chapter 17

Soft Tissue

Few areas in cytology arouse such passionate discourse as fine-needle aspiration (FNA) of soft tissue masses. Critics jump at the opportunity to point out the limitations of the technique but pepper their few valid arguments with outdated fallacies and academic concerns that do not necessarily consider the best interests of individual patients. Over the years, as needle core biopsy has largely replaced incisional biopsy as the primary diagnostic modality for soft tissue lesions, FNA has begun to be accepted as an adjuvant tool to assess the adequacy of the needle core biopsy, as an essential element for proper triage of precious small biopsy tissue and as an alternative diagnotic tool in certain settings.

One undeniable difficulty with soft tissue FNA is the lack of experience most cytopathologists have with the greater than 130 different soft tissue lesions, including more than 30 types of sarcoma.¹ Because the morphologic heterogeneity is so astounding, it has been suggested that at least five soft tissue lesions ought to be sampled weekly to maintain practitioner experience.² With sarcomas constituting only 0.6% of all malignancies in the United States,³ this recommendation is unrealistic for most cytopathologists. Although it might be desirable to restrict FNA of primary soft tissue lesions to centers with cytologic expertise and the capability for a multidisciplinary approach, most patients are first seen in community practices. A systematic approach to differential diagnosis, specimen triage, recommendations, and referrals can enhance the efforts of the cytopathologist, regardless of his or her level of experience. For this reason, this chapter focuses on pattern recognition and is organized into sections on adipocytic, myxoid, spindle cell, fibrohistiocytic, round cell, epithelioid, and pleomorphic neoplasms.

Sampling error is a problem FNA shares with core and even incisional biopsies.4–6 Ancillary techniques such as immunohistochemistry and cytogenetics are often indispensable for definitive classification.7–10 Needle tract seeding, a concern in the era of larger needles, is exceedingly rare with FNA.¹¹ To prevent this potential complication, use of a single needle insertion point is recommended for several passes in cases with a high suspicion for sarcoma.¹²

FNA is a useful screening tool for evaluating soft tissue masses. It is the least invasive method for sampling a heterogeneous lesion, does not compromise tissue planes for a subsequent excision, and is cost-effective.13,14 Although not widely accepted by most soft tissue pathologists as the sole sampling modality for the primary classification of sarcomas, it plays an important role in triaging patients—the initial clinical differential diagnosis for many soft tissue masses is broad in scope and might include lymphoma and even metastatic carcinoma. FNA can be indispensible as the initial step toward narrowing the possibilities. FNA is especially valuable for confirming a recurrence or metastasis of a sarcoma.^15–17 With recent advances in our understanding of mesenchymal lesions and the availability of an increasing number of ancillary diagnostic tests, FNA is suitable for sampling a growing number of primary soft tissue tumors, including most small round cell tumors, which have well-defined cytomorphologic, immunophenotypic, and/or cytogenetic features.¹⁸

The location, size, and degree of invasiveness of a lesion and its relationship to surrounding structures are important clues to the correct diagnosis. With a few exceptions, benign lesions are generally small, circumscribed, and subcutaneous or superficial masses, whereas malignant lesions are more often large (greater than 5 cm), infiltrative, and deep-seated (inter- or intramuscular or retroperitoneal).2,13–15,19–22 Patients with a benign mesenchymal mass lesion, which is at least 100 times more common than a sarcoma, benefit the most from a definitive benign dignosis provided by FNA, the fastest, least expensive, and least invasive diagnostic procedure.

Advantages of fine-needle aspiration for soft tissue lesions

• technical ease

• less invasive; less morbidity

• allows for immediate assessment of specimen adequacy and allocation of tissue for special studies

• provides suitable material for ancillary studies

• no contamination of tissue planes

• cost-effective

Most (87% to 100%) cases are correctly diagnosed as benign or malignant, with an average sensitivity and specificity for malignancy of approximately 95%.2,13,14,16,19,21,23,24 The distinction among lymphoma, carcinoma, and sarcoma has specificity rates for sarcoma of 54% to 98% and a positive predictive value of 91% to 99%.^16,23,25 Owing to the rarity of most soft tissue tumors, data on sensitivity and specificity for a specific entity are just emerging.^18,26 The three main reasons for a false diagnosis are sampling error, a technically limited specimen, and misinterpretation.¹³ The false-positive rate for soft tissue FNA is relatively low (0% to 5%). False-negative rates vary a little more (2% to 15%), but any clinically suspicious lesion should be further evaluated by biopsy.^{2,6,13,14,16,17,21,23}

