table 2). These tumours are more often the high-grade spindle cell and pleo-

Table 1

morphic sarcomas of later adulthood.4,5 In addition, this subset of sarcomas is characterized by the almost invariable presence of alterations in the p53 pathway, a feature that is much less common in the tumours listed in Table 1.

Molecular genetics as a diagnostic tool Molecular genetic and cytogenetic testing have become increasingly important diagnostic tools. There are a number of different diagnostic situations where identification of a specific translocation can be an important adjunctive study to immunohistochemical stains. First, it can help confirm the diagnosis of tumours with a variety of morphological/histological appearances and assist in differentiating between tumours that are morphologically similar. For example, distinguishing between Ewing’s sarcoma/malignant peripheral primitive neuroectodermal tumour (MPNET) and poorly differentiated synovial sarcoma (see Fig. 1), or between clear cell sarcoma and metastatic melanoma can sometimes be difficult without

Sarcomas with specific karyotypic aberrations.

Tumour

Translocation

Fusion gene

Alveolar rhabdomyosarcoma

t(2;13)(q35;q14) t(1;13)(p36;q14)

PAX3-FKHR PAX7-FKHR

Alveolar soft-part sarcoma

t(X;17)(p11.2;q25)

ASPL-TFE3

Clear cell sarcoma

t(12;22)(q13;q12)

ATF1-EWS

Dermatofibrosarcoma protuberans (giant-cell fibroblastoma)

t(17;22)(q22;13)

COL1A1-PDGFB

Desmoplastic small round cell tumour

t(11;22)(p13;q12)

WT1-EWS

Ewing sarcoma and MPNET

t(21;22)(q22;q12) t(7;22)(p22;q12) t(17;22)(q12;q12) t(2;22)(q33;q12) t(11;22)(q24;q12)

EWS-ERG EWS-ETV1 EWS-E1AF FEV-EWS FLI1-EWS

Extraskeletal myxoid chondrosarcoma

t(9;22)(q22;q12)

TEC-EWS

Inflammatory myofibroblastic tumour

t(2;19)(p23;p13.1) t(1;2)(q22-23;p23)

ALK-TPM4 TPM3-ALK

Infantile fibrosarcoma

t(12;15)(p13;q25)

ETV6-NTRK3

Low-grade fibromyxoid sarcoma

t(7;16)(q33;p11)

FUS-CREB3L2

Myxoid/round cell liposarcoma

t(12;16)(q13;p11) t(12;22)(q13;q12)

TLS(FUS)-CHOP EWS-CHOP

Synovial sarcoma

t(X;18)(p11;q11)

SYT-SSX1 SYT-SSX2

 The primary references for most translocations are provided in Ladanyi and Bridge.2

ARTICLE IN PRESS The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal 363 identification of the Ewing’s/MPNET t(11;22), the synovial sarcoma-specific t(X;18) translocation or the t(12;22) translocation of clear cell sarcoma. Secondly, these techniques can also be useful in making diagnoses on small core biopsy specimens where the amount of tumour tissue is limited and when positivity of relevant immunohistochemical stains may be limited or focal in extent. Thirdly, molecular genetic analysis can assist in the classification of lesions in which morphology may have been altered by neoadjuvant therapy prior to excision. Molecular and cytogenetic diagnostics has also expanded our knowledge of the epidemiological features of soft tissue tumours. In addition to proving that Ewing’s sarcoma and MPNET are part of a single disease spectrum (see below), the demonstration of the EWS gene rearrangement confirmed that Ewing’s/MPNET could arise in previously unrecognized and unexpected sites (e.g. kidney,

Table 2 Sarcomas with non-specific cytogenetic alterations. High grade pleomorphic sarcomas in adults Leiomyosarcoma Malignant peripheral nerve sheath tumour Angiosarcoma Osteosarcoma Listed in order of frequency are sarcomas with complex karyotypes and non-specific cytogenetic alterations.

skin) and in older age groups.2,6 Similarly, molecular techniques have also supported the diagnosis of desmoplastic small round cell tumour (DSRCT) and synovial sarcoma in novel primary sites.2,7,8 Although most of the translocations listed in Table 1 appear to be tumour-specific, there have been published reports in which the detected fusion gene transcript did not match the morphological appearance of the lesion. For example, EWS-FLI1 transcripts were found in two polyphenotypic tumours and in two rhabdomyosarcomas.9 The significance of such exceptional cases remains poorly understood.

