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Alveolar soft part sarcoma and granular cell tumor: an immunohistochemical comparison study
Human Pathology (2014) 45, 1039–1044
www.elsevier.com/locate/humpath
Original contribution
Alveolar soft part sarcoma and granular cell tumor: an immunohistochemical comparison study☆ Benjamin K. Chamberlain MD, Colt M. McClain MD, Raul S. Gonzalez MD, Cheryl M. Coffin MD, Justin M.M. Cates MD, PhD ⁎ Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232 Received 20 November 2013; revised 24 December 2013; accepted 27 December 2013
Keywords: Alveolar soft part sarcoma; Granular cell tumor; S-100 protein; Inhibin; SOX10; Nestin
Summary Although the histologic features of alveolar soft part sarcoma and granular cell tumor are typically distinctive, occasional cases show a significant morphologic overlap. Differentiating these entities is crucial because granular cell tumor is almost always benign and alveolar soft part sarcoma is invariably malignant. We evaluated a panel of immunohistochemical stains (S-100 protein, inhibin, SOX10, nestin, calretinin, and TFE3) in 13 alveolar soft part sarcomas and 11 granular cell tumors. Tissue sections were also stained by the periodic acid–Schiff method after diastase digestion (PAS-D) and evaluated for coarse cytoplasmic granularity or crystalline cytoplasmic inclusions. S-100 protein, inhibin, SOX10, and nestin each distinguished granular cell tumor and alveolar soft part sarcoma with 100% sensitivity and specificity. PAS-D staining also distinguished cases with 100% accuracy, as granular cell tumor consistently demonstrated coarsely granular, PAS-D–positive cytoplasm and alveolar soft part sarcoma showed only focal intracytoplasmic crystalline inclusions. Although all granular cell tumors were calretinin positive, so were 46% of alveolar soft part sarcomas. TFE3 was positive in 91% of granular cell tumors and all alveolar soft part sarcomas. Together with PAS-D, immunohistochemical stains for S-100 protein, inhibin, SOX10, and nestin accurately identify alveolar soft part sarcoma and granular cell tumor. Although TFE3 has been reported as a relatively specific marker for alveolar soft part sarcoma, it should be recalled that it is also expressed in most granular cell tumors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction In general, the characteristic histologic patterns of both alveolar soft part sarcoma and granular cell tumor are sufficiently distinctive to establish the diagnosis of either entity by routine hematoxylin and eosin (H&E) staining. However, ☆ Disclosures: The authors disclose no conflicts of interest and source of funding. ⁎ Corresponding author. Department of Pathology, Microbiology and Immunology, Medical Center North, Vanderbilt University Medical Center, C-3322, Nashville, TN 37232-2561. E-mail address: [email protected] (J. M. M. Cates).
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.12.021
occasional cases show histomorphologic overlap such that a definitive diagnosis requires the use of special and immunohistochemical (IHC) stains [1-3]. Whereas almost all granular cell tumors are benign and adequately treated with simple excision, alveolar soft part sarcoma is invariably malignant [4]. Although the clinical course of alveolar soft part sarcoma is often slow and indolent, these patients need to be followed up indefinitely for local recurrence and metastatic disease [3-5]. Although alveolar soft part sarcoma responds poorly to radiotherapy and conventional chemotherapy, molecularly targeted therapies hold promise for the systemic treatment of this tumor [6-8]. Granular cell tumor has a wide anatomical distribution and is composed of a uniform population of polygonal to
1040 spindle-shaped cells with abundant eosinophilic cytoplasm containing namesake lysosomal granules that impart a coarsely granular appearance to the cytoplasm upon staining with the periodic acid–Schiff reagent after diastase digestion (PAS-D). The nuclei are usually small and pyknotic. Some tumors may demonstrate stromal elastosis, and secondary epithelial hyperplasia may be seen in submucosal tumors. Alveolar soft part sarcoma also has a wide anatomical distribution and is characterized by well-defined nests of cells separated by thin fibrovascular septa. Like granular cell tumor, the constituent cells are large and polygonal, with finely granular, eosinophilic cytoplasm. However, they are generally discohesive and typically have large, vesicular nuclei with prominent nucleoli. Rod-shaped or globular crystalline inclusions are often noted PAS-D stains, which are morphologically distinct from the coarsely granular cytoplasmic staining seen in granular cell tumors [4,9]. Given the dramatically different prognoses and recommended treatment of these 2 tumors, correct pathologic diagnosis is critical. We recently encountered a case of alveolar soft part sarcoma with small, pyknotic nuclei, focal spindling of tumor cells and a sheet-like, cohesive growth pattern that closely resembled granular cell tumor (Fig. 1), which prompted us to evaluate a panel of IHC stains and the PAS-D stain in resolving this differential diagnosis.
