Cytopathology of alveolar soft part sarcoma

A report of 10 cases




Alveolar soft part sarcoma (ASPS) rarely is subjected to cytopathologic evaluation. With the exception of some very small series, the literature is limited to case reports. The objectives of the current study were to evaluate the cytomorphology of 10 ASPS cases on fine-needle aspiration (FNA) or imprint cytology, review the literature, and highlight potential diagnostic pitfalls.


The authors searched their files for all lesions that were signed out as ASPS or suspicious for ASPS and searched the surgical pathology files for any cases of ASPS that had corresponding cytology. FNA was performed using the standard technique. Scrape preparations were performed on tissues that were sent fresh for frozen section examination.


Ten cases of ASPS were retrieved from 7 patients (male-to-female ratio, 4:3; mean age, 22 years). All had subsequent tissue confirmation. Six specimens were from primary lesions, 3 specimens were from metastases, and 1 intraoperative smear was from a previously aspirated primary mass in a newly diagnosed patient. Anatomic sites of involvement included extremities (5 specimens), trunk/pelvis (3 specimens), oral cavity (1 specimen), and lung (1 specimen). Eight specimens were aspirates, and 2 specimens were intraoperative smears. Nine cases were diagnosed correctly as ASPS/consistent with ASPS, and 1 specimen was diagnosed as “tumor, not otherwise specified”. Cytomorphology included variably cellular smears composed of large cells with an enormous amount of finely granular or vacuolated cytoplasm, markedly enlarged nuclei/nucleoli, and bare nuclei. The cytomorphology of some cases revealed a strong similarity to renal cell carcinoma, clear cell type.


The results of the current study indicated that ASPS has cytomorphology that overlaps with several other neoplasms, including renal cell carcinoma. Nonetheless, the morphologic features, when combined with the clinical presentation, radiologic findings, and ancillary testing, may allow for a specific diagnosis. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.

The cytopathology of alveolar soft part sarcoma (ASPS), a rare sarcoma that primarily involves the deep soft tissues, has only infrequently been described. ASPS principally affects young adults in the second and third decades of life and is associated with an unbalanced translocation, der(17)t(X;17)(p11;q25), resulting in the formation of an ASPL-transcription factor binding to immunoglobulin heavy constant μ enhancer 3 (ASPL-TFE3) fusion gene. As a consequence of an active soft tissue fine-needle aspiration (FNA) biopsy service, we have been able to accumulate 10 examples of this rare tumor from our combined files. Because the cytopathology literature is extremely limited regarding ASPS, our purpose is to provide a detailed report of the cytomorphology of ASPS that examined both its differential diagnosis and ancillary testing that may assist in diagnosis using the cytologic method.


We reviewed our cytology files for cases diagnosed as either ASPS or consistent with ASPS. In addition, our surgical pathology files were examined for any cases of ASPS that may have had corresponding cytopathology.

Percutaneous FNA was performed using standard technique with 22-gauge and 23-gauge needles. Conventional direct smears were performed in all cases. Aspirate material for cell block preparation was obtained in 5 cases. A pathologist performed all aspirates. Slides were stained using both Papanicolaou (P) and Romanowsky (R) stains with alcohol fixation or air drying, respectively. Some P-stained slides were first air-dried and then underwent rehydration before staining. Imprint/scrape preparations of fresh tissue were stained with R stain and were examined along with the frozen section. Formalin-fixed, paraffin-embedded tissue sections from cell block, core needle biopsy, or subsequent wide excision specimens were stained with hematoxylin and eosin. Immunostaining with vimentin, pan-cytokeratin, S-100 protein, and HMB-45 was performed using the standard methodology.


