Histology means examination of the tissue on a thin slide stained with hematoxylin and eosin (HE) and remains the gold standard for diagnosis. Histology provides information about the morphology and the architecture of the lesion. Soft tissue tumors in most cases infiltrate diffusely, which is quite consistent for those tumors, in contrast to epithelioid neoplasms, where the cells usually form aggregates. There are of course exceptions to the rules, for instance, a biphasic synovial sarcoma contains also an epithelioid component that is composed of cell groups. On the other hand, an epithelioid neoplasm may also grow diffusely. Some tumors have a very characteristic architecture; examples are solitary fibrous tumor that shows a distinctive “patternless pattern,” schwannoma with also the distinctive Antoni A and Antoni B tissue, representing compact areas alternating with loosely arranged foci of spindle cells (Fig. 6.3) and alveolar rhabdomyosarcoma with discrete nests with discohesive cells. As expected there are atypical forms of these tumors making their diagnosis more difficult and challenging. Of utmost importance is that lymphomas can also infiltrate diffusely; however, lymphomas have other morphological characteristics and a different clinico-radiological presentation.

Cell type can be also recognized on histology. In general soft tissue tumors are composed of cells that are elongated or spindled and have eosinophilic cytoplasm and indistinctive cell borders (Fig. 6.4). The nucleus differs from case to case with the more aggressive and malignant tumors that demonstrate more pleomorphism with enlarged nuclei, abnormal nuclear membrane, and sometimes an evident (macro)nucleolus. Multinucleation may indicate an aggressive cell type, but one has to bear in mind that giant cells are also multinucleated and that some benign tumor cells can also merged into giant forms. Desmoid tumors exhibit at the edge of the lesion multinucleated cells with strong eosinophilic cytoplasm, which is nothing more than degeneration of entrapped muscle cells when the tumor infiltrates between striated muscle (Fig. 6.5).

Not all soft tissue tumors are composed of spindle cells. Some neoplasms have an epithelioid morphology, such as epithelioid sarcoma, making the diagnosis more complicated.

It is evident that beyond the morphology of the tumor and the cellular composition, there are also other elements that can lead to the diagnosis. Many neoplasms show a vascular pattern that in many cases is diagnostic for this neoplasm. Thus, a “chicken wire” vascular pattern is described in myxoid lipomas and liposarcomas and a hemangiopericytoma-like pattern in solitary fibrous tumor, while schwannomas show hyalinization of the vascular wall.

Histology serves not only in tissue-specific diagnosis but plays also an important role in grading the tumor by recognizing and counting, for instance, the mitotic activity of the cells as well as identifying necrotic areas (Fig. 6.6). On histology one can also estimate the resection margins of a specimen.

Special histochemical techniques can be used to reveal material in the cell itself as well as in the tumor background (e.g., mucin). In alveolar soft part sarcoma (Fig. 6.7), the cells contain rod-shaped intracytoplasmic crystals that can be easily demonstrated with PAS staining (Fig. 6.8), which is pathognomonic of the lesion.

As it is already mentioned, morphology is the gold standard of diagnosis. For many years the diagnosis is made solely on basis of the morphology. It is apparent that many of the subtypes that exist nowadays would not be recognized if there was no possibility of further examination. Immunohistochemistry uses antibodies that are specific to epitopes located on the cells of interest. The antibodies are not specific for malignancy with some exceptions that will be discussed further. That means antibodies indicate the type of cell differentiation in nonmalignant cells but also in tumor cells arising from this type, which will also stain for the same antibody. For instance, endothelial cells stain for CD31; thus, angiosarcomas will stain for the same marker. Combining morphology and immunohistochemistry may allow a more precise diagnosis. However, many tumors present nonspecific positive staining for markers of another differentiation lineage, and unfortunately most antibodies are not specific to one differentiation line. A typical example is S100, an antibody that is widely used in everyday practice. In soft tissue pathology, S100 is known for its strong and diffuse positivity in schwannomas (Fig. 6.9). It stains in neural crest-derived tissue (such as Schwann cells, melanocytes, glial cells), in adipocytes, chondrocytes, dendritic cells, Langerhans cells, macrophages, and myoepithelial and melanocytic cells. This makes things complicated because an undifferentiated tumor with S100 positivity comprises a wide differential diagnosis. Furthermore, tumor cells can lose the antibody expression that reveals the differentiation line, making sometimes the diagnosis impossible or one of exclusion. Less than half of the malignant peripheral nerve sheath tumors (MPNSTs) stain positive for S100, and the staining is usually focal. Diffuse staining for S100 is rarely compatible with conventional MPNSTs and should raise the possibility of other tumors [2].

It is thus of paramount importance to have immunohistochemical markers that are specific and sensitive for the different lines of cell differentiation or for the different tumor types. Nowadays, new antibodies have been added in the armamentarium of a soft tissue pathologist. Some of the most important ones are anti-MDM2 and CDK4. Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) display amplification of MDM2 and CDK4 genes that are located in chromosome 12q13-15. By immunohistochemistry, overexpression of MDM2 and CDK4 is indicated by nuclear staining for the corresponding antibodies (Fig. 6.10). This positivity is very sensitive for ALT/WDLPS and DDLPS and shows a strong correlation with the gene amplification status [33]. Still, as the majority of antibodies are used in pathology, they are not specific to these entities. It has been shown that intimal sarcomas of pulmonary artery show consistent genetic alteration (gains and amplifications in the 12q13-14 region) and overexpression of the MDM2 gene [34]. Very recently it has been demonstrated that almost half of the MPNSTs exhibit loss of histone H3K27 trimethylation (H3K27me3) which can be highlighted immunohistochemically by loss of nuclear staining for the corresponding antibody. H3K27me3 loss although not very sensitive is a highly specific marker for malignant peripheral nerve sheath tumor, and immunohistochemistry may be useful in differential diagnosis from other high-grade spindle cell sarcomas [35].

