Percutaneous guided biopsy for diagnosing suspected primary malignant bone tumors in pediatric patients: a safe, accurate, and cost-saving procedure.

BACKGROUND: Percutaneous biopsy is the reference diagnostic procedure for adult musculoskeletal tumors. Its place in pediatrics is controversial and open biopsy remains recommended. OBJECTIVE: To assess diagnostic performance and feasibility of percutaneous biopsy performed on children and young adults for suspected malignant bone tumors. MATERIALS AND METHODS: We conducted a 5-year retrospective study including patients ≤21 years who underwent a bone biopsy for a suspected malignant bone tumor. We assessed diagnostic yield (percentage of analyzable biopsies), accuracy (percentage of accurate diagnoses among all analyzable biopsies) and efficacy (percentage of accurate diagnoses among all biopsies), costs, anesthetic requirements and sample availability for biomedical research. Patients diagnosed with an open biopsy were used to compare diagnostic performances, anesthetic requirements and costs. RESULTS: We included 90 percutaneous and 27 open biopsies in 117 patients. For percutaneous biopsy, diagnostic yield was 95.5% (95% confidence interval [CI] 88.8-98.7%), accuracy was 96.2% (95% CI 86.8-99.5%) and efficacy was 89.3% (95% CI 78.1-96.0%). There was no statistical difference with open biopsy (Fisher exact test, P > 0.05). Mean costs were reduced with percutaneous biopsy: €1,937 (standard deviation [SD] €2,408) versus €6,362 (SD €5,033; Mann-Whitney, P < 0.0001). Thirty-two of the 48 (67%) patients included in clinical trials and diagnosed with percutaneous biopsy had suitable samples for ancillary analyses. CONCLUSION: Percutaneous biopsy is a valid alternative to open biopsy for diagnosing pediatric and young adult primary malignant bone tumors.

Prediction of desmoid tumor progression using miRNA expression profiling.

Desmoid tumor is a rare connective tissue tumor with locoregional aggressiveness but unpredictable behavior. The miRNA profile was ascertained for 26 patients included in the Desminib phase II trial and an independent validation cohort of 15 patients. Predictive and prognostic supervised analysis on the Desminib cohort failed to identify miRNAs differentially expressed between progressive and non-progressive patients under imatinib treatment or between progressive and non-progressive patients after discontinuation of imatinib. However, an unsupervised hierarchical clustering of the Desminib cohort identified two groups (A and B) of 13 patients each, where only the number of previous lines of treatment before inclusion in the study differed significantly between the two groups. Time to progression after discontinuation of imatinib was longer in group B than in group A. Fifteen miRNAs were highly statistically differentially expressed between groups A and B, targeting more than 3000 genes, including AGO1, BCL2, CDK6, SMAD4, PTEN, CCND1, VEGFA, and RB1. These results were confirmed in the independent validation cohort: hierarchical clustering of these 15 miRNAs identified two groups, in which time to recurrence was statistically different (28.8 months vs 68.8 months). These results provide the first indication of the prognostic value of miRNA expression profiling with a possible direct impact on patient management. A more precise miRNA signature must now be determined to select patients who would not benefit from surgical resection of their tumor and who ought to be monitored without treatment.

Chromosome 12 long arm rearrangement covering MDM2 and RASAL1 is associated with aggressive craniofacial juvenile ossifying fibroma and extracranial psammomatoid fibro-osseous lesions.

