Primary synovial sarcoma of bone: a retrospective analysis of 25 patients.

AIMS: To evaluate the diagnostic accuracy of SSX and SSX::SS18 antibodies in decalcified surgical specimens and outcome of synovial sarcomas (SS) of bone. METHODS AND RESULTS: Twenty-five cases were classified as bone SS (prevalence 0.32% among malignant primary bone sarcoma). Median age was 34 years (range = 9-79). Twenty-four of 25 patients presented with non-metastatic tumours, one with lung metastases. The majority of tumours involved the long bones of extremities with metaphyseal origin. Mean size of the tumour was 7.1 cm. Twenty cases (80%) were monophasic and five (20%) were biphasic. SS18::SSX fusion-specific antibody had 92% sensitivity and 99% specificity for primary bone SS, whereas SSX C-terminus antibody had 100% sensitivity and 94% specificity. Fluorescence in-situ hybridisation (FISH) analysis was feasible in nine (36%) cases and detected SS18 rearrangement in all nine cases. All patients underwent surgical removal of their primary tumour, with adequate margins in 18 (72%) patients. Chemotherapy with metothrexate, cisplatin, doxorubicin and ifosfamide was used in the seven patients. Two patients with inadequate surgical margins received radiotherapy. With a median follow-up of 80 months (range = 6-428), 5- and 10-year overall survival (OS) was 66.6% and 47.9%, respectively, and 5 and 10 years' disease-free survival (DFS) was 36.8% [95% confidence interval (CI) = 18.0-55.7%] and 32.2% (95% CI = 14.6-51.2%), respectively. A significant improvement in 10 years' DFS in patients undergoing chemotherapy compared with patients who did not was observed (P = 0.039). CONCLUSIONS: Our series highlights the utility of SS18::SSX fusion-specific and SSX C-terminus antibodies to support the diagnosis of SS. Adjustment chemotherapy was associated with improved prognosis in this series.

The Efficacy of Molecular Analysis in the Diagnosis of Bone and Soft Tissue Sarcoma: A 15-Year Mono-Institutional Study.

The histological diagnosis of sarcoma can be difficult as it sometimes requires the combination of morphological and immunophenotypic analyses with molecular tests. A total of 2705 tissue samples of sarcoma consecutively collected from 2006 until 2020 that had undergone molecular analysis were assessed to evaluate their diagnostic utility compared with histological assessments. A total of 3051 molecular analyses were performed, including 1484 gene fusions tested by c/qRT-PCR, 992 gene rearrangements analysed by FISH, 433 analyses of the gene status of MDM2, 126 mutational analyses and 16 NGS analysis. Of the samples analysed, 68% were from formalin-fixed, paraffin-embedded tissue and 32% were from frozen tissue. C/qRT-PCR and FISH analyses were conclusive on formalin-fixed, paraffin-embedded tissue in 74% and 76% of samples, respectively, but the combination of the two methods gave us conclusive results in 96% and 89% of frozen and formalin-fixed, paraffin-embedded tissues, respectively. We demonstrate the utility of c/qRT-PCR and FISH for sarcoma diagnosis and that each has advantages in specific contexts. We conclude that it is possible to accurately predict the sarcoma subtype using a panel of different subtype-specific FISH probes and c/qRT-PCR assays, thereby greatly facilitating the differential diagnosis of these tumours.

Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression.

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low-molecular-weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high-grade STS sera using hydrogel core-shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty-two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non-metastatic tumors. Cox's regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H-related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.

Excision repair cross-complementation group 1 protein expression predicts survival in patients with high-grade, non-metastatic osteosarcoma treated with neoadjuvant chemotherapy.

