Oropharyngeal cancer is an important, understudied cancer affecting Aboriginal and Torres Strait Islander Australians. The human papillomavirus (HPV) is a significant risk factor for oropharyngeal cancer. Current generation HPV vaccines are effective against the 2 most common types of high-risk HPVs in cancer (hrHPVs 16/18).This study aims (1) to yield population estimates of oncogenic genotypes of HPV in the mouth and oropharynx of defined Aboriginal and Torres Strait Islander populations; (2) to estimate the proportion of oropharyngeal cancer attributable to HPV among these Australian citizens; (3) to estimate the impact of HPV vaccination as currently implemented on rates of oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians; and (4) taking into account impact on oropharyngeal as well as cervical cancer, to evaluate efficacy and cost-effectiveness of targeted extended HPV vaccination to older ages, among our study population.Our study design and operation is straightforward, with minimal impost on participants. It involves testing for carriage of hrHPV in the mouth and oropharynx among 1000 Aboriginal South Australians by simple saliva collection and with follow-up at 12 and 24 months, collection of sexual history at baseline, collection of information for estimating health state  utilities at baseline, genotyping of viruses, predictive outcome and cost-effectiveness modeling, data interpretation and development of vaccination, and follow-up management strategies driven by the Aboriginal community.Participant recruitment for this study commenced in February 2018 and enrollment is ongoing. The first results are expected to be submitted for publication in 2019.The project will have a number of important outcomes. Synthesis of evidence will enable generation of estimates of the burden of oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians and indicate the likely effectiveness and cost-effectiveness of prevention. This will be important for health services planning, and for Aboriginal health worker and patient education. The results will also point to important areas where research efforts should be focused to improve outcomes in Aboriginal and Torres Strait Islander Australians with oropharyngeal cancer. There will be a strong focus on community engagement and accounting for the preferences of individuals and the community in control of HPV-related cancers. The project has international relevance in that it will be the first to systematically evaluate prevention of both cervical and oropharyngeal cancer in a high-risk Indigenous population taking into account all population, testing, and surveillance options.RR1-10.2196/10503 Human papillomaviruses (HPVs) are a heterogeneous group of over 100 genotypes, being circular, double-stranded DNA viruses that grow in stratified epithelia of skin and mucous membranes. There are approximately 15 HPV types that have potential to cause cancer. Before implementation of vaccination, a restricted number of these genotypes, known as high risk or oncogenic types, were the most common sexually transmitted infection in Australia, with an estimated 4 out of 5 Australians having a high-risk HPV (hrHPV) infection at some point in their lives . PreventThe rate of carriage of hrHPV at a population level at sites relevant to cancer  in Australia is difficult to characterize; most surveys have not been national or representative, and almost all have focused on females only. Although there are some general population oral HPV DNA prevalence data , the bigCervical cancer is the fourth most common cancer of women internationally . VirtualCandida or a pro-inflammatory bacteriale, or a compromised immune system [Oropharyngeal cancers include cancer of the middle part of the throat: the tonsils, posterior one-third of the tongue, and lateral and posterior walls of the oropharynx . Approxie system ,15. Surve system . Once the system .In addition to tobacco and alcohol, HPV has been increasingly identified as a significant risk factor for oropharyngeal cancer . Both orHead and neck cancers  have been described as being more emotionally traumatic than any other form of cancer ,24. TreaThere is little documented evidence on the incidence of oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians at a national level. Johnson and colleagues reported that Indigenous persons living in Queensland (who comprise 4% of the Queensland population) were more likely than the total Queensland population to be diagnosed with certain head and neck cancers between 1997 and 2012, specifically base of tongue or tonsil or oropharynx  . Five-yeAs with the general population, alcohol and tobacco use are often cited to be significant risk factors for oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians, although the population attributable fraction for HPV-related oropharyngeal cancers in this group is unknown . AborigiThere are currently no population estimates of carriage of hrHPV in the upper aero-digestive tract among Aboriginal and Torres Strait Islander Australians. This is a substantial deficit in the contemporary knowledge base, particularly given the higher risk for oropharyngeal cancer among this population. To determine prevalence of hrHPV, and risk factors associated with the infection among this population, data on prevalence using sensitive HPV detection methods are necessary.Prevention of acquiring a persistent infection with a hrHPV through vaccination is a cost-effective and life-saving intervention to decrease the burden of HPV-related cancers in Australia. Current bi- or trivalent vaccines are effective against the 2 genotypes most strongly associated with cancer (types 16 and 18), which are detected in approximately 95% of HPV-positive oropharyngeal tumors in the United States ,33, 94% There are no national-level data available for the uptake of HPV vaccination among Aboriginal and Torres Strait Islander Australians. Data from the first stage of the National HPV Vaccination Program (NHVP) suggest that, in Queensland, coverage among Indigenous girls aged 12-17 years compared with all girls aged 12-17 years was lower with each dose . This pattern was not seen in the Northern Territory, where initial coverage was 17% lower among Indigenous girls, but the course completion rate among those who started vaccination was identical (84%) . Both usWheeler and colleagues  conducteAlthough population-level impact and herd effects following HPV vaccination have been widely documented, cost-effectiveness evaluations in Australia are scarce. Kulasingam et al reported on the cost-effectiveness for females, which supported the original Commonwealth Serum Laboratories application for the vaccine to be included on the National Immunization Program . The cosUtilities are fundamental values that represent the strength of an individual’s preferences for specific health-related outcomes. Measuring health utilities involves 2 main steps: defining a set of health states of interest and valuing those health states. It is important to estimate utilities in relation to HPV, cervical cancer, and oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians because the frame of reference regarding prevention, screening, and burden of cancer treatment is likely to differ in meaningful ways compared with the non-Indigenous population. Differences may be because of the substantial travel required for many Indigenous Australians and because of time away from family and country. There may be inherent distrust and fear of hospital systems not apparent in non-Indigenous populations, and the specific treatment-associated morbidity may be valued differently. It is important to capture this information that can be used to directly calculate quality-adjusted life years and to, in turn, be translated into health policy regarding Aboriginal patient journeys with primary and secondary prevention for cervical, other genital, and oropharyngeal cancer. Although health state valuations appropriate for modeled economic evaluations have been undertaken for cervical HPV disease including cancer and precancerous lesions ,50 and fThe aims of this study were as follows:Hypothesis: The prevalence of oral HPV among Aboriginal and Torres Strait Islander Australians will be high compared with national-level estimates.To yield population estimates of the age-specific prevalence of oncogenic genotypes of HPV in the mouth and oropharynx of defined Aboriginal and Torres Strait Islander populations . Hypothesis: The burden among Aboriginal and Torres Strait Islanders of HPV and related oropharyngeal cancer will be high.Using preliminary data from Aim 1, and information on the prevalence of other risk factors for oropharyngeal cancer in the Aboriginal and Torres Strait Islander population, to estimate burden of HPV-related oropharyngeal cancer among Aboriginal and Torres Strait Islander men and women. Hypothesis: HPV vaccination will, over time, reduce the burden of cervical and oropharyngealcancer among Aboriginals and Torres Strait Islanders.To estimate the impact of HPV vaccination as currently implemented on rates of cervical and oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians. Hypothesis: Age-extended HPV vaccination for Aboriginal and Torres Strait Islander Australians will be efficacious; we will estimate an upper age limit at which it would be cost-effective.To evaluate efficacy and cost-effectiveness of targeted extended HPV vaccination among Aboriginal and Torres Strait Islander Australians, incorporating the effectiveness against both cervical cancer  and oropharyngeal cancer. Different upper age thresholds for targeted extension will be considered. Our overall study design will impose minimal impost on Aboriginal and Torres Strait Islander participants. It involves testing for carriage of hrHPV in the mouth and oropharynx among 1000 Aboriginal South Australians by simple saliva collection, with follow-up at 12 and 24 months, collection of sexual history at baseline, collection of information for estimating utilities at baseline, genotyping the viruses, statistical analysis (including cost-effectiveness modeling), data interpretation and development of vaccination and clinical therapeutic strategies to better communicate the benefits of HPV vaccination and lifestyle changes in cancer prevention, and approaches to take into account the preferences of Indigenous people in prevention and management of cancer driven by the Aboriginal community.Ethics approval has been obtained from the University of Adelaide Human Research Ethics Committee (H-2016-246). Before being recruited, all participants will be required to sign an informed consent form, which includes consent for the authors to publish the findings in the peer-reviewed scientific literature. The authors confirm that supporting data and material in the study will be made available through Springer Nature’s Data Support Services.We will recruit 1000 Aboriginal South Australian male and female adults, with a focus on Port Augusta, Whyalla, Port Lincoln, Mount Gambier, Ceduna, and Adelaide. Census data indicate approximately 22,000 Aboriginal adults reside in these areas. The investigators have a 13-year relationship with key Aboriginal stakeholder groups in these locations, who are willing and excited to be part of the study. Recruitment strategies will be based on those successfully implemented in the past, including the following: establishing service agreements with key Aboriginal community-controlled health organizations, liaising with community champions previously involved in our research, and encouraging word-of-mouth spread of knowledge.Participants will be aged 18+ years, identify as being Aboriginal or Torres Strait Islander, and planning to live in South Australia for the next 3 years. Participants not enrolled during the original recruitment period will not be eligible to participate in the follow-up phases.Permission to obtain sensitive information from participants will be sought, with relevant information related to alcohol and tobacco use, HPV diagnosis, health behaviors , and social determinants asked through a self-report questionnaire. Data will be collected by experienced Aboriginal research officers.We will design a questionnaire for the utility study, in which all 1000 participants taking part in the oral HPV prevalence study will be asked to indicate preferences (rank and utility scores) for 6 hypothetical states