2. Materials and methods Nine cases of alveolar soft part sarcoma and 11 cases of granular cell tumor were identified through a search of the surgical pathology archives. Four additional cases of alveolar soft part sarcoma were retrieved from the personal consult
B. K. Chamberlain et al. files of one of the authors (C. M. C.). After obtaining institutional review board approval, H&E-stained slides were reviewed to confirm the histologic diagnosis and select blocks for further study. For IHC analysis, formalin-fixed, paraffin-embedded tissue sections were deparaffinized and subjected to heatinduced epitope retrieval for 20 minutes in Epitope Retrieval 2 solution (Leica Microsystems, Buffalo Grove, IL) on the Leica Bond Max IHC autostainer. Sections were then incubated with the following antibodies for 1 hour: rabbit antihuman S100 (1:6000; Dako, Carpinteria, CA), antiinhibin (1:200; Leica Microsystems), anti-SOX10 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), antinestin (1:5000; Sigma-Aldrich Co, St Louis, MO), anticalretinin (1:20,000; Leica Microsystems), or anti-TFE3 (1:20,000; Santa Cruz Biotechnology). The Bond Polymer Refine detection system was used for immunolocalization. PAS stains were performed on the Dako Artisan Link Pro Special Staining System (Dako North America) after pretreatment with diastase for 30 minutes. Appropriate positive and negative control slides were included for each assay run. All stains were evaluated independently by 2 different pathologists (B. K. C. and C. M. M.) and scored as either positive or negative based on the presence of any specific staining in the appropriate subcellular compartments for each marker. Nuclear staining was evaluated for SOX10 and TFE3, cytoplasmic staining for nestin and inhibin, and nuclear and/or cytoplasmic staining for S-100 protein and calretinin. PAS-D stains were recorded as either coarsely granular or demonstrating focal rod-shaped or globular crystalline cytoplasmic inclusions. Discrepancies were resolved by a senior pathologist. Sensitivity and specificity for each marker were determined and 95% confidence intervals
Fig. 1 Alveolar soft part sarcoma with a solid growth pattern, spindled cells, and banal nuclear features, mimicking granular cell tumor. A, H&E, ×100; B, H&E, ×200.
Alveolar soft part sarcoma and granular cell tumor (95% CIs) were calculated using Jeffreys' [10] method. Twosample tests of proportions were performed using the Stata software package (v12.1; StataCorp, College Station, TX).
3. Results Representative results for each marker are demonstrated in Figs. 2 and 3, and the results are summarized in the Table. S100 protein, inhibin, SOX10, and nestin were positive in each
1041 case of granular cell tumor and negative in each case of alveolar soft part sarcoma (100% accuracy). PAS-D staining demonstrated large, coarse cytoplasmic granules in all cases of granular cell tumor, whereas each alveolar soft part sarcoma showed at least focal intracytoplasmic crystalline inclusions. Although calretinin stained all granular cell tumors, 6 (46%) of 13 alveolar soft part sarcomas were also positive for this marker. It was also noted that TFE3 is not specific for alveolar soft part sarcoma in this diagnostic setting, as 10 (91%) of 11 cases of granular cell tumor were
Fig. 2 Representative images of alveolar soft part sarcoma and granular cell tumor stained with H&E and by IHC for S-100 protein, SOX10, and inhibin (×200).
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Fig. 3 Representative images of alveolar soft part sarcoma and granular cell tumor stained by IHC for nestin, calretinin, and TFE3 (×200) and stained with PAS after diastase digestion (×400).
