Korelacija ekspresije estrogenskih i progesteronskih receptora, ciklina D1 i Bcl-2 sa relapsom kolorektalnih karcinoma Dukes B i C stadijuma
Correlation of expression of estrogen and progesterone receptors, cyclin D1 and Bcl-2 with relapse of colorectal cancers in Dukes' B and C stages
Author
Babić, AleksandraMentor
Milenković, SanjaCommittee members
Zdravković, NatašaCvetković, Aleksandar
Janković, Radmila
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Uvod
Prema podacima Svetske zdravstvene organizacije i bazi podataka GLOBOCAN,
kolorektalni karcinom (KRK) je treći karcinom po učestalosti kod muškaraca a drugi
kod žena, i drugi karcinom po smrtnosti u svetu. Oko 70% obolelih biva
dijagnostikovano sa lokalizovanom bolešću i potom radikalno operisano. Nažalost,
kod oko 1/3 ovih pacijenata se javlja relaps bolesti, a 73% njih umre. Zbog toga je pojava
relapsa jedan od najvažnijih faktora preživljavanja obolelih od KRK. Uprkos
opsežnom znanju o faktorima rizika za pojavu relapsa, ona se ne može u potpunosti
predvideti.
Sve je više dokaza da ekspresije ERα, ERβ i PR mogu biti faktori koji utiču na
prognozu KRK. Postoje dokazi da je gubitak ekpresije ERβ u tumorskom tkivu povezan je
sa višim stadijumima bolesti i predstavlja loš prognostički znak. Prisustvo ERα je
otkriveno u debelom crevu najpre na nivou mRNK, ali su nedavne studije potvrdile i
njegovu merljivu imunohistohemijsku ekspresiju i prognostiki potencijal....
Istraživanja koja su se bavila ekpresijom i ulogom ciklina D1 u patogenezi i prognozi
KRK dala su potpuno kontradiktorne rezultate. Prema nekim autorima. povećana
ekspresija ciklina D1 je dobar, a prema drugima loš prognostički znak. S druge strane,
neki autori su pokazali da se prognostička vrednost ovog markera razlikuje kod
muškaraca i žena. Slično je i sa rezultatima studija koje su se bavile značajem
ekspresije Bcl-2 u prognozi KRK-a. Neki autori sugerišu da je visoka ekspresija Bcl-2
povezana sa povoljnom prognozom kod pacijenata sa KRK iz Evrope i Amerike, ali su
razlike u ekspresiji bile povezane sa primenom neoadjuvantne terapije. Studije koje su
ispitivale ekspresiju gore pomenutih markera na tkivu KRK dale su kontradiktorne
rezultate u pogledu velikih varijacija nivoa ekspresije i korelacije sa kliničkim
ishodom.
Cilj naše studije je bio da ispita nivoe ekspresije ERα, ERβ, PR, ciklina D1 i
Bcl-2 na tkivu KRK u "visoko rizičnom" Dukes B i Dukes C stadijumu i utvrdi da li
postoji njihova korelacija sa ranim relapsom KRK u periodu od dve godine nakon
radikalnog hirurškog lečenja.
Materijali i metode
Dizajn studije i populacija koja se istražuje
Ova retrospektivna studija obuhvatila je 101 pacijenta sa dijagnozom
kolorektalnog karcinoma. U studiju su bili uključeni pacijenti sa potvrđenim Dukes
C i „visoko rizičnom“ Dukes B stadijumom (stadijum pT4 i/ili N2; slabo
diferencirani tumor; perforacija; limfovaskularna invazija; perineuralna invazija;
<12 pregledanih limfnih čvorova; pozitivne margine nakon operacije). Svi pacijenti
su nakon operativnog lečenja primili adjuvantnu hemiterapiju. Iz studije su
isključeni pacijenti lečeni od karcinoma rektuma koji su primili neoadjuvatnu
hemio/radio terapiju.
Pacijenti su podeljeni u dve grupe. Grupu A su činili oni kojima je relaps bolesti
dijagnostikovan u prva 24 meseca od operacije (DFS ≤ 24 meseca), a grupu B oni koji
nisu imali relaps bolesti najmanje 48 meseci od operativnog lečenja (DFS ≥ 48 meseci).