relating to oral HPV testing, precursor oropharyngeal cancer, and early-stage oropharyngeal cancer (including examinations and treatment). On the basis of standard methods used in the development of utilities, preferences for health states will be measured through ranking (1 through to 6), followed by a 2-stage standard gamble. We will focus on valuing the long-term oropharyngeal cancer health state of the average patient who survives up to 5 years after diagnosis and treatment, which is the most appropriate state for modeling cost-effectiveness of prophylactic HPV vaccination. We will seek to generate utility values for all cancer stages at diagnosis. The process for developing the health states will involve the following: (1) the most common stage(s) of HPV-associated oropharyngeal cancer at diagnosis identified from the literature; (2) the recommended treatment for the relevant stages(s) of oropharyngeal cancer identified and confirmed from published studies; and (3) the more common long-term consequences (applying to ≥50% patients) in patients surviving the initial treatment phase described based on the literature, and subsequent refinement by clinical experts involved in managing oropharyngeal cancer ,53.All participants will be asked to provide a saliva sample using a commercially available kit  from which microbial DNA for genotyping will be extracted. This involves the participant: (a) not eating or drinking for 30 min before collection; (b) spitting until 2 ml reaches the fill line on the container (takes 2-3 min); (c) closing the lid on the funnel (to release preservative liquid into tube); (d) removing the funnel lid on the container; and (e) placing the small cap on the tube and shaking the tube for 5 seconds. This results in over 100 μg of DNA collection, which is sufficient for the testing required. The sample can be kept at room temperature (for up to 12 months) until collection by the Aboriginal research assistants, who will send it to an appropriate laboratory for analysis. Saliva samples will be collected at baseline, 12 months, and 24 months.In brief, the analysis plan for each aim is described below.Antonsson and colleagues have evaluated 3 different kits  for DNA extraction, namely, Promega’s Maxwell-16 Viral Total Nucleic Acid Purification Kit, QIAGEN’s QIAamp Mini Elute Virus Spin Kit, and QIAamp Blood DNA Mini Kit (QIAcube). We will use the Promega Maxwell viral kit for DNA extraction as the DNA yield and quality was superior compared with the 2 other kits. β-globin polymerase chain reaction (PCR) with the primers PCO3 and PCO4 will be carried out on all samples to ensure that they contain enough cells to detect human DNA, and that no PCR inhibiting agents are present .We will analyze all samples with the optimized general primer (GP)+PCR system that detects most mucosal HPV types and all hrHPV types that have oncogenic potential in mucosal tissue . All HPVHPV status and genotypes found will be analyzed. The genotypes will also be divided into low-risk  and hrHPV types . As multiple HPV infections are likely, incident infection will be defined as a new type-specific HPV infection not detected in a previous sample. We will determine the precision of HPV prevalence estimates obtainable with the sample of 1000 in age- and sex-specific subgroups. We will base these estimates on measurements in other populations by age and sex, and then characterize 95% CIs obtainable in the given sample size.Sufficiently detailed information on oropharyngeal cancer rates overall, or the proportion of oropharyngeal cancers which are HPV-positive, are not available for the Indigenous population. Therefore, these rates will be estimated using published data on HPV-positive and HPV-negative oropharyngeal cancers in the general population ,58. EachWe will perform systematic reviews of utilities for oropharyngeal cancer diagnosis, surveillance, surgery, and the diagnosis/treatment of associated cancers. We will also perform systematic reviews of complication rates and associated utilities for surveillance and surgery. Aggregate costs for each step involved in screening, diagnosis, family counseling, referral and management pathways, and cancer diagnosis and treatment will be collated using methods previously employed by Canfell and colleagues for other cancer-related applications ,60. BrieThe results of Aim 2 will feed into an existing model of HPV transmission, vaccination, and natural history developed by Canfell and colleagues through previous grants from Australia’s National Health and Medical Research Council (NHMRC). This model has been used extensively for evaluations around HPV and cervical cancer prevention in government-commissioned reports and in 20 journal publications. In the proposed study, this model will be further tailored to the Aboriginal and Torres Strait Islander population. The developed model will be used to make detailed predictions of the impact of HPV vaccination over time on oropharyngeal and cervical cancer among Aboriginal and Torres Strait Islander Australians, including under a range of age ranges for vaccination and dose/uptake assumptions. This will be based on the burden of HPV-attributable oropharyngeal and cervical cancer in the Indigenous population estimated in Aim 2, which will take into account different tobacco smoking prevalence or varied attributable fraction to allow for the higher proportion of HPV-negative tumors in the Indigenous population. Estimates of HPV vaccine uptake in Aboriginal and Torres Strait Islander Australians will be used, in conjunction with available data on HPV vaccine impact in Aboriginal and Torres Strait Islander populations or precursor/proxy outcomes, such as prevalence of infection with vaccine-included types and anogenital warts. We will validate model predictions against these previously reported outcomes ,42.We will use data from the literature review and utility estimations in Aim 2 to inform model assumptions on the demographics and risk profile of the population. To achieve higher coverage in this group, we will estimate impact and cost-effectiveness of funding extended catch-up vaccination for Aboriginal and Torres Strait Islander Australians (for those who did not receive the vaccine through the school-based program). A range of potential extended catch-up strategies will be considered, eg, funding HPV vaccination for females ± males aged up to 18, 25, 30, or 45 years who have not already received a full course. We will consider both first and second generation HPV vaccines and also consider strategies involving revaccination of individuals who have already received the first generation vaccine with the second generation vaccine. We will also consider different potential delivery mechanisms, eg, via Aboriginal health services and/or other community providers. For each analysis, a large “virtual” sample of the Aboriginal and Torres Strait Islander population will be simulated. Each evaluation will then use all fitted parameter sets to derive a baseline result and 95% CI. These evaluations will simulate 10,000 individuals. All evaluations will be accompanied by extensive sensitivity analysis, using one-way and probabilistic sensitivity analysis techniques that will take into account the full range of identified fitted parameter sets. For each strategy, we will calculate a number of measures of effectiveness, including change in cancer incidence, mortality, life years saved, and quality-adjusted life years gained. We will also estimate absolute case numbers based on population projections available by age, sex, and calendar year for ASTI Australians. We will assess morbidity via calculation of quality-adjusted life years and calculate complication numbers from surveillance and surgery. We will take into account varying rates of attendance for treatment in the Indigenous population , but conEthical approval for this study has been obtained by the University of Adelaide Human Research Ethics Committee (H-2016-246).Participant recruitment for this study commenced in February 2018 and enrollment is ongoing. The first results are expected to be submitted for publication in 2019.Oropharyngeal cancer is an important cancer affecting Aboriginal and Torres Strait Islander Australians at a higher rate than other Australians. Infection with hrHPV is a significant risk factor for oropharyngeal cancer. HPV vaccination is effective against the 2 most common types of hrHPV, with some promise that current vaccines may prevent oral infections . Given the elevated risk of HPV-related cancers in this group, it may be reasonable to extend the comparatively brief timeframe (when aged 12-13 years) in which Aboriginal and Torres Strait Islander Australians can access an otherwise costly vaccine via public funding. The project will have a number of important outcomes. Synthesis of evidence will directly support estimates of the burden of oropharyngeal cancer among Aboriginal and Torres Strait Islander Australians and the effectiveness and cost-effectiveness of prevention. This will be important for health services planning, and for Aboriginal health worker and patient education. The project provides a key example of how carefully calibrated, data-driven disease models can integrate with Aboriginal community views and expectations to estimate disease burden and to guide policy decisions.The strengths of the study include it being the first to obtain and link all the information on cervical and oropharyngeal cancers via modeling  and the focus on engagement, enabling community and individual preferences to play a large role in decision making for Aboriginal and Torres Strait Islander Australians. Burger and colleagues investigated the impact of HPV on 6 HPV-associated cancers, including cervical and oropharyngeal cancer, among 5 ethnic groups in the United States, one of which included Native American/Alaskan Natives .The limitations include the sample frame for oral HPV prevalence assessment being pragmatic, ie, utilizing convenience sampling methodology rather than attempting to be representative. Although there is a risk of sampling bias, the efforts required to obtain a representative sample are recognized as being both expensive and time-consuming . Additio


This study presents the case of a man who developed a temporary and asymptomatic blue tongue. The dyschromia occurred following topical contact with gelato that contained Food, Drug, and Cosmetic (FD&C) blue dye no. 1. The etiology of a blue tongue is either congenital  or acquired. Acquired blue dyschromia of the tongue results from either endogenous conditions or exogenous agents. The endogenous conditions include not only benign  and malignant (ovarian carcinoma) tumors but also reactive lesions  and systemic disorders . Exposure to the exogenous agents can either be systemic , traumatic , or topical (contact with FD&C blue dye no. 1). Clinical clues to the topical exogenous etiology in the reported individual included not only the fact that the dyschromia spared both the lateral aspects and the tip of the tongue but also the observation that the blue color focally appeared on his upper lip. Lingual dyschromia can be congenital or acquired. It can result from exogenous agents , inherited syndromes, systemic conditions, benign melanocytic or vascular lesions, and reactive conditions. It is also rarely associated with visceral tumors -17. ThisA 60-year-old man presented with the new onset of an asymptomatic blue tongue. His friends had noted the change in the color of his tongue. He had been eating Cookie Monster simple gelato . The dry mix powder consists of not only FD&C blue no. 1 dye, but also several other ingredients: sugar, non-dairy creamer , chocolate cookies based cake {enriched flour , sugar, palm oil, and cocoa [processed with alkali]}, crystalline fructose, nonfat milk, sweet whey, cocoa powder , guar gum salt, natural and artificial flavors, silicon dioxide (anti-caking agent).An examination of his tongue showed that the dorsal surface had a confluent blue color Figure . The disThe blue appearance of his tongue and upper lip spontaneously resolved within six hours. The dyschromia did not recur. Subsequent investigation determined that the composition of the gelato included FD&C blue dye no. 1.A blue tongue is very uncommon. It is usually associated with an acquired condition. Endogenous and exogenous etiologies for blue lingual dyschromia are described below Table  1-17].