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Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

Liang WS, Aldrich J, Nasser S, Kurdoglu A, Phillips L, Reiman R, McDonald J, Izatt T, Christoforides A, Baker A, Craig C, Egan JB, Chase DM, Farley JH, Bryce AH, Stewart AK, Borad MJ, Carpten JD, Craig DW, Monk BJ - Int. J. Gynecol. Cancer (2014)

Bottom Line: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma.Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration.This integration site has not been reported for HPV-18.

View Article: PubMed Central - PubMed

Affiliation: *Translational Genomics Research Institute; †Comprehensive Cancer Care, St Joseph's Hospital and Medical Center, Phoenix; ‡Mayo Clinic, Scottsdale; §Department of Obstetrics and Gynecology, St Joseph's Hospital and Medical Center, and ∥Division of Gynecologic Oncology, Creighton University School of Medicine, St Joseph's Hospital and Medical Center, University of Arizona Cancer Center, Phoenix, AZ.

ABSTRACT

Objective: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient's cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample.

Materials and methods: We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient's lung metastasis.

Results: We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient's tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18.

Conclusions: We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

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Related in: MedlinePlus

HPV-18 integration analysis. This schematic summarizes the major integration events that were detected using RNA data. Integrated Genomics Viewer was used to visualize RNA reads mapping to HPV-18 (A). A coverage track at the top of (A) illustrates HPV-18 expression levels. Orange and dark red coloring mark discordant reads that fall at breakpoints, whereas gray reads mapped without discordance. B, The figure shows the contigs assembled from the reads such that peach-colored portions of the contigs correspond to chromosome 6 sequences and blue-colored portions of the contigs correspond to HPV-18 sequences. The red contig spans the LCR and E6 regions of HPV-18 to indicate the presence of episomal DNA. The locations of identified breakpoints are shown at the top of panel B with green, purple, and pink arrows, and lineup with breakpoints identified in panel A. C, The figure shows a linearized view of the HPV-18 genome (blue) and the chromosome 6 region (peach) where HPV-18 reads were aligned. As shown in panel B and C, 3 separate events were identified (demarcated by green, purple, and pink arrows in panel B and corresponding green, purple, and pink connecting lines in panel C).
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Figure 2: HPV-18 integration analysis. This schematic summarizes the major integration events that were detected using RNA data. Integrated Genomics Viewer was used to visualize RNA reads mapping to HPV-18 (A). A coverage track at the top of (A) illustrates HPV-18 expression levels. Orange and dark red coloring mark discordant reads that fall at breakpoints, whereas gray reads mapped without discordance. B, The figure shows the contigs assembled from the reads such that peach-colored portions of the contigs correspond to chromosome 6 sequences and blue-colored portions of the contigs correspond to HPV-18 sequences. The red contig spans the LCR and E6 regions of HPV-18 to indicate the presence of episomal DNA. The locations of identified breakpoints are shown at the top of panel B with green, purple, and pink arrows, and lineup with breakpoints identified in panel A. C, The figure shows a linearized view of the HPV-18 genome (blue) and the chromosome 6 region (peach) where HPV-18 reads were aligned. As shown in panel B and C, 3 separate events were identified (demarcated by green, purple, and pink arrows in panel B and corresponding green, purple, and pink connecting lines in panel C).

Mentions: Visual inspection of DNA and RNA reads suggested that multiple integration events occurred. Because of the multicellularity of the sequenced sample, this phenotype suggests that different cells may have undergone different integration events. We assembled RNA reads from HPV-18 and discordant reads from chr6p25.1 that mapped to HPV-18 to pinpoint the major breakage events in expressed transcripts. Figure 2 illustrates the mapped HPV-18 RNA reads (A), assembled contigs generated from the HPV-18 RNA reads (B), and a linearized schematic of the HPV-18 genome and the chr6 region into which HPV-18 integrated (C). Overall, we assembled 155,884 paired reads to generate 20 contigs that ranged in size from 220 to 11,616 base pairs. Contigs that fully or partially aligned to HPV-18 are also shown.


Simultaneous characterization of somatic events and HPV-18 integration in a metastatic cervical carcinoma patient using DNA and RNA sequencing.

