Integration of high-risk human papillomavirus DNA correlates with HLA genotype aberration and reduced HLA class I molecule expression in human cervical carcinoma

In human cervical cancer (CC), local immunity against this human papilloma virus (HPV)-associated neoplasia has been signified. To stratify the possibility of HPV integration on HLA mutations, we measured the genotypic and phenotypic integrity of all available HLA class I loci in 30 cases of CC. Paired normal and cancer genomic DNA was analyzed with DNA typing trays, including 57 subtypes of HLA-A, 120 subtypes of HLA-B, and 60 subtypes of HLA-C. We demonstrated significant mutations of HLA genotype with reduced HLA molecule expression in CC. HPV coincide in > 70% cases of aberrant HLA genes. Southern blot analysis revealed the presence of HPV DNA within the mutated HLA foci. Our study reveals a plausible role of HPV integration in the contexts of aberrant HLA genotypes in CC cells. Disruptions of the HLA genes can be possible tactics of HPV to attain the potential carcinogenetic purposes, and thus the cancer immune escape.     

Therapeutic vaccination for HPV induced cervical cancers

Cervical Cancer is the second leading cause of cancer-related deaths in women worldwide and is associated with Human Papillomavirus (HPV) infection, creating a unique opportunity to treat cervical cancer through anti-viral vaccination. Although a prophylactic vaccine may be available within a year, millions of women, already infected, will continue to suffer from HPV-related disease, emphasizing the need to develop therapeutic vaccination strategies. A majority of clinical trials examining therapeutic vaccination have shown limited efficacy due to examining patients with more advanced-stage cancer who tend to have decreased immune function. Current trends in clinical trials with therapeutic agents examine patients with pre-invasive lesions in order to prevent invasive cervical cancer. However, longer follow-up is necessary to correlate immune responses to lesion regression. Meanwhile, preclinical studies in this field include further exploration of peptide or protein vaccination, and the delivery of HPV antigens in DNA-based vaccines or in viral vectors. As long as pre-clinical studies continue to advance, the prospect of therapeutic vaccination to treat existing lesions seem good in the near future. Positive consequences of therapeutic vaccination would include less disfiguring treatment options and fewer instances of recurrent or progressive lesions leading to a reduction in cervical cancer incidence.     

Immunomanipulative strategies for the control of human papillomavirus associated cervical disease

Three vaccine strategies that target human papillomavirus (HPV) are likely to be effective in the control of HPV-associated preneoplastic and neoplastic lesions of the uterine cervix.

1. Immunotherapy for HPV-associated cervical cancer targeted at two nonstructural PV proteins expressed in cancer cells (E6 and E7).
2. Vaccines against existing HPV infection and early premalignant lesions targeted at early viral proteins expressed in suprabasal stem cells of infected anogenital epithelium.
3. Prophylactic vaccines to prevent HPV infection involving immunization with genetically engineered virus-like particles to elicit neutralizing antibody.

Strategies 1 and 2 will need to evoke cytotoxic T-cell (CTL) mediated responses.     

人乳头瘤病毒感染与头颈癌关系的研究进展

人乳头瘤病毒(HPV)与部分头颈癌的发病相关,HPV阳性头颈癌在流行病学、病因学、病理学、分子特征等方面与HPV阴性头颈癌存在明显差异.并且头颈癌中HPV基因组存在形式及转录活性对头颈癌的预后都会产生影响.生物信息手段分析头颈癌中HPV的存在形式成为热点,而采集患者脱落细胞进行HPV检测是另一种简单易行的检测方法.     

Human papillomavirus types 16 and 18 and the prognosis of patients with stage I cervical cancer

OBJECTIVE:

This study sought to evaluate the prevalence of human papillomavirus (HPV) types 16 and 18 in women with clinical stage IB cervical cancer treated by radical hysterectomy with pelvic lymphadenectomy as well as to establish a correlation between HPV type and cancer prognosis.

METHODS:

A single-center cohort study was conducted with 86 patients who had undergone radical hysterectomy for stage I cervical cancer. Prognostic factors and the presence of HPV 16 and 18 were analyzed using a polymerase chain reaction assay. A univariate analysis using Kaplan-Meier curves was conducted to estimate survival.