-17.1-17]Blue tongues are not limited to humans. The tongues of several animals are also blue or blue-black, e.g., blue-tongued skink Figure , chow-chCyanosis of the tongue, with a blue appearance, is a characteristic finding of bluetongue disease; other major features of this condition include facial and lingual swelling, fever, and hypersalivation. Bluetongue disease is a viral condition of ruminant animals-mammals that chew and regurgitate their food more than once as they digest it several times in their multi-chambered stomach. This arthropod-transmitted, non-contagious disease caused by bluetongue virus is most commonly observed in sheep and less frequently in antelopes, buffalo, camels, cattle, deer, elk, and goats .Systemic conditions associated with a blue tongue include argyria, blue rubber bleb nevus syndrome, cyanosis, methemoglobinemia, and primary adrenal insufficiency Table  1,9-13],9-131,9-Benign melanocytic lesions (melanocytic macules) and vascular tumors  can present as blue patches or nodules on the tongue. Menni et al. have described the case of a 3-day old White boy with three blue-black, smooth, non-blanchable, and non-palpable macules noted on the left side of the dorsal tongue. Biopsy, at one month of age, showed a focal increase of melanin in the basal cells and some melanophages of the lamina propria. The lesions were unchanged at a follow-up examination five months later .Oral angioleiomyoma most commonly occurs on the lip and palate. However, 9% of the tumors occur on the tongue. Brooks et al. have described the case of a 54-year-old Black woman who presented with a 10-mm rubbery bilobed raised tongue tumor of ten years' duration that was whitish on the dorsum with a bluish ventral mucosa. Biopsy showed bundles of smooth muscle cells surrounding numerous vascular spaces, establishing the diagnosis of angioleiomyoma .A hemangioma is a benign developmental anomaly consisting of vascular channels lined by endothelial cells. They are asymptomatic red or blue lesions that typically appear in infancy, grow rapidly and regress spontaneously as the patient gets older. Tongue hemangiomas usually extend deeply between tongue’s muscles and can cause clinical problems such as bleeding, chewing difficulty, recurrent trauma (from tongue biting and toothbrushing), and upper-airway obstruction .Varicosities are more common in older individuals. They present as blue, red, or purple serpiginous elevations on the ventrolateral surface of the tongue. However, it can appear as a bluish-purple nodule if a thrombus is present within varix .Blue reactive lesions of the tongue include intravascular papillary endothelial hyperplasia and mucocele. An intravascular papillary endothelial hyperplasia is a non-neoplastic reactive intravascular proliferation of endothelial cells from the walls of veins; the benign vascular lesion is associated with a thrombus that is undergoing organization and recanalization. Clinically, intravascular papillary endothelial hyperplasia of the tongue present as nodules that are bluish to red in color and covered by normal mucosa .Dental amalgam consists of a mixture of copper, mercury, silver, tin, and zinc. Amalgam tattoos appear blue, blue-gray, gray, or black and result from the traumatic implantation of the metal into the oral soft tissue. Hence, amalgam tattoos have been observed on the buccal mucosa, the gingiva, the palate, and the tongue .Systemic medications can cause a blue tongue; these include minocycline and some of the drugs typically used to treat psychiatry patients -17. SeveBlue tongues have been observed in individuals treated with haloperidol or dopamine antagonists. The tongue of a patient receiving large doses of haloperidol (a butyrophenone agent) was observed to be blue . In addiAlroe et al. have described the case of a 19-year-old woman with nausea and headache who received intramuscular prochlorperazine (a phenothiazine dopamine-receptor antagonist) along with intramuscular metoclopramide (a selective D2 dopamine antagonist). Her tongue became blue and swollen with partial obstruction of her upper airway. However, her tongue returned to its normal color and size with a resolution of the respiratory distress after she was treated with benztropine .Yun et al. have described the case of a 23-year-old man with schizophrenia who also developed the blue-tongue sign. He initially received risperidone (a selective D2 dopamine antagonist) while hospitalized; amisulpride (a dopamine D2-receptor antagonist and a dopamine D3-receptor antagonist) was subsequently added. His tongue was noticed to be blue at three weeks of discharge; however, its normal color spontaneously returned without the cessation of his antipsychotic medications .Systemic absorption of FD&C blue dye no. 1 has previously been observed to cause blue discoloration of the skin and nails in a critically ill 79-year-old woman who received total parenteral nutrition through a percutaneous gastrojejunal tube. Blue dye had been added to enteral feeding bags prior to starting the infusion in order to determine if there was an aspiration of gastric contents. The blue skin and nails appeared within two-and-a-half weeks after starting the dye. The dye was discontinued, and the blue color disappeared during the next month .The reported patient developed a blue tongue secondary to exposure to the FD&C blue dye no. 1 used in a gelato. The diagnostic clues to the exogenous etiology included not only the focal blue staining of the upper lip but also the sparing the tip and lateral aspects of the tongue. His lingual dyschromia resolved spontaneously.Lingual dyschromia can present as a blue tongue. The features of a man who developed asymptomatic and temporary blue dyschromia of his tongue after contact with FD&C blue dye no. 1 are described in this study. The differential diagnosis of blue dyschromia of the tongue includes not only endogenous and exogenous acquired conditions but also congenital etiologies.


Shobha S Krishnan is the Founder and President of the Global Initiative Against HPV and Cervical Cancer (GIAHC). She is a board-certified family physician and gynecologist and has been involved in the areas of primary care and women's health for over 30 years. She has extensive experience both nationally and internationally in the field of HPV and HPV-related diseases. Her vast experience in this field has given her a unique perspective on various issues related to HPV and its disease consequences.The HPV Vaccine Controversy: Sex, Cancer, God and Politics . Her book chapter on ‘Challenges in Screening for Cervical Cancer’ was recently published in Cancer Screening in the Developing World .She is the author of the national and international award-winning book, She is the chair of the HPV and Cervical Cancer Task Force at the American Medical Women's Association. She is also a member on the expert's panel of the American Sexual Health Association, Medical Advisory Board of the National Cervical Cancer Coalition and on the scientific committee at the Medical Women's International Association. In addition, she has served on the sexually transmitted disease research working group at Columbia University  and as a surveillance physician for the Centers for Disease Control.After completing her residency training, Krishnan went into private practice for 10 years before joining Columbia University, where she worked for several years. In December 2015, she received her master's certification in Non-Profit Leadership and Management from the University of Florida . I have had a pretty adventurous career. First, I completed a residency in OBGYN and then in family medicine. Soon after, I realized I could combine the two and focus on women's health. I started off with a private practice in Indianapolis , and then moved to New York (USA), where I was at Columbia University for 13 years focusing on women's health and adolescent medicine. In 2006, a publisher approached me based on my background and work performed in both the developed and developing parts of the world. At the time they asked me to speak about the new Human Papillomavirus Virus (HPV) vaccine, which had become somewhat controversial due to it being a sexually transmitted virus. There seemed to be a lot of promise in the vaccine but because it was being recommended for girls around the age of 11 or 12, parents were concerned it was going to promote promiscuity.The vaccine was marketed to prevent cervical cancer only, and cervical cancer is one of the biggest killers today in rural parts of the developing world. Cervical cancer rates 50–60 years ago were the same in the USA, but now it is not even on the top 10 list because of our fantastic screening program. However, in the developing world, as simple as a pap test is, it is laborious and not widely available. Therefore, women do not present with the disease until they have symptoms, usually at an advanced stage.HPV Vaccine Controversy: Sex, Cancer, God and Politics.In order to address these important concerns and questions, in 2008 Praeger published my book titled A Yale professor  contacted me after reading my book and was interested in my experiences. She had a student who was very interested in cultural anthropology and healthcare. As a result of the introduction, we sent this student to India, working with her remotely as a team to see if we could do something about the cervical cancer in a rural community I had visited previously.While pap smears in the developed world are considered the standard of care, we have other tests available for less fortunate areas. In the developing world, we use a simple method called VIA, ‘visual inspection with acetic acid’, which involves painting the cervix with household vinegar. The nice thing about this method is that we can visualize the cervix with the naked eye and if something abnormal is detected we then take a freezing gun and ‘zap’ the cervix for a few minutes to reduce the chance of further growth. This somewhat crude approach has been shown to reduce morbidity and mortality by nearly 40% according to WHO.Additionally important is the one-stop screen and treat program is done in the same visit. In rural settings if you send women away and then have them come back for treatment, you can lose more than half of them for follow-up care.This humble small project, headed by the Yale student armed with a group of community health workers subsequently trained at the cancer center in Mumbai (India), was the beginning of our organization, the Global Initiative Against HPV and Cervical Cancer (GIAHC).Our mission is to save lives from cervical cancer one woman at a time, one day at a time. Our focus is on building awareness, education, prevention through vaccination, screening and early treatment.Although we are focused on cervical cancer, we are also educating providers, parents, the public, politicians and the research community that HPV does not just cause cervical cancer, but other cancers in both men and women. By 2020 we believe that in the developed world oropharyngeal cancer in men, caused by HPV, will surpass cervical cancers in women. This is because screening tests for these cancers have not yet been developed. What is more alarming, is that HPV is a virus that is ubiquitous. It is believed that by the age of 50, almost 80–90% of people would have had HPV exposure at some point in their lives. Although 90% of these infections clear up on their own, 10% persist owing to contributing factors such as a weakened immune system and cigarette smoking. These are just some of the other topics GIAHC is trying to educate people about.In the developing world we also act as a coordinating platform; we identify partners and places where need is high and provide them with tools to develop and build local programs to serve both women and children.Cervical cancer is something that we can almost completely eliminate by vaccination and screening. However, in the USA, for example, both the vaccination and screening are covered by most insurance. In spite of this, we continue to have about 12,500 cases annually with a reported 4000 deaths. The key message here is that we should not even have one death, because we have the tools to prevent it.At GIAHC we believe the root of the problem is education. Core to our efforts is to educate providers to recommend the vaccine to parents and patients and to be able to answer any questions they may