Jou nal o
Clinical Medicine
A icle
In luence o Se um Vi amin D Le el in he Response
o Ac inic Ke a osis o Pho odynamic The apy
wi h Me hylaminole ulina e
Rica do Mo eno 1,* , Lau a Náje a 2, Ma a Masca aque 3,Ángeles Jua anz 3,
Sal ado González 4and Yolanda Gilabe e 5
1De ma ology Se ice, Hospi al Uni . del Hena es, Coslada, 28822 Mad id, Spain
2Pa hology Se ice, Hospi al Pue a de Hie o, Majadahonda, 28222 Mad id, Spain;
[email p o ec ed]
3Depa men o Cellula Biology, Uni e sidad Au ónoma de Mad id, 28049 Mad id, Spain;
[email p o ec ed] (M.M.); [email p o ec ed] (A.J.)
4
Medicine and Medical Special ies Depa men , Ins i u o Ram
ó
n y Cajal de In es igaci
ó
n Sani a ia (IRYCIS),
Uni e sidad de Alcalá, 28034 Mad id, Spain; [email p o ec ed]
5De ma ology Se ice, Hospi al Uni . Miguel Se e , 50009 Za agoza, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +34-911-912-000
Recei ed: 16 Decembe 2019; Accep ed: 25 Janua y 2020; Published: 1 Feb ua y 2020
Abs ac :
In mouse models o squamous cell ca cinoma, p e- ea men wi h calci iol p io o
pho odynamic he apy wi h aminole ulinic acid (ALA) enhances umo cell dea h. We ha e e alua ed
he associa ion be ween i amin D s a us and he esponse o ac inic ke a oses o pho odynamic
he apy wi h me hylaminole ulina e. Twen y- i e pa ien s wi h ac inic ke a oses on he head ecei ed
one session o pho odynamic he apy wi h me hylaminole ulina e. Biopsies we e aken a baseline
and six weeks a e ea men . Immuno-his ochemical s aining was pe o med o VDR, P53, Ki67
and
β
-ca enin. Basal se um 25(OH)D le els we e de e mined. Cases wi h a posi i e his ological
esponse o ea men had signi ican ly highe se um 25(OH)D le els (26.96 (SD 7.49) ng /mL) han
hose wi hou esponse (18.60 (SE 7.49) ng /mL) (p=0.05). Pa ien s wi h a comple e clinical esponse
displayed lowe basal VDR exp ession (35.71% (SD 19.88)) han pa ial esponde s (62.78% (SD
16.735)), (p=0.002). Ou esul s suppo a ela ionship be ween i amin D s a us and he esponse o
ac inic ke a oses o pho odynamic he apy wi h me hylaminole ulina e.
Keywo ds:
pho ochemo he apy; me hylaminole ulina e; ac inic ke a osis; i amin d; calci iol;
i amin d ecep o
1. In oduc ion
Vi amin D (VD) is a p oho mone in ol ed in a wide a ie y o unc ions in he o ganism, and has
been ela ed wi h se e al ypes o cance [
1
]. I has se e al known e ec s on epide mal ca cinogenesis [
2
]:
i egula es ke a inocy e p oli e a ion p omo ing i s di e en ia ion [
3
], and i p e en s UV-induced
mu a ions [4], enhancing mu a ion epai .
In humans, i amin D is ob ained mainly h ough exposu e o sunligh which, in he epide mis,
p omo es ans o ma ion o 7-dehyd ocholes e ol in o cholecalci e ol o p e i amin D3. Seconda ily,
cholecalci e ol is hyd oxyla ed in he li e o become 25(OH)D o calcidiol, hen u he hyd oxyla ed
in he kidney in o 1,25(OH)D o calci iol, he biologically ac i e o m o i amin D [
1
]. Calci iol ac s
on i s in acellula ecep o (VDR), which is p esen in almos all cell ypes in humans, and i s signaling
exe s an ip oli e a i e, an iangiogenic, p o-di e en ia ing and an iapop o ic e ec s [5].
