MDM2 gene amplification in metastatic osteosarcoma.

The human homologue of the murine double minute 2 gene (MDM2), a p53-binding protein which may act as a regulator of p53 protein function, has recently been cloned. Initial studies of this gene in a variety of human tumors have shown frequent gene amplification in most types of sarcomas, including osteosarcomas. Amplification of the MDM2 gene may produce a functional inactivation of the p53 protein. To examine possible clinical or pathological correlates of MDM2 gene amplification in osteosarcoma, we studied 28 specimens on 26 patients with high grade osteosarcoma (16 primary, 11 metastatic, and 1 local recurrence) for MDM2 gene amplification by Southern blot analysis, using two MDM2 complementary DNA probes isolated by polymerase chain reaction. Four specimens (14%) showed amplification, including 3 metastases and 1 local recurrence. None of the primary osteosarcoma specimens had detectable MDM2 gene amplification. None of the specimens tested showed MDM2 gene rearrangement. In the present series, MDM2 gene amplification was detected significantly more frequently in metastatic or recurrent osteosarcomas than it was in primary osteosarcomas (P = 0.02). Our data suggest that MDM2 gene amplification may be associated with tumor progression and metastasis in osteosarcoma. Further investigation is warranted on the potential clinicopathological correlates of MDM2 gene amplification in osteosarcoma.

Differential transactivation by alternative EWS-FLI1 fusion proteins correlates with clinical heterogeneity in Ewing's sarcoma.

The t(11;22)(q24;q12) translocation is present in up to 95% of cases of Ewing's sarcoma and results in the formation of an EWS-FLI1 fusion gene which encodes a chimeric transcription factor. The proximate role of EWS-FLI1 in the pathogenesis of Ewing's sarcoma is thought to involve the activation of as yet largely unknown target genes. Many alternative forms of EWS-FLI1 exist because of variations in the locations of the EWS and FLI1 genomic breakpoints. The most common form, designated "type 1," consists of the first seven exons of EWS joined to exons 6-9 of FLI1 and accounts for approximately 60% of cases. The "type 2" EWS-FLI1 fusion also includes FLI1 exon 5 and is present in another 25%. We and others have observed previously that the type 1 fusion is associated with a significantly better prognosis than the other fusion types. Because EWS-FLI1 is an aberrant transcription factor, we investigated whether these differences in clinical behavior may be correlated to functional differences by comparing transactivation by the type 1 EWS-FLI1 with other types in both heterologous cells (HeLa, NIH3T3) and homologous cells (Ewing's sarcoma cell lines). In a panel of seven Ewing's sarcoma cell lines, we found transactivation of a transiently transfected FLI1-responsive reporter construct to be significantly lower in cell lines with the type 1 fusion than in cell lines with the type 2 fusion (P = 0.003). Cotransfection of the same reporter construct with each of a series of seven EWS-FLI1 expression constructs (corresponding to the two major fusion types and five less common types) also showed that type 1 EWS-FLI1 was a significantly weaker transactivator than the type 2 product in both HeLa and NIH3T3 cells (P = 0.003, and P = 0.033, respectively). Electromobility shift assays showed equivalent binding of the type 1 and type 2 EWS-FLI1 to the consensus FLI1-responsive binding site, indicating that differences in transactivation were not due simply to differences in DNA binding affinity. The finding that the type 1 EWS-FLI1 fusion, associated with less aggressive clinical behavior, encodes a less active chimeric transcription factor may provide the basis for a molecular explanation of clinical heterogeneity in Ewing's sarcoma.

Regulation and heteromeric structure of the fibroblast activation protein in normal and transformed cells of mesenchymal and neuroectodermal origin.

