REZUMATUL TEZEI DE DOCTORAT

Factori prognostici n cancerul

colorectal operat

Doctorand Bogdan Vasile Micu

Conductor de doctorat Prof. dr. Aurel Andercou

CUPRINS
INTRODUCERE
STADIUL ACTUAL AL CUNOATERII

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1. Factori prognostici n cancerul colorectal

1. 1. Factori clinici
1.1.1. Vrsta
1.1.2. Sexul
1.1.3. Simptomatologia
1.1.3.1. Durata simptomatologiei
1.1.4. Obstrucia i perforaia
1.1.5. Localizarea tumorii primare
1.1.6. Anemia i transfuziile sanguine perioperatorii
1. 2. Factori histopatologici
1.2.1. Stadiul tumoral
1.2.2. Factori aparinnd tumorii primare
1.2.2.1. Profunzimea invaziei tumorale
1.2.2.2. Dimensiunea tumorii primare
1.2.2.3. Aspectul macroscopic al tumorii primare
1.2.2.4. Aspectul histopatologic al tumorii primare
1.2.2.5. Marginile de rezecie - tumora rezidual
1.2.2.6. Configuraia marginilor de invazie tumoral
1.2.2.7. Budding-ul tumoral
1.2.2.8. Necroza tumoral
1.2.2.9. Gradul de difereniere al tumorii
1.2.3. Ganglionii limfatici regionali
1.2.3.1. Numrul nodulilor limfatici
1.2.3.2. Topografia nodulilor limfatici invadai
1.2.3.3. Limph node ratio (LNR)
1.2.3.4. Micrometastazele
1.2.3.5. Depozite tumorale izolate n nodulii limfatici
1.2.3.6. Invazia extracapsular a nodulilor limfatici
1.2.3.7. Ganglionul santinel
1.2.3.8. Adenopatia limfatic reactiv
1.2.4. Invazia vascular
1.2.4.1. Invazia venoas
1.2.4.1. Invazia venoas
1.2.5. Invazia perineural
1.2.6. Depozitele tumorale extramurale
1.2.7. Angiogeneza tumoral
1.2.8. Reacia imun peritumoral (rspunsul imun local al gazdei)
1. 3. Factori biologici
1.3.1. Markeri serici tumorali
1.3.1.1. Antigenul carcinoembrionar (ACE)
1.3.1.2. Antigenul carbohidrat 19-9 (CA 19-9)
1.3.1.3. Antigenul carbohidrat 242 (CA 242)

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1.3.1.4. Antigenul carbohidrat 50 (CA50)

1.3.1.5. Antigenul carbohidrat 125 (CA 125)
1.3.2. Markeri biochimici tumorali
1.3.2.1. Timidilat sintetaza
1.3.3. Coninutul de ADN
1.4. Factori moleculari
1.4.1. Gene de supresie tumoral
1.4.1.1. Gena p53
1.4.1.2. Gena p27
1.4.1.3. 18qLOH (pierderea heterozigozitii) i Gena Deleiei n cancerul
colorectal (DCC)
1.4.1.4. Instabilitatea microsatelitar
1.4.1.5. Hipermetilarea ADN-ului
1.4.2. Oncogenele
1.4.2.1. Ras
1.4.2.2. BRAF

2. Rspunsul inflamator al gazdei n cancerul colorectal

2. 1. Rspunsul inflamator sistemic
2. 2. Rspunsul inflamator local

3. Instabilitatea microsateliilor n cancerul colorectal

3. 1. Patogeneza apariiei instabilitii microsatelitare
3. 2. Metode de identificare a instabilitii microsatelitare
3. 3. Fenotipul cancerelor colorectale instabile microsatelitar
3. 4. Noi abordri n managementul cancerelor colorectale instabile microsatelitar

4. Ganglionul santinel n cancerul colorectal

4. 1. Argumente pentru identificarea ganglionului santinel n cancerul colorectal
4. 2. Tehnici de identificare a ganglionului santinel
4. 3 Examenul histopatologic
4. 4. Perspectivele identificrii ganglionului santinel n cancerul colorectal

