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Human Papillomaviruses in Head and Neck Squamous cell carcinomas : the relevance of HPV16 variants

COCHICHO, DANIELA · 2023

Abstract

ABSTRACT Head and Neck Carcinoma is considered the sixth most common cancer worldwide and is responsible for 450,000 deaths/year. These tumours appear in various locations of the upper aero-digestive tract. Most of these neoplasms originate in the squamous epithelium and correspond to 90 to 95% of all squamous cell carcinomas of the head and neck region. Despite their common origin in the squamous epithelium, tumours from different locations within the head and neck region show high heterogeneous phenotypic, etiological, and biological characteristics. The registered risk factors are tobacco and alcohol consumption, described as the most common causes for the appearance of these tumours, and Human Papillomavirus (HPV) infection as a risk factor associated with tumours that arise in the oropharynx. The developed project aimed to obtain information about some specific aspects of squamous cell carcinoma of the head and neck region through a detailed molecular analysis of a case series. The study design was developed in distinct and structured tasks to: 1- obtain data related to clinical and pathological variables, 2- identify the different HPV16 variants present and 3- determine the frequency of PIK3CA gene mutations. The cohort included in the study was selected from a universe of patients diagnosed with squamous cell carcinoma of the head and neck region at the Instituto Português de Oncologia Francisco Gentil de Lisboa IPOLFG, being composed mostly of male patients with oropharyngeal cancer, HPV-positive and with active consumption habits. Additionally, a series of samples from a murine model of oropharyngeal carcinogenesis induced by HPV16 was studied to identify the virus variant present in this experimental model. The sequencing of the complete genome of HPV16 was performed using Next Generation Sequencing methodology, developed from previously described primers, which were adapted to optimize a laboratory workflow that allows the identification of HPV16 variants from multiple tissues. Finally, the frequency of the PIK3CA mutation strongly associated with squamous cell carcinoma of the head and neck region was calculated based on the detection of the presence of four different mutations (E545D, E545K, E542K and H1047L) by real-time PCR methodology. The main data obtained express the clinical characterization of the pathology in the series of patients, describe the different HPV16 strains identified in the total cohort of patients, as well as the mutational profile for each HPV16 gene. Phylogenetic analysis shows lineage A as the most prevalent in our cohort. In the exploratory study of the respective strains, it was not possible to establish any reliable association with clinicopathological variables, including survival variables, due to the small number of cases included in the analysis (n=35). Regarding the genomic comparative analysis of the 35 patients, 243 nucleotide variations were reported, 84 of which integrate an amino acid alteration. The mutational profile agrees with that previously described; a total of 41% (100/243) single nucleotide variation (SNV) were published by other authors based on cervix pathology and/or in the pathology of the head-neck region. The remaining SNV that were not described in the systematic review contribute with new data in different genes of the viral genome, including those genes that until now are less explored, such as the E4, E5 and L2 gene. From the exploratory analysis of biological significance, a total of 23 variants were described, the majority studied in cervical carcinoma. Overall, the greatest variability was observed in the E5 and L2 genes that were not previously studied. Oppositely, the most conserved gene was E7, as previously reported by other authors. The frequency of PIK3CA gene mutations found in our cohort support the high occurrence of these mutations among head and neck tumours. In our cohort, we identified the presence of at least one mutation in the PIK3CA gene in 39% of the patients included, and the E545D mutation was the most frequently detected. It should also be noted that the data from the analysis of the murine model genome allowed for the first time to identify the HPV16 lineage present as A1 (European) sublineage, which was the most prevalent in patients, supporting the hypothesis that this is a lineage with high oncogenic potential in the oropharynx and making this model even more interesting. Overall, our data corroborate those previously reported by other groups, highlighting the importance of molecular analysis to better understand the distribution of HPV16 variants and PIK3CA mutations and their potential influence on carcinogenesis, diagnosis and clinical approach in head and neck cancer.

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Human Papillomaviruses in Head and Neck Squamous cell carcinomas : the relevance of HPV16 variants

Author: COCHICHO, DANIELA
Year: 2023
Source: https://run.unl.pt/bitstream/10362/160556/1/Tese_Danela%20Cochicho.pdf
1
HUMAN PAPILLOMAVIRUSES IN HEAD AND
NECK SQUAMOUS CELL CARCINOMAS
THE RELEVANCE OF HPV16 VARIANTS
DANIELA COCHICHO TAVARES DE SOUSA
Tese pa a ob enção do g au de Dou o em Biomedicina
Dou o amen o em associação en e:
Faculdade de Ciências Médicas | NOVA Medical School - Uni e sidade NOVA de
Lisboa
Uni e sidade de A ei o
Janei o, 2023
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HUMAN PAPILLOMAVIRUSES IN HEAD AND NECK SQUAMOUS CELL
CARCINOMAS: THE RELEVANCE OF HPV16 VARIANTS
Daniela Cochicho Ta a es de Sousa
Supe iso : Ana Félix MD PhD
Faculdade de Ciências Médicas | NOVA Medical School da Uni e sidade NOVA de
Lisboa,
Se iço de Ana omia Pa ológica do Ins i u o Po uguês de Oncologia de Lisboa
F ancisco Gen il, (IPOLFG)
Co-Supe iso : Rui M. Gil da Cos a PhD
LEPABE, Labo a o y o P ocess Enginee ing, En i onmen , Bio echnology and
Ene gy, Facul y o Enginee ing, Uni e si y o Po o
ALiCE - Associa e Labo a o y in Chemical Enginee ing, Facul y o Enginee ing,
Uni e si y o Po o
Molecula Oncology and Vi al Pa hology G oup, Resea ch Cen e o IPO Po o (CI-
IPOP) / RISE@CI-IPOP (Heal h Resea ch Ne wo k), Po uguese Oncology Ins i u e
o Po o (IPO Po o), Po o Comp ehensi e Cance Cen e (Po o.CCC)
Pos -g adua e P og amme in Adul Heal h (PPGSAD), Uni e si y Hospi al
(HUUFMA) and Mo phology Depa men , Fede al Uni e si y o Ma anhão, B azil
TESE PARA OBTENÇÃO DO GRAU DE DOUTOR NA ESPECIALIDADE EM
BIOMEDICINA
Janei o, 2023
3
HOST INSTITUTIONS
4
FINANCIAL SUPPORT
This esea ch was no di ec ly inanced bu some expe imen al asks and
pe sonal included in some a icles we e pa o p ojec s wi h unds and he cos
we e pa ially co e ed, and o ha we b ie ly men ion he e:
• Fundação pa a a Ciência e a Tecnologia (FCT) pa a a Ciência e
Tecnologia/Minis é io da Ciência, Tecnologia e Ensino Supe io (FCT/MCTES,
Po ugal) h ough na ional unds o iNOVA4Heal h.
(UIDB/04462/2020 and UIDP/04462/2020) – IPO Lisboa
• GenomePT p ojec suppo ed by COMPETE 2020 Ope a ional P og amme o
Compe i i eness and In e na ionalisa ion (POCI), Lisboa Po ugal Regional
Ope a ional P og amme (Lisboa2020), Alga e Po ugal Regional Ope a ional
P og amme (CRESC Alga e2020), unde he PORTUGAL 2020 Pa ne ship
Ag eemen , h ough he Eu opean Regional De elopmen Fund (ERDF) and by
Fundação pa a a Ciência e a Tecnologia (FCT).
POCI-01-0145-FEDER-022184) - INSA
• FEDER unds h ough he P og ama Ope acional Fac o es de Compe i i idade
– COMPETE and by Na ional unds h ough he Fundação pa a a Ciência e a
Tecnologia wi hin he scope o he p ojec Cen e o Toxicogenomics and
Human Heal h -ToxOmics).
(UID/BIM/00009/2019) – INSA
• Na ional unds h ough FCT/MCTES (PIDDAC) o 2SMART (NORTE-01-0145-
FEDER-000054) suppo ed by No e Po ugal Regional Ope a ional
P og amme (NORTE 2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen ,
h ough he Eu opean Regional De elopmen Fund (ERDF).
(LA/P/0045/2020 ALICE) (UIDB/00511/2020 and UIDP/00511/2020 LEPABE) – IPO
Po o
5
LIST OF SCIENTIFIC PUBLICATIONS
These a e he main scien i ic a icles published by he au ho whose con en was
o ally o pa ially used in he elabo a ion o he hesis:
I. De ecção e Geno ipagem do HPV em es egaços e biópsias buco‐
a ingeos.
Daniela Cochicho, Luís Ma ins, Ped o Mon al ão, Lígia Fe ei a, Luís Oli ei a, Rui
Fino, Hugo Es ibei o, Rica do Pacheco, Ana Hebe, Má io Cunha, Miguel
Magalhães.
Re is a Po uguesa de O o inola ingologia e Ci u gia Ce ico- acial, ol.55, nº3
Se emb o 2017.h ps://www.jou nalspo l.com/index.php/spo l/a icle/ iew/414
The au ho con ibu ed o he s udy's idea and design, pe o med all
labo a o y echnical p ocedu es. Pe o med da a collec ion and
compila ion. Di ec ly con ibu e o analysing and in e p e ing da a.
P epa e all he w i ing, edi ing, and ew i ing he inal manusc ip .
II. Explo ing he oles o HPV16 a ian s in head and neck squamous cell
ca cinoma: Cu en challenges and oppo uni ies.
Daniela Cochicho; Rui Gil da Cos a; Ana Félix.
Vi ol J 18, 217 (2021). h ps://doi.o g/10.1186/s12985-021-01688-9
The au ho con ibu ed o he s udy´s concep s and s udy design, including
he li e a u e sea ch using h ee da abases (me a-analysis), he da a
collec ion, analysis, and in e p e a ion. Also conduc ed he p epa a ion o
he manusc ip , in i s edi ing and e iewed en i ely.
III. Dis ibu ion and clinical signi icance o HPV16 Va ian s in Head and Neck
Squamous Cell Ca cinomas: Da a om a Po uguese coho and
sys ema ic e iew.
Daniela Cochicho, Alexand a Nunes, João Paulo, Susana Es e es, Joana
Mendonça, Luís Viei a, Luís Ma ins, Má io Cunha, Ped o Mon al ão, Miguel
Magalhães, Rui Gil da Cos a, Ana Félix.
Pa hobiology - DOI: 10.1159/000529723
The au ho con ibu ed o he s udy´s concep s and s udy design,

6
including he sys ema ic e iew o he li e a u e using wo da abases. Also
pe o med he da a collec ion, analysis, and in e p e a ion. Design and
de eloped he en i e NGS wo k low, di ec ly con ibu e o he analysing and
in e p e ing o he molecula da a. Conduc ed he p epa a ion and edi ing
he inal manusc ip .
IV. Cha ac e iza ion o he Human papilloma i us 16 oncogenes in
K14HPV16 mice: Sublineage A1 d i es mul i-o gan ca cinogenesis.
Daniela Cochicho, Alexand a Nunes, João Paulo Gomes, Luís Ma ins, Má io
Cunha, Bea iz Medei os-Fonseca, Paula Oli ei a, Ma ga ida Bas os, Rui
Medei os, Joana Mendonça, Luís Viei a, Rui M. Gil da Cos a, Ana Félix.
In . J. Mol. Sci. 2022, 23, 12371. h ps://doi.o g/10.3390/ijms232012371
The au ho con ibu ed on he s udy's design, pe o med all labo a o y
echnical p ocedu es o DNA HPV molecula analysis. Pe o med da a
collec ion and compila ion. Di ec ly con ibu e o molecula analysing and
in e p e ing da a. P epa e all he w i ing, edi ing, and ew i ing he inal
manusc ip .
V. PIK3CA Gene Mu a ions in HNSCC: Sys ema ic Re iew and Co ela ions
wi h HPV S a us and Pa ien Su i al.
Cochicho D, Es e es S, Ri o M, Sil a F, Ma ins L, Mon al ão P, Cunha M,
Magalhães M, Gil da Cos a RM, Félix A.
Cance s. 2022; 14(5):1286. h ps://doi.o g/10.3390/cance s14051286
The au ho con ibu ed o he s udy´s concep s and s udy design, including
he li e a u e sea ch using h ee da abases (me a-analysis), he da a
collec ion, analysis, and in e p e a ion. Pe o med all labo a o y echnical
op imiza ion and p ocedu es. Conduc ed he p epa a ion o he
manusc ip , in i s edi ing and e iewed en i ely.
7
Ful illing he Po uguese Law, he Candida e decla es o ha e ac i ely pa icipa ed
in he collec ion and s udy o all ma e ial and da a included in his Thesis, ha ing
w i en all pape s.
This Thesis and he all he wo k included in i ha e been app o ed by he:
• E hics Resea ch Commi ee o NOVA Medical School (nº 70/2019/CEFCM)
• Scien i ic boa d o Ins i u o Po uguês de Oncologia de Lisboa and he
E hics Commi ee (UIC/1168)
• The animal model expe imen s we e pe o med and app o ed by he
Uni e si y o T ás-os-Mon es and Al o Dou o E hics Commi ee (App o al
Numbe 10/2013) and by he Po uguese Ve e ina y Di ec o a e (App o al
Numbe 0421000/000/2014).
8
9
TABLE OF CONTENTS
THESIS OUTLINE ........................................................................................................................................ 14
SUMÁRIO ...................................................................................................................................................... 15
ABSTRACT .................................................................................................................................................... 17
ACRONYMS AND ABBREVIATIONS ..................................................................................................... 19
FOREWORD ................................................................................................................................................ 21
CHAPTER 1 ........................................................................................................................................................ 22
Backg ound ..................................................................................................................................................... 22
1. Human Papilloma i uses .................................................................................................................... 23
1.1 S uc u e and egula ion o he i al genome ........................................................................ 23
1.2 In ec ion and Replica ion cycle ................................................................................................... 24
1.3 Ca cinogenesis ................................................................................................................................. 28
1.4 Classi ica ion ..................................................................................................................................... 30
1.5 HPV Va ian s ...................................................................................................................................... 32
2. Head and Neck Squamous cell ca cinoma .................................................................................. 37
2.1 Epidemiology .................................................................................................................................... 37
2.2 Risk ac o s ........................................................................................................................................ 38
2.3 Clinical-pa hological cha ac e iza ion ..................................................................................... 39
2.4 Molecula cha ac e iza ion .......................................................................................................... 40
2.4.1 Non-HPV associa ed HNSCC ............................................................................................... 42
2.4.2 HPV associa ed HNSCC ......................................................................................................... 43
2.5 HNSCC ca cinogenesis, HPV in ec ion and PI3K/AKT/mTOR pa hway ........................ 45
2.5 T ea men .......................................................................................................................................... 49
3. K14HPV16 Animal Model ..................................................................................................................... 51
CHAPTER 2 ....................................................................................................................................................... 54
Aim .................................................................................................................................................................. 55
Gene al hypo hesis ................................................................................................................................... 55
Speci ic objec i es ..................................................................................................................................... 55
CHAPTER 3 ....................................................................................................................................................... 56
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No es udo explo a ó io das espec i as linhagens não oi possí el es abelece
nenhuma associação de con iança com as a iá eis clinico-pa ológicas, incluindo as
a iá eis de sob e i ência, de ido ao eduzido núme o de casos incluídos na análise
(n=35). Na análise compa a i a do genoma dos 35 pacien es o am epo adas 243
a iações nucleo ídicas, 84 das quais a al e ação esul a num aminoácido di e en e. O
pe il mu acional ap esen ado oi conco dan e com o p e iamen e desc i o, em 41%
(100/243) das a iações nucleo ídicas o am publicadas po di e en es au o es na
pa ologia do colo e/ou na pa ologia egião de cabeça-pescoço. As es an es SNV
con ibuem com no os dados em di e en es genes do genoma i al, incluindo os
genes que a é à da a se encon am menos explo ados, nomeadamen e o gene E4, E5
e L2. Da análise explo a ó ia do signi icado biológico o o al de 23% (23/100) a ian es
o am desc i as, a maio ia já es udada no ca cinoma do colo do ú e o.
No ge al, os nossos esul ados e elam exis i uma maio a iabilidade, p ecisamen e
nos genes E5 e L2, que não es a am ão es udados, e con i ma am que o gene mais
conse ado é o E7, o que es á de aco do com o epo ado p e iamen e po ou os
au o es.
Os nossos esul ados no es udo da equência de mu ações do gene PIK3CA
sus en am a ele ada oco ência des as mu ações nos umo es de cabeça-pescoço. Na
nossa coo e iden i icamos a p esença de pelo menos uma mu ação no gene PIK3CA
em 39% dos pacien es incluídos e a mu ação E545D oi a mais equen emen e
de ec ada.
A análise do genoma do modelo mu ino que e e uámos pe mi iu pela p imei a ez
iden i ica qual a é linhagem do HPV16 p esen e nes e modelo que é a sublinhagem
A1 (Eu opeia). Es e dado no o é mui o in e essan e po que es a é a mais p e alen e em
doen es e que e o ça a hipó ese de que es a é uma linhagem com ele ado po encial
oncogénico na o o a inge, o nando es e modelo ainda mais in e essan e.
Globalmen e, os nossos dados co obo am os p e iamen e epo ados po ou os
g upos, des acando a impo ância da análise molecula pa a melho comp eende a
dis ibuição das a ian es do HPV16 e das mu ações do PIK3CA e a sua possí el
in luência na ca cinogénese, diagnós ico e abo dagem clínica no canc o de cabeça e
pescoço.
PALAVRAS-CHAVE: CARCINOMA, OROFARINGE, HPV16 VARIANTES, PIK3CA, GENOMA, NGS

17
ABSTRACT
Head and Neck Ca cinoma is conside ed he six h mos common cance
wo ldwide and is esponsible o 450,000 dea hs/yea . These umou s appea in
a ious loca ions o he uppe ae o-diges i e ac . Mos o hese neoplasms
o igina e in he squamous epi helium and co espond o 90 o 95% o all squamous
cell ca cinomas o he head and neck egion. Despi e hei common o igin in he
squamous epi helium, umou s om di e en loca ions wi hin he head and neck
egion show high he e ogeneous pheno ypic, e iological, and biological
cha ac e is ics. The egis e ed isk ac o s a e obacco and alcohol consump ion,
desc ibed as he mos common causes o he appea ance o hese umou s, and
Human Papilloma i us (HPV) in ec ion as a isk ac o associa ed wi h umou s ha
a ise in he o opha ynx.
The de eloped p ojec aimed o ob ain in o ma ion abou some speci ic aspec s o
squamous cell ca cinoma o he head and neck egion h ough a de ailed
molecula analysis o a case se ies. The s udy design was de eloped in dis inc and
s uc u ed asks o: 1- ob ain da a ela ed o clinical and pa hological a iables, 2-
iden i y he di e en HPV16 a ian s p esen and 3- de e mine he equency o
PIK3CA gene mu a ions.
The coho included in he s udy was selec ed om a uni e se o pa ien s
diagnosed wi h squamous cell ca cinoma o he head and neck egion a he
Ins i u o Po uguês de Oncologia F ancisco Gen il de Lisboa IPOLFG, being
composed mos ly o male pa ien s wi h o opha yngeal cance , HPV-posi i e and
wi h ac i e consump ion habi s. Addi ionally, a se ies o samples om a mu ine
model o o opha yngeal ca cinogenesis induced by HPV16 was s udied o iden i y
he i us a ian p esen in his expe imen al model. The sequencing o he
comple e genome o HPV16 was pe o med using Nex Gene a ion Sequencing
me hodology, de eloped om p e iously desc ibed p ime s, which we e adap ed
o op imize a labo a o y wo k low ha allows he iden i ica ion o HPV16 a ian s
om mul iple issues. Finally, he equency o he PIK3CA mu a ion s ongly
associa ed wi h squamous cell ca cinoma o he head and neck egion was
calcula ed based on he de ec ion o he p esence o ou di e en mu a ions
(E545D, E545K, E542K and H1047L) by eal- ime PCR me hodology.
The main da a ob ained exp ess he clinical cha ac e iza ion o he pa hology in
he se ies o pa ien s, desc ibe he di e en HPV16 s ains iden i ied in he o al
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coho o pa ien s, as well as he mu a ional p o ile o each HPV16 gene.
Phylogene ic analysis shows lineage A as he mos p e alen in ou coho . In he
explo a o y s udy o he espec i e s ains, i was no possible o es ablish any
eliable associa ion wi h clinicopa hological a iables, including su i al a iables,
due o he small numbe o cases included in he analysis (n=35). Rega ding he
genomic compa a i e analysis o he 35 pa ien s, 243 nucleo ide a ia ions we e
epo ed, 84 o which in eg a e an amino acid al e a ion. The mu a ional p o ile
ag ees wi h ha p e iously desc ibed; a o al o 41% (100/243) single nucleo ide
a ia ion (SNV) we e published by o he au ho s based on ce ix pa hology and/o
in he pa hology o he head-neck egion. The emaining SNV ha we e no
desc ibed in he sys ema ic e iew con ibu e wi h new da a in di e en genes o
he i al genome, including hose genes ha un il now a e less explo ed, such as
he E4, E5 and L2 gene. F om he explo a o y analysis o biological signi icance, a
o al o 23 a ian s we e desc ibed, he majo i y s udied in ce ical ca cinoma.
O e all, he g ea es a iabili y was obse ed in he E5 and L2 genes ha we e no
p e iously s udied. Opposi ely, he mos conse ed gene was E7, as p e iously
epo ed by o he au ho s.
The equency o PIK3CA gene mu a ions ound in ou coho suppo he high
occu ence o hese mu a ions among head and neck umou s. In ou coho , we
iden i ied he p esence o a leas one mu a ion in he PIK3CA gene in 39% o he
pa ien s included, and he E545D mu a ion was he mos equen ly de ec ed. I
should also be no ed ha he da a om he analysis o he mu ine model genome
allowed o he i s ime o iden i y he HPV16 lineage p esen as A1 (Eu opean)
sublineage, which was he mos p e alen in pa ien s, suppo ing he hypo hesis
ha his is a lineage wi h high oncogenic po en ial in he o opha ynx and making
his model e en mo e in e es ing.
O e all, ou da a co obo a e hose p e iously epo ed by o he g oups,
highligh ing he impo ance o molecula analysis o be e unde s and he
dis ibu ion o HPV16 a ian s and PIK3CA mu a ions and hei po en ial in luence
on ca cinogenesis, diagnosis and clinical app oach in head and neck cance .
KEYWORDS: HNSCC, HPV16 VARIANTS, PIK3CA, WHOLE-GENOME, NGS, SNVS
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ACRONYMS AND ABBREVIATIONS
AA - Asia-Ame ican HPV16 Va ian
AF - A ican HPV16 Va ian
AJCC - Ame ican Join Commi ee on Cance
AS - Asian HPV16 Va ian
A260- Abso bance a 260 nm
A280 - Abso bance a 280 nm
bp - Base pai
cDNA - Complemen a y DNA
CDKN2A - Cyclin-dependen kinase inhibi o 2A
CT - Chemo he apy
CRT - Chemo adio he apy
DFS - Disease- ee su i al
DNA - Deoxy ibonucleic acid
E - Eu opean HPV16 a ian
E HPV - Ea ly p o ein
EU - Eu opean Union
FFPE - Fo malin- ixed pa a in-embedded
H&E - Hema oxylin and eosin s aining
HN – Head and Neck
HNSCC - Head and Neck Squamous cell ca cinoma
HPV - Human Papilloma i us
HNSCC-HPV - Head and Neck Squamous cell ca cinoma associa ed o Human
Papilloma i us
HNSCC-nonHPV - Head and Neck Squamous cell ca cinoma wi hou Human
Papilloma i us associa ion
IARC - In e na ional Agency o Resea ch on cance
ICTV - In e na ional Commi ee on Taxonomy o Vi uses
ISO - In e na ional O ganiza ion o S anda diza ion
K14HPV16 - Ea ly HPV16 genes unde he con ol o he cy oke a in 14
LCR - Long con ol Region
MUT - Mu a ed
mRNA - Messenge RNA
NA - No h Ame ican HPV16 Va ian
20
NGS - Nex -gene a ion sequencing
NTC – No Ta ge Con ol
O.C.T. - Compound Op imal cu ing empe a u e compound
OD - Odds a io
ORF - Open eading ame
OS - O e all su i al
PCR - Polyme ase chain eac ion
PIK3CA - Phospha idylinosi ol-4,5-biphospha e 3-kinase
PI3K - Phosphoinosi ide 3-kinases
PI3-K-AKT - Phospha idylinosi ol-3-kinase-AK
PIP2 - Phospha idylinosi ol-(4,5)-bisphospha e
PIP3 - Phospha idylinosi ol-(3,4,5)- isphospha e
PRB - Re inoblas oma p o ein
PTEN - Phospha ase and ensing homolog
PV - Papilloma i us
P16 - p16INK4a p o ein, cyclin-dependen kinase inhibi o 2A, o CDKN2A
P53 – Tumou supp esso p o ein 53
qPCR - Real- ime PCR
RNA - Ribonucleic acid
RT – Radio he apy
SR – Sys ema ic e iew
TNM - Tumou , lymph nodes, me as asis
TCGA - Tumou Cance Genome A las
UICC - Union o In e na ional Cance Con ol's
USA - Uni ed S a es o Ame ica
WT - Wild ype
WHO - Wo ld heal h o ganiza ion
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FOREWORD
Head and neck ca cinoma is he six h leading cance by incidence wo ldwide and
is esponsible o mo e han 800,000 new cases yea ly. HPV is p esen in
app oxima ely 35% o HNSCC, eme ges in he lingual and pala ine onsils, and
HPV16 is he mos de ec ed ype. The epidemiological, e iological, and molecula
da a sugges ha in a- ype HPV a ian s a e biologically dis inc and may be
associa ed wi h di e en isks in u e ine ce ical cance p og ession. None heless,
HPV16 a ian s may also play an impo an ole in head and neck ca cinogenesis,
bu ew ha e es ablished hei ele ance. The genomic p o ile o HNSCC published
by he Cance Genome A las in 2015 b oadened he de elopmen o mu a ion
s udies and highligh ed a high equency o changes. The iden i ica ion o
bioma ke s o p edic esponse o he apy will allow he selec ion o pa ien s mo e
likely o espond o new he apeu ic combina ions making a aluable con ibu ion
o clinical ea men s a egies. Essen ially, i is c i ical o e ine he mu a ional
p o ile o HPV-posi i e and HPV-nega i e wi hin HNSCC pa ien s. This p ojec
add esses a deep molecula analysis o a Po uguese coho o HNSCC pa ien s and
explo es i s co ela ion wi h clinicopa hological a iables and hei po en ial
con ibu ion o clinical p ac ice.

22
CHAPTER 1
BACKGROUND
23
1. HUMAN PAPILLOMAVIRUSES
The Papilloma Vi us (PV) a membe o he Papilloma i idae amily (De Villie s e
al., 2004), a e ubiqui ous i uses ha in ec he epide mis and mucosa o se e al
mammals, including humans. PV in ec ions a e usually ansien and
asymp oma ic, wi h no ele an clinical mani es a ions. Howe e , hey can cause
benign umou s (wa s, papilloma) ha usually eg ess o es ablish pe sis en
in ec ions ha can lead o umou s (zu Hausen, 1996). The e a e o e 300 dis inc
ypes o PVs isola ed. Abou 200 can in ec humans – human papilloma i us (HPV),
whe e 40 ypes in ec he anogeni al egion (Wo ld Heal h O ganiza ion, 2010).
Only a small g oup is associa ed wi h he de elopmen o neoplas ic pa hologies.
Al hough he wide numbe o HPV ypes isola ed and opism di e ences, hey all
sha e he same genome a angemen (Be na d, Calleja-Macias and Dunn, 2006).
1.1 STRUCTURE AND REGULATION OF THE VIRAL GENOME
The i us pa icle is small (55nm-diame e ), non-en eloped, sui able only o a
p o ein capsid o icosahed al symme y ha encloses he i al DNA genome
(Münge e al., 2004). The i al DNA, wi h a leng h o 7,906 base pai s (bp), is ci cula ,
double-s anded and s uc u ed in o h ee unc ional egions: Ea ly Region, La e
Region, and Long Con ol Region. These h ee egions a e sepa a ed by wo
polyadenyla ion si es (pA) as shown in Figu e 1 (Zheng and Bake , 2006). The
gene ic in o ma ion is dis ibu ed among he eigh coding sequences o p o eins
classi ied as ea ly (E) and la e (L) acco ding o hei empo al exp ession. The Ea ly
egion occupies mo e han 50% o he i us genome and encodes six open eading
ames (ORFs): E1, E2, E4, E5, E6 and E7. The la e egion con ains he L1 and L2 ORFs
ha encode wo s uc u al p o eins. In addi ion, he e is a long con ol egion (LCR)
si ua ed be ween he E6 and L1 genes, which does no con ain ORFs bu includes
he o igin o eplica ion (o i) as well as mul iple elemen s (enhance s) equi ed o
ansc ip ion (Münge e al., 2004).
24
Figu e 1. HPV16 comple e genome ep esen a ion adap ed om (Zheng and Bake , 2006). The
colou ed line illus a es he linea o m o he i us genome (≈8000 pb), p esen ing he h ee
unc ional egions LCR (blue), Ea ly ( ed) and La e (g ey); he p omo e s P (a ows) and
polyadenyla ion si es (pA); he ea ly (AE) and la e (AL). Abo e he line, whi e boxes illus a e he ORFs
o each coding egion ( ed and g ey): he Ea ly E6, E7, E1, E2, E4 and E5 genes; and he la e genes L2
and L1. The numbe s abo e ep esen he i s nucleo ide in he s a codon and he las nucleo ide
posi ion in he s op codon o each ORF.
The ansc ip ion o he genome occu s om a single s and o DNA conside ed o
be polycis onic, wi h mul iple in ons ha unde go ex ensi e al e na i e RNA
splicing p oducing mul iple RNAs wi h se e al open eading ames. The
ansc ip ion o he Ea ly and La e genes occu s unidi ec ionally om p ime s and
p omo e s. I is ini ia ed a p97 p omo e loca ed ups eam o he ORF o he E6
gene and is esponsible o almos all ea ly gene exp ession (Smo kin and
We s ein, 1986). Nex is ollowed by he p670 p omo e loca ed wi hin he ORF o
he E7 gene, which is esponsible o he ansc ip ion o la e genes. This
mechanism gi es o HPV he abili y o exp ess di e en p o eins a a gi en ime
and in he di e en ia ion s age o he in ec ed epi helium (Zheng and Bake , 2006).
I also ensu es he smoo h p og ession o key e en s o he i us, such as he
eplica ion cycle, along wi h immune e asion.
1.2 INFECTION AND REPLICATION CYCLE
HPV accesses o he basal laye h ough mic o ab asions in he epi helium and
binds o hos cells using cell su ace molecules (Sha i-Ke ama e al., 2003;
Pa e son, Smi h and Ozbun, 2005). The i s e en s a e induced by he in e ac ion
be ween he L1 s uc u al p o ein and he cell su ace ecep o (hepa in sulpha e
25
p o eoglycan) (Gi oglou e al., 2001). Followed by he in e ac ion o he L2 p o ein
wi h he cell su ace ec ui ing a second ecep o ha allows he en ance o he
i us (Kawana e al., 1999), which as a non-en eloped pa icle, adop s mechanisms
h ough cla h inid-media ed endocy osis (Day, Lowy and Schille , 2003). Once
inside he cell, he i al pa icle is anspo ed o he nucleus, no be o e losing he
capsid i s . This p ocess allows he uncoa ing o he ci cula genome p o ec ed in
he endosome (Me le e al., 1999; Nelson, Rose and Mo oianu, 2002) and is
media ed by he L2 p o ein, which can dis up he endosome memb ane, and he
i al genome is eleased in o he cy osol. Then he i al genome (8000 bp) di uses
h ough he cy osol, media ed he eby L2 p o ein, which in e ac s wi h bo h ac in
and ubulin (Yang e al., 2003). Upon eaching he nucleus, he i al DNA en e s
h ough he nuclea po es.
The PVs, as DNA i uses, eplica e and a e assembled exclusi ely in he nucleus. In
issues, he eplica ion begins in undi e en ia ed cells, loca ed in he basemen
laye , in ol ing hos cell ac o s ha in e ac wi h he LCR egion and hus ini ia e
he ansc ip ion o i al genes (Bu d, 2003). The i s p o eins o be exp essed a e
E1 and E2, necessa y o i al DNA eplica ion and o es ablish genomes wi h low
copy numbe s o 20-100 copies pe cell (Ruesch, S uben auch and Laimins, 1998;
Ozbun, 2002). The i al genomes eplica e simul aneously wi h he eplica ion o
cellula DNA (You e al., 2004). The in ec ed cell, when i di ides, o igina es new
cells ha emain in he basal laye , hus se ing as a ese oi o he i us. Jus he
same hese in ec ed cells p oceed wi h exp ession and eplica ion in a igh ly
con olled and egula ed manne by ke a inocy e di e en ia ion (zu Hausen,
1996).
The comple e i al genome encodes only 8 o 10 i al p o eins, which equi e hos
cell ac o s o egula e i al ansc ip ion and eplica ion. The E1 and E2 p o eins
accompany his p ocess, by ec ui ing he cellula machine y in ol ed in DNA
eplica ion. The E1 p o ein is an ATP-dependen helicase which opens he
eplica ion o igin, allowing i al genome eplica ion o begin (Wilson e al., 2002),
while E2 in e ac as a ansc ip ion ac o h ough i s unc ions o egula e he
exp ession o ea ly ORF p omo e s (Ham e al., 1991). Wi h he epi helial
di e en ia ion, cells s op di iding and he di e en ia ed ke a inocy es do no
exp ess he c ucial ac o s in ol ed in DNA eplica ion. The i us sol es his
p oblem by using i al oncop o eins E6 and E7 ha ac on he egula ion o he cell
cycle p og ess and blockage o apop osis o hose cells ha en e he cell cycle
32
Figu e 4. Compa ison o Epidemiological and Phylogene ic classi ica ion. The colou diag am
p esen s he Taxonomy by phylogene ic classi ica ion and epidemiological classi ica ion, wi h he
dis ibu ion o he HPV Types in o high- ( ed) and low- isk (g een) g oups. Abo e all, excep (yellow)
HPV 70 and HPV73, obse e good ag eemen be ween he wo classi ica ions. This co ela ion is
help ul o p edic whe he a new geno ype may be o a high- isk sequence (Muñoz e al., 2003).
1.5 HPV VARIANTS
The e is also clea e idence o a wide sequence di e si y wi hin each HPV ype (De
Villie s e al., 2004; Bu k, Ha a i and Chen, 2013), whe e he genome sequencing
e ealed in a- ype a ian s wi h gene ic di e ences anging be ween 0.5 and 1%
(Ho e al., 1991; Mi abello e al., 2016). HPVs, being a double-s anded DNA i us, uses
he e icien e ision o he hos 's DNA polyme ase o i s eplica ion, which a oids
high mu a ion a es; he e o e, changes in HPV genomes a e acqui ed slowly, he
andom mu a ions ha e en ually occu wi hin i al ypes gene a e he nucleo ide
polymo phisms con ained in in a ype a ian s (Gillison e al., 2000; Muñoz e al.,
2003).
Ini ially, in a ype a ian s we e essen ially s udied in HPV16 and HPV18 and we e
named acco ding o hei geog aphical dis ibu ion depending on he o igin and
e hnici y o he in ec ed popula ion (Muñoz e al., 2003). Thus, six dis inc
phylogene ic clus e s we e de ined o HPV16: Eu opean (E), Asian (As), Asian-
Ame ican (AA), A ican 1 (A 1), A ican 2 (A 2) (Be na d e al., 1993; Ho e al., 1993;

