MyoD1 expression in fibroepithelial stromal polyps.

Fibroepithelial stromal polyps (FESPs) are benign polypoid mesenchymal lesions thought to arise from desmin-positive specialized stromal cells of the female genital tract. Although most cases are easily diagnosed by morphology alone, the morphology of FESPs is variable and in some instances can contain hypercellular stroma with numerous atypical desmin-positive cells, simulating botryoid embryonal rhabdomyosarcoma (ERMS). Recently, we encountered a cellular FESP showing desmin expression as well as nuclear immunoreactivity for the skeletal muscle-associated transcription factor MyoD1. Although these lesions are widely known to express desmin, there are very few studies examining expression of the more specific markers of skeletal muscle differentiation, myogenin and MyoD1. The aim of our study was to examine desmin, MyoD1, and myogenin expression in a series of 25 FESPs. Of the 25 cases, desmin expression was present in 23 (92%), at least focal MyoD1 expression was present in 10 (40%), and all cases were negative for myogenin. Follow-up data were available for all 25 cases, and none recurred or behaved in a malignant fashion. Awareness of this potential immunohistochemical pitfall and careful morphologic evaluation should allow for the confident distinction of MyoD1-positive FESP from botyroid ERMS in almost all instances.

Chemoradiotherapy-induced Cytodifferentiation in Bladder/Prostate Rhabdomyosarcoma With Genetic Downregulation of Myogenin and MyoD1 Gene Expression: A Case Study and Review of the Literature.

Rhabdomyosarcoma is the most common sarcoma diagnosed in childhood and adolescence, arising from the bladder/prostate in only 5%-10% of cases. Treatment-induced cytodifferention of tumor cells into mature rhabdomyoblasts has been reported following chemoradiation and is thought to suggest a more favorable outcome. We report a case of embryonal rhabdomyosarcoma of the bladder/prostate that exhibited extensive cytodifferentiation with downregulation of myogenin and MyoD1 gene expression in rhabdomyoblasts following treatment with chemoradiation therapy. The downregulation of myogenin and MyoD1 expression in rhabdomyoblasts following chemoradiation treatment has not previously been described in the literature and its significant remains uncertain.

Clinicopathologic and Molecular Features of a Series of 41 Biphenotypic Sinonasal Sarcomas Expanding Their Molecular Spectrum.

Biphenotypic sinonasal sarcoma (BSNS) is a locally aggressive tumor occurring in the sinonasal region. It harbors both myogenic and neural differentiation and is characterized by PAX3 rearrangement with MAML3 as the most frequent fusion partner, but the partner of PAX3 remains unidentified in a subset of cases. About 70 cases have been reported so far. In this study, we report a series of 41 cases with clinical, pathologic, and molecular description. Twenty-five (61%) patients were female individuals, and the median age was 49 years. Tumors arose predominantly in the nasal cavity and ethmoidal sinuses. Local recurrences occurred in 8 cases of the 25 (32%). Histologic features were characteristic of BSNS, with 5 cases showing focal rhabdomyoblastic differentiation. Immunohistochemistry showed a constant positivity of S100 protein and PAX3 and negativity of SOX10. MyoD1 was focally positive in 91% of cases, whereas only 20% were positive for myogenin. Molecular analysis showed a PAX3-MAML3 transcript in 37 cases (90%). RNA sequencing was performed in the 4 negative cases for PAX3-MAML3 fusion, and it showed that 1 case harbored a PAX3-FOXO1 fusion, as previously described in the literature, and 2 novel fusions: PAX3-WWTR1 fusion in 2 cases and PAX3-NCOA2 fusion in 1 case. RNA sequencing results were confirmed by fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and Sanger sequencing. The PAX3-NCOA2-positive case showed focal rhabdomyoblastic differentiation. In conclusion, we report 2 novel fusions (PAX3-WWTR1 and PAX3-NCOA2) in BSNS and show that MyoD1 is more sensitive than myogenin for demonstrating myogenic differentiation in this tumor.

Muscle stem cell activation in a mouse model of rotator cuff injury.

