ANATOMICAL PATHOLOGY
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
BHARAT REKHI*, ABHIJEET INGLE*, MANISH AGARWAL§, AJAY PURIT, SIDDHARTH LASKARĘ AND NIRMALA A. JAMBHEKAR*
Departments of *Pathology, ¡Surgical Oncology (Bone and Soft Tissues), and Radiation Oncology, Tata Memorial Hospital, Parel, and §Department of Orthopaedics, P.D. Hinduja National Hospital and Medical Research Centre, Mumbai, India
Summary
Background: Alveolar soft part sarcoma accounts for 0.5- 1.0% of soft tissue sarcomas in the United States. At our Hospital, it constitutes 1.8% of the newly diagnosed soft tissue sarcomas. Lately, TFE3 has been found to be a useful immunohistochemical marker for diagnosing this sarcoma. Methods: We reviewed 47 cases of alveolar soft part sarcoma that were either treated at Tata Memorial Hospital, Mumbai, India, or were referred in consultation from various parts of India. TFE3 immunohistochemical staining was performed on 22 alveolar soft part sarcomas and on 21 other tumours.
Results: Unlike most other large series, 58% of patients were males and 40% were females. The ages ranged from 2 to 54 years (median 24 years). Tumours were located in the deep soft tissues of lower extremities (54%), upper extremities (13%), head and neck (11%), retroperitoneum (10%), chest wall (6%), pelvis (4%), and were positive for TFE3 (20/22, 91%), desmin (3/18, 16%), myoglobin (1/6, 17%) and smooth muscle actin (1/9, 11%). TFE3 was positive in tumour controls that comprised paragangliomas (3/4), translocation related renal cell carcinoma (1/1), adrenocortical carcinoma (1/3) and granular cell tumour (1/3). Treatment consisted of primary surgical excision, metastatectomy, chemotherapy and radiotherapy. Seven tumours (24%) recurred locally and 21 of 29 (72%) metastasised, mainly to the lungs. Follow- up information (5-108 months, median 27.5 months) was available for 22 patients. No patients died in the relatively short follow-up period.
Conclusions: TFE3 is a useful immunohistochemical marker for diagnosis of an alveolar soft part sarcoma. Awareness of other tumours expressing TFE3 is vital. Alveolar soft part sarcoma has a high metastasis rate but relatively good short-term survival. Surgical excision with follow-up forms the present management.
Key words: Alveolar soft part sarcoma, immunohistochemistry of sarcomas, new sarcoma markers, soft tissue tumours, TFE3, uncommon sarcomas.
Received 24 April, revised 18 June, accepted 21 June 2011
INTRODUCTION
Alveolar soft part sarcoma is an uncommon high grade soft tissue sarcoma, first described by Christopherson et al.1 It accounts for 0.5-1.0% of soft tissue sarcomas in the United States.2 In a study, Bu and Bernstein3 identified 87 cases of alveolar soft part sarcoma from the United States population based, National Cancer Institute sponsored Surveillance, Epidemiology, and End Results (SEER) registries over 26 years. At our tertiary cancer referral centre, on average six to seven new cases of alveolar soft part sarcoma are diagnosed annually, forming 1.8% of the newly diagnosed soft tissue sarcomas.4 This histologically characteristic tumour of unknown histo- genesis mostly occurs in the deeper soft tissues of the lower extremities of adolescents and young adults. It has an indolent clinical course featuring recurrences, delayed metastases, late stage presentation and a high death rate.5-
Initially, it was proposed that alveolar soft part sarcomas show myogenic differentiation, in view of immunohistochemi- cal expression of skeletal muscle markers like MyoD1.10,11 However, subsequent studies disproved this theory by showing that the expression of these markers within the tumour cells was cytoplasmic, rather than nuclear which is considered as the ‘true positive’ expression.12,13 Recently, results from a comprehensive study of alveolar soft part sarcoma gene expression profiling have shown elevated expression of certain transcripts within this tumour, including some muscle-specific transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) that are suggestive of a muscle cell progenitor for this tumour.14 Ladyani et al.15 identified monocarboxylate transporter 1 (MCT1) protein and CD147 within the intracytoplasmic crystals of tumour cells of an alveolar soft part sarcoma.
Further, it has been found that alveolar soft part sarcoma exhibits an unbalanced translocation der(17)t(X;17)(p11;q25) that fuses the N-terminal region of the ASPL gene, located on 17q25, to the C-terminal of transcription factor E3 (TFE3), located at Xp11, leading to two alternate fusion transcripts, ASPL-TFE3 type 1 and type 2, that result in tumorigenesis by transcriptional deregulation.16 As one portion of protein from this fusion gene is overexpressed (transcription factorE3/
Print ISSN 0031-3025/Online ISSN 1465-3931 @ 2011 Royal College of Pathologists of Australasia DOI: 10.1097/PAT.0b013e32834d7ba4
TFE3), antibody for TFE3 has become commercially available for immunohistochemistry. TFE3 has been utilised as a diag- nostic marker for alveolar soft part sarcoma, but in only a few studies.17-19 Herein, we present a series of 47 cases of alveolar soft part sarcoma, diagnosed over period of 7 years at a tertiary cancer referral centre in India. The study involves a review of clinical profile, histopathological features, immunohistochemical results, including TFE3 expression in 22 tumours, and clinical outcomes in cases wherever available.