Rapid evaluation of specimen adequacy at the time of the procedure, along with triage of tissue for ancillary studies, will decrease the rate of inadequate specimens.2,13–15,19,27

Summary of statistical performance characteristics of soft tissue fine-needle aspiration

• sensitivity rates: as high as 95%

• specificity for sarcoma: 54% to 98%

• false-positive rate: 0% to 5%

• false-negative rate: 2% to 15%

Specimen Collection and Preparation

Cytomorphologic evaluation should be based on both alcohol-fixed, Papanicolaou-stained preparations (for nuclear details) and air-dried, Romanowsky-type stained preparations (for cytoplasmic details and matrix material). Thinlayer preparations offer good nuclear detail, optimize results from aspirates carried out without the benefit of rapid evaluation, and can be used for adjunct studies, but cells can appear smaller, rounder, and falsely epithelioid, and useful background information (e.g., vascular patterns, chrondromyxoid material) is sometimes lost.2,14,24,28–32 Cell block preparations are especially helpful for immunohistochemical studies and for mini-tissue architecture. Unstained smears or cytospin slides are suitable for immunocytochemistry, fluorescence in situ hybridization (FISH), and even molecular studies. A concurrent core biopsy, taken with a larger gauge needle (18G or larger), is commonly obtained when imaging characteristics or the rapid, on-site evaluation of smears suggests a primary soft tissue tumor.

Specimen collection and preparation

• 22 to 25 gauge needles

• separate passes for ancillary studies

• alcohol-fixed, Papanicolaou-stained smears for nuclear detail

• air-dried, Romanowsky-stained smears for cytoplasmic and matrix details

• cell block and core needle sample for immunohistochemical studies

• air-dried, unstained cytospin slides for FISH studies

• limited use of thinlayer preparations for morphologic evaluation

Ancillary Studies

Ancillary techniques like immunohistochemistry, cytogenetics, and molecular genetics are often indispensable for the classification of soft tissue lesions. Electron microscopy is only occasionally used for assistance in classifying a poorly differentiated neoplasm or soft tissue tumor that exhibits specific ultrastructural features, like the Weibel-Palade bodies of vascular tumors.2,33,34 Cell block sections are preferred for immunohistochemical studies, but air-dried or alcohol-fixed direct smears and cytocentrifuge or thinlayer preparations can also be used. Flow cytometry is useful when lymphoma is in the differential diagnosis. Ideally, one makes a dedicated pass for each desired ancillary study.^23,34–37

An increasing number of reproducible, relatively specific cytogenetic abnormalities can be identified with conventional chromosomal analysis (karyotyping), FISH, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques (Table 17.1).^{10,21,23,38,39} Conventional chromosomal analysis offers the advantages of a full karyotype, but this is counterbalanced by its low sensitivity and longer turnaround time as compared with molecular methods such as FISH.^9,21,38,40

