Persistent infections by high-risk HPVs (human papillomavirus) are associated with the development of cervical and vulvar cancer in women, penile cancer in men, and anal and oropharyngeal tumors in both gender. Among this group, HPV-16 is worldwide the most prevalent type in cervical carcinomas. In neoplasias of other anogenital sites and the oropharynx, HPV-16 is detected in almost all the tumors attributable to HPV. We and others have shown that intra-typical nucleotide variability of HPV-16 impacts not only upon the risk of infection persistence and disease development in both men and women, but also upon the biological functions of viral E6 and E7 oncoproteins. These issues will be discussed in the seminar.
I’m very honored to be here and to give this talk and shared some of our our our research um in in the lab which is to analyze the epidemiological and the functional impact of HPV genetic heterogenity which is a a research area for which I have been studying since my
Master degree but uh we I’ll show you some data that we had by that time and also more recent data to complement this so as Georgia told you I am located at the Cancer Institute in the city of s Paulo our will building is here in the
Map and we are part of the hospital of clinics from the University of s Paulo the univers the faculty of medicine of the university in s Paulo it’s here and we are the part of the hospital of clinics that is totally dedicated to cancer to cancer so this is a hospital
We have we are in this very beautiful building this is a 23 floor uh building it’s a hospital and we are over 4,000 employees in this Hospital in the eighth floor it’s located the center for translational research in oncology and this is where all groups uh that dedicated to basic science are so
This is our building during uh the campaign for the prevention of of of breast cancer it’s very beautiful so since uh uh since long since I started my career I have been interested in in the the in the study of HPV HPV stands for human papiloma virus and this virus
Is associated with almost all cervical and anal Canal cancers so they respond for over 90% of cancers at this an atomical sites and for a less extent of cancers at other an anogenital regions like the fva the penis and the vagina and is also Associated to the development of aaral cancer and these
Ranges uh this is varies extensively in the globe where uh autopal cancers Associated to HPV infection are more prevalent in the United States and in Europe in comparison to other parts of the world so the burden of HPV Associated cancers yearly is about 700,000 cases per year and the majority
Of these cases occurs in the undeveloped world and developing countries where 80% of these cases are observers so this is a very important uh health health issue and for which now we have a vaccine a prophylactic vaccine and so we are able to eliminate these diseases
So um HPV 16 and other 12 HPV types are are considered carcinogenics to human are considered carcinogens type ones uh in the association with cervical cancer and there’s a sufficient evidence epidemiological and biological evidence to consider these viral types uh Associated to the development of cervical cancer however
Hpv16 is the is respon for the majority of a of HPV Associated cancers at other extra cervical cancer sites uh including the oral fings so this is hpv16 is very prevalent not only in the normal population I mean normal uh with normal smears but also in uh disease sbls so
Hpv16 uh is a small virus it here I show the particle of the viral it has about uh 50 00 nanometers and this is an unenveloped virus that is composed by a a protein a proteic capsid that is composed by L1 L L2 proteins that are uh
Coated by the virus and it Globes a double stranded circular uh molecule of about 8,000 pair base The genome is divided in three regions it’s the long control region which does not code for any protein but is very important because it contains uh binding sides for viral and cellular transcription factors
That regulate replication and transcription an early region and a late region and the designations of early and late corresponds uh to the phase of the viral cycle in which these proteins are expressed E6 and E7 proteins are considered oncogenic proteins because they play a critical role in driving
Cell cycle entry for V to to to to they they need the cells need to be in cycling in the cell cycle so there is a genome amplification but if there is a disregulation of the expression of V6 and E7 this may lead to Cellular transformation so this is why this these
Proteins are cons viral onco proteines so in this slide I show The Natural History of HPV infections and this is a uh the history at the cervix but it may be translated to the anal canal and also to the oral fings and so uh women are exposed to an HPV infection
And the highest prevalence of HPV infection in women is observed in young women that uh coincident with the initiation of the sexual Debo of this women so risk factors are associated to sexual activities like having multiple sex partners early sexual debut Hy parity and Nos and concision most of the infections they
Clear within one or two years but however some infections May persist for a longer time and may lead to the development of of of cervical lesions then low grade SOS lesions high grade SOS lesions and finally SE cervical cancer and this may take over 20 years to
Happen so as as most associated uh cancers of cancers Associated to viral infections it takes a lot of time to develop the cancer and this is associated to viral factors like V HPV variability which I’m going to go through in more details but also to host factors and the micro
Environment so to date over 200 HPV types have been fully characterized and sequencing so uh these are considered genotypes because the classification of HPV types relies on HPV sequence variability and here it’s shown a phenetic tree which was constructed based in the sequence of the L1 L Gene
