Impact of tumor viability at second-look procedures performed before completing treatment on the Intergroup Rhabdomyosarcoma Study Group protocol IRS-IV, 1991-1997: a report from the children's oncology group.

PURPOSES: The aims of the study were to compare results of clinical/radiographic studies before second-look procedures (SLP) with SLP specimens from patients with gross residual sarcoma at diagnosis and to relate tumor viability to outcome. PATIENTS: Seventy-three patients underwent SLP before completing chemotherapy, with (n = 59) or without (n = 14) radiotherapy. Tumor sites were bladder/prostate (n = 27), head/orbit/parameningeal (n = 22), extremity/trunk (n = 14), and retroperitoneum/pelvis (n = 10). RESULTS: Of 14 patients, 1 (7%) with clinical/radiographic complete response (CR) had viable tumor. Of 59 patients, 35 (59%) without CR had viable tumor. Five-year failure-free survival (FFS) rates were 81% in 37 patients without viable tumor and 53% in 36 patients with viable tumor (Cox proportional hazards adjusted P = .05). Five-year FFS rates were 67% in 15 patients with clear margins and 43% in 21 patients with tumor-involved margins (n = 18) or viable gross tumor (n = 3) (Cox proportional hazards adjusted P = .04). Five-year survival was 78% to 79% among 73 patients with and 333 patients without SLP during treatment. CONCLUSIONS: Second-look procedures can show whether viable tumor is present and may be beneficial in selected patients with rhabdomyosarcoma. Disappearance of tumor (CR) usually correlated with no viable tumor at SLP. However, 41% of patients without CR had no viable tumor. Those without viable tumor had increased FFS but not survival compared to those with viable tumor.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models.

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

The coinheritance of beta- and alpha- thalassemia: a review of one patient and her family.

The diagnosis and management of alpha-thalassemia may be complicated by the variability of the phenotype, which is due to the interaction of coinherited alpha-thalassemia and the variable severity of beta-thalassemia mutations. A well-documented case of complex beta- and alpha-thalassemia coinheritance is described. Laboratory and clinical data for the patient and her family are reviewed. The patient is an asymptomatic girl, one of identical twins. She presented at 1 month of age for follow-up of an abnormal newborn-screening result (hemoglobin F only), which initially suggested homozygosity for beta-thalassemia. Extensive studies on the patient and family revealed that she had coinherited alpha-thalassemia traits and homozygous beta-thalassemia. This case demonstrates the interaction of coinherited alpha- and beta-thalassemia with the resultant amelioration of the clinical phenotype. It also highlights the importance of family studies and close follow-up in diagnosing complex hemoglobinopathies.

Credentialing a preclinical mouse model of alveolar rhabdomyosarcoma.

The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 "druggable" kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model.

Bortezomib reverses a post-translational mechanism of tumorigenesis for patched1 haploinsufficiency in medulloblastoma.

BACKGROUND: Tumor initiation has been attributed to haploinsufficiency at a single locus for a large number of cancers. Patched1 (Ptc1) was one of the first such loci, and Ptc1 haploinsufficiency has been asserted to lead to medulloblastoma and rhabdomyosarcoma in mice. PROCEDURE: To study the role of Ptc1 in cerebellar tumor development and to create a preclinical therapeutic platform, we have generated a conditional Ptc1 haploinsufficiency model of medulloblastoma by inactivating Ptc1 in Pax7-expressing cells of the cerebellum. RESULTS: These mice developed exclusively medulloblastoma. We show that despite the presence of transcription of Ptc1, Ptc1 protein is nearly undetectable or absent in tumors. Our results suggest that Ptc1 loss of function is complete, but achieved at the protein level rather than by the classic genetic two-hit mechanism or a strict half-dosage genetic haploinsufficiency mechanism. Furthermore, we found that bortezomib, a 26S proteasome inhibitor, had a significant anti-tumor activity in vitro and in vivo, which was accompanied by restoration of Ptc1 protein and downregulation of the hedgehog signaling pathway. The same effect was seen for both human and mouse medulloblastoma tumor cell growth. CONCLUSIONS: These results suggest that proteasome inhibition is a potential new therapeutic approach in medulloblastoma.

Molecular classification of rhabdomyosarcoma--genotypic and phenotypic determinants of diagnosis: a report from the Children's Oncology Group.