33
Yamada e al., 1995) and No h Ame ican 1 (NA1) (Yamada e al., 1997; Bu k, Ha a i
and Chen, 2013; Jackson e al., 2016). Pos e io ly, i was p oposed a mo e p ecise
nomencla u e, es ablishing lineages and sub-lineages (Bu k, Ha a i and Chen,
2013) as shown in Figu e 5. Rega ding HPV16 geno ype, whole-genome analysis
allowed he cha ac e iza ion o ou main a ian lineages (A, B, C, D) and six een
sublineages (A1, A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, C4, D1, D2, D3, D4). Acco ding
o hese indings, lineage A g ouped Eu opean clus e wi h sublineage A1, A2 and
A3, and Asia ic clus e wi h sublineage A4. The lineage B encloses he A ican (A 1)
clus e wi h sublineage B1 and he Lineage C encloses he A ican (A 2) clus e wi h
app op ia e sublineage (C1 o C4); Lineage D encloses he Ame ican clus e wi h
sublineage D1, o No h Ame ican clus e sublineage D2 and o Asian-Ame ican
clus e sublineage D3 (Bu k, Ha a i and Chen, 2013).
34
Figu e 5. Phylogene ic ee om HPV16 in a- ype a ian s. The phylogeny ee om (Bu k, Ha a i
and Chen, 2013; Mi abello e al., 2018) illus a es human papilloma i us 16 (HPV16) lineages (A-D) and
sublineage (A1-4, B1-4, C1-4, D1-4) ela ionships, highligh ed on di e en colou s, indica ing he main
lineage b anches.
35
The eme ging epidemiological, e iological, and molecula da a sugges ha he
in a- ype a ian s o HPV a e biologically dis inc and may be associa ed wi h
di e en isk o cance p og ession. As shown by in i o s udies using 3D
o gano ypic epi helial cell cul u es, he HPV16 E6 a ian s di e in hei abili y o
abolish ke a inocy e di e en ia ion and o induce p53 deg ada ion (Ho mann e
al., 2004; Ag awal e al., 2008; Cance Genome A las Ne wo k, 2015). The e o e, i
was pos ula ed ha he occu ence o nucleo ide a ia ions be ween in a ype
HPV a ian s e lec s in hei unc ional di e ences and pa hogenici y (De la C uz-
He nández e al., 2005; Hochmann e al., 2016; Zhao e al., 2020; Hadami e al., 2021).
In addi ion, se e al isola es o in a ype a ian s o HPV, p e iously desc ibed by
di e en clinical s udies om coho s o ca cinoma o he ce ix, anal canal,
o opha ynx and o al ca i y, con ibu e o a ca e ul classi ica ion o a ian s o he
mos equen HPV ypes in cance (Be na d e al., 2010; Bu k, Ha a i and Chen,
2013; Chen e al., 2015). The HPV16 T350G (L83V) a ian is he mos equen ly ound
among in asi e ce ical cance s (Hassani e al., 2015; Be iol e al., 2016) and has
been associa ed wi h an inc eased oncogenic po en ial han he p o o ype (Pakdel
e al., 2021), possibly by acili a ing pe sis en i al in ec ion (Boscolo-Rizzo e al.,
2009; Joseph e al., 2013; Zhang e al., 2015).
Following his, se e al s udies ha e epo ed ha in a- ype a ian s o HPV16 and
HPV18 al e hos cells in a di e en ly way, which ansla es in di e en isks o
pe sis ence and p og ession o cance (Yamada e al., 1995; Siche o e al., 2007;
Co ne e al., 2013). Fu he unc ional s udies explo ed di e ences in biological
beha iou conce ning he clinical e olu ion o pa ien s wi h HPV- ela ed cance s.
Decoding he impac ha hese a ia ions may be impo an o know he impac
in he pa ien 's p ognosis and in he de elopmen o he apeu ic s a egies.
All hese a ian s’ impac we e es ablished o ce ical cance as he ocus o mos
s udies, compa ed o s udies pe o med on o he HPV-induced pa hologies. In ac ,
in HNSCC cance , wha is known abou dis ibu ion HPV16 a ian s esemble he
knowledge acqui ed in ce ical cance . Indeed, au ho s desc ibed bo h sha e
common biological ea u es. A p esen ing da a om a coho s udy ha compa es
genomic cha ac e is ics o HPV associa ed wi h ce ical e sus o opha yngeal
umou s using DNA sequence analysis showed no signi ican di e ences be ween
dis ibu ion in HPV16 a ian s (LeCon e e al., 2018) whe e bo h p esen ing majo
p e alence o he Eu opean a ian .
36
Unlike he HPV E6 gene ampli ied om o opha yngeal samples epo ed o e
mo e nonsynonymous mu a ions, also o he E7 gene, no di e ences we e ound
in mu a ion a es be ween he wo ana omical loca ions (LeCon e e al., 2018; Zhu
e al., 2020). No ably, he E7 gene is conse ed in bo h loca ions co obo a ing ha
he appa en es ic ion on E7 mu a ions seems o be p esen in he o opha ynx,
as well. O he wise i is s ill poo ly unde s ood he po en ial con ibu ion o hose
speci ic a ian s in cance isk inc easing, o p ognosis (Gillison e al., 2000) o he
clinicopa hological ele ance o he iden i ied a ian s (Ho mann e al., 2004;
Bada acco e al., 2007). The main eason behind his lack o conclusions in his ield
o knowledge may be he ela i ely low numbe o s udies pe o med and he small
size o he HNSCC HPV-associa ed coho s (Hassani e al., 2015). The e o e, ecen ly
his pic u e is s a ing o change, and some mo e s udies on HPV16 a ian s ha e
explo ed he HN egion ca cinomas. In 2022, a la ge s udy was published using
HNSCC cases om wo e ia y cance cen es. The s udy pe o med a NGS analysis
o nea ly 400 pa ien s and ound ha speci ic a ia ions ha e di e en clinical
implica ions, namely eigh HPV16 SNPs we e signi ican ly associa ed wi h wo se
su i al (E1 gene posi ion 1053, L2 gene posi ions 4410, 4539, 5050, 5254, L1 gene
posi ions 5962, 6025, and 7173 in he ups eam egula o y egion) in a US
popula ion (Lang Kuhs e al., 2022).
Al hough he e is s ill da a limi a ion, c ucial s eps ha e been achie ed, and
hope ully new s udies wi h la ge coho s o pa ien s and o he HPV-associa ed
umou s, le e aged due o he in oduc ion o NGS echniques ha ha e op imized
wo k lows ha as e and mo e obus sequencing esul s (Be na d e al., 1993;
Yamada e al., 1995; De la C uz-He nández e al., 2005).
37
2. HEAD AND NECK SQUAMOUS CELL CARCINOMA
Head and neck squamous cell ca cinoma (HNSCC) a e a g oup o umou s loca ed
in se e al dis inc s uc u es in he o al ca i y, pha ynx, and la ynx subsi es o he
uppe ae odiges i e ac (Figu e 6).
Figu e 6. Illus a ion o he Head and Neck ana omic egion adap ed om (Johnson e al., 2020). The
c oss-sec ion (le ) and on sec ion ( igh ) classi ica ion by each subloca ion.
2.1 EPIDEMIOLOGY
Wo ldwide HNSCC is conside ed o be he six h leading cance ega ding
incidence and es ima ed o ha e an impac o ~890,000 new pa ien s and
esponsible o 450,000 dea hs/yea (Global cance Obse a o y, 2021). The
incidences a e ising and expec ed o con inue o inc ease o e he nex decade
(Global cance Obse a o y, 2021). The age a diagnosis usually anges be ween 50
and 70 yea s o age, and men a e signi ican ly mo e likely o de elop he disease
han women (up o a 4:1 p opo ion) (B ay e al., 2018). Al hough his disease bu den
a e a ies ac oss coun ies, o e he pas decade, se e al epidemiologic s udies
epo ed highe p e alence in egions such as Sou heas Asia and Aus alia, he
majo i y associa ed wi h speci ic ca cinogen subs ances such as ch onic

38
consump ion o alcohol and obacco (Mo k e al., 2010; Cha u edi e al., 2013;
Hwang e al., 2015; McDe mo and Bowles, 2019). Con as ing o his, he inc ease
ending incidence, in high-income coun ies such as he USA and Wes e n
Eu ope, o HNSCC is associa ed wi h o opha yngeal HPV ela ed cance s
(Cha u edi e al., 2011; Cen e s o Disease Con ol and P e en ion, 2018).
In Po ugal, acco ding o da a om he Na ional Cance Regis y, he o al ca i y
and pha ynx a e conside ed o be he se en h mos diagnosed malignan umou
(Regis o Oncológico Regional do No e, 2010; Mi anda e al., 2018). E e y yea
app oxima ely 3,000 new cance cases in he ae odiges i e ac (Mi anda e al.,
2018) a e diagnosed, bu he mo ali y a e om hese malignan umou s is low
compa ed o he EU a e age.
In conclusion, his is a ela i ely common human cance , cha ac e ized by high
mo bidi y, high mo ali y, and ew he apeu ic op ions, ou side o s anda d
schemes ha may include su ge y, chemo he apy, and adia ion a e a ailable.
2.2 RISK FACTORS
The wide geog aphic a iabili y in he incidence o hese umou s e lec s he
p e alence o ce ain isk ac o s in speci ic egions (Johnson e al., 2020). In
addi ion, HNSCC can also occu in young pa ien s wi hou associa ion wi h known
isk ac o s (Ro henbe g and Ellisen, 2012). In gene al, he mos ep esen ed isk
ac o s a e he obacco use and alcohol consump ion, as well as in ec ion wi h
oncogenic i us such he HPV (Ka abi and Lewis, 2017).
The associa ion es ablished be ween isk ac o s and Head and Neck subsi es
epo ed in epidemiological s udies, ha e linked he o opha ynx umou s o a
p e ious in ec ion wi h human papilloma i us (HPV) and he o he HNSCC
subsi es, which majo i y a e a consequence o he ha m ul e ec s o smoking and
alcohol consump ion and collec i ely e e ed o as HNSCC-nonHPV (Ka abi and
Lewis, 2017).
In hese wo g oups (HPV and nonHPV) posi i e HNSCC, gene ic and p ognos ic
di e ences (Hwang e al., 2015) e lec he c i ical ole o he molecula p o ile
igge ed by each isk ac o s ha con ibu e o i s ca cinogenesis (Mo k e al.,
2010). Cu en ly, hese wo g oups a e ecognised as majo ca cinogenic pa hways
leading o HNSCC (Ka abi and Lewis, 2017).
39
2.3 CLINICAL-PATHOLOGICAL CHARACTERIZATION
Head and neck cance comp ises e y di e en ana omical a eas and in ol es wide
his ological issues. Howe e , mos o hese umou s o igina e om he squamous
epi helium, co esponding o 90 o 95% o all lesions (Ka abi and Lewis, 2017). This
cance is cha ac e ized by i s mul i ocal de elopmen , coupled wi h slow and silen
g ow h. Despi e e idence o his ological p og ession om cellula a ypia h ough
a ious deg ees o dysplasia, mos pa ien s a e diagnosed in ad anced s age
disease wi hou clinical e idence o he an eceden p emalignan lesion (Nelson,
Rose and Mo oianu, 2002). The main clinical mani es a ions o he disease a y
acco ding o he si e o o igin o he umou , o en cha ac e ized by mucosal ulce s
o umou , dysphagia, odynophagia o o algia, ch onic cough, and neck nodes
ume ac ion (Johnson e al., 2020).
The diagnosis is usually es ablished based on he clinical o o hinola yngological
exam ha is complemen ed wi h imaging exams o e alua e he loco egional and
dis an ex ension o he disease classi ied using he TNM (Tumou , Node,
Me as ases s aging sys em) classi ica ion o he Ame ican Join Commi ee on
Cance and he In e na ional Union o Cance Con ol (Ka abi and Lewis, 2017;
Johnson e al., 2020), and his ological e alua ion om umou issue (El-Nagga AK
e al., 2017). The his opa hological spec um o squamous cell ca cinoma is
cha ac e ized by he e alua ion o cellula a ypia and squamous di e en ia ion. I
de ines a well-di e en ia ed umou , a s a i ied epi helium ha con ains ma u e
cells o ganized in laye s wi h i egula ke a iniza ion, called a “ke a in pea l”. On he
con a y, he poo ly di e en ia ed umou p esen s mos ly imma u e cells wi h
nuclea pleomo phism and a ypical mi oses, wi h a poo ly de ined s a i ica ion
and wi hou ke a iniza ion (Johnson e al., 2020) Figu e 7.
40
Figu e 7. Squamous cell ca cinoma o he o opha ynx, wi h HPV16 DNA iden i ied. In asi e cell nes s
wi h e y i egula bo de s wi h s omal eac ion (A). High powe a ea showing poo ly di e en ia ed
cells wi h mi o ic igu es (a ow) and ocal nec osis (*), in il a ing s ia ed muscle cell issue (B);
neoplas ic cells show in ense p16 nuclea (and also cy oplasmic) s aining, a su oga e ma ke o HPV.
2.4 MOLECULAR CHARACTERIZATION
The HNSCC molecula analysis e eals subs an ial gene ic ins abili y wi h equen
loss o gain o ch omosomal egions as no ed by copy numbe al e a ions and
ch omosomal usions (Cance Genome A las Ne wo k, 2015). Cho and colleagues
desc ibe he comp ehensi e gene ic p o ile o hese umou s, cha ac e ising TP53,
CDKN2A, PIK3CA, HRAS, PTEN, and NOTCH1 as being changed di e en ially ac oss
all Head and Neck loca ions in con as o CASP8, which is also con ined wi hin o al
ca i y (Loyo e al., 2013; Fa ah, 2021). Mu a ions in RAS oncogenes a e less equen ,
han in o he solid umou malignancies, cha ac e izing HNSCC as neoplasms
p edomina ely associa ed wi h loss o umou supp esso s genes (Cance Genome
A las Ne wo k, 2015).
41
Genes ha e been g ouped in o ca ego ies including cell su i al and p oli e a ion
- TP53, EGFR, PIK3CA, and HRAS, cell-cycle con ol genes - CDKN2A and CCND1,
cellula di e en ia ion genes - NOTCH1, and cellula adhesion and in asion
signalling - FAT1, ha e been classi ied (Ag awal e al., 2011; S ansky e al., 2011;
Cance Genome A las Ne wo k, 2015).
The o e iew o he gene ic p o ile o HNSCC umou s (Figu e 8) g ouped
acco ding o Copy Numbe Al e a ions, gene mu a ions and gene exp ession
p o iles in en classi y dis inc classes o umou s based on pa e ns o p o ein
exp ession ha co ela e wi h clinical beha iou (Ro henbe g and Ellisen, 2012). The
biological a iabili y shown wi hin HNSCC ansla es he di ision o he wo main
ca cinogenesis pa hways es ablished o HNSCC-HPV and HNSCC-nonHPV (Fa ah,
2021).
Figu e 8. Molecula cha ac e iza ion o he HNSCC. Scheme adap ed om (Fa ah, 2021) p esen ing
he main p o iles g ouped acco ding o copy numbe al e a ion (CNA), gene ic mu a ions and gene
exp ession p o iles. Acco dingly, he mu a ional analysis di ided he HNSCC-nonHPV in o CNA-silen
and CNA-high umou s. No mally he TP53 and RB pa hways a e ab oga ed in HNSCC-nonHPV
umou s bu emain ac i e in CNA-silen umou s. These CNA-silen a e conside ed a subg oup o
umou s ha , al hough hei ae iology is no well es ablished, age appa en ly is he mos isk ac o
associa ed and exhibi ing mu a ions in he HRAS and CASP8 genes and o en ound in umou s o
he o al ca i y. On he o he hand, HNSCC-nonHPV umou s (CNA-high), in which obacco is he
associa ed isk ac o , exhibi ed changes in oncogenes such FAT1 and NOTCH1 pa hway (WNT-B-
ca enin).
48
Pa icula ly, in HPV-posi i e HNSCC umou s, oncop o eins E6 and E7 in e ac
di ec ly in signalling pa hways such PI3K/Ak /mTOR. This is hough o play a e y
impo an ole in HPV-induced ca cinogenesis by ac ing h ough mul iple cellula
and molecula e en s (Pim e al., 2005; Con e as-Pa edes e al., 2009). These i al
oncop o eins inc ease PI3K/AKT/mTOR signalling in se e al ways: he E7 was
shown o signi ican ly up egula e AKT ac i i y no only h ough deg ada ion o RB
bu also in an RB and PI3K independen manne (Figu e 10). I in e ac s wi h
p o ein phospha ase 2A (PP2A) and p e en s dephospho yla ion o AKT ha
causes signi ican ly highe le els o phospho yla ed Bcl-2 an agonis o cell dea h
(BAD) (Ma qua d and Jücke , 2020). The E6 can ac i a e Ak as well, o bind TSC2,
leading o i s deg ada ion and esul ing in s imula ion o mTORC1.
The PI3K pa hway is unique, in ha all he majo componen s o his pa hway a e
equen ly ound ampli ied o mu a ed in HPV-induced cance s (Zhang e al., 2017).
The mu a ion equency among HNSCC-HPV umou s was epo ed o be
app oxima ely hal o ha ound in HNSCC-nonHPV (S ansky e al., 2011). E en so,
he PIK3CA gene emains one o he mos mu a ed in HNSCC-HPV (Sewell e al.,
2014; Cance Genome A las Ne wo k, 2015). Gene mu a ions a ge ing he p110α
ca aly ic subuni we e ound in 56% o HPV HNSCC umou s and in 34% o HPV-
nega i e HNSCC umou s (Cance Genome A las Ne wo k, 2015).
Among he PIK3CA mu a ions obse ed in HNSCC, 63% occu a h ee speci ic
loca ions encoding he p110α subuni , namely E542, E545, and H1047, known as
canonical mu a ions (Hende son e al., 2014; Miao e al., 2018). Among hese h ee
ac i a ing mu a ions he E542 and E545 a ec s he helical domain o p110α,
inducing PI3K hype ac i i y by dis up ing he egula o y ac i i y o p85 on p110α
(Miled e al., 2007), and he H1047 a ec s he p110α kinase domain and is hough
o cause a con o ma ional change allowing easie access o he phospholipid
subs a e (Mandelke e al., 2009).
Also, mu a ions a ge ing he p110α subuni ha e been associa ed wi h ad e se
ou comes in solid umou s al hough he p ognos ic signi icance in o opha yngeal
SCC is s ill unclea . To be p ecise, he speci ic su i al da a did no di e be ween
PIK3CA wild- ype (WT) and mu a ed (MUT) lesions, howe e , he WT-PIK3CA
pa ien s had a signi ican ly highe 3-yea disease- ee su i al (DFS) compa ed o
PIK3CA mu a ed pa ien s. So, he p ognos ic alue o PIK3CA mu a ions is s ill a
ma e o deba e (Alexand o and S a on, 2014).

49
Figu e 10. The PI3K pa hway and HPV HNSCC. Rep esen a i e scheme o he PI3K–AKT–mTOR
pa hway adap a ion om (Medda, Duca and Chiocca, 2021). The HPV16 E7 p o ein binds o p o ein
phospha ase 2A (PP2A) subuni s, p e en ing hei in e ac ion wi h p-Ak and keeping i ac i e. The
E6 can ac i a e Ak , o bind TSC2, leading o i s deg ada ion and esul ing in he s imula ion o
mTORC1.
2.5 TREATMENT
The desc ip ion o cu en ea men managemen o hese pa ien s is complex
due o he a ie y o umou o igin si es, pa icula ly in he de ini ion o he su gical
op ions, wi h he in en ion o p ese a ion o he in eg i y o he su ounding
ana omical s uc u es and o ensu e ha he in ol ed o gans s ill main ain is
unc ions. Nowadays, he apeu ic decisions a e made in a mul idisciplina y
app oach, ha includes se e al speciali ies and medical a eas such as
o o hinola yngologis s, oncologis s, adia ion oncologis s, den is s, die icians,
psychologis s, and a ious he apis s in ol ed in he ehabili a ion a ea. The
a ying p esen a ions de ined o speci ic subsi e p o iles e ol e a ange o
he apies including su ge y, adio he apy, chemo he apy and mo e ecen ly
immuno he apy (Fa ah, 2021). The he apeu ic scheme mus conside he umou
ea u es in e ms o i s size, g ow h pa e n, dep h o in asion, p esence and
50
loca ion o in aded lymph nodes, p esence o ex acapsula nodal ex ension, and
pe ineu al and lympha ic in il a ion: all hese cha ac e is ics a e impo an o he
umou s aging and p ognosis, which help o de e mine su gical ma gins and he
need o pos ope a i e adju an he apy (Machiels e al., 2020). The e o e, in
gene al, ca cinomas diagnosed a an ea ly s age o he disease (I and II) a e usually
ea ed wi h isola ed he apy (su ge y o adio he apy). Howe e , o ca cinomas
diagnosed in mo e ad anced s ages (III, IVA and IVB), combined he apy (su ge y,
adio he apy and/o chemo he apy o o he s) is usually used.
A good he apeu ic esponse in low s ages o disease occu s and he majo i y o
pa ien s ha e a good p ognosis wi h a 5-yea su i al a e o 70 o 90%, in con as
o high s age disease whe e he 5-yea su i al is be ween 30 o 60% (Johnson e
al., 2020).
All pa ien s, a he end o ac i e ea men phase, equi e egula su eillance and
ollow-up, as he a e o ecu ence and appea ance o a new p ima y neoplasm is
equen wi hin umou s wi h he wo s p ognosis (Fa ah, 2021). Recu ences occu
in 80 o 90% o cases wi hin he i s 2 o 4 yea s o ollow-up. Fo ha , his pe iod
o ollow-up mus be mo e equen and ho ough. Disease’s ecu ence has a
dismal p ognosis, since he apeu ic op ions a e limi ed by he p e ious ea men
and umou biological agg essi eness esul ing ha mos o pa ien s ha e a low
mean su i al, a ound 6 o 9 mon hs.
I should be no ed, ha despi e all ea men s like su ge y, adia ion, and
chemo he apy, app oxima ely hal o he pa ien s will die o he disease.
The isk s a i ica ion o HNSCC by i s ana omic si e, s age, and his ologic
cha ac e is ics o he umou , has been con inuously in es iga ed aiming o
imp o e s a egies ega ding be e ea men o hese pa ien s.
Howe e , i should be no ed ha he inpu s o he cu en knowledge ela i e o
genomic al e a ions o gene exp ession p o iles p e iously desc ibed ha e limi ed
clinical u ili y, and managemen o mos pa ien s is s ill p edominan ly he “classic”
(Cance Genome A las Ne wo k, 2015).
In he las decades esea ch is being ac i ely explo ed on he mu a ional, genomic,
and ansc ip omic landscape and mos ecen a ailable da a show ha HPV
posi i i y is an indica o o be e o e all su i al and has a subs an ially be e
p ognosis a e he apy compa ed wi h non-HPV+ HNSCC (Mehanna e al., 2019).
The ini ial da a om HPV-posi i e H&N cance cell lines ha e shown ha cance
cells ha e an inc eased sensi i i y o adia ion, which may explain he enhanced
51
su i al o HPV-posi i e pa ien s (Nul on e al., 2017). Howe e , da a ob ained in
se e al clinical ials p o ide e idence o conside de-in ensi ied he apy o hese
pa ien s (A gi is e al., 2008; Smi h e al., 2010), bu un il now, da a do no suppo
any modi ica ion in con empo a y ea men p o ocols, educing he associa ed
mo bidi y while main aining umou con ol (Ho mann and T ibius, 2019). A la ge
p opo ion o HNSCCs is s ill le wi h limi ed ea men op ions, unde sco ing he
u gen need o iden i y no el clinical s a egies and mo e impo an ly, bioma ke s
ha could help o selec pa ien s o cu en and u u e he apies.
A be e unde s anding o he mu a ional, genomic, and ansc ip omic landscape
o HNSCC has become he p omise le e aged o explo e new he apeu ic
app oaches o manage his g oup o diseases, and i is hoped ha hese addi ional
insigh s will u he d i e he co ec s a i ica ion o pa ien s enabling hem o
apply mo e speci ic ea men s o each g oup o umou s (Fa ah, 2021).
3. K14HPV16 ANIMAL MODEL
The associa ion o HPV wi h human cance has been he subjec o ex ensi e
esea ch and conside able e o has been expended o model he diseases hey
cause (Doo ba , 2016). Cu en ly, he e a e se e al use ul in i o and in i o models
ha allow s udying he p ocess o cance p og ession and he ole o i al p o eins
in his p ocess. O e all, in i o models o epi helial cell di e en ia ion can suppo
he en i e p oduc i e li ecycle o high- isk HPV ypes (Doo ba , 2016), and in i o
models a e use ul o s udying and modula ing he unc ion o di e en cell
popula ions, which se e o p omo e an i umo ac i i y and educe p o umou
signs (San os e al., 2017).
Despi e hese a ious e o s, he cu en models o HPV-associa ed disease ha e a
ew limi a ions ha a e linked o he ac ha hese i uses only eplica e and
comple e hei li e cycle in humans, and cause cance s a disc e e epi helial si es
ha a e no s aigh o wa d o he model (Doo ba , 2016). Ne e heless, each
model has i s own po en ial and limi a ions, and adequa e models should be
chosen ca e ully (San os e al., 2017).
The K14-HPV16 in i o model is aluable o explo e componen s o he immune
esponse agains HPV-induced lesions and is p omising in he de elopmen o
no el, p e en i e and he apeu ic, s a egies (San os e al., 2017). This model o
52
HPV induced cance elies on he cy oke a in 14 (CK14) gene p omo e o d i e he
exp ession o all HPV16 ea ly oncogenes and ep oduces he mul i-s age
ca cinogenic p ocess occu ing in human disease, sha ing his ological ea u es
wi h he human lesions (Du e al., 2012).
K14-HPV16 ansgenic mice we e i s de eloped in he 1990s using s anda d
echniques by mic oinjec ion o B6D2/F2 emb yos. A K14 exp ession casse e
de eloped by E. Fuchs a he Uni e si y o Chicago was p epa ed, which con ains 2
kb o he K14 p omo e /enhance and 500 bp o a 3' lanking sequence ha
includes he K14 polyadenyla ion signal. Ins ead o he endogenous long con ol
egion, he i al genes a e egula ed by his human K14 enhance /p omo e (A bei ,
Howley and Hanahan, 1996). Th ee o ms o HPV16 DNA o he ea ly egion we e
used by cons uc ed plasmids p1203, p16N and p16P , which con ain espec i ely
a genome o wild- ype HPV16 (p1203), a genome wi h a ansla ion e mina ion
ligand (TTL) in ORF E1 a nucleo ide 1311 (p16N ) and a genome ha con ains a TTL
in ORF E2 a nucleo ide 2922 (p16P ). The agmen s we e hen excised and cloned
in o he BamHI si e o he K14 exp ession plasmid o gene a e plasmids pK14-1203
(K14-w ), pK14-16N (K14-El l) and pK14-16P (K14-E2 l). The e o e, he K14-HPV16
ansgenic mice con ain, wi hin hei genomes, he en i e HPV16 ea ly egion
(A bei , Howley and Hanahan, 1996), howe e , since he wo ea ly- egion a ian s
con ain speci ic mu a ions (E1 l and E2 l), he unc ions o he E1 and E2 genes a e
abolished and he E6 and E7 genes emained in ac in hese mu an s (A bei e al.,
1994). Fo he c ea ion o he ollowing ansgenic lines se e al inb ed backg ounds
we e ca ied ou o se e al gene a ions (minimum o i e) and main ained in
he e ozygo es s a e (A bei e al., 1994), including C57BL/6, BALB/c, and
SSIN/SENCAR gene ic backg ounds, howe e , only mice backc ossed in o he
FVB/n backg ound p og ess o malignan squamous cell ca cinomas (Coussens,
Hanahan and A bei , 1996).
The success ul a ge ing o HPV DNA o basal ke a inocy es (A bei e al., 1994)
gene a ed lesions in he same si es obse ed in cance pa ien s allowed a be e
unde s anding o he unc ions o oncop o eins in i o (A bei e al., 1993; Yang, Liu
and Iannaccone, 1995). Fo ins ance, when emale E5+/− ansgenic mice we e
ea ed wi h oes ogen, his gene was capable o inducing ce ical cance (Smi h-
McCune e al., 1997). Bu , on he o he hand, he absence o he la e i al coding
egion (L1 and L2) and he ac ha gene exp ession is egula ed by he cy oke a in
14 p omo e , in oduce signi ican di e ences compa ed o human ke a inocy es
53
in ec ed by HPV and cons i u e signi ican limi a ions o hese models (San os e
al., 2017).
Fu he mo e, his model has p o ed o be e y p o i able in se e al epi helial
cance s, and p e-malignan lesions. Mos o he ea ly s udies we e p edominan ly
pe o med on u e ine ce ix ca cinomas. Bu la e o he epo s ha e also used in
i o animal models in he s udy o o he HPV-associa ed malignancies, such as
head and neck (Jabba e al., 2010), penile (Dias e al., 2022) and anus cance s
(A bei e al., 1994).
In Head and Neck umou s, Jabba (Jabba e al., 2010) demons a ed he po en ial
impo ance o s udying cance g ow h associa ed wi h HPV a a ious s ages. They
used a chemical ca cinogen 4-ni oquinoline-1-oxide (4-NQO) o induce head and
neck cance in mice K14E6 and K14E7 ansgenic models and demons a e he E7
gene as a dominan oncogene, syne gising wi h 4-NQO. In con as , unde he
condi ion used in he p e ious s udies, E6 ansgenic mice ea ed simila ly did no
de elop HNSCC (S a i and Lambe , 2007). These esul s a e consis en wi h he
dominan oncogenic p ope ies o E7 gene in he head and neck egion.
Recen da a s udied he dis ibu ion o o al and pha yngeal lesions in K14HPV16
ansgenic mice (Mes e e al., 2020). These indings p o ided expe imen al
e idence ha HPV16 is su icien o d i e HNSCC in he absence o chemical
ca cinogens and is obse ed a di e ence in hei dis ibu ion. The HPV-d i en
lesions in he model p e e en ially a ge a clinically ele an a ea a he base o he
ongue namely a squamocolumna junc ion in he ci cum alla e papilla (Mes e e
al., 2020). Addi ionally, a di e en incidence o ongue base cance in male and
emale HPV16- ansgenic mice was ound being hese umou s mo e common in
emale mice (Ne o e al., 2021).
The K14HPV16 animal model can be qui e use ul o he s udy o o al ca cinogenesis,
Howe e , i should be poin ed ou , ha his model lacks a deepe molecula s udy,
a aluable issue o explo e he biological signi icance o he HPV16 a ian based
on clinical and pa hological obse a ions. The cha ac e iza ion o he mu a ional
p o ile wi h he iden i ica ion o he speci ic lineage and sublineage in ol ed in
K14HPV16 mice (phylogene ic analysis) would be use ul o b oaden
clinicopa hologic da a associa ion, in addi ion o imp o ing basic and ansla ional
s udies in his ield.

54
CHAPTER 2
AIM
55
AIM
The main pu pose o his s udy is o explo e he hypo hesis ha speci ic HPV16
E6E7 a ian s in luence he occu ence, he mo phological and molecula
pheno ypes o HPV-induced HNSCC in a selec popula ion and o alida e his
molecula se ing in a mouse model o HPV-induced HNSCC.
GENERAL HYPOTHESIS
Speci ic HPV16 E6 E7 a ian s iden i ied in he ca cinomas o he Head and Neck
egion may be associa ed wi h di e en mo phological and molecula pheno ypes
and wi h clinical-pa hologic se ings and molecula ma ke s o p ognosis and
esponse o he apy.
SPECIFIC OBJECTIVES
1- To iden i y and o s udy he equency o HPV16 a ian s in squamous cell
ca cinomas o he head and neck egion in a selec ed popula ion, and o desc ibe
he clinical-pa hological cha ac e is ics o HPV associa ed ca cinomas, including
umou loca ion, his ological ype, biological beha iou , and esponse o he apy.
2- To s udy gene mu a ions ypically associa ed wi h HNSCC-HPV posi i e, as well
as he exp ession o po en ial p ognos ic and he apeu ic ma ke s, ega ding HPV
a ian s iden i ied in each pa ien sample.
3- Cha ac e iza ion o o al cance in an animal model o de elop a ool o
expe imen al esea ch:
• To iden i y he HPV16 a ian (E6/E7) p esen in he K14HPV16 animal model
unde CK14 egula ion o elucida e o a mo e accu a e use o he animal
model.
56
CHAPTER 3
MATERIALS AND METHODS
57
1. CHARACTERIZATION OF THE BIOLOGICAL SAMPLES
1.1 SQUAMOUS CELL CARCINOMA OF HEAD AND NECK SAMPLES
A de ailed cha ac e iza ion o his ma e ial was done in he di e en a icles ha
we e published o submi ed o publica ion and a e p esen ed in Chap e 4-
Resul s.
The umou samples a e om a uni e se o pa ien s diagnosed wi h HNSCC,
ea ed in he Ins i u o Po uguês de Oncologia de Lisboa F ancisco Gen il
(IPOLFG) be ween 2008 and 2019 and we e included wi hin he scope o he
esea ch p ojec ( e . IPOLFG's UIC/1092), o s udy he p e alence o HPV in hese
umou ypes. The pa ien s we e clinically ollowed up by he medical s a o he
o o hinola yngology and head and neck su ge y depa men s o IPOLFG. The
diagnosis was es ablished by his ological analysis o umou issue in he Pa hology
depa men . The molecula analysis and HPV DNA de ec ion was pe o med in he
win umou sample.
A o al o 457 cases we e selec ed using ollowing selec ion c i e ia:
● His ological con i ma ion o squamous cell ca cinoma in he Head and Neck
egion (HNSCC), namely he o opha ynx (base o he ongue, so pala e,
onsils, wall) and o al ca i y (a lip, o al mucosa, mobile ongue, loo , jaw, and
ha d pala e)
● HPV DNA s a us iden i ied
● Pa ien s wi hou a p e ious diagnosis o a malignan umou ( elapse) o
synch onous diagnosis o a malignan neoplasm in ano he loca ion besides
he uppe espi a o y/diges i e ai way
● Only cases o pa ien s aged o e 18 yea s we e included
64
2.4.1.1 LABORATORY PROTOCOLS
P oduc Na u e: Sample P epa a ion
All biological samples used o he iden i ica ion o HPV by molecula
me hodologies can be used o NGS. The p e-analy ical sample p ocessing is
applied wi h he same speci ic p ocedu es o he a ious biospecimen ypes
(p oduc na u e) such as esh biopsy samples conse ed: liquid-based cy ology,
d y ozen swab samples (-80ºC), o issue de i ed om o malin- ixed pa a in-
embedded (FFPE). In gene al, all samples we e esh biopsies om umou s in he
Head and Neck egion. The DNA ex ac ion was pe o med in Au oma ic
equipmen o Manual ex ac ion, ollowing he manu ac u es speci ici ies. The
quali y and sui abili y o DNA ex ac ed was spec opho ome ically con olled
(A260/A280).
This checkpoin allows o de e mine he sui abili y o he samples: only hose wi h
an accep able ange o quan i y, pu i y and in eg i y alues a e alida ed o he
nex s ep o he NGS wo k low.
Ta ge en ichmen : PCR design and op imiza ion
Ou app oach was he ampli ica ion o he comple e HPV16 DNA genome
(~8000pb) and he p ime design was based on 47 p ime s se p e iously desc ibed
by Cullen e al 2015, which makes possible o co e he en i e genome based on
agmen ed ampli ica ion p oducing amplicons wi h a size ha anged be ween
120-359 nucleo ides. This s a egy con ibu es o a be e sensi i i y o he assay, as
opposed o op ing o a long- agmen s a egy ha lowe s he sensi i i y alues.
The sensibili y o he assay is an impo an pa ame e ha we mus o e come since
he main a ge ( i al DNA) is in a small quan i y wi hin he abundan hos genomic
DNA. An ex ensi ely e alua ion om p ime cha ac e is ics and in silico alida ion
we e pe o med. Fo he 103 p ime s ha composes he 47 p ime s se , i was
desc ibed he espec i e sequence, size, mel empe a u e, a ge egion, and
agmen size p oduced and e alua e seconda y ampli ica ions o o he o ganisms
wi h he Blas ool om PubMed (Supplemen Table 2 in silico alida ion). Fo he
pool p ime concep ion was conside he in silico alida ion and in en ion o
p omo e he bes balance be ween speci ici y and sensibili y o he assay. The bes
o 3 combina ion es ed was an app oach wi h 4 pools wi h 7 o 10 p ime each.