You remember as I told you I one codes for the for the major protein of the capsid and this is a very conserved Gene so we can observe that HPV clusters within five different geneus that is Alpha mu new GMA and beta papiloma virus and here shown in red are those HPV
Types that are classified by the ark as carcinogenic to humans so we see that they clustered together these smaller grouments are considered a species and each of these are HPV types so the classification in family genus species and types relies on the sequence of the L1 Gene where HPV types are
Classified whenever they have more than 10% Divergence in the sequence of the L1 Gene with all other HPV types known and characteriz it however since early 90s it has been observed that hpvs uh within there is an HPV in type variability that is if you analyze different isolates
From different samples you can see that there is small variations in the Genome of these isolates so hpvs Tripes they also cluster within lineages and sublineages but this classification this taxonomic classification is based on the analysis of the whole genome where HPV lineages are uh defined by 1 to 2% variability
Within the whole genome and they are named after letters so HPV 16a is a lineage of HPV 16 and sub lineages are defined by 0.1.5 to 1% Divergence within the whole genome and these are named after numbers this is the modern nomenclature for for hpv16 variants and in some
Slides I I will have to show you the old nomenclature however I will always make the correlation for make the to make the the presentation more clear so this was a very Pioneer studies that was conducted in the early 90s you imagine that by that time there was no automatic
Sequencing every every sequencing was made by hands and so in this study uh performed in the group of of Olie Bernards with at the time was at the University of Singapore they gathered together over 300 isolates from all over the world from more than 25 different geographic region
Uh countries and they analyzed a small fragment of the LCR and they use this fragment to construct a philogenetic tree so here is the phenetic tree of hpv16 variant and we can observe that this cluster within different uh uh branches and these branches they were named after the the uh the origin of
Most of the isolates with compos these branches so we can see here two African branches an Asian-American branch and two European uh uh branches and also an an Asian sublineage of the European Branch so uh we see here in this Slide the distribution of hpv16 barant within this cervical uh within uh cervical
Carcinomas and we see that in the African continent there is a high prevalence of African variants which are now named C but I will show you always when I talk about the associations I will show you the the the the correlation with the modern nomenclature
I just want to make a point that the European variant which is now considered an Al lineage variant was the first Proto was the first hpv16 isolates to be fully sequenc it and it’s not considered uh uh A Primitive uh isolate but it’s considered the Prototype because it was the first
Hpv6 genome to be fully sequenced and it was obtained from a German patient so in Europe we can see a high prevalence of European variants and here are shown in green and in Orange and I will not go within details of this classification and European varant are detected with
High frequency also in other parts parts of the world and uh in the Asia there is a high prevalence of Asian variants which are now categorized as as A4 and in the you and in the uh Americas you can see that uh especially in s in Central and South America we we detect
Varant from all branches of geographical and phenetic Rel relation and this is this uh transduces into the history of the colonization of these countries so this is the correspondence of hpv16 virant classification this is the modern nomenclature and this is the old nomenclature so as I told you the
European variant uh which was the first sequence to be fully sequenced it clusters within the H1 sublineage Asian variant is an A4 sub lineage African variants now are defined by B and C lineages and asan American variant as D lineage so uh HPV V inra type variability has been used successfully
To study the philogyny and evolution of this virus but also it has been used as an important tool to better understand the natural history of hpv6 infections because it was s that uh maybe some persistent infections by hpv16 were not uh were not were not classified as persistent but this could be different
Transient infections by different variants and we also sought to analyze if these genomes uh isolate AER are associated to differences in the risk of of of leading to to clinical outcomes to disease and Cancers and Etc so by the time I was doing my PhD uh this study was being conducted uh
With the coordin of my supervisor at the moment which was Louisa Villa and this study was conducted uh with also uh the coordination of Dr Eduardo Franco from the M University in Canada so the ludic magil cohort study was a prospective study which was conducted in women from
Brazil and these women were enrolled in s Paulo it was a very important study and in this study over 2,500 women were followed for four years and then there was an extension of this study and uh these women they came for a visit or follow up every four months in
The first year of followup and every six months in the subsequent four years so each visit a sample uh blood samples wrong in addition to to cervical swaps which were used not only for pop analysis to analyze the cytology to see if there was cervical alterations
Because because these women had to be at the at this Baseline visits these women had to be pychological normally and also these PS mes were used to to extract um DNA to analyze different markers viral and host markers and for viral markers this included HPV typing assessment of viral load and
Variant analysis for some of the HPV types these women were also submitted to a CER where we were able to collect information social demographic smoking and and other characteristics Associated to reproductive health and sexual behavior so all hpv16 positive samples from this study uh were separated for variant analysis and they were sequenced