Lečenje i praćenje pacijenata
Nakon radikalnog operativnog lečenja i histopatološke potvrde tumora, pacijenti
su lečenje nastavili primenom adjuvantne hemioterapije u skladu sa indikacijama
Pravilnika o standardima za primenu citostatika Republičkog fonda za zdravstveno
osiguranje Republike Srbije, i praćeni na redovnim kontrolama prema preporukama
Nacionalnog vodiča dobre kliničke prakse za dijagnostikovanje i lečenje raka kolona i
rektuma.
Radikalno hirurško lečenje sprovedeno je na Hirurškoj klinici KBC Zemun, a
klinički i dijagnostički podaci dobijeni su iz medicinske evidencije koja je
formirana tokom adjuvantnog lečenja i redovnog praćenja pacijenata na Službi
medikalne onkologije KBC Zemun u periodu od 1. aprila 2008. do 1. januara 2018.
Sprovođenje studije odobrila je lokalna Etička komisije za sprovođenje kliničkih
studija KBC Zemun.
Patohistološko ispitivanje i evaluacija
Uzorci tumorskih su preuzeti su iz arhive Službe kliničke patologije KBC Zemun.
Semikvantitativna procena ekspresije svih imunohistohemijskih markera izvršena je
na osnovu intenziteta imunohistohemijskog bojenja, uzimajući u obzir broj obojenih
struktura tkiva i intenzitet imunohistohemijskog bojenja prema sistemu skorovanja
datom odvojeno za pojedine vrste markera.
Statistička obrada podataka
Deskriptivna statistika izračunata je za osnovne demografske i kliničke
karakteristike, kao i ishode lečenja. Kategoričke promenljive su predstavljene kao broj
i procenat. Kontinuirana distribucija podataka testirana je matematičkim i
grafičkim metodama. Kontinuirane promenljive su predstavljene kao srednja vrednost
sa standardnom devijacijom (engl. Standard Deviation, SD) ili medijana od 25-75
percentila, prema distribuciji podataka. Razlike između grupa analizirane su pomoću
Student T-testa i/ili Man-Whitney-jevog testa za kontinuirane promenljive i Pirsonovog χ2 testa za kategoričke promenljive. Višestruka logistička regresija korišćena
je za analizu prediktora za kolorektalni recidiv. Uključene su demografske i kliničke
karakteristike od značaja (p < 0.05) i korišćen je unapred uslovni odabir modela kako
bi se izbeglo njegovo precenjivanje. Hosmer–Lemeshow test je korišćen za dobro
prilagođavanje modelu logističke regresije. Nagelkerke koeficijent je korišćen kao
procenat varijanse u zavisnoj promenljivoj povezanoj sa prediktorskom (nezavisnom)
promenljivom. Za sve statističke proračune nivo značajnosti (α) iznosio je 0.05. Za
statističku obradu dobijenih rezultata koristili smo programski paket SPSS (verzija
23.0, SPSS Inc., Chicago, IL, USA).
Rezultati
Ova studija je obuhvatila 101 pacijenta radikalno operisanih od kolorektalnog
karcinoma u „visoko rizičnom“ Dukes B i Dukes C stadijumu. Prosečna starost
pacijenata bila je 64.3 ± 9.9 (DFS ≤ 24 grupa) i 61.1 ± 9.5 (DFS ≥ 48 grupa); nije bilo
statistički značajne razlike među grupama (p = 0.105). Medijana praćenja u grupi A bila
je 10.0 (3.0-24.0), a u grupi B 69.3 (48.0-115.0) meseci. Učestalost muškaraca i žena bila
je slična među grupama; nije uočena značajnija razlika. Nije bilo značajne razlike u
lokalizaciji rektuma između grupa. Leva strana debelog creva bila je češće pogođena u
grupi sa DFS ≤ 24 meseci; razlika je bila blizu konvencionalnog nivoa značajnosti (p
= 0.054). Dukes C stadijum i uznapredovali T stadijum bili su statistički češći u
grupi sa DFS ≤ 24 (p = 0.045; p = 0.037). Nije bilo značajne razlike između grupa u N
stadijumu i broju pozitivnih žlezda. Limfovaskularna invazija je češće bila
prisutna u grupi sa DFS ≤ 24 meseca; razlika je bila blizu konvencionalnog nivoa
značajnosti (p = 0.087). Nije bilo razlike između učestalosti perivaskularne invazije
i histološkog gradusa među grupama. Učestalost hipertenzije i dijabetesa bila je
slične među grupama.