Rotator cuff (RC) tears are frequently complicated by muscle atrophy. Muscle stem cells (MuSCs) repair damaged myofibers following injury, but their role in the prevention or pathogenesis of atrophy following RC tears remains undefined. We hypothesized that the RC MuSC population would be affected by supraspinatus (SS) and infraspinatus (IS) tendon transection (TT) compared to uninjured muscle in a mouse model of RC tear. C57BL6/J mice underwent unilateral SS and IS TT and contralateral sham surgery. At 3, 8, or 14 weeks after injury, mice were euthanized, and SS and IS were harvested for FACS sorting of CD31-/CD45-/Sca1-/ITGa7+/VCAM+ MuSCs or histological analysis. Ki-67+ MuSCs from injured muscle increased 3.4-fold at 3 weeks (p = 0.03) and 8.1-fold at 8 weeks (p = 0.04) following TT injury, but returned to baseline by 14 weeks (p = 0.91). Myod1 remained upregulated 3.3-fold at 3 weeks (p = 0.03) and 2.0-fold at 14 weeks (p = 0.0003), respectively. Myofiber cross-sectional area was decreased at both 3 and 14 weeks after injury, but the number of MuSCs per fiber remained relatively constant at 3 (p = 0.3) and 14 (p = 0.6) weeks after TT. In this study, we characterized the longitudinal effect of RC tendon injury on the MuSC population in supraspinatus and infraspinatus muscles. MuSCs are transiently activated, and are not depleted, in spite of persistent muscle atrophy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1370-1376, 2018.

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

Changes in calsequestrin, TNF-α, TGF-ß and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles.

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-ß; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-ß and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-ß serve as markers of dystrophy primarily for the diaphragm.

Low-level laser therapy modulates musculoskeletal loss in a skin burn model in rats.

PURPOSE: To investigate the effectiveness of low-level laser therapy (LLLT) on gastrocnemius muscle morphology and Myod immunoexpression in a model of dorsal burn in rats. METHODS: Sixteen male Wistar rats were distributed into two groups: control group (CG): rats submitted to scald burn injury without treatment and laser treated group (LG): rats submitted to scald burn injury and treated with laser therapy. Fourteen days post-surgery, gastrocnemius muscle was evaluated being the specimens stained with HE and morphometric data was evaluated. MyoD expression was assessed by immunohistochemistry. RESULTS: The results showed that laser treated animals presented more organized tissue morphology compared to the non-treated animals, with a higher number of nucleus in the fibers. Also, the cross sectional area of the fibers and the MyoD immunoexpression in the laser treated groups was higher. CONCLUSION: Low-level laser therapy had positive effects on gastrocnemius muscle, improving tissue muscle morphology, increasing cross sectional area and MyoD immunoexpression.

Low-level laser therapy modulates musculoskeletal loss in a skin burn model in rats

PURPOSE: To investigate the effectiveness of low-level laser therapy (LLLT) on gastrocnemius muscle morphology and Myod imunoexpression in a model of dorsal burn in rats. METHODS: Sixteen male Wistar rats were distributed into two groups: control group (CG): rats submitted to scald burn injury without treatment and laser treated group (LG): rats submitted to scald burn injury and treated with laser therapy. Fourteen days post-surgery, gastrocnemius muscle was evaluated being the specimens stained with HE and morphometric data was evaluated. MyoD expression was assessed by immunohistochemistry. RESULTS: The results showed that laser treated animals presented more organized tissue morphology compared to the non-treated animals, with a higher number of nucleus in the fibers. Also, the cross sectional area of the fibers and the MyoD immunoexpression in the laser treated groups was higher. CONCLUSION: Low-level laser therapy had positive effects on gastrocnemius muscle, improving tissue muscle morphology, increasing cross sectional area and MyoD immunoexpression. .

Clinicopathologic analysis of spindle cell/sclerosing rhabdomyosarcoma.

