A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and
Rhabdomyosarcoma (RMS) is subdivided into three major variants: embryonal, alveolar, and pleomorphic. Embryonal and alveolar subtypes are commonest sarcomas of childhood and adolescence. Better clinical outcome is associated with botryoid and spindle cell variants of embryonal RMS. In particular, the spindle cell variant in childhood is considered to be of low malignant potential with excellent overall patient survival. Pleomorphic RMS is rare and highly aggressive adult sarcomas typically arising in the deep soft tissue of the extremities. Even rarer are recently described spindle cell and sclerosing variants of RMS in adults. Due to their rarity, the experience with the newer subsets is limited but appears to show poor outcome in adults. Sclerosing variant of RMS as a distinct entity was initially reported in three cases by Mentzel and Katenkamp in 2000 [
Representative 5-
Fresh tumor in RPMI tissue culture medium (Invitrogen, Carlsbad, CA, USA) was sent to the Cytogenetics Laboratory within 2 hours from time of biopsy. The sample was enzymatically dissociated using Type I Collagenase (cat. no. LS004196; Worthington Biochemical, Lakewood, NJ, USA) at a final concentration of 1 unit/mL for 90 minutes. The dissociated cells were cultured in RPMI plus 15% fetal bovine serum (Irvine Scientific, Santa Ana, CA, USA) or a combination of this with Chang (Irvine Scientific) complete medium in closed tissue culture flasks for 5 and 7 days. Cell harvest and preparation of slides were performed according to standard laboratory protocol. Twenty-four G-banded (trypsin/Wright stain) metaphase cells were analyzed at approximately the 300 band level.
Fluorescence in situ hybridization was performed using Abbott probes for
Multiplex mutation analysis was performed on the Mass Array system (Sequenom, San Diego, CA, USA) using a panel that screens for 647 mutations across 53 genes, exactly as previously described [
10T1/2 cells and 10T1/2-H1047R cells have been previously described [
The
10T1/2 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer or NP40 buffer containing both protease and phosphatase inhibitor (Sigma-Aldrich). The lysates were homogenized and centrifuged at 8000 g for 10 minutes. The resulting supernatants were used for immunoblot analysis by mouse anti-
10T1/2 cells were plated at 6 × 103 cells per well in a 6-well plate with soft-agar with DMEM and 10% fetal bovine serum (SeePlaqueAgarose; cat. 50101, Lonza, Allendale, NJ, USA).
10T1/2 cells were plated at 2 × 103 cells of each cohort per well in a 96-well plate.
An otherwise healthy 23 year-old man presented with a 6-month history of an enlarging mass within the plantar aspect of his left foot. MRI revealed an 8 cm mass infiltrating the plantar surface, extending through the dorsal surface, and circumferentially involving the 2nd through 4th metatarsals (Figure
(a) MRI imaging. (b) Diagnostichistology of the tumor demonstrating round hyperchromatic to spindled cells surrounded by a densely hyalinized stroma. Hematoxylin-eosin, original magnification ×200. (c) Desmin immunohistochemical stain showing strong diffuse cytoplasmic expression of desmin. (d) Although sparse, individual cells demonstrated strong nuclear staining with myogenin. Myogenin immunohistochemical stain. (e) Several cells demonstrated strong nuclear staining with MDM2 immunohistochemical stain. (f) Representative G-banded karyogram demonstrating a complex abnormal hyperdiploid clone. These cells contained from 20 to 50 double minutes, shown by fluorescence in situ hybridization (FISH) to be amplification of the
Cytogenetic analysis of tumor from the original biopsy revealed fourteen of twenty-four metaphase cells to have a complex hyperdiploid (near-triploid) karyotype. These cells contained approximately 20 to 50 double minutes, shown by FISH (below) to include amplification of the
Interphase fluorescence in situ hybridization (FISH) showed that 15.5% of 200 nuclei had amplification of
Staging studies, including operative sampling of left inguinal and iliac lymph nodes due to an abnormal PET scan, did not reveal evidence of metastatic disease. The patient underwent systemic chemotherapy with vincristine, dactinomycin, and cyclophosphamide as a part of a clinical trial. He was offered a below-the-knee amputation, but he declined in favor of radiation. He received a total dose of 50.4 Gy/28 fractions and had an excellent response in the foot. Unfortunately, he developed pulmonary metastases immediately upon the completion of chemotherapy. Subsequent therapies included irinotecan with temozolomide, doxorubicin with ifosfamide, and sorafenib, none of which resulted in clinical response. He also underwent surgical resection of a massive and symptomatic right lower lung metastasis and received several courses of palliative radiation therapy. The patient expired 25 months after diagnosis.
