A p ospec i e coho s udy o HPV-media ed
o al and o opha yngeal cance and e icien
de ec ion me hods
Zd a ka Pasho a-Tasse a1, Vessela Rayko a2, Milena I ano a-Shi a o a3, S anisla Yo dano 4,
S e osla Sla ko 5, Daniel Ma ko 5, Vik o Lenko 6, Eli sa Deli e ska7
1 Depa men o Pe iodon ology, Facul y o Den al Medicine, Medical Uni e si y-So ia, So ia, Bulga ia
2 Medical Uni e si y-So ia, So ia, Bulga ia
3 Depa men o Clinical immunology, Uni e si y hospi al Alexand o ska, Medical Uni e si y-So ia, So ia, Bulga ia
4 Depa men o ENT diseases, Uni e si y hospi al S . Anna, So ia, So ia, Bulga ia
5 Depa men o Maxillo acial Su ge y, Pi ogo Hospi al; Bulga ia., So ia, Bulga ia
6 FDM, MU – So ia, So ia, Bulga ia
7 Depa men o O al and Maxillo acial su ge y, Medical Uni e si y-So ia, So ia, Bulga ia
Co esponding au ho :
Eli sa Deli e ska (eli sadeli e [email p o ec ed]m)
Recei ed
14 Augus 2025♦
Accep ed
11 Sep embe 2025♦
Published
7 Oc obe 2025
Ci a ion:
Pasho a-Tasse a Z, Rayko a V, I ano a-Shi a o a M, Yo dano S, Sla ko S, Ma ko D, Lenko V, Deli e ska E (2025)
A p ospec i e coho s udy o HPV-media ed o al and o opha yngeal cance and e icien de ec ion me hods. Pha macia 72: 1–10.
h ps://doi.o g/10.3897/pha macia.72.e168813
Abs ac
The ole o HPV is accep ed in pa hogenesis o o al and o opha yngeal squamous cell ca cinoma (OPSCC) .
The aim o his s udy is o in es iga e he associa ion be ween HPV and o al and o opha yngeal ca cinoma, o analyze he oncoana-
omical localiza ions and e ec i e de ec ion me hods.
We in es iga ed 89 pa ien s-50 pa ien s wi h his ologically p o en OPSCC and 39 people as a es g oup. Samples o HPV de ec ion
we e aken wi h smea es and o al inse o each pa ien . In 30% o cases wi h OPSSC we ound an associa ion wi h HPV in he es
g oup. S ains we e isola ed om samples by PCR me hod. Combina ion o o al inse and b ush has he highes HPV de ec ion a e
(77.8%). Tumou s in o opha ynx ha e a highe isk o HPV in ec ion. In he s udy, HPV16 was ound o be p edominan , as well as
o he s ains 51,52,58,66,6,31,39, alone o in combina ion.
O impo an clinical impo ance is he accu a e de e mina ion o he HPV s a us o umo , which is impo an o managemen
and p ognosis.
Keywo ds
de ec ion me hods, Human papilloma i us, O al ca cinoma, O opha yngeal Squamous cell ca cinoma, p e alence
Copy igh Pasho a-Tasse a Z e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License (CC-BY 4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho
and sou ce a e c edi ed.
Pha macia 72: 1–10
DOI 10.3897/pha macia.72.e168813
Resea ch A icle
Pasho a-Tasse a Z e al.: A p ospec i e coho s udy o hp -media ed o a and o opha yngeal cance s2
In oduc ion
O al ca cinoma anks among he en mos common ma-
lignan diseases wo ldwide, which emphasizes i s impo -
ance in he con ex o public heal h. No ably, he inci-
dence o o opha yngeal squamous cell ca cinoma (OSCC)
in Wes e n Eu ope and he Uni ed S a es has inc eased
d ama ically o e he pas wo decades (Cha u edi e al.
2011; Roman and A agones 2021). In 2020, 94,412 new
cases o (OSCC) and 48,143 dea hs wo ldwide we e egis-
e ed (Sung e al. 2021; Khan e al. 2025).
This inc ease in incidence is la gely due o human pap-
illoma i us (HPV) in ec ion, as he incidence o HPV –
media ed OPSCC (HPV + OPC) has inc eased bu wi h
clea geog aphic a ia ions (Mehanna e al. 2013, 2016;
Van Dyne e al. 2015).
In Bulga ia o 2020, he incidence is 3.3/100,000, wi h
a di e ence be ween he sexes, espec i ely 5.0/100,000
in men and 1.7/100,000 in women (Fe lay e al. 2020).
The e is also high a iabili y in he incidence o in ec ion
by ana omical loca ion (Ye e e al. 2018). HPV-media ed
o opha yngeal cance is a dis inc disease en i y wi h dis-
inc epidemiological and molecula ea u es and biological
beha iou and is cha ac e ized by be e esponse o ea -
men and su i al han HPV-un ela ed o opha yngeal can-
ce (Ang e al. 2010; Lechne e al. 2022; Jung e al. 2010).
Oncogenesis in HPV-associa ed cance s is media ed by
i al oncop o eins E6 and E7, which inhibi he umo sup-
p esso p o eins p53 and e inoblas oma (Rb) p o ein, e-
spec i ely (Hebne and Laimins 2006; Lechne e al. 2022).
In con as , HPV-nega i e HNSCC is due o DNA damage
by ca cinogens wi h subsequen mu a ions in umo sup-
p esso genes such as TP53 and CDKN2A (Khan e al. 2025).