The human fibroblast activation protein (FAP), defined by monoclonal antibody F19, is expressed in vivo in reactive stromal fibroblasts of epithelial cancers, subsets of bone and soft tissue sarcomas, and granulation tissue of healing wounds. FAP is generally absent from the stroma of benign epithelial tumors and normal adult tissues. In vitro FAP induction is observed in proliferating cultured fibroblasts and in melanocytes grown with fibroblast growth factor and phorbol ester. In the present study, we show that fibroblast and melanocyte FAP is a cell surface protein comprising noncovalently linked M(r) 95,000 (p95) and M(r) 105,000 (p105) subunits. In contrast, cultured sarcoma and melanoma cell lines express only p95 or are FAP negative. Immunoblot experiments show that p95, but not p105, carries the epitope defined by monoclonal antibody F19. Furthermore, peptide maps of purified p95 and p105 differ, suggesting that they may be distinct gene products. Loss of FAP or a change from p95/p105 to p95 expression accompanies the acquisition of growth factor independence and tumorigenicity in several in vitro test systems, including simian virus 40 transformation of normal fibroblasts, Ha-ras transformation of normal melanocytes, supertransformation of osteosarcoma cells, and enhanced N-MYC expression in variant neuroblastoma cells, whereas serum-starved normal fibroblasts continue to express p95/p105. Thus, fAP expression appears to be linked to the growth factor-dependent proliferative capacity of normal cells and is not merely a secondary event in proliferating cells; furthermore, FAP expression is inversely correlated with growth factor independence and tumorigenicity in transformed cell lines. This distribution pattern is consistent with a role for p95/p105 in mediating extrinsic, growth regulatory signals in normal cells, possibly as a heteromeric cell surface receptor. Such a physiological function may be obviated when oncogenes with cytoplasmic or nuclear sites of action are activated, reducing extrinsic growth factor dependence and permitting down-regulation of FAP in certain transformed cells.

The der(17)t(X;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25.

Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.

Intensification of preoperative chemotherapy for osteogenic sarcoma: results of the Memorial Sloan-Kettering (T12) protocol.

PURPOSE: It has been observed previously in osteosarcoma (OS) that the degree of necrosis of the resected primary tumor following a period of preoperative chemotherapy is predictive of subsequent event-free survival (EFS). The aim of this study was to determine if more intensive preoperative chemotherapy would increase the proportion of patients with a good histologic response and improve EFS. PATIENTS AND METHODS: Seventy-three patients with OS were treated at Memorial-Sloan Kettering Cancer Center (MSKCC) on the T12 protocol between 1986 and 1993. Patients were randomized between therapy based on the T10 protocol and therapy with more intensive preoperative chemotherapy. The more intensive preoperative regimen consisted of two courses of cisplatin (CDDP) and doxorubicin (DOX) in addition to the usual preoperative regimen of high-dose methotrexate (HD MTX) and bleomycin, cyclophosphamide, and dactinomycin (BCD). RESULTS: The regimen with more intensive preoperative chemotherapy achieved a modest increase in the proportion of patients with a good histologic response (44% with a grade III or IV histologic response v 37% in the control arm, 33% with grade IV histologic response v 13% in the control arm). EFS continued to correlate with histologic response. The actuarial 5-year EFS in patients with localized disease was 78% for the regimen with more intensive preoperative chemotherapy and 73% for the control arm. CONCLUSION: Despite modest increases in the proportion of patients with good histologic response with intensified preoperative chemotherapy, no improvement in EFS was observed.

Osteogenic sarcoma with clinically detectable metastasis at initial presentation.

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.

Synovial sarcoma: a multivariate analysis of prognostic factors in 112 patients with primary localized tumors of the extremity.