CONTRIBUIA PERSONAL
1. Ipoteza de lucru/obiective
2. Metodologie general
3. Studiu 1. Evaluarea factorilor prognostici clinici i
morfopatologici n cancerul colorectal operat
3.1. Introducere
3.2. Ipoteza de lucru
3.3. Material i metod
3.4. Rezultate
3.5. Discuii
3.6. Concluzii

4. Studiu 2. Analiza factorilor prognostici ai recidivei n cancerul

colorectal operat
4.1. Introducere
4.2. Ipoteza de lucru

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4.3. Material i metod

4.4. Rezultate
4.5. Discuii
4.6. Concluzii

5. Studiu 3. Analiza i evaluarea rolului prognostic al genelor

MLH1, MSH2, MSH6 i PMS2 n cancerul colorectal operat
5.1. Introducere
5.2. Ipoteza de lucru
5.3 Material i metod
5.4 Rezultate
5.5 Discuii
5.6 Concluzii

6. Studiu 4. Rolul analizei ganglionului santinel n cancerul

colorectal operat
6.1 Introducere
6.2 Ipoteza de lucru
6.3 Material i metod
6.4 Rezultate
6.5 Discuii
6.6 Concluzii

7. Concluzii generale
8. Originalitatea i contribuiile inovative ale tezei
REFERINE

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Cuvinte cheie: cancer colorectal, factori prognostici, supravietuire, recidiva, raspuns

inflamator, instabilitate microsatelitara, ganglion santinela

Introducere
Cancerul colorectal a devenit, la nivel mondial, o problem major de sntate
public datorit prevalenei, mortalitii i morbiditii ridicate. n Romnia, cancerul
colorectal se situeaz pe locul al treilea n ceea ce privete incidena i mortalitatea
prin tumori maligne, iar rata supravieuirii nu s-a mbuntit substanial n ultimii 5
ani.
Dei procedeele terapeutice sunt bine stabilite i sunt aplicate unitar la toi
pacienii, rezultatele n ceea ce privete supravieuirea i incidena recidivelor locale
sau la distan difer, mai probabil datorit unor factori prognostici care depind de
pacient, dar i de caracteristicile tumorii.
Dup intervenia chirurgical cu viz curativ putem stabili cu exactitate stadiul
tumoral al pacienilor cu cancer colorectal. De multe ori pacienii ncadrai n stadii
incipiente prezint ulterior recidive locale i/sau la distan, iar ali pacieni ncadrai
iniial n stadii avansate vor supravieui fr recidiv, mai probabil datorita implicarii
unor factori care intervin n evoluia pacienilor cu cancer colorectal operat radical.
Aproximativ 15-20% din cancerele colorectale se dezvolt printr-o cale
alternativ a genezei tumorale caracterizat prin instabilitate microsatelitar (MSI). n

cursul replicrii ADN-ului pot apare unele erori n secvenele de microsatelii. Sistemul
MMR (missmatchrepair) al ADN-ului, sistem alctuit din gene de reparare a
mperecherilor nepotrivite de baze azotate, are rolul de a repara erorile aprute n
timpul replicrii. Dac sistemul MMR funcioneaz defectuos, apar erori la nivelul
microsateliilor, fenomen denumit instabilitate microsatelitar, responsabil de
acumularea unor mutaii somatice la nivelul unor oncogene sau gene supresoare cu rol
important n procesul de iniiere i progresie tumoral. Aceste tumori sunt clasificate
ca MSI (instabile microsatelitar), iar cele care nu prezint acest fenomen sunt
considerate cu stabilitate microsatelitar (MSS).
Rata de recidiv este de peste 30% la pacienii cu cancer colorectal stadiul I i II
supui rezeciilor cu viz curativ.Pentru o stadializare ct mai exact ar fi nevoie de
examinri multiseriate cu hematoxilin-eozin, tehnici de imunohistochimie i,
respectiv, de reacia de polimerizare n lan. Conceptul de ganglion santinel ar putea
oferi o soluie, astfel ar permite, redefinirea stadializrii i identificarea unui grup de
pacieni care ar beneficia de chimioterapie adjuvant. Procedeele de identificare a
ganglionului santinel nu sunt standardizate, metodele, materialele, tehnicile i
selecia pacienilor diferind ntre instituii i chirurgi.

