Serum, cell-free, HPV-human DNA junction detection and HPV typing for predicting and monitoring cervical cancer recurrence
- Department of Genetics, Albert Einstein College of Medicine, United States
- Department of Obstetrics Gynecology and Women’s Health, Albert Einstein College of Medicine, United States
- Department of Pathology, Albert Einstein College of Medicine, United States
- School of Animal and Comparative Biomedical Sciences, College of Agriculture and Life Sciences BIO5 Institute, University of Arizona, United States
- Broad Institute, United States
- Rutgers Cancer Institute, United States
Figures
Figure 1
Time course characterizations of the 16 patients in the cohort with individual patients (P), type of cancer (C) either squamous carcinoma (S) or adenocarcinoma (A), diagnostic stage (stage), and human papillomavirus (HPV) type identified in the tumor tissue (HPV).
(A) Analysis of HPV-human DNA junctions in serum cell-free DNA (cfDNA) at initial examination and at the 6 month post-treatment follow-up. (B) Detection of the cognate HPV E7 DNA in serum cfDNA at the same time points. Descriptions of the symbols are in the box included in each panel. (C) Integrated Genome Viewer (IGV) (Deleage et al., 2016) plots of sequence reads obtained by HPV DNA hybridization capture for each sample as a function of the standard HPV genome numbering system (Van Doorslaer et al., 2017). (D) The integrated HPV genomic structures for the 10 tumors that contained only subgenomic segments of the viral genome. Viral DNAs were centered on the URR-E6-E7 stretch of the viral genomes. HPV open reading frame structure is shown at the top. The 5’ splice site (5’ss) shown in the viral genome is just inside the 5’ end of the E1 open reading frame. As ORF sizes and precise positions vary slightly among HPV types, integrated DNA segments may be slightly off-scale. Some of the HPV DNA insertions had more than two junctions with human DNA clustered within stretches up to tens of kilobase pairs of the human genome, a well-established phenomenon most likely due to instability of inserted HPV DNA, including extra-chromosomal circularization of HPV-human DNA heterocatemer segments, and nearby reintegration of such structures (Rossi et al., 2023; Van Arsdale et al., 2024; Akagi et al., 2023; Akagi et al., 2014; Holmes et al., 2016; Hu et al., 2015; Xu et al., 2013; Liu et al., 2016). In those instances, the junctions shown were evident as read gap borders in Panel C, and they also had the largest number of PCR-confirmed, hybridization capture +sequencing reads. (E) The presumed genomic structures of integrated HPV DNAs in six tumors containing viral segments that were longer than unit length. All were from phylogenetic clade α9, five HPV16 and one HPV35. Arrows show the viral 5’ to 3’ transcriptional orientation of the HPV protein-coding strands.
Figure 2
PCR detection of human papillomavirus (HPV)-human DNA junctions in human genomic DNAs isolated from biopsy tissue of the cancer recurrences that occurred in the five indicated patients.
PCRs for each patient included a sample of DNA from the recurrence and a positive control using the primary tumor tissue DNA from the individual patients. The junctions in the five recurrences were also detected by hybridization capture plus Illumina sequencing on the recurrence biopsies, which likewise confirmed clonal derivation from the cognate primary tumors.
-
Figure 2—source data 1
Original gels.
- https://cdn.elifesciences.org/articles/105741/elife-105741-fig2-data1-v1.zip
-
Figure 2—source data 2
Original gels with relevant bands identified.
- https://cdn.elifesciences.org/articles/105741/elife-105741-fig2-data2-v1.zip
Figure 3
DNA concentration determinations and Kaplan–Meier analyses performed using cell-free DNA (cfDNA) from patients.
(A) Total cfDNA concentrations in early- vs. late-stage cancers. (B) The same DNA concentration determinations plotted from patients who did not have a recurrence vs. those that did. (C, D) Kaplan–Meier analyses of recurrence-free survival in patients where human papillomavirus (HPV)-human DNA junctions were detected in serum vs. those where it was not detected at initial examination (C) or at 6 months post-treatment (D). (E, F) Kaplan–Meier analyses of recurrence-free survival in patients where HPV E7 DNA was detected in serum vs. those where it was not detected at initial examination (E) or at 6 months post-treatment (F). In all four plots, squares show left-censoring at the last patient visits.
Figure 4
Analyses of human papillomavirus (HPV) DNAs in Patient 16.
(A) PCR analysis for HPV16 E7 DNA in serum cell-free DNA (cfDNA) plus DNA from the primary tumor as a positive control. (B) PCR analysis for HPV18 E7 DNA in serum cfDNA plus DNA from the primary tumor as a positive control. (C) The Integrated Genome Viewer (IGV) plots of sequence reads following HPV DNA hybridization capture from the primary tumor and cfDNA. Sequence reads for HPV16 and HPV18 in serum cfDNA were plotted separately.
-
Figure 4—source data 1
Original gels.
- https://cdn.elifesciences.org/articles/105741/elife-105741-fig4-data1-v1.zip
-
Figure 4—source data 2
Original gels with relevant bands identified.
- https://cdn.elifesciences.org/articles/105741/elife-105741-fig4-data2-v1.zip
Figure 5
Kaplan–Meier recurrence-free survival plot of 297 The Cancer Genome Atlas (TCGA) cervical cancer patients.
