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.

NTRK rearranged sarcoma of the bone. Role for larotrectinib in the neoadjuvant setting of an ultra-rare tumor: a case report.

BACKGROUND: Neurotrophic tyrosine receptor kinase (NTRK) gene-fusion targeted molecules revolutionized the paradigm of treatment of a limited subgroup of cancers of various histologies. Entrectinib and larotrectinib obtained unprecedented response rates in patients with cancer harboring NTRK rearrangements. This evidence recently led to the agnostic approval of these drugs, and evidence (confirmation) of their activity in a broader disease setting is emerging. Here, we report the case of a patient affected by EML4-NTRK3 rearranged undifferentiated spindle cell bone sarcoma treated with larotrectinib, and we argue (discuss about) the incidence and clinical presentation of NTRK gene-fusion positive bone sarcomas, the potential use of upfront treatment with NTRK inhibitors in neoadjuvant setting, and the role of a multidisciplinary tumor board. Despite the rarity of these rearrangements in patients with primitive bone sarcomas, the therapy with NTRK inhibitors represents a highly effective strategy to be pursued in selected cases even in neoadjuvant settings. The management of these very rare cancers should always be discussed in a multidisciplinary board of reference centers.

Fusion transcriptome profiling defines the monoclonal origin of multifocal epithelioid haemangioma of bone.

AIMS: Epithelioid haemangioma (EH) of bone remains a highly controversial entity. Indeed, the WHO classifies EHs of soft tissues as benign tumours, whereas bone EHs are considered intermediate-locally aggressive tumours due to common multifocal presentation and local destructive growth. To gain insights into the clinical behaviour and biology of EH of bone we retrospectively analysed 42 patients treated in a single institution from 1978 to 2021. METHODS AND RESULTS: Multifocal presentation was detected in 17 of 42 patients (40%) primarily as synchronous lesions. Patients were treated with curettage (57%), resection (29%) or biopsy, followed by radiotherapy or embolisation (14%). Follow-up (minimum 24 months) was available for 38 patients, with only five local recurrences (13%) and no death of disease. To clarify whether the synchronous bone lesions in multifocal EH represent multicentric disease or clonal dissemination, four cases were profiled by RNA-sequencing. Separate lesions from the same patient, which showed a similar transcriptional profile, expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints. CONCLUSIONS: These results indicate that, in EH of bone, multifocal lesions are clonally related and therefore represent the spread of a same neoplastic clone rather than simultaneous independent tumours. This finding is in apparent contradiction with the benign clinical course of the disease, and suggests that tumour dissemination in bone EH probably reflects a phenomenon of passive spreading, with tumour cells colonising distal sites while maintaining their benign biological nature.

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.

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.

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.

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 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.

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.

Primary sclerosing epithelioid fibrosarcoma of the spine: a single-institution experience.

AIMS: To present our experience on spinal sclerosing epithelioid fibrosarcoma (SEF) and review the existing literature pertaining to SEF of the spine. METHODS AND RESULTS: Six cases of spinal SEF were reviewed, and a literature search of all primary SEFs of the spine was performed. All tumours occurred in adults (median age, 41 years) and were located all along the spine, the lumbar vertebrae being the most commonly involved. All patients presented with pain that they had experienced for months. The mean tumour size at diagnosis was 52 mm. Five tumours showed a spectrum of microscopic features consistent with pure SEF, and one showed a hybrid morphology with areas of low-grade fibromyxoid sarcoma. All were diffusely and strongly positive for mucin 4. Two cases were initially misdiagnosed as epithelioid haemangioendothelioma and aggressive chondroblastoma. Fluorescence in-situ hybridisation showed rearrangements of either FUS or EWSR1 in four cases. Reverse transcription polymerase chain reaction showed the presence of FUS-CREB3L1 and EWSR1-CREB3L1 fusion transcripts in two cases and one case, respectively. Of five patients with follow-up data available, two developed one or more local recurrences and three patients had metastatic disease. Distant metastases were mainly to other osseous locations, followed by lungs and lymph nodes. At last follow-up, three patients had died of disease and one was alive with multiple metastases. CONCLUSIONS: SEF is an aggressive sarcoma that can involve the spine. It is important to recognise the spine as the primary location of SEF, in order to avoid misdiagnosis as more common primary spinal neoplasms, which can impact on therapeutic approaches.