Gi en he p ognos ic ad an age o HPV- ela ed o o-
pha yngeal cance , he Ame ican Join Commi ee on
Cance (AJCC) and he Union o In e na ional Cance
Con ol (UICC) (UICC) de eloped sepa a e classi ica-
ions o HPV- ela ed and non-HPV- ela ed diseases in
he new TNM 8 h edi ion s aging sys em (TNM 8) o
HPV- ela ed o opha yngeal cance (C aig e al. 2019).
Da a on he p e alence o HPV in he o al ca i y a e
impo an o se e al easons – o unde s and he p o ile
o he umo lesion – HPV posi i e o nega i e, as HPV
posi i es ha e a be e p ognosis; o de ine isk g oups; o
be used as a measu e o he esul s and e icacy o acci-
na ion p og ams. The e o e, a ious e ec i e and non-in-
asi e me hods o esea ch a e sough (Cha u edi e al.
2011; C aig e al. 2019; De Ma el e al. 2020).
HPV media ion o o opha yngeal cance can be es ab-
lished by es ing o he p esence o HPV DNA o mRNA
in he umo using PCR-based me hods o in-si u hyb id-
iza ion(ISH). O e exp ession o he p16 p o ein se es as
an excellen su oga e bioma ke o HPV causa ion in
OPSSC (Smee s e al. 2007; Tang e al. 2020). The posi-
i e p ognosis is also mo e p onounced in HPV-posi i e
pa ien s who a e also p16 posi i e (Mehanna e al. 2023).
The wo ld li e a u e discusses con en ional me hods
o HPV de ec ion (p16 immunohis ochemis y, HPV
DNA ISH, HPV DNA PCR, HPV E6/E7 mRNA RT-PCR,
HPV RNA ISH, as well as eme ging new app oaches (HPV
ci cula ing umo DNA, HPV16 E6 an ibodies, o al HPV
DNA/mRNA PCR, NGS). Cu en ly, a combined es ing
app oach using sequen ial sc eening wi h p16 immuno-
his ochemis y and con i ma ion wi h HPV DNA PCR is
p e e ed by many au ho s(Smee s e al. 2007; Tang e al.
2020; Mehanna e al. 2023). HPV RNA in-si u hyb idiza-
ion could po en ially se e as a single es due o i s good
sensi i i y and speci ici y, bu is no ou inely used (Jung
e al. 2010; Mena e al. 2022; Khan e al. 2025).
The use o liquid biopsies such as HPV ci cula ing u-
mo DNA could be success ully used o ea ly de ec ion
o HPV-associa ed o opha yngeal ca cinomas. HPV16 E6
an ibodies and o al HPV DNA PCR can be used as addi-
ional es s o aid diagnosis (Tang e al. 2020; Mehanna e
al. 2023). Howe e , he e a e limi a ions o he use o PCR
o HPV DNA de ec ion – he es can be e y sensi i e; i
is impossible o dis inguish whe he HPV DNA de ec ed by
his me hod is om malignan umo issue o he i us is
only p esen in adjacen , non-neoplas ic issues. De ec ion
o HPV DNA by PCR, like DNA ISH, does no p o ide in-
o ma ion on whe he he i us is ansc ip ionally ac i e.
To add ess his ques ion, se e al s udies ha e shown ha e-
e se ansc ip ion PCR can be used on o malin- ixed spec-
imens o de ec E6 and E7 mRNA ansc ip s, al hough his
me hod is no cu en ly a ailable o clinical use and has no
been compa ed wi h o he me hodologies ( an Hou en e al.
2001; Smee s e al. 2007; Pal e e al. 2018; Tang e al. 2020;
Mehanna e al. 2023; Hillie e al. 2025; Khan e al. 2025).
I is assumed ha o be biologically and clinically ele an ,
HPV mus be ansc ip ionally ac i e, and some in es iga o s
ha e sugges ed ha de ec ion o HPV RNA should be he
gold s anda d o HPV es ing. Fo example, pa ien s wi h
HPV DNA+/RNA+ ha e imp o ed o e all su i al com-
pa ed wi h hose wi h HPV DNA+/RNA– (Jung e al. 2010).
In he dynamic en i onmen o new es s and de elop-
men s in molecula biology, clinicians mus ha e access
o and choose cos -e ec i e, accu a e, and e ec i e HPV
es s, as he e is s ill no s anda d o HPV i us de ec ion.
The aim o his s udy is o in es iga e he ela ionship
be ween HPV and o al and o opha yngeal ca cinoma, o
analyze he oncoana omical localiza ions, and o discuss
e ec i e de ec ion me hods.
Design o he s udy
This s udy ep esen s a cen alized analysis o indi idual
da a o pa ien s diagnosed wi h o al and o opha yngeal ca -
cinoma based on da a om h ee hospi als in So ia, Bulga ia.
Ma e ial and me hods
A o al o 89 subjec s pa icipa ed in he s udy – 50 cance
pa ien s and 39 heal hy con ols. All pa icipan s ag eed o
pa icipa e in he s udy and signed an in o med consen .
Pha macia 72: 1–10 3
The pa ien s we e ec ui ed om Alexand o ska Hospi-
al, Pi ogo Hospi al and S . Anna Hospi al in So ia, while
heal hy olun ee s we e andomly selec ed.
