and cells and are not reflected in other somatic cells, e.g., blood cells. The establishment of chromosomal changes in a tumor requires fresh (not fixed) tissue; fol- lowing short-term culture, dividing cells can be exam- ined in late prophase or metaphase, when the chromo- somes are morphologically well defined and readily rec- ognized. These chromosomal changes may be either numerical (gain or loss or chromosomes) or structural (morphological; Fig. 7.4).
Since space limitations for this chapter preclude de- tailed presentations of cytogenetic terminology (Figs.
7.1–7.4) and of the genetics of soft tissue and bone tu- mors, proportionally more space has been given to pic- torial presentations (Figs. 1–10). The salient cytogenetic changes in these tumors are listed in Tables 1 and 2, ac- companied by short discussions of particular tumors.
Human tumors are primarily caused by anomalies af- fecting two types of genes: (1) Dominantly acting onco- genes, whose protein products serve to accelerate cell growth and whose functions are altered by increased gene dosage (amplification) or by activating mutations or participation in fusion genes, resulting from chro- mosomal translocations, inversions, or insertions; and (2) tumor-suppressor genes (TSG), whose products normally serve as brakes on cell growth and runaway cell proliferation and whose inactivation leads to un- controlled cell proliferation and downregulation of apoptosis (programmed cell death). Such inactivation is typically altered by physical elimination of TSG or by inactivating mutations (Fig. 7.7).
The recurrent and specific translocations in many soft tissue and bone tumors are unique in that they are diagnostic of the tumor and usually affect the onco- genes that have been identified in almost all of these conditions (Table 7.1). The translocations lead to the genesis of abnormal fusion genes of varying parts of the oncogenes involved and result in the mutation and/or overexpression of components of the fused genes.
The occurrence of specific chromosome changes in benign tumors (e.g., lipoma, leiomyoma; Table 7.2), i.e., translocations, as well as nonspecific changes in a num- ber of others, bears witness to the role of genetic events in cellular proliferation but without malignant aspects. In 7.1 Introduction
This chapter presents the genetic changes (cytogenetic and molecular) in bone tumors and soft tissue tumors applicable to fuller understanding and evaluation of their in the clinical setting. Thus, in keeping with the aims and nature of this volume, the genetic changes are addressed primarily to radiologists, but also to orthope- dists, oncologists, and surgeons.
The genetic changes in tumors can be established by a number of methodologies: cytogenetics, fluorescence in situ hybridization (FISH), and molecular approaches [1, 2]. Chromosomal (karyotypic, cytogenetic) changes in human tumors are confined to the involved tissues
Cytogenetics
and Molecular Genetics of Soft Tissue Tumors and Bone Tumors
A.A. Sandberg
7
7.1 Introduction . . . 93
7.2 Soft Tissue Tumors and Bone Tumors with Specific and Diagnostic Translocations . . . 99
7.2.1 Synovial Sarcoma . . . 99
7.2.2 Liposarcoma . . . 100
7.2.3 Ewing Tumors . . . 102
7.2.4 Rhabdomyosarcoma . . . 102
7.2.5 Clear Cell Sarcoma (Malignant Melanoma of Soft Parts) . . . 102
7.2.6 Desmoplastic Round-Cell Tumor . . . 102
7.2.7 Dermatofibrosarcoma Protuberans . . . 102
7.2.8 Congenital (Infantile) Fibrosarcoma and Mesoblastic Nephroma . . . 102
7.2.9 Inflammatory Myofibroblastic Tumor . . . 103
7.2.10 Chondrosarcoma . . . 103
7.2.11 Alveolar Soft-Part Sarcoma . . . 103
7.3 Soft Tissue Tumors and Bone Tumors Without Specific Cytogenetic Changes . . . 103
7.3.1 Malignant Peripheral Nerve Sheath Tumor . . . 103
7.3.2 Gastrointestinal Stromal Tumors . . . 103
7.3.3 Desmoid Tumors . . . 104
7.3.4 Rhabdoid Tumors . . . 104
7.3.5 Leiomyosarcomas . . . 104
7.3.6 Neuroblastoma . . . 104
7.3.7 Chondroma . . . 104
References . . . 105 Contents
Fig. 7.1Ia, b. Metaphase spread (a) consisting of 46 unbanded chromosomes (22 pairs of somatic chromosomes or autosomes and 1 pair of sex chromosomes, XY in males and XX in females).
The variations in chromosome length, location of the centromere (structures holding the two chromatids together prior to cell divi- sion into daughter cells), and particularly the banding pattern of the chromosomes (see Fig. 7.2) are used to arrange and identify individual chromosomes (b). The normal set of the 46 chromo-
somes is called diploid. Cells with less than 46 chromosomes are hypodiploid and those with a higher number than 46 are hyper- diploid. Cells with 69 or 92 chromosomes are triploid and tetraploid, respectively. Cells with the above numbers of chromo- somes but with numerical or structural anomalies are labeled pseudodiploid, etc. b Shown is a karyotype of a normal male cell, containing 22 pairs of autosomes and one set of sex chromosomes (XY)
a b
Fig. 7.2. Schematic karyotype of a normal cell showing the unique banding pattern of each chromosome The banding patterns de- scrubed this chapter are based on Giemsa staining and hence are called G-banding. Other banding techniques include R-banding, C-banding, and T-banding [19]. The centromeres are shown as heavily hatched areas and heterochromatin as lightly hatched areas. The acrocentric chromosomes (13–15, 21, and 22) are char-
acterized by satellites. Chromosomes with the centromeres located centrally are called metacentric, those with the centromere away from the center are called submetacentric, and those with the cen- tromeres at the end are called telocentric or acrocentric. For fur- ther information and details regarding cytogenetic terms, nota- tions, definitions, etc., the reader should consult ISCN 1995 [19]
chronologies in tumor development and associated with different genetic changes and milieus (Fig. 7.10).
