Mutations affecting a single nucleotide (SNV) or insertion/deletion (indel) of up to 10,000 nucleotides are often referred to as small genetic variants. Such mutations are very common in neoplasia, as well as in constitutional DNA. Actually, any individual is thought to deviate from the reference genome at >4 million sites and to have close to 10,000 larger structural variants, including >5000 deletions and duplications, affecting hundreds of genes. ^, Some SNVs are fairly frequent (at least 1% of the population), so-called single nucleotide polymorphisms (SNPs), and may confer increased risk for certain diseases. Not unexpectedly, neoplastic cells may harbor thousands of SNVs and indels that are not seen in the corresponding constitutional DNA. These neoplasia-restricted mutations can be subgrouped into distinct signatures, correlating with different types of DNA damage and/or deficient DNA repair or replication mechanisms. The vast majority of these mutations are located outside the coding regions which, however, does not exclude that they may have a significant impact on tumorigenesis; good examples are the SNVs in the promoter region of the TERT gene that increase the affinity for certain transcription factors, a common finding in myxoid liposarcoma and solitary fibrous tumor. ^, Still, it is mutations that lead to protein-level changes that have attracted the most attention. SNVs could result in the exchange of a single amino acid (so-called nonsynonymous exonic variants), introduce a premature stop codon, or abolish a splice recognition site. Similarly, indels could lead to frameshift mutations, truncated proteins, or proteins with novel amino acid sequences. Consequently, SNVs and indels can activate oncogenes, as well as inactivate tumor suppressor genes. Only a minority of all the exonic nonsynonymous SNVs and indels that are found in neoplasms have a major impact on tumor development (driver mutations), while the remaining mutational load represents mutations occurring prior to neoplastic transformation or noise caused by increased genetic instability (passenger mutations). Several bioinformatic tools to predict the functional outcome of a given mutation have been developed and numerous databases, such as COSMIC ( http://cancer.sanger.ac.uk/cosmic ), list reported mutations by gene and/or disease, greatly improving the interpretation of detected mutations. There are also a number of databases in which the therapeutic aspects of a given variant can be evaluated, such as OncoKB. However, many mutations can still be difficult to evaluate (variants of unknown significance, VUS) and may require functional analysis in an experimental system.

Genome-wide sequencing efforts at the exome and genome levels initially focused on common epithelial malignancies, such as carcinomas of the breast, colon, and lung. When similar large-scale studies were done on soft tissue sarcomas, some interesting differences were noticed. First, the tumor mutation burden (TMB) is in general much lower in sarcomas than in carcinomas, ^, a difference that could reflect that many carcinomas develop through a gradual transformation of a normal cell, whereas most sarcomas seem to start as malignant lesions, without a recognizable dysplastic or benign precursor lesion. Another possible explanation is that carcinomas develop from stem-like progenitor cells that have undergone numerous cell divisions, each of which may generate SNVs and indels, prior to transformation while the turnover of putative mesenchymal progenitor cells is much slower. Second, many sarcomas seem to have few and infrequent recurrent mutations, a phenomenon that may be explained by the presence of other strong driver mutations (see below). However, there are many important exceptions, such as the frequent KIT and PDGFRA mutations in gastrointestinal stromal tumors, RAS signaling pathway mutations in embryonal rhabdomyosarcoma, and MYOD1 mutations in spindle cell rhabdomyosarcoma. It should be kept in mind, though, that both whole-exome and whole-genome sequencing analyses of sarcomas are still comparatively few. Benign soft tissue tumors are even less extensively investigated with regard to somatic SNVs and indels. Frequent and recurrent mutations have so far been observed in only a handful of benign lesions, e.g., of CTNNB1 in desmoid fibromatosis, NF2 and SOX10 in schwannoma, and PDGFRB and NOTCH3 in various pericytic neoplasms.