AIMS: To evaluate the clinical impact of excision repair cross-complementation group 1 (ERCC1) expression in high-grade osteosarcoma (OS). METHODS AND RESULTS: Immunohistochemistry was performed on biopsies from 99 OS patients enrolled in the ISG/OS-Oss training set or ISG/SSG1 validation set neoadjuvant chemotherapy protocols, based on the use of cisplatin, adriamycin, methotrexate, and ifosfamide. In the training set, ERCC1 positivity was found in eight of 31 (26%) patients, and was significantly associated with worse event-free survival (EFS) (P = 0.042) and overall survival (OVS) (P = 0.001). In the validation set, ERCC1 positivity was found in 22 of 68 (32%) patients, and its significant associations with poorer EFS (P = 0.028) and OVS (P = 0.022) were confirmed. Multivariate analyses performed on the whole patient series indicated that ERCC1 positivity was the only marker that was significantly associated with a higher risk of worse prognosis, in terms of both EFS and OVS (P = 0.013). Co-evaluation of ERCC1 and ABCB1 expression showed that patients who were positive for both markers had a significantly worse prognosis. CONCLUSIONS: The ERCC1 level at diagnosis is predictive for the outcome of patients with non-metastatic, high-grade OS treated with neoadjuvant chemotherapy, and co-evaluation with ABCB1 can identify high-risk groups of OS patients who are refractory to standard regimens.

The utility of FISH analysis in the diagnosis of BCOR-rearranged sarcomas.

BACKGROUND: A BCL6 corepressor (BCOR) gene alteration is a genetic signature of rare subsets of sarcomas. The identification of this alteration has recently contributed to the definition of new entities in the current WHO (2020) classification of soft tissue and bone tumours. We retrospectively examined cases of BCOR-rearranged sarcoma (BRS) to assess the reliability of the BCOR FISH analysis using an IVD (in vitro diagnostic) probe. METHODS: We investigated and compared the molecular diagnostic strategies and features by collecting 17 data from patients with a BCOR gene rearrangement detected using quantitative-Reverse Transcription-Polymerase Chain Reaction (qRTPCR), Next-Generation Sequencing (NGS) and Fluorescence in situ hybridization (FISH). RESULTS: We describe fourteen BCOR::CCNB3 sarcomas, one spindle cell sarcoma with a novel BCOR::MAML1 fusion, one spindle cell sarcoma with a novel BCOR::AHR fusion, and one ossifying fibromyxoid tumour with a BCOR::ZC3H7B fusion. FISH analysis of all, except one, BCOR::CCNB3 sarcoma, showed a FISH break-apart pattern with mild signal separation. The MAML1::BCOR sarcoma showed large-space split signals, while in the two patients with AHR::BCOR and ZC3H7B::BCOR fusions, no BCOR rearrangement was observed using FISH. CONCLUSIONS: Our study indicates that BCOR FISH analysis using an IVD probe, may be useful to detect the presence of a BCOR rearrangement, including both translocations and inversions; however, negative results, in some cases, can occur.

Conventional Spinal Chordomas: Investigation of SMARCB1/INI1 Protein Expression, Genetic Alterations in SMARCB1 Gene, and Clinicopathological Features in 89 Patients.

The partial loss of SMARCB1/INI1 expression has recently been reported in skull base conventional chordomas, with possible therapeutic implications. We retrospectively analyzed 89 patients with conventional spinal chordomas to investigate the differences in the immunohistochemical expression of SMARCB1/INI1 and the underlying genetic alterations in the SMARCB1 gene. Moreover, we assessed the correlation of clinicopathological features (age, gender, tumor size, tumor location, surgical margins, Ki67 labelling index, SMARCB1/INI1 pattern, previous surgery, previous treatment, type of surgery, and the Charlson Comorbidity Index) with patient survival. Our cohort included 51 males and 38 females, with a median age at diagnosis of 61 years. The median tumor size at presentation was 5.9 cm. The 5-year overall survival (OS) and 5-year disease-free survival (DFS) rates were 90.8% and 54.9%, respectively. Partial SMARCB1/INI1 loss was identified in 37 (41.6%) patients with conventional spinal chordomas (27 mosaic and 10 clonal). The most frequent genetic alteration detected was the monoallelic deletion of a portion of the long arm of chromosome 22, which includes the SMARCB1 gene. Partial loss of SMARCB1/INI1 was correlated with cervical-thoracic-lumbar tumor location (p = 0.033) and inadequate surgical margins (p = 0.007), possibly due to the high degree of tumor invasiveness in this site. Among all the considered clinicopathological features related to patient survival, only tumor location in the sacrococcygeal region and adequate surgical margins positively impacted DFS. In conclusion, partial SMARCB1/INI1 loss, mostly due to 22q deletion, was detected in a significant number of patients with conventional spinal chordomas and was correlated with mobile spine location and inadequate surgical margins.