The preponderant number of human cancers, in- cluding tumors of the bones and soft tissues, are not characterized by specific translocations affecting onco- genes, but develop through a stepwise and orchestrated sequence of genetic events, primarily loss of heterozy- gosity (LOH) of TSG (Fig. 7.10). Some of these losses are evident as deletions of chromosomal material estab- lished microscopically, ranging from partial loss of a band to loss of the whole arm of a chromosome or a whole chromosome. Other LOH changes are submicro- scopic.
Advantage has been taken of the composition and structure of fusion genes by tailoring therapies affect- ing the function of these genes, e.g., blocking of expres- sion of the mutated tyrosine kinase present in the fu- sion gene of chronic myelocytic leukemia and in the mutated KIT gene in gastrointestinal stromal tumors (GIST). The uniqueness of such therapy is reflected by the successful treatment of GIST with imatinib, which inhibits the tyrosine kinase of KIT, but only if the muta- tion occurs at exon 11 and not, for example, at exon 17.
In many tumors specific translocations may be the only alterations; however, in a significant number of cases, additional karyotypic changes appear and are possibly responsible (or at least associated with) pro- gression of the disease. This is also reflected by alter- ations in the expression of a number of genes (not evi- dent microscopically and hence cytogenetically) aside from those involved in the translocation. The exact cause(s) for these alterations is not known, i.e., whether the translocation per se is responsible or the process leading to the translocation or other factors. In some of these conditions, e.g., Ewing-type tumors (Table 7.1), variant translocations may occur, but they always in- volve the EWS gene located on chromosome 22.
The genetic and molecular consequences of inver- sions and insertions, quite rare events in soft tissue tumors and bone tumors, are probably similar to those associated with translocations in that they lead to the genesis of fusion genes.
The specific translocations shown in Table 7.1 are di- agnostic of the tumors in which they are found; they have not been observed in other tumor types and can be of crucial value in establishing the correct diagnosis in confusing cases.As mentioned above, fresh tumor tissue is required for cytogenetic analysis and, hence, both the surgeon and pathologist must be alert to the possibility of a tumor requiring cytogenetic analysis and obtain appropriate tissue for such an analysis. Such an alert could originate with the radiologist (see Things to re- member).
Having failed to obtain fresh tissue for cytogenetic analysis, the presence of specific translocations can be established by several interphase FISH techniques, par- fact, the specific chromosome alterations in benign tu-
mors not only serve diagnostic purposes, but also serve as a means of differentiating them from their malignant counterparts (e.g., liposarcomas, leiomyosarcoma).
Some of the genes (particularly TSG) affected in ma- lignant tumors may be involved in the genesis of benign tumors. In fact, the same genes can be altered in a num- ber of different tumors, but apparently at varying
Fig. 7.3. For a full appreciation of the chromosome changes in tu- mors, one must understand the nomenclature used to describe normal and abnormal chromosomes. The nomenclature for band- ed chromosomes is based on a system in which each chromosome (chromosome 9 is used as an example here) is divided in relation to the centromere (heavily hatched area) into its upper (usually short) arm labeled p and lower (usually long) arm labeled q. Each arm is divided into regions: in chromosome 9 into two regions in the p arm and into three regions in the q arm. The bands are then assigned to each region, the numbering system consisting of the region and then band number . Thus, for example, 9q23 refers to band number 3 on chromosome 9 in region 2. In the higher-reso- lution system, the sub-band number is shown following a period.
A band overlapping two regions is assigned to the more distal re- gion. The centromere per se is designated as 10. The total set of chromosomes contains about 400 bands (chromosome 9 on the left) demonstrated with usual banding techniques; more refined banding increases the number to 550, so that sub-bands can be vi- sualized, as shown with the chromosome 9 on the right [19]
ticularly with cosmid probes, which can be applied to frozen or archival tissues. In fact, results have been ob- tained in fixed specimens a number of years old.
The presence of translocations may also be ascer- tained by molecular analysis (usually reverse transcrip- tase polymerase chain reaction, RT-PCR), based on messenger ribonucleic acid (mRNA) or deoxyribonu- cleic acid (DNA) extracted from fresh or archival tissues and in which the products of fusion genes can be iden- tified. This approach may also detect varying tran- scripts of such fusion genes.
Examples of genetic changes not reflected in recog- nizable cytogenetic anomalies but determinable with molecular techniques or FISH are the KIT mutation in GIST, amplification of HER2/neu in breast cancer, and NMYC in neuroblastoma.
The genetic findings in Ewing tumors based on a number of techniques (Figs. 7.5 and 7.6) are examples of the approaches available in the diagnosis of the tu- mors associated with specific translocations shown in Table 7.1. The translocation t(11;12)(q24;q12) in Ewing sarcoma and related tumors leads to the genesis of an abnormal fusion gene containing elements of the EWS
and FUL genes involved by the breaks in the t(11;12).
However, the products of this translocation show vari- ability in the breaks in these genes occurring at different exons (but still in the chromosomal bands indicated), leading to variable transcripts. The clinical conse- quences of such variability is the demonstration that patients with tumors with type 1 transcript do much better than those with type 2. Though as many as 18 dif- ferent transcripts have been identified as a result of the EWS-FUL fusion gene, insufficient numbers of cases with the other fusion products have been examined and hence clinical significance of these varying transcripts is unknown.