To evaluate the diagnostic value of MDM2 status in craniofacial fibro-osseous lesions, we investigated MDM2 expression by immunohistochemistry and analyzed MDM2 amplification by qPCR in 30 cases of ossifying fibroma (including 13 cases of the juvenile variant) and 17 cases of fibrous dysplasia. Two cases of uncommon extragnathic psammomatoid fibrous dysplasia and a mixed control group of 15 cases of low-grade osteosarcoma and 15 cases of well-differentiated/dedifferentiated liposarcoma were included. MDM2 amplification was found in 33% of ossifying fibromas (peak of 69% for the juvenile variant) and in 12% of fibrous dysplasia, in none of which was MDM2 overexpressed. All control cases exhibited MDM2 amplification and overexpression. To investigate possible polysomy of chromosome 12, we studied RASAL1 amplification, a gene telomeric to MDM2 on the long arm of chromosome 12. RASAL1 amplification was reported in all benign fibro-osseous lesions exhibiting MDM2 amplification but not in controls. Simultaneous amplification of these two genes was significantly higher in juvenile ossifying fibromas compared with fibrous dysplasia (P=0.004), non-juvenile ossifying fibromas (P=0.001), and all other benign craniofacial fibro-osseous lesions combined (P=0.0001). Of the nine cases of juvenile ossifying fibroma exhibiting amplification, three were locally invasive and four were recurrent, suggesting aggressive disease. The two cases of extragnathic psammomatoid fibrous dysplasia also showed MDM2 and RASAL1 amplification with no MDM2 overexpression. This large chromosome 12 rearrangement, spanning MDM2 and RASAL1, is the first recurrent molecular abnormality to be reported in juvenile ossifying fibroma. It may represent both a molecular diagnostic marker and a characteristic of more aggressive forms with a higher risk of recurrence. Finally, the presence of this rearrangement in extragnathic psammomatoid fibro-osseous lesions mimicking ossifying fibromas might reflect a common molecular pathway in their pathogenesis and calls into question the classification of such lesions within fibrous dysplasia.

KIT exon 10 variant (c.1621 A > C) single nucleotide polymorphism as predictor of GIST patient outcome.

BACKGROUND: Tumor genotype plays a crucial role in clinical management of GIST. Whether genetic polymorphism of KIT may influence GIST patient outcome is unclear. METHODS: We investigated the biological and clinical significance of the presence of KIT exon 10 variant (c.1621 A > C), KIT (L541), in a transfected cell line (3 T3 L541) and in two retrospectively collected series of 109 GIST patients in total. The control group consisted of 60 healthy donors collected at the French department of blood transfusion. RESULTS: In the 3 T3 L541 cell line, KIT(L541) protein exhibited a spontaneous phosphorylation status comparable to that of wild-type KIT but displayed a phosphorylation pattern of AKT and ERK1/2 that was found similar to that of the classical mutated forms of the KIT receptor. Of 109 patients enrolled in this retrospective translational research study, 24 (22%) harboured KIT (L541), similarly to the control group of healthy donors (n = 10 of 60, 17%). A higher prevalence of the variant KIT (L541) was observed in patients with metastatic status at diagnosis (KIT (L541) correlated nine of 22 versus 15 of 87, p = 0.02). In addition, patients with KIT (L541) and localized GIST had a higher rate of relapse at 5 years and lower relapse free survival at 5 years in univariate, as well as in multivariate analysis. Response rate and duration of response to imatinib was similar in KIT (L541) and KIT (M541) patients. CONCLUSION: KIT (L541) genotype is associated with a higher risk of metastasis at diagnosis and a higher risk of relapse in GIST patients.

[Immunohistochemistry in the diagnosis of sarcomas]. / Apport de l'immuno-histochimie dans le diagnostic des sarcomes.

Immunohistochemistry (IHC) is essential in the diagnosis of soft tissue tumor and must rely on good quality technic. Among useful antibodies, it is important to distinguish those with a poor specificity required in order to establish the broad lineage, from those with high specificity, which may lead straightforward towards the entity. Diagnostically useful antibodies such as myogenin, ALK1 and DOG1 have been recently completed by MUC4 and STAT6 which show good sensitivity and specificity in the diagnosis of low-grade fibromyxoid sarcoma and solitary fibrous tumor respectively. ERG is also an interesting antibody. However, it is not completely specific of vascular tumors. Moreover, available material is often limited because of the increase of microbiopsy specimens. Therefore, it is mandatory to optimize this precious tissue by using these new antibodies, especially because molecular technics are increasingly performed in addition to IHC.

Epstein-Barr virus infection induces an increase of T regulatory type 1 cells in Hodgkin lymphoma patients.