Clarification of tumour classification Classification of soft tissue tumours has also been impacted significantly by molecular and cytogenetic analysis. The prototypical example of this effect is the reclassification of a group of neoplastic entities as Ewing’s/MPNET after common genetic aberrations were found. Ewing’s sarcoma, malignant peripheral neuroectodermal tumour, malignant small-cell tumour of the thoracopulmonary region (Askin tumour) and peripheral neuroepithelioma, were reclassified as a single entity with a spectrum of neuroectodermal differentiation.10 Approximately 85% of these tumours have a detectable t(11;22)(q24;q12) translocation involving the EWS gene on chromosome 22 and the FLI-1

Figure 1 FISH can aid in the discrimination between morphologically similar tumours: (A) on histological examination, poorly differentiated synovial sarcoma (left) and Ewing’s sarcoma (right) are both ‘small round blue cell tumours’ and may not be easily distinguishable; (B) FISH analysis using ‘split-apart’ probes targeting either the centromeric (c) or telomeric (t) portions of a specific gene, indicate the presence of a translocation by the physical separation of one pair of the c and t probes. When FISH was performed on these two cases, identification of a SYT 18q11.2 translocation (left) and an EWSR 1.22q12 translocation (right) confirmed the diagnoses of poorly differentiated synovial sarcoma and Ewing’s sarcoma, respectively. (courtesy of Dr. Paola Dal Cin.).

ARTICLE IN PRESS 364

H.P. Hahn, C.D.M. Fletcher breakpoint region

FLI1 (11q24)

1 2

EWS-FLI1 der(22) Type 1

3

5

6

78

9

breakpoint region

EWS (22q12)

1 23 4

4

56

78

9 1011

12 13 1415 1617

1 23 4

56

7

6

78

9

EWS-FLI1 der(22) Type 2

1 23 4

56

7

5

6

78

9

Figure 2 EWS-FLI1 chimeric transcripts. Eighty-five percent of Ewing’s sarcomas contain translocations involving the EWS gene (red) and FLI-1 gene (blue). However, breakpoints in both genes are spread among several different exons (indicated in grey), producing a variety of chimeric transcripts. Thus far, 14 different types of EWS-FLI1 chimeric transcripts have been described. The most commonly identified are the type 1 and type 2 translocations. Type 1 translocations, found in 60% of Ewing sarcomas, result from the fusion of exon 7 of EWS to exon 6 of FLI1. Type 2 translocations are the next most common (25%) and result from the fusion of exon 7 of EWS to exon 5 of FLI1.

gene on chromosome 11 (see Fig. 2).2,11 Many variant translocations have also been identified (Table 1), resulting in the fusion of the EWS gene to other erythroblastosis virus-transforming sequence (ETS)-related genes, including ERG, ETV1, E1AF and FEV. Classification of lipomatous neoplasms, has also been clarified by these techniques.12 The presence of a ring and/or giant marker chromosome composed of material from the long arm of chromosome 12 in both well-differentiated liposarcomas and atypical lipomas helped to prove that they represent the same tumour type (see Fig. 3)13 for which the alternative term ‘atypical lipomatous tumour’ is now generally used, at least in surgically amenable locations. Furthermore, the association of dedifferentiated liposarcomas with well-differentiated liposarcomas has been confirmed by the finding that they share several karyotypic alterations. Identification of an identical t(12;22)(q13;q11-12) translocation in both myxoid and round cell liposarcoma demonstrated that they represent a single entity with a continuum of morphological appearances and a range of cellularity.14 These karyotypic findings are also a substantial diagnostic aid when faced with a differential diagnosis of lipoma, spindle cell/ pleomorphic lipoma, hibernoma and atypical lipoma in adipocytic neoplasms or when considering a differential diagnosis of myxofibrosarcoma, myxoid chondrosarcoma, or aggressive angiomyxoma in myxoid lesions. Many proposed associations between apparently different types of soft tissue tumours have also been confirmed by cytogenetic studies. After careful examination of the clinical and morphological features of dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF), it was originally suggested that GCF represents a juvenile

