Viral Integration Status Research Articles

Multi-feature next-generation liquid biopsy for diagnosis and prognosis in HPV-associated head and neck cancer.

6064 Background: Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+HNSCC) releases circulating tumor HPV DNA (ctHPVDNA) into the blood which is a highly accurate biomarker of disease status. Existing approaches based on droplet digital PCR (ddPCR) have limitations in sensitivity, genotype coverage, and do not annotate additional prognostic genomic features. In this study, we tested the diagnostic and prognostic performance of a custom HPV whole-genome next-generation sequencing (NGS) liquid biopsy, termed HPV-DeepSeek, at the time of diagnosis with HPV+HNSCC, compared to ctHPVDNA ddPCR, HPV serology, and clinical work-up. Methods: 304 participants (152 untreated HPV+HNSCC patients, 152 population-level controls) were prospectively enrolled in this single-center prospective cohort study. Plasma samples were analyzed using HPV-DeepSeek, a multiplexed serology assay, single-plex ddPCR, and a 5-genotype multiplexed ddPCR assay. We tested the hypothesis that HPV-DeepSeek would have the highest diagnostic accuracy. We secondarily examined the accuracy of HPV-DeepSeek for prognostic feature annotation including high-risk HPV16 single nucleotide polymorphisms (SNPs), integration events, and PIK3CA mutations. Results: Of 152 cases, 114 (75%) were AJCC stage 1 and 138/152 (91%) were oropharynx primary site cancers. The sensitivity (98.7%), specificity (98.7%), and diagnostic accuracy (0.974) of HPV-DeepSeek was superior (P<0.001) to singleplex ddPCR (94.2%, 98.6%, 0.928), multiplex ddPCR (90.6%, 96.3%, 0.869), HPV serology (86.4%, 96.3%, 0.827), and first attempt clinical diagnostic biopsy (78% sensitive). Utilizing HPV-DeepSeek, 8 different genotypes were detected. 4 patients were found to have multi-genotype infections. 137/152 cases were classified as HPV16, with A1 the most common sub-lineage (90/137). 21 of 137 HPV+HNSCC HPV16 cases (15%) displayed high risk SNPs. Head-to-head comparison of HPV SNP genotyping by HPV-DeepSeek, with the gold-standard tissue-based HPV whole genome sequencing assay, demonstrated 89% concordance in tissue and 85% in blood. The internal concordance rate for HPV-DeepSeek between tissue and blood was 91%. 48 of 152 cases (32%) had at least one viral integration event detected and 36 of 152 cases (24%) had a PIK3CA mutation detected by HPV-DeepSeek. Fragmentomic analysis demonstrated a mean ctHPVDNA fragment size of 148bp for ctHPVDNA versus 167bp for human cell free (cf)DNA from HPV+HNSCC patients and 168bp for control patient cfDNA. Baseline ctHPVDNA quantity was strongly correlated with viral integration status, T stage and N stage (P<0.001). Conclusions: HPV-DeepSeek has improved diagnostic accuracy relative to ddPCR, HPV serology and clinical work-up at the time of diagnosis and demonstrates detection of prognostic features including high-risk HPV16 SNPs, viral integration events, and PIK3CA mutations.

