Identification of potential biomarkers for giant cell tumor of bone using comparative proteomics analysis.

Giant cell tumor of bone can be locally aggressive and occasionally can metastasize in the lungs. To identify new markers predictive of aggressive behavior, we analyzed five patients who developed lung metastasis and five who remained disease free for a minimum of 5 years. Using two-dimensional electrophoresis, we detected 28 differentially expressed spots. Fourteen spots were identified using mass spectrometry, including seven up-regulated and seven down-regulated in metastatic samples and classified according to functional categories. We then selected five proteins involved in cell cycle or apoptosis. Thioredoxin peroxidase, allograft inflammatory factor 1, and ubiquitin E2N had more than threefold up-regulation; glutathione peroxidase 1 had 1.9-fold up-regulation; and heat shock protein 27 showed down-regulation in metastatic samples with a very low P value. After validation and analysis of protein levels, evaluation of clinical impact was assessed in a much wider cohort of primary archival specimens. Immunodetection showed a higher frequency of thioredoxin peroxidase, allograft inflammatory factor 1, ubiquitin E2N, and glutathione peroxidase 1 overexpression in primary tumors that developed into lung metastases or that locally relapsed than in the disease-free group, with variable stain intensity and distribution. Kaplan-Meier analysis showed that high expression of glutathione peroxidase 1 was strongly related to local recurrence and metastasis, suggesting that its up-regulation may identify a subset of high-risk patients with giant cell tumor prone to receive diverse clinical management.

YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma.

BACKGROUND: The polycomb transcription factor Yin Yang 1 (YY1) overexpression can be causally implicated in experimental tumor growth and metastasization. To date, there is no clinical evidence of YY1 involvement in outcome of patients with osteosarcoma. Prognosis of osteosarcoma is still severe and only few patients survive beyond five years. We performed a prospective immunohistochemistry analysis to correlate YY1 immunostaining with metastatic development and survival in a selected homogeneous group of patients with osteosarcoma. METHODS: We studied 41 patients suffering from osteosarcoma (stage II-IVa). Multivariate analysis was performed using Cox proportional hazard regression to evaluate the correlation between YY1 expression and both metastasis development and mortality. RESULTS: YY1 protein is not usually present in normal bone; in contrast, a high number of patients (61%) showed a high score of YY1 positive cells (51-100%) and 39% had a low score (10-50% positive cells). No statistical difference was found in histology, anatomic sites, or response to chemotherapy between the two degrees of YY1 expression. Cox regression analysis demonstrated that the highest score of YY1 expression was predictive of both low metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) and poor overall survival (HR = 8.353, 95%CI = 1.863-37.451 p = 0.006) regardless of the effects of covariates such as age, gender, histology and chemonecrosis. CONCLUSION: Overexpression of YY1 in primary site of osteosarcoma is associated with the occurrence of metastasis and poor clinical outcome.

Chordoma of the mobile spine and sacrum: a retrospective analysis of a series of patients surgically treated at two referral centers.

BACKGROUND: Chordoma is a rare tumor, and its natural history is still not well known. MATERIALS AND METHODS: All patients affected by localized chordoma surgically treated at Istituto Ortopedico Rizzoli, Bologna, and Istituto Nazionale Tumori, Milan, Italy, between 1980 and 2008 were reviewed. Local recurrence, distant metastasis, and overall survival (OS) were analyzed both from time of diagnosis and from time of local recurrence/distant metastasis. A multivariable analysis to identify independent prognostic factors was carried out. RESULTS: A total of 138 consecutive patients were identified (sacrum 78%, lumbar spine 15%, cervical-dorsal spine 7%). Of these, 130 underwent surgical resection. Median follow-up was 142 months. The 5- and 10-year OS, local relapse-free survival (LRFS), and distant relapse-free survival (DRFS) were, respectively, 78% and 54%, 52% and 33%, and 86% and 72%. Size was an independent prognostic factor for OS (P value < .001), LRFS (P value: .038), and DRFS (P value: .004), while surgical margins independently predicted LRFS (P value: .003) with a trend for OS. The 5- and 10-year OS, LRFS, and DRFS after the first local relapse were 50% and 26%, 47% and 31%, and 64% and 61%. The size of the recurrence and quality of surgical margins did not influence postrelapse OS. The 5-year OS after the second local relapse was 19%. 22% of patients developed distant metastases with a 5-year post-metastases OS of 33%. CONCLUSIONS: Tumor size and surgical margins affected outcome only on initial presentation. However, wide surgery was feasible in a minority of cases. Most patients died of local-regional disease even when metastases occurred. Indeed, long-term prognosis was such that disease-free survival at 10 years was only 26%.