65
(Supplemen Table 2 Pool P ime Design): hese we e s uc u ed o ampli y he
i al whole-genome, a oiding he de elopmen o p ime -dime . Fo ha , each
p ime was single ampli ied and e i y wi hin each p ime pool o alida e
speci ici y. The PCR condi ions we e op imized o a eal- ime PCR sys em (60ºC
hyb idiza ion empe a u e) in he Applied Biosys ems Quan S udio 5 Real-Time
PCR Sys em, o a eac ion olume o 25uL. The mixed compounds we e accu a e
o wo k wi h High-Fideli y DNA Polyme ase, a p oo eading ho -s a DNA
polyme ase o high yield sensi i i y and speci ici y, being o e 300x mo e accu a e
han Taq-enzyme, esul ing in ul a-low e o a es. Valida ion was ca ied ou o
each pool p ime wi h inpu concen a ion se o 1 pmol and no malised each DNA
empla e in o ~100-250ng. The eac ion is pe o med in 3 s ages o comple e
ampli ica ion: (1) 95ºC Dena u a ion; ollowing 40 cycles o segmen (2) 60ºC
hyb idiza ion; and (3) 72ºC elonga ion. The de ec ion me hod by aga ose gel was
used o iden i y he PCR p oduc s. The 4 independen PCR p oduc s o each
sample we e pooled oge he in equal mola amoun s based on he signal
in ensi y.
Lib a y P epa a ion and Sequencing
Lib a y p epa a ion p o ocols we e based on Ampli ica ion-based NGS. The e a e
se e al di e en eac ion eagen s a ailable o speci ic p o ocols due o he
di e si y o pla o ms. A p esen , he e is no a ecommend p o ocol o any HPV
whole-genome applica ions. Ou in-house assay has been op imized o Illumina
echnology wi h he collabo a ion o he Co e NGS om INSA (Na ional Ins i u e o
Heal h). The op imiza ion o he quali y, quan i y, and agmen leng h includes
con i ma ion o expec ed esul s om posi i e con ol and alida es he
measu emen s da a o he speci ic analy ical pu pose. The pooled amplicons we e
pu i ied om small agmen s and p ime s using he AMPu e XP beads (Beckman
Coul e ) acco ding o he manu ac u e ´s ins uc ions be o e lib a y p epa a ion.
The Nex e a XT DNA lib a y p ep ki was used o p epa e bead-based no malized
dual-indexed lib a ies o sequencing. The alida ion conside ed agmen s size o
clus e e iciency and concen a ion o he p epa ed lib a ies since low
concen a ion may esul in o a low numbe o sequenced eads. The lib a y pool
was sequenced on a MiSeq bench op sequence using a eagen ki V3 (illumina
MS-102-3001). A sequencing s a egy wi h 250-bp pai ed-end eads was used.
66
2.4.1.2 BIOINFORMATIC DATA ANALYSIS
NGS bioin o ma ics pipelines consis s in a speci ic pla o m o analyse sequence
consensus single nucleo ide a ian (SNV) and may be cus omizable based on
labo a o y needs. The Bioin o ma ics Analysis o NGS Da a we e de eloped wi h
Co e bioin o ma ic INSA collabo a ion. P ima y analyses we e pe o med on
INSaFLU (h ps://insa lu.insa.p /) an online pla o m o amplicon-based nex -
gene a ion sequencing da a analysis (Bo ges e al., 2018) and used o eads’ quali y
con ol, a ian de ec ion/inspec ion and sequence consensus gene a ion. The
genome sequence o a ep esen a i e o HPV16 sublineage A1 (GenBank accession
numbe NC001526.4) was used as a e e ence o mapping and SNV anno a ion.
Regions wi h a dep h o co e age below 10- old we e au oma ically masked in he
INSaFLU pipeline by placing unde ined bases “N” in he consensus sequence. Low-
co e age egions we e isually inspec ed using In eg a i e Genomics Viewe (IGV).
SNVs we e assumed in consensus when hey displayed mo e han 50% in a-
sample equency. MEGA11 so wa e (h p://www.megaso wa e.ne ) (Tamu a, Nei
and Kuma , 2004) was applied o calcula e ma ices o nucleo ide dis ances and o
pe o m phylogene ic econs uc ions.
2.4.2 ANALYTICAL VALIDATION
The pe o mance cha ac e is ics o his me hod we e alida ed based on da a
measu emen s om Posi i e Con ol, Nega i e Con ol, and In e nal Con ol. The
cell line SiHa, posi i e o HPV16, was used as posi i e con ol wi h 100%
conco dance and we e included in each un o e alua e PCR e iciency (HPV16 A
lineage (GenBank:AF001600.1). The nega i e con ol (No Ta ge Con ol) p o ides
insigh ega ding chemical eagen s con amina ion; Albumin ha was also used
as an in e nal con ol o dis inguish ue a ge nega i es om PCR ailu e o
inhibi ion.
To e alua e he assay pe o mance: he p ecision o he me hod was es ablished
by pe o ming posi i e e e ence con ol o 10 epea s and he assay pe o mance
esul s we e analysed by aga ose gel. To es ima e e iciency sensibili y he posi i e
con ol was quan i ied and dilu ed 10x se ies in o 4 poin s be ween 5 o 5000
copies. The assays demons a ed a wide dynamic ange e iciency and can
67
analy ically de ec a 5 HPV16 copies/PCR. The speci ici y was accu a e o each
pool p ime , which he speci ic a ge was analysed h ough agmen size in
aga ose gel wi h Bio ad geldocX ool. The ep oducibili y was based on 10
eplica es o he posi i e con ol and has demons a ed excellen ep oducibili y
in e and in a-assay (Supplemen Table 3).
2.4.3 IMPLEMENTATION AS ROUTINE PROCEDURE
Fo he assay implemen a ion as a ou ine p ocedu e, se e al ac ions we e
es ablished based on speci ic equi emen s ace o quali y con ol me ic as
desc ibed in Table 2 o each sec ion o he wo k low.
Table 2. Implemen a ion and Quali y Con ol Me ics
NGS Wo k low sec ions
Requi emen s
Ac ion
Sample P epa a ion
Biological Specimen
P e-analy ical sample p ocessing
DNA
Moni o ing quan i y and pu i y
PCR en ichmen
Reac ion pe o mance
Posi i e con ol ampli ica ion HPV16 in ec ed
cell line Siha o CaSki
Pool P ime E iciency
Moni o ing amplicons concen a ion (signal
in ensi y)
HPV16 Whole-genome
Speci ici y
Moni o ing F agmen size (mig a ion posi ion)
Ta ge in eg i y
In e nal con ol ampli ica ion albumin gene
Lib a y P epa a ion and
Sequencing
Lib a y Quali ica ion and
Quan i ica ion
Moni o ing numbe o eads
Da a analysis
Bioin o ma ic pipeline
S anda diza ion o analy ical pa ame e s
Biologic signi icance
Da a sys ema iza ion in o a da abase
68
2.5 DETECTION OF MUTATIONS OF THE PIK3CA GENE
Assessmen o he PIK3CA gene mu a ions we e pe o med by eal- ime PCR assay
(Amoy Dx) o he quali a i e de ec ion o ou mu a ions (H1047L; E542K, E545K
and E545D) a ge in exon nine and exon wen y om he human genomic DNA
ex ac ed om umou issue. The assay comp ises speci ic p ime s and
luo escen p obes. Du ing he nucleic acid ampli ica ion, he a ge ed mu an
DNA is de ec ed by FAM-labelled p obes. I used an in e nal con ol sys em
con aining p ime s and a HEX-labelled p obe o a egion o genomic DNA wi hou
known mu a ions and polymo phisms. All he p ocedu es o p e-analy ical,
analy ical and da a in e p e a ion we e pe o med acco ding o he
manu ac u e 's speci ica ions.
Fo he PCR condi ions, he p epa a ion o he eac ion componen s (mas e mix)
was op imized o he elu ed DNA e alua ed. This amoun was no malized o 15 ng
o empla e DNA o all samples. The di e en compounds PIK3CA Reac ion Mixes,
Mixed S anda d and Taq DNA polyme ase we e combined a di e en
concen a ions (ng/µL) o a inal olume o 20µl. The e o e ampli ica ion we e
pe o med on qPCR BioRad CFX96 equipmen wi h Cycling Pa ame e s
p og ammed o 3 s ages de ined by Tempe a u e, Time and Cycles: S age 1
de ined by 1 cycle a 95 ℃ 5 min 1; S age (2) de ined in 15 cycles a 95 ℃ 25 s, ollow
dec ease o 64 ℃ 20 s 15 and inc ease o 72 ℃ 20 s; and S age (3) de ined in 31 cycles
a 93 ℃ 25 s, ollow dec ease 60 ℃ 35 s (Da a collec ion o FAM and HEX/VIC) and
inc ease o 72 ℃ 20 s. Analysis da a un au oma ically in so wa e o check he FAM
and VIC C alue o each sample. Based on di e en mu an C alues, he
de ec ion esul s a e di ided in o s ong posi i e, weak posi i e o nega i e.
69
CHAPTER 4
RESULTS

70
1. DESCRIPTIVE ANALYSIS OF THE STUDY SERIES
1.1. IDENTIFICATION OF HPV DNA IN HNSCC SAMPLES
F om a o al o 457 pa ien ´s wi h he diagnosis o HNSCC, HPV was ound in 34.4%
(n=157) o he cases. The HPV geno ype was achie ed o 101 HNSCC-HPV despi e
56 cases whe e geno yping was inconclusi e. Single in ec ion was p esen in 93
cases which he mos equen ype was HPV16 (n=68), ollowed by HPV18 (n=4),
HPV35 (n=4), HPV31 (n=2), HPV53 (n=3), HPV33 (n=2), HPV51 (n=2), HPV56 (n=2),
HPV59 (n=2), HPV6 (n=2), HPV52 (n=2) and HPV26 (n=1) (G aphic 1). In 5.7% (n=8) o
he cases, mo e han one ype o HPV was de ec ed - coin ec ion, whe e HPV16 was
p esen wi h o he HPV ypes bo h low- isk and/o high- isk ypes.
G aphic 1. F equency HPV ypes o single-in ec ion cases (n=93).
Legend: colou ed ed - High-Risk Types; colou ed g ey - Low-Risk Types
71
1.1.1 DEMOGRAPHIC CHARACTERISTICS
The age a diagnosis o HNSCC-HPV pa ien s ange be ween 36 and 91 yea s wi h
a median o 61 yea s. Bo h gende s a e p esen wi h 76.4% (120/157) males and 23.6%
(37/157) emales (G aphic.2-A). In ou coho , he gende dis ibu ion is simila in
bo h g oups HNSCC-HPV and HNSCC-nonHPV wi h a male o emale a io o 3:1.
The median age o 61 yea s was also eco ded o HNSCC-nonHPV g oup, which
anged om 27 o 93 in HNSCC-nonHPV, wi h 76.3% (229/300) o males and 23.7%
(71/300) emales (G aphic.2-B).
G aphic 2. Age a diagnosis pe g oup HNSCC-HPV (A) and HNSCC-nonHPV (B) s a i ied by
pa ien s' gende . The da a e e s o he o al numbe o pa ien s diagnosed wi h HNSCC in las
decades (2008 and 2019) in IPOLFG.
1.1.2 CONSUMPTION HABITS
The consump ion habi s we e e alua ed acco ding o sel - epo ed in he clinical
iles. Da a analysis was based on s a i ica ion by ne e d ank, ex-d inke , spo adic,
mode a e, and se e e d inke o Alcohol exposu e. Tobacco consump ion was
de ined in uni s as one pack/yea (equal o one pack o ciga e es/day/yea , wi h 20
ciga e es/pack) and classi ied as cu en o hea y smoke s: cu en obacco use s
included hose who used obacco ≤ 30 pack/yea s and hea y use s we e hose who
smoked > 30 pack/yea . Pa ien s wi hou consump ion habi s we e de ined as no
ha ing consumed none o hese subs ances p io o cance diagnosis. O he o al
o he 457 pa ien s included, 41 pa ien s had missing in o ma ion in iles. The
analysis om he emaining 416 pa ien s, conside ed wo g oups ac i e
consump ion s. no consump ion. Mos pa ien s ha e an ac i e consump ion (82%;
72
n=341/416) wi h a consump ion p ac ice o bo h obacco and alcohol. On he o he
hand, 18% (n=75/416) o he pa ien s sel - epo ed no consump ion habi s. The
analysis o he dis ibu ion o HPV by consump ion habi s (G aphic 3) shows ha a
highe ac i e consump ion among HNSCC-nonHPV pa ien s [65% (n=223/341)]
compa ed o he consump ion obse ed among HNSCC-HPV pa ien s [35%
(n=118/341)]. The analysis o consump ion by gende e eals ha he no-
consump ion g oup is mos ly ep esen ed by emales (Table 3).
G aphic 3. Cha ac e iza ion o HNSCC pa ien s' consump ion habi s by HPV de ec ion
Table 3. Cha ac e iza ion o HNSCC pa ien s’ consump ion habi s by HPV and by gende
HNSCC-HPV
HNSCC-nonHPV
Consump ion
n
male
emale
male
emale
no consump ion
75
7
20
21
27
ac i e
341
105
13
190
33
no da a
41
8
4
18
11
To al
457
120
37
229
71
73
1.1.3 TUMOUR SITES
The Table 4 shows he dis ibu ion o he di e en umou loca ions. Tumou s we e
in di e en subsi es o he o opha ynx [45.3% (207/457)] and o al ca i y [54.7%
(250/457)]. The si e o he umou om HNSCC-HPV pa ien s is mo e equen in
he o opha ynx wi h 55% cases (86/157), unlike HNSCC-nonHPV pa ien s which he
umou si e is mo e equen in he o al ca i y wi h 60% cases (179/300).
Table 4. Dis ibu ion o umou si e by HPV de ec ion
Tumou si e
HNSCC-HPV
HNSCC-nonHPV
o opha ynx
207
86
55%
121
40%
base ongue
25
15
17%
10
8%
so pala e
46
10
12%
36
30%
onsil
118
56
65%
62
51%
o opha ynx wall
18
5
6%
13
11%
o al ca i y
250
71
45%
179
60%
ha d pala e
5
2
3%
3
2%
o al mucosa
42
11
15%
31
17%
o al loo
52
13
18%
39
22%
ongue
151
45
63%
106
59%
To al
457
157
300
1.1.4 TUMOUR HISTOLOGY
All cases included in his se ies we e con i med as in asi e squamous cell
ca cinoma in he his ological analysis. Tumou we e diagnosed in di e en s ages
o disease a ime o diagnosis (Table 5). The dis ibu ion ega ding s age o disease
e ealed ha umou s we e p edomina ely in s age II and I, ollowed by s age III
and ew cases we e diagnosed a s age IV.
80
Cochichoe al. Vi ol J (2021) 18:217
h ps://doi.o g/10.1186/s12985-021-01688-9
REVIEW
Explo ing he oles o HPV16 a ian s inhead
andneck squamous cell ca cinoma: cu en
challenges andoppo uni ies
Daniela Cochicho1,2†, Rui Gil da Cos a3,4,5† and Ana Felix1,6*†
Abs ac
The incidence o squamous cell ca cinomas o he head and neck (HNSCC) is consis en ly inc easing, in associa ion
wi h human papilloma i us (HPV) in ec ion, especially HPV16. HPV a ian s show he e ogenei y in he pa hogenici y
o ce ical cance , bu li le has been es ablished abou hei ele ance on HNSCC. This e iew add esses he dis ibu-
ion o HPV16 a ian s in HNSCC and hei po en ial con ibu ion o clinical p ac ice. A sea ch was pe o med in Pub-
Med using he keywo ds HNSCC HPV16 a ian s. Six y a icles we e iden i ied be ween 2000 and 2020 and 9 a icles
we e selec ed o a sys ema ic analysis. Clinical coho s comp ised 4 o 253 pa ien s aged be ween 17 and 91 yea s
wi h con i med HPV16-posi i e HNSCC. Samples we e collec ed om esh biopsies o he umou , o al inse o o mol
ixed/pa a in embedded issue, om he o al ca i y, o opha ynx, hypopha ynx, la ynx and Waldeye ’s onsilla ing.
HPV16 a ian s we e iden i ied using Sange sequencing echniques. Se en s udies add essed he HPV16 E6 gene,
one s udied E6 and E7, ano he s udied L1 and one ocused on he long con ol egion. Eu opean a ian s ep e-
sen 25–95%, Asian-Ame ican 5–57% and A ican 2–4% o he o al isola es, sugges ing a ma ked p edominance o
Eu opean s ains. No co ela ions could be d awn wi h pa ien p ognosis, pa ly because many s udies elied on small
pa ien coho s. Addi ional s udies a e needed, pa icula ly hose employing nex gene a ion sequencing echniques
(NGS), which will allow as e and accu a e analysis o la ge numbe s o samples.
Keywo ds: HNSCC, HPV16, HPV16 a ian s, Nex gene a ion sequencing
© The Au ho (s) 2021. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which
pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he
o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o
o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line
o he ma e ial. I ma e ial is no included in he a icle’s C ea i e Commons licence and you in ended use is no pe mi ed by s a u o y
egula ion o exceeds he pe mi ed use, you will need o ob ain pe mission di ec ly om he copy igh holde . To iew a copy o his
licence, isi h p:// c ea i eco mmons. o g/ licen ses/ by/4. 0/. The C ea i e Commons Public Domain Dedica ion wai e (h p:// c ea i eco
mmons. o g/ publi cdoma in/ ze o/1. 0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed in a c edi line o he da a.
Backg ound
Head and neck cance is he six h leading cance by
incidence wo ldwide [1] and comp ises many di e en
pa hological en i ies. The diagnosis is o en cha ac e -
ized by mul i ocal de elopmen and p esen a ion a an
ad anced s age. [2] The age a diagnosis usually anges
be ween 50 and 70yea s o age and men a e signi ican ly
mo e likely o de elop he disease han women (up o a
4:1 p opo ion, depending on geog aphical localiza ion)
[3]. Cu en ly, wo majo ca cinogenic pa hways leading
o head and neck squamous cell ca cinomas a e ecog-
nized: he i s is associa ed wi h isk ac o s like smok-
ing and abusi e alcohol consump ion while he second is
associa ed wi h human papilloma i us (HPV) in ec ion
[4, 5]. O e he pas decade, se e al epidemiologic s ud-
ies epo ed a 36.5% inc ease in he incidence o head and
neck squamous cell ca cinomas (HNSCC), pa icula ly
in high-income coun ies and among men < 60yea s [4,
6–8]. A ailable da a indica es ha hese changes spe-
ci ically in ol e o opha yngeal cance s [9, 10]. Al hough
obacco consump ion has dec eased, he incidence o
HPV-posi i e o opha yngeal cance s has inc eased [11],
indica ing ha HPV in ec ion is he unde lying cause
o he o e all inc ease in HNSCC incidence [12–14].
Open Access
*Co espondence: ana. [email protected]
†Daniela Cochicho, Rui Gil da Cos a and Ana Felix con ibu ed equally o
his wo k
1 NOVA Medical School, NOVA Uni e si y o Lisbon, CEDOC, Campo
Má i es da Pá ia 130, 1169-056 Lisbon, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle

81
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Cochichoe al. Vi ol J (2021) 18:217
Pa ien s showing o al HPV in ec ion a e 53 imes mo e
likely o de elop HNSCC [15]. P esen ly, HPV is p esen
in app oxima ely 35% o HNSCC and mos HPV-posi i e
cases eme ge in lingual and pala ine onsils [16]. The
p esence o HPV DNA in umou cells de ines a speci ic
pa hologic en i y wi hin HNSCC wi h speci ic epidemi-
ology, molecula cha ac e is ics, and biological beha -
iou [17]. HPV in e e es wi h key signaling pa hways
o p omo e ca cinogenesis ia i s i al oncop o eins E6
and E7, which lead o he inac i a ion o umou p o ein
53 (p53) and he e inoblas oma p o ein (pRB), espec-
i ely [18]. HPV is a g oup o small, double-s anded
DNA i uses ha in ec he epide mis and ke a inizing
mucosae. Mo e han 100 HPV ypes ha e been iden i ied
[19, 20]. P esen ly, 40 di e en HPV ypes a e known o
in ec mucosal epi helia and a e ca ego ized in o low- isk
and high- isk HPV ypes acco ding o hei epidemio-
logic associa ion wi h ce ical cance [21]. In HNSCC,
he majo i y o HPV ypes belong o his g oup includ-
ing HPV16, HPV18, HPV39 and HPV45 [22]. HPV16 is
by a , he mos de ec ed ype, accoun ing o 90% o all
HPV-posi i e HNSCC cases [23], a signi ican ly g ea e
p opo ion han in ce ical cance whe e i accoun s
o li le o e 50% o cases [24]. Sequencing o he HPV
genome also e ealed in a- ype a ian s wi h gene ic di -
e ences anging be ween 0.5 and 1% [25, 26]. HPV a i-
an s a ise mainly om nucleo ide subs i u ions in some
es ic ed posi ions in he genome coding egion o in he
noncoding egion [27, 28]. The p e alence o a ian s o
each HPV ype a ies signi ican ly in di e en geog aphi-
cal a eas [27]. Rega ding HPV16, whole-genome analysis
allowed he cha ac e iza ion o i e dis inc phylogene ic
clus e s named acco ding o hei o iginal geog aphical
dis ibu ion: Eu opean (E), Asian (As), Asian-Ame ican
(AA), A ican 1 (A 1) and A ican 2 (A 2) [29–31]. Sub-
sequen ly, a new b anch, No h Ame ican 1 (NA1) was
iden i ied [32, 33]. Cu en ly, eme ging epidemiological,
e iological, and molecula da a sugges ha in a- ype
HPV a ian s a e biologically dis inc and may be asso-
cia ed wi h di e en isk o ce ical cance p og ession
[28]. In i o s udies using 3D o gano ypic epi helial
cell cul u es showed ha HPV16 E6 a ian s di e in
hei abili y o abolish ke a inocy e di e en ia ion and
o induce p53 deg ada ion [34–36]. Those expe imen al
esul s a e co obo a ed by clinical s udies on ce ical
cance . The T350G (L83V) HPV16 a ian is he mos
equen ly ound among in asi e ce ical cance s [21,
37] and has been linked o a highe oncogenic po en ial
han he p o o ype [38], possibly by acili a ing pe sis-
en i al in ec ion, a c i ical ac o o cance de elop-
men [39–41]. Despi e he g owing numbe o s udies
conce ning HPV16 a ian s on ce ical cance , he e a e
cu en ly e y ew epo s desc ibing hei dis ibu ion
in HNSCC and no hing is es ablished abou hei impac
on he de elopmen , ea men esponse and impac on
disease ou come. None heless, HPV16 a ian s may also
play an impo an ole in head and neck ca cinogenesis
and s udying hei impac is needed due o he ising
incidence o HPV-posi i e HNSCC [18, 23, 25, 42]. The
pu pose o his wo k is o sys ema ically e iew he dis-
ibu ion o HPV16 a ian s in HNSCC and o assess he
a ailable knowledge conce ning hei po en ial con ibu-
ion o HNSCC pa hologic he e ogenei y in published
s udies.
Main ex
Re iew compila ion da a
A sys ema ic e iew was pe o med on he PubMed elec-
onic da abase (h ps:// pubmed. ncbi. nlm. nih. go ) using
he ollowing keywo ds sea ch c i e ia: HNSCC HPV16
a ian s. Obse a ional s udies epo ing he dis ibu ion
o HPV16 a ian s in HNSCC and published be ween
2000 and 2020 we e included. Re iew a icles, case
epo s and a icles dealing wi h o he ypes o cance
we e excluded (Fig.1). The selec ion sea ch c i e ia we e
based on umou localiza ion, na u e o sample and back-
g ound o he a ian s udy ield. The s udies we e inde-
penden ly assessed o iden i y he p e alence o HPV16
a ian s in HNSCC and e alua e in espec i e coho s o
co ela ion wi h clinicopa hological pa ame e s. HNSCC
Fig. 1 Analy ical low o sys ema ically e iewing published a icles
dealing wi h HPV16 a ian s in HNSCC
82
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Cochichoe al. Vi ol J (2021) 18:217
pa ien s we e ca ego ized in o di e en si es (o al ca i y
and o opha ynx) and u he in o sub coho s based on
he demog aphic and clinical in o ma ion p o ided. The
published da a we e summa ized using equencies and
pe cen ages o HPV16 a ian s, s a i ied by umou
loca ion, ypes o umou samples, pa ien coho size,
sequencing me hodology, and clinical a iables such as
age and gende (Fig.2.)
Gene al indings
F om 61 eco ds ound using he sea ch c i e ia HNSCC
HPV16 DNA cell ca cinoma a ian s equency pa ien s’
coho s, we selec ed 9 s udies while 52 s udies we e
excluded om u he analysis, including e iew a i-
cles and pape s which i he exclusion c i e ia (Fig.1).
Selec ed a icles epo da a om a o al o 945 pa ien s
in he pe iod be ween 2000 and 2020, dis ibu ed by 6
coun ies. (Table1) Th ee s udies we e pe o med in
Eu ope, h eein he USA, wo in Asia and Middle Eas
(Japan and I an) and one in Sou h Ame ica (B azil).
HPV16 a ian s we e e alua ed in nine di e en clini-
cal coho s. All s udies we e di e en and did no con-
ain he e alua ion o he same cases. O e all, he s udy
popula ion consis ed o pa ien s whose age a diagnosis
anged be ween 17 and 91yea s. The gende a io was 1:3
o 1:4 ( emale o male), and all cases we e his ologically
con i med as HNSCC. The specimens used o e alua e
HPV16 we e p ima y umou s loca ed in he o al ca -
i y, o opha ynx, hypopha ynx, la ynx and Waldeye ’s
onsilla ing. Regional lymph node me as ases we e also
included in one s udy. The samples used o analyze we e
di e se and consis ed o esh issue, o al inse, o o ma-
lin ixed/pa a in embedded issue.
The use o he p16INK4a ma ke is o clinically de ec
an oncogenically ac i e HPV in ec ion and is conside ed
as a su oga e ma ke o HPV in ec ion [43, 44]. F om
he 9 s udies e alua ed h ee desc ibed he exp ession
pa e n o p16INK4a among nega i e HNSCC HPV(−)
and HNSCC HPV16 cases. Fo all s udies he e was a
signi ican di e ence in p16INK4a exp ession be ween
HNSCC HPV(−) and HNSCC HPV16(+). Pakdel e al.
2020 obse ed he exp ession o p16INK4a among HPV
a ian s and showed ha a s ong p16INK4a exp es-
sion was p esen in poo ly di e en ia ed umou issues
in ec ed wi h HPV16 sublineage A2 [45].
HPV de ec ion andgeno yping
HPV was de ec ed using PCR-based echniques
employing consensus degene a e p ime s: MY09/11 (4
s udies) MY09/11 and/o GP05/06 (2 s udies), PGMY
(one s udy) and SPF1/2 (one s udy); all o which a e
complemen a y o he conse ed L1 egion. One s udy
de ec ed HPV using insi u hyb idiza ion. HPV geno-
yping was pe o med using di e en me hods. Two
s udies using a comme cial ki (INNO LIPA) which
iden i ies 28 di e en HPV ypes (6, 11, 16, 18, 26, 31,
33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68,
69, 70, 71, 73, 74, 82) by e e se blo hyb idiza ion. Two
o he s udies used TaqMan PCR me hods a ge ing he
E6 o E6/LCR. One s udy used es ic ion agmen
leng h polymo phism (RFLP) analysis. Two s udies pe -
o med di ec Sange sequencing o he PCR p oduc s.
The HPV equency in he di e en coho s anged
om 10 o 100% o he cases, and he HPV16 dis i-
bu ion anged be ween 67 and 100% o all HPV posi-
i e cases (Table1). Excep he s udy done in 2020 in
Fig. 2 Analy ical a iables low o included a icles dealing wi h HPV16 a ian s in HNSCC sea ch c i e ia
83
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Table 1 Iden i ica ion o HPV16 a ian s in HNSCC in he li e a u e (PubMed 2000–2020)
E6 Ea ly gene 6; E7 ea ly gene 7; LCR long con ol egion; L1 la e gene 1; FF/PE Fo malin ixed Pa a in embedded
Yea
publica ion Re e ence Coun y Mean age
pa ien s
(y s)
Gende (%
male) Consump ion
(% ac i e) Samples To al cases
(n) To al
cases HPV
de ec ed
%(n)
To al cases
HPV16
de ec ed
%(n)
To al
isola es
o HPV
sequencing
Genome
egion
loca ion
Amplicon
size (pb) Sequencing
2000 [48] USA 63 no da a
a ailable 87% F esh issue 253 24% (62) 90% (56) 52 E6 455 Sange
2004 [49] Ge many no da a
a ailable no da a
a ailable no da a a ail-
able FF/PE 24 100% (24) 100% (24) 21 E6 and E7 793 Sange
2007 [50] I aly 63 74% 67% F esh issue
FF/PE 115 18% (21) 67% (14/21) 13 L1 150 Sange
2008 [51] USA 57 77% 76% O al inse
FF/PE 135 32% (44) 100% (44/44) 19 E6 609 Sange
2009 [52] Japan 64 86% 79% F esh issue 77 10% (8) 100% (8/8) 8 E6 323 Sange
2013 [53] B azil 59 100% 50% FF/PE 4 100% (4) 100% (4/4) 4 E6 no da a
a ailable Sange
2015 [54] I aly 65 88% 63% FF/PE 24 100% (10) 100% (10/10) 10 E6 no da a
a ailable Sange
2016 [55] USA 60 86% No da a a ail-
able FF/PE 205 18% (36) 70% (25) 21 LCR 193 Sange
2020 [56] I an 56 58% No da a a ail-
able FF/PE 108 23% (25) 16% (17) 13 E6 no da a
a ailable Sange
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Cochichoe al. Vi ol J (2021) 18:217
I an, whe e jus 16% o he HPV de ec ed we e HPV16.
The mos common HPV in hei se ies was HPV16 bu
also ollowed by HPV18 and HPV11 and he cases we e
almos loca ed in O al ca i y and La ynx; only 6 onsils
in all 108 cases we e e alua ed, explaining he low HPV
equency o e all.
Iden i ica ion HPV16 a ian s
O he 9 s udies, se en s udies iden i ied HPV16 a i-
an s by sequencing he E6 gene, one add essed a ian s
in he L1 gene and ano he he long con ol egion. In
one s udy he E7 gene was also sequenced along wi h
E6 (Table1). In all s udies, he iden i ica ion o HPV16
a ian s was pe o med by Sange sequencing ech-
niques and by compa ison o he e e ence sequence
(p o o ype). Nex gene a ion sequencing (NGS) was
no used in any o he s udies e alua ed. Howe e , he
eliance on con en ional Sange sequencing is likely
o limi he numbe o cases ha can be analyzed and
he eliabili y o esul s. [26, 46, 47] Sange sequenc-
ing echniques show limi a ions conce ning he size o
amplicons, which may comp omise he assay sensi i -
i y. Addi ionally, o o e come polyme ase e o s and
gua an ee he in eg i y and obus ness o he esul s,
i is necessa y o pe o m eplica es o each case o
ec o molecula assays [48]. Such eplica es add sig-
ni ican ly o he wo kload and cos s and cons i u e
a limi a ion o analyze la ge pa ien coho s. In ac ,
only wo ou o nine s udies included o e 200 HNSCC
pa ien s and none analyzed mo e han 52 HPV-posi i e
pa ien s o HPV16 a ian s. Fu u e s udies aiming o
s udy la ge pa ien coho s a e likely o bene i om
NGS echniques, which p o ide as e and eliable
sequencing esul s o la ge amplicons [25, 26, 46].
P e alence o HPV16 a ian s
The equency o each HPV16 a ian (E, NA, AF, AS and
AA) was es ima ed o each o he 9 s udies included in
he sys ema ic e iew (Table2). Gillison e al. [37] i s
epo ed da a conce ning he dis ibu ion o HPV16 E6
a ian s in HNSCC. The au ho s e alua ed 52 HPV16-
posi i e pa ien s om an o e all HNSCC coho com-
p ising 259 pa ien s. The age a diagnosis anged be ween
17 and 91yea s-old (median, 63yea s-old) and he
majo i y we e smoke s, wi h o wi hou alcohol con-
sump ion (87%). Tumou s we e localized in he naso-
pha ynx (n = 2), o al ca i y (n = 84), o opha ynx (n = 60),
hypopha ynx (n = 21), la ynx (n = 86). HPV-posi i e
pa ien s showed signi ican ly imp o ed disease ou comes
compa ed wi h HPV-nega i e pa ien s. All samples used
we e HNSCC esh umou specimens. The HPV16 a i-
an s we e classi ied in o he same phylogene ic g oup as
he Eu opean p o o ype in 75% o cases, Asian in 17%,
No h Ame ican in 4.0% and A ican 1 in 4.0% o cases.
Six no el a ian s no p e iously epo ed (E-G315T,
E-G315G, E-C395G, E-A478T, E-A132T, A 1-C311,
A 1-A389) we e also iden i ied. The au ho s ema ked
ha he dis ibu ion o a ian s in HNSCC esembled
ha obse ed in ce ical cance . Howe e , no conclu-
sions we e d awn conce ning he po en ial con ibu-
ion o speci ic HPV16 E6 a ian s o inc easing cance
isk o modi ying umou biopa hology and p ognosis.
Fou yea s la e , Ho mann e al. [38] iden i ied HPV16
a ian s in 7 ou o 21 umou specimens o HNSCC.
The au ho s analyzed he E6 and E7 ORFs. Al oge he ,
he DNA samples ca ied HPV16 p o o ype Eu opean
Table 2 F equency o HPV16 a ian s
(–) no e alua ed; AA Asia-Ame ican HPV16 Va ian ; AF A ican HPV16 Va ian ; AS Asian HPV16 a ian ; E Eu opean HPV16 a ian ; E6 HPV Ea ly p o ein 6; E7 HPV Ea ly
p o ein 7; LCR long con ol egion; L1 la e gene; NA No h Ame ican HPV16 Va ian
Re e ence To al cases
HPV16
de ec ed (n)
To al
isola es HPV
sequencing (n)
HPV16
a ian egion
loca ion
Eu opean (E) No h
Ame ican A ican Asian Asia Ame ican
All
lineages
(n)
E-350-G (n) (NA) (n) (AF1|AF2) (n) (AS) (n) (AA) (n)
[48] 56 52 E6 39 6 2 2 9 0
[49] 24 21 E6 E7 15 8 0 0 0 0
[50] 14 13 L1 9 0 1 2 0 1
[51] 44 19 E6 18 4 0 0 1 0
[52] 8 8 E6 5 5 0 0 0 0
[53] 4 4 E6 2 1 0 0 0 2
[54] 10 10 E6 8 8 0 0 0 0
[55] 25 21 LCR 9 – 0 0 0 12
[56] 17 13 E6 11 – 0 0 0 2
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Cochichoe al. Vi ol J (2021) 18:217
a ian (29%), he Eu opean a ian T350G (38%) and
33% Eu o-Ge man a ian (A131G + C712A). Again,
no conclusions could be d awn conce ning he clinical-
pa hological ele ance o he HPV16 a ian s iden i ied,
pa ly because o he coho ’s small size. Bada acco e al.
[49], published molecula analysis da a on he HPV16 L1
ORF, om an I alian coho ( o al n = 115, o which only
13 we e es ed o HPV a ian s), composed o 86 men
and 29 women, wi h a mean age o 63.21yea s old. A high
pe cen age o pa ien s we e smoke s (67%) and alcohol
d inke s 43%. Tumou s we e localized mos ly in he o al
ca i y (n = 60), ollowed by he la ynx (n = 30), he o o-
pha ynx (n = 10), he onsil a ea (n = 8), he hypopha -
ynx (n = 5) and he sinus/nose (n = 2). In his s udy, he
p esence o HPV was no signi ican ly associa ed wi h
disease- ee su i al a 2yea s. Six y nine pe cen o he
cases (13 cases) analyzed showed Eu opean-Ge man
a ian s (9 cases), 15% we e A ican ype 2 (2 cases),
8% Asian-Ame ican (1 case), and he emaining 8% (1
case) had an unclassi ied a ian . The p edominance
o he Eu opean a ian was unsu p ising conside ing
he I alian o igin o he pa ien s. No co ela ions we e
d awn be ween he p esence o HPV16 a ian s and any
epidemiological, pa hological, o clinical da a. Ag wal
e al. [50], s udied 19 HPV16 isola es om a uni e se o
135 HNSCC samples. The median age a diagnosis was
57yea s-old, 77% o he pa ien s we e men, mos pa ien s
had a his o y o smoking (n = 62 smoke s e sus 51 non-
smoke s) bu we e non-d inke s (73 non-d inke s e sus
41 d inke s). All analyzed cases showed Eu opean a i-
an s and a single case ca ied an Asian a ian . The mos
common Eu opean a ian was E-350T (n = 6), ollowed
by E-350G (n = 4) and E-T131G (n = 2). Impo an ly,
eigh o he 19 isola es con ained Eu opean a ian s
wi h sequences unique o a single indi idual. Boscolo-
Rizzo e al. [51] analyzed HPV E6 a ian s in a sho
(n = 8) case se ies. The au ho s showed he p esence o
he T350G mu a ion in 5 cases loca ed in he o al ca -
i y and o opha ynx, while h ee umou s loca ed in he
la ynx and hypopha ynx con ained HPV 16 p o o ype
sequences. Joseph e al. [52] compa ed HPV16 a ian s
p esen in ou pa ien s wi h bila e al onsilla HNSCC.
Two cases ca ied Eu opean a ian s while wo o he s
ca ied Asian-Ame ican a ian s. The esul s show ha ,
in all 4 pa ien s, he same HPV16 a ian was p esen in
he bila e al umou s, suppo ing he hypo hesis ha a
single HPV in ec ion, a he han independen in ec ions
wi h dis inc agen s, is esponsible o hose bila e al
umou s. Hassani e al. [53] s udied he HPV16 a ian s
p esen in 10 cases o onsilla HNSCC. The au ho s
ound ha he E-350G- a ian was p esen in 80% o
cases while he Eu opean p o o ype was iden i ied in he
o he 20%. Again, his sho case se ies did no p o ide
da a conce ning he pa hobiological ele ance o HPV16
a ian s. In he ollowing yea , Be iol e al. [54] epo ed
he dis ibu ion o HPV16 a ian s in a B azilian coho
o 21 HNSCC pa ien s. The au ho s analyzed he HPV16
LCR and ound ha 12 (57.1%) pa ien s ca ied Eu o-
pean and 9 ca ied Asian-Ame ican (42.9%) a ian s.
The au ho s ema ked ha he sligh p edominance o
Eu opean a ian s accompanied obse a ion om hei
no mal ce ical samples, sugges ing ha he dis ibu-
ion o HPV16 a ian s e lec s he o e all equency in
each s udied popula ion. The mos ecen s udy was pub-
lished by Pakdel e al. [45]. Thi een HNSCC issue speci-
mens es ed posi i e o HPV16 using o e lapping PCR
assays and we e analyzed o he p esence o E6 a ian s.
The e was a ma ked p edominance o Eu opean a ian s
(84.6%) ollowed by Asian-Ame ican (15.4%) a ian s.
Discussion
We e iewed he published li e a u e on HPV16 a ian s
in HNSCC, aiming o e alua e he biological meaning
o hose a ian s in his pa icula loca ion. The clini-
cal managemen o HNSCC imp o ed g ea ly since he
ecogni ion o HPV-posi i e and HPV-nega i e lesions.
Al hough umou ecu ence s ill occu s in 10–20% o
HPV-posi i e HNSCC pa ien s, he majo i y o hese
pa ien s clea ly bene i om he apeu ic de-escala ion
[55]. The lack o adequa e bioma ke s o de ine mo e ai-
lo ed app oaches is s ill necessa y in SCC HPV associ-
a ed umou s [56]. The la ge majo i y o hese umou s
a e associa ed wi h HPV16 and a deepe unde s anding
o he bio pa hological implica ions o dis inc HPV16
a ian s would con ibu e o he molecula cha ac e i-
za ion o HPV-posi i e HNSCC and may help o de ine
pa ien ’s subg oups ha would bene i om speci ic he -
apeu ic app oaches. Da a om ce ical cance pa ien s
showed an associa ion be ween he p esence o non-
Eu opean HPV16 lineages, a longe i al pe sis ence [57,
58] and an inc eased isk o de eloping high-g ade ce i-
cal in aepi helial neoplasia [59]. Appa en ly, wi hin indi-
iduals, HPV genomes ha bou high le els o a iabili y
in HPV16 genome sequence upon no mal o p e-cance /
cance [60] e ealing se e al undamen al disco e ies
and sugges ing a pa adigm shi om HPV16 as a single
i al en i y o heo ize each HPV16 isola ed o be a sepa-
a e i us wi h dis inc ca cinogenic po en ial [61]. This
would imply ha wi hin HPV16, he gene ic a ia ion
pa ly p edic s he isk o p e-cance and cance [62]. In
pa icula , speci ic sublineages (A4, C, D2, and D3) ha e
shown a signi ican ly inc eased isk compa ed o he
mos common A1/A2 sublineages [26] and assu ed D2,
o he s onges isk o cance wi hin glandula epi he-
lium (adenoca cinomas) [26].