Abstract 6062: Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma

Abstract Integration of viral DNA into the host genome has been implicated in human papillomavirus (HPV) associated tumorigenesis, and studies in HPV associated head and neck squamous cell carcinoma (HNSCC) attempting to classify viral integration status have reported associations among HPV status, viral load, and overall survival. However, few genomic studies have correlated HPV structure with commonly occurring somatic mutations in HNSCC. Here, we conducted hybrid-capture DNA sequencing using a panel of over 800 cancer related genomic targets, as well as probes specific for the full HPV16 and HPV18 genomes, in a novel cohort of 525 HNSCC cases. Paired reads were aligned to a human reference containing complete viral genomes, including all high-risk HPV types, which allowed for robust identification of HPV(+) tumors and specific HPV type. Variant calling and filtering, followed by pathway analysis of significantly mutated genes, defined the catalog of somatic driver alterations in HNSCC. Viral genome structure, integration status, and viral load were determined via initial copy number analysis of HPV-specific reads, followed by identification of viral/human breakpoints and indels with variant calling algorithms. Manual review was required in some cases due to variability in read coverage and alignment to HPV genome. We identified 252 HPV(+) tumors, the majority of which were HPV16 (n=228, 90%). Analysis of HPV16(+) tumors revealed five classes with variable viral loads: pure episome (EPI, n=54), episomal rearranged (EPI-R, n=51), episomal chimeric (EPI-C, n=22), integrated (INT, n=92), and integrated deleted (INT-A, n=12). Integrated tumors had at least one integration event (INT) and a few exhibited recurrent deletions in the viral genome (INT-A). Episomal samples had either complete HPV genomes (EPI), circular genomes with one or more indels (EPI-R), or circular genomes with HPV and human DNA (EPI-C). Interestingly, PIK3CA, the most frequently mutated gene in HPV(+) HNSCC, was mutated at a higher frequency in all integrated samples compared to all episomal types (44% vs 23%, p=0.005). We also noticed more deleterious mutations in innate immunity genes (HLA-A/B, B2M, TRAF3, BIRC3) in the episomal samples (16% vs 8%), which may allow cells with intact HPV genomes to evade anti-viral immune responses. Additionally, mutations in oxidative stress response genes that lead to the constitutive activation of NRF2 (NFE2L2, CUL3, KEAP1) were more frequent in integrated samples (11% vs 4%), which could confer radiotherapy resistance and a worse prognosis. Overall, using DNA sequencing and integrative genomic analysis in the largest cohort of HPV(+) HNSCC to date, we developed a novel classification method based on viral genome structure which associates with host somatic alterations that may contribute to variable outcomes in HNSCCs. Citation Format: Jeremiah Ray Holt, Xiaobei Zhao, Hyo Young Choi, Paul Little, Angela L. Mazul, Benjamin Wahle, Vonn Walter, Jose P. Zevallos, David Neil Hayes. Viral genome structure correlates with patterns of somatic alteration in human papillomavirus associated head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6062.

Viral Integration Analysis Reveals Likely Common Clonal Origin of Bilateral HPV16-Positive, p16-Positive Tonsil Tumors.

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and CD36 as targets of viral integration are involved in cell migration and ECM-receptor interactions, which provides a possible mechanism for clonal migration from one tonsil to another.

Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis.

Different studies have shown that HPV16-positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome-wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty-three fresh-frozen HPV-16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo-keto-reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV-positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non-hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV-positive (p ≤0.001) and -negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV-negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.

Racial/ethnic differences in HPV 16/18 genotypes and integration status among women with a history of cytological abnormalities

ObjectiveHPV genotype distribution varies by race/ethnicity, but is unclear whether there are racial/ethnic variations in HPV 16/18 integration in the host genome. We describe HPV16/18 infection and integration status in a racially/ethnically diverse sample of women with a recent abnormal Pap test. MethodsPatients (n=640) represent a subset of women participating in a clinical trial. Cervical swabs were tested for HPV16/18 DNA using type-specific polymerase chain reaction assays. Viral integration status was assessed using type-specific integration assays and categorized as fully integrated, fully non-integrated, or mixed. Unconditional logistic regression was used to generate unadjusted (OR) and adjusted odds ratios (aOR) to assess the association between self-reported race/ethnicity and risk of these outcomes. ResultsHispanic and non-Hispanic black women had half the odds of prevalent HPV16 compared to non-Hispanic white women (aORs: 0.43 and 0.45, respectively). The prevalence odds of HPV18 was less than half among Hispanic women (aOR: 0.48), but not significantly different between black and white women (aOR: 0.72). Among women with prevalent HPV16, the odds of fully integrated viral DNA were significantly higher among black women (aORs: 2.78) and marginally higher among Hispanic women (aOR: 1.93). No racial/ethnic differences were observed for HPV18 DNA integration. ConclusionsWhile HPV16 and 18 infections were less prevalent among Hispanic and black women compared to whites, their HPV16 DNA was more likely to be present in a fully integrated state. This could potentially contribute to the higher rates of abnormal cytology and cervical dysplasia observed among Hispanic and black women.

