p53 and mdm-2 Expression in Rhabdomyosarcoma of Childhood and Adolescence: Clinicopathologic Study by the Kiel Pediatric Tumor Registry and the German Cooperative Soft Tissue Sarcoma Study

Rhabdomyosarcomas (RMS) are the most common malignant soft tissue sarcomas in childhood and adolescence. Despite a large number of publications about this heterogeneous group of tumors, little is known about proliferation, p53 and mdm-2 in relation to histological subtype, clinical parameter, and prognosis of patients. We studied 150 cases of RMS treated in the German Cooperative Soft Tissue Sarcoma Study (CWS) by immunohistochemistry on paraffin-embedded tissue, using antibodies against p53, mdm-2, and Ki-67. The results were correlated with histological subtype, mitotic count, and various clinical parameters. Both p53 and mdm-2 were expressed at low levels and did not show differences between embryonal and alveolar RMS. Tumors of patients with metastatic embryonal RMS showed significantly higher levels of p53 protein than nonmetastatic tumors. This might be a clue to an important role of p53 in metastatic embryonal RMS. Nevertheless, neither p53 nor mdm-2 showed any correlation to prognosis. Proliferation measured by Ki-67 immunostaining (KiS5 antibody) or mitotic count did not show significant differences between embryonal and alveolar RMS. In addition, these parameters did not correlate with response to therapy or prognosis. In conclusion, we could not demonstrate that any of the investigated parameters had an influence on prognosis of RMS. p53 protein overexpression might be a crucial step in metastatic disease for patients with embryonal RMS.     

Effects of standard chemotherapy on tumor growth and regulation of multidrug resistance genes and proteins in childhood rhabdomyosarcoma

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrified after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunhistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunhistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.     

Association of Alveolar Rhabdomyosarcoma with the Beckwith-Wiedemann Syndrome

Rhabdomyosarcoma (RMS) is a soft tissue tumor of childhood frequently diagnosed between the first and fifth year of life. Children with the Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth syndrome characterized by exomphalos, macroglossia, and macrosomia, have an increased risk of developing childhood tumors including Wilms tumor, hepatoblastoma, neuroblastoma, and RMS. Although an association between RMS and the BWS is well accepted, only four cases have been reported to date, and of these, three were reported as embryonal RMS. Based on these data, an association between BWS and embryonal RMS has been proposed. We report three additional cases of BWS with RMS and review the clinical data for each patient as well as the pathology of their tumors. All three cases of BWS had histology consistent with alveolar RMS and were diagnosed at 6 weeks and 5 and 13 years of age. In two of these BWS cases, constitutional defects of 11p15 imprinting were demonstrated. Furthermore, cytogenetic analysis of the tumors did not detect the t(2;13) or t(1;13) translocations that generate the PAX3- or PAX7-FKHR fusion proteins common to alveolar RMS. These observations suggest that the development of alveolar RMS tumors in BWS may occur without the chromosomal rearrangement producing the PAX-FKHR fusion protein. In summary, we present three new cases of RMS demonstrating a new association between BWS and an uncommon subtype of alveolar RMS. The absence of the translocations commonly associated with alveolar rhabdomyosarcoma suggests a common 11p15 pathway for alveolar RMS and BWS. Received May 25, 2001; accepted July 11, 2001.     

Primary and metastatic rhabdomyosarcoma in the breast: Neoplasms of adolescent females,a report from the Intergroup Rhabdomyosarcoma Study

The occurrence of rhabdomyosarcoma (RMS) primary in or metastatic to breast has been regarded as an uncommon event, associated with an unfavorable outcome. Records of 26 patients with diagnoses of breast RMS, either primary or secondary, entered in the Intergroup Rhabdomyosarcoma Study (IRS) (1972–1992) were reviewed and compared with data regarding 47 similar patients in published reports. Of the 26 IRS cases, the histologic subtype was alveolar in 24, embryonal in 1, and not determined in 1. All were female with ages ranging from 11.5 to 20.2 years (median, 15.2 years; mode, 14–16 years). This compact age distribution of both primary (n = 7) and metastatic (n = 19) breast RMS was seen in previously reported series. Among the 19 cases of RMS with initial dissemination to breast, primary tumor sites were: extremity (n = 8), nasopharynx/paranasal sinuses (n = 7), and trunk (n = 4). IRS treatment was risk-based according to site and extent of disease. Four of 7 patients with primary RMS remain disease free 2.9 to 7 years post diagnosis. Among 19 patients with RMS initially metastatic to breast, including 7 in IRS clinical group IV at original diagnosis, three are disease free at 7.6, 15.7 and 17.0 years. Conclusions: primary or metastatic RMS in breast is almost confined to adolescent females having tumors with alveolar histology. Approximately one-half of the patients with primary breast disease and 15% of those with metastatic breast disease as an initial recurrence are long-term survivors. Med. Pediatr. Oncol. 29:181–189, 1997. © 1997 Wiley-Liss, Inc.     