U adjuvantnom režimu 84% pacijenata je primilo Mayo hemioterapijski protokol,
13% De Gramont i 3% njih FOLFOX4 protokol. Najčešći komorbiditeti su bili
hipertenzija (oko 2/3 pacijenata) i dijabetes (oko 1/5 pacijenata).
ERα nije bio ekprimiran na tumorskim tkivima ni jednog pacijenta. ERβ je bio
umerereno eksprimiran kod oko 25% pacijenata, češće u grupi sa DFS ≥ 48 meseci u
poređenju sa grupom sa DFS ≤ 24 meseca. ERβ je bio je prisutan kod 21 (28.4%) pacijenata
sa tumorima levog kolona, i kod 4 (16.7%) pacijenta sa tumorima desnog kolona, razlika
nije bila statistički značajna (p = 0.253). Umerena ekspresija PR bila je prisutna kod
1/5 pacijenata, a jaka ekspresija bila je prisutna samo kod dva pacijenta, bez značajne
razlike među grupama A i B (p = 0.145). Bcl-2 nije bio izražen kod više od 2/3
pacijenata; 1/3 pacijenata je imalo umerenu ekspresiju ovog receptora, sa sličnom
ekspresijom među grupama (p = 0.566). Ciklin D1 je eksprimiran na svim tumorskim
uzorcima pacijenata; 15% (14.9) je imalo slabu, 37% (36.6) umerenu, 30% (29.7) jaku i
19% (18.8) vrlo jaku ekpresiju. Jaka i veoma jaka ekspresija ciklina D1 > 50% bila je
statistički češća u grupi DFS ≤ 24 nego u grupi DFS ≥ 48 (p = 0.021).
Nivoi hemoglobina bili su slični među grupama. CRP je bio značajno veći u grupi
sa DFS ≤ 24 (p < 0.001). Nije bilo razlike u NLR i PLR između grupa. Nivo albumina
bio je značajno veći u grupi DFS ≥ 48.
Pacijenti sa ekspresijom ciklina D1 > 50% su imali 5.2 a sa odmaklim T
stadijumom 11.3 puta veću šansu da imaju relaps u toku prve dve godine od radikalne
operacije. Umerena ekspresija ERβ je bila udružena sa 79.2% manjom šansom za pojavu
relapsa u ovom periodu. Povišene vrednosti CRP, iznad referentnih, bile su
udružene sa 5.9 puta većom šansom za kraće vreme do pojave relapsa.
Zaključak
Pojava ranog relapsa KRK u prve dve godine nakon radikalnog operativnog lečenja
u "visoko rizičnom" Dukes B i Dukes C stadijumu povezana je sa smanjenom ekspresijom
ERβ i visokom ekspresijom ciklina D1. Ekspresija ERα, PR i Bcl-2 na tkivu
kolorektalnog karcinoma nije u korelaciji sa ranim relapsima.
Rezultati naše studije ukazuju su povećana ekspresija ciklina D1 i smanjena
ekspresija ERβ pokazatelji loše prognoze obolelih od kolorektalnog karcinoma,
posebno kod pacijenata sa uznapredovalim T stadijumom. Stoga bi se ciklin D1 i ERβ
mogli smatrati značajnim, nezavisnim prognostičkim faktorima za pacijente obolele
od ove bolesti i potencijalnim terapijskim metama.
Ključne reči: kolorektalni karcinom, relaps, ERα, ERβ, PR, ciklin D1, Bcl-2
Introduction
According to the World Health Organization and the GLOBOCAN database, colorectal
cancer (CRC) is the third most common cancer in men and the second in women. It is the second
most deadly cancer worldwide. About 70% of the patients undergo surgery with curative intent.