Notwithstanding that SNVs and indels presently seem to have a limited impact on soft tissue tumor diagnostics, they may provide important information on treatment response. The prime example is, of course, gastrointestinal stromal tumor. Here, not only the absence or presence of mutations, but also their precise location, in the KIT and PDGFRA genes show strong correlation with response to treatment with different tyrosine kinase inhibitors. ^, Even if actionable mutations are otherwise rare in sarcomas, targeted treatment based on the presence of certain mutations could potentially improve outcome. ^{, ,} Hence, in patients responding poorly to conventional treatment mutational screening should be considered.

The term chromosomal imbalance is here used to refer to a structural or numerical rearrangement resulting in a quantitative deviation from the normal diploid state. Such chromosomal imbalances may theoretically range from gain or loss of a single nucleotide to whole chromosomes, but it is reasonable to reserve the term for rearrangements affecting at least an entire gene; in molecular genetic studies of constitutional variants, copy number alterations ranging in size from 1000 (1 Kb) to 1 million (1 Mb) nucleotides are often referred to as copy number variations (CNVs). Whereas the effect on the expression level of an individual gene may be the same irrespective of whether it was affected by a numerical or structural rearrangement, the overall pattern of chromosomal imbalances in a tumor is clearly of both diagnostic and biological interest. It is well known from both cytogenetic and molecular analyses that certain tumors preferentially display numerical changes, while others usually have segmental losses and gains. The reasons for this diversity are largely unknown, but an important step in the efforts to understand the causes of these differences was the recent introduction of a model for describing copy number changes in a reproducible way, on the basis of copy number segments in the tumor genome: their lengths and copy number states together with their allele status (i.e., whether one of the parental alleles is lost or not, also known as loss of heterozygosity, LOH; Fig. 4.1 ). Using these parameters, both sequencing and genomic array data can be used to define a set of copy number signatures which, in turn, are nonrandomly distributed among different types of neoplasia.

Copy Number Changes.

Multiple chromosomal imbalances detected by high-resolution SNP array analysis of DNA from an embryonal rhabdomyosarcoma with a near-triploid chromosome number. The upper part of the image shows the distribution of tumor DNA with regard to allelic ratio (y-axis) and copy number (x-axis). The lower part shows the average log ratios and allele frequencies for all chromosomes, starting with the end of the short arm of chromosome 1 to the left and ending with the end of the long arm of the X-chromosome to the right. By combining the data, it can be concluded that there are either two (e.g., chromosomes 3 and 4) or four (e.g., chromosomes 2 and 8) copies of most chromosomes, that there are two maternal and two paternal copies of the tetrasomic chromosomes, but uniparental isodisomy for the disomic chromosomes, and that larger structural rearrangements are restricted to chromosomes 2, 15, and 17.

While numerical chromosomal aberrations are poorly tolerated at the organism level, they are common in tumors, ranging from gain or loss of individual chromosomes (aneusomy) to gain or loss of one or more copies of the entire genome (aneuploidy) ; chromosome counts as low as 23 and as high as >400 have been detected at chromosome banding analysis of tumors, and a similar range in modal chromosome number has been found using genomic arrays or MPS-based approaches. In soft tissue tumors, numerical chromosome aberrations are found in close to two-thirds of all cases that have been subjected to chromosome banding analysis, being much more common among sarcomas than among benign soft tissue tumors—around 90% versus one-third. The difference between benign and malignant soft tissue tumors becomes even more obvious when considering only tumors with chromosome numbers below 45 or above 47; such chromosome numbers are seen in only 10% of the benign lesions but in two-thirds of sarcomas. Aneusomies probably arise through irregular/incomplete segregation of sister chromatids at anaphase, a phenomenon facilitated by a variety of neoplasia-associated disturbances of e.g., centrioles, centromeres, and telomere status, and tolerated through mutations in various caretaker genes. ^, Gain of one or more complete sets of all chromosomes, so-called polyploidization or whole-genome doubling, is strongly associated with malignancy and is most likely caused by accidental events such as abortive mitoses, cell fusions, or endoreduplication. ^, One or more whole-genome doubling events are particularly frequent in sarcomas with complex genomes, notably myxofibrosarcoma, malignant peripheral nerve sheath tumors, and undifferentiated pleomorphic sarcoma, but are occasionally seen also in fusion-associated sarcomas. ^, Also more extensive, meiosis-like, loss of chromosomes (near haploidization) has been observed in soft tissue tumors, notably inflammatory leiomyosarcoma/rhabdomyoblastic sarcoma, malignant peripheral nerve sheath tumor, and undifferentiated pleomorphic sarcoma ^, ; the mechanisms behind this phenomenon remain unknown.