Massive bone marrow involvement by clear cell variant of rhabdomyosarcoma

A 16-year-old boy with generalized myalgia and petechial hemorrhage was found to have a diffuse infiltrative disease in his bone marrow (BM). The BM aspirate contained a dense population of vacuolated blast-like cells. The BM biopsy displayed compact sheets of small round cells with clear cytoplasm, reminiscent of Ewing sarcoma. Immunostains were not diagnostically conclusive while transmission electron microscopy on the BM cells demonstrated a clear skeletal muscle differentiation. The morphologic findings led to a tentative designation of metastatic embryonal rhabdomyosarcoma (RMS). It was not until cytogenetic analysis revealed the specific translocation t(2;13)(q35;q14) did the alveolar RMS finally get confirmed. Despite an exhaustive search by imaging studies, a primary tumor was not detected. This case illustrates that the massive BM involvement by atypical alveolar RMS of unknown origin may pose serious diagnostic challenges. Multidisciplinary studies are required to reach a definitive diagnosis     

CELLULAR EXPRESSION OF ADHESION FACTORS IN CHILDHOOD RHABDOMYOSARCOMA

Tumor invasion and metastasis are believed to involve the adhesive interaction of tumor cells with extracellular matrix ECM . This study reports the expression of laminin, fibronectin, thrombospondin, tenascin, and CD44 in rhabdomyosarcoma cells taken from tumors derived from 12 pediatric patients. Twelve paraffin-embedded rhabdomyosarcomas consisting of five alveolar, six embryonal, and one botryoid subtype were examined. All tumors demonstrated positive staining for tenascin and thrombospondin, 10 were positive for fibronectin, 5 positive for laminin, and 4 positive for CD44. Both specimens without cellular staining for fibronectin were of embryonal subtype and no alveolar rhabdomyosarcomas were positive for CD44. No association of the expression of the studied ECM proteins and receptor with the presence of metastatic disease at clinical presentation or with the level of tumor differentiation was found.     

Orbital rhabdomyosarcomas and related tumors in childhood: Relationship of morphology to prognosis—an intergroup rhabdomyosarcoma study

Children and adolescents who develop rhabdomyosarcoma (RMS) and related sarcomas in the orbit and treated on Intergroup Rhabdomyosarcoma protocols have had an extremely high cure rate. This study evaluates the possible relationship between their tumor morphologic subtypes and this high cure rate. The histology of tumors was re-reviewed from 229 of the 264 patients with tumors of the orbit, conjunctiva, and eyelids treated on Intergroup Rhabdomyosarcoma Studies (IRS) I, II, III, and IV pilot protocols, and followed through July, 1992. Immunohistochemistry was applied in selected cases. Clinical correlations were done on all 264 cases including both the re-reviewed cases and those reviewed only by the IRS Pathology committee. The 5-year survival rate of 24 children with alveolar RMS was 74% (p < .001). All five infants diagnosed to have an alveolar RMS died before the age of one. Two hundred and twenty-one patients (84%) had embryonal RMS. About three-fourths of the re-reviewed embryonal RMS tumors showed only minimal rhabdomyoblastic differentiation. Thirty-one had a spindle cell RMS, two were anaplastic variants. The 5-year survival rate for patients with embryonal RMS subtypes combined was 94%, and 97% for the 144 patients with poorly differentiated embryonal RMS. In contrast, 190 of 432 IRS II patients treated for poorly differentiated embryonal RMS located in extraocular sites had a 66% survival estimate. Med. Pediatr. Oncol. 29:51–60, 1997. © 1997 Wiley-Liss, Inc.     