TFE3 positive. Two-sample tests of proportions confirmed statistically significant differences in the percentage of positive granular cell tumor cases compared with alveolar soft part sarcoma for S-100 protein, inhibin, SOX10, nestin (P b .0001), and calretinin (P = .004), but not for TFE3 (P = .27). There were very few interobserver discrepancies among the markers studied. Weak background staining for calretinin was present in 5 alveolar soft part sarcomas, resulting in 2 discordant interpretations that were resolved as nonspecific staining through consensus review with a senior pathologist.
One alveolar soft part sarcoma showed focal cytoplasmic background staining for S-100 protein (but no nuclear staining) that was interpreted differently by the reviewing pathologists. Consensus review confirmed a negative result. For nestin, background cytoplasmic staining was noted in 2 alveolar soft part sarcomas, one of which resulted in a diagnostic discrepancy. Consensus review confirmed the interpretation of background staining. There were no discrepancies in interpretation of inhibin, TFE3, SOX10, and PAS-D stains.
Alveolar soft part sarcoma and granular cell tumor
1043
Table IHC profiles of granular cell tumor and alveolar soft part sarcoma a Granular cell tumor
Alveolar soft part sarcoma
PAS-D b S100 protein Inhibin SOX10 Nestin Calretinin
11/11 11/11 11/11 11/11 11/11 11/11
TFE3
10/11 (91%; 65%-99%)
0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 6/13 (46%; 22%-72%) 13/13 (100%; 83%-100%)
(100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%)
a Data presented as number positive/number tested (% positive; 95% CI). b PAS-D stains were considered positive if cytoplasm was coarsely granular, without crystalline cytoplasmic inclusions.
Most of these antibodies performed well in our laboratory. Two notable exceptions were SOX10 and calretinin. The former showed heavy cytoplasmic background and focal weak nuclear staining in 8 of 13 alveolar soft part sarcomas that complicated diagnostic interpretation but did not result in diagnostic discrepancy. Focal, weak calretinin immunoreactivity was seen in 7 of 24 cases overall. Although this pattern was resolved uniformly in 5 cases, one reviewer misinterpreted this staining as positive in 2 alveolar soft part sarcomas. One particular alveolar soft part sarcoma showed results that were difficult to interpret for S-100 protein, SOX10, and nestin and accounted for 2 of the 4 diagnostic discrepancies observed in this study. This was the most recent in-house case, and review of the H&E-stained slides suggested that the tissue was fixed inadequately in formalin prior to histologic processing.
4. Discussion Occasionally, alveolar soft part sarcoma and granular cell tumor show significant overlap in their histologic features on H&E-stained sections, rendering definitive diagnosis particularly challenging. A correct diagnosis is crucial in this setting because the prognosis and management of these soft tissue tumors are significantly different. A number of previous studies have examined the IHC profiles of each of these tumors separately (results summarized in Fig. 4 and Supplementary Table S1), but relatively few have compared them directly. Furthermore, the staining patterns of inhibin, SOX10, nestin, or calretinin have not been reported previously for alveolar soft part sarcoma. It is well established that granular cell tumor is positive for S-100 protein and SOX10 (Fig. 4; Supplementary Table S1) [11-15]. Results with inhibin have been more variable. Whereas most investigators find that nearly 100% of granular cell tumors are inhibin positive, others have
Fig. 4 Compilation of reported IHC results in alveolar soft part sarcoma and granular cell tumor. Error bars indicate 95% CIs.
