Coinfections with additional oncoviruses in HPV+ individuals: Status, function and potential clinical implications.

Oncovirus infections account for an estimated 12%-20% of human cancers worldwide. High-risk human papillomavirus (HPV) infection is the etiological agent of some malignancies such as cervical, oropharyngeal, anal, penile, vaginal, and vulvar cancers. However, HPV infection is not the only cause of these cancers or may not be sufficient to initiate cancer development. Actually, certain other risk factors including additional oncoviruses coinfections have been reported to increase the risk of patients exposed to HPV for developing different HPV-related cancers. In the current review, we summarize recent findings about coinfections with different oncoviruses in HPV+ patients from both clinical and mechanistic studies. We believe such efforts may lead to an interesting direction for improving our understanding and developing new treatments for virus-induced cancers.

An Online Survey and Focus Groups for Promoting Cancer Prevention Measures.

In order to design a cancer prevention promotion program in the region, suggestions were solicited at a medical center. We hypothesized that a majority would be native to state, and would be able to articulate about the barriers that may exist. Through online survey and focus groups, suggestions were sought, and the knowledge and the compliance with cancer prevention recommendations were assessed to determine the participants' qualifications as potential educators. Sixty-five point two percent of participants (n = 1018) graduated from high school in Arkansas. The most commonly given suggestions were to provide education to increase awareness, to use social media for promotion, to improve access, and to reduce costs. Self-reported adherence rates to breast, cervical, and colorectal cancer screening were 82.6% (n = 954), 75.8% (n = 541), and 76.7% (n = 453), respectively. Having a personal history of cancer significantly increased colorectal cancer screening uptake (p = 0.04), but paradoxically decreased mammography uptake (p = 0.007). Salary of $40,000 and more and having a Bachelor's degree or higher were associated with higher compliance of Papanicolaou test only (p = 0.007 and p = 0.001, respectively). A majority (67.7%, n = 1056) of respondents expressed willingness to contribute to promoting cancer prevention measures, and 38.3% (n = 559) were willing to participate in focus groups. However, only 6.3% (n = 35) actually participated. The participants' knowledge and compliance appeared to be sufficient, but their follow through in focus group participation was poor.

Assessing the Feasibility of an Online Module for Promoting Cancer Prevention Measures.

OBJECTIVES: The aims of this pilot study were (1) to develop a cancer prevention module consisting of an animated video and a short questionnaire, (2) to assess new knowledge gained by the participants, and (3) to solicit feedback for improving the cancer prevention module. METHODS: Volunteers who previously agreed to be contacted regarding research studies were approached via email. After completing the cancer prevention module, a list of cancer prevention recommendations was provided. Newly gained knowledge was assessed, and feedback was solicited. RESULTS: Overall, 290 of 3165 individuals contacted completed the online module (9.2%), and 38.6% of the participants indicated that they learned something new about cancer prevention measures. A similar proportion, 41.4%, mentioned that they learned about measures that were recommended and due. Paradoxically, response rate was the lowest in the ≥50 year old age group although this group reported the highest rate of learning about new cancer prevention measures. Feedback was favorable in that 70.7% mentioned that the recommendations were helpful to them personally, 69.3% felt motivated to take action to reduce their risk of cancers, and 67% would recommend the online module to their friends and family. CONCLUSION: We developed an online cancer prevention module which seems to be suitable for promoting cancer prevention measures as feedback was favorable, and new knowledge was gained. Future efforts will focus on using the module to promote cancer prevention measures to the general public particularly for the ≥50 year age group.

Expansion of Human Papillomavirus-Specific T Cells in Periphery and Cervix in a Therapeutic Vaccine Recipient Whose Cervical High-Grade Squamous Intraepithelial Lesion Regressed.

Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) ß deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. In order to verify the vaccine-specificity of these clonotypes, T cells with specificity to a region, HPV 16 E6 91-115, previously identified to be vaccine-induced using an interferon-γ enzyme-linked immunospot assay, were sorted and analyzed using single-cell RNA-seq and TCR sequencing. HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR ß bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells not only in the periphery but also in the cervix. Such an approach should be useful as a novel approach to assess vaccine-specific responses in various anatomical areas.

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota.

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

Examining aspects of successful community-based programs promoting cancer screening uptake to reduce cancer health disparity: A systematic review.