Abstract 562: Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing

Abstract Viruses are a major contributor to oncogenesis, causing 10-15% of human cancers. Molecular pathways involved in malignant transformation are frequently activated by genetic alterations, including but not limited to, somatic mutations, copy number aberrations, structural variants, and oncoviruses. Precision cancer medicine aims to classify tumors by site, histology, and molecular tests to determine an “individualized” profile of cancer alterations. However, clinical tests for these various alterations are sequential, time consuming, and use a lot of material, which is often quite limited (e.g., biopsies). Moreover, tests for the presence of viral sequence are generally performed separately to tests (such as massively parallel sequencing) to detect human genomic alterations. Here we present a hybrid capture and massively parallel sequencing approach to detect viral infection concurrently with targeted genomic analysis, which may decrease assay costs, increase sensitivity and scalability, and detect many types of alterations, thereby providing a more complete tumor genetic profile all from a single sample. We have created a custom hybrid capture probeset for targeted Illumina sequencing to determine whether oncoviruses are present in tissue samples and also determine if the virus has integrated into the host’s genome. We have created both ‘detection’ and ‘integration’ baits for several oncoviruses, including polyomaviruses, human papilloma viruses, Epstein-Barr virus, human cytomegalovirus, Kaposi sarcoma herpesvirus, human T-lymphotropic virus, and hepatitis B virus. To distinguish between different strains of a single virus, strain-specific detection baits were created to bind to variable regions of viral genomes. The integration bait was designed to bind to regions of the viral genomes that are commonly integrated into the human genome. This baitset can also be combined with other capture panels targeting oncogenes to simultaneously determine infection and integration statuses, as well as somatic mutations, copy number and structural variants. To detect virus presence, reads were aligned to a hybrid reference of both the human, and targeted virus genomes. Viral integration status and integration loci were determined by leveraging discordant read pairs that aligned to both the human genome and a viral genome. We have tested our techniques on tissue samples that were infected with either Merkel Cell Polyomavirus or Epstein-Barr virus, as determined using quantitative polymerase chain reaction (qPCR) or immunohistochemistry (IHC) techniques, and have successfully detected these viruses and identified viral integration loci. Overall, this viral hybrid capture probeset provides the ability to simultaneously determine a tissue sample’s infection and viral integration status alongside other somatic genomic analyses, saving both time and sample material. Citation Format: Robert T. Burns, Samuel S. Hunter, Matthew D. Ducar, Aaron R. Thorner, James A. Decaprio, Paul Van Hummelen, Alexander Frieden, Anwesha Nag, Haley A. Coleman, Michael K. Slevin, Andrea Clapp, Samantha D. Drinan, Suzanne R. McShane, Edwin Thai, Priyanka Shivdasani, Joshua Bohannon, Johann Hoeftberger, Reuben Jacobs, Bruce M. Wollison, Neil A. Patel, Monica D. Manam, Phani Davineni, Matthew Meyerson, Laura E. MacConaill. Oncovirus detection and integration analysis from human tumor samples using targeted massively parallel sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2017-562

HPV16 DNA and integration in normal and malignant epithelium: implications for the etiology of laryngeal squamous cell carcinoma

BackgroundMolecular evidence suggests that human papillomavirus (HPV) has a role in the etiology of oropharyngeal squamous cell carcinoma. However, the role of HPV in laryngeal squamous cell carcinoma (LSCC) is unclear.Patients and methodsWe conducted a case–control study using tumor tissue specimens from 300 LSCC patients and vocal cord polyp specimens from 300 cancer-free controls. HPV genotype, HPV16 viral load and viral integration status, and p16 expression were determined.ResultsThe prevalence of HPV (all types) was higher in cases than controls [21 (7.0%) versus 10 (3.3%), adjusted odds ratio (aOR) 2.37, 95% CI 1.08–5.21]. The prevalence of HPV16 was higher in cases than controls [20 (6.7%) versus 8 (2.7%), aOR 2.84, 95% CI 1.21–6.68]. The risk of LSCC associated with HPV16 DNA positivity was even higher in patients aged 55 years or younger (aOR 3.52, 95% CI 1.07–11.54), males (aOR 4.74, 95% CI 1.33–16.90), never-smokers (aOR 5.57, 95% CI 1.41–22.10), and never-drinkers (aOR 3.72, 95% CI 1.09–12.72). HPV DNA was partly or fully integrated in all 20 HPV16-positive cases but was episomal in all 8 HPV16-positive controls; however, the HPV16-positive cases and controls had similar viral loads (P = 0.28). P16 immunostaining was positive in 31 of the 300 cases (10.3%) and negative in all 300 controls.ConclusionThese results suggest that prior infection with HPV16 may play a role in the etiology of some LSCC. This larger case–control study will offer for the first time the possibility to address in depth the understanding of a tissue-specific role of HPV in laryngeal carcinogenesis. Further studies with larger samples are needed to confirm these findings.