The appearance of chromosomal changes, numerical and/or structural, in addition to the translocations seen in bone tumors and soft tissue tumors is usually associ- ated with biological progression, manifested by inva- sion and metastases. These additional changes are usu- ally variable from tumor to tumor, even those with the same diagnosis. With or without additional chromo- some changes, tumors with specific translocations may show a variety of anomalies at the molecular level which may involve a number of genes.
Fig. 7.4Ia–e.Schematic presentation of the most common struc- tural chromosomal changes seen in tumors. a A translocation showing an exchange of materials in a balanced fashion between two chromosomes. Translocations usually involve oncogenes and lead to the genesis of abnormal fusion genes resulting from the juxtaposition of the oncogenes involved. A translocation may in- clude additional chromosomes leading to a more complex translo- cation but retaining the molecular effects of the basic transloca- tion. b Deletion of material from one chromosome. Deletions may affect varying segments of a chromosome, ranging from submi- croscopic deletions to loss of a whole arm or whole chromosome.
Deletions are often responsible for loss of heterozygosity (LOH).
cFormation of isochromosomes consisting of two identical arms.
Thus, i(17q) would describe an isochromosome of the long arm of chromosome 17. d Paracentric inversions (top) do not involve the centromere, e.g., inv(3)(q21q26); pericentric inversion (bottom) include the centromere, e.g., inv(3)(p13q21). Note that, in contrast to the notation of translocations, no semicolons are placed be- tween the two breakpoints in inversions. As in translocations, in- versions may result in fusion genes resulting from the juxtaposi- tion of parts of genes. e Ring chromosomes. Though shown as originating from one chromosome, rings may contain material from a number of chromosomes. A r(12) would describe a ring chromosome originating from chromosome 12
Fig. 7.5Ia–c. The cytogenetic changes in Ewing sarcomas will be presented as exam- ples for the range of genetic tests available in diagnosing certain tumors. a A transloca- tion, t(11;22)(q24;q12), seen in most Ewing tumors and involving the oncogenes EWSR1 on 22q12 and FLI1 on 11q24. The notation for translocations consists of two sets of parentheses: the first shows the chromo- somes involved, and the second, the break- points. As shown here for Ewing tumors and based on the ISCN guidelines, e.g., in the notation t(11;12)(q24;q12), t indicates a translocation with involvement of chromo- somes 11 and 12 shown in the first set of parentheses and the breakpoints on chromo- some 11 at q24 and on chromosome 12 at q12 in the second set of parantheses. All the translocations shown in Table 7.1 follow this notation. The resulting fusion chromosome shown in b, i.e., EWSR1/FLI1, and its protein products are probably responsible for the genesis of Ewing tumors. An “alternative splice” area is shown in the EWSR1 gene as- sociated with variability in the breakpoints and leading to the genesis of fusion products of varying nature and of clinical significance regarding tumor aggressiveness. c A kary- otype (containing 50 chromosomes) of a Ewing tumor with the t(11;22) (horizontal arrows). Four extra chromosomes are indi- cated by vertical arrows. The presence of chromosomes (normal and/or abnormal) in addition to the translocation is usually asso- ciated with more aggressive tumors [20, 21]
Fig. 7.6. Results obtained with reverse transcriptase polymerase chain reaction (RT-PCR) on the DNA of Ewing tumors. The differ- ence in the mobility of type 1 and type 2 tumors is due to the dif- ferences (mentioned in Fig 7.5) in the breakpoint in the EWSR1 gene. Patients with type 1 have a more favorable prognosis than those with other types of translocation breaks. Note that the pa- tient tested here had the unfavorable type 2 results [21]
a
b
c
Fig. 7.7. Schematic presentation of loss of heterozygosity (LOH) often affecting tumor suppressor genes and responsible for the genesis of most human tumors. M, a mutated normal gene; N, a normal gene.
The various mechanisms responsible for LOH are shown: submicroscopic mutation and chromosomal deletion are the two most common mechanisms
Fig. 7.8Ia, b. An example of the use of FISH with cosmid probes in a metaphase (a) and two interphase nuclei (b) for ascertaining a translocation. The green probe includes one gene and the red probe another gene. Arrow- heads point to the fusion gene. The findings in B show the feasibility of ascertaining translocations in interphase nuclei, such as in archival tissues [22]
a
b
7.2.1 Synovial Sarcoma
Histologically, synovial sarcoma (SS) can be either monophasic (containing preponderantly spindle cells) or biphasic (containing both spindle cell and epithelial elements), and the molecular findings seen in SS bear a distinct relationship to the tumor histology [3]. A spe- cific and diagnostic translocation in SS consists of t(X;18)(p11.2;q11.2) (Table 7.1), seen in almost all of these tumors. An oncogene at 18q11.2 (SS18) is either fused to the SSX1 or to its neighboring SSX2 gene. Cyto- genetically, it is not possible to distinguish SS18-SSX1 from SS18-SSX2; however, these fusions can be demon- strated with either FISH or RT-PCR.
SS with the SS18-SSX1 are invariably biphasic, where- as SS with SS18-SSX2 can be either biphasic or mono- phasic and have a longer metastasis-free survival than patients with the SS18-SSX1 fusion gene.
The t(X;18)(p11.2;q11.2) translocation is unique to SS and serves to differentiate it from confusing tumors such as hemangiopericytoma, mesothelioma, leiomyosarco- ma, or malignant peripheral nerve sheath tumors.