A clinical ques ionnai e was p epa ed o he pa ien
and he ea ing eam o doc o s o ill in in o ma ion –
in o ma ion on age, gende , obacco use and use o elec-
onic ciga e es and alcohol; TNM 7 h edi ion s aging;
como bidi y; local o al s a us in ela ion o he p esence
o pe iodon al disease and local i i an s, loca ion o he
lesion, biological beha iou o lesion,clinical cha ac e is-
ic o lesion, and his opa hological indings.
Inclusion c i e ia we e a pa ien wi h his ologically e -
i ied p ima y o opha yngeal squamous cell ca cinoma.
Pa ien s unde wen imaging (CT o MRI) and his ologi-
cal con i ma ion by biopsy – o malin- ixed, pa a in-em-
bedded issue. Pa ien s we e ea ed wi h su ge y, adio-
he apy, chemo he apy o a combina ion o hese, o we e
e e ed o pallia i e ea men .
HPV es ing was pe o med using o al inse and o al
b ush samples o HPV DNA PCR de ec ion. – o al inse
15 – o 30-second insing/swishing, ollowed by 15 – o
30-second ga gling. B ush samples a e aken om he
a ea o he umo lesion. Samples we e collec ed om all
pa icipan s in he s udy using p e-de ined p o ocols by
he ea ing clinicians and s o ed and anspo ed acco d-
ing o s ic guidelines.
In he collec ed samples, geno yping and quan i ica ion
o HPV we e pe o med by mul iplex Real Time PCR us-
ing alida ed comme cial ki s HPV Quan 21 on he es
g oup – 50 pa ien s wi h o opha yngeal ca cinoma and on
he heal hy con ols – 39. Mul iplex se ologic es ing was
pe o med a Depa men o Mic obiology – Facul y o
Medicine – MU – So ia. Ki s o Real-Time PCR quan i a-
i e de ec ion and geno yping o 21 HPV geno ypes we e
used, including high- isk 16, 18, 26, 31, 33, 35, 39, 45, 51,
52, 53, 56, 58, 59, 66, 68, 73, 82 and low- isk 6, 11 and 44 .
Molecula me hods o HPV de ec ion, speci ical-
ly PCR-based i al DNA assays in ou s udy we e pe -
o med using o al inse and b ush biopsy es samples in
ou s udy is consis en wi h molecula me hods o HPV
de ec ion, speci ically PCR-based assays o i al DNA.
These nonin asi e app oaches a e sui able o iden i ying
he p esence o HPV in ex olia ed o al epi helial cells and
sali a, al hough hey do no dis inguish be ween an-
sc ip ionally ac i e in ec ions, bu hey allow us o com-
pa e he esul s o he wo me hods and de e mine which
is mo e accu a e.
Da abase, e hics app o al and consen
o pa icipa e
This s udy was pe o med in acco dance wi h he Decla-
a ion o Helsinki. In o med consen was ob ained o pa-
ien s diagnosed in 2024–2025. A s anda dized Mic oso
Excel o m was used o collec and ha monize da a. E hics
app o al o he s udy was ob ained by he e hics commi -
ee (KENIMUS) o he Medical Uni e si y – So ia. No.
03/23.02.2024., e hic app o al da e – №985-29/02/2024.
S a is ical analysis
SPSS (S a is ical P og am o S a is ics) was used o p o-
cess he da a om he s udy. Package o he Social Scienc-
es ) was e sion 20.0.
1. Desc ip i e s a is ics
– Quan i a i e a iables a e ep esen ed by sum-
ma y s a is ical cha ac e is ics – a i hme ic mean
(Mean), s anda d de ia ion (SD); minimum and
maximum alue.
– Ca ego ical a iables a e ep esen ed by absolu e
(N) and ela i e (%) equencies.
2. One-Sample Kolmogo o -Smi no es o check he
shape o equency dis ibu ions o quan i a i e
a iables.
3. Chi-squa e es o Fishe ’s exac es – when exam-
ining ela ionships be ween desc ip i e (ca ego ical)
da a wi h wo o mo e ca ego ies.
4. T- es – when compa ing wo independen g oups
when he dis ibu ion o he a iable unde s udy
is no mal.
The adop ed signi icance le el is α=0.05. S a is ical
signi icance is accep ed when he p alue is less han α
(p < 0.05).
Resul s
The esul s o ou s udy show ha he a e age age o pa-
ien s wi h HPV posi i e (+) is 60 yea s, and HPV nega-
i e (-) is 62 yea s. No s a is ically signi ican ela ionship
was obse ed (Table 1).
Males a e mo e a ec ed, accoun ing o 80% o pa-
ien s, wi h nea ly 1/3 o HPV (+).
In ou s udy 70% o he es g oup – pa ien s wi h OSSC
– we e HPV (-), while in he con ol g oup 95% we e HPV
(-). In 12% o he es g oup mo e han one s ain was de-
ec ed (Table 3).
Table 1. Mean age o pa ien s wi h OSCC ca cinoma depending
on HPV s a us.
HPV s a us N Mean SD Min Max p
Age No 35 61.97 10.93 38.00 83.00 0.783
Yes 15 60.17 10.15 44.00 78.00
Table 2. Dis ibu ion o pa ien s by gende depending on HPV
s a us.
