Cancer-related diseases represent the second overall cause of death worldwide. Human papilloma virus (HPV) is an infectious agent which is mainly sexually transmitted and may lead to HPV-associated cancers in both men and women. Almost all cervical cancers are HPV-associated, however, an increasing number of head and neck cancers (HNCs), especially oropharyngeal cancer, can be linked to HPV infection. Moreover, anogenital cancers, including vaginal, vulvar, penial, and anal cancers, represent a subset of HPV-related cancers. Whereas testing and prevention of cervical cancer have significantly improved over past decades, anogenital cancers remain more difficult to confirm. Current clinical trials including patients with HPV-related cancers focus on finding proper testing for all HPV-associated cancers as well as improve the currently applied treatments. The HPV viral oncoproteins, E6 and E7, lead to degradation of, respectively, p53 and pRb resulting in entering the S phase without G1 arrest. These high-risk HPV viral oncogenes alter numerous cellular processes, including DNA repair, angiogenesis, and/or apoptosis, which eventually result in carcinogenesis. Additionally, a comprehensive analysis of gene expression and alteration among a panel of DNA double strand breaks (DSB) repair genes in HPV-negative and HPV-positive HNC cancers reveals differences pointing to HPV-dependent modifications of DNA repair processes in these cancers. In this review, we discuss the current knowledge regarding HPV-related cancers, current screening, and treatment options as well as DNA damage response-related biological aspects of the HPV infection and clinical trials. As of 2016, the latest year for which incidence data are available, 1,658,716 new cases of cancer were reported, and 598,031 people died of cancer in the US. According to the American Cancer Society (ACS), more than 1.8 million new cases of cancer are estimated for 20203. Worldwide, lung, breast, and colorectum cancer show the highest numbers of new cases; on the other hand, the highest mortality is caused by lung, colorectum, stomach, and liver cancer1 . In the US, the cancer type-related incidence rate resembles the global pattern with most new cases for breast and lung cancers and the highest mortality in patients suffering from lung, colorectum, and pancreas cancers2 . Importantly, the high incidence of lung and colorectal cancers was very high in both men and women2 . Fortunately, the overall cancer-associated death rate in the US has dropped by 29% since 1991 saving more than 2.9 million lives, as of 20173. This remarkable result is due to prevention and education, early detection, and the enormous improvement in cancer treatment. Although awareness across the population increased, the behavioral factors like alcohol use, unhealthy diet, and/or lack of physical activity, or tobacco use are still the most relevant cancer-associated risk factors4. Of note, tobacco use causes the majority of diverse cancer types with approximately 22% of all cancer deaths worldwide1. Interestingly, in the US the distribution of cancer-related risk factors is gender-dependent2 . In men, the most cancer deaths were related to tobacco use, whereas in women most were related to obesity followed by alcohol use and poor physical activity2 . Besides these lifestyle and diet-related risk factors, infection agents like the human papilloma virus (HPV) may lead to HPV-associated cancers in both men in women. In this review, we strive to give an overview of the available statistics, current testing and treatment options, clinical trials, and we also discuss the biological aspects of the HPV infection.Cancer-related diseases represent the second leading cause of death worldwide and in the United States (US) behind cardiovascular diseases and stroke, and led to around 9.6 million deaths globally in 2018Helicobacter pylori (36.3%) and HPV (31.1%), followed by hepatitis B (16.3%) and C (7.1%)4 . Despite lower numbers of cancer related to infection with other agents, including Epstein–Barr virus (EBV), human herpesvirus type 8 , and human T-cell lymphotropic virus type 1 (HTLV-1) as well as three parasites: Opisthorchis viverrini, Clonorchis sinensis, and Schistosoma haematobium, are a cause of nearly 10% of all infection-mediated cancers .Despite the diversity of cancer-related risk factors, infections with bacteria and viruses are known risk factors for cancer. Worldwide, the most prominent infectious agents causing cancer are Papillomaviridae. The virons are non-enveloped and contain a double-stranded DNA genome. The genetic material is enclosed by an icosahedral capsid composed of major and minor structural proteins, L1 and L2, respectively. These viruses are highly tissue-specific and infect both cutaneous and mucosal epithelium. Based on the genomic sequence of L1, the gene encoding the principal capsid protein, over 200 HPV types have been identified and characterized5, with at least 14 high risk types which may cause cancer6. Two HPV types (16 and 18) cause the most HPV-related cancers and about 70% of cervical cancers and pre-cancerous cervical lesions6 . Importantly, HPV is mainly transmitted through sexual activity but people can also be infected by skin-to skin contact6. Notably, about 80% of people – both men and women – will get a HPV infection at some point in their lives. All sexually active persons can be infected with low- or high-risk HPV and usually become infected with HPV shortly after the onset of sexual activity. However, most often the immune system clears the virus, but for some, the infection persists and can lead to precancerous changes. In more detail, cells infected with high-risk HPV increase their proliferation rate and, over time, if not controlled by the immune system, they may form precancerous changes or a tumor. Factors like long-term usage of oral contraceptives, many pregnancies and smoking, a compromised immune system and coinfection with another sexually transmitted disease as well as the HPV type (high risk) may increase the risk of precancerous cervical cells leading to cancer2. Of note, nearly all cervical cancer cases are related to HPV infection. Nevertheless, HPV infection can be linked to other types of cancer, including anogenital cancer , head and neck cancers (HNCs) or genital warts in both men and women. The estimated median annual incidence of new anogenital warts is 137 per 100,000 men and 121 per 100,000 women and the prevalence ranges from 0.15% to 0.18% in men and women, respectively8. In general, HPV is thought to be responsible for more than 90% of anal and cervical cancers, about 70% of vaginal and vulvar cancers, and more than 60% of penile cancers. In contrast, oropharyngeal cancers traditionally have been caused by tobacco and alcohol, but recent studies show that about 70% of cancers of the oropharynx may be linked to HPV. Many cancers of the oropharynx may be caused by a combination of tobacco, alcohol, and HPV infection2. Worldwide, high-risk HPVs cause about 5% of all cancer cases, with an estimated number of infections of 570,000 in women and 60,000 in men each year. As of 2018, approximately 311,000 women died from cervical cancer worldwide and more than 85% of these deaths occurred in low- and middle-income countries where cervical cancer represents the leading cancer-related death in women1. The reason being that in these parts of world there is very limited access to screening tests, which would highly increase chances of early detection and allow more timely application of treatment1. In the US, high-risk HPVs cause 3% of all cancers in women and 2% of all cancers in men, and about 44,000 new cases of HPV infection-associated cancers are found annually, and about 34,800 of these cancers are terminal2. Importantly, nearly 50% of all HPV-associated cancers in women are cervical cancers, whereas in men oropharyngeal cancer was mostly related to HPV infection9 . In males, the highest prevalence of becoming infected with a HPV was observed in HIV-infected men who have sex with other men11. Across heterosexual men, the highest risk of infection was found in African men having at least three female sexual partners12. In addition, the average prevalence of infection with any type of HPV was significantly higher in HIV-positive vs HIV-negative men, 78.2% vs 49.4%, respectively13.HPVs belong to the family Saccharomyces cerevisiae, whereas for Cervarix’s production the recombinant baculovirus expression system is applied14. The nonavalent provides additional protection against HPV types 31, 33, 45, 52, and 58. Data from clinical trials and initial post-marketing surveillance conducted in several continents show all three vaccines to be safe. However, rare cases of anaphylaxis (1.7 cases in 1 million doses), a life threatening allergic reaction, have been reported15. HPV-related post-vaccination syndromes with focus on the adjuvants, which increase the immune response to a certain antigen, have been investigated. The autoimmune/inflammatory syndrome Induced by adjuvants (AIAS) have been reported in 3.6 cases per 100,000 vaccine doses17. Moreover, it remains controversial if the rare death cases after HPV vaccine could be linked to the adjuvant or the vaccine itself17. Despite the rare cases of post-vaccination syndromes, HPV vaccines are reported as being very safe and are the most effective preventive option. However, HPV vaccine is not recommended in pregnant women because of a lack of scientific proof from clinical studies. Nevertheless, previous clinical trials including in pregnant women showed no increase in spontaneous abortions if compared to non-pregnant women18.Comprehensive cervical cancer control includes primary prevention (vaccination against HPV), secondary prevention (screening and treatment of pre-cancerous lesions), tertiary prevention , and palliative care. Vaccines that protect against HPV 16 