7.2 Soft Tissue Tumors and Bone Tumors with Specific and Diagnostic Translocations
Emphasis is put on those tumors for which the cytoge- netic and/or molecular changes are diagnostic (Table 7.1); though the changes shown in Table 7.2 may not be specific in most of these tumors, these can be of diag- nostic help in confusing and complicated cases [2].
Fig. 7.9. Metaphase of a tumor cell containing double minutes (dms), a finding usually associated with gene amplification and a poor prognosis, e.g., in neuroblastoma
Fig. 7.10. Stepwise process of genetic changes leading to full tumor development. Thus, an initial genetic change affects cell proliferation, followed by further stepwise changes which lead to neoplastic transformation and ultimately to an aggressive tumor.
This process probably accounts for the bulk of tumors, particular- ly cancers of epithelial origin. Most of the genetic changes consist of LOH (Fig. 7.7), often through chromosomal deletions seen microscopically, but may also consist of translocations and muta- tions of varying origin as well as of altered expression of some
genes. The same genes may be involved in different types of tumors. For examples, the step at which the p53 gene is involved may be genetic event 2 in one type of tumor and genetic event 5 in another type. Furthermore, in one tumor the p53 may be overex- pressed, whereas in another it may be underexpressed or totally silent. This process of involvement of multiple genes in an orches- trated and progressive succession is quite different from the process of fusion genes resulting from a translocation and appar- ently sufficient by itself to cause tumor development
7.2.2 Liposarcoma
Liposarcoma (LPS) is characterized by cytogenetic anomalies unique to each histological type [4]. Myxoid or round cell LPS is associated with a recurrent and di- agnostic chromosomal change consisting of t(12;16) (q13;p11.2) and much less frequently t(12;22)(q13;q12).
The t(12;16) translocation results in the fusion of the DDIT3 gene at 12q13 to the FUS gene at 16p11.2. In t(12;22) the DDIT3 gene is fused with the EWSR1 gene.
These translocations are retained in myxoid LPS ac–
quiring round-cell features. The translocations just de- scribed have not been found in any other types of LPS or in other types of myxoid tumors.
Well-differentiated LPS are characterized by large to gi- ant marker and/or ring chromosomes [4], which are usu- ally comprised of chromosome 12 material, as well as that from several other chromosomes. The abnormal chromo- somes in well-differentiated LPS contain various ampli- fied genes which are retained when these LPS progress to the much more aggressive dedifferentiated LPS.
Lipomas, tumor types which may on occasion pre- sent diagnostic dilemmas in differentiating from LPS, are associated with several distinct cytogenetic profiles.
A significant proportion of lipomas show involvement of 12q14-q15, often as translocations with one of the other chromosomes [5]. These translocations involve the HMGA2 gene, resulting in fusion genes. Another group of lipomas is associated with rearrangements of the short arm of chromosome 6 or deletion of the long arm of chromosome 13. Lipomas with deletions of the long arm of chromosome 16, often accompanied by deletions of the long arm of chromosome 13, often have a spindle-cell or pleomorphic histology.
Lipoblastomas contain translocations involving the long arm of chromosome 8 at bands 8q11–12, leading to rearrangement of the PLAG1 oncogene. Hibernomas, consisting of brown fat, usually have rearrangements of the long arm of chromosome 11. Angiolipomas usually have normal karyotypes.
Table 7.1. Specific chromosomal translocations established cytogenetically and the corresponding gene changes in bone and soft tissue tumors
Tumors Translocation Gene changes
Aggressive angiomyxoma t(8;12)(p12;q15) HMGA2
Alveolar rhabdomyosarcoma t(2;13)(q35;q14) PAX3-FOXO1A
t(1;13)(p36;q14) PAX7-FOXO1A
Alveolar soft part sarcoma t(X;17)(p11.2;q25) ASPSCR1-TFE3
Aneurysmal bone cyst t(16;17)(q22;p13) CDH11-DUSP6
Angiomatoid fibrous histiocytomaa t(12;16)(q13;p11) FUS-ATF1
Clear cell sarcoma (malignant melanoma of soft parts) t(12;22)(q13;q12) ATF1-EWSR1
Congenital fibrosarcoma and mesoblastic nephroma t(12;15)(p13;q25) ETV6-NTRK3
Dermatofibrosarcoma protuberans (giant cell fibroblastoma) t(17;22)(q22;q13) COL1A1-PDGFB
Desmoplastic round cell tumor t(11;22)(p13;q12) WT1-EWSR1
Endometrial stromal sarcoma t(7;17)(p15;q21) JAZF1-SUZ12
t(10;17)(q22;p13)
Ewing tumor and peripheral primitive neuroectodermal tumors t(11;22)(q24;q12) EWSR1-FLI1
t(21;22)(q22;q12) EWSR1-ERG
t(7;22)(p22;q12) EWSR1-ETV1
t(17;22)(q12;q12) EWSR1-ETV4
t(2;22)(q33;q12) FEV-EWSR1
Hemangioendothelioma, epithelioida t(1;3)(p36.3;q25)
Hemangiopericytomaa t(12;19)(q13;q13)
Inflammatory myofibroblastic tumor t(2;19)(p23;p13.1) ALK-TPM4
t(1;2)(q22–23;p23) TPM3-ALK