MATERIALS AND METHODS
We accessioned 47 cases of alveolar soft part sarcoma from the pathology database of the Tata Memorial Hospital, Mumbai, India, over a 7 year period (2002-2009) for inclusion in the present study. These cases were included after critical review by three authors (NAJ and BR with AI), who followed established histopathological diagnostic criteria for alveolar soft part sarcoma.1,5,7,9 Clinical information was obtained from the hospital case files and electronic medical records. Radiological details were obtained from the Picture Archiving and Communications System (PACS) at Centricity (GE Healthcare, USA). There were 34 registered cases in form of biopsies (12) and resection specimens (22) and 13 pathology consultation cases, referred from other hospitals across various parts of the country and adjoining countries, in the form of paraffin blocks with or without slides (10) and only slides (3). The tumour resection specimens included cases operated at our hospital, as well as from other hospitals and were defined as follows: wide excision specimens included tumours with grossly and microscopically free tumour resection margins; marginal excision included grossly free, but microscopically involved resection margin, passing through the tumour pseudocapsule; intracapsular excision included grossly as well as microscopically involved tumour resection margin.
Haematoxylin and eosin (H&E) stained microsections were available in all cases.
Immunohistochemical results were available in 34 cases (72.3%). TFE3 immunostaining was performed in 22 cases of alveolar soft part sarcoma and in 21 other tumours.
Immunohistochemical staining
Immunohistochemical staining was performed by immunoperxoidase method using the MACH 2 Universal HRP-Polymer Detection Kit (Biocare, USA) including 3’-3’-diaminobenzidine tetrahydrochloride (DAB) as the chromogen. The details of the various antibody markers are listed in Table 1. TFE3 (ab70008) rabbit polyclonal to TFE3 antibody (Abcam, USA) was utilised in the present study. The antigen retrieval method used was heat (as per the manufacturer’s recommendations), with Pascal using Tris-EDTA as the buffer. The antibody results in positive cases were validated with 1:250 dilution (as per the manufacturer’s recommendations).
Interpretation of immunohistochemical staining of TFE3
TFE3 nuclear staining was scored as 0, 1+ (mild, focal), 2+ (moderate, diffuse) and 3+ (intense, diffuse). Cytoplasmic staining was ignored. Only 2+ and 3+ nuclear staining was considered as positive. Cases showing focal/equivocal nuclear staining (1+) were considered negative, along with (0) nil staining. Cases with cytoplasmic staining were considered negative, unless nuclear staining was clearly more intense than cytoplasmic staining. Positive cases with classic histopathological features with 3+ and 2+ TFE3 immunostaining were included as positive controls. Positive and negative controls (lacking primary antibody) were included in all cases.18
RESULTS
Of 47 cases, 28 (58.3%) were males and 19 (40.4%) were females, with a male:female ratio of 1.47:1. The ages ranged from 2 to 54 years (mean and median 24 years). Tumours were located in the deep soft tissues of the lower extremities (26, 54.1%), including the thigh (14), leg (7), gluteal region (3), groin (1) and foot (1), followed by the upper extremities (arm and forearm) (6, 12.5%), retroperitoneum (5, 10.4%), chest wall (3, 6.3%), pelvis (2, 4.1%), orbit (2, 4.1%), tongue (2, 4.1%) and cheek (1).
Grossly, tumour size (21 cases) ranged from 3 to 21 cm (mean 8 cm, median 7 cm). Tumour thickness/depth ranged from 1.0 to 6.9 cm (mean and median 3.6 cm). The cut surface was lobulated and fleshy with areas of haemorrhage.
On microscopy, the various tumour patterns noted were ‘classic’ patterns composed of uniform organoid nests of polygonal cells, separated by fibrovascular septae that imparted a nodular pattern and delicate capillary sized vascular channels and focal pseudoalveolar arrangement (42, 89.3%). Within classic patterns, there were small uniform tumour nests (11), large uniform nests (4) and solid growth pattern (12). Certain cases revealed prominent haemangiopericytomatous vascula- ture. Intravascular tumour extension was a prominent feature in several tumours. Tumour cells revealed prominent dyscohe- sion, were large in size and commonly showed eosinophilic, granular cytoplasm and round nuclei with vesicular chromatin and prominent nucleoli. Variable features included cytoplasmic vacuolation (7), multinucleation (6), rhabdoid morphology, anaplasia (5), along with focal mucinous change, cystic change and chronic inflammatory infiltrate. Certain cases showed periodic acid-Schiff with diastase positivity in the form of granules and/or rod-like crystalline structures in sheaf-like or stacked configuration (Fig. 1 and 2).