and 18 are recommended by the World Health Organization (WHO) and have been approved for use in many countries. All males and females ages 9–26 years should get the HPV vaccine. It is most effective when given at ages 11–12 years. The vaccine is given in two doses for males and females ages 9–14 years. Beginning at age 15 through age 45, three doses are required for full immunity. The HPV vaccine prevents HPV-related genital warts, most cervical, anal, vaginal, and vulvar cancers and reduces the risk of most HPV-related cancers of the throat and the penis. These vaccines consist of recombinant viral vaccine proteins containing purified virus-like particles for each virus genotype, thus they protect against different HPV types. Importantly, they are free of viral DNA so they are inactive and cannot infect cells. Three vaccines, Gardasil 4v , Gardasil 9v , and Cervarix 2v , are now being marketed in many countries throughout the world – a quadrivalent, a nonavalent, and a bivalent vaccine, respectively. Both Gardasil vaccines are produced in recombinant 19 (NCT03638427).Most importantly, HPV vaccine is not protective against all types of HPV and therefore women still need to be screened despite previous vaccinations. It is recommended for women between the ages of 21 and 29 years to be Papanicolaou (Pap) tested every 3 years. For women between the ages of 30 and 65 years, additional HPV testing or HPV/Pap testing every 5 years is recommended. Women older than 65 years who were frequently tested and diagnosed negative may be advised to no longer be screened for cervical cancer. However, continuing screening is recommended if recent tests were abnormal. Whereas Pap tests screen for precancerous and cancerous processes in the cervix, HPV tests check for the occurrence of the HPV in the tissue. It is important to consider that a positive test after years of negative testing is not necessarily caused by a new infection but can be a result of virus reactivation years later. Currently, it is not possible to distinguish between a new infection and viral reactivation, nor can we compare the risk of both to cause cancer. A different approach of testing for cervical cancer is currently under investigation within a clinical trial which proposes using self-collected menstrual blood for HPV DNA and HPV E6/E7 mRNA analysis19 (NCT03061435). Additionally, a current clinical study measures the amount of HPV DNA in blood samples from patients who have recently been diagnosed with cervical cancer, trying to correlate the DNA amount of HPV with the stage of disease or the applied treatment19 (NCT03749720). This approach may help to uncover a potential recurrence earlier than is currently possible.Currently, screening can only be performed in cervical tissue as the U.S. Food and Drug Administration (FDA)-approved tests for other types of HPV-related cancers are not available yet. Nevertheless, an ongoing study is testing anal Pap smears and conducting high resolution anoscopy (HRA) to find a proper testing method for anal cancer21. However, the incidence among older and non-White people has increased according to other studies23. Indeed, a recent comprehensive study investigated the differences in HPV genotype distribution in an age-dependent manner showing higher levels of HPV in younger HPV-positive oropharynx cancer patients in comparison to older patients6. Interestingly, 100% of the younger individuals were HPV-positive in comparison to 76% among the older tested patients. The investigators claim that the HPV-positivity in oropharynx cancer depends on the intensity of sexual exposure explaining the lower numbers of HPV-positive cancers in older patients. HNC can include oral cavity, larynx, pharynx, paranasal sinuses, and nasal cavity and salivary glands, so the symptoms can vary. Nevertheless, most of the HNCs cause a sore throat that does not heal, problems with swallowing and changes in the voice. Depending on the specific location, it may also include difficulties in breathing, pain in the neck area, headaches, earache or chronic sinus infections6. While nowadays the number of HNC cases has decreased due to reduced use of alcohol and/or tobacco, the HPV infection-related incidence has increased6. Since the symptoms may be very dependent on the localization of the cancerous changes, different diagnostic tests may be necessary. Additionally, to confirm a cancerous lesion, a tissue sample needs to be examined under a microscope. To confirm a HPV infection, the National Comprehensive Cancer Network (NCCN) guidelines note that p16 protein expression is highly correlated with HPV status and recommend assessment of p16 expression by immunohistochemistry (IHC) or detection of HPV DNA in tumor cell nuclei by in situ hybridization (ISH)6.Patients with HPV-positive HNC were previously described as younger, White, non-smokers with a high number of sexual partners6. According to the ACS6, the 5-year overall survival for cervical cancer is relatively high. Of note, HPV infection is not treatable but therapies for HPV-related precancerous cellular alternations and cancers are available. In most women with precancerous lesions, the loop electrosurgical excision procedure (LEEP) is applied6. During LEEP, the abnormal cells can be removed using an electrical current. Moreover, laser therapy, cryotherapy – freezing the abnormal tissue, and cold knife conization are the treatment options for precancerous lesions. External genital warts caused by HPV infection can be treated by topical medicine7.Presumably, access to Pap tests and HPV vaccine contributed to a decline in cervical cancer cases over decades which was also associated with a lower mortality6. A new type of surgery, transoral robotic surgery, is supported by three-dimensional (3D)-imaging which helps to remove tumors at places that are difficult to reach. Stereotactic body radiation applies high doses of radiation into the tumor by positioning the patient in the same way in each session which reduces the chance of damaging surrounding healthy tissue. Intensity-modulated radiation therapy (IMRT) uses 3D-imaging to precisely irradiate the tumor, which also reduces damage to healthy tissue. For oropharyngeal cancer, cetuximab, a monoclonal antibody for epidermal growth factor receptor (EGFR), which stops the tumor cell proliferation, is applied as a targeted therapy for HNC patients6. However, patients with HPV-related cancers receive the same treatment as patients with HPV-unrelated cancers at the same anatomical site. An FDA-approved anti-PD-1 antibody, pembrolizumab, became a first-line treatment option for all PD-L1 expressing tumors including HPV-positive and -negative HNC25. It can be used as a monotherapy or in combination with chemotherapy.As the testing for HNC depends on the type and location of the HNC, the treatment is also not unified for all HNCs, but it may include surgery, radiotherapy, chemotherapy, targeted therapy, or a combination of those26. Current treatments for both cervical cancer and HNC cover a wide spectrum of options, which not only kill the tumor but also consider the protection of healthy tissue.Because HPV-associated HNC respond vastly better to treatment than the HPV-negative ones, they also show a better survival rate. Using data obtained from the Surveillance, Epidemiology, and End Results HPV Status Database, a recent study reported that from 2013 to 2014, the US incidence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) was 4.62  vs 1.82  per 100,000 persons for HPV-negative OPSCC and the incidence of HPV-positive vs negative non-OPSCC of the head and neck was 0.62  vs 1.38 in situ. Most CIN lesions regress spontaneously, although over a number of years, lesions on the cervix can slowly become cancerous28. A diagnosis of CIN is based primarily on the presence of nuclear atypia and loss of normal squamous maturation (polarity). Accurate grading of CIN lesions is essential as the treatment and clinical follow-up algorithms are very different for the low-grade (CIN 1) and high-grade lesions (CIN 2 and 3)29. Although the likelihood of progression clearly increases with the increasing grade of cervical intraepithelial neoplasia, a proportion of high-grade lesions could still regress. Infection with high-risk HPV subtypes has been shown to be a risk factor for persistent and/or progressive cervical dysplasia. It has also been proposed that HPV DNA integration into host DNA is critical in cervical carcinogenesis31 mediated by disruption of the E1/E2 open reading frames of the HPV genome and subsequent loss of the E2-controlled regulation of E6 and E731. This leads to strongly decreased expression levels of p53 in HPV-associated cancer cells32. The inactivation of the host p53 and retinoblastoma protein (pRb) by E6 and E7, respectively, results in suppressed cell cycle checkpoints and uncontrolled cell proliferation36 . In detail, E6 binds p53 leading to its degradation via E6-associated protein (E6AP)-mediated ubiquitination37. Interestingly, E6 first needs to bind to the protein E3-ligase E6AP as neither E6 or E6AP can bind to p53 alone38. Additionally, only high-risk HPV E6 and E7 can bind to p53 or pRb39 confirming that HPV-related cancers originate only from high-risk HPV types. Degradation of p53 leads to inactivation of one of its targets – p21 40, a cyclin-dependent kinase inhibitor, which prevents cells from entering the S phase by promoting cell cycle arrest in the G1 phase in response to many stimuli44 . E7 targets pRb for ubiquitination, leading to the release of E2F transcription factors, which transcribe cyclin E, cyclin A, and p16INK4A, an inhibitor of CDK4/6, forcing the cells through premature S phase entry45 . To date, it remains unclear how E7 mediates the pRb ubiquitination. However, it has been proposed that Cullin2 can bind to E7 and be responsible for the ubiquitination of pRb46. Of note, E7 was found to