86
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Cochichoe al. Vi ol J (2021) 18:217
The majo da a om whole-sequences ob ained om
indi idual clinical specimens ag ees ha he gene ic a i-
a ion occu s mo e commonly on low-g ade o benign
HPV16 in ec ions [61] and co ec ed explained In
case–con ol analyses desc ibing he highes amino acid
changing a ian s HPV16 in he con ols h oughou he
genome wi h ce ix cance . [62] The e o e, E7 oncogene
lacks nonsynonymous (amino acid changing) a ian s in
ce ical cance s, sugges ing he E7 conse a ion is admi -
ed o ca cinogenici y. [61] The speci ic conse a ion o
he 98 amino acids o E7, ha di ec ly dis up s Rb unc-
ion, was shown o be c ucial o igge ca cinogenesis,
owing o be commen ed as a highly speci ic a ge o
e iologic and he apeu ic esea ch [26].
P esen ing da a om a coho s udy ha compa es
genomic cha ac e is ics o HPV associa ed wi h ce i-
cal e sus o opha yngeal umou s using DNA sequence
analysis showed no signi ican di e ences be ween dis-
ibu ion in HPV16 a ian s [24], bo h p esen ing majo
p e alence o he Eu opean a ian . Ins ead, he HPV
E6 gene ampli ied om o opha yngeal samples epo ed
o e mo e nonsynonymous mu a ions, bu also o he
E7 gene, no di e ences we e ound in mu a ion a es
be ween he wo ana omical loca ions [24, 62]. No ably,
he E7 gene is conse ed in bo h loca ions co obo a ing
he ecen indings on ce ical cance s udies. The appa -
en es ic ion on E7 mu a ions seems o be p esen in
he o opha ynx, as well. Indeed, desc ibed all bo h sha e
common biological ea u es, bu he impo an di e -
ences p esen in HPV-genome may explain hei dis inc
pa hophysiological mechanisms and suscep ibili y o
ea men . Ne e heless, he in a iabili y o E7 p esen s
an a ac i e po en ial a ge o he apy a bo h loca-
ions. [24]
In all he s udies e alua ed in his analysis, he mos
p e alen a ian was he Eu opean Va ian , which
belongs o he Eu opean lineage and wi hin his line-
age he E-350-G HPV16 a ian was he mos equen
in majo i y o he s udies sequencing based E6 and/o
E7 egions wi h 29% and 53% espec i ely, al hough he
coho s had o no an Eu opean o igin, (coho s we e
om B azil, Uni ed S a es o Ame ica, Japan, I aly and
Ge many (Table3).
As i was es ablished om molecula analysis s ud-
ies in ce ical cance , ha he Eu opean a ian T350G,
was he a ian equen ly ound in ce ical in aepi-
helial neoplasms and cance s, and has been associa ed
wi h p og ession o ce ical cance pa icula ly in No h
Eu opean women. The de ec ion o he T350G a ian in
a la ge p opo ion o HNSCC pa ien s, he e o e, indi-
ca es ha his a ian migh also play an impo an ole
in HN ca cinogenesis. Hassani e al.in 2015 suppo ed
his hypo hesis, elucida ing ha he HPV-16 E-350G
a ian has a polymo phism in esidue 83, a leucine o
aline (L83V), p obably esponsible o he inc eased
cance isk [53].
Conclusions
In HNSCC di e en lineages o HPV16 a ian s can
be iden i ied and di e geog aphically. Al hough mos
e iews desc ibed only he dis ibu ion o HPV16 a i-
an s in HNSCC, LeCon e in a mo e de ailed analysis o
hose s udies ound impo an di e ences. They ound
he dis ibu ion o wo HPV16 a ian g oups di e sig-
ni ican ly in o opha yngeal cance and ce ical cance .
The Eu opean + Sou h Ame ica (E + AS) a ian g oups
showed a highe p e alence in he o opha yngeal sam-
ples, ep esen ing 90.2%, han in ce ical ca cinomas
(71.4%). Mo eo e , he Asia-Ame ican (AA1 + AA2) a -
ian g oups we e p esen in 22.5% o ce ical cance s in
con as o 4.4% in he o opha yngeal cance s [24]. The
numbe o cases s udied does no allow us o explo e he
impo ance o hose di e ences no o unde s and he
po en ial ole de e mining he clinical beha iou and
po en ial use o selec ea men and p ognosis. Hope-
ully, he new sequencing e a will en ich he s udy o
HPV and ela ed cance s. The ad ances in HPV whole-
genome sequencing [46] p o ided echnically achie able
la ge-scale longi udinal s udies on HPV whole-genomic
sequences and p omo es an exhaus i e unde s anding o
Table 3 F equency HPV16 Va ian s by egion genome loca ion
(–) no e alua ed; AA Asia-Ame ican HPV16 Va ian ; AF A ican HPV16 Va ian ; AS Asian HPV16 Va ian ; E Eu opean HPV16 a ian ; E6 HPV Ea ly p o ein 6; E7 HPV Ea ly
p o ein 7; LCR long con ol egion; L1 la e gene; NA No h Ame ican HPV16 a ian
Re e ence To al
numbe o
cases
To al isola es o
HPV sequencing HPV16 a ian
egion loca ion Eu opean No h
Ame ican
(NA)
A ican
(AF1|AF2) Asian (AS) Asia
Ame ican
(AA)
All lineages E-350-G
[50] 115 115 L1 69% No De ec ed - 15% - 8%
[48, 51–54, 56] 621 106 E6 78% 29% 2% 2% 9% 4%
[49] 24 21 E6E7 71% 53% - - - -
[55] 205 21 LCR 42.8% - - - - 57.2%
87
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Cochichoe al. Vi ol J (2021) 18:217
he i al gene ic di e si y wi hin and be ween in ec ed
indi iduals and will make he link be ween a ian s and
cance isk, comp ehensi ely [46, 61].
Abb e ia ions
AA: Asia-Ame ican HPV16 a ian ; AF: A ican HPV16 a ian ; AS: Asian HPV16
a ian ; E: Eu opean HPV16 a ian ; E6: HPV Ea ly p o ein 6; E7: HPV Ea ly
p o ein 7; HNSCC: Head and neck squamous cell ca cinoma; HPV: Human
papilloma i us; NA: No h Ame ican HPV16 VARIANT; NGS: Nex gene a ion
sequencing.
Acknowledgemen s
No applicable.
Au ho s’ con ibu ions
These au ho s con ibu ed equally o his wo k. S udy concep s: AF, DC, RGC.
S udy design: AF, DC, RGC. Da a acquisi ion: DC, AF, RGC. Da a analysis and
in e p e a ion: RGC, DC, AF. Manusc ip p epa a ion: DC. Manusc ip edi ing:
AF, DC, RGC. Manusc ip e iew: AF, DC, RGC. All au ho s ead and app o ed
he inal manusc ip .
Funding
No applicable.
A ailabili y o da a and ma e ials
No applicable.
Decla a ions
E hics app o al and consen o pa icipa e
No applicable.
Consen o publica ion
No applicable.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho de ails
1 NOVA Medical School, NOVA Uni e si y o Lisbon, CEDOC, Campo Má i es da
Pá ia 130, 1169-056 Lisbon, Po ugal. 2 Vi ology Labo a o y IPOLFG, Rua P o es-
so Lima Bas os, 1099-023 Lisbon, Po ugal. 3 LEPABE, Labo a o y o P ocess
Enginee ing, En i onmen , Bio echnology and Ene gy, Uni e si y o Po o,
Rua D . Robe o F ias, 4200-465 Po o, Po ugal. 4 Pos -g adua e P og amme
in Adul Heal h (PPGSAD), Uni e si y Hospi al (HUUFMA) and Mo phology
Depa men , Fede al Uni e si y o Ma anhão, A . dos Po ugueses 1966 - Vila
Bacanga, São Luís, MA 65080-805, B azil. 5 Molecula Oncology and Vi al Pa hol-
ogy G oup, Resea ch Cen e o IPO Po o (CI-IPOP) / RISE@CI-IPOP (Heal h
Resea ch Ne wo k), Po uguese Oncology Ins i u e o Po o (IPO Po o) /
Po o Comp ehensi e Cance Cen e (Po o.CCC), Rua D . An ónio Be na dino
de Almeida, 4200-072 Po o, Po ugal. 6 Pa hology Depa men IPOLFG, Rua
P o esso Lima Bas os, 1099-023 Lisbon, Po ugal.
Recei ed: 27 July 2021 Accep ed: 28 Oc obe 2021
Re e ences
1. Fe lay J, Soe joma a am I, e al. Cance incidence and mo ali y wo ld-
wide: sou ces, me hods and majo pa e ns in GLOBOCAN 2012. In J
Cance . 2015. h ps:// doi. o g/ 10. 1002/ ijc. 29210.
2. De B ee R, Leemans CR. Recen ad ances in su ge y o head and neck
cance . Cu Opin Oncol. 2010. h ps:// doi. o g/ 10. 1097/ CCO. 0b013 e3283
380009.
3. B ay F, Fe lay J, e al. Global cance s a is ics 2018: GLOBOCAN es ima es
o incidence and mo ali y wo ldwide o 36 cance s in 185 coun ies. CA
Cance J Clin. 2018. h ps:// doi. o g/ 10. 3322/ caac. 21492.
4. McDe mo JD, Bowles DW. Epidemiology o head and neck squamous
cell ca cinomas: impac on s aging and p e en ion s a egies. Cu T ea
Op ions Oncol. 2019. h ps:// doi. o g/ 10. 1007/ s11864- 019- 0650-5.
5. Gillison ML. Human papilloma i us-associa ed head and neck cance is a
dis inc epidemiologic, clinical, and molecula en i y. Semin Oncol. 2004.
h ps:// doi. o g/ 10. 1053/j. semin oncol. 2004. 09. 011.
6. Cha u edi AK, Ande son WF, e al. Wo ldwide ends in incidence a es
o o al ca i y and o opha yngeal cance s. J Clin Oncol. 2013. h ps:// doi.
o g/ 10. 1200/ JCO. 2013. 50. 3870.
7. Hwang TZ, Hsiao JR, e al. Incidence ends o human papilloma i us
ela ed head and neck cance in Taiwan. In J Can. 2015. h ps:// doi. o g/
10. 1002/ ijc. 29330.
8. Mo k J, Molle B, e al. Time ends in pha yngeal cance incidence in
No way 1981–2005: a subsi e analysis based on a e-abs ac ion and
ecoding o egis e ed cases. Can Causes Con ol. 2010. h ps:// doi. o g/
10. 1007/ s10552- 010- 9567-9.
9. Zums eg ZS, Cook-Wiens G, e al. Incidence o o opha yngeal cance
among elde ly pa ien s in he Uni ed S a es. JAMA Oncol. 2016. h ps://
doi. o g/ 10. 1001/ jamao ncol. 2016. 1804.
10. To a JE, Bes AF, e al. E olu ion o he o opha ynx cance epidemic in he
Uni ed S a es: mode a ion o inc easing incidence in younge indi iduals
and shi in he bu den o olde indi iduals. J Clin Oncol. 2019. h ps:// doi.
o g/ 10. 1200/ JCO. 19. 00370.
11. Fa aji F, Eisele DW, e al. Eme ging insigh s in o ecu en and me as a ic
human papilloma i us- ela ed o opha yngeal squamous cell ca cinoma.
La yngoscope In es ig O ola yngol. 2017. h ps:// doi. o g/ 10. 1002/ lio2. 37.
12. Cha u edi AK, Engels EA, e al. Human papilloma i us and ising
o opha yngeal cance incidence in he Uni ed S a es. J Clin Oncol. 2011.
h ps:// doi. o g/ 10. 1200/ JCO. 2011. 36. 4596.
13. Hamma s ed L, Lindquis D, e al. Human papilloma i us as a isk ac o
o he inc ease in incidence o onsilla cance . In J Can. 2006. h ps://
doi. o g/ 10. 1002/ ijc. 22177.
14. Hocking JS, S ein A, e al. Head and neck cance in Aus alia be ween
1982 and 2005 show inc easing incidence o po en ially HPV-associa ed
o opha yngeal cance s. B J Can. 2011. h ps:// doi. o g/ 10. 1038/ sj. bjc.
66060 91.
15. Gillison ML, D’Souza G, e al. Dis inc isk ac o p o iles o human papil-
loma i us ype 6-posi i e and human papilloma i us ype 6-nega i e
head and neck cance s. J Na l Cance Ins . 2008. h ps:// doi. o g/ 10. 1093/
jnci/ djn025.
16. El-Mo y SK. HPV- ela ed squamous cell ca cinoma a ian s in he
head and neck. Head Neck Pa hol. 2012;6:55. h ps:// doi. o g/ 10. 1007/
s12105- 012- 0363-6.
17. WHO, Wo ld Heal h O ganiza ion. In e na ional agency o esea ch on
cance (IARC) monog aphs on he e alua ion o ca cinogenic isks o
humans. 2007;90(Human Papilloma Vi us):689. h ps:// monog aphs. ia c.
who. in / wp- con e n / uploa ds/ 2018/ 06/ mono90. pd
18. Polanska H, Raudenska M, e al. Clinical signi icance o head and neck
squamous cell cance bioma ke s. O al Oncol. 2014. h ps:// doi. o g/ 10.
1016/j. o alo ncolo gy. 2013. 12. 008.
19. De Villie s EM, Fauque C, e al. Classi ica ion o papilloma i uses. In: Vi ol-
ogy. Academic P ess. 2004; h ps:// doi. o g/ 10. 1016/j. i ol. 2004. 03. 033
20. De Villie s EM, Guns K. Cha ac e iza ion o se en no el human papil-
loma i us ypes isola ed om cu aneous issue, bu also p esen in
mucosal lesions. J Gen Vi ol. 2009. h ps:// doi. o g/ 10. 1099/ i .0. 011478-0.
21. Muñoz N, Bosch FX, e al. Epidemiologic classi ica ion o human papil-
loma i us ypes associa ed wi h ce ical cance . N Engl J Med. 2003.
h ps:// doi. o g/ 10. 1056/ NEJMo a0216 41.
22. K eime AR, Cli o d GM, e al. Human papilloma i us ypes in head and
neck squamous cell ca cinomas wo ldwide: a sys emic e iew. Cance
Epidemiol Bioma k P e . 2005. h ps:// doi. o g/ 10. 1158/ 1055- 9965.
EPI- 04- 0551.
23. Hue as-Salgado A, Ma ín-Gámez DC, e al. E6 molecula a ian s o
human papilloma i us HPV ype 16: an upda ed and uni ied c i e ion o
clus e ing and nomencla u e. Vi ology. 2011. h ps:// doi. o g/ 10. 1016/j.
i ol. 2010. 10. 039.
24. LeCon e BA, Szaniszlo P, e al. Di e ences in he i al genome be ween
HPV-posi i e ce ical and o opha yngeal cance . PLOS ONE. 2018. h ps://
doi. o g/ 10. 1371/ jou n al. pone. 02034 03.
25. Bu k RD, Ha a i A, e al. Human papilloma i us genome a ian s. Vi ology.
2013. h ps:// doi. o g/ 10. 1016/j. i ol. 2013. 07. 018. Human.
88
Page 9 o 9
Cochichoe al. Vi ol J (2021) 18:217
26. Mi abello L, Yeage M, e al. HPV16 sublineage associa ions wi h his ol-
ogy-speci ic cance isk using HPV whole-genome sequences in 3200
women. J Na l Cance Ins . 2016. h ps:// doi. o g/ 10. 1093/ jnci/ djw100.
27. Siche o L, Simão Sob inho J, e al. Oncogenic po en ial di e ge among
human papilloma i us ype 16 na u al a ian s. Vi ology. 2012. h ps:// doi.
o g/ 10. 1016/j. i ol. 2012. 06. 011(8).
28. Be na d HU, Calleja-Macias IE, e al. Genome a ia ion o human papillo-
ma i us ypes: phylogene ic and medical implica ions. In J Cance . 2006.
h ps:// doi. o g/ 10. 1002/ ijc. 21655.
29. Ho L, Chan SY, e al. Sequence a ian s o human papilloma i us ype 16
in clinical samples pe mi e i ica ion and ex ension o epidemiologi-
cal s udies and cons uc ion o a phylogene ic ee. J Clin Mic obiol.
1991;29:1765–72. h ps:// doi. o g/ 10. 1128/ jcm. 29.9. 1765- 1772. 1991.
30. Ho L, Chan SY, e al. The gene ic d i o human papilloma i us ype 16 is
a means o econs uc ing p ehis o ic i al sp ead and he mo emen o
ancien human popula ions. J Vi ol. 1993;67:6413–23. h ps:// doi. o g/ 10.
1128/ j i. 67. 11. 6413- 6423. 1993.
31. Ho L, Tay SK, e al. Sequence a ian s o human papilloma i us ype 16
om couples sugges sexual ansmission wi h low in ec i i y and poly-
clonali y in geni al neoplasia. J In ec Dis. 1993. h ps:// doi. o g/ 10. 1093/
in dis/ 168.4. 803.
32. Yamada T, Wheele CM, e al. Human papilloma i us ype 16 a ian line-
ages in Uni ed S a es popula ions cha ac e ized by nucleo ide sequence
analysis o he E6, L2, and L1 coding segmen s. J Vi ol. 1995;69:7743–53.
33. Yamada T, Manos MM, e al. Human papilloma i us ype 16 sequence a -
ia ion in ce ical cance s: a wo ldwide pe spec i e. J Vi ol. 1997;71:2463–
72. h ps:// doi. o g/ 10. 1128/ j i. 71.3. 2463- 2472. 1997.
34. Jackson R, Rosa BA, e al. Func ional a ian s o human papilloma i us
ype 16 demons a e hos genome in eg a ion and ansc ip ional al e a-
ions co esponding o hei unique cance epidemiology. BMC Genom-
ics. 2016. h ps:// doi. o g/ 10. 1186/ s12864- 016- 3379-6.
35. No d o s C, Sobkowiak M, e al. Human papilloma i us (HPV) 16 E6
a ian s in onsilla cance in compa ison o hose in ce ical cance in
S ockholm, Sweden. PLOS ONE. 2012. h ps:// doi. o g/ 10. 1371/ jou n al.
pone. 00362 39.
36. S öpple MC, Ching K, e al. Na u al a ian s o he human papilloma i us
ype 16 E6 p o ein di e in hei abili ies o al e ke a inocy e di e en ia-
ion and o induce p53 deg ada ion. J Vi ol. 1996. h ps:// doi. o g/ 10. 1128/
j i. 70. 10. 6987- 6993. 1996.
37. Gillison ML, Koch WM, e al. E idence o a causal associa ion be ween
human papilloma i us and a subse o head and neck cance s. J Na l
Cance Ins . 2000;92:709–20. h ps:// doi. o g/ 10. 1093/ jnci/ 92.9. 709.
38. Ho mann M, Loh ey C, e al. Human papilloma i us ype 16 E6 and E7
geno ypes in head-and-neck ca cinomas. O al Oncol. 2004. h ps:// doi.
o g/ 10. 1016/j. o alo ncolo gy. 2003. 10. 011.
39. Xi LF, Deme s GW, e al. Analysis o human papilloma i us ype 16 a i-
an s indica es he es ablishmen o pe sis en in ec ion. J In ec Dis. 1995.
h ps:// doi. o g/ 10. 1093/ in dis/ 172.3. 747(3).
40. Van Belkum A, Ju e mans L, e al. Geno yping human papilloma i us
ype 16 isola es om pe sis en ly in ec ed p omiscuous indi iduals and
ce ical neoplasia pa ien s. J Clin Mic obiol. 1995. h ps:// doi. o g/ 10.
1128/ jcm. 33. 11. 2957- 2962. 1995.
41. Zhang L, Liao H, e al. Va ian s o human papilloma i us ype 16 p edis-
pose owa d pe sis en in ec ion. In J Clin Exp Pa hol. 2015;87:8453.
42. The Cance Genome A las Ne wo k Comp ehensi e genomic cha ac e i-
za ion o head and neck squamous cell ca cinomas. Na u e. 2015; doi:
h ps:// doi. o g/ 10. 1038/ na u e14129
43. Cas ellsagué X, Alemany L, e al. HPV in ol emen in head and neck
cance s: comp ehensi e assessmen o bioma ke s in 3680 pa ien s. J
Na l Cance Ins . 2016. h ps:// doi. o g/ 10. 1093/ jnci/ dj 403.
44. Dok R, Nuy s S. HPV posi i e head and neck cance s: Molecula pa ho-
genesis and e ol ing ea men s a egies. Cance s (Basel). 2016. h ps://
doi. o g/ 10. 3390/ cance s804 0041.
45. Pakdel F, Fa hadi A, e al. The equency o high- isk human papillo-
ma i us ypes, HPV16 lineages, and hei ela ionship wi h p16INK4a
and NF-κB exp ession in head and neck squamous cell ca cinomas
in Sou hwes e n I an. J Mic obiol. 2020. h ps:// doi. o g/ 10. 1007/
s42770- 020- 00391-1.
46. Cullen M, Boland JF, e al. Deep sequencing o HPV16 genomes: A new
high- h oughpu ool o explo ing he ca cinogenici y and na u al
his o y o HPV16 in ec ion. Papilloma i us Res. 2015. h ps:// doi. o g/ 10.
1016/j. p . 2015. 05. 004.
47. La ezzo E, Masi G, e al. Cha ac e iza ion o in a- ype a ian s o onco-
genic human papilloma i uses by nex -gene a ion deep sequencing o
he E6/E7 egion. Vi uses. 2016. h ps:// doi. o g/ 10. 3390/ 8030 079.
48. an de Weele P, Meije CJLM, e al. Whole-Genome Sequencing and Va i-
an Analysis o Hp 16 In ec ions. J Vi ol. 2017. h ps:// doi. o g/ 10. 1128/ JVI.
00844- 17.
49. Bada acco G, Rizzo C, e al. Molecula analyses and p ognos ic ele ance
o HPV in head and neck umou s. Oncol Rep. 2007
50. Ag awal Y, Koch WM, e al. O al HPV in ec ion be o e and a e ea men
o HPV16-posi i e and nega i e head and neck squamous cell ca ci-
noma. O al Oncol. 2008. h ps:// doi. o g/ 10. 1158/ 1078- 0432. CCR- 08- 0498.
O al.
51. Boscolo-Rizzo P, Da Mos o MC, e al. HPV-16 E6 L83V a ian in squamous
cell ca cinomas o he uppe ae odiges i e ac . J Cance Res Clin Oncol.
2009. h ps:// doi. o g/ 10. 1007/ s00432- 008- 0490-3.
52. Joseph AW, Ogawa T, e al. Molecula e iology o second p ima y umou s
in con ala e al onsils o human papilloma i us-associa ed index onsilla
ca cinomas. O al Oncol. 2013. h ps:// doi. o g/ 10. 1016/j. o alo ncolo gy.
2012. 09. 009.
53. Hassani S, Cas illo A, Oho i J-I, Higashi M, Ku ono Y, Akiba S, Ko iyama C,
Molecula pa hogenesis o human papilloma i us ype 16 in Tonsilla
squamous cell ca cinoma. An icance Res. 2015
54. Be iol JC, de Ma os LL, e al. P e alence o human papilloma i us ypes
and a ian s and p16INK4a exp ession in head and neck squamous cells
ca cinomas in São Paulo, B azil. In ec ious Agen s Cance . 2016. h ps://
doi. o g/ 10. 1186/ s13027- 016- 0067-8.
55. Ang KK, Ha is J, e al. Human papilloma i us and su i al o pa ien s
wi h o opha yngeal cance . N Engl J Med. 2010. h ps:// doi. o g/ 10. 1056/
nejmo a0912 217.
56. Bea y BT, Moon DH, e al. PIK3CA mu a ion in HPV-associa ed OPSCC
pa ien s ecei ing dein ensi ied chemo adia ion. JNCI J Na l Cance Ins .
2019;112:855–8. h ps:// doi. o g/ 10. 1093/ jnci/ djz224.
57. Villa LL, Siche o L, e al. Molecula a ian s o human papilloma i us ypes
16 and 18 p e e en ially associa ed wi h ce ical neoplasia. J Gen Vi ol.
2000. h ps:// doi. o g/ 10. 1099/ 0022- 1317- 81- 12- 2959.
58. Schi man M, Rod iguez AC, e al. A popula ion-based p ospec i e s udy
o ca cinogenic human papilloma i us (HPV) a ian lineages, i al pe sis-
ence, and ce ical neoplasia. NIH Public Access. 2010. h ps:// doi. o g/ 10.
1158/ 0008- 5472. CAN- 09- 4179.A.
59. Siche o L, Fe ei a S, e al. High g ade ce ical lesions a e caused p e e -
en ially by non-Eu opean a ian s o HPVs 16 and 18. In J Cance . 2007.
h ps:// doi. o g/ 10. 1002/ ijc. 22481.
60. Hi ose Y, Onuki M, e al. Wi hin-hos a ia ions o human papilloma i us
e eal APOBEC signa u e mu agenesis in he i al genome. J Vi ol. 2018.
h ps:// doi. o g/ 10. 1128/ j i. 00017- 18.
61. Mi abello L, Cla ke MA, e al. The in e sec ion o HPV epidemiology,
genomics and mechanis ic s udies o HPV-media ed ca cinogenesis.
Vi uses. 2018. h ps:// doi. o g/ 10. 3390/ 1002 0080.
62. Zhu B, Xiao Y, e al. Mu a ions in he HPV16 genome induced by
APOBEC3 a e associa ed wi h i al clea ance. Na Commun. 2020. h ps://
doi. o g/ 10. 1038/ s41467- 020- 14730-1.
Publishe ’s No e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in pub-
lished maps and ins i u ional a ilia ions.
89
2.2 THE RELEVANCE OF HPV16 VARIANTS IN HEAD AND NECK
SQUAMOUS CELL CARCINOMAS
Accep ed o publica ion in Pa hobiology.
Abs ac
The a ian s o he human papilloma i us (HPV) a e belie ed o play an impo an
ole in he ca cinogenesis p ocess o head and neck squamous cell ca cinomas
(HNSCC). This s udy aims o es ablish he p e alence o HPV16 a ian s in a
Po uguese HNSCC coho , desc ibe clinical-pa hological cha ac e is ics and
de e mine he associa ion wi h pa ien su i al. We e ie ed 68 HNSCC pa ien s
om he Ins i u e o Oncology in Lisboa. DNA samples we e a ailable om umou
biopsy a he ime o he p ima y diagnosis. Ta ge ed NGS was used o analyse
whole-genome sequences and a ian s we e es ablished based on phylogene ics
classi ica ion. The majo i y o he HPV16 samples clus e ed in lineage A and he
emaining clus e ing wi hin sublineages B, C and D. Compa a i e genome analysis
e ealed a o al o 1356 SNV all wi hin he coding egions E1, E2, E4, E5, E6, E7 ea ly
genes and L1 and L2 la e genes. Ou in eg a ed app oach epo s a deep molecula
HNSCC HPV16 mu a ional p o ile cha ac e iza ion, in ended o desc ibe molecula
cha ac e is ics o HPV16 a ian s and hei biological signi icance in HN cance .
Keywo ds: K14HPV16, Va ian
96
excluded due o pe sis ence). In he emaining 27 eco ds wi h HPV16 lineage A
(n=20), Lineage B (n=2) and Lineage D (n=5) a 3 yea s o ollow-up he e we e 4
e en s ( elapse o dea h). Pa ien s wi h lineage A and D had o e lapping DFS wi h
100% a 3 yea s (no e en s). The Lineage B pa ien s wi h comple e esponse (n=2)
died wi hou elapse a median ime 1.37-yea s.
HPV16 a ian s iden i ied and clinical p esen a ion o HNSCC
A ecen publica ion iden i ied eigh SNVs as ma ke s o poo p ognosis [23]. Ou
s udy epo s ou o hem indi idually p esen in hi een pa ien s (genome posi ion
4410 n=1; posi ion 4539 n=9; posi ion 5962 n=1 and posi ion 7173 n=2). Among
hese, we ha e en men and h ee women, wi h an a e age age ≥65 and umou s in
s ages II-III (n=11). Mos cases p esen ed a comple e esponse, while wo cases
showed disease pe sis ence. In o al, ou dea h e en s we e egis e ed. The
emaining pa ien s a e well wi hou e idence o disease.
Discussion
The di e en oncogenic po en ial o HPV lineages in he de elopmen o ce ical
cance has been ecognized [37]. Con e sely, in head-and-neck pa hology, he
mu a ional p o ile o HPV16 is s ill li le explo ed and ew s udies p esen a whole-
genome analysis o HPV-associa ed cance s [31]. In his con ex , ou s udy o 35
cases p o ides a aluable con ibu ion o a be e cha ac e iza ion o he molecula
p o ile o HPV-d i en SCC in he head-and-neck egion o a Eu opean coun y.
The clinical-pa hological cha ac e is ics o ou coho a e in line wi h published
li e a u e. Male gende and o opha ynx loca ion a e he p edominan clinical-
pa hological aspec s o his HPV16 posi i e squamous cell ca cinoma se ies.
Rega ding HPV16 analysis mos samples clus e ed in lineage A (n=26), he mos
common lineage desc ibed in Eu ope. This obse a ion ag ees wi h p e ious s udies
[38–41] showing a p eponde ance o lineage A s ains, as ecen ly e iewed [17].
This is also he case wi h ce ical cance [11,13,18], e ealing an in e es ing
simila i y be ween he HPV16 s ains in ol ed in ca cinogenesis a hese wo
ana omical si es.
In he p esen s udy, we analysed and compa ed he equency o di e en HPV16
a ian s. The mu a ion p o ile desc ibes 243 a iable si es, o which 100 ha e been
p e iously epo ed in ce ical pa hology, nine in SCC head-and-neck pa hology, and
143 a e possible new changes. The la ges s udy on HNSCC HPV16 a ian s and
SNV was e y ecen ly published and eigh HPV16 SNVs we e signi ican ly
associa ed wi h wo se su i al in a US popula ion [23]. This was now compa ed wi h
ou Po uguese se ies, and we ound ou a ian s (one missense and 3
synonymous) in common (posi ions 4410, 4539, 5962 and 7173). Howe e , ou da a
do no allow us o d aw conclusions ega ding he po en ial o hese a ian s o
in luence he umou 's biological beha iou . Al oge he , 25 pa ien s we e ali e a he
las ollow-up, and he emaining en had dea h e en s.
Se e al s udies ha e shown ha in he E6 coding egion o HPV16 in he Eu opean
lineage he E31G, L83V and D25E ansi ions we e signi ican ly associa ed wi h he
de elopmen o ce ical ca cinoma [22,38–40]. S ikingly, none o hese h ee
subs i u ions was ound in ou da a, highligh ing he need o a mo e de ailed