The pathogenetic consequences of aneusomies are difficult to assess, as they affect hundreds to thousands of genes. In general, though, gene expression levels vary with the number of copies, i.e., trisomies and monosomies result in increased and decreased, respectively, expression of many, but not all, genes located on these chromosomes. In some cases the outcome of monosomies can be reduced to represent one step in the biallelic inactivation of one or more tumor suppressor genes. A good example is schwannoma, where inactivation of the NF2 gene, which maps to chromosome band 22q11, could be achieved through any combination of monosomy 22, partial deletions of chromosome arm 22q, and structural variants targeting NF2 specifically. In other tumors in which monosomy and partial deletions alternate, such as spindle cell lipomas, which display either monosomy 13 or partial deletions, with 13q14 as a minimal shared target, the remaining copy of chromosome 13 is typically intact, suggesting that loss of one copy (haploinsufficiency) is enough for tumorigenesis. Finally, some inactivating mutations, such as those affecting the CDKN2A , CDKN2B , and MTAP loci in chromosome band 9p22, typically do not affect larger segments of chromosome 9, indicating that loss of adjacent segments is deleterious. Gain of entire chromosomes is even more difficult to reduce to single gene effects, but the as yet limited data on this topic suggest that the salient effect(s) of a trisomy may indeed sometimes be attributable to small increases in gene expression levels of one or a few genes. Still, the significance of most characteristic numerical aberrations in soft tissue tumors, such as extra copies of chromosome 8 in embryonal rhabdomyosarcoma or trisomies occurring as secondary changes in, e.g., myxoid liposarcoma, synovial sarcoma, or infantile fibrosarcoma, remains elusive.

Also chromosomal imbalances resulting from structural rearrangements are very common in soft tissue tumors, especially in sarcomas. ^, For several subtypes, such as GIST, synovial sarcoma, and malignant peripheral nerve sheath tumor, there is compelling evidence for the notion that the overall number of structural rearrangements leading to imbalances could be used to predict patient outcome. Two types of chromosomal imbalance have attracted particular attention: homozygous deletions and gene amplifications. Homozygous deletions are relatively rare and do not always have to affect bona fide tumor suppressor genes ; some homozygous deletions are constitutional variants without phenotypic consequences. However, the pinpointing of homozygous deletions in tumors has been instrumental in detecting many of the classical tumor suppressor genes, such as RB1 and SMARCB1 . As men usually have only one X-chromosome, a single deletion event on this chromosome will have the same effect as a homozygous deletion on an autosome, i.e., complete loss of one or more genes. Several potential targets for such deletions on the X-chromosome have been described in soft tissue tumors, such as the DMD gene in myogenic sarcomas and sclerosing epithelioid fibrosarcoma, or the ATRX gene in undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma. Genes frequently deleted in soft tissue tumors are listed in Table 4.2 .

Gene amplification is a term that is poorly defined, but usually refers to a selective ≥3- to 5-fold gain of a DNA sequence relative to adjacent sequences on the same chromosome. Different types of chromosome rearrangement are associated with gene amplification, the most common being double minutes (dmin), homogeneously staining regions (hsr), and ring chromosomes, all arising through different mechanisms. The presence of hsr or dmin is strongly associated with a malignant phenotype, whereas ring chromosomes are found in both high-grade malignant and benign/low-grade malignant soft tissue tumors; the latter tumors, however, have a clear propensity for transforming into high-grade tumors, probably due to the intrinsic mitotic instability of ring chromosomes. Ring chromosomes are particularly common among atypical lipomatous tumor/well-differentiated liposarcoma/dedifferentiated liposarcoma (ALT/WDLS/DDLS) and dermatofibrosarcoma protuberans, where they occur in 75% and 50% of the cases, respectively, and often are seen as the sole cytogenetic aberration. ^, The chromosomal organization of amplicons is not always readily apparent from sequencing data, making direct comparisons with cytogenetic findings difficult. However, so-called seismic amplification, which likely corresponds to amplification through ring formation, has been described in the vast majority of ALT/WDLS/DDLS.