BACKGROUND: Clinical characteristics and optimal treatment strategies for spindle cell/sclerosing rhabdomyosarcoma (ssRMS) have not been well established because of its rarity. PROCEDURE: Retrospective re-evaluation of sarcoma specimens (1997-2014) identified 16 ssRMSs (median age 20 years, range 7-39 years). Clinicopathological features, clinical course, and outcome were analyzed. RESULTS: Primary disease sites were the head and neck (10 cases) and other regions (6 cases). Nine cases were at Intergroup Rhabdomyosarcoma Study preoperative stage 3. The primary tumors were >5 cm in 13 cases. Two patients had lymph node metastases, but none had distant metastases at presentation. At follow-up (median period 39 months, range 4.6-201), seven patients were alive without disease. Among nine patients treated with the vincristine, actinomycin, and cyclophosphamide (VAC) regimen, five responded well, with four surviving free of disease. Among ten patients with recurrent or progressive disease, three experienced local recurrence, four had distant metastases, and three had both. None exhibited bone marrow invasion. Eight of the ten patients died in median time from relapse to death of 18 months (range 11-56). CONCLUSIONS: Although most ssRMSs present as a bulky tumor, nodal or distant metastases are rare at presentation. ssRMSs initially show good response to VAC, but >50% of tumors recur or progress; these data suggest a worse prognosis of ssRMS compared to the pediatric embryonal variant. As relapse typically occurs as local or distant solitary lesion without bone marrow invasion, localized treatment combined with chemotherapy would contribute to improve the prognosis of recurrent ssRMS.

Skeletal and soft tissue response to automated, continuous, curvilinear distraction osteogenesis.

PURPOSE: To document the bone formation and soft tissue changes in response to automated, continuous, curvilinear distraction osteogenesis (DO) at rates greater than 1 mm/day in a minipig model. MATERIALS AND METHODS: Two groups of Yucatan minipigs underwent automated, continuous, curvilinear DO of the right mandible: group A, 1.5 mm/day (n = 5); and group B, 3.0 mm/day (n = 5). Each minipig underwent 12 mm of distraction followed by 24 days of fixation. The distracted and contralateral mandibles were harvested at the end of fixation. The percentage of surface area (PSA) of the regenerate occupied by bone, fibrous tissue, cartilage, and hematoma was determined using computerized histomorphometric analysis. The control groups consisted of DO wounds distracted discontinuously at 1 mm/day and the nonoperated contralateral mandible. The ipsilateral and contralateral digastric muscles were harvested and stained for proliferating cell nuclear antigen (PCNA), myogenic differentiation-1 (MyoD), and paired Box 7 protein (PAX7). RESULTS: All 10 minipigs completed the distraction and fixation period. The PSA occupied by bone was similar for groups A (PSA 64.36% ± 5.87%) and B (PSA 63.83% ± 3.37%) and the control group (1 mm/day; PSA 64.89% ± 0.56%) but was less than that on the nonoperated side (PSA 84.67% ± 0.86%). The PSA occupied by cartilage and hematoma in all groups was minimal (<1.1%). The digastric muscles had no abnormal tissue or inflammation, and PAX7, MyoD, and PCNA expression had returned to the baseline levels. CONCLUSIONS: The results of the present study indicate that bone formation in response to automated, continuous, and curvilinear DO at a rate of 1.5 and 3.0 mm/day is nearly identical to that with discontinuous DO at 1 mm/day. In addition, no deleterious effects were found on the digastric muscles.

Rhabdomyosarcoma of the duodenum: report of a case.

This report presents a case of primary pleomorphic rhabdomyosarcoma arising in the duodenum. A 63-year-old male with persistent melena was referred for a solid tumor in his right upper abdomen detected using ultrasonography. Gastrofiberscopy revealed a protrusion in the upper part of the duodenum, with a large ulcer on the top of it. Enhanced computed tomography showed that the tumor extended to the pancreas. Pancreaticoduodenectomy was performed, despite the absence of malignant cells in the biopsy specimen, with a preoperative diagnosis of duodenal cancer. The tumor consisted of multiple cell types, and immunohistochemical staining was positive for desmin, HHF-35 and alpha smooth muscle actin. Electron microscopy revealed primitive Z-band structures in the tumor. The final diagnosis was pleomorphic rhabdomyosarcoma of the duodenum. This is the first report of primary rhabdomyosarcoma occurring in the duodenum, confirmed by immunohistochemical staining and electron microscopy.