reported significantly lower sensitivity [16-20]. These discrepancies do not appear to be related to the antibodies used and likely are the result of other methodological differences between laboratories. In corroboration with a recent study by Parfitt and coworkers [21], we show that nestin is positive in granular cell tumor. Our results and those of other investigators demonstrate that S-100 protein, SOX10, nestin, and inhibin accurately identifies granular cell tumor with high sensitivity and specificity. Together with a coarsely granular pattern of PAS-D staining, this panel of IHC markers is useful in distinguishing granular cell tumor from alveolar soft part sarcoma with high interobserver concordance. The absence of SOX10, inhibin, or nestin immunoreactivity or coarse granular cytoplasmic staining with PAS-D is particularly helpful in identifying unusual alveolar soft part sarcomas that are reactive for S-100 protein (Fig. 4; Supplementary Table S1) [13]. Although none of the IHC markers tested were specific for alveolar soft part sarcoma, PAS-D–positive, crystalline cytoplasmic inclusions were seen exclusively in alveolar soft part sarcoma. Other investigators have shown that TFE3 is almost always positive in alveolar soft part sarcoma; however, this marker is not entirely specific because it is frequently positive in granular cell tumor [22-25]. This pitfall seriously limits its diagnostic use in this clinical setting. Calretinin has previously been reported as a marker for granular cell tumor and was positive in all examined cases of granular cell tumor in our study [16,18]. However, it was also positive in approximately half of alveolar soft part sarcomas and is not informative in this differential diagnosis. In summary, although granular cell tumor and alveolar soft part sarcoma are generally distinguishable by assessment of H&E-stained sections, occasional cases may show significant histomorphologic overlap. That only one case of all alveolar soft part sarcoma and granular cell tumor evaluated in this study posed a differential diagnostic dilemma suggests that this is a relatively uncommon occurrence. Regardless, the clinical implications of distinguishing these tumors are critical because there are
1044 significant differences in their prognosis and treatment. Here we define a panel of special stains (PAS-D, S-100 protein, inhibin, SOX10, and nestin) that are reliably positive in granular cell tumor and negative in alveolar soft part sarcoma and are therefore useful in the resolution of this differential diagnosis.
Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.humpath.2013.12.021.
References [1] Weiss SW, Goldblum JR. Enzinger and Weiss's soft tissue tumors. Mosby Elsevier: Philadelphia; 2008. [2] Cates JM, Coffin CM. Neurogenic tumors of soft tissue. Pediatr Dev Pathol 2012;15:62-107. [3] Zarrin-Khameh N, Kaye KS. Alveolar soft part sarcoma. Arch Pathol Lab Med 2007;131:488-91. [4] Folpe AL, Deyrup AT. Alveolar soft-part sarcoma: a review and update. J Clin Pathol 2006;59:1127-32. [5] Portera Jr CA, Ho V, Patel SR, et al. Alveolar soft part sarcoma: clinical course and patterns of metastasis in 70 patients treated at a single institution. Cancer 2001;91:585-91. [6] Tsuda M, Davis IJ, Argani P, et al. TFE3 fusions activate MET signaling by transcriptional up-regulation, defining another class of tumors as candidates for therapeutic MET inhibition. Cancer Res 2007;67:919-29. [7] Azizi AA, Haberler C, Czech T, et al. Vascular-endothelial-growthfactor (VEGF) expression and possible response to angiogenesis inhibitor bevacizumab in metastatic alveolar soft part sarcoma. Lancet Oncol 2006;7:521-3. [8] Vistica DT, Hollingshead M, Borgel SD, et al. Therapeutic vulnerability of an in vivo model of alveolar soft part sarcoma (ASPS) to antiangiogenic therapy. J Pediatr Hematol Oncol 2009;31:561-70. [9] Masson P. Human tumors: histology, diagnosis, and technique. Detroit: Wayne State University Press; 1970. [10] Jeffreys H. Theory of probability. Oxford: Clarendon Press; 1961. [11] Mukai M, Iri H, Nakajima T, et al. Alveolar soft-part sarcoma: a review on its histogenesis and further studies based on electron microscopy, immunohistochemistry, and biochemistry. Am J Surg Pathol 1983;7:679-89.
