Complex genomic rearrangement of ALK loci associated with integrated human Epstein-Barr virus in a post-transplant myogenic liver tumor

Abstract

Epstein-Barr virus (EBV) is a ubiquitous viral agent, well known to be associated with lymphoid, epithelial, and smooth-muscle malignancies in immunocompromised individuals. This report describes a 10-year-old patient with an EBV-related liver tumor occurring after kidney transplantation. The neoplasm presented a phenotypic spectrum, ranging from a smooth-muscle tumor to an inflammatory pseudotumor (IPT). The neoplastic cells failed to disclose CD21, CD35, or ALK expression, the latter confirmed by reverse-transcription polymerase chain reaction. Cytogenetic analysis revealed a single clonal cell population showing 46,XY,del (2)(p23),der(3)t (2;3)(p23;q29),der(21) t(Y;21)(q12;p13) karyotype. By metaphase FISH analysis, the neoplastic cells demonstrated the presence of two molecularly different but related aberrant clones, one with the loss of one ALK allele and the second with translocation of the 3'end of ALK kinase domain on the der(3) chromosome. Using FISH with an EBV-specific and 3'end ALK DNA probes, a co-localization of the viral DNA and the ALK sequences was found on the der(3) chromosome. Metaphases with loss of rearranged ALK did not show integrated virus; instead, viral particles together with an associated 3'end ALK domain formed an ex-chromosomal, episomal-like type configuration. The interphase study, using dual-color 5'/3' end ALK FISH assay, revealed 30% of nuclei with only one fused signal, confirming the total loss of one ALK allele in the subset of tumor cells. A combined immunofluorescence and FISH study indicated this separate clonal variant to correspond to desmin-positive smooth-muscle cells. In contrast, desmin-negative myofibroblasts showed the presence of both normal and rearranged ALK alleles. Our results indicate that ALK locus may be a target of EBV integration, a hitherto unreported finding. Although the sustained clonal expansion in EBV-related smooth-muscle tumors/IPTs may depend on functions provided by the EBV oncogenic proteins, the tumor phenotype may be further modified by the secondary genomic rearrangements imposed by the virus during and/or after the integration event. In this respect, the observed phenotypic heterogeneity most likely reflects divergence during neoplastic progression, with the subsequent expansion of morphologically and molecularly distinct but cytogenetically related clones.

Figure 1.

Map of ALK gene on 2p23 with localization of the FISH probes used in the study.

Figure 2.

Overview of the histopathological and immunohistochemical findings. In many areas, the transition between the smooth-muscle component (left) and the IPT component (right) is easily seen (H&E stain, ×25) (a). The tumor is relatively well delineated from the surrounding parenchyma (arrows) (H&E stain, ×25) (b). Higher magnification of the smooth-muscle component shows bundles of eosinophilic spindle cells with cigar-shaped nuclei (H&E stain, ×160) (c). Higher power of the IPT component discloses vessels, lymphocytes, plasma cells (arrowheads), and spindle cells in a myxoid background (H&E stain, ×160) (d). The smooth-muscle component is strongly desmin-positive (indirect immunoperoxidase stain, ×400) (e). The spindle cells of the IPT component only express α-SMA (indirect immunoperoxidase stain, ×400) (f).

Figure 3.

Electron micrograph showing tumor cells with smooth muscle (asterisks) and fibroblast-like (wide arrow) characteristic (×9.200). Inset: detail of EBV virus particles in a cytoplasmic cell fragment (arrow) (×72.500).

Figure 4.

G-banded karyogram of abnormal tumor cells showing 46,XY,del(2)(p23),der(3)t(2;3)(p23;q29),der(21)t(Y;21)(q12;p13) karyotype. Arrows point to the breakpoints.

Figure 5.

Molecular cytogenetic analysis of abnormal tumor cells carrying del(2)(p23) and der(3)t(2;3)(p23;q29) chromosomes. A: Metaphase FISH using LSI ALK (Vysis) probe: dual-color signals are present only on normal chromosome 2p23 (arrow), indicating clone 1 with the loss of ALK allele (arrowhead). B: Metaphase FISH using LSI ALK probe: dual-color signal is present on normal chromosome 2p23 (arrow) and the red signal is present on der(3) chromosome, indicating clone 2 with the rearranged ALK gene (arrowhead). C: Dual-color hybridization to interphase nuclei from tumor specimen using LSI ALK probe: two separate clones, one with loss (arrow) and the second with rearranged ALK allele (arrowheads) are evident. D: Co-hybridization of digoxigenin-labeled ALKP1 and biotin-labeled EBV probes: a single red and overlapping red/green (arrows) signals in interphase nuclei indicate the integration of EBV at the site of ALK in subset of cells; metaphase cell reveals only red signal on normal chromosome 2. E: Co-hybridization of digoxigenin-labeled ALKP1 and biotin-labeled EBV probes: metaphase cell showing single red signal on normal chromosome 2p23 (arrowhead) and fused green/red signal on der(3) chromosome (arrow). F: Co-hybridization of digoxigenin-labeled ALKP1 and biotin-labeled EBV probes: metaphase cell showing single red signal on normal chromosome 2p23 (arrow) and fused red/green signal outside chromosome, indicating the presence of EBV together with 3′ end ALK sequences in the episomal form (arrowhead). G: Identification of desmin-positive cells by immunostaining (arrow). Nuclei are counterstained with DAPI. H: Double-color interphase FISH analysis of cells corresponding to E, using double-color LSI ALK probe. Loss of rearranged ALK allele was revealed only in the nuclei that corresponded to desmin-positive cells (arrow).

Figure 6.

RT-PCR analysis of ALK cDNA. RT-PCR was performed with primers designed to amplify respectively the 3′-end (lanes 1 and 2) or the 5′-end (lanes 3 and 4) of ALK. Lanes 1 and 3 show the absence of any product using cDNA of the tumor. Lanes 2 and 4: PCR was performed with cDNA from an ATIC-ALK positive control case. Lane 5 is a positive control for the cDNA synthesis showing amplification of the CIZ gene, starting from cDNA of the tumor.