A complete autopsy was performed twenty seven hours after death. Tumor masses were present in all lung lobes, nearly replacing the right lung. There were multiple metastases in both liver lobes, adjacent to esophagus, in the right adrenal, and in the right frontal cortex of the brain, as well as invading the chest wall, diaphragm, and multiple vertebrae. Fresh tissue was obtained sterilely for culture and frozen aliquots from the right chest, left upper lobe, and liver as well as normal skin and skeletal muscle (frozen only) immediately after opening of the body. Histologically the tumor was similar to that in the prior tumor biopsy with somewhat more cytologicatypia and a range of 30 to 90% necrosis (Figure
Disseminated disease at necropsy. (a) Cross-section of liver demonstrating metastatic nodules. (b) Histological section of lung with metastatic sclerosing rhabdomyosarcoma. Hematoxylin-eosin, insert original magnifications ×200.
Genotyping performed on tumor from the autopsy revealed a
To investigate the effect of combined
(a) Western blotting of MDM2, Akt, phosphoAkt, and
In contrast to childhood RMS, the adult variants are believed to be highly aggressive irrespective of the morphologic subtype. The pleomorphic variant of RMS, exclusively seen in adults, has been extensively studied. Recently described variants in adults include spindle cell and sclerosing RMS. One of the limitations of understanding biology of these new entities is the overall rarity of such cases. It has also been noted that about 15–20% cases of adult spindle cell RMS show morphological overlap with the sclerosing variant [
The sclerosing variant of rhabdomyosarcoma presents a significant diagnostic challenge. From its hyalinizing pseudovascular appearance and sometimes focal and dot-like desmin and myogenin expression, these tumors could easily be mistaken for variants of angiosarcoma, extraskeletal myxoid chondrosarcoma, osteosarcoma, or sclerosing epithelioid fibrosarcoma.
Sclerosing rhabdomyosarcoma is an aggressive sarcoma. Among the original three cases reported by Mentzel and Katenkamp, one exhibited progressive pulmonary metastases despite systemic chemotherapy [
Among the few karyotypes reported, no characteristic numerical alteration of the chromosomal complement has emerged (Table
Summary of reported cytogenetic abnormalities in sclerosing rhabdomyosarcoma.
Author | Year | Cases | Notes |
---|---|---|---|
Mentzel and Katenkamp [ |
2000 | 3 | No ARMS fusion transcripts due to t(1;13) or t(2;13) found by RT-PCR |
Folpe et al. [ |
2002 | 4 | Cases 1 and 3: inadequate RNA for RT-PCR. Case 2: no evidence of |
Vadgama et al. [ |
2004 | 1 | No ARMS fusion transcripts by RT-PCR |
Chiles et al. [ |
2004 | 13 | 4 cases negative for ARMS fusion transcripts ( |
Croes et al. [ |
2005 | 1 | Tumor cells negative for |
Zambrano et al. [ |
2006 | 3 | Case 1: normal. Case 2: complex structural and numerical abnormalities with numerous double minutes in most cells; case 3: less complex, with balanced translocation 46,XX,t(5;20)(q31;pl3) |
Kuhnen et al. [ |
2006 | 1 | CGH: loss of 10q22, loss of chromosome Y, gain of chromosome 18 (trisomy) |
Bouron-Dal Soglio et al. [ |
2009 | 1 | SNP array: complex aneuploid pattern including gains and losses of whole chromosome and an amplification of 12q13-15. FISH analysis showed amplification of |
Genes screened by solid tumor sequenom panel.
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Bouron-Dal Soglio et al. reported a highly complex aneuploid pattern by SNP array, including whole chromosome trisomy 5, 7, 8, 11, 15, 16, 20, and 22; tetrasomy 4 and 18; and monosomy 1, 2, 3, 9, 10, 13, 14 [
Ours is the second reported case of sclerosing rhabdomyosarcoma in which
Somatic mutations of
To investigate the effect of combination with
Given the discussion above that
The authors declare that they do not have conflict of interests.
C. Keller and A. Mansoor made the overall conception and design. C. Ryan and A. Hung made case history. G. Wettach, C. Beadling, A. Warrick, S. Olson, C. Corless, J. Hooper, and A. Mansoor wrote down the acquisition of clinical test data. C. Corless, S. Olson, C. Keller, and A. Mansoor accomplished analysis and interpretation of clinical data (e.g., cytogenetics and histopathology). K. Kikuchi and C. Keller performed development of experimental methodology. K. Kikuchi made the acquisition of experimental data (
This work was supported in part by NIH/NCI Grant 1R01CA133229 to Charles Keller as well as an anonymous donor. The authors acknowledge Mingkui Chen MD, OHSU Department of Pathology, for autopsy photomicrographs. They thank the family of the patient for their selfless contributions made possible by the autopsy for research. They also thank Jonathan Hart and Peter Vogt for wildtype and H1047R 10T1/2 cells.