97
molecula cha ac e iza ion o HPV-associa ed cance s in di e en ana omic
loca ions. In his con ex , i is also ema kable ha he E7 N29S a ian , associa ed
wi h a highe oncogenic isk o ce ical cance [41], was only de ec ed in a single
HNSCC case, u he highligh ing he molecula di e ences associa ed wi h ce ical
and head-and-neck ca cinogenesis. Simila ly, p e ious obse a ions om he
HPV16- ansgenic mouse model show di e ences in cance pene ance and
mic oRNA exp ession p o iles be ween di e en ana omical si es [42–46], his is in
he same line as wha is obse ed in HPV-d i en cance s. Taken oge he , hese
clinical and expe imen al da a sugges ha he in e play be ween di e en HPV16
s ains and issue-speci ic hos ac o s di e en ially modula es ca cinogenesis in
di e en o gans.
The E7 gene was shown o be he mos conse ed, co obo a ing ecen indings on
he appa en es ic ion on E7 gene mu a ions in ce ical cance and in he
o opha ynx, sus aining he hypo hesis ha ca cinogenici y depends on a highly
conse ed E7 p o ein [11,23]. In e es ingly, he indica o o selec i e p essu e
e ealed ha none o he genes eached he posi i e p essu e cu -o , and his was
especially no iceable o he E7 gene, owing o pu i ying selec ion agains
nonsynonymous changes.
We acknowledge ha one o he limi a ions o he p esen s udy is he low numbe o
cases. Howe e , we p o ide high-quali y whole-genome da a on he HNSCC HPV16
mu a ional p o ile in 35 new cases o a Po uguese coho , adding o he a ailable
knowledge o HPV16 a ian s and mu a ion si e and amino acid changes ha allow
he unde s anding o he biological signi icance o each a ian .
In conclusion, he p esen da a p o ide an in-dep h genomic cha ac e iza ion o
HPV16 p esen in HNSCC samples, e ealing a ma ked p edominance o lineage A
s ains. A o al o 243 genome a iable si es we e iden i ied, o which 143 a e
po en ially new a ia ions. Majo di e ences we e obse ed conce ning he
dis ibu ion o SNVs in ou HSNCC coho and in ano he HPV-associa ed
malignancy, ce ical cance . O e all, hese da a con ibu e o a deepe
unde s anding o he in luence o hese mu a ions on ca cinogenesis, diagnos ics,
and clinical managemen .
S a emen s
Acknowledgemen (op ional)
In he Acknowledgemen sec ion, au ho s mus include indi iduals and o ganiza ions
ha ha e made subs an i e con ibu ions o he esea ch o he manusc ip . An
excep ion is whe e unding was p o ided, which should be included in Funding
Sou ces. Please e e o he Guidelines issued by he ICMJE o de e mine non-
au ho con ibu o s ha should be included in he Acknowledgemen sec ion.
S a emen o E hics
All he da a we e anonymized, and he s udy was conduc ed in acco dance wi h he
Helsinki Decla a ion and app o ed by he E hics Commi ee o he Po uguese
Oncologic Ins i u e F ancisco Gen il, Lisbon (Re . UIC/2019/1168). A w i en
in o med consen was ob ained om all pa icipan s o he s udy.
Con lic o In e es S a emen
98
The au ho s ha e no con lic s o in e es o decla e.
Funding Sou ces
This s udy was inancially suppo ed by he Vi ology Labo a o y Pa hology
Depa men om he Po uguese Oncology Ins i u e o Lisboa IUIC/1168, wi h
con ibu ions by he GenomePT p ojec (POCI-01-0145-FEDER-022184), suppo ed
by COMPETE 2020 - Ope a ional P og amme o Compe i i eness and
In e na ionalisa ion (POCI), Lisboa Po ugal Regional Ope a ional P og amme
(Lisboa2020), Alga e Po ugal Regional Ope a ional P og amme (CRESC
Alga e2020), unde he PORTUGAL 2020 Pa ne ship Ag eemen , h ough he
Eu opean Regional De elopmen Fund (ERDF), and by Fundação pa a a Ciência e
a Tecnologia (FCT).
Au ho Con ibu ions
Con ibu ion au ho s AF, DC and RGC o s udy concep s and s udy design; he
au ho s AN, DC, DS, JPG, JM, LM, LV, MC, MM, and PM pe o med and da a
acquisi ion; he au ho 's AN, DC, LV and JPG analyse he genomic da a; he au ho s
AF, AN, DC, LM, MC, RGC and SE we e esponsible o analysis and in e p e a ion;
he au ho DC o manusc ip p epa a ion; he au ho s AF, AN, DC, DS, LM, MC,
RGC and SE o manusc ip edi ing and e iew. C i ical e ision o he a icle o
impo an in ellec ual con en was pe o med by AF, AN, DC, JPG, LV, MC, MM, PM
and RGC. All he au ho s e iewed and app o ed he inal e sion o he manusc ip .
Da a A ailabili y S a emen
The au ho s con i m ha he da a suppo ing he indings o his s udy a e a ailable
wi hin he a icle and i s supplemen a y ma e ials. Fu he enqui ies can be di ec ed
o he co esponding au ho .
Re e ences
1 Fe lay J, Soe joma a am I, Rajesh Dikshi , Sul an Ese , Ma he s C, Rebelo M, e al. Cance
incidence and mo ali y - Majo pa e ns in GLOBOCAN 2012, wo ldwide and Geo gia.
In e na ional Jou nal o Cance . 2015;136:E359–86.
2 Sung H, Fe lay J, Siegel RL, La e sanne M, Soe joma a am I, Jemal A, e al. Global Cance
S a is ics 2020: GLOBOCAN Es ima es o Incidence and Mo ali y Wo ldwide o 36 Cance s
in 185 Coun ies. CA: A Cance Jou nal o Clinicians. 2021;71(3):209–49.
3 El-Mo y SK. HPV-Rela ed Squamous Cell Ca cinoma Va ian s in he Head and Neck. Head
and Neck Pa hology. 2012 Jul;6(SUPPL. 1):55–62.
4 De B ee R, Leemans CR. Recen ad ances in su ge y o head and neck cance . Cu en
Opinion in Oncology. 2010;22(3):186–93.
5 McDe mo JD, Bowles DW. Epidemiology o Head and Neck Squamous Cell Ca cinomas:
Impac on S aging and P e en ion S a egies. Cu en T ea men Op ions in Oncology.
2019;20(5):1–13.
6 Gillison ML. Human papilloma i us-associa ed head and neck cance is a dis inc
epidemiologic, clinical, and molecula en i y. Semina s in Oncology. 2004 Dec;31(6):744–
54.
99
7 B ay F, Fe lay J, Soe joma a am I, Siegel RL, To e LA, Jemal A. Global cance s a is ics
2018: GLOBOCAN es ima es o incidence and mo ali y wo ldwide o 36 cance s in 185
coun ies. CA: a cance jou nal o clinicians. 2018 No ;68(6):394–424.
8 Ang KK, Ha is J, Wheele R, Webe R, Rosen hal DI, Nguyen-Tân PF, e al. Human
Papilloma i us and Su i al o Pa ien s wi h O opha yngeal Cance . New England Jou nal o
Medicine. 2010 Jul;363(1):24–35.
9 IARC. Human Papilloma i uses. [ci ed 2023 Jan 10].A ailable om:
h ps://publica ions.ia c. /Book-And-Repo -Se ies/Ia c-Monog aphs-On-The-Iden i ica ion-
O -Ca cinogenic-Haza ds-To-Humans/Human-Papilloma i uses-2007
10 Hue as-Salgado A, Ma ín-Gámez DC, Mo eno P, Mu illo R, B a o MM, Villa L, e al. E6
molecula a ian s o human papilloma i us (HPV) ype 16: An upda ed and uni ied c i e ion
o clus e ing and nomencla u e. Vi ology. 2011 Feb;410(1):201–15.
11 LeCon e BA, Szaniszlo P, Fennewald SM, Lou DI, Qiu S, Chen NW, e al. Di e ences in he
i al genome be ween HPV-posi i e ce ical and o opha yngeal cance . PLoS ONE. 2018
Aug;13(8):e0203403.
12 Mes e VF, Medei os-Fonseca B, Es ê ão D, Casaca F, Sil a S, Félix A, e al. HPV16 is
su icien o induce squamous cell ca cinoma speci ically in he ongue base in ansgenic
mice. Jou nal o Pa hology. 2020 May;251(1):4–11.
13 Mi abello L, Yeage M, Cullen M, Boland JF, Chen Z, Wen zensen N, e al. HPV16
Sublineage Associa ions wi h His ology-Speci ic Cance Risk Using HPV Whole-Genome
Sequences in 3200 Women. Jou nal o he Na ional Cance Ins i u e. 2016
Sep;108(9):djw100.
14 Ho L, Chan SY, Bu k RD, Das BC, Fujinaga K, Icenogle JP, e al. The gene ic d i o human
papilloma i us ype 16 is a means o econs uc ing p ehis o ic i al sp ead and he
mo emen o ancien human popula ions. Jou nal o Vi ology. 1993 No ;67(11):6413–23.
15 Gillison ML, Koch WM, Capone RB, Spa o d M, Wes a WH, Wu L, e al. E idence o a
causal associa ion be ween human papilloma i us and a subse o head and neck cance s.
Jou nal o he Na ional Cance Ins i u e. 2000 May;92(9):709–20.
16 Muñoz N, Bosch FX, de Sanjosé S, He e o R, Cas ellsagué X, Shah KV, e al.
Epidemiologic classi ica ion o human papilloma i us ypes associa ed wi h ce ical cance .
The New England jou nal o medicine. 2003 Feb;348(6):518–27.
17 Cochicho D, Gil da Cos a R, Felix A. Explo ing he oles o HPV16 a ian s in head and neck
squamous cell ca cinoma: cu en challenges and oppo uni ies. Vi ol J. 2021 No ;18:217.
18 Bu k RD, Ha a i A, Chen Z. Human papilloma i us genome a ian s. Vi ology. 2013
Oc ;445(1–2):232–43.
19 Ho L, Chan SY, Chow V, Chong T, Tay SK, Villa LL, e al. Sequence a ian s o human
papilloma i us ype 16 in clinical samples pe mi e i ica ion and ex ension o
100
epidemiological s udies and cons uc ion o a phylogene ic ee. Jou nal o Clinical
Mic obiology. 1991 Sep;29(9):1765–72.
20 Yamada T, Wheele CM, Halpe n AL, S ewa AC, Hildesheim A, Jenison SA. Human
papilloma i us ype 16 a ian lineages in Uni ed S a es popula ions cha ac e ized by
nucleo ide sequence analysis o he E6, L2, and L1 coding segmen s. Jou nal o i ology.
1995 Dec;69(12):7743–53.
21 Yamada T, Manos MM, Pe o J, G ee CE, Munoz N, Bosch FX, e al. Human papilloma i us
ype 16 sequence a ia ion in ce ical cance s: a wo ldwide pe spec i e. Jou nal o i ology.
1997 Ma ;71(3):2463–72.
22 Jackson R, Rosa BA, Lamei as S, Cuninghame S, Be na d J, Flo iano WB, e al. Func ional
a ian s o human papilloma i us ype 16 demons a e hos genome in eg a ion and
ansc ip ional al e a ions co esponding o hei unique cance epidemiology. BMC
Genomics. 2016 Dec;17(1):851.
23 Lang Kuhs KA, Faden DL, Chen L, Smi h DK, Pinhei o M, Wood CB, e al. Gene ic a ia ion
wi hin he human papilloma i us ype 16 genome is associa ed wi h o opha yngeal cance
p ognosis. Annals o Oncology. 2022 DOI: 10.1016/j.annonc.2022.03.005
24 B eich YT, Hos i JE, Wehbi JA, Daou LR. Cu en landscape o clinical ials o HPV-posi i e
head and neck squamous cell ca cinoma (HNSCC). Ecance medicalscience. 2022;16:1447.
25 Julian R, Sa ani M, Bauman JE. Immuno he apy App oaches in HPV-Associa ed Head and
Neck Cance . Cance s. 2021 Jan;13(23):5889.
26 Lechne M, Liu J, Mas e son L, Fen on TR. HPV-associa ed o opha yngeal cance :
epidemiology, molecula biology and clinical managemen . Na Re Clin Oncol. 2022
May;19(5):306–27.
27 Che a BS, Amdu RJ, Teppe JE, Tan X, Weiss J, G illey-Olson JE, e al. Ma u e esul s o a
p ospec i e s udy o dein ensi ied chemo adio he apy o low- isk human papilloma i us-
associa ed o opha yngeal squamous cell ca cinoma. Cance . 2018 Jun;124(11):2347–54.
28 Bea y BT, Moon DH, Shen CJ, Amdu RJ, Weiss J, G illey-Olson J, e al. PIK3CA Mu a ion
in HPV-Associa ed OPSCC Pa ien s Recei ing Dein ensi ied Chemo adia ion. J Na l Cance
Ins . 2020 Aug;112(8):855–8.
29 Mehanna H, Robinson M, Ha ley A, Kong A, Fo an B, Ful on-Lieuw T, e al. Radio he apy
plus cispla in o ce uximab in low- isk human papilloma i us-posi i e o opha yngeal cance
(De-ESCALaTE HPV): an open-label andomised con olled phase 3 ial. Lance (London,
England). 2019 Jan;393(10166):51–60.
30 Qin T, Li S, Hen y LE, Liu S, Sa o MA. Molecula umo sub ypes o hp -posi i e head and
neck cance s: Biological cha ac e is ics and implica ions o clinical ou comes. Cance s.
2021;13(11):1–21.
31 Cance Genome A las Ne wo k. Comp ehensi e genomic cha ac e iza ion o head and neck
squamous cell ca cinomas. Na u e. 2015 Jan;517(7536):576–82.
101
32 Fa ah CS. Molecula landscape o head and neck cance and implica ions o he apy.
Annals o T ansla ional Medicine. 2021 May;9(10):915–915.
33 Amin MB, G eene FL, Edge SB, Comp on CC, Ge shenwald JE, B ookland RK, e al. The
Eigh h Edi ion AJCC Cance S aging Manual: Con inuing o build a b idge om a popula ion-
based o a mo e “pe sonalized” app oach o cance s aging. CA Cance J Clin. 2017
Ma ;67(2):93–9.
34 Bo ges V, Pinhei o M, Pechi a P, Guioma R, Gomes JP. INSaFLU: an au oma ed open
web-based bioin o ma ics sui e “ om- eads” o in luenza whole-genome-sequencing-based
su eillance. Genome Med. 2018 Jun;10(1):46.
35 Tamu a K, Nei M, Kuma S. P ospec s o in e ing e y la ge phylogenies by using he
neighbo -joining me hod. P oceedings o he Na ional Academy o Sciences o he Uni ed
S a es o Ame ica. 2004;101(30):11030–5.
36 Sai ou N, Nei M. The neighbo -joining me hod: a new me hod o econs uc ing phylogene ic
ees. Molecula biology and e olu ion. 1987 Jul;4(4):406–25.
37 Gillison ML, D’Souza G, Wes a W, Suga E, Xiao W, Begum S, e al. Dis inc isk ac o
p o iles o human papilloma i us ype 16-posi i e and human papilloma i us ype 16-
nega i e head and neck cance s. J Na l Cance Ins . 2008 Ma ;100(6):407–20.
38 Hassani S, Cas illo A, Oho i JI, Higashi M, Ku ono Y, Akiba S, e al. Molecula Pa hogenesis
o Human Papilloma i us Type 16 in Tonsilla Squamous Cell Ca cinoma. An icance
esea ch. 2015 Dec;35(12):6633–8.
39 Be na d HU, Calleja-Macias IE, Dunn ST. Genome a ia ion o human papilloma i us ypes:
Phylogene ic and medical implica ions. In e na ional Jou nal o Cance . 2006
Ma ;118(5):1071–6.
40 Siche o L, Villa LL. Epidemiological and unc ional implica ions o molecula a ian s o
human papilloma i us. B az J Med Biol Res. 2006 Jun;39(6):707–17.
41 Song YS, Kee SH, Kim JW, Pa k NH, Kang SB, Chang WH, e al. Majo sequence a ian s
in E7 gene o human papilloma i us ype 16 om ce ical cance ous and noncance ous
lesions o Ko ean women. Gynecol Oncol. 1997 Aug;66(2):275–81.
42 San os C, Vilano a M, Medei os R, Gil da Cos a RM. HPV- ansgenic mouse models: Tools
o s udying he cance -associa ed immune esponse. Vi us Resea ch. 2017 May;235:49–
57.
43 Cos a AC, San os JMO, Medei os-Fonseca B, Oli ei a PA, Bas os MMSM, B i o HO, e al.
Cha ac e izing he In lamma o y Mic oen i onmen in K14-HPV16 T ansgenic Mice: Mas
Cell In il a ion and Mic oRNA Exp ession. Cance s (Basel). 2022 Ap ;14(9):2216.
44 A aújo R, San os JMO, Fe nandes M, Dias F, Sousa H, Ribei o J, e al. Exp ession p o ile o
mic oRNA-146a along HPV-induced mul is ep ca cinogenesis: a s udy in HPV16 ansgenic
mice. J Cance Res Clin Oncol. 2018 Feb;144(2):241–8.

102
45 Pai a I, Gil da Cos a RM, Ribei o J, Sousa H, Bas os MMSM, Faus ino-Rocha A, e al.
Mic oRNA-21 exp ession and suscep ibili y o HPV-induced ca cinogenesis - ole o
mic oen i onmen in K14-HPV16 mice model. Li e Sci. 2015 May;128:8–14.
46 Pai a I, Gil da Cos a RM, Ribei o J, Sousa H, Bas os M, Faus ino-Rocha A, e al. A ole o
mic oRNA-155 exp ession in mic oen i onmen associa ed o HPV-induced ca cinogenesis
in K14-HPV16 ansgenic mice. PLoS One. 2015;10(1):e0116868.
Figu e Legends
Fig.1 HPV16 phylogeny in HNSCC samples. The phylogeny ee was econs uc ed
using he Neighbo -Joining me hod [38] wi h he Maximum Composi e Likelihood
model [37]. Numbe s nex o he b anch nodes indica e he boo s ap alues (1000
eplica es).
103
Fig.2 Non-synonymous a ian si es in HPV16 genome. Fo each genome posi ion o
he HPV16 A1 sublineage ep esen a i e (NC_001526.4), all non-synonymous
mu a ions displayed by he 35 HNSCC umou samples a e shown. Vi al coding
egions a e ep esen ed on he op o he igu e, while on he le , he phylogene ic
ee wi h he pa ien samples shown in Figu e 1. Nucleo ide a ian s a e colou ed-
coded, wi h nucleo ides C, G, T, and A ep esen ed by yellow, ed, blue and g een
(colou ed-coded), espec i ely.
Fig.3 Nonsynonymous- o-synonymous mu a ion a io o each HPV16 gene. Fo
each gene, es ima es we e based on he p-dis ance model. Minimum and maximum
alues ep esen lowe and uppe limi s o he s anda d e o (SE) o he es ima e
( e ical ba ). Fo be e isualiza ion, he posi i e p essu e h eshold is shown
(ho izon al ed line).
104
Fig.4 Kaplan–Meie plo s o o e all su i al by each HPV lineage.
105
2.3 CHARACTERIZATION OF THE HPV16 E6 AND E7 ONCOGENES IN
K14HPV16 MICE: SUBLINEAGE A1 DRIVES MULTI-ORGAN
CARCINOGENESIS.
In . J. Mol. Sci. DOI: h ps://doi.o g/10.3390/ijms232012371
Abs ac
The s udy o HPV-induced ca cinogenesis elies on mul iple in i o mouse models,
one o which elies on he cy oke a in 14 gene (CK14) p omo e o d i e he
exp ession o all HPV ea ly oncogenes. Ou s udy e alua ed HPV DNA om 17
samples om 4 animals, wild- ype (WT, n=2) and HPV16- ansgenic mice (MUT,
n=2). To al DNA was ex ac ed and de ec ion o HPV16 was pe o med using a qPCR
mul iplex. HPV16-posi i e samples we e subsequen ly whole-genome sequenced
by NGS echniques. Compa a i e genome analysis o K14HPV16 samples e ealed
a o al o 12 mu a ions o he HPV16 sublineage A1 ep esen a i e s ain. Mos o he
mu a ions all wi hin he coding egions o E1, E2, E4 and E5 ea ly genes and o L1
and L2 la e genes. The phylogene ic posi ioning clea ly shows K14HPV16 samples
clus e ing oge he in he sub-lineage A1 (NC001526.4). The knowledge o he
a ian is e y impo an , and hese indings will allow he a ional use o his
animal model o explo e he ole o he A1 sublineage in HPV-d i en cance .
Keywo ds: K14HPV16, Va ian
112
In . J. Mol. Sci. 2022,23, 12371 5 o 10
exhibi ing di e en ial in e media e equencies ac oss he nine K14HPV16 samples ( om
12.3% o K14HPV16_15 o 22.1% o K14HPV16_11).
3. Discussion
HPV16 a ian s show a di e en ial p e alence in di e se geog aphical loca ions and
a e associa ed wi h di e en ial oncogenic po en ial [
5
–
8
]. Caucasian women in ec ed wi h
he A1/A2 a ian a e a a highe isk o CIN3+ compa ed o women o o he gene ic
backg ounds [
19
]. This is also he mos common a ian ound in head and neck o al
cance s in Caucasians and ce ical ca cinomas [
20
], sugges ing a heigh ened oncogenic
po en ial compa ed wi h o he HPV16 a ian s.
Fo he i s ime, he p esen s udy de e mined he a ian lineage p esen in he
K14HPV16 ansgenic mouse model, based on he ela edness o e e ence sequences
including en HPV16 A, B, C, and D a ian lineages. The K14HPV16 mouse model was
shown o ca y he HPV16 A1 sublineage. Despi e hei isola ion om dis inc issues,
he K14HPV16 samples we e ound o be highly gene ically ela ed among hem, only
di e ing by a mean o 1.0
±
0.8 nucleo ides and showing a mean dis ance o 3.1
±
1.7
nucleo ides om he phylogene ically closes sublineage A1 ep esen a i e s ain. On he
o he hand, K14HPV16 samples exhibi ed a mean dis ance o o he sublineages ha anged
om 22.0
±
4.5 (sublineage A3) o 112.0
±
10.3 (sublineage D2). Conside ing ha a ian
HPV sublineages a e empi ically de ined as exhibi ing genome di e ences in he 0.5–1.0%
ange [
21
], espec i ely, hese esul s poin o a clonal o igin o K14HPV16 samples, in
ag eemen wi h he congenic na u e o his mouse s ain [12].
Ha ing de e mined he a ian lineage and sublineage p esen in K14HPV16 mice,
i is now possible o discuss i s associa ion wi h he a ious kinds o neoplas ic lesions
obse ed in his mouse s ain. K14HPV16 mice ha e been used o mimic he de elopmen
o ce ical cance in he 1990s [
13
]. In his s udy, he HPV16 oncogenes we e necessa y bu
insu icien o induce ce ical cance and ch onic es ogen supplemen a ion was equi ed
o ca cinogenesis. Based on he p esen esul s, i is in e es ing o specula e ha , al hough
he A1 sublineage is clinically associa ed wi h a heigh ened isk o ce ical cance , i may
equi e ho monal co- ac o s o e icien ca cinogenesis. Recen ly, ou g oup employed
K14HPV16 mice o p oducing he i s mouse model o HPV- ela ed penile cance [
22
].
Again, he A1 lineage was necessa y bu insu icien o induce in asi e squamous cell
ca cinoma and a obacco- ela ed co-ca cinogen was needed. Howe e , he HPV16 A1
lineage was able o induce a ange o penile in aepi helial lesions in his model wi hou
addi ional co- ac o s. In e es ingly, K14HPV16 mice de elop squamous cell ca cinomas
speci ically loca ed a he ongue base, wi hou he need o any chemical o ho monal
co-ca cinogens—al hough umo s we e mo e equen in emale mice [
15
]. O opha yngeal
cance incidence could be inc eased in his model by exposu e o p aquiloside, a b acken
e n ca cinogen, sugges ing a syne gis ic e ec [
23
]. Based on hese obse a ions, we
specula e ha he oncogenic po en ial o he HPV16 A1 a ian lineage in his mouse
model is dependen on he ana omic si e: ce ical and penile ca cinogenesis equi e he
p esence o co- ac o s while o opha yngeal cance may be solely induced by he i al
oncogenes, al hough he p edominan incidence in emale mice sugges s a ole o he highe
physiological s a e o es ogen. Fu he s udies a e wa an ed o examine he in e play
be ween di e en HPV16 lineages and ho monal co- ac o s. Rema kably, ano he s udy
using mice ca ying only he HPV16 E6 and E7 ansgenes wi hou a ian assignmen
ound ha o opha yngeal ca cinogenesis depended on a chemical co-ca cinogen [24].
The K14HPV16 mouse model was also used o es new he apies and cance p e en-
i e s a egies by ou g oup and o he s [
14
,
16
,
25
,
26
]. In gene al, he de elopmen o lesions
in his animal model is s ongly associa ed wi h modula ion o he immune esponse wi hin
he umo mic oen i onmen , and combined immune he apy app oaches a e equi ed
o p e en lesion de elopmen [
26
]. Now, i will be possible o assess he speci ic impac
o hose app oaches on he HPV16 A1 sublineage, and i would be desi able o de elop
animal models ha ep esen ed o he HPV16 s ains as well.