have regarding cervical cancer or HPV. When it comes to politicians, we talk about how cost effective the vaccine is in reducing overall healthcare cost and how important it is to both men and women's health.In terms of the developing world where the health infrastructure is not the best, conducting clinical training and creating local care sites are the two big requirements. We have to have proper diagnostic treatment facilities and affordable therapeutics tailored to the local community. Another issue for clinicians and researchers alike is that globally we do not maintain cancer registries, and only when we have this, will we fully understand the incidence, prevalence and mortality rates.Focusing on the HPV vaccine itself, specifically in the developing world we have to make it affordable, accessible and acceptable. In the USA the vaccine is USD140 a shot, and is a series of two or three depending on age – that is a lot of money, but it is mostly covered by insurance. In the developing world where insurance systems are poor or not existent, the Global Alliance for Vaccines and Immunization (GAVI) has been able to bring down the price of the vaccine to $9 for a whole series of two shots. Even then there are obstacles: you have to be in a GAVI-eligible country; there are quotas; a series of two to three shots/visits reduces compliance; and storage is a problem because the vaccine requires refrigeration. These are just some of the obstacles we are fighting, and that is not to mention the social and cultural norms and all of the political innuendos that go along with it.Once again it is important to restate, the biggest obstacle is the fact that we have to educate and advise the public. Educational tools and healthcare materials must be designed by listening to locals, addressing their questions, and working with them to make all materials culturally sensitive and linguistically appropriate. We have to come up with inspirational and aspirational messages that strike a chord with patients and the public like the Lady Ganga story. One of GIAHC's communication strategy is to use film and arts in medicine to change minds and lives.Ruth Frazier approached GIAHC regarding her daughter, Michele Baldwin, who eventually becomes Lady Ganga, the focus of the film. Michele's cancer had come back for the third time, and was diagnosed as incurable. With this diagnosis in hand she wanted to do something to raise awareness and support our organization and work. Michele had been to India when she was young and had a passion for the country. Knowing that India had the highest number of cervical cancer cases recorded, she wanted to do something. Her intention was to paddle board from the foot hills of the Himalayas all the way to Varanasi to raise awareness of cervical cancer while a friend filmed the journey. This amazing journey was made into a short film for educational use.It was shown in remote area of India, including a place called Ladakh where a teacher called Nilza saw it and showed it to her friends. The result was just what Michele had envisioned. The group went to their local camp for cervical cancer screening, where, in 3 days, 1700 women were screened and treated. Nilza was diagnosed with cervical precancer. She was treated in a simple procedure, and her story was later added to Michele's original story. The film, Lady Ganga: Nilza's story was later screened on World Cancer Day 2016 at the United Nations in New York City (USA).Today GIAHC's goal is to help the Global Film Fund (GFF) take the original English film version and  dub it in various languages. Although currently there are some translations available, in the written form, this is not ideal for the developing world. So today we are working with GFF to dub the film in as many languages as possible. We started with Indian languages and are also planning to work on languages commonly spoken in Latin and South American countries.The next thing to focus on would be the HPV vaccine. How do we make the vaccine available to people who need it the most? This is not only about education, but are we able to bring the price of the vaccine down? For example, the hepatitis B vaccine was over $100 a shot in the USA and then, albeit 20 years later, it came down to <$1 a shot. Now the number of cases of hepatitis B are very low. We need to do the same for HPV. GIAHC and its partners are looking for ways of making this vaccine affordable, acceptable and accessible to all, both men and women, all over the world.The other thing we are also interested in is self-sampling. Another good example is colon cancer. Often, getting people to attend colon cancer screening is difficult. Now there is a home test where you send a sample of your fecal material through the mail and if the tests positive it gives you the incentive to get more extensive screenings done. This approach would be really useful for cervical cancer and HPV screening; especially in remote rural areas.GIAHC and the American Medical Women's Association have partnered to kick off 2019 with a January ‘HPV and Cervical Cancer Awareness Month’ webinar series called: USA vs HPV. This 5-day series is aimed at educating professional and the public on various topics related to cervical cancer and HPV. The program will feature several thought leaders in this field. Also, the Medical Women's International Association is celebrating their centennial in July 2019. We are hoping this conference in NYC will be a good platform to provide educational materials to doctors from all around the world, including regions where cervical cancer cases are high. We are looking forward to this association event in the hope of exchanging knowledge and new ideas so we can end cervical cancer on a global level.


Head and neck squamous cell carcinomas (HNSCC), emerging in the mucosa of the upper aerodigestive tract, are associated with either the classical risk factors, tobacco and alcohol consumption, or with infections with high-risk types of the human papillomavirus (HPV). Depending on the involvement of HPV, HNSCC follow different pathways of carcinogenesis and show distinct clinical presentations regarding survival, prognosis and treatment response. For instance, HPV-driven HNSCC exhibit an enhanced radiation response compared to their typically radioresistant HPV-negative counterparts. Although radiosensitivity of HNSCC has been studied by many research groups, the major causes for the difference in radiation responses between HPV-driven and HPV-negative HNSCC are still an open question. In this mini review, we discuss the reported cellular and immunological factors involved in the enhanced radiation response in HPV-driven HNSCC, focusing on the vital role of the immune response in the outcome of HNSCC radiotherapy. It has response , 19. Theresponse . In thisresponse .Numerous research groups have investigated the cellular basis of the observed differential radiosensitivity of HPV-driven and HPV-negative HNSCC, hypothesizing that viral proteins may affect the cellular radiation response. Indeed, recent work has shown that HPV inhibits the anti-viral cGAS-STING pathway, influences the cellular DNA repair machinery, alters cell cycle distribution, affects apoptosis as well as DNA replication and mediates unique kinetics of hypoxia during radiotherapy , 21–25. DNA repair system is crucial for the sensitivity of cancer cells toward irradiation. Liu et al. reported that abrogation of TGFβ signaling by HPV results in DNA repair deficiencies, which consequently cause elevated radiosensitivity in HPV-driven HNSCC  was associated with radiosensitivity in HPV-driven OPSCC ((CDKN2A) and TP53, leads to radioresistance in HPV-negative OPSCC  in HPV-negative and HPV-driven HNSCC  data, that showed a higher level of T cell signatures in HPV-positive compared to virus-negative tumors  , 57. Espen HNSCC , specifien HNSCC –62, and en HNSCC . Analysipression , despitepression . The corsurvival , 61. Thee tumors , 65–67, e tumors , 65, 66.tumor associated macrophages (TAMs) and regulatory T cells (Tregs). Lee et al. defined the number of CD68+ TAMs and the distribution of Tregs as negative factors that determine the outcome of concurrent chemoradiotherapy in HPV-driven tonsillar cancer  receptor CD11b were also defined to be a pivotal cell population in HPV-driven HNSCC that moderates inflammatory responses and immune suppression  expression of HPV-driven tonsillar squamous cell carcinoma was significantly associated with a favorable clinical outcome . This une cancer . As desce cancer . NeverthCD8, ICOS and HLA-DRA; decreased expression of epithelial markers and upregulation of mesenchymal markers . The “classical” subtype was represented by enrichment of components of the polyamine degradation pathway, related to an even higher proliferation rate. Associated with HPV infection, both subtypes displayed high cell cycle-related activities (Considering all the factors enhancing the radiation response that have been reviewed above and in the light of the research certifying that HPV-positivity predicts a better outcome , 10, 11,tivities . The auttivities . This sttivities .In summary, characterization of HNSCCs according to their immune-related markers, independent from HPV status, will most likely contribute to tailor therapies more efficiently in the future, and help understand the difference in radiotherapy response between HPV-driven and HPV-negative HNSCC.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila homolog (APPL) in the adult giant fiber (GF) neurons. We find that endogenous APPL is transported from the synapse to the soma in the adult. Live-imaging revealed that retrograde moving APPL vesicles co-traffic with L1-type cell adhesion molecule Neuroglian (Nrg). In APPL null mutants, stationary Nrg vesicles were increased along the axon, and the number of Nrg vesicles moving in retrograde but not anterograde direction was reduced. In contrast, trafficking of endo-lysosomal vesicles, which did not co-localize with APPL in GF axons, was not affected. This suggests that APPL loss of function does not generally disrupt axonal transport but that APPL has a selective role in the effectiveness of retrograde transport of proteins it co-traffics with. While the GF terminals of APPL loss of function animals exhibited pruning defects, APPL gain of function had no disruptive effect on GF morphology and function, or on retrograde axonal transport of Nrg. However, cell-autonomous developmental expression of a secretion-deficient form of APPL (APPL-SD), lacking the α-, β-, and, γ-secretase cleavage sites, resulted in progressive retraction of the GF terminals. Conditional expression of APPL-SD in mature GFs caused accumulation of Nrg in normal sized synaptic terminals, which was associated with severely reduced retrograde flux of Nrg labeled vesicles in the axons. Albeit β-secretase null mutants developed GF terminals they also exhibited Nrg accumulations. This suggests that cleavage defective APPL has a toxic effect on retrograde trafficking and that β-secretase cleavage has a function in Nrg sorting in endosomal compartments at the synapse. In summary, our results suggest a role for APPL and its proteolytic cleavage sites in retrograde trafficking, thus our findings are of relevance to the understanding of the endogenous role of APP as well as to the development of therapeutic treatments of Alzheimer’s disease.The role of the Amyloid Precursor Protein (APP) in the pathology of Alzheimer’s disease (AD) has been well studied. However, the normal function of APP in the nervous system is poorly understood. Here, we characterized the role of the  Drosophila homolog of APP, the β-amyloid protein precursor-like (APPL), in an adult central nervous system neuron.Amyloid Precursor Protein (APP) as well as APP-like protein 1 and 2 (APLP1 and APLP2) are a highly conserved family of type 1 transmembrane proteins. Human APP and its proteolytic cleavage has been extensively studied due to its relevance in Alzheimer’s disease (AD). APP can either be cleaved by α-secretase at the plasma membrane (non-amyloidogenic pathway) or by β-site APP-cleaving enzyme 1 (BACE-1) in early endosomes (amyloidogenic pathway) resulting in soluble extracellular sAPPα and sAPPβ fragments as well as C83 and C99 transmembrane fragments, respectively . SubsequdAPPL) are viable, exhibit learning, and other behavioral defects  neuron model for live imaging of axonal