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After observing metaphyseal changes in pediatric and young adult patients with osteosarcoma who were treated with pamidronate, we initiated a retrospective study to identify bone findings on computed radiography of the chest and extremities, chest CT, extremity MRI, and radionuclide bone scans in this population.To review the generalized skeletal imaging findings on computed radiography, CT, and MR examinations in patients with osteosarcoma (OS) receiving pamidronate.A retrospective review of 40 patients with newly diagnosed OS (mean age, 16.5 years) receiving pamidronate with chemotherapy were identified at one institution. Computed radiography, CT, MR, and bone scans on 36 patients were reviewed for osseous changes.Dense metaphyseal bands at the growth plates in long bones, epiphyseal ossification centers, anterior rib ends, sternum and spine were observed. Osseous changes occurred on CT in 69% of patients, and on computed chest radiography in 53%. Bone-within-bone appearance in the spine and ossification centers was identified on computed radiography in 36%.OS patients treated with pamidronate can develop metaphyseal sclerotic bands as well as epiphyseal and vertebral endplate sclerosis progressing to a bone-within-bone appearance. Findings occur more frequently in younger patients with open epiphyses.

BACKGROUND: Histologic response to preoperative chemotherapy is a strong prognostic factor for osteosarcoma (OS). Thallium-201 (Tl-201) scintigraphic response to initial chemotherapy has previously been described as a predictor of histologic response. In the current study, the authors re-examined a series of patients studied using Tl-201 scintigraphy to determine the correlation between changes observed on Tl-201 scintigraphy and event-free survival (EFS). METHODS: A total of 22 patients with biopsy-proven OS of the extremity underwent Tl-201 imaging before and immediately after preoperative chemotherapy. The maximum pixel counts taken over the tumor divided by those taken of a background region yielded a tumor-to-background ratio (TBR). The percentage of change in the TBR before and after adjuvant chemotherapy, defined as the alteration ratio (AR), was correlated with EFS. RESULTS: The median AR was 85% (range, 28-100%). The 3-year EFS was 0.72 (95% confidence interval [95% CI], 0.48-0.86) and the 5-year EFS was 0.67 (95% CI, 0.43-0.86). There was no association between AR and EFS detected in this cohort (hazard ratio, 0.99; 95% CI, 0.95-1.02 [Somers rank correlation coefficient, 0.15]). CONCLUSIONS: Although Tl-201 scintigraphy was used as a tool for the assessment of response to chemotherapy in patients with OS, the AR did not appear to be predictive of EFS in this small group of patients. It is necessary to use the outcome variables of ultimate interest–EFS and overall survival– and not rely on surrogates for outcome to evaluate potential prognostic factors. Cancer, 2010. © 2010 American Cancer Society.

Rephosgenation of N-carboxyanhydrides of γ-benzyl and y-methyl L-glutamates, vs multiple recrystallizations, is a very efficient method for obtaining highly purified N-carboxyanhydrides, from which very high MW (0.98-1.5 × 106 Daltons) polymers can be derived.

The pesticides diazinon, trifluralin, and alachlor were successfully encapsulated into temperature-responsive Intelimer microcapsules. The pesticides were differentially released into aqueous solutions at predictable rates in response to temperature. The response of corn (Zea may L.) insect pests Diabrotica balteata LeConte (banded cucumber beetle) and Diabrotica virgifera LeConte (western corn rootworm) with the insecticide diazinon confirmed the temperature activity relationship of the encapsulated formulations compared to a commercial formulation

Recent studies have identified germline mutations of the p53 tumor-suppressor gene in families with the Li-Fraumeni syndrome, a rare inherited disorder characterized by a high risk of sarcomas of bone and soft tissue, breast cancer, and other tumors. In this report, we address the possibility that some sporadic sarcomas may be associated with new germline mutations of the p53 gene, which would not be manifested as familial cancer unless the patient survived to reproduce.We studied DNA from peripheral leukocytes of 196 patients with sarcoma and from 200 controls. Of the 196 patients with sarcoma, 15 were selected because they had had multiple primary cancers or had a family history of cancer. The entire coding sequence and splice junctions of the p53 gene were analyzed for mutations.Eight germline mutations were found, three in patients with no known family history of cancer and five in patients with an unusual personal or family history of cancer. Four mutations caused amino acid substitutions, and four caused stop codons. These mutations were not present in any of the 200 controls.New germline mutations of the p53 gene are rare among patients with "sporadic" sarcoma but may be common in patients with sarcoma whose background includes either multiple primary cancers or a family history of cancer. Diverse mutations of this gene were associated with an increased likelihood of cancer; hence, the entire gene should be considered a target for heritable mutation. It appears that the group of patients with cancer who carry germline mutations of the p53 gene is more diverse than is suggested by the clinical definition of the Li-Fraumeni syndrome. The identification of carriers could be of substantial clinical importance.

The addition of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) to chemotherapy has been shown to improve overall survival in patients with nonmetastatic osteosarcoma (OS). The authors report the results of addition of liposomal MTP-PE to chemotherapy for patients with metastatic OS.Intergroup-0133 was a prospective randomized phase 3 trial for the treatment of newly diagnosed patients with OS. The authors compared 3-drug chemotherapy with cisplatin, doxorubicin, and high-dose methotrexate (Regimen A) to the same 3 drugs with the addition of ifosfamide (Regimen B). The addition of liposomal MTP-PE to chemotherapy was evaluated.Five-year event-free survival (EFS) for patients who received liposomal MTP-PE (n = 46) was 42% versus 26% for those who did not (n = 45) (relative risk for liposomal MTP-PE, 0.72; P = .23; 95% confidence interval [CI], 0.42-1.2). The 5-year overall survival for patients who received MTP-PE versus no MTP-PE was 53% and 40%, respectively (relative risk for liposomal MTP-PE, 0.72; P = 0.27; 95% CI, 0.40-1.3). The comparison of Regimen A with Regimen B did not suggest a difference for EFS (35% vs 34%, respectively; relative risk for Regimen B, 1.07; P = .79; 95% CI, 0.62-1.8) or overall survival (52% vs 43%, respectively; relative risk for Regimen B, 1.1, P = .75; 95% CI, 0.61-2.0).When the metastatic cohort was considered in isolation, the addition of liposomal MTP-PE to chemotherapy did not achieve a statistically significant improvement in outcome. However, the pattern of outcome is similar to the pattern in nonmetastatic patients.