Figure 3 Karyotypes of well differentiated liposarcoma/ atypical lipomatous tumours. Shown are the metaphase spreads of two well differentiated liposarcoma/atypical lipomatous tumours. These tumours commonly demonstrate either a ring chromosome (A, arrow) or giant marker chromosome (B, arrow) composed of material from chromosome 12 (courtesy of Dr. Paola Dal Cin.).

form of DFSP.15 Supporting this relationship was the finding that both tumours contained ring chromosomes characterized by a complex alternating translocation t(17;22)(q22;q13).16 Likewise, an association between infantile fibrosarcoma and congenital mesoblastic nephroma was hypothesized based on their morphological and epidemiological features. The discovery that they had a common t(12;15)(p13;q25) translocation and CTV6-NTRK3 fusion gene product17,18 demonstrated that these tumours are a single entity, occurring either in the kidney or in soft tissue. Surprisingly, cloning of chromosomal translocations has also revealed connections between tumours of very different cellular lineages. Identical translocations involving the anaplastic lymphoma kinase (ALK) gene have been identified in both inflammatory myofibroblastic tumour (IMT)19 and anaplastic large cell lymphoma (ALCL).20 Five translocations have been identified in both tumours

ARTICLE IN PRESS The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal 365 thus far, resulting in the fusion genes TPM4-ALK, TPM3-ALK, CLTC-ALK, CARS-ALK and RANBPS-ALK with a constitutively activated ALK. Thus, although these mutational events occurred in cells of different lineages, the pathogenesis of both IMT and ALCL may occur through a common oncogenetic mechanism.

Molecular prognostication Obviously, accurate diagnosis of soft tissue tumours, by whatever means, often allows better prediction of patient prognosis. Similarly, using molecular genetic testing, detection of t(2;13) (q35;q14) or t(1;13)(p36;q14) can assist in discriminating between embryonal rhabdomyosarcoma and alveolar rhabdomyosarcoma.21 The distinction between these two lesions is important, since alveolar rhabdomyosarcoma has a more aggressive course than embryonal rhabdomyosarcoma and thus confers a worse prognosis. Proper classification of lesions also allows a better estimation of response to treatment regimens. Because synovial sarcoma is more chemosensitive than other fascicular spindle cell sarcomas,22 accurate diagnosis assists in predicting treatment response. Similarly, in gastrointestinal stromal tumours (GISTs), c-kit mutational analysis correlates well with response to treatment with imatinib mesylate.23 There is increasing interest in determining whether or not the type of fusion gene present in a single tumour entity has prognostic significance. In Ewing’s/primitive neuroectodermal tumour (PNET), fusion of EWS to FLI1 can occur in a variety of ways. Although the breakpoints in EWS are fairly closely clustered, the FLI1 breakpoints are spread among several different exons, resulting in as many as 14 different chimeric transcripts (Fig. 2). Furthermore, in addition to the FLI1 gene partners, a number of variant translocations have also been described, resulting in the fusion of EWS to other members of the ETS family of transcription factors (Table 1). Reports regarding the association of fusion gene type and patient prognosis have been conflicting. Initial studies found that Ewing’s/PNET containing the EWS-FLI1 type I fusion gene (fusion of EWS exon 7 to FLI1 exon 6) was associated with better patient prognosis, when compared to other fusion gene types.24,25 However, a later study did not find an association between type of fusion gene and event-free and overall survival.26 Analogous studies have also examined the prognostic significance of fusion gene type in synovial sarcoma. As described above, in synovial sarcoma, the SYT gene