J. Clin. Med. 2020,9, 398; doi:10.3390/jcm9020398 www.mdpi.com/jou nal/jcm
J. Clin. Med. 2020,9, 398 2 o 9
Ac inic ke a oses (AKs) a e skin a eas o ke a inocy ic dysplasia ep esen ing a p eneoplas ic
s a e—o acco ding o some au ho s, an in si u o m—o cu aneous squamous cell ca cinoma (SCC).
In AK, he se e i y o ke a inocy ic dysplasia is classi ied, as in o he in aepide mal ca cinomas
(CIN o ce ical, VIN o ul a , AIN o anal in aepi helial neoplasia) in o KIN (ke a inocy ic
in aepide mal neoplasia) g ade 1, 2 o 3 acco ding o he p esence o dysplas ic ke a inocy es in one
hi d, wo hi ds o he comple e hickness o he epide mis [6].
Pho odynamic he apy (PDT) wi h aminole ulinic acid (ALA) o me hyl-aminole ulina e (MAL) is
e ec i e in clea ing ke a inocy ic dysplasia and e e sing some o he molecula ea u es o AK, such as
he exp ession o mu an P53 [
7
]. In his he apy, AKs a e ea ed wi h men ioned pho osensi ize s
and exposed o speci ic wa eleng h ligh sou ces. ALA and MAL a e p ecu so s o p o opo phy in
IX (PpIX), a molecule ha selec i ely accumula es in dysplas ic ke a inocy es. I adia ion induces
pho obleaching o PpIX which is esponsible o umo cell dea h.
In SCC mu ine models, p e- ea men wi h opical i amin D p io o ALA-PDT has been shown
o enhance PpIX accumula ion and umo cell dea h [
8
]. This has also been obse ed in o he oden
models wi h o al [
9
] o in ape i oneal [
10
] adminis a ion o calci iol. In humans, he clinical esponse
o AK o PDT, in a spli -scalp ial compa ing MAL-PDT alone s. MAL-PDT wi h a p e- ea men
o 15 days wi h opical calcipo iol (a syn he ic de i a i e o calci iol ma ke ed o ea pso iasis),
imp o ed in he p e ea ed g oup [
11
]. Galimbe i also demons a ed supe io e icacy o dayligh
media ed MAL-PDT a e p e- ea men wi h calcipo iol oin men [12].
We in ended o explo e i VD o i s ecep o play a ole in he esponse o AK o PDT. The e o e,
we designed a s udy o e alua e he associa ion be ween he se um 25(OH)D le el and he skin
exp ession o VD ecep o (VDR) in AK wi h he esponse o MAL-PDT a clinical, his ological and
immuno-his ochemical le els.
2. Pa ien s and Me hods
2.1. Design
A p ospec i e obse a ional pilo s udy was designed o es ablish whe he se um 25(OH)D le el
in luences he esponse o AK o MAL-PDT in pa ien s.
2.2. E hics
The s udy was app o ed by he local E hical Commi ee a Hospi al Uni e si a io Pue a de
Hie o in Mad id (Spain). The w i en in o med consen was ob ained om all he subjec s be o e
being ec ui ed o he s udy.
2.3. Subjec s
Twen y- i e pa ien s we e en olled in he s udy. Inclusion c i e ia we e as ollows: ha ing i e
o mo e neighbo ing AKs suscep ible o be ea ed wi h MAL-PDT, loca ed on he ace o he scalp.
Exclusion c i e ia we e: uns able heal h condi ions, such as cance o immunosupp ession; medical
con aindica ions o he ea men , such as p egnancy o pho osensi i i y; alle gy o he MAL o any
o he excipien s o he c eam; and ha ing ecei ed any ea men o ace o scalp AKs wi hin he las
six mon hs. Pa ien s we e ec ui ed om Ma ch 2014 o Sep embe 2016. Va iables such as age, gende ,
body mass index (BMI) and Fi zpa ick pho o ype we e collec ed o each pa ien .