The gene content of amplicons is of considerable clinical interest, both with regard to diagnostics and treatment. For instance, the amplicons in ALT/WDLS/DDLS always contain material from chromosome arm 12q, and invariably include the MDM2 gene in band 12q15. Although MDM2 is amplified in other sarcomas, notably intimal sarcoma, its consistent amplification and overexpression in ALT/WDLS/DDLS makes it an excellent marker in the differential diagnostics of lipomatous tumors. Furthermore, CDK4 , which is typically coamplified, is a putative treatment target. Many other sarcomas, especially those with complex genomes, display amplicons through dmin and/or hsr formation. ^{, ,} Recurrently (at least 5%–10% of the cases) amplified segments and genes in soft tissue sarcomas are shown in Table 4.2 .

Another type of chromosomal imbalance that does not change the gene copy number is uniparental isodisomy (UPiD), which may affect entire chromosomes or parts of chromosomes. For UPiDs to occur, one normal copy has to be lost, followed by duplication of the remaining copy. The effect could be the unmasking of a recessive mutation or, if imprinted genetic loci are involved, deregulated expression of genes inherited from the mother or father. The most commonly affected chromosomal region in soft tissue tumors, as well as in a variety of pediatric malignancies, is 11p. This chromosome arm contains a set of paternally and maternally imprinted loci in band 11p15, including the IGF2 gene, which is expressed exclusively on the copy inherited from the father. Various combinations of loss of the maternal copy of 11p, with or without duplication of the paternal copy, are common in, e.g., embryonal rhabdomyosarcoma.

Finally, it should be emphasized that the functional outcome of some imbalances, in particular deletions, does not necessarily have to be the copy number change as such. Each structural rearrangement resulting in loss or gain of chromosomal material could also lead to the juxtaposition of two genes, one in each breakpoint. Indeed, the HAS2 :: PLAG1 fusion in lipoblastoma is typically created through an interstitial deletion of the intervening sequences on chromosome 8.

Structural chromosome rearrangements reshuffle the genetic material and may thus result in the juxtaposition of (parts of) two genes. This, in turn, may lead to the translation of a deregulated and/or chimeric protein ( Fig. 4.2 ). Such gene fusions have been described in all types of neoplasia, including benign as well as malignant lesions. ^{, ,} The pathogenetic impact is unknown for most of the gene fusions that have been reported in the literature, the vast majority of which were detected through large-scale deep sequencing studies. Many have only been described once and are accompanied by numerous other chromosome-level mutations and thus likely represent passenger events. Other fusions are repeatedly detected, are often restricted to one or a few morphological subtypes, and are associated with relatively few other mutations, all suggesting that they constitute strong driver mutations; this is especially true for soft tissue tumors, where characteristic gene fusions abound. The indirect support for an important role in tumorigenesis has, in a few cases, been further supported by results from in vitro studies and from experimental animal models, showing that the gene fusion, at least if it occurs in a permissive cellular context, is sometimes sufficient for malignant transformation. ^, It is important to emphasize, however, that several pathogenetically important gene fusions are present also in tumors with highly complex genomes, suggesting either that those gene fusions are weak transformers or that they facilitate the occurrence of other mutations. Finally, the strong impact that gene fusions can have on the tumor cells coupled with the fact that chimeric genes are specific for tumor cells, make them very attractive as potential targets for treatment. Indeed, pharmacological treatment of sarcomas displaying fusions that activate protein kinases, such as ALK or NTRK proteins, is already in clinical use. ^,

Gene Fusions.