Prognostic significance of immunohistochemical expression of ezrin in non-metastatic high-grade osteosarcoma.

BACKGROUND: Ezrin is a membrane-cytoskeleton linker protein involved in regulating growth and metastatic behaviour of cancer cells. The study evaluated ezrin expression and its prognostic value in patients with non-metastatic osteosarcoma. PROCEDURES: Ezrin expression and pattern of staining (cytoplasmic or membraneous and cytoplasmic) were assessed using immunohistochemistry on slides from tumour biopsy. We studied 95 patients (median 16 years, range: 4-39 years) with primary non-metastatic osteosarcoma of the extremity treated by neoadjuvant chemotherapy based on methotrexate, cisplatin, doxorubicin and ifosfamide. RESULTS: Seventy-six patients (80%) showed ezrin immunoreactivity: in cytoplasm (37, 47%) and in cytoplasm and membrane (42, 53%) of tumour cells. Immunohistochemical staining score was: 1+ (16, 24%), 2+ (10, 13%), 3+ (17, 21.5%) and 4+ (36, 45.5%). Ezrin and score expression were not related to gender, site, alkaline phosphatase (AP), LDH serum levels, chemotherapy-induced tumour necrosis or patient outcome. A significant association was seen between expression pattern and prognosis. The 3-year probability of disease-free survival was 80% for patients with only cytoplasmic immunostaining and 54% for patients with cytoplasmic and membranous immunostaining (P < 0.02). CONCLUSION: Ezrin immunoreactivity can be detected in the majority of patients with non-metastatic osteosarcoma of the extremity. The pattern of ezrin staining can identify patients with different risks of relapse. In patients who only have ezrin cytoplasmic expression, a probability of EFS >80% at 5 years can be expected. These results suggest further investigations to define the relation between expression pattern, ezrin functional status and outcome in patients with non-metastatic osteosarcoma.

Growth inhibition and sensitization to cisplatin by zoledronic acid in osteosarcoma cells.

Since osteosarcoma is a drug-resistant disease, the aim of the present study was to explore the possible interest of therapeutic approaches including nitrogen-containing biphosphonate zoledronic acid using osteosarcoma cell lines with different genetic backgrounds. Parental p53+/pRb+ U2-OS, p53-mutant U2-OS (U2-OS/175) and p53-/pRb- SAOS were sensitive to zoledronic acid with no significant differences in IC50 values. Analysis of cell cycle distribution revealed a time-dependent shifting of U2-OS cells towards G2 phase with cell cycle arrest in G2 phase at 96 h of exposure to the compound. Conversely, U2-OS/175 and SAOS cells responded to treatment with transient cell accumulation in S phase up to 48-72 h, respectively. Cell lines were exposed to increasing concentrations of cisplatin alone or combined with sub-toxic doses of zoledronic acid. A growth inhibitory effect was seen after combined treatment in U2-OS, otherwise resistant to cisplatin up to 100 ng/ml. Zoledronic acid did not efficiently sensitized U2-OS/175 and SAOS to cisplatin, thereby suggesting that different behavior may depend on p53 mutation. This data was confirmed in U2-OS cells where p53 expression was downregulated by RNA interference. Present findings indicate occurrence of sensitization to cisplatin by zoledronic acid in wild-type p53 osteosarcoma cells but not in p53-null cells nor in cells expressing a dominant-negative form of p53, supporting that wild-type p53 is required for synergistic interaction of cisplatin and zoledronic acid.

Prognostic and therapeutic relevance of HER2 expression in osteosarcoma and Ewing's sarcoma.

Expression of HER2 was evaluated by immunohistochemical techniques in 84 osteosarcoma (OS) and 113 Ewing's sarcoma (ES) paraffin-embedded tumour biopsies. HER2 gene status was also assessed in a panel of cell lines as well as in vitro efficacy of trastuzumab (a humanised antibody directed against HER2) as single agent or in combination with the insulin-like growth factor I receptor (IGF-IR) IR3 antibody. Overexpression of HER2 was present in 32% of OS and 16% of ES and was significantly associated with the increased expression of P-glycoprotein, a surface molecule responsible for multidrug resistance. Event-free survival analyses revealed a prognostic value for HER2 and/or P-glycoprotein expression in OS, but not in ES. However, despite its prognostic relevance, no therapeutic effectiveness was observed pre-clinically for trastuzumab-driven therapy, in both OS or ES cell lines, unless the antibody was associated with anti-IGF-IR targeting strategies. Therefore, the therapeutic potential of trastuzumab in these neoplasms may be better exploited in combined treatments with anti-IGF-IR approaches.

Primary diffuse meningeal melanomatosis: radiologic-pathologic correlation.