Osteosarcoma is an ultraorphan disease. There are approximately 1000 new patients diagnosed with osteosarcoma each year in the USA and Europe. Current treatment for osteosarcoma utilizes multiagent chemotherapy and surgical resection of all clinically detectable disease. Current treatments for osteosarcoma achieve 60–70% event-free survival (EFS) for patients with localized disease and approximately 20% EFS for patients who present with metastasis. These results have been stable for two decades. The addition of muramyl tripeptide (mifamurtide) to chemotherapy resulted in a trend towards improved EFS and a one-third reduction in the risk of death from osteosarcoma. Mifamurtide has been approved in Europe for the treatment of newly diagnosed osteosarcoma in combination with chemotherapy.

A novel actinomycete, strain TVU1T, was isolated from leaves of the indigenous South African plant Tulbaghia violacea. Applying a polyphasic approach, the isolate was identified as a member of the genus Micromonospora. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain TVU1T was most closely related to Micromonospora echinospora DSM 43816T. However, phylogenetic analysis based on gyrB gene sequences showed that strain TVU1T was most closely related to the type strains of Micromonospora aurantiaca and Micromonospora chalcea. DNA–DNA relatedness values between strain TVU1T and the type strains of M. echinospora, M. aurantiaca and M. chalcea were 7.6±4.5, 45.9±2.0 and 60.9±4.5 %, respectively. Strain TVU1T could be distinguished from the type strains of all three of these species by several physiological characteristics, such as colony colour, NaCl tolerance, growth temperature range and sole carbon source utilization pattern. Strain TVU1T (=DSM 45142T=NRRL B-24576T) therefore represents a novel species for which the name Micromonospora tulbaghiae sp. nov. is proposed.

Background The prognosis for recurrent/progressive Ewing sarcoma (ES) remains poor. Pre-clinical, adult phase I and II trials have demonstrated the combination of irinotecan and temozolomide to have schedule-dependent synergy and significant antitumor activity. A pediatric phase I trial has shown this regimen to be safe and active in advanced ES. Procedure We conducted a retrospective chart review to identify patients with recurrent/progressive ES treated with irinotecan [20 mg/m2/day × 5(×2)] and temozolomide (100 mg/m2/day × 5) in our institution. The best response achieved, time to progression (TTP), and associated toxicities were recorded. Results Twenty patients received a total of 154 cycles of therapy. Of 19 evaluable patients, there were 5 complete and 7 partial responses (a 63% overall objective response). Median TTP for 20 evaluable patients with recurrent/progressive ES was 8.3 months; for the subset of 14 patients with recurrent ES, it was 16.2 months. Median TTP was better for patients who sustained a 2-year first remission than for those who relapsed

As part of an enzyme-screening programme, an actinobacterium, strain HSM#10T, was isolated from a sample collected from the base of a translucent quartz rock in Miers Valley, eastern Antarctica. The isolate produced branching vegetative mycelium that was characteristic of filamentous actinobacteria. The chemotaxonomic characteristics of the strain suggested that HSM#10T should be classified as a member of the genus Streptomyces. Furthermore, phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was closely related to members of the genus Streptomyces, which supports the classification of this strain within the family Streptomycetaceae. Phenotypic and phylogenetic results allowed strain HSM#10T to be differentiated from known streptomycetes. DNA-DNA hybridization data also showed that strain HSM#10T could be differentiated from its nearest phylogenetic neighbours Streptomyces chryseus DSM 40420T (53.55+/-3.15% DNA relatedness), Streptomyces helvaticus DSM 40431T (38.75+/-2.75%), Streptomyces flavidovirens DSM 40150T (30.7+/-2.90%) and Streptomyces albidochromogenes DSM 41800T (33.9+/-0.10%). Therefore, the name Streptomyces hypolithicus sp. nov. is proposed, with HSM#10T (=DSM 41950T=NRRL B-24669T) as the type strain.

As part of an antibiotic-screening programme, an actinomycete, designated strain SPR(T), was isolated from soil collected from the banks of the Umgeni River, KwaZulu-Natal Province, South Africa. The isolate produced branching vegetative mycelia with sporangiophores bearing sporangia developing at a late stage of growth. The sporangia contained smooth, almond-shaped, non-motile spores. Strain SPR(T) exhibited antibiosis against various Gram-positive and Gram-negative bacteria, including Enterococcus faecium VanA (a vancomycin-resistant strain), Mycobacterium aurum A+ and Escherichia coli ATCC 25922. The chemotaxonomic characteristics of the strain, with the exception of the phospholipid pattern, corresponded with those of the members of the family Streptomycetaceae Waksman and Henrici 1943. Furthermore, phylogenetic analysis based on 16S rRNA genes showed that the strain was closely related to members of the genus Streptomyces, which supports its classification in the family Streptomycetaceae. Thus strain SPR(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces polyantibioticus sp. nov. is proposed. The type strain is SPR(T) (=DSM 44925(T)=NRRL B-24448(T)).

Purpose The purpose of this trial was to assess the efficacy of imatinib in patients with one of 10 different subtypes of advanced sarcoma. Patients and Methods Eligible patients were treated daily with imatinib dosed at 300 mg twice a day (for body-surface area ≥ 1.5 m2). The primary end point was response (clinical benefit response [CBR]), defined as complete (CR) or partial response (PR) at 2 months, or stable disease, CR, or PR at 4 months. Rules for early termination within each disease type were based on a Bayesian hierarchical probability model (BHM) accounting for correlation of the responses of the 10 subtypes. Available tissue samples were analyzed for molecules within the KIT/platelet-derived growth factor receptor (PDGFR) signal transduction pathway. Results One hundred eighty-five assessable patients with one of 10 subtypes of sarcoma were treated. One CR and three PRs were achieved. A CBR was achieved in 28 patients treated overall and by subtype: two angiosarcomas (n = 16), 0 Ewing (n = 13), one fibrosarcoma (n = 12), six leiomyosarcomas (n = 29), seven liposarcomas (n = 31), three malignant fibrous histiocytomas (n = 30), five osteosarcomas (n = 27), one malignant peripheral-nerve sheath tumor (n = 7), 0 rhabdomyosarcoma (n = 2), and three synovial sarcomas (n = 22). Variable expression and mutations within the KIT/PDGFR pathway were observed. Conclusion This is the first phase II study of a new agent in sarcoma to include sufficient patients with each of the common histologic subtypes to permit generalizable conclusions. The BHM is an effective method for studying rare diseases and their subtypes, when it is reasonable to assume that their response rates are exchangeable. Although rare dramatic responses were seen, imatinib is not an active agent in advanced sarcoma in these subtypes.

