PMC9643764_01
Clinicopathological findings of the tumor in case 1. The tumor was composed of the spindle and epithelioid cells embedded in myxedematous and hyalinized stroma (A). The spindle cells were focally arranged in the intersecting fascicles with frequently observed mitotic figures (B), while the epithelioid cells were arranged in the nest- or cord-like pattern in the myohyaline background (C). In some areas, the tumor cells were more cellular with relatively brisk mitoses (D). Focal necrosis was recognized (E). Tumor cells infiltrating the surrounding adipose tissues and thin-walled branching vessels were seen (F). Inflammatory cell infiltration was observed locally (G). Tumor cells were positive for CD34 (H), S100 (I), and ALK-D5F3 (J).
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Magnetic resonance imaging (MRI) suggested an intramuscular mass between the anterior rectus and vastus lateralis muscles in the left thigh ( Supplementary Figure 1 ). Microscopically, the tumor was composed of spindle cells juxtaposed with epithelioid cells embedded in myxedematous and hyalinized stroma ( Figure 1A ), partially infiltrating surrounding striated muscles and adipose tissue. The spindle cells were arranged in sheet-like, intersecting fascicles, or in patternless patterns, showing indistinct cytoplasmic borders and moderate nuclear pleomorphism ( Figure 1B ). The epithelioid cells were arranged in a nest- or cord-like pattern in a myohyaline background with ample eosinophilic cytoplasm and round to ovoid nuclei ( Figure 1C ). Figure 1D ). Focal hemorrhage and necrosis were also observed in the spindle cell area ( Figure 1E ). Prominent branching of thin-walled blood vessels of different sizes was also found ( Figure 1F ). At the periphery, some spindle tumor cells admixed with infiltrating inflammatory cells, closely resembling IMT ( Figure 1G ). The tumor cells were immunohistochemically positive for CD34 ( Figure 1H ), S100 ( Figure 1I ), ALK-D5F3 ( Figure 1J ), H3K27me3, vimentin, and CD99 (paranuclear dot-like staining), and they were negative for STAT6, CK-pan, EMA, desmin, SMA, CD31, WT-1, and pan-TRK.
PMC9643764_02
Clinicopathological findings of the tumor in case 2. The tumor consisted of spindle-shaped mesenchymal cells with stromal and perivascular hyalinization (A, B). Focal staghorn vessels and clusters of clear cytoplasmic cells were observed (C). Giant multinucleated tumor cells and inflammatory infiltration were also seen (D). The tumor cells were diffusely positive for CD34 (E), S100 (F), and ALK-D5F3 (G). The relapsed tumor showed diffuse proliferation of compact spindle cells, also infiltrating striated muscles (H).
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Microscopically, the lesion was composed of spindle-shaped mesenchymal cells infiltrating the fat tissue and striated muscle, with stromal and perivascular hyalinization ( Figures 2A, B ). Focal clusters of cells showed clear cytoplasm ( Figure 2C ). Sparse inflammatory cell infiltration was also observed ( Figure 2D ). The tumor cells were immunohistochemically positive for CD34 ( Figure 2E ), S100 ( Figure 2F ), and ALK-D5F3 ( Figure 2G ), and negative for AE1/3, SMA, desmin, STAT6, and SOX10. Pathologically, the relapsed tumor showed similar morphology and immunophenotype to the original tumor, with more compact tumor cells ( Figure 2H ).
Molecular tests to validate ALK fusions. The presence of the PLEKHH2::ALK fusion transcript was validated by Sanger sequencing (A). Direct sequencing of the purified RT-PCR products revealed the chimeric transcripts between exon 6 of EML4 and exon 20 of ALK
(B). Schematic representation of the predicted chimeric proteins (C, D). By ALK break-apart FISH test, most tumor cells in both cases demonstrated a signal pattern consisting of isolated 5' (green) and isolated 3' (orange), along with fused 3'/5' signals (E, F). TD, trimerization domain; HELP, hydrophobic motif in EML proteins; TAPE, tandem atypical propeller domain; ECD-LB, extracellular ligand-binding domain; TM, transmembrane domain; TK, tyrosine kinase domain; CC, a-helical coiled-coil domain; PH, Pleckstrin homology domain; MyTH, MyTH4 domain; FERM, four-point-one ezrin radxin mesoin family.
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NGS test identified a transcript comprising intron 6 of PLEKHH2 and intron 20 of ALK in case 1, which was validated by RT-PCR and Sanger sequencing ( Figure 3A ). ARMS test detected a transcript comprising exon 6 of EML4 and exon 20 of ALK in case 2 ( Figure 3B ). FISH results confirm ALK rearrangements in both cases ( Figures 3E, F ).