PURPOSE: Synovial sarcoma is a high-grade tumor that is associated with poor prognosis. Previous studies analyzing prognostic factors are limited because of inclusion of heterogeneous cohorts of patients with nonextremity and recurrent tumors. The objective of this study was to determine independent prognostic factors of primary synovial sarcoma localized to the extremity. PATIENTS AND METHODS: Between July 1, 1982, and June 30, 1996, 112 patients underwent surgical resection for cure at our institution and then were followed-up prospectively. Clinical and pathologic factors examined for prognostic value included age, sex, tumor site and location, depth, size, microscopic status of surgical margins, invasion of bone or neurovascular structures, and monophasic or biphasic histology. The end points analyzed were the time to first local recurrence that was not preceded by a distant recurrence, time to any distant recurrence, and time to disease-related mortality. These end points were modeled using the method of Kaplan and Meier and analyzed by the log-rank test and Cox regression. RESULTS: The median duration of follow-up among survivors in this cohort of 112 patients was 72 months. The 5-year local-recurrence, distant-recurrence, and mortality rates were 12%, 39%, and 25%, respectively. Tumor size > or = 5 cm (P =.001; relative risk [RR] = 2. 7; 95% confidence interval [CI], 1.5 to 5.2) and the presence of bone or neurovascular invasion (P =.04; RR = 2.3; 95% CI, 1.0 to 5. 3) were independent adverse predictors of distant recurrence. Tumor size > or= 5 cm (P =.003; RR = 2.3; 95% CI, 1.4 to 6.3) and the presence of bone or neurovascular invasion (P =.03; RR = 2.7; 95% CI, 1.0 to 6.5) were also independent adverse predictors of mortality. CONCLUSION: The natural history of primary synovial sarcoma of the extremity is related to tumor size and invasion of bone and neurovascular structures.

Very-high-dose short-term chemotherapy for poor-risk peripheral primitive neuroectodermal tumors, including Ewing's sarcoma, in children and young adults.

PURPOSE: To improve the prognosis of patients with poor-risk peripheral primitive neuroectodermal tumors (pPNETs; including peripheral neuroepithelioma and Ewing's sarcoma), while testing the feasibility of intensive use in adolescents and young adults of high-dose cyclophosphamide, doxorubicin, and vincristine (HD-CAV). PATIENTS AND METHODS: This report concerns previously untreated patients with newly diagnosed pPNET deemed poor-risk because of a tumor volume more than 100 cm3 or metastases to bone or bone marrow. The P6 protocol consists of seven courses of chemotherapy. Courses 1, 2, 3, and 6 include 6-hour infusions of cyclophosphamide on days 1 and 2 for a total of 4,200 mg/m2 per course (140 mg/kg per course for patients < 10 years old), plus 72-hour infusions of doxorubicin 75 mg/m2 and vincristine 2.0 mg/m2 beginning on day 1 (HD-CAV). Courses 4, 5, and 7 consist of 1-hour infusions of ifosfamide 1.8 g/m2/d and etoposide (VP-16) 100 mg/m2/d, for 5 days. Granulocyte colony-stimulating factor (G-CSF) and mesna are used. Courses start after neutrophil counts reach 500/microL and platelet counts reach 100,000/uL. Surgical resection follows course 3 and radiotherapy follows completion of all chemotherapy. RESULTS: Among the first 36 consecutive assessable patients (median age, 17 years), HD-CAV achieved excellent histopathologic or clinical responses in 34 patients and partial responses (PRs) in two patients. For 24 patients with locoregional disease, the 2-year event-free survival rate was 77%; adverse events were two locoregional relapses, one distant relapse, and one secondary leukemia. All six patients with metastatic disease limited to lungs achieved a complete response (CR) and did not relapse; one is in remission 36+ months from diagnosis, but the other patients are not assessable in terms of long-term efficacy of the P6 protocol because of short follow-up time (n = 3), additional systemic therapy (bone marrow transplantation), or septic death (autopsy showed no residual pPNET). All six patients with widespread metastases had major responses, including eradication of extensive bone marrow involvement, but distant relapses ensued. Myelosuppression was severe, but most patients received the first three courses of HD-CAV within 6 to 7 weeks. Major nonhematologic toxicities were mucositis and peripheral neuropathy. CONCLUSION: Excellent antitumor efficacy and manageable toxicity support the dose-intensive use of HD-CAV for pPNET in children, as well as in young adults. Consolidation of remissions of pPNET metastatic to bone and bone marrow remains a therapeutic challenge.

Peripheral primitive neuroectodermal tumors in adults: documentation by molecular analysis.