TABLE 17.1

SOFT TISSUE TUMORS, THEIR ASSOCIATED CHROMOSOMAL CHANGES, AND FLUORESCENCE IN SITU HYBRIDIZATION (FISH) PROBES

TUMOR	CYTOGENETIC ABERRATION(S)	FLUORESCENCE IN SITU HYBRIDIZATION PROBE(S)
Angiofibroma of soft tissue	t(5;8)(p15;q13)	N/A
Angiomatoid fibrous histiocytoma	t(12;16)(q13;p11) t(12;22)(q13;q12) t(2;22)(q34;q12)	FUS EWSR1 EWSR1
Alveolar soft part sarcoma	der(X)t(X;17)(p11;q25)	TFE3
Clear cell sarcoma	t(12;22)(q13;q12) t(2;22)(q33;q12)	EWSR1 EWSR1
Dermatofibrosarcoma protuberans/giant cell fibroblastoma	r(17;22)(q21;q13)/t(17;22)(q21;q13)	PDGFB
Desmoid fibromatosis	Trisomy 8 and 20	CEP8/CEP20
Desmoplastic small round cell tumor	t(11;22)(p13;q12)	EWSR1
Epithelioid hemangioendothelioma	t(1;3)(p36.3;q25)	N/A
Epithelioid sarcoma	t/der(22)(q11.2)	N/A
Ewing sarcoma	t(11;22)(q24;q12) t(21;22)(q22;q12) t(2;22)(q33;q12) t(7;22)(p22;q12) t(17;22)(q12;q12) t(20;22)(q13;q12)	EWSR1 EWSR1 EWSR1 EWSR1 EWSR1 EWSR1
Extraskeletal myxoid chondrosarcoma	t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;17)(q22;q11) t(3;9)(q11;q22)	EWSR1 NR4A3 NR4A3 NR4A3
Hibernoma	11q13 rearrangement	N/A
Inflammatory myofibroblastic tumor	2p23 rearrangement	ALK
Infantile fibrosarcoma	t(12;15)(p13;q26)	ETV6
Lipoblastoma	8q12 rearrangement or polysomy 8	N/A
Lipoma, chondroid	t(11;16)(9q13;p12-13)	N/A
Lipoma, ordinary	12q14.3 rearrangement	HMGA2
Lipoma, spindle cell and pleomorphic	Deletions of 13q and 16q	N/A
Liposarcoma, well-differentiated/dedifferentiated	Ring/giant marker chromosomes from chromosome 12q	HMGA2/MDM2
Liposarcoma, myxoid	t(12;16)(q13;p11) t(12;22)(q13;q12)	DDIT3/FUS DDIT3/EWSR1
Low-grade fibromyxoid sarcoma	t(7;16)(q34;p11) t(1;16)(p11;p11)	FUS FUS
Myoepthelioma, soft tissue	t(19;22)(q13;q12) t(1;22)(q23;q12) t(6;22)(p21;q12)	EWSR1 EWSR1 EWSR1
Myxoinflammatory fibroblastic sarcoma	der(10) t(1;10)(p22;q24)	N/A
Nodular fasciitis	t(17;22)(p13.1;q12.3)	N/A
Rhabdomyosarcoma, alveolar	t(2;13)(q35;q14) t(1;13)(p36;q14), double minutes 2q35 rearrangement	FOXO1 FOXO1 N /A
Rhabdomyosarcoma, embryonal	Trisomies 2q, 8, and 20 Loss of heterozygosity at 11p15	N/A
Schwannoma	Deletion of 22q	N/A
Synovial sarcoma	t(X;18)(p11;q11)	SS18
Tenosynovial giant cell tumor	t(1;2)(p13;q37)	N/A

N/A, Not available, i.e., either the probe is not commercially available or the gene is unknown.

From: Dal Cin P, Qian X, Cibas ES. The marriage of cytology and cytogenetics. Cancer Cytopathol 2013, with permission from Wiley.

FISH accurately labels targeted chromosomal regions, and virtually all cytologic preparations (cytocentrifuge preparations, thinlayer slides, and smears) are ideal substrates for FISH, because they contain intact cells and nuclei free of sectioning artifact and truncation. When the diagnostic chromosomal aberration is known for a suspected soft tissue tumor, FISH is preferred because the number of cells needed is far less than for conventional karyotyping.2,41 A FISH test is especially useful in the differential diagnosis of lipomatous tumors, myxoid tumors, and small round cell tumors. Because only a specified chromosomal abnormality is targeted, however, a negative result provides minimal information.^9,42

Reporting Terminology

As with other cytologic specimens, the use of general category headings on the report (e.g., benign, atypical, suspicious, positive, nondiagnostic), along with a descriptive interpretation, is useful for clarity of communication. Although histologic typing is desired whenever possible, when the findings are not conclusive for a specific entity, a descriptive interpretation with differential diagnosis is appropriate.2,15,16,21,23,43