Ipoteza de lucru/obiective
Evaluarea i analiza factorilor prognostici implicai n cancerul colorectal
operat;
Identificarea i analiza unor noi factori prognostici n cancerul colorectal
operat;
Valoarea i rolul prognostic al rspunsului inflamator local i general n cancerul
colorectal operat;
Analiza i rolul prognostic al limph node ratio (LNR) n cancerul colorectal
stadiul III operat;
Analiza si evaluarea diferenelor fenotipice i prognostice existente ntre
tumorile colorectale instabile microsatelitar aprute prin mecanism sporadic
sau transmis ereditar i tumorile colorectale stabile microsatelitar, n cancerul
colorectal operat;
Identificarea ganglionului santinel n cancerul colorectal prin metoda in vivo i
ex vivo;
Evaluarea si analiza rolului prognostic al ganglionului santinel n chirurgia
cancerul colorectal;

Metodologie general
Pentru realizarea obiectivelor propuse am luat n studiu pacieni internai i
operai n Clinica Chirurgie a Spitalului Clinic Municipal Cluj-Napoca, Catedra Chirurgie
V a Universitii de Medicin i Farmacie Iuliu Haieganu Cluj-Napoca i am colaborat
cu Serviciul de Anatomie Patologic a Spitalului Clinic Municipal Cluj-Napoca i
Laboratorul de Anatomie Patologic Santomar Cluj-Napoca.
Toi pacienii inclui n studiu au semnat consimmntul informat, iar studiul a
fost aprobat de Comisia de Etic a Spitalului Clinic Municipal Cluj-Napoca. Diagnosticul
de cancer colorectal s-a stabilit preoperator prin examen clinic, examinri de laborator

i paraclinic (radiografie toracic, ecografie abdominal, endoscopie digestiv

inferioar cu biopsie). Au fost exclui din studiu pacienii care au prezentat: metastaze
la distan decelate pre sau intraoperator, tumori sincrone primare, afeciuni
intestinale inflamatorii, pacienii cu alte tipuri histopatologice de cancer n afar de
adenocarcinom, pacienii operai de urgen, crora li s-a administrat radioterapie
preoperatorie, pacienii decedai la mai puin de 30 de zile postoperator, cei cu date
incomplete i cei care nu au semnat consimmntul informat.
Datele au fost analizate folosind foile de observaie, protocoalele operatorii i
rapoartele anatomo-patologice. S-a realizat, astfel, o baz de date cu datele
demografice, date clinico-anamnestice, examinri de laborator, paraclinice, aspecte
descrise intraoperator, examen morfopatologic, numr de ganglioni limfatici
examinai, raportul dintre numr de ganglioni limfatici invadai i numr de ganglioni
limfatici examinai definit ca i limph node ratio (LNR).
Am luat n studiu: numrul de leucocite definite n trei intervale
(<8500/mm3;8500-11.000/mm3;>11.000/mm3),
numrul
de
limfocite
(<1000/mm3;1000-3000/mm3;>3000/mm3),
numr
de
neutrofile
(<7500/mm3;>7500/mm3), raportul dintre neutrofile i limfocite, hemoglobina,
hematocrit, numr de trombocite (150000-370000/mm3), prezena anemiei.
S-a efectuat o nou analiz anatomo-patologic microscopic cu analiza marginii
de invazie tumoral i redefinirea stadializrii TNM, conform ultimei ediii a AJCC
Cancer Staging Manual, a aptea, operaional din 1 ianuarie 2010.
S-a calculat indicele Petersen alctuit prin utilizarea a patru variabile
morfopatologice, fiecreia atribuindu-se un punctaj. Punctajul total s-a calculat
nsumnd aceste scoruri, rezultnd un indice Petersen cuprins ntre 0 i 5, submprit
n risc sczut (0-1) i risc ridicat (2-5).
Rspunsul inflamator local s-a calculat folosind criteriile Klintrup la nivelul
marginii de invazie tumoral, cuantificndu-se infiltratul inflamator local. Se consider
rspuns inflamator local de grad redus pentru punctajele 0 i 1, iar grad nalt pentru
punctajele 3 i 4.
Necroza tumoral a fost i ea cuantificat, astfel: punctajul 0 pentru necroza
absent; 1 pentru necroza focal, sub 10%; 2 pentru necroza moderat, ntre 1030%; 3 pentru necroza extensiv, peste 30%. Componenta mucinoas a fost i ea
cuantificat, astfel nct 0 se atribuie absenei componentei mucinoase; 1 cnd
componenta mucinoas este minim, sub 10 %; 2 cnd componenta mucinoas este
moderat, ntre 10-50%, iar 3 se atribuie componentei mucinoase importante, peste
50%. Punctajul desmoplaziei: 0 pentru absena, 1 pentru desmoplazie redus sau
minim, 2 cnd desmoplazia este medie i 3 cnd desmoplazia este
marcat/important/extins. Pentru pacienii n stadiul III s-a calculat limph node
ratio (LNR) prin divizarea numrului de limfoganglioni invadai tumoral la numrul
total de limfoganglioni excizai. Pe baza acestui criteriu, pacienii au fost divizai n 5
grupe, valorile de interval fiind stabilite la<0,10, 0,11-0,21, 0,22-0,36, 0,37-0,6 i
respectiv >0,61;