Metastatic rhabdomyosarcoma and histologically similar tumors in childhood: A retrospective european multi-center analysis

This is a retrospective analysis of children with metastatic soft tissue sarcoma (STS) registered in the major European STS studies: the SIOP MMT 75, the German CWS 1981 and 1986 studies, the Italian STS study, and the United Kingdom Children's Cancer Study Group centres in Britain. One hundred forty-six patients out of 164 were evaluable in this analysis. The median age was 7 years (1–18) and the male/female ratio was 1.3:1. The median follow-up was 80 months (7–171). The most common site of the primary tumor was the extremity (28%), which correlated well with the high preponderance (39%) of the alveolar type of RMS (aRMS). There was no dominant combination of metastatic sites and the most common single organ with metastasis was the lung (41%). Since the therapy depended on the country and period in which the patient was treated, we did not analyse the outcome and therapy together. Complete remission was achieved in 50% of the cases. Those with aRMS had a good chance of achieving CR (61%) but the majority of these patients relapsed (78%). The median time needed to relapse was 243 days (53 days to 47 months) for all patients. Analysis of the site of the primary tumor showed that the CR rate was best in the GU non-BP site (62%) and worst in PM cases (35%). The overall survival and DFS rate were 18% and 15%, respectively. The GU non-BP patients had a DFS rate of 50%, while the rate for all other sites varied between 12% and 18%. A total of 24 patients remained in CCR. The majority of them had embryonal type of RMS and metastases in only one anatomic site. Our analysis indicates that the best chance of prolonged survival was in metastatic GU non-BP cases, patients with embryonal type RMS, and those with metastases in only one organ. To try to improve the treatment of metastatic STS, a prospective European cooperative study was started in 1989. © 1992 Wiley-Liss, Inc.     

Chromosome 2 Abnormalities (+ Der 2) in Two Cases of Childhood Rhabdomyosarcoma

Chromosome analysis was performed in two cases of rhabdomyosarcoma (RMS): one embryonal RMS and one alveolar RMS. Analysis showed number and structural abnormalities of chromosome 2. The relationship between these findings and published reports of karyotypes from RMS is discussed.     

Clinicopathologic Study of Ectomesenchymomas from Intergroup Rhabdomyosarcoma Study Groups III and IV

Ectomesenchymomas (EM) are rare malignant neoplasms usually consisting of rhabdomyosarcoma (RMS) with a neural component. Only 21 cases have been previously reported. Here we extend the clinicopathologic spectrum of EM by describing our findings in 15 cases. Only 5 patients were infants; 10 were ≤3 years old and 5 were ≥6 years old. No male predilection was observed; 7 were female. The originating institutional diagnoses were; RMS (12), undifferentiated sarcoma (1), or EM (2), suggesting underdiagnosis of this entity. The primary tumor sites included external genital (5), pelvis/abdomen (6), head and neck (3), and extremity (1). The size of the primary neoplasm was usually ≥5 cm at diagnosis but dissemination only occurred in a minority. Local infiltration was not uncommon. These neoplasms were typically multilobate, thinly encapsulated, hemorrhagic, and necrotic. Light microscopic features were highly variable, but embryonal RMS with scattered or clustered ganglion cells, often in lacunae, was characteristic. In some cases, primitive neuroblastic or neuroectodermal areas were found and/or a component of alveolar RMS was seen. Focal anaplasia was occasionally observed. Mitotic activity appears higher than previously appreciated and some necrosis was invariably present. Electron microscopy was performed in 11 cases, which confirmed skeletal muscle ± neural differentiation. Cytogenetic studies performed in five cases revealed no specific abnormality. Monoclonal neuron-specific enolase was the best marker of ganglion cells and primitive neural elements. MIC-2 (CD99) membrane expression was not definitively present in any of the six cases examined. A number of the above parameters appear to be of some prognostic significance, but overall, these neoplasms appear to have a similar outcome as would be predicted for their RMS element alone (exclusive of any neural component), with respect to the RMS subtype, age of the patient, and anatomic location of the neoplasm.     

小儿睾旁横纹肌肉瘤的诊治

目的 提高小儿睾旁横纹肌肉瘤(PARA RMS)的诊治水平.方法 回顾性分析我院1977至2008年6月收治的14例睾旁横纹肌肉瘤病例.年龄1～15岁,其中≤5岁6例,平均年龄6岁.左侧9例,右侧5例.全组病例均因单侧阴囊肿大就诊,经影像检查都属Ⅰ期病变.14例中有4例曾在外院手术,经患侧阴囊切口;1例做睾丸切除,3例保留睾丸、做肿物切除.这4例都再次经腹股沟切口,高位精索离断,3例切除曾被保留的睾丸;并切除原阴囊皮肤切口及其周闱组织;余10例也是经腹股沟切口,根治性睾丸切除.病理检查除1例是腺泡型外,其余均为胚胎型中的梭形细胞型.术后化疗主要用VAC(长春新碱+放线菌素D+环磷铣胺).结果 14例中13例获得随访,随访时间1～24年,平均6年.随访≥2年者11例,10例(91%)无瘤存活,1例于手术后近2年时复诊,腹膜后转移瘤的最大径达8 cm,家长放弃治疗,该例仅接受化疗6次.1例术后10年复查,精液内无精子.结论 小儿睾旁横纹肌肉瘤易于被检出,故多为Ⅰ期病变,且梭形细胞属良好型,经腹股沟切口高位精索离断、睾丸切除+VAC化疗,存活率达91%.由于环磷铣胺可致精液内无精子,故近年对Ⅰ期良好型病变可不用环磷铣胺.     