Liang WS, Aldrich J, Nasser S, Kurdoglu A, Phillips L, Reiman R, McDonald J, Izatt T, Christoforides A, Baker A, Craig C, Egan JB, Chase DM, Farley JH, Bryce AH, Stewart AK, Borad MJ, Carpten JD, Craig DW, Monk BJ - Int. J. Gynecol. Cancer (2014)

HPV-18 integration analysis. This schematic summarizes the major integration events that were detected using RNA data. Integrated Genomics Viewer was used to visualize RNA reads mapping to HPV-18 (A). A coverage track at the top of (A) illustrates HPV-18 expression levels. Orange and dark red coloring mark discordant reads that fall at breakpoints, whereas gray reads mapped without discordance. B, The figure shows the contigs assembled from the reads such that peach-colored portions of the contigs correspond to chromosome 6 sequences and blue-colored portions of the contigs correspond to HPV-18 sequences. The red contig spans the LCR and E6 regions of HPV-18 to indicate the presence of episomal DNA. The locations of identified breakpoints are shown at the top of panel B with green, purple, and pink arrows, and lineup with breakpoints identified in panel A. C, The figure shows a linearized view of the HPV-18 genome (blue) and the chromosome 6 region (peach) where HPV-18 reads were aligned. As shown in panel B and C, 3 separate events were identified (demarcated by green, purple, and pink arrows in panel B and corresponding green, purple, and pink connecting lines in panel C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921261&req=5

Figure 2: HPV-18 integration analysis. This schematic summarizes the major integration events that were detected using RNA data. Integrated Genomics Viewer was used to visualize RNA reads mapping to HPV-18 (A). A coverage track at the top of (A) illustrates HPV-18 expression levels. Orange and dark red coloring mark discordant reads that fall at breakpoints, whereas gray reads mapped without discordance. B, The figure shows the contigs assembled from the reads such that peach-colored portions of the contigs correspond to chromosome 6 sequences and blue-colored portions of the contigs correspond to HPV-18 sequences. The red contig spans the LCR and E6 regions of HPV-18 to indicate the presence of episomal DNA. The locations of identified breakpoints are shown at the top of panel B with green, purple, and pink arrows, and lineup with breakpoints identified in panel A. C, The figure shows a linearized view of the HPV-18 genome (blue) and the chromosome 6 region (peach) where HPV-18 reads were aligned. As shown in panel B and C, 3 separate events were identified (demarcated by green, purple, and pink arrows in panel B and corresponding green, purple, and pink connecting lines in panel C).
Mentions: Visual inspection of DNA and RNA reads suggested that multiple integration events occurred. Because of the multicellularity of the sequenced sample, this phenotype suggests that different cells may have undergone different integration events. We assembled RNA reads from HPV-18 and discordant reads from chr6p25.1 that mapped to HPV-18 to pinpoint the major breakage events in expressed transcripts. Figure 2 illustrates the mapped HPV-18 RNA reads (A), assembled contigs generated from the HPV-18 RNA reads (B), and a linearized schematic of the HPV-18 genome and the chr6 region into which HPV-18 integrated (C). Overall, we assembled 155,884 paired reads to generate 20 contigs that ranged in size from 220 to 11,616 base pairs. Contigs that fully or partially aligned to HPV-18 are also shown.

Bottom Line: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma.Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration.This integration site has not been reported for HPV-18.

View Article: PubMed Central - PubMed

Affiliation: *Translational Genomics Research Institute; †Comprehensive Cancer Care, St Joseph's Hospital and Medical Center, Phoenix; ‡Mayo Clinic, Scottsdale; §Department of Obstetrics and Gynecology, St Joseph's Hospital and Medical Center, and ∥Division of Gynecologic Oncology, Creighton University School of Medicine, St Joseph's Hospital and Medical Center, University of Arizona Cancer Center, Phoenix, AZ.

ABSTRACT

Objective: Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient's cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample.

Materials and methods: We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient's lung metastasis.

Results: We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient's tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18.

Conclusions: We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis.

Show MeSH
Related in: MedlinePlus