Calcium ions released from mineral trioxide aggregate convert the differentiation pathway of C2C12 cells into osteoblast lineage.

INTRODUCTION: The purpose of this study was to examine the effect of mineral trioxide aggregate (MTA) on pluripotent-mesenchymal cell differentiation. METHODS: The pluripotent-mesenchymal cell line C2C12 was cultured in a 5% serum medium to induce cell differentiation with or without MTA. The differentiation to myoblasts was analyzed by the immunocytochemical staining of myosin heavy chains. The cellular phenotype-specific markers characterizing the osteoblasts (Runx2 and osterix), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (LPL) were estimated with mRNA and protein levels by using real-time polymerase chain reaction and Western blot analysis, respectively. To verify that the effect of MTA was caused by the released calcium ions, the mRNA levels were analyzed in the presence or absence of MTA with ethylene glycol tetraacetic acid, calcium chloride, or verapamil. RESULTS: C2C12 cells cultured without MTA altered their phenotype to myoblasts, exhibiting positive reactions to myosin heavy chains. However, the cells cultured with MTA were strongly inhibited from developing into myoblasts. The mRNA and protein expressions of Runx2, osterix, and Sox9 significantly increased with MTA; the expressions of MyoD and LPL decreased significantly. Calcium chloride addition without MTA presented a significant increase of mRNA levels of Runx2, osterix, and Sox9; ethylene glycol tetraacetic acid addition with MTA presented a significant increase of mRNA levels of MyoD and LPL. Verapamil blocked the stimulating or suppressing effect of MTA on these transcription factors. CONCLUSIONS: Our study showed that MTA converted the differentiation pathway of C2C12 cells into osteoblast and/or chondroblast lineages as a result of elution components such as calcium ions from MTA.

Calpain 3 is important for muscle regeneration: evidence from patients with limb girdle muscular dystrophies.

BACKGROUND: Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in the CAPN3 gene and complete lack of functional calpain 3 leads to the most severe muscle wasting. Calpain 3 is suggested to be involved in maturation of contractile elements after muscle degeneration. The aim of this study was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration. METHODS: We studied muscle regeneration in 22 patients with LGMD2A with calpain 3 deficiency, in five patients with LGMD2I, with a secondary reduction in calpain 3, and in five patients with Becker muscular dystrophy (BMD) with normal calpain 3 levels. Regeneration was assessed by using the developmental markers neonatal myosin heavy chain (nMHC), vimentin, MyoD and myogenin and counting internally nucleated fibers. RESULTS: We found that the recent regeneration as determined by the number of nMHC/vimentin-positive fibers was greatly diminished in severely affected LGMD2A patients compared to similarly affected patients with LGMD2I and BMD. Whorled fibers, a sign of aberrant regeneration, was highly elevated in patients with a complete lack of calpain 3 compared to patients with residual calpain 3. Regeneration is not affected by location of the mutation in the CAPN3 gene. CONCLUSIONS: Our findings suggest that calpain 3 is needed for the regenerative process probably during sarcomere remodeling as the complete lack of functional calpain 3 leads to the most severe phenotypes.

Myogenic capacity of muscle progenitor cells from head and limb muscles.