be ubiquitinated by the complex Cullin 1/Skp247. Although pRb also interacts with Skp2, it is not involved in its ubiquitination47. Overall, degradation of both p53 and pRb mediated by high-risk HPV results in cellular immortalization which presumably leads to the development of high-grade dysplasia (CIN 2 and 3), with a potential to progress to invasive carcinoma48.Persistent cervical HPV infections may progress towards premalignant glandular or squamous intra-epithelial lesions, classified histopathologically as cervical intraepithelial neoplasia (CIN), and to cancer. CIN is further classified as: CIN 1: mild dysplasia; CIN 2: moderate to marked dysplasia; and CIN 3: severe dysplasia to carcinoma in vitro report suggested applying antisense HPV RNA transcripts in combination with cisplatin for HPV-positive HNC cells49. Here, the investigators infected a HPV-positive HNC cell line, UM-SCC-47 harboring HPV 16, and the HPV-negative HNC cancer cell line YCU-T892, with antisense RNA transcripts of HPV 16 E6/E7 using a recombinant adenoviral vector Ad-E6/E7-AS. They observed a significantly suppressed cell growth in the HPV-positive but not in the HPV-negative cells which correlated with increased expression of p53 and pRb in the HPV-positive cell line. Finally, a combinatory treatment with cisplatin evoked a cell growth reduction in vitro and suppressed tumor growth significantly if compared to monotherapy of either cisplatin or transfection with an Ad-E6/E7-AS construct. Another study also reported suppressed cell growth in HPV-related cervical cancer cells after downregulating HPV 16 E6 and E7 using an adenovirus-mediated transfection which correlated with higher expression of p53 and/or pRb proteins51. Additionally, a similar effect of increased sensitization to cisplatin treatment has been shown in cervical cancer cells after treatment with siRNA against the HPV 16 and HPV 18 E6 and E752. Furthermore, downregulation of HPV 16 and HPV 18 E6 and E7 led to radiosensitizing effects and suppressed growth in cervical cancer cells and xenograft mouse tumors53. Targeting HPV 16 E6/E7 by a gRNA induced apoptosis in HPV-positive cervical cancer cells SiHa and an additional treatment with a gRNA against PD1 significantly reduced the growth of SiHa cell-xenografted humanized SCID mice54. Taken together, downregulation of HPV E6 and E7 significantly suppresses cell growth and reconstitutes p53 and pRb expression. Moreover, downregulation of HPV makes tumor cells more susceptible to chemotherapy.A recent 55. Moreover, four cytokines, small molecular messengers between cells, are approved for the treatment of patients with kidney cancer, leukemia, lymphoma, melanoma, and sarcoma. Since 1997 the FDA has approved multiple monoclonal antibodies for the treatment of several solid tumors and hematological cancers56. For HPV-related cancers, cervical and HNC, an anti-CTLA4 monoclonal antibody ipilimumab is being tested within phase II clinical studies alone or in combination with radiotherapy19 . CUE-101 is a novel E7-pHLA-IL-2-Fc fusion protein and its Fc domain is linked to the protein of interest. Thus, it is designed to activate and expand a population of tumor specific T cells to eradicate HPV-driven malignancies. It is currently being tested in a new phase I clinical study recruiting patients to verify the toxicity and estimate the optimal dosage19 (NCT03978689). Avelumab is a monoclonal antibody which targets protein programmed death-ligand 1 (PDL-1) and has been recently approved by the FDA for a specific type of skin cancer – Merkel cell carcinoma, renal, and bladder cancer. Currently, over 200 clinical trials are testing avelumab’s effect on different types of cancer. One of these trials combines avelumab with a newly developed vaccine TG4001 with HPV 16 antigens in HPV-related cancers19 (NCT03260023). Durvalumab is another FDA-approved human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody which blocks the interaction of PD-L1 with the PD-1 and is used in treatment of different types of lung and bladder cancers. Durvalumab is now being tested with a vaccine MEDI0457 in HPV-positive OPSCC in a phase 2 clinical trial19 (NCT04001413). MEDI0457 is composed of DNA plasmids encoding the E6/E7 genes of HPV 16 and HPV 18 (VGX-3100) and DNA plasmid encoding the human interleukin-12 (IL-12), INO-9012, which triggers the immune system6. A recent paper revealed that MEDI0457 was well-tolerated by patients with cervical cancer after receiving chemoradiation and raised the idea of combining tumor-specific vaccines with radiotherapy57. MEDI0457 was also well tolerated by patients suffering from HPV-associated HNC cancer showing elevated antigen-specific T cell activity58. However, despite the promising safety studies, the final efficacy of a vaccine can only be evaluated after phase 2 and 3 clinical trials. Moreover, durvalumab is also being tested in combination with radiation in patients with HPV-associated OPSCC19 (NCT03623646).Immunotherapy is nowadays listed as a treatment option for cancer patients. On the one hand, immunotherapy can be used as a booster in the form of checkpoint inhibitors or vaccines to trigger a patient’s immune system. On the other hand, immunotherapy uses monoclonal engineered T-cells or antibodies as substances mimicking the compounds of the immune system. Of note, the FDA has approved 14 different immunomodulators including seven checkpoint inhibitors disturbing the PD-1/PD-L1 interaction or blocking the CTLA-4 which can support the immune system to recognize the cancer cells and to induce cell death19 (NCT03795610) or in combination with PD-1 blocking antibody nivolumab in a panel of HPV-related cancers19 (NCT02379520). The underlying working hypothesis is that mRNA signatures of immune response will be increased in IPI-549-treated patients and therefore this combination may enhance anti-PD-1 therapy.Many kinase inhibitors have now entered cancer treatment. A phosphoinositide-3-kinase gamma (PI3Kγ)-inhibitor, IPI-549, is currently under investigation in a phase II clinical study including HNC cancer patients either as a monotherapy19 (NCT02379520). HPVST cells originate from the patients who previously suffered from HPV-related cancer. The goal is to validate whether these cells can affect the tumors and whether they can develop resistance against transforming growth factor-beta (TGF-β) produced by HPV-mediated tumors. After the optimal dose of HPVST cells is established, an FDA-approved human IgG4 monoclonal antibody, nivolumab, that blocks PD-1, will be applied in combination with HPVST. The overall purpose of this study is to potentially increase the effectiveness of HPVST cells using the nivolumab in patients suffering from HPV-related cancers. A 6-month application of nivolumab is a final step of a phase II study including patients diagnosed with HPV-positive OPSCC who will first receive chemotherapy  followed by radiotherapy or surgery19 .Interestingly, a novel treatment involving HPVST cells, HPV-specific T cells, is currently under investigation in one clinical study including patients with recurring HPV-related cancers19 (NCT03634267). Furthermore, proton therapy has attracted attention and is now being considered as treatment option for many cancer patients and can be applied in a variety of tumor types. An ongoing clinical trial is using the traditional radiotherapy and/or proton therapy in patients with HPV-related OPSCC to achieve a reduction of toxicity by maintaining the efficiency of the treatment19 (NCT03621696). The limitation of radiotherapy-induced side effects in patients suffering from HPV-associated HNC is a focus of a phase II studies applying IMRT and chemotherapy19 .Another phase I clinical study uses magnetic resonance imaging (MRI)-guided brachytherapy  to verify whether this approach can improve the high-risk clinical target volume (HR-CTV) D90  rate compared to conventional guidance using ultrasound and a freehand technique in stage IB2-IV cervical or stage II-IVA vaginal cancerin vitro and in vivo studies. The usage of this genome editing tool is currently under investigation in a phase I clinical trial19 (NCT03747965). In more detail, the clinical study includes patients with different solid tumors, especially pancreatic cancer, cholangiocarcinoma cancer, and ovarian cancer, and monitors the safety of CRISPR-Cas9-mediated PD-1 gene-knocked out chimeric antigen receptor (CAR) T cells. Another study is addressing the safety and efficacy of CRISPR/Cas9-HPV E6/E7, which can disrupt the DNA of HPV 16 and HPV 18, in patients with HPV-related cervical intra-epithelial neoplasia (CIN)19 (NCT03057912). Zinc finger nucleases, called ZFN-603 and ZFN-758, cleave HPV 16 and HPV 18 E7 oncogene and are currently under investigation including in patients suffering from CIN19 (NCT02800369).CRISPR/Cas9 technology is now widely used for Altogether, there are many ongoing clinical trials including patients suffering from HPV-related cancers which address different aspects and methods of treatment, but these trials mainly focus on unique HPV antigens for vaccination as well as in combination with immune checkpoint agents.SHKBP1, ERBB3, CASP8, HLA-A, and TGFBR2 as novel significantly mutated genes in cervical cancer and confirmed mutations in PIK3CA, EP300, FBXW7, HLA-B, PTEN, NFE2L2, ARID1A, KRAS, and MAPK1 in these patients59. In contrast, HPV-negative cancers, mostly including HNC and a few endometrial-like cervical cancers, showed an increased mutation rate in ARID1A, KRAS, and PTEN. Moreover, amplifications in PD-L1 and PD-L2, and the BCAR4 long non-coding RNA which has previously been linked to respond to the chemotherapy drug lapatinib, an EGFR/HER2 inhibitor, have also been observed59. Importantly, over 70% of cervical cancers exhibited genomic alterations in either one or both of PI3K–MAPK and TGF-β signaling pathways, suggesting compounds of these pathways