transport in the adult nervous (Studies in  nervous . The GFs nervous . Due to n = 8) of Lis-RNAi animals but not in wildtype control animals , a regulatory protein of the dynein motor using the R68A06 or GF-Split Gal4-lines . Both Gas n = 8, . This suN = 8) of all APPL vesicles co-localized with Nrg (N = 8) vesicles co-localized with APPL-V5  is transported from GF terminals in retrograde direction , while vwith Nrg . Similar APPL-V5 . We prev APPL-V5 . To dete APPL-V5 . We foun APPL-V5 .dAPPL null mutants and live imaged axons of 1–5-day old animals. dAPPL axons contained a significantly higher amount of stationary Nrg vesicles than in dAPPL heterozygote control animals  did not co-localize with V5-tagged APPL in GF axons  of the GF terminals in 1–5-day old dAPPL null mutants exhibited pruning defects  and a secretion-defective form of APPL (APPL-SD), lacking the α-, β-, and, γ-secretase cleavage sites, resulted in similar phenotypes, such as the formation of additional boutons . Here, wdAPPL null mutants, and determined if expression of APPL-WT and APPL-SD have similar or distinctive effects on axonal transport of Nrg-GFP vesicles.We and others previously showed that inhibition of retrograde transport during development results in GF terminal retraction and that when inhibited in mature terminals leads to accumulation of retrogradely transported proteins in GF terminals, such as Nrg . This sun = 12). However, Nrg-GFP did not accumulate in GF terminals when co-expressed with APPL-WT (n = 10) or when expressed in dAPPL null mutants (n = 12) or in wildtype (n = 10) background (n = 12) but not of APPL-WT (n = 16) with either the R68A06 or the GF-Split Gal4-line resulted in accumulation of endogenous Nrg in all dye-injected GFs as well  or the Go-binding motifs (APPL-SD-ΔCg) in the intracellular domain were deleted as well. Deletion of either motif, suppressed the ability of APPL-SD to increase boutons at the larval neuromuscular junction (g (n = 12) but not in animals that expressed APPL-SD-ΔNPTY (n = 10) with the R68A06 or GF-Split Gal4-line . These results suggest that the lack of GYENPTY directly or indirectly suppresses the disruptive effect of APPL-SD on retrograde receptor trafficking at the synapse.The finding that Nrg did not selectively accumulate in the GF terminals of junction . Howeveral4-line . SimilarThe finding that Nrg accumulated in vesicular compartment in animals expressing APPL-SD, suggests that β- but not the α-secretase cleavage has a role in sorting of Nrg in endosomal compartments at GF terminals. To test this hypothesis, we assessed localization of endogenous Nrg in GFs of viable BACE mutants  as well n = 14) of 3–5 days old BACE-SK3 and BACE-SK5 null mutants had GF terminals that did not exhibit any retraction bulbs. However, in the majority (9 out of 14 GFs) of the GFs had Nrg accumulated in their terminals, albeit less extensive than seen with expression of APPL-SD (n = 12). This strongly suggest a role for β-secretase cleavage in regulating Nrg trafficking at GF synapses , while the amount of APPL present in endosomal compartments is also dependent on the amount of full-length APPL normally endocytosed. In addition, neuronal activity has a role in converging APP and BACE to the same vesicle as well . If duriThe lack of the endocytic GYENPTY motif suppressed the disruptive effect of APPL-SD . Recent Expression of APPL-SD did not cause retraction or degeneration of larval motoneurons, albeit like APPL-WT, it causes axonal transport defects , suggestIn summary, our results support a direct role for APPL in the efficacy of retrograde axonal transport of select vesicles suggesting that reduced retrograde signaling of associated cargo protein contributes to loss of function phenotypes, such as pruning, synapse formation, and neurite growth. In addition, we provide evidence that proteolytic cleavage of APPL is essential for proper trafficking of Nrg proteins at the synapse. The finding that APPL gain and loss of function as well as expression of APPL incapable of being cleaved, have distinctive effects in the GFs than in larval motoneurons, while the latter causes degenerative phenotypes in the GFs and in the retina , highlig1118w , dAppl , UAS-APPL-WT , UAS-APPL-SD , UAS-APPL-SD-ΔNPTY , UAS-APPL-SD-ΔCg , UAS-APPL-V5 , UAS-LAMP1-GFP , UAS-CD8-GFP , R91H05-Gal4 , R68A06-Gal4, , UAS-Lis1-RNAi , and UAS-Dicer . UAS-BACE-RNAi (Cat#v15541) was obtained from the Vienna Drosophila Resource Center. UAS-Nrg-GFP and UAS-Nrg-mCherry were obtained from the Jan Pielage lab and the trafficking of the constructs in the GFs has been previously characterized (RRID:BDSC_79602) was obtained from the Gwyneth Card lab : cterized . GF-SpliCard lab . DrosophTM vector  and subsequently recombined into the pTWG vector  using the Invitrogen GatewayTM Cloning System. All cloning products were confirmed by Sanger sequencing . Transgenic UAS-APPL-GFP lines were established on the second and third chromosomes .UAS-APPL-GFP was generated by cloning the APPL cDNA  into the pENTRR91H05-Gal4 was used to drive cell-autonomous expression of UAS-constructs throughout GF development, whereas R68A06-Gal4 and the GF-Split Gal4-line were used to drive expression after GF terminal development . Flies wL-Lysine  coated glass bottom dishes containing saline  . The GF axons were imaged at the dorsal side in the cervical connective with an upright Nikon A1 or an inverted Nikon A1 plus Confocal with GaAsP multi-detector units using a CFl Plan 100 × /1.1 NA water immersion lens or a CFI Plan APO lamba 60 × /NA1.4 oil objective at room temperature (20–23°C). Live-recordings were obtained at 1–2 frames/second, 512 or 1024 resolution, 2–10% of 488 nm and/or 561 nm excitation laser power, and with a 90% open pinhole for up to 10 min. When necessary, photo-bleaching was performed at 40–50% excitation laser power for 0.5–1 min. Nikon Elements Advanced Research 4.4 was used for video recordings, kymograph generation, and analysis. Flux (vesicles/min) was determined as the number of vesicles passing through an arbitrary vertical line along the GF axon per minute. Stationary vesicles were defined as vesicles that remain within their initial position ± 0.1 μm for at least 1 min of recording. The average number of stationary vesicles per 40 μm length of unbleached axons were determined. Statistical significance between groups was calculated using Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test using GraphPad’s Prism 7.03 (RRID:SCR_010279). Data are shown as the mean ± standard error of the mean (SEM) where number of asterisks indicate degree of significance .Live imaging was performed similar as previously described . IsolateDrosophila nervous system dissection, dye-injection, and immunolabeling have been previously described in detail (RRID:AB_1563367), chicken anti-GFP , mouse anti-V5 , and anti-Neuroglian . The following secondary antibodies were used: anti-Rabbit Cy3 , anti-Chicken DyLight 488 , anti-mouse Cy3 , and anti-mouse DyLight 649 . Samples were scanned at a resolution of 1024 × 1024 pixels, 2.4-pixel dwell, and 0.2 μm step size with a Nikon A1R plus confocal microscope using a 60 × /1.4 NA oil immersion objective lens. Images were processed using Nikon Elements Advance Research 4.4 (RRID:SCR_014329) and Adobe Illustrator (RRID:SCR_010279) software. For images in Giant Fibers were labeled by injections of tetramethylrhodamine isothiocyanate-Dextran (Cat# 42874 Sigma) into the GF axons or by expression of membrane-bound GFP (UAS-CD8-GFP). The procedures for adult n detail ,b. In brRRID:SCR_011323) software. Statistical significance between genotypes was calculated using Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test using GraphPad’s Prism 7.03 (RRID:SCR_002798). Data are shown as the mean ± SEM where number of asterisks indicate degree of significance .Electrophysiological recordings were obtained as previously described in detail . To deteThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.TP conducted the experiments, prepared the figures, analyzed and interpreted the data, and wrote and edited the manuscript. SK conducted the experiments, prepared the figures, analyzed the data, edited the manuscript. RP conducted the experiments, analyzed the data. TG conceived, designed, planned, and supervised the entire study, secured the funding, interpreted the data, prepared the figures, wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both multivariable logistic and linear regression models estimated the association between race, gender, and sexual risk factors. About 63% of participants were males, and 65% identified as Blacks. Compared to females, males were more likely to have a higher number of oral sexual partners . Males were also more likely to have earlier oral sexual  and vaginal sexual  debuts compared to females. Blacks were more likely to have higher number of vaginal sexual partners  and earlier vaginal sex  but less likely to have earlier oral sexual debuts compared with Whites . Because HPV is associated with several cancers, our findings provide impetus for the development of targeted educational interventions aimed at improving the knowledge of these sexual risk factors, especially among men and across race groups.This study assessed if race and gender predict known sexual risk factors associated with HPV. Data ( Notably, the human papillomavirus (HPV), with its more than 150 documented strains, is the most prevalent STI in the United States, and virtually all sexually active adults will acquire HPV infection in their lifetimes2. Although most of these strains remain asymptomatic, a few oncogenic types, particularly HPV-16 and HPV-18, are associated with an increased risk of several anogenital cancers, including cervical, vaginal, vulvar, penile, anal, as well as oropharyngeal cancers3. Each year, over 43,000 newly diagnosed cancers are thought to be associated with HPV, and nearly half of these new cancer cases are HPV-positive oropharyngeal cancer4. HPV-positive oropharyngeal cancer has emerged as a leading HPV-associated cancer in the United States5, and its incidence has increased by 225% in the last three decades6.Sexually transmitted infections (STIs) remain a source of significant morbidity and mortality in the United States and are strongly associated with racial and gender disparities7. These sexual risk behaviors include age of first vaginal sex, age of first oral sex, and number of oral and vaginal sexual partners7. Previous studies have shown that these HPV-associated sexual risk factors vary by socioeconomic status, age, race, and education level8. However, there is a dearth of information about how individuals engage in these sexual behaviors in the context of HPV, based on their race and gender9. Efforts to investigate predictors of HPV infection in populations at high risk for HPV infection are critical to addressing healthcare disparities, especially understanding the differences in sexual behavior between males and females and between individuals from different racial backgrounds. This is important nationally as STIs continue to contribute significantly to the healthcare expenditure of the United States and remain a significant cause of mortality and morbidity10. It is even more important in the St. Louis region, which has consistently reported higher STI rates than more than half of major metropolitan areas in the United States reporting to the CDC11.High-risk sexual behavior is the main risk factor associated with the acquisition and persistence of HPV infection and development of HPV-associated cancersThe goal of this study was to assess racial and gender differences in the patterns of high-risk sexual behaviors among a predominantly Black population surveyed at an event hosted by the United Black Drag Racers Association (UBDRA). This event draws participants from communities throughout the St. Louis metropolitan area and the Midwest region. Study aims were to assess number of oral sexual partners, number of vaginal sexual partners, age of first oral sex, and age of first vaginal sex, in association with HPV infection in this population.p = 0.0021) and vaginal (p = 0.0108) sexual partners, initiated both oral sex (p = 0.0181) and vaginal sex (p = 0.0002) at an earlier age, and were less educated (p = 0.0079). There was no statistically significant difference between males and females in terms of race, marital status, income, drinking and cigarette