De asemenea, pentru un subgrup de pacieni s-a calculat i rspunsul inflamator

sistemic preoperator, prin utilizarea scorului Glasgow modificat (mGPS), calculat n
funcie de valorile proteinei C reactive i a albuminemiei.
Pacienii au fost urmrii pe o perioad de 5 ani, n primul an la 3 i 6 luni
postoperator, iar n urmtorii ani, anual.
Analiza statistic a fost realizat cu ajutorul programului SPSS versiunea 20.
Datele au fost considerate ca fiind nominale, ordinale sau cantitative. Datele cantitative
au fost testate pentru normalitatea distribuiei cu testul Kolmogorov-Smirnov.
Variabilele cantitative au fost decrise prin median i percentilele 25 i 75 (distribuie
non-normal). Variabilele nominale i ordinale au fost caracterizate prin frecven i
procent.

Studiu1.
Evaluarea
factorilor
prognostici
morfopatologici n cancerul colorectal operat

clinici

Ipoteza de lucru
Pornind de la datele existente n literatur, ne-am propus s evalum i s analizm
factorii prognostici implicai n supravieuire la pacienii cu cancerul colorectal operat
cu viz curativ. De asemenea, ne-am propus s identificm i s analizm noi factori
prognostici implicai n cancerul colorectal.

Material i metod
Pentru realizarea obiectivelor propuse am efectuat un studiu retrospectiv
observaional care a inclus un numr de 301 pacieni diagnosticai cu cancer colorectal
stadiul I-III, internai i operai cu viz radical n Clinica Chirurgie V a Spitalului Clinic
Municipal Cluj-Napoca, n perioada ianuarie 1999-decembrie 2008.

Rezultate
n studiu au fost 197 de pacieni (66,4%), reprezentnd pe cei care au
supravieuit pe o perioad de urmrire de 5 ani i 104 pacieni (34,6%) care au
decedat.
Numrul de leucocite nregistrat la pacienii decedai a fost nalt, semnificativ
statistic mai mare 8700 (6925; 11000/mm3) vs. 7050 (6100; 8275)/mm3, comparativ
cu cel determinat la supravieuitori (p<0,001).
Raportul neutrofile/limfocite calculat la pacienii decedai a fost nalt,
semnificativ statistic mai mare dect la supravieuitori [3,3 (1,5; 6) vs. 2,7 (2,1; 3,4)]
(p<0,001).
Dintre pacienii supravieuitori 83 (42,1%) au avut cancer rectal, iar 114
(57,9%) cancer colonic. Dintre pacienii decedai 59 (56,7%) au avut cancer rectal, iar
45 (43,3%) cancer colonic. Dintre pacienii cu neoplasm de colon 54 (34%) au avut
localizare pe dreapta. La aceti pacieni s-au nregistrat 21 (46,7%) decese, o frecven
semnificativ statistic mai mare comparativ cu celelalte localizri (p=0,03).
Supravieuirea pe stadii a fost de 87,8% pentru stadiul I, 81,5% pentru stadiul
IIA, 63,33% pentru stadiul IIB, 44,4% pentru stadiul IIC. Pentru stadiul III am obinut
urmtoarele rezultate: 80% pentru stadiul IIIA, 51,38% pentru stadiul IIIB, respectiv
34,88% pentru stadiul IIIC.