A patient with fatal severe thrombocytopenia and acute hepatic necrosis complicating carboplatin (JM8, CBDCA, NSC 241240) therapy is described. The patient, an 18-year-old man with acute lymphocytic leukemia, was given high-dose carboplatin as a part of a phase I trial of this agent for the treatment of leukemia. Carboplatin (270 mg/m2/day) was administered as an intravenous infusion on five consecutive days, and the patient died 10 days after his last dose of carboplatin from complications of thrombocytopenia and acute liver necrosis. Autopsy revealed hemorrhage into the substance of the myocardium and hemorrhagic centrilobular liver necrosis. The temporal relationship between the initial rise in this patient's liver function tests and treatment with carboplatin suggests that this patient's liver failure was in part due to carboplatin. The autopsy findings of hemorrhage into the substance of the myocardium and centrolobular liver necrosis suggest that, in addition to its direct effects, carboplatin may have also contributed indirectly to this patient's liver failure through the complications of thrombocytopenia.

Preclinical testing suggests the combination of carboplatin and irinotecan has at least additive antitumor activity. The primary objectives of the current study were to determine the maximum tolerated doses (MTDs) and recommended phase 2 doses of carboplatin administered with irinotecan to pediatric patients with refractory solid tumors.This was a multicenter, open-label, single-arm dose escalation study in which subjects with refractory solid tumors received 21-day treatment cycles of intravenous carboplatin on Day 1 followed by intravenous irinotecan administered daily for 10 days within 2 consecutive weeks. The plasma pharmacokinetics of ultrafiltrable platinum, irinotecan, and 2 irinotecan metabolites were determined during Cycle 1. The interpatient plan for dose escalation at study initiation was to increase irinotecan first followed by increases in carboplatin.Twenty-eight patients with a median age of 8.5 years (range, 1-21 years) were enrolled with a variety of solid tumors. Two of 6 subjects at the first dose level (carboplatin target area under the curve [AUC], 4.0 mg/mL*min; irinotecan, 18 mg/m2/dose) experienced dose-limiting gastrointestinal toxicities requiring a dose de-escalation scheme (carboplatin AUC, 4.0 mg/mL*min; irinotecan, 15 mg/m2/dose). Three of 6 subjects at the second dose level experienced dose-limiting gastrointestinal complications and bone marrow suppression. A further dose de-escalation to carboplatin AUC of 4.0 mg/mL*min and irinotecan of 12 mg/m2/dose resulted in dose-limiting bone marrow suppression in 1 of 13 patients treated at that dose, and therefore was determined to be the MTD. One complete response (in a patient with medulloblastoma) and 3 partial responses (in patients with neuroblastoma, medulloblastoma, and lymphoendothelial carcinoma, respectively) were observed.The recommended phase 2 dose in heavily pretreated pediatric patients is carboplatin (AUC, 4 mg/mL*min on Day 1) and irinotecan (12 mg/m2/dayx10 days) given every 21 days.

The collection of multi-ligament knee reconstruction procedure data generates long tabulated lists of featureless abbreviations, which are often difficult to interpret and present. As demonstrated with the launch of the Scandinavian anterior cruciate ligament registries, such data are under increasing scrutiny. When developing a visual tool to improve the interpretation, presentation, and ongoing collection of data within this field, much can be learnt from the historical teachings of Florence Nightingale and John Venn.Unknown to many, Florence Nightingale was a pioneer of graphic data illustration, becoming a Fellow of the Royal Statistical Society in 1858. Further advances in the visualization of complex data relations were made by John Venn, who introduced the Venn diagram in 1880. With this background in mind, the present work has been based upon the senior author's case series of 70 patients undergoing complex knee-ligament reconstruction at Warrington Hospital, from 2001 to the present time.Although obviously not negating the need for tabulated data, the graphic representation put forward here successfully supplements featureless tabulated lists of abbreviations and can be updated easily and regularly. Providing a clear, bright illustration that is free from patient identifiers, it can be used in presentations and publications, and freely accessed by a multidisciplinary team. It assists in the identification of injury patterns, can accommodate illustration of associated factors such as meniscal injury, and clearly demonstrates each hospital's multi-ligament knee reconstruction experience. This facilitates comparison and collaboration between hospitals and promotes research.

Background Methotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines. Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

BACKGROUND: Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. METHODS: Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. RESULTS: G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). CONCLUSIONS: The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further Fil: Morales Arias, Jaime. University of Texas; Estados Unidos Fil: Meyers, Paul A.. Memorial Sloan Ket tering Cancer Center; Estados Unidos Fil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. University of Texas; Estados Unidos Fil: Rodriguez, Nidra. University of Texas; Estados Unidos Fil: Zhou, Zhichao. University of Texas; Estados Unidos Fil: Reddy, Krishna. University of Texas; Estados Unidos Fil: Chou, Alexander J.. Memorial Sloan Ket tering Cancer Center; Estados Unidos Fil: Koshkina, Nadezhda V.. University of Texas; Estados Unidos Fil: Kleinerman, Eugenie S.. University of Texas; Estados Unidos

Purpose Children diagnosed with osteosarcoma (OS) and Ewing sarcoma (ES) have greatly benefited from the addition of alkylator therapy. However, with greater numbers of long-term survivors, the rising incidence of secondary malignant neoplasms (SMNs) is concerning. Herein we report on 2 patients with sarcoma who developed a case of secondary mucoepidermoid carcinoma after chemotherapy treatment without associated radiation therapy. To our knowledge, this is the first series of mucoepidermoid carcinomas arising in pediatric patients treated for sarcoma without radiotherapy. Methods Long-term survivors of OS and ES currently undergoing routine follow-up care were reviewed and noted for the development of a new secondary malignancy. Details of their initial evaluation, previous therapies, resection techniques, pathologic findings, and follow-up compose this report. Results Two patients, a 17-year-old adolescent boy with OS and 16-year-old adolescent girl with ES, with secondary mucoepidermoid carcinoma of the parotid gland were identified. Both patients underwent primary resection and chemotherapy including alkylating agents, but neither received radiation. The mucoepidermoid carcinomas developed 27 months and 132 months after completion of therapy, respectively, and were noted on routine yearly follow-up. Fine-needle aspiration was nondiagnostic on each, and parotidectomy with preservation of the facial nerve was performed. Pathology revealed low-grade mucoepidermoid carcinoma with tumor extending to the deep margins for both lesions, and radiotherapy to the parotid bed was administered. There were no surgical complications. One patient is alive, without evidence of recurrent mucoepidermoid carcinoma after 4 years; the other recently completed radiotherapy and is disease-free after 12 months. Conclusion Primary mucoepidermoid carcinoma of the parotid gland accounts for less than 10% of all head and neck tumors in childhood. Previous series of secondary mucoepidermoid carcinoma have demonstrated an increased risk in patients with leukemia/lymphoma. This is the first reported series of parotid mucoepidermoid carcinomas occurring after sarcoma treatment without radiotherapy. A common link between the 2 patients may be the use of alkylating therapy.