Rhabdomyosarcoma (RMS) in children occurs as two major histological subtypes, embryonal (ERMS) and alveolar (ARMS). ERMS is associated with an 11p15.5 loss of heterozygosity (LOH) and may be confused with nonmyogenic, non-RMS soft tissue sarcomas. ARMS expresses the product of a genomic translocation that fuses FOXO1 (FKHR) with either PAX3 or PAX7 (P-F); however, at least 25% of cases lack these translocations. Here, we describe a genomic-based classification scheme that is derived from the combined gene expression profiling and LOH analysis of 160 cases of RMS and non-RMS soft tissue sarcomas that is at variance with conventional histopathological schemes. We found that gene expression profiles and patterns of LOH of ARMS cases lacking P-F translocations are indistinguishable from conventional ERMS cases. A subset of tumors that has been histologically classified as RMS lack myogenic gene expression. However, classification based on gene expression is possible using as few as five genes with an estimated error rate of less than 5%. Using immunohistochemistry, we characterized two markers, HMGA2 and TFAP2ss, which facilitate the differential diagnoses of ERMS and P-F RMS, respectively, using clinical material. These objectively derived molecular classes are based solely on genomic analysis at the time of diagnosis and are highly reproducible. Adoption of these molecular criteria may offer a more clinically relevant diagnostic scheme, thus potentially improving patient management and therapeutic RMS outcomes.

Juvenile myelomonocytic leukemia: report of seven cases and review of literature.

Juvenile myelomonocytic leukemia (JMML) is a rare, aggressive, clonal hematopoietic disorder of childhood with features of both myelodysplasia (thrombocytopenia, anemia) and myeloproliferation (leukocytosis, monocytosis). In most cases there is marrow hypercellularity, splenomegaly, and extramedullary involvement. In 1997 an international consensus on terminology was reached and guidelines/criteria for diagnosis were proposed. A recent World Health Organization classification described the current diagnostic criteria of JMML. Although the diagnosis of JMML has been facilitated, it can be challenging, especially in the early stages or when it 1st presents as an extramedullary tumor. We report a series of 7 cases diagnosed over a period of 10 years (from January 1, 1996, to December 31, 2005). Two cases had interesting associated findings that would potentially lead to delay in diagnosis or misdiagnosis. Two other cases had extramedullary involvement with symptoms referable to the organs of involvement at presentation. Clinical and pathologic findings are summarized with a review of relevant literature.

Defining the cooperative genetic changes that temporally drive alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of RMS known as alveolar (aRMS) have a 5-year survival rate of <30%. aRMS tissues exhibit a number of genetic changes, including loss-of-function of the p53 and Rb tumor suppressor pathways, amplification of MYCN, stabilization of telomeres, and most characteristically, reciprocal translocation of loci involving the PAX and FKHR genes, generating the PAX7-FKHR or PAX3-FKHR fusion proteins. We previously showed that PAX3-FKHR expression in primary human myoblasts, cells that can give rise to RMS, cooperated with loss of p16INK4A to promote extended proliferation. To better understand the genetic events required for aRMS formation, we then stepwise converted these cells to their transformed counterpart. PAX3-FKHR, the catalytic unit of telomerase hTERT, and MycN, in cooperation with down-regulation of p16INK4A/p14ARF expression, were necessary and sufficient to convert normal human myoblasts into tumorigenic cells that gave rise to aRMS tumors. However, the order of expression of these transgenes was critical, as only those cells expressing PAX3-FKHR early could form tumors. We therefore suggest that the translocation of PAX3 to FKHR drives proliferation of myoblasts, and a selection for loss of p16INK4A/p14ARF. These early steps, coupled with MycN amplification and telomere stabilization, then drive the cells to a fully tumorigenic state.

Prevalence and clinical impact of anaplasia in childhood rhabdomyosarcoma : a report from the Soft Tissue Sarcoma Committee of the Children's Oncology Group.