Unfortunately, around 1/3 of these patients experience disease recurrence, and approximately
73% of them die. Therefore, CRC recurrence is one of the most important factors influencing
patient survival. Despite the extensive knowledge about the risk factors for CRC recurrence, its
occurrence cannot be fully predicted.
There has been growing evidence that the expression of ERα and ERβ, as well as PR, may
be factors that influence CRC prognosis. The loss of ERβ expression in tumor tissue is
associated with advanced cancer stages and is a poor prognostic sign. ERα was found in the
colon at the mRNA level, but recent studies also confirm its measurable immunohistochemical
expression and prog...nostic potential. Researches that have addressed the role of cyclin D1 in the
pathogenesis of CRC and its value as a prognostic marker has yielded entirely contradictory
results. According to some authors, increased cyclin D1 expression is a good, and according to
others, a poor prognostic sign. On the other hand, some authors showed that this marker's
prognostic value differs between men and women. The similar is with the results of studies on
the importance of Bcl-2 expression in CRC prognosis. Some of them suggest that high
expression of Bcl-2 is associated with favorable prognosis in CRC patients
from Europe and America, but differences in expression depended on neoadjuvant therapy. The
studies examining the expression of the receptors mentioned above on CRC tissue are
contradictory in terms of large variations in expression levels and correlations with the clinical
outcome.
This study aimed to examine the expression levels of ERα, ERβ, PR, Cyclin D1 and Bcl2 on the tissue of CRC in "high risk" Dukes’ B and Dukes’ C stages and determine whether the
expression correlates with early CRC recurrence within two years after surgery.
Material and methods
Study design and study population
This retrospective study included 101 patients diagnosed with colorectal cancer in Dukes’
C and "high-risk" Dukes’ B stages (stage pT4 and/or N2; a poorly differentiated tumor;
perforation; lymphovascular invasion; perineural invasion; <12 examined lymph nodes;
positive margins after surgery). All patients received adjuvant chemotherapy after surgery.
Patients treated for rectal cancer who received neoadjuvant chemo/radiotherapy were excluded
from the study.
Patients were divided into two groups. Group A consisted of those diagnosed with CRC
relapse in the first 24 months from surgery (DFS ≤ 24 months), and group B consisted of those
who did not relapse for at least 48 months from surgical treatment (DFS ≥ 48 months).
Treatment and follow up
After radical surgical treatment and histopathological confirmation of tumor, all patients
were treated with adjuvant chemotherapy according to the indications of the Ordinance on
Standards for the Cytostatic Use of the Republic Health Insurance Fund of the Republic of
Serbia. Follow-up was performed according to the recommendations of the National Guidelines
for Treatment and Follow-up of Colorectal Cancer Patients.
Surgical treatment was carried out at the Surgery Clinic of the Clinical Hospital Center
Zemun. The additional adjuvant therapy and follow-up were conducted at the Department of
Medical Oncology of the Clinical Hospital Center Zemun from April 1st, 2008. to January 1st,
2018. The study received ethical approval from the Local Research Ethics Committee of the
University Clinical Hospital Center of Zemun.
Immunohistochemical staining and evaluation
Tumor samples were obtained from the archive of the Department of Clinical Pathology
of Clinical Hospital Center Zemun. Semiquantitative assessment of all immunohistochemical
markers' expression was performed based on the intensity of immunohistochemical staining,
considering the number of stained tissue structures and the intensity of immunohistochemical
staining according to the assessment system given separately for certain types of markers.
Statistical analysis
Descriptive statistics were calculated for the baseline demographic and clinical features, as
well as treatment outcomes. Categorical variables were presented as numbers and percentages.