Ten examples of ASPS from 7 patients (male-to-female ratio, 4:3) were identified from our combined cytology files. Patient ranged in age from 8 years to 28 years (mean age, 22 years). FNA was performed on 5 cases that presented as primary soft tissue masses and on 3 cases that represented metastatic deposits (lung, chest wall, and arm; 1 each), and 2 cases were intraoperative smears that were made at the time of frozen section examination. One of those intraoperative smears occurred in an undiagnosed girl aged 8 years who had an intraoral mass. The other smear was from a soft tissue mass that had been diagnosed as ASPS by FNA biopsy 2 weeks earlier. The scrape was performed on an incisional biopsy at the time of frozen section confirmation before radical resection (Table 1). Anatomic sites of involvement included the extremities (5 cases), trunk/pelvis (3 cases), oral cavity (1 case), and lung (1 case).

Table 1. Clinical Features of Alveolar Soft Part Sarcoma: 10 Aspirates/Imprints From 7 Patients*
Case No.Age, ySexNeoplasm TypeLocationProcedureCytologic DiagnosisCell BlockTissue Diagnosis
  • M indicates metastatic neoplasm; FNA, fine-needle aspiration biopsy; ASPS, alveolar soft part sarcoma; P, primary neoplasm; NA, not applicable; CNB, accompanying core needle biopsy at the time of FNA.

  • *

    Patients 1 and 2 are the same patient, Patients 4 and 5 are the same patient, and Patients 6 and 7 are the same patient.

38FemalePMouthIntraoperative smearTumorNAASPS
421MalePLeft thighIntraoperative smearASPSNASame
521MalePLeft thighFNAProbable ASPSYesASPS
626MalePInstep, right footFNAASPSYesSame
726MaleMLeft lungFNAASPSYesNone
828FemalePPosterior left shoulderFNAASPSYesSame
920FemalePLeft iliac wingFNAASPSYesSame
1026MalePRight anterior calfFNAConsistent with ASPSCNBSame

Cytologic features were similar in all cases. Smears were variably cellular, because cellularity was highly dependent on the amount of blood aspirated. In 2 cases that the first author aspirated, such a copious amount of blood was encountered immediately with the initial excursion of the needle that it was believed clinically that the mass represented a hemangioma or some other highly vascular neoplasm before microscopic examination. No capillary fragments were noticeable in any of those smears. Cells were arranged in syncytial groups and also were distributed as single forms. Unlike its tissue counterpart, only rarely were cells in a “pseudoalveolar” or acinar-type arrangement (Fig. 1). Common to nearly all cases was a detachment of cytoplasm from nuclei, resulting in numerous bare nuclei and an amorphous, granular background (Fig. 2). This background did not assume a linear or reticulated (so-called “tigroid”) pattern. Malignant cells harbored an enormous amount of cytoplasm that was fine to coarsely granular and variably vacuolated (Fig. 3). Extensive vacuole formation that produced clear cell change was particularly noticeable in Case 8 and, to a lesser degree, in Cases 1 and 6 (Fig. 4). Nuclei were nearly always rounded and large but were of similar size and contained easily visible, single, circular macronucleoli. It is interesting to note that, regardless of the massive size of some nuclei, irregularities of nuclear contour rarely were observed. These sometimes were placed eccentrically within the cell and occasionally were coupled with mirror-image binucleation (Fig. 5). Only rarely was a nuclear pseudoinclusion encountered. The slide background lacked a necrotic diathesis in all cases, but contained a variable amount of fibrillar metachromatic-staining, basement membrane-like material that could be fully appreciated only on R-stained slides. When observed, this material typically was fine and thread-like and often interdigitated among cells, but it also could be thick and ropey (Fig. 6). Mitotic figures were exceedingly uncommon. Cell blocks were obtained in 5 aspirates, but only 2 had any immunohistochemical staining performed. These revealed positive staining with vimentin and negative results with pan-cytokeratin, S-100 protein, and HMB-45. No staining was performed using the TFE3 antibody, because it was not available.

Figure 1.

Cells are distributed in a vague acinar arrangement with a circular strand of metachromatic staining basement membrane-like stroma (Romanowsky stain, original magnification × 40).

Figure 2.

Cell fragility leads to a sheet of numerous, dissociated, large, bare nuclei with discrete acidophilic macronucleoli. Note the fragmented granular cytoplasm in the smear background (Papanicolaou stain, original magnification ×20).

Figure 3.