2.4. T ea men P o ocol
Pa ien s we e ea ed wi h con en ional MAL-PDT as ollows. AKs we e p epa ed o he
ea men by emo al o hype ke osis h ough gen le use o sandpape o cu e age. Then, a 1 mm laye
o MAL 160 mg/g c eam (Me ix
®
, Galde ma, Pa is, F ance) was applied o e each AK, sp eading he
emaining c eam on he su ounding cance iza ion a ea. A e incuba ion unde occlusion o h ee
hou s, he whole a ea was exposed o a ed LED de ice emi ing a 630 nm (Ak ili e
®
; Pho oCu e,
J. Clin. Med. 2020,9, 398 3 o 9
Oslo, No way) wi h a luence o 37 J/cm
2
. A e he ea men , pa ien s we e ins uc ed o a oid sun
exposu e using he same SPF 50 sunsc een c eam (Helioca e Ai gel, IFC, Spain) when ou doo s un il
he end o he s udy.
2.5. Clinical E alua ion
Clinical e alua ion was assessed using digi al pho og aphs be o e and six weeks a e he ea men .
Clinical lesion esponse was e alua ed by wo de ma ologis s who measu ed he educ ion in he AK
numbe and he Olsen g ade in he ea ed a ea. Pa ien esponse was classi ied in h ee ca ego ies:
comple e esponse, de ined by a 75% o 100% educ ion in numbe and imp o emen in he g ade o
Olsen o he AKs; pa ial esponse when he o e all imp o emen in numbe and g ade was lowe
han 75% and highe han 25%; and imp o emen was lowe han 25% pe cen .
2.6. Biochemical Va iables
Two blood es s we e pe o med on each pa ien , he i s one on he day o he PDT ea men
and he second one 6 weeks la e , a he end o he ollow-up. Se um le els o 25(OH)D (ng/mL)
(elec ochemiluminescence, Roche Diagnos ics, Mad id, Spain) we e de e mined in he Cen al Labo a o y
o Mad id. VD de iciency was de ined as se um 25(OH)D o 20 ng/mL o less, VD insu iciency as alues
o 20–30 ng/mL, and su iciency o e i [13].
2.7. His ological and Immune-His ochemical Va iables
A 3 mm punch biopsy o he index lesion ( he mos se e e AK in he a ea) was pe o med 1 week
be o e he ea men and 6 weeks a e i . The second biopsy was aken a a minimal dis ance om he
i s biopsy sca .
The skin samples we e ixed (10% o malin), embedded in pa a in, sec ioned (3
µ
m hickness)
and s ained by haema oxylin and eosin, and hen simul aneously subjec ed o immunohis ochemis y
using he co esponding an ibodies o de ec ion o Ki67 (p edilu ed; Ven ana Medical Sys ems Tucson,
AZ, USA), i amin D ecep o (VDR), P53, and
β
-ca enin an ibodies (Cell Signaling Technologies,
Leiden, The Nede lands). Rep esen a i e sec ions we e examined using posi i e and nega i e con ols.
Immuno-his ochemical e alua ion o P53, Ki-67,
β
-ca enin and VDR was pe o med by iden i ying,
in each sec ion, he a ea wi h he highes le els o immunoexp ession (“ho spo s”) and es ima ing
he pe cen age o cells wi h nuclea posi i i y in a high-powe ield (
×
400). In ensi y o VDR s aining
was semi-quan i a i ely assessed by classi ying exp ession in ensi y in o 4 ca ego ies: 0, absence o
s aining; 1, mild s aining (0%–33% umo al cell s aining); 2, mode a e s aining (>33%–66%) and 3,
in ense s aining (>66%–100% umo al cell s aining).
His ological and immuno-his ochemical a iables (his ological diagnosis, his ological sub ype
o AK, KIN g ade as de ined by Röwe -Hube e al. [
6
],
β
-ca enin, P53, Ki67, VDR exp essions and
VDR in ensi y we e e alua ed by a pa hologis , blind o he iden i y o he samples. His ological
esponse o he AK index, assessed on hema oxylin-eosin s ained sec ions, was de ined as posi i e
when comple e clea ance o a leas a dec ease in wo KIN g ades was achie ed, and nega i e when
absence o his ological esponse o dec ease o only one KIN g ade was shown.