HPV s a us To al p
No Yes
Gende Male N 29 11 40 0.462
% 82.9% 73.3% 80.0%
Female N 6 4 10
% 17.1% 26.7% 20.0%
Pasho a-Tasse a Z e al.: A p ospec i e coho s udy o hp -media ed o a and o opha yngeal cance s4
In he conduc ed s udy, HPV16 was p edominan ly
ound bo h alone and in combina ion wi h o he s ains
– 51, 52, 58, 66, 6, 31, 39 (Table 4) n=1.0717 close o s a-
is ical signi icance. I is in e es ing ha o he s ains we e
also ound bo h alone –HPV 6 and in combina ion –HPV
39 and 58. In he con ol g oup – 5.1% posi i e o HPV,
only single s ains we e ound – HPV16 and 51.
The oncoana omical localiza ion o he s udied pa ien s
(Fig. 1) om he es g oup shows ha 20% o he pa ien s
ha e ca cinoma o he ongue and 34% ha e ca cinoma o
he la ynx, 10% ha e loo o he mou h, 8% ha e localiza-
ion o he ongue and loo o he mou h, and 10% ha e
pha yngeal in ol emen . In 22% o he pa ien s, in ol e-
men o mo e han one oncoana omical a ea was ound.
The esul s o ou s udy show ha pha ynx ca cinomas
a e HPV (+) in a highe pe cen age o cases, as well as pha -
ynx and oo o he ongue, wi hou a s a is ically signi ican
ela ionship. Rega ding he o al ca i y, he loo o he mou h
and ongue a e mo e a ec ed, bu again wi hou a s a is ical-
ly signi ican ela ionship, bu weakly exp essed ends a e
isible. In he localiza ion o ca cinoma-la ynx, o e hal o
he cases a e HPV (-). In 25 cases wi h he loo o he mou h
in ol ed, he ca cinoma is nega i e o HPV in isola ion o
in a ious combina ions , in 4 cases i is HPV (+). In all o h-
e localiza ions, 25 cases a e HPV nega i e and 11 a e HPV
posi i e wi hou a s a is ically signi ican ela ionship.
In he 50 cases o malignan umo s in di e en a eas o
he o al ca i y, la ynx and pha ynx s udied, HPV was no
de ec ed in mos cases (70%, 35/50), HPV posi i es we e in
a o al 30% (15/50). De ec ion was ound in:1 HPV s ain
in 9 cases (18%) and mo e han 1 HPV s ain in 6 cases
(12%)(Table 5, Fig. 2). This means ha mos umo s do
no con ain HPV, bu in some pa ien s he i us is p esen ,
which may be a ac o in he de elopmen o he disease.
Dis ibu ion by localiza ion in he s udy as he mos com-
monly a ec ed a ea is he La ynx – 17 cases (34% o all), wi h
29% o hem being HPV posi i e.The second mos common
is Tongue – 10 cases (20%), hal o which a e HPV posi i e.
O he mo e common loca ions: Floo o he mou h – 5 cases
(40% HPV posi i e), Pha ynx – 5 cases (60% HPV posi i e).
Loca ions wi h he highes HPV equency.
A 100% HPV posi i i y (all cases es ed posi i e): i
is ound ha by localiza ion – Gingi a/Floo o mou h/
Tongue (1 case, >1 s ain) , Floo o mou h/Tongue (2 cas-
es, 1 s ain) and Pha ynx/Tongue (1 case, >1 s ain).
Al hough he numbe o cases in hese g oups is small,
he comple e HPV posi i i y sugges s a s ong associa ion
be ween his ana omical combina ion and HPV in ec ion.
In he o al ca i y, HPV is p obably no a majo ac o in
umo de elopmen .
The conclusions ha can be made a e ha localiza ions
ha in ol e he ongue o pha ynx ha e a highe isk o
HPV in ec ion. This is suppo ed by he high posi i i y
a es. The la ynx is he mos commonly a ec ed a ea, bu
Table 3. HPV s a us in es g oup and con ol g oup.
G oups HPV s a us N %
Pa ien s wi h OSSC HPV (-) 35 70.0
1 s ain 9 18.0
Mo e han 1 s ain 6 12.0
To al 50 100.0
Con ol g oup HPV (-) 37 94.9
s ain 2 5.1
To al 39 100.0
Table 4. HPV s ains in es g oup and con ol g oup.
G oup HPV s a us N %
Tes g oup No 35 70.0
16 8 16.0
16;51;66 1 2.0
16;52 1 2.0
16;6 1 2.0
16;6;31 1 2.0
39;58 2 4.0
6 1 2.0
To al 50 100.0
Con ol g oup No 37 94.9
16 1 2.6
51 1 2.6
To al 39 100.0
Figu e 1. Oncoana omical localiza ion o OSSC.
2,0%
2,0%
2,0%
2,0%
2,0%
2,0%
2,0%
2,0%
2,0%
8,0%
10,0%
10,0%
20,0%
34,0%
gingi a/bucal mucosa
gingi a/pala e
gingi a/floo o he mou h
gingi a/ ongue/floo o he mou h
ha d pala e
floo o he mou h/gingi a/buccal mucosa
floo o he mou h/ ongue
ongue/floo o he mou h/pala e
pha ynx/ ongue
ongue/floo o he mou h
floo o he mou h
pha ynx
ongue
la ynx
Oncoana omical localiza ion
Pha macia 72: 1–10 5
Table 5. Oncoana omical localiza ion o OSSC and HPV s a us.