Epstein-Barr Virus (EBV) is present in the neoplastic cells of around 20-30% of patients with Hodgkin Lymphoma (HL). Although, an immunosuppressive environment is currently described in HL patients, little is known concerning the regulatory mechanism induced by EBV proteins expression in tumour cells. This study aimed to investigate an association between regulatory Type 1 cells (Tr1) and EBV tissue positivity in HL patients. Transcriptomic analysis of both EBV-positive and EBV-negative tumours showed that EBV infection increased gene expression of Tr1-related markers (ITGA2, ITGB2, LAG3) and associated-immunosuppressive cytokines (IL10). This up-regulation was associated with an over-expression of several chemokine markers known to attract T-helper type 2 (Th2) and regulatory T cells thus contributing to immune suppression. This Tr1 cells recruitment in EBV-positive HL was confirmed by immunohistochemical analysis of frozen nodes biopsies and by flow cytometric analysis of peripheral blood mononuclear cells of EBV-positive patients. Additionally, we showed that IL10 production was significantly enhanced in tumours and blood of EBV-positive HL patients. Our results propose a new model in which EBV can recruit Tr1 cells to the nodes' microenvironment, suggesting that the expression of EBV proteins in tumour cells could enable the escape of EBV-infected tumour cells from the virus-specific CTL response.

Comprehensive genome characterization of solitary fibrous tumors using high-resolution array-based comparative genomic hybridization.

Solitary fibrous tumors (SFTs) are rare spindle cell tumors with limited therapeutic options. Their molecular basis is poorly known. No consistent cytogenetic abnormality has been reported. We used high-resolution whole-genome array-based comparative genomic hybridization (Agilent 244K oligonucleotide chips) to profile 47 samples, meningeal in >75% of cases. Few copy number aberrations (CNAs) were observed. Sixty-eight percent of samples did not show any gene CNA after exclusion of probes located in regions with referenced copy number variation (CNV). Only low-level CNAs were observed. The genomic profiles were very homogeneous among samples. No molecular class was revealed by clustering of DNA copy numbers. All cases displayed a "simplex" profile. No recurrent CNA was identified. Imbalances occurring in >20%, such as the gain of 8p11.23-11.22 region, contained known CNVs. The 13q14.11-13q31.1 region (lost in 4% of cases) was the largest altered region and contained the lowest percentage of genes with referenced CNVs. A total of 425 genes without CNV showed copy number transition in at least one sample, but only but only 1 in at least 10% of samples. The genomic profiles of meningeal and extra-meningeal cases did not show any differences.

Diagnostic value of investigating GNAS mutations in fibro-osseous lesions: a retrospective study of 91 cases of fibrous dysplasia and 40 other fibro-osseous lesions.

GNAS (guanine nucleotide-binding protein/α-subunit) mutations that induce the activation of G-protein α-subunit participate in the pathogenesis of fibrous dysplasia. The aim of this study was to evaluate the sensitivity and specificity of GNAS mutations in fibrous dysplasia and other fibro-osseous lesions, to assess the value of investigating this mutation in the diagnosis of fibro-osseous lesions. We studied 91 cases of fibrous dysplasia. The quality and/or quantity of genomic DNA were suitable for molecular analysis for 51 cases of fibrous dysplasia. GNAS mutations were investigated by three techniques: high-resolution melting (exon 8), allele-specific PCR (exons 8 and 9) and/or direct DNA sequencing (exons 8 and 9). Fibrous dysplasia samples were classified blind to the GNAS mutation status into six histological subtypes as conventional, fibro-involutive, osteosclerosing, cementifying, osteocartilaginous and with prominent aneurysmal cystic changes. We also studied 14 cases of low-grade osteosarcoma, 21 cases of ossifying fibroma, 3 cases of osteofibrous dysplasia, 1 case of osseous dysplasia of the jawbone and 1 post-traumatic lesion of the ribs. Twenty-three cases of fibrous dysplasia (45%) showed mutations of codon 201 (exon 8, p.R201H or p.R201C). No mutation was found on codon 227 (exon 9). GNAS mutations in conventional fibrous dysplasia were detected in the same proportion (47%) as in the other histological subtypes (47%, P=0.96), regardless of sex (P=0.44), age (P=0.90) and location (P=1). GNAS mutations were not detected in any other fibro-osseous lesions. The GNAS mutation was thus specific to fibrous dysplasia in the context of fibro-osseous lesions. The particular mosaicism of mutant and non-mutant cells within the lesion or the existence of other mutations not already described could explain the lack of GNAS mutation in cases of fibrous dysplasia. Investigating this mutation may constitute a valuable complementary diagnostic tool, despite its low sensitivity, particularly in unconventional morphologically different subtypes of fibrous dysplasia.

Clinicians' adherence versus non adherence to practice guidelines in the management of patients with sarcoma: a cost-effectiveness assessment in two European regions.