SMARCB1/INI1 loss in skull base conventional chordomas: a clinicopathological and molecular analysis.

Introduction: The loss of SMARCB1/INI1 protein has been recently described in poorly differentiated chordoma, an aggressive and rare disease variant typically arising from the skull base. Methods: Retrospective study aimed at 1) examining the differential immunohistochemical expression of SMARCB1/INI1 in conventional skull base chordomas, including the chondroid subtype; 2) evaluating SMARCB1 gene deletions/copy number gain; and 3) analyzing the association of SMARCB1/INI1 expression with clinicopathological parameters and patient survival. Results: 65 patients (35 men and 30 women) affected by conventional skull base chordoma, 15 with chondroid subtype, followed for >48 months after surgery were collected. Median age at surgery was 50 years old (range 9-79). Mean tumor size was 3.6 cm (range 2-9.5). At immunohistochemical evaluation, a partial loss of SMARCB1/INI1 (>10% of neoplastic examined cells) was observed in 21 (32.3%) cases; the remaining 43 showed a strong nuclear expression. Fluorescence in situ hybridization (FISH) analysis was performed in 15/21 (71.4%) cases of the chordomas with partial SMARCB1/INI1 loss of expression. Heterozygous deletion of SMARCB1 was identified in 9/15 (60%) cases and was associated to copy number gain in one case; no deletion was found in the other 6 (40%) cases, 3 of which presenting with a copy number gain. No correlations were found between partial loss of SMARCB1/INI1 and the clinicopathological parameters evaluated (i.e., age, tumor size, gender, tumor size and histotype). Overall 5-year survival and 5-year disease-free rates were 82% and 59%, respectively. According to log-rank test analysis the various clinico-pathological parameters and SMARCB1/INI1 expression did not impact on overall and disease free-survival. Discussion: Partial loss of SMARCB1/INI1, secondary to heterozygous deletion and/or copy number gain of SMARCB1, is not peculiar of aggressive forms, but can be identified by immunohistochemistry in a significant portion of conventional skull base chordomas, including the chondroid subtype. The variable protein expression does not appear to correlate with clinicopathological parameters, nor survival outcomes, but still, it could have therapeutic implications.

Osteogenic sarcomas of the hands: A case series with emphasis in its peculiarities and literature review.

AIM: To present our experience on osteosarcomas of the hands and review the existing literature pertaining osteosarcomas in this extremely rare location. METHODS: and results: Seven cases of osteosarcomas of the hands were reviewed, and a literature search of all primary osteosarcomas of the hands was performed. All tumors occurred in adults (mean age, 41 years) and were located mainly around the metacarpophalangeal joints. All patients presented with localized long-lasting pain as main symptom. The mean size at diagnosis was 33 mm. Three tumors were low-grade central osteosarcomas, 1 low-grade central chondroblastoma-like osteosarcoma and 3 high-grade osteosarcomas. All tumors were positive for mouse double-minute 2 homolog (MDM2) immunohistochemistry. Three cases yielded results with fluorescence in-situ amplification for MDM2 (12q15)/CEP12. At last follow-up, one patient with a high-grade osteosarcoma was dead of disease. The literature review revealed similar demographic and site distribution of osteosarcomas within the hands than our series and an unusually high proportion of low-grade central and parosteal osteosarcomas when compared to the proportion of these infrequent neoplasms in the whole skeleton. CONCLUSIONS: osteosarcomas of hands present in older individuals compared to the population affected by conventional osteosarcomas of all sites. Importantly from a diagnostic, therapeutic and prognostic points of view, around 40% of osteosarcomas of the hands are low-grade osteosarcomas of the central or parosteal types.

