Exome sequence analysis of Kaposiform hemangioendothelioma: identification of putative driver mutations*

BACKGROUND Kaposiform hemangioendothelioma is a rare, intermediate, malignant tumor. The tumor's etiology remains unknown and there are no specific treatments. OBJECTIVE In this study, we performed exome sequencing using DNA from a Kaposiform hemangioendothelioma patient, and found putative candidates for the responsible mutations. METHOD The genomic DNA for exome sequencing was obtained from the tumor tissue and matched normal tissue from the same individual. Exome sequencing was performed on HiSeq2000 sequencer platform. RESULTS Among oncogenes, germline missense single nucleotide variants were observed in the TP53 and APC genes in both the tumor and normal tissue. As tumor-specific somatic mutations, we identified 81 candidate genes, including 4 nonsense changes, 68 missense changes and 9 insertions/deletions. The mutations in ITGB2, IL-32 and DIDO1 were included in them. CONCLUSION This is a pilot study, and future analysis with more patients is needed to clarify: the detailed pathogenesis of this tumor, the novel diagnostic methods by detecting specific mutations, and the new therapeutic strategies targeting the mutation.


INTRODUCTION
According to the International Society for the Study of Vas- The disease concept of KHE was first proposed by Zukerberg in 1993. 1  On physical examination at the first visit, multiple, indurated nodules and erythemas merged to become geographical Based on the above findings, a diagnosis of KHE was made.
Since the tumor was limited to the lower abdomen without muscle infiltration or distant metastasis, and because the patient was not accompanied with KMS, the decision was taken to treat the tumor by serial excision. Three times resection removes almost all the lesions and the pain disappears.

DNA purification
The genomic DNA for exome sequencing was obtained from the tumor tissue and matched normal tissue from the same individual, using the DNA mini kit (Qiagen, Valencia, CA). Institutional review board approval and informed consent were obtained, conforming to the Declaration of Helsinki.        However, since this SNV is seen in about 60% of the population, there is a possibility that "second-hit" somatic mutation is also necessary for tumorigenesis. Another vascular anomaly, mucocutaneous venous malformation is caused by the combination of germline substitutions in the endothelial cell tyrosine kinase receptor TIE2 and the somatic 'second hit' lesion-restricted mutation of TIE2. 9 Similarly, we previously indicated germline heterozygous SNV in KDR and TEM8 as the risk mutations for infantile hemangioma. 10 As infantile hemangioma typically appears on the head or face around the second week of life, the hypothesis proposed is that the clonal expansion of endothelial cells within the lesions may be a consequence of somatic events such as microvessel trauma during delivery.

Library preparation and sequencing
We also identified 81 genes as candidates for somatic mutation. For example, ITGB2 has been strongly implicated in angiogenesis, suggesting the possible association with the pathogenesis of KHE. IL-32, which possesses heterozygous frameshift in KHE tissue, has also been known as a angiogenesis-related cytokine. 11,12 Furthermore, we found 4 nonsense mutations only in tumor tissue, including the DIDO1 gene, which is up-regulated by apoptotic signals and encodes a cytoplasmic protein that translocates to the nucleus upon apoptotic signal activation. DIDO1 is considered a tumor suppressor gene in myeloid cells, and thought to be significantly involved in the pathogenesis of myelodysplastic syndrome, a malignant blood disorder. 13 Accordingly, the DIDO1 gene may also be involved in the tumorigenesis of KHE.

CONCLUSION
These mutations have not been reported so far, which is consistent as driver mutations of the rare tumor. Potentially, multiple genetic abnormalities -rather than a single abnormality -may be involved in KHE. For instance, germline TP53 SNV and the somatic DIDO1 gene change may cooperate to induce tumorigenesis.
As limitations to this study, 'germline' mutation of TP53 may be due to the contamination of normal tissue and tumor tissue, because the parent DNA is not determined. In addition, the result of exome analysis was not confirmed by the sanger method. Since KHE is a rare tumor, the team was unable to collect other samples. This is a pilot study, and future analysis with more patients is need-