2.8. S a is ical Analysis
Quan i a i e a iables a e exp essed as mean and s anda d de ia ion (SD) and dicho omous
a iables as p opo ions. Associa ions be ween quali a i e a iables we e assessed using Pea son’s
Chi-squa ed es o Fishe ’s exac es . Mann–Whi ney U- es o S uden ’s - es o pai ed da a was
used o e alua e associa ions be ween quan i a i e a iables. Pea son co ela ion coe icien was
calcula ed o e alua e he linea co ela ion be ween wo a iables. S a is ical signi icance was se a
p≤0.05. Analyses we e pe o med using SPSS S a is ics ( e sion 19.0: IBM, A monk, NY, USA).
J. Clin. Med. 2020,9, 398 4 o 9
3. Resul s
3.1. Demog aphic Cha ac e is ics o he Sample
All wen y- i e pa ien s comple ed he s udy. Howe e , one case was excluded om he his ological
analysis since he pos - ea men biopsy e ealed a collision o an ac inic and a sebo heic ke a osis.
The mean age was 70.1 yea s ( ange 61–81) and 76% we e males wi h Fi zpa ick pho o ype 3 (60%) o
pho o ype 2 (40%). Mos o he ea ed AKs we e loca ed on he scalp (64%) and 36% on he acial a ea
(Table 1). The mean basal 25(OH)D se um le els we e 25.37 (SD 9.86) ng/mL.
The se e i y o ke a inocy ic dysplasia was conside ed KIN3 in 7 lesions (29.17%), KIN2 in 10
(41.66%) and KIN1 in 7 (29.17%) AK.
Table 1.
Sociodemog aphic and biochemical a iables o he sample. (SD: s anda d de ia ion; BMI:
body mass index.).
Va iables (N=25) F ecuency Mean (Range o SD)
Age (yea s) 70.1 (61–81)
Gende Male
Female
19/25 (76%)
6/25 (24%)
Pho o ype II
III
10/25 (40%)
15/25 (60%)
B.M.I. (kg/m2)30.1 (23.30–42.40)
Loca ion o ea ed AK Face
Scalp
9/25 (36%)
16/25 (64%)
Se um 25(OH)D3(ng/mL) 25.37 (SD 9.86)
3.2. Clinical and His ological Response o PDT Pe Lesion
As expec ed, PDT induced a signi ican educ ion in he mean numbe o AKs pe pa ien ,
om 7.80 (SD 2.79) o 2.8 (SD 1.61) (p=0.005) (Figu e 1). O e all clinical esponse was comple e in
16 pa ien s (64%) and pa ial in 9 (36%); he e we e no cases wi hou esponse.
J. Clin. Med. 2019, 8, x FOR PEER REVIEW 4 o 10
3.1. Demog aphic Cha ac e is ics o he Sample
All wen y- i e pa ien s comple ed he s udy. Howe e , one case was excluded om he
his ological analysis since he pos - ea men biopsy e ealed a collision o an ac inic and a sebo heic
ke a osis. The mean age was 70.1 yea s ( ange 61–81) and 76% we e males wi h Fi zpa ick pho o ype
3 (60%) o pho o ype 2 (40%). Mos o he ea ed AKs we e loca ed on he scalp (64%) and 36% on
he acial a ea (Table 1). The mean basal 25(OH)D se um le els we e 25.37 (SD 9.86) ng/mL.
The se e i y o ke a inocy ic dysplasia was conside ed KIN3 in 7 lesions (29.17%), KIN2 in 10
(41.66%) and KIN1 in 7 (29.17%) AK.
Table 1. Sociodemog aphic and biochemical a iables o he sample. (SD: s anda d de ia ion; BMI:
body mass index.).
Va iables (N = 25) F ecuency Mean ( ange o SD)
Age (yea s) 70.1 (61–81)
Gende Male
Female
19/25 (76%)
6/25 (24%)
Pho o ype II
III
10/25 (40%)
15/25 (60%)
B.M.I. (kg/m2) 30.1 (23.30–42.40)
Loca ion o ea ed AK Face
Scalp
9/25 (36%)
16/25 (64%)
Se um 25(OH)D3 (ng/mL) 25.37 (SD 9.86)
3.2. Clinical and His ological Response o PDT Pe Lesion
As expec ed, PDT induced a signi ican educ ion in he mean numbe o AKs pe pa ien , om
7.80 (SD 2.79) o 2.8 (SD 1.61) (p = 0.005) (Figu e 1). O e all clinical esponse was comple e in 16
pa ien s (64%) and pa ial in 9 (36%); he e we e no cases wi hou esponse.