CIC rearranged sarcomas: A single institution experience of the potential pitfalls in interpreting CIC FISH results.

AIM: The aim of this study was to establish how reliable FISH CIC analysis using an IVD (in vitro diagnostic) commercial probe is. METHODS AND RESULTS: A series of 19 CIC-DUX4 sarcomas were evaluated. The samples presenting CIC-DUX4 fusion transcript detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger sequencing and/or Next Generation Sequencing were selected for Fluorescent in Situ Hybridization (FISH) CIC analysis with CIC break-apart IVD probe and compared to molecular analysis. CIC FISH analysis showed 26% of false negatives. CONCLUSION: Our results indicate that, in the setting of CIC-DUX4 fusion positive small round cell sarcomas, CIC FISH using IVD commercial probe may lead to false-negative results. This novel study evaluates the diagnostic use of a commercial IVD CIC probe for FISH.

Primary malignant peripheral nerve sheath tumors of bone: a clinicopathologic reappraisal of 8 cases.

Primary spindle cell and pleomorphic sarcomas of bone represent an exceedingly rare group of mesenchymal malignancies that include soft tissue histotypes, as malignant peripheral nerve sheath tumor. Outside the head and neck region, only 36 cases of primary malignant peripheral nerve sheath tumor of bone have been described. We retrieved from our archives eight cases of primary malignant peripheral nerve sheath tumor of bone arising outside the head and neck region, describing their clinical, radiological, and morphologic features. Our series, in which all but one patient died of diseases after a median of seven months, confirms that primary malignant peripheral nerve sheath tumors of bone are aggressive tumors. Pathologists should be aware of this rare histotype. More aggressive and active adjuvant treatments should be investigated.

Periosteal chondrosarcoma: A case series in a referral center with survivorship analysis.

BACKGROUND: Periosteal chondrosarcomas are among the rarest types of chondrosarcomas dealt with in few small series of cases. In this study, we aimed to present our experience with this chondrosarcoma, seek for prognostic factors for OS and DFS and survey the status of IDH1 and IDH2. RESULTS: 55 periosteal chondrosarcomas were retrospectively identified. Median age was 37 years, there was a male predominance (62%). The great majority of cases involved the metaphysis of long bones of the extremities. The median size of the tumors was 7.5 cm. Thirty patients underwent to subtotal surgical resection, 22 to tangential resection and the remaining 3 to amputation. The margins, reported in 54 cases, were wide/radical in 38 patients (70.4%), marginal in 9 (16.7%) and intralesional in 7 (12.9%). Histologically, 23 (42%) were grade 1; 27 (49%), grade 2; 3 (5%), grade 3 and 2 (4%) were dedifferentiated. A third of cases in which mutational analysis was feasible harbored heterozygous mutations in codon 132 of IDH1. Fifty-four cases were included for follow-up (median, 137 months). Four patients had local recurrences and six patients developed metastasis to the lungs. All patients that developed metastasis died of disease, two died of unrelated causes and 46 were alive without disease. OS and DFS was not found to be statistically associated with clinical and pathological parameters considered. CONCLUSIONS: periosteal chondrosarcomas exhibit a low-grade behavior that can be adequately treated with marginal excisions. Clinical and morphologic parameters do not seem to predict their outcome.

ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis.

PURPOSE: The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. METHODS: The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. RESULTS: We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. CONCLUSIONS: Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

Giant Cell Tumor of Bone in Patients under 16 Years Old: A Single-Institution Case Series.