Background Little is known about the effects of chemotherapy on patient antibody titers to vaccine-preventable infectious diseases; thus, there is no standard protocol for revaccinating post-chemotherapy patients. Procedures To assess losses of detectable antibody titers due to chemotherapy, we retrospectively examined antibody titers for tetanus, varicella, measles, mumps, rubella, hepatitis B, and polio in 109 pediatric sarcoma patients. We also evaluated revaccination data to determine current practices and efficacy of revaccination. We limited our sample to osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma patients to control for the chemotherapy regimen patients received. Results Patients had pre-treatment detectable antibody titer that fell within the range of healthy children's antibody titers. However, 71% of patients had post-chemotherapy negative titers for at least one infectious disease. Patients most commonly had negative titers for hepatitis B (64%). Few patients had negative titers for measles (14%), mumps (9%), rubella (4%), polio 1 (0%), polio 2 (2.9%), polio 3 (4.8%), tetanus (5%), or varicella (11%). Revaccinations most frequently administered were hepatitis B and polio. Conclusions Our findings suggest that post-chemotherapy patients may need to be revaccinated against certain vaccine-preventable diseases including hepatitis B, tetanus, varicella, polio, measles, mumps, and rubella. Larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients. Pediatr Blood Cancer 2007;49:656–660. © 2007 Wiley-Liss, Inc.

BACKGROUND. Patients with metastatic osteosarcoma have a poor prognosis. The objectives of the study were to determine the antitumor activity and toxicity of topotecan (daily ×5) in newly diagnosed patients with metastatic osteosarcoma followed by chemotherapy (ifosfamide, carboplatin, etoposide [ICE], alternating with cisplatin and doxorubicin [CD]). METHODS. Newly diagnosed patients (≤30 years of age) with extensive metastatic disease (primary and ≥5 pulmonary nodules and/or bone metastases) with normal hepatic, renal, and cardiac function were eligible. Patients were eligible to receive further topotecan after standard chemotherapy if they exhibited a response. Twenty-eight patients were enrolled. Seventeen had metastases to the lung only and 11 had metastases to the bone or multiple sites. Of 28 patients enrolled, 27 could be evaluated for response. A limited dose escalation was incorporated. RESULTS. No responses were seen in the 11 patients treated at 3 mg/m2/day. One partial response (PR) and 1 clinical response (CLR) were reported among 15 patients who received topotecan at 3.5 mg/m2/day. No dose-limiting toxicity was observed. Principal nondose-limiting toxicities were hematologic and gastrointestinal. The 2- and 5-year event-free survival rates were low, 7% and 4%, respectively, but the 2- and 5-year overall survival rates were 44% and 22%, respectively. CONCLUSIONS. Topotecan at dose of 3.5 mg/m2/day can be safely administered upfront to newly diagnosed patients without excessive toxicity. Insufficient activity was seen with topotecan in this schedule to warrant further studies in osteosarcoma. The combination of ICE and CD was tolerable when delivered after initial topotecan therapy. Cancer 2007. © 2007 American Cancer Society.

BACKGROUND Complete staging of rhabdomyosarcoma is critical to deliver appropriate therapy. We evaluated the role of F-fluorodeoxyglucose positron emission tomography (PET) in the staging of patients with rhabdomyosarcoma. METHODS Twenty-four patients with rhabdomyosarcoma had a PET scan during staging evaluation, before or within 13 days of initiation of therapy. PET was compared with computed tomography (CT), magnetic resonance imaging (MRI), bone scan, and pathology. RESULTS Ninety-six sites were evaluated. All patients had positive PET scans at the primary site. Thirty-one PET positive sites at primary, regional, or distant sites were biopsied. Pathology in all 31 confirmed disease. Standardized uptake value for the primary site at diagnosis ranged from 2.4 to 12.7 (mean 6.4). At 23 sites, CT or MRI was equivocal for the detection of regional or distant spread. In these cases, a negative PET helped to exclude disease in 21 of 23 patients. PET failed to capture sites of disease visualized by CT, MRI, or bone scan at 10 sites. When comparing PET with the final clinical determination of disease extent, PET was 77% sensitive and 95% specific. CONCLUSIONS These preliminary data indicate that PET is a useful adjunct in staging rhabdomyosarcoma. A prospective study of PET for staging of rhabdomyosarcoma is warranted.

As part of an antibiotic-screening programme, the actinomycete strain HMC1T was isolated from soil collected from the banks of the Gamka River in the Swartberg Nature Reserve, Western Cape Province, South Africa. The isolate produced branching vegetative mycelia, which entwined to form rope-like structures. It exhibited antibiosis against Enterococcus faecium VanA (a vancomycin-resistant clinical strain) and Mycobacterium aurum A+. Organic solvent extracts of the culture filtrate and mycelial mass of strain HMC1T exhibited moderate antibiosis against Mycobacterium aurum A+, Mycobacterium bovis BCG (Tokyo) and Mycobacterium tuberculosis H37RvT. The chemotaxonomic characteristics of strain HMC1T corresponded with those of members of the genus Actinomadura. Furthermore, phylogenetic analysis based on the 16S rRNA gene showed that the strain was closely related to members of the genus Actinomadura. Phenotypic and 16S rRNA gene sequence analyses indicated that strain HMC1T represents a novel species of the genus Actinomadura, for which the name Actinomadura rudentiformis sp. nov. is proposed. The type strain is HMC1T (=DSM 44962T=NRRL B-24458T).