Integration of human papillomavirus type 16 in cervical cancer cells.

Cervical cancer remains an important cause of women morbidity and mortality. The progression of cervical pathology correlates with the HPV integration into the host genome. However, the data on the viral integration status in cervical dysplasias are controversial. The aim of the current study was to evaluate the status of HPV integration in two types of cervical pathology – invasive and non invasive cervical cancer (e.g. carcinoma in situ). 156 women were included in the study: 66 women were diagnosed with invasive cervical cancer (CC) and 90 with non invasive cervical cancer (carcinoma in situ, CIS). 74.2% [95% PI: 63.64÷84.76] of specimens collected from women with diagnosed CC and 85.6% [95% PI: 85.53÷92.85] of CIS specimens were positive for HPV. The most prevalent HPV genotype in both groups was HPV16. To evaluate HPV integration, three selected HPV16 E2 gene fragments were analyzed by PCR. In the majority of CC and CIS specimens the amplification of all three HPV16 E2 gene fragments was observed. The episomal HPV16 form was detected in the majority of CC and CIS specimens. The deletion of all three HPV16 E2 gene fragments was detected in 9.4% of CC specimens and 2.2% of CIS specimens. Finally, integration status could not be used as diagnostical additional test to distinguish between invasive and non invasive cervical cancer.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

Abstract 2623: The role of polymorphisms in DNA repair genes and HPV 16 integration status in cervical dysplasia

Abstract BACKGROUND: Cervical cancer is the second leading cause of death in women worldwide, and yet, little is understood about why some HPV-infected women develop cancerous lesions while others are able to clear the infection with no sequelae. Previous evidence has indicated that HPV integration status may be associated with cervical cancer development and progression. However, host genetic variation within genes that may play important roles in the viral integration process has been understudied. The purpose of this study was to examine the association between both HPV 16 viral integration status and single nucleotide polymorphisms (SNPs) in non-homologous end-joining (NHEJ) DNA repair pathway genes on cervical dysplasia, comparing women with no dysplasia to those with low-grade or high-grade squamous intraepithelial lesions. METHODS: A total of 765 women were selected from two large trials designed to evaluate optical technologies for cervical cancer, being conducted at MD Anderson Cancer Center, Lyndon B Johnson General Hospital, and the British Columbia Cancer Agency. Genotyping was conducted using the Illumina Golden Gate platform; individuals with call rates &amp;lt;85% were excluded from the analyses. HPV integration status was determined using an assay described in the literature. Chi-square and Fisher's exact tests were conducted to determine which SNPs were significantly associated with normal cytology, low-grade, or high-grade lesions. Among participants with cervical dysplasia, chi-square tests and polytomous logistic regression models were used to evaluate the effect of each polymorphism on viral integration status. We used an additive genetic model for all tests. P-values were adjusted using the false discovery rate method and FDR-adjusted q values are reported. RESULTS: Women with high-grade lesions were significantly younger than women with low-grade or no lesions. Tag-SNPs in 13 DNA repair genes, including RAD50, WRN, and XRCC5, were significantly associated with cervical dysplasia. HPV16 integration status was differential across cervical cytology, but overall, most participants had a mix of both episomal and integrated HPV 16. Four tag-SNPs in the XRCC4 gene were found to be significantly associated with HPV 16 integration status. CONCLUSION: Our findings indicate that host genetic variation in NHEJ DNA repair pathway genes, specifically XRCC4, are significantly associated with HPV integration, and that these genes may play an important role in determining cervical cancer development and progression. This is the first time that host genetic variation has been linked to the viral integration event. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2623. doi:1538-7445.AM2012-2623

The Role of Polymorphisms in DNA Repair Genes and HPV 18 Integration Status in Cervical Dysplasia