may be targets for anticancer therapy. Moreover, a cross-cancer analysis in this study highlighted the similarity among HPV-positive squamous cancers [cervical and HPV-related head and neck squamous cell carcinoma (HNSCC)] independent of the anatomical site. Interestingly, HPV 18-associated tumors exhibited a significantly higher ratio of unspliced vs spliced versions of the E6 protein than the HPV 16-related cancers showing the diversity between different HPV types. This suggests different functional aspects of E6 and E7 in tumors infected with different HPV types. Taken together, this study provides a comprehensive analysis of gene deregulations in cervical cancers including the dependence on HPV subtypes which underlines the importance of targeted therapy for certain cancer associated with particular HPV subtypes.A comprehensive analysis of 228 primary cervical cancers revealed an upregulation of in silico analysis including HPV-positive and HPV-negative HNC revealed that TP53 was the most frequently mutated (over 80% of samples) gene in HPV-negative HNC60 . In contrast, HPV-positive HNC samples were mostly harboring a mutation in TTN which encodes the protein Titin responsible for the passive elasticity of muscles. The second highly mutated gene was PIK3CA which correlates with findings of Burk59. Additionally, we performed the same analysis with cervical cancer samples and found that TTN and PIK3CA were also highly mutated in these samples .Our own n = 38) and HPV-negative (n = 75) HNC61. Our data reveals significant mRNA expression differences in NHEJ1, BRCA2, PRKDC, RAD51, H2AFX, XRCC6, XRCC4, DCLRE1C, UIMC1, and RNF8 . Interestingly, most of the genes, except for PRKDC and RNF168, showed higher expression in HPV-positive HNC. However, 50% of all HPV-positive HNC samples were altered in the RNF168 gene which overlaps with a study from Sitz and colleagues62 . Our analysis uncovered additionally high rates of alterations in the BRCA1 gene in HPV-positive HNC samples . As BRCA1 and RNF168 both promote homologous recombination repair (HRR)64, the high alteration rate of both suggests a HPV-mediated facilitated HRR in HPV-associated HNC cancers. In contrast, HPV-negative HNC samples showed alterations in the LIG4 and XRCC6 genes involved in non-homologous end joining (NHEJ), suggesting an affected DNA double strand breaks (DSB) repair by NHEJ . However, comparing the co-expression of DNA DSB repair genes in HPV-positive and HPV-negative HNC has not shown striking differences60 , indicating that co-expression between those genes cannot be significantly affected by the virus, despite the affected DNA repair processes described.As DNA repair processes in tumor cells are affected, we performed an additional mRNA expression analysis including DNA repair genes within HPV-positive . Moreover, the decreased expression of DNAPKcs inversely correlated with the increased expression of the viral oncoproteins E6 and E7, and was associated with higher radiosensitivity66. Furthermore, cervical cancer cell lines and HPV-positive HNSCC, but not the HPV-negative HNSCC, were associated with higher amount of persistent infrared (IR)-induced γH2AX, a marker for DNA DSBs indicating a HPV-dependent impaired removal of DNA DSBs68. In line with this observation, it has been suggested that the lack of DNAPK and BRCA2 in HPV-positive HNSCC has led to a reduced DNA repair capacity by NHEJ and/or HRR, despite sustained activity of 53BP1 and BRCA1, which are independent of HPV status66. Moreover, it has been proposed that HPV decreases the efficacy of HRR by hindering RAD51 to localize to DNA DSBs69. A new report presented that E6-transduced U2OS cells failed to show colocalization of the Fanconi anemia complementation group D2 (FancD2),a protein involved in interstrand crosslink repair, with DNA DSBs, which subsequently hindered the recruitment of the downstream repair protein RAD51 to DSBs70. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective interstrand crosslink repair, resulting in its increased chromatin retention. Finally, E6 could suppress USP1 de-ubiquitinating activity, and led to persistent activation of ATR/CHK-1/pS565 FancI signaling72. Thus, a prediction to sensitivity to DNA crosslinking agents in HPV-negative HNSCC based on an interstrand crosslink repair gene expression has been introduced and confirmed that defects in these DNA repair genes correlated with a poor prognosis71. Indeed, ATR or CHK1 inhibitors suppress both amplification and late viral gene expression in differentiated cells and reduce stable copy numbers of the viral genome in undifferentiated cells73. Moreover, application of the ATR inhibitor in both HPV- and HPV+ HNSCC cell lines led to enhanced sensitivity to cisplatin treatment74. Interestingly, ATM- and ATR-mediated signaling is constitutively active in HPV-associated cells unexposed to any DNA damaging agent76, suggesting that application of ATR and ATM inhibitors could represent a promising approach in the treatment of HPV-related carcinoma.DNA damaging agents leading to generation of diverse lesions, for example, DNA DSBs, induce DNA damage response and DNA repair. DSBs represent the most severe type of DNA lesions repaired by NHEJ or HRR, which enforce the maintenance of genomic stability. NHEJ is a fast but error-prone DNA repair pathway with DNA-dependent protein kinase (DNAPKcs) as a key protein kinase important for recruitment of downstream factors necessary to join and ligate the broken DNA ends62. It has also been observed that expression of an exonuclease with a proofreading function, Three prime repair exonuclease 1 (TREX1), is upregulated exclusively in HPV-transformed primary keratinocytes expressing HPV 16 E6 and E777. Downregulation of TREX1 resulted in inhibition of cell growth, which was associated with p53 upregulation.As the viral oncoproteins, E6 and E7, are crucial in HPV-mediated tumorigenesis, researchers are focusing on their mechanistic functions, partially in DNA repair, in promoting cancer. A recent study proposed E7 as the oncoprotein which hampers the DNA damage response to DSBs by interacting with the E3 ligase RNF168 involved in DNA DSB repair80 suggesting that HPV-positive cancer cells are more sensitive to therapy by interfering with DNA DSB repair. In line with this observation, HPV-positive OPSCCs are more sensitive to radiotherapy than the HPV-negative ones, correlating with the reduced DNA DSB repair capacity in HPV-positive cancers81. Several studies showed that HPV-positive HNSCC cells treated with ionizing radiation show a significantly higher cell death than the HPV-negative HNSCC85. Importantly, a higher radiosensitizing effect of olaparib was observed in HPV-negative OPSCCs than in HPV-positive OPSCC, suggesting this combined treatment to be applied in relatively more radioresistant HPV-negative cancers85. In addition, roscovitine, a cyclin-dependent kinase inhibitor targeting CDK2, CDK7, and CDK9, has been proposed as an olaparib-enhancer towards radiosensitization for HPV-negative HNSCC86. In contrast, a study using a panel of HPV-positive and HPV-negative OPSCC revealed that only a subset of HPV-positive OPSCCs were susceptible for olaparib treatment at a therapeutic concentration (0.1–0.5 μM), whereas other HPV-positive and HPV-negative OPSCCs were either sensitive to the PARP inhibition at higher doses or not at all87.Remarkably, HPV infection can block TGF-β resulting in more prominent activation of alternative end joining  instead of HRR, enhancing the cellular sensitivity to cisplatin or PARP inhibitors89. Furthermore, a comprehensive analysis of protein expression in HPV-positive and HPV-negative HNC cell lines revealed different expression patterns between proton and photon irradiation90. In brief, they found differences in expression of IL-6, GSK3, Src, AMPK, cyclooxygenase (COX)-2, vascular endothelial growth factor (VEGF) pathways in HNC cells treated with proton or photon irradiation90, suggesting varying benefit from both radiotherapy modulations in combination with targeted therapy or immunotherapy.The wide spectrum of cancer treatment includes proton beam irradiation which uses particles to irradiate tumors. Despite a relatively low number of studies including proton irradiation and HPV-related cancers, most reports claim an advantage of proton beam therapy especially for relatively radioresistant HPV-negative cancers. For instance, proton irradiation-induced reduction of cell growth in HPV-negative HNC cells was significantly higher than the standard photon-based radiation92. However, it has also been shown that HPV 31 needs an accumulation of ATM and HR-related factors, for example, MRN complex composed of RAD50-MRE11-NBS1 proteins, to its replication sites92. Furthermore, HPV abolishes the apoptosis upon agents inducing UV-mediated lesions95 suggesting that HPV can induce resistance to DNA crosslinking agents, for example, cisplatin. To predict a potential cisplatin resistance in cervical cancer, different proteins have been identified, for example, p5398, excision repair cross-complementation group 1 (ERCC1)99, an apoptosis regulator B-cell lymphoma 2 (Bcl-2), p21100. This approach supports screenings and personalized treatment for HPV-related cancers to avoid resistances and to apply the best suitable treatment following diagnosis.On the other hand, HPV infection can activate different DNA repair proteins involved in response to ultraviolet (UV)-mediated DNA damage, including nucleotide end resection (NER), the Fanconi anemia (FA) pathway, and translesion synthesis (TLS) because HPV requires these DNA repair mechanisms to properly replicate its genome