PURPOSE: The Ewing tumor (ET) family of peripheral primitive neuroectodermal tumors (pPNETs) are primitive small round-cell tumors (SRCTs) of the bone and soft tissue that occur predominantly in children and adolescents. However, pPNETs only rarely enter the differential diagnosis of bone and soft tissue SRCTs in adults. Recently, gene fusions between the EWS gene and different members of the ETS transcription factor family have been shown to occur in virtually all pPNETs and thus constitute a pathognomonic marker for this tumor subclass. The aim of the present study was to document EWS/ETS fusion gene expression in suspected pPNETs of adults as objective evidence for the existence of this tumor family in older patients. PATIENTS AND METHODS: The three contributing molecular diagnostic laboratories retrospectively compiled a cohort of all SRCT cases in which EWS/ETS gene fusions had been shown by molecular analysis. This cohort was surveyed for cases that occurred in patients aged 40 years or older, which were then analyzed for their clinical and pathologic features. RESULTS: Nine patients between 40 and 65 years of age were found to have tumors positive for EWS/ETS gene fusions. Standard histopathologic and clinical features of these cases, other than age, were similar to those of childhood pPNETs. Patients were initiated on appropriate therapy after molecular analysis confirmed the diagnosis of pPNET. CONCLUSION: Identification of an EWS/ETS gene fusion is useful in providing objective evidence of the diagnosis of pPNET in patients over the age of 40 years. This diagnosis should be considered in adults who present with bone and soft tissue SRCTs and appropriate biopsy specimens should be collected for molecular analysis at the time of diagnosis.

Expression of HER2/erbB-2 correlates with survival in osteosarcoma.

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.

MDR1 gene expression and outcome in osteosarcoma: a prospective, multicenter study.

PURPOSE: Increased expression of the multidrug resistance gene (MDR1) has been implicated in osteosarcoma prognosis. This study represents the first prospective assessment of the prognostic value of MDR1 mRNA expression in patients with newly diagnosed extremity osteosarcoma. PATIENTS AND METHODS: A series of patients with high-grade, nonmetastatic extremity osteosarcoma were enrolled from six tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 30 months). All patients were treated with (neo)adjuvant chemotherapy and surgery. Tumors from 123 patients were analyzed for MDR1 mRNA expression. The association of the level of MDR1 expression with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors. RESULTS: Using the highest MDR1 value for each patient, a dose-response relationship was not identified between the level of MDR1 expression and systemic relapse (relative risk, 1.15; P =.44). Analyses based on biopsy or resection values alone gave similar results (P =.11 and.41, respectively, log rank test). In multivariate analysis, large tumor size (> 9 cm) was the only significant independent predictor of systemic outcome (relative risk, 2.8; P =.002). CONCLUSION: We did not identify any correlation between MDR1 mRNA expression and disease progression in patients with osteosarcoma. It is likely that alterations in other genes are involved in resistance to chemotherapy in osteosarcoma and that they play a more critical role than MDR1 in this disease.

EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.

PURPOSE: More than 90% of Ewing's sarcomas (ES) contain a fusion of the EWS and FLI1 genes, due to the t(11;22)(q24;q12) translocation. At the molecular level, the EWS-FLI1 rearrangements show great diversity. Specifically, many different combinations of exons from EWS and FLI1 encode in-frame fusion transcripts and result in differences in the length and composition of the chimeric protein, which functions as an oncogenic aberrant transcription factor. In the most common fusion type (type 1), EWS exon 7 is linked in frame with exon 6 of FLI1. As the fundamental pathogenetic lesion in ES, the molecular heterogeneity of these fusion transcripts may have functional and clinical significance. PATIENTS AND METHODS: We performed a clinical and pathologic analysis of 112 patients with ES in which EWS-FLI1 fusion transcripts were identified by reverse-transcriptase polymerase chain reaction (RT-PCR). Adequate treatment and follow-up data were available in 99 patients treated with curative intent. Median follow-up in these 99 patients was 26 months (range, 1 to 140 months). Univariate and multivariate survival analyses were performed that included other prognostic factors, such as age, tumor location, size, and stage. RESULTS: Among the 99 patients suitable for survival analysis, the tumors in 64 patients contained the type 1 fusion and in 35 patients contained less common fusion types. Stage at presentation was localized in 74 patients and metastatic in 25. Metastases (relative risk [RR] = 2.6; P = .008), and type 1 EWS-FLI1 fusion (RR = 0.37; P = .014) were, respectively, independent negative and positive prognostic factors for overall survival by multivariate analysis. Among 74 patients with localized tumors, the type 1 EWS-FLI1 fusion was also a significant positive predictor of overall survival (RR = 0.32; P = .034) by multivariate analysis. CONCLUSION: EWS-FLI1 fusion type appears to be prognostically relevant in ES, independent of tumor site, stage, and size. Further studies are needed to clarify the biologic basis of this phenomenon.

