Balkan Med J 2019;36:283-6 B ie Repo 283
O6-me hylguanine-DNA Me hyl ans e ase P omo e Me hyla ion
in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy
T ea men : Clinical Implica ions
1Cen e o Cance Resea ch and Cell Biology, Queen’s Uni e si y Bel as , Bel as , UK
2Ins i u e o Biopa hology and Regene a i e Medicine, Cen e o Biomedical Resea ch, Uni e si y o G anada, G anada, Spain
3Depa men o Radia ion Oncology, Uni e si a y Hospi al Vi gen de la Vic o ia, Málaga, Spain
4Depa men o Ana omy and Emb yology, Uni e si y o G anada, G anada, Spain
5Biosani a y Ins i u e o G anada (ibs. GRANADA), SAS-Uni e sidad de G anada, G anada, Spain
6Medical Oncology Se ice, Uni e si a y Hospi al Vi gen de las Nie es, G anada, Spain
Jaime A. Oli e 1,2*, Jaime Gómez-Millán3*, Jose A. Medina3, Lau a Cabeza2,4,5, Glo ia Pe azzoli2,5,
C is ina Jimenez-Luna2, Ke in Doello6, Raúl O iz2,4,5
Aims: To analyze he clinical ele ance o O6-me hylguanine-
DNA me hyl ans e ase in ec al adenoca cinoma ea ed wi h
chemo adio he apy ollowed by su ge y.
Me hods: Tissue samples om 29 ec al adenoca cinoma pa ien s
we e ob ained a e chemo adio he apy. O6-me hylguanine-DNA
me hyl ans e ase p omo e me hyla ion s a us was es ablished by
me hyla ion-speci ic polyme ase chain eac ion. O6-me hylguanine-
DNA me hyl ans e ase p o ein le els we e de e mined by
immunohis ochemis y. Clinicopa hologic a iables, including
ea men eg ession g ade, ecu ence, lymph node in asion, and
s age and di e en ia ion g ade o he umo , we e de e mined.
Resul s: The O6-me hylguanine-DNA me hyl ans e ase gene
p omo e was me hyla ed in 81.5% o samples. Mos pa ien s (88.9%)
showed low O6-me hylguanine-DNA me hyl ans e ase p o ein
exp ession. O6-me hylguanine-DNA me hyl ans e ase me hyla ion
s a us was no co ela ed wi h any o he clinicopa hological a iables
de e mined in ec al adenoca cinomas selec ed o chemo adio he apy.
Conclusion: O6-me hylguanine-DNA me hyl ans e ase me hyla ion
s a us is no co ela ed wi h clinicopa hologic a iables examined in
ec al adenoca cinoma selec ed o chemo adio he apy, al hough i s
ole as a bioma ke awai s u he in es iga ion.
Keywo ds: Chemo adio he apy, O6-me hylguanine-DNA
me hyl ans e ase, ec al adenoca cinoma
Pa ien s wi h ec al adenoca cinoma s age II-III a e usually ea ed
wi h p eope a i e chemo adio he apy based on 5- luo ou acil
o capeci abine. Howe e , li le da a on molecula bioma ke s
o he p ognosis and ea men esponse in colo ec al cance
has been ob ained (1). The enzyme O6-me hylguanine-DNA
me hyl ans e ase (MGMT), which elimina es me hyl g oups in
he O6-guanine posi ion a oiding G:C o A:T ansi ions, has also
been ela ed o colo ec al cance (2,3). MGMT p e en s cell dea h
due o cy o oxic d ugs by epai ing DNA, bu i can be silenced
by epigene ic me hyla ion (4). Loss o MGMT exp ession has
been de ec ed in colo ec al cance and associa ed wi h G o A
ansi ion in he p53, K- as, and PIK3CA genes (5). P e ious s udies
sugges ed ha MGMT p omo e me hyla ion s a us was ela ed o
glioblas oma ea men ailu e (6). In his s udy, MGMT exp ession
and MGMT p omo e me hyla ion s a us we e e alua ed in ec al
adenoca cinoma pa ien s a e chemo adio he apy ea men in o de
o de e mine hei s a us and ele ance as p ognos ic bioma ke s.
