Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

<div><p>Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.</p></div

Tumor T2548 with six HPV16 DNA integration sites.

<p>The six integration sites are distributed on the chromosomes 3, 19 and 20 (two DJs on each). All six integration sites are intragenic, but none of them is transcriptionally active. The cellular genes directly targeted by HPV16 DNA integration in T2548 include <i>FHIT</i> (transcript 002, Ensembl ID: ENST00000468189, minus strand, 9 exons), <i>ARID3A</i> (transcript 001, Ensembl ID: ENST00000263620, plus strand, 9 exons), <i>MOB3A</i> (transcript 001, Ensembl ID: ENST00000357066, minus strand, 5 exons), <i>MACROD2</i> (transcript 010, Ensembl ID: ENST00000217246, plus strand, 17 exons) and <i>CBFA2T2</i> (transcript 003, Ensembl ID: ENST00000375279, plus strand, 12 exons).</p

Examples of intragenic HPV16 DNA integration sites.

<p>(A) Tumor T182e has one HPV16 integration site 0182_DJ1, which is transcriptionally active (TA-group 1). The integrated HPV16 DNA is located in the intron (i) between exons (e) 5 and 6 of the cellular gene <i>LIPC</i> (transcript 003, Ensembl ID: ENST00000414170, plus strand, 10 exons), and has the same orientation as <i>LIPC</i>. APOT analysis identified an HPV16-cellular fusion transcript (0182_RJ) in which the viral exon is spliced to the downstream <i>LIPC</i> exon 6. (B) Tumor T892 (TA-group 2) has two HPV16 integration sites (0892_DJ1 and 0892_DJ2), which are both located in the intron between exons 12 and 13 of the cellular gene <i>GPN1</i> (transcript 001, Ensembl ID: ENST00000264718, plus strand, 14 exons). While 0892_DJ1 has the same orientation as <i>GPN1</i>, the transcriptionally active 0892_DJ2 has the opposite orientation. In the transcript 0892_RJ the viral exon is spliced to an alternative cellular exon. (C) In tumor T2319 (TA-group 2), all three identified HPV16 integration sites are located within the cellular gene <i>CASZ1</i> (transcript 003, Ensembl ID: ENST00000377022, 21 exons). Since the <i>CASZ1</i> gene is located on the minus strand, the sense orientation of the gene is from right to left. Junction 2319_DJ1 is located in an intron in opposite direction to <i>CASZ1</i>. Junctions 2319_DJ2 and 2319_DJ3 are located in the terminal exon 21 in the same direction as the <i>CASZ1</i> gene, DJ2 in the terminal part of the translated region and DJ3 in the 3′ untranslated region (3′-UTR). Both are possible templates for the HPV16-cellular fusion transcript 2319_RJ. – In the DJs and RJs, the open boxes denote the HPV16 part and the black boxes/arrows the fused cellular part. The arrow of the DJs indicates the sense orientation of the HPV16 oncogenes. Transcribed DJs and the RJs are shown in red, non-transcribed DJs in blue letters.</p

Genomic structure and transcription of episomal and integrated HPV16 DNA.

<p>(A) The circular HPV16 genome becomes linearized and inserted into the host cell genome upon integration. The breakpoints of integrated HPV16 DNA can be located anywhere in the L2-L1 region (5′ breakpoint) and E1-E2-E5 region (3′ breakpoint), respectively. The breakpoint regions are indicated by dotted underlines. The circular HPV16 genome is reproduced with slight modifications with permission, from Doorbar, (2006), (Clinical Science), (110), (525–541). © the Biochemical Society. (B) In case of episomal HPV16 DNA, early transcription is initiated at the early promoter P97 and terminated at the early poly-A signal (PAE at pos. 4215). The early transcript shown contains two exons (e) with ORFs for E6, E7, E1?E4 and E5. An intron (i) of 2477 nucleotides is removed by splicing at the indicated donor and acceptor positions. Amplification of HPV16 oncogene transcripts by the APOT assay gives rise to a constant-size RT-PCR amplicon of ∼1 kb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Klaes1" target="_blank">[21]</a>. The APOT primers are indicated by the arrows. (C) Early transcription from integrated HPV16 DNA will lead to HPV16-cellular fusion transcripts, because the viral PAE signal is missing and instead a cellular poly-A signal (PA) is adopted. If the 3′ breakpoint is located upstream of the splice acceptor at position 3358, an alternative cellular splice acceptor (Ac) will be used, and the HPV16-cellular DNA junction sequence (red bar) will be spliced out as part of a viral-cellular intron (upper part). If the 3′ breakpoint is located downstream of the splice acceptor 3358, the HPV16-cellular DNA junction sequence (blue bar) will remain as part of a viral-cellular exon, and the DNA and RNA junction sequences are colinear (lower part). The HPV16-cellular fusion transcripts are amplified in APOT assays as RT-PCR products that are shorter or longer than the ∼1-kb amplicon derived from episomal HPV16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Klaes1" target="_blank">[21]</a>.</p

HPV16 E6 variants.

<p>E-p = European prototype; E-T350G = European T>G at position 350; NA1 = North-American type 1; AA = Asian-American; As = Asian; Af1 = African type 1; Af2 = African type 2.</p>*<p>Underlined are those tumors which seem to contain episomal HPV16 DNA, because no DNA junctions of integrated HPV16 DNA could be identified by the TEN16 analysis.</p

Comparison of HPV16-cellular DNA junctions and RNA junctions.