Low-grade fibromyxoid sarcoma t(7;16)(q34;p11) CREB3L2-FUS
Myxoid chondrosarcoma, extraskeletal t(9;22)(q22;q12) EWSR1-NR4A3
t(9;17)(q22;q11) TAF15-NR4A3
t(9;15)(q22;q21) TEC-TCF12
Myxoid liposarcoma t(12;16)(q13;p11) FUS-DDIT3
t(12;22)(q13;q12) EWSR1-DDIT3
Synovial sarcoma t(X;18)(p11;q11) SS18-SSX1
SS18-SSX2
aSmall number of cases analyzed
Table 7.2. Recurrent but not specific chromosome changes in soft tissue tumors and bone tumors
Tumors Chromosome changes Molecular findings
Atypical fibroxanthomas TP53 mutations
(due to UV radiation?) Cardiac myxoma 12p12 rearrangements; telomeric association
Chondromatous, synovial Chromosome 6 and 1p abnormalities
Chondromyxoid fibroma del(6q)
Collagenous fibroma
(desmoplastic fibroblastoma) 11q12 involvement
Elastofibroma 1p rearrangements
Fibroma of tendon sheath 11q12 involvement
Gastrointestinal stromal tumor del(1p), –14, –22 KIT mutations
Giant cell tumor of bone Telomere association
Giant cell tumor of tendon sheath Cathepsins B and K expressed
Hamartoma
Pulmonary 12q14-q15 rearrangements
6p21 HMGA1 rearrangement
t(6;14)(p21;q23-q24) RAD51L1 involvement
Hepatic 19q13.4 rearrangements
Hibernoma 11q13 rearrangements
Inflammatory myofibroblastic tumors 2p23 rearrangements ALK fusion genes
Leiomyoma, extrauterine del(1p)
Uterine t(12;14)(q15;q24) or del(7q) HMGA2 rearrangement
Leiomyosarcoma del(1p)
Lipoblastoma 8p12 rearrangement, polysomy 8 PLAG1 oncogene changes
Lipoma 12q14-q15 rearrangements
(often translocations
with various chromosomes) HMGA2 rearrangement
Spindle cell or pleomorphic del(13q) or del(16q)
Chondroid t(11;16)(q13;p12–13)
Parosteal 12q14 rearrangements
Malignant fibrous histiocytoma
myxofibrosarcoma r(12)
Malignant mesenchymoma Ring markers containing amplified 1q21-q23 and 12q14-q15 sequences
Mesothelioma del(1p), del(9p) BCL10 inactivation, CDKN2A
inactivation
Neurinoma –22
Neuroblastoma Hyperdiploid, no del(1p) MYCN amplification
Poor prognosis del(1p), dms
Neurofibroma –22 and other changes
Osteochondroma del(8q) EXT1 inactivation
Osteosarcoma
Low-grade Ring chromosomes
High-grade RB1 and TP53 inactivation
Paraganglioma (nonfamilial) –11 SDHD mutations
Pigmented villonodular synovitis +5, +7
Rhabdoid tumor del(22q) SMARCB1 inactivation
Rhabdomyosarcoma, embryonal +2q, +8, +20, LOH at 11p15
Schwannoma, benign del(22q) NF2 inactivation
Sclerosing epithelioid fibrosarcoma Amplification of 12q13 and 12a15
Sclerosing hemangioma of lung Possible TSG at 5q TTF1 expression
and other hemangiomas
The notation del(1q) or any other chromosome arm means that the deletion may involve varying sections of the arms. Extra chromosomes are designated by the + sign, e.g., +5, and loss by a – sign, e.g., –22.The terms “rearrangements,” “abnormalities,” and
“involvement” refer to a variety of chromosomal changes, which may include translocations, deletions, inversions, additional material on a chromosome, and other structural changes. Telomeric association results from the fusion of the telomere ends of two or more chromosomes
7.2.3 Ewing Tumors
Ewing sarcomas contain chromosome translocations involving the Ewing sarcoma gene (EWSR1), located at 22q12 with a number of partner genes (Table 7.1). The most common rearrangement is t(11;22)(q24;q12), which leads to the fusion of the EWSR1 with the FLI1 gene located at 11q24, i.e., the EWSR1-FLI1 abnormal fusion gene, diagnostic of and probably responsible for Ewing sarcoma [6]. In the other types of translocations, other genes fuse with EWSR1; these belong to the ETS family of transcription factor genes (Table 7.1). The EWSR1 gene is apparently essential to the development of Ewing tumors, with the varying partner genes play- ing roles which have not been clearly defined. When tu- mor material is not available for cytogenetic analysis, FISH studies using cosmid probes for EWSR1 and part- ner genes, most often FLI1, or RT-PCR analysis readily establish the diagnosis of Ewing sarcoma. Clinical cor- relates with the various translocations seen in Ewing tu- mors have not been established. On the other hand, variability in the breakpoint location in the exons, par- ticularly of the EWSR1 gene, as determined by RT-PCR, may have a prognostic significance.
Askin tumor of the thoracopulmonary region has the same cytogenetic changes as those seen in Ewing sarco- ma; esthesioneuroblastoma only rarely shows such changes.
7.2.4 Rhabdomyosarcoma
Cytogenetic findings have revealed specific and diag- nostic translocations in alveolar rhabdomyosarcoma (RMS), t(2;13)(q35;q14) being the most common and t(1;13)(p36;q14) much less common [2]. The former translocation results in a fusion gene of the FOXO1A gene located at 13q14 and of the PAX3 gene located at 2q35.
The translocation t(1;13) results in a fusion gene involv- ing the PAX7 gene at 1p36 with the FOXO1A gene. The PAX7-FOXO1A fusion gene is often highly amplified, par- ticularly in the form of double minutes (dms) (Fig. 7.9.).