Certain minorities in the US are disproportionately burdened with higher cancer incidence and mortality rates. Programs encouraging timely uptake of cancer screening measures serve to reduce cancer health disparities. A systematic literature review was conducted to assess the effectiveness and the qualities of these programs, and to elucidate characteristics of success programs to aid in designing of future ones. We focused on community-based programs rather than clinic-based programs as the former are more likely to reach disadvantaged populations, and on prevention programs for breast, cervical, and/or colon cancers as longstanding screening recommendations for these cancers exist. PubMed, CINAHL and EBSCO databases were searched for articles that utilized community organizations and community health workers. Fourteen programs described in 34 manuscripts were identified. While 10 of 14 programs reported statistically significant increases in cancer prevention knowledge and/or increase in screening rates, only 7 of them enrolled large numbers of participants (defined as ≥1000). Only 7 programs had control groups, only 4 programs independently verified screening uptake, and 2 programs had long-term follow-up (defined as more than one screening cycle). Only one program demonstrated elimination of cancer health disparity at a population level. While most community-based cancer prevention programs have demonstrated efficacy in terms of increased knowledge and/or screening uptake, scalability and demonstration in reduction at a population level remain a challenge.

Cervical Microbiome and Response to a Human Papillomavirus Therapeutic Vaccine for Treating High-Grade Cervical Squamous Intraepithelial Lesion.

Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.

An assessment of Oxford Nanopore sequencing for human gut metagenome profiling: A pilot study of head and neck cancer patients.

Gut metagenome profiling using the Oxford Nanopore Technologies (ONT) sequencer was assessed in a pilot-sized study of 10 subjects. The taxonomic abundance of gut microbiota derived from ONT was comparable with Illumina Technology (IT) for the high-abundance species. IT better detected low-abundance species through amplification, when material was limited.

The promise of combining cancer vaccine and checkpoint blockade for treating HPV-related cancer.

Human papillomavirus (HPV)-associated intraepithelial neoplasia or cancers are ideal candidates for cancer immunotherapy since HPV oncoproteins, such as E6 and E7 proteins of high-risk HPVs, could be utilized as foreign antigens. In HPV-associated cancers as well as nonviral cancers, the cancer cells may evade host immunity through the expression of immune checkpoint molecules, downregulation of human leukocyte antigen, and activation of immune regulatory cells. Because of these immune suppressive mechanisms, HPV therapeutic vaccines have shown little efficacy against HPV-associated cancers, although they have shown efficacy in treating HPV-associated intraepithelial neoplasias. Recently, checkpoint blockade emerged as a promising new treatment for solid cancers; however, these therapies have shown only modest efficacy against HPV-associated cancers. Here we reviewed literature analyzing a combinatory therapy using an immune checkpoint inhibitor and an HPV therapeutic vaccine for treating HPV-associated cancers to compensate for shortfalls of each monotherapy. Complimentary modes of T cell activation would be deployed; as vaccines would directly stimulate the T cells, while checkpoint inhibitors would do so by releasing inhibition. Some promising studies using animal models and early human clinical trials raised a possibility that such combinations may be efficacious in regressing HPV-associated cancers. Epitope spreading (the phenomenon in which non-targeted antigens become new targets of immune response) may play a critical role mechanistically. Currently ongoing studies will shed light as to whether such combination therapy would indeed be a promising new treatment paradigm. Current and future studies must also determine the adverse effect profile of such a combination treatment.

Natural history of human papillomavirus and vaccinations in men: A literature review.

BACKGROUND AND AIMS: Infection with high-risk (HR) genotypes of the human papillomavirus (HPV) is necessary for and causative of almost all cervical cancers and their precursor condition, cervical intraepithelial neoplasia. These conditions have been sharply reduced by cervical cytology screening, and a further decrease is expected because of the recent introduction of prophylactic HPV vaccinations. While significant attention has been given to gynecologic HPV disease, men can be affected by HPV-related cancers of the anus, penis, and oropharynx. This literature review aims to address disparities in HPV-related disease in men, and certain HR male subpopulations, compared with women. DISCUSSION: Overall, immunocompetent men are far less likely than women to develop anogenital HPV-related cancers, despite harboring HR HPV infections at anogenital sites. On the other hand, men who have sex with men and men living with human immunodeficiency virus infection are at considerably higher risk of HPV-related disease. Historic rates of prophylactic HPV vaccination in males have trailed those of females due to numerous multilevel factors, although, in recent years, this sex gap in vaccination coverage has been closing. In the absence of routine HPV screening in males, therapeutic vaccinations have emerged as a potential treatment modality for preinvasive neoplasia and are in various phases of clinical testing. CONCLUSION: Successful reductions in HPV disease morbidity at the population level must acknowledge and target HPV infections in men.