MATERIALS AND METHODS
Clinical his o y and issue samples
Twen y-nine ec al adenoca cinoma pa ien s (s age II-III) who
we e candida es o p eope a i e chemo adio he apy we e ec ui ed
Add ess o Co espondence: Raúl O iz, Ins i u e o Biopa hology and Regene a i e Medicine (IBIMER), School o Medicine, Uni e si y o G anada, 18100
G anada, Spain
Phone: +34-958-249322 e-mail: oquesa@ug .es ORCID: o cid.o g/0000-0001-8409-5235
Recei ed: 25 Decembe 2018 Accep ed: 11 June 2019 • Bo h au ho s con ibu ed equally o his a icle • DOI: 10.4274/balkanmedj.galenos.2019.2018.12.93
A ailable a www.balkanmedicaljou nal.o g
Ci e his a icle as:
Oli e JA, Gómez-Millán J, Medina JA, Cabeza L, Pe azzoli G, Jimenez-Luna C, e al. O6-me hylguanine-DNA Me hyl ans e ase P omo e Me hyla ion in Pa ien s
wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men : Clinical Implica ions. Balkan Med J 2019;36:283-6
©Copy igh 2019 by T akya Uni e si y Facul y o Medicine / The Balkan Medical Jou nal published by Galenos Publishing House.
a e hey ga e in o med consen (Biomedical In es iga ion
E hic Commi ee; Se icio Andaluz de Salud). All pa ien s
we e e alua ed be o e ea men (physical examina ion wi h a
digi al ec al examina ion, colonoscopy and biopsy, ches X- ay,
abdominopel ic scan and/o endo ec al ul asound, and magne ic
esonance image o he pel is). These pa ien s we e ea ed wi h
pel ic adio he apy (46-50 Gy in 2 Gy ac ions) and in a enous
5- luo ou acil (5-day cycles o 500 mg/m2 5- luo ou acil e e y 21
days) o capeci abine (4 cycles o 1250 mg/m2 capeci abine e e y
12 h o 14 days) ollowed by su ge y ( o al meso ec al excision) 6
weeks a e chemo adio he apy. Tumo samples we e ob ained om
each pa ien om endoscopic biopsy be o e chemo adio he apy.
The chemo adio he apy esponse was s aged his opa hologically on
he basis o umo eg ession g ade (Manda d’s classi ica ion: g ade
I and II = comple e/pa ial eg ession and g ade III, IV, o V = no
eg ession) (7). Two expe pa hologis s e alua ed an in a-ope a i e
sample a e chemo adio he apy. Demog aphic da a (sex and age)
we e ob ained. In addi ion, clinicopa hological a iables, including
umo di e en ia ion g ade, umo s age, ea men eg ession
g ade, ecu ence, and lymph node in asion, we e analyzed.
Me hyla ion-speci ic polyme ase chain eac ion and
immunohis ochemis y
DNA was ex ac ed om pa a in-embedded issues by using
a Chemagic MSM I obo (Chemagen, Ge many, Baesweile ).
Me hyla ion pa e ns in CpG islands o he MGMT p omo e
we e de e mined by me hyla ion-speci ic polyme ase chain
eac ion as p e iously desc ibed (8). Samples we e classi ied as
me hyla ed (ampli ica ion p oduc wi h M o bo h M and UM
p ime s) and unme hyla ed (ampli ica ion wi h UM p ime s
only). Immunohis ochemical analysis was pe o med wi h a Dako
Au os aine EnVision™ FLEX Sys em ki (Agilen Technologies)
and he esul s e alua ed by wo expe ienced pa hologis s. MGMT
(1:50, San a C uz Bio echnology, Inc., Heidelbe g, Ge many) mAb
was used as he label and 3.3'-diaminobenzidine as he ch omogen
subs a e. Coun e s aining was pe o med wi h hema oxylin (blue).
As p e iously desc ibed by Oli e e al. (2), MGMT s aining we e
sco ed and g ouped as low (<50%) and high exp ession (≥50%).
S a is ical analysis
SPSS e sion 15.0 (IBM, Chicago, IL) was used o da a analyses.
Associa ions be ween p omo e gene me hyla ion s a us and
clinicopa hologic a iables we e analyzed by Fishe ’s exac es .