on chromosome 18 can be fused with either the SSX1 or SSX2 gene on chromosome X.2 Unfortunately, although early studies described an association between the SYT-SSX1 fusion gene and a poorer prognosis,27–29 a later large study found no such association.30 In preliminary studies examining alveolar rhabdomyosarcomas, which can carry either the PAX3-FKHR or PAX7-FKHR fusion genes, the presence of the PAX-7-FKHR fusion gene was associated with a better prognosis.31,32 By contrast, initial studies indicate that the type of fusion gene present in myxoid and round cell liposarcoma has no relationship with clinical outcome.33 Thus, molecular genetic and cytogenetic techniques are not ready for clinical use as an independent prognostic indicator. The reasons for the discrepant findings described above are probably multifactorial (for a review, see Oliveira and Fletcher34) and underscore the critical importance of study design in generating results that reflect biological truth and compare cost-effectiveness to variables currently used as prognostic factors. Therefore, in current practice, once a diagnosis has been established, the identification of specific fusion gene type is not usually warranted, as it has an unclear association with patient prognosis and has no further affect on treatment planning. However, this situation may evolve in the coming years. In addition to experiments examining the association of fusion gene type with prognosis, the utility of molecular genetic techniques in the detection of clinically occult residual disease or ‘micrometastases’ is also being evaluated. In Ewing’s/PNET, initial studies demonstrated that reverse transcriptase-polymerase chain reaction (RT-PCR) could detect EWS–FLI1 transcripts in the bone marrow or peripheral blood of clinically ‘disease-free’ patients.35 As a prognostic tool, the presence of EWSFLI1 transcripts in the bone marrow at the time of diagnosis has not been linked clearly to patient outcome.36,37 However, prospective studies using RT-PCR as a screening tool for disease progression have yielded encouraging results. Presence of the EWS-FLI1 transcript during and following treatment was associated with disease progression and often preceded clinical recurrence,38,39 but additional studies are needed before this can be utilized as a routine clinical assay.

Sarcoma pathogenesis Although most of the relevant fusion genes have been cloned, it has been difficult to determine the precise role of these chimeric proteins in tumour

ARTICLE IN PRESS 366 pathogenesis or their downstream signalling pathways. At least in part, this reflects our usual lack of understanding as to the cell type in which these gene fusions should be modelled. However, occasional insight has been yielded during examination of the individual genes involved in a translocation. For example, the translocation that occurs in DFSP results in the joining of platelet-derived growth factor-beta (PDGF-beta), a mitogen in many cell types, with type I collagen, a component of connective tissue.40 However, in most sarcomas, little is known about the function of genes involved in tumour translocations. Most commonly, chromosomal translocations in sarcomas appear to induce transcription factor activation and aberrant triggering of unknown downstream pathways. With the exception of transgenic mouse models of myxoid liposarcoma41 and alveolar rhabdomyosarcoma,42 attempts to model the molecular mechanisms of sarcomagenesis have been relatively unsuccessful. In most cases, transgenic expression of fusion genes is lethal. Furthermore, the cell type needed to express the fusion gene, the effects of the cellular environment and the effects of developmental stage are still not known. However, molecular genetic and cytogenetic techniques are clarifying the differences between the pathogenesis of benign versus malignant mesenchymal neoplasms. Two genes often involved in translocations in benign mesenchymal neoplasms are the high-mobility group (HMGA) genes HMGA2, located at 12q14-15, and HMGA1, located at 6q21. Surprisingly, chromosomal rearrangements involving these genes are more commonly identified in a wide range of benign neoplasms.43,44 In general, the function of HMGA proteins is to regulate chromatin architecture and regulate gene transcription. In benign lesions, there is increased transcription of these genes, but there are no structural alterations. Benign nerve sheath tumours, despite their differing biology, also share a distinct chromosomal aberration in many cases. Monosomy 22 is shared among some examples of schwannoma, neurofibroma and perineurioma, resulting in inactivation of one of the NF-2 alleles.45 Thus, pathogenetic mechanisms may be shared by a variety of benign mesenchymal tumours and these pathways may be quite distinct from those involved in the development of malignant mesenchymal neoplasms.

Development of new treatments Finally, it is hoped that molecular genetic analysis will assist in the development of new therapies that

H.P. Hahn, C.D.M. Fletcher are tailored to specific genetic alterations. This is best exemplified in the treatment of GISTs. Mutation of the KIT protein, resulting in constitutive activation, is known to have an important role in the pathogenesis of GISTs.46,47 However, the c-kit mutations in these tumours are heterogeneous, involving several different exons. Furthermore, GISTs lacking detectable c-kit mutations quite often contain mutations in kinase platelet-derived growth factor receptor alpha (PDGFRA).48 Interestingly, mutation type was found to correlate with treatment response. Although blocking the KIT enzymatic domain with imatinib mesylate induced a good clinical response in many patients with GIST,49 patients whose tumours contained an exon 11 c-kit mutation had a longer event-free and overall survival than patients whose tumours carried other types of mutations.23 Furthermore, in patients with imatinib-refractory GIST, presence of an exon 9 c-kit mutation was associated with a greater response rate to SU11248, a multi-targeted tyrosine kinase inhibitor.50 Thus, this represents an evolving paradigm in which mutational analysis leads not only to the development of new small molecule inhibitors, but also to the selection of genotype-specific therapies.