(Top) Cell nuclei are placed randomly in a syncytium of abundant, finely granular cytoplasm without distinct cell borders (Papanicolaou stain, original magnification ×60). (Bottom) A massive, intact cell contains granular cytoplasm and a “fuzzy” border. Note the huge, rounded nucleus and nucleolus and numerous surrounding bare nuclei with enlarged nucleoli (Romanowsky stain, original magnification ×60).

Figure 4.

(Top) Case 8. Florid cytoplasmic microvacuolization and macrovacuolization create the appearance of a clear cell neoplasm (Romanowsky stain, original magnification ×40). (Bottom) Case 1. Clear cell change is less extensive than that noted in the top view and is mixed with cytoplasmic granularity. Note thin, stringy basal lamina surrounding some cells (Romanowsky stain, original magnification ×60).

Figure 5.

Several binucleated cells with enlarged mirror-image nuclei and nucleoli that are placed eccentrically within the cell, mimicking melanoma cells or Reed-Sternberg cells, are noted (Top: Papanicolaou stain, original magnification ×60; Bottom: Romanowsky stain, original magnification ×60).

Figure 6.

The quality of metachromatic staining stroma varies from (Top) thick and ropy to (Bottom) thin and lacelike (Romanowsky stain, original magnification ×40).

Eight aspirates were diagnosed definitively as ASPS (however, 1 of those cases, Case 10, had a concomitant core needle biopsy specimen available for examination before sign out); 1 aspirate was diagnosed as “probable” ASPS; and 1 aspirate had a noncommittal diagnosis of tumor, not otherwise specified. Histologic examination confirmed the diagnosis of ASPS in all cases.


Since its initial description and acceptance as a clinicopathologic entity more than 50 years ago, numerous case reports, case series, and comprehensive reviews have been written about ASPS—a rare sarcoma that largely affects the deep soft tissue of young adults and adolescents.1, 2 The histopathology of this neoplasm has been well documented, with the classic example demonstrating a nested or pseudoalveolar pattern of large polygonal cells with rounded nuclei; single, markedly enlarged nucleoli; and a voluminous amount of finely granular or, less commonly, clear cytoplasm. Cells typically (but not universally) contain periodic acid–Schiff (PAS)-positive, diastase-resistant cytoplasmic crystals that usually are needle-shaped on light microscopy and that, ultrastructurally, have a rhomboid, cross-hatched periodicity. Recent cytogenetic evidence has demonstrated an association of ASPS with an unbalanced translocation, der(17)t(X;17)(p11;q25).3 This results in the formation of an ASPL-TFE3 fusion gene and aberrant expression of transcription factor (TFE3) protein that can be demonstrated by immunohistochemical staining.

Because of its rarity, to the best of our knowledge, no large series describing the detailed cytopathology of this neoplasm exists. To our knowledge, 22 cases of ASPS cytology have been reported previously (1 case was reported twice) (Table 2). The majority represents single case reports, and a few of those were only in the form of letters to the editor.4-20 The largest series before ours was comprised of 3 cases, all of which were diagnosed correctly by using FNA biopsy.17 The distinctive pseudoalveolar architecture observed in tissue specimens of ASPS largely is absent from cytologic smears. Published images from most of those reports describing such a pattern in FNA cytology are not convincing to us. In the current series, we rarely encountered a cell aggregate that attempted to recapture this pseudoalveolar pattern (Fig. 1). ASPS from the oral cavity in children is noteworthy, in that it frequently lacks this pseudoalveolar architecture even in tissue sections,21 and this is what we observed in smears and histologic sections from our Case 3.

Table 2. Diagnosis of Alveolar Soft Part Sarcoma Using Cytopathology: 22 Reported Patients
StudyAge, ySexSiteSpecimen TypeNeoplasm TypePseudoalveolar PatternCytoplasmic Features
  1. I indicates imprint cytology; P, primary neoplasm; CR, cytoplasmic crystals; EC, extracellular crystals; G, granular; FNA, fine-needle aspiration biopsy; M, metastatic neoplasm; V, vacuoles.