| Sr no. | Antibody marker | Clonality, clone | Dilution | Antigen retrieval | Manufacturer |
|---|---|---|---|---|---|
| 1 | Epithelial membrane antigen (EMA) | Monoclonal, E 29 | 1:200 | Heat (Tris-EDTA) Pascal | Dako |
| 2 | Cytokeratin (CK) | Monoclonal, MNF116 | 1:200 | Heat (Tris-EDTA) Pascal | Dako |
| 3 | Myoglobin | Polyclonal | 1:800 | Enzymatic (Pepsin) | Dako |
| 4 | MyoD1 | Monoclonal | 1:20 | Heat, pressure cooker | Dako |
| 5 | Myogenin | Monoclonal, MyF4 | 1:50 | Heat (Tris-NaCitrate) | Novocastra |
| 6 | CD10 | Monoclonal | 1:40 | Heat (Tris-EDTA) Pascal | Biocare |
| 7 | Vimentin | Monoclonal, V9 | 1:400 | Heat (Tris-EDTA) microwave | Dako |
| 8 | Synaptophysin | Monoclonal | 1:100 | Heat (Tris-EDTA) Pascal | Dako |
| 9 | Chromogranin | Monoclonal | 1:400 DAK3 | Heat (Tris-EDTA) Pascal | Dako |
| 10 | S-100P | Polyclonal | 1:1500 | Heat (Tris-EDTA) Pascal | Dako |
| 11 | HMB45 | Monoclonal | 1:50 | Heat, Microwave | Dako |
| 12 | Smooth muscle actin (SMA) | Monoclonal, 1A4 | 1:400 | Heat (Tris-EDTA) Pascal | Dako |
| 13 | Desmin | Monoclonal, D33 | 1:200 | Heat (Tris-EDTA) Pascal | Dako |
| 14 | Inhibin | Monoclonal, R1 | 1:50 | Heat (Tris-EDTA) microwave | Serotec |
| 15 | CKIT/CD117 | Polyclonal | 1:100 | Heat (Tris-EDTA) microwave | Dako |
| 16 | TFE3 (ab70008) | Polyclonal | 1:250 | Heat (Tris-EDTA) Pascal | Abcam |
Abcam, USA; Biocare, USA; Dako, Denmark; Novocastra, UK; Serotec, UK; Sr, serial.
A
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On immunohistochemistry, tumours were focally positive for desmin (3/18, 16.1%), myoglobin (1/6, 16.6%), smooth muscle actin (1/9, 11.1%) and vimentin (2/10, 20%). Tumours were negative for cytokeratin (20), epithelial membrane anti- gen (19), S100-P (21), synaptophysin (9), chromogranin (9), HMB45 (11), CD10(8), «-inhibin (4) and CKIT (3), wherever performed. Nine tumours were negative for nuclear expression of MyoD1 and myogenin (MyF4) and showed cytoplasmic reactivity for these two markers.
Further, tumours were positive for TFE3 (20/22, 90.9%), including 3+ (11), 2+ (9), 1+ (1) and nil staining (Fig. 3).
TFE3 was positive in tumour controls (6/21, 28.5%) that comprised paragangliomas (3/4), translocation related renal cell carcinoma (1/1), adrenocortical carcinoma (1/3) and granular cell tumour (1/3) (Fig. 4). All three paragangliomas were diffusely positive for neuroendocrine markers (synapto- physin and chromogranin), including two that, in addition, displayed focal S100-P positivity in sustentacular cells.
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F
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9
TFE3 was negative in phaeochromocytoma (1), embryonal rhabdomyosarcoma (1), conventional renal cell carcinoma (1), papillary renal cell carcinoma (1) hepatocellular carcinomas (3), synovial sarcomas (2) and metastatic adenocarcinoma (1).
Treatment (25 patients, 53.1%) comprised primary surgical excision (24, 96%), including wide excision (12), marginal excision (1), intracapsular excision (1) and excision with unclear margins (9); adjuvant radiotherapy (6, 24%), including external beam radiotherapy or brachytherapy; palliative radio- therapy (1, 4%); adjuvant chemotherapy (3, 12%) and pulmon- ary metastatectomy (2, 8%).
Seven of 29 tumours (24%) recurred locally, including two tumours that presented as recurrences. Twenty-one tumours (72.4%) metastasised, mainly to the lung (18, 81.8%), followed by bone (4), suprapancreatic region (1) and liver (1). Whereas both lungs were involved in 13 cases, a single case presented with bilateral lungs, bone and liver metastases. Thirteen patients (48.1%) presented with metastasis and eight cases developed tumour metastasis subsequently.
Overall, outcome was available in 22 patients (46.8%) (5-108 months, median 27.5, mean 31). Five patients were
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D
free of disease over 5, 13, 22, 34 and 43 months. Four were treated by wide excision, including two who underwent adjuvant radiotherapy. Seventeen patients were alive with disease (5-108 months, median 31, mean 33.2). Of the 17 patients, five underwent wide excision, four underwent excisions with unclear margins and one underwent marginal excision. Details of the surgery performed on the remaining patients are not available.
Follow-up was available for 16 of the 21 patients with metastases. All were alive with disease in the limited period of follow-up (Table 2).