B. K. Chamberlain et al. [12] Ogawa K, Nakashima Y, Yamabe H, Hamashima Y. Alveolar soft part sarcoma, granular cell tumor, and paraganglioma. An immunohistochemical comparative study. Acta Pathol Jpn 1986;36:895-904. [13] Miettinen M, Ekfors T. Alveolar soft part sarcoma: immunohistochemical evidence for muscle cell differentiation. Am J Clin Pathol 1990;93:32-8. [14] Karamchandani JR, Nielsen TO, van de Rijn M, West RB. Sox10 and S100 in the diagnosis of soft-tissue neoplasms. Appl Immunohistochem Mol Morphol 2012;20:445-50. [15] Heerema MG, Suurmeijer AJ. Sox10 immunohistochemistry allows the pathologist to differentiate between prototypical granular cell tumors and other granular cell lesions. Histopathology 2012;61:997-9. [16] Fine SW, Li M. Expression of calretinin and the alpha-subunit of inhibin in granular cell tumors. Am J Clin Pathol 2003;119:259-64. [17] Le BH, Boyer PJ, Lewis JE, Kapadia SB. Granular cell tumor: immunohistochemical assessment of inhibin-alpha, protein gene product 9.5, S100 protein, CD68, and Ki-67 proliferative index with clinical correlation. Arch Pathol Lab Med 2004;128:771-5. [18] Bellezza G, Colella R, Sidoni A, et al. Immunohistochemical expression of Galectin-3 and HBME-1 in granular cell tumors: a new finding. Histol Histopathol 2008;23:1127-30. [19] Zamecnik M, Michal M, Mukensnabl P. Reactivity of granular cell tumors for inhibin and other markers of sex cord and steroid cell differentiation. Am J Surg Pathol 2003;27:413-4. [20] Murakata LA, Ishak KG. Expression of inhibin-alpha by granular cell tumors of the gallbladder and extrahepatic bile ducts. Am J Surg Pathol 2001;25:1200-3. [21] Parfitt JR, McLean CA, Joseph MG, Streutker CJ, Al-Haddad S, Driman DK. Granular cell tumours of the gastrointestinal tract: expression of nestin and clinicopathological evaluation of 11 patients. Histopathology 2006;48:424-30. [22] Argani P, Lal P, Hutchinson B, Lui MY, Reuter VE, Ladanyi M. Aberrant nuclear immunoreactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay. Am J Surg Pathol 2003;27:750-61. [23] Williams A, Bartle G, Sumathi VP, et al. Detection of ASPL/TFE3 fusion transcripts and the TFE3 antigen in formalin-fixed, paraffinembedded tissue in a series of 18 cases of alveolar soft part sarcoma: useful diagnostic tools in cases with unusual histological features. Virchows Arch 2011;458:291-300. [24] Tsuji K, Ishikawa Y, Imamura T. Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for ASPSCR1TFE3 fusion transcript. HUM PATHOL 2012;43:356-63. [25] Rekhi B, Ingle A, Agarwal M, Puri A, Laskar S, Jambhekar NA. Alveolar soft part sarcoma ‘revisited’: clinicopathological review of 47 cases from a tertiary cancer referral centre, including immunohistochemical expression of TFE3 in 22 cases and 21 other tumours. Pathology 2012;44:11-7.
www.elsevier.com/locate/humpath
Original contribution
Alveolar soft part sarcoma and granular cell tumor: an immunohistochemical comparison study☆ Benjamin K. Chamberlain MD, Colt M. McClain MD, Raul S. Gonzalez MD, Cheryl M. Coffin MD, Justin M.M. Cates MD, PhD ⁎ Department of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232 Received 20 November 2013; revised 24 December 2013; accepted 27 December 2013
Keywords: Alveolar soft part sarcoma; Granular cell tumor; S-100 protein; Inhibin; SOX10; Nestin
Summary Although the histologic features of alveolar soft part sarcoma and granular cell tumor are typically distinctive, occasional cases show a significant morphologic overlap. Differentiating these entities is crucial because granular cell tumor is almost always benign and alveolar soft part sarcoma is invariably malignant. We evaluated a panel of immunohistochemical stains (S-100 protein, inhibin, SOX10, nestin, calretinin, and TFE3) in 13 alveolar soft part sarcomas and 11 granular cell tumors. Tissue sections were also stained by the periodic acid–Schiff method after diastase digestion (PAS-D) and evaluated for coarse cytoplasmic granularity or crystalline cytoplasmic inclusions. S-100 protein, inhibin, SOX10, and nestin each distinguished granular cell tumor and alveolar soft part sarcoma with 100% sensitivity and specificity. PAS-D staining also distinguished cases with 100% accuracy, as granular cell tumor consistently demonstrated coarsely granular, PAS-D–positive cytoplasm and alveolar soft part sarcoma showed only focal intracytoplasmic crystalline inclusions. Although all granular cell tumors were calretinin positive, so were 46% of alveolar soft part sarcomas. TFE3 was positive in 91% of granular cell tumors and all alveolar soft part sarcomas. Together with PAS-D, immunohistochemical stains for S-100 protein, inhibin, SOX10, and nestin accurately identify alveolar soft part sarcoma and granular cell tumor. Although TFE3 has been reported as a relatively specific marker for alveolar soft part sarcoma, it should be recalled that it is also expressed in most granular cell tumors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction In general, the characteristic histologic patterns of both alveolar soft part sarcoma and granular cell tumor are sufficiently distinctive to establish the diagnosis of either entity by routine hematoxylin and eosin (H&E) staining. However, ☆ Disclosures: The authors disclose no conflicts of interest and source of funding. ⁎ Corresponding author. Department of Pathology, Microbiology and Immunology, Medical Center North, Vanderbilt University Medical Center, C-3322, Nashville, TN 37232-2561. E-mail address: [email protected] (J. M. M. Cates).