smoking status.Characteristics of the study participants are summarized in Table Multivariable logistic regression, summarized in Table In multivariable linear regression analysis, summarized in Table 11. The data derived from this study has importance for educating members of the community about cancer prevention through HPV vaccination.This study aimed at understanding the racial and gender differences in sexual risk-taking behaviors among individuals in a primarily Black community, and our findings indicate that there are considerable racial and gender differences in high-risk sexual behaviors that are associated with HPV infection. While male participants were more likely to initiate both vaginal and oral sexual intercourse earlier than women, Whites were more likely than Blacks in general to initiate oral sex. These racial and gender patterns shown in our study align with current trends in the incidence of HPV-associated cancers of the oropharynx and have important implications in their prevention. Recent data suggest that the rate of HPV-associated oropharyngeal cancer ranged from 3.5 to 4.8 times higher for men than for women, but significantly lower among Blacks than Whites12. On average, males in the United States report sexual debut at 16.8 years, females at 17.2 years12. Of note, much of the published data on sexual debut limits the definition of sexual intercourse to vaginal penetration, forgoing an important, yet commonly overlooked, practice that is oral sexual intercourse12. This study extends the scope of the literature by classifying sexual debut as age at either first vaginal or first oral intercourse. It is important to distinguish the role of gender differences in not only vaginal but also in oral sexual practices, because studies have reported that men are at a three-fold higher risk of developing HPV-associated head and neck cancers compared to females13. Oral HPV is a major etiological factor in the development of head and neck squamous cell cancers, particularly oropharyngeal cancer14, and linked to oral sexual practices14. Because males are more likely to initiate oral sex and they also have a higher burden of HPV-associated oropharyngeal cancer, it is important that males in the community be educated about their oral sex lifestyles and their higher risk of developing HPV-associated oropharyngeal cancer.Findings that demonstrated a significant difference in age of oral and vaginal sexual debut based on gender are consistent with previous studies that have shown that males, more than females, report earlier initiation of sexual activity15. Because individuals who initiate oral and vaginal sex at an early age, and/or have a greater number of oral sexual partners have higher risks of developing oropharyngeal cancer13, our findings have important implications for the community, especially among males. While cervical cancer continues to contribute to the overall cancer burden of the United States, the incidence and mortality rates have decreased tremendously in the last five decades14. Cervical cancer is also limited to females, whereas HPV-associated oropharyngeal cancers not only affect both genders, they, in fact, disproportionately affect males16. Cervical cancer prevention and control has also benefitted strongly from the availability of an early detection tool, the Papanicolaou (Pap) test14. Its introduction in the 1940’s allowed for earlier detection of cervical precancerous lesions and has greatly contributed to the dramatic decrease in both the incidence and mortality rates related to cervical cancer14. However, similar screening mechanisms for precancerous lesions of the oropharynx, as part of a periodic health examination, have not been established, although there have been calls for such a mechanism to be established17. For this reason, greater focus on oropharyngeal cancer prevention by means of increasing awareness about risky sexual activities is warranted as the next best prevention strategy.Oral sex behavior is well-established as a central driver of the increasing incidence of HPV-associated oropharyngeal cancer, which has now replaced cervical cancer as the leading HPV-associated cancer16. This lack of clear evidence of benefits of mass oral cancer screening places greater value on HPV vaccination as an important means of preventing HPV-associated oropharyngeal cancer. All three HPV vaccines  presently in use are highly efficacious in providing long-term immunity against various strains of HPV, including the oncogenic types 16 and 18 which represent 90–95% of HPV-associated oropharyngeal cancers18. Previous HPV vaccine trials have demonstrated nearly 100% response by oral HPV strains to existing HPV vaccines19. However, vaccination coverage among American youth remains low. Currently, the percentage of female adolescents receiving all three doses of HPV vaccine by age 13–15 years was documented at 37.1%, whereas only 27.1% of males in the same age category completed the full three-dose series20. Although the complete dose of HPV vaccine has since been updated from three doses to two if taken before 15 years of age21, the persistently low HPV vaccine uptake render the Healthy People 2020 objective of achieving 80% uptake highly unlikely22. Not only would increasing vaccination coverage of males protect against transmission of oral HPV infection and effectively reduce the incidence of HPV-associated oropharyngeal cancers, but it would also imply a secondary protective effect for individuals who have not been vaccinated, through a phenomenon known as herd immunity23. Therefore, the results of our study identify an objective need for male-targeted HPV vaccination campaigns, as well as guided sexual health programs that are more conscious of the role that gender plays in sexual debut at a younger age.Currently, the position of the United States Preventive Services Task Force (USPSTF) is that there is inadequate evidence to establish mortality benefits for mass oral cancer screening for asymptomatic individuals6. Thus, while the USPSTF position on mass oral cancer screening is clear, this lack of knowledge about risk factors for head and neck cancer, such as HPV, has been cited as a rationale for providing educational opportunities for lay members of the public through oral cancer screening.Besides males having lower HPV vaccination uptake, other studies have shown that males have poorer knowledge of the association between HPV and oropharyngeal cancer, despite the fact that males are indeed at significantly higher risk for developing head and neck cancer than females24. In the present study, our results found that Blacks, when compared to Whites, were more likely to have a higher number of vaginal sexual partners rather than oral sexual partners. These findings are consistent with previous studies which found that White youths tend to engage in oral sex more often than Black youths and that Black girls and boys tend to initiate oral sex at a later age than Whites, after having initiated vaginal sex at an earlier age25. There may be cultural or societal norms driving this racial pattern in initiating oral and vaginal sex shown in our study. A previous nationally representative study showed that Whites were more likely than Blacks to engage in oral sex and perceive it as nonsexual, less intimate, or serious26. Additionally, authors found that only 55% of Black males and 25% of Black females reported having oral sex as appealing, compared to 66% of White males and 55% of White females26. Because the increasing incidence of HPV-associated oropharyngeal cancer has been driven largely by White males6, this finding that Whites initiate oral sex earlier highlights the need to continue to increase knowledge about the association between oral sex, oral HPV, and HPV-associated oropharyngeal cancer.Differences in sexual practices vary by race and ethnicity and may shift over time as a reflection of societal influences on sexual behavior27. Results of this study parallel those of previous studies, having revealed that current smokers were more likely to engage in earlier sexual behavior28. These findings are perhaps suggestive that smoking behavior is a marker for other high-risk behaviors, including sexual activity. High-risk sexual behaviors, if established in adolescence, are likely to extend into adulthood and have serious implications for the risk of acquiring HPV infection. Given this established association between early sexual debut and substance abuse behaviors, interventions that address both risk domains are more likely to result in greater influence on positive behavior change than would focusing on either high-risk behavior.It is well known that other high-risk behaviors impact an individuals’ risk for acquiring HPV infection, including cigarette smokingFinally, this study adds value to the current literature through its innovative methodology. Survey of community members in a non-traditional setting proved useful for carrying out this research effectively. For some interventions, particularly those targeting high-risk populations, selection of an appropriate setting may seem less obvious. Therefore, identification of a potential setting that was both convenient and comfortable for our study participants was a strategic action employed in the planning phase of this investigation.30. Thus, integration of electronic data capture for this study purpose offered an attractive alternative within this population.Additionally, application of electronic data capture for survey distribution offered logic features not otherwise available with the use of paper surveys. These design capabilities permitted customization of the survey to each participant and prevented submission of incomplete responses. Moreover, use of this questionnaire platform was less time-consuming and less costly compared with the use of a paper-based questionnaire. Prior studies have reported that use of Web-based questionnaires maintains similar, if not greater, accuracy of reported information, as well as fewer response errors and socially desirable responses compared to paper-based surveys34. Second, the data was collected from a convenience sample within a narrow population; therefore, our results are susceptible to selection bias. The possibility of recall and social desirability bias also deserves mention, as this study asked participants to recall sexual risk behaviors from their late adolescence to young adulthood – nearly three decades prior. As such, applicability of this survey to the current population that is at sexual debut will be more beneficial from an interventional and cancer prevention standpoint. Additionally, data regarding anal sex and oral sex, stratified by sexual orientation and practice (receptive vs. insertive) was not collected; however, future studies should include this information for the purpose of designing effective interventions in this population. This type of data concerning minority sexual practices, which are highly stigmatized in this population, may best be collected in a qualitative setting using focus groups or structured interviews35. Importantly, specific sexual practices  may have different implications for enhancing the risk of HPV acquisition within the oral cavity.This study has several limitations that should be noted. First, the relatively small sample size of our study may have prevented the detection of other significant differences among the participants. However, our sample size is consistent with other community-based studies at race eventsFurthermore, most participants in our study were Black males; therefore, limiting the generalizability of our findings to the US population. However, because studies on demographic factors as predictors of risk of HPV infection are limited and predominantly focus on women, information from male participants, who are more susceptible to HPV-associated oropharyngeal cancer, is invaluable in identifying gender gaps to tailor methods to improve behavioral interventions. Though the generalizability of this study is limited by characteristics of the study sample, its conclusions prompt the need for future studies to include measures of HPV vaccination status, as well as knowledge and awareness of HPV and HPV-associated oropharyngeal cancer to better identify racial-gender gaps that may exist.Our study showed that race and gender are independent predictors of HPV risk factors, and being male, and White were found to be risk factors for initiating earlier oral sex, a sexual behavior closely associated with HPV infection and HPV-associated oropharyngeal cancer. Our findings provide impetus for the development of targeted educational interventions that are sensitive to gender and racial differences in sexual risk behaviors. The need for preventive programs to improve knowledge of males about sexual risk factors and their link to cancer is paramount in the community.36. The questionnaire was administered using research electronic data capture (REDCap) methods and software tools hosted at Saint Louis University35. REDCap is a secure, web-based application designed to support data capture for research studies providing (1) an intuitive interface for validated data entry, (2) audit trails for tracking data manipulation and export procedures, (3) automated export procedures for seamless data downloads to common statistical packages, and (4) procedures for importing data from external sources35. A total of 15 trained volunteer survey administrators approached all participants around the racing area, including spectators in the bleachers, vendors at concession stands, and drag racers in the pit area. Potential enrollees were informed of the anonymous nature of the survey and the objective of the study, and oral informed consent was obtained from all participants. The data collected represents a convenience sample of 301 participants, 18 years or older. In exchange for participation, subjects were offered the opportunity to be entered anonymously into a raffle drawing of 15 various prizes, valued at no more than $20 each.The study was approved by the Saint Louis University Institutional Review Board. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This was a cross-sectional study conducted at the 2015 United Black Drag Racers Association event on September 12 and 13, 2015, in Madison, Illinois. Established in 1994, the UBDRA has gained national recognition as one of the premier Black organizations in the world of motorsports. Study participants consisted of UBDRA drag racers, vendors, and spectators from across the Midwest region who attended the drag race event. Participants responded to a 78-item questionnaire survey that was based on previously validated surveysQuestions elicited sociodemographic factors including age, gender, race, marital status, education, income, drinking status, cigarette smoking status, as well as sexual factors including number of oral and vaginal sexual partners, age of first vaginal sex, and age at first oral sex. The independent variables assessed in this study included gender and race and were categorized as follows: race , and gender .1) At what age did you FIRST have vaginal sexual intercourse? (Please list age in years) 2) How many vaginal sexual partners have you had in your lifetime? 3) At what age did you FIRST have oral sexual intercourse? (Please list age in years) 4) How many oral sexual partners have you had in your lifetime? Number of sexual partners was categorized as follows: number of oral and vaginal sexual partners was classified as high vs. low with five or more representing a ‘high’ number, according to measures described by the National Health Statistics Report.The outcome variables assessed in this study included number of oral and vaginal sexual partners, age of first vaginal sex, and age of first oral sex. To collect this data, the following questions were asked: Covariates adjusted for included: age; marital status ; education level ; income level ; drinking status ; and cigarette smoking status [excluding e-cigarettes] .2 tests for categorical variables and independent sample t tests for continuous variables. Multivariable logistic regression was used to examine the association between race and gender, and number of oral and vaginal sexual partners. Multivariable linear regression analysis assessed the association between race and gender, and age of first vaginal sex, and age of first oral sex. Analyses were 2-tailed, and statistical significance was set at p < 0.05.Analyses were performed using statistical software . Descriptive statistics (frequency and percentage) were used to analyze characteristics of the respondents. We compared sociodemographic characteristics and sexual behavioral factors by using χ


LncRNA SRA1 plays important roles in several types of human diseases. The present study aimed to explore the role of SRA1 in cervical squamous cell carcinoma (CSCC). In the present study, we showed that plasma SRA1 was down-regulated in human papillomavirus (HPV)-negative CSCC patients but not in HPV-positive CSCC patients compared with healthy females. Down-regulated SRA1 distinguished HPV-negative CSCC patients from HPV-positive CSCC patients and healthy females. In HPV-negative CSCC patients, miR-9 was up-regulated and inversely correlated with SRA1. In HPV-negative CSCC cells, SRA1 overexpression caused the down-regulated miR-9, while miR-9 overexpression failed to affect SRA1. Moreover, SRA1 overexpression caused decreased, while miR-9 overexpression caused increased proliferation, migration and invasion rates of cancer cells. In addition, miR-9 overexpression attenuated the effects of SRA1 overexpression. Therefore, SRA1 is down-regulated in HPV-negative CSCC and regulates cancer cell behaviors possibly by down-regulating miR-9. In both developing and developed countries, cervical cancer is a common female malignancy and causes unacceptable high mortality rate . Due to Pathogenesis of HPV-negative cervical cancer has not been well studied. However, previous studies have revealed the different gene expression patterns of HPV-negative and HPV-positive subtypes of this disease ,8, indicOur study selected 72 CSCC patients  from the 133 CSCC patients who were admitted to the Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute between December 2015 and December 2018. Inclusion criteria: (i) new CSCC cases diagnosed by histopathological examinations and (ii) patients willing to join the study. Exclusion criteria: (i) recurrent CSCC; (ii) any therapies performed within 3 months before admission; (iii) family history of malignancies and (iv) previous history of malignancies. Sensitive PCR was performed to detect the HPV infections. The results revealed 16 HPV-11 positive cases, 20 HPV-16 positive cases, 14 HPV-18 positive cases and 22 HPV-negative cases. During the same time period, 72 healthy females  were also selected at the Physical Health Center of Cancer Hospital of China Medical University, Liaoning Cancer Hospital and Institute to serve as controls. These two groups of participants showed similar age distributions. Ethics Committee of the aforementioned hospital approved the present study.g for 10 min to collect plasma.Fasting blood (5 ml) was extracted from each CSCC patient and healthy control on the day of admission before the initiation of any therapies. Blood was centrifuged in EDTA-treated tubes and was centrifuged at 1200×2.HPV-negative human CSCC cell line C-33 A. Eagle’s Minimum Essential Medium containing 10% fetal bovine serum (FBS) was used as cell culture medium and cell culture conditions were 37°C and 5% COTRIzol reagent  was mixed with plasma and C-33 A cells to extract total RNAs. Following DNase I digestion, RNA samples were subjected to reverse transcription using AMV Reverse Transcriptase  to synthesize cDNA. SYBR Green for qPCR  was used to prepare PCR mixtures with 18S rRNA as endogenous control to detect the expression of SRA1.MiRNAs were extracted from plasma and C-33 A cells using microRNA Purification Kit . qScript microRNA cDNA Synthesis Kit  was used to perform miRNA reverse transcriptions and miScript SYBR Green PCR Kit  was used to prepare PCR with U6 as endogenous control to detect the expression of miR-9.Ct values were processed using 2C−ΔΔT method.All PCR were repeated three times and all 5 cells. Cells with no transfections were also included to serve as control group (C). Following studies were performed using cells harvested at 24 h after transfection.SRA1 expression vector was constructed using pcDNA3.1 vector . Negative control (NC) miRNA and miR-9 mimic were bought from Sigma–Aldrich (U.S.A.). C-33 A cells were collected when confluence reached 70–80%. Lipofectamine 2000 reagent  was used to transfect 10 nM SRA1 expression vector, 10 nM empty pcDNA3.1 vector (NC), 30 nM miR-9 mimic or 30 nM NC miRNA (NC) into 104 cells were mixed with 1 ml Eagle’s Minimum Essential Medium containing  to prepare single cell suspensions. A 96-well cell culture plate was used to cultivate the cells with 0.1 ml per well. Cells were cultivated under the conditions of 37°C and 5% CO2. CCK-8 solution (10 μl) was added 22, 46, 70 and 94 h later, and cells were harvested 24, 48, 72 and 96 h later. After that, DMSO (10 μl) was added into each well and OD values were measured at 450 nm to reflect cell proliferation.C-33 A cells were harvested at 24 h after transfection, and 4 × 104 cells were mixed with 1 ml non-serum Eagle’s Minimum Essential Medium to prepare single cell suspensions. The upper Transwell chamber was filled with 0.1 ml cell suspension and the lower chamber was added with Eagle’s Minimum Essential Medium containing 20% FBS. Matrigel  was used to coat the upper chamber membrane before invasion assay to mimic in vivo invasion conditions. Transwell chambers were cultivated under conditions of 37°C and 5% CO2 for 4 h and staining with 0.5% Crystal Violet  was performed for 20 min at 22°C. Stained cells were observed and counted under an optical microscope.C-33 A cells were harvested at 24 h after transfections, and 4 × 10P<0.05 was statistically significant.Data presented in the present paper were mean values of three biological replicates. Differences among different groups of participants or among different cell transfection groups were analyzed using one-way ANOVA and Tukey’s test. Linear regression was used for correlation analysis. Diagnostic analyses were performed using ROC curve analysis. n=72) was detected by performing RT-qPCR. Plasma levels of SRA1 among those groups of participants were compared by performing one-way ANOVA and Tukey’s test. Compared with Control group, significantly lower plasma levels of SRA1 were found in the 22 HPV-negative patients but not in other groups of patients (P<0.05).The 72 CSCC patients included 16 HPV-11 positive cases, 20 HPV-16 positive cases, 14 HPV-18 positive cases and 22 HPV-negative cases. SRA1 in plasma of all CSCC patients and healthy controls  was 0.86, with standard error of 0.050 and 95% confidence interval of 0.76–0.96 A. With HP<0.05). Moreover, SRA1 overexpression caused the down-regulated miR-9, while miR-9 overexpression failed to affect SRA1 expression (P<0.05).MiR-9 in plasma of HPV-negative patients was detected by performing RT-qPCR. Correlation between miR-9 and SRA1 was analyzed by performing linear regression. It was observed that miR-9 and SRA1 were inversely and significantly correlated in HPV-negative CSCC patients A. To furpression C, P<0.05P<0.05). In addition, miR-9 overexpression attenuated the effects of SRA1 overexpression.Cell proliferation, migration and invasion abilities of C-33 A were tested 24 h after transfections. Comparing with C and NC groups, SRA1 overexpression caused decreased, while miR-9 overexpression caused increased proliferation A, migratP<0.05).The expression levels of E6 A, E7 FiB, NF-κb In the present study, we investigated the involvement of SRA1 in two types of CSCC. We found that SRA1 was only down-regulated in HPV-negative CSCC and overexpression of SRA1 caused the inhibited cancer cell behaviors. We also showed that the actions of SRA1 in HPV-negative CSCC are likely mediated by miR-9, which is up-regulated in CSCC .Previous studies have identified a considerable number of differentially expressed lncRNAs in CSCC . LncRNA Interestingly, our study showed that SRA1 down-regulated miR-9 to suppress HPV-negative CSCC cell proliferation, migration and invasion. It has been reported that miR-9 was up-regulated in both HPV-negative and -positive CSCC . TherefoIn addition, SRA1 was down-regulated specifically in HPV-negative CSCC. SRA1 may suppress HPV-negative CSCC behaviors by down-regulating miR-9.E6 and E7 are critical players in HPV-associated cervical carcinogenesis . ConsistThe analyzed datasets generated during the study are available from the corresponding author on reasonable request.