NY-ESO-1 is expressed in ∼70% of synovial sarcomas and not expressed in vital tissues. We report interim results of NCT01343043 evaluating safety and activity of autologous T cells engineered to express an HLA-A2+ restricted, affinity-enhanced T cell receptor (TCR) targeting NY-ESO-1. HLA-A2+ patients with unresectable, metastatic, or recurrent synovial sarcoma were eligible if tumors expressed NY-ESO-1 by IHC. Lymphocytes were activated using anti-CD3/28 microbeads, genetically modified with a lentivector, then cryopreserved. Subjects received fludarabine 30mg/m2/d (D-6 to -2) and cyclophosphamide 1800mg/m2/d (D-3,-2), and infusion of engineered T cells. Systemic IL-2 was not administered. Nine subjects have received NY-ESO-1 cell infusions. Median transduced T cell dose was 3.4 × 109 cells (range 0.4-14.4), 60×106 cells/kg (range 5.7-165.5), and median transduction efficiency was 45.8%. Toxicity likely attributable to the T cells included fever, and grade 1-2 cytokine release syndrome. No autoimmune toxicity has been observed. Circulating engineered NY-ESO-1 cells were detected in all patients, peaking 3-21 days post-infusion. Persistence has been evaluated beyond 3 months in 4 subjects, all of whom had detectable NY-ESO-1 cells at 4 mos, 6 mos+, 12 mos+ and 12 mos+. We identified persisting NY-ESO-1 T cells using dextamer and/or anti-vβ13.1 mAbs, and observed that a high fraction of CD4+ and CD8+ NY-ESO-1 TCR expressing T cells were CD45RA+CCR7+CD95+, consistent with a stem cell memory phenotype. Persisting cells also demonstrate a polyfunctional (IFN-γ and TNF-α) and cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Of 8 patients whose follow-up is sufficient to assess response, 4 experienced objective responses (1CR x 9 mos, 1PR x 9 mos, 2PR x 6 mos). Tumor shrinkage could not be attributed to chemotherapy alone as progressive decreases in tumor size were observed over several months following completion of the lymphodepleting regimen. All PR patients (2 upon signs of progression and 1 still responding to therapy) ultimately underwent resection for residual disease and two remain without evidence of disease. Adoptive immunotherapy with NY-ESO-1 engineered T cells shows promising results in synovial sarcoma with acceptable toxicity. High dose IL-2 is not required for therapeutic benefit with this regimen. Citation Format: Melinda S. Merchant, Sandra P. D'Angelo, Hua Zhang, Donna Bernstein, Gwen Binder-Scholl, Tom Holdich, Luca Melchiori, Dan Williams, Marylene Fortin, Yoav Peretz, Jason Howe, Michael Mehler, Bruce A. Hug, Matthew Wright, Stephen Grupp, Paul A. Meyers, William Tap, Bent Jakobsen, Crystal L. Mackall. Genetically engineered NY-ESO-1-specific T cells in HLA-A2+ patients with synovial sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4707. doi:10.1158/1538-7445.AM2015-4707

321 Background: Whole exome sequencing in WD pNETs demonstrated an increased number of mutations in genes implicated in chromatin remodeling and in the PI3K/Akt/mTOR pathway. Next-generation sequencing (NGS) allows us to bring this technology to the clinic to better characterize WD pNETs and potentially offer tailored therapies. Methods: A NGS platform developed at Memorial Sloan Kettering Cancer Center (MSKCC) termed MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) was used. This platform screens for 341 cancer genes commonly altered across tumor types. Thus far, 14 patients with WD pNETs have consented to MSK-IMPACT. Results: To date, mostpatients harbor no clinically validated or actionable mutations. Two patients, however, harbor different and potentially actionable BRAF mutations. The first patient is a 39 year-old woman with a BRAF V600E mutation. She presented with somatostatin receptor-negative, aggressive disease involving the liver, lymph nodes, adnexa, and peritoneum. She was initially diagnosed elsewhere as having a signet ring cell adenocarcinoma, and responded to FOLFOX and bevacizumab. Pathology review at MSKCC, however, identified the tumor as metastatic WD pNET. Upon disease progression, a repeat biopsy confirmed metastatic WD pNET. She is currently on capecitabine and temozolomide with clinical improvement. The second patient is a 22 year-old woman with metastatic WD pNET with a BRAF K601E mutation. Her tumor also has an aggressive behavior; she has progressed on all conventional and nonconventional therapies. Given the finding of a BRAF K601E mutation, she received trametinib and dabrafenib, but developed disease progression after 3 months. Conclusions: Two potentially actionable BRAF mutations were identified from NGS of WD pNETs. BRAF mutations have not been previously reported in pNETs. It is unclear whether BRAF mutations are oncogenic drivers, and whether BRAF-targeted treatments would be beneficial. Both patients show an atypical, aggressive presentation and course. Further testing is warranted to determine the role and clinical significance of BRAF in pNETs. Evaluation using pNET tissue microarrays is ongoing and will be reported at the meeting.

Purpose: The authors introduced a successful trial of a chemotherapy regimen to treat refractory thrombocytopenia and pain caused by a rare kaposiform hemangioendothelioma that had been refractory to corticosteroids. Patient and Method: A 6-year-old Latina girl with diffuse petechiae, constant pain and edema in her right lower extremity, and severe thrombocytopenia was brought for treatment. Physical examination revealed a large mass in the right lower extremity. The surgical specimen resected earlier in Mexico showed kaposiform hemangioendothelioma, an extremely rare vascular tumor that is frequently associated with consumptive coagulopathy and lymphangiomatosis. Corticosteroid therapy in Mexico had been ineffective in controlling the tumor size, pain, or thrombocytopenia. The patient was brought to the United States because of a rapidly enlarging tumor and intractable leg pain, causing inability to ambulate. Laboratory examinations revealed profound thrombocytopenia and evidence of consumptive coagulopathy. Because of the rapidly enlarging lesion, it was decided to give six cycles of a chemotherapy regimen consisting of vincristine, cyclophosphamide, and actinomycin D (VAC). Result: The patient responded to the first cycle of chemotherapy with partial reversal of the thrombocytopenia. The additional five cycles of the same chemotherapy regimen resulted in complete resolution of thrombocytopenia and significant improvement of the pain and edema in the right lower extremity. A follow-up imaging study 9 months later revealed a marked decrease in size of the vascular tumor. Conclusion: Inoperable kaposiform hemangioendothelioma unresponsive to corticosteroids can be successfully treated with chemotherapy.

We implanted 6 Common Murres (Uria aalge) and 10 Thick-billed Murres (Uria lomvia) with satellite transmitters and compared subsequent presence at the colony, nesting status, and provisioning to a control group that underwent a simple surgical procedure. In the 10 days following implantation, we resighted 10 of 11 control birds at the colony and 6 of 16 implanted birds. Of the birds that returned, 7 of 10 control birds retained breeding status, whereas zero of six implanted birds retained breeding status. We conclude that abdominal implantations alter murre nesting behavior.

Background Previous clinical and pre-clinical research has demonstrated synergy between anti-angiogenic agents and cytotoxic chemotherapy. This trial was undertaken to investigate whether the combination of cyclophosphamide and thalidomide would be active against pediatric tumors. Procedure Patients with pediatric malignancies who had no remaining conventional therapeutic options were recruited from January 1999 to May 2001. They received thalidomide (6–12 mg/kg po every day; maximum daily dose 800 mg) and cyclophosphamide (1,200 mg/m2 IV every 28 days). Results Twenty-seven patients were enrolled on the study. Seventeen were male and 10 were female. Median age at the time of registration was 15 years (range 1–54 years). The median number of prior treatment regimens was four. Twenty-one patients were evaluable for response; 1 had a partial response (Hodgkin disease), 1 demonstrated stable disease (neuroendocrine tumor), and 19 had progressive disease. The most common toxicities were hematological (leukocytopenia and neutropenia) and gastrointestinal. One patient experienced a grade 3 rash. Fatigue and daytime somnolence were variable. No peripheral neuropathy was observed. Conclusion The combination of thalidomide and cyclophosphamide as described herein has a modest and tolerable toxicity profile but little evidence of efficacy. Pediatr Blood Cancer 2007;49:261–265. © 2006 Wiley-Liss, Inc.