The Japanese government suspended proactive recommendations for the HPV vaccine in June 2013. The suspension is now in its seventh year, despite all the data pointing to the safety of the HPV vaccine. We reported a high vaccine effectiveness in the group of women vaccinated before their first intercourse (93.9%). The prevalence of cross-protected types of HPV 31/45/52 was also lower in the vaccinated group, and the vaccine effectiveness was 67.7%. Furthermore, prevalence of HPV16, 31 and 52 infection rates in the vaccinated group were obviously lower than that in the unvaccinated group, and no one had HPV18 or 45 infection in the vaccinated group. The addition of a cross-protective effect toward HPV types 31/45/52 to HPV types 16/18, which is the direct target of the bivalent HPV vaccine, may possibly prevent around 82% of invasive cervical cancer cases in Japan. With regard to the preventive effect of histological abnormalities, we also reported significant reduction in incidence of cervical intraepithelial neoplasia (CIN)3 or worse. Thus, the efficacy of the vaccine has been demonstrated for precancerous disease, and the diverse symptoms after HPV vaccination are likely functional somatic. For the future of Japanese girls, there is a need to resume the proactive recommendation of HPV vaccination and for immediate action to be taken by the Japanese government. There are more than 10,000 cases of cervical cancer, and nearly 3000 women die from the disease annually in Japan . A pap sThe bivalent HPV vaccine was licensed in October 2009, and the quadrivalent vaccine in July 2011. Public funding became available from 2010, and from April 2013, both the bivalent and quadrivalent HPV vaccines were included in the Japanese National Immunization Program (NIP) for girls aged 12–16 years. However, after unfounded sensational reports of so-called ‘adverse events’ were published by the Japanese media, the Japanese Ministry of Health, Labour, and Welfare (MHLW) suspended proactive recommendations for the HPV vaccine in June 2013, only two months after the vaccine had been introduced into the NIP . After tIn 2014, the generation of girls who received the HPV vaccination during the first year of its introduction under public funding turned 20 years of age and thereby became eligible for cervical cancer screening. Accordingly, Japanese data on the preventive effects of HPV vaccination against HPV infection and cervical intraepithelial neoplasia (CIN) have been published one after another.We have conducted two studies with different designs to evaluate the efficacy of the HPV vaccine and published the results of the interim analysis ,8. This The NIIGATA STUDY is a cross-sectional study that examined the effect of the HPV vaccine on the reduction of HPV infections, cytological abnormalities, and histological abnormalities in women who underwent cervical cancer screening in Niigata, Japan . In addiThe OCEAN STUDY is a prospective study that enrolled and tracked individuals who were vaccinated between 12 and 18 years of age, who would undergo HPV testing and cytological and histological examinations at the ages of 20 and 25 years, and compared their results to unvaccinated individuals. The interim analysis showed that in the cervical cancer screening for individuals aged 20 and 21 years, the infection rate of high-risk type HPV was significantly lower in vaccinated individuals at 12.9%, relative to 19.7% in unvaccinated individuals . Th. Th17]. If the suspension of proactive recommendations of the HPV vaccine is not lifted and the administration of the vaccine remains halted, we may continue to face a reality wherein the vaccination rates vary significantly according to the year of birth. Whereas women in the OHPV (pre-suspension) generation, born between 1994 and 1999, have a vaccination rate of approximately 70%, among women born after the year 2000, in the post-suspension generation, the vaccination rate reduced to almost 0%. As such, the risk of HPV infection and development of cervical cancer in Japanese women born after 2000 (post-suspension generation) may return to the same state as that in the pre-OHPV generation .Approximately 80% of women develop localized symptoms, such as temporary pain and inflammation of the injection site, during HPV vaccination. Additionally, there have been reports of cases of women that developed vagal reflex due to pain and anxiety from the injection. The Vaccine Adverse Reactions Review Committee (VARRC) of the MHLW in Japan has expressed its opinion that there has been no evidence relating “The diverse symptoms reported after HPV vaccination” and “HPV vaccine,” and that the symptoms are likely functional somatic . The CocIn their national epidemiological study, Sobue et al. reported the following: “It is estimated that among girls between the ages of 12 and 18 years, 40.3  exhibit “diverse symptoms” after vaccination, similar to those that develop after HPV vaccination, and 20.4 develop “diverse symptoms” without a history of HPV vaccination,” which indicated that a certain number of girls exhibit such “diverse symptoms” even with no history of HPV vaccination . A questAustralia has published a report indicating that cervical cancer can be eradicated and has proposed a timeline toward its realization . They esThe MHLW in Japan approved the manufacturing and marketing of the nonavalent vaccine on 20 May 2020. In Japan, the application for the vaccine was submitted on 3 July 2015. However, the approval reviews took an unusually long period of about five years. After approval by the MHLW, further discussions will be conducted in the subcommittee for use in NIP. For the nonavalent vaccine, the Japanese Society of Obstetrics and Gynecology has repeatedly requested that the Japanese government obtain early approval for a routine immunization program. A strong opposition movement against HPV vaccination and lawsuits against the Japanese government and pharmaceutical companies seem to be the cause of the delay in approval. The nonavalent HPV vaccine may be able to prevent approximately 90% of all HPV-related cervical cancer cases  and is a


Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed ‘isocarbostyril alkaloid’. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in  Upon diagnosis with PEL, the median survival time of patients is only 3 to 6 months2. Therefore, there is an immediate need to identify novel treatment options for PEL.Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma with extremely poor prognosis when treated with conventional chemotherapy. The presence of KSHV in all tumor cells is the defining feature of PELAmaryllidaceae (amaryllis) family. Narciclasine has been shown to possess potent anticancer activity against tumors of brain, skin and breast3. Earlier studies have shown that translation elongation factor eEF1A is the direct target of narciclasine5. Further, it has been found that narciclasine triggers actin stress fiber formation by activation of a small GTPase, RhoA6. Recently, narciclasine was named ‘Molecule of the Week’ by American Chemical Society (ACS) for its potential as a cancer drug.Narciclasine  is a natural product found in daffodils and other flowering plants belonging to the 7. Even though, the Myc genomic locus is structurally intact in PEL, they modestly overexpress MYC and we have shown that compounds that down regulate MYC expression are effective and selective against PEL8.MYC regulates numerous cellular activities, including signal transduction, cell cycle, proliferation, differentiation and apoptosis. MYC is deregulated in many cancers, and has been implicated in almost a third of all cancersIn this study, we tested the effect of narciclasine and its structural analogs on a panel of cell lines comprising five hematological malignancies. We show that while all the cancer cell lines in our panel were susceptible to narciclasine and its structural analogs, the PEL derived cell lines displayed preferential sensitivity. We further show that preferential activity of narciclasine against PEL is associated with its ability to downregulate MYC.50 ranging from 82 to 162 nM  and 50 nM (8 to 10-fold), as compared with cells that were treated with a vehicle control Fig. . In cont9. Since, narciclasine also display preferential activity against PEL, we tested the effect of narciclasine on the expression of MYC in PEL. BC-1, BC-3 and JSC-1 cells were treated with narciclasine (25 nM) for 48 hours followed by immunoblotting for MYC and GAPDH (loading control). As shown in Fig. TERT, SLC19A1, MYB and PMM2. Narciclasine treatment induced a significant reduction in expression of all four MYC target genes in both BC-1 and BC-3 cell lines  narciclasine did not induce stress fiber formation as observed by F-actin staining (data not shown). Further, two known inhibitors of Rho pathway, CCG1423 and Rhosin-HCl11, did not have any effect on the activity of narciclasine in PEL cells  were incubated with 293A-PAN-Luc cells for 48 hours in the presence of polybrene, followed by measurement of firefly luciferase activity. A striking increase in luciferase activity was observed in wells that were incubated with supernatants from doxycycline treated BCBL-TREx-RTA cells , a master regulator and marker for lytic reactivationing Fig. . As a poing Fig. . Earlierlls Fig. . In contlls Fig. . These rin vivo efficacy of narciclasine against PEL, we employed two distinct mouse models of PEL – a Cell Line Xenograft (CLX) model using BC-1 cells expressing an RSV-promoter-driven firefly luciferase (BC-1-Fluc) and a Patient Derived Xenograft (PDX) model using UMPEL-1 cells15. In the CLX model, 1 × 107 BC-1-Fluc cells were intraperitoneally injected into NSG mice on day 1. Bioluminescence imaging (BLI) was performed on day 5 to confirm the presence of tumors . As shown in Fig. in vivo is in clear contrast to the striking effect of narciclasine at similar treatment doses, which confirms the in vitro cytotoxicity data suggesting the superior activity narciclasine over its structural analogs.For PDX model, 1 × 1016. The median overall survival of PEL is only 4.8 months, indicating an urgent need to identify