Figu e 1. Comple e clinical esponse o pho odynamic he apy (PDT), as clea ance o ac inic ke a oses
in he nasal a ea o a pa ien six weeks a e ea men .
His ological esponse was posi i e in 17 AK (70.8%) and nega i e in 7 AK (29.2%). Index AK
exhibi ed basal KIN g ade 3 in 29.17%, KIN 2 in 41.66%, and KIN 1 in 29.17% o he samples, and
a e ea men KIN g ade was 3 in 8.33%, KIN 2 in 12.50%, KIN 1 in 16.67% and KIN 0 in 62.50% o
he lesions, showing a signi ican imp o emen o he KIN g ade (p = 0.004) Conside ing he KIN
g ade as a quan i a i e a iable, PDT induced a signi ican dec ease in he mean KIN g ade, om
1.88 (SD 0.85) o 0.67 (SD 1.01) (p = 0.000).
PDT also induced a signi ican dec ease in he mean o he immunos aining o Ki67 (57.08 (SD
27.10) o 26.88 (SD 19.27), p = 0.001) and P53 exp ession (59.17 (SD 27.72) o 26.39 (SD 24.54), p = 0.001).
VDR exp ession inc eased a e PDT bu he di e ences we e no s a is ically signi ican (56.67 (SD
20.36) o 66.67 (SD 22.00), p = 0.062) (Figu e 2). No ele an di e ences we e ound in he es o he
immunological ma ke s a e PDT (Table 2).
Figu e 1.
Comple e clinical esponse o pho odynamic he apy (PDT), as clea ance o ac inic ke a oses
in he nasal a ea o a pa ien six weeks a e ea men .
His ological esponse was posi i e in 17 AK (70.8%) and nega i e in 7 AK (29.2%). Index AK
exhibi ed basal KIN g ade 3 in 29.17%, KIN 2 in 41.66%, and KIN 1 in 29.17% o he samples, and a e
ea men KIN g ade was 3 in 8.33%, KIN 2 in 12.50%, KIN 1 in 16.67% and KIN 0 in 62.50% o he
lesions, showing a signi ican imp o emen o he KIN g ade (p=0.004) Conside ing he KIN g ade as
a quan i a i e a iable, PDT induced a signi ican dec ease in he mean KIN g ade, om 1.88 (SD 0.85)
o 0.67 (SD 1.01) (p=0.000).
J. Clin. Med. 2020,9, 398 5 o 9
PDT also induced a signi ican dec ease in he mean o he immunos aining o Ki67 (57.08
(SD 27.10) o 26.88 (SD 19.27), p=0.001) and P53 exp ession (59.17 (SD 27.72) o 26.39 (SD 24.54),
p=0.001
). VDR exp ession inc eased a e PDT bu he di e ences we e no s a is ically signi ican
(56.67 (SD 20.36) o 66.67 (SD 22.00), p=0.062) (Figu e 2). No ele an di e ences we e ound in he
es o he immunological ma ke s a e PDT (Table 2).
J. Clin. Med. 2019, 8, x FOR PEER REVIEW 5 o 10
Figu e 2. Ac inic ke a oses: immuno-his ochemical esponse o MAL-PDT (me hyl-aminole ulina e
pho odynamic he apy). Baseline i amin D ecep o (VDR) exp ession (A) did no signi ican ly
change a e ea men (B). Baseline P53 (C) and Ki67 (E) exp ession signi ican ly dec eased (D and F,
espec i ely) a e PDT.
Table 2. Clinical, his ological and immuno-his ochemical a iables o he sample, be o e and a e
MAL-PDT (me hyl-aminole ulina e pho odynamic he apy).