113
In . J. Mol. Sci. 2022,23, 12371 4 o 10
samples we e no included in he phylogene ic analysis. Ne e heless, hey sha e he same
majo mu a ions as he emaining K14HPV16 samples agains he HPV16 sublineage A1
ep esen a i e s ain (see de ails below). The phylogene ic analysis (Figu e 3) clea ly shows
K14HPV16 samples clus e ing oge he in he sub-lineage A1 (NC001526.4).
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 o 11
2.3. K14HPV16 Lineage Classi ica ion
HPV16 a ian s ha e been classi ied in o ou majo lineages (A–D) and sublineages
(A1–A4, B1, B2, and D1–D3), based on hei genome sequence di e si y [17]. To de e mine
he phylogene ic posi ioning o he HPV16 ansgenes in K14HPV16 samples, he
espec i e consensus sequences we e aligned agains he genomes o lineage/sublineage
ep esen a i e sequences (Supplemen a y Table S1). Fo K14HPV16-3 and K14HPV16-6,
he gene a ion o a consensus sequence ailed (% o genome co e ed <70%), and hose wo
samples we e no included in he phylogene ic analysis. Ne e heless, hey sha e he same
majo mu a ions as he emaining K14HPV16 samples agains he HPV16 sublineage A1
ep esen a i e s ain (see de ails below). The phylogene ic analysis (Figu e 3) clea ly
shows K14HPV16 samples clus e ing oge he in he sub-lineage A1 (NC001526.4).
Figu e 3. Phylogene ic posi ioning o K14HPV16 samples wi hin he majo HPV16 lineages (A–D).
Legend: he phylogene ic ee was gene a ed using he neighbo -joining me hod [18] wi h he
maximum composi e likelihood model [17] and depic s he gene ic ela ionships o he ob ained
K14HPV16 genome consensus sequences o each ep esen a i e genome. Numbe s nex o he
b anch nodes indica e he boo s ap alues (1000 eplica es). HPV16 lineages a e shown in g ey
Figu e 3.
Phylogene ic posi ioning o K14HPV16 samples wi hin he majo HPV16 lineages
(A–D)
.
Legend: he phylogene ic ee was gene a ed using he neighbo -joining me hod [
18
] wi h he
maximum composi e likelihood model [
17
] and depic s he gene ic ela ionships o he ob ained
K14HPV16 genome consensus sequences o each ep esen a i e genome. Numbe s nex o he b anch
nodes indica e he boo s ap alues (1000 eplica es). HPV16 lineages a e shown in g ey boxes nex o
he main ee b anches, while sublineages (A1–A4, B1, B2, and D1–D3) a e illus a ed in black boxes.
2.4. HPV16 Sequencing
Compa a i e genome analysis o K14HPV16 samples e ealed a o al o h ee mu-
a ions o he HPV16 sublineage A1 ep esen a i e s ain. All mu a ions all wi hin he
coding egion o he E1 ea ly gene, wi h mo e han hal yielding amino acid changes. Two
o hese (330A > G|Leu110Leu and 978A > G|Ile326Me ) ha e become e olu iona ily
ixed ( equency o 100%) based on he obse ed deep co e age suppo ing each al e a ion.
Indeed, 100% o he eads mapping each posi ion ( o pos 330: anging om 1580x o
sample 6 o 15456x o sample 8; and o pos. 978; anging om 636x o sample 6 o 11951x
o sample 8) suppo he wo a ian s ound in all K14HPV16 samples. The las mu a ion
is a non-synonymous mino in a-pa ien single nucleo ide a ian (166A > T|Asn56Ty )
114
In . J. Mol. Sci. 2022,23, 12371 3 o 10
2. Resul s
2.1. Sample Cha ac e iza ion
His ological samples om wild- ype (WT) animals showed no mal his ology and
HPV16- ansgenic (MUT) mice samples showed ypical p oli e a i e epi helial lesions
o he skin and ongue, while he li e and lymph node samples om WT and HPV16-
angenic mice showed mild in lamma o y changes o none a all. The o al umo ound
in he HPV16- angenic mice was iden i ied as a squamous cell ca cinoma, wi h minimal
in asion o he ongue (Figu e 2).
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 o 11
Figu e 1. Schema ic o he HPV-16R genome. The genome o ganiza ion o 7906 base pai s (bp) a -
anged in a ci cula o m ep esen s he h ee unc ional egions, ea ly, la e, and long con ol (LCR);
and a non-coding egion (NCR). These a e sepa a ed by wo polyadenyla ion si es, desc ibed as
ea ly (pAE) and la e (pAL). In o al, he genome encodes eigh open eading ames (ORFs), o which
E1, E2, E4, E5, E6, and E7 a e om he ea ly egion; and L1 and L2 a e om he la e egion. The LCR,
loca ed be ween he E6 and L1 gene, is a egula o y egion ha includes he o igin o eplica ion
(o i) and he p97 p omo e .
2. Resul s
2.1. Sample Cha ac e iza ion
His ological samples om wild- ype (WT) animals showed no mal his ology and
HPV16- ansgenic (MUT) mice samples showed ypical p oli e a i e epi helial lesions o
he skin and ongue, while he li e and lymph node samples om WT and HPV16-
angenic mice showed mild in lamma o y changes o none a all. The o al umo ound
in he HPV16- angenic mice was iden i ied as a squamous cell ca cinoma, wi h minimal
in asion o he ongue (Figu e 2).
Figu e 2. His ological indings o WT and MUT ongue and o al cance . Legend: His ological sam-
ples om wild- ype and ansgenic K14HPV16 mice s ained wi h H&E. (A) low powe iew o no -
mal squamous cell epi helium o he ongue om a wild- ype mouse; (B) low powe iew o mild
dysplas ic squamous cell epi helium wi h enla ged hype ch oma ic nuclei in he uppe laye s o he
epi helium om a ansgenic K14HPV16 mouse and (C) low powe iew o a ocally in asi e squa-
mous cell ca cinoma wi h hickened e e idges and dysplas ic cells wi h hype ch oma ic nuclei,
i egula basemen memb ane and ocally dis up ed associa ed wi h in lamma o y cells in he
s oma om a ansgenic K14HPV16 mouse; he inse shows he in asi e on . (D) low powe iew
o he li e o one ansgenic K14HPV16 mouse showing no mal s uc u e and cy ology.
2.2. HPV16 Genome Co e age and Quali y
Among he nine HPV16-posi i e samples, he mean dep h o co e age was 2080- old,
anging be ween 870- old o he node sample and 3726- old o he umo sample. On
a e age, samples had 75% o he HPV16 genome co e ed, wi h 95% o i co e ed by a
leas 10- old. The only excep ion was a ~1900 bp egion comp ising mos o he L1 gene,
he ups eam egula o y egion (URR) and he beginning o he E6 gene o which no am-
pli ica ion signal was obse ed o all samples. This is in ag eemen wi h he model design
[12] which encompasses he en i e HPV16 ea ly coding egion om bp 97 o 6152, al -
hough he gap be ween he L1 and E6 egion (~1945 pb) comp omises he hyb idiza ion
e iciency o speci ic p ime s wi hin he p ime pool o ha egion.
Figu e 2.
His ological indings o WT and MUT ongue and o al cance . Legend: His ological
samples om wild- ype and ansgenic K14HPV16 mice s ained wi h H&E. (
A
) low powe iew o
no mal squamous cell epi helium o he ongue om a wild- ype mouse; (
B
) low powe iew o mild
dysplas ic squamous cell epi helium wi h enla ged hype ch oma ic nuclei in he uppe laye s o
he epi helium om a ansgenic K14HPV16 mouse and (
C
) low powe iew o a ocally in asi e
squamous cell ca cinoma wi h hickened e e idges and dysplas ic cells wi h hype ch oma ic nuclei,
i egula basemen memb ane and ocally dis up ed associa ed wi h in lamma o y cells in he s oma
om a ansgenic K14HPV16 mouse; he inse shows he in asi e on . (
D
) low powe iew o he
li e o one ansgenic K14HPV16 mouse showing no mal s uc u e and cy ology.
2.2. HPV16 Genome Co e age and Quali y
Among he nine HPV16-posi i e samples, he mean dep h o co e age was 2080- old,
anging be ween 870- old o he node sample and 3726- old o he umo sample. On
a e age, samples had 75% o he HPV16 genome co e ed, wi h 95% o i co e ed by a
leas 10- old. The only excep ion was a ~1900 bp egion comp ising mos o he L1 gene,
he ups eam egula o y egion (URR) and he beginning o he E6 gene o which no
ampli ica ion signal was obse ed o all samples. This is in ag eemen wi h he model
design [
12
] which encompasses he en i e HPV16 ea ly coding egion om bp 97 o 6152,
al hough he gap be ween he L1 and E6 egion (~1945 pb) comp omises he hyb idiza ion
e iciency o speci ic p ime s wi hin he p ime pool o ha egion.
2.3. K14HPV16 Lineage Classi ica ion
HPV16 a ian s ha e been classi ied in o ou majo lineages (A–D) and sublineages
(A1–A4, B1, B2, and D1–D3), based on hei genome sequence di e si y [
17
]. To de e -
mine he phylogene ic posi ioning o he HPV16 ansgenes in K14HPV16 samples, he
espec i e consensus sequences we e aligned agains he genomes o lineage/sublineage
ep esen a i e sequences (Supplemen a y Table S1). Fo K14HPV16-3 and K14HPV16-6,
he gene a ion o a consensus sequence ailed (% o genome co e ed <70%), and hose wo
115
In . J. Mol. Sci. 2022,23, 12371 2 o 10
1. In oduc ion
Papilloma i uses a e species-speci ic double-s anded DNA i uses o 8000 base pai s
(bp) leng h (Figu e 1) ha ha e p e e en ial opism o epi helial cells. In ec ion wi h
human papilloma i us (HPV) is he mos common sexually ansmissible in ec ion and
induces a ange o benign (e.g., condylomas) and malignan lesions, such as ce ical cance
and o he anogeni al squamous cell ca cinomas and a g owing subse o o opha yngeal
squamous cell ca cinomas [
1
]. HPV can be classi ied in o geno ypes de ined by a g ea e
han 10.0% a ia ion in hei L1 gene sequence [
2
]. Cu en ly, o e 200 HPV ypes a e
ecognized and g ouped as high- isk (e.g., HPV16 and HPV18, associa ed wi h malignan
ce ical lesions) o low- isk (HPV6 and HPV11, associa ed wi h benign lesions) [
2
–
4
].
Accumula ing da a indica e ha HPV in a- ype a ian s, lineages (de ined by a 1.0–10.0%
genomic a ia ion), and sub-lineages (0.5–1.0% a ia ion) may di e in hei ca cinogenic
po en ial [
5
–
8
], as ecen ly e iewed [
9
,
10
]. The s udy o HPV-induced ca cinogenesis
uses mul iple
in i o
mouse models [
11
], one o which elies on he K 14 (cy oke a in
14) gene p omo e o d i e he exp ession o all HPV16 ea ly oncogenes and speci ically
a ge basal ke a inocy es (known as K14HPV16 mice) [
12
]. This widely used model was
i s de eloped in he 1990s and has since been used o model ce ical cance [
13
] and
o he HPV16-associa ed malignancies [
14
–
16
]. This animal model has also p o ed use ul o
elucida e he immune-modula o y mechanisms in ol ed in HPV16-induced cance s and
o es po en ial new he apies. Howe e , he speci ic a ian and sub-lineage in ol ed
in K14HPV16 mice emain unknown [
12
]. De e mining which a ian and sublineage
a e p esen in his animal model would help es hei po en ial associa ions wi h he
de elopmen o cance a speci ic loca ions. Addi ionally, his in o ma ion would help
esea che s. This s udy aimed o de e mine he HPV16 a ian and sublineage p esen
in he K14HPV16 mouse model, u he cha ac e izing his impo an esea ch ool and
po en ia ing i s use o basic and ansla ional s udies.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 o 11
Keywo ds: K14HPV16; ca cinogenesis; HPV16; a ian ; lineage
1. In oduc ion
Papilloma i uses a e species-speci ic double-s anded DNA i uses o 8000 base
pai s (bp) leng h (Figu e 1) ha ha e p e e en ial opism o epi helial cells. In ec ion
wi h human papilloma i us (HPV) is he mos common sexually ansmissible in ec ion
and induces a ange o benign (e.g., condylomas) and malignan lesions, such as ce ical
cance and o he anogeni al squamous cell ca cinomas and a g owing subse o o opha-
yngeal squamous cell ca cinomas [1]. HPV can be classi ied in o geno ypes de ined by a
g ea e han 10.0% a ia ion in hei L1 gene sequence [2]. Cu en ly, o e 200 HPV ypes
a e ecognized and g ouped as high- isk (e.g., HPV16 and HPV18, associa ed wi h malig-
nan ce ical lesions) o low- isk (HPV6 and HPV11, associa ed wi h benign lesions) [2–
4]. Accumula ing da a indica e ha HPV in a- ype a ian s, lineages (de ined by a 1.0–
10.0% genomic a ia ion), and sub-lineages (0.5–1.0% a ia ion) may di e in hei ca -
cinogenic po en ial [5–8], as ecen ly e iewed [9,10]. The s udy o HPV-induced ca cino-
genesis uses mul iple in i o mouse models [11], one o which elies on he K 14 (cy-
oke a in 14) gene p omo e o d i e he exp ession o all HPV16 ea ly oncogenes and
speci ically a ge basal ke a inocy es (known as K14HPV16 mice) [12]. This widely used
model was i s de eloped in he 1990s and has since been used o model ce ical cance
[13] and o he HPV16-associa ed malignancies [14–16]. This animal model has also p o ed
use ul o elucida e he immune-modula o y mechanisms in ol ed in HPV16-induced can-
ce s and o es po en ial new he apies. Howe e , he speci ic a ian and sub-lineage
in ol ed in K14HPV16 mice emain unknown [12]. De e mining which a ian and sub-
lineage a e p esen in his animal model would help es hei po en ial associa ions wi h
he de elopmen o cance a speci ic loca ions. Addi ionally, his in o ma ion would help
esea che s. This s udy aimed o de e mine he HPV16 a ian and sublineage p esen in
he K14HPV16 mouse model, u he cha ac e izing his impo an esea ch ool and po-
en ia ing i s use o basic and ansla ional s udies.
Figu e 1.
Schema ic o he HPV-16R genome. The genome o ganiza ion o 7906 base pai s (bp)
a anged in a ci cula o m ep esen s he h ee unc ional egions, ea ly, la e, and long con ol (LCR);
and a non-coding egion (NCR). These a e sepa a ed by wo polyadenyla ion si es, desc ibed as ea ly
(pAE) and la e (pAL). In o al, he genome encodes eigh open eading ames (ORFs), o which E1,
E2, E4, E5, E6, and E7 a e om he ea ly egion; and L1 and L2 a e om he la e egion. The LCR,
loca ed be ween he E6 and L1 gene, is a egula o y egion ha includes he o igin o eplica ion (o i)
and he p97 p omo e .
116
2.4 PIK3CA GENE MUTATIONS IN HNSCC: SYSTEMATIC REVIEW AND
CORRELATIONS WITH HPV STATUS AND PATIENT SURVIVAL
Cance s. DOI: 10.3390/cance s14051286
Abs ac
PIK3CA mu a ions a e belie ed o con ibu e o he pa hogenesis o human
papilloma i us (HPV)-associa ed head and neck squamous cell ca cinomas
(HNSCC). This s udy aims o es ablish he equency o PIK3CA mu a ions in a
Po uguese HNSCC coho and o de e mine hei associa ion wi h HPV s a us and
pa ien su i al. A me a-analysis o scien i ic li e a u e also done e ealed widely
di e en mu a ion a es in coho s om di e en wo ld egions and a end
owa ds imp o ed p ognosis among pa ien s wi h PIK3CA mu a ions. DNA
samples we e a ailable om 95 pa ien s diagnosed wi h HNSCC a he Po uguese
Ins i u e o Oncology in Lisbon, be ween 2010 and 2019. HPV s a us was es ablished
based on i al DNA de ec ed using eal- ime PCR. E alua ion o PIK3CA gene
mu a ions was pe o med by eal- ime PCR o 4 mu a ions (H1047L; E542K, E545K,
E545D). 37 cases we e ound o ha bou PIK3CA mu a ions (39%) wi h he E545D
mu a ion (73%) mo e equen de ec ed. The e we e no signi ican associa ions
be ween mu a ional s a us and HPV s a us (74% WT and 68% MUT we e HPV (+);
p=0.489) o o e all su i al (OS) (3-y OS: WT 54% and MUT 65%; p= 0.090). HPV
s a us was he only ac o signi ican ly associa ed wi h bo h OS and disease- ee
su i al (DFS), wi h HNSCC-HPV pa ien s ha ing consis en ly be e ou comes (3-
y OS: HPV (+) 65% and HPV (-) 36%; p=0.007; DFS HPV (+) 83% and HPV (-) 43%;
p=0.001). The e was a s a is ically signi ican in e ac ion e ec be ween HPV s a us
and PIK3CA mu a ion ega ding DFS (In e ac ion es : p=0.026). In HNSCC-HPV
pa ien s, PIK3CA wild ype is associa ed wi h a signi ican 4.64 imes inc ease in he
haza d o ecu ence o dea h (HR=4.64; 95% CI 1.02-20.99; p=0.047). O e all, PIK3CA
gene mu a ions a e p esen in many pa ien s and may help de ine pa ien subse s
who can bene i om he apies a ge ing he PI3K pa hway. The sys ema ic
assessmen o PIK3CA gene mu a ions in HNSCC pa ien s will equi e u he
me hodological s anda diza ion.
Keywo ds: HNSCC; HPV; p16 INK4a; PIK3CA
117
!"#!$%&'(!
!"#$%&'
Ci a ion: Cochicho, D.; Es e es, S.;
Ri o, M.; Sil a, F.; Ma ins, L.;
Mon al ão, P.; Cunha, M.; Magalhães,
M.; Gil da Cos a, R.M.; Felix, A.
PIK3CA Gene Mu a ions in HNSCC:
Sys ema ic Re iew and Co ela ions
wi h HPV S a us and Pa ien Su i al.
Cance s 2022,14, 1286. h ps://
doi.o g/10.3390/cance s14051286
Academic Edi o : Da id Wong
Recei ed: 5 Feb ua y 2022
Accep ed: 25 Feb ua y 2022
Published: 2 Ma ch 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
cance s
A icle
PIK3CA Gene Mu a ions in HNSCC: Sys ema ic Re iew and
Co ela ions wi h HPV S a us and Pa ien Su i al
Daniela Cochicho 1,2, Susana Es e es 3, Miguel Ri o 4, Fe nanda Sil a 1, Luís Ma ins 2, Ped o Mon al ão5,
Má io Cunha 2, Miguel Magalhães 5, Rui M. Gil da Cos a 6,7,8,9,† and Ana Felix 1,4,*,†
1NOVA Medical School, NOVA Uni e si y o Lisbon, 1099-085 Lisbon, Po ugal;
[email p o ec ed] (D.C.); [email p o ec ed] (F.S.)
2Vi ology Labo a o y om Clinical Pa hology Depa men , IPOLFG, 1099-023 Lisbon, Po ugal;
[email p o ec ed] (L.M.); [email p o ec ed] (M.C.)
3Clinical Resea ch Uni , IPOLFG, 1099-023 Lisbon, Po ugal; [email p o ec ed]
4Pa hology Depa men , IPOLFG, 1099-023 Lisbon, Po ugal; [email p o ec ed]
5O o hinola yngology Depa men , IPOLFG, 1099-023 Lisbon, Po ugal;
[email p o ec ed] (P.M.); [email p o ec ed] (M.M.)
6Pos -G adua e P og amme in Adul Heal h (PPGSAD), Mo phology Depa men ,
Uni e si y Hospi al (HUUFMA), Fede al Uni e si y o Ma anhão, São Luís 65080-805, B azil;
[email p o ec ed]
7LEPABE, Labo a o y o P ocess Enginee ing, En i onmen , Bio echnology and Ene gy,
Facul y o Enginee ing, Uni e si y o Po o, Rua Robe o F ias, 4200-465 Po o, Po ugal
8Cen e o he Resea ch and Technology o Ag o-En i onmen al and Biological Sciences (CITAB),
Ino 4Ag o, Uni e si y o T ás-os-Mon es e Al o Dou o (UTAD), Quin a de P ados,
5000-801 Vila Real, Po ugal
9Molecula Oncology and Vi al Pa hology G oup, Resea ch Cen e o IPO Po o (CI-IPOP)/RISE@CI-IPOP
(Heal h Resea ch Ne wo k), Po uguese Oncology Ins i u e o Po o (IPO Po o)/Po o Comp ehensi e
Cance Cen e (Po o.CCC), 4200-162 Po o, Po ugal
*Co espondence: [email p o ec ed]
† These au ho s con ibu ed equally o his wo k.
Simple Summa y:
Mu a ions o he PIK3CA gene a e hough o con ibu e o he de elopmen
o head and neck squamous cell ca cinomas (HNSCC), especially hose associa ed wi h human
papilloma i us in ec ion. Fu he mo e, hese mu a ions may help iden i y pa ien s who can bene i
om speci ic a ge ed he apies. This s udy p esen s a sys ema ic e iew o he PIK3CA mu a ions
p o ile in HNSCC. The esul s a e compa ed wi h a coho o Po uguese pa ien s o s udy he
possible associa ions wi h HPV s a us and pa ien su i al. The Po uguese coho ha bou ed
PIK3CA mu a ions in 39% o pa ien s, and he e we e no signi ican associa ions wi h he HPV
s a us o wi h he OS. In his o iginal case se ies, he e was a s a is ically signi ican in e ac ion
e ec be ween HPV s a us and PIK3CA mu a ion ega ding disease- ee su i al. In HPV-posi i e
pa ien s, he PIK3CA wild- ype is associa ed wi h a signi ican 4.64 imes inc ease in he haza d o
ecu ence o dea h. Addi ional s udies a e needed o cla i y he implica ions o PIK3CA mu a ions
o pa ien p ognosis.
Abs ac :
PIK3CA mu a ions a e belie ed o con ibu e o he pa hogenesis o human papilloma i us
(HPV)-associa ed head and neck squamous cell ca cinomas (HNSCC). This s udy aims o es ablish he
equency o PIK3CA mu a ions in a Po uguese HNSCC coho and o de e mine hei associa ion
wi h he HPV s a us and pa ien su i al. A me a-analysis o scien i ic li e a u e also e ealed widely
di e en mu a ion a es in coho s om di e en wo ld egions and a end owa ds imp o ed
p ognosis among pa ien s wi h PIK3CA mu a ions. DNA samples we e a ailable om 95 pa ien s
diagnosed wi h HNSCC a he Po uguese Ins i u e o Oncology in Lisbon be ween 2010 and 2019.
HPV s a us was es ablished based on i al DNA de ec ed using eal- ime PCR. The e alua ion
o PIK3CA gene mu a ions was pe o med by eal- ime PCR o ou mu a ions (H1047L; E542K,
E545K, and E545D). Thi y-se en cases we e ound o ha bou PIK3CA mu a ions (39%), wi h he
E545D mu a ion (73%) mo e equen ly de ec ed. The e we e no signi ican associa ions be ween he
mu a ional s a us and HPV s a us (74% WT and 68% MUT we e HPV (+); p= 0.489) o o e all su i al
Cance s 2022,14, 1286. h ps://doi.o g/10.3390/cance s14051286 h ps://www.mdpi.com/jou nal/cance s

118
Cance s 2022,14, 1286 19 o 19
44.
Shigaki, H.; Baba, Y.; Wa anabe, M.; Mu a a, A.; Ishimo o, T.; Iwa suki, M.; Iwagami, S.; Nosho, K.; Baba, H. PIK3CA mu a ion is
associa ed wi h a a o able p ognosis among pa ien s wi h cu a i ely esec ed esophageal squamous cell ca cinoma. Clin. Cance
Res. 2013,19, 2451–2459. [C ossRe ][PubMed]
45.
Zi˛eba, S.; Chechli´nska, M.; Kowalik, A.; Kowalewska, M. Genes, pa hways and ul a ca cinoma-New insigh s om nex -
gene a ion sequencing s udies. Gynecol. Oncol. 2020,158, 498–506. [C ossRe ][PubMed]
46.
Qiu, W.; Tong, G.X.; Manolidis, S.; Close, L.G.; Assaad, A.M.; Su, G.H. No el Mu an -En iched Sequencing Iden i ied High
F equency o PIK3CA Mu a ions in Pha yngeal Cance . In . J. Cance 2008,122, 1189–1194. [C ossRe ][PubMed]
47.
R Co e Team. A Language and En i onmen o S a is ical Compu ing; R Founda ion o S a is ical Compu ing: Vienna, Aus ia, 2021.
A ailable online: h p://www.R-p ojec .o g/ (accessed on 19 No embe 2021).
119
Cance s 2022,14, 1286 18 o 19
21.
C ook, T.; Mo gens e n, J.P.; C aw o d, L.; Banks, L. Con inued exp ession o HPV-16 E7 p o ein is equi ed o main enance o
he ans o med pheno ype o cells co- ans o med by HPV-16 plus EJ- as. EMBO J. 1989,8, 513–519. [C ossRe ][PubMed]
22.
Vee a agha alu, K.; Subbaiah, V.K.; S i as a a, S.; Chak aba i, O.; Syal, R.; K ishna, S. Complemen a ion o human papillo-
ma i us ype 16 E6 and E7 by Jagged1-speci ic No ch1-phospha idylinosi ol 3-kinase signalling in ol es pleio opic oncogenic
unc ions independen o CBF1;Su(H);Lag-1 ac i a ion. J. Vi ol. 2005,79, 7889–7898. [C ossRe ]
23.
Spangle, J.M.; Munge , K. The HPV16 E6 Oncop o ein Causes P olonged Recep o P o ein Ty osine Kinase Signaling and
Enhances In e naliza ion o Phospho yla ed Recep o Species. PLoS Pa hog. 2013,9, e1003237. [C ossRe ][PubMed]
24.
Mo gan, E.L.; Macdonald, A. Au oc ine STAT3 ac i a ion in HPV posi i e ce ical cance h ough a i us-d i en Rac1-NF

B-IL-6
signalling axis. PLoS Pa hog. 2019,15, e1007835. [C ossRe ]
25.
Menges, C.W.; Baglia, L.A.; Lapoin , R.; McCance, D.J. Human papilloma i us ype 16 E7 up- egula es AKT ac i i y h ough he
e inoblas oma p o ein. Cance Res. 2006,66, 5555–5559. [C ossRe ]
26.
Lui, V.W.Y.; Hedbe g, M.L.; Li, H.; Vanga a, B.S.; Pendle on, K.; Zeng, Y.; Lu, Y.; Zhang, Q.; Du, Y.; Gilbe , B.R.; e al. F equen
mu a ion o he PI3K pa hway in head and neck cance de ines p edic i e bioma ke s. Cance Disco .
2013
,3, 761–769. [C ossRe ]
27.
Hende son, S.; Chak a a hy, A.; Su, X.; Bosho , C.; Fen on, T.R. APOBEC-media ed cy osine deamina ion links PIK3CA helical
domain mu a ions o human papilloma i us-d i en umo de elopmen . Cell Rep. 2014,7, 1833–1834. [C ossRe ]
28.
Miao, D.; Ma golis, C.A.; Vokes, N.I.; Liu, D.; Taylo -Weine , A.; Wankowicz, S.M.; Adeegbe, D.; Kelihe , D.; Schilling, B.; T acy,
A.; e al. Genomic co ela es o esponse o immune checkpoin blockade in mic osa elli e-s able solid umo s. Na . Gene .
2018
,
50, 1271–1281. [C ossRe ]
29. S ansky, N.; Eglo , A.M.; Twa d, A.D.; Kos ic, A.D.; Cibulskis, K.; Si achenko, A.; K yuko , G.V.; Law ence, M.S.; Sougnez, C.;
McKenna, A.; e al. The mu a ional landscape o head and neck squamous cell ca cinoma. Science
2011
,333, 1157–1160. [C ossRe ]
30.
Sewell, A.; B own, B.; Bik aso a, A.; Mills, G.B.; Lu, Y.; Tyson, D.R.; Issae a, N.; Ya b ough, W.G. Re e se-phase p o ein a ay
p o iling o o opha yngeal cance and signi icance o PIK3CA mu a ions in HPV-associa ed head and neck cance . Clin. Cance
Res. 2014,20, 2300–2311. [C ossRe ][PubMed]
31.
Bea y, B.T.; Moon, D.H.; Shen, C.J.; Amdu , R.J.; Weiss, J.; G illey-Olson, J.; Pa el, S.; Zana ion, A.; Hackman, T.G.; Tho p, B.; e al.
PIK3CA Mu a ion in HPV-Associa ed OPSCC Pa ien s Recei ing Dein ensi ied Chemo adia ion. J. Na l. Cance Ins .
2020
,112,
855–858. [C ossRe ][PubMed]
32.
Mazumda , T.; Bye s, L.A.; Ng, P.K.; Mills, G.B.; Peng, S.; Diao, L.; Fan, Y.H.; S emke-Hale, K.; Heymach, J.V.; Mye s, J.N.; e al. A
comp ehensi e e alua ion o bioma ke s p edic i e o esponse o PI3K inhibi o s and o esis ance mechanisms in head and
neck squamous cell ca cinoma. Mol. Cance The . 2014,13, 2738–2750. [C ossRe ][PubMed]
33.
Holzhause , S.; Wild, N.; Zupancic, M.; U su, R.G.; Be sani, C.; Näsman, A.; Kos opoulou, O.N.; Dalianis, T. Ta ge ed The apy
wi h PI3K and FGFR Inhibi o s on Human Papilloma i us Posi i e and Nega i e Tonsilla and Base o Tongue Cance Lines wi h
and Wi hou Co esponding Mu a ions. F on . Oncol. 2021,11, 640490. [C ossRe ][PubMed]
34.
Young, N.R.; Liu, J.; Pie ce, C.; Wei, T.F.; G ushko, T.; Olopade, O.I.; Liu, W.; Shen, C.; Seiwe , T.Y.; Cohen, E.E.W. Molecula
pheno ype p edic s sensi i i y o squamous cell ca cinoma o he head and neck o epide mal g ow h ac o ecep o inhibi ion.
Mol. Oncol. 2013,7, 359–368. [C ossRe ][PubMed]
35.
Michme huizen, N.L.; Leona d, E.; Kulka ni, A.; B enne , J.C. Di e en ial compensa ion mechanisms de ine esis ance o PI3K
inhibi o s in PIK3CA ampli ied HNSCC. O o hinola yngol. Head Neck Su g. 2016,1, 44–50. [C ossRe ][PubMed]
36.
Nichols, A.C.; Palma, D.A.; Chow, W.; Tan, S.; Rajakuma , C.; Gianan hony Rizzo, G.; Ke in Fung, K.; Kei h Kwan, K.; B e Weh li,
E.W.; Ko opa nick, J.; e al. High equency o ac i a ing PIK3CA mu a ions in human papilloma i us-posi i e o opha yngeal
cance . JAMA O ola yngol. Head Neck Su g. 2013,139, 617–622. [C ossRe ][PubMed]
37.
Samuels, Y.; Wang, Z.; Ba delli, A.; Silliman, N.; P ak, J.; Szabo, S.; Yan, H.; Gazda , A.; Powell, S.M.; Riggins, G.J.; e al. High
equency o mu a ions o he PIK3CA gene in human cance s. Science 2004,304, 554. [C ossRe ]
38.
Miled, N.; Yan, Y.; Hon, W.C.; Pe isic, O.; Z elebil, M.; Inba , Y.; Schneidman-Duho ny, D.; Wol son, H.J.; Backe , J.M.; Williams,
R.L. Mechanism o wo classes o cance mu a ions in he phosphoinosi ide 3-kinase ca aly ic subuni . Science
2007
,317, 239–242.
[C ossRe ][PubMed]
39.
Mandelke , D.; Gabelli, S.B.; Schmid -Ki le , O.; Zhu, J.; Cheong, I.; Huang, C.H.; Kinzle , K.W.; Vogels ein, B.; Amzel, L.M. A
equen kinase domain mu a ion ha changes he in e ac ion be ween PI3K
↵
and he memb ane. P oc. Na l. Acad. Sci. USA
2009
,
106, 16996–17001. [C ossRe ][PubMed]
40.
LeCon e, B.A.; Szaniszlo, P.; Fennewald, S.M.; Lou, D.I.; Qiu, S.; Chen, N.W.; Lee, J.H.; Res o, V.A. Di e ences in he i al genome
be ween HPV-posi i e ce ical and o opha yngeal cance . PLoS ONE 2018,13, e0203403. [C ossRe ]
41.
Samuels, Y.; Diaz, L.A., J .; Schmid -Ki le , O.; Cummins, J.M.; Delong, L.; Cheong, I.; Rago, C.; Huso, D.L.; Lengaue , C.; Kinzle ,
K.W.; e al. Mu an PIK3CA p omo es cell g ow h and in asion o human cance cells. Cance Cell 2005,7, 561–573. [C ossRe ]
42.
Ogino, S.; Nosho, K.; Ki kne , G.J.; Shima, K.; I aha a, N.; Ku e, S.; Chan, A.T.; Engelman, J.A.; K a , P.; Can ley, L.C.; e al.
PIK3CA mu a ion is associa ed wi h poo p ognosis among pa ien s wi h cu a i ely esec ed colon cance . J. Clin. Oncol.
2009
,27,
1477–1484. [C ossRe ]
43.
Kalinsky, K.; Jacks, L.M.; Heguy, A.; Pa il, S.; D obnjak, M.; Bhano , U.K.; Hed a , C.V.; T aina, T.A.; Soli , D.; Ge ald, W.; e al.
PIK3CA mu a ion associa es wi h imp o ed ou come in b eas cance . Clin. Cance Res.
2009
,15, 5049–5059. [C ossRe ][PubMed]
120
Cance s 2022,14, 1286 17 o 19
Ins i u ional Re iew Boa d S a emen :
The s udy was conduc ed acco ding o he guidelines o he
Decla a ion o Helsinki and app o ed by he Ins i u ional E hics Commi ee o IPOLFG (p o ocol code
UIC/1168, app o ed on 29-10-2019) and E hics Commi ee o NMS|FCM-UNL (CEFCM) (p o ocol
code 70/2019/CEFCM, app o ed on 23-01-2020).
In o med Consen S a emen : In o med consen was ob ained om all subjec s in ol ed in he s udy.
Da a A ailabili y S a emen :
The da a p esen ed in his s udy a e a ailable on eques om he
co esponding au ho . The da a a e no publicly a ailable due o p i acy.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Fe lay, J.; Soe joma a am, I.; Dikshi , R.; Ese , S.; Ma he s, C.; Rebelo, M.; Pa kin, D.M.; Fo man, D.; B ay, F. Cance incidence and
mo ali y-Majo pa e ns in GLOBOCAN 2012, wo ldwide and Geo gia. In . J. Cance
2015
,136, E359–E386. [C ossRe ][PubMed]
2.
B ay, F.; Fe lay, J.; Soe joma a am, I.; Siegel, R.L.; To e, L.A.; Jemal, A. Global cance s a is ics 2018: GLOBOCAN es ima es o
incidence and mo ali y wo ldwide o 36 cance s in 185 coun ies. CA Cance J. Clin. 2018,68, 394–424. [C ossRe ][PubMed]
3.
El-Mo y, S.K. HPV- ela ed squamous cell ca cinoma a ian s in he head and neck. Head Neck Pa hol.
2012
,6, S55–S62. [C ossRe ]
[PubMed]
4.
Ko oulakis, A.; Aga wal, M. La yngeal Cance ; S a Pea ls [In e ne ]; S a Pea ls Publishing: T easu e Island, FL, USA, 2021.
[PubMed]
5.
Pa mo e, H.S.; Ashman, J.N.E.; S a o d, N.D.; Be ieman, H.K.; MacDonald, A.; G eenman, J.; Cawkwell, L. Gene ic analysis o
head and neck squamous cell ca cinoma using compa a i e genomic hyb idisa ion iden i ies speci ic abe a ions associa ed wi h
la yngeal o igin. Cance Le . 2007,258, 55–62. [C ossRe ][PubMed]
6.
Zanoni, D.K.; Pa el, S.G.; Shah, J.P. Changes in he 8 h Edi ion o he Ame ican Join Commi ee on Cance (AJCC) S aging o
Head and Neck Cance : Ra ionale and Implica ions. Cu . Oncol. Rep. 2019,21, 52. [C ossRe ][PubMed]
7.
Cance Genome A las Ne wo k. Comp ehensi e genomic cha ac e iza ion o head and neck squamous cell ca cinomas. Na u e
2015,517, 576–582. [C ossRe ]
8.
Gillison, M.L.; Koch, W.M.; Capone, R.B.; Spa o d, M.; Wes a, W.H.; Wu, L.; Zahu ak, M.L.; Daniel, R.W.; Viglione, M.; Syme ,
D.E.; e al. E idence o a causal associa ion be ween human papilloma i us and a subse o head and neck cance s. J. Na l.
Cance Ins . 2000,92, 709–720. [C ossRe ]
9.
Cen es o Disease Con ol and P e en ion. Cance s associa ed wi h human papilloma i us, Uni ed S a es—2011–2015. A ailable
online: www.cdc.go /cance /hp /pd /USCS-Da aB ie -No4-Augus 2018-508.pd (accessed on 10 July 2019).
10.
Ang, K.K.; Ha is, J.; Wheele , R.; Webe , R.; Rosen hal, D.I.; Nguyen-Tân, P.F.; Wes a, W.H.; Chung, C.H.; Jo dan, R.C.; Lu,
C.; e al. Human Papilloma i us and Su i al o Pa ien s wi h O opha yngeal Cance . N. Engl. J. Med.
2010
,363, 24–35. [C ossRe ]
11.
Gillison, M.L.; T o i, A.M.; Ha is, J.; Eisb uch, A.; Ha a i, P.M.; Adels ein, D.J.; Jo dan, R.C.K.; Zhao, W.; S u gis, E.M.; Bu ness,
B.; e al. Radio he apy plus ce uximab o cispla in in human papilloma i us-posi i e o opha yngeal cance (NRG Oncology
RTOG 1016): A andomised, mul icen e, non-in e io i y ial. Lance 2019,393, 40–50. [C ossRe ]
12.
Che a, B.S.; Amdu , R.J.; Teppe , J.; Qaqish, B.; G een, R.; Aume , S.L.; Hayes, N.; Weiss, J.; G illey-Olson, J.; Zana ion, A.; e al.
Phase 2 T ial o De-in ensi ied Chemo adia ion The apy o Fa o able-Risk Human Papilloma i us-Associa ed O opha yngeal
Squamous Cell Ca cinoma. In . J. Radia . Oncol. Biol. Phys. 2015,93, 976–985. [C ossRe ][PubMed]
13.
Lee, M.J.; Jin, N.; G andis, J.R.; Johnson, D.E. Al e a ions and molecula a ge ing o he GSK-3 egula o , PI3K, in head and neck
cance . BBA Mol. Cell Res. 2020,1867, 118679. [C ossRe ][PubMed]
14.
Chang, F.; Lee, J.T.; Na olanic, P.M.; S eelman, L.S.; Shel on, J.G.; Blalock, W.L.; F anklin, R.A.; McCub ey, J.A. In ol emen
o PI3K/Ak pa hway in cell cycle p og ession, apop osis, and neoplas ic ans o ma ion: A a ge o cance chemo he apy.
Leukemia 2003,17, 590–603. [C ossRe ]
15.
Xue, G.; Hemmings, B.A. PKB/ak -dependen egula ion o cell mo ili y. J. Na l. Cance Ins .
2013
,105, 393–404. [C ossRe ]
[PubMed]
16. Ka a , J.; Mai y, A. PI3K/AKT/mTOR pa hway in angiogenesis. F on . Mol. Neu osci. 2011,4, 1–8. [C ossRe ][PubMed]
17. Van Doo slae , K.; Bu k, R.D. E olu ion o human papilloma i us ca cinogenici y. Ad . Vi us Res. 2010,77, 41–62. [C ossRe ]
18.
Zhang, S.; Wu, X.; Jiang, T.; Lu, Y.; Ma, L.; Liang, M.; Sun, X. The up- egula ion o KCC1 gene exp ession in ce ical cance cells
by IGF-II h ough he ERK1/2MAPK and PI3K/AKT pa hways and i s signi icance. Eu . J. Gynaecol. Oncol.
2009
,30, 29–34.
[PubMed]
19.
Tang, X.; Zhang, Q.; Nishi ani, J.; B own, J.; Shi, S.; Le, A.D. O e exp ession o human papilloma i us ype 16 oncop o eins
enhances hypoxia-inducible ac o 1 alpha p o ein accumula ion and ascula endo helial g ow h ac o exp ession in human
ce ical ca cinoma cells. Clin. Cance Res. 2007,13, 2568–2576. [C ossRe ]
20.
Hyland, P.L.; McDade, S.S.; McCloskey, R.; Dickson, G.J.; A hu , K.; McCance, D.J.; Pa el, D. E idence o al e a ion o EZH2,
BMI1, and KDM6A and epigene ic ep og amming in human papilloma i us ype 16 E6/E7-exp essing ke a inocy es. J. Vi ol.
2011,85, 10999–11006. [C ossRe ]
121
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odds a io ac oss s a a). We e alua ed he p ognos ic impac o PIK3CA mu a ions and
p16 o e exp ession on he o e all su i al (OS) and disease- ee su i al (DFS). O e all
su i al was de ined as he pe iod o ime (in yea s) om he da e o diagnosis o he da e o
dea h om any cause, pa ien s ali e we e censo ed a he da e o las ollow-up assessmen .
Disease- ee su i al was e alua ed in he pa ien s wi h a comple e esponse a e he i s
ea men and was de ined as he ime om he end o he i s ea men o disease elapse
o dea h om any cause; pa ien s ali e wi hou disease ecu ence we e censo ed a he da e
o he las ollow-up assessmen . We used Kaplan–Meie cu es o isualize he di e ences
in su i al be ween subg oups de ined by PIK3CA mu a ion, p16 o e exp ession, and HPV
s a us and he log- ank es o g oup compa isons. Cox p opo ional haza ds eg ession
analysis was used o compu e he haza d a ios (HRs) and 95% Con idence In e als based
on Wald s a is ics, wi h OS and DFS as he ou come a iables and adjus ing o age, HPV
s a us, and d inking/smoking habi s as po en ial con ounding ac o s. Age and HPV s a us
we e chosen a p io i as he mos impo an con ounding ac o s based on clinical c i e ia,
and d inking/smoking habi s we e also included due o he imbalances obse ed be ween
he PIK3CA MUT and WT g oups in ou coho . The p opo ional haza ds assump ion
was checked using s a is ical es s and g aphical diagnosis based on Schoen eld esiduals.
We also g aphically assessed he unc ional o m o he age a iable in he models using
Ma ingale esiduals. As his analysis showed ha he linea i y assump ion was no
accep able in he OS model, we adjus ed he nonlinea e ec o age wi h a smoo hing spline
using he “pspline” unc ion o he R package “su i al”. The po en ial in e ac ion be ween
HPV s a us and PIK3CA mu a ions on su i al was es ed using he log-likelihood a io
es be ween he i ed Cox eg ession models wi h and wi hou he in e ac ion e m. The
analyses we e conduc ed in he comple e case da ase . All s a is ical es s we e wo-sided,
and we conside ed a signi icance le el o 5%. As his was an explo a o y s udy, no p- alue
co ec ion o mul iple es ing was done. We used he so wa e R package e sion 4.1.0
(h p://www.R-p ojec .o g, accessed on 1 Oc obe 2021) [47].
5. Conclusions
O e all, hese esul s con i med a high equency o canonical PIK3CA mu a ions
(subs i u ions H1047L and E542K, E545K, and E545D) in HNSCC, including in HPV (