Tables
Table 1
Human papillomavirus (HPV) types, viral phylogenetic clades, human genome integration sites, serum cell-free DNA (cfDNA) coverage by HC + SEQ, and serum cfDNA type-specific E7 PCR.
| Serum cfDNA HPV type-specific | |||||||
|---|---|---|---|---|---|---|---|
| Patient | Stage | Cancer* | HPV type† | Clade | Human genome integration | genome coverage | HPV E7 PCR |
| 1 | 2B | SCC | HPV70 | α7 | Chr14:chr14:99,216,417–99,217,370 | 0.506 | Positive |
| 2 | 2B | SCC | HPV16 | α9 | Chr11:119,699,940–119,699,962 | 0.238 | Positive |
| 3 | 1B3 | AC | HPV16 | α9 | ChrX:137,024,646 ‡ | 0.237 | Positive |
| 4 | 3C1r | SCC | HPV18 | α7 | Chr8:126,992,703–127,083,002 | 0 | Positive |
| 5 | 2B | SCC | HPV16 | α9 | Chr5:1,346,104–1,386,461§ | 1 | Positive |
| Chr6:106,847,095–106,849,161§ | |||||||
| 6 | 1B3 | SCC | HPV39 | α7 | Chr14:102,789,751–102,790,038 | 0.33 | Positive |
| 7 | 2B | AC | HPV18 | α7 | Chr16:71,705,032–71,732,359 | 0.33 | Positive |
| 8 | 2B | SCC | HPV69 | α5 | Chr9:87,266,100–87,266,133 | 0.903 | Positive |
| 9 | 1B2 | SCC | HPV45 | α7 | ChrX:13,185,650–13,391,674 | ND¶ | Positive |
| 10 | 1B1 | SCC | HPV35 | α9 | Chr8:59,436,141–59,436,178 | ND | Positive |
| 11 | 1B1 | AC | HPV16 | α9 | Chr7:117,590,654–117,605,110 | 0.726 | Negative |
| 12 | 1B1 | SCC | HPV16 | α9 | Chr2:63,048,331–63,048,344 | 0 | Positive |
| 13 | 1B1 | AC | HPV18 | α7 | Chr6:134,482,206–134,527,589 | 0.818 | Negative |
| 14 | 1B1 | SCC | HPV18 | α7 | Chr15:89,108,581–89,144,328 | 0.049 | Positive |
| 15 | 1B1 | SCC | HPV73 | α11 | Chr4:67,867,259–68,002,635 | 0.087 | Negative |
| 16 | 1B1 | SCC | HPV16 | α9 | ChrX:35,045,995–35,046,002 | 0.982/0.919** | Positive |
-
*
SCC, squamous cell carcinoma; AC, adenocarcinoma.
-
†
HPV type determined by HC + SEQ on tumor tissue.
-
‡
Only one junction between HPV16 and human DNA was detected by hybridization capture for which effective PCR primers were developed for Patient 3.
-
§
Tumor 5 had two DNA insertions of the identical HPV16 variant, one on chromosome 5 and the other on chromosome 6 at the indicated positions.
-
¶
ND, not done.
-
**
Both HPV16 and HPV18 were present in serum cfDNA of Patient 16 with respective genome coverages shown.
Table 2
A priori clinicopathologic variables of the The Cancer Genome Atlas (TCGA) cohort by human papillomavirus (HPV) phylogenetic clade.
| Variable | Cohort (n=247) | Clade 9 (n=83) | Clade 7 or other | p-value |
|---|---|---|---|---|
| Age (years) | 47.8 (13.6) | 48.6 (13.8) | 45.8 (13.0) | 0.12 |
| Histology | ||||
| Squamous cell | 254 (84.4) | 183 (85.9) | 67 (83.8) | 0.71 |
| Adenocarcinoma | 47 (15.6) | 30 (14.1) | 13 (16.2) | |
| Stage at Dx | 0.57 | |||
| Stage I | 156 (54.6) | 113 (54.6) | 43 (54.4) | |
| Stage II | 65 (22.7) | 44 (21.3) | 21 (26.6) | |
| Stage III | 46 (16.1) | 34 (16.4) | 12 (15.2) | |
| Stage IV | 19 (6.6) | 16 (7.7) | 3 (3.8) |
-
Continuous variables are presented as mean ± standard deviation and compared using Student’s t-test, while categorical variables are summarized as frequencies with percentages and compared using Fisher’s exact test or Pearson’s chi-square test, as appropriate.
Table 3
Multivariable Cox proportional hazard regression model for recurrence-free survival.
| Variable | Multivariable hazard ratio | 95% CI | p-value |
|---|---|---|---|
| Clade | |||
| Clade 9 | 1.00 | -- | -- |
| Clade 7 | 2.48 | 1.34–4.49 | 0.004 |
| Age (years) | 1.01 | 0.99–1.04 | 0.28 |
| Histology | |||
| Squamous | 1.00 | -- | -- |
| Adenocarcinoma | 1.08 | 0.51–2.28 | 0.84 |
| Stage at Dx | |||
| Stage I | 1.00 | -- | -- |
| Stage II | 0.63 | 0.28–1.41 | 0.26 |
| Stage III | 0.43 | 0.13–1.46 | 0.18 |
| Stage IV | 4.40 | 1.89–10.18 | 0.001 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/105741/elife-105741-mdarchecklist1-v1.pdf
-
Supplementary file 1
PCR primers used for HPV type and junction validation.
- https://cdn.elifesciences.org/articles/105741/elife-105741-supp1-v1.docx
-
Supplementary file 2
Patient demographics and clinical parameters.
- https://cdn.elifesciences.org/articles/105741/elife-105741-supp2-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Serum, cell-free, HPV-human DNA junction detection and HPV typing for predicting and monitoring cervical cancer recurrence
eLife 14:RP105741.
https://doi.org/10.7554/eLife.105741.2