Low Body Mass Index and Myeloablative Conditioning Regimen Prolong the Duration of Parenteral Nutrition During Hematopoietic Stem Cell Transplantation.

BACKGROUND/AIMS: Patients undergoing hematopoietic stem cell transplantation (HSCT) often receive total parenteral nutrition (TPN) due to poor oral intake. In clinical practice, it is difficult to predict adequate nutritional management, especially the duration of parenteral nutrition (PN), because of inter-individual variability in the conditions and types of treatment regimens. This study investigated the relationship between patient factors and the duration of TPN and the duration of PN. METHODS: Data on clinical features, patient characteristics, and the duration of TPN and PN were collected from medical records of 61 of 73 patients who underwent HSCT between April 2010 and December 2014 and were analyzed by multiple linear regression analysis. RESULTS: Forty-nine patients (80.3%) received TPN and 53 (86.9%) received PN. Sixty patients were affected by poor oral intake soon after pretreatment. Body mass index (BMI) was significantly correlated with the duration of TPN (ß = -2.733; 95% CI -4.679 to -0.787). BMI (ß = -2.260; 95% CI -4.304 to -0.213) and conditioning regimen (ß = 12.726; 95% CI 0.692-24.76) were significantly correlated with the duration of PN. CONCLUSIONS: BMI at admission and the type of conditioning regimen should be considered in choosing the nutritional management plan in patients with HSCT with poor oral intake.

A novel prostate cancer immunotherapy using prostate-specific antigen peptides and Candida skin test reagent as an adjuvant.

OBJECTIVES: Our group developed the use of the Candida skin test reagent as an adjuvant of cell-mediated immunity in designing a human papillomavirus therapeutic vaccine. Here, this technology is being applied for designing a prostate cancer immunotherapy. METHODS: Peptides based on the prostate-specific antigen amino acid sequences were selected, synthesized, and evaluated in terms of their (1) solubility, (2) maturation effects on Langerhans cells by fluorescence-activated cell sorter analysis, and (3) recognition by peripheral immune cells from prostate cancer patients using interferon-γ enzyme-linked immunospot assay. RESULTS: The peptides were soluble in 10 mM succinate at pH of 5 with 5% glycine, and they demonstrated no maturation effects on Langerhans cells from healthy donors. On the other hand, peripheral immune cells from 4 of 10 prostate cancer patients examined had positive responses in enzyme-linked immunospot assay to one or more prostate-specific antigen peptides. CONCLUSION: In summary, a design and a formulation of a novel prostate cancer immunotherapy are described. The immunogenicity of prostate-specific antigen peptides in some prostate cancer patients supports further development of this immunotherapy.

Japaneseplex: A forensic SNP assay for identification of Japanese people using Japanese-specific alleles.

It is sometimes necessary to determine whether a forensic biological sample came from a Japanese person. In this study, we developed a 60-locus SNP assay designed for the differentiation of Japanese people from other East Asians using entirely and nearly Japanese-specific alleles. This multiplex assay consisted of 6 independent PCR reactions followed by single nucleotide extension. The average number and standard deviation of Japanese-specific alleles possessed by an individual were 0.81 ±â€¯0.93 in 108 Koreans from Seoul, 8.87 ±â€¯2.89 in 103 Japanese from Tottori, 17.20 ±â€¯3.80 in 88 Japanese from Okinawa, and 0 in 220 Han Chinese from Wuxi and Changsha. The Koreans had 0-4 Japanese-specific alleles per individual, whereas the Japanese had 4-26 Japanese-specific alleles. Almost all Japanese were distinguished from the Koreans and other people by the factorial correspondence and principal component analyses. The Snipper program was also useful to estimate the degree of Japaneseness. The method described here was successfully applied to the differentiation of Japanese from non-Japanese people in forensic cases. This Japanese-specific SNP assay was named Japaneseplex.