)
cases, sugges ing ha sc eening o PIK3CA mu a ions should no be es ic ed o HPV (+)
pa ien s. Addi ional s udies a e needed o cla i y he implica ions o PIK3CA mu a ions o
HNSCC pa ien p ognosis.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a h ps:
//www.mdpi.com/a icle/10.3390/cance s14051286/s1: Figu e S1: Pa icipan low cha .
Figu e S2
:
Kaplan–Meie o e all su i al cu es o he included and excluded g oups. Figu e S3: Flow cha
om he me a-analysis inclusion c i e ia. Table S1: Sys ema ic Re iew HNSCC PIK3CA qPCR.
Table S2: O e all, he 17 a icles selec ed o HNSCC PIK3CA mu a ion. Table S3: HNSCC PIK3CA
mu a ional p o ile. Table S4: Clinical and demog aphic cha ac e isa ion o he 295 excluded cases.
Au ho Con ibu ions:
Concep ualisa ion, A.F. and R.M.G.d.C.; Da a cu a ion: D.C., M.M., P.M.,
M.R., F.S., M.C. and L.M.; Fo mal analysis, D.C., S.E., M.R. and A.F.; W i ing—o iginal d a p epa a-
ion, D.C., R.M.G.d.C. and A.F.; and W i ing— e iew and edi ing, R.M.G.d.C. and A.F. All au ho s
ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This s udy was inancially suppo ed by he Vi ology Labo a o y om he Pa hology
Depa men o he Po uguese Oncology Ins i u e o Lisboa IUIC/1168, wi h con ibu ions by he
Resea ch Cen e o he Po uguese Oncology Ins i u e o Po o (p ojec no. PI86-CI-IPOP-66-2017), by
Base Funding-UIDB/00511/2020 o he Labo a o y o P ocess Enginee ing, En i onmen , Bio ech-
nology, and Ene gy—LEPABE— unded by na ional unds h ough he FCT/MCTES (PIDDAC),
and P ojec 2SMART-enginee ed Sma ma e ials o Sma ci izens, wi h e e ence NORTE-01-
0145-FEDER-000054, suppo ed by No e Po ugal Regional Ope a ional P og amme (NORTE 2020)
unde he PORTUGAL 2020 Pa ne ship Ag eemen h ough he Eu opean Regional De elopmen
Fund (ERDF).
128
Cance s 2022,14, 1286 9 o 19
Figu e 1.
Kaplan–Meie cu es compa ing pa ien s wi h PIK3CA-mu a ed s. he wild- ype a
diagnosis (
A
). O e all su i al (Log- ank es chi-squa e = 2.9 wi h 1 deg ee o eedom, p= 0.090).
(B). Disease- ee su i al (Log- ank es chi-squa e = 1.7 wi h 1 deg ee o eedom, p= 0.198).
Figu e 2. Con .

129
Cance s 2022,14, 1286 8 o 19
Analysis PIK3CA, HPV S a us, and p16 Immunohis ochemis y
OS and DFS we e de e mined sepa a ely o pa ien s g ouped by he PIK3CA mu a-
ional s a us, HPV DNA, and p16
INK4a
(Table 4and Figu es 1and 2). Du ing he uni a iable
analysis, we could no demons a e a signi ican associa ion be ween he PIK3CA mu a-
ion s a us and OS o DFS (Table 4and Figu e 1A,B). The HPV s a us was he only ac o
signi ican ly associa ed wi h bo h OS and DFS, wi h pa ien s wi h posi i e HPV ha ing
consis en ly be e ou comes compa ed wi h he ones wi h non-de ec ed HPV (Table 4and
Figu e 2A,B). Pa ien s wi h p16 o e exp ession had signi ican ly longe DFS han pa ien s
wi h no p16 o e exp ession, bu no signi ican di e ence could be demons a ed conce ning
OS (Table 4).
Table 4.
PIK3CA mu a ion, HPV, and p16 s a us associa ion wi h he o e all and disease- ee su i al
by uni a iable analysis.
O e all Su i al Disease-F ee Su i al
Median
(Yea s)
3-Yea
% (95% CI)
HR
(95%CI) p*Median,
(Yea s)
3-Yea
% (95% CI)
HR (95%
CI) p*
PIK3CA
MUT 6.2 65 (50–83) 1 0.090 NR 77 (63–95) 1 0.198
WT 4.0 54 (41–70) 1.71
(0.91–3.18) 4.8 72 (58–90) 1.80
(0.73–4.42)
HPV s a us
Posi i e 5.8 65 (54–78) 1 0.007 NR 83 (72–94) 1 0.001
No de ec ed 2.4 36 (19–69) 2.40
(1.15–4.61) 2.2 43 (21–90) 4.81
(1.74–13.29)
p16 o e exp ession
No 2.6 46 (33–64) 1 0.089 4.8 59 (44–80) 1 0.029
Yes 6.9 73 (60–90) 0.58
(0.31–1.10) NR 91 (81–100) 0.34
(0.13–0.94)
* Log- ank es ; HR = Haza d Ra io; 95% CI = 95% Con idence In e al; NR = No Reached; MUT = mu a ed;
W = wild- ype.
Figu e 1. Con .
130
Cance s 2022,14, 1286 7 o 19
2.2.2. P e alence o PIK3CA Mu a ions
The PIK3CA mu a ions we e p esen in 39% o HNSCC cases (n= 37, 95% CI:
29–49%
).
The p e alence o PIK3CA mu a ions in HNSCC HPV (+) (n= 68) and HNSCC HPV
(

)(n= 27) was, espec i ely, 36.8% (n= 25) and 44.4% (n= 12). Nex , we analysed he
dis ibu ion o PIK3CA mu a ions in pa ien subg oups de ined by bo h he HPV DNA
and p16
INK4a
s a us, as summa ised in Table 2. We could no demons a e a s a is ically
signi ican associa ion be ween he p esence o PIK3CA mu a ions and HPV DNA de ec ion
(p= 0.489, Table 1). When e alua ing a possible associa ion be ween PIK3CA mu a ion
and he p16
INK4a
s a us wi hou s a i ica ion by he HPV DNA s a us, no s a is ically
signi ican associa ion was ound (p= 0.163, Table 1). Simila ly, he e was no e idence o
an associa ion be ween he p16
INK4a
s a us and PIK3CA mu a ion when conside ing HPV
s a us s a i ica ion (p= 0.245).
Table 2.
F equency o PIK3CA mu a ions in HNSCC wi h and wi hou HPV DNA and p16
INK4a
o e exp ession (n= 89; p16 missing da a n= 6).
HNSCC PIK3CA Gene
WT
n(%)
MUT
n(%)
HPV (+) p16(+) 22 (68.8%) 10 (31.3%)
p16() 17 (56.7%) 13 (43.3%)
HPV ()p16(+) 3 (75%) 1 (25%)
p16() 12 (52.2%) 11 (47.8%)
2.2.3. Classi ica ion o PIK3CA Gene Mu a ions: H1047L, E542K, E545K, and E545D
HNSCC cases we e sc eened o ou di e en single subs i u ions, known as canonical
PIK3CA mu a ions. Among he cases ha bou ing PIK3CA mu a ions (n= 37), he majo i y
ca ied E545D (73%; n= 27), ollowed by E545K (5%; n= 2), E542K (3%; n= 1), and H1047R
(3%; n= 1). We obse ed he occu ence o combined subs i u ions E545D|E542K (5.4%;
n= 2), E545D|E545K (8.1%; n= 3), and H1047R|E545D (2.7%; n= 1). The dis ibu ion o
PIK3CA mu a ions in he HPV-posi i e and -nega i e subg oups is summa ised in Table 3.
Table 3.
Classi ica ion o PIK3CA gene mu a ions (subs i u ions H1047L and E542K, E545K,
and E545D).
HPV S a us
PIK3CA Gene MUT
One Subs i u ion Two Subs i u ions
E545D
%(n)
E545K
%(n)
E542K
%(n)
H1047R
%(n)
E545D|E545K
%(n)
E545D|E542K
%(n)
E545D|H1047R
%(n)
HPV (+) 76% (19) 8% (2) 4% (1) 4% (1) 8% (2) ND ND
HPV () 67% (8) ND ND ND 8% (1) 17% (2) 8% (1)
To al 73% (27) 5% (2) 3% (1) 3% (1) 8% (3) 5% (2) 3% (1)
Legend: ND = no de ec ed.
2.2.4. PIK3CA Mu a ions and Pa ien P ognosis
The median ollow-up de e mined using he e e se Kaplan–Meie me hod was
4.12 yea s
(95% CI 3.1–5.5 yea s). A o al o 46 dea hs we e epo ed du ing he ollow-up
pe iod. The median o e all su i al (OS) in he whole sample (n= 95) was 4.75 yea s (95%
CI 2.6–6.9 yea s). The 3-yea OS was 58% (95% CI 48–70%). Disease- ee su i al (DFS) was
e alua ed in he 66 pa ien s wi h a comple e esponse o he i s -line ea men (
17 pa ien s
wi h pe sis en disease and 12 wi h missing in o ma ion conce ning esponse o ea men
we e excluded om he DFS analysis). O e all, he median DFS was 6.16 yea s (95% CI
4.6–NA), and he 3-yea DFS was 75% (64–87%).
131
Cance s 2022,14, 1286 6 o 19
Table 1.
Clinical, pa hological, and demog aphic cha ac e is ics and associa ion wi h PIK3CA mu a ion.
Va iable Ca ego ies
PIK3CA E alua ion To al Cases
n= 95 p-Value
WT (n= 58) MUT (n= 37)
n(%) n(%) n(%)
Gende
Male 45 (77.6%) 25 (67.6%) 70 (73.7%) 0.2795
Female 13 (22.4%) 12 (32.4%) 25 (26.3%)
Age a diagnosis
Mean (S anda d De ia ion) 62 (10.7) 62 (12.7) 62 (11.5) 0.9740
65 Yea s 24 (41.4%) 12 (32.4%) 36 (37.9%) 0.3807
<65 Yea s 34 (58.6%) 25 (67.6%) 59 (62.1%)
Consump ion habi s ( obacco and/o alcohol)
Ac i e consump ion 44 (75.9%) 22 (59.5%) 66 (69.5%)
0.0325 *
Alcohol ac i e 18 (31.0%) 10 (27%) 28 (29.5%)
Tobacco ac i e 7 (12.1%) 1 (2.7%) 8 (8.4%)
Bo h ac i e 19 (32.8%) 11 (29.7%) 30 (39.6%)
No consump ion 9 (15.5%) 13 (35.1%) 22 (23.2%)
Ne e 5 (8.6%) 10 (27%) 15 (15.8%)
Nonac i e 4 (6.9%) 3 (8.1%) 7 (7.4%)
No da a 5 (8.6%) 2 (5.4%) 7 (7.4%)
Tumou Ana omic egion
O opha ynx 46 (79.3%) 29 (78.4%) 75 (78.9%) 0.9135
O al ca i y 12 (20.7%) 8 (21.6%) 20 (21.1%)
Tumou s age
I 4 (6.9%) 4 (10.8%) 8 (8.4%)
0.9342
II 11 (19%) 8 (21.6%) 19 (20.0%)
III 16 (27.6%) 10 (27.0%) 26 (27.4%)
IV 25 (43.1%) 15 (40.5%) 40 (42.1%)
No da a 2 (3.4%) 0 2 (2.1%)
p16 IHQ
HNSCC p16() 29 (50.0%) 24 (64.9%) 53 (55.8%) 0.1627
HNSCC p16(+) 25 (43.1%) 11 (29.7%) 36 (37.9%)
No da a 4 (6.9%) 2 (5.4%) 6 (6.3%)
HPV in ec ion
HPV16 DNA() 15 (25.9%) 12 (32.4%) 27(28.4%) 0.4887
HPV16 DNA(+) 43 (74.1%) 25 (67.6%) 68 (71.6%)
Single in ec ion 39 (90.7%) 23 (92.0%) 62 (91.2%)
Co in ec ion wi h o he
HR/LR HPV 4 (9.3%) 2 (8%) 6 (8.8%)
P ima y T ea men
Su ge y 1 (1.7%) 2 (5.4%) 3 (3.2%)
0.7376
Radio he apy (RT) 10 (17.2%) 6 (16.2%) 16 (16.8%)
Chemo he apy (CTX) 46 (79.3%) 29 (78.4%) 75 (78.9%)
CTX + RT 26 (56.5%) 12 (41.4%) 38 (50.7%)
Su ge y + RT 9 (19.6%) 11 (37.9%) 20 (26.7%)
Su ge y + CTX 11 (23.9%) 6 (20.7%) 17(22.7%)
No da a 1 (1.7%) 0 1 (1.1%)
T ea men esponse
Comple e 38 (65.5%) 28 (75.7%) 66 (69.5%)
0.5190
Pe sis ence 11 (19.0%) 6 (16.2%) 17 (17.9%)
No da a 9 (15.5%) 3 (8.1%) 12 (12.6%)
Legend: No da a = no in o ma ion a ailable; RT = Radio he apy; CTX = Chemo he apy; * p- alue calcula e o
ac i e s. no consump ion g oup.
132
Cance s 2022,14, 1286 5 o 19
ma ke (n= 208) and all cases associa ed wi h HPV in ec ion o he han HPV16 (n= 79); he
emaining 103 cases we e included in he s udy and analysed o PIK3CA s a us (Figu e S1).
Eigh cases we e u he excluded om he s udy due o echnical ailu e in he PIK3CA
mu a ional s a us e alua ion. As such, ou s udy sample comp ised 95 pa ien s. The
demog aphic and clinical ea u es o he excluded pa ien s we e simila o he included
pa ien s (Table S4), and bo h g oups showed o e lapping su i al cu es (Figu e S2), which
sugges s he absence o selec ion bias.
The demog aphic and clinical–pa hological cha ac e is ics o he 95 HNSCC pa ien s
included in he analysis a e summa ized in Table 1. The a e age age a diagnosis was
62 yea s
old and anged be ween 37 and 90 yea s old. Mos pa ien s we e men (73.7%,
n= 70) and, acco ding o sel - epo ed consume habi s, he majo i y had ac i e obacco
and/o alcohol consump ion (70%,
n= 66)
. S a i ica ion was pe o med o each consump-
ion habi . Alcohol exposu e was cha ac e ised acco ding o quali a i e da a in clinical
eco ds: ne e d ank, ex-d inke , spo adic, mode a e, and se e e d inke s. The classi-
ica ion o obacco consump ion was de ined as one pack/yea (equal o one pack o
ciga e es/day/yea , wi h 20 ciga e es in a pack) dis ibu ed among cu en and hea y
smoke s. Cu en obacco use s included hose who used obacco

30 pack/yea s. Hea y
use s we e hose who smoked > 30 pack/yea s. The ex-smoke g oup included smoke s
who s opped un il he da e o diagnosis. Cases whe e he pack-a-yea uni in o ma ion was
no a ailable we e classi ied as un a ed smoke s, and he da a only epo ed consump ion
in a quali a i ely way. Ne e use s o obacco o alcohol we e de ined as no ha ing con-
sumed ei he o hese subs ances p io o cance diagnosis. The analysis conside ed he
wo g oups o ac i e consump ion s. no consump ion and did no conside he missing
alues. In 75 cases (79%), he p ima y umou was loca ed in he o opha ynx, including he
pala ine onsil (n= 47), he base o he ongue (n= 12), u ula (n= 2), so pala e (
n= 10
),
igone e omola (n= 1), and o opha ynx wall (n= 3). In he emaining 20 cases, he
p ima y umou was loca ed in he o al ca i y, including he ongue body (n= 13), buccal
loo (n= 4), o al mucosa (n= 2), and mandible (n= 1). HPV in ec ion was de ec ed in
68 HNSCC cases
, including 62 cases o HPV16 single-in ec ion and six cases o coin ec ion
wi h HPV16 and o he HR o LR HPV ypes (HPV6, HPV18, HPV53, and HPV58). Da a
om p16
INK4a
immunohis ochemical assays we e a ailable o 89 pa ien s. Pa ien s we e
ea ed wi h di e en schemes in ol ing su ge y and adio he apy (RT) as single he apies
o in associa ion wi h sys emic an ineoplas ic ea men (Table 1). This ea men he e o-
genei y was expec ed, aking in o conside a ion he clinical cha ac e is ics and umou
s age dis ibu ion obse ed in ou coho . Mos pa ien s showed comple e esponse o
he applied ea men (n= 66), bu cases wi h disease pe sis ence (n= 17) and elapse
(
n=6
) we e also epo ed. In 12 cases, he ea men esponse could no be e ospec i ely
e alua ed (Table 1).
The e we e no di e ences be ween he wild- ype (WT) and mu an (MUT) PIK3CA
g oups ega ding age, p ima y umou loca ion, umou s age a diagnosis, HPV16 in ec ion
s a us, and p ima y ea men adminis e ed (Table 1). The PIK3CA WT g oup had a
nume ically highe p opo ion o males (78% s. 68%), p16 o e exp ession (43% s. 30%),
and comple e esponses o ea men (65% s. 76%) compa ed o MUT PIK3CA, al hough
none o hese di e ences was s a is ically signi ican . The e was a s a is ically signi ican ly
highe p opo ion o pa ien s wi h ac i e obacco/alcoholic consump ion habi s among he
WT compa ed o he MUT PIK3CA (76% s. 60%) g oup; as such, his a iable was also
conside ed in he mul i a iable analysis o he p ognos ic impac o PIK3CA mu a ions.
133
Cance s 2022,14, 1286 4 o 19
( wo cases) in exon 20). The highes epo ed equency (32%) was obse ed in a Japanese
s udy (Suda 2012) wi h a coho o 115 cases, equally dis ibu ed ac oss di e en subsi es o
he HN egion (o al ca i y n= 31, o opha ynx n= 25, la ynx n= 23, hypopha ynx n= 25,
and nasopha ynx n= 11). Fi e mu a ions spo s we e e alua ed in exon 9 and exon 20. The
dis ibu ion o mu a ions by loca ion e ealed a p edominance o o opha yngeal si es (o al
ca i y n= 9 (24%), o opha ynx n= 10 (27%), la ynx n= 8 (22%), hypopha ynx n= 9 (24%),
and nasopha ynx n= 1 (3%) da a om T Suda 2012).
In addi ion, we pe o med a second analysis o explo e he HNSCC PIK3CA mu a-
ion p o ile in g ea e dep h. Fo his second analysis, we assessed 17 selec ed a icles
comp ising da a om 1286 HNSCC pa ien s published be ween 2006 and 2021 whe e
mu a ions we e de ec ed using DNA sequencing echniques ins ead o PCR-based me hods
(
Supplemen al Da a Table S2
). The mos used sequencing me hodology was he classic
Sange assay (
n= 10 s udies
), ollowed by cu ing-edge NGS echnology (n= 7). All s udies
e alua ed umou issue, and he majo i y o samples we e collec ed a he ime o diagnos-
ic (i.e., we e ea men -nai e samples). P ima y umou s we e loca ed a di e en subsi es
om he HN egion s a i ied by he o al ca i y, o opha ynx, la ynx, hypopha ynx, and
nasopha ynx; all da a ha did no speci ically desc ibe hese loca ions we e conside ed
as no s a i ied. Th ee ou o 17 s udies e alua ed SCC exclusi ely om he o al ca i y,
h ee o he s om he o opha ynx, and one s udy om he hypopha ynx. The emaining
eigh s udies include da a om di e en subsi es. O e all, he mos ep esen ed subsi e
was he o al ca i y (n= 678 cases, epo ed by 11 a icles), ollowed by he o opha ynx
(
n= 340
, epo ed by se en a icles), la ynx (n= 101, epo ed by i e a icles), hypopha -
ynx (n= 94, epo ed by six a icles), nasopha ynx (n= 40, epo ed by i e a icles), and
32 non-s a i ied cases
( epo ed by ou a icles). The HPV s a us was desc ibed in nine
ou o 17 s udies: h ee s udies e alua ed single HPV s a us coho s HPV (+) o HPV (

),
and a leas six s udies p esen ed bo h HPV (+) and HPV (

) cases. HPV (

) HNSCC
was he mos ep esen ed subg oup (n= 330 cases), ollowed by HNSCC HPV (+) (n= 244
cases). O e all, he equency o PIK3CA mu a ions anged be ween 3% and 36%. Only
wo s udies epo ed he lowes equency (3%) (B uckman 2010 and Co elazzi 2015), a
leas i e s udies p esen ed equencies abo e 20%, and h ee s udies abo e 32%. O e all,
45 mu a ions we e epo ed, dis ibu ed by bo h exon 9 and exon 20, and no el mu a ions
we e epo ed in a ew s udies. All mu a ions we e s a i ied by exon, excep o 30 cases
epo ed in h ee s udies.
The analysis based on gene al da a o he mu a ional p o ile was suppo ed by da a
epo ed in each o he s udies. The h ee mos equen ly encoun e ed mu a ions we e he
E545K mu a ion (n= 31 cases; da a om 15 s udies), ollowed by E542K (n= 21; nine s udies)
on exon 9 and H1047R (n= 16; nine s udies), T1025T (n= 16; wo s udies), M10431 (n= 8;
wo s udies), and G1049 (n= 2; h ee s udies) on exon 20 (Supplemen a y Table S3). The
PIK3CA-mu a ed cases pe HN subsi e we e accu a e; he highes numbe was in he o al
ca i y (n= 115, epo ed by se en s udies), ollowed by o opha ynx (n= 32, h ee s udies),
hypopha ynx (n= 10, h ee s udies), la ynx (n= 8, h ee s udies), and nasopha ynx wi h no
cases epo ed. We also e alua ed he PIK3CA mu a ion (da a epo ed om six s udies) o
subg oups o he HPV s a us. A o al o 33 cases o HNSCC HPV (+) and 19 cases o HNSCC
HPV (

) ha bou ed PIK3CA mu a ions. In i e o he 17 s udies, he p esence equency
o PIK3CA mu a ions was associa ed wi h he clinical pa ame e s and co ela ed wi h he
espec i e ou come (OS and DFS). Fou s udies es ima ed a be e p ognosis associa ed
wi h he p esence o he mu a ion (Also yania 2020, Lim 2019, Ga cía-Ca acedo 2016, and
Cohen 2011), and in one s udy, no co ela ion was ound (Chau 2016).
2.2. Po uguese HNSCC S udy Popula ion
2.2.1. Clinical and Demog aphic Cha ac e is ics
Cases we e selec ed om a uni e se o 390 consecu i e p ima y HNSCC cases loca ed
in he o opha ynx o o al ca i y a ou e ia y cance cen e be ween 2010 and 2019. We
excluded all cases wi h HPV-nega i e and missing in o ma ion conce ning he p16INK4a

134
Cance s 2022,14, 1286 3 o 19
pa ien s had signi ican ly highe 3-yea disease- ee su i al (DFS) compa ed o PIK3CA
MUT pa ien s, and a mul i a iable analysis o age, sex, smoking, TNM s age, and ea -
men showed associa ions be ween PIK3CA s a us and disease ecu ence [
31
]. A g ea e
unde s anding o he apies a ge ing he PI3K pa hway has been achie ed, de eloping new
he apeu ic combina ions o imp o e he su i al o HNSCC pa ien s, bu hei e icacy
is a iable [
32
,
33
]. The iden i ica ion o bioma ke s o p edic he esponse o he apy
will allow he selec ion o pa ien s who a e mo e likely o espond o new he apeu ic
combina ions [
32
], sugges ing ha di e en ea men s a egies need o be conside ed
based on he molecula pheno ype o each umou [
34
,
35
]. In his con ex , i is c i ical o
e ine he p o ile o PIK3CA mu a ions in HPV-posi i e and HPV-nega i e HNSCC pa ien s
and i s associa ion wi h p ognosis.
To add ess hese issues, he p esen s udy includes a sys ema ic e iew o he equency
o PIK3CA mu a ions in HNSCC and he impac o he di e en de ec ion echniques. The
s udy also epo s he p e alence o canonical PIK3CA gene mu a ions (subs i u ions o
H1047L and E542K, E545K, and E545D) wi hin an HNSCC Po uguese pa ien se ies a he
ime o diagnosis and conduc ed an explo a o y analysis o es he hypo heses: (1) whe he
PIK3CA (canonical) mu a ions a e associa ed wi h HPV-posi i e HNSCC, as e alua ed by
HPV DNA and p16INK4a and (2) whe he PIK3CA mu a ions a e independen ly associa ed
wi h o e all and disease- ee su i al.
2. Resul s
2.1. Sys ema ic Re iew
We pe o med a sys ema ic e iew o s udy he PIK3CA mu a ion equency in di -
e en HNSCC coho s ( o al case numbe ange be ween 25 and 115 cases; o al num-
be o cases was 479) in scien i ic a icles published om 2012 o 2021. The PIK3CA
gene mu a ion analysis was pe o med by qPCR designed o speci ic a ge s in he e-
gions known as ho spo s o he PIK3CA gene exon 9 and exon 20, such E542K, E545K,
E545D, H1047R, and H1047L, espec i ely. This analysis co e s h ee di e en geog aph-
ical egions: Eu ope ( h ee s udies), Asia ( wo s udies), and No h Ame ica (one s udy)
(Supplemen al Da a Table S1).
The majo i y o he s udies pe o med a mu a ional analysis using un ea ed umou
biopsy issue, he male gende was he mos ep esen ed in all s udies, always ep esen ing
mo e han hal o he sample (55–93%), he mean age anged be ween 63 and 65 yea s, and
all he coho s p esen ed he ac i e consump ion o obacco and/o alcohol. Almos all he
s udies epo ed a s a i ica ion by HN subsi es (o al ca i y, o opha ynx, la ynx, hypopha -
ynx, and nasopha ynx), and he majo i y o he cases o igina ed om he o opha ynx ( i e
ou o six s udies, a o al o 138 cases), ollowed by he o al ca i y ( ou s udies, a o al
o 97 cases), la ynx ( ou s udies, a o al o 80 cases), hypopha ynx ( ou s udies, a o al
o 80 cases), and nasopha ynx ( h ee s udies, a o al 16 cases). All pa ien s p esen ed a
his ological diagnosis o SCC, mos o hem a ad anced s ages (III and IV). Only h ee
o he six s udies epo ed he HPV DNA s a us o he coho . In o al, 54 HNSCC HPV
(+) and 219 HNSCC HPV (

) we e e alua ed. In h ee s udies, he p16INK4 s a us was
de e mined by immunohis ochemis y wi h a o al o 45 posi i e cases. The mu a ion
equency was es ima ed in a quali a i e analysis o he numbe o cases wi h a mu a ion
p esen . All s udies epo ed PIK3CA gene mu a ions, and hei equency anged om
8 o 32%. The lowes equency (8%) was epo ed in a coho o 113 cases (Bo kowska
2021), mos ly consis ing o la yngeal umou s (43%) and, la gely, HPV (

) lesions (77%).
This low equency o mu a ion de ec ion can be ela ed o he s udy’s design a ge ing
only H1047R. Fou o he s udies p esen ed equencies anging be ween 16% and 18%
and mainly consis ed o lesions om he o opha ynx and o al ca i y. Among hese ou
s udies, HPV (

) HNSCC cases p e ailed in he wo s udies whe e he HPV s a us was
de e mined (Ga cía-Escude o 2018; SB Pa le 2017). All bu one o hese s udies e alua ed
a leas ou mu a ions dis ibu ed bo h in he exon 9 and exon 20. The highes equency
was ound ( aking in o accoun only ( h ee cases) E545D ( wo cases) in exon 9 and H1047R
135
Cance s 2022,14, 1286 2 o 19
(OS) (3-yea OS: WT 54% and MUT 65%; p= 0.090). HPV s a us was he only ac o signi ican ly
associa ed wi h bo h OS and disease- ee su i al (DFS), wi h HPV (+) pa ien s ha ing consis en ly
be e ou comes (3-yea OS: HPV (+) 65% and HPV (