DISCUSSION
Alveolar soft part sarcoma is an uncommon tumour and constitutes 1.8% of soft tissue sarcomas at our tertiary cancer referral centre. The present study is a clinicopathological analysis of 47 cases of alveolar soft part sarcoma diagnosed over a 7 year period. A wide age range, along with a mean and median age of occurrence of 24 years, noted in the present study, is in keeping with earlier studies.5-7 The male prepon- derance is in contrast to the earlier studies showing a female predisposition. This has been attributed to the female’s extra X chromosome, which theoretically would increase the risk of
| Sr no. | Age/sex | Site | Specimen | T-size (cm) | Treatment resection | TFE3 staining | Recurrence/Metastasis | Outcome |
|---|---|---|---|---|---|---|---|---|
| 1 | 17/M | Leg | BS | NK | – | 2 | – | FOD (22 m) |
| 2 | 26/M | Thigh | BS | 6.5×5.5×3.2 | RO+RT | 3 | – | FOD (34 m) |
| 3 | 31/F | Forearm | EXC | NK | RX | 2 | – | NK |
| 4 | 25/M | Leg | BS | NK | – | 0 | – | NK |
| 5 | 6//M | Pelvis | EXC | 11×7.5×5 | R2 | 3 | – | NK |
| 6 | 26/F | Gluteal | BX | NK | – | 3 | Mets: spine, radius (3 m) | AWD (14 m) |
| 7 | 43/M | Thigh | EXC | 7×6.2×4.4 | R1 | 2 | Recurr (96 m); mets: B/L lungs (108 m) | AWD (108 m) |
| 8 | 27/F | Thigh | EXC | 7.5×5×4 | RX | 2 | Mets: B/L lungs | NK |
| 9 | 24/F | Tongue | EXC | NK | R0 | 3 | – | NK |
| 10 | 13/F | RP | EXC | 4×3×2 | R2 | 2 | Recurrence: duration NK | NK |
| 11 | 19/M | Arm | BS | NK | – | 3 | – | NK |
| 12 | 16/M | Chest wall | BS | NK | – | 3 | – | NK |
| 13 | 2/F | Thigh | EXC | 6×2.5×1.5 | RO+RT+ | 3 | Mets: lung (24 m) | AWD (36 m) |
| metastatectomy | ||||||||
| 14* | 49/M | Arm | EXC | 14×6.5×4.3 | R0 | 1 | Recurrence; mets: lung | NK |
| 15 | 46/F | Thigh | BX | NK | – | 3 | Mets: humerus | AWD (15 m) |
| 16 | 19/M | Thigh | BX | 12×5.6×3.6 | – | 3 | Mets: B/L lungs | AWD (6 m) |
| 17 | 5/M | Cheek | BX | NK | – | 3 | – | NK |
| 18 | 19/M | Orbit | EXC | 3×3×2 | R0 | 2 | Mets: lung | AWD (39 m) |
| 19 | 21/M | Thigh | BX | 10×6.5×4 | RT (palliative) | 2 | Mets: B/L lungs (60 m) | AWD (64 m) |
| 20 | 22/M | Back | BX | 6.5×5.5×3.5 | – | 3 | – | NK |
| 21 | 14/M | Leg | EXC | 6×2×1 | RO+metastatectomy+ RT+CT | 2 | Mets: B/L lungs, bone (36 m) | AWD (54 m) |
| 22 | 14/M | Thigh | EXC | NK | RX | – | Recurr ence (12 m); mets: lung (24 m) | AWD (24 m) |
| 23 | 6/M | Gluteal | BS | NK | – | 2 | – | NK |
| 24* | 27/F | Gluteal | BS | 6×4.5×4 | RX+RT+CT | – | Recurr ence (12 m); mets: B/L lungs | AWD (17 m) |
| 25* | 25/M | Forearm | BX | NK | – | – | Mets: B/L lungs | NK |
| 26 | 27/M | Pelvis | BX | 7.6×7.3×6.9 | – | – | Mets: suprapancreatic | NK |
| 27 | 13/F | Leg | EXC | NK | R0+CT | – | Mets: B/L lungs, recurrence (9 m) | AWD (48 m) |
| 28 | 16/F | Arm | EXC | 5×4×3 | RO+RT | – | – | FOD (43 m) |
| 29 | 22/F | Leg | EXC | 9×4.8×3.5 | R0 | – | Mets: B/L lungs | NK |
| 30 | 25/F | RP | S | NK | – | – | – | NK |
| 31 | 26/F | Thigh | EXC | 8×5×5 | R0 | – | – | FOD (13 m) |
| 32 | 18/M | Leg | EXC | 3×3×1 | R0 | – | – | FOD (5 m) |
| 33 | 24/M | Leg | BX | 21×6.4×5.6 | – | – | Mets: B/L lungs (24 m) | AWD (12 m) |
| 34 | 26/F | Thigh | S | NK | – | Mets: lung | AWD (5 m) | |
| 35 | 50/M | Arm | BS | NK | – | – | – | NK |
| 36 | 31/M | Orbit | S | NK | – | – | – | NK |
| 37 | 28/F | RP | EXC | 9×8.5×5.5 | RX | – | – | NK |
| 38 | 8/F | Thigh | EXC | NK | R0 | – | – | AWD (36 m) |
| 39 | 35/M | Foot | EXC | 7×4.2×3 | RX+EBRT | – | Recurrence; mets: B/L lungs, bone, liver (48 m) | AWD (48 m) |
| 40 | 52/M | RP | BS | NK | – | – | – | NK |
| 41 | 18/F | Tongue | BX | NK | – | – | – | NK |
| 42 | 15/M | Chest wall | EXC | NK | RX | – | Mets: B/L lungs | AWD (31 m) |
| 43 | 26/M | Thigh | EXC | NK | RX | – | Mets: B/L lungs (6 m) | AWD (8 m) |
| 44 | 54/M | Thigh | BX | NK | – | – | – | NK |
| 45 | 45/M | Thigh | BX | NK | – | – | – | NK |
| 46 | 12/F | RP | EXC | NK | RX | – | – | NK |
| 47 | 30/F | Groin | BS | NK | – | – | – | NK |
Cases with events at the time of presentation.