The restoration of muscles in the soft palate of patients with cleft lip and/or palate is accompanied by fibrosis, which leads to speech and feeding problems. Treatment strategies that improve muscle regeneration have only been tested in limb muscles. Therefore, in the present study the myogenic potential of muscle progenitor cells (MPCs) isolated from head muscles was compared with that of limb muscles. Muscle progenitor cells were isolated from the head muscles and limb muscles of rats and cultured. The proliferation of MPCs was analysed by DNA quantification. The differentiation capacity was analysed by quantifying the numbers of fused cells, and by measuring the mRNA levels of differentiation markers. Muscle progenitor cells were stained to quantify the expression of paired box protein Pax 7 (Pax-7), myoblast determination protein 1 (MyoD), and myogenin. Proliferation was similar in the head MPCs and the limb MPCs. Differentiating head and limb MPCs showed a comparable number of fused cells and mRNA expression levels of myosin-1 (Myh1), myosin-3 (Myh3), and myosin-4 (Myh4). During proliferation and differentiation, the number of Pax-7(+), MyoD(+), and myogenin(+) cells in head and limb MPCs was equal. It was concluded that head and limb MPCs show similar myogenic capacities in vitro. Therefore, in vivo myogenic differences between those muscles might rely on the local microenvironment. Thus, regenerative strategies for limb muscles might also be used for head muscles.

Serial histologic and immunohistochemical changes in anterior digastric myocytes in response to distraction osteogenesis.

PURPOSE: To document histologic and immunohistochemical changes in the anterior digastric muscle during distraction osteogenesis (DO). MATERIALS AND METHODS: Nineteen Yucatan minipigs with mixed dentition were used for these experiments. Group A (n = 16) underwent unilateral mandibular distraction at a rate of 1 mm/day (no latency) for 12 days. Animals were killed at mid-DO (n = 5), end-DO (n = 5), mid-fixation (n = 4), and end-fixation (n = 2). Group B (n = 2) underwent acute 12-mm advancement, and group C (n = 1) dissection and osteotomy. Animals from groups B and C were killed at the end-DO time point. Digastric muscles from treatment and contralateral sides of all animals were harvested and embedded in paraffin. Specimens were stained with hematoxylin/eosin or immunohistochemically for proliferating cell nuclear antigen (PCNA; total cell proliferation), paired Box-7 gene protein (Pax7; satellite cells), or myogenic differentiation 1 protein (MyoD; differentiating myoblasts). Descriptive and bivariate statistics were computed to compare groups (P ≤ .05 statistically significant). RESULTS: All animals survived the operation and observation period; there were no device failures. Two animals (1 at mid-DO, 1 at mid-fixation) were eliminated from the study because of postoperative infection. There was minimal digastric inflammation, fibrosis, and muscle fiber size variability during active DO. Immunohistochemical analysis showed statistically significant increases in PCNA (cellular proliferation), Pax7 (satellite cells), and MyoD (differentiating myoblasts) positive nuclei in digastrics at mid-DO and end-DO. CONCLUSIONS: Results of this study indicate that there are minimal pathologic changes but significant increases in PCNA, Pax7, and MyoD positive nuclei during active distraction. This supports the hypothesis that the digastric muscle response to DO consists of proliferation and hypertrophy.

Embryonal rhabdomyosarcoma of the adult soft palate.

Embryonal rhabdomyosarcoma is the most common soft tissue sarcoma in children. We report a rare case of embryonal rhabdomyosarcoma of the soft palate in a 32-year-old Caucasian female. Detailed histology of the tumor is described. Positive staining with desmin, myogenin and myoD1 confirmed the tumor to be embryonal rhabdomyosarcoma. A genetic association between rhabdomyosarcoma, polycystic ovary syndrome and the FEM1A gene on the human chromosome is speculated upon.

Effects of preexercise feeding on markers of satellite cell activation.

