Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor

Abstract Background Clear cell sarcoma of soft tissue (CCSST) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT) are malignant mesenchymal tumors that share some pathological features, but they also have several different characteristics. They are well known to express chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) and cAMP response element-binding protein (CREB) family members; namely, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1. In addition, recent studies have suggested the presence of other fusions. Methods We used fluorescence in situ hybridization to detect specific rearrangements including EWSR1, ATF1, CREB1, and cAMP response element modulator (CREM) in 16 CCSST and 6 CCSLGT cases. We also used reverse transcription polymerase chain reaction (RT-PCR) to detect specific chimeric fusions of EWSR1-ATF1 and EWSR1-CREB1 using fresh tumor samples in available cases. Results A total of 15 of 16 CCSST cases (93.8%) had EWSR1 rearrangement, of which 11 (68.8%) also had ATF1 rearrangement, suggestive of the presence of EWSR1-ATF1 fusions. One CCSST case (6.3%) was found to have EWSR1 and CREM rearrangements, and 4 of 6 CCSLGT cases (66.7%) had EWSR1 rearrangement, of which 2 (33.3%) showed ATF1 rearrangement and the other 2 cases (33.3%) showed CREB1 rearrangement. These cases most likely had EWSR1-ATF1 and EWSR1-CREB1 fusions, respectively. RT-PCR was performed in 8 available cases, including 6 CCSSTs and 2 CCSLGTs. All CCSSTs showed EWSR1-ATF1 fusions. Among the 2 CCSLGT cases, one had EWSR1-ATF1 fusion and the other had EWSR1-CREB1 fusion. Conclusions Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. A novel CREM fusion was also detected in a few cases of CCSST and CCSLGT. The cases in which EWSR1 rearrangement was detected without definitive partner genes should be considered for the presence of CREM rearrangement

An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

<div><p>The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg²⁴⁷ is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis.</p></div

Characterization of C7 scFv purified from the <i>E</i>. <i>coli</i> extract.

<p>(A) Purification of C7 scFv from <i>E</i>. <i>coli</i> extracts. The purified protein was subjected to SDS-PAGE on a 10–20% gradient gel under reducing conditions and was stained with Coomassie brilliant blue. (B) ELISA using the purified C7 scFv. C7 scFv diluted at 1 μg/ml was incubated in wells coated with 1 μg/ml of Lu-Fc. (C) SPR analysis of C7 scFv. The sensorgrams represent the association and dissociation upon injecting different concentrations of C7scFv (6.25, 12.5, 25, 50, and 100 nM) to immobilized Lu-Fc. The rate constants, Ka and Kd, and KD are shown in the sensorgram. (D) Migration of A549 cells on LM-511 in the presence of C7 scFv. After the cells adhered to substrata coated with LM-511 (0.8 nM), control or C7 scFv was added to the media (30 μg/ml). Cell movements were evaluated as described above.</p

Mapping the amino acid residue recognized by C7 scFv on a three-dimensional model of Lu D1 and D2 domains.

<p>An amino acid residue of the C7 scFv epitope mapped on the crystal structure of Lu D1 and D2 domains (Protein Data Bank code 2PET) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167860#pone.0167860.ref016" target="_blank">16</a>]. The location of Arg²⁴⁷ is not very close to the epitope of the mAb87207 epitope. The binding sites of laminin α5 on Lu D2 domain are located on the opposite side of Arg²⁴⁷.</p

Isolation of human Lu-specific phage clones.

<p>(A) Biopanning was performed against Sol-Lu immobilized on wells using HCC patient tissues (left) or peripheral blood cells (right) -derived phage libraries. Enrichment for human Lu-specific phages was only observed from the peripheral blood cell-derived phage library using phage ELISA. (B) Cloning of human Lu-specific phage. After the third round of biopanning, phages were cloned by colony formation. The binding specificities of each phage clone were examined by monoclonal phage ELISA. Thirteen phage clones specific to human Lu were obtained.</p

Effects of anti-Lu phage antibodies on tumor cell migration.

<p>(A) Flow cytometric analyses using anti-Lu phage antibodies. The transfectants expressing Lu and HuH-7 cells were incubated with the indicated primary antibodies (A7, 2.0 × 10¹⁰ pfu/2.0 × 10⁵ cells; C7, 2.0 × 10¹⁰ pfu/2.0 × 10⁵ cells; BRIC221, 10μg/2.0 × 10⁵ cells) and then with Alexa 488-conjugated secondary antibody for flow cytometric analysis. The expression of Lu is shown as a solid line. The gray area indicates the negative control. (B) Migration of A549 cells on LM-511 in the presence of either mAb87207 or the anti-Lu phage antibodies. After the cells adhered to substrata coated with LM-511 (0.8 nM), antibodies were added to the media (A7, 1.2 × 10¹¹ pfu/ml; C7, 5 × 10¹⁰ pfu/ml; mAb87207, 10 μg/ml). Cell movements were monitored by time-lapse video microscopy and were quantified as described in Materials and Methods. Ten cells were randomly selected in each field. *, P < 0.01 by <i>t</i> test.</p

Fine mapping of the epitope recognized by C7 scFv.

<p>(A) Alignment of the human and mouse Lu amino acid sequences. Amino acids that are identical across species are connected with vertical lines. Ser²⁴², Glu²⁴⁵, Arg²⁴⁷, and His²⁵⁷ in Lu-Fc were substituted with the corresponding mouse residues, Asp, Ser, Gln, and Arg, respectively. Gln²⁴⁰ in MsLu-Fc was substituted with Arg, the corresponding human residue. (B) Purification of the mutant proteins. The mutant proteins purified from conditioned media were subjected to SDS-PAGE on a 7.5% gel. (C) ELISA using C7 scFv mixed with various recombinant proteins. The mutant proteins were mixed with the diluted C7 scFv and were added to the wells coated with Lu-Fc. C7 scFv did not react with R247Q-Fc, indicating that Arg²⁴⁷ is required for immunoreactivity.</p