) 36%; p= 0.007; DFS HPV (+) 83% and HPV
(

) 43%; p= 0.001). The e was a s a is ically signi ican in e ac ion e ec be ween HPV s a us and
PIK3CA mu a ion ega ding DFS (In e ac ion es : p= 0.026). In HPV (+) pa ien s, PIK3CA wild- ype
is associa ed wi h a signi ican 4.64 imes inc ease in he haza d o ecu ence o dea h (HR = 4.64;
95% CI 1.02–20.99; p= 0.047). O e all, PIK3CA gene mu a ions a e p esen in a la ge numbe o
pa ien s and may help de ine pa ien subse s who can bene i om he apies a ge ing he PI3K
pa hway. The sys ema ic assessmen o PIK3CA gene mu a ions in HNSCC pa ien s will equi e
u he me hodological s anda disa ion.
Keywo ds: HNSCC; HPV; p16 INK4a; PIK3CA
1. In oduc ion
Head and neck ca cinoma (HN) is he six h leading cance by incidence wo ldwide [
1
],
acco ding o da a published in 2018, and is esponsible o mo e han 800,000 new cases
yea ly and 450,000 dea hs/yea wo ldwide [
2
]. I comp ises many di e en common and
a e en i ies, he la ge majo i y (90%) being squamous cell ca cinomas (SCC) [
3
]. The e is
a wide a ia ion in disease p og ession and o e all pa ien su i al a di e en ana omic
subsi es [
4
,
5
], which may be explained by i s mul i ac o ial ae iology, in which en i on-
men al ac o s ha e a s ong con ibu ion, such as smoking and alcohol consump ion.
In ec ion wi h human papilloma i us (HPV), mainly he high- isk (HR) ype HPV16, also
plays an impo an ole in a subg oup o hese umou s, pa icula ly in o opha yngeal
cance s [
6
]. HPV-posi i e and HPV-nega i e umou s a e clinically dis inc [
7
], and wo
sepa a e ca cinogenesis ou es a e ecognized [
8
]. O e he pas decades, he incidence
o HPV-associa ed o opha yngeal SCC has inc eased, as epo ed by he Uni ed S a es
Cen e o Disease Con ol (CDC) in 2018 [
9
]. HPV-associa ed HNSCC has a subs an ially
be e p ognosis a e he apy compa ed wi h HPV-nega i e cases [
10
]. De-in ensi ied
he apy o hese pa ien s is being ac i ely explo ed o educe he associa ed mo bidi y
while main aining umou con ol [11,12].
The genomic p o ile o HNSCC published by The Cance Genome A las (TCGA) in
2015 allowed he de elopmen o new mu a ion s udies and highligh ed a high equency
o changes in componen s o he phospha idylinosi ol-3-kinase (PI3K) signalling pa hway,
poin ing ou PIK3CA as he mos equen ly al e ed gene [
13
]. Ac i a ing PIK3CA gene mu-
a ions up egula e in acellula signalling ia he PI3K–p o ein kinase T (Ak )–mammalian
a ge o apamycin (mTOR) pa hway, con ibu ing o mul iple hallma ks o cance , such as
esis ing cell dea h and uncon olled p oli e a ion [
14
–
16
]. As desc ibed p e iously, HPV
genome in eg a ion leads o ke a inocy e immo aliza ion and ans o ma ion by inhibi ing
he umou supp esso p53 and e inoblas oma p o ein (pRb) bu also by in e ac ing wi h
o he pa hways, including PI3K/Ak /mTOR [
17
–
20
]. Se e al s udies ha e add essed
he ole o he PIK3CA pa hway in ce ical cance and o he ypes o HPV-associa ed
cance s [
21
–
25
]. In he e alua ion o 151 HNSCC whole-exome sequences, he PI3K pa h-
way was ound o be he mos commonly al e ed mi ogenic pa hway (30.5% o umou s)
compa ed o he JAK/STAT pa hway (9.3%) and MAPK pa hway (8.0%) [
26
]. Among he
PIK3CA mu a ions obse ed in HNSCC, 63% occu a h ee speci ic loca ions encoding he
p110
↵
subuni , namely E542, E545, and H1047, known as canonical mu a ions [
27
,
28
]. The
PIK3CA mu a ion equency among HPV-posi i e umou s was epo ed o be app oxi-
ma ely hal o ha ound in HPV-nega i e cance s [
29
]. E en so, he PIK3CA gene emains
one o he mos mu a ed in HPV-associa ed HNSCC [
7
,
30
]. Addi ionally, mu a ions ha e
been associa ed wi h ad e se ou comes in solid umou s, bu hei p ognos ic signi icance
in o opha yngeal SCC is s ill unclea . In p e ious s udies, he speci ic su i al da a did no
di e be ween PIK3CA wild- ype (WT) and mu a ed (MUT) lesions. Howe e , WT-PIK3CA
136
CHAPTER 5
GENERAL DISCUSSION
137
HNSCC and HPV ype, lineage and NGS
The HNSCC associa ed wi h HPV ypically a ises in he o opha ynx and is
conside ed a dis inc en i y compa ed wi h o he HN umou s named HNSCC-
nonHPV. The ecogni ion o his di e ence inci ed he de elopmen o s udies in
his ield, o gain insigh s in o pa ien isk s a i ica ion and mo e pe sonalized
ea men s. In he ea ly 2000s wi h he in oduc ion o new molecula ools aided
se e al esea ch g oups o explo e molecula mechanism-based app oaches o
de ine gene ic signa u es ha may play a p ime ole in pa ien s a i ica ion
(Alexand o and S a on, 2014; Chai, Lim and Cheong, 2020).
In his con ex , ou esea ch p ojec led us o explo e i speci ic HPV16 a ian s could
in luence he occu ence, he mo phology, and he molecula pheno ype o
HNSCC-HPV. Appa en ly, wi hin each hos , HPV genomes ha bou high a iabili y
le els in he e olu ion om low-g ade lesions o high-g ade lesions and cance ,
which may e eal se e al gene ic signa u es (Nache e al., 2020).
The g owing numbe o s udies on HPV16 a ian s, was mainly done in u e ine
ce ical cance and has exposed a e y la ge numbe o di e en papilloma i us
genomes. Mo eo e , he esea ch on HPV phylogene ic lineages wi h clinical hos
speci ici y epo s se e al di e ences wi hin i s oncogenic po en ial (Siche o e al.,
2007; Zhang e al., 2015).
Ou gene al hypo hesis was ha he speci ic mo phological and molecula
pheno ype HPV16 a ian s iden i ied in HNSCC umou could be associa ed wi h
di e en clinical pa hologic se ings. In his manne , ou s udy was concei ed o
iden i y he equency o HPV16 a ian s in squamous cell ca cinomas o he head
and neck egion and o e alua e hei co ela ion wi h clinical a iables.
A he onse , we ca ied ou a sys ema ic e iew (SR) o he li e a u e o sc u inize
which sequencing me hodologies we e pe o med. We concluded ha in mos
s udies in he HN egion, a limi ed numbe o samples we e examined, mainly
because o he labou -in ensi e echniques used (PCR). Sange sequencing
echniques we e applied o iden i y HPV16 a ian s in all nine HN s udies included
in he analysis; no Nex Gene a ion Sequencing (NGS) was used, mos likely due o
di icul ies on he access o he echnique and he limi a ion on suppo ing cos s
o applying his new sequencing me hodology. The use o con en ional Sange
sequencing likely limi ed he numbe o cases analysed and in oduced limi a ions
in a whole genome analysis wi h espec o he size o he amplicons, which may
comp omise he sensi i i y o he assay (Ho mann e al., 2004; Pa k e al., 2016;
144
and su i al o cance cells and is associa ed wi h a poo pa ien p ognosis.
De e mining which pa ien s ca y PIK3CA mu a ions is o g ea clinical impo ance
since i can help o de ine subg oups o pa ien s wi h speci ic needs and who may
bene i om p omising a ge ed he apies agains he PI3K-Ak -mTOR signalling
pa hway.
Ou s udy p oposed a quali i e analysis o he mu a ions commonly associa ed
wi h HNSCC umou s. The p esen esul s con ibu e o be e de ining his
complex scena io, de e mining he equencies o canonical PIK3CA mu a ions in
a Po uguese coho o pa ien s wi h HNSCC and es ing he pu a i e associa ions
be ween PIK3CA mu a ions and mul iple clinicopa hological pa ame e s. In ou
da ase , mos pa ien s wi h HNSCC ca ying canonical PIK3CA mu a ions had
ad anced disease s ages (III, IV), especially among HNSCC-HPV, sugges ing a
nega i e impac on pa ien p ognosis and co obo a ing p e iously published da a
(Zieba e al., 2020). Howe e , he e was no signi ican associa ion be ween
mu a ion equency and HPV s a us, and no co ela ion be ween PIK3CA and
o e all su i al be ween uni a ia e o mul i a ia e co ela ions. Impo an ly, ou
da a e idence a high equency o canonical PIK3CA mu a ions in HNSCC,
including in HNSCC-nonHPV cases, sugges ing ha sc eening o PIK3CA
mu a ions should no be es ic ed o he HNSCC-HPV g oup bu can also be
ele an o HNSCC-nonHPV pa ien s, which con as s wi h p e ious s udies
ha indica ed a highe equency in HNSCC-HPV umou s.
O e all, ou esul s con ibu e wi h new da a in he ield o molecula analysis in
oncological pa hology applied o HPV16-posi i e o opha yngeal umou s.
Cu en ly, i is aluable o conside ha umou ecu ence s ill occu s in 10-20% o
HPV-posi i e HNSCC pa ien s. Al hough he clinical managemen o HNSCC has
imp o ed since he ecogni ion o HPV-posi i e and HPV-nega i e lesions, u he
he apeu ic ad ances will equi e e ec i e clinical alida ion o all hese p omising
bioma ke s in la ge-scale popula ion s udies.
Cu en ly, in esponse o he clinical diagnosis, he HPV s a us is de e mined in
suspicious lesions o he o opha ynx by quali a i e PCR me hodologies ha
ansla e i s p esence o no . These me hodologies designed o de ec i al DNA
only iden i y he geno ype p esen . The new molecula ools de eloped in he
p esen wo k magni y he in o ma ion on his diagnosis and en ich he in o ma ion
on he Vi us-Hos dynamics, p omo ing be e pa ien isk s a i ica ion and mo e

145
a ge ed ea men s in clinical p ac ice. Pa icula ly, one o he s eng hs o his
p ojec was he de elopmen o he NGS wo k low, alida ed agains he ISO
s anda d 15189 o ce i y i s implemen a ion in a clinical labo a o y, hus
con ibu ing wi h new molecula inpu s o he clinical p ac ice among head and
neck umou s.
Finally, ou u u e pe spec i e is o apply his wo k low o clinically e e ed
casuis ic associa ed o HPV umou s p e ending o expand, sus ain and alida e
bioma ke s o answe he clinical p ac ice needs. Hope ully, he new sequencing
e a will inno a e he s udy o HPV and ela ed cance s.
146
CHAPTER 6
CONCLUDING REMARKS
147
Molecula analysis using nex -gene a ion sequencing me hodology ep esen s a
aluable ool o consolida e gene ic ma ke s wi h hei espec i e
clinicopa hological cha ac e is ics. The s udies published in his ield ein o ce i s
applica ion on a la ge scale o imp o e he clinical implica ions o ea ly diagnosis,
p ognosis, and he apeu ics. The NGS me hodologies implemen a ion a he
ou ine labo a o y wi hin he scope o HPV-associa ed pa hology po en ially
inc eases he s udies o hese coho s, p o ide a de ailed desc ip ion o mu a ional
p o iles associa ed wi h he espec i e clinicopa hological cha ac e is ics and
assesses g ea e obus ness as a complemen a y o diagnosis.
A s ong poin o his p ojec was he de elopmen o an NGS wo k low o he
iden i ica ion o HPV16 a ian s in HNSCC umou s, op imized o analyse DNA om
di e en ypes o p oduc s, and he e o e also p omising o i s applica ion in he
con ex o o he associa ed pa hologies, such as he ce ix and anal canal.
The analy ical alida ion de ines he cha ac e is ics o he assay based on s udies
he sensi i i y and speci ici y o cell lines in ec HPV16 (SiHa cell), issue sample
lesions om he K14HPV16 model and issue umou s samples om a coho o
pa ien s diagnosed wi h HNSCC.
The weaknesses a e highligh ed mainly by he unsa is ac o y esul s o 33 samples
o de e mine HPV16 a ian s, di ec ly comp omising he expec ed coho o he
phylogene ic analysis and associa ion wi h he clinicopa hological cha ac e is ics
and su i al. Howe e , he ulne able poin s iden i ied in he global p ocess and
hei esolu ion make i possible o e ine echnical laws and con ibu e o inc ease
he success o he NGS wo k low.
Finally, in gene al, i is a echnically consis en s udy ha e eals o iginal da a om
genomic analysis ha di ec ly con ibu e o deepening he s udy o he ole o
a ian s in he pa hology o HNSCC and ein o cing i s p omisso y pu pose as a
complemen a y ool o diagnosis.
In ou u u e pe spec i es, we in end o expand ou s udy, inc easing he numbe
o cases included, and in es in he imp o emen o bioin o ma ics analysis. We
p e end o de elop an HPV a ian Da abase pla o m allowing clinical
classi ica ion o he di e en HPV a ian s ound. The implemen a ion o s anda d
me hods allows his da abase o se e as a ool o u he s udies and con ibu e
o a be e ansla ional unde s anding o he biologic signi icance o each a ian .
148
BIBLIOGRAPHIC REFERENCES
Ag awal, N. e al. (2011) ‘Exome sequencing o head and neck squamous cell ca cinoma
e eals inac i a ing mu a ions in NOTCH1’, Science (New Yo k, N.Y.), 333(6046), pp. 1154–
1157. A ailable a : h ps://doi.o g/10.1126/science.1206923.
Ag awal, Y. e al. (2008) ‘O al human papilloma i us in ec ion be o e and a e ea men
o human papilloma i us 16-posi i e and human papilloma i us 16-nega i e head and
neck squamous cell ca cinoma.’, Clinical cance esea ch : an o icial jou nal o he
Ame ican Associa ion o Cance Resea ch, 14(21), pp. 7143–50. A ailable a :
h ps://doi.o g/10.1158/1078-0432.CCR-08-0498.
Alexand o , L.B. and S a on, M.R. (2014) ‘Mu a ional signa u es: The pa e ns o soma ic
mu a ions hidden in cance genomes’, Cu en Opinion in Gene ics and De elopmen ,
24(1), pp. 52–60. A ailable a : h ps://doi.o g/10.1016/j.gde.2013.11.014.
Amin, M.B. e al. (2017) ‘The Eigh h Edi ion AJCC Cance S aging Manual: Con inuing o
build a b idge om a popula ion-based o a mo e “pe sonalized” app oach o cance
s aging’, CA: a cance jou nal o clinicians, 67(2), pp. 93–99. A ailable a :
h ps://doi.o g/10.3322/caac.21388.
And ophy, E.J., Schille , J.T. and Lowy, D.R. (1985) ‘Iden i ica ion o he P o ein Encoded by
he E6 T ans o ming Gene o Bo ine Papilloma i us’, Science, 230(4724), pp. 442–445.
A ailable a : h ps://doi.o g/10.1126/science.2996134.
A bei , J.M. e al. (1993) ‘Neu oepi helial ca cinomas in mice ansgenic wi h human
papilloma i us ype 16 E6/E7 ORFs’, Ame ican Jou nal o Pa hology, 142(4), pp. 1187–1197.
A bei , J.M. e al. (1994) ‘P og essi e squamous epi helial neoplasia in K14-human
papilloma i us ype 16 ansgenic mice’, Jou nal o Vi ology, 68(7), pp. 4358–4368.
A ailable a : h ps://doi.o g/10.1128/j i.68.7.4358-4368.1994.
A bei , J.M., Howley, P.M. and Hanahan, D. (1996) ‘Ch onic es ogen-induced ce ical and
aginal squamous ca cinogenesis in human papilloma i us ype 16 ansgenic mice’,
P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica, 93(7),
pp. 2930–2935. A ailable a : h ps://doi.o g/10.1073/pnas.93.7.2930.
A gi is, A. e al. (2008) ‘Head and neck cance ’, The Lance , 371(9625), pp. 1695–1709.
A ailable a : h ps://doi.o g/10.1016/S0140-6736(08)60728-X.
Bada acco, G. e al. (2007) ‘Molecula analyses and p ognos ic ele ance o HPV in head
and neck umou s’, Oncology Repo s, 17(4), pp. 931–939. A ailable a :
h ps://doi.o g/10.3892/o .17.4.931.
Bake , C.C. e al. (1987) ‘S uc u al and ansc ip ional analysis o human papilloma i us
ype 16 sequences in ce ical ca cinoma cell lines’, Jou nal o Vi ology, 61(4), pp. 962–971.
A ailable a : h ps://doi.o g/10.1128/j i.61.4.962-971.1987.
Balbo, S. e al. (2012) ‘Kine ics o DNA Adduc Fo ma ion in he O al Ca i y a e D inking
Alcohol’, Cance Epidemiology, Bioma ke s & P e en ion, 21(4), pp. 601–608. A ailable a :
h ps://doi.o g/10.1158/1055-9965.EPI-11-1175.
Bech old, V., Bea d, P. and Raj, K. (2003) ‘Human Papilloma i us Type 16 E2 P o ein Has No
E ec on T ansc ip ion om Episomal Vi al DNA’, Jou nal o Vi ology, 77(3), pp. 2021–2028.
149
A ailable a : h ps://doi.o g/10.1128/JVI.77.3.2021-2028.2003.
Be na d, H.U. e al. (1993) ‘Sequence Va ian s o Human Papilloma i us Type 16 om
Couples Sugges Sexual T ansmission wi h Low In ec i i y and Polyclonali y in Geni al
Neoplasia’, Jou nal o In ec ious Diseases, 168(4), pp. 803–809. A ailable a :
h ps://doi.o g/10.1093/in dis/168.4.803.
Be na d, H.-U. e al. (2010) ‘Classi ica ion o papilloma i uses (PVs) based on 189 PV ypes
and p oposal o axonomic amendmen s’, Vi ology, 401(1), pp. 70–79. A ailable a :
h ps://doi.o g/10.1016/j. i ol.2010.02.002.
Be na d, H.U., Calleja-Macias, I.E. and Dunn, S.T. (2006) ‘Genome a ia ion o human
papilloma i us ypes: Phylogene ic and medical implica ions’, In e na ional Jou nal o
Cance , 118(5), pp. 1071–1076. A ailable a : h ps://doi.o g/10.1002/ijc.21655.
Be iol, J.C. e al. (2016) ‘P e alence o human papilloma i us ypes and a ian s and
p16(INK4a) exp ession in head and neck squamous cells ca cinomas in São Paulo, B azil.’,
In ec ious agen s and cance , 11(1), p. 20. A ailable a : h ps://doi.o g/10.1186/s13027-016-
0067-8.
Bhaijee, F. e al. (2012) ‘Cance s em cells in head and neck squamous cell ca cinoma: a
e iew o cu en knowledge and u u e applica ions’, Head & Neck, 34(6), pp. 894–899.
A ailable a : h ps://doi.o g/10.1002/hed.21801.
Bo ges, V. e al. (2018) ‘INSaFLU: an au oma ed open web-based bioin o ma ics sui e
“ om- eads” o in luenza whole-genome-sequencing-based su eillance’, Genome
Medicine, 10(1), p. 46. A ailable a : h ps://doi.o g/10.1186/s13073-018-0555-0.
Boscolo-Rizzo, P. e al. (2009) ‘HPV-16 E6 L83V a ian in squamous cell ca cinomas o he
uppe ae odiges i e ac ’, Jou nal o Cance Resea ch and Clinical Oncology, 135(4), pp.
559–566. A ailable a : h ps://doi.o g/10.1007/s00432-008-0490-3.
B ay, F. e al. (2018) ‘Global cance s a is ics 2018: GLOBOCAN es ima es o incidence and
mo ali y wo ldwide o 36 cance s in 185 coun ies.’, CA: a cance jou nal o clinicians,
68(6), pp. 394–424. A ailable a : h ps://doi.o g/10.3322/caac.21492.
Bu d, E.M. (2003) ‘Human papilloma i us and ce ical cance ’, Clinical Mic obiology
Re iews, 16(1), pp. 1–17. A ailable a : h ps://doi.o g/10.1128/CMR.16.1.1-17.2003.
Bu k, R.D., Chen, Z. and Van Doo slae , K. (2009) ‘Human papilloma i uses: Gene ic basis o
ca cinogenici y’, Public Heal h Genomics, 12(5–6), pp. 281–290. A ailable a :
h ps://doi.o g/10.1159/000214919.
Bu k, R.D., Ha a i, A. and Chen, Z. (2013) ‘Human papilloma i us genome a ian s’, Vi ology,
445(1–2), pp. 232–243. A ailable a : h ps://doi.o g/10.1016/j. i ol.2013.07.018.
Campo, L., Zhang, C. and B eue , E.-K. (2015) ‘EMT-Inducing Molecula Fac o s in
Gynecological Cance s’, BioMed Resea ch In e na ional, 2015, p. 420891. A ailable a :
h ps://doi.o g/10.1155/2015/420891.
Cance Genome A las Ne wo k (2015) ‘Comp ehensi e genomic cha ac e iza ion o head
and neck squamous cell ca cinomas.’, Na u e, 517(7536), pp. 576–82. A ailable a :
h ps://doi.o g/10.1038/na u e14129.
Ca e , J.J. e al. (1995) ‘Use o human papilloma i us ype 6 capsids o de ec an ibodies in
people wi h geni al wa s’, Jou nal o In ec ious Diseases, 172(1), pp. 11–18. A ailable a :
h ps://doi.o g/10.1093/in dis/172.1.11.

150
Ca e , J.J. e al. (1996) ‘The na u al his o y o human papilloma i us ype 16 capsid
an ibodies among a coho o uni e si y women’, Jou nal o In ec ious Diseases, 174(5), pp.
927–936. A ailable a : h ps://doi.o g/10.1093/in dis/174.5.927.
Cen e s o Disease Con ol and P e en ion (2018) ‘Cance s Associa ed wi h Human
Papilloma i us, Uni ed S a es, 2011–2015’, USCS DATA BRIEF, 4, pp. 1–2.
Chai, A.W.Y., Lim, K.P. and Cheong, S.C. (2020) ‘T ansla ional genomics and ecen
ad ances in o al squamous cell ca cinoma’, Semina s in Cance Biology, 61(1), pp. 71–83.
A ailable a : h ps://doi.o g/10.1016/j.semcance .2019.09.011.
Cha u edi, A.K. e al. (2011) ‘Human papilloma i us and ising o opha yngeal cance
incidence in he Uni ed S a es’, Jou nal o Clinical Oncology, 29(32), pp. 4294–4301.
A ailable a : h ps://doi.o g/10.1200/JCO.2011.36.4596.
Cha u edi, A.K. e al. (2013) ‘Wo ldwide ends in incidence a es o o al ca i y and
o opha yngeal cance s’, Jou nal o Clinical Oncology, 31(36), pp. 4550–4559. A ailable a :
h ps://doi.o g/10.1200/JCO.2013.50.3870.
Chen, A.A. e al. (2015) ‘Human Papilloma i us 18 Gene ic Va ia ion and Ce ical Cance
Risk Wo ldwide’, Jou nal o Vi ology. Edi ed by L. Banks, 89(20), pp. 10680–10687. A ailable
a : h ps://doi.o g/10.1128/j i.01747-15.
Che a, B.S. e al. (2015) ‘Phase 2 ial o de-in ensi ied chemo adia ion he apy o
a o able- isk human papilloma i us-associa ed o opha yngeal squamous cell
ca cinoma’, In e na ional Jou nal o Radia ion Oncology Biology Physics, 93(5), pp. 976–
985. A ailable a : h ps://doi.o g/10.1016/j.ij obp.2015.08.033.
Chien, M.-H. e al. (2012) ‘E ec s o E-cadhe in (CDH1) gene p omo e polymo phisms on
he isk and clinicopa hologic de elopmen o o al cance ’, Head & Neck, 34(3), pp. 405–
411. A ailable a : h ps://doi.o g/10.1002/hed.21746.
Chi gwin, K.D. e al. (1995) ‘Incidence o ene eal wa s in human immunode iciency i us-
in ec ed and unin ec ed women’, Jou nal o In ec ious Diseases, 172(1), pp. 235–238.
A ailable a : h ps://doi.o g/10.1093/in dis/172.1.235.
Chung, C.H. e al. (2004) ‘Molecula classi ica ion o head and neck squamous cell
ca cinomas using pa e ns o gene exp ession’, Cance Cell, 5(5), pp. 489–500. A ailable a :
h ps://doi.o g/10.1016/s1535-6108(04)00112-6.
Con ad, M., Bubb, V.J. and Schlegel, R. (1993) ‘The human papilloma i us ype 6 and 16 E5
p o eins a e memb ane-associa ed p o eins which associa e wi h he 16-kilodal on po e-
o ming p o ein’, Jou nal o Vi ology, 67(10), pp. 6170–6178. A ailable a :
h ps://doi.o g/10.1128/j i.67.10.6170-6178.1993.
Con e as-Pa edes, A. e al. (2009) ‘E6 a ian s o human papilloma i us 18 di e en ially
modula e he p o ein kinase B/phospha idylinosi ol 3-kinase (ak /PI3K) signaling
pa hway’, Vi ology, 383(1), pp. 78–85. A ailable a : h ps://doi.o g/10.1016/j. i ol.2008.09.040.
Co ne , I. e al. (2012) ‘Human Papilloma i us Type 16 Gene ic Va ian s: Phylogeny and
Classi ica ion Based on E6 and LCR’, Jou nal o Vi ology, 86(12), pp. 6855–6861. A ailable a :
h ps://doi.o g/10.1128/j i.00483-12.
Co ne , I. e al. (2013) ‘HPV16 gene ic a ia ion and he de elopmen o ce ical cance
wo ldwide’, B i ish Jou nal o Cance , 108(1), pp. 240–244. A ailable a :
h ps://doi.o g/10.1038/bjc.2012.508.
151
Coussens, L.M., Hanahan, D. and A bei , J.M. (1996) ‘Gene ic p edisposi ion and pa ame e s
o malignan p og ession in K14- HPV16 ansgenic mice’, Ame ican Jou nal o Pa hology,
149(6), pp. 1899–1917.
Cullen, M. e al. (2015) ‘Deep sequencing o HPV16 genomes: A new high- h oughpu ool
o explo ing he ca cinogenici y and na u al his o y o HPV16 in ec ion’, Papilloma i us
esea ch (Ams e dam, Ne he lands), 1, pp. 3–11. A ailable a :
h ps://doi.o g/10.1016/j.p .2015.05.004.
Day, P.M., Lowy, D.R. and Schille , J.T. (2003) ‘Papilloma i uses in ec cells ia a cla h in-
dependen pa hway’, Vi ology, 307(1), pp. 1–11. A ailable a : h ps://doi.o g/10.1016/S0042-
6822(02)00143-5.
De B ee, R. and Leemans, C.R. (2010) ‘Recen ad ances in su ge y o head and neck
cance ’, Cu en Opinion in Oncology, 22(3), pp. 186–193. A ailable a :
h ps://doi.o g/10.1097/CCO.0b013e3283380009.
De la C uz-He nández, E. e al. (2005) ‘Di e en ial splicing o E6 wi hin human
papilloma i us ype 18 a ian s and unc ional consequences’, Jou nal o Gene al Vi ology,
86(9), pp. 2459–2468. A ailable a : h ps://doi.o g/10.1099/ i .0.80945-0.
De Villie s, E.M. e al. (2004) ‘Classi ica ion o papilloma i uses’, Vi ology, 324(1), pp. 17–27.
A ailable a : h ps://doi.o g/10.1016/j. i ol.2004.03.033.
Del Mis o, A. e al. (2006) ‘Human papilloma i us yping o in asi e ce ical cance s in
I aly.’, In ec ious agen s and cance , 1(1), p. 9. A ailable a : h ps://doi.o g/10.1186/1750-9378-
1-9.
Dias, T.R. e al. (2022) ‘Exp ession o LncRNAs in HPV-induced Ca cinogenesis and Cance
Cachexia: A S udy in K14-HPV16 Mice.’, An icance esea ch, 42(5), pp. 2443–2460.
A ailable a : h ps://doi.o g/10.21873/an ican es.15723.
Dillne , J. (1999) ‘The se ological esponse o papilloma i uses’, Semina s in Cance
Biology, 9(6), pp. 423–430. A ailable a : h ps://doi.o g/10.1006/scbi.1999.0146.
Doo ba , J. e al. (1991) ‘Speci ic in e ac ion be ween HPV-16 E1-E4 and cy oke a ins esul s
in collapse o he epi helial cell in e media e ilamen ne wo k’, Na u e. A ailable a :
h ps://doi.o g/10.1038/352824a0.
Doo ba , J. (2006) ‘Molecula biology o human papilloma i us in ec ion and ce ical
cance ’, Clinical Science, 110(5), pp. 525–541. A ailable a :
h ps://doi.o g/10.1042/CS20050369.
Doo ba , J. (2016) ‘Model sys ems o human papilloma i us-associa ed disease:
Papilloma i us disease models’, The Jou nal o Pa hology, 238(2), pp. 166–179. A ailable a :
h ps://doi.o g/10.1002/pa h.4656.
Du, J. e al. (2012) ‘Human papilloma i us (HPV) 16 E6 a ian s in onsilla cance in
compa ison o hose in ce ical cance in S ockholm, Sweden.’, PloS one. Edi ed by R.
Medei os, 7(4), p. e36239. A ailable a : h ps://doi.o g/10.1371/jou nal.pone.0036239.
El-Mo y, S.K. (2012) ‘HPV-Rela ed Squamous Cell Ca cinoma Va ian s in he Head and
Neck’, Head and Neck Pa hology, 6(SUPPL. 1), pp. 55–62. A ailable a :
h ps://doi.o g/10.1007/s12105-012-0363-6.
El-Nagga AK e al. (2017) WHO Classi ica ion o Head and Neck Tumou s. 4 h edn. Wo ld
Heal h O ganiza ion. A ailable a : h ps://publica ions.ia c. /Book-And-Repo -
152
Se ies/Who-Classi ica ion-O -Tumou s/WHO-Classi ica ion-O -Head-And-Neck-Tumou s-
2017.
E ande , M. e al. (1995) ‘Human papilloma i us in ec ion is ansien in young women: A
popula ionbased coho s udy’, Jou nal o In ec ious Diseases, 171(4), pp. 1026–1030.
A ailable a : h ps://doi.o g/10.1093/in dis/171.4.1026.
Fan, C.-C. e al. (2013) ‘Exp ession o E-cadhe in, Twis , and p53 and hei p ognos ic alue
in pa ien s wi h o al squamous cell ca cinoma’, Jou nal o Cance Resea ch and Clinical
Oncology, 139(10), pp. 1735–1744. A ailable a : h ps://doi.o g/10.1007/s00432-013-1499-9.
Fa ah, C.S. (2021) ‘Molecula landscape o head and neck cance and implica ions o
he apy’, Annals o T ansla ional Medicine, 9(10), pp. 915–915. A ailable a :
h ps://doi.o g/10.21037/a m-20-6264.
Faulkne Valle, G. and Banks, L. (1995) ‘The human papilloma i us (HPV)-6 and HPV-16 E5
p o eins co-ope a e wi h HPV-16 E7 in he ans o ma ion o p ima y oden cells’, Jou nal
o Gene al Vi ology, 76(5), pp. 1239–1245. A ailable a : h ps://doi.o g/10.1099/0022-1317-76-5-
1239.
Flo in, L. e al. (2002) ‘Assembly and T ansloca ion o Papilloma i us Capsid P o eins’,
Jou nal o Vi ology, 76(19), pp. 10009–10014. A ailable a :
h ps://doi.o g/10.1128/JVI.76.19.10009-10014.2002.
Fo bes, D. (2007) Eu oguide: on he accommoda ion and ca e o animals used o
expe imen al and o he scien i ic pu poses; based o he e ised appendix A o he
Eu opean Copn en ion ETS 123. Edi ed by Fede a ion o Eu opean Labo a o y Animal
Science Associa ions. London: Felasa.
Gillison, M.L. e al. (2000) ‘E idence o a causal associa ion be ween human
papilloma i us and a subse o head and neck cance s.’, Jou nal o he Na ional Cance
Ins i u e, 92(9), pp. 709–20. A ailable a : h ps://doi.o g/10.1093/jnci/92.9.709.
Gillison, M.L. e al. (2008) ‘Dis inc isk ac o p o iles o human papilloma i us ype 16-
posi i e and human papilloma i us ype 16-nega i e head and neck cance s’, Jou nal o
he Na ional Cance Ins i u e, 100(6), pp. 407–420. A ailable a :
h ps://doi.o g/10.1093/jnci/djn025.
Gillison, M.L. e al. (2019) ‘Radio he apy plus ce uximab o cispla in in human
papilloma i us-posi i e o opha yngeal cance (NRG Oncology RTOG 1016): a andomised,
mul icen e, non-in e io i y ial’, The Lance , 393(10166), pp. 40–50. A ailable a :
h ps://doi.o g/10.1016/S0140-6736(18)32779-X.
Gillison, M.L. and Shah, K.V. (2003) ‘Chap e 9: Role o mucosal human papilloma i us in
nongeni al cance s.’, Jou nal o he Na ional Cance Ins i u e. Monog aphs, 2003(31), pp.
57–65. A ailable a : h ps://doi.o g/10.1093/ox o djou nals.jncimonog aphs.a003484.
Gi oglou, T. e al. (2001) ‘Human Papilloma i us In ec ion Requi es Cell Su ace Hepa an
Sul a e’, Jou nal o Vi ology, 75(3), pp. 1565–1570. A ailable a :
h ps://doi.o g/10.1128/j i.75.3.1565-1570.2001.
Global cance Obse a o y (no da e) ‘Global Cance Obse a o y: Cance Today.’,
In e na ional Agency o Resea ch on Cance . A ailable a : h ps://gco.ia c. / oday
(Accessed: 17 June 2022).
G a es, C.A. e al. (2014) ‘The ansla ional signi icance o epi helial-mesenchymal
ansi ion in head and neck cance ’, Clinical and T ansla ional Medicine, 3(1), p. 60.
153
A ailable a : h ps://doi.o g/10.1186/s40169-014-0039-9.
Hadami, K. e al. (2021) ‘Deg ada ion o p53 by HPV16-E6 a ian s isola ed om ce ical
cance specimens o Mo occan women.’, Gene, 791, p. 145709. A ailable a :
h ps://doi.o g/10.1016/j.gene.2021.145709.
Ham, J. e al. (1991) ‘The papilloma i us E2 p o ein: a ac o wi h many alen s’, T ends in
Biochemical Sciences, 16(No embe ), pp. 440–444. A ailable a :
h ps://doi.o g/10.1016/0968-0004(91)90172-R.
Hamma s ed , L. e al. (2006) ‘Human papilloma i us as a isk ac o o he inc ease in
incidence o onsilla cance ’, In e na ional Jou nal o Cance , 119(11), pp. 2620–2623.
A ailable a : h ps://doi.o g/10.1002/ijc.22177.
Hassani, S. e al. (2015) ‘Molecula Pa hogenesis o Human Papilloma i us Type 16 in
Tonsilla Squamous Cell Ca cinoma’, An icance esea ch, 35(12), pp. 6633–6638.
Ha akeyama, H. e al. (2014) ‘Epi helial-mesenchymal ansi ion in human papilloma i us-
posi i e and -nega i e o opha yngeal squamous cell ca cinoma’, Oncology Repo s, 32(6),
pp. 2673–2679. A ailable a : h ps://doi.o g/10.3892/o .2014.3509.
zu Hausen, H. (1996) ‘Papilloma i us in ec ions — a majo cause o human cance s’,
Biochimica e Biophysica Ac a (BBA) - Re iews on Cance , 1288(2), pp. F55–F78. A ailable
a : h ps://doi.o g/10.1016/0304-419X(96)00020-0.
Haw ho n, R.J.S. e al. (1988) ‘Lange hans’ cells and sub ypes o human papilloma i us in
ce ical in aepi helial neoplasia.’, BMJ, 297(6649), pp. 643–646. A ailable a :
h ps://doi.o g/10.1136/bmj.297.6649.643.
an de Heijden, M. e al. (2020) ‘Epi helial- o-mesenchymal ansi ion is a p ognos ic
ma ke o pa ien ou come in ad anced s age HNSCC pa ien s ea ed wi h
chemo adio he apy’, Radio he apy and Oncology: Jou nal o he Eu opean Socie y o
The apeu ic Radiology and Oncology, 147, pp. 186–194. A ailable a :
h ps://doi.o g/10.1016/j. adonc.2020.05.013.
Hende son, S. e al. (2014) ‘APOBEC-Media ed Cy osine Deamina ion Links PIK3CA Helical
Domain Mu a ions o Human Papilloma i us-D i en Tumo De elopmen ’, Cell Repo s,
7(6), pp. 1833–1841. A ailable a : h ps://doi.o g/10.1016/j.cel ep.2014.05.012.
He e o, R. e al. (2000) ‘Popula ion-Based S udy o Human Papilloma i us In ec ion and
Ce ical Neoplasia in Ru al Cos a Rica’, JNCI: Jou nal o he Na ional Cance Ins i u e,
92(6), pp. 464–474. A ailable a : h ps://doi.o g/10.1093/jnci/92.6.464.
Hi ose, Y. e al. (2018) ‘Wi hin-Hos Va ia ions o Human Papilloma i us Re eal APOBEC
Signa u e Mu agenesis in he Vi al Genome’, Jou nal o Vi ology, 92(12), pp. e00017-18.
A ailable a : h ps://doi.o g/10.1128/j i.00017-18.
Ho, G.Y.F. e al. (1998) ‘Na u al His o y o Ce ico aginal Papilloma i us In ec ion in Young
Women’, New England Jou nal o Medicine, 338(7), pp. 423–428. A ailable a :
h ps://doi.o g/10.1056/nejm199802123380703.
Ho, L. e al. (1991) ‘Sequence a ian s o human papilloma i us ype 16 in clinical samples
pe mi e i ica ion and ex ension o epidemiological s udies and cons uc ion o a
phylogene ic ee’, Jou nal o Clinical Mic obiology, 29(9), pp. 1765–1772. A ailable a :
h ps://doi.o g/10.1128/jcm.29.9.1765-1772.1991.
Ho, L. e al. (1993) ‘The gene ic d i o human papilloma i us ype 16 is a means o