Figure 3 | Modern Pathology

Figure 3

From: Renal cell carcinoma with novel VCL–ALK fusion: new representative of ALK-associated tumor spectrum

Figure 3

Breakpoint of the VCL–ALK fusion gene and expression of the VCLALK chimeric protein in case 1. (a) Electrophoresis of products from RT-PCR analysis of the tumor cDNA with the VCL exon 16 forward and ALK exon 20 reverse primers shows an expected 159-bp product (column 2, arrow). In column 1, GAPDH was amplified as a positive control of tumor cDNA, and in column 3, a negative RT-PCR control reaction was run. (b) Sequence of the VCL–ALK fusion breakpoint with in-frame translation (medium line); the 811 residue on VCL (proline) is immediately followed by the 1058 residue on ALK (valine). The breakpoint (marked by a vertical black line in the center of the sequence) is located at the beginning of the ALK exon 20. (c) Western blot analysis of protein lysates. Column 1, neuroblastoma line IMR32; column 2, tumor tissue, column 3, normal kidney tissue. In line 1, immunoblotting with the anti-VCL antibody hVIN1 shows an 117-kDa product in all three samples, consistent with the wild-type VCL expression. An 150-kDa additional product is seen in the tumor (arrow), consistent with the expression of abnormally sized fusion protein. In line 2, immunoblotting with the ALK/p80 antibody shows an 180-kDa band consistent with full-length ALK in the neuroblastoma line IMR32. An aberrantly sized (150 kDa) product (arrow), consistent with the ALK-VCL fusion is seen in the tumor. No ALK expression is observed in normal renal tissue. In line 3, immunoblotting with the anti-β-actin antibody shows sample-loading control. (d) Immunohistochemistry with the anti-ALK antibody ALK11 shows cytoplasmic and subplasmalemmal staining of tumor cells. (e) Immunohistochemistry with the anti-VCL antibody hVIN1 shows subplasmalemmal and cytoplasmic staining identical to that observed with the ALK11 antibody (d), which is consistent with colocalization of VCL and ALK reaction products to the VCL subcellular domains. Magnification (panels d and e) × 600.

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