BACKGROUND: Anapalsia is rare in childhood rhabdomyosarcoma and has not been included in the International Classification of Rhabdomyosarcoma (ICR). A recent review of cases from the Soft Tissue Sarcoma Committee of the Children's Oncology Group (COG) suggests that anaplasia might be more common than previously reported and may impact clinical outcome. METHODS: The prevalence of anaplasia (focal or diffuse) was prospectively assessed in 546 eligible cases who were registered in an Intergroup Rhabdomyosarcoma Study Group (IRSG) or COG therapeutic trial from 1995 through 1998. The incidence of anaplasia in tumor samples and its impact in predicting clinical outcome was assessed. RESULTS: Overall, 71 (13%) of all samples analyzed had anaplasia. Anaplasia was more common in patients with tumors in favorable sites and was less commonly observed in younger patients and in those with stage II, III, or clinical group III disease. Regardless of its distribution (focal or diffuse), on univariate analysis the presence of anaplasia negatively influenced the failure-free survival rate (63% vs 77% at 5 years) and overall survival (68% vs 82% at 5 years) rates in patients with embryonal rhabdomyosarcoma. This effect was most pronounced in children with intermediate-risk tumors. Anaplasia did not affect outcome in patients with alveolar tumors. CONCLUSIONS: The incidence of anaplasia in patients with rhabdomyosarcoma is higher than previously described and may be of prognostic significance in children with intermediate-risk embryonal rhabdomyosarcoma.

NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma.

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

Primary renal sarcomas in the Intergroup Rhabdomyosarcoma Study Group (IRSG) experience, 1972-2005: A report from the Children's Oncology Group.

PURPOSE: To describe clinical and pathologic characteristics and outcome of patients with renal sarcomas. PATIENTS/METHODS: The IRSG database includes newly diagnosed patients <21 years old with rhabdomyosarcoma (RMS) or undifferentiated sarcoma (UDS). We identified patients with renal sarcoma and reviewed their charts. RESULTS: Ten of the 5,746 eligible IRSG patients enrolled from 1972 to 2005 had primary renal embryonal RMS (N = 6) or UDS (N = 4). Anaplasia was present in six (60%) of the tumors. Patients' ages ranged from 2.6 to 17.8 years. Tumor diameters ranged from 7 to 15 cm (median, 12 cm). At diagnosis, seven patients had localized disease: four underwent complete removal of tumor (Group I), two had microscopic residual (Group II), and one had gross residual tumor (Group III). Three patients had distant metastases (Group IV) in lungs and bone. Nine patients received vincristine, actinomycin D and cyclophosphamide (VAC). Two Group I patients received no radiation therapy (XRT); others received XRT to the primary tumor and to some metastatic sites. Nine patients achieved complete disappearance of tumor, six due to the initial operation. Tumors recurred in lung (N = 2) or brain (N = 1) in Group IV patients; each died within 16 months. The Group III patient died of Aspergillus pneumonia. The six Group I and II patients survive, continuously disease-free, at 2.7-17.3 years (median, 4.7 years). CONCLUSIONS: Patients with renal sarcomas often present with large tumors, many of them containing anaplastic features. Removing all gross disease at diagnosis, if feasible, is a critical component of treatment to curing patients with renal sarcoma.

Primary rhabdomyosarcoma of the sacrum: a case report and review of the literature.

We describe herein a rare case of primary rhabdomyosarcoma (RMS) occurring in the sacrum. A 16-year-old woman presented with a 2-month history of pain in bilateral buttocks and posterior thighs. Computed tomography showed a primary tumor with bone destruction in the 2nd sacral vertebra and invasion to the 1st to 3rd vertebrae and retroperitoneal space. Histological examination of the tumor showed proliferation of spindle-shaped cells intermingled with rhabdomyoblasts in a fascicular and storiform growth pattern. Tumor cells showed immunoreactivity for vimentin, desmin, muscle-specific actin, sarcomeric actin, alpha-smooth muscle actin and CD99, and partial immunoreactivity for myoD1, myf-4, myogenin and myoglobin. Reverse transcription polymerase chain reaction demonstrated expression of myoD1. On the basis of the aforementioned findings, a poorly differentiated spindle cell variant of embryonal RMS was diagnosed. The patient underwent combined therapy with chemotherapy and radiotherapy, but died 17 months after incisional biopsy. The present case is instructive in differential diagnosis of primary bone tumors, and the possibility of skeletal RMS needs to be considered.

Results of treatment of patients with superficial facial rhabdomyosarcomas on protocols of the Intergroup Rhabdomyosarcoma Study Group (IRSG), 1984-1997.