Uehara 1978424FemaleRetroperitoneumIPAbsent, not convincingCR, EC
McDonald 1981522FemaleRight thighIPAbsent, not convincingG
Nieberg 1984622MaleRight lungFNAMAbsent, not convincingG
Kapila 1985725FemaleRight thighFNAPAbsent, not convincingG
Zaleski 1986826FemaleVaginaSmearPAbsentG, rare CR
Ordonez 1988947MaleForearmFNAPAbsentV, G, CR
 28MaleForearmFNAPAbsentV, G, CR
Persson 19881026FemaleRight thighFNAPAbsentV, G
Shabb 19911124MaleRight thighFNAPVagueV, G
 29FemaleLeft legFNAPVagueV, G
 22FemaleLungFNAMVagueV, G
Drachenberg 19911217MaleLeft glutealFNAPRareV, G
Husain & Nguyen 19951315FemaleLeft lower legFNAPAbsentV, G
Tsou 19971419FemaleRight breastIPAbsentG, CR, EC
Logrono 19991523MaleLeft lungFNAMAbsent, not convincingG
Fukuda 19991622FemaleLumbarIPAbsent, not convincingV, CR, EC
Lopez-Ferrer 20021721FemaleCalfFNAPAbsentG
Carter 20081854MaleLeft legFNAPAbsentG, CR
Van Buren & Stewart 20091913FemaleRight breastFNAPAbsentG
Machhi 20022029FemaleLeft buttockFNAPAbsentV, G, CR, EC

Crystalline cytoplasmic structures also typify ASPS. In tissue sections, these are observed best with special histochemical staining, because their rod-shaped structure resists diastase digestion after PAS stain. We were not able to convincingly identify cytoplasmic crystals in any of our smears using either R stain or P stain. Others have reported success in identifying such crystals with Giemsa stain16 and even with P stain.18, 20 Because PAS staining was not attempted on any of these 10 cases, it is possible that cytoplasmic crystals were overlooked. PAS staining also has been used to successfully identify the coarse granules of ASPS on destained P smears.11 A single letter to the editor emphasized the use of Riu stain (an uncommon stain for North American laboratories) as an adjunct that would easily highlight these needle-shaped crystals.14 Of the 22 previously reported cytologic cases of ASPS, only 7 (33%) reported or illustrated the presence of cytoplasmic or extracellular rod-shaped crystals (Table 2). Thus, although they are helpful when present, we believe that rod-shaped or rhomboid-shaped cytoplasmic crystals (not nonspecific coarse granularity) are an uncommon finding in aspirates or imprint smears of ASPS, and their absence should not persuade against the diagnosis. Other than the absence of a pseudoalveolar pattern and cytoplasmic crystals, the cytopathology of ASPS in this series faithfully replicated its tissue counterpart. Similar to previous cytopathologic descriptions, cells were large but were relatively uniform in nuclear size and were devoid of obvious irregularities in nuclear contour and anisonucleosis. Common to most cells were cytoplasmic and nuclear enlargement and the constant presence of a single, markedly enlarged nucleolus. The fragility of cells was noticeable in most slides, because bare nuclei were a common finding. Although intranuclear cytoplasmic inclusions have been described in several case reports, we only rarely encountered a cell nucleus with this change. However, 2 features that are not particularly emphasized regarding ASPS cytomorphology that we encountered were the presence of florid cytoplasmic vacuolization in some aspirates and delicate threads of metachromatic-staining stromal material. The latter varied in quantity and was visible only in R-stained slides frequently distributed in a delicate, lace-like pattern. Although cytoplasmic vacuoles were present to some degree in most cases, they were particularly noticeable in both R-stained and P-stained smears in 3 cases. Case 8 had sufficient vacuolization to be considered a clear cell neoplasm (Fig. 4, Top).