BACKGROUND: Giant cell tumor of bone is a locally aggressive, rarely metastasizing tumor that accounts for about 5% of bone tumors and generally occurs in patients between 20 and 45 years old. A driver mutation in the histone 3.3 (H3.3) gene H3F3A has been identified in as many as 96% of giant cell tumors of bone. The immunohistochemical expression of H3F3A H3.3 G34 expression was found in 97.8% of cases. In the present study, we describe our series of cases of giant cell tumor of bone in pediatric patients <16 years old. METHODS: All cases of giant cell tumor of bone in pediatric patients <16 years old treated in our institute between 1982 and 2018 were reviewed. Immunohistochemistry and/or molecular analysis for H3F3A gene mutations was performed to confirm the diagnosis. A group of aneurysmal bone cysts in patients <16 years old was used as a control group. RESULTS: Fifteen cases were retrieved. A pronounced female predominance (93%) was observed. A pure metaphyseal central location occurs in 2 skeletally immature patients. CONCLUSIONS: Giant cell tumor of bone should be distinguished from its mimickers due to differences in prognosis and treatment. Immunohistochemical and molecular detection of H3F3A gene mutation represents a reliable diagnostic tool.

Usefulness of ß-catenin expression in the differential diagnosis of osteosarcoma, osteoblastoma, and chondroblastoma.

The aim of this study is to assess the usefulness of beta-catenin immunohistochemical expression in the differential diagnosis of osteoid-producing primary tumors of bone. Seventy cases of osteoid-producing tumors of bone (24 conventional osteosarcomas, 18 osteoblastomas, 13 osteoblastoma-like osteosarcomas, 10 chondroblastomas, and 5 chondroblastoma-like osteosarcomas) diagnosed at Istituto Ortopedico Rizzoli were reviewed and evaluated for the intensity, extension, and subcellular distribution of immunohistochemical expression of beta-catenin. A majority of cases (73%, 51 cases) exhibited cytoplasmic and/or membranous positivity in varied degrees of intensity and proportion of positive cells, in the absence of nuclear staining. Fifteen cases (21%) were completely negative, including two osteoblastomas, five chondroblastomas, three conventional osteosarcomas, four osteoblastoma-like osteosarcomas, and one chondroblastoma-like osteosarcoma. A minority of cases (6%) including three osteoblastoma-like osteosarcomas and one osteoblastoma showed focal nuclear beta-catenin positivity with or without concomitant cytoplasmic staining. In the current series, beta-catenin showed not to be useful in the differential diagnosis of osteoid-producing primary bone tumors.

Histological response to neoadjuvant chemotherapy in localized Ewing sarcoma of the bone: A retrospective analysis of available scoring tools.

AIM: The aim is to evaluate which of the existing scoring systems of histological response to neoadjuvant chemotherapy best stratifies the clinical outcome of patients with localized Ewing sarcoma of bone. METHODS: 474 patients with diagnosis of localized Ewing sarcoma of bone were included. The median follow-up was 13.5 years. RESULTS: The overall survival and the disease-free survival (DFS) were 70.8% and 63.9% at 5 years. The percentage of histological response to neoadjuvant chemotherapy ranged between 5% and 100% (mean 83%). The agreement between Bologna System and the different percentual cut-offs of histological response to neoadjuvant chemotherapy was high, with kappa statistics of 0.83 for a cut-off of ≥90%; 0.86 for a cut-off of ≥95%; 0.79 for a cut-off of ≥96% and 0.61 for a cut-off of 100%. Statistically higher DFS rates for good responders compared to poor responders were found when using each given system. Model performance indicators showed that Bologna system had a lower AIC score and a higher c-statistics to predict DFS. When the patients classified as good responders using the different percentual cut-offs of histological response to neoadjuvant chemotherapy, were instead re-classified using the Bologna system, statistical differences were noted in DFS within each specific group. CONCLUSIONS: All scoring tools to evaluate histological response to neoadjuvant chemotherapy offer good predictive value for DFS in localized Ewing's sarcoma of bone. The Bologna system better stratifies those patients with histological response to neoadjuvant chemotherapy between 90 and 99%, representing a more reliable scoring tool in this subset.

Lipoblastoma-like tumor of the spermatic cord: case report and review of the literature.