BACKGROUND: Although the management of sarcoma is improving, non adherence to clinical practice guidelines (CPGs) remains high, mainly because of the low incidence of the disease and the variety of histological subtypes. Since little is known about the health economics of sarcoma, we undertook a cost-effectiveness analysis (within the CONnective TIssue CAncer NETwork, CONTICANET) comparing costs and outcomes when clinicians adhered to CPGs and when they did not. METHODS: Patients studied had a histological diagnosis of sarcoma, were older than 15 years, and had been treated in the Rhône-Alpes region of France (in 2005/2006) or in the Veneto region of Italy (in 2007). Data collected retrospectively for the three years after diagnosis were used to determine relapse free survival and health costs (adopting the hospital's perspective and a microcosting approach). All costs were expressed in euros (€) at their 2009 value. A 4% annual discount rate was applied to both costs and effects. The incremental cost-effectiveness ratio (ICER) was expressed as cost per relapse-free year gained when management was compliant with CPGs compared with when it was not. To capture uncertainty surrounding ICER, a probabilistic sensitivity analysis was performed based on a non-parametric bootstrap method. RESULTS: A total of 219 patients were included in the study. Compliance with CPGs was observed for 118 patients (54%). Average total costs reached 23,571 euros when treatment was in accordance with CPGs and 27,313 euros when it was not. In relation to relapse-free survival, compliance with CPGs strictly dominates non compliance, i.e. it is both less costly and more effective. Taking uncertainty into account, the probability that compliance with CPGs still strictly dominates was 75%. CONCLUSIONS: Our findings should encourage physicians to increase their compliance with CPGs and healthcare administrators to invest in the implementation of CPGs in the management of sarcoma.

[Place of the pathologist in the management of primary bone tumors (osteosarcoma and Ewing's family tumors after neoadjuvant treatment)]. / Rôle du pathologiste dans la prise en charge des tumeurs osseuses primitives malignes : ostéosarcomes et tumeurs de la famille Ewing après traitement néoadjuvant.

The survival of osteosarcoma and Ewing family tumours has been improved by the introduction of neoadjuvant chemotherapy. The response to preoperative chemotherapy is evaluated on the microscopic analysis of the surgical resection, by the percentage of tumour necrosis according to the Huvos and Rosen's grading. It remains the only reliable prognostic factor for patients and is used to guide the choice of post-operative chemotherapy. The macroscopic and microscopic management of the surgical resection (cf. supra) is essential and is the subject of a specific protocol. Several studies have been conducted to identify news factors able to predict the response to chemotherapy, the tumour aggressiveness and its ability to develop metastases. Inhibitors of mTOR and/or regulators of the balance RANKL/OPG are promising therapeutics. The study's expression of these new factors could be performed on the biopsy and will offer new therapeutic strategy.

miRNA Profiling: How to Bypass the Current Difficulties in the Diagnosis and Treatment of Sarcomas.

Sarcomas are divided into a group with specific alterations and a second presenting a complex karyotype, sometimes difficult to diagnose or with few therapeutic options available. We assessed if miRNA profiling by TaqMan low density arrays could predict the response of undifferentiated rhabdomyosarcoma (RMS) and osteosarcoma to treatment. We showed that miRNA signatures in response to a therapeutic agent (chemotherapy or the mTOR inhibitor RAD-001) were cell and drug specific on cell lines and a rat osteosarcoma model. This miRNA signature was related to cell or tumour sensitivity to this treatment and might be not due to chromosomal aberrations, as revealed by a CGH array analysis of rat tumours. Strikingly, miRNA profiling gave promising results for patient rhabdomyosarcoma, discriminating all types of RMS: (Pax+) or undifferentiated alveolar RMS as well as embryonal RMS. As highlighted by these results, miRNA profiling emerges as a potent molecular diagnostic tool for complex karyotype sarcomas.

Epidemiological evaluation of concordance between initial diagnosis and central pathology review in a comprehensive and prospective series of sarcoma patients in the Rhone-Alpes region.