new therapeutic options for this disease17. In this study, we found that narciclasine and its structural analogs have preferential activity against PEL. Preclinical studies of narciclasine in other cancers have shown that its mechanism of action involves inhibition of translation elongation factor eEF1A4, activation of RhoA6, and induction of autophagy18. It is possible that all these pathways play a role in the activity of the narciclasine against PEL. However, all those pathways are essential for the survival of both cancer cells and normal cells. In this study, we found that all the cancer cells in the panel were susceptible to narciclasine induced cell death at higher doses, while cell lines representing PEL succumbed to narciclasine at doses that were approximately 2.5-fold lower than the cell lines representing other cancers. This led us to hypothesize that narciclasine may downregulate MYC in PEL as MYC is essential for the survival of PEL and molecules that target MYC are effective and selective against PEL9. Indeed, we found that narciclasine downregulated the expression of MYC with a concomitant decrease in the expression of its direct target genes. However, time-course experiments in PEL upon treatment with narciclasine revealed that apoptosis was initiated before the downregulation of MYC, suggesting that MYC is not a direct target of narciclasine in PEL.PEL accounts for approximately 4% of all HIV-related non-Hodgkin lymphoma and is universally associated with KSHV infection13. As lytic reactivation of KSHV is believed to promote KSHV tumorigenesis, it can be a potential safety issue with the use of narciclasine in PEL19. However, we found that narciclasine treatment neither activated RTA nor induced any infectious KSHV particles, thereby allaying those safety concerns. These results are consistent with our observation that MYC is not a direct target of narciclassine.Downregulating MYC expression in PEL using shRNA leads to lytic reactivation of KSHV by activating RTA/ORF50, the master regulator of KSHV lytic cycle6. However, our study suggests that Rho pathway has no role in the activity of narciclasine in PEL. The KSHV genome contains several genes that mimics the activity of cellular genes, thereby activating multiple oncogenic signaling pathways 20. At present, it is not known whether narciclasine has any effect on any of the above-mentioned oncogenic signaling pathways present in PEL. Therefore, further studies are needed to identify the primary target of narciclasine in PEL. Apart from its potent anticancer activities, narciclasine has been shown to possess anti-inflammatory properties by inhibiting NF-κB pathway in murine models of inflammation21. NF-κB pathway is constitutively active in a number of PEL cell lines24 and has been shown to be essential for their survival and proliferation25. Therefore, it is conceivable that inhibition of survival pathways, such as NF-κB, also contributes to the activity of narciclasine against PEL cells.Narciclasine has been shown to activate Rho signaling and induce stress fiber formation in glioblastoma cellsnarcissus. However, recent studies have successfully demonstrated organic routes for chemical synthesis of narciclasine and its analogs27. In this study, we show that compounds that are structurally related to narciclasine also display similar activity, albeit at higher doses. Therefore, structural modifications of narciclasine can be made to improve its efficacy and bio-availability. Finally, it is pertinent to note that concentrations as high as ~250 nM of narciclasine were readily achievable in murine pharmacokinetics studies, indicating the presence of a significant therapeutic window, which can be exploited for the treatment of PEL3.Even though narciclasine has shown impressive activity in various pre-clinical studies of cancers, it has never been tested in a human clinical trial. A potential bottleneck for the clinical testing of narciclasine is its limited availability. Until now, narciclasine has been isolated from the bulbs of 28. 293 A cells (Invitrogen) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum. The cells were maintained at 37 °C and 5% CO2. After the cell lines were received they were expanded and multiple vials were frozen in liquid nitrogen. None of the cell lines used in this study were cultured continuously for longer than 3 months. After 3 months, a new frozen vial was thawed and used for experiments. PEL cells were authenticated by their expression of KSHV-LANA. No further authentication of cell lines characteristics was done.BC-1, BC-3, BCBL-1, JSC-1, BCP-1, VG-1 and APK-1 were obtained from Dr. Jae Jung . UMPEL-1 and UMPEL-3 were provided by Dr. Izidore Lossos and Dr. Juan Ramos, respectively . DG-75 was obtained from Dr. Alan Epstein . RS4.11 was purchased from American type culture collection, Manassas, VA, USA. L428 and L1236 cells were obtained from Dr. Markus Mapara . MM.1 S was obtained from Dr. Alan Lichenstein  and U266 was provided by Dr. Gregor Adams . All the cells were grown in conditions as described previouslyNarciclasine (NSC 266535) and lycoricidine (NSC 349155) were obtained from the Developmental Therapeutics Program (DTP) of National Cancer Institute (NCI). Lycorine was obtained from Enzo Life Sciences (BML-GR313). Phenazine methosulfate (P9625); Dimethyl sulfoxide (D2650); Doxycycline (D9891) and 2-Hydoxypropyl-β-cyclodextrin (HPCD – 332607) were obtained from Sigma-Aldrich. Apo-ONE® Homogeneous Caspase-3/7 assay kit (G7792) and Cell Titer 96® Aqueous MTS reagent powder (G1111) were obtained from Promega. FITC Annexin V apoptosis detection kit was obtained from BD biosciences (556547). Human IL-6 (DY206) and IL-10 (DY217B) were measured using the Duoset ELISA kit from R&D systems. CCG1423 (5233) and Rhosin.HCl (5033) was obtained from Tocris. Phalloidin-iFluor 488 reagent for F-actin staining was obtained from Abcam (ab176753).4 cells/well, treated with an increasing concentration of the drugs and subsequently assessed for cell viability using the MTS reagent -5-(3-carboxy -methoxyphenyl)-2-(4-sulfophenyl)-2H-tet razolium, inner salt) following the manufacturer’s instructions. Percent cell survival was calculated based on the reading of cells grown in the presence of DMSO control, as described previously29. IC50 values were calculated using Graphpad prism. Experiments were performed in triplicate. Cell-cycle analysis was done as described previously30. Apoptosis was analyzed using Apo-ONE® Homogeneous Caspase-3/7 Assay kit and FITC AnnexinV apoptosis detection kit I as per manufacturer instructions, as described previously28.Cells from exponentially growing cultures were plated in untreated flat-bottom 96 well plates at a density of 1 × 1031. Primary antibodies used in these experiments were from the following sources: PARP ; MYC ; Caspase-3 ; GAPDH .Cells were lysed in lysis buffer containing 20 mM sodium phosphate (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 0.2 M PMSF, and 10% glycerol supplement with a protease inhibitor mixture tablet (Roche). Whole cell extracts were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies, as described previously−∆∆ CT method as described earlier14. The qRT-PCR data was presented as fold change in target gene expression ± standard error.PEL cells treated with narciclasine were harvested to extract total RNA using the RNeasy mini kit (Qiagen) and cDNA was synthesized using reverse transcriptase enzyme Superscript II (Invitrogen). Real-time quantitative reverse transcript-polymerase chain reaction (qRT-PCR) was performed with SYBR Green using gene-specific PCR primers listed in Supplementary Table 32.293A-PAN-Luc cells engineered to express a stably integrated copy of PAN promoter-driven luciferase reporter construct were plated in 24 well plates and treated with cell-free supernatants from PEL cell lines treated with narciclasine or doxycycline (500 ng/ml) treated BCBL-TREx-RTA supernatants for 48 hours in the presence of polybrene (8 µg/ml). Post incubation, cells were lysed to assay the firefly luciferase activity as described earlier7 BC-1 cells stably expressing firefly luc were injected intraperitoneally into approximately 6-week-old female NOD.Scid-Gamma (NSG) mice (purchased from Jackson laboratory or from the breeding colony maintained in house). To assess establishment of tumors, mice were imaged 5 days post inoculation using an IVIS Spectrum Imaging system . Animals were randomly assigned into two groups that were treated with once daily intraperitoneal injections of vehicle control (10% HPCD) or narciclasine (1 mg/kg) for 12 days, continuously. The animals were monitored for survival and body weight gain was measured every 3 days as a surrogate measure of tumor progression9. All animal handling procedures were performed with the approval of the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC), in accordance with ethical guidelines and regulations.A total of 1 × 107 patient derived UMPEL-1 cells that were passaged only in mice were injected intraperitoneally into approximately 6-week-old female NSG mice15. 4 days after tumor cells inoculation, mice were randomly divided into three groups and were treated with a once daily intraperitoneal injections of vehicle control (10% HPCD) or narciclasine (1 mg/kg) or lycoricidine (1 mg/kg) for 9 days. The animals were monitored for survival and body weight gain over the experimental period. All animal handling procedures were performed with the approval of the University of Southern California (USC) Institutional Animal Care and Use Committee (IACUC), in accordance with ethical guidelines and regulations.A total of 1 × 10t test was used to test for differences between 2 groups. Differences with a P ≤ 0.05 were considered statistically significant. All experiments were repeated a minimum of two times.Two-tailed unpaired Student Supplementary Information-Narciclasine in PEL.