Association of Clinicopathological Features With Outcome in Chondrosarcomas of the Head and Neck.

OBJECTIVE: The aim of this study is to assess the association between clinical and radiological features as well as of isocitrate dehydrogenase 1 and 2 (IDH 1,2) mutations with outcome in head and neck chondrosarcomas. STUDY DESIGN: Retrospective study. SETTING: Tertiary referral center. METHODS: Clinical, histological, and molecular data of patients with head and neck chondrosarcomas treated by surgery were collected. RESULTS: Forty-six patients were included. The mean age at diagnosis was 56 years (range, 17-78). The tumor originated from the skull base (52.2%), facial bones (28.2%), or laryngotracheal area (19.6%). At last follow-up (median 52.5 months), 38 patients were alive, 30 of which were disease free, whereas 8 had died, 4 of disease progression and 4 of other causes. Fourteen (30.4%) had local recurrence and 2 (4.3%) had lung metastasis. All cases were negative for cytokeratin AE1/AE3, brachyury, and IDH1 at immunohistochemistry, while Sanger sequencing identified IDH1/2 point mutations, typically IDH1 R132C, in 9 (37.5%) tumors arising from the skull base. Margin infiltration on the surgical specimen negatively affected the outcome, whereas no correlation was identified with IDH mutation status. CONCLUSIONS: An adequate margin positively affects survival. IDH mutation status does not affect patient outcome.

Extraskeletal Myxoid Chondrosarcoma with Molecularly Confirmed Diagnosis: A Multicenter Retrospective Study Within the Italian Sarcoma Group.

BACKGROUND: Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain origin, marked by specific chromosomal translocations involving the NR4A3 gene, and usually characterized by an indolent course. Surgery (with or without radiotherapy) is the treatment of choice in localized disease. The treatment for advanced disease remains uncertain. In order to better evaluate prognostic factors and outcome, a retrospective pooled analysis of patients with EMC treated at three Italian Sarcoma Group (ISG) referral centers was carried out. METHODS: All patients with localized EMC surgically treated from 1989 to 2016 were identified. Diagnosis was centrally reviewed according to WHO 2013. Only patients with NR4A3 rearrangement were included. RESULTS: Sixty-seven patients were identified: 13 (20%) female, 54 (80%) male. Median age was 56 years (range 18-84). Numbers and type of translocation were: 50 (80%) NR4A3-EWS, 10 (16%) NR4A3-TAF15, 1 (2%) NR4A3-TCF12, and 1 (2%) NR4A3-TFG. Median follow-up was 55 months (range 2-312). Five- and ten-year overall survival rates were 94% (86-100 95%CI) and 84% (69-98 95%CI). Thirty-five (52%) patients relapsed: 9 had local recurrence (LR) and 26 had distant metastasis (5 with concomitant LR). The 5- and 10-year disease-free survival rates (DFS) were 51% (38-65 95%CI) and 20% (7-33 95%CI). Size of the primary tumor was significantly related to distant metastasis-free survival (DMFS) (p = 0.004). Patients carrying the NR4A3-EWS translocation had a trend in favor of better DFS (p = 0.08) and DMFS (p = 0.09) compared with the patients with NR4A3-TAF15. CONCLUSIONS: Prolonged survival can be expected in patients with EMC, in spite of a high rate of recurrence. Size is significantly associated with distant relapse. The type of NR4A3 translocation could influence outcome.

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.

Synovial chondrosarcoma: a single-institution experience with molecular investigations and review of the literature.

