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.

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.

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.

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.

Correlation between histology and PAX/FKHR fusion status in alveolar rhabdomyosarcoma: a report from the Children's Oncology Group.

At the molecular level, alveolar rhabdomyosarcomas (ARMS) are characterized by 3 mutually exclusive PAX/FKHR conditions: PAX3/FKHR fusion (present in 60% of cases), PAX7/FKHR fusion (present in 20%), and PAX/FKHR fusion-negativity (present in 20%). The possibility of morphologic variation among these molecular subtypes has not been investigated. We undertook a blinded retrospective study of 65 cases of ARMS (16 PAX/FKHR fusion-negative, 36 PAX3/FKHR-positive, and 13 PAX7/FKHR-positive by routine reverse transcription-polymerase chain reaction). We evaluated cytohistologic parameters such as microcyst formation, solid foci, differentiation, giant cell formation, anaplasia, nuclear grade, mitosis/karyorrhexis index, rosette formation, geographic necrosis, presence and amount of rhabdomyoblastic differentiation, and the presence of foci resembling embryonal rhabdomyosarcoma. We analyzed the results using a simple chi formula. Of these features, only totally solid alveolar architecture reached significance (P=0.00014), with 7 of 16 PAX/FKHR-negative cases lacking this feature, compared with 0 of 36 PAX3/FKHR cases and 2/13 PAX7/FKHR cases. These preliminary results indicate that in general, only totally solid alveolar architecture in ARMS may predict the absence of a PAX/FKHR fusion. No features seemed to predict the presence of a particular fusion type. Our results suggest that histologic assessment of ARMS has limited correlation with PAX/FKHR fusion status.

Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells.

BACKGROUND: Stat3 has been classified as a proto-oncogene and constitutive Stat3 signaling appears to be involved in oncogenesis of human cancers. However, whether constitutive Stat3 signaling plays a role in the survival and growth of osteosarcomas, rhabdomyosarcomas, and soft-tissue sarcomas is still unclear. METHODS: To examine whether Stat3 is activated in osteosarcomas, rhabdomyosarcomas and other soft-tissue sarcomas we analyzed sarcoma tissue microarray slides and sarcoma cell lines using immunohistochemistry and Western blot analysis, respectively, with a phospho-specific Stat3 antibody. To examine whether the activated Stat3 pathway is important for sarcoma cell growth and survival, adenovirus-mediated expression of a dominant-negative Stat3 (Y705F) and a small molecule inhibitor (termed STA-21) were used to inhibit constitutive Stat3 signaling in human sarcoma cell lines expressing elevated levels of Stat3 phosphorylation. Cell viability was determined by MTT assays and induction of apoptosis was analyzed by western blotting using antibodies that specifically recognize cleaved caspases-3, 8, and 9. RESULTS: Stat3 phosphorylation is elevated in 19% (21/113) of osteosarcoma, 27% (17/64) of rhabdomyosarcoma, and 15% (22/151) of other soft-tissue sarcoma tissues as well as in sarcoma cell lines. Expression of the dominant-negative Stat3 and treatment of STA-21 inhibited cell viability and growth and induced apoptosis through caspases 3, 8 and 9 pathways in human sarcoma cell lines expressing elevated levels of phosphorylated Stat3. CONCLUSION: This study demonstrates that Stat3 phosphorylation is elevated in human rhabdomyosarcoma, osteosarcomas and soft-tissue sarcomas. Furthermore, the activated Stat3 pathway is important for cell growth and survival of human sarcoma cells.

Embryonal rhabdomyosarcoma with a der(16)t(1;16) translocation.

Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of RMS that predominantly involves the genitourinary tract and the head and neck regions in children younger than 10 years of age. Cytogenetically, ERMS is most frequently hyperdiploid, with extra copies of chromosomes 2, 7, 8, 11, 12, 13, and 20. No consistent structural chromosomal alteration has been identified in ERMS. In contrast, a t(2;13)(q35;q14) or t(1;13)(q36;q14) corresponding to PAX3-FOXO1A (previously FKHR) and PAX7-FOXO1A gene fusions are considered tumor-specific anomalies for alveolar RMS (ARMS). Occasionally, a recurrent secondary structural rearrangement involving chromosomes 1 and 16 is seen in translocation-positive ARMS, a der(16)t(1;16) resulting in an imbalance of 1q and 16q material. Conventional cytogenetic analysis of an ERMS arising in the urinary bladder of a 22-month-old male child revealed this nonrandom secondary chromosomal aberration, der(16)(1;16)(q22;q24), in a hyperdiploid complement with extra copies of chromosomes 2, 7, 8, 10, 12, 13, 19, and 20. Subsequent analyses showed tumor cells to be negative for FOXO1A, PAX3, or PAX7 gene locus rearrangements (by fluorescence in situ hybridization) and also negative for PAX3-FOXO1A and PAX7-FOXO1A fusion transcripts (by reverse transcriptase-polymerase chain reaction). These results suggest that the unbalanced t(1;16) translocation may be seen in RMSs lacking a primary genetic rearrangement.

Examination of gene fusion status in archival samples of alveolar rhabdomyosarcoma entered on the Intergroup Rhabdomyosarcoma Study-III trial: a report from the Children's Oncology Group.

Alveolar rhabdomyosarcoma (ARMS) is a soft tissue cancer in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR gene fusions. To improve the approach for fusion detection in archival samples, we developed a real-time reverse transcriptase-polymerase chain reaction assay for these fusion transcripts. By incorporating consensus primers and gene-specific probes, both presence and subtype of the fusion were determined in one assay. We applied this approach to a convenience sample of 78 formalin-fixed, paraffin-embedded ARMS tumors from the Intergroup Rhabdomyosarcoma Study (IRS)-III clinical trial and obtained satisfactory results in 59 (76%) cases. The distribution of fusion types was 35 (59%) PAX3-FKHR, 11 (19%) PAX7-FKHR, and 13 fusion-negative (22%). In a subsequent clinical analysis, we found that IRS-III ARMS cases analyzed for fusion status had a significantly improved outcome compared to IRS-III ARMS cases that were not available for fusion analysis. The basis of this outcome could not be explained by known prognostic clinical factors, and multivariate analysis confirmed that our convenience sample was not representative of the whole IRS-III cohort. In conclusion, although these robust assays provide new opportunities for correlative studies of archival material, our first application illustrates an important limitation of using a convenience sample for molecular-clinical correlative studies.

An immunohistochemical algorithm to facilitate diagnosis and subtyping of rhabdomyosarcoma: the Children's Oncology Group experience.

Immunohistochemistry remains the current ancillary method of choice in the pathologic evaluation of small blue round-cell tumors. In at least 20% of cases of rhabdomyosarcoma (RMS), it is considered an essential factor in the final and/or differential diagnosis of the malignancy. Newer immunostains (antimyogenin, MyoD1) generated against intranuclear myogenic transcription factors offer pathologists the best hope for improving the sensitivity and specificity of RMS diagnosis. A large series of RMS (956) were studied consecutively from the intergroup rhabdomyosarcoma study and children's oncology group files, along with multiple other malignant, benign or reactive lesions. A panel of antibodies to muscle-related antigens (myogenin, MyoD1, desmin, muscle-specific actin) was studied using formalin-fixed, paraffin-embedded tissue, an avidin-biotin/peroxidase complex immunohistochemical technique, antigen retrieval technique as appropriate, and automated immunostaining. Myogenin and MyoD1 were equally sensitive (positive for 97% of RMS cases), with both also showing similar specificity (90% vs. 91% of cases) for the diagnosis of RMS. Myogenin and MyoD1 staining were sometimes intact in areas of coagulative tumor necrosis, but negated by B5 fixation. Isolated, rare benign myogenin-positive nuclei were seen infrequently in reactive lymph nodes. Specifically, both myogenin and MyoD1 had significantly greater extent of expression for alveolar RMS (ARMS) than embryonal RMS (ERMS) (both with P < 0.001). Similarly, both myogenin (P = 0.001) and MyoD1 (P < 0.001) had significantly higher expression for ARMS than RMS, not otherwise specified (NOS). They were never expressed in undifferentiated sarcomas; however, reactive or regenerative myocytes did show expression. Immunostains against intranuclear myogenic transcription factors are, at present, the best available markers for confirming the diagnosis of RMS. Their differential expression in reactive myogenic lesions, variability in ARMS versus ERMS, and absence in undifferentiated sarcomas suggest new biologic questions to be explored in future studies.

Genetic heterogeneity in the alveolar rhabdomyosarcoma subset without typical gene fusions.

Previous studies of the PAX3-FKHR and PAX7-FKHR gene fusions in alveolar rhabdomyosarcoma (ARMS) indicated that the corresponding fusiontranscripts are not detectable in 20% of ARMS cases. To investigate the genetic features of this ARMS subset, we identified 23 ARMS cases in which PAX3-FKHR and PAX7-FKHR transcripts were not detected by a standard sensitivity reverse transcription-PCR (RT-PCR) assay. Subsequent analysis with a high sensitivity RT-PCR assay identified low-level expression of PAX3-FKHR or PAX7-FKHR in three cases. Analysis with a Southern blot assay for PAX3 and PAX7 rearrangements and a fluorescence in situ hybridization assay for FKHR rearrangements identified three cases with variant fusions in which PAX3 or PAX7 is postulated to be joined to novel genomic loci. In one such case, RT-PCR analysis of candidate partners identified a fusion of PAX3 to AFX, which is highly similar in structure and function to FKHR. Additional fluorescence in situ hybridization analysis identified two cases in which a PAX3-FKHR or PAX7-FKHR genomic fusion is present but is not associated with a fusion transcript detectable by RT-PCR. Finally, our analyses of the PAX3, PAX7, and FKHR loci did not identify rearrangements in >50% of cases, consistent with the possibility that there is a true fusion-negative subset. In summary, our analysis of ARMS cases without characteristic PAX3-FKHR or PAX7-FKHR transcripts identified several genetically distinct subsets including low expression or atypical presentation of standard fusions, variant fusions with other genes, and possibly true fusion-negative cases.

Treatment of localized nonorbital, nonparameningeal head and neck rhabdomyosarcoma: lessons learned from intergroup rhabdomyosarcoma studies III and IV.

PURPOSE: The characteristics and clinical outcomes of children and adolescents with localized nonorbital, nonparameningeal head and neck rhabdomyosarcoma (RMS) treated on national protocols from the Intergroup Rhabdomyosarcoma Group are reported. PATIENTS AND METHODS: We conducted a retrospective review of 164 children and adolescents enrolled in the third and fourth Intergroup Rhabdomyosarcoma Studies. Variables analyzed included age, sex, primary tumor site, histologic subtype, clinical group, therapy, site and rate of treatment failure, and time to initial recurrence. RESULTS: Localized nonorbital, nonparameningeal RMS accounted for 9% of all cases of RMS. The median age at diagnosis was 5 years; the median follow-up was 6.6 years. Estimated 5-year failure-free survival (FFS) and survival (S) rates were 76% (95% CI, 69% to 83%) and 83% (95% CI, 77% to 89%), respectively. In multivariate analysis, patients with clinically involved regional nodes (N1) had worse FFS (P =.02). For patients with embryonal tumors, FFS was significantly improved, especially among patients with Group I/II without nodal disease clinical Group I, II N0. For patients with alveolar/undifferentiated histology, FFS was significantly worse in children under the age of 1 year. Actuarial estimates of recurrences at 15 years were local (19%), regional (5%), and distant (9%). CONCLUSION: More than 80% of patients with RMS of the head and neck are cured of their disease using surgery and vincristine, dactinomycin +/- cyclophosphamide with or without radiotherapy. Our results indicate that early, limited exposure to cyclophosphamide might reduce recurrence in low-risk embryonal patients and that reduced dosages might achieve comparable results with improved toxicity profiles. These hypotheses will be tested in the next generation of trials from the Soft Tissue Committee of the Children's Oncology Group.

Efficacy of topotecan and cyclophosphamide given in a phase II window trial in children with newly diagnosed metastatic rhabdomyosarcoma: a Children's Oncology Group study.

PURPOSE: To determine the antitumor activity and toxicity of topotecan given immediately after cyclophosphamide as window therapy, then in combination with conventional agents in pediatric patients with newly diagnosed metastatic rhabdomyosarcoma (RMS). PATIENTS AND METHODS: Sixty-one patients younger than 21 years with newly diagnosed metastatic RMS or undifferentiated sarcoma were assigned window therapy (weeks 0 to 6) with topotecan (0.75 mg/m(2) daily x 5 every 21 days) immediately after cyclophosphamide (250 mg/m(2) daily x 5 every 21 days; TC). We continued to give these agents in combination with vincristine (VTC) to patients who showed objective improvement, partial response (PR), or complete response (CR) to TC and alternated courses of VTC with vincristine, dactinomycin and cyclophosphamide (VAC) during weeks 6 to 41 (VTC/VAC). Those who showed no response or progressive disease after TC received only VAC. All patients received radiotherapy to sites of unresected disease (weeks 15 to 21). RESULTS: The overall response rate (CR + PR) to TC was 47% (95% CI, 35% to 60%). Tumor size < or = 5 cm was associated with early response. Myelosuppression was the primary toxicity to TC. Overall 3-year disease-free survival and survival were estimated to be 10% (95% CI, 2% to 19%) and 20% (95% CI, 8% to 32%), respectively. Toxicity profiles for patients who received VTC/VAC or VAC alone were comparable. CONCLUSION: Topotecan after cyclophosphamide is a combination that is active against newly diagnosed RMS, with an acceptable toxicity profile. Disease-free survival and overall survival, however, remain disappointing for children with metastatic RMS at diagnosis.

Children from ethnic minorities have benefited equally as other children from contemporary therapy for rhabdomyosarcoma: a report from the Intergroup Rhabdomyosarcoma Study Group.

PURPOSE: To define the clinical characteristics of rhabdomyosarcoma (RMS) occurring in children from ethnic minorities and determine whether these children have benefited equally from advances in therapy. PATIENTS AND METHODS: This was a retrospective cohort analysis of children treated on the Intergroup Rhabdomyosarcoma Study Group protocols between 1984 and 1997. The clinical features and outcomes of 336 African-American children and 286 children from other ethnic minorities were compared with those of white children (n = 1,721). RESULTS: African-American, other ethnic group, and white children enjoyed similar 5-year failure-free survivals (FFS) of 61%, 61%, and 66%, respectively, P =.15. Compared with white children, nonwhite patients more often had (1) invasive, T2 tumors (P =.03); (2) stage 2 or 3 tumors (P =.003); (3) large tumors (more than 5 cm, P <.006); and/or (4) tumors with positive regional nodes (ie, N1, P =.002). Using Cox proportional hazards analysis, seven patient risk categories were defined with significant differences in outcome. This model was then used to search for other factors associated with FFS after adjusting for these risk categories. Only T stage and age remained associated with FFS (P =.001 and P <.001, respectively). After adjusting for T stage, risk category, and age, we explored the relationship of ethnic group to FFS and found that, compared with whites, the relative risk of failure was 1.14 for African-American patients and 1.2 for other ethnic minority patients, values that are not significantly different. CONCLUSION: Patients from ethnic minority groups more often have larger, invasive tumors with positive lymph nodes. Nevertheless, they have benefited as equally as white children from the dramatic progress in therapy of RMS.

PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children's oncology group.

PURPOSE: Alveolar rhabdomyosarcoma (ARMS) is an aggressive soft tissue malignancy of children and adolescents. Most ARMS patients express PAX3-FKHR or PAX7-FKHR gene fusions resulting from t(2;13) or t(1;13) translocations, respectively. We wished to confirm the diagnostic specificity of gene fusion detection in a large cohort of RMS patients and to evaluate whether these alterations influence clinical outcome in ARMS. PATIENTS AND METHODS: We determined PAX3-FKHR or PAX7-FKHR fusion status in 171 childhood rhabdomyosarcoma (RMS) patients entered onto the Intergroup Rhabdomyosarcoma Study IV, including 78 ARMS patients, using established reverse transcriptase polymerase chain reaction assays. All patients received central pathologic review and were treated using uniform protocols, allowing for meaningful outcome analysis. We examined the relationship between gene fusion status and clinical outcome in the ARMS cohort. RESULTS: PAX3-FKHR and PAX7-FKHR fusion transcripts were detected in 55% and 22% of ARMS patients, respectively; 23% were fusion-negative. All other RMS patients lacked transcripts, confirming the specificity of these alterations for ARMS. Fusion status was not associated with outcome differences in patients with locoregional ARMS. However, in patients presenting with metastatic disease, there was a striking difference in outcome between PAX7-FKHR and PAX3-FKHR patient groups (estimated 4-year overall survival rate of 75% for PAX7-FKHR v 8% for PAX3-FKHR; P =.0015). Multivariate analysis demonstrated a significantly increased risk of failure (P =.025) and death (P =.019) in patients with metastatic disease if their tumors expressed PAX3-FKHR. Among metastatic ARMS, bone marrow involvement was significantly higher in PAX3-FKHR-positive patients. CONCLUSION: Not only are PAX-FKHR fusion transcripts specific for ARMS, but expression of PAX3-FKHR and PAX7-FKHR identifies a very high-risk subgroup and a favorable outcome subgroup, respectively, among patients presenting with metastatic ARMS.

Prognostic factors and clinical outcomes in children and adolescents with metastatic rhabdomyosarcoma--a report from the Intergroup Rhabdomyosarcoma Study IV.

PURPOSE: To identify risk factors associated with outcomes in children with metastatic rhabdomyosarcoma (RMS) treated on the fourth Intergroup Rhabdomyosarcoma Study (IRS-IV). PATIENTS AND METHODS: Patients with metastatic RMS were treated with one of two regimens that incorporated a window of either ifosfamide and etoposide (IE) with vincristine, dactinomycin, and cyclophosphamide (VAC) or vincristine, melphalan (VM) and VAC. Study end points were failure-free survival (FFS) and overall survival (OS). Clinical factors including age, histology, sites of primary and metastatic disease, and number of sites of metastatic disease were correlated with those end points. RESULTS: One hundred twenty-seven patients were eligible for analysis. The estimated 3-year OS and FFS for all patients were 39% and 25%, respectively. By univariate analysis, 3-year OS was significantly influenced by histology (47% for embryonal v 34% for all others, P =.026) and increasing number of metastatic sites (P =.028). By multivariate analysis, the presence of two or fewer metastatic sites was the only significant predictor (P =.007 and.006, respectively). The combination of embryonal histology with two or fewer metastatic sites identified a subgroup with 3-year FFS of 40% and OS of 47%. CONCLUSION: Children with group IV RMS treated on the IRS-IV study had improved OS and FFS if they had two or fewer metastatic sites and embryonal histology. This favorable subset of patients has outcomes approaching those observed in selected patients with localized, nonmetastatic disease. Thus, these patients might not be appropriate candidates for regimens that include experimental agents with substantial toxicities or unproven antitumor activity.

Baalc, a marker of mesoderm and muscle.

Transcripts of the Brain and Acute Leukemia, Cytoplasmic (BAALC) gene are expressed in human neuroectodermal tissues and in CD34-positive bone marrow cells. High transcript levels occur in leukemic blasts from some patients with acute myeloid leukemia (AML), where high expression is an independent marker of poor prognosis. To gain insight into the hitherto unknown function of BAALC/Baalc, we studied its protein expression in embryonic and adult mouse tissue by immunohistochemical analysis. Baalc protein was mainly expressed in developing and mature muscle cells (cardiac, skeletal, and smooth) beginning on day E9 (heart). Signal was seen in the pre-muscle mesodermal cells of the dermatomyotome regions, and the derivatives of the lateral plate and intermediate mesoderm such as smooth muscle wall of the esophagus, stomach, the gut tube, bronchi, small blood vessels, and urinary bladder. This pattern continued through the late embryonic stages into adulthood. Baalc appeared to localize in the cytoplasm, adjacent to the cell membrane. This is distinctly observed in adult skeletal muscle cells. Baalc co-localized with known muscle-associated proteins but not with neural crest or neuronal markers. Scattered expression in adult bone marrow hematopoietic cells and weak expression in the brain neuropil also occurred. In conclusion, BAALC/Baalc is a marker of the mesodermal lineage, especially muscle.

Cyclophosphamide dose intensification during induction therapy for intermediate-risk pediatric rhabdomyosarcoma is feasible but does not improve outcome: a report from the soft tissue sarcoma committee of the children's oncology group.

PURPOSE: More than half of pediatric rhabdomyosarcoma cases have intermediate-risk features and suboptimal outcome (3-year failure-free survival estimates, 55 to 76%). Dose intensification of known active agents may improve outcome. EXPERIMENTAL DESIGN: This pilot study evaluated the feasibility of dose intensification of cyclophosphamide in previously untreated patients ages < 21 years with intermediate-risk rhabdomyosarcoma. Induction therapy comprised four 3-week cycles of VAC: vincristine (V) 1.5 mg/m2 on days 0, 7, and 14; actinomycin D (A) 1.35 mg/m2 on day 0; and dose-intensified cyclophosphamide (C) on days 0, 1, and 2. The three cyclophosphamide dose levels tested were as follows: (a) 1.2 g/m2/dose; (b) 1.5 g/m2/dose; and (c) 1.8 g/m2/dose. Continuation therapy comprised nine additional cycles of VAC with 2.2 g/m2/cycle of C. Radiotherapy was administered at week 0 (parameningeal tumors with intracranial extension) or week 12 or 15 (all others). RESULTS: Between October 1996 and August 1999, 115 eligible patients were enrolled. Three of 15 patients treated at dose level 2 experienced life-threatening dose-limiting toxicity (typhlitis +/- other severe toxicity). Dose level 1 was the maximum-tolerated dose, and 91 evaluable patients were treated at this level. The 3-year failure-free and overall survival estimates for patients treated at the maximum-tolerated dose were 52% (95% confidence interval, 41-64%) and 67% (95% confidence interval, 56-77%), respectively, at a median follow-up of 3 years. CONCLUSIONS: A 64% increase in the standard cyclophosphamide dosage during induction (to 3.6 g/m2/cycle) was tolerated. However, outcomes were similar to those observed at lower dosages, suggesting that alkylator dose intensification does not benefit patients with intermediate-risk rhabdomyosarcoma.

Suppression of tumorigenicity in neuroblastoma cells by upregulation of human vasoactive intestinal peptide receptor type 1.

We hypothesize that vasoactive intestinal peptide (VIP) promotes neural crest differentiation through VIP receptor type I (VPAC1). In order to test this hypothesis, SKNSH neuroblastoma cells were stably transfected with VPAC1 and receptor expression was verified by real-time RT-PCR. Overexpression of VPAC1 in SKNSH cells resulted in upregulation of endogenous retinoic acid receptor expression for both RARalpha and RXRalpha with no change in expression of RARbeta. Transfected cells demonstrated high affinity binding of VIP (K(D)=0.2 nM) and VIP-mediated stimulation of adenylate cyclase and a shift in cell cycle kinetics to a near triploid DNA index in G1. SKNSH/VPAC1 cells treated with VIP were observed to express a more differentiated phenotype compared to wild type cells as characterized by an increase in tissue transglutaminase II and a decrease in bcl-2 immunostaining. VIP-induced differentiation effects were potentiated by retinoic acid. This differentiation resulted in decreased proliferative potential in a xenograft model. Whereas, wild type SKNSH cells induced tumor growth in 100% of nude mice within 13 days post-injection, SKNSH transfected with VPAC1 demonstrated no tumor formation in xenografts followed for 6 months. Taken together, these data support the hypothesis that VIP modulation of neural crest differentiation is mediated via VPAC1 and that high expression of VPAC1 induces differentiation in and decreases tumorigenicity of neuroblastoma cells.

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.

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.