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.12.021
occasional cases show histomorphologic overlap such that a definitive diagnosis requires the use of special and immunohistochemical (IHC) stains [1-3]. Whereas almost all granular cell tumors are benign and adequately treated with simple excision, alveolar soft part sarcoma is invariably malignant [4]. Although the clinical course of alveolar soft part sarcoma is often slow and indolent, these patients need to be followed up indefinitely for local recurrence and metastatic disease [3-5]. Although alveolar soft part sarcoma responds poorly to radiotherapy and conventional chemotherapy, molecularly targeted therapies hold promise for the systemic treatment of this tumor [6-8]. Granular cell tumor has a wide anatomical distribution and is composed of a uniform population of polygonal to
1040 spindle-shaped cells with abundant eosinophilic cytoplasm containing namesake lysosomal granules that impart a coarsely granular appearance to the cytoplasm upon staining with the periodic acid–Schiff reagent after diastase digestion (PAS-D). The nuclei are usually small and pyknotic. Some tumors may demonstrate stromal elastosis, and secondary epithelial hyperplasia may be seen in submucosal tumors. Alveolar soft part sarcoma also has a wide anatomical distribution and is characterized by well-defined nests of cells separated by thin fibrovascular septa. Like granular cell tumor, the constituent cells are large and polygonal, with finely granular, eosinophilic cytoplasm. However, they are generally discohesive and typically have large, vesicular nuclei with prominent nucleoli. Rod-shaped or globular crystalline inclusions are often noted PAS-D stains, which are morphologically distinct from the coarsely granular cytoplasmic staining seen in granular cell tumors [4,9]. Given the dramatically different prognoses and recommended treatment of these 2 tumors, correct pathologic diagnosis is critical. We recently encountered a case of alveolar soft part sarcoma with small, pyknotic nuclei, focal spindling of tumor cells and a sheet-like, cohesive growth pattern that closely resembled granular cell tumor (Fig. 1), which prompted us to evaluate a panel of IHC stains and the PAS-D stain in resolving this differential diagnosis.