Alveolar rhabdomyosarcoma of the extremity and nodal metastasis: Is the in‐transit lymphatic system at risk?

Alveolar rhabdomyosarcoma (RMS) of the extremity is not infrequently associated with regional node metastasis. Knowledge of lymphatic drainage of extremity RMSs is important to determine radiotherapy fields. In this report we describe two patients with alveolar RMS of the lower extremity with inguinal metastasis at presentation. Both the distal lower extremity and inguinal region received local therapy consisting of surgery and postoperative radiotherapy. Both patients later developed in‐transit lymphatic metastasis outside of the irradiated field. The in‐transit lymphatics can be a site of failure in children with alveolar RMS of the extremity and nodal involvement. Pediatr Blood Cancer 2009; 53:1332–1333. © 2009 Wiley‐Liss, Inc.     

Metastatic rhabdomyosarcoma and histologically similar tumors in childhood: a retrospective European multi-center analysis.

This is a retrospective analysis of children with metastatic soft tissue sarcoma (STS) registered in the major European STS studies: the SIOP MMT 75, the German CWS 1981 and 1986 studies, the Italian STS study, and the United Kingdom Children's Cancer Study Group centres in Britain. One hundred forty-six patients out of 164 were evaluable in this analysis. The median age was 7 years (1-18) and the male/female ratio was 1.3:1. The median follow-up was 80 months (7-171). The most common site of the primary tumor was the extremity (28%), which correlated well with the high preponderance (39%) of the alveolar type of RMS (aRMS). There was no dominant combination of metastatic sites and the most common single organ with metastasis was the lung (41%). Since the therapy depended on the country and period in which the patient was treated, we did not analyse the outcome and therapy together. Complete remission was achieved in 50% of the cases. Those with aRMS had a good chance of achieving CR (61%) but the majority of these patients relapsed (78%). The median time needed to relapse was 243 days (53 days to 47 months) for all patients. Analysis of the site of the primary tumor showed that the CR rate was best in the GU non-BP site (62%) and worst in PM cases (35%). The overall survival and DFS rate were 18% and 15%, respectively. The GU non-BP patients had a DFS rate of 50%, while the rate for all other sites varied between 12% and 18%. A total of 24 patients remained in CCR. The majority of them had embryonal type of RMS and metastases in only one anatomic site. Our analysis indicates that the best chance of prolonged survival was in metastatic GU non-BP cases, patients with embryonal type RMS, and those with metastases in only one organ. To try to improve the treatment of metastatic STS, a prospective European cooperative study was started in 1989.     

Allogeneic hematopoietic stem cell transplantation against recurrent rhabdomyosarcoma

The prognosis of high-risk rhabdomyosarcoma (RMS) with metastatic or recurrent disease remains poor. We report a 6-year-old girl who successfully underwent allogeneic hematopoietic stem cell transplantation against recurrent metastatic alveolar RMS. The disease recurred at distant lymph node metastasis with bone marrow involvement. After chemotherapy and radiotherapy for the metastatic site, she underwent allogeneic bone marrow transplantation during complete remission from her 5/8 HLA-matched father. She developed acute graft-versus-host disease after preemptive donor lymphocyte infusion and remains in a disease-free condition for 31 months after transplantation. A graft-versus-tumor effect through allogeneic immune cells might produce a beneficial effect for high-risk RMS.     

Results of treatment of fifty-six patients with localized retroperitoneal and pelvic rhabdomyosarcoma: a report from The Intergroup Rhabdomyosarcoma Study-IV, 1991-1997

BACKGROUND: We reviewed 56 IRS-IV patients with localized rhabdomyosarcoma [RMS] of the retroperitoneum/pelvis to assess outcome and prognostic factors, including the value of initially excising >or=50% of the tumor (debulking) before chemotherapy. METHODS: Patients had embryonal RMS [N=38], alveolar RMS [N = 7], RMS not otherwise specified [NOS, N = 7], or undifferentiated sarcoma [N = 4]. Fifteen patients were debulked; 41 patients were biopsied. All received VAC; most received radiotherapy. RESULTS: Estimated 5-year failure-free survival [FFS] and overall survival rates were 70 and 75%, respectively. FFS rates were better for patients <10 years old and those with embryonal RMS compared to alveolar RMS/undifferentiated sarcoma. After adjusting for age and histological differences, FFS was better for patients whose tumor was debulked prior to beginning therapy [P = 0.02]. CONCLUSIONS: These results are superior to those of previous protocols for patients with RMS of the retroperitoneum/pelvis. Initial excision of >or=50% of the tumor may be associated with increased FFS.     