The most important prognostic consideration with soft tissue tumors is the histologic grade.⁴⁴ Grading should only be attempted when a specific diagnosis can be unequivocally established, and the tumor is gradable based on cellularity, pleomorphism, mitoses, and necrosis. For many sarcomas, additional grading is not necessary, as a specific diagnosis itself is indicative of a grade: Ewing sarcoma, synovial sarcoma, and angiosarcma are high-grade malignancies, whereas well-differentiated liposarcoma and dematofibrosarcoma protuberans are low-grade ones. Some sarcomas are not readily gradeable, such as epithelioid sarcoma and clear cell sarcoma. If grading is applied, a two-tiered grading system of low versus high is probably sufficient in a majority of cases.^{2,13,17,44,45} Low-grade lesions exhibit mild nuclear atypia, minimal or absent necrosis, low cellularity with minimal nuclear overlap, and rare or absent mitoses (less than 3 per 10 high-power fields [hpf]). High-grade lesions show moderate to marked nuclear atypia, intermediate to high cellularity with conspicuous to prominent nuclear overlap, definite necrosis, and frequent mitoses.^17,45

The presence or absence of mitoses and necrosis is an objective finding worthy of separate mention in the report. A mitotic count based on field unit is rarely possible on cytologic preparations. A statement such as “mitoses are absent” or “mitoses are numerous” suffices to avoid falsely high or low counts.17,44

Reporting soft tissue fine-needle aspiration results

• general category

• histologic subtype or description with differential diagnosis

• grade

• mitoses

• necrosis

• ancillary study results

• notes and recommendations

Adipocytic and Lipogenic Neoplasms

Neoplasms with lipogenic differentiation occur over a broad age range, but malignant tumors occur almost exclusively in adults. Lesions with well-developed adipocytic morphology are difficult to classify accurately.⁴⁶ Some authors discourage the use of FNA for large, deep-seated, intramuscular or retroperitoneal lesions that radiographically appear predominantly fatty.²³ The diagnosis of a well-differentiated liposarcoma can be missed if the (focal) diagnostic areas are not sampled, and this has led to the suggestion that potentially lipogenic lesions need to be excised and thoroughly sampled for definitive diagnosis. This approach does not take into consideration the newer therapeutic options (tumors pretreated before surgery) or the value of ancillary studies (e.g., cytogenetics), which are useful in the classification of most fatty lesions (see Table 17.1).^{14,21–23,47}

Lipoma

Benign lipomas, occurring mainly in adults older than 30 years of age, are very common and account for about half of all soft tissue tumors. Most are slowly growing, subcutaneous or intramuscular tumors that occur over a wide anatomic distribution but spare the hands and feet; rarely exceed 10 cm in size; and manifest as soft, painless lumps. Retroperitoneal lipomas are exceedingly rare and usually need molecular/genetic confirmation. Benign-appearing adipose tissue in a retroperitoneal mass aspirate should evoke the differential diagnosis of well-differentiated liposarcoma, myelolipoma, angiomyolipoma, and retroperitoneal fibrosis.

Cytomorphology of lipoma

• small tissue fragments

• large, univacuolate adipocytes of uniform size

• small, bland nuclei without atypia

Smears are composed of small tissue fragments; isolated cells are rarely present. Larger fragments contain thin capillaries, and striated or atrophic muscle fibers can be seen in juxta- or intramuscular tumors. The large, univacuolate adipocytes are of uniform size and shape (Fig. 17.1). Their nuclei are small, round, and regular, with evenly distributed chromatin.^29,48 Scattered foamy macrophages with ingested lipid (called “lipophages”) are common in tumors with regressive changes (Fig. 17.2). Occasionally, myxoid stroma and metaplastic bone are seen.

Figure 17.1 Lipoma.
Benign lipomas show occasional capillaries, but they are not as numerous as in hibernomas (Papanicolaou stain).

Figure 17.2 Fat necrosis.
Multivacuolate histiocytes accompanying areas of fat necrosis or regressive change lack the more distinct, larger cytoplasmic vacuoles, well-demarcated cytoplasmic membranes, and scalloped nuclei of true lipoblasts (Papanicolaou stain).