2. Materials and methods Nine cases of alveolar soft part sarcoma and 11 cases of granular cell tumor were identified through a search of the surgical pathology archives. Four additional cases of alveolar soft part sarcoma were retrieved from the personal consult
B. K. Chamberlain et al. files of one of the authors (C. M. C.). After obtaining institutional review board approval, H&E-stained slides were reviewed to confirm the histologic diagnosis and select blocks for further study. For IHC analysis, formalin-fixed, paraffin-embedded tissue sections were deparaffinized and subjected to heatinduced epitope retrieval for 20 minutes in Epitope Retrieval 2 solution (Leica Microsystems, Buffalo Grove, IL) on the Leica Bond Max IHC autostainer. Sections were then incubated with the following antibodies for 1 hour: rabbit antihuman S100 (1:6000; Dako, Carpinteria, CA), antiinhibin (1:200; Leica Microsystems), anti-SOX10 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), antinestin (1:5000; Sigma-Aldrich Co, St Louis, MO), anticalretinin (1:20,000; Leica Microsystems), or anti-TFE3 (1:20,000; Santa Cruz Biotechnology). The Bond Polymer Refine detection system was used for immunolocalization. PAS stains were performed on the Dako Artisan Link Pro Special Staining System (Dako North America) after pretreatment with diastase for 30 minutes. Appropriate positive and negative control slides were included for each assay run. All stains were evaluated independently by 2 different pathologists (B. K. C. and C. M. M.) and scored as either positive or negative based on the presence of any specific staining in the appropriate subcellular compartments for each marker. Nuclear staining was evaluated for SOX10 and TFE3, cytoplasmic staining for nestin and inhibin, and nuclear and/or cytoplasmic staining for S-100 protein and calretinin. PAS-D stains were recorded as either coarsely granular or demonstrating focal rod-shaped or globular crystalline cytoplasmic inclusions. Discrepancies were resolved by a senior pathologist. Sensitivity and specificity for each marker were determined and 95% confidence intervals
Fig. 1 Alveolar soft part sarcoma with a solid growth pattern, spindled cells, and banal nuclear features, mimicking granular cell tumor. A, H&E, ×100; B, H&E, ×200.
Alveolar soft part sarcoma and granular cell tumor (95% CIs) were calculated using Jeffreys' [10] method. Twosample tests of proportions were performed using the Stata software package (v12.1; StataCorp, College Station, TX).
3. Results Representative results for each marker are demonstrated in Figs. 2 and 3, and the results are summarized in the Table. S100 protein, inhibin, SOX10, and nestin were positive in each
1041 case of granular cell tumor and negative in each case of alveolar soft part sarcoma (100% accuracy). PAS-D staining demonstrated large, coarse cytoplasmic granules in all cases of granular cell tumor, whereas each alveolar soft part sarcoma showed at least focal intracytoplasmic crystalline inclusions. Although calretinin stained all granular cell tumors, 6 (46%) of 13 alveolar soft part sarcomas were also positive for this marker. It was also noted that TFE3 is not specific for alveolar soft part sarcoma in this diagnostic setting, as 10 (91%) of 11 cases of granular cell tumor were
Fig. 2 Representative images of alveolar soft part sarcoma and granular cell tumor stained with H&E and by IHC for S-100 protein, SOX10, and inhibin (×200).
1042
B. K. Chamberlain et al.
Fig. 3 Representative images of alveolar soft part sarcoma and granular cell tumor stained by IHC for nestin, calretinin, and TFE3 (×200) and stained with PAS after diastase digestion (×400).
TFE3 positive. Two-sample tests of proportions confirmed statistically significant differences in the percentage of positive granular cell tumor cases compared with alveolar soft part sarcoma for S-100 protein, inhibin, SOX10, nestin (P b .0001), and calretinin (P = .004), but not for TFE3 (P = .27). There were very few interobserver discrepancies among the markers studied. Weak background staining for calretinin was present in 5 alveolar soft part sarcomas, resulting in 2 discordant interpretations that were resolved as nonspecific staining through consensus review with a senior pathologist.
One alveolar soft part sarcoma showed focal cytoplasmic background staining for S-100 protein (but no nuclear staining) that was interpreted differently by the reviewing pathologists. Consensus review confirmed a negative result. For nestin, background cytoplasmic staining was noted in 2 alveolar soft part sarcomas, one of which resulted in a diagnostic discrepancy. Consensus review confirmed the interpretation of background staining. There were no discrepancies in interpretation of inhibin, TFE3, SOX10, and PAS-D stains.
Alveolar soft part sarcoma and granular cell tumor
1043
Table IHC profiles of granular cell tumor and alveolar soft part sarcoma a Granular cell tumor
Alveolar soft part sarcoma
PAS-D b S100 protein Inhibin SOX10 Nestin Calretinin
11/11 11/11 11/11 11/11 11/11 11/11
TFE3
10/11 (91%; 65%-99%)
0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 0/13 (0%; 0-17%) 6/13 (46%; 22%-72%) 13/13 (100%; 83%-100%)
(100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%) (100%; 80%-100%)
a Data presented as number positive/number tested (% positive; 95% CI). b PAS-D stains were considered positive if cytoplasm was coarsely granular, without crystalline cytoplasmic inclusions.