RESULTS:

The prevalence of HPV 16 in the study group was 65.3%, and the prevalence of HPV 18 was 33.3%. The prevalence of infection with both viruses was 26.9%. Overall survival at 5 years was 91% among women with HPV 18 and 96% among those without this virus type (p = 0.133). Among the women with HPV 16, the overall survival was 94%, whereas this rate was 96% among those without this virus type (p = 0.663). Disease-free survival was unaffected by the presence of HPV type 16 or 18.

CONCLUSION:

In the present study, despite the high prevalence of HPV types 16 and 18, the presence of these virus types did not affect the prognosis of patients with stage I cervical cancer who underwent radical hysterectomy.     

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures.From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied.CD123⁺ pDCs as well as CD1c⁺, CD141⁺, and CD1c^-CD141^- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123⁺ pDC being dominant and CD141⁺ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles.We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.     

2020 list of human papillomavirus assays suitable for primary cervical cancer screening

BackgroundOnly clinically validated HPV assays can be accepted in cervical cancer screening.ObjectivesTo update the list of high-risk HPV assays that fulfil the 2009 international validation criteria (Meijer-2009).Data SourcesPubMed/Medline, Embase, Scopus, references from selected studies; published in January 2014 to August 2020.Study eligibility criteriaHPV test validation studies and primary screening studies, involving testing with an index HPV test and a comparator HPV test with reporting of disease outcome (occurrence of histologically confirmed cervical precancer; CIN2+).ParticipantsWomen participating in cervical cancer screening.InterventionsTesting with an index and a comparator HPV test of clinician-collected cervical specimens and assessment of disease outcome (<CIN2, CIN2+). Comparator HPV assays were HC2, GP5+/6+ PCR-EIA, recommended in validation guidelines, or tests with consistent previous validations.MethodsAssessment of relative clinical accuracy (including non-inferiority statistics index vs comparator assay) and test reproducibility in individual studies; random effects meta-analyses of the relative clinical sensitivity and specificity of index vs comparator tests.ResultsSeven hrHPV DNA tests consistently fulfilled all validation criteria in multiple studies using predefined test positivity cut-offs (Abbott RealTime High Risk HPV, Anyplex II HPV HR Detection, BD Onclarity HPV Assay, Cobas 4800 HPV Test, HPV-Risk Assay, PapilloCheck HPV-Screening Test and Xpert HPV). Another assay (Alinity m HR HPV Assay) was fully validated in one validation study. The newer Cobas 6800 HPV Test, was validated in two studies against Cobas 4800. Other tests partially fulfilled the international validation criteria (Cervista HPV HR Test, EUROArray HPV, Hybribio's 14 High-Risk HPV, LMNX Genotyping Kit GP HPV, MALDI-TOF, RIATOL qPCR and a number of other in-house developed assays) since the non-inferior accuracy was reached after a posteriori cut-off optimization, inconsistent accuracy findings in different studies, and/or insufficient reproducibility assessment. The APTIMA HPV Assay targeting E6/E7 mRNA of hrHPV was fully validated in one formal validation study and showed slightly lower pooled sensitivity but higher specificity than the standard comparator tests in seven screening studies. However, the current international validation criteria relate to DNA assays. The additional requirement for longitudinal performance data required for non-DNA based HPV assays was not assessed in this review.ConclusionsEleven hrHPV DNA assays fulfil all requirements for use in cervical cancer screening using clinician-collected specimens.     

The application of human papillomavirus testing to cervical cancer screening

Although cytologic screening has considerably reduced the incidence of cervical cancer, there are some problems which remain to be solved, such as the low sensitivity of this procedure. HPV testing is fundamentally different from conventional cytologic testing, because it evaluates the HPV infection itself, the most important causative factor for cervical cancer. In this study, the roles and clinical applications of HPV testing in cervical cancer screening are examined from 3 standpoints: in primary screening, in the management of women with low-grade cytologic abnormalities, and in the follow-up after treatment of pre-invasive or early invasive lesions.     

