Maria Fernanda Amary

Genome-wide association study identifies two susceptibility loci for osteosarcoma

Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. To better understand the genetic etiology of osteosarcoma, we performed a multistage genome-wide association study consisting of 941 individuals with osteosarcoma (cases) and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: a locus in the GRM4 gene at 6p21.3 (encoding glutamate receptor metabotropic 4; rs1906953; P = 8.1 × 10−9) and a locus in the gene desert at 2p25.2 (rs7591996 and rs10208273; P = 1.0 × 10−8 and 2.9 × 10−7, respectively). These two loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.

Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma

Synovial Sarcoma consistently harbors t(X;18) resulting in SS18-SSX1, SS18-SSX2 and rarely SS18-SSX4 fusion transcripts. Of 328 cases included in our study, synovial sarcoma was either the primary diagnosis or was very high in the differential diagnosis in 134 cases: of these, amplifiable cDNA was obtained from 131. SS18-SSX fusion products were found in 126 (96%) cases (74 SS18-SSX1, 52 SS18-SSX2), using quantitative and 120 by conventional reverse transcriptase–polymerase chain reaction (RT-PCR). One hundred and one cases in a tissue microarray, analyzed by fluorescence in situ hybridization (FISH), revealed that 87 (86%) showed SS18 rearrangement: four RT-PCR positive cases, reported as negative for FISH, showed loss of one spectrum green signal, and 15 cases had multiple copies of the SS18 gene: both findings are potentially problematic when interpreting results. One of three cases, not analyzed by RT-PCR reaction owing to poor quality RNA, was positive by FISH. SS18-SSX1 was present in 56 monophasic and 18 biphasic synovial sarcoma: SS18-SSX2 was detected in 41 monophasic and 11 biphasic synovial sarcoma. Poorly differentiated areas were identified in 44 cases (31%). There was no statistically significant association between biphasic, monophasic and fusion type. Five cases were negative for SS18 rearrangement by all methods, three of which were pleural-sited neoplasms. Following clinical input, a diagnosis of mesothelioma was favored in one case, a sarcoma, not otherwise specified in another and a solitary fibrous tumor in the third case. The possibility of a malignant peripheral nerve sheath tumor could not be excluded in the other two cases. We concluded that the employment of a combination of molecular approaches is a powerful aid to diagnosing synovial sarcoma giving at least 96% sensitivity and 100% specificity but results must be interpreted in the light of other modalities such as clinical findings and immunohistochemical data.

The role of epidermal growth factor receptor in chordoma pathogenesis: a potential therapeutic target.

Chordoma, the molecular hallmark of which is T (brachyury), is a rare malignant bone tumour with a high risk of local recurrence and a tumour from which metastatic disease is a common late event. Currently, there is no effective drug therapy for treating chordomas, although there is evidence that some patients respond to the empirical use of epidermal growth factor receptor (EGFR) antagonists. The aim of this study was to determine the role of EGFR in the pathogenesis of chordoma. Paraffin‐embedded material from 173 chordomas from 160 patients [sacro‐coccygeal (n = 94), skull‐based (n = 50), and mobile spine (n = 16)] was analysed by immunohistochemistry and revealed total EGFR expression in 69% of cases analysed. Of 147 informative chordomas analysed by FISH, 38% revealed high‐level EGFR polysomy, 4% high‐level polysomy with focal amplification, 18% low‐level polysomy, and 39% disomy. Phospho‐receptor tyrosine kinase array membranes showed EGFR activation in the chordoma cell line U‐CH1 and all of the three chordomas analysed. Direct sequencing of EGFR (exons 18–21), KRAS, NRAS, HRAS (exons 2, 3), and BRAF (exons 11, 15) using DNA from 62 chordomas failed to reveal mutations. PTEN expression was absent by immunohistochemistry in 19 of 147 (13%) analysed chordomas, only one of which revealed high‐level polysomy of EGFR. The EGFR inhibitor tyrphostin (AG 1478) markedly inhibited proliferation of the chordoma cell line U‐CH1 in vitro and diminished EGFR phosphorylation in a dose‐dependant manner, a finding supported by inhibition of phosphorylated Erk1/2. p‐Akt was suppressed to a much lesser degree in these experiments. There was no reduction of T as assessed by western blotting. These data implicate aberrant EGFR signalling in the pathogenesis of chordoma. This study provides a strategy for patient stratification for treatment with EGFR antagonists. Copyright

Detection of USP6 gene rearrangement in nodular fasciitis: an important diagnostic tool