BACKGROUND: Sarcomas are rare malignant tumors. Accurate initial histological diagnosis is essential for adequate management. We prospectively assessed the medical management of all patients diagnosed with sarcoma in a European region over a one-year period to identify the quantity of first diagnosis compared to central expert review (CER). METHODS: Histological data of all patients diagnosed with sarcoma in Rhone-Alpes between March 2005 and Feb 2006 were collected. Primary diagnoses were systematically compared with second opinion from regional and national experts. RESULTS: Of 448 patients included, 366 (82%) matched the inclusion criteria and were analyzed. Of these, 199 (54%) had full concordance between primary diagnosis and second opinion (the first pathologist and the expert reached identical conclusions), 97 (27%) had partial concordance (identical diagnosis of conjonctive tumor but different grade or subtype), and 70 (19%) had complete discordance (different histological type or invalidation of the diagnosis of sarcoma). The major discrepancies were related to histological grade (n = 68, 19%), histological type (n = 39, 11%), subtype (n = 17, 5%), and grade plus subtype or grade plus histological type (n = 43, 12%). CONCLUSIONS: Over 45% of first histological diagnoses were modified at second reading, possibly resulting in different treatment decisions. Systematic second expert opinion improves the quality of diagnosis and possibly the management of patients.

Efficacy of trabectedin for advanced sarcomas in clinical trials versus compassionate use programs: analysis of 92 patients treated in a single institution.

Trabectedin was recently approved for patients failing doxorubicin, the standard treatment for advanced/metastatic sarcoma. This retrospective study aimed to compare trabectedin efficacy between compassionate use in unselected patients and clinical trials. From May 1999 to January 2006, 92 patients were treated at the Centre Léon Bérard, either in phase II studies or on a named patient compassionate basis. All cases were retrospectively analyzed to assess trabectedin efficacy in terms of response, progression-free, and overall survival.The objective response rate was 10% (N=9): 4% (N=2) for patients treated in compassionate use program and 16% (N=7) for those in clinical trials (P=0.18); 26 (28%) patients had stable disease for at least 6 months, 11 (23%) in the compassionate group and 15 (33%) in clinical trials. Median progression-free and overall survivals were, respectively, 2.2 [95% confidence interval (CI): 1.9-3.6] and 8.9 (95% CI: 6.4-14.2) months for all patients, 2.3 (95% CI: 1.9-4.3) and 10.4 (95% CI: 6.9-24.2) months for patients in clinical trials and 1.8 (95% CI: 1.4-3.4) and 6.4 (95% CI: 3.3-14.2) months for patients under compassionate treatment. In this retrospective analysis, the reported grade 3-4 toxicities were increased transaminase (34 patients, 37%) and neutropenia (38 patients; 42%). Higher efficacy was observed in phase II studies than with compassionate treatment, but no significant difference remained after adjustment in multivariate analysis for performance status, a well-established prognosis factor. The safety and tolerability of trabectedin shown in clinical trials is confirmed for patients in real-life situation treated in compassionate use programs, but its benefit is higher for patients with performance status 0-1.

18F-FDG PET SUVmax correlates with osteosarcoma histologic response to neoadjuvant chemotherapy: preclinical evaluation in an orthotopic rat model.

UNLABELLED: Assessment of osteosarcoma response to neoadjuvant chemotherapy is performed by histopathologic analysis after surgical resection of the primary tumor. The purpose of this study was to evaluate whether (18)F-FDG PET could be a noninvasive surrogate to histopathologic analysis and allow for earlier response evaluation to neoadjuvant chemotherapy in osteosarcoma. METHODS: Metabolic response to neoadjuvant chemotherapy was assessed in immunocompetent rats with a preestablished orthotopic osteosarcoma using (18)F-FDG PET before and after receiving 2 doses of ifosfamide. Comparison was then made by assessing histologic responses on euthanized animals. RESULTS: Maximum standardized uptake value (SUVmax) measured by (18)F-FDG PET after 2 doses of chemotherapy was correlated to histologic classification (P < 0.01). An SUVmax less than 15 corresponded to good responders, whereas an SUVmax greater than 15 but less than 20 and an SUVmax greater than 20 corresponded to partial responders or nonresponders, respectively. A 40% decrease in SUVmax between the first and second (18)F-FDG PET scans distinguished between partial and good response to chemotherapy. CONCLUSION: Determination of SUVmax using semiquantitative (18)F-FDG PET predicts response to neoadjuvant chemotherapy earlier than does histologic analysis.