Extrarenal extracranial malignant rhabdoid tumors (MRT) are extremely rare and typically lethal. No consensus exists on an optimal treatment approach, and, in particular, the role of radiation therapy (RT) is poorly defined. We report on three children who underwent multimodality treatment including surgery, chemotherapy (CMT), and RT. Two of these patients are alive and without evidence of disease 6(1/2) and 7 years after diagnosis and one has expired. Our limited experience suggests that RT in the setting of CMT and surgery has the potential to prolong survival with acceptable toxicity, but outcomes are inconsistent and further study is necessary.

Leydig cell tumors account for 3% of testicular tumors and have never been reported after treatment for Ewing's sarcoma. We report the unusual occurrence of a patient who developed a Leydig cell tumor of the testis 18 years after successful treatment for Ewing's sarcoma. Additional monitoring for second malignancies may become appropriate as long-term survival continues to improve for patients with Ewing's sarcoma. Pediatr Blood Cancer 2007;49:1008–1010. © 2006 Wiley-Liss, Inc.

Purpose Prognosis is poor for Ewing sarcoma patients with metastasis at diagnosis. We intensified a five-drug therapy (ifosfamide, etoposide alternated with vincristine, doxorubicin, and cyclophosphamide) using filgrastim but not stem-cell support. We studied topotecan alone and combined with cyclophosphamide in therapeutic windows before the five-drug therapy. A randomly assigned proportion of patients received amifostine as a cytoprotective agent. Patients and Methods Eligible patients were ≤ 30 years old and had histologically proven Ewing sarcoma or primitive neuroectodermal tumor (PNET) and metastasis at diagnosis. Chemotherapeutic cycles began every 21 days, after recovery from toxicities. Results One hundred ten of the 117 patients enrolled were eligible. Thirty-six patients received initial topotecan. Three had partial responses (PRs), and 17 had progressive disease (PD). Thirty-seven patients were administered topotecan and cyclophosphamide; 21 of these patients achieved PR, and one patient had PD. In a randomly assigned group of 69 patients, amifostine did not provide myeloprotection, which was measured by absolute neutrophil count, platelet count, or cycle intervals. The best responses to the overall therapy included 45 complete responses, 41 PRs, stable disease in 14 patients, and PD in five patients. For all patients, the 2-year event-free survival (EFS) rate was 24% (± 4%), and the overall survival rate was 46% (± 5%). For the 39 patients with isolated pulmonary metastases, the 2-year EFS rate was 31% (± 7%) compared with 20% (± 5%) for patients with more widespread disease. Conclusion Topotecan had limited activity in patients with Ewing sarcoma or PNET metastatic at diagnosis. The topotecan-cyclophosphamide combination was active. Amifostine was not myeloprotective. Overall results showed no improvement compared with previous studies.

A novel actinomycete, strain CZH20(T), was isolated from a soil sample taken from the banks of the Gamka River in the Swartberg Nature Reserve, Western Cape Province, South Africa. Strain CZH20(T) was identified as a member of the genus Nocardia by a polyphasic approach. Strain CZH20(T) could be differentiated from other members of the genus Nocardia on the basis of physiology and 16S-rRNA gene sequence analysis. It exhibited weak antibiosis against Mycobacterium aurum A+. Organic solvent extracts of the culture filtrate and mycelial mass of CZH20(T) exhibited moderate antibiotic activity against Mycobacterium smegmatis LR222 and Mycobacterium tuberculosis H37Rv. The name Nocardia gamkensis is proposed, with the type strain CZH20(T) (=DSM 44956(T) =NRRL B-24450(T)).

An actinomycete, strain CZA14 T , was isolated from Worcester in the Western Cape province of South Africa. Based on rapid genus identification, 16S-rDNA sequence similarities and chemotaxonomy, strain CZA14 T was identified as a member of the genus Streptomyces . It exhibited weak antibiosis against Bacillus coagulans ATCC 7050 T , Mycobacterium aurum A+ and Acinetobacter calcoaceticus C91. The results of physiological tests and analysis of the 16S-rDNA sequence allowed for the differentiation of strain CZA14 T from other species of the genus Streptomyces . Strain CZA14 T therefore represents a new species for which the name Streptomyces pharetrae is proposed, with the type strain CZA14 T (=DSM 41856 T =NRRL 24333 T ).

Purpose: To evaluate the outcomes of patients with Ewing's sarcoma family of tumors (ESFT) treated with modern radiotherapy techniques with MRI along with optimal chemotherapy. Methods and Materials: The records of all 60 patients with ESFT who received radiation to the primary site between 1990 and 2004 were reviewed. All patients received chemotherapy, including vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide. Radiation was used as the sole modality for local control in 31 patients and was given either before ( n = 3) or after surgical resection ( n = 26) in the remainder. All patients had MRI and CT scan–based treatment planning, and 43% received intensity-modulated radiation therapy. Radiation doses ranged from 30 Gy to 60 Gy (median, 51 Gy), and 35% received hyperfractionated radiotherapy. Results: Median age was 16 years (range, 2–40 years). Because of selection bias for radiotherapy, the majority of primary tumors were centrally located (72%): spine ( n = 18), pelvis ( n = 15), extremities ( n = 12), chest wall ( n = 5), head and neck ( n = 5), and other ( n = 5). Thirty-eight percent of patients presented with metastatic disease, and 52% of primary tumors were ≥8 cm. Actuarial 3-year local control was 77%. The presence of metastases at diagnosis was an adverse prognostic factor for local control (84% vs. 61%, p = 0.036). No other predictive factors for local failure were identified. In patients without metastatic disease, 3-year disease-free and overall survival rates were 70% and 86%, respectively, whereas in patients with metastases they were both 21%. Follow-up of surviving patients was 6–178 months (median, 41 months). Conclusion: In this unfavorable cohort of ESFT patients, radiation therapy was an effective modality for local control, especially for patients without metastases. The presence of metastases at diagnosis is a predictive factor not only for death but also for local failure.