AWD, alive with disease; B/L, bilateral; BS, paraffin blocks and/or slides; BX, biopsy; CT, chemotherapy; EBRT, external beam radiotherapy; EXC, excision; F, female; FOD, free of disease; IHC, immunohistochemistry; M, male; m, months; Mets, metastasis; NK, not known; R, type of resection; RP, retroperitoneum; RT, radiotherapy; S, slide(s); Sr, serial; T, tumour; UP, unknown primary; W/E, wide excision.
developing the X;17 translocation of alveolar soft part sarcoma.3,16 The reason for the male preponderance in the present study is unknown. As in earlier studies, the lower extremities including the thigh were most the common sites of origin.5-7
Previously described histopathological features such as cytoplasmic clearing, multinucleation, rhabdoid-like cells, anaplasia, inflammatory reaction and cystic mucoid degene- ration were seen in some tumours.1,5-7,9,20-24 Special stains highlighted intracytoplasmic granules and various forms of crystals in many of the tumours. The granules that are pre- cursors of crystals and the crystals have been noted in 22% of alveolar soft part sarcomas.3 Recently, with ultrastructural immunohistochemistry, including immunogold labelling and Western blot analysis, Ladyani et al.15 have shown that mono- carboxylate transporter protein MCT1, one of the family of transporter proteins and chaperone CD147 are localised on the cell surface and cytoplasm of cells of alveolar soft part sarcoma, especially within the electron-dense granules and crystals, where they form complexes. Functions of these proteins, especially CD147 that induces matrix metalloprotei- nases, have been possibly related to the loss of matrix, leading to alveolar morphology and propensity of vascular invasion and metastasis in this tumour.25
Based on morphology, the differential diagnoses considered in the present study were paraganglioma, adrenocortical carcinoma, hepatocelluar carcinoma, rhabdomyosarcoma, melanoma, metastatic renal cell carcinoma and granular cell tumour. Prominent nucleoli within tumour cells, instead of ‘speckled’ chromatin pattern and negative immunohistochem- ical staining for neuroendocrine markers, objectively ruled out a paraganglioma. Negative immunostaining for vimentin and Melan-A ruled out an adrenocortical carcinoma in certain cases. S100-P, HMB45 and Melan-A negativity in tumour cells ruled out a melanoma in some cases. CD10 negativity in eight tumours was useful in ruling out a metastatic renal cell carcinoma.26 In these patients, absence of a tumour mass in the kidney on intra-abdominal imaging findings was further helpful in ruling out a renal cell carcinoma. Overall, immuno- histochemical results showed focal desmin (3/18, 16.1%), myoglobin (1/6, 16.6%), smooth muscle actin (1/9, 11.1%) and vimentin positivity (2/10, 20%). Despite desmin positivity in few tumours, along with focal expression of other myogenic markers, lack of nuclear positivity for MyoD1 and myogenin ruled out a rhabdomyosarcoma.5,10-13 Like previous authors, we observed cytoplasmic positivity for MyoD1 and myogenin in certain tumours.13 Desmin positivity, suggestive for myo- genic differentiation in alveolar soft part sarcomas, has been noted in earlier studies.27,28 Besides, predilection of MCT1 protein that is an integral part of the characteristic cytoplasmic granules of tumour cells of an alveolar soft part sarcoma, for skeletal and cardiac muscle fibres, indirectly suggests myo- genic differentiation in this tumour.29,30 Unlike Lieberman et al.,” we did not identify S100-P positivity in any of the tumours, wherever it was performed. S100-P negativity also ruled out a granular cell tumour. In our recently documented series of granular cell tumours, we observed S100-P positivity in 100% tumours.31 In this way, the present study and our earlier study reinforce the current view that alveolar soft part sarcoma and granular cell tumour are two distinct tumour entities.32,33 Focal vimentin positivity in few tumours and lack of epithelial markers in any of the tumours studied was similarly noted in an earlier study.” In this way, there is
lack of a consistent immunohistochemical marker that could indicate either an epithelial, neural, neuroendocrine, melano- cytic or a conclusive mesenchymal differentiation within this tumour. Therefore, alveolar soft part sarcoma has been included as a tumour with uncertain histogenesis. The earlier theories speculating alveolar soft part sarcoma as a non-chromaffin paraganglia or a malignant counterpart of angioreninoma have also been disproved.34,35
In view of its variable histological features that overlap with other tumours, there has been a need for identification of a specific and sensitive diagnostic marker for alveolar soft part sarcoma, especially in limited biopsy material and in cases with uncommon sites of occurrence. Lately, a characteristic, unbalanced translocation der(17)t(X;17)(p11;q25) has been identified in alveolar soft part sarcomas that includes fusion of the N-terminal region of ASPL gene, located on 17q25, to the C-terminal of transcription factor E3 (TFE3), located at Xp11, leading to two alternate fusion transcripts, ASPL-TFE3 type 1 and type 2, thereby resulting in tumorigenesis by transcriptional deregulation.16 TFE3 is a ubiquitous basic helix-loop-helix (bHLH) leucine zipper transcription factor, which specifically recognises and binds to MUE boxes, a subset of E-box sequences (3’- CANNTG-5’). Efficient DNA binding by TFE3 relies on homodimerisation or heterodimerisation with another miT/TFE family member such as TEFB, TEFC and MITF. On the other hand ASPL has only been partially characterised as a tethering protein that forces retention of GLUT-4 containing vesicles when insulin is absent.36 TFE3 contains a nuclear localisation signal that maps to a portion of TFE3 retained within all known TFE3 fusion proteins.37 It has been accepted that aberrant TFE3 transcriptional activity modulated by the ASPL fusion partner leads to pathogenesis of alveolar soft part sarcoma. Although the function of ASPL remains unknown, TFE3 gene has also been