We report a case of primary diffuse meningeal melanomatosis, a rare variant of primary malignant melanoma of the CNS, in a 68-year-old woman. The disease mimicked intracranial hypotension syndrome and was diagnosed only at autopsy (CSF cytologic results were negative). CT revealed hydrocephalus with effacement of the cerebral convexity sulci and abnormal contrast enhancement in the right sylvian and frontoparietal fissures, whereas MR imaging showed diffuse marked dural and leptomeningeal contrast enhancement. In retrospect, these nonspecific findings correlated with the extensive leptomeningeal invasion in the cerebral hemispheres, brain stem and spinal cord. The clinical, radiologic, and pathologic features of diffuse meningeal melanomatosis are reviewed.

Leiomyosarcoma of the spermatic cord: a light and ultrastructural description of one case.

A case of leiomyosarcoma arising in the spermatic cord is described. A 83-year-old man required medical care for an irreducible inguinal hernia. The patient underwent herniorraphy and transinguinal radical orchiectomy. Macroscopically, the spermatic cord was enlarged by a gray-tan and ill-defined neoplasm measuring 4 x 4 x 3 cm. Histologically, this proliferation was composed of atypical spindle cells with blunted end nuclei and eosinophilic cytoplasm. Immunohistochemistry and ultrastructure confirmed the smooth muscle nature of the neoplastic cells. The diagnosis of leiomyosarcoma of the spermatic cord was made. To improve the assignment of this rare lesion to its specific anatomic location, we analyzed the immunohistochemical and ultrastructural characteristics of the smooth muscle tumoral cells and in particular those of the intracellular filament aggregates.

Fibrosarcomatous changes and expression of CD34+ and apolipoprotein-D in dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a relatively common soft-tissue tumor. A more aggressive appearing fibrosarcoma may arise in DFSP, changing its biological behavior. CD34 and apolipoprotein-D are highly expressed in DFSP, but their prognostic significance is uncertain. METHODS: DFSP and fibrosarcomatous-DFSP (FS-DFSP) patients referred to our institute between 1982 and 2009 were identified. Fibrosarcomatous changes, expression of CD34 and apolipoprotein-D were evaluated. RESULTS: 40 patients, (median age 43 years, 55% males) were identified. Tumor was located in the limbs in 60%, in the trunk in 40%. Thirty-seven patients had localized and 3 had metastatic disease. Thirteen (32%) patients were FS-DFSP. All but one underwent surgery with adequate surgical margins in 72%. 7 FS-DFSP received also radiotherapy (RT). Chemotherapy was administered to 3 patients with FS-DFSP. With a median follow-up of 49 months, the 5-OS was 90%. Local recurrence rate was 23%: 42% FS-DFSP, 15% DFSP. Metastases developed in three FS-DFSP patients. The 5-year EFS was 70% in localized patients. Histology (DFSP 75% vs. FS-DFSP 52%, p = 0.002), surgical margins (adequate 74% vs. inadequate 55%, p = 0.02), site (limb 47% vs. trunk 100%), CD34 expression (CD34 positive: 70% vs. CD34 negative: 33%, p = 0.05), and apolipoprotein-D expression (Apo-D positive: 73% vs. Apo-D negative: 33%, p = 0.02) influenced the 5-year EFS, whereas sex, use of RT or number of previous surgical treatments did not. CONCLUSIONS: Patients with DFSP have a high survival probability. Site, adequate surgical margins, presence of the fibrosarcomatous component, lack of CD34 expression and apolipoprotein-D influence outcome.

Molecular diagnosis in Ewing family tumors: the Rizzoli experience--222 consecutive cases in four years.

The Ewing's family of tumors (EFTs) are characterized by chimeric transcripts generated by specific chromosomal rearrangements. The most common fusions are between the EWSR1 gene on chromosome 22 and the ETS family of transcription factors; rarely, FUS (on chromosome 16) substitutes for EWSR1. The detection of specific translocations using molecular analysis is now a routine part of the pathological examination of EFT. Here, we report our experience with molecular diagnosis of EFT during the 4 years (2006-2009) at the Rizzoli Institute. We analyzed 222 consecutive tumors with a presumptive diagnosis of EFT using molecular techniques and IHC. We found five distinct types of EWSR1-FLI1 fusion transcripts resulting from translocation t(11;22), three types of EWSR1-ERG transcripts resulting from t(21;22), and one type of t(2;22) resulting in EWSR1-FEV fusion. Molecular investigation validated 92% of cases ultimately diagnosed as EFT; IHC validated 76% of the cases. Thus, despite the difficulties and limitations associated with both molecular and IHC analysis on fresh and formalin-fixed, paraffin-embedded tissue, a combination of these techniques is the best approach to enhancing the accuracy of EFT diagnosis. We also present our method for choosing which molecular techniques to apply. Finally, we collected the most prevalent breakpoints reported in the literature, indicating which exons are involved, the sequence breakpoints, and the NCBI reference sequences.