Embryonal RMS lack specific translocations, but do have a recurrent cytogenetic profile, including extra copies of chromosomes 2, 8, and 20. The role of 11p deletions in embryonal RMS has not been clearly de- fined. Clinical or pathological correlates with these two types of diagnostic translocations in RMS have not been published.
7.2.5 Clear Cell Sarcoma
(Malignant Melanoma of Soft Parts)
The bulk of clear cell sarcomas are associated with a di- agnostic translocation, t(12;22)(q13;q12) [7]. Though clear cell sarcoma may resemble malignant melanoma phenotypically and histologically, the t(12;22) translo- cation has not been seen in malignant melanomas and serves as a means of differentiating these two entities.
The t(12;22) translocation leads to the creation of a fu- sion gene consisting of ATF1 located at 12q13 and the EWSR1 located at 22q12, i.e., ATF1-EWSR1.
7.2.6 Desmoplastic Round-Cell Tumor
These tumors are usually found in intra-abdominal soft tissues and are quite aggressive in nature. Almost all cases are associated with a diagnostic translocation, t(11;22)(p13;q22) [8]. This results in the fusion gene WT1-EWSR1, which upregulates the expression of platelet-derived growth factor-a (PDGFA), the latter be- ing an activator of mitogenic signaling pathways in fi- broblasts.
7.2.7 Dermatofibrosarcoma Protuberans
Dermatofibrosarcoma protuberans (DFSP) and Bednar tumors may be characterized by a diagnostic transloca- tion, t(17;22)(q23;q13), or by ring chromosome contain- ing sequences of chromosomes 17 and 22 related to multiple copies of the fusion gene COL1A1-PDGFB, the former located at 17q22 and the latter at 22q13 [9]. The COL1A1-PDGFB fusion results in the overexpression of PDGFB (platelet-derived growth factor-b), which is a growth factor that activates platelet-derived growth fac- tor-b receptor and platelet-derived growth factor recep- tor-a. The drug imatinib is an inhibitor of the PDGFB receptor and has been used successfully in the treat- ment of DFSP.
7.2.8 Congenital (Infantile) Fibrosarcoma and Mesoblastic Nephroma
These two tumors are characterized by the diagnostic translocation t(12;15)(p13;q25), which may be difficult to ascertain cytogenetically [10]. However, the fusion gene ETV6-NTRK3 is readily determined by RT-PCR or FISH. Those tumors that do not contain the t(12;15) translocation may have trisomies of chromosomes 8, 11, 17, and 20.
molecular aspects of these changes. Low-grade fibroid sarcoma and its closely related hyalinizing spindle-cell tumor, both thought to be distinct variants of fibrosar- coma, have been described to be associated with t(7;16)(q34;p11.2) [13].
7.3 Soft Tissue Tumors and Bone Tumors Without Specific Cytogenetic Changes
The genetic events shown in Table 7.2 are related to tu- mors of the bone and soft tissues which are not charac- terized by specific translocations (or other karyotypic changes) but may contain some anomalies which are more frequent than others [2, 14, 15]. In some tumors the changes shown in Table 7.2 are part of complex karyotypes seen in these tumors, e.g., skeletal chon- drosarcoma, osteosarcoma, leiomyosarcoma, malignant fibrous histiocytoma (MFH), and malignant peripheral nerve sheath tumors, in which the malignant process probably developed through a stepwise mechanism (Fig. 7.10).
7.3.1 Malignant Peripheral Nerve Sheath Tumor
Malignant peripheral nerve sheath tumors (MPNST) of- ten have a deletion of the NF1 gene, which can be demonstrated with FISH, and are usually accompanied by complex karyotypes. Characterization of the neurofi- bromatoses genes (NF1 located on 17q/1.2 and NF2 on 22q/2.2) has shed light on MPNST pathogenesis. Both these genes encode tumor-suppressor proteins. Neurofi- bromas and MPNST are common in individuals with neurofibromatosis type 1, whereas schwannomas are as- sociated with type 2 [2].
7.3.2 Gastrointestinal Stromal Tumors
These tumors deserve special attention because of mol- ecular facets that have played a key role in their therapy.
GIST contain activating mutation of the KIT or BGFRA oncogenes [16]. If the mutation is in exon 13 (present in approx. 70% of cases) therapy with imatinib is quite ef- fective; whereas if the mutation is in exon 17 (present in approx. 30% of cases) the drug is ineffective. These point mutations can only be ascertained by RT-PCR, since they are not detectable cytogenetically. GIST is probably caused by a KIT mutation; cytogenetic changes, when present, may play a role in tumor pro- gression. Loss of 14q, often accompanied by loss of 1p, 9p, 11p, or 22q, is common in GIST of various stages, though 9p deletion, 8q amplification, and 17 amplifica- tion are seen only in malignant tumors.
7.2.9 Inflammatory Myofibroblastic Tumor
Some of these tumors are characterized by either a t(2;19)(p23;p13.1) or t(1;2)(q22–23;p23) translocation.
The former results in the fusion gene ALK-TPM4 and the latter in the fusion gene TPM3-ALK, where ALK is located at 2p23, TPM4 at 19p13.1, and TPM3 at 1q21–22.
Cytogenetic or FISH analysis for ALK rearrangements is helpful in differentiating inflammatory myofibroblastic tumors from other similar spindle-cell proliferations [2].