This preliminary study aimed to examine the feasibility of sentinel lymph node biopsy (SLNB) using contrast-enhanced ultrasound (CEUS) vs. indocyanine green fluorescence (ICG), combined with blue dye in patients with breast cancer.This was a retrospective study of consecutive female patients with invasive stage I-III  primary breast cancer at the Peking Union Medical College Hospital between 01/2013 and 01/2015 who underwent preoperative SLNB by ICG + blue dye or CEUS + blue dye. The numbers of detected SLNs, detection rates, and recurrence-free survival (RFS) rates were compared between the two groups.P = 0.814). The average numbers of SLNs detected per patient showed no significant difference between the two groups . After a median follow-up of 46 months, five patients in the CEUS + blue dye group and 15 in the ICG + blue dye group had recurrence. RFS rates showed no significant difference (P = 0.55).A total of 443 patients were included. The detection rates of SLNs in the CEUS + blue dye and ICG + blue dye groups were 98.4 and 98.1%, respectively (This preliminary study suggests that CEUS + blue dye and ICG + blue dye are both feasible for SLN detection in breast cancer. Breast cancer is currently the most common malignancy in Chinese women , 2. ReceThe current standard SLNB method involves the injection of a technetium-labeled nanocolloid and blue dye interstitially into the breast, either around the tumor via the periareolar procedure . Use of In China, using blue dye alone is common in SLNB. Although SLNs are dyed, blue dye cannot indicate their localization prior to skin incision. As a result, the identification rate is not as high as that of the dual method (radiotracer and blue dye) , 8. TherTherefore, new techniques are being developed for SLNB. Among them, indocyanine green fluorescence (ICG) and contrast-enhanced ultrasound (CEUS) have some advantages , 6, 9–21Therefore, the aim of the present preliminary study was to examine the effectiveness of SLN identification using CEUS vs. ICG, in combination with blue dye. In addition, we attempted to compare breast cancer recurrence rates between both techniques. The present results provide a proof-of-concept for designing prospective trials.This study was approved by the independent ethical committee/institutional review board of Peking Union Medical College Hospital (PUMCH). We obtained permission from PUMCH to collect data from the Breast Surgery Department Database. As this was a retrospective study of anonymized data without any contact with the patients, individual consent was not required. The study was performed in accordance with the relevant guidelines and regulations.A retrospective review of the Breast Surgery Department database of PUMCH was performed. Consecutive female patients aged ≥18 years, with invasive primary breast cancer , who underwent preoperative SLNB using ICG or CEUS combined with blue dye between January 2013 and January 2015 were included for analysis. Preoperatively, these patients had no clinical  or radiological signs of lymph node invasion. Patients who received neoadjuvant systemic therapy (including chemotherapy and endocrine therapy) were excluded, as well as those with bilateral breast cancer or a history of axillary surgery. All eligible patients in the database had complete medical information. No patient was lost to follow-up. Follow-up was censored on January 19, 2018.The choice of the SLNB procedure was based on the surgeon’s experience and preference at the time of surgery. All SLNB procedures were performed by the same team of senior and skilled breast surgeons. Undiluted methylene blue  was used for both SLNB procedures.For ICG + blue dye, ICG  was first dissolved in 5.0 ml sterile water (5.0 mg/ml stock solution). Then, 1.25 ml of the stock solution was diluted in 5.0 ml sterile water for injection (1.0 mg/ml). Before surgery, 0.2 ml of methylene blue (10 mg/ml) and 0.2 ml of ICG (1.0 mg/ml) were injected intradermally into the periareolar region. The breast was gently massaged for 5 min. Next, the lights were turned off and a photodynamic eye (PDE) camera  was used to trace the lymphatic flow. The location of the skin incision for the SLNB was selected as the point where the fluorescent signal disappeared  with 18L6HD and 9 L4 high-frequency linear array probes, using contrast pulse sequences (CPS). Low mechanical index (MI) values were used (MI = 0.06) to reduce cavitation and microbubble destruction. Sonovue  was used as the contrast agent. The Sonovue powder was mixed with 2.0 ml of sterile saline. The ultrasound contrast agent (0.4 ml) was injected intradermally into the periareolar area, and the injection area was gently massaged for 10-30 s. Subcutaneous lymphatic channels could be visualized immediately on CPS. Enhanced lymph nodes were detected by moving the probe along the channels .The detection rate of SLNs was defined as the number of patients with SLNs identified by the labeling technique divided by the total number of patients administered the technique. Categorical data were compared by the two-tailed chi-square test. Quantitative data were compared by Student’s t-test. Recurrence-free survival (RFS) was estimated by the Kaplan-Meier method. A two-sided log-rank test for time-to-event endpoint was used. Differences were considered statistically significant at P > 0.05). There were also no significant differences between the two groups in tumor size, tumor grade, stage, lymphovascular invasion, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) . No adverse reactions or complications related to the ICG procedure, microbubbles, or blue dye injection were recorded.Between January 2013 and January 2015, a total of 443 patients were operated and included in this study. The ICG + blue dye technique was used in 316 (71.3%) individuals, and CEUS + blue dye in 127 (28.7%) patients. Table P = 0.814). The numbers of SLNs identified showed no significant difference between the two groups . There were no significant differences in the positive SLN rate between the two groups. Precisely, there were 13 (10.2%) patients  with positive SLNs in the CEUS + blue dye group, and 36 (11.4%)  in the ICG + blue dye group (P = 0.726) . Of the 316 patients administered ICG + blue dye, SLN detection was successful in 310 (98.1%). The SLN detection rate showed no significant difference (P = 0.105)  in the CEUS + blue dye group, and 5  in the ICG + blue dye group.P = 0.55)  months. Among the 443 patients, 20 (4.5%) had tumor recurrence. Five (3.9%) individuals in the CEUS + blue dye group had recurrence, including 1, 2 and 2 with axillary recurrence, ipsilateral breast/chest wall recurrence and bone metastasis, respectively. A total of 15 (4.7%) patients in the ICG + blue dye group showed recurrence, including 3, 4, 4 and 4 with axillary recurrence, ipsilateral breast/chest wall recurrence, bone metastasis and lung metastasis, respectively. The 3-year RFS was 95.6% in the CEUS + blue dye group versus 94.3% in the ICG + blue dye group  had 0/3 and 0/4 positive SLN, respectively; the third patient, a 38-year old woman, had 1/4 positive SLN for a macrometastasis and underwent ALND, with 0/18 positive lymph node.ICG, a novel technique for SLNB, is increasingly used in clinical practice. The SLNB detection rate with ICG alone ranges from 93.1 to 100%, for 1.5-5.4 sentinel lymph nodes sampled per patient , 20, 21.CEUS is another new technique for SLNB and has been validated in a pig melanoma model , 26. SubIn the present preliminary study, the detection rates of SLNs for the two techniques were high and comparable: 98.4% for CEUS + blue dye, and 98.1% for ICG + blue dye. These rates are similar to that (96%) reported in the literature , 28. TheWithout performing ALND in all patients, the real false-negative rate could not be determined, but the rate of ipsilateral axillary recurrence could be used as an imperfect adjunct. In the present study, the recurrence rates in the ipsilateral axilla were low [1/127 (0.8%) and 3/316 (1.0%)], suggesting that false-negative rates for both approaches were most likely low. The reported false-negative rate for SLNB is 5-13%, depending upon the number of SLNs sampled, the SLNB method applied, and the cancer type , 30. In In addition to axillary recurrence cases, six (1.4%) patients had ipsilateral breast/chest wall recurrence and 10 (2.3%) developed distant metastasis during the 46-month follow-up. These rates were similar to those reported previously –35. HoweThis study had limitations. ALND was not performed in all patients, and the false-negative rates of the two novel techniques could not be evaluated. Even though there were no significant differences in baseline patient and tumor characteristics between the two groups, a retrospective analysis inevitably has some biases, e.g. we were limited to the data available in medical charts. Furthermore, the surgeons were free to select the preferred method for different patients, and the exact reasons for method selection were usually not indicated in patient charts. Finally, patients assessed by the radiotracer + blue dye technique could not be included because our center does not use radiotracers.Overall, the present preliminary study suggested that CEUS + blue dye and ICG + blue dye are both feasible techniques for SLNB in breast cancer. Randomized controlled trials including the radiotracer + blue dye gold standard technique are required to confirm the feasibility, efficacy, and safety of these two novel techniques before their introduction into mainstream clinical practice.