Osteosarcoma is the most common malignant bone tumor in children. Osteosarcoma patients who respond poorly to chemotherapy are at a higher risk of relapse and adverse outcome. Therefore, it was the aim of this study to identify prognostic factors at the time of diagnosis to characterize the genes predictive of poor survival outcome and to identify potential novel therapeutic targets. Expression profiling of 30 osteosarcoma diagnostic biopsy samples, 15 with inferior necrosis following induction chemotherapy (Huvos I/II) and 15 with superior necrosis following induction chemotherapy (Huvos III/IV), was conducted using Affymetrix U95Av2 oligonucleotide microarrays. One hundred and four genes were found to be statistically significant and highly differentially expressed between Huvos I/II and III/IV patients. Statistically significant genes were validated on a small independent cohort comprised of osteosarcoma xenograft tumor samples. Markers of Huvos I/II response predominantly were gene products involved in extracellular matrix (ECM) microenvironment remodeling and osteoclast differentiation. A striking finding was the significant decrease in osteoprotegerin, an osteoclastogenesis inhibitory factor. Additional genes involved in osteoclastogenesis and bone resorption, which were statistically different, include annexin 2, SMAD, PLA2G2A, and TGFβ1. ECM remodeling genes include desmoplakin, SPARCL1, biglycan, and PECAM. Gene expression of select genes involved in tumor progression, ECM remodeling, and osteoclastogenesis were validated via quantitative reverse transcription-PCR in an independent cohort. We propose that osteosarcoma tumor–driven changes in the bone microenvironment contribute to the chemotherapy-resistant phenotype and offer testable hypotheses to potentially enhance therapeutic response.

Peak serum concentrations of methotrexate (MTX) have been reported to correlate with outcome in osteosarcoma (OS). Modification of the MTX dose to achieve peak levels between 700 and 1,000 micromol/L has been recommended. The goal of the study was to assess whether there is a correlation between histologic necrosis of the tumor and/or prognosis with peak MTX serum concentration.Treatment included multi-agent adjuvant chemotherapy, including high-dose MTX (12 g/m2). Peak MTX levels were drawn following a 4-hr infusion. Histologic evaluation for percent necrosis was done at the time of definitive resection.The median peak MTX level (n = 52 patients) was 1,060 micromol/L (range: 410-4,700 micromol/L), with significant intra-patient and inter-patient variability. Fifty-eight percent of the levels were 1,000 micromol/L or higher. Response to pre-operative chemotherapy was: 18% Grade I necrosis, 35% Grade II, 31% Grade III, and 16% Grade IV. No significant association was found between the mean peak MTX levels and necrosis (P = 0.44). Event-free survival (EFS) for the 48 patients with non-metastatic disease at diagnosis was 76% at 4 years of follow-up, with no association between the mean peak MTX level and EFS (P = 0.24).The absence of a demonstrable correlation between peak MTX levels and histologic necrosis or EFS may suggest that most patients achieve therapeutic levels when MTX is given at a dose of 12 g/m(2). The significant degree of intra-patient variability in peak levels poses a dilemma for pharmacokinetic adjustment. Continued use of HD-MTX in all patients, rather than dose adapted therapy, may be justified.

Two types of telomere maintenance mechanisms (TMMs) have been described in human tumors: telomerase activation and alternative lengthening of telomeres (ALT). Although the vast majority of epithelial tumors rely on telomerase activation, many mesenchymal tumors rely on ALT for telomere maintenance, but within this tumor group, the TMMs used by translocation-associated sarcomas have not been systematically studied. We studied telomere lengths and telomerase expression and activity in 30 uncultured tumor samples and in 10 cell lines of Ewing's sarcoma, a prototypical translocation-associated sarcoma, and compared the data to an identical analysis of 60 osteosarcomas, the most common type of sarcoma lacking a specific translocation. Telomerase activity was demonstrated in 21 Ewing's sarcoma tumor samples (70%) and in 9 of 10 Ewing's sarcoma cell lines. Evidence of ALT, indicated by the presence of long and heterogeneous telomeres, was observed only in the cell line without telomerase activity and in none of the 30 Ewing's sarcoma tumor samples. The 9 Ewing's sarcoma patients whose tumors lacked detectable telomerase activity did not differ significantly from the remaining patients in age, stage, EWSR1-FLI1 fusion type, prevalence of TP53 point mutations, or overall survival. The low prevalence of ALT in Ewing's sarcoma contrasted sharply with our data on TMMs in 60 osteosarcomas, which showed ALT in 38 of 60 cases (P

Osteosarcoma (OS) is a primary malignancy of bone with a tendency to metastasize early. Despite intensive chemotherapy and surgical resection, ∼30% of patients still develop distant metastasis. Our previous work using clinical OS samples suggested that expression of the Wnt receptor LRP5 might be associated with tumor metastasis. In the present study, we used a Dickkopf (Dkk) family member and a dominant-negative LRP5 receptor construct to modulate Wnt signaling in OS cells. Saos-2 cells, which ectopically express Dkk-3, do not undergo apoptosis and exhibit enhanced resistance to serum starvation and chemotherapy-induced cytotoxicity. Transfection of Dkk-3 and dominant-negative LRP5 into Saos-2 cells significantly reduces invasion capacity and cell motility. This blockade is associated with changes in cell morphology consistent with a less invasive phenotype. In addition, Dkk-3 and dominant-negative LRP5 also induce changes in β-catenin localization consistent with an increase in cell-cell adhesion. Taken together, these results support a possible role for Wnt signaling in the pathobiology and progression of human OS.

Purpose: To determine the preliminary results of intensity-modulated radiotherapy (IMRT) for head-and-neck rhabdomyosarcoma. Methods and Materials: Twenty-eight patients underwent IMRT as a part of multimodality therapy. Twenty-one tumors were parameningeal, three were orbital, and four were in other sites. The median age was 8 years (range, 1–29 years). Most (89%) had Group III disease. Intracranial extension was present in 71% of parameningeal tumors. A 1.5-cm margin was used, and the median dose was 50.4 Gy (range, 30–55.8 Gy). Results: The actuarial 3-year survival rate for patients with parameningeal tumors was 65%. The 3-year actuarial freedom from failure rate was 95% locally, 90% in regional nodes, 88% in the central nervous system, and 80% at distant sites. No failures occurred among patients with orbit tumors; a single central nervous system failure occurred in 1 patient with a lip/cheek tumor. Disease-free survival was significantly worse for patients with alveolar histologic features ( p = 0.01). Acute radiation toxicity was similar to that reported by the Intergroup Rhabdomyosarcoma Study Group. Late radiation toxicity was recorded and was mild. Conclusion: IMRT with image fusion results in outstanding local control despite the use of a reduced margin. However, survival among patients with alveolar histologic findings or intracranial extension remains unacceptably low.