Molecular genetic and cytogenetic techniques Numerous techniques can be utilized in the molecular genetic and cytogenetic analysis of tumours. However, not every technique is equally efficacious in every clinical situation. Here, we will discuss some of the advantages, limitations and diagnostic pitfalls of each technique.

Conventional cytogenetics and karyotyping Cytogenetic analysis involves culture of fresh tissue and karyotypic analysis of a metaphase spread (Fig. 3). An advantage of karyotypic analysis is that it evaluates multiple chromosomes and allows for a wide survey of potential abnormalities. However, because of its relatively low level of resolution, karyotypic analysis is best used to evaluate large chromosomal aberrations and may not detect small rearrangements. Technical disadvantages of karyotype analysis include the necessity to procure fresh viable, non-contaminated tissue and the difficulty of culturing tumour cells in vitro. Depending upon the quality of tissue submitted by the pathologist,

ARTICLE IN PRESS The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal 367 few laboratories achieve a successful culture rate of more than 60% from solid tumours. Even with both trained personnel and specialized facilities, tumour cells may require several days to grow or may not grow at all. In addition, overgrowth of tissue cultures by normal stromal cells can make identification of lesional cells difficult. Indeed, misinterpretation of a stromal cell karyotype as a tumour cell karyotype is potentially an important source of error.

Fluorescence in situ hybridization (FISH) In situ hybridization of interphase cells with fluorescently labelled nucleic acid probes can be used to detect chromosomal rearrangements, numerical changes in chromosome number, or map loci on specific chromosomes. The probes that are most commonly used in clinical practice are ‘split-apart’ probes, which target specific breakpoint regions in chromosomal translocations (Fig. 1). The main advantage of FISH is that it does not require fresh tissue and can be performed on a variety of preparations including touch preps, aspirate smears and paraffin embedded tissue. Compared to standard cytogenetic analysis, FISH assays are faster and cheaper to perform. In addition, FISH is better than PCR-based technologies in detecting translocations that have heterogeneous and diffuse breakpoint regions. However, successful FISH requires sufficient concentrations of neoplastic cells for analysis and a targeted chromosomal alteration that is larger than tens of kilobases (kB). FISH is best used for the directed analysis of a specific alteration and not for broad screening of all possible chromosomal abnormalities. In addition, although FISH analysis is good for the detection of chromosomal translocations, it is not the ideal method for the detection of deletions or duplications. Because variations in tissue fixation and processing can affect the success of hybridization, inhibition of probe access or binding can result in erroneous results. Another limiting factor is the small number of commercially available probes. Many probes were created ‘in house’ in specialized treatment centres and can only be utilized in those laboratories. FISH performed on histological sections allows the correlation of hybridization results with cell morphology. Chromogenic in situ hybridization (CISH) is particularly promising in this regard, as the use of chromogenic probes would allow simultaneous visualization of hybridization results and tissue morphology. However, CISH

techniques have not been as well developed and are currently less sensitive than standard FISH. Unfortunately, because the thickness of a histological section is less than the size of an average nucleus, the entire nucleus is not available for hybridization, possibly leading to errors of omission. This problem can be abrogated by making thick sections (50 mm) of paraffin embedded material, disaggregating the cells and performing FISH on the intact nuclei. However, this technique is not routinely performed in all laboratories.