PURPOSE: We analyzed the outcome of 47 patients with superficial facial rhabdomyosarcoma (RMS) treated on Intergroup Rhabdomyosarcoma Study Group (IRSG) Protocols-III, -IV-Pilot, and -IV. METHODS: We reviewed patients' records. Clinico-pathologic features, treatment, and outcome were examined to identify prognostic factors. RESULTS: Thirty-two patients were males; 35 patients were 1-9 years old at diagnosis. Tumor sites were buccal/cheek (N = 21), external nasal/nasolabial (N = 12), lip/chin (N = 9), and masseter (N = 5). Patients (46/47) had localized disease: 18 biopsy only (Group III), 17 microscopic residual tumor (Group II), and 11 complete resection without residual tumor (Group I). Eight-year estimated event-free survival (EFS) and overall survival (OAS) rates were 61% and 65%. Patients <12 months old had inferior EFS, 21%, compared to approximately 68% in older patients (P = 0.077). Eight-year EFS rates were 80% for females and 50% for males (P = 0.096). Eight-year EFS rates were 72% in 33 patients without regional lymph-nodal tumor and 39% in 14 patients with regional nodal tumor (P = 0.07). Eight-year EFS rates were 72% for 22 patients with embryonal RMS and 53% for 23 patients with alveolar RMS (P = 0.28). Location of the primary tumor was not significantly related to outcome. CONCLUSIONS: Patients with superficial facial RMS often have localized, grossly resectable lesions at the time of presentation. Favorable prognostic factors include age >12 months, female gender, embryonal histology, and no lymph-nodal tumor.

Results in patients with cranial parameningeal sarcoma and metastases (Stage 4) treated on Intergroup Rhabdomyosarcoma Study Group (IRSG) Protocols II-IV, 1978-1997: report from the Children's Oncology Group.

PURPOSE: Determine outcome of patients with cranial parameningeal sarcoma and concurrent metastases treated on Intergroup Rhabdomyosarcoma Study Group (IRSG) Protocols II-IV. PATIENTS: We identified 91 patients in the database, which includes newly diagnosed subjects <21 years old with rhabdomyosarcoma (RMS) and undifferentiated sarcoma, and reviewed their charts in detail. RESULTS: The 54 males and 37 females were <1-19 years at diagnosis. Primary sites were nasopharynx-nasal cavity, middle ear/mastoid and parapharyngeal area ("better" sites, 55%), paranasal sinus and infratemporal-pterygopalatine area ("worse" sites, 42%), and other (3%). Sixty-eight percent of informative patients had direct intracranial extension. Major metastatic sites at diagnosis were lung (63%), bone marrow (33%), and bone (27%). Treatment included vincristine, actinomycin D, and cyclophosphamide (VAC) chemotherapy and radiotherapy to the primary tumor and up to five metastatic sites/tissues. OUTCOME: The estimated 10-year failure-free survival (FFS) rate was 32% (95% confidence interval [CI]: 22%, 42%). Sixty patients had progressive disease (N = 49) or death as a first event (N = 11); another developed myelodysplastic syndrome and died. Sites of first progression/relapse were distant (55%), local (12%), CNS extension (8%), mixed (6%), and uncertain (18%). Factors indicating likelihood of 10-year FFS included tumor arising in "better" versus "worse" sites (FFS 46% vs. 18%, P = 0.02) and embryonal versus other histology (FFS 37% vs. 19%, P = 0.06). CONCLUSIONS: Cure was possible for some patients with metastatic cranial parameningeal sarcoma. Patients with the best outlook had embryonal RMS located in the nasopharynx/nasal cavity, middle ear/mastoid, or parapharyngeal region. Distant metastases were the most frequent type of recurrence, indicating that more effective systemic agents are needed to eliminate residual disease.

A standards based ontological approach to information handling for use by organizations providing human tissue for research.