Continuous data distribution was tested with mathematical and graphical methods. According
to data distribution, continuous variables were presented as mean with standard deviation (SD)
or median with 25-75 the percentile. Differences between groups were analysed using Student's
t-test (or Mann Whitney) for continuous variables and Pearson's Chi-squared test for
categorical variables. Multiple logistic regression was used for the analysis of predictors for
colorectal recurrence. Significant demographic and clinical features (p < 0.05) were included,
and forward conditional model selection was used to avoid model overestimation. Hosmer–
Lemeshow test was used for goodness of fit for the logistic regression model. Nagelkerke
coefficient was used as a proportion of variance in the dependent variable associated with the
predictor (independent) variable. For all statistical calculations, the significance level (α) was
0.05. For statistical processing of the obtained results, we used the SPSS software package
(version 23.0, SPSS Inc., Chicago, IL).
Results
This study included 101 patients with "high risk" Dukes’ B and Dukes’ C stages of
colorectal carcinoma. Mean age was 64.3±9.9 (DFS ≤ 24 group) and 61.1±9.5 (DFS ≥ 48
group) without significant difference between groups (p = 0.105). Median follow up in DFS
≤ 24 group was 10.0 (3.0-24.0) and in DFS ≥ 48 group was 69.3 (48.0-115.0) months. The
frequency of males and females was similar between groups; no significant difference was
observed. There was no significant difference in rectal localization between groups. The left
side of the colon was more often affected in DFS ≤ 24 group; the difference was close to the
conventional level of significance (p=0.054). Dukes’ stage C and advanced T stage were
statistically more frequent in DFS ≤ 24 group (p = 0.045; p = 0.037). There was no significant
difference in the N stage and number of positive glands between groups. Lymphovascular
invasion was more often present in DFS ≤ 24 group; the difference was close to the conventional
level of significance (p = 0.087). There was no difference between perivascular invasion and
histological grade between groups. The frequency of hypertension and diabetes were similar
between groups.
In the adjuvant regimen, 84% of patients received the Mayo chemotherapy protocol, 13%
the De Gramont protocol, and 3% of them the FOLFOX4 protocol. The most common
comorbidities were hypertension (about 2/3 of patients) and diabetes (about 1/5 of patients).
Overall, ERα was not expressed in all patients. ERβ moderate expression was present in
25% of all patients, more often in DFS ≥ 48 group when compare to DFS ≤ 24 group (p
= 0.001). Positive ERβ was present in 21 (28.4%) patients with left colon tumors, and in 4
(16.7%) with the right side of the colon, the difference was not significant (p = 0.253). Moderate
PR expression was present in 1/5 of patients, and strong expression was present only in two
patients, without significant difference between groups (p = 0.145). Bcl-2 was not expressed in
more than 2/3 of the patients; 1/3 of the patients had the moderate expression of this receptor,
with a similar expression between groups (p = 0.566). Cyclin D1 was expressed in the whole
sample of patients; 15% (14.9) had a weak, 37% (36.6) moderate, 30 % (29.7) strong, and 19%
(18.8) very strong expression. Strong and very strong expression >50% was statistically more
often in DFS ≤ 24 group than in the DFS ≥ 48 group (p = 0.021).
Levels of hemoglobin were similar between groups. CRP was significantly higher in the
DFS ≤ 24 group (p < 0.001). There was no difference in NLR and PLR between groups. The
level of albumins was significantly higher in the DFS ≥ 48 group.
Patients with an expression of cyclin D1 > 50% had 5.2, and an advanced T stadium had
11, 3 times higher odds of having a DFS ≤ 24 months. Moderate expression of ERβ was joined
with 79.2 % smaller odds for DFS ≤ 24 months. Higher levels of CRP (above referent range)
were joined with 5,9 higher odds for DFS ≤ 24 months.
Conclusion
The occurrence of early relapse of CRC in the first two years after radical surgical treatment
in the “high-risk” Dukes’ B and Dukes’ C stages is associated with decreased ERβ expression
and high cyclin D1 expression. The expression of ERα, PR, and Bcl-2 on colorectal cancer
tissue is not correlated with early recurrences.
Our findings indicate the increased expression of cyclin D1 and reduced expression of ERβ
are indicators of poor prognosis in CRC, especially in patients with advanced T stage.
Therefore, cyclin D1 and ERβ may be considered significant, independent prognostic factors in
CRC patients and possible therapeutic targets.