PCR and RT-PCR PCR is a technique that yields rapid results and utilizes equipment that is more easily accessed by many laboratories. Similar to the technique of in situ hybridization, PCR and even RT-PCR can be performed on both fresh and formalin-fixed tissues. Another advantage of PCR is the variety of chromosomal alterations that can be detected, including translocations, deletions and point mutations. Under ideal conditions, PCR is extremely sensitive, detecting one abnormal cell out of 100 000. This sensitivity has been exploited in assays detecting minimal residual disease or early relapse in patients with leukaemias. However, there are also several disadvantages in employing PCR as the sole molecular genetic technique. First, PCR is a directed assay and not a wide survey; the flanking sequences of the chromosomal alteration must be cloned before a test can be performed. Secondly, if there are large variations in the location of the breakpoint regions, or large intervening sequences, then PCR may fail to detect some translocations. This problem has been addressed in two ways: multiple primer sets can be used to cover various possible breakpoint regions and RT-PCR. Unfortunately, RT-PCR is not always an option, since extraction of RNA from formalin-fixed materials is not reliable. Most importantly, a common cause of false positive results is crosscontamination. If an unexpected result occurs, in addition to reassessment of the morphological diagnosis and re-evaluation of negative controls, PCR results should be confirmed by a different technique such as FISH.51

Realities in molecular and cytogenetic analyses of soft tissue tumours Knowledge of the genetics of soft tissue tumours has increased tremendously over the past 15–20

ARTICLE IN PRESS 368 years. However, it is not always clear how this rapidly accumulating data can be utilized in current practice. Here, based on our own experience in a centre dealing with a large number of cases each year, we briefly discuss some of the general limitations of molecular genetic and cytogenetic techniques in research as well as in clinical settings and, given these limitations, how we believe molecular genetic and cytogenetic techniques can be best utilized. In general, the limitations of the role of cytogenetics and molecular genetics in soft tissue tumour examination and diagnosis can be divided into one of two categories: technological and biological. An inherent technical limitation of research studies examining genetic alterations in soft tissue tumours is the rarity of many of these entities. Analysis of statistically significant numbers of cases is extremely difficult. In addition, many soft tissue tumours are morphologically heterogeneous and misdiagnoses can confound the results of research studies. In clinical practice, apart from issues of rarity of tumour types and relative infrequency of test requests, there are often an even greater number of technical difficulties. Currently, most tests are available only in specialized centres and are not widely available. In addition, because methods of tissue handling, fixation and processing in different labs are variable, there may be inconsistent results when samples are sent to a centralized laboratory. However, there is often little incentive to develop these tests outside of central laboratories, in smaller centres where costs must be covered by the laboratory because reimbursements or institutional subsidies are often poor. Furthermore, there are no guidelines or regulations for quality control or standardization of molecular genetic laboratory testing of soft tissue tumours. It has not been established what controls should be run, how test results should be validated, or whether PCR results should be confirmed with a second assay. The biology of soft tissue tumours is also an intrinsic limitation to the widespread utilization of molecular genetic and cytogenetic techniques for diagnostic purposes. High grade pleomorphic sarcomas and spindle cell sarcomas in older patients, which are overall the commonest sarcomas, have complex karyotypes that are not tumour specific.4 Therefore, cytogenetic and molecular genetic analysis is not helpful in the diagnosis of this group of tumours, nor in prognostication. Furthermore, not all acquired chromosomal alterations are tumour type specific. Primary genetic aberrations are characteristic of a specific entity and often the

H.P. Hahn, C.D.M. Fletcher only anomaly present. Secondary genetic changes are additional mutations that reflect the progression of the neoplasm and can be common to more than one tumour type. Thus, although mutations are present, detecting the presence or absence of some of these chromosomal changes cannot confirm specific diagnoses. Finally, soft tissue tumourigenesis may involve epigenetic changes, such as DNA methylation, which cannot be detected by the methods described above.

Recommendations regarding the use of molecular genetic and cytogenetic analysis of soft tissue tumours Despite the problems listed above, there are situations where molecular genetic or cytogenetic analysis of soft tissue tumours is clearly beneficial. Of course, as mentioned above, these techniques are a valuable adjunctive study in difficult cases where diagnosis confirmation would alter treatment protocols, predicted response to treatment, or overall prognosis. This is especially true among round cell malignant neoplasms. In addition, certain cases definitely benefit from ‘up front’ cytogenetic analysis. Fresh tissue should ideally be sent for cytogenetic analysis in cases such as: (1) soft tissue tumours suspicious for sarcoma in patients less than 40 years old; (2) fatty neoplasms that are deep seated; and (3) fatty neoplasms that are larger than 10 cm in greatest dimension. These are the tumours that are most likely to contain characteristic molecular genetic abnormalities and are most likely to be difficult to classify based on morphological appearance alone. In conclusion, continued research and exploratory testing in major treatment centres is important for soft tissue diagnosis, classification and understanding of tumour biology. However, at the present time, the benefits of molecular genetic and cytogenetic testing in a routine, non-specialist, clinical setting are relatively limited and are sharply defined in specific disease types. The value of establishing such testing in centres other than those with a dedicated sarcoma clinic handling a significant volume of cases each year is questionable.