Oncoana omical localiza ion o OSSC HPV s a us To al
No Yes
Gingi a/buccal mucosa N 1 0 1
% 2.9% 0.0% 2.0%
Gingi a/ ha d pala e N 1 0 1
% 2.9% 0.0% 2.0%
Gingi a/ loo o he mou h N 1 0 1
% 2.9% 0.0% 2.0%
Gingi a/ loo o he mou h / ongue N 0 1 1
% 0.0% 6.7% 2.0%
Tongue N 8 2 10
% 22.9% 13.3% 20.0%
Tongue/ loo o he mou h N 4 0 4
% 11.4% 0.0% 8.0%
La ynx N 12 5 17
% 34.3% 33.3% 34.0%
Ha d pala e N 1 0 1
% 2.9% 0.0% 2.0%
Floo o he mou h N 3 2 5
% 8.6% 13.3% 10.0%
Floo o he mou h/buccal mucosa N 1 0 1
% 2.9% 0.0% 2.0%
Floo o he mou h/ ongue N 0 1 1
% 0.0% 6.7% 2.0%
Floo o he mou h/ ongue/pala e N 1 0 1
% 2.9% 0.0% 2.0%
Pha ynx N 2 3 5
% 5.7% 20.0% 10.0%
Pha ynx/ ongue N 0 1 1
% 0.0% 6.7% 2.0%
To al N 35 15 50
% 100.0% 100.0% 100.0%
Table 6. HPVs ains in di e en oncoana omical loca ions.
Oncoana omical
localiza ion
HPV inding To al
No inding 1 s ain Mo e han 1 s ain
Gingi a/buccal
mucosa
N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Gingi a/Pala e N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Gingi a/Floo o
mou h
N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Gingi a/Floo o
mou h/Tongue
N 0 0 1 1
% 0.0% 0.0% 16.7% 2.0%
Language N 8 2 0 10
% 22.9% 22.2% 0.0% 20.0%
Tongue/Floo o
mou h
N 4 0 0 4
% 11.4% 0.0% 0.0% 8.0%
La ynx N 12 4 1 17
% 34.3% 44.4% 16.7% 34.0%
Pala e N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Floo o he
mou h
N 3 1 1 5
% 8.6% 11.1% 16.7% 10.0%
Floo o mou h/
buccal mucosa
N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Floo o mou h/
Tongue
N 0 1 0 1
% 0.0% 11.1% 0.0% 2.0%
Floo o mou h/
Tongue/Pala e
N 1 0 0 1
% 2.9% 0.0% 0.0% 2.0%
Pha ynx N 2 1 2 5
% 5.7% 11.1% 33.3% 10.0%
Pha ynx/Tongue N 0 0 1 1
% 0.0% 0.0% 16.7% 2.0%
To al N 35 9 6 50
% 100.0% 100.0% 100.0% 100.0%
Figu e 2. Oncoana omical localiza ion o OSSC and HPV s a us.
0,0% 5,0% 10,0% 15,0% 20,0% 25,0% 30,0% 35,0% 40,0%
gingi a/buccal mucosa
gingi a/ha d pala e
gingi a/floo o he mou h
gingi a/floo o he mou h/ ongue
ongue
ongue/floo o he mou h
la ynx
ha d palа e
floo o he mou h
floo o he mou h/buccal mucosa
floo o he mou h/ ongue
floo o he mou h/ ongue/pala e
pha ynx
pha ynx/ ongue
Oncoana omical localiza ion
To al HPV finding - HPV finding -
no always associa ed wi h HPV (mos a e HPV nega i e).
In combined ana omical a eas (e.g. Tongue + Pha ynx)
he incidence o HPV is highe . Mo e han 1 HPV s ain
is less common (only 12%), bu is o en ound in a eas
wi h a mo e agg essi e umo p o ile (pha ynx, ongue)
(Table 6, Fig. 3).
Loca ions such as he gingi a and pala e a e less asso-
cia ed wi h HPV and o he ac o s p obably play a g ea e
ole he e (smoking, alcohol, e c.).
The oncoana omic loca ion as Gingi a/Floo o mou h/
Tongue, Floo o mou h/Tongue, and Pha ynx/Tongue all
ha e 100% HPV posi i i y, while se e al loca ions ha e 0%.
Pasho a-Tasse a Z e al.: A p ospec i e coho s udy o hp -media ed o a and o opha yngeal cance s6
In ou s udy, i was ound ha he p edominan local-
iza ion o he pha ynx and la ynx demons a ed a highe
equency o HPV (+) – in 11 pa ien s HPV (+) ca cinoma
was de ec ed , wi h 7 cases ha ing one s ain, and 4 cases
ha ing mo e han one s ain (Table 7).
Combina ion pa ien s o o al inse and b ush has he
highes de ec ion a e o HPV (77.8%). And wi h s a is ical
signi icance o he esul s (Table 8, Fig. 4). And o al inse
and b ush es sepa a ely de ec HPV in abou 61% o cas-
es, bu he combined me hod inc eases he likelihood o
de ec ing mo e han one s ain. In he case o a pap smea
in he o al ca i y, 2 cases (40%) we e diagnosed, and in
he uppe espi a o y ac 3 cases (60%). The same a io
was obse ed in a mou hwash sample. When using bo h
me hods oge he , cases we e iden i ied only in he uppe
Table 8. Compa ison o HPV de ec ion me hods in pa ien s wi h OSSC.