Tissue resources have become an important component of the infrastructure of institutions as well as companies performing biomedical research. Such tissue resources may be in the model of a bank, collecting a limited type of tissues and processing and storing them following a specific protocol. Such banks or archives may be associated with a clinical study or may function indepedently. An alternative type of tissue resource is utilized by many institutions and cancer centers. In this model, the investigator specifies the methods by which selected tissues are to be collected, processed and stored. In such a "prospective model", initially developed at the University of Alabama at Birmingham and the Ohio State University in the late 1970's and adopted by the Cooperative Human Tissue Network in 1986, specific types of tissues are not collected unless requested by an investigator. At some sites, both a prospective and an archival (bank) model are followed. This article describes an informatics approach needed to support a prospective tissue resource. It is by necessity more complicated than a model which supports a tissue bank but also can be used by a tissue bank. Of great importance is the approach to vocabulary and common data elements needed to support the informatics system of a prospective tissue resource, especially if the informatics system is to be used by a variety of personnel with greatly varying educational backgrounds.

Primary renal botryoid rhabdomyosarcoma: diagnosis and outcome.

Primary renal rhabdomyosarcoma is a rare entity. We report on a pediatric patient who, despite having multiple metastases to the lung on presentation, is free of disease 28 months after radical nephrectomy combined with chemotherapy and radiation therapy.

Assessment of response to induction therapy and its influence on 5-year failure-free survival in group III rhabdomyosarcoma: the Intergroup Rhabdomyosarcoma Study-IV experience--a report from the Soft Tissue Sarcoma Committee of the Children's Oncology Group.

PURPOSE: Initial response to induction chemotherapy predicts failure-free survival (FFS) in osteosarcoma and Ewing's sarcoma. For Intergroup Rhabdomyosarcoma Study (IRS) IV patients with group III rhabdomyosarcoma, we assessed whether reported response assessed by anatomic imaging at week 8 predicted FFS. PATIENTS AND METHODS: We studied 444 group III patients who received induction therapy, had response assessed at week 8 by anatomic imaging, and continued with protocol therapy. Induction chemotherapy was generally followed by radiation therapy (RT) starting after week 9. Response to induction therapy was determined at weeks 0 and 8. Local institutions coded response. RESULTS: Response rate for the entire cohort at week 8 was 77% (95% CI, 73% to 81%; complete response [CR], 21%; partial response [PR], 56%) but response had no influence on FFS (P = .57). Two hundred seventy-two patients received standard-timing RT at week 9 and thus only chemotherapy during induction. Response rate was 81% (95% CI, 76% to 86%; CR, 22%; PR, 59%). In these patients, response did not influence FFS except for those with alveolar histology. One hundred thirty-two other patients received chemotherapy and RT during induction (up-front RT). Response rate was 65% (95% CI, 57% to 73%; CR, 12%; PR, 53%), but response had no influence on FFS (P = .69). Forty patients received no RT at all (protocol violation) and response to induction therapy had no effect on FFS. CONCLUSION: In IRS-IV, response rate to induction therapy was 77% in group III patients, was independent of histology, and had no influence on FFS overall.

The PAX3-FKHR fusion gene of rhabdomyosarcoma cooperates with loss of p16INK4A to promote bypass of cellular senescence.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of <30%. ARMS is characterized by a chromosomal translocation generating the PAX3-FKHR fusion gene. However, ectopic expression of PAX3-FKHR often induces inhibition of cell proliferation, or cell death, when expressed in nonmuscle cells. This prompted us to explore the effect of expressing PAX3-FKHR in more relevant cells, specifically primary human skeletal muscle cells because these cells can be converted to a tumorigenic state that mimics rhabdomyosarcoma. PAX3-FKHR expression promoted both fetal and postnatal primary human skeletal muscle cell precursors to bypass the senescence growth arrest checkpoint. This bypass was accompanied by epigenetic DNA methylation of the p16(INK4A) promoter and correspondingly a loss of expression of this tumor suppressor. Knockdown of p16(INK4A) cooperated with PAX3-FKHR to drive proliferation past senescence, whereas reintroduction of wild-type p16(INK4A) in post-senescent cells caused growth arrest. Thus, PAX3-FKHR acts in concert with loss of p16(INK4A) to promote inappropriate proliferation of skeletal muscle cells. This association between PAX3-FKHR expression and p16(INK4A) loss was seen in human ARMS tumor tissue, as both human rhabdomyosarcoma cell lines and tissue microarrays showed a trend toward down-regulation of p16(INK4A) protein in alveolar subsets. We surmise that the generation of the PAX3-FKHR fusion protein may require loss of p16(INK4A) to promote malignant proliferation of skeletal muscle cells as an early step in ARMS tumorigenesis.