implicated in a subset of renal cell carcinomas that mostly occur in paediatric cases and young adults, characterised by t(X;1)(p11.2; q21) translocation that leads to fusion of TFE3 gene with PRCC gene.38-40 TFE3 immunostaining has been found to be useful in diagnosis of alveolar soft part sarcoma and other neoplasms characterised by TFE3 gene fusions, but in only a few published studies.17-19,41 Although translocation analysis is the diagnos- tic ‘gold’ standard, validation of this immunohistochemical marker would enhance diagnostic objectivity, especially in limited resource settings like ours. Argani et al.18 observed TFE3 positivity in 19 (100%) alveolar soft part sarcomas, in 20 of 21 (95.2%) renal cell carcinomas with Xp11.2 transloca- tions and in few other tumours. They documented 97.5% sensitivity and 99.6% specificity of TFE3 for diagnosis of alveolar soft part sarcomas. Pang et al.17 observed TFE3 positivity in 16 (100%) alveolar soft part sarcomas, along with variable expression of myogenic markers like myoglobin (81.2%), actin (62.5%) and desmin (12.5%) in those tumours. Vistica et al.41 generated polyclonal antibodies to 25 mer peptides encompassing the junctional regions of ASPL-TFE3 type 1 and ASPL-TFE3 type 2. They observed TFE3 immuno- staining on alveolar soft part sarcomas and documented its utility in differentiation between tumours that express the type 1 and type 2 fusion proteins. Williams et al.19 documented TFE3 positivity in 18 (100%) alveolar soft part sarcomas. We observed TFE3 positivity in 20 of 22 (91%) alveolar soft part sarcomas, with focal expression of myogenic markers in some cases, barring positivity for skeletal muscle specific markers. In addition, we observed TFE3 positivity in 28.5%
tumour controls that included granular cell tumour (1/3), as was also noted by Argani et al.18 Williams et al.19 documented TFE3 positivity in four granular cell tumours. However, none of those cases displayed ASPL/TFE3 fusion transcripts on molecular analysis, indicating lack of ‘link’ between an alveo- lar soft part sarcoma and a granular cell tumour. We observed TFE3 positivity in our single documented case of metastatic renal cell carcinoma, reinforcing diagnosis of translocation related renal cell carcinoma.42 We also observed TFE3 positivity in paragangliomas (3/4) that were positive for neuro- endocrine markers. Besides, we noted TFE3 positivity in a single case of adrenocortical carcinoma, as noted earlier.18,19 Therefore, it is important to employ a panel of markers while differentiating an alveolar soft part sarcoma from its differen- tial diagnoses. The reasons for TFE3 false positivity include detection of up-regulated native TFE3 protein.18 Despite initial standardisation with optimal temperature for antigen retrieval and antibody concentration, variability in processing of tissues, including referral material, could enhance native TFE3 expres- sion in these tumours. Ideally, such cases should be tested with molecular and/or cytogenetic analysis that was not performed in the present study. Nonetheless, other histopathological features and immunohistochemical findings were supportive of diagnoses of tumour controls. Conversely, the reasons for TFE3 negativity in two tumours included referral biopsy material from mofussil areas, wherein antigen retrieval is compromised as a result of suboptimal fixation. Immunostain- ing was repeated on two referral tumours that initially showed TFE3 negativity; with repeat staining on re-biopsied sections they revealed positive staining. It has been stated that despite high sensitivity, TFE3 negative staining does not refute diagnosis of an alveolar soft part sarcoma in cases with classic histopathological features. The reasons for negative expression in such cases include suboptimal fixation, inadequate sampling and relatively higher immunoexpression at tumour edges, as a result of differences in oxygenation.18,43 At the same time, TFE3 expression in older paraffin blocks and already cut slides merits its usage as a marker within archival specimens, as noted in our study.18
Therapeutically, alveolar soft part sarcoma is optimally treated with complete surgical excision. The role of adjuvant radiotherapy and chemotherapy has not been found to be significant.5,44,45 Treatment details were available in 53% of our patients, wherein most patients were treated with wide excisions. Seven tumours (24%) recurred locally and 21 of 29 (72%) metastasised, mainly to the lungs. Of these, 48.1% of patients presented with distant metastasis. Earlier, Lieberman et al.3 observed tumour metastasis in 66.6% of patients, includ- ing 22.5% of patients initially presenting with metastasis. A larger number of patients presenting with metastasis is attributed to the fact that ours is a tertiary referral centre, where more patients present with higher tumour stage. Similar observations were made in a study conducted at another cancer referral centre, wherein 65% patients presented with stage IV disease.6 In both of these studies and the present study, lung was observed as the most common site of metastasis, followed by bone, brain and liver as other sites. In view of occurrence of brain metastasis, practice guidelines by the Society of Surgical Oncology (SSO) recommend routine intracranial imaging in patients with alveolar soft part sarcoma; however, the present study is in agreement with observations by Portera et al., who stated that the possibility of isolated brain metastasis is exceedingly rare in such patients. In the present study, brain
metastasis was not identified in any of the few patients in whom intracranial imaging was performed, considering financial constraints among our patients. Further, metastatectomy is known to enhance median survival.5,6 In our study, two patients underwent metastatectomy and were alive with disease over 36 and 54 months. Outcomes were accessible in 46.8% patients (median 27.5 months). Most of them (77.2%) were alive with disease and five were free of disease, including four patients who underwent wide excision.