Cervical cancer is caused by Human Papilloma viruses (HPV) and is preceded by precursor stages: Cervical Intraepithelial Neoplasia (CIN). CIN is mostly found in women in their reproductive age and treated with a Loop Electrosurgical Excision Procedure (LEEP). The recurrence or residual disease rate after treatment is up to 17%. These women have a lifelong increased risk of recurrent CIN, cervical cancer and other HPV related malignancies. Furthermore, LEEP treatments are associated with complications such as premature birth. Limited data show that prophylactic HPV vaccination at the time of LEEP reduces recurrence rates, therefore leading to a reduction in repeated surgical interventions and side effect like preterm birth.The primary study objective is to evaluate the efficacy of the nonavalent HPV vaccination in women with a CIN II-III  lesion who will undergo a LEEP in preventing recurrent CIN II-III after 24 months.This study is a randomised, double blinded, placebo controlled trial in 750 patients without prior HPV vaccination or prior treatment for CIN and with histologically proven CIN II-III (independent of their hrHPV status) for whom a LEEP is planned. Included patients will be randomised to receive either three injections with nonavalent (9 HPV types) HPV vaccine or placebo injections (NaCL 0.9%) as a comparator. Treatment and follow-up will be according the current Dutch guidelines. Primary outcome is recurrence of a CIN II or CIN III lesion at 24 months. A normal PAP smear with negative hrHPV test serves as surrogate for absence of CIN. At the start and throughout the study HPV typing, quality of life and cost effectiveness will be tested.Although prophylactic HPV vaccines are highly effective, little is known about the effectivity of HPV vaccines on women with CIN. Multiple LEEP treatments are associated with complications. We would like to evaluate the efficacy of HPV vaccination in addition to LEEP treatment to prevent residual or recurrent cervical dysplasia and decrease risks of repeated surgical treatment.NL 7938. Date of registration 2019-08-05.Medical Ethical Committee approval number: NL66775.078.18. Affiliation: Erasmus Medical Centre. Dutch trial register:  Worldwide, cervical cancer is diagnosed annually in more than 500,000 women and is still a major health problem among women worldwide . CervicaThe most commonly used method to treat CIN II-III is Loop Electrosurgical Excision Procedure (LEEP) to prevent possible progression to invasive cancer. Data on recurrent disease after LEEP vary in the literature. Up to 17% of women treated for CIN II-III have residual or may develop recurrent CIN II-III , 6. TreaP < 0.001) related to the vaccine HPV types. Irrespective of HPV vaccine type the recurrence rate was 2.5% (9/360) in the vaccinated group versus 7.2% (27/377) in the unvaccinated group. Multivariate analysis showed that no vaccination after LEEP was an independent risk factor for recurrent CIN II-III  [Many studies have proven the efficacy and safety of the prophylactic HPV vaccine against the development of cervical intraepithelial neoplasia in HPV naïve women –16. In 2 < 0.01) . Previou < 0.01) . There i < 0.01) , 19–22.  < 0.01) , 24. A rIn this study, we will compare HPV vaccination with the nonavalent HPV vaccine  versus placebo  vaccination subsequently to LEEP treatment in women without previous treatment for CIN or HPV vaccination. This study is a randomised, double blinded, placebo controlled trial to evaluate the efficacy of the nonavalent HPV vaccine in preventing recurrent CIN II-III after 24 months.The VACCIN study is a randomised multicenter, double blinded, placebo controlled trial in female patients with histological diagnosed CIN II or CIN III and treated with LEEP. Recruitment of participants will be done from colposcopy clinics in participating centers. After histologic diagnosis of CIN II-III for which a LEEP is necessary, patients will be counselled for participating in this study.In the Netherlands most women with CIN are detected via population based cervical cancer screening starting at the age of 30 years.In addition, general practitioners may refer women with intermenstrual or postcoital bleeding. This also includes women younger than those in the national screening program for cervical cancer. When abnormal Pap smears are found, they are referred for colposcopy.Women are eligible for participation if they are eighteen years old or above, histologically proven CIN II or III and will be treated with a LEEP. Inclusion must be within four weeks after LEEP. Exclusion criteria are: a prior HPV vaccination, invasive carcinoma, immune-compromised women, pregnant women, prior treatment for CIN-lesions, insufficient understanding of the Dutch language or allergic to vaccine components.https://castoredc.com). Patients will be randomised to either active study medication, or placebo in a 1:1 ratio. Randomisation will be stratified by age, categorized as being 18 to 29 years, 30 to 44 years, or 45 years and older. Women aged 18 to 29 years are not eligible for routine screening under the national screening program. The second stratum includes women of 30 to 44 years who are of reproductive age and invited for routine testing under the national screening program. The third stratum women of 45 years and older, who are less likely to be fertile. Randomisation will use random permuted blocks of sizes 2 and 4.Randomisation will be performed through the secure web-based interface Castor EDC  and productivity cost questionnaire (iPCQ) will be performed at the start of the study, at 2 months and after 24 months.Quality of life will be assessed with quality of life questionnaire: Euroqol 5D-5 L at the start of the study, at 2 months, at 6 months and after 24 months.Side effects and adverse events. One week after each vaccination, a telephonic interview will be performed to ask for side effects after the injection.The secondary objectives are:Cytology result after 5 years by retrieving HPV-test and Pap smear results from the national database of the population based cervical cancer screening program.Cytology result after 10 years by retrieving HPV-test and Pap smear results from the national database of the population based cervical cancer screening program.Vaccination effect on premature delivery and obstetrical complications by comparing the results to a national database on childbirth data.The tertiary and long-term objectives are:Nonavalent HPV-vaccine (Gardasil-9®) orPlacebo vaccine Patients eligible for participation will be counselled and receive written information. All patients must provide written informed consent prior to participation. After written consent patients will be randomised to eitherThe LEEP should be performed according to guideline for regular care. Preferably, at the day of LEEP the first vaccination should be administered. The first vaccination should be administered at least within 4 weeks after the LEEP. This time window was chosen in particular for logistical reasons, for example with the see and treat method. The second dose should be administered at least one month after the first dose and the third dose should be administered at least 3 months after the second dose. All three doses should be given within a 1-year period. Although studies do not indicate adverse pregnancy outcomes, patients will be asked to use reliable contraceptive during the vaccination period (6 months). Follow-up will be according the Dutch guideline for cervical and vaginal dysplasia  , . With a target power of 0.8 and an alpha of 0.050, a total number of 646 patients needs to be recruited into the study if the incidence of CIN II-III at two-year follow-up is 3% in the group receiving active treatment, and 8% in the group receiving placebo. Clinical practice shows that the proportion of women who do not attend follow-up visits in this population is high. Therefore, a loss to follow-up is anticipated of 10 to 15%. To compensate for such attrition, the total number of patients that is to be recruited for the study is rounded to 750 patients in total.All data will be collected using an eCRF and by means of electronic questionnaires. The eCRF and questionnaire data will be collected and stored using Castor EDC, which is also used for randomisation. Subject numbers will be assigned sequentially to subjects enrolled in the study. Stored data is only accessible to the principal and coordinating investigator.Patient characteristics; age, obstetrical history, contraceptive use, condom use complementary to contraceptive, HPV related history, smoking, family planning.Indication for testing, cytology results, KOPAC-classification, HPV genotype, biopsy result.Quality of life questionnaire (Euroqol 5D-5 L) and societal economical costs questionnaire .Initial Pap smear and HPV test resultsThe following data and outcome parameters are collected  and LEEP margins (clear margins or CIN in margins).1 week follow-up:Side effects of the injection.QoL (Euroqol 5D-5 L) and societal economical costs questionnaire .2 months follow up:Cytology results and HPV genotype, QoL (Euroqol 5D-5 L) questionnaire.Side effects of the injection.6 months follow-up:Cytology results and HPV genotype. When either test is abnormal, colposcopy will be performed and biopsies taken for histologic study endpoint.QoL and societal economical costs questionnaire .All other cytology test, colposcopy results, LEEP result and HPV genotype test between appointment 6 months and 24 months follow-up if appropriate.Pregnancy during follow-up with obstetrical outcome.24 month follow-up:Collection of HPV test and Pap smear result via population screening program and sub sequential treatment.Collection of national database on childbirth data on subsequent pregnancies and the following pregnancy outcomes: gestational age, miscarriages, preterm rupture of membranes.60 and 120 months follow-up:2 test. Outcome measure for the primary outcome is a relative risk with 95% confidence interval.The main analysis for the primary outcome testing the hypothesis of superiority of nonavalent vaccine as compared to placebo, for preventing CIN II-III recurrence after 24 months will be conducted using a χ2 or Fisher’s exact test for significance. Continuous data will be described using means with standard deviation, or medians with interquartile ranges; t-tests or Mann-Whitney U tests will be used as appropriate. Kaplan-Meier curves will be made for time between LEEP, vaccination with active or placebo and CIN II-III recurrence. The curves will be compared using the log-rank test. Quality of Life will be measured at every visit. They will be assessed with Euroqol 5D-5 L questionnaire. Patients from both groups will be analyzed using generalized linear mixed models.Relative risks will also be estimated for secondary categorical outcomes, with 95% confidence intervals and χThe cost-effectiveness will be assessed by calculating the incremental cost-effectiveness ratio (ICER), defined as the difference in costs, divided by the average change in effectiveness of the vaccine versus a placebo following LEEP treatment. A Budget impact analysis (BIA) will be performed, in accordance with the principles for good practice for BIA, to address the financial stream of consequences in order to assess the affordability of offering the vaccine following LEEP treatment. BIA will compare the likely impact of replacing LEEP by HPV vaccination on national health plan budgets, on hospital level and on assurance level. From the viewpoint of the government, the broad societal perspective and the ‘budgetary framework for care’ will be highlighted. A valid framework (Markov model) will give insight into the budget consequences. We will also perform sensitivity analyses and a ‘most optimistic versus pessimistic’ scenario. If the vaccine has a preventive effect, it will be included in the new national guideline. As a result, the health care insurance can compensate this as regular care.Prophylactic HPV vaccines are very effective in reducing premalignant lesions of the cervix in HPV negative young women and less so in women up to 25 years of age . Less cl


