Fitim Berisha

IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours.

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in gliomas and acute myeloid leukaemia (AML). Since patients with multiple enchondromas have occasionally been reported to have these conditions, we hypothesized that the same mutations would occur in cartilaginous neoplasms. Approximately 1200 mesenchymal tumours, including 220 cartilaginous tumours, 222 osteosarcomas and another ∼750 bone and soft tissue tumours, were screened for IDH1 R132 mutations, using Sequenom® mass spectrometry. Cartilaginous tumours and chondroblastic osteosarcomas, wild‐type for IDH1 R132, were analysed for IDH2 (R172, R140) mutations. Validation was performed by capillary sequencing and restriction enzyme digestion. Heterozygous somatic IDH1/IDH2 mutations, which result in the production of a potential oncometabolite, 2‐hydroxyglutarate, were only detected in central and periosteal cartilaginous tumours, and were found in at least 56% of these, ∼40% of which were represented by R132C. IDH1 R132H mutations were confirmed by immunoreactivity for this mutant allele. The ratio of IDH1:IDH2 mutation was 10.6 : 1. No IDH2 R140 mutations were detected. Mutations were detected in enchondromas through to conventional central and dedifferentiated chondrosarcomas, in patients with both solitary and multiple neoplasms. No germline mutations were detected. No mutations were detected in peripheral chondrosarcomas and osteochondromas. In conclusion, IDH1 and IDH2 mutations represent the first common genetic abnormalities to be identified in conventional central and periosteal cartilaginous tumours. As in gliomas and AML, the mutations appear to occur early in tumourigenesis. We speculate that a mosaic pattern of IDH‐mutation‐bearing cells explains the reports of diverse tumours (gliomas, AML, multiple cartilaginous neoplasms, haemangiomas) occurring in the same patient. Copyright

Ollier disease and Maffucci syndrome are caused by somatic mosaic mutations of IDH1 and IDH2.

Ollier disease and Maffucci syndrome are characterized by multiple central cartilaginous tumors that are accompanied by soft tissue hemangiomas in Maffucci syndrome. We show that in 37 of 40 individuals with these syndromes, at least one tumor has a mutation in isocitrate dehydrogenase 1 (IDH1) or in IDH2, 65% of which result in a R132C substitution in the protein. In 18 of 19 individuals with more than one tumor analyzed, all tumors from a given individual shared the same IDH1 mutation affecting Arg132. In 2 of 12 subjects, a low level of mutated DNA was identified in non-neoplastic tissue. The levels of the metabolite 2HG were measured in a series of central cartilaginous and vascular tumors, including samples from syndromic and nonsyndromic subjects, and these levels correlated strongly with the presence of IDH1 mutations. The findings are compatible with a model in which IDH1 or IDH2 mutations represent early post-zygotic occurrences in individuals with these syndromes.

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

GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabrauds syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

A common single-nucleotide variant in T is strongly associated with chordoma

Chordoma is a rare malignant bone tumor that expresses the transcription factor T. We conducted an association study of 40 individuals with chordoma and 358 ancestry-matched controls, with replication in an independent cohort. Whole-exome and Sanger sequencing of T exons showed strong association of the common nonsynonymous SNP rs2305089 with chordoma risk (allelic odds ratio (OR) = 6.1, 95% confidence interval (CI) = 3.1–12.1; P = 4.4 × 10−9), a finding that is exceptional in cancers with a non-Mendelian mode of inheritance.

An integrated functional genomics approach identifies the regulatory network directed by brachyury (T) in chordoma

Chordoma is a rare malignant tumour of bone, the molecular marker of which is the expression of the transcription factor, brachyury. Having recently demonstrated that silencing brachyury induces growth arrest in a chordoma cell line, we now seek to identify its downstream target genes. Here we use an integrated functional genomics approach involving shRNA‐mediated brachyury knockdown, gene expression microarray, ChIP‐seq experiments, and bioinformatics analysis to achieve this goal. We confirm that the T‐box binding motif of human brachyury is identical to that found in mouse, Xenopus, and zebrafish development, and that brachyury acts primarily as an activator of transcription. Using human chordoma samples for validation purposes, we show that brachyury binds 99 direct targets and indirectly influences the expression of 64 other genes, thereby acting as a master regulator of an elaborate oncogenic transcriptional network encompassing diverse signalling pathways including components of the cell cycle, and extracellular matrix components. Given the wide repertoire of its active binding and the relative specific localization of brachyury to the tumour cells, we propose that an RNA interference‐based gene therapy approach is a plausible therapeutic avenue worthy of investigation. 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.

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.

H3f3a (histone 3.3) G34w Immunohistochemistry: A Reliable Marker Defining Benign and Malignant Giant Cell Tumor of Bone

Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component.