G oup Sample HPV s a us To al p
HPV(-) HPV(+) 1
s ain
HPV(+) Mo e
han 1 s ain
Tes g oup Sample No N 35 0 0 35 <0.001
% 100.0% 0.0% 0.0% 70.0%
N 0 4 1 5
% 0.0% 44.4% 16.7% 10.0%
N 0 4 1 5
% 0.0% 44.4% 16.7% 10.0%
Combined me hod (b ush es and o al inse) N 0 1 4 5
% 0.0% 11.1% 66.7% 10.0%
Con ol g oup Sample No N 37 0 37 0.001
% 100.0% 0.0% 94.9%
B ush es N 0 2 2
% 0.0% 100.0% 5.1%
Figu e 3. The pe cen age o HPV-posi i e cases o each umo loca ion.
Table 7. HPV s a us in o al ca i y ca cinoma and pha yngeal and la yngeal ca cinoma.
HPV s a us To al p
Nega i e 1 s ain Mo e han one s ain
Localiza ion O al mucosa N 21 2 2 25 0.104
% 60.0% 22.2% 33.3% 50.0%
Pha yngeal/la yngeal cance N 14 7 4 25
% 40.0% 77.8% 66.7% 50.0%
Figu e 4. Compa ison o HPV de ec ion me hods in pa ien s
wi h OSSC.
Pha macia 72: 1–10 7
espi a o y ac – 5 cases (100%). Fo g ea e accu acy,
we ecommend combining he wo HPV de ec ion es s.
In summa y, he esul s highligh he impo ance o
HPV as an e iological ac o in o opha yngeal and o al le-
sions, which may ha e se ious and impo an diagnos ic,
clinical and he apeu ic implica ions.
Discussion
Human papilloma i us (HPV)-posi i e o al and o o-
pha yngeal squamous cell ca cinomas (OSCCs) exhib-
i dis inc molecula cha ac e is ics compa ed o hei
HPV-nega i e coun e pa s. A ma ked geog aphic he e o-
genei y in HPV-a ibu able ac ions (HPV-AFs) ac oss
head and neck cance s (HNCs), pa icula ly in o opha-
yngeal ca cinoma (OPC), has been consis en ly epo ed
(Mehanna e al. 2013, 2016; Anan ha aman e al. 2017;
Algudaibi e al. 2021). Wi hin his con ex , ou s udy p o-
ides an upda ed assessmen o he epidemiological p o-
ile o HPV-associa ed OPC in he Bulga ian popula ion.
In ou coho , he mean age o HPV-posi i e pa ien s was
60 yea s, compa ed o 62 yea s o HPV-nega i e pa ien s.
This di e ence was no s a is ically signi ican , and ou
indings align wi h hose o o he in es iga o s who epo -
ed simila age dis ibu ions (Re ig e al. 2018a, b; Windon
e al. 2018). These obse a ions challenge he ea lie pe cep-
ion ha HPV-posi i e OPC p edominan ly a ec s younge
indi iduals and ins ead highligh an inc easing p e alence
among olde pa ien s, a end ha has also been linked o
less a o able su i al ou comes. Con e sely, se e al s udies
ha e documen ed a highe incidence o HPV-posi i e OPC
in younge popula ions (Gillison e al. 2008; Ang e al. 2010;
Mehanna e al. 2013; Mena e al. 2020), sugges ing po en ial
popula ion – and egion-speci ic di e ences.
Rega ding sex dis ibu ion, ou indings con i m he
p edominance o males, wi h 80% o cases occu ing in
men, i espec i e o HPV s a us. This male p edominance
has also been consis en ly documen ed in p e ious epide-
miological s udies (D‘Souza e al. 2007; Gillison e al. 2015).
Globally, HPV is es ima ed o accoun o app ox-
ima ely 25–30% o OPCs, compa ed wi h only 2–4% o
o al ca i y ca cinomas and 2–4% o la yngeal ca cinomas
(Alemany and Mena 2021; Algudaibi e al. 2021; Roman
and A agones 2021; Mena e al. 2022).
Analysis o he oncoana omical dis ibu ion o umo s in
he s udy g oup e ealed ha 20% o pa ien s p esen ed wi h
ca cinoma o he ongue, 34% wi h ca cinoma o he la -
ynx, 10% wi h ca cinoma o he loo o he mou h, 8% wi h
combined in ol emen o he ongue and loo o he mou h,
and 10% wi h pha yngeal ca cinoma. In 22% o cases, mo e
han one ana omical si e was a ec ed. These indings a e
consis en wi h p e ious epo s showing ha app oxi-
ma ely 30% o new cases in ol e o opha yngeal ca cinoma
(OPC), compa ed wi h 2.1% o o al ca i y ca cinoma (OC)
and 2.3% o la yngeal ca cinoma (LC) (Cha u edi e al.
2011; De Ma el e al. 2020; Mena e al. 2022). Mo eo e ,
HPV-posi i e OPC emains subs an ially mo e common in
males, a end epea edly con i med ac oss mul iple la ge-
scale s udies (Gillison e al. 2015; Mena e al. 2020; Lechne
e al. 2022; Fonsêca e al. 2023; Mehanna e al. 2023).