Evaluation of immune responses induced by a novel human papillomavirus type 16 E7 peptide-based vaccine with Candida skin test reagent as an adjuvant in C57BL/6 mice.

Cell mediated immune (CMI) responses are crucial for the clearance of human papillomavirus (HPV) infection and HPV-associated lesions. Activated CD8 T cells are critical effector cells in recognizing and killing HPV-infected or HPV-transformed cells. CD4 T cells provide help for priming the generation and maintenance of CD8 T cells as well as for tumors immunity. An ideal therapeutic HPV peptide-based vaccine should induce both a robust CD8 T-cell response as well as a CD4 T-cell response for ensuring their efficiency. Candida skin test reagent was demonstrated to be able to induce the secretion of IL-12 by Langerhans cells and T-cell proliferation in vitro by our group, which indicated the potential of Candida to enhance CMI response. In this current study, we designed a novel HPV peptide-based vaccine which includes HPV16 E7 peptides and Candida as an adjuvant. The immune responses induced by the vaccine were comprehensively evaluated. The results showed that the vaccine induced significant HPV-specific CD8 T-cell and Th1 CD4 T-cell responses as well as humoral immune response. It is interesting that Candida alone induced a significant polarization of Th1 response an production of IFN-γ, which indicated Candida alone may be used as a potential immunotherapeutic reagent not only for HPV-associated lesions but also for other viral infection or even cancers.

Regulatory T Cells in Gynecologic Cancer.

Increasing evidence supports that regulatory T cells (Tregs) within the tumor, tumor draining lymph nodes, ascites and peripheral blood of patients with cancer are associated with poor prognosis. Tregs are important mediators of active immune evasion in cancer. In this review, the potential mechanisms of Treg actions and the roles of Tregs specifically in the tumor microenvironment derived from three types of gynecological cancers, cervical, vulvar and ovarian, are described. The correlations between Tregs and clinical immunotherapeutic study outcomes are discussed. Successful modulation of Tregs would likely have significant impact on the effectiveness of immunotherapeutic treatments in cancer patients.

Genotyping of the c.1423C>T (p.P475S) polymorphism in the ADAMTS13 gene by APLP and HRM assays: Northeastern Asian origin of the mutant.

ADAMTS13 is a von Willebrand factor-cleaving protease. The mutant types of p.P475S (c.1423C>T) polymorphism in ADAMTS13 have a reduced activity in comparison with the wild type. In the present study, we investigated the frequency of the C-to-T substitution in 2584 genomic DNA samples from 25 Asian, European, and African populations using APLP (amplified product length polymorphism) and/or HRM (high-resolution melting) assays. Allele T (ADAMTS13(∗)T) was detected only in Asian populations and its frequency was observed to decrease gradually from north to south in 24 East Asian populations. Almost all ADAMTS13(∗)T were associated with ABO(∗)O. These results suggested that ADAMTS13(∗)T had occurred on a chromosome with ABO(∗)O in a northern part of East Asia. This SNP is useful as an ancestry-informative marker, and the present genotyping techniques are applicable to the investigation of an association between this SNP and aortic dissection (Kobayashi et al., 2012).

Human papillomavirus type 16 viral load is decreased following a therapeutic vaccination.

In the dose-escalation phase of a Phase I clinical trial in which six subjects each were vaccinated with PepCan at the 50, 100, 250, and 500 µg per peptide dose, the 50 µg dose showed the best histological regression rate. Ten additional subjects were vaccinated at this dose in the final dose phase. As with the dose-escalation phase, no dose-limiting toxicities were observed. Overall, the histological regression rates were 50% at the 50 µg dose (7 of 14) and 100 µg dose (3 of 6), and 45 % overall (14 of 31). Of subjects in whom HPV type 16 (HPV 16) was detected at entry, it became undetectable in three subjects after vaccination, and the viral loads significantly decreased in nine subjects in whom HPV 16 infection was detected at entry and exit (p = 0.008). Immune profiling revealed increased T-helper type 1 cells after vaccinations (p = 0.02 and 0.0004 after 2 and 4 vaccinations, respectively). T-helper type 2 cells initially increased after two vaccinations (p = 0.01), but decreased below the baseline level after four vaccinations although not significantly. Pre-vaccination regulatory T cell levels were significantly lower in histological responders compared to non-responders (p = 0.03). Feasibility of testing plasma for multiplex cytokine/chemokine analysis and of performing proteomic analysis of PBMCs was examined for potentially identifying biomarkers in the future. While these analyses are feasible to perform, attention needs to be given to how soon the blood samples would be processed after phlebotomy. As sufficient safety of PepCan has been demonstrated, enrollment for the Phase II clinical trial has been opened.