Monoclonality of multifocal myxoid liposarcoma: confirmation by analysis of TLS-CHOP or EWS-CHOP rearrangements.

Multifocal presentation, defined as the presence of tumor at two or more anatomically separate sites, before the manifestation of disease in sites where sarcomas usually metastasize (e.g., lungs) occurs in about 1% of extremity soft tissue sarcomas (STSs). Debate still persists whether multifocal STSs represent an unusual pattern of metastasis or multiple separate primary tumors. Among STSs with multifocal presentation, myxoid liposarcoma is the predominant histological type. This subtype of liposarcoma contains the specific t(12;16) chromosomal translocation, which results in rearrangement of the TLS and CHOP genes that is clone specific at the DNA level. We, therefore, sought to address the question of clonality by molecular analysis in six patients who presented with either synchronous or metachronous multifocal myxoid liposarcoma. In all six cases, adequate frozen tumor was available for DNA extraction from at least two distinct anatomical sites. Southern blot analysis using CHOP, TLS, and EWS cDNA probes was performed on genomic DNA. Five cases contained a TLS-CHOP rearrangement, and one case had the variant EWS-CHOP fusion (seen in <5% of cases). The size of the rearranged CHOP fragment differed among the six patients, as expected, but was identical in all anatomically separate tumor samples from each patient. Likewise, the sizes of the rearranged bands observed with either the TLS or EWS probes supported the monoclonality of all cases. Our results confirm the monoclonal origin of multifocal myxoid liposarcoma, establishing the metastatic nature of distant soft tissue lesions in these cases. It remains unclear whether this unusual pattern of metastasis represents an intrinsic property of this subset of myxoid liposarcoma or merely a rare chance occurrence. The clinical outcomes observed in this small series suggest that the prognosis of multifocal myxoid liposarcoma is poor, regardless of its often bland or "low-grade" histological appearance.

Mechanisms of methotrexate resistance in osteosarcoma.

High-dose methotrexate is a major component of current protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resistance include decreased transport through the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantitative reverse transcription-PCR. The RFC and DHFR genes were studied for deletions and amplification by Southern blot. Thirteen of 20 (65%) osteosarcoma samples were found to have decreased RFC expression at the time of initial biopsy. At definitive surgery and relapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (P = 0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at initial biopsy. The frequency of increased DHFR expression was significantly higher in metastatic or recurrent tumors (62%, P = 0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy material suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions.

Lack of correlation of functional scintigraphy with (99m)technetium-methoxyisobutylisonitrile with histological necrosis following induction chemotherapy or measures of P-glycoprotein expression in high-grade osteosarcoma.