Resul s we e conside ed s a is ically signi ican i p<0.05.
RESULTS
The clinical pa ien cha ac e is ics a e summa ized in Table 1.
The mean age was 64.43±12.24 yea s ( ange, 33-83 yea s); 75.9%
(22/29) o pa ien s we e male and 24.1% (7/29) we e emale. The
median ollow-up pe iod was 20.53±9.07 mon hs. No pa ien
died due o ec al cance , and disease ecu ence was obse ed
in 13.8% (4/29). MGMT p omo e me hyla ion s a us could be
de e mined in 93.1% o specimens (27/29). O he 27 pa ien s, he
MGMT gene p omo e was me hyla ed 81.5% (22/27) (Figu e 1).
Immunohis ochemical analysis (Figu e 2) showed low MGMT
p o ein exp ession in mos pa ien s (88.9%). Only 11.1% o
pa ien s showed high exp ession o MGMT. We also examined
he associa ion be ween MGMT p omo e me hyla ion and
clinicopa hologic ea u es. MGMT p omo e me hyla ion s a us
was no associa ed wi h sex, umo di e en ia ion, o umo s age.
Fu he mo e, no associa ion be ween MGMT me hyla ion and he
clinicopa hologic a iables examined was de ec ed (Table 2).
284
Balkan Med J, Vol. 36, No.5, 2019
Oli e e al. MGMT P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men
TABLE 1. Cha ac e is ics o ec al cance pa ien s
All pa ien s (n=29)
Sex
Male 22 (75.9%)
Female 7 (24.1%)
Age
≥50 yea s 27 (93.1%)
<50 yea s 2 (6.9%)
Tumo di e en ia ion g ade
Well-mode a ely 27 (93.1%)
Poo ly 2 (6.9%)
Tumo s age
II 10 (34.5%)
III 19 (65.5%)
Lymph node me as asis
Yes 19 (65.5%)
No 10 (34.5%)
Recu ence
Yes 4 (13.8%)
No 25 (86.2%)
FIG. 1. Me hyla ion-speci ic polyme ase chain eac ion analysis o he O6-me hylguanine-
DNA me hyl ans e ase p omo e in ec al adenoca cinoma issue samples. DNA was
ex ac ed by using a Chemagic MSM I obo (Chemagen, Ge many, Baesweile ), dena u ed,
and pu i ied wi h an EpiTec Bisul i e ki (Qiagen, USA, Ma yland). P ime sequences
o he unme hyla ed eac ion we e 5`-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3`
( o wa d p ime ) and 5`-AACTCCACACTCTTCCAAAAACAAAACA-3` ( e e se p ime )
and o he me hyla ed eac ion we e 5`-TTTCGACGTTCTAGGTTTTCGC-3` ( o wa d
p ime ) and 5`-GCACTCTTCCGAAAACGAAACG-3` ( e e se p ime ). Polyme ase chain
eac ion-ampli ied p oduc s we e elec opho esed on 3% aga ose gels, isualized by s aining
wi h e hidium b omide, and examined unde ul a iole illumina ion. The ep esen a i e
image depic s O6-me hylguanine-DNA me hyl ans e ase p omo e me hyla ion analysis o
18 samples. Pa ien s wi h me hyla ed p omo e s showed ampli ica ion in bo h unme hyla ed
and me hyla ed lanes o he me hyla ed lane alone. The lack o a band in he lane
co esponding o me hyla ion-speci ic p ime s o ec al cance sample 2, 6 o 18 e lec s
he absence o O6-me hylguanine-DNA me hyl ans e ase p omo e me hyla ion.
M: me hyla ed; UM: unme hyla ed
DISCUSSION
The ela ionship be ween MGMT and colo ec al cance emains
unclea , and esul s ha e been con adic o y. Whe eas Nilsson e
al. (9) ound a lowe isk o ecu ence in 5- luo ou acil- ea ed
colo ec al cance pa ien s wi h a me hyla ed e sus unme hyla ed
MGMT p omo e , Shima e al. (3) concluded ha nei he MGMT
p omo e me hyla ion no loss o MGMT exp ession is a use ul
p ognos ic bioma ke . Sinha e al. (10) obse ed ha MGMT
me hyla ion was associa ed wi h s age III in spo adic colo ec al
cance cases. Recen ly, he me hyla ion s a us o MGMT has been
co ela ed wi h pa hologic comple e esponse in colo ec al cance
pa ien s (11).