N = 24 Basal (mean, SD) A e PDT (mean, SD) p
Clinical and his ological a iables
AK numbe pe pa ien 7.84 (SD 2.79) 2.80 (SD 1.61) 0.005
KIN g ade (quan i a i e) 1.88 (0.85) 0.67 (1.01) <0.001
KIN g ade (quali a i e)
0.004
KIN 3 7 (29.17 %) 2 (8.33%)
KIN 2 10 (41.66%) 3 (12.50%)
KIN 1 7 (29.17%) 4 ( 16.67%)
KIN 0 0 15 (62.50%)
Immunoma ke s
VDR exp ession (%) 56.67 (20.36) 66.67 (22.00) 0.062
VDR in ensi y (0–3) 1.96 (0.81) 2.08 (0.93) 0.479
β-ca enin exp ession (%) 4.17 (5.69) 2.61 (4.59) 0.191
Figu e 2.
Ac inic ke a oses: immuno-his ochemical esponse o MAL-PDT (me hyl-aminole ulina e
pho odynamic he apy). Baseline i amin D ecep o (VDR) exp ession (
A
) did no signi ican ly
change a e ea men (
B
). Baseline P53 (
C
) and Ki67 (
E
) exp ession signi ican ly dec eased (
D
and
F
,
espec i ely) a e PDT.
J. Clin. Med. 2020,9, 398 6 o 9
Table 2.
Clinical, his ological and immuno-his ochemical a iables o he sample, be o e and a e
MAL-PDT (me hyl-aminole ulina e pho odynamic he apy).
N=24 Basal (mean, SD) A e PDT (mean, SD) p
Clinical and his ological a iables
AK numbe pe pa ien 7.84 (SD 2.79) 2.80 (SD 1.61) 0.005
KIN g ade (quan i a i e) 1.88 (0.85) 0.67 (1.01) <0.001
KIN g ade (quali a i e)
0.004
KIN 3 7 (29.17 %) 2 (8.33%)
KIN 2 10 (41.66%) 3 (12.50%)
KIN 1 7 (29.17%) 4 ( 16.67%)
KIN 0 0 15 (62.50%)
Immunoma ke s
VDR exp ession (%) 56.67 (20.36) 66.67 (22.00) 0.062
VDR in ensi y (0–3) 1.96 (0.81) 2.08 (0.93) 0.479
β-ca enin exp ession (%) 4.17 (5.69) 2.61 (4.59) 0.191
Ki67 exp ession (%) 57.08 (27.10) 26.88 (19.27) 0.000
P53 exp ession (%) 59.17 (27.72) 26.39 (24.54) 0.000
SD: S anda d de ia ion; VDR: i amin D ecep o ; KIN: ke a inocy ic in aepi helial neoplasia; AK: ac inic ke a osis.
3.3. Associa ion o Va iables wi h O e all Clinical Response
No s a is ically signi ican ela ionship was ound be ween pa ien clinical esponse and age,
gende , pho o ype, AK loca ion and se um 25(OH)D. Howe e , hose pa ien s wi h comple e clinical
esponse showed lowe VDR exp ession (35.71, SD 19.88) han hose wi h pa ial esponse (62.78,
SD 16.74) (p=0.002). Basal
β
-ca enin, Ki67 and P53 exp essions we e no associa ed wi h he o e all
clinical ou come (Table 3).
3.4. Associa ion o he Va iables wi h he His ological Response
No s a is ically signi ican ela ionship was ound be ween age, gende , pho o ype and loca ion o he
AK and he his ological esponse. Pa ien s who esponded o PDT had signi ican ly highe se um 25(OH)D
le els (26.96 ng/mL, SD 7.49) han hose wi hou esponse (18.60, SD 7.49) (p=0.05). Baseline exp ession o
he explo ed immunoma ke s was no associa ed wi h he his ological esponse o PDT (Table 3).
J. Clin. Med. 2020,9, 398 7 o 9
Table 3.
In luence o clinical and his ological a iables on o e all clinical esponse o pa ien s and
his ological esponse o AK o MAL-PDT.