Abstract Despite the fact that cervical cancer is one of the leading causes of death in women worldwide, research has yet to elucidate why some HPV-infected women develop cancerous lesions while others are able to clear the infection. Previous studies have shown that HPV integration status may be associated with cervical cancer development, and yet, host genetic factors that may be involved in the viral integration process have not yet been identified. The purpose of this study was to examine the association between both HPV 18 viral integration status and single nucleotide polymorphisms (SNPs) in non-homologous end-joining (NHEJ) DNA repair pathway genes on cervical dysplasia. Specifically, we sought to compare women with no dysplasia to those with low-grade or high-grade squamous intraepithelial lesions. Methods: A total of 765 women were selected from two large trials designed to evaluate optical technologies for cervical cancer. Genotyping was performed using the Illumina Golden Gate platform. HPV 18 integration status was determined using a previously established protocol. Chi-square tests were conducted to determine which SNPs were associated with normal cytology, low-grade, or high-grade lesions. Among participants with cervical dysplasia, polytomous logistic regression models were used to evaluate the effect of each polymorphism on viral integration status. An additive genetic model was used for all tests. P-values were adjusted using the false discovery rate method. Results: Women with high-grade lesions were significantly younger than women with low-grade or no lesions. Tag-SNPs in 13 DNA repair genes, including MRE11A, ATM, and XRCC4, were significantly associated with cervical dysplasia. Most participants had a mix of both episomal and integrated HPV 18. Tag-SNPs in the XRCC4, PRKCH, and MRE11A genes were found to be significantly associated with HPV 18 integration status. Conclusion: Our study indicates that host genetic variation in NHEJ DNA repair pathway genes, including MRE11A and XRCC4, are significantly associated with HPV 18 integration, and that these genes may play a key role in determining cervical cancer development and progression. This is the first study to examine host genetic variation in association with the viral integration event.

Herpesviruses as possible cofactors in HPV-16-related oncogenesis.

Cervical carcinogenesis is a complex problem with papillomavirus widely accepted as a causative agent. Integration of a human papillomavirus (HPV) of the high-risk type into the host cell genome is one of the major contributing factors to cervical malignant transformation. In this study, the correlation of CMV, EBV, HSV-1, HSV-2, HHV-6 and HHV-7 infections with the physical status of the HPV genome in cervical cancer and precancerous cervical lesions was investigated in sixty HPV-16-positive women. Cervical secretion samples were submitted to DNA extraction and analyzed by PCR. HPV-16 DNA was confirmed in genotyping with the reverse hybridization line probe assay. Multiplex PCR with specific primers for the E2/E6 genes was used to assess the viral integration status of HPV-16. Our results show that CMV DNA was more frequently present in samples with mixed forms of HPV-16 than in the episomal form (P < 0.025). Such a correlation was also observed in the case of EBV (P < 0.005). The presence of CMV resulted in a six-fold (OR 6.069; 95% CI 1.91-19.22; P = 0.002), while EBV caused a seven-fold (OR 7.11; 95% CI 1.70-29.67; P = 0.007) increase in the risk of the integrated or mixed HPV-16 genome occurrence. Our data suggest that coinfection with herpesviruses, especially CMV and EBV, may be involved in the integration of the HPV-16 genome and may contribute to the development of cervical cancer.

Physical State of Human Papillomavirus Type 16 in Cervical Cell DNA

Multiplex PCR with specific primers for E2/E6 genes was used to assess the viral integration status of HPV-16 in women with low and high grade squamous intraepithelial lesions (LSIL and HSIL, respectively) in comparison to cervical cancer patients. Women with confirmed HPV-16 infection were examined: 30 with LSIL, 12 with HSIL and 23 with cervical cancer. The PCR products were separated electrophoretically in agarose gels and densitometric analysis was performed using Bio-Rad Quantity One software. E2 and E6 sequences of HPV-16 were detected in 91% of the women. The free episomal viral genome was not detected in the cervical carcinoma group. Twenty six percent of the samples obtained from this group harboured the integrated form, whereas the remaining samples possessed a mixture, i.e. episomal and integrated forms of viral DNA. The free episomal form dominated in women with LSIL and HSIL. In 6 cases the episomal and integrated forms were detected simultaneously. HPV-16 integration occurred in a subset of LSILs and HSILs, not only in the cervical cancer patients and correlated with progression of cytological changes. The assessment of the status of HPV-16 may be the molecular factor preceding the morphological features leading to malignancy.