7.2.10 Chondrosarcoma
Only extraskeletal myxoid chondrosarcoma (CS) is characterized by a diagnostic translocation, t(9;22) (q22;q12), including less common variants thereof, i.e., t(9;17)(q22;q11) and t(9;15)(q22;q21) [11]. Since ex- traskeletal myxoid CS, often occurring in the deep tis- sues of the extremities, particularly the musculature of the thigh and popliteal fossa, is not associated with a distinctive radiographic picture to separate it from oth- er types of soft tissue tumors, the chromosome changes mentioned above may be of diagnostic help. The t(9;22) translocation results in the fusion of the EWSR1 gene located at 22q12 and the NR4A3 gene located at 9q22.
The t(9;22) translocation and the variants mentioned above can be ascertained in extraskeletal myxoid CS by cytogenetics or FISH and spectrokaryotyping (SKY) and the products of the fusion gene by RT-PCR. In oth- er types of CS there is considerable heterogeneity in the cytogenetic findings, ranging from simple numerical changes to very complex karyotypes with many numer- ical and structural changes.
7.2.11 Alveolar Soft-Part Sarcoma
This is a rare, malignant neoplasm found predominant- ly in adolescents and young adults with a rather poor prognosis. Cytogenetically it has been established that alveolar soft-part sarcoma (ASPS) is characterized by a specific change, i.e., der(17)t(X;17)(p11.2;q25) [12]. The translocation fuses the TFE3 gene at Xp11 to a gene at 17p25 designated as ASPSCR1; the fusion gene leads to transcriptional deregulation in the pathogenesis of ASPS. It should be remembered that a small group of re- nal cell carcinomas (primarily in infants and children) display a t(X;17)(p11.2;q25) translocation and molecu- lar findings identical to those seen in ASPS.
Several kinds of tumor in Table 7.1 (aggressive an- giomyxoma, angiomatoid fibrous histiocytoma, epithe- lioid hemangioendothelioma) are probably associated with specific chromosome changes (translocations), but these remain to be more firmly established, as are the
7.3.3 Desmoid Tumors
Desmoid tumors (deep fibromatoses) may be associated with a number of cytogenetic changes [2], e.g., tri- somies 8 and 20, and del(5q). Mutations of APC and the b-catenin genes may precede the chromosome changes.
The latter would then be responsible for, or at least be associated with, progression.
7.3.4 Rhabdoid Tumors
Rhabdoid tumors of various locations (kidney, CNS, or soft tissue) usually have a deletion of 22q, involving a TSG, SMARCB1, which encodes a protein involved in chromatin remodeling. The 22q deletion is often the on- ly anomaly in rhabdoid tumors, suggesting that SMAR- CB1 inactivation is an early event in rhabdoid tumori- genesis.
7.3.5 Leiomyosarcomas
Leiomyosarcomas (LMS) usually have a complex kary- otype, with deletion of 1p occurring with some consis- tency [17]. Since a similar deletion may occur in other tumors (MFH, MPNST, and GIST), the finding of del(1p) lacks specificity for LMS. Karyotypic complexity is pre- sent even in low-grade LMS.
Leiomyomas, benign counterparts of LMS, often have translocations and deletions with rather simple karyo- types, though 50% of leiomyomas lack evident karyo- typic abnormalities [18]. A distinctive cytogenetic ab- normality in uterine leiomyoma is a translocation, t(12;14)(q15;q23), seen in about 20% of these tumors.
This translocation leads to overexpression of the HMGA2 gene, located at 12q15, through its fusion with the RAD51 gene, located at 14q23.
7.3.6 Neuroblastoma
Cytogenetics and molecular studies can be of consider- able clinical value in evaluating the prognosis in neu- roblastoma [2]. The tumors prompting a favorable prognosis are usually near-triploid, without 1p dele- tions or N-MYCN amplification. Neuroblastomas carry- ing an unfavorable prognosis have near-diploid or near- tetraploid karyotypes, 1p deletion and MYCN amplifica- tion, often manifest as dms (Fig. 7.9). In cases where cytogenetic results are not obtained, the MYCN amplifi- cation and the del(1p) can be determined in interphase cells by FISH with appropriate probes.
7.3.7 Chondroma
The chromosome changes seen in chondroma cannot differentiate those tumors which arise in bone from those in the periosteum or soft tissues [11]. Rearrange- ments of chromosome 6 and 12p(q13-q15) appear to be recurrent. Maffucci syndrome and Ollier disease, men- tioned elsewhere in this book, are conditions associated with multiple enchondromatosis.
Plantar fibromatosis (Ledderhose disease) has been shown to be associated with +8 and +14 as has been shown for other fibromatosis subtypes, e.g., Dupuytren contracture. Though clonal chromosome abnormalities have been reported for nodular fasciitis, proliferative myositis, Dupuytren contracture, Kaposi sarcoma, and Peyronie disease, no convincing recurrent changes have been described in these conditions.
Things to remember:
1. The radiologist is in a unique position for deter- mining which soft tissue tumor or bone tumor may require cytogenetic and/or molecular diag- nostic studies.
2. When the radiological findings are confusing and raise uncertainty regarding the exact diagnosis, the radiologist is in a position to alert the respon- sible surgeons and physicians before surgical or therapeutic procedures are initiated to the possi- bility that genetic studies may be indicated. This is particularly true if cytogenetic analysis is con- templated, since fresh (not fixed) tissue is re- quired for such an analysis and may be obtained at the time of surgery or biopsy. Though a similar uncertainty regarding the exact diagnosis may be encountered by the pathologist, usually fresh tis- sue is not available at that time, though interphase FISH and/or molecular studies can be performed on fixed specimens.