Lipoblastoma-like tumor is a very rare mesenchymal tumor believed to be restricted to female patients and only recently reported in the spermatic cord of a male patient. We describe herein an additional case of lipoblastoma-like tumor occurring in the spermatic cord, describing its histopathological, immunohistochemical, and molecular features.

Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.

Differential gene expression in classic giant cell tumours of bone: Tenascin C as biological risk factor for local relapses and metastases.

AIMS: To identify candidate prognostic biological markers useful in selecting high-risk patients with classic primary giant cell tumours (GCT). GCT specimens with different behaviour associated with an increased risk of local and/or distant relapses were studied. METHODS AND RESULTS: Screening mRNA microarray analysis followed by real-time polymerase chain reaction and immunohistochemistry on tissue microarray sections was used to validate the prognostic role of differentially expressed genes on a larger series by statistical analysis tests. Global gene expression profiling identified 109 differentially expressed genes according to prognosis. A correlation was found between mRNA levels and clinical outcome identifying Tenascin C (TNC) as the most significant prognostic biological marker. By means of backward Wald logistic regression, TNC overexpression defined an eightfold increased risk for metastasis and fourfold for local recurrence. At the protein level, TNC immunoreactivity resulted in a significant difference in the disease-free survival probability curves, providing a stratification for GCT patients, useful for predicting relapse. CONCLUSIONS: Our study provides important data for the selection of biomarkers with a significant clinical impact and suggests the importance of TNC expression in identifying GCT patients at a higher risk of relapse for closer follow-up and adjuvant medical therapy.

Detection of H3F3A p.G35W and p.G35R in giant cell tumor of bone by Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR).

Giant Cell Tumor (GCT) represents about 20% of benign bone tumors, is locally aggressive although malignant transformation is extremely rare, <1% of cases but 2-3% give pulmonary metastasis. Age at onset is between 20 and 40 years with a slight predominance for the female gender. GCT is characterized by specific mutations in H3F3A gene encoding the protein histone 3.3. The study of these mutations is important for the differential diagnosis with giant cell rich sarcomas, chondroblastoma and aneurysmal bone cyst. To identify the most frequent H3F3A mutations we developed a novel allele specific Real Time Polymerase Chain Reaction method, based on Allele Specific Locked Nucleic Acid (ASLNAqPCR) that is here described. Molecular analyses were performed on 20 GCT and 2 osteosarcoma arising on a previous GCT. All cases were verified by Sanger sequencing. We demonstrated that ASLNAqPCR is a quick, sensitive and reliable method to identify mutations of the H3F3A gene, in giant cell tumor of bone, to support diagnosis in morphologically ambiguous cases.

Circulating Candidate Biomarkers in Giant Cell Tumors of Bone.

PURPOSE: Approximately 5% of giant cell tumors (GCT) of bone develop pulmonary metastases. Although many biomarkers have been proposed, identification of circulating low abundance molecules may be useful to predict malignant progression. EXPERIMENTAL DESIGN: The hydrogel nanoparticle technique followed by MS was used to detect low molecular weight serum proteins or protein fragments in serum of 20 GCT patients with different clinical course and in ten healthy sera used as control. The most representative low-abundant de novo or differentially abundant proteins were submitted to String database that recognized interconnected activated pathways including protein activation cascade, wound healing, cell-substrate adhesion, and response to stress. Statistics were performed for identification of candidate prognostic factors. RESULTS: Proteome cluster analysis separated metastasis-free from metastatic GCT patients in two well-defined groups where serum levels of signaling transduction mediators and regulators of kinase activity presented a high discriminatory power. Increased expression of proteins STAT5B, GRB2, and OXSR1 was related to a higher probability of metastasis. Multivariate analysis demonstrated that tumor grade and STAT5B were independent prognostic factors. CONCLUSIONS AND CLINICAL RELEVANCE: By using a noninvasive technique, we identified differentially abundant serum candidate biomarkers, also providing prognostic information in patients with GCT of bone.