Ezrin immunohistochemical expression in cartilaginous tumours: a useful tool for differential diagnosis between chondroblastic osteosarcoma and chondrosarcoma.

Ezrin is a cytoskeleton linker protein that is actively involved in the metastatic process of cancer cells. We have recently reported that ezrin expression in conventional osteosarcoma was an independent prognostic factor for event-free survival and overall survival. In this work, ezrin expression was found in all histological subtypes. Especially cartilaginous areas in chondroblastic osteosarcomas were immunopositive for ezrin. We wanted to know if ezrin could be a useful diagnostic marker in bone pathology. We have searched for ezrin expression in 208 cartilaginous tumours by immunohistochemistry and in 16 chondroblastic osteosarcomas. All conventional chondrosarcomas, whatever their grade, were negative, while ten of 16 chondroblastic osteosarcomas were positive. In contrast, dedifferentiated (five of 14) and mesenchymal chondrosarcomas (five of ten) showed ezrin positivity. Some chondroblastomas and more rarely chondromyxoid fibromas also exhibited ezrin expression. These data suggest that ezrin is a useful immunohistochemical marker for differential diagnosis between chondroblastic osteosarcomas and conventional chondrosarcomas with a specificity of 100%. Ezrin expression in dedifferentiated and mesenchymal chondrosarcomas which are aggressive neoplasms resistant to conventional treatment means that ezrin could be a therapeutic target. Ezrin expression in chondroblastomas is more intriguing and requires further study to assess prognostic value.

T cell receptor genotyping and HOXA/TLX1 expression define three T lymphoblastic lymphoma subsets which might affect clinical outcome.

PURPOSE: T lymphoblastic lymphomas (T-LBL) are rare disorders of immature T cells which predominantly involve the mediastinum. Their oncogenic pathways and prognostic variables are not clear. EXPERIMENTAL DESIGN: We undertook a retrospective study of 41 cytoplasmic CD3+ T-LBL (nine cases aged <16 years) by assessing stage of maturation arrest based on T cell receptor (TCR) immunogenotyping, immunohistochemistry, and quantification of the oncogenes thought to be important in immature T cell malignancies. RESULTS: Application of a TCR-based immunogenetic classification allowed the identification of three subcategories: 11 immature IM0/D-LBL showed no TCR or only incomplete TCRD DJ rearrangement and corresponded to cytoplasmic CD3+ precursors of uncertain lineage. Sixteen mature TCRD(del)-LBL showed biallelic TCRD deletion and both TCRG and TCRB rearrangement, consistent with TCRalphabeta lineage restriction. Fourteen intermediate LBL (Int-LBL) showed complete TCRD VDJ and TCRG VJ rearrangement, with TCRB VDJ rearrangement in the majority. All Int-LBL expressed HOX11/TLX1 or HOXA9 transcripts and a proportion of the latter were associated with CALM-AF10 or NUP214-ABL fusion transcripts. IM0/D-LBL were restricted to adults with extrathymic disease and bone marrow involvement, whereas Int-LBL and TCRD(del)-LBL were found in children and adults with predominantly thymic disease. In adults, the Int-LBL subgroup was associated with a significantly superior clinical outcome. This subgroup can be identified either by TCR immunogenotyping or HOXA9/TLX1 transcript quantification. CONCLUSION: Application of this molecular classification will allow the prospective evaluation of prognostic effects within pediatric and adult protocols.

Integrated molecular characterization of chondrosarcoma reveals critical determinants of disease progression.

Chondrosarcomas are primary cancers of cartilaginous tissue with highly contrasting prognoses. These tumors are defined by recurrent mutations in the IDH genes and other genetic alterations including inactivation of CDKN2A and COL2A1; however, these have no clinical value. Here we use multi-omics molecular profiles from a series of cartilage tumors and find an mRNA classification that identifies two subtypes of chondrosarcomas defined by a balance in tumor differentiation and cell cycle activation. The microRNA classification reveals the importance of the loss of expression of the 14q32 locus in defining the level of malignancy. Finally, DNA methylation is associated with IDH mutations. We can use the multi-omics classifications to predict outcome. We propose an mRNA-only classifier to reproduce the integrated multi-omics classification, and its application to relapsed tumor samples shows the progressive nature of the classification. Thus, it may be possible to use mRNA-based signatures to detect patients with high-risk chondrosarcomas.