PMC5848058_01
Characterization of the lesions on MRI, macroscopic, microscopic, and immunohistochemical level. (a) MRI gadolinium-enhanced fat-saturated T1-weighted image in the sagittal plane showing an intraosseous lesion of the calcaneus and a plantar soft tissue lesion of the forefoot (arrows). (b) Macroscopic appearance of lesions in the calcaneus and in the cuneiform bone (arrows). (c-e) Lobulated infiltrative pattern made of plasmacytoid cells (H and E). (d) Staining of the chondromyxoid stroma by alcian blue. (f-h) Focal expression of the tumor cells for EMA (f), diffuse expression for cytokeratin AE1/AE3 (g), and diffuse expression for S100 protein (h). Sections of bone were performed using the diamond band saw (EXAKT312, Germany) and decalcified with a formamid solution (DC1, V.W.R.) after formalin fixation. 5 microm thick sections of the paraffin-embedded material were stained with H and E (Symphony 5-Plus, Roche). Immunohistochemistry experiments were performed according to standard procedures. Primary antibodies used on XT benchmark platform (Ventana) were CKAE1/AE3 (cloneAE1/AE3; 1.8 mg/L), EMA (clone E29; 2.4 mg/L), protein S100 (rabbit polyclonal; 1/100), GFAP (rabbit polyclonal; 1/500), alpha-smooth muscle actin (clone 1A4; 0.4 mg/L), desmin (clone D33; 2.05 mg/L), INI1/BAF47 (clone 25/BAF47; 2.5 mg/L), and vimentin (clone V9; 0.5 mg/L).
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MRI and plain radiography demonstrated a main lesion in the calcaneus, two others in the navicular bone, and a last one in the plantar soft tissue (Figure 1). The tumor of the calcaneus measured 4.4 x 3.4 cm (Figure 1), and the other lesions in the medial cuneiform measured 2.2 x 0.9 cm (Figure 1) and 0.5 mm. On histology, the different tumors appeared lobulated and contained plasmacytoid cells arranged in lobules and in large cellular sheets, intermixed with areas of fibrous and chondromyxoid stroma positive for alcian blue (Figure 1). These cells showed mild nuclear pleomorphism and some cysts were observed, but no convincing ductular differentiation (Figure 1). Immunohistochemistry demonstrated an intense and diffuse staining of plasmacytoid cells for the broad-spectrum cytokeratin, S100 protein (Figures 1 and 1), and vimentin. EMA was focally positive in the cytoplasm, and a very focal membranous apical staining was also present (Figure 1).
Cytogenetic characterization. (a) Conventional karyotype on the bone tumor (calcaneum): 46,XY,add(2)(q?21),der(3)t(2;3)(q?21;q?21),-8,-19,+mar1,+mar2[8]. The soft tissue tumor harbor the same chromosomal aberrations: 46,XY, add(2)(q?21),der(3)t(2;3)(q?21;q?21),-8,-19,+mar1,+mar2[2]. (b) FISH experiments with break-apart probes on fixed cells: deletion of the BAC probe located 5'/centromeric (RP11-1144D2 labelled in green) to the LPP (lipoma preferred partner or LIM Domain Containing Preferred Translocation Partner In Lipoma) locus in a metaphasic cell and in a nucleus (soft tissue tumor): ish der(?)t(3,?)(RP11-1144D2-,RP11-67E18-;RP11-67E18+[5].nuc ish(RP11-1144D2x1,RP11-67E18x2)(RP11-1144D2 con RP11-67E18x1)[334/400]. (c, d) Molecular karyotyping (soft tissue tumor): 2 interstitial deletions within the long arm of chromosome 3 (c) and the telomeric one delete the 5' part of the LPP locus (d) arr[hg19] 3q22.1q26.2(133,374,187-169,925,119),3q27.2q28(185,628,780-188,411,171)x1. Culturing, harvesting, and G-banding of the tumor samples for karyotyping were performed according to standard procedures. Culturing, harvesting, and G-banding of the tumor samples for karyotyping were performed according to standard procedures. Dual-color FISH experiments were performed on fixed nuclei and on formalin-fixed paraffin-embedded tissue sections (4 mum-thick), using commercial probes (LSI-EWSR1, LSI-FUS, LSI-TP53/CEP17, LSI-9p21/CEP9, LSI-TP53/CEP17 from Abbott Molecular/Vysis; ON-TFE3 from Kreatech) and bacterial artificial chromosome (BACs) probes. The BAC clones were purchased from the Chori BACPAC Resources Center (Oakland, USA) to study the following loci: NR4A3/9q31.1 (RP11-412F16, RP11-47M15, RP11-266D8, and RP11-282C24); HMGA2/12q14.3 (RP11-317J13, RP11-412I20, RP11-945G8, and RP-347J7); and LPP/3q27.3-q28 (RP11-1144D2 and RP11-67E18). Extraction, labeling, and hybridization were performed, as previously reported. Two hundred interphasic cells and all hybridized metaphases were analysed. Molecular karyotyping was performed with Cytoscan 750K SNP-arrays according to the manufacturer's instructions (Affymetrix). Results were analysed as previously reported. Aberrations greater than 100 kb involving at least 20 consecutive SNPs were considered for copy number variant (CNV) analysis. Constitutional CNV polymorphisms were excluded based on comparisons with the Database of Genome Variants (hg19). The quality control metrics were within the normal range (SnpQC = 17.656, Mapd = 0.195, and Waviness = 0.088).
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Molecular karyotyping detected 2 main deletions on the long arm of chromosome 3 located at 3q22.1-3q26.2 (37Mb) (Figure 2) and 3q27.2-3q28 (3Mb). The latter includes the 5' part of the LPP gene, probably the 8 first exons (Figure 2).