A phase I dose-escalation clinical trial of a peptide-based human papillomavirus therapeutic vaccine with Candida skin test reagent as a novel vaccine adjuvant for treating women with biopsy-proven cervical intraepithelial neoplasia 2/3.

PURPOSE: Non-surgical treatments for cervical intraepithelial neoplasia 2/3 (CIN2/3) are needed as surgical treatments have been shown to double preterm delivery rate. The goal of this study was to demonstrate safety of a human papillomavirus (HPV) therapeutic vaccine called PepCan, which consists of four current good-manufacturing production-grade peptides covering the HPV type 16 E6 protein and Candida skin test reagent as a novel adjuvant. PATIENTS AND METHODS: The study was a single-arm, single-institution, dose-escalation phase I clinical trial, and the patients (n = 24) were women with biopsy-proven CIN2/3. Four injections were administered intradermally every 3 weeks in limbs. Loop electrical excision procedure (LEEP) was performed 12 weeks after the last injection for treatment and histological analysis. Six subjects each were enrolled (50, 100, 250, and 500 µg per peptide). RESULTS: The most common adverse events (AEs) were injection site reactions, and none of the patients experienced dose-limiting toxicities. The best histological response was seen at the 50 µg dose level with a regression rate of 83% (n = 6), and the overall rate was 52% (n = 23). Vaccine-induced immune responses to E6 were detected in 65% of recipients (significantly in 43%). Systemic T-helper type 1 (Th1) cells were significantly increased after four vaccinations (P = 0.02). CONCLUSION: This study demonstrated that PepCan is safe. A significantly increased systemic level of Th1 cells suggests that Candida, which induces interleukin-12 (IL-12) in vitro, may have a Th1 promoting effect. A phase II clinical trial to assess the full effect of this vaccine is warranted.

Early Defensive Mechanisms against Human Papillomavirus Infection.

Cervical cancer is the fourth most common cancer in women and is almost exclusively caused by human papillomavirus (HPV) infection. HPV is also frequently associated with other cancers arising from mucosal epithelium, including anal and oropharyngeal cancers, which are becoming more common in both men and women. Viral persistence and progression through precancerous lesion stages are prerequisites for HPV-associated cancer and reflect the inability of cell-mediated immune mechanisms to clear infections and eliminate abnormal cells in some individuals. Cell-mediated immune responses are initiated by innate pathogen sensing and subsequent secretion of soluble immune mediators and amplified by the recruitment and activation of effector T lymphocytes. This review discusses early defensive mechanisms of innate responders to natural HPV infection, their influence on response polarization, and the underappreciated role of keratinocytes in this process.

Cross-Reactivity, Epitope Spreading, and De Novo Immune Stimulation Are Possible Mechanisms of Cross-Protection of Nonvaccine Human Papillomavirus (HPV) Types in Recipients of HPV Therapeutic Vaccines.

Numerous versions of human papillomavirus (HPV) therapeutic vaccines designed to treat individuals with established HPV infection, including those with cervical intraepithelial neoplasia (CIN), are in development because approved prophylactic vaccines are not effective once HPV infection is established. As human papillomavirus 16 (HPV-16) is the most commonly detected type worldwide, all versions of HPV therapeutic vaccines contain HPV-16, and some also contain HPV-18. While these two HPV types are responsible for approximately 70% of cervical cancer cases, there are other high-risk HPV types known to cause malignancy. Therefore, it would be of interest to assess whether these HPV therapeutic vaccines may confer cross-protection against other high-risk HPV types. Data available from a few clinical trials that enrolled subjects with CINs regardless of the HPV type(s) present demonstrated clinical responses, as measured by CIN regression, in subjects with both vaccine-matched and nonvaccine HPV types. The currently available evidence demonstrating cross-reactivity, epitope spreading, and de novo immune stimulation as possible mechanisms of cross-protection conferred by investigational HPV therapeutic vaccines is discussed.