The p esen s udy u he highligh s he p e alence o
HPV in lesions localized o he o al ca i y, o opha ynx,
and la ynx. These indings a e consis en wi h in e na ion-
al e idence suppo ing HPV as a key isk ac o in he de-
elopmen o malignan and p emalignan lesions ac oss
hese ana omical egions. In ou coho , HPV posi i i y
was mos equen ly obse ed in o opha yngeal ca cino-
mas, in line wi h global ends. Wi hin he o al ca i y, he
loo o he mou h and ongue we e mo e commonly a -
ec ed; howe e , hese di e ences did no each s a is ical
signi icance, hough weakly exp essed ends could be ob-
se ed. In con as , mo e han hal o la yngeal ca cinomas
we e HPV nega i e. Speci ically, among 25 cases in ol ing
he loo o he mou h, ca cinoma – whe he isola ed o in
combina ion wi h o he localiza ions – was HPV nega i e
in he majo i y, wi h only 4 cases es ing HPV posi i e.
The highe equency o HPV in o opha yngeal lesions
likely e lec s a g ea e p opensi y o i al in eg a ion and
malignan ans o ma ion in his egion. This may be ex-
plained by dis inc ea u es o he immune mic oen i on-
men and si e-speci ic issue cha ac e is ics, which appea
o acili a e i al a achmen and pe sis ence o a g ea e
ex en han in he o al epi helium.
When compa ing he localiza ion o HPV-posi i e le-
sions be ween he o al ca i y and o opha ynx, ou da a
indica e ha , al hough HPV is less equen in he o al
ca i y, i s p esence is clinically ele an and should no be
o e looked. This unde sco es he impo ance o inco po-
a ing ou ine HPV es ing in o he diagnos ic algo i hm
o pa ien s wi h o al neoplasia, pa icula ly in ligh o he
ising incidence o HPV-associa ed OPC obse ed global-
ly o e he pas wo decades (Cha u edi e al. 2011; Pal e
e al. 2018; Mena e al. 2022; Fonsêca e al. 2023).
Ano he key conside a ion is he ole o di e en HPV
geno ypes, as high- isk ypes such as HPV16 a e s ongly
associa ed wi h mo e agg essi e disease and poo e p og-
nosis (Mehanna e al. 2019). In ou s udy, 70% o pa ien s
in he es g oup wi h OSCC we e HPV nega i e, com-
pa ed wi h 95% in he con ol g oup. In e es ingly, 12%
o es g oup pa ien s ha bo ed mul iple HPV s ains. Al-
hough ou sample size was limi ed, we iden i ied mul iple
s ains in cases o la yngeal ca cinoma, loo o he mou h
ca cinoma, and pha yngeal ca cinoma.
HPV16 was he mos commonly de ec ed geno ype,
ound bo h alone and in combina ion wi h o he s ains,
including HPV51, HPV52, HPV66, HPV6, and HPV31.
Addi ional s ains such as HPV6, HPV39, and HPV58 we e
also iden i ied, ei he indi idually o in combina ion. In
con as , only single s ains – HPV16 and HPV51 – we e
de ec ed in he con ol g oup. No ably, pha yngeal and la-
yngeal ca cinomas demons a ed he highes a es o HPV
posi i i y in ou se ies: HPV-posi i e umo s we e iden i ied
in 11 pa ien s, o whom se en ca ied a single s ain and ou
had mul iple s ains. Ne e heless, he la yngeal epi helium
is gene ally ega ded as less suscep ible o HPV in ec ion,
owing o ana omical and immunological ba ie s (D’Souza
e al. 2007; Roman and A agones 2021; Mena e al. 2022).
Pasho a-Tasse a Z e al.: A p ospec i e coho s udy o hp -media ed o a and o opha yngeal cance s8
The geno ypic p o ile obse ed in ou s udy la gely
pa allels pa e ns epo ed in e na ionally, whe e HPV31,
HPV33, and HPV52 equen ly eme ge as addi ional
oncogenic s ains ( an Hou en e al. 2001; Gillison e al.
2008; Mehanna e al. 2016; Schache e al. 2016). While
we did no de ec HPV18 o HPV33 in ou coho , we
iden i ied o he oncogenic ypes such as HPV51, HPV66,
HPV6, HPV39, and HPV58. This di e si y emphasizes he
ubiqui y o HPV and ein o ces he necessi y o e ec i e
p e en i e s a egies, including accina ion, o educe
he bu den o HPV-associa ed malignancies (Bha ia e al.
2015; Mehanna e al. 2019; Tsen emeidou e al. 2021).
F om a clinical s andpoin , he iden i ica ion o
HPV-posi i e lesions has a di ec impac on he apeu-
ic decision-making and pa ien ollow-up (Fakh y e
al. 2008). HPV-posi i e o opha yngeal ca cinomas, o
ins ance, a e cha ac e ized by an enhanced esponse o
adio he apy and chemo he apy, which ansla es in o im-
p o ed o e all su i al a es (B enne e al. 2020).