Differential diagnosis of lipoma

• subcutaneous adipose tissue

• pleomorphic lipoma

• well-differentiated liposarcoma

Normal subcutaneous adipose tissue lacks the masslike clinical presentation of a lipoma, thus the distinction between the two is most confidently achieved when the cytopathologist performs the aspiration, especially under ultrasound guidance. Atrophic muscle fibers in some lipoma aspirates can mimic the floret cells of a pleomorphic lipoma, but atypical mononucleated stromal cells are absent. Lipomas lack the variable cell size and atypical stromal cells of well-differentiated liposarcoma.21,29,48–50

Hibernoma

The hibernoma is a rare benign tumor of brown fat that occurs predominantly in adults aged 20 to 50 years. The most common site is the thigh, followed by the trunk/chest, upper extremity, and head and neck. Although most often subcutaneous, it can be intramuscular and large (greater than 10 cm), mimicking an atypical lipomatous tumor radiologically.⁵¹ It can also occur in the intra-abdominal cavity, mediastinum, and retroperitoneum.

Cytomorphology of hibernoma

• large tissue fragments containing many delicate capillaries

• numerous hibernoma cells with multiple small cytoplasmic vacuoles

• variable cytoplasm ranging from granular to microvesicular to containing larger fat vacuoles

• small, bland nuclei

Hibernomas yield moderately cellular aspirates of fatty tissue fragments containing many delicate capillaries and numerous hibernoma (brown fat) cells of variable size. These round to polygonal cells have finely multivacuolate to granular cytoplasm and small, bland nuclei. Some large cells with two or more larger fat vacuoles indenting the small nucleus may resemble lipoblasts (Fig. 17.3). Regular fat, myxoid stroma, and bland spindle cells may also be present.

Figure 17.3 Hibernoma.
Delicate capillaries (arrows) can be numerous and are closely associated with tumor cells that have distinct granular and multivacuolate cytoplasm. (Papanicolaou stain).

Differential diagnosis of hibernoma

• well-differentiated liposarcoma and atypical lipomatous tumor

• myxoid liposarcoma

• spindle cell/pleomorphic lipoma

• chondroid lipoma

• lipoma with fat necrosis

• granular cell tumor

• adult rhabdomyoma

• pilosebaceous units

Although lipoblast-like cells are seen in a hibernoma, the atypical lipoblasts (see Fig. 17.7) characteristic of well-differentiated liposarcoma and atypical lipomatous tumor are not present. Capillary vessels can be numerous in hibernomas, but they are less prominent than in a myxoid liposarcoma. Spindle cell lipomas lack the characteristic microvacuolate hibernoma cells. The myxoid stroma and chondromyxoid matrix of a chondroid lipoma are not prominent in a hibernoma. Lipophages in a lipoma generally have a larger, plumper nucleus, and their fat vacuoles are more variable in size (see Fig. 17.2). Granular cell tumors lack adipocytic differentiation, and rhabdomyomas have larger cells with dense, granular cytoplasm.^49,51,52 Normal pilosebaceous units can contaminate any FNA that traverses hair-bearing skin. They are recognized by the admixture of finely vacuolated sebaceous cells with ductlike epithelial cells that have nonvacuolated cytoplasm.

Spindle Cell Lipoma and Pleomorphic Lipoma

Spindle cell and pleomorphic lipomas are benign lipoma variants with overlapping clinical, morphologic, and cytogenetic features; they likely represent a single entity with variable morphology. Both are solitary, painless, well-circumscribed, and slowly growing lesions that often arise in the subcutis or dermis of the upper back, shoulder, neck or anterior head and neck regions in middle-aged to older men. They rarely exceed 5 cm in greatest dimension. Recent studies have shown that spindle cell lipoma is related to cellular angiofibroma and mammary-type myofibroblastoma, with overlapping morphology and an association with deletions involving 13q.⁵³

Cytomorphology of spindle cell and pleomorphic lipoma

• fragments of mature adipose tissue

• often myxoid background

• occasional multivacuolate lipoblast-like cells

Spindle cell lipoma

• bland and uniform spindle cells

• ropy collagen fibers

The spindle cell lipoma is characterized by a mixture of mature adipocytes and bland spindle cells in short fascicles. Hyaline, ropy collagen fibers and mast cells are typically seen (Fig. 17.4).⁵⁴ The pleomorphic lipoma shows predominantly mature adipocytes admixed with pleomorphic large, atypical stromal cells that have multiple hyperchromatic, floret-type nuclei (Fig. 17.5).^29,55 Hybrids of spindle cell and pleomorphic lipomas are quite common.