Pa ien Clinical Response
p
His ological Response
p
Pa ial Response
(mean, SD) n=6
Comple e esponse
(mean, SD) n=19
Posi i e (mean,
SD) n=17
Nega i e (mean,
SD) n=7
Age (mean, SD) 71.47 (6.66) 69.67 (3.20) 0.53 69.88 (6.19) 73.86 (5.37) 0.153
Gende
10.608
Male 14 (73.37%) 5 (26.30%) 14 (73.70%) 5 (26.30%)
Female 5 (83.30%) 1 (16.70%) 3 (60.00%) 2 (40.00%)
Pho o ype
0.175 0.356
II 6 (60.00%) 4 (40.00%) 5 (55.60%) 4 (44.4%)
III 13 (86.70%) 2 (13.30%) 12 (80.00%) 3 (20.00%)
Loca ion
10.352
-Face 7 (77.80%) 2 (22.20%) 7 (87.50%) 1 (12.50%)
-Scalp 12 (75.00%) 4 (25.00%) 10 (62.50%) 6 (37.50%)
Vi amin D (ng/ml) 24.42 (9.67) 27.67 (9.86) 0.483 26.96 (9.49) 18.60 (7.49) 0.05
VDR exp ession (%) 62.78 (16.74) 35.71 (19.88) 0.002 59.41 (18.53) 53.33 (25.03) 0.535
VDR in ensi y (0–3) 2.00 (0.77) 1.71 (0.95) 0.442 2.00 (0.79) 1.83 (0.98) 0.68
β-ca . exp ession (%) 4.39 (5.62) 3.86 (5.40) 0.832 5.53 (6.06) 17 (2.04) 0.103
Ki67 exp ession(%) 56.39 (28.12) 51.00 (32.20) 0.683 55.00 (29.42) 59.17 (22.00) 0.756
P53 exp ession (%) 64.41 (23.51) 59.00 (38.79) 0.701 64.69 (27.35) 52.00 (22.80) 0.361
4. Discussion
This s udy suppo s he ela ionship be ween 25(OH)D se um le els and he esponse o AK
o MAL-PDT: VD de icien le els we e ound o be signi ican ly associa ed o a lack o esponse in
he educ ion o he KIN g ade o ac inic ke a oses, and pa ien s whose AK exhibi ed a signi ican ly
lowe VDR basal exp ession showed a comple e clinical esponse o he ea men . Compa ing he
his ological samples o AK in e e y pa ien be o e and a e MAL-PDT, we obse ed a ma ginally
signi ican inc ease in VDR exp ession a e he ea men in addi ion o he al eady know educ ion in
P53 and Ki67 exp ession [7].
Thus, ou indings sugges ha a poo e esponse o AK o MAL-PDT is likely o be expec ed
unde a de icien VD s a us. The mechanisms by which VD may exe an e ec on he esponse o
AK o MAL-PDT a e unknown. I has been demons a ed ha VD p omo es UV-induced mu a ion
epai in ke a inocy es h ough an up- egula ion o unc ional P53 [
14
] and has se e al an i umo al
e ec s on epide mal neoplasms h ough he immune sys em [
15
,
16
]. The ansc ip ional p o ile o
heal hy ke a inocy es ea ed wi h 1,25(OH)D has been s udied, showing he up- egula ion o some
82 genes and down- egula ion o 16 o he genes; among hose up- egula ed we e pep idila ginine
deaminases, calic eins, se in-p o ease inhibi o s, c- os o K uppel-like ac o 4, all o which a e in ol ed
in ke a inocy e di e en ia ion [
17
]. These indings illus a e a poo ly unde s ood p o-di e en ia ion
ne wo k o e ke a inocy es sus ained by VD.
The inc ease on PpIX accumula ion and consequen enhanced umo al cell dea h induced by
exposu e o calci iol o calcipo iol p io o ALA-PDT has been p oposed as a possible mechanism
in se e al p e iously published s udies on mu ine models [
8
–
10
]. Howe e , Bay e al. [
18
] exposed
hai less mice o ca cinogenic doses o UV- adia ion, and ound ha p e- ea men wi h calcipo iol
p io o MAL-PDT nei he inc eased PpIX accumula ion, as measu ed by luo escence, no delayed he
onse o SCC compa ed o MAL-PDT wi hou p e- ea men . Acco dingly, VD abili y o enhance AK
esponse o PDT may no be ela ed o an inc eased PpIX accumula ion in ke a inocy es, bu o o he
mechanisms, pe haps in ol ing i s complex e ec s on ke a inocy e p oli e a ion and di e en ia ion.