In summary, alveolar soft part sarcoma is an uncommon soft tissue sarcoma. Intense, intranuclear TFE3 positivity is useful in diagnosing this sarcoma. Awareness of TFE3 positivity in other tumours is vital. A panel of markers should be employed in order to identify an alveolar soft part sarcoma from its differential diagnoses. This sarcoma is optimally managed with a wide excision, complete metastatic ‘work-up’ and follow- up.14 Identification of TFE3 as a useful marker seems to open a new avenue for putative therapeutic target for this sarcoma that is invariably chemoresistant.
Acknowledgements: We acknowledge the technical support of Mr Mahendra Palker, Mrs Rekha Thorat, Mrs Jyoti, Mr Amir Khan and Mr Pritam Shinde from the immunohistochemistry laboratory.
Conflicts of interest and sources of funding: The authors have no conflicts of interest to declare.
Address for correspondence: Dr B. Rekhi, Department of Pathology, Tata Memorial Hospital, Dr E. B. Road, Parel, Mumbai, India, 400012. E-mail: rekhi.bharat@gmail.com
References
1. Christopherson WM, Foote FW Jr, Stewart FW. Alveolar soft-part sarcomas; structurally characteristic tumours of uncertain histogenesis. Cancer 1952; 5: 100-11.
2. Enzinger FM, Weiss SW. Malignant tumours of uncertain type. In: Enzinger FM, Weiss SW, editors. Soft Tissue Tumours. St Louis: Mosby, 1995; 1067-74.
3. Bu X, Bernstein L. A proposed explanation for female predominance in alveolar soft part sarcoma. Noninactivation of X; autosome translocation fusion gene? Cancer 2005; 103: 1245-53.
4. Dinshaw KA, Ganesh B. Hospital Based Cancer Registry. Annual Report 2002-2005, Tata Memorial Hospital, Mumbai, India. Mumbai: Tata Memorial Hospital, 2005; 1-110.
5. Lieberman PH, Brennan MF, Kimmel M, Erlandson RA, Garin-Chesa P, Flehinger BY. Alveolar soft-part sarcoma. A clinico-pathologic study of half a century. Cancer 1989; 63: 1-13.
6. Portera CA Jr, Ho V, Patel SR, et al. Alveolar soft part sarcoma: clinical course and patterns of metastasis in 70 patients treated at a single institu- tion. Cancer 2001; 91: 585-91.
7. Ogose A, Yazawa Y, Ueda T, et al. Japanese Musculoskeletal Oncology GroupAlveolar soft part sarcoma in Japan: multi-institutional study of 57 patients from the Japanese Musculoskeletal Oncology Group. Oncology 2003; 65: 7-13.
8. Weiss SW. Alveolar soft part sarcoma. Are we at the end or just the beginning of our quest? (Commentary.) Am J Pathol 2002; 160: 1197-9.
9. Folpe AL, Deyrup AT. Alveolar soft-part sarcoma: a review and update. J Clin Pathol 2006; 59: 1127-32.
10. Rosai J, Dias P, Parham DM, Shapiro DN, Houghton P. MyoD1 protein expression in alveolar soft part sarcoma as confirmatory evidence of its skeletal muscle nature. Am J Surg Pathol 1991; 15: 974-81.
11. Foschini MP, Eusebi V. Alveolar soft-part sarcoma: a new type of rhabdomyosarcoma? Semin Diagn Pathol 1994; 11: 58-68.
12. Wang NP, Bacchi CE, Jiang JJ, McNutt MA, Gown AM. Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immuno- cytochemical and biochemical study of myogenic regulatory protein expression. Mod Pathol 1996; 9: 496-506.
13. Gómez JA, Amin MB, Ro JY, Linden MD, Lee MW, Zarbo RJ. Immuno- histochemical profile of myogenin and MyoD1 does not support skeletal muscle lineage in alveolar soft part sarcoma. Arch Pathol Lab Med 1999; 123: 503-7.
14. Stockwin LH, Vistica DT, Kenney S, et al. Gene expression profiling of alveolar soft-part sarcoma (ASPS). BMC Cancer 2009; 9: 22.
15. Ladanyi M, Antonescu CR, Drobnjak M, et al. The precrystalline cyto- plasmic granules of alveolar soft part sarcoma contain monocarboxylate transporter 1 and CD147. Am J Pathol 2002; 160: 1215-21.
16. Ladanyi M, Lui MY, Antonescu CR, et al. The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25. Oncogene 2001; 20: 48-57.
17. Pang LJ, Chang B, Zou H, et al. Alveolar soft part sarcoma: a biomarker diagnostic strategy using TFE3 immunoassay and ASPL-TFE3 fusion transcripts in paraffin-embedded tumour tissues. Diagn Mol Pathol 2008; 17: 245-52.
18. 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.
19. Williams A, Bartle G, Sumathi VP, et al. Detection of ASPL/TFE3 fusion transcripts and the TFE3 antigen in formalin-fixed, paraffin-embedded 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.
20. Fanburg-Smith JC, Miettinen M, Folpe AL, Weiss SW, Childers EL. Lingual alveolar soft part sarcoma; 14 cases: novel clinical and morpho- logical observations. Histopathology 2004; 45: 526-37.
