Multiple ancillary molecular techniques are available to pathologists. Each technique carries unique strengths and weaknesses. It is important to be familiar with these features as they may influence which assay is best for a given situation. Cytogenetic karyotyping has been long considered the gold standard in detecting genetic aberrations , and has given rise to many of the discoveries today [6]. Fresh tissue is obtained so that tumor cells can be grown and then processed so that metaphase chromosomes can be examined (Fig. 3.2). Fluorescently labeled probes can sometimes be applied to identify the chromosomes (also known as spectral karyotyping) or to detect rearrangements of specific genes (see below for fluorescence in situ hybridization). An advantage of this technique is that it requires no prior knowledge of the genetic aberration and thus can detect a wide range of translocations and chromosomal abnormalities. This is particularly useful when dealing with difficult-to-diagnose mesenchymal tumors and where the identification of an unexpected translocation may provide clues to further classification, such as unusual histological or immunohistochemical variants. However, this technique requires fresh tissue, which is not always available. Lengthy processing time, expert technical staff, complex interpretation, bacterial contamination or normal tissue overgrowth, and associated costs can be negative issues. Finally, the technique cannot detect small mutations and translocations where the genes are in close proximity to each other (also known as cryptic translocations.) As a result, conventional karyotyping has largely given way to faster molecular techniques which have been optimized for formalin-fixed paraffin-embedded (FFPE) tissue .

One of the most widely used molecular technique for detecting translocations is fluorescence in situ hybridization (FISH) because of its robustness and broad utility in frozen, FFPE and cytological tissues. In this technique, fluorescently labelled DNA probes are applied to unstained slides. Interphase nuclei are examined under fluorescence microscope [6]. The most commonly available FISH assays are break-apart assays. In break-apart assays, two DNA probes flank a single gene of interest (Fig. 3.3). For example, in the EWSR1 break-apart assay, two DNA probes flank the EWSR1, one in the 5ʹ end labeled with a probe which will fluorescence orange and one in the 3ʹ end with a probe which will fluorescence green. In tumors where EWSR1 is not rearranged, both orange- and green-labeled probes co-localize to produce a yellow signal because of their spectral overlap when in close proximity to each other. In contrast, tumors where EWSR1 is rearranged will have split signals, one separate orange and one separate green signal. FISH assays with fusion probes will also have two DNA probes but one for every involved genes. For example, in a fusion FISH assay for Ewing sarcoma, one probe will be bound to the 5ʹ EWSR1 end, while a second probe will be bound to the 3ʹ end of FLI1. In contrast to the break-apart assays, tumors without rearrangement will have split signals, while tumors with rearrangements will have both probes next to other, co-localizing into a single yellow signal. In practice, break-apart FISH is a much more common approach as it covers a wider range of events with a single set of probes. The strengths of using FISH include its robustness in multiple tissues; most notably in FFPE tissue, the most commonly available tissue. Because it is in situ, the test is interpreted within tumor nuclei, something not possible in polymerase chain reactions (PCRs; see below). Furthermore, it is much faster than karyotyping as it does not require time for cells to grow. One disadvantage of FISH is that prior knowledge of a gene to target is needed. Furthermore, most commercially available FISH are break-apart assays that do not provide information on partner. Therefore, it cannot be used to differentiate similar appearing entities which have the same gene involved. For example, Ewing sarcoma and desmoplastic small round cell sarcoma are both small round cell tumors and both have rearrangements involving EWSR1, though with different partners. Break-apart FISH for EWSR1 cannot be used to distinguish between these two entities. Another disadvantage is that multiplexing (ability to screen multiple gene aberrations at the same time) is limited as each assay typically requires an unstained slide, and is compounded by the fact that only a few DNA probes are commercially available .

Polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) have become common ancillary diagnostic assays for mesenchymal tumors [6]. DNA primers flank the gene or fusion gene of interest, are amplified several fold and sequenced to determine if mutation is present or not (Fig. 3.4). In RT-PCR , fusion transcript RNA is converted to DNA (cDNA) first. The major advantage of this technique is that it detects a specific gene fusion, providing information on both gene partners. This is important when knowledge of partner genes is needed to differentiate between two similar appearing tumors. As mentioned earlier, break-apart FISH for EWSR1 would not be useful to distinguish between Ewing sarcoma and desmoplastic small round cell tumor. However, RT-PCR would be able to discriminate between Ewing sarcoma (EWSR1-FLI1 or EWSR1-ERG) and desmoplastic small round cell tumor (EWSR1-WT1), because it can specify fusion partners. Also, RNA extracted from unstained slides can be used to do multiple reactions. The major disadvantage of this technique is that it is not as robust as FISH. RNA is susceptible to degradation particularly in older FFPE blocks and decalcification. In addition, the majority of laboratories have assays which only detect the most common fusion transcript types and variants. A fusion transcript type is created between variations in breakpoints between the same genes, each of which is designated numerically as a type. For example, in Ewing sarcoma, there are multiple EWSR1-FLI1 fusion types, with types I and II being most common. A fusion transcript variant is created with different genes. In Ewing sarcoma, EWSR1 is most commonly paired with FLI1; however, it can also be paired with ERG, FTV1, and FEV among others to yield other variants. Therefore, it is possible that a less common variant or type is missed because the assay is not designed to detect them. One particular problematic tumor is dermatofibrosarcoma protuberans (DFSP) because it has multiple types due to substantial variation in break points within COL1A1 which comprise more than 50 exons. Therefore, multiple primers are needed to cover these many types [26]. Similar to FISH, commercial laboratories only offer a selected few RT-PCR assays. Finally, lack of in situ testing, raises the possibility that contamination can occur. Therefore, it is important to be confident that the laboratory chosen for such studies has solid protocols and impeccable procedures .

An exciting development in molecular diagnostics is the increasing utilization of next generation sequencing techniques. While global RNA sequencing (RNAseq) is not ready for broad clinical application, semi-anchored approaches are being explored where primers specific for a common known gene is used with opposing nonspecific primers to theoretically identify any partner gene (Fig. 3.5). The bioinformatics for interpreting fusions is rapidly evolving and this is a growth area in molecular testing for translocations. Next generation sequencing can also be used to examine for broad panels of genes for point mutations, insertions, and deletions (and increasingly copy number alterations). These approaches have limited applications in sarcoma as the genomic properties of most sarcomas are poorly examined, but this is rapidly changing as new genomic analyses of sarcomas are being published regularly.