Most studies regarding the FNA cytology of ASPS have concentrated on a differential diagnosis that includes epithelioid sarcoma, clear cell sarcoma, melanoma, paraganglioma, and granular cell tumor (GCT). The distinguishing morphologic features among these different neoplasms and ASPS have been described in detail in a recent review.22 In brief, the cell shape of ASPS is more epithelioid than that in GCT, which may have an elongated, rectangular configuration. In addition, the cytoplasm of ASPS generally is not as coarsely granular as that in GCT and is more vacuolated than the cytoplasm typically encountered in melanoma, epithelioid sarcoma, and paraganglioma. Paraganglioma tends to have more striking anisonucleosis without the impressive nucleoli of ASPS, epithelioid sarcoma has more nuclear pleomorphism, and clear cell sarcoma has smaller nuclei (also without the marked nucleolomegaly) compared with the large but relatively uniform size and shape of ASPS nuclei. Though binucleation was present in ASPS, it was less than we typically experience in soft tissue aspirates of metastatic melanoma and clear cell sarcoma of soft parts (unpublished data). ASPS nuclei also do not have a consistent eccentric position within the cell compared with these melanocytic neoplasms, and intranuclear inclusions are uncommon but are not completely absent. Multinucleation may be a feature of clear cell sarcoma but was not observed in our ASPS cases.

In addition, an immunohistochemical panel that includes positive staining for pan-cytokeratin (epithelioid sarcoma), S-100 protein (GCT), S-100/HMB-45/melan-A (melanoma and clear cell sarcoma), and neuroendocrine markers (paraganglioma) would help exclude these malignancies. In the current series, we were struck by the similarity of some ASPS clusters to those of renal cell carcinoma (RCC), clear cell type. Though the peak incidence at the time of presentation for patients with clear cell RCC is approximately 2 to 4 decades older than the typical ASPS patient, overlapping age ranges occur between these neoplasms. It has been stated that a main difference between RCC and ASPS is that clear or finely vacuolated cytoplasm is observed more often in the former.12 Although this may be true in general, it is not always the case. This was emphasized particularly to us by a recent example of RCC metastatic to the forearm (19 years after removal of the kidney tumor) in a middle-aged individual who presented clinically with a soft tissue sarcoma (Fig. 7). Immunoreactivity with pan-cytokeratin and epithelial membrane antigen helps to exclude ASPS and to confirm a diagnosis of RCC.

Figure 7.

Metastatic renal cell carcinoma of the clear cell type is shown. Note the strong resemblance between this soft tissue aspirate from the forearm and the clear cell neoplasm shown in the top view of Figure 4 (Romanowsky stain, original magnification ×40).

TFE3 staining is an extremely helpful antibody in confirming the diagnosis of ASPS; however, it is not yet widely available in most hospital laboratories. Because we did not have it accessible to us for 9 of our cases (and, in Case 10 with a concurrent core needle biopsy, it was not needed), we cannot specifically comment on its efficacy in this series. However, others have documented its utility in cell block and histologic sections.3, 23, 24 Positive nuclear staining with TFE3 is specific for ASPS when dealing with a soft tissue sarcoma, but it must be remembered that a specific subtype of renal carcinoma—Xp11.2 renal carcinoma—also expresses this nuclear marker. Because patients who have Xp11.2 renal carcinoma (primarily children) share an overlapping age group with ASPS, possible metastases from this neoplasm must be considered whenever positive TFE3 staining occurs. Nonetheless, a distinction between the 2 may be possible by cytomorphology alone. Of the 2 case reports that have described the cytologic morphology of this unusual renal neoplasm, 1 demonstrated clear cell change with numerous psammoma bodies and hyaline globules on tissue imprints,23 whereas the other exhibited follicular and papillary structures with globules reminiscent of adenoid cystic carcinoma in an aspirate from a pulmonary metastasis.24 Neither of these features is typical of ASPS cytology.

The results of the current series demonstrate that a diagnosis of ASPS is possible using FNA even without the use of immunohistochemical staining and that the cytomorphology of this neoplasm is relatively (although not completely) distinctive. In addition, an accumulation of sufficient cells to produce a cell block and the addition of TFE3 staining to a panel of immunomarkers may allow for diagnostic accuracy in the cytologic recognition of ASPS.

Conflict of Interest Disclosures

The authors made no disclosures.