Background With the introduction of intensive high-dose chemotherapy for the treatment of osteosarcoma, changes in the pattern of metastases observed at relapse have been reported. To further investigate this hypothesis, the relapse patterns among groups receiving chemotherapy regimens of variable intensity at Memorial Sloan-Kettering Cancer Center were analyzed. Procedure All patients treated with the protocols T4, T5, T7, T10, and T12 were included. Patients were divided into two groups, one including those patients treated with less intense therapy (T4, T5, and T7) and the other, those treated with current regimens (T10 and T12). Results Of the 25 patients who relapsed on the earlier protocols, 76% relapsed to the lungs, 8% had local recurrences, and 16% distant metastases to the bone. The median time to first relapse was 12 months. Of the 69 patients who relapsed on the T10 and T12 protocols, 75% relapsed in the lungs, 9% had local recurrences, and 16% distant bone metastases. The median time to first relapse was 17 months. There was no statistically significant difference in the timing of relapse between the two groups studied, although a longer median time to relapse was observed for patients treated on the later protocols. The range of time to relapse was also wider in the later protocols. Conclusions These data do not support the hypothesis that patterns of relapse are changing with alterations in osteosarcoma treatment. This limited single institutional experience can be explored further in the context of a multi-institutional effort. © 2003 Wiley-Liss, Inc.

A rapid method for identifying filamentous actinomycete genera was developed based on 16S rRNA gene restriction fragment patterns. The patterns were generated by using specific restriction endonucleases to perform in silico digestions on the 16S rRNA gene sequences of all validly published filamentous actinomycete species. The method was applied to identifying actinomycete isolates from soil. Amplified 16S rDNA of soil actinomycetes was restricted with selected endonucleases and electrophoresed on agarose gels. The restriction fragment patterns of the unknown isolates were easily compared to the established patterns. Significantly, the genus Streptomyces could be differentiated from all other actinomycete genera by using only four restriction endonucleases, Sau3AI, AsnI, KpnI and SphI. This could be achieved in a time period of as little as a week, following PCR-template DNA isolation by a simple method. The identification method allowed unknown, non-Streptomyces soil isolates to be identified to a genus or small subgroup of genera. The genera in these subgroups could, in some cases, be distinguished by virtue of colony-morphology differences.

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.

Osteosarcoma (OS) is a highly malignant bone neoplasm of children and young adults. It is characterized by chaotic karyotypes with complex marker chromosomes. We applied a combination of molecular cytogenetic techniques including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH) to decipher the chromosomal complexity in a panel of 25 tumors. Combined SKY and G-banding analysis identified several novel recurrent breakpoint clusters and 9 nonrecurrent reciprocal translocations. CGH identified several recurrent chromosomal losses including 2q, 3p, 9, 10p, 12q, 13q, 14q, 15q, 16, 17p, and 18q, gains including Xp, Xq, 5q, 6p, 8q, 17p, and 20q, and high-level chromosomal amplifications at Xp11.2, 1q21-q22, 4p11, 4q12, 5p15, 6p12.1, 8q13, 8q23, 10q11, 10q22, 11q13, 11q23, 12q13-q14, 13q21-q34, 16q22, 17p11.2, 17q21-q22, 18q22, 20p11.2, and 20q12. Frequent amplification and rearrangement involving chromosomal bands at 6p12-p21 and 17p11.2 were found in 28% and 32% of cases, respectively. In an attempt to identify the genes involved in these amplicons, we used three nonoverlapping BAC clones contained within each amplicon as probes for FISH analysis, leading to a more detailed characterization and quantification of the 6p and 17p amplicons.

The cyanide dihydratase in Bacillus pumilus was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in Escherichia coli . Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has K m and V max values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative Pseudomonas stutzeri strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of B . pumilus (strain 8A3) was cloned and expressed. This suggests that cynD , the gene coding for the cyanide dihydratase, is not unique to the C1 strain of B . pumilus and is not a reflection of its origin at a mining waste site.

Dynamic contrast material-enhanced magnetic resonance (MR) images of primary osteogenic sarcoma (n = 19) and Ewing sarcoma (n = 10) were reviewed in 29 patients undergoing induction chemotherapy before surgery. Histogram distributions containing the initial slope and pharmacokinetic model parameters from individual voxels within each tumor were fitted for each patient. The histogram analysis of initial slope from the tumor correlated well with percentage necrosis as determined at pathologic examination (r = 0.60, P.001), as did a two-compartment pharmacokinetic model (r = 0.64, P.001). Both methods predicted tumors with clinically important degrees of necrosis (ie,or =90%) in a large majority of cases. The ability to determine response to induction chemotherapy by means of noninvasive monitoring of necrotic fraction with perfusion MR imaging methods may provide useful prognostic information and help surgical planning.

Previous studies have shown that decreased expression of the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR) are associated with intrinsic and acquired methotrexate resistance, respectively, in osteosarcoma (OS). It has also been shown in colorectal cancer that E2F-1 expression correlates with thymidylate synthase (TS) and, to a lesser extent, DHFR expression. To begin to investigate the regulation of DHFR and RFC expression in OS samples, mRNA expression of E2F-1 and E2F-4 were measured in OS tumor samples and related to DHFR, RFC, and TS mRNA expression. Using fluorescent quantitative real-time PCR, 112 human OS patient samples were investigated for potential E2F-1/E2F-4:DHFR, E2F-1/E2F-4:RFC, and E2F-1/E2F-4:TS correlations. The expression ranges for each gene are as follows: DHFR, 0.02-33.13 (median = 0.20); RFC, 0.02-229.13 (median = 1.91); TS, 0.01-9.99 (median = 0.15); E2F-1, 0.05-69.07 (median = 0.52); and E2F-4, 0.24-52.35 (median = 1.45). Spearman correlation coefficients (r(s)) for E2F-1:DHFR, E2F-1:RFC, E2F-1:TS, E2F-4:DHFR, E2F-4:RFC, and E2F-4:TS were calculated to be 0.53, 0.63, 0.60, 0.41, 0.58, and 0.33, respectively (P0.001). On the basis of this data, moderate correlations exist between E2F-1/E2F-4 and DHFR, RFC, and TS. These results suggest E2F-1/E2F-4 may play a role in the regulation of RFC expression, which has not been reported previously. The E2F transcription factors are also related to DHFR and TS expression in OS samples, suggesting a possible involvement in methotrexate resistance. Although E2F mRNA levels correlate with DHFR, RFC, and TS mRNA expression, additional experiments are necessary to determine the direct effects of these transcription factors and identify other proteins that may influence this relationship.