There are many novel antibodies with very promising results in defining diagnosis, but one has to be aware of their limitations, as most of them are not entirely specific to one entity or differentiation line.

Jason Hornick et al. separated the novel antibodies into three categories: (1) lineage-restricted transcription factors, (2) protein correlates of molecular alterations, and (3) diagnostic markers identified by gene expression profiling [36], emphasizing on a close correlation of the immunohistochemical profile to the cytogenetic and molecular events in the tumors (Table 6.1) (Figs. 6.9, 6.10, 6.11 and 6.12).

Over the last few decades, remarkable advances are made in the understanding of molecular biology of the tumors. This paved the way for improving our diagnostic effectiveness, by defining the underlying genes and the corresponding pathways involved in tumorigenesis. Already in the previous WHO edition of 2002, 11 % of all benign and malignant tumors presented with a karyotypic abnormality, whereas an additional 19 % was also described in the corresponding molecular findings [37].

Many soft tissue tumors harbor a recurrent chromosomal translocation caused by rearrangement of part of genes between nonhomologous chromosomes. This event lead to development of fusion genes which in turn encodes altered proteins that are oncogenic. The most extensively studied translocations relate to Ewing sarcoma. The majority of these sarcomas demonstrate rearrangement between chromosomes 11 and 22, namely, a t(11;22)(q24;q12) [38] or a t(11;22)(q22;q12) [39] rearrangement that results in EWSR1-FLI1 or EWSR1-ERG fusion gene, respectively. The EWSR1 gene located in the q12 domain of chromosome 22 can rarely present translocations with different partner genes located at different chromosomes resulting in a number of other fusion proteins which are also described in Ewing sarcoma [40]. In addition, EWSR1 gene translocations are involved in a variety of non-Ewing sarcomas. For instance, myxoid liposarcoma can show a t(12;22)(q13;q12) reciprocal translocation resulting in a EWSR1-CHOP fusion protein [41].

Another molecular event that is observed in sarcomas is gene amplification. The most illustrative examples are atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS). These entities are characterized by amplified sequences in the region q14-15 of chromosome 12 where are located the murine double minute (MDM2) and the cyclin-dependent kinase 4 (CDK4) genes. Co-amplification of those genes represents the hallmark for ALT/WDLPS and DDLPS [42], although recently it has also been described in other entities as well, such as intimal sarcoma to name one [43].

A gene mutation is a permanent alteration in the DNA sequence that makes up a gene. It can affect a single base pair or larger segments of the DNA band. Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm that is believed to arise from or is differentiated toward interstitial cell of Cajal. It is proven that these neoplasms can harbor a somatic oncogenetically activating mutation. The vast majority demonstrate KIT (which is a proto-oncogene receptor tyrosine kinase) mutations on exon 11. This can be an insertion, a deletion, or a missense mutation. After this initial discovery, three other less frequent hot spots came into light, regarding exons 9, 13, and 17 [44]. PDGFRA gene encodes platelet-derived growth factor receptor A. A small percentage of GIST that does not show KIT mutations can demonstrate a PDGFRA mutation. The exons involved in this case are 12, 14, and 18 with the last being the most common one [44]. Both KIT and PDGFRA are driver mutations and are presumed to be the initiating oncogenic event. These mutations are mutually exclusive, namely, when one happens, the other is not present. The overall mutation frequency for KIT and PDGFA is 86 % with 14 % being wild type. Nowadays it is known that many of those wild-type GISTs contain another mutation, with most extensively described the BRAF V600E mutation [44].

From a genetical point of view, sarcomas are divided in two groups. First is the group with a simple karyotype that presents one main genetic alteration. As previously described, this alteration can be either a somatic mutation, or a gene amplification, or a recurrent translocation, or even an intergene deletion. Second are those presenting with more complex karyotypes [37, 45–48]. This last group represents almost two-thirds of soft tissue sarcomas and shows aberrant chromosomal events but no recurrent reciprocal translocations. Most demonstrate mutations involving p53 gene and retinoblastoma gene [45, 46, 48].

Three main technical approaches are nowadays used in clinical practice regarding soft tissue tumors. Those are conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) [37, 45].

Conventional cytogenetic analysis or simply karyotyping aims to detect numerical and/or large structural chromosome abnormalities in metaphase cells. Both primary and secondary changes can be identified. This study is limited to fresh, not fixated, sterile tumor tissue and demands special culture for the tumor cells to grow.

FISH detects the presence, absence, relative positioning, and/or the copy number of specific DNA segments by fluorescence microscopy. It can be performed on either fresh or formalin-fixed and paraffin-embedded (FFPE) tissue. In contrast to the karyotyping, this method requires the knowledge of the specific target examined. FISH testing uses dual-color/fusion or dual-color/break-apart probes to detect specific rearrangements, involving a variety of translocation events (Fig. 6.13). There are also the locus-specific probes coupled with a control probe usually pointing the centromere of the chromosome and aim to identify gene amplifications or losses.

RT-PCR uses specific primers to copy or amplify a small section of a DNA or RNA sequence. It is quick and simple and can be performed on either snap-frozen or FFPE material. Its role in soft tissue pathology is to identify chimeric or fusion genes as well as oncogenic mutations.

Next-generation sequencing (NGS) takes nowadays more and more part in molecular biology of human tissue. It is a high-throughput DNA sequencing technique that aims to investigate simultaneously in the same specimen a large number of mutation genes that are proven to cause tumorigenesis. It can be performed on FFPE tissue which makes this technique even more applicable. The advantage of this technique is that different genetic aberrations can be tested simultaneously. This technique requires high-quality DNA/RNA.