Accu a e de ec ion and geno yping o HPV s ains a e
he e o e c ucial o diagnosis, p ognosis, and long- e m
managemen o pa ien s wi h HPV- ela ed lesions. A a i-
e y o labo a o y me hods a e cu en ly employed in clini-
cal p ac ice, each wi h dis inc ad an ages and limi a ions
in e ms o sensi i i y, speci ici y, complexi y, and appli-
cabili y. Polyme ase chain eac ion (PCR) emains one o
he mos widely used app oaches, o e ing high sensi i i y
and he capaci y o de ec bo h gene al HPV p esence and
indi idual geno ypes. In ou s udy, we employed PCR, in-
cluding mul iplex PCR, which enables he simul aneous
de ec ion o mul iple HPV ypes – a aluable ad an age in
umo s wi h po en ial coin ec ion. Two ypes o samples
we e analyzed, umo issue ob ained by b ush sampling
and o al inse specimens. Ou indings demons a ed a
s a is ically signi ican ad an age o using bo h app oach-
es in combina ion, yielding he mos eliable and accu-
a e esul sл The choice o diagnos ic me hod con inues
o be deba ed in he li e a u e, wi h inc easing in e es in
non-in asi e, highly sensi i e app oaches. While PCR is
apid and sensi i e, i does no p o ide in o ma ion e-
ga ding i al in eg a ion. Immunohis ochemis y (IHC)
o p16 is cos -e ec i e and simple ye lacks speci ici y. In
si u hyb idiza ion (ISH) o e s localiza ion o i al DNA
bu is echnically mo e demanding and cos ly. Nex -gen-
e a ion sequencing (NGS) ep esen s he mos in o ma-
i e ool, capable o iden i ying in eg a ion e en s and i-
al a ian s, hough i equi es subs an ial esou ces and
echnical expe ise (Gillison e al. 2008; Tang e al. 2020;
Hillie e al. 2025). Based on ou esul s, we conclude ha
combined b ush and o al inse sampling wi h PCR p o-
ides a p ac ical, sensi i e, and easible s a egy ha could
be in eg a ed in o diagnos ic and sc eening p o ocols.
Despi e hese p omising indings, se e al limi a ions
o ou s udy mus be acknowledged. The ela i ely small
sample size and he diagnos ic me hods applied es ic
he gene alizabili y o ou conclusions. Fu u e esea ch
should ocus on la ge and mo e he e ogeneous popula-
ions, employing mo e ad anced molecula echniques o
imp o e HPV de ec ion and cha ac e iza ion.
Gi en he sensi i i y o HPV-d i en umo s o im-
mune-media ed mechanisms, no el he apeu ic s a egies
such as immuno he apy and molecula ly a ge ed he -
apies hold conside able p omises o imp o ing pa ien
p ognosis and su i al (Wakeham e al. 2019).
In conclusion, ou s udy ein o ces he pi o al ole o
HPV in he pa hogenesis o lesions o he o al ca i y and
o opha ynx and highligh s he necessi y o an in eg a -
ed diagnos ic and he apeu ic app oach ha inco po-
a es HPV s a us assessmen . Such s a egies will acili-
a e mo e e ec i e p e en ion, p o ide a mo e accu a e
p ognosis, and suppo he de elopmen o pe sonalized
ea men egimens.
Conclusion
HPV-media ed o al and o opha yngeal ca cinomas
ha e di e en pa hogenesis and a be e p ognosis han
HPV-nega i e o opha yngealca cinomas, which a ec
s aging andp ognosis. The e o e, accu a e de e mina ion
o he HPV s a us o he umo lesion is o impo an clin-
ical impo ance, especially in o opha yngeal ca cinomas.
Acknowledgemen
We hank he medical and den al p ac i ione s who pa -
icipa ed in he s udy. W i en consen o m was consid-
e ed as an ag eemen o pa icipa ion in he s udy.
Addi ional in o ma ion
Con lic o in e es
The au ho s ha e decla ed ha no compe ing in e es s exis .
E hical s a emen s
Clinical ials: An e hics s a emen was conduc ed in ull ac-
co dance wi h he Wo ld Medical Associa ion Decla a ion
o Helsinki. The s udy was app o ed by The Ins i u ional E h-
ic Commi ee o Medical Uni e si y - So ia, KENIMUS. No.
03/23.02.2024., e hic app o al da e- №985-29/02/2024.
The au ho s decla ed ha expe imen s on humans o human
issues we e pe o med o he p esen s udy.
In o med consen om he humans, dono s o dono s’ ep e-
sen a i es: Medical Uni e si y- So ia
The au ho s decla ed ha no expe imen s on animals we e
pe o med o he p esen s udy.
The au ho s decla ed ha no comme cially a ailable immo -
alised human and animal cell lines we e used in he p esen s udy.
Use o AI
No use o AI was epo ed.
Funding
This esea ch was unded by Scien i ic Resea ch Fund, Minis y
o Educa ion and Science, Bulga ia, con ac No. KP-06-N73/8
da ed 5.12.23.
Pha macia 72: 1–10 9
Au ho con ibu ions
All au ho s had ull access o he da a in he s udy and ook e-
sponsibili y o he in eg i y o he da a and he accu acy o he
da a analysis.
Au ho ORCIDs
Zd a ka Pasho a-Tasse a h ps://o cid.o g/0000-0002-2635-
083X
Vessela Rayko a h ps://o cid.o g/0000-0002-9199-9600
Milena I ano a-Shi a o a h ps://o cid.o g/0000-0003-4128-
9915
S anisla Yo dano h ps://o cid.o g/0009-0002-9145-7235
S e osla Sla ko h ps://o cid.o g/0000-0001-9088-5532
Daniel Ma ko h ps://o cid.o g/0000-0001-9169-6987
Vik o Lenko h ps://o cid.o g/0009-0000-8164-7559
Eli sa Deli e ska h ps://o cid.o g/0000-0002-2356-9932
Da a a ailabili y
All o he da a ha suppo he indings o his s udy a e a ailable
in he main ex .
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