A, Structural chromosome rearrangements, such as translocations or inversions, may result in the fusion or juxtaposition of two separate genes. B, Many gene fusions are due to intronic breakpoints in two genes, yielding in-frame fusions at the transcript level. Green boxes represent introns. C, A common pathogenetic mechanism, here illustrated by a fusion of exon 18 of the VCL gene with exon 12 of the RET gene in a case of lipofibromatosis, is transcriptional up-regulation of the kinase domain-encoding part of the 3′-partner. During RNA sequencing, the increased expression, driven by the new promoter from the VCL gene, of the distal part of RET is evident from the increased number of reads ( yellow spikes) for the exons included in the fusion.

The first gene fusions to be detected in soft tissue tumors were EWSR1 :: FLI1 in Ewing sarcoma in 1992 and FUS :: DDIT3 in myxoid liposarcomas in 1993. Since then, close to 800 different gene fusions have been found, involving almost 1000 different genes; of these, however, less than 75 have been confirmed, i.e., described in more than one publication. Gene fusions estimated to occur in at least 10% of a specific tumor type, or being of particular clinical relevance, are shown in Table 4.3 .

Table 4.3

Recurrent Gene Fusions in Soft Tissue Tumors ^a

Gene Fusion	Tumor type b	Frequency	Comment
ACTB :: FOSB	Pseudomyogenic hemangioendothelioma	50%
ACTB :: GLI1	Distinctive nested glomoid neoplasm Pericytoma	>20% >90%	Variant 5′-genes described Variant 5′-genes described
AHRR :: NCOA2	Soft tissue angiofibroma	65%	Rare variant with NCOA3 as 3′-gene
ASPSCR1 :: TFE3	Alveolar soft part sarcoma	>95%	Variant 5′-genes described
BCOR :: CCNB3	BCOR -rearranged sarcoma	20%	Rare variant with MAML3 as 3′-gene
CIC :: DUX4	CIC -rearranged sarcoma	20%–30%
CIC :: FOXO4	CIC -rearranged sarcoma	<10%
CIC :: NUTM1	CIC -rearranged sarcoma	10%–20%
CITED2 :: PRDM10	SCFT	>40%
COL1A1 :: PDGFB	Dermatofibrosarcoma protuberans b	>95%
COL3A1 :: PLAG1	Lipoblastoma	>10%
COL6A3 :: PDGFD	Dermatofibrosarcoma protuberans	<10%	More frequent in breast lesions
CSF1 ::	Tenosynovial giant cell tumor	>70%	Multiple variant 3′-genes, incl COL6A3
EP400 :: PHF1	Ossifying fibromyxoid tumor	45%	Variant PHF1 fusions described
ETV6 :: NTRK3	Infantile fibrosarcoma	>90%	Variant 5′-genes described
EWSR1 :: ATF1	Clear cell sarcoma Angiomatoid fibrous histiocytoma	>80% <10%	Rare variant with FUS as 5′-gene
EWSR1 :: CREB1	Clear cell sarcoma Angiomatoid fibrous histiocytoma Primary pulmonary myxoid sarcoma	>10% >80% >65%	More frequent in intestinal lesions
EWSR1 :: CREB3L1	SEF	>70%	Rare variants with FUS as 5′-gene or CREB3L3 or CREM as 3′-gene
EWSR1 :: CREB3L2	SEF	>10%
EWSR1 :: CREM	Angiomatoid fibrous histiocytoma AFH-like mesenchymal neoplasms	<10% Unknown	More frequent in intracranial lesions
EWSR1 :: FLI1	Ewing sarcoma	85%	Rare variants with ERG, ETV1or ETV4 as 3′-gene