High-risk human papillomavirus (HR-HPV) load is thought to be influenced by many factors, and the relationship between viral load and the degree of cervical lesion is controversial. This study explored the possible influencing factors of HR-HPV viral load in the uterine cervix.A total of 605 women who needed colposcopic evaluation for abnormal cervical screening at the Affiliated Hospital of Weifang Medical University, China, between November 2017 and September 2018 were enrolled. Cervical specimens were collected from the endo- and ectocervix separately using two different cervical brushes. The hybrid capture II test was used to measure HR-HPV load. Age, histological severity, number of viral types, and area and location of cervical lesions were recorded. The correlations between viral load and influencing factors were analysed using univariate and multivariate analyses.P < 0.05). Viral load with the combination of endo- and ectocervical sampling was significantly higher than simple endocervical sampling (P < 0.001). Multivariate analysis showed that age, multiple HPV types and area of cervical lesions were independent factors for HR-HPV load with a combination of endo- and ectocervical sampling (P < 0.05). However, only age and area of cervical lesions were independent factors for viral load with simple endocervical sampling (P < 0.05). No significant association was found between viral load and lesion severity in multivariate analysis (P > 0.05).HR-HPV load was positively correlated with age, histological severity, multiple HPV types and area of cervical lesions (HR-HPV load is influenced by age, histological severity, multiple viral types, area of cervical lesion and sampling methods. Age and area of cervical lesions are independent factors for viral load. Cervical cancer is the second most common gynaecological cancer worldwide. It is well established that almost all precancerous and cancerous lesions of the cervix are caused by persistent oncogenic high-risk human papillomavirus (HR-HPV) infection. Therefore, HPV DNA testing was recently recommended as an alternative to cytology-based cervical cancer screening by the American updated screening guidelines . HoweverMany epidemiologic studies have provided strong and consistent evidence that persistent infection with HR-HPV is responsible for the development, maintenance and progression of cervical intraepithelial lesions (SILs) and cervical cancer (CC) , 5. PersBased on the abovementioned findings, we aim to explore the possible influencing factors of HR-HPV viral load in the uterine cervix. The results of this study are expected to contribute to an understanding of the association of viral load and the severity of cervical lesions.All clinical samples in this study were organized using the Weifang Cervical Lesions Screening cohorts, Shandong, P. R. China. Detection of the 21 HPV genotypes was performed by the HPV GenoArray Test Kit  before enrolment of patients. A total of 605 women who needed colposcopic evaluation for abnormal cervical screening at the Affiliated Hospital of Weifang Medical University, China, between November 2017 and September 2018 were enrolled. The inclusion criteria were as follows: (1) agreement to participate; (2) HR-HPV positivity; (3) indication for colposcopy ; (4) no At first, one physician (T. T. Wang) who was trained used a cytobrush  to collect exfoliated cells from the endocervix . Then, a different cervical brush  was used to collect exfoliated cells of the ectocervix . Both specimens were separately transferred to specimen preserving medium , stored at − 4 °C and sent to the laboratory for viral load testing using the commercially available Hybrid Capture II system . Based on the anatomical sites of sampling, sampling methods were stratified into two groups: a simple endocervical sampling group and a combination of endo- and ectocervical sampling group.2) and location  of the lesions were quantified using a colposcopy image processor. Based on the location of lesions, women were stratified into two groups: involvement of the area bounded below by the ectocervix within a 0.5-cm radius of the external orifice of the uterus (≤ 0.5) and involvement of the area bounded below by the ectocervix over a 0.5-cm radius of the external orifice of the uterus (> 0.5). For pathology diagnosis, colposcopy with a directed biopsy was performed as indicated, and biopsy results were evaluated by two pathologists who were blind to the colposcopy results or clinical information. All cases with disagreement were reanalysed by another experienced pathologist. Based on colposcopy and pathology findings, the patients were stratified into three groups: negative , LSIL , and HSIL/CC .A standard colposcopic examination was performed by the same professional colposcopist (Y.Z. Liu). Cervical lesions showing aceto-white epithelium and/or atypical vessel changes  were measured under 15X magnification of colposcopy. The area . This assay can determine 21 HPV types, including 14 high-risk HPV types , five low-risk HPV types , and two unknown-risk types (53 and CP8304), by the flow-through hybridization technique using HPV DNA amplified by PCR as described before [A hybrid capture II assay  was performed on cervical scrapings collected from the endo- and ectocervix  separately using two different cervical brushes. Light measurements were quantified using a luminometer and were expressed by comparing the relative light units (RLU) of clinical samples with a positive control (PC). RLU/PC ratios (RLUs) were calculated as the ratio of the specimen luminescence to the luminescence of the 1.0 pg/mL HPV cut-off standard, which is known to represent a semiquantitative value for the cumulative viral burden of one or more of the 13 oncogenic HPV genotypes . An RLU/PC ratio of 1 or higher was considered a positive result as proposed by the manufacturer .rs) was applied to measure the association between viral load and variables. Multiple linear regression analysis was applied to relate the viral load with variables. The variables included in the model were age, histological severity, number of HPV types, area and location of the cervical lesions. A paired t-test was used to compare viral load with different sampling methods. Two-tailed P values less than 0.05 were considered statistically significant.All statistical analyses were performed using SPSS version 22.0 for Windows. Means, medians, ranges, standard deviations, 95% confidence intervals, and standard errors of means were used as descriptive statistics. Viral load was measured as RLU/PC ratios (RLUs) and agreed with previous specifications in the hybrid capture assay. Viral quantification data in RLUs were initially continuous measurements. RLUs were transformed into their logarithm (Log10). An index of the Spearman correlation coefficient . A distinct upward trend of viral load paralleled increasing age . A significant association between viral load and age was found  from the mean value of 0.79 (± 0.39) in the pathologically negative group to 0.87 (± 0.91) and 1.41 (± 0.81) in the LSIL and HSIL/CC groups, respectively . Similarly, in combination with endo- and ectocervical sampling methods, distinct upward trends of log10-transformed viral load (log10RLUs) were observed from a mean value of 0.88 (± 0.35) in the pathologically negative group to 1.03 (± 0.89) and 1.66 (± 0.70) in the LSIL and HSIL/CC groups, respectively  . In the combination of endo- and ectocervical sampling methods, the multiple HPV type infection group showed a significantly higher viral load than the single HPV type infection group   of the women were positive for a single viral type. A total of 26.7% (73/273) were positive for multiple viral types. In the simple endocervical sampling method, no significant association between viral load and multiple HPV types was found . A distinct upward trend of viral load paralleled the increasing area of cervical lesions   and bounded below by the ectocervix over a 0.5-cm radius (> 0.5) of the external orifice of uterus, respectively. No statistical association was identified between viral load and location of the cervical lesions  showed a significant difference between simple endocervical sampling and the combination of endo- and ectocervical sampling (1.19 ± 0.76 vs. 1.02 ± 0.790). Viral load with the combination of endo- and ectocervical sampling was significantly higher than with simple endocervical sampling  . In combination with endo- and ectocervical sampling methods, this analysis found that age, multiple HPV types, and area of cervical lesions were independent influencing factors for HR-HPV load   infection is the main cause of precancerous and cancerous lesions of the cervix , 5. ViraIn this study, the results showed that age was an independent factor for HR-HPV load and that a significantly increasing age correlated with a higher viral load. This result agrees with previous population-based studies , 18, whiSeveral studies have reported the association of high viral load with the risk for cervical cancer and its precursors. A large number of studies used the Hybrid Capture II assay to measure viral load, and while some found viral load to be positively associated with increased severity of cervical lesions , 7, otheThe prevalence rate of concurrent multiple HPV genotype infections was reported to be approximately 20%-25% in several large studies , 23, whiCurrent data showed that a distinct upward trend of viral load paralleled the increasing area of cervical lesions in both univariate and multivariate analyses. This result corroborates previous studies that have reported that more severe lesions tended to be larger and to harbour more HPV viral copies –28. DiffHPV viral load estimated from cervical scrapings can be easily affected by sampling . The radTo our knowledge, our study is the first to demonstrate that age and area of cervical lesions are independent factors determining viral load, which may contribute valuable data for future discussions related to the potential application of viral load measurement. Nevertheless, several limitations also need to be addressed. First, the number of women with cervical lesions involving the area of the external orifice of the uterus over a 0.5-cm radius is too limited to draw a definitive conclusion for the effect of location of the cervical lesions on viral load and specimen collection methods. In addition, clinical data collected in one institution limit our statistical power to draw definitive conclusions for populations worldwide. Finally, the sample size in our study hinders our capacity to evaluate the association of HR-HPV load with other potential confounders, such as sampling physicians and individual HPV types. Further intensive clinical setup and laboratory investigations are therefore needed.High-risk human papillomavirus (HR-HPV) load is influenced by age, histological severity, multiple viral types, area of cervical lesions and sampling methods. Age and area of cervical lesions are independent factors for viral load. Comprehensive sampling is needed, especially when the cervical lesions are located far from the cervical orifice of the uterus.