A fragment also of the LCR to categorize these variants and so there were eight 187 women who had at least one positive smear uh that was positive for hpv1 16 we here there is uh I show the prevalence of each of each lineage variant and we see that most of these
Variants cluster within the A1 A2 uh lineage which were prevalently named as European we also see that uh there was about 25% of these women which had a a an infection by other lineage variants including D1 D2 lineage the African variants and Asian variants so this correlates with the
High mixture level of our population so uh we also performed analyses of the hpv16 variants and the risk for cervical lesions because some of these women develop low grade and highgrade lesions during this study they entered their study with normal mirors but during the time of they study they
Develop a cervical lesions so we did this analysis at different time points including two years of followup five years of followup and the entire followup and taking as a reference HPV negative women which considered the reference we see that women infected with high risk types and also with HPV
16 they were at a higher risk to develop cervical lesions in comparison to HPV negative we when we when we stratifi the hpv16 infection by the different variants we see that non lineage variant that is non-european variants were associated to an even higher risk of developing cervical
Lesions so we showed for the first time we were Pioneer and showing prospectively the association between hpv16 variants and the development of cervical disease in then following this study in other studies conducted in other AD mixture populations in North America Costa Rica and Mexico we also uh observed that European non-european
Varant that mean non a lineage virus were associated to a higher risk of Le of of cervical disease and they were also more prevalent in cervical cancer in comparison to A- lineage variants so most recently uh this is study conducted by Lisa marello at the NCI and the National
Cancer Institute in the United States this study uh was uh hpv16 varant classification was built on whole genome sequencing and they analyzed uh over 3,000 women and similar to our studies a similar observations were B were were obtained even when analyzing the whole genome so here I showed the AL ratios
For different disease outcomes that is highr cervical lesion esal cell carcinoma adenocarcinoma Ino and adenocarcinoma and these are the different lineages so in comparison to the A1 lineage which is prev denominated the European they also observed that Asian uh variants and asan American varant are associated to a higher risk
Of all of this lesions so very similar to what we observed before but this was uh based on a on H genome sequencing so regarding women we can conclude that globally hpv16 varant distribution is uneven and varies with the ad mixture level of the population and we also show together with other
Studies that A4 and D2 D3 variants are at a higher risk of cervical lesion development in comparison to women infected with the A1 and A2 virant preval is defined as European and also with increased risk of adenocarcinoma so what about men so uh uh the study of HPV natural history in
Man was uh was conducted uh um more recently in comparison to studies in women and so we recently performed a a a static review followed by meta analysis in which we observed that there were only very few studies uh regarding the hpv16 variability in man and furthermore
These studies were only uh restricted to very few samples so we decided to analyze in more details the HPV in type uh variability impact upon the natural history of HPV infection in man that is the association to persistence infection which is uh necessary for the development of genital disease and also
To association with disease outcome in man so we took advantage that the hymn study was also uh was also being conducted in s Pao this was a study the himm study stands for HPV in man this was one of the most important studies conducted worldwide to analyze the
Natural history of hpv16 infection in men this was a prospective multicenter international study and women were enrolled in three different countries this study the pi of this study is Dr Anna Juliano which is based at the a morphit Cancer Center and Research Institute in uh in Tampa Florida so this
Is a cohort uh which uh were enrolled over 4,000 men uh 105,000 men at each geographical region and these mens were enrolled uh between 2005 and 20 9 they were followed for uh for up to four years and every and these visits of followup were conducted at every six
Months and also this men completed a a self- minister co uh CER so we could see the sexual and soci damic risk factors that could be Associated to the different uh outcomes of infection and of disease so we collected exfoliated cells from the genital region and also
For the ano canal and also in this study a gargo sample from the Oro cavity was also collected and so HPV was assessed by a protocol of PCR followed by reverse line ization and all hpv6 variant uh positives mirrors were separated to analyze the variant so initially so what we did in
This study is that HPV positive samples were uh DNA was extracted and they were submitted to a PCR to analyze a fragment of the LCR which contains sufficient uh polymorphisms that we are able to Define all classified variants so I’ll first show you uh some analysis which we conducted regarding the ano
Canal and uh so this analysis was based in 227 samples which were collected for 124 men and so we can observe uh that regarding the population most of the men which were hpv16 positive were young men with a me age of 30 years most of these men
Were single they reported over 12 12 years of of Education reported being non-smokers and only regarding uh the race there were some differences in the in between countries because uh most men’s uh from Mexico they classified themselves a Mexican and Hispanic we also see some difference regarding the