PURPOSE: The purpose of this study was to determine if consuming isoenergetic (25 g) doses of carbohydrate or protein versus a noncaloric placebo before conventional resistance training affected the myogenic expression of cell cycle-regulating genes as well as the muscle [DNA] acutely after exercise. METHODS: Ten untrained men (mean +/- SD: age = 22 +/- 4 yr, body mass = 77.8 +/- 8.3 kg, percent body fat = 17.8 +/- 4.0) participated in three resistance exercise sessions (three sets of 10 repetitions at 80% one-repetition maximum for the bilateral hack squat, leg press, and leg extension exercises) in a crossover fashion, which were preceded by carbohydrate, protein, or placebo ingestion 30 min before training. Presupplement/preexercise and 2- and 6-h postexercise muscle biopsies were obtained during each session and analyzed for fold changes in CDK4, CYCLIN D1, MGF, MYOD, P21(CIP1), and P27(KIP1) messenger RNA expression using real-time reverse transcriptase-polymerase chain reaction as well as muscle [DNA] using cuvette-based fluorometric methods. RESULTS: Nonparametric statistics were completed, and no conditions x time interaction effects were revealed. Several exercise-mediated responses were found to occur independent of condition: 1) muscle [DNA] increased at 6 h (+40%, P < 0.05), 2) CDK4 expression increased at 6 h (+86%, P < 0.05), 3) MYOD expression increased at 6 h (+98%, P < 0.05), 4) P27(KIP1) expression decreased at 2 h (j35%, P < 0.05) and 6 h (-59%, P < 0.001), and 5) P21(CIP1) expression substantially increased 2 and 6 h postexercise (+1.250% and +4.670%, respectively, P < 0.001). CONCLUSIONS: The tandem DNA and cell cycle regulator gene expression analyses provide preliminary evidence to suggest that satellite cell activation and proliferation may be occurring at early post-exercise time points after a conventional resistance exercise bout, a phenomenon that may seemingly be independent of preexercise macronutrient ingestion.

Diffuse myogenin expression by immunohistochemistry is an independent marker of poor survival in pediatric rhabdomyosarcoma: a tissue microarray study of 71 primary tumors including correlation with molecular phenotype.

The pathologic classification of rhabdomyosarcoma (RMS) into embryonal or alveolar subtype is an important prognostic factor guiding the therapeutic protocol chosen for an individual patient. Unfortunately, this classification is not always straightforward, and the diagnostic criteria are controversial in a subset of cases. Ancillary studies are used to aid in the classification, but their potential use as independent prognostic factors is rarely studied. The aim of this study is to identify immunohistochemical markers of potential prognostic significance in pediatric RMS and to correlate their expression with PAX-3/FKHR and PAX-7/FKHR fusion status. A single tissue microarray containing 71 paraffin-embedded pediatric RMSs was immunostained with antibodies against p53, bcl-2, Ki-67, CD44, myogenin, and MyoD1. The tissue microarray and whole paraffin blocks were studied for PAX-3/FKHR and PAX-7/FKHR gene fusions by fluorescence in situ hybridization and reverse transcription-polymerase chain reaction. Clinical follow-up data were available for each patient. Immunohistochemical staining results and translocation status were correlated with recurrence-free interval (RFI) and overall survival (OS) using the Kaplan-Meier method, the log-rank test, and Cox proportional hazard regression. The minimum clinical follow-up interval was 24 months (median follow-up=57 mo). On univariable analysis, immunohistochemical expression of myogenin, bcl-2, and identification of a gene fusion were associated with decreased 5-year RFI and 10-year OS (myogenin RFI P=0.0028, OS P=0.0021; bcl-2 RFI P=0.037, OS P=0.032; gene fusion RFI P=0.0001, OS P=0.0058). After adjustment for Intergroup Rhabdomyosarcoma Study-TNM stage, tumor site, age, tumor histology, and translocation status by multivariable analysis, only myogenin retained an independent association with RFI (P=0.034) and OS (P=0.0069). In this retrospective analysis, diffuse immunohistochemical reactivity for myogenin in RMS correlates with decreased RFI and OS, independent of histologic subtype, translocation status, tumor site, or stage.

Rhabdomyosarcoma of the urinary bladder in adults: predilection for alveolar morphology with anaplasia and significant morphologic overlap with small cell carcinoma.

