While approximately 98% of malignant rhabdoid tumors harbor bi-allelic SMARCB1
gene abnormalities with few additional karyotypic abnormalities, there is conflicting data for epithelioid sarcoma (5
). The first major study to evaluate SMARCB1
in epithelioid sarcoma was by Modena et al in 2005 (19
). Of 7 (5 proximal and 2 classical) immunohistochemistry negative cases, 5 (71%) proximal epithelioid sarcomas demonstrated homozygous deletions of SMARCB1
using bacterial artificial chromosome probes for dual-color fluorescence in situ hybridization. Two of the cases with homozygous deletions were also tested using comparative genomic hybridization, with one case showing a heterozygous deletion and the other failing to demonstrate any deletions. The discrepancy in the fluorescence in situ hybridization and comparative genomic hybridization results was likely due to the comparative genomic hybridization resolution of 2–4 Mb in this study. Semi-quantitative polymerase chain reaction of SMARCB1
exon 1was performed on 4 of the cases with homozygous deletions and showed concordant results. No mutations were identified by sequencing.
In 2009 Kohashi et al. studied 39 immunohistochemistry-negative epithelioid sarcomas (19 proximal and 27 classical) (20
). Deletions were identified by quantitative real-time polymerase chain reaction for exons 1 to 9 of the SMARCB1
gene. Only 2 (5%) proximal epithelioid sarcoma cases showed homozygous deletions. Sequencing was performed for mutation analysis and 2 (5%) proximal epithelioid sarcomas showed homozygous frameshift mutations consisting of a one base pair deletion in exon 9 and a two base pair deletion in exon 3. Following this article, a correspondence was written by Flucke et al. describing a case of proximal epithelioid sarcoma with a c.769C>T mutation in exon 6 resulting in the generation of an in-frame stopcodon (22
Most recently in 2011, Gasparini et al. evaluated 19 immunohistochemistry-negative epithelioid sarcomas (5 proximal and 14 classical) (21
). As tabulated in the article, a total of 11 cases (58%, 4 proximal and 7 classical) were shown to harbor homozygous deletions of SMARCB1
with 9 identified through fluorescence in situ hybridization and an additional 2 by quantitative polymerase chain reaction for exon 4. No mutations were identified through sequencing. This was the first series to demonstrate SMARCB1
deletions in both proximal and classical epithelioid sarcoma.
Taken together, the above studies demonstrate that SMARCB1
deletions are far more common than gene mutations in epithelioid sarcoma; however, the exact number of cases with primary gene abnormalities varies dramatically between published reports. In the present study, 12 immunohistochemistry-negative epithelioid sarcomas (7 classical, 5 proximal) had at least 50% tumor in the sample and adequate DNA recovery for multiplex ligation dependent probe amplification and sequencing. Multiplex ligation dependent probe amplification revealed that 10/12 (83%, 6 classical, 4 proximal) harbored homozygous deletions. This percentage is similar to what we reported for extrarenal rhabdoid tumors but higher than that seen in CNS atypical teratoid/rhabdoid tumor or renal malignant rhabdoid tumor (6
). Of note, 2 of the 10 cases (20%) had a homozygous deletion spanning a small portion of the gene, either exons 4 to 5 or exons 6 to 9. These cases would likely have been missed using traditional fluorescence in situ hybridization techniques or limited quantitative polymerase chain reaction including only a subset of exons. Of the 2 remaining cases (17%, 1 classical, 1 proximal), multiplex ligation dependent probe amplification showed heterozygous deletions involving all exons of the SMARCB1
gene. No mutations were identified through sequencing.
The finding of this high rate of bilallelic loss or inactivation of SMARCB1 in a high percentage of both proximal and classical epithelioid sarcoma tumors tested is consistent with the two hit model of carcinogenesis associated with loss of function of a tumor suppressor gene, and correlates with the near universal biallelic loss or inactivation of SMARCB1 seen in malignant rhabdoid tumor. Although 2 of our cases only showed heterozygous deletions, the loss of SMARCB1 protein expression suggests the presence of an undetected abnormality of SMARCB1 in the other allele. Possible explanations for the failure to demonstrate this may be due to the high percentage of normal stromal cells in the samples, because a high percentage of normal cell contamination can mask a deletion using multiplex ligation dependent probe amplification. Despite the fact that hematoxylin and eosin stained sections of each tumor were evaluated for percentage of tumor present, increased proportions of normal cells could have been present in the tissue scrolls used for DNA extraction. As a result, the 2 cases with heterozygous deletions may represent false negative results in tumors with true homozygous deletions. One of the 2 cases had additional formalin-fixed, paraffin-embedded tissue available for fluorescence in situ hybridization testing, which would have allowed for evaluation of SMARCB1 copy number limited exclusively to the tumor tissue. However, the fluorescence in situ hybridization assay was unsuccessful. Alternatively, there may have been an intronic mutation present that would have been missed by only analyzing the exons and intron/exon boundaries.