Primary ovarian rhabdomyosarcoma in children

Although rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, primary rhabdomyosarcomas of the ovary are extremely rare, with only eight well-documented pediatric cases previously reported in the literature. We present two additional cases: an alveolar RMS originating in the right ovary with metastatic spread to the splenic flexure of the colon and to both lungs in a 13-year-old African American girl, and an embryonal RMS arising in the right ovary of a 6-year-old Caucasian girl with pre-operative intra-abdominal rupture and a malignant right pleural effusion. Both patients had complete resection of their primary tumors and received chemotherapy including vincristine, doxorubicin and cyclophosphamide with good response to therapy. Both are alive 8 and 9 months post-operatively.     

Cellular expression of MDM2 and p53 in childhood leukemias with poor prognosis

BACKGROUND: Previous studies have suggested that altered expression or dysfunction of the tumor suppressor gene p53 or the oncogene MDM2 could indicate disease progression in children with leukemia who would fail to achieve complete remission or who would relapse. While these studies mainly have described aberrations of MDM2 and p53 function at the DNA and mRNA-level, we have examined p53 and MDM2 expression at the protein level. Mutation of the p53 tumor suppressor gene may result in cellular accumulation of the p53 protein, due to prolonged half-life of the abnormal protein. The p53 protein can also be rendered nonfunctional by overexpression of proteins that bind to p53, such as MDM2. Both pathways have been proposed to disrupt cell cycle regulation in humans. Recent studies have shown that increased expressions of MDM2 as well as of p53 can be detected at the protein level in the absence of gene amplification. PROCEDURE: Forty-three bone marrow samples were analyzed immunohistochemically for p53 and MDM2. Twenty-nine bone marrow samples were obtained in children with active, prognostically unfavorable leukemia and MDS. Fourteen bone marrow samples were from children with non-malignant hematological disorders. RESULTS: p53 protein was expressed in 12 patients and MDM2 in 17 patients with leukemia. In the control group MDM2 expression was detected in one child, while p53 was not found in any of the samples. CONCLUSIONS: Our findings of p53 or MDM2 positive cells in a majority of children with unfavorable prognostic features suggests that dysfunction of the p53-dependent cell growth control have a role in the development of high risk leukemias.     

Expression of cell cycle-related gene products in Langerhans cell histiocytosis

BACKGROUND The pathogenesis of Langerhans cell histiocytosis (LCH), a disease characterized by an abnormal accumulation of the dendritic Langerhans cells, is still unknown. Based on the monoclonality of the CD1a+ cell and reports of familial clustering, it is hypothesized that a genetic alteration at a cellular level may be causative. This genetic change may have an effect on the cellular mechanisms controlling proliferation and apoptosis.MATERIALS AND METHODS LCH-lesions were studied for the expression of Ki-67, present in the nucleus of proliferating cells. Furthermore, the expression of cell cycle-related gene products TGF-beta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 were studied. The TGF-betaR genes play a role in tumor suppression, whereas Bcl2 inhibits apoptosis. The remaining genes are part of either the p53-p21 and/or p16-Rb pathways, which induce cell cycle arrest or apoptosis in response to DNA damage.RESULTS In 30 biopsies the diagnosis of LCH could be confirmed on the basis of CD1a positivity (27 bone and 3 skin). All cases showed scattered nuclear-positive staining for the proliferation marker Ki-67. In more than 90% (n >/=27) of these cases, expression of TGFbeta receptor I and II, MDM2, p53, p21, p16, Rb, and Bcl2 was detected in lesional LCH cells. The overexpression was in general heterogeneous, ranging from limited focal staining of scattered cells within the lesion to strong diffuse staining.CONCLUSIONS These findings suggest that the cellular mechanisms that sense and respond to DNA-damage, namely the p53-p21 pathway and the p16-Rb pathway, are activated. The expression of Ki-67 indicates that the cells in LCH are proliferating. The observed overexpression of Bcl2 may play a role in the activation of p53 and p16 and/or the arrest of apoptosis.