The sexual orientation whereas most men from the US and Mexico reported being men who have sex with women and men from the Brazil reported being men who have sex with men or men who has sex with both men and women this is not the the full population but uh we have to
Remember remember that the this population are only those men who were uh who from which we detected an HPV 16 atano Canal at one of the visits and uh the mean number of of sexual partners of female sexual partners were lower in Mexico also in comparison to the US and
Brazil and so regarding the lineage prevalence here we have the lineages a BC aan with exception of A4 that was prev classified Asian BC which were prev characterized as African variant and Asian-American variants and we can see that regarding the distribution of lineages in the US Brazil and Mexico
Most of the infections were of a lineage variants however we detected some non- aage varant both in the US and in Brazil but not in Mexico Meo and we detected the lineage variants in all of the countries so regarding the persistence Theo Canal I will show you some
Conclusions because these are very big tables and I don’t want to go through this in this presentation so this was the first study to prospectively evaluate the association between hpv16 virant and and viral persistence in the Mayo all and qual and uh we found not the significant difference regarding the distribution of
Hpv6 varant among cases of persistent and transient infections even when men were stratified by countri S reported ethnicity or age but concerning the sexual orientation we observed a significant higher prevalence of non- Environ lineage among men who had sex with men with a transient hpv16 infection in the
Ano Canal we don’t have we do not have a plausible explication for this and uh but however we think this warrant is further studies now we think that we were not able to to address to find any association between variance and Persistence of infection which are necessary for the development of lesions
Because uh our cohort uh although bigger than the other studies it’s not big enough to find significant associations so we believe larger studies are necessary to address these issues we also analyzed hpv16 variability among these mens in the genital region and but this was a a much
Higher sample size because uh uh more man had at least one hpv16 positives near at the genital in any of these visits so this analysis uh enclosed uh 852 men so 150 uh uh so 222 men from the US 3 40 56 men from Brazil and two 214 men from Mexico
And these men contributed with at least one genital ismere sample H but overall uh we analyzed almost 2,000 2,000 samples uh that is because uh although the most men had had only one visit positive for hpv16 which categorized Str and infections some of the men contributed to more than two genital Ms
Because they had a persistent infection with hpv16 and they were positive in more than one visit so uh similar to what uh we observed for the ano can now most of these men were young were never smokers were men who have sex with women which which reported over to the more than 12
Years of education and having a steady sexual partner uh Mo in the same as the anoc canal most of the men detected with at least one HPV 16 positive dianal sample were men who have sex with men or men who have sex with men and women in
The Brazilian Cort in comparison to the United States and Mexico so in this very nice slide I show you the distribution and the prevalence of hpv16 variants by country so this is the overall prevalence the prevalence in Mexico in the United States and in Brazil we can observe that in all
Countries a lineage variants were the most prevalent ones uh with uh being detected in 92% of the M uh from Mexican men we also observed that no n lineage varant were more prevalent in the in Brazil and then in United States and in Mexico these were less
Disected but we see a very uh uh very interesting thing that uh regarding BC varant which were prev defined as as African uh where in Brazil most of these variants were from the sea lineage in braz in the United States uh the majority were from the B
Lineage so we also sought to analyze if there was a difference in the distribution of this of this variants among men who have persistent and transient infections and so in this table I show you the association of hpv16 variants and the Persistence of infection at the genital region so here
Are lineage a and Nona variants we have to group this this Varian like this because uh because uh we do not have too much uh persistent infections but we can observe here in this slide that infection with non a lineage virence was associated with a higher relative risk
Of having a persistent infection so this if you remember when we analyzed uh women aine non Al lineage variants were associated with a higher risk and in men we see some difference because Al lineage variants are associated with a higher risk we also analyzed the risk uh regarding long-term persistent
Infections and in this analysis men with the a persistent infection of less than 12 months were categorized together with transient infections and we can observe that a lineage variants were also associated with a 2.69 increased risk for having a persistent infection in this study some men develop
Uh some genital warts and pen intian neoplasia and pen in neoplasia we know that this is associated with HPV in scen infection and these seven men who had developed a pen inal lesion two from the United States two from Brazil and three from Mexico here we describe the HPV
Type detected in biopsy of the samples of the of these lesions and also at the Swap of the suace of the swab and we see that hpv16 is uh is the cause of the development of these lesions and in all these cases we detected uh uh A1 sublineage variant and here uh where