Shalaby e al. (12) showed a good co ela ion be ween MGMT
me hyla ion and down egula ion o i s mRNA exp ession. Al hough
hese au ho s p oposed MGMT me hyla ion as a new bioma ke
o di e en ia e benign and malignan ec al umo s, no ela ion
be ween MGMT me hyla ion and clinicopa hological ea u es was
de ec ed. Sun e al. (13) showed ha e en he MGMT p omo e
me hyla ion s a us o plasma-cell- ee DNA was associa ed wi h
a be e umo esponse. In addi ion, MGMT me hyla ion le els in
he blood we e simila o hose in ec al cance issues (12). Ou
esul s showed ha he me hyla ion s a us o he MGMT p omo e
was no associa ed wi h a be e ea men esponse. Howe e , he
ole o MGMT p omo e me hyla ion s a us as an ea ly bioma ke
o colo ec al cance has no ye been es ablished, despi e se e al
s udies in colo ec al adenoma and adenoca cinoma (14,15). In ac ,
Balkan Med J, Vol. 36, No.5, 2019
Oli e e al. MGMT P omo e Me hyla ion in Pa ien s wi h Rec al Adenoca cinoma A e Chemo adio he apy T ea men 285
TABLE 2. Co ela ion be ween MGMT me hyla ion s a us and demog aphic and
clinicopa hologic a iables
Va iables
MGMT me hyla ion s a us: n
(% o pa ien s)
Unme hyla ed Me hyla ed
Sex
Male 3 (11.1) 17 (63)
Female 2 (7.4) 5 (18.5)
Tumo di e en ia ion g ade
Well-mode a ely 4 (14.8) 21 (77.8)
Poo ly 1 (3.8) 1 (3.8)
Tumo s age
II 1 (3.8) 9 (33.3)
III 4 (14.8) 13 (48.1)
Recu ence
Yes 3 (11.1) 20 (74)
No 2 (7.4) 2 (7.4)
T ea men eg ession g ade
I, II 1 (3.7) 9 (33.3)
III, IV, V 4 (14.8) 13 (48.1)
Lymph node me as asis
Yes 4 (14.8) 13 (48.1)
No 1 (3.8) 9 (33.3)
MGMT: O6-me hylguanine-DNA me hyl ans e ase
FIG. 2. a-c. Immunohis ochemical s aining o ec al adenoca cinoma issue samples wi h
a mouse monoclonal an ibody agains human O6-me hylguanine-DNA me hyl ans e ase
p o ein. Fo malin- ixed, pa a in-embedded ec al cance samples we e s ained wi h
an an ibody agains O6-me hylguanine-DNA me hyl ans e ase (see Me hods). O6-
me hylguanine-DNA me hyl ans e ase s aining o umo cells was sco ed and g ouped as
low exp ession (<50%) (-, +, and ++ sco es) and high exp ession (≥50%) (+++ and ++++
sco es). The in ensi y o O6-me hylguanine-DNA me hyl ans e ase s aining was sco ed
as low o high. The igu e shows ep esen a i e pho omic og aphs o slides illus a ing
di e en pe cen ages o O6-me hylguanine-DNA me hyl ans e ase exp ession. A umo
wi h no de ec able O6-me hylguanine-DNA me hyl ans e ase exp ession (a); A posi i e
umo wi h low O6-me hylguanine-DNA me hyl ans e ase exp ession (˂50%) (b); A
posi i e umo wi h high O6-me hylguanine-DNA me hyl ans e ase exp ession (>50%)
(20× magni ica ion) (c).
Side is e al. (16) showed ecen ly ha he e was no associa ion
be ween he s a us o MGMT exp ession and pa hological ea u es,
including esponse o neo-adju an he apy. Fu u e esea ch will be
needed o elucida e he ela ionship be ween hese bioma ke s and
ec al cance ea men .
Con lic o In e es : No con lic o in e es was decla ed by he au ho s.
Financial Disclosu e: No inancial disclosu e was decla ed by he au ho s.
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