3. Emphasis must be placed again on the combined use of cytogenetic (including various FISH methodologies) and molecular techniques in ob- taining an optimal and full picture of diagnostic value of the genetic changes in tumors, due to the fact that tumors may have molecular changes ex- ceeding in number that of the cytogenetic anom- alies and at the same time present cytogenetic changes not reflected in the molecular abnormal- ities. Particularly useful in that regard are FISH and RT-PCR.
4. FISH can be based on a number of methodolo- gies, depending on the probes employed, i.e., cen- tromeric probes unique for the centromeric area of each chromosome are hence very useful in es- tablishing numerical changes of individual chro- mosomes. This approach is applicable not only to
7. Sandberg AA, Bridge JA (2001) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. Clear cell sarcoma (malignant melanoma of soft parts). Cancer Genet Cytogenet 130:1–7
8. Sandberg AA, Bridge JA (2002) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: desmo- plastic small round-cell tumors. Cancer Genet Cytogenet 138:1–10
9. Sandberg AA, Bridge JA (2003) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: der- matofibrosarcoma protuberans and giant cell fibroblastoma.
Cancer Genet Cytogenet 140:1–12
10. Sandberg AA, Bridge JA (2002) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: con- genital (infantile) fibrosarcoma and mesoblastic nephroma.
Cancer Genet Cytogenet 132:1–13
11. Sandberg AA, Bridge JA (2003) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. chon- drosarcoma and other cartilaginous neoplasms. Cancer Genet Cytogenet 143:1–31
12. Sandberg AA, Bridge JA (2002) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: alveo- lar soft part sarcoma. Cancer Genet Cytogenet 136:1–9 13. Reid R, Chandu de Silva MV, Paterson L, Ryan E, Fisher C
(2003) Low-grade fibromyxoid sarcoma and hyalinizing spindle cell tumor with giant rosettes share a common t(7;16)(q34;p11) translocation. Am J Surg Pathol 27:1229–1236 14. Sandberg AA, Bridge JA (2003) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. os- teosarcoma and related tumors. Cancer Genet Cytogenet 145:1–30
15. Sandberg AA, Bridge JA (2001) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors.
Mesothelioma. Cancer Genet Cytogenet 127:93–110
16. Sandberg AA, Bridge JA (2002) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors:
gastrointestinal stromal tumors. Cancer Genet Cytogenet 135:
1–22
17. Sandberg AA (2005) Updates on the cytogenetics and molecu- lar genetics of bone and soft tissue tumors. Leiomyosarcoma.
Cancer Genet Cytogenet 161:1–19
18. Sandberg AA (2005) Updates on the cytogenetics and molecu- lar genetics of bone and soft tissue tumors. leiomyoma. Cancer Genet Cytogenet 158:1–26
19. Mitelman F (ed) (1995) ISCN: an international system for hu- man cytogenetic nomenclature. Karger, Basel
20. Alava E de, Gerald WL (2000) Molecular biology of the Ewing’s sarcoma/primitive neuroectodermal tumor family. J Clin On- col 18:204–213
21. Alava E de, Kawai A, Healey JH, Fligman I, Meyers PA, Huvos AG, Gerald WL, Jhanwar SC, Argani P, Antonescu CR, Pardo- Mindán FJ, Ginsberg J, Womer R, Lawlor ER, Wunder J, An- drulis I, Sorensen PHB, Barr FG, Ladanyi M (1998) EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing’s sarcoma. J Clin Oncol 16:1248–1255 22. Knezevich SR, McFadden DE, Tao W, Lim JF, Sorenson PHB
(1998) A novel ETV6-NTRK3 gene fusion in congenital fi- brosarcoma. Nature Genet 18:184–187
metaphase but also to interphase chromosomes;
cosmid dual-color probes for unique genetic or chromosomal sequences (of genes or various chromosome bands or areas) are particularly use- ful in establishing the presence of translocations both in metaphase and interphase nuclei; and SKY and M-FISH by which each chromosome is uniquely labeled and hence are useful in the es- tablishment of esoteric or complex translocations or changes not deciphered by cytogenetics.
5. For diagnostic purposes, the use of FISH in inter- phase nuclei, i.e., in fixed or archival tissues, must be stressed, since this approach affords an oppor- tunity to establish genetic changes when cytoge- netic studies are not available.
6. A number of methodologies are available for the determination of genetic molecular changes in tu- mors, e.g., PCR amplification of tumor DNA, RT- PCR in which tumor mRNA is converted to cDNA, which is then amplified, and nested PCR, which can effectively amplify low copy-number tem- plates.
7. Molecular studies are useful when cytogenetics and FISH have yielded inconclusive results. Fur- thermore, the molecular techniques require small amounts of tissue, can be performed on archival specimens, and require a relatively short time for analysis.
References
1. Sandberg AA (1990) The chromosomes in human cancer and leukemia, 2nd edn. Elsevier Science, New York
2. Sandberg AA, Bridge JA (1994) The cytogenetics of bone and soft tissue tumors. Landes, Austin
3. Sandberg AA, Bridge JA (2002) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. Syn- ovial sarcoma. Cancer Genet Cytogenet 133:1–23
4. Sandberg AA (2004). Updates on the cytogenetics and molecu- lar genetics of bone and soft tissue tumors. Liposarcoma. Can- cer Genet Cytogenet 155:1–24
5. Sandberg AA (2004) Updates on the cytogenetics and molecu- lar genetics of bone and soft tissue tumors. Lipoma. Cancer Genet Cytogenet 150:93–115
6. Sandberg AA, Bridge JA (2000) Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors. Ewing sarcoma and peripheral primitive neuroectodermal tumors.
Cancer Genet Cytogenet 123:1–26