The biomarkers of human papillomavirus infection in tonsillar squamous cell carcinoma—molecular basis and predicting favorable outcome

Presence of human papillomavirus (HPV) in variable proportions in tonsillar squamous cell carcinoma tissues has been demonstrated by several worldwide studies. Some reports emphasized the significance of HPV in predicting a better prognosis, as well as ethnic differences between Chinese and Caucasians. In order to understand the biological role of HPV and find out clinically accessible methods to determine its prognostic significance in primary tonsillar squamous cell carcinoma, we collected 92 patients with primary tonsillar squamous cell carcinoma diagnosed or treated in National Taiwan University Hospital, for whom archival tumor tissue were available. Immunohistochemical stains of p16INK4A, high-risk HPV in situ hybridization, and nested polymerase chain reaction (PCR)-based genechips were performed to detect HPV infection and determine its genotype. Clinical data were compared with HPV infection detected by the different methods mentioned above. Real-time PCR was also performed on the HPV16-positive [HPV16(+)] lesions to understand viral integration status. The positive rates of nested PCR-based genechips, overexpression of p16INK4A, and high-risk HPV in situ hybridization were 75% (6 9/92), 53% (4 9/92), and 44% (4 0/92), respectively. Both overexpression of P16INK4A and high-risk HPV in situ hybridization positivity were associated with favorable prognoses (P=0.004 and 0.001, respectively) and also independent prognostic factors in multivariate analyses (P=0.01 and 0.01, respectively). The positivity of nested PCR-based genechips was not statistically significant. From our data, primary tonsillar squamous cell carcinoma with positive immunohistochemical stains of p16INK4A and/or high-risk HPV in situ hybridization is associated with a better outcome, and both methods may serve as clinically accessible markers.

Evaluation of a Commercialized In Situ Hybridization Assay for Detecting Human Papillomavirus DNA in Tissue Specimens from Patients with Cervical Intraepithelial Neoplasia and Cervical Carcinoma

To evaluate a commercialized in situ hybridization (ISH) assay for detecting human papillomavirus (HPV) DNA, we compared the ability of a new ISH probe, Inform HPV III (Ventana Medical Systems, Tucson, AZ), to that of PCR assays to detect HPV DNA in cervical tissue specimens with normal cervix (20 cases), cervical intraepithelial neoplasia (CIN; CIN 1, 27 cases; CIN 2, 28 cases; and CIN 3, 33 cases), and cervical carcinoma (29 cases). General HPV DNA was detected using consensus primer-mediated PCR assays. HPV genotyping was performed by using EasyChip HPV blot (King Car Yuan Shan Institute, I-Lan, Taiwan). HPV16 integration status (E2/E6 ratio) was determined by using quantitative real-time PCR. Our findings showed that the ISH and PCR had fair to good agreements in detecting HPV DNA across all CIN categories without significant differences (Kappa coefficient, 0.34 to 0.63; P = 0.13 to 1.0). However, ISH detected significantly fewer HPV-positive cases in carcinoma than PCR did (Kappa coefficient, 0.2; P = 0.03). Eleven cases with ISH- PCR+ results had HPV types that can be detected by Inform HPV III. Five carcinoma cases with ISH- PCR+ results showed a significantly higher level of integrated HPV16 (P = 0.008) than did the ISH+ cases. As a consequence, lower copy numbers of episomal HPV16 in carcinoma might be the cause for the false-negative ISH results. Although the punctate signal pattern of HPV significantly increased with the severity of disease (P trend = 0.01), no significant difference in the HPV16 integration status was observed between the cases with a punctate signal only and the cases with mixed punctate and diffuse signals (P = 0.4). In conclusion, ISH using the Inform HPV III probe seems comparable to PCR for detecting HPV DNA in cervical tissue with CINs. False-negative ISH results appear to be associated with the lower copy numbers of the episomal HPV16 but not with the ability of the Inform HPV III probe to detect specific HPV types. In addition, signal patterns, especially a mixed punctate and diffuse pattern of HPV, cannot be reliably used to predict viral integration status.

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma

AbstractCurrent human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67–39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89–9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories. Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.