Type-specific persistence of human papillomavirus DNA before the development of invasive cervical cancer

BACKGROUND: Infection with the human papillomavirus (HPV) has been established as a cause of cervical cancer, but the association between a positive test for HPV DNA and the risk of the subsequent development of invasive cervical cancer is unknown. METHODS: In a study of women who participated in a population-based screening program for cancer of the cervix in Sweden from 1969 to 1995, we compared the proportion of normal cervical smears (Pap smears) that were positive for HPV DNA among 118 women in whom invasive cervical cancer developed an average of 5.6 years later (range, 0.5 month to 26.2 years) with the proportion of HPV DNA-positive smears from 118 women who remained healthy during a similar length of follow-up (controls). The control women were matched for age to the women with cancer, and they had had two normal Pap smears obtained at time points that were similar to the times of the baseline smear and the diagnosis of cancer confirmed by biopsy in the women with cancer. RESULTS: At baseline, 35 of the women with cancer (30 percent) and 3 of the control women (3 percent) were positive for HPV DNA (odds ratio, 16.4; 95 percent confidence interval, 4.4 to 75.1). At the time of diagnosis, 80 of the 104 women with cancer for whom tissue samples were available (77 percent) and 4 of the 104 matched control women (4 percent) were positive for HPV DNA. The HPV DNA type was the same in the base-line smear and the biopsy specimen in all of the women with cancer in whom HPV DNA was detected at base line. None of the control women had the same type of HPV in both smears. CONCLUSIONS: A single positive finding of HPV DNA in a Pap smear confers an increased risk of future invasive cervical cancer that is positive for the same type of virus as identified earlier.     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Detection of human papillomavirus DNA in breast cancer of patients with cervical cancer history.

BACKGROUND: Recent studies have revealed a possible role for the human papillomavirus (HPV) in the pathogenesis of breast cancer. In this study, patients having both a history of invasive cervical cancer and breast cancer as second primary cancer were selected for enrolment in a study of breast carcinomas for the presence of HPV. METHODS: Paraffin-embedded tissue from cervical cancer, pelvic lymph nodes, breast cancer and axillary lymph nodes of eleven patients were examined for the presence of HPV DNA using a polymerase chain reaction - enzyme immuno assay. DNA extraction was performed with the "QIAamp Tissue Kit" according to the manufacturer's instructions. Additionally, serum samples taken between diagnosis of cervical and breast cancer, were analyzed for the presence of HPV DNA to examine a possible haematogenous spread of oncogenic HPV DNA. RESULTS: All cervical carcinomas were HPV-positive. HPV DNA was detected in seven out of eleven cases in breast cancer and/or axillary lymph node tissue. Six patients had the same HPV type (HPV-16) in cervical cancer and in the corresponding breast cancer/lymph node tissue. In one case, the same HPV DNA type (HPV 16) was detected in cervical cancer, breast cancer and serum sample. CONCLUSION: These results suggest that HPV DNA might be transported from the original site of infection to the breast tissue by the bloodstream, and that it is possibly involved in the carcinogenesis of breast neoplasia in some patients.     

Human papillomavirus DNA in cervical lesions from Morocco and its implications for cancer control

To determine the types of human papillomaviruses (HPV) in northern Morocco, information which is needed for the design and use of HPV vaccines, we have analysed 129 cervical biopsies from this region. In our study, 91 cases were HPV positive, 45 cases had HPV-16 DNA, and 20 cases had HPV-18 DNA. This distribution of virus type was similar in inflammatory cervical lesions and in invasive cervical carcinomas. In conclusion, the HPV type distribution in Morocco is similar to that in other African Mediterranean countries, where the proportion of HPV-18 cases is significantly higher than in Europe. Determination of virus-type distribution is essential for vaccination programs.     

DNA vaccine encoding endosome-targeted human papillomavirus type 16 E7 protein generates CD4+ T cell-dependent protection

Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. We describe in this work a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo. Plasmids allowing the expression of the E7 protein in distinct cellular compartments were generated and assayed in an in vivo model of tumor growth. Our data demonstrate that mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8+ CTL and required the presence of CD4+ cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment.     

Concordant testing results between various human papillomavirus assays in primary cervical cancer screening: systematic review

ObjectivesHuman papillomavirus (HPV) assays are increasingly used for primary cervical screening and HPV-vaccination-effect monitoring. We undertook a systematic literature review to determine the concordance in positive test results (i.e. detection of HPV infections) between Hybrid Capture 2 (HC2) and other assays.MethodsWe searched PubMed, Embase and Scopus for studies of primary screening with HC2 and one or more assays, with cross-tabulated testing results for the assays. Two authors applied inclusion criteria and three authors extracted data from included studies. For each inter-assay comparison, we calculated the concordance by comparing the number of concordant samples with the number of samples that tested positive on at least one assay.ResultsSixteen studies fulfilled inclusion criteria, comparing nine assays to HC2, and including 392 to 9451 patients each. The calculated concordance varied between 48% and 69% for HC2 and APTIMA, Cobas, Abbott RealTime, Cervista, GP5+/6+, CLART, BD HPV test, Amplicor and Linear Array, i.e. 31%–52% of all positive tests on any pair of compared assays were discordant. Although modest variation in the degree of concordance with HC2 was suggested for particular assays, the numbers of studies per assay were generally low. No pronounced systematic patterns were observed by study (e.g. liquid medium) or population characteristics.ConclusionsThe ten commercially available assays do not detect the same HPV infections. Even in the most favourable case, the two assays provided discordant test results in 31% of all detected infections.     

Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction

A method for detecting HPV DNA in cervical cancer tissue was developed without using isotopes. The DNA samples from the cancer tissues were first subjected to amplification by PCR, followed by polyacrylamide gel electrophoresis to identify the specific amplified fragment. The specificity and sensitivity of the PCR method are described. Compared with the dot hybridization technique, it is shown that the method is able to detect HPV DNA in cervical cancer tissues.     

A novel peptide motif binding to and blocking the intracellular activity of the human papillomavirus E6 oncoprotein

Specific types of human papillomaviruses (HPVs) cause cervical cancer. The viral E6 oncogene is a critical factor for maintaining the malignant phenotype of HPV-positive tumour cells. By yeast two-hybrid screening of a randomised peptide expression library, we isolated linear short peptides, which specifically bind to the HPV16 E6 oncoprotein. Sequence alignments and mutational analyses of the peptides identified a hitherto undiscovered E6-binding motif. Intracellular expression of a peptide containing the novel E6-binding motif resulted in inhibition of colony formation capacity, specifically of HPV16-positive cancer cells. A solubility-optimised variant of the peptide was created, which binds to HPV16 E6 with high affinity. Its intracellular expression efficiently induced apoptosis in HPV16-positive cancer cells. This was linked to restoration of intracellular p53 activities. Thus, this newly identified E6-binding motif could form a novel basis for the development of rational strategies for the treatment of HPV16-positive preneoplastic and neoplastic lesions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transfer of tumor specific immunity with “immune” RNA: prospects for the treatment of human cancer

Summary Ribonucleic acids extracted from specifically sensitized lymphoid cells (I-RNA) have been shown to transfer specific immunoreactivity to normal non-immune lymphoid cells. Evidence for the transfer by I-RNA, of immune responses to tumor-associated antigens of animal and human neoplasms, in vivo and in vitro, is reviewed. Results obtained in our laboratory and in other laboratories indicate that xenogeneic, allogeneic and syngeneic I-RNA extracts mediate specific cytotoxicity to tumor cells, in vitro, and mediate transplantation resistance and tumor rejection responses in vivo. Our results suggest that I-RNA preparations fail to elicit immune responses directed against self antigens. By contrast, I-RNA's directed against non-self tumor-associated antigens appear to induce lymphocytes to effect specific anti-tumor immune responses. The mechanisms responsible for the failure of I-RNA to initiate immune responses against self antigens are not known at present and demand investigation.Preliminary results of a clinical Phase I trial of immunotherapy with xenogeneic I-RNA in selected cancer patients are reviewed. I-RNA might offer promise as a new modality for the immunotherapy of human cancer.Recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft     

DNA fusion gene vaccines induce cytotoxic T-cell attack on naturally processed peptides of human prostate-specific membrane antigen

For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.     

98例宫颈癌人乳头状瘤病毒16型E6基因突变及多态性分析

目的 研究湖北地区宫颈癌患者人乳头状瘤病毒16型(HPV16)E6基因点突变分布及频率,与国内外其他研究人员发现的E6突变进行对比,分析E6突变在宫颈癌发生中的意义.方法 从宫颈癌患者手术切除标本中提取组织DNA,用HPV16 E6特异性引物进行PCR扩增,对扩增的E6基因片段进行测序分析.结果 在35例宫颈癌组织DNA中有18例发生E6基因178位核苷酸的突变,变突频率为51.43%,相应核苷酸改变为Asp→Glu,442位核苷酸有4例发生核苷酸序列的改变,突变频率为11.43%,另有10例发生1～2个核苷酸序列的改变.结论 湖北地区98例宫颈癌患者人乳头状瘤病毒E6基因存在高频率的178和442位核苷酸突变,一些为新发现的核苷酸序列的突变,这些突变在宫颈癌发生中的作用有待于进一步研究.