AIMS: To evaluate the available diagnostic histological criteria for synovial chondrosarcoma and to screen for the presence of IDH1/IDH2 mutations in a series of cases of this malignant cartilaginous neoplasm. METHODS AND RESULTS: Ten cases of synovial chondrosarcoma diagnosed at our institute were reviewed. At presentation, all tumours occurred in adults (median age, 62 years). The most common location was the knee joint (five cases), and the size at diagnosis ranged from 30 mm to 170 mm. Eight patients had secondary synovial chondrosarcomas associated with pre-existing/recurrent or concomitant synovial chondromatosis. Five patients had local recurrences and three had lung metastases. All patients with intralesional excisions developed local recurrences, whereas those who underwent wide resections did not. At last follow-up (mean, 91 months), available for nine patients, seven patients were alive and disease-free, one patient had died of disease, and one was alive with paravertebral metastases. Frequent histological features observed included loss of clustering of chondrocytes (nine cases), the presence of variable amounts of myxoid matrix (eight cases), peripheral hypercellularity (eight cases), tumour necrosis (six cases), and spindling of chondrocytes (four cases). Of the seven cases for which it was possible to evaluate bone permeation, six showed infiltration of bone marrow. All seven cases screened for mutations of exon 4 of IDH1 and IDH2 were found to be wild-type. CONCLUSIONS: Histological criteria in correlation with clinical and radiological features allow the recognition of synovial chondrosarcoma. IDH1/IDH2 mutations were not present in synovial chondrosarcoma. Adequate surgical margins are important for disease control.

Primary Vascular Tumors of Bone: A Monoinstitutional Morphologic and Molecular Analysis of 427 Cases With Emphasis on Epithelioid Variants.

Recent molecular discoveries have refined vascular bone tumor classification. To investigate the clinical relevance of these refinements, we reviewed all cases of primary vascular bone tumors treated at our Institute. On the basis of morphology, cases were assessed immunohistochemically and molecularly. A total of 427 cases of primary vascular tumor of bone with available follow-up and histologic material were retrieved and reclassified according to the most recent diagnostic criteria as follows: 289 hemangiomas, 38 epithelioid hemangiomas, 21 epithelioid hemangioendotheliomas, 2 retiform hemangioendotheliomas, 1 intraosseous papillary intralymphatic angioendothelioma, 24 pseudomyogenic hemangioendotheliomas, and 52 angiosarcomas (of these, 45 were epithelioid angiosarcomas and 7 spindle cell secondary angiosarcoma). Both epithelioid and classic hemangiomas behave as benign tumors with excellent prognosis. The distinction between cellular and conventional type of epithelioid hemangioma was not associated with a different clinical course. Conversely, epithelioid hemangioendothelioma exhibited a more aggressive clinical behavior than hemangioma, with higher rates of multifocality and distant spread. Immunohistochemical positivity for CAMTA1 or TFE3 did not have a prognostic implication. In epithelioid hemangioendothelioma, the presence of morphologic malignant features was associated with reduced disease-free (P=0.064) and overall survival (P=0.055). Pseudomyogenic hemangioendothelioma featured local aggressiveness in 5/24 patients exhibiting a clinical behavior closer to epithelioid hemangioma than epithelioid hemangioendothelioma. Last, 32/45 patients with epithelioid angiosarcoma died of disease with a median survival time of 10 months from diagnosis. In conclusion, the integration of morphologic, immunohistochemical, and molecular features allows a better stratification of primary vascular tumors of bone with significant prognostic and therapeutic implications.

Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth, migration, and invasion.

Osteosarcoma (OS) is a rare, insidious tumor of mesenchymal origin that most often affects children, adolescents, and young adults. While the primary tumor can be controlled with chemotherapy and surgery, it is the lung metastases that are eventually fatal. Multiple studies into the initial drivers of OS development have been undertaken, but few of these have examined innate immune/inflammatory signaling. A central figure in inflammatory signaling is the innate immune/stress-activated kinase double-stranded RNA-dependent protein kinase (PKR). To characterize the role of PKR in OS, U2OS, and SaOS-2 osteosarcoma cell lines were stably transfected with wild-type or dominant-negative (DN) PKR. Overexpression of PKR enhanced colony formation in soft agar (U2OS and SaOS-2), enhanced cellular migration (U2OS), and invasive migration (SaOS-2). In contrast, overexpression of DN-PKR inhibited attachment-independent growth, migration and/or invasion. These data demonstrate a role for inflammatory signaling in OS formation and migration/invasion and suggest the status of PKR expression/activation may have prognostic value.