21. Auerbach HE, Brooks JJ. Alveolar soft part sarcoma. A clinicopathologic and immunohistochemical study. Cancer 1987; 60: 66-73.
22. Jong R, Kandel R, Fornasier V, Bell R, Bedard Y. Alveolar soft part sarcoma: review of nine cases including two cases with unusual histology. Histopathology 1998; 32: 63-8.
23. Ordonez NG, Mackay B. Alveolar soft-part sarcoma: a review of the pathology and histogenesis. Ultrastruct Pathol 1998; 22: 275-92.
24. Evans HL. Alveolar soft-part sarcoma. A study of 13 typical examples and one with a histologically atypical component. Cancer 1985; 55: 912-7.
25. Zucker S, Hymowitz M, Rollo EE, et al. Tumourigenic potential of extracellular matrix metalloproteinase inducer. Am J Pathol 2001; 158: 1921-8.
26. Avery AK, Beckstead J, Renshaw AA, Corless CL. Use of antibodies to RCC and CD10 in the differential diagnosis of renal neoplasms. Am J Surg Pathol 2000; 24: 203-10.
27. Mukai M, Torikata C, Shimoda T, Iri H. Alveolar soft part sarcoma. Assessment of immunohistochemical demonstration of desmin using paraffin sections and frozen sections. Virchows Arch A Pathol Anat His- topathol 1989; 414: 503-9.
28. Sciot R, Dal Cin P, De Vos R, et al. Alveolar soft-part sarcoma: evidence for its myogenic origin and for the involvement of 17q25. Histopathology 1993; 23: 439-44.
29. Kirk P, Wilson MC, Heddle C, Brown MH, Barclay AN, Halestrap AP. CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J 2000; 19: 3896-904.
30. Daigeler A, Kuhnen C, Hauser J, et al. Alveolar soft part sarcoma: clinicopathological findings in a series of 11 cases. World J Surg Oncol 2008; 6: 71.
31. Rekhi B, Jambhekar NA. Morphologic spectrum, immunohistochemical analysis, and clinical features of a series of granular cell tumours of soft tissues: a study from a tertiary referral cancer center. Ann Diagn Pathol 2010; 14: 162-7.
32. Ekfors TO, Kalimo H, Rantakokko V, Latvala M, Parvinen M. Alveolar soft part sarcoma: a report of two cases with some histochemical and ultra- structural observations. Cancer 1979; 43: 1672-7.
33. Ordonez N, Ladyani M. Alveolar soft part sarcoma. In: Fletcher CDM, Unni KK, Mertens F, et al., editors. Pathology and Genetics of Tumours of Soft Tissue and Bone. Lyon: IARC Press, 2002.
34. Smetana HF, Scott WF Jr. Malignant tumours of nonchromaffin para- ganglia. Mil Surg 1951; 109: 330-49.
35. 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.
36. Kuiper RP, Schepens M, Thijssen J, Schoenmakers EF, van Kessel AG. Regulation of the MiTF/TFE bHLH-LZ transcription factors through restricted spatial expression and alternative splicing of functional domains. Nucleic Acids Res 2004; 32: 2315-22.
37. Weterman MAJ, van Groningen JJM, Jansen A, van Kessel AG. Nuclear localization and transactivating capacities of the papillary renal cell carcinoma-associated TFE3 and PRCC (fusion) proteins. Oncogene 2000; 19: 69-74.
38. Sidhar SK, Clark J, Gill S, et al. The t(X; 1)(p11.2;q21.2) translocation in papillary renal cell carcinoma fuses a novel gene PRCC to the TFE3 transcription factor gene. Hum Mol Genet 1996; 5: 1333-8.
39. Weterman MAJ, Wilbrink M, Janssen I, et al. Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)- positive papillary renal cell carcinomas. Proc Natl Acad Sci USA 1996; 93: 15294-8.
40. Argani P, Antonescu CR, Illei PB, et al. Primary renal neoplasms with the ASPL-TFE3 gene fusion of alveolar soft part sarcoma: a distinctive tumour entity previously included among renal cell carcinomas of children and adolescents. Am J Pathol 2001; 159: 179-92.
41. Vistica DT, Krosky PM, Kenney S, Raffeld M, Shoemaker RH. Immunohistochemical discrimination between the ASPL-TFE3 fusion proteins of alveolar soft part sarcoma. J Pediatr Hematol Oncol 2008; 30: 46-52.
42. Rekhi B, Kumar R, Menon S, Medhi S, Desai SB. Calvarial metastasis of a renal cell carcinoma, mimicking a primary alveolar soft part sarcoma, in a young girl-a rare case report. Pathol Oncol Res 2009; 15: 137-41.
43. Camp RL, Charette LA, Rimm DL. Validation of tissue microarray technology in breast carcinoma. Lab Invest 2000; 80: 1943-9.
44. Kayton ML, Meyers P, Wexler LH, Gerald WL, La Quaglia MP. Clinical presentation, treatment, and outcome of alveolar soft part sarcoma in children, adolescents, and young adults. J Pediatr Surg 2006; 41: 187-93.
45. Casanova M, Ferrari A, Bisogno G, et al. Alveolar soft part sarcoma in children and adolescents: A report from the Soft-Tissue Sarcoma Italian Cooperative Group. Ann Oncol 2000; 11: 1445-9.