The ela ionship be ween 25(OH)D se um le els and VDR exp ession in ke a inocy es has no been
s udied in humans. In swine models ed wi h a VD-de icien die , VD insu iciency s a us esul ed in a
di use p esence o VDR in he ke a inocy ic cy oplasm, whils supplemen a ion wi h VD up o se um
le els o 25(OH)D su iciency induced a p e e en ially nuclea loca ion o he VDR [
19
]. Mo eo e ,
unpublished da a o ou g oup in se e al
in i o
expe iences wi h skin and ul a squamous cell
ca cinoma cell lines (SCC-13 and A-431) e eal ha hese umo cells exhibi a di use exp ession o
J. Clin. Med. 2020,9, 398 8 o 9
VDR in he cy oplasm ha changes d ama ically o a p edominan ly nuclea exp ession upon he
addi ion o calci iol o he cell cul u es.
As o he disc epancy be ween he in luence o a iables on clinical and on his ological esponses
o AK o PDT, i has al eady been p o en ha clinical and his ological classi ica ions o AK do no
accu a ely ma ch and ha conclusions mus no be d awn abou he his ology o AK lesions om hei
clinical appea ance [
20
]. Hence, we can in e ha pa allel indings should no necessa ily be expec ed
be ween clinical and his ological app oaches.
Acco ding o ou esul s compa ing immunohys ochemical ea u es o ou samples be o e and a e
he ea men , MAL-PDT migh be able o inc ease VDR exp ession in he nucleus o he ke a inocy es
in AK. This may imp o e ke a inocy e sensi i i y o se um VD, hus p o iding an addi ional plausible
way h ough which PDT and VD in e ac o gene a e a syne gic an i umo al e ec on ke a inocy ic
neoplasms such as AK.
An impo an limi a ion o his is s udy is he small sample. A la ge sample is wa an ed o
s eng hen he e idence p o ided by ou indings.
In summa y, ou indings imply ha se um VD could be conside ed a modula o o he esponse
o AK o MAL-PDT. De e mina ion o se um 25(OH)D migh be app op ia e in pa ien s wi h AK
eligible o ea men wi h MAL-PDT, in o de o p edic hei in insic abili y o espond o i , and o
selec hose pa ien s who could bene i om VD supplemen a ion p io o ea men . Mo e esea ch
is needed, i s ly o con i m ou esul s and secondly o es ablish i VD supplemen a ion in de icien
pa ien s p e ious o PDT migh imp o e i s e icacy.
Au ho Con ibu ions:
Concep ualiza ion, S.G., A.J. and Y.G.; Me hodology, Y.G.; Valida ion, Y.G.; Fo mal
Analysis, R.M. and Y.G.; In es iga ion, R.M., M.M., A.J. and L.N.; Resou ces, M.M., L.N.,
Á
.J. and R.M.; Da a
Cu a ion, R.M.; W i ing—O iginal D a P epa a ion, R.M; W i ing—Re iew & Edi ing, Y.G. and S.G.; P ojec
Adminis a ion,
Á
.J. and Y.G.; Funding Acquisi ion,
Á
.J., S.G. and Y.G. All au ho s ha e ead and ag eed o he
published e sion o he manusc ip .
Funding:
This esea ch has been pa ially suppo ed by a g an om he Ca los III Heal h Ins i u e, Minis y o
Science, Inno a ion and Uni e si y, Spain (PI18/00858), and by Galde ma.
Acknowledgmen s:
The au ho s wan o acknowledge o Galde ma o hei economic unding and scien i ic
suppo in he de elopmen o his esea ch line, o hei help wi h English edi ing o his a icle and o he
Me ix c eam employed in he s udy. Ou wo k was also suppo ed by Can ab ia Labs who p o ided he Helioca e
Ai gel sunsc een o he subjec s o he s udy.
Con lic s o In e es : The sponso s had no ole in he design, execu ion, in e p e a ion, o w i ing o he s udy.
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