Rhabdomyosarcoma (RMS) represents the most common malignant soft tissue tumor in children and adolescents with the urinary bladder representing a frequent site. Most of these urinary bladder tumors are embryonal RMS, predominantly the botryoid subtype. RMSs of the urinary bladder in adults are distinctively rare and the subject of only case reports. We report the clinicopathologic features of 5 bladder neoplasms with rhabdomyosarcomatous differentiation in adults and emphasize the differential diagnosis in the adult setting. The patients, 4 men and 1 woman, ranged in age from 23 to 85 years (mean 65.4 y). Gross hematuria was the most common initial symptom, although 2 patients had metastatic disease at presentation. Four cases were pure primary RMSs of the bladder and 1 case was a sarcomatoid urothelial carcinoma with RMS representing the extensive heterologous component. All 5 cases demonstrated a diffuse growth pattern (ie, non-nested), of which 4 cases had nuclear anaplasia (Wilms criteria without the atypical mitotic figure requirement); only 1 case (the sarcomatoid carcinoma) showed obvious rhabdomyoblastic differentiation (ie, strap cells). Three cases were of the alveolar subtype (1 admixed with embryonal histology) and 2 were RMS, not further classified. Microscopically, all tumors had a primitive undifferentiated morphology with cells containing scant cytoplasm, varying round to fusiform nuclei with even chromatin distribution, and frequent mitoses. The degree of morphologic overlap with small cell carcinoma of the bladder, a relatively more common round cell tumor in adults, was striking. The epithelial component of the sarcomatoid carcinoma was high-grade invasive urothelial carcinoma with glandular differentiation. No other case had previous history of bladder cancer or concurrent carcinoma in situ or invasive urothelial carcinoma. All tumors showed immunohistochemical expression for desmin, myogenin, and/or MyoD1. Synaptophysin was performed in 4 cases, and 3 showed weak cytoplasmic immunoreactivity. Two patients received chemotherapy, 2 underwent cystectomy, and 1 had transurethral resection alone. Outcome data were available in 4 cases, and all 4 died of disease (1, 4, 8, and 8 mo). In conclusion, (1) RMS of the urinary bladder in adults more commonly presents as a primitive round blue cell neoplasm that has significant morphologic and immunohistochemical overlap with small cell carcinoma of the bladder. (2) Although RMS in children generally have a botryoid embryonal histology with favorable outcome, bladder RMS in adults frequently demonstrates alveolar or unclassified histology, commonly with anaplasia, and have a uniformly aggressive clinical course.

Oestrogen receptors mediate oestrogen-induced increases in post-exercise rat skeletal muscle satellite cells.

AIM: Our laboratory recently demonstrated that increases in post-exercise muscle satellite cell numbers are augmented by oestrogen. We investigated whether muscle oestrogen receptors (ORs) mediate this effect through administration of an OR antagonist, ICI 182,780. METHODS: Ovariectomized female rats were divided into three groups: sham, oestrogen (0.25 mg pellet) and oestrogen plus OR blocker (ICI 182,780). Each group was divided into control and exercised groups. ICI 182,780 (5 mg kg(-1) sc) was administered 1 day prior to and 6 days following oestrogen pellet implantation. After 8 days of oestrogen exposure, animals ran downhill for 90 min (17 m min(-1), -13.5 degrees grade) on a treadmill. Soleus and white vastus muscles were removed 24 and 72 h post-exercise and immunostained for total (Pax7), activated (MyoD) and proliferating (BrdU) satellite cells. Muscle damage was indirectly assessed by measuring beta-glucuronidase activity. Two markers (His48 and ED1) of leucocyte infiltration were also examined. RESULTS: beta-Glucuronidase activities and His48+ and ED1+ leucocytes increased post-exercise, and these increases were attenuated with oestrogen. ICI 182,780 did not influence the attenuating effect of oestrogen on leucocyte infiltration or beta-glucuronidase activities in muscle. Total (Pax7+), activated (MyoD+) and proliferating (BrdU+) satellite cells increased post-exercise, and these increases were augmented with oestrogen. Interestingly, ICI 182,780 abolished both exercise- and oestrogen-mediated increases in these satellite cell markers. CONCLUSION: Oestrogen may augment increases in muscle satellite cells following exercise through OR-mediated mechanisms; furthermore, the attenuation of post-exercise muscle damage and leucocyte infiltration by oestrogen appears to be a non-OR-mediated process.