Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis

Abstract

Background: Engineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3' end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results.

Results: We collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients. By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported. We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients.

Conclusions: Our findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

Keywords: Adeno-associated virus; Gene therapy; Hepatocellular carcinoma; Intrahepatic cholangiocarcinoma; Liver cancer; Sequence analysis; Viral capture sequencing; Viral oncogenesis; Virus integration.

Fig. 1

Visualization of the adeno-associated virus strain 2 (AAV2) genome including the X gene [20] and a promoter-enhancer region [36] that may be important to carcinogenesis but are not typically shown in schematic illustrations of AAV2

Fig. 2

Design of the study and patient selection. The RNA sequencing can detect expression of transcripts containing viral genome segments as explained in the flowchart of Fig. 3 A. The viral capture DNA sequencing was designed to detect AAV sequences that are present and AAV sequences that are integrated into the host genome, whether the sequences are expressed or not

Fig. 3

A Workflow of RNAseq analysis (B) AAV2/13 detected in Thai patients with HCC and iCCA (C) Counts of host genome integrations of AAV2/13 in tumor vs. non-tumor tissue in viral capture DNAseq samples (D) RNA expression of CCNA2 measured in transcripts per million (TPM) by AAV2/13 integration status in tumor vs. non-tumor

Fig. 4

The distribution of chimeric reads in the samples that have at least one AAV integration. These boxplots visualize part of the tabular data in Supplementary Table S1

Fig. 5

Comparison of AAV integrations into new Thai samples and published AAV integrations with respect to the AAV2 genome as the reference. In both panels, the X axis represents the base pair positions in the AAV2 reference genome and overlapping integrated segments are stacked on top of one another; there is no Y axis, but the number of rectangles stacked on top of one another indicate the number of overlapping integrations from that region of AAV. A Thirteen out of 17 Thai patients with samples subjected to viral capture sequencing had at least one integration of AAV. Ten had integrations only in the NT sample; two had integrations only in the T sample. Only one patient had a possibly clonal integration into the gene CCNA2. That integration, shown in maroon, is the only one that overlaps the X gene. B AAV intervals spanned by previously published integrations of AAV into liver cancer samples. The data for panel B are adapted from the supplementary information of reference 5; see also our Supplementary Table S3

Fig. 6

Flowchart describing viral capture DNA sequencing and its analysis