It’s uh showned in bod it’s the moment that this lesion was collected and we see that all of these lesions they develop uh in in the context of long-term persistent hpv16 infection so in conclusion in man we observing that hpv16 lineage a variants are the most prevalent independent of the
Country we also see an uneven geographical distribution of hpv16 varant in men and in the ano Canal we were unable to detect a significant Association of hpv16 variants and Persistence of infection however genital samples we show for the first time that among men genital hpv1 16 Linea J
Variant confer a higher risk for the develop for low grade for long-term infections for persistent infections and that all hpv16 palian neoplas had a sublineage A1 variant with were cured in the cost of long-term infections so we see there are some difference uh Associated to gender
In the in the natural history of hpv16 variants and uh we we want we think this should be analyze it in more details but because although the HPV carcinogenesis is also all is often assumed to be similar between men and women and between different mucosal and no mucosal
Sites it should be noted that the the keratinous keratinize epithelia of the penis may be different from that of the mucosal EPO of the cervix and indeed this study showed that there was difference in the global splash of hpv16 productive infections in at the cervix at the foreskin and the tonsillar
Epithelial so should this should be further studied I also want to finalize my presentation I will talk uh the next five minutes about functional studies to which we have contributed in which we show there are also difference uh in the different molecular variants of hpv16 so as I told you earlier in this
Presentation hpv16 E6 and E7 they do not have an enzimatic activity but however they interact with different cellular proteins and the association of this of both C6 and E7 with so with several cellular proteins they impact upon uh the different hm marks of cancer so we T to analyze the difference
Of the lineage a and lineage G two variants I will not go in details of in these variants and uh we see that the E6 and E7 genes are very conserved all the difference between A1 a lineage variants and D2 variants uh in the A7 genes are
Conservative that is they don’t lead to an alteration in the amino acid however these variants defer in three amino acid positions uh within the E6 protein so we what we did is that uh we constructed uh we constructed uh vectors and we transduced keros sites with this with
These E6 and E7 from these different variants and initially we performed a colony formation aays so these are three different Colony formations ofays and the mean number of colonies that we counted and we can see that regarding normal keratinocytes which were non transduced uh in in one of the the
Experiments we were able to observe nine colonies however none of these colonies they were able to grow until passion 30 where we considered the cells immortalized they scied much before and regarding uh sample Str said uh cartino transed with the EPT Vector we do not observe any colony and regarding a
Variant a lineage variants and D2 lineage variants uh we observed that these variants prev is classified as Asian-American that mean uh that is G2 variants they were able to form a higher number of colonies in comparison to alage variants and also we observe that all of these colonies which were formed
They were able to to to be mortalized they were grown over passage P30 and we at where we consider these cells as immortalized on the other hand a aen transduced kinoy two of these colonies they were not able to immortalize and they sessed near passage 20 we also
Assess the transformation ability of the karatinos sites and these cells were grown in soft AAR medium and we observed in three D experiments that compared to the a lineage variants nonion varant were associated uh with a higher oncogenic potential because they were able to form more more colon in comparison to a lineage
Variant we also assessed uh map K silently in the samples in the keratinocytes and by Western blots uh uh we observe that in cervical that in extract from from keratinocytes transduced with the D2 variant we observe a higher levels of map kyes one phosphorilation which also trans uced in
Higher levels of a to phosphorilation and this was was assessed by means of proteomic uh uh Pro an antibody array and also uh B Anala say we see higher levels of a phosphorilation so we also perform finally we perform migrations a Say by means of one healing aay and we observed
That the D2 variant was able to migrate uh more faster in comparison to the a lineage variants and we also observed that when when these cells were treated uh with a mapk inhibitor uh we observed that this uh was attenuated suggesting the involvement of this Sally passway in
The migration so we we we we have made uh some contributions to the analysis of HPV 16 variants also also epidem technological but also functionally and uh this is one of the lines of research in our landb so finally I would like to acknowledge my funding agencies which is
Fesi sen and capis I want to take all the collaborators uh of these studies in The Institute of cancer of the state of s Paulo especially Louisa Villa who was my sub advisor and was involved uh in the coordination of the him study and
The m l m study in s Pao also to Mela gonalves who performed all the analysis regarding the association of hpv16 variants IND theano canal and Matthew FAA who de who made the analysis regarding genital samples and also Rosana who performed the statistical analysis and uh svan who was a previous technician in
Our lab and also to Hima hosman who was a PhD student in my life involved in some of this functional assays I will also like to thank my collaborators in the in the magio university and the morit cancer center I’m going to leave here my email so if you have any
Questions or if you come to Brazil and have the opportunity to come here and would like to to to see our institution and to know the lab I am available please do not exit to write to me I thank you very much for hearing me for