<p>TA = TEN16/APOT comparison; DJ = DNA junction; RJ = RNA junction; Chr. = chromosome; n.a. = not applicable; Do = splice donor; Ac = splice acceptor.</p>(1)<p>TA-group 1: samples with one DJ and a corresponding RJ.</p>(2)<p>TA-group 2: samples with one corresponding DJ/RJ pair and additional DJs without RJ counterpart.</p>(3)<p>TA-group 3: samples without corresponding DJ/RJ.</p>&<p>The 22 RNA junctions (APOT) are part of a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Schmitz2" target="_blank">[50]</a> in which information on chromosomal locations, cellular genes and splicing is given, but without the exact position numbers of the cellular breakpoints.</p>§<p>Genomic distance between the cellular breakpoints of DJ and RJ.</p>$<p>Distance from the HPV16 splice donor (position 880) to the cellular splice acceptor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone-0066693-g001" target="_blank">Figure 1</a>).</p>#<p>Discovered by searching in the TEN16 sequence library for DJs located within 1 Mb upstream of the respective RJs.</p>*<p>For sample T186e, the cellular sequences of DJ1 and RJ were both mapped to chromosome 9, but in opposite orientation to each other.</p>a)<p>In the fusion transcript, the viral E6/E7 exon is spliced to the next downstream exon of the cellular gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone-0066693-g003" target="_blank">Figure 3</a>).</p

Frequency distribution of HPV16 3′-breakpoints in different segments of the HPV16 early region.

<p>The distribution of HPV16 3′-breakpoints of viral-cellular DNA junctions (n = 74) was analyzed within the five different segments E1, E2, E5, E1-Ac and Ac-PAE of the HPV16 early region. The positions of each segment in the HPV16 genome are given in parentheses. The E1-PAE segment of the HPV16 early region (pos. 865–4215, 3351 bp) was taken as reference. The relative length of each segment is shown by the white bars. The relative frequency of HPV16 3′-breakpoints within each segment is shown by the grey bars for all DNA junctions (DJ_all, n = 74, middle-grey bar), the transcribed DNA junctions (DJ_tr., n = 21, light-grey bar) and the non-transcribed DNA junctions (DJ_n.tr., n = 53, dark-grey bar). The exact two-tailed one-sample binomial test was used for statistical analysis by comparing the relative frequency of HPV16 3′-breakpoints in each segment to the relative segment length. Bars marked with asterisks indicate statistically significant results (P<0.05). Data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693.s005" target="_blank">Table S4</a>.</p

Examples of intergenic HPV16 DNA integration sites.

<p>(A) Tumor T3966 has one transcriptionally active HPV16 integration site 3966_DJ1 (TA-group 1), which is located between the cellular genes <i>RASSF6</i> and <i>IL8</i>, and has the same orientation as the downstream gene <i>IL8</i>. In the fusion transcript 3966_RJ, the viral exon is spliced to an intergenic alternative cellular exon. (B) Tumor T3256 (TA-group 2) has three HPV16 integration sites, one (3256_DJ1) on chromosome 3 (not shown) and two (DJ2 and DJ3) on chromosome 10q24.2 between the cellular genes <i>LOXL4</i> and <i>PYROXD2.</i> Only 3256_DJ3 is transcribed. The fusion transcript 3256_RJ is in opposite orientation to the downstream gene <i>PYROXD2</i>. (C) In the chromosome region 13q22.1-2, five HPV16 integration sites identified in four independent DNA samples are located in the large intergenic region between the cellular genes <i>KLF5</i> and <i>KLF12</i>. Only one integrated HPV16 DNA (4046_DJ1) is transcribed.</p

Tumors with integrated HPV16 DNA, but without detected APOT fusion transcripts (TA-group 4).

<p>Tumors with integrated HPV16 DNA, but without detected APOT fusion transcripts (TA-group 4).</p

The TEN16 strategy for simultaneous determination of HPV16 DNA integration sites in multiple samples.

<p>(A) The TEN16 workflow. Details of the individual steps are given in parentheses. (B) Structure of DNA fragments produced by Nextera reaction and blocking of the DNA 3′-ends. DNA fragmented by Nextera transposition carries a 19-nt universal adapter (striated arrow; referred to as Nextera adapter) covalently coupled to the 5′-end of each strand, whereas a 9-nt gap separates the 3′-end from the complementary oligonucleotide (open arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066693#pone.0066693-Caruccio1" target="_blank">[52]</a>. In a PCR using Nextera DNA fragments as templates, DNA polymerase will repair the 9-nt gap. This will lead to whole-genome amplification if the Nextera adapter is used as a primer. For this reason, a blocking reaction with ddNTP was conceived to make the 3′-OH groups inaccessible for gap repair to reduce the amplification primed by Nextera adapter alone. (C) Enrichment of the HPV16 early region by multiplex PCR. Sixteen HPV16 forward primers assembled in two mixtures (HPM-A and -B) are used to cover the E1-E2-E5 region, where the viral 3′ breakpoints can occur. The approximate primer locations are marked by arrows. (D) Categories of HiSeq2000 read pairs after sorting and mapping. In category 1 both reads of the same pair contain HPV16 sequences (open arrow) only, and in category 2 cellular sequences (filled arrow) only. Category 3 contains pairs with one HPV16 read and a cellular mate. Category 4 contains pairs with one junction read (combined arrow) plus an HPV16, a cellular, or a junction mate. Categories 3 and 4 are important for HPV16 integration site determination. (E) Validation of potential HPV16-cellular junctions by junction-specific PCR. The five DNA samples sharing the same barcode are used individually as PCR templates, and only one of them should be positive for the junction being tested. Shown here is the junction-PCR for the five DNA samples of barcode 08 and the positive result for 3256_DJ1.</p