Most of these antibodies performed well in our laboratory. Two notable exceptions were SOX10 and calretinin. The former showed heavy cytoplasmic background and focal weak nuclear staining in 8 of 13 alveolar soft part sarcomas that complicated diagnostic interpretation but did not result in diagnostic discrepancy. Focal, weak calretinin immunoreactivity was seen in 7 of 24 cases overall. Although this pattern was resolved uniformly in 5 cases, one reviewer misinterpreted this staining as positive in 2 alveolar soft part sarcomas. One particular alveolar soft part sarcoma showed results that were difficult to interpret for S-100 protein, SOX10, and nestin and accounted for 2 of the 4 diagnostic discrepancies observed in this study. This was the most recent in-house case, and review of the H&E-stained slides suggested that the tissue was fixed inadequately in formalin prior to histologic processing.
4. Discussion Occasionally, alveolar soft part sarcoma and granular cell tumor show significant overlap in their histologic features on H&E-stained sections, rendering definitive diagnosis particularly challenging. A correct diagnosis is crucial in this setting because the prognosis and management of these soft tissue tumors are significantly different. A number of previous studies have examined the IHC profiles of each of these tumors separately (results summarized in Fig. 4 and Supplementary Table S1), but relatively few have compared them directly. Furthermore, the staining patterns of inhibin, SOX10, nestin, or calretinin have not been reported previously for alveolar soft part sarcoma. It is well established that granular cell tumor is positive for S-100 protein and SOX10 (Fig. 4; Supplementary Table S1) [11-15]. Results with inhibin have been more variable. Whereas most investigators find that nearly 100% of granular cell tumors are inhibin positive, others have
Fig. 4 Compilation of reported IHC results in alveolar soft part sarcoma and granular cell tumor. Error bars indicate 95% CIs.
reported significantly lower sensitivity [16-20]. These discrepancies do not appear to be related to the antibodies used and likely are the result of other methodological differences between laboratories. In corroboration with a recent study by Parfitt and coworkers [21], we show that nestin is positive in granular cell tumor. Our results and those of other investigators demonstrate that S-100 protein, SOX10, nestin, and inhibin accurately identifies granular cell tumor with high sensitivity and specificity. Together with a coarsely granular pattern of PAS-D staining, this panel of IHC markers is useful in distinguishing granular cell tumor from alveolar soft part sarcoma with high interobserver concordance. The absence of SOX10, inhibin, or nestin immunoreactivity or coarse granular cytoplasmic staining with PAS-D is particularly helpful in identifying unusual alveolar soft part sarcomas that are reactive for S-100 protein (Fig. 4; Supplementary Table S1) [13]. Although none of the IHC markers tested were specific for alveolar soft part sarcoma, PAS-D–positive, crystalline cytoplasmic inclusions were seen exclusively in alveolar soft part sarcoma. Other investigators have shown that TFE3 is almost always positive in alveolar soft part sarcoma; however, this marker is not entirely specific because it is frequently positive in granular cell tumor [22-25]. This pitfall seriously limits its diagnostic use in this clinical setting. Calretinin has previously been reported as a marker for granular cell tumor and was positive in all examined cases of granular cell tumor in our study [16,18]. However, it was also positive in approximately half of alveolar soft part sarcomas and is not informative in this differential diagnosis. In summary, although granular cell tumor and alveolar soft part sarcoma are generally distinguishable by assessment of H&E-stained sections, occasional cases may show significant histomorphologic overlap. That only one case of all alveolar soft part sarcoma and granular cell tumor evaluated in this study posed a differential diagnostic dilemma suggests that this is a relatively uncommon occurrence. Regardless, the clinical implications of distinguishing these tumors are critical because there are
1044 significant differences in their prognosis and treatment. Here we define a panel of special stains (PAS-D, S-100 protein, inhibin, SOX10, and nestin) that are reliably positive in granular cell tumor and negative in alveolar soft part sarcoma and are therefore useful in the resolution of this differential diagnosis.
Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.humpath.2013.12.021.
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