In osteosarcoma, some studies have suggested P-glycoprotein expression is a prognostic factor. The clearance of (99m)technetium hexakis-2-methoxyisobutylisonitrile ((99m)Tc-MIBI) has been used in some tumor systems as an in vivo measure of P-glycoprotein-mediated efflux. In this study we explored the correlation between (99m)Tc-MIBI clearance and histological necrosis following induction chemotherapy and P-glycoprotein expression in osteosarcoma. The primary tumors of 20 patients with high-grade osteosarcoma were imaged at diagnosis with (99m)Tc-MIBI, and the uptake ratios and biological half-lives were calculated. P-Glycoprotein expression in the tumor tissue was determined immunohistochemically and by measuring mRNA expression of the multidrug resistance-1 gene. The histological necrosis following induction chemotherapy was assessed by the Huvos grading system. The biological half-life of (99m)Tc-MIBI ranged from 1.4 to 52.5 h. Seven of the 20 tumor samples had a favorable extent of necrosis following induction chemotherapy. The (99m)Tc-MIBI half-life and uptake ratio showed no correlation with histological necrosis following induction chemotherapy. The (99m)Tc-MIBI half-life and uptake ratio did not correlate with either measure of P-glycoprotein expression. The results of this pilot study indicate that (99m)Tc-MIBI imaging is not an effective predictor of histological necrosis following induction chemotherapy in high-grade osteosarcoma. (99m)Tc-MIBI imaging did not correlate with measures of P-glycoprotein expression in the tumor tissue.

Acetabular development after bipolar hemiarthroplasty for osteosarcoma in children.

A retrospective analysis was performed of eight patients with an open triradiate cartilage, who underwent resection for osteosarcoma and reconstruction of the proximal femur with a hemiarthroplasty, in order to identify changes of acetabular development. An analysis of the centre-edge angle, teardrop-to-medial prosthesis distance, superior joint space, teardrop-to-superior prosthesis distance, degree of lateral translation, and arthritic changes, was performed on serial radiographs. The median age at the time of the initial surgery was 11 years (5 to 14). All patients developed progressive superior and lateral migration of the prosthetic femoral head. Following hemiarthroplasty in the immature acetabulum, the normal deepening and enlargement of the acetabulum is arrested. The degree of superior and lateral migration of the prosthetic head depends on the age at diagnosis and the length of follow-up.

Orthopedic emergencies in cancer patients.

The most frequent orthopedic emergency in oncology patients is fracture. Stabilization of the entire fractured bone restores function and relieves pain. The site, quality, and extent of the lesion can identify impending fractures that should be stabilized. New methods of pelvic stabilization effectively bypass periacetabular bone deficiency. Spinal cord decompression is important to maintain neurologic function. Advances in segmental fixation of the spine have improved the outcome over what was achieved by radiation alone. Infection is common in neutropenic patients, and should be treated aggressively with antibiotics and drainage of abscesses of the musculoskeletal system. Extravasation of doxorubicin requires prompt local debridement to limit the extent of necrosis propagation. These treatments can effectively improve the quality of life of patients with metastatic cancer. They should be included as "best supportive care" for patients with more than 1 month to live.

Specificity of TLS-CHOP rearrangement for classic myxoid/round cell liposarcoma: absence in predominantly myxoid well-differentiated liposarcomas.