Am determinat o diferen nalt semnificativ statistic n ceea ce privete

supravieuirea pacienilor cu T>2 (93 (89,4%)), comparativ cu cei cu T=<2 (11
(10,6%) (p<0,001).
Am determinat o diferen nalt semnificativ statistic n ceea ce privete
supravieuirea pacienilor cu N>0 [67 (64,4%)], comparativ cu cei cu N=0 (37 (35,6%))
(p<0,001). Rata de supravieuire a fost mai mare semnificativ statistic pentru grupul
N1, comparativ cu N2 (p<0,001).
Am determinat un HR de 12,3 (IC 95% 4,9-31) pentru pacienii aflai n LNR
>0,61,comparativ cu cei din LNR <0,1 (p<0,001).
Gradul nalt al scorului Klintrup a fost stabilit la 9 (8,7%) pacieni din grupul
deceselor, n timp ce 136 (69%) pacieni supravieuitori au avut grad nalt al scorului
Klintrup(p<0,001). Riscul nalt al scorului Petersen a fost stabilit la 52 (50%) pacieni
din grupul deceselor i la 11 (5,6%) pacieni supravieuitori (p<0,001).
Invazia venoas a fost descris la 14 (7,1%) pacieni supravieuitori i la 60
(57,7%) pacieni decedai (p<0,001). Invazia perineural a fost descris la 32 de
pacieni, din care 8 (4,1%) pacieni supravieuitori i la 24 (23,1%) pacieni decedai
(p<0,001).
Pacienii care au prezentat un scor de necroz mai mare au avut o rat mai mare
a deceselor. (p<0,001). Pacienii care au prezentat un scor al componentei mucinoase
mai mare au avut o rat mai mare a deceselor (p<0,001). Pacienii care au prezentat un
scor de desmoplazie mai mic au avut o rat mai mare a deceselor (p<0,001).
La analiza multivariata, localizarea cancerului la nivelul colonului a fost asociat
cu o supravieuire mai bun dect cea rectal (HR 0,57; IC 95% 0,35-0,94; p=0,02).
Pacienii cu T>2 au avut un prognostic infaust comparativ cu cei cu T=<2 (HR - 2,23; IC
95% 1,11-4,47; p=0,02). Pacienii cu scor Kintrup >1 prognostic mai bun, comparativ
cu cei cu Klintrup=<1 (HR - 0,20; IC 95% 0,09-0,44; p<0,001). Pacienii cu invazie
venoas au avut un prognostic mai prost comparativ cu cei fr invazie venoas (HR 2,26; IC 95% 1,31-3,91; p=0,003).Pacienii cu scor desmoplazic 3 au avut o rat a
deceselor mai mic dect cei cu scor 1 (HR - 0,42; IC 95% 0,22-0,85; p=0,01).

Studiu 2. Analiza factorilor prognostici ai recidivei n cancerul

colorectal operat
Ipoteza de lucru
Pornind de la datele din literatur, ne-am propus s evalum i s analizm
factorii prognostici implicai n recidiva local i la distan la pacienii cu cancer
colorectal operat cu viz curativ. De asemenea, ne-am propus s identificm i s
analizm noi factori prognostici implicai n recidiva cancerului colorectal operat cu
intenie curativ.

Material i metod
Pentru realizarea obiectivelor propuse am efectuat un studiu retrospectiv
observaional care a inclