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High-dose methotrexate is a standard component of therapy for high-grade osteosarcoma. Its effectiveness may be limited by intrinsic and acquired resistance. Decreased reduced folate carrier (RFC) expression has been shown in approximately half of osteosarcomas at diagnosis. Mutations and polymorphisms in the RFC gene have been reported in various cell lines. The purpose of this study was to investigate sequence alterations in the RFC gene in osteosarcoma tumor samples. The entire coding region of the RFC gene in samples from 162 osteosarcoma patients was screened by DNA single-stranded conformational polymorphism, followed by direct sequencing of any region with altered mobility. A previously identified polymorphism at cDNA position number 174 of RFC exon 2 was observed. Sixty-one samples (37.6%) were heterozygous with both A/G at this position (His(27)/Arg(27)), 52 samples (32.2%) were homozygous with G (Arg(27)), and 49 samples (30.2%) were homozygous with A (His(27)). Fifteen (9.2%) samples were identified with other RFC sequence variants in exon 2, none of which have been reported. The sequence variants in exon 2 included a G to A substitution at cDNA position 231, a G to A substitution at cDNA position 155, a C to T substitution at cDNA position 114, and a T to C substitution at cDNA position 104, resulting in a serine to asparagine substitution at amino acid 46, a glutamate to lysine substitution at amino acid 21, an alanine to valine substitution at amino acid 7, and a serine to proline substitution at amino acid 4, respectively. A deletion of A at cDNA position 126 resulting in a frameshift was also observed. Some of these variants were observed in multiple samples. Eight samples had altered single-stranded conformational polymorphism patterns in exon 3 that were associated with nucleotide changes that altered the amino acid sequence. All of these RFC sequence variants appeared to be heterozygous. Heterozygous C/T and homozygous C also were observed at RFC cDNA position 790 in exon 3, which does not alter the amino acid coding sequence. This study shows that RFC sequence alterations are frequent in samples from osteosarcoma patients. Additional studies are under way to determine the clinical significance of these sequence alterations and their effect on methotrexate transport and resistance.

BACKGROUND The authors investigated the incidence and relative risk of secondary malignant neoplasms in long-term survivors of osteosarcoma. METHODS A comprehensive list of 509 patients with primary osteosarcoma treated at our institution between February 1973 and March 2000 was identified. All study patients received chemotherapy and/or surgery on one of six different protocols (T4, 5, 7, 10, 12, and CCG-7921/POG-9351). Chemotherapy was scheduled for up to 40 weeks with some variations in the actual treatment period and consisted of various combinations of the following agents: high-dose methotrexate, doxorubicin, bleomycin, cyclophosphamide, dactinomycin, vincristine, cisplatin, and ifosfamide. RESULTS Secondary malignant neoplasms (SMN) occurred in 14 of 509 patients. Only one had pulmonary metastasis at diagnosis and subsequent multiple recurrences that required thoracotomies and further modification of the chemotherapy regimen. The median age at diagnosis for osteosarcoma was 16.6 years (range, 3.1–74.4 years). The median follow-up was 5.2 years (range, 0.1–25.0 years). The time interval from diagnosis of the primary osteosarcoma to the development of SMN was 1.3–13.1 years (median, 5.5; 95% confidence interval [CI], 3.6–9.6). The most common SMN occurred in the central nervous system (n = 4): anaplastic glioma, meningioma, high-grade glioma, and maxillary astrocytoma. There were two cases of acute myeloid leukemia and one case each of myelodysplastic syndrome, non-Hodgkin lymphoma, high-grade pleomorphic sarcoma, leiomyosarcoma, fibrosarcoma, breast carcinoma, and mucoepidermoid carcinoma. The overall 5 and 10-year cumulative incidences of SMNs were 1.4% ± 1.1% and 3.1% ± 1.8%. The standardized incidence ratio was 4.6 (95% CI, 2.53–7.78, P = 0.00001) for the cohort and 3.64 (95% CI, 1.82–6.52, P = 0.0007) when patients with a history of retinoblastoma or Rothmund-Thomson syndrome were excluded. CONCLUSIONS The overall incidence of secondary malignancies in long-term survivors of osteosarcoma was significantly higher than the expected incidence of cancer in the general population. However, the standardized incidence ratios were much lower than those reported for Hodgkin disease and retinoblastoma. Although additional follow-up is warranted, the successes of current treatment regimens consisting of intensive, high-dose chemotherapy in combination with topoisomerase II inhibitors outweigh the risks. Cancer 2002;95:1728–34. © 2002 American Cancer Society. DOI 10.1002/cncr.10861

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma diagnosed in children. To identify novel and recurrent oncogenic mutations in RMS, we performed whole exome sequencing on 20 RMS samples, including 9 alveolar RMS, and 11 embryonal RMS (ERMS). Two ERMS samples showed the same c.365T>G point mutation in MYOD1, leading to an L122R substitution. Both were primary tumor samples and, in each case, normal tissue did not harbor the mutation, confirming its somatic nature. The L122R mutation occurs in the conserved basic region of MYOD1, at a highly specific residue that is a leucine in all myogenic bHLH transcription factors (MYOD1, MYF5, myogenin, MRF4) but is an arginine in MYC. Interestingly, previous studies of artificial mutants of the MYOD1 basic region found that MYOD1 L122R competes wild-type MYOD1 but without leading to target gene transactivation and also binds to MYC consensus sequences, regulating target genes similarly to MYC. We genotyped an additional 97 ERMS samples for the MYOD1 L122R point mutation using a mass spectrometry-based platform. The mutation was detected in 8 additional cases, resulting in an overall prevalence of 10% (10/103) in our ERMS samples. Corresponding normal tissue was available in 6 of the 8 additional cases, and, again, no mutations were identified in germline DNA. The 10 ERMS tumors harboring the MYOD1 L122R mutation displayed highly cellularity and frequent spindle cell morphology with uniform strong positivity for MYOD1 by IHC, features associated with the adult spindle cell variant of ERMS. Notably, these ERMS with MYOD1 mutations were from patients diagnosed in later childhood or adulthood (mean age = 25; median age = 28), and were more likely to arise in the head/neck (8/10 vs 16/80; p=0.0003). Furthermore, the overall survival of this subgroup of patients was significantly poorer than for ERMS lacking this mutation (0% vs 48% at 10 yrs; p=0.02). Transfection of MYOD1 L122R or wild type MYOD1 into C2C12 mouse myoblasts showed that MYOD1 L122R favors proliferation and transformation over myogenic differentiation. In a previous report, we had identified activating PIK3CA mutations in 3 of 60 ERMS samples. Remarkably, all 3 of these PIK3CA-mutated tumor samples are among the 10 MYOD1 L122R cases identified here. Therefore, we examined the combined effect of PIK3CA H1047R and MYOD1 L122R in soft-agar colony formation assays in C2C12 cells; this resulted in an increased number of colonies compared to either PIK3CA H1047R alone or PIK3CA H1047R + wild type MYOD1. Finally, expression profiling experiments in C2C12 cells and in BJ human fibroblasts comparing MYOD1 L122R, wild type MYOD1, and MYC showed that MYOD1 L122R shifts gene expression from a myogenic program to a MYC-like program. In summary, we have identified a distinctive subset of ERMS with recurrent somatic mutations in MYOD1, a transcription factor that functions as a critical regulator of skeletal muscle differentiation, altering its function resulting in impaired myogenic differentiation and enhanced proliferation. The tumors tend to present in older patients, usually arise in the head/neck, overlap pathologically with the recently described spindle cell variant of ERMS, and have especially poor outcomes. This represents a novel, molecularly defined subset of RMS that should be considered for high risk protocols and targeted therapeutic development. Citation Format: Neerav Shukla, Shinji Kohsaka, Nabahet Ameur, Angela Yannes, Agnes Viale, Li-Xuan Qin, Snjezana Dogan, Raf Sciot, Julia Bridge, Paul Meyers, Leonard Wexler, Jonathan Fletcher, Marc Ladanyi. A recurrent neomorphic mutation in MYOD1 defines a clinically aggressive subset of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A35.

Extraosseous Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) is a rare soft tissue tumor of childhood usually found in the extremities. The authors present the case of a 17-year-old girl who presented with right flank pain and hematuria and during operation was found to have a right ureteral mass. The histopathologic, immunohistochemical, ultrastructural, and cytogenetic characteristics of the excised mass were consistent with extraosseous ES/PNET. This is the first known reported case of extraosseous ES/ PNET of the ureter. The pathologic features and clinical management of this case, as well as a review of the literature, are presented.

An 8-year-old Hispanic boy with a hypoplastic left thumb, absent right thumb, and short stature experienced right leg pain and limp. A right tibial lesion was imaged and found to be osteosarcoma on biopsy. A 6-year-old Hispanic girl with congenitally absent thumbs experienced a pathologic fracture of her left femur after a minor sports injury. The radiologic abnormality seen was diagnosed as osteosarcoma on biopsy. Both patients continue to do well after intensive preoperative and postoperative high-dose chemotherapy and definitive reconstructive limb surgery. Osteosarcoma has been linked to several congenital syndromes in which absent thumbs are a feature. These two patients with absent thumbs and no definable syndrome experiencing osteosarcoma suggest that congenitally absent thumbs might be a risk factor for osteosarcoma in the absence of a syndrome.

PURPOSE: In osteosarcoma, prognostic factors at diagnosis other than clinical stage have not been clearly identified. The aim of this study was to determine whether human epidermal growth factor receptor 2 (HER2)/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or event-free survival. PATIENTS AND METHODS: We performed a retrospective immunohistochemical study on material obtained from patients treated on the Memorial Sloan-Kettering Cancer Center T12 protocol between 1986 and 1993. Paraffin-embedded tissue was identified from 53 patients (73% of patients enrolled onto protocol) and stained for HER2/erbB-2, p53, and p-glycoprotein expression using standard monoclonal antibodies and methods. RESULTS: At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Higher frequencies of expression were observed in samples from patients with metastatic disease at presentation and at the time of relapse. Expression of HER2/erbB-2 correlated with a significantly worse histologic response (P = .03). In patients presenting with nonmetastatic disease, expression of HER2/erbB-2 at the time of initial biopsy was associated with a significantly decreased event-free survival (47% v 79% at 5 years, P = .05). p53 and p-glycoprotein expression did not correlate with histologic response or patient event-free survival. CONCLUSION: The correlation of HER2/erbB-2 expression with histologic response to preoperative chemotherapy and event-free survival in this study suggests that HER2/erbB-2 should be evaluated prospectively as a prognostic indicator. The correlation also suggests that clinical trials of antibodies that target this receptor, such as recombinant humanized anti-HER2 monoclonal antibody (Herceptin; Genentech, San Francisco, CA), should be considered for the treatment of osteosarcoma.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis , Mycobacterium bovis , and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Seventy-four patients who had a Ewing sarcoma of bone were managed with preoperative and postoperative chemotherapy and operative resection, with or without postoperative irradiation. The primary objectives of the study were to determine the histological response to preoperative chemotherapy in terms of the percentage of tumor necrosis and to assess the relationship between the histological response and the oncological outcome. The minimum duration of follow-up of the surviving patients who were continuously free of disease was five years. Sections of each operative specimen were examined, and the histological response to chemotherapy was graded semiquantitatively. Grade I indicated necrosis of 50 per cent of the tumor or less; grade II, necrosis of more than 50 per cent but less than 90 per cent; grade III, necrosis of 90 to 99 per cent; and grade IV, necrosis of 100 per cent of the tumor. Of the seventy-four tumors, forty-four (59 per cent) were exquisitely sensitive to chemotherapy and had complete (grade-IV) or nearly complete (grade-III) necrosis. In contrast, fourteen tumors (19 per cent) had little or no response to chemotherapy (grade I) and sixteen (22 per cent) had a moderate degree of necrosis (grade II). The histological response to preoperative chemotherapy (p = 0.0001), followed by the size of the tumor (p = 0.001), were the most important predictors of event-free survival. At five years, the rate of event-free survival was zero of fourteen patients who had had a grade-I response, six of sixteen who had had a grade-II response, and thirty-seven (84 per cent) of forty-four who had had a grade-III or IV response. The risk of local recurrence was most strongly associated with the operative margins; there were only four local recurrences (6 per cent) after sixty-seven resections with negative margins. Local recurrence may also have been influenced by the histological response and the use of local radiation. There were no local recurrences after operative treatment of six tumors that had been associated with pathological fracture. The histological response to preoperative chemotherapy and the size of the primary tumor are the most important clinical predictors of the outcome of operative treatment of non-metastatic Ewing sarcoma. These indicators should be used to identify patients who are at high risk for metastasis as such patients may be candidates for more intensive or novel therapies.

3101 Background: Heat shock protein (HSP) 90 regulates the disposition and activity of a large number of deregulated proteins in Ewing sarcoma. We have shown pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor developed at MSKCC, in Ewing sarcoma. Unfolded proteins as a result of HSP90 inhibition are degraded via the ubiquitin-proteasome pathway or the autophagy pathway. We investigated the effects of combined inhibition of HSP90 and the proteasome pathway. Methods: We studied the effects of PU-H71 and bortezomib alone and in combination on cell proliferation and viability in multiple Ewing cell lines, benign stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis, reverse phase protein array and in vivo experiments in NOD/SCID IL2R gamma null (NSG) mice using the A673 cell line transduced with GFP luciferase. Using A673 metastatic model in NSG mice, we investigated the disease burden when treated with PU-H71, bortezomib and in combination. Results: In vitro, PU-H71 and bortezomib treatment resulted in caspase mediated cell death in a dose-dependent and time-dependent manner. Using PU-H71 beads we showed that some of the critical proteins, including IGF1R, hTERT and EWS-FLI1, are stabilized by HSP90. Exposure to PU-H71 resulted in depletion of AKT, ERK, MYC, C-kit, IGF1R and EWS-FLI1. Combination index (CI)-Fa plots and normalized isobolograms indicated synergism between PU-H71 and bortezomib. We noted increased expression of cleaved PARP and cleaved caspase 3 when Ewing cell lines were exposed to a combination of PU-H71 and bortezomib. Based on PK studies we treated mice (4 groups) using vehicle, PU-H71 75mg/kg i.p three times/wk, bortezomib 0.8mg/kg i.v twice/wk or a combination of PU-H71 and bortezomib for 4 wk. Ewing xenografts were significantly inhibited when treated with combination of PU-H71 and bortezomib compared to vehicle or either drug alone. Conclusions: Both PU-H71 and bortezomib have single agent activity against Ewing sarcoma. However they exhibit synergism when combined in in vitro and in vivo models providing a strong rationale for clinical evaluation in Ewing sarcoma.

Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein-90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins responsible for the assembly and regulation of cell-signaling systems. We tested the efficacy of PU-H71, a novel purine scaffold HSP90 inhibitor developed at Memorial Sloan-Kettering Cancer Center in several Ewing sarcoma cell lines and primary cultures established from patient samples. We investigated its effect on cell proliferation and cell viability on multiple Ewing cell lines, benign epithelial, endothelial and mesenchymal stromal cells and its effect on colony forming assay using CD34 hematopoietic stem cells . We performed cell cycle analysis, clonogenic assay, immunoblot analysis and in vivo experiments in NOD scid gamma (NSG) mice using A673 cell line transduced with GFP luciferase for bioimaging. We also established a metastatic model by calculating the mean fluorescence intensity (MFI) in various organs. Our results show that there is a significant therapeutic window in its activity against Ewing cell lines and benign cells. PU-H71 treatment resulted in arrest in G2 phase and diminished S phase in Ewing cell lines. We also noted that many of the deregulated pathway proteins including IGF1R, AKT, ERK, MYC, hTERT and EWS-FLI1 are stabilized by HSP90 and exposure to PU-H71 resulted in depletion of the above proteins. We also noted an increase in cleaved PARP and proapoptotic proteins and a decrease in antiapoptotic proteins on immunoblot analysis. Based on earlier PK and MTD studies, we treated mice at a dose of 75mg/kg by intraperitoneal injection, three times a week for 4 weeks. Our results indicated that Ewing's sarcoma tumor growth was significantly inhibited and metastatic burden was significantly decreased in the mice injected with PU-H71 compared to the mice injected with vehicle, based on MFI, weight and size of the tumors. Immunohistochemical staining showed that CD31 expression was significantly reduced in PU-H71 treated mice tumors. Our results suggest that PU-H71 has the ability to affect multiple critical oncogenic pathways simultaneously resulting in decreased tumor growth and metastases and has a strong rationale for clinical evaluation in Ewing sarcoma. Citation Format: Srikanth R. Ambati, Eloisi Caldas Lopes, Ullas Mony, Kohji Kosugi, Andre Moreira, Paul Meyers, Gabriela Chiosis, Malcolm Moore. Novel inhibitor of HSP90- PU-H71 exhibits significant activity in Ewing sarcoma pre-clinical models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2752. doi:10.1158/1538-7445.AM2013-2752

Examined predictors of depressive symptoms among 59 parents providing primary care to children newly diagnosed with cancer. Parents were studied for a 3-month period. The parent providing primary care to the child during medical treatment completed measures of depressive symptoms, endorsement of family routines, family functioning, amount of assistance from the spouse in providing care to the child, child behavior problems, as well as measures of the severity of the child's treatment regimen. A strong relationship was found between child behavior problems and parent depressive symptomatology. Although disease-related factors such as the child's functional impairment played a role in the parent's depressive symptoms, results revealed that the child's behavior problems were most strongly associated with parent depressive symptoms and that family cohesiveness also had a contributory role in the maintenance of parent depressive symptoms.

Introduction and research objectives Liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE; mifamurtide) is approved for treatment of non-metastatic osteosarcoma (OS) in Europe, Israel, and Mexico. The primary objective of MTP-OS-403 was to collect information regarding the safety and tolerability of MTP in patients with high-risk OS. The open label access study called for L-MTP-PE 2mg/m2 twice /week x 24 doses, then weekly (total doses= 48 in 9 months). OS patients could receive MTP as a single agent or with appropriate chemotherapy. Results As of April 2012, 202 patients were registered at 8 participating centers. Most had metastases at initial presentation or recurrent OS. Median age was 16 yr. Time from initial diagnosis to L-MTP-PE treatment was 29 +/- 23 months (median 24 mo). Although >88% had metastases, 27% had been resected to no evidence of disease (NED) before study entry. The most common Infusion related AE (IRAE) within 24 hours of infusion were chills (37%), fever (25%), nausea (21%), headache (19%), and fatigue (15%). The most common AE (grade 3-4) other than IRAE were chemotherapy-related and included thrombocytopenia (22%), neutropenia (19%), leukopenia (18%), febrile neutropenia (16%), lymphopenia (15%), and anaemia (15%). At MDACC, there was 1 episode of pleural effusion and one episode of pericardial effusion that were possibly L-MTP-PE related. The median number of L-MTP-PE doses given was 34 (mean 30.1 +/- 17.5); 32% of these high risk OS patients received 48 doses. As of April 2012 45% (90/202) of these high risk OS patients continue to be followed for survival. Conclusion The safety profile of L-MTP-PE in high-risk OS patients is consistent with the known toxicity of the product. The infusion related AEs were generally mild to moderate. Since survival benefit in high-grade OS from MTP may be related to disease burden (i.e. achieving NED status using local control measures such as surgery and/or radiotherapy), L-MTP-PE may be more effective when used in the adjuvant setting. A randomized multi-institutional clinical trial in OS patients presenting with metastases at diagnosis should be considered. Disclosure C. Oliva: Cristina Oliva is a Takeda employee. Y. Liu: I am a Millennium employee. All other authors have declared no conflicts of interest.

(1994). Limb-Sparing Surgery for Extremity Sarcoma. Cancer Investigation: Vol. 12, No. 5, pp. 497-504.

TO THEEDITOR: Widemann et al 1 report the use of glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction. When patients develop delayed excretion of methotrexate and impaired renal function after the administration of high-dose methotrexate there are two equally important imperatives. First, we must protect the patients from the potential toxicity of prolonged exposure to the antifolate drug methotrexate. Potential toxicities include myelosuppression, mucositis, and hepatoxicity, and can be lethal. The second imperative is to have the patient’s renal function return to normal to allow continuation of the chemotherapy needed to treat the underlying malignancy. The authors performed an analysis to determine which factors predicted greater than or equal to grade 4 toxicity. They noted that inappropriate increase in leucovorin predicted perfectly for development of greater than or equal to grade 4 toxicity and therefore could not be included in their multiple factor logistic regression analysis. We have reported our experience treating patients who have delayed methotrexate excretion and renal failure after high-dose methotrexate administration using high-dose leucovorin as the sole form of rescue. 2 All patients in our experience were salvaged. None of our patients died. The authors report that the median time to recovery of renal function for their cohort was 22 days. Our experience, albeit considerably smaller, achieved at least as good a time to recovery of normal renal function. One possible interpretation of the results that the authors report is that glucarpidase was not an essential component of rescue for patients experiencing high-dose methotrexate-related renal dysfunction and that similar results could have been achieved with the use of high-doses of leucovorin alone. Glucarpidase is unquestionably an excellent way to lower the plasma methotrexate concentration. This effect, however, has no impact on intracellular concentrations of methotrexate or on renal function. Protection of cells from intracellular methotrexate still requires the administration of high-doses of leucovorin and renal recovery appears to take place independently of glucarpidase administration. The authors’ conclusion that early intervention with the combination of leucovorin and glucarpidase is highly effective in patients who develop high-dose methotrexate-related renal dysfunction may be true but the authors should consider the possibility that similar results could be achieved with leucovorin alone.

PURPOSE Chemotherapy and surgery have improved the length of survival for patients with osteogenic sarcoma (OS) who present without metastatic disease. We reviewed our experience with patients with OS who presented with clinically detectable metastasis to determine the prognostic factors and the effects of surgery on the primary tumor and on metastatic disease. PATIENTS AND METHODS From 1975 to 1984 we treated 62 patients who had previously untreated OS with metastasis detected at presentation. All of these patients received intensive chemotherapy that included high-dose methotrexate; doxorubicin; and bleomycin, cyclophosphamide, and dactinomycin (BCD). Selected patients also received cisplatin. The intent of surgery was resection of the primary tumor and metastatic disease. RESULTS Survival was extremely poor; only 11% of patients survived, with a median survival of 20 months. Survival was not affected by use of preoperative chemotherapy versus immediate surgery, and did not correlate with serum lactate dehydrogenase (LDH) level, alkaline phosphatase level, or the site of the primary tumor. Survival did correlate with age, location of metastatic disease, histologic response to preoperative chemotherapy, and completeness of surgical resection of all sites of tumor. Resection of all sites of tumor identified at initial presentation was necessary for survival. CONCLUSION OS that presents with metastatic disease has a very poor prognosis with therapy, although therapy has achieved good results for patients without metastasis detected at diagnosis. Aggressive surgical resection of tumor is necessary for survival. The use of novel therapies at initial presentation is justified with this group of patients.

At the Memorial Sloan-Kettering Cancer Center (MSKCC) multiagent chemotherapy for the treatment of osteosarcoma (OS) was introduced by Dr. Gerald Rosen in 1973[1]. Rosen and Marcove developed the concept of an initial period of chemotherapy prior to definitive surgery of the primary tumor (neoadjuvant chemotherapy). The initial impetus for this strategy came from the need to create custom prostheses for limb-sparing surgery. Rosen and Huvos examined the tumors removed at definitive surgery for the degree of necrosis present following neoadjuvant chemotherapy [2]. They demonstrated a strong correlation between the histologically evaluated response and subsequent disease-free survival (DFS).

10637 Background: Several sarcoma subtypes are characterized by well-defined genetic events such as chromosomal translocations or gene amplification. In translocation-associated sarcomas, the prese...

9500 Background: Lexatumumab is an agonist, fully-human monoclonal antibody against TNF-Related Apoptosis Inducing Ligand Receptor 2 (TRAIL-R2) with preclinical evidence of activity in several pedi...

Twenty patients with known primary untreated and recurrent bone and soft tissue tumors underwent thallium imaging and three-phase bone imaging in the same session. The ratio of thallium uptake in the tumor tissue to the contralateral normal tissue areas was compared with the same ratio for phase 1 (blood flow or arterial phase), phase 2 (blood pool), and phase 3 (delayed medroxy-diphosphonate, MDP, uptake). There was poor correlation between Tl uptake and phases 1 and 3 of the bone scan ratios; r = 0.37 and 0.46; P = 0.097 and 0.047, respectively. The thallium uptake ratios correlated well with blood pool ratios (phase 2) (r = 0.84 and P less than 0.01). In contrast to uptake into normal muscle, Tl-201 uptake into tumor is not highly dependent on blood flow alone and other factors predominate in determining its magnitude.

PURPOSE Adjuvant chemotherapy improves disease-free survival (DFS) for patients with osteogenic sarcoma (OS). We reviewed our experience with OS to determine prognostic factors, the role of preoperative chemotherapy and subsequent histologic response, and the role of salvage chemotherapy after poor initial response. METHODS From 1975 to 1984, we saw 279 patients with previously untreated OS without metastasis. All patients received intensive chemotherapy and underwent surgical resection of primary tumor. Chemotherapy included high-dose methotrexate; Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH); and bleomycin, cyclophosphamide, and dactinomycin (BCD). Selected patients also received cisplatin. RESULTS DFS was not affected by use of preoperative chemotherapy versus immediate surgery, by use of limb-sparing surgery versus amputation, age, sex, or dose intensity of chemotherapy. DFS did correlate with serum lactate dehydrogenase (LDH), alkaline phosphatase, primary tumor site, race, and histologic response to preoperative chemotherapy. There was no difference in DFS for patients with a poor histologic response who did or did not receive cisplatin, although patients who did receive cisplatin had a longer time to relapse. The 5-year DFS was 76% for patients aged less than or equal to 21 years who had extremity primary tumor and were treated with the T10 protocol. CONCLUSIONS Intensive chemotherapy can achieve DFS for a high proportion of patients with OS. Although it is a powerful predictor of DFS, histologic response to preoperative chemotherapy cannot be assessed at diagnosis. We have not shown an ability to salvage patients with an unfavorable response. We need to increase the proportion of patients with a favorable response, identify the patients who will have an unfavorable response, and develop novel treatments to salvage poor responders.

Summary: A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg I-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide I-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ I-1 and did not occur at Mn2+ concentrations below 0.002 mg I-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.

10018 Background: With longer followup, the addition of muramyl tripeptide (MTP) to chemotherapy has been shown to improve overall survival in patients with non-metastatic osteosarcoma (OS). We rep...

19530 Background: Currently between 80–90% of patients with lower extremity sarcoma undergo limb salvage procedures. A few trials show that the functional quality of life is significantly higher for limb salvage surgery compared to patients with amputation. While taking quality of life and psychological consequences into account it would seem that limb salvage would be the procedure of preference; however, this difference has yet to be demonstrated in previous studies. Methods: Eighty-two long-term survivors of lower extremity sarcoma were studied to make a comparison of the overall quality of life in limb salvage and amputation patients. Forty-eight patients with limb salvage (age 14–49 years) and thirty-four patients with amputations (age 15–49 years) were studied who were at least one-year post- surgical procedure. Sensitive psychometric measures such as self-report questionnaires and visual analog scales were used to assess psychological consequences and quality of life. Results: The overall quality of life of patients with the limb salvage was significantly higher than patients with amputation (p-value < 0.01). For the quality of life questionnaire, the limb salvage patients had a higher mean value in every domain except parent-child relations. The domains that showed a significant difference between patients with limb salvage and patients with amputations are material well being, occupational relations, creative-aesthetic behavior, and sports activity. Conclusions: For patients with lower extremity sarcoma, the psychological quality of life is better for those who received the limb salvage procedure. The significance of this study is that it one of the first to show that there is a direct benefit to limb salvage surgery as compared to amputation in regards to several psychological factors and total quality of life. No significant financial relationships to disclose.

9525 Background: Pulmonary relapse is the most common site of relapse for patients with osteosarcoma (OS). Few therapeutic options are available for these patients. By encapsulating the drug within liposomes, sustained release lipid inhalation targeting (SLIT) cisplatin is a novel sustained-release formulation of cisplatin designed for administration via inhalation, thereby targeting the lungs with little systemic exposure. Methods: A two-center, open-label, phase Ib/IIa study was designed to characterize the safety and efficacy of SLIT cisplatin in patients with OS metastatic to the lung. Two dose levels were utilized: 24 mg/m2/dose, and 36 mg/m2/dose. Study drug was administered via nebulizer once every two weeks. OS patients with only pulmonary relapse were eligible. Toxicities were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events v3.0. Response was evaluated radiographically according to the World Health Organization criteria after every 2 cycles. When clinically indicated, metastasectomy was undertaken in patients on study after 2 cycles, and levels of SLIT cisplatin were measured in tumor samples upon metastasectomy. Results: Fourteen patients have been treated on study. SLIT cisplatin has been well tolerated in heavily pre-treated patients who have received multiple cycles of therapy, including one patient, approaching cumulative doses of 840 mg of SLIT cisplatin over one year and a second patient receiving 1,020 mg. Specifically, no patients have experienced hematologic toxicity, nephrotoxicity or peripheral neuropathy. Nausea (= grade 3) was attributed to study drug in 5 patients. Respiratory symptoms (= grade 3) were attributable to study drug in 4 patients. No adverse events required discontinuation of study drug. Two patients are pulmonary disease free one year after initiation of therapy. Conclusions: SLIT cisplatin is well tolerated in patients with pulmonary relapse of OS. Two of 14 patients are pulmonary disease free one year after initiation of therapy. SLIT cisplatin provides a novel therapeutic modality for patients with pulmonary relapse of OS, and warrants further study. No significant financial relationships to disclose.

An actinomycete with blue aerial mycelium and yellow substrate mycelium was isolated from a suburban soil sample collected in Cape Town, South Africa and named strain CPJVR-HT. The colour of the substrate mycelium was not sensitive to changes in pH. The organism produced spiny spores in Spirales spore chains. Chemical taxonomy indicated that it is a member of the genus Streptomyces. Strain CPJVR-HT grew at 45 °C and did not produce melanin or any diffusible pigments. It exhibited weak antibacterial activity against a clinical isolate of Enterococcus faecium, but no antibacterial activity against Escherichia coli ATCC 25922 or Pseudomonas aeruginosa ATCC 27853. Analysis of its 16S rRNA gene sequence, DNA–DNA hybridization studies and the results of physiological tests showed that this strain represents a novel species of Streptomyces, for which the name Streptomyces africanus sp. nov. is proposed. The type strain is CPJVR-HT (=NRRL B-24143T=DSM 41829T).

9515 Background: Aggressive fibromatosis (desmoid tumors, AF) are uncommon, locally aggressive, connective tissue neoplasms. Existing literature on systemic treatment of AF is sparse and consists mostly of case reports and small case-series. Based on previous observation of regression of AF treated with IM and tumoral expression of IM targets, SARC (Sarcoma Alliance for Research through Collaboration) included the treatment of AF onto a multi-institution phase II trial of IM in sarcoma. Here we report early clinical and laboratory results of the AF group. Methods: Eligible patients had histologically proven AF, unresectable or difficult to resect without considerable functional impairment. Patients were treated with IM 300 mg po BID (BSA≥1.5m2). The primary endpoint was complete (CR) or partial response(PR) at two months or stable disease (SD) or better at four months. Tumor DNA was extracted from available formalin fixed paraffin embedded tissue specimens and analyzed via allelic PCR and genomic DNA sequence analysis for specific point mutations in PDGFRα exons 12/14/18, PDGFRβ exons 12/18, KIT exons 9/11/13/17, and bRAF. Results: 51 patients were enrolled from 10/02 to 12/05 at 5 institutions, with 45 patients currently evaluable. The median age is 37 (range 14–67), and median number of prior therapies is 1 (range 0–3). 36 patients (80%) reached the primary endpoint of CR/PR at 2 months or SD or better at 4 months. The median time to treatment failure is 6.8 months (95% C.I. 5.8–17.1). Thus far, the maximum change in the largest dimension of the tumor ranged from a 21% increase to a 45% decrease. In 22 available tumor specimens, deletions within PDGFRαE12 and E18 were noted in 1 and 3 patients, respectively, while a wildtype genotype was found in other regions. Conclusions: IM has activity in AF, the mechanism of which remains unclear. While this is the largest reported phase II trial of AF, further improvement in evaluating clinical efficacy in this disease is clearly necessary. We plan an analysis of the maximum change in largest tumor dimension for each patient, which will be particularly beneficial in AF as responses often occur late. We have not as yet identified a laboratory predictor of clinical benefit. Further investigation of other potential targets in fresh tissue is warranted. [Table: see text]

8514 Background: Limited information exists regarding risk of t-MDS/AML after Ewing’s sarcoma. Methods: We followed a cohort of 578 individuals less than 30 years of age, diagnosed with Ewing’s sarcoma, PNET or primitive sarcoma of bone between 1988 and 1994 (treated according to a therapeutic protocol: CCG-7881/POG-8850) to determine the incidence of t-MDS/AML and associated risk factors. Between 1988 and 1992, all patients were assigned randomly to standard chemotherapy with doxorubicin (dox), vincristine (VCR), cyclophosphamide (CPM), and dactinomycin (DACT) (Regimen A: n=262) or experimental therapy consisting of these four drugs in alternation with Ifosfamide (I) and etoposide (E) (Regimen B: n=256). Between 1992 and 1994, patients with metastatic disease were nonrandomly assigned to Regimen C (n=60). Cumulative therapeutic exposures were: Regimen A: CPM:20.4 g/2, dox:375 mg/m2; Regimen B: CPM:9.6 g/m2, dox:375 mg/2, E:5 g/m2, I:90 mg/2; Regimen C: CPM:17.6 g/m2, dox:450 mg/m2, E:5 g/m2, I:140 g/m2. ...

8555 Purpose: To evaluate the outcome of patients with Ewing’s sarcoma family of tumors (ESFT) treated with modern radiotherapy techniques using MRI imaging along with optimal chemotherapy. Materia...

8545 Background: Complete staging of rhabdomyosarcoma is critical for delivery of appropriate therapy. Current diagnostic tools have known limitations. We have evaluated the role of 18F fluorodeoxyglucose positron emission tomography (PET) in the staging of patients with rhabdomyosarcoma. Methods: Since 1997, 60 patients with rhabdomyosarcoma underwent 159 PET scans. Of these, 21 patients with a median age of 11 years had a PET scan as part of their initial staging evaluation, before or within 13 days of initiation of therapy. Twelve patients had alveolar and 9 had embryonal histology. Primary sites included parameningeal (11), extremity (4), trunk (3), bladder/prostate (2), and head and neck (1). Results from CT, MRI, bone scan, and pathology within 1 month of the PET were compared. Results: A total of 83 sites were evaluated. All 21 patients had positive PET scans at the primary site. Twenty six PET positive sites at primary or nodal regions were biopsied and in all sites disease was confirmed. Standard...

9003 Background: We describe the safety and feasibility and incidence of leucovorin (LCV) dose modification for an outpatient high dose methotrexate (HDMTX) regimen. Methods: HD-MTX (12 g/m2) is administered in a pediatric day hospital (PDH) intravenously (IV) over 4 hours. Urinary alkalinization is achieved using an IV bolus of sodium bicarbonate followed by scheduled oral bicarbonate tablets, with supplemental dosing for any urine pH less than 7. Daily visits to the PDH follow and include IV hydration and MTX level monitoring. Overnight hydration is administered via an ambulatory infusion pump. LCV is begun 24 hours after MTX at a standard dose of 10 mg po q6h. The LCV dose is escalated between a range of 20 mg po q6h to 1 gm as a continuous IV drip over 24 hours according to an institutional algorithm for levels above 10,1, and 0.1 μmol/L at 24, 48, and 72 hours post MTX. Patients at risk of methotrexate toxicity including MTX levels >50 μmol/L at 24 hours, nephrotoxicity, dehydration, severe mucositis...

8514 Background: Outcome in osteosarcoma (OS) is predicted by percent necrosis (% NEC) after pre-operative chemotherapy. Efforts to improve outcome by post-operative therapeutic intensification have had limited success. Historically, 70% are standard responders (SR: < 98% NEC) whose outcomes justify efforts to intensify therapy. Methods: Patients

9013 Background: Desmoid tumors (aggressive fibromatosis) are rare clonal neoplastic proliferations of connective tissues. Although classified as benign, local aggressiveness can lead to significant impairment. Standard treatment involves wide surgical resection and/or radiation therapy. In cases of unresectable or recurrent disease, tamoxifen, chemotherapy, and NSAIDs have been used with varying success. We and others have previously reported desmoid tumors expressing c-kit, PDGFRα, and/or PDGFR. We reported two patients with extraabdominal desmoid tumors treated with the selective tyrosine kinase inhibitor imatinib (Gleevec) with significant shrinkage. SARC, in association with the Connective Tissue Oncology Society, initiated a prospective phase II trial in patients with desmoid tumors, or one of nine sarcoma subtypes. Here, we report specifically on patients with desmoid tumors. Methods: Patients ≥ 10 years old with desmoid tumors that were not curable by surgical management or in whom curative surger...

PURPOSE: Hemodialysis, hemoperfusion, thymidine, and carboxypeptidase have been recommended together with high-dose (HD) leucovorin (LV) to treat patients at risk for methotrexate (MTX) toxicity. To elucidate the efficacy of high LV rescue as the sole salvage modality for severe MTX intoxication, we studied 13 patients who were treated in this fashion at Memorial Sloan-Kettering Cancer Center (New York, NY). PATIENTS AND METHODS: To identify patients at high risk for severe MTX toxicity, we performed a retrospective review of all patients with MTX levels greater than 100 μmol/L at 24 hours and greater than 10 μmol/L at 48 hours after HD MTX. RESULTS: A total of 13 patients were identified. The median MTX concentration was 164 μmol/L at 24 hours (range, 102 to 940 μmol/L), 16.3 μmol/L at 48 hours (range, 10.5 to 190 μmol/L), and 6.2 μmol/L at 72 hours (range, 1.35 to 39 μmol/L). MTX levels remained greater than 0.1 μmol/L for an average of 11 ± 3 days (mean ± SD) (range, 7 to 17 days). In addition to supportive treatment with hydration and sodium bicarbonate administration, all patients were treated solely with HD LV, which was started within the first 24 hours in nine patients, 48 hours in three patients, and 72 hours in one patient in doses that varied from 0.24 to 8 g/d. Significant neutropenia (neutrophil count < 1,000/μL) occurred in eight patients and lasted for 1 to 5 days. Thrombocytopenia (platelet count < 100,000/μL) occurred in seven patients and lasted for 5 to 10 days. Other toxic manifestations included mucositis of varying degrees, diarrhea, and neutropenic fever, but all patients recovered. CONCLUSION: In the range of MTX levels observed, HD LV can be used as a sole therapy for MTX toxicity without the need for extracorporeal removal and with tolerable morbidity.