Myxoid liposarcoma (LS), the most common subtype of LS, is known to be characterized by the specific t(12;16) resulting in a TLS-CHOP fusion in almost all cases. We wished to address the following questions: (i) Is this genetic hallmark also present in other types of LS with predominant myxoid change? (ii) What is the proportion of cases with the variant EWS-CHOP fusion? (iii) What is the optimal approach for Southern blot detection of TLS breakpoints? We identified 59 LS characterized histologically by >90% myxoid component, in which frozen tissue tumor was available for DNA extraction. These 59 LS with myxoid features were divided into 2 groups: 42 LS with classic myxoid/round cell appearance (myxoid LS) and 17 well-differentiated LS (WDLS) with a predominant (>90%) myxoid component. Within the myxoid LS group, 29 tumors were low grade and 13 high grade (>20% round cell component). Among the 17 predominantly myxoid WDLS, there were 15 low grade and 2 focally high grade tumors. In addition, we selected as control group, 20 LS of other histological types with minimal or no myxoid change (17 WDLS and 3 pleomorphic LS) and 13 myxofibrosarcomas. Southern blot analysis was performed in all cases using a CHOP cDNA probe, and in all CHOP rearranged cases using a TLS cDNA probe. Probe/enzyme combinations for Southern blot analysis were CHOP exon 3-4 cDNA probe with BamHI or SacI, TLS exon 3-6 cDNA probe with BclI. All 42 cases of myxoid LS showed a CHOP rearrangement and 38 of them also had a TLS rearrangement. Among the 4 myxoid LS without Southern blot evidence of TLS rearrangement, 1 showed an EWS-CHOP fusion by Southern blotting and reverse transcriptase-polymerase chain reaction and in another case, reverse transcriptase-polymerase chain reaction detected a TLS-CHOP fusion transcript. None of the predominantly myxoid WDLS and none of the tumors included in the control group showed rearranegements with CHOP probe. In addition, 12 predominantly myxoid WDLS, 10 other LS, and 5 myxofibrosarcoma from the control group were also tested for TLS rearrangement; all were negative. The TLS-CHOP fusion is highly sensitive and specific for the entity of classic myxoid/round cell LS. Other types of LS, even with a predominant myxoid component, lack the TLS-CHOP rearrangement, confirming that they represent a genetically distinct group of LS. The prevalence of the EWS-CHOP variant fusion was approximately 2% in this series. The optimal enzyme for TLS genomic breakpoint detection is BclI.

Ewing sarcoma of the rib: results of an intergroup study with analysis of outcome by timing of resection.

OBJECTIVE: We sought to establish the outcome and optimal therapeutic sequence for patients with nonmetastatic Ewing sarcoma/primitive neuroectodermal tumor of the chest wall. METHODS: Patients 30 years of age or younger with nonmetastatic Ewing sarcoma/primitive neuroectodermal tumor of the bone were randomly assigned to receive vincristine, doxorubicin, cyclophosphamide, and dactinomycin or those drugs alternating with ifosfamide and etoposide. Local control was obtained with an operation, radiotherapy, or both. RESULTS: Fifty-three (13.4%) of 393 patients had primary tumors of the chest wall (all rib). Event-free survival at 5 years was 57% for the chest wall compared with 61% for other sites (P >.2). Ifosfamide and etoposide improved outcome in the overall group (5-year event-free survival, 68% vs 54%; P =.002), and a similar trend occurred in chest wall lesions (5-year event-free survival, 64% vs 51%). Patients with chest wall lesions had more attempts at initial surgical resection (30%) than those with other primary tumor sites (8%, P <.01). The attempt at initial resection for chest wall lesions did not correlate with size. Initial resections at other sites were restricted to smaller tumors. Initial resection resulted in negative pathologic margins in 6 of 16 patients, whereas the delayed resection resulted in negative margins in 17 of 24 patients (P =.05). Although there was no difference in survival by timing of the operation in rib lesions, a higher percentage of patients with initial surgical resection received radiation than those with resection after initial chemotherapy (P =. 13). CONCLUSIONS: Although rib primary tumors are significantly larger than tumors found in other sites, their outcome is similar. We favor delayed resection whenever possible to minimize the number of patients requiring radiation therapy.

p53 mutations in chondrosarcoma.

Chondrosarcoma is a primary bone tumor that has several different grades and variants. We evaluated 48 chondrosarcomas for p53 overexpression and p53 mutations. p53 expression was evaluated with immunohistochemistry using monoclonal antibodies PAb421, PAb1801, and PAb240. p53 mutations were identified with single-strand conformational polymorphism (SSCP) and DNA sequencing in selected cases. Immunohistochemistry revealed nuclear staining with PAb421 and PAb1801 in the spindle cell portion of one dedifferentiated chondrosarcoma. SSCP analysis was abnormal only in the case with positive immunostaining and localized the mutation to exons 7 and 8. DNA sequence analysis identified a point mutation of G to C in codon 276, resulting in an amino acid substitution of proline for alanine. This point mutation has been reported previously in other tumors but not in chondrosarcoma. Assimilation of our results with previous studies suggests that p53 mutations are present in a minority of chondrosarcomas but when present, are in higher grade chondrosarcomas and their variants.