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ARTICLE IN PRESS The role of cytogenetics and molecular genetics in soft tissue tumour diagnosis—a realistic appraisal 369 2. Ladanyi M, Bridge JA. Contribution of molecular genetic data to the classification of sarcomas. Hum Pathol 2000;31: 532–8. 3. Panagopoulos I, Storlazzi CT, Fletcher CDM, et al. The chimeric FUS/CREB312 gene is specific for low-grade fibromyxoid sarcoma. Gene Chromosome Cancer 2004;40: 218–28. 4. Mertens F, Fletcher CDM, Dal Cin P, et al. Cytogenetic analysis of 46 pleomorphic soft tissue sarcomas and correlation with morphologic and clinical features. A report of the CHAMP study group. Gene Chromosome Cancer 1998; 22:16–25. 5. Fletcher CDM, Dal Cin P, De Wever I, et al. Correlation between clinicopathological features and karyotype in spindle cell sarcomas: a report of 130 cases from the CHAMP study group. Am J Pathol 1999;154:1841–7. 6. O’Sullivan MJ, Perlman EJ, Furman J, et al. Visceral primitive peripheral neuroectodermal tumours: a clinicopathologic and molecular study. Hum Pathol 2001;32: 1109–15. 7. Gerald WL, Miller HK, Battifora H, et al. Intra-abdominal desmoplastic small round-cell tumor: report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. Am J Surg Pathol 1991;15: 499–513. 8. Roberts CA, Seemayer TA, Neff JR, et al. Translocation (X;18) in primary synovial sarcoma of the lung. Cancer Genet Cytogenet 1996;88:49–52. 9. Thorner P, Squire J, Chilton-MacNeil S, et al. Is the EWS/FLI1 fusion transcript specific for Ewing sarcoma and peripheral primitive neuroectodermal tumor ? A report of four cases showing this transcript in a wider range of tumor types. Am J Pathol 1996;148:1125–38. 10. Delattre O, Zucman J, Melot T, et al. The Ewing family of tumours—a subgroup of small round cell tumors defined by specific chimeric transcripts. N Engl J Med 1994;331:294–9. 11. de Alava E, Gerald WL. Molecular biology of the Ewing’s sarcoma/primitive neuroectodermal tumor family. J Clin Oncol 2000;18:204–13. 12. Fletcher CDM, Akerman M, Dal Cin P, et al. Correlation between clinicopathological features and karyotype in lipomatous tumors. A report of 178 cases from the Chromosomes and Morphology (CHAMP) Collaborative Study Group. Am J Pathol 1996;148:623–63. 13. Rosai J, Akerman M, Dal Cin P, et al. Combined morphologic and karyotypic study of 59 atypical lipomatous tumors. Evaluation of their relationship and differential diagnosis. Am J Surg Pathol 1996;20:1182–9. 14. Tallini G, Akerman M, Dal Cin P, et al. Combined morphologic and karyotypic study of 28 myxoid liposarcomas. Implications for a revised morphologic typing (a report from the CHAMP group). Am J Surg Pathol 1996;20:1047–55. 15. Shmookler BM, Enzinger FM, Weiss SW. Giant cell fibroblastoma. A juvenile form of dermatofibrosarcoma protuberans. Cancer 1989;64:2154–61. 16. Rubin BP, Fletcher JA, Fletcher CDM. The histologic, genetic, and biological relationships between dermatofibrosarcoma protuberans and giant cell fibroblastoma: an unexpected story. Adv Anat Pathol 1997;4:336–41. 17. Knezevich SR, Garnett MJ, Pysher TJ, et al. ETV6-NTRK3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma. Cancer Res 1998;58:5046–8. 18. Rubin BP, Chen C-J, Morgan TW, et al. Congenital mesoblastic nephroma t(12;15) is associated with ETV6-NTRK3 gene fusion: cytogenetic and molecular relationship to

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