Figure 17.4 Spindle cell lipoma.
This tumor is characterized by a mixture of dispersed bland spindle cells, mature adipocytes, and long, ropy collagen fibers (arrows) in a myxoid background (Romanowsky stain).

Figure 17.5 “Floret” cell.
Multinucleated giant cells with nuclei in a wreathlike configuration are a characteristic but nonspecific finding in pleomorphic lipomas (Papanicolaou stain).

Differential diagnosis of spindle cell and pleomorphic lipoma

• well-differentiated liposarcoma and atypical lipomatous tumor

• schwannoma

• myxoid liposarcoma

• myxofibrosarcoma, low grade

• solitary fibrous tumor

• dematofibrosarcoma protuberans

A myxoid background and multivacuolate lipoblast-like cells are well-recognized diagnostic pitfalls associated with both variants, particularly the pleomorphic lipoma, that can lead to an erroneous diagnosis of a myxoid or well-differentiated liposarcoma or other myxoid or spindle cell neoplasm.49,55 This is particularly likely in cases that lack the classic floret cells and/or mature adipose tissue component. Spindle cell and pleomorphic lipomas are distinguished from a liposarcoma by their typical anatomic distribution and superficial location, along with supportive immunohistochemistry (CD34 positivity in spindle cells) and cytogenetic findings (chromosomal aberrations of 13q and 16q) (see Table 17.1). The positivity for CD34 and lack of S-100 protein in the spindle cells also help to distinguish it from a schwannoma. Myxoid liposarcoma and myxofibrosarcoma have more prominent blood vessels and lack mature adipose tissue and collagen fibers. With a spindle cell–predominant variant, the distinction from a dermatofibrosarcoma protuberans (DFSP) and a solitary fibrous tumor, especially the fat-forming variant, can be difficult, because both mimics can have mature adipose tissue and CD34-positive spindle cells. Clinical correlation and cytogenetic findings are essential in difficult cases.^29,54,56

Well-Differentiated Liposarcoma and Atypical Lipomatous Tumor

Liposarcoma is the most common soft tissue sarcoma of adults. It has three major forms that are biologically distinct: (1) well-differentiated and dedifferentiated; (2) myxoid; and (3) pleomorphic. The terms well-differentiated liposarcoma and atypical lipomatous tumor are essentially synonymous. Well-differentiated liposarcoma applies to deep-seated lesions in the retroperitoneum, spermatic cord, and mediastinum, where recurrence and local aggressiveness are common because a resection with clear margins is difficult to achieve at these locations; atypical lipomatous tumor applies to lesions of the limbs and trunk that do not recur if adequately excised.

Cytomorphology of well-differentiated liposarcoma and atypical lipomatous tumor

• clusters of lipogenic cells with lipid vacuoles of varying size

• atypical stromal cell nuclei

• lipoblasts

• occasional floret cells

• nonlipogenic pleomorphic or spindle-cell tissue fragments suggestive of dedifferentiation

In general, tissue fragments of large but variably sized, univacuolate adipocytes (Fig. 17.6) are admixed with blood and extruded lipid. Smaller multivacuolate cells with atypical, scalloped nuclei (lipoblasts) are present in some cases (Fig. 17.7). Lipoblasts are not necessary for the diagnosis of liposarcoma, with the exception of pleomorphic liposarcoma. The nuclei of larger adipocytes range from small and compressed to large, round to oval, and hyperchromatic (Fig. 17.8). They are often multilobated or convoluted. Similar atypical nuclei are seen in fragments of collagenous stromal tissue.^46,48,57,58