Identification of a novel fusion transcript EWSR1-VEZF1 by anchored multiplex PCR in malignant peripheral nerve sheath tumor.

The aim of the study is to describe a novel genetic finding examining the molecular and pathological features of a case of malignant peripheral nerve sheath tumor occurring in the thigh of a 17-year-old male. Fusion gene detection using a next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) was used to identify the novel fusion of EWSR1-VEZF1 from the frozen tumor sample. EWSR1-VEZF1 fusion is a novel molecular gene rearrangement involving exon 8 of the EWSR1 gene and exon 2 of the VEZF1 gene. Data were validated with gene sequencing and fluorescent in situ hybridization (FISH) analysis. This case report describes a novel rearrangement involving EWSR1 on chromosome 22 and VEZF1 on chromosome 17. The result obtained demonstrates the value of the next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) both in diagnosis and patient care and might become a helpful diagnostic tool for this tumor type.

Unlocking bone for proteomic analysis and FISH.

Bone tissue is critically lagging behind soft tissues and biofluids in our effort to advance precision medicine. The main challenges have been accessibility and the requirement for deleterious decalcification processes that impact the fidelity of diagnostic histomorphology and hinder downstream analyses such as fluorescence in-situ hybridization (FISH). We have developed an alternative fixation chemistry that simultaneously fixes and decalcifies bone tissue. We compared tissue morphology, immunohistochemistry (IHC), cell signal phosphoprotein analysis, and FISH in 50 patient matched primary bone cancer cases that were either formalin fixed and decalcified, or theralin fixed with and without decalcification. Use of theralin improved tissue histomorphology, whereas overall IHC was comparable to formalin fixed, decalcified samples. Theralin-fixed samples showed a significant increase in protein and DNA extractability, supporting technologies such as laser-capture microdissection and reverse phase protein microarrays. Formalin-fixed bone samples suffered from a fixation artifact where protein quantification of ß-actin directly correlated with fixation time. Theralin-fixed samples were not affected by this artifact. Moreover, theralin fixation enabled standard FISH staining in bone cancer samples, whereas no FISH staining was observed in formalin-fixed samples. We conclude that the use of theralin fixation unlocks the molecular archive within bone tissue allowing bone to enter the standard tissue analysis pipeline. This will have significant implications for bone cancer patients, in whom personalized medicine has yet to be implemented.

miR­494.3p expression in synovial sarcoma: Role of CXCR4 as a potential target gene.

Synovial sarcoma (SS) is a rare tumour, with dismal survival when metastasis occurs. SS contains a characteristic translocation (X;18)(p11;q11) and the fusion genes appear to be mutually exclusive and concordant in primary and metastatic tumours. Novel prognostic and predictive factors are required. The C­X­C motif chemokine ligand 12 (CXCL12)/C­X­C chemokine receptor 4 (CXCR4) axis is involved in tumour development and metastatic spread in many types of cancer and previous data have demonstrated a pivotal role of CXCR4 in SS cell migration and invasion. Bioinformatics and biological data indicated CXCR4 is a possible candidate target of miR­494.3p, known to be involved in tumour progression. In this study, we analysed the expression of miR­494.3p and its potential target, CXCR4, in a series of SS specimens. A significantly lower miR­494.3p expression was found in the tumour compared to normal tissue associated with higher levels of CXCR4 both at the gene and protein level. The role of CXCR4 as a potential target of miR­494.3p was assessed in two SS cell lines (SW982 and SYO­I). Transfection with miR­494.3p expression plasmid led to a marked decrease in CXCR4 gene and protein expression, concomitant with a transitory decrease in cell proliferation and migration. The SYO­I cells also responded with an increased apoptotic fraction. The data of this study also demonstrate that the downregulation of miR­494.3p in SS surgical specimens, concomitant with an increased expression of its potential target, CXCR4, was more evident in the metastatic subset. In vitro experiments confirmed that miR­494.3p functioned as a tumour suppressor through the involvement of CXCR4 and ongoing studies are directed to better clarify its role in SS therapeutic strategies.