Dear Editor, Nodular fasciitis is a lesion that is well known for posing a diagnostic challenge to pathologists. The clinical presentation of sudden onset and fast growing rate of this soft tissue lesion along with histological features, comprising a poorly demarcated cellular myofibroblastic mitotically active lesion, frequently raises concern of malignancy. There are no specific immunohistochemical markers for nodular fasciitis and the diagnosis, until recently, was based on histological features alone. The age of occurrence and anatomical site are unhelpful in making the diagnosis as these lesions can occur at any age and at any anatomic soft tissue site, although the most frequent site is the forearm and the trunk and is more common in young adults [5]. These lesions have traditionally been considered reactive in nature because if unresected, they may involute and are not associated with malignant transformation. If the patient is quizzed, a history of trauma to the site of tumour may be obtained. In 2004, a USP6 gene rearrangement (chromosome 17p13) was identified in aneurysmal bone cysts [2, 3]. This rearrangement has been associated with a variety of fusion partners including CDH11, ZNF9, COL1A1, TRAP150 and OMD [3]. In 2011, it was also reported that nodular fasciitis harboured a USP6 rearrangement; however the most common fusion partner, MYH9 in chromosome 22q12.3, found in 65 % of cases of nodular fasciitis has not been reported in aneurysmal bone cysts [1]. We therefore set out to determine the incidence of USP6 gene rearrangements in a series of cases of nodular fasciitis diagnosed at our service in order to incorporate this test as an ancillary diagnostic tool for this lesion. Thirty-four cases, previously diagnosed as nodular fasciitis, were selected from our files. All cases were analysed by fluorescence in situ hybridisation (FISH) using custom-made break-apart BAC probes as previously described [1]. Material was available from 28 of these cases for analysis by RT-PCR for MYH-USP6 fusion transcript using previously published primers sets [1]. Thirty-one cases including 1 reactive tonsil, 5 B cell lymphomas, 9 PNET/Ewing sarcoma, 5 desmoidtype fibromatoses, 5 myxoid liposarcomas and 6 synovial sarcomas were selected as negative controls. All control samples were diagnosed in conjunction with their characteristic genetic alteration. In addition, 16 cases of primary aneurysmal bone cyst were tested. The presence of break-apart signals was assessed in ‘hot spots’ tumour-rich areas where 50 nonoverlapping nuclei were scored. Cases were classified as positive for USP6 gene rearrangement when 15 %, or above, of cells harboured the break-apart signal. All 34 cases of nodular fasciitis analysed by FISH, and 21 of the 28 cases tested by RT-PCR were informative. Ninety-one percent (31/34) of the cases initially diagnosed as nodular fasciitis showed a clear USP6 gene rearrangement by FISH in more than 15 % of the cells (range, 15–80 %; mean, 35.6 %) (Fig. 1). This result confirms the data of Erickson-Johnson et al. reporting USP6 gene rearrangement in 92 % of the cases [1]. M Fernanda Amary and Hongtao Ye contributed equally to this study.

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma

UNLABELLED Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. SIGNIFICANCE Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.

The H3F3 K36M mutant antibody is a sensitive and specific marker for the diagnosis of chondroblastoma.

We recently reported that 95% of chondroblastomas harbour a p.K36M mutation in either H3F3A (chromosome 1) or H3F3B (chromosome 17), with the majority involving H3F3B. The aim of this study was to assess the expression of the K36M‐mutated protein by immunohistochemistry in a large group of tumours.

Molecular characterization of a novel variant of a SYT-SSX1 fusion transcript in synovial sarcoma

including carcinoma of the lung, breast, gastrointestinal tract, urinary tract and female reproductive system, and affects tumour growth. Horiguchi et al. have reported that the expression of VEGF by tumour cells causes angiogenesis in angiomyofibroblastoma in the vaginal wall. They have also suggested that stem cell factor expressed by the tumour cells might induce recruitment of abundant mast cells into the tumour. Recently, mast cells have been shown to express VEGF and induce angiogenesis in cutaneous malignancies and ⁄ or other tumours. Taking these data together, it has been speculated that stem cell factor expressed by tumour cells might recruit abundant mast cells and infiltrating mast cells rather than tumour cells might be responsible for angiogenesis in this tumour. To our knowledge, this is the first case suggesting that mast cells may induce angiogenesis in angiomyofibroblastoma of the vaginal wall.

Polymerase chain reaction-Based clonality analysis of cutaneous B-cell lymphoproliferative processes

INTRODUCTION: The differential diagnosis of B-cell lymphoproliferative processes remains a challenge for pathologists, dermatologists and oncologists, despite advances in histology, immunohistochemistry and molecular biology. OBJECTIVE: Evaluate aid and limitations of clonality analysis in the diagnosis of primary cutaneous B-cell lymphomas and B-cell pseudolymphomas. METHODS: This study included 29 cases of B-cell lymphoproliferative processes classified as primary cutaneous B-cell lymphomas (13), B-cell pseudolymphomas (6) and inconclusive cases (10) using histology and immunohistochemistry. The clonality analysis was performed by polymerase chain reaction analysis of immunoglobulin light chain and heavy chain rearrangements. RESULTS: DNA quality was shown to be generally poor; eight samples were inadequate for polymerase chain reaction analysis. The results showed monoclonality in eight of the primary cutaneous B-cell lymphomas and polyclonality in four of the B-cell pseudolymphomas. In addition, monoclonality was shown in two of the inconclusive cases by histology and immunohistochemistry, demonstrating the utility of polymerase chain reaction as an ancillary diagnostic tool for primary cutaneous B-cell lymphomas. DISCUSSION: The low quality DNA extracted from these cases demanded the use of an IgH protocol that yielded small fragments and IgK. Both methods used together improved detection. CONCLUSION: Use of the two protocols, immunoglobulin heavy chain FR3-trad and immunoglobulin light chain-Kappa Biomed protocols for clonality analysis improved diagnostic accuracy.

