Josephine Issakov

Prediction of high risk Ewing's sarcoma by gene expression profiling

Ewings sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. Currently accepted clinical prognostic factors fail to classify ES patients risk to relapse at diagnosis. We aimed to find a new strategy to distinguish between poor and good prognosis ES patients already at diagnosis. We analysed the gene expression profiles of 14 primary tumor specimens and six metastases from ES patients, using oligonucleotide microarray analysis. The over-expression of two genes was validated by quantitative PCR using the LightCycler system. We identified two distinct gene expression signatures distinguishing high-risk ES patients that are likely to progress from low-risk ES patients with a favorable prognosis of long-term progression-free survival. The microarray-based classification was superior to currently used prognostic parameters. Over-expressed genes in the poor prognosis patients included genes regulating the cell cycle and genes associated with invasion and metastasis, while among the downregulated genes were tumor suppressor genes and inducers of apoptosis. Our results indicate the existence of a specific gene expression signature of outcome in ES already at diagnosis, and provide a strategy to select patients who would benefit from risk-adapted improved therapy.

Synovial sarcoma of bone delineated by spectral karyotyping

Representative karyogram of a metaphase after spectral karyotyping (SKY) after hybridisation with a 24-chromosome-specific DNA probe cocktail The chromosomes are visualised in pseudocolours according to their spectrum signatures. Note presence of t(X;18) (arrows) and der (1)t(1;22) (arrow head). nucleoli. There was a lobular growth pattern and evidence of a few Homer-Wright rosettes with a fibrillary intercellular background. Spindle cells were noted in many areas. Immunohistochemical staining was positive for MIC-2 neuron-specific enolase, keratin, synaptophysin and vimentin, and focally positive for S-100. Cells were negative for smooth muscle actin, muscle actin (HHF-35), desmin, LCA, EMA, Leu-7, and PAS. Electron microscopy showed no glycogen particles and sheets of fusiform cells with scanty cytoplasm, rudimentary cell junctions and cytoplasmic (neuritic) processes that contained microtubules and dense neurosecretory-type core granules of average diameter 100 nm. Findings were consistent with a primitive neuroectodermal variant of Ewing sarcoma. Conventional chromosome analysis showed a karyotype of 45 chromosomes with an unbalanced translocation from chromosome 22 to the short arm of chromosome 1 (confirmed by FISH) and unidentified chromosomal material on the short arm of chromosome X. SKY on 15 metaphase spreads unequivocally confirmed the presence of der (1)t(1;22) and the unidentified extra material added to the short arm of chromosome X was a balanced translocation t(X;18)(p;q11), typical of synovial sarcoma (figure). The karyotype was defined as 45, Y, t(X;18) (p11;q11), der (1)t(1;22)(p13;q12?), –22. The 583 base pair SYT/SSX chimeric transcript resulting from the t(X;18)(p11.2;q11.2) was identified by molecular techniques, but the EWS-FL-1 and EWS-ERG transcripts of Ewing sarcoma were not. No evidence of the typical Ewing sarcoma translocations t(11;22), t(21;22), or t(7;22) was found. The t(X;18)(p11.2;q11.2) translocation disclosed by SKY and confirmed by molecular biology has only been found in synovial-cell sarcomas, a tumour that occurs primarily in para-articular regions, commonly in the area of the knee in young adults but never previously described as a bone tumour.

Short telomeres: a novel potential predictor of relapse in Ewing sarcoma.

Purpose: Despite advances in therapy, >50% of patients with Ewing sarcoma will relapse. The current prognostic factors are not optimal for risk prediction. Studies have shown that telomere length could predict outcome in different malignancies. Our aim was to evaluate whether telomere length could be a better prognostic factor in Ewing sarcoma and correlate the results with clinical variables, outcome, and chromosomal instability. Experimental Design: Telomere length was determined in the primary tumor and peripheral blood of 32 patients with Ewing sarcoma. Chromosomal instability was evaluated by combining classical cytogenetics, comparative genomic hybridization and random aneuploidy. Telomere length was correlated to clinical variables, chromosomal instability, and outcome. Results: In 75% of the tumors, changes in telomere length, when compared with the corresponding peripheral blood lymphocytes, were noted. The majority of changes consisted of a reduction in telomere length. Patients harboring shorter telomeres had a significantly adverse outcome (P = 0.015). Chromosomal instability was identified in 65% of tumors, significantly correlating with short telomeres (P = 0.0094). Using multivariate analysis, telomere length remained the only significant prognostic variable (P = 0.034). Patients with short telomeres had a 5.3-fold risk of relapse as compared to those with unchanged or longer telomeres. Conclusion: We have shown that tumors with telomere length reduction result in genomic instability. In addition, telomere length reduction was the only significant predictor of outcome. We suggest that reduction of telomere length in tumor cells at diagnosis could serve as a prognostic marker in Ewing sarcoma.

Primary chronic sclerosing (Garré's) osteomyelitis in children.

Three children with unifocal nonpyogenic inflammatory bony lesions with a prolonged, fluctuating course are reported. The lesions were located at the metaphyseal region of long bones. Three was progressive sclerosis and hyperostosis in the tibia or femur, such as the changes described in Garrés osteomyelitis. No pus was released by exploration of the lesions. Tissue and blood cultures were negative. The histology was typical of chronic osteomyelitis: the symptoms returned intermittently over several years, together with the development of sclerosis but without disturbance of bone growth. It is not clear whether Garrés chronic sclerosing osteomyelitis is a different entity from chronic recurrent multifocal osteomyelitis.