It is important to note that the high percentage of cases with homozygous deletions, relatively low number of cases with intragenic deletions and subset of cases where only a single gene defect could be identified raises the possibility that loss of SMARCB1 protein expression may be a secondary event in epithelioid sarcoma. The driving force for chromosome 22 deletions seen in epithelioid sarcoma may actually result from selection of a nearby gene instead of SMARCB1. Additionally, an intronic or upstream change that affects expression of the SMARCB1 protein also cannot be excluded based upon our data.
The results from this study and others suggest that somatic SMARCB1
status alone is not sufficient to differentiate malignant rhabdoid tumor from epithelioid sarcoma. As a result, the finding of bi-allelic mutations or deletions in SMARCB1
should not be used as criteria to support reclassifying epithelioid sarcoma as malignant rhabdoid tumor. Being able to reliably differentiate epithelioid sarcoma and malignant rhabdoid tumor is important due to differences in treatment, prognosis and the association of malignant rhabdoid tumor with germline mutations necessitating additional genetic testing and family counseling (6
). Despite intensive treatment for patients with malignant rhabdoid tumor, many patients die of widespread metastatic disease, especially those with germline mutations and second primary lesions. For example, one recent study limited to extra-renal non-cerebral rhabdoid tumors demonstrated a median time to recurrence of 5 months with only 1 of 26 (4%) patients alive and without evidence of disease at 7 years (28
). Currently, many patients with soft tissue malignant rhabdoid tumor are enrolled in Children’s Oncology Group protocol AREN0321 which employee a highly aggressive initial multimodality approach including surgery, radiation therapy and multiagent chemotherapy using vincristine, doxorubicin, cyclophospamide, carboplatin and etoposide, but results of patient outcome are unknown at this time.
In contrast, the overall 5-year survival rates for classical epithelioid sarcoma range from 50–85% and 10-yr survival rates range from 42–55% (18
). The propensity of epithelioid sarcoma to grow along fascial planes, tendons and nerves often results in initial resections with positive margins and a protracted clinical course including multiple local recurrences and metastasis (26
). Adequate treatment for epithelioid sarcoma requires early radical local excision or amputation with regional lymph node dissection. The specific regimen for radiation therapy and adjuvant chemotherapy is unclear, but is often similar to treatment plans for other adult sarcomas (29
The survival rate for proximal epithelioid sarcoma is challenging to define due to difficulties in separating these lesions from malignant rhabdoid tumor in many studies and the fact that many of the reported adverse prognostic features for classical epithelioid sarcoma are often seen in proximal epithelioid sarcoma, including non-distal extremity location, large tumor size, increased tumor depth and inadequate initial excision (26
). Until recently, many used SMARCB1
deletions or absence of SMARCB1 protein expression by immunohistochemistry as evidence for the diagnosis of malignant rhabdoid tumor, especially in children. However, studies of firmly diagnosed epithelioid sarcoma, including those in older patients, have shown SMARCB1
abnormalities as well, suggesting that SMARCB1
status alone cannot reliably distinguish epithelioid sarcoma from malignant rhabdoid tumor (19
Our study demonstrates that the majority of both proximal and classical epithelioid sarcoma have homozygous deletions of SMARCB1
, which can be identified with multiplex ligation dependent probe amplification. Given the high percentage of SMARCB1
alterations in epithelioid sarcoma, these findings argue against using SMARCB1
gene deletion to distinguish epithelioid sarcoma from malignant rhabdoid tumor (20
). Moreover, these findings suggest an underlying genetic relationship between epithelioid sarcoma and malignant rhabdoid tumor. Further study is warranted to explore why these two tumors which so closely share phenotypic and genetic features behave in clinically distinctive ways.