Clear cell sarcoma of the mediastinum

A 59-year-old woman presented with a large mediastinal mass. At thoracotomy, the mass was found tightly adherent to the esophageal wall and right lower lobe of the lung. Histological examination showed a solid tumor composed of closely packed nests of cells with clear and eosinophilic cytoplasm, which were strongly and diffusely positive for S100 protein but negative for HMB45 and Melan-A. The diagnosis of clear cell sarcoma was supported by demonstrating the presence of an EWS gene rearrangement by fluorescence in situ hybridization. There was no evidence that this lesion represented metastatic disease. To the best of our knowledge, primary mediastinal clear cell sarcoma has not been previously reported in the literature. We present the case and discuss the differential diagnosis.

Abstract 4593: Genome-wide association study identifies novel loci associated with osteosarcoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Osteosarcoma (OS), the most common primary malignant bone tumor, has a peak incidence during the pubertal growth spurt. OS occurs at increased frequency in several inherited cancer syndromes (e.g., Li-Fraumeni and Rothmund Thomson syndromes). A limited number of common SNPs associated with OS have been identified in biologically plausible pathways (e.g., growth or DNA repair). We developed an international, multi-institutional study in order to conduct the first genome-wide association study (GWAS) of OS. Participating subjects provided informed consent through local IRBs. Control subjects were cancer free adults derived from large studies in prior GWAS at the NCI. Genotyping of all cases was conducted using the Illumina OmniExpress SNP microarray chip platform in 2 phases. The first phase analyzed 910 osteosarcoma cases. After quality control filtering and assessment of population substructure, 596 cases of European (EUR) ancestry were advanced to the primary GWAS analysis (Phase 1A). We combined data from 2,703 previously genotyped controls drawn from 3 EUR cohorts. There was no evidence for significant population substructure differences between OS cases and controls. The association analysis (1-degree of freedom trend test) was adjusted for study, sex and 4 eigenvectors. The top 30 SNPs (P <10-5) were genotyped by TaqMan in an additional 247 OS cases and 588 EUR controls. DNA from an additional 218 OS cases were subsequently scanned and, after the same quality filtering and population substructure analyses were applied, the data from 98 EUR cases were merged with the initial scan for the final analysis (Phase1B). The fixed-effects meta-analysis of all 941 OS cases and 3,291 controls identified 2 regions that achieved genome-wide significance. The first, rs1906953 on 6p21.3, is associated with susceptibility to OS (P = 8.1x10-9, odds ratio [OR] 1.57, 95% confidence interval [CI] 1.35-1.83). rs1906953 is in intron 7 of GRM4. GRM4 is expressed in osteoblasts and osteoclasts and involved in glutamate signaling, which is suggested to be involved in cell differentiation and regulation of bone formation. The second signal resides in an intergenic region on chromosome 2p25.2; rs7591996 (P = 1.0 x 10-8, OR 0.72, 95% CI 0.65-0.81). Another SNP in this region, rs10208273, is moderately correlated with the first signal (r2= 0.32) and approached genome-wide significance (P = 2.9 x 10-7, OR 1.35, 95% CI 1.21-1.52). A third locus in the ADAMTS6 gene on 5q12.3 is promising but does not yet achieve genome-wide significance: rs17206779 (P = 5.1x10-7, OR 0.75, 95% CI 0.68-0.84). Notably, variants in genes encoding members of the ADAMTS protein family have been associated with height, a known OS risk factor. We have conducted the first GWAS of OS and identified several novel loci associated with OS risk. Further investigation of these loci will advance understanding of OS etiology and biology. Citation Format: Sharon A. Savage, Lisa Mirabello, Zhaoming Wang, Julie Gastier-Foster, Richard Gorlick, Chand Khanna, Adrienne M. Flanagan, Roberto Tirabosco, Irene L. Andrulis, Jay Wunder, Nalan Gokgoz, Ana Patino-Garcia, Luis Sierrasesumaga, Fernando Lecanda, Nilgun Kurucu, Inci Ergurhan Ilhan, Neriman Sari, Massimo Serra, Claudia Hattinger, Piero Picci, Logan Spector, Donald A. Barkauskas, Neyssa Marina, Siliva Caminada de Toledo, Sergio Petrilli, Maria Fernanda Amary, Dina Halai, David Thomas, Chester Douglass, Paul Meltzer, Kevin Jacobs, Charles C. Chung, Sonja I. Berndt, Mark P. Purdue, Neil E. Caporaso, Margaret Tucker, Nathaniel Rothman, Maria Teresa Landi, Debra T. Silverman, Peter Kraft, David J. Hunter, Nuria Malats, Manolis Kogevinas, Sholom Wacholder, Rebecca Troisi, Lee Helman, Joseph F. Fraumeni, Jr., Meredith Yeager, Robert Hoover, Stephen J. Chanock. Genome-wide association study identifies novel loci associated with osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2013-4593