Synovial sarcoma: retrospective analysis of 250 patients treated at a single institution.

BACKGROUND: The optimal treatment for synovial sarcoma remains controversial. Treatment, outcome, and prognostic factors in patients treated in a single institution were examined. METHODS: Synovial sarcoma patients who underwent surgery at the Rizzoli Institute between 1976 and 2006 were identified and analyzed. RESULTS: Characteristics of the 250 patients (128 female; 122 male) included: median age, 37 years (range, 7-83 years); 177 (71%) with tumors in the lower extremity, 40 (16%) with tumors in the upper extremity, and 33 with tumors in the trunk (13%); primary lesion size >5 cm in 121 patients (55%); and 204 (82%) patients with localized disease and 46 (18%) with metastatic disease at the time of presentation. All patients with localized disease underwent surgery. Twenty-four percent of patients underwent amputation. Adequate surgical margins were achieved in 88% patients. In patients with localized disease, radiotherapy was administered to 103 (50%) patients, and chemotherapy to 98 (48%). With a median follow-up of 5.5 years (range, 1-30 years), the 5-year overall survival rate was 10% for patients with metastatic disease and 76% for patients with localized disease (P = .0001). The 5-year event-free survival was 58% in patients with localized disease. Multivariate analysis indicated that size, age, histologic subtype, and the use of radiotherapy were independent factors for event-free survival. CONCLUSIONS: In those patients with localized disease, a good rate of cure can be achieved. Age, size, histology, and use of radiotherapy influence prognosis, whereas to the authors' knowledge, the role of adjuvant chemotherapy remains unproven.

Desmoplastic fibroblastoma: a light and ultrastructural description of two cases.

The authors report 2 cases of collagenous fibroma in which ultrastructural analysis revealed the presence of fibronexus junctions, markers of myofibroblastic differentiation, never described in this rare lesion before. The tumors occurred in the trapezius muscle and in the right arms of a 41 -year-old and a 25-year-old man. They were both intramuscular and showed sharp edges. Grossly, the excised masses were whitish and firm. Microscopically, they were both composed of stellate or spindle-shaped cells separated by a collagenous hypovascular and focally myxoid stroma. Mitotic figures and necrotic areas were not identified. Immunohistochemistry showed positivity for vimentin and focal positivity for smooth and human muscle actin, and flow cytometry showed the tumoral cells to be diploid.

Evaluation of P-glycoprotein, HER-2/ErbB-2, p53, and Bcl-2 in primary tumor and metachronous lung metastases in patients with high-grade osteosarcoma.

BACKGROUND: Investigation of the relation between primary tumor and metastatic disease is necessary for the identification of predictive factors for postrecurrence survival (PRS) in patients with recurrent osteosarcoma. METHODS: Cellular levels of P-glycoprotein, ErbB-2, p53, and Bcl-2 expression were evaluated in primary tumor biopsy and metachronous pulmonary metastasis specimens from 19 patients with high-grade osteosarcoma. Results were analyzed for differences between primary tumor and pulmonary metastases and for correlations between expression patterns and survival. RESULTS: Positive staining in lung metastases was noted in 68%, 53%, 32%, and 84% of patients for P-glycoprotein, ErbB-2, p53, and Bcl-2, respectively. These percentages were higher than those observed in primary tumor specimens for all genetic markers evaluated, with a significant difference in the percentage of patients with positive staining for P-glycoprotein (68% vs. 32%; P = 0.05) and a near-significant difference in the percentage of patients with positive staining for Bcl-2 (84% vs. 53%; P = 0.08). Patients with ErbB-2 expression in the primary tumor were more likely to have multiple metastases and shorter recurrence-free intervals compared with patients in whom ErbB-2 expression was not observed, whereas differences in P-glycoprotein, p53, and Bcl-2 expression were not related to differences in metastatic pattern. PRS was influenced by p53 expression levels in pulmonary metastases, with patients who had negative staining for p53 having a significantly better PRS rate relative to patients with positive staining for p53 (3-year PRS rate: p53-negative, 64%; p53-positive, 17%; P = 0.008). CONCLUSIONS: In the current study of patients with high-grade osteosarcoma, most patients exhibited increased cellular expression of P-glycoprotein, ErbB-2, and Bcl-2 in recurrent pulmonary metastases compared with primary tumor. Further studies aimed at investigating the relation between altered p53 expression in lung metastases and postrecurrence survival are recommended.