EWSR1 :: NR4A3	Extraskeletal myxoid chondrosarcoma	70%	Variant 5′-genes described
EWSR1 :: SMAD3	Fibroblastic spindle cell tumors	Unknown
EWSR1 :: TFCP2	Rhabdomyosarcoma	Rare
EWSR1 :: WT1	Desmoplastic small round cell tumor	>95%
FN1 :: ACVR2A	Synovial chondromatosis	>50%
FN1 :: EGF	Calcifying aponeurotic fibroma	>80%
FN1 :: FGF1	Phosphaturic mesenchymal tumor	<10%
FN1 :: FGFR1	Phosphaturic mesenchymal tumor	40%
FUS :: CREB3L2	LGFMS Hybrid LGFMS/SEF	90% >90%	Rare variants with EWSR1 as 5′-gene or CREB3L1 as 3′-gene
FUS :: DDIT3	Myxoid liposarcoma	95%	Rare variant with EWSR1 as 5′-gene
FUS :: TFCP2	Rhabdomyosarcoma	Rare
GTF2I :: NCOA2	Angiofibroma of soft tissue	>10%
HAS2 :: PLAG1	Lipoblastoma	>10%	Multiple variant 5′-genes, incl COL1A2 , COL3A1 , RAB2A , and SRSF3
HEY1 :: NCOA2	Mesenchymal chondrosarcoma	>80%
HMGA2 ::	Lipoma	>50%	Multiple variant 3′-genes, incl ACKR3 , LPP , and NFIB
MED12 :: PRDM10	SCFT	>40%
MIR143 :: NOTCH2	Glomus tumor	35%	Variants with NOTCH1 /- 3 as 3′-gene or other 5′-genes described. More common in benign lesions
MYH9 :: USP6	Nodular fasciitis	75%
NAB2 :: STAT6	Solitary fibrous tumor	>95%
PAX3 :: FOXO1	Alveolar rhabdomyosarcoma	65%	Variants with NCOA1 , NCOA2 or other 3′-gene described
PAX3 :: MAML3	Biphenotypic sinonasal sarcoma	>50%	Rare variants with FOXO1 , NCOA1 , or other 3′-gene described
PAX7 :: FOXO1	Alveolar rhabdomyosarcoma	20%
PDGFRA :: USP8	Calcified chondroid neoplasms	Unknown
PRRX1 :: NCOA1	Benign fibroblastic tumors	Unknown	Rare variants with NCOA2 as 3′-partner
SERPINE1 :: FOSB	Pseudomyogenic hemangioendothelioma	50%	Rare variant with EGFL7 as 5′-partner
SH3PXDA :: HTRA1	Schwannoma	5%–10%
SRF :: RELA	Cellular myofibroma	50%	Multiple variant 3′-genes described
SS18 :: SSX1	Synovial sarcoma	60%	Rare variants with SSX4 or other 3′-gene described
SS18 :: SSX2	Synovial sarcoma	35%
TAF15 :: NR4A3	Extraskeletal myxoid chondrosarcoma	20%
TEAD1 :: NCOA2	Congenital spindle cell rhabdomyosarcoma	20%
TRIO :: TERT	Dedifferentiated liposarcoma	10%	Also described in pleomorphic sarcomas
:: USP6	Cellular fibroma of tendon sheath Nodular fasciitis	>80% >90%	Multiple variant 5′-genes, incl COL3A1 , MYH9 , and PPP6R3 Multiple variant 5′-genes, incl ASPN and MYH9
VGLL2 ::	Spindle cell rhabdomyosarcoma	30%	Multiple variant 3′-genes, incl CITED2 and NCOA2
:: VGLL3	Hybrid schwannoma-perineurioma Spindle cell rhabdomyosarcoma	Unknown Unknown	Variant 5′-genes described Frequent in head and neck lesions. Variant 5′-genes described
WWTR1 :: CAMTA1	Epithelioid hemangioendothelioma	>90%	Rare variant 3′-genes, incl MAML2
YAP1 :: KMT2A :: YAP1	SEF-like sarcoma	Unknown
YAP1 :: TFE3	Epithelioid hemangioendothelioma Clear cell stromal tumor of the lung	Rare 90%
ZFP36 :: FOSB	Epithelioid hemangioma	15%	Variant 5′-genes described
ZFTA :: MRTFB	Chondroid lipoma	>90%	Variant 3′-gene described

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