Excellent Prognosis in a Subset of Patients with Ewing Sarcoma Identified at Diagnosis by CD56 Using Flow Cytometry

Purpose: Ewing sarcoma (ES) is considered a systemic disease with the majority of patients harboring micrometastases at diagnosis. Multiparameter flow cytometry (MPFC) was used to detect ES cells in bone marrow (BM) of ES patients at diagnosis and to evaluate the prognostic significance of CD56 expression in BM samples. Experimental Design: BM samples from 46 ES patients, 6 tumor aspirates, 2 ES cell lines, and 10 control BM samples were analyzed by MPFC. ES cells were identified by the combination of CD45−/CD90+/CD99+. CD56 was evaluated on these cells by a cutoff of 22%. Results: BM samples obtained from all patients at diagnosis were found to be positive for micrometastatic tumor cells assessed by CD99+/CD90+/CD45− expression. A total of 60% of the BM samples harbored high CD56 expression. There was a highly significant correlation between CD56 expression and progression-free survival (PFS; 69% in low/negative expression versus 30% in high expression groups, P = 0.024). In patients with localized nonpelvic disease, those expressing low/negative CD56 had 100% PFS versus 40% in the high expressing group (P = 0.02). By Cox regression analysis, CD56 was found to be an independent prognostic marker with an 11-fold increased risk for relapse in patients with localized disease (P = 0.006). Conclusion: All samples contained cells that are positive for the CD99+/CD90+/CD45− combination at diagnosis, indicating that ES is a systemic disease. CD56 expression could be used to reveal ES patients with excellent prognosis or patients predisposed to relapse, thus improving treatment stratification and implementation of personalized therapy. Clin Cancer Res; 17(9); 2900–7. ©2011 AACR.

EFFECT OF P-GLYCOPROTEIN EXPRESSION ON OUTCOME IN THE EWING FAMILY OF TUMORS

This study was designed to determine the prognostic significance of multidrug resistance, mediated by P-glycoprotein (Pgp) expression, in Ewing sarcoma. The clinical and laboratory features, treatment protocol, and outcome of 75 patients with Ewing sarcoma or peripheral neuroectodermal tumor treated between 1972 and 1997 were reviewed. Pgp expression was tested with the monoclonal antibody JSB-1. Thirty-four (64%) of the 53 tissue samples from untreated patients stained positive for Pgp. Progression-free and overall survival were 44 and 59%, respectively, in patients with negative findings, and 28 and 41% in those with positive findings; neither difference was significant. Of the 12 relapsed patients, 6 (50%) expressed more Pgp after chemotherapy than at diagnosis and 4 (33%) expressed less. Within these subgroups, 5 out of 6 and 3 out of 4 died from the disease. No correlation was found between Pgp and known prognostic factors of Ewing tumors. Pgp expression is probably an intrinsic factor of Ewing tumors but has no correlation to prognosis.

Carcinoid tumor of the coccyx: case report and review of the literature.

Study Design. Report of a patient with a carcinoid tumor of the coccyx. Objectives. To describe the clinical presentation, diagnosis, and treatment of a patient with a carcinoid tumor of the coccyx and to review the relevant medical literature in English. Summary of Background Data. No reports of a carcinoid tumor of the coccyx were found in the literature. Seven reports of carcinoid of the sacrum are described. Methods. Clinical history, magnetic resonance imaging studies, and light and electronic microscope micrographs are reviewed. Results. A coccygeal mass was detected during evaluation of coccygodynia in a 40-year-old woman. Four years after extended coccygectomy, there are no signs of local tumor recurrence. Conclusions. Carcinoid tumor of the coccyx is extremely rare. An extended coccygectomy may lead to a cure or at least to a prolonged disease-free interval.

The complexity of management of pregnancy-associated malignant soft tissue and bone tumors.

Objective: The incidence of musculoskeletal tumors during pregnancy is very low. The aim of this study was to summarize our experience in treating a large cohort of pregnant patients diagnosed with these rare tumors. Methods: Women diagnosed with musculoskeletal tumors during pregnancy or immediately after delivery were identified retrospectively in our database between 1996 and 2006. Relevant maternal and neonatal data were collected. Results: Twenty patients, 8 with bone sarcomas (BS) and 12 with soft tissue sarcomas (STS) were identified. Two women were treated by wide excision of mass during pregnancy. In all other cases oncological treatment was delayed until delivery or termination of pregnancy. Vaginal delivery was possible in 9 patients, cesarean section was performed in 7, spontaneous abortion occurred in 1, and 3 underwent termination of pregnancy. Three newborns were premature, but normal growth and development were observed. Different techniques of fertility preservation were used in our patients. Five patients with BS and 5 patients with STS received preoperative chemotherapy, with different grades of toxicity. The degree of tumor necrosis tended to correlate with dose-intensity of chemotherapy. Seven patients with BS received adjuvant chemotherapy. Two patients with STS received adjuvant chemotherapy, two – radiotherapy, and four – both modalities. Median disease-free survival was 15.1 months, median overall survival – 25.4 months. Conclusions: Musculoskeletal tumors diagnosed during pregnancy, or after delivery, do not appear to have a significant impact on the prognosis. A multidisciplinary team should tailor the oncological approach individually.

The association between let-7, RAS and HIF-1α in Ewing Sarcoma tumor growth.

Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family. We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment. Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.

Cryosurgery in fibrous dysplasia: Good result of a multimodality protocol in 16 patients

The indications for surgical treatment of x8e ber dysplasia are: chronic pain, increasing swelling and deformity, imminent fracture, the development of malignancy and cosmetic considerations (Feintuch 1973, De Smet et al. 1981, Yabut et al. 1988, Simpson et al. 1989). To reduce the rate of local recurrence, we used cryosurgery as an adjuvant treatment to curettage, cementing and/or bone grafting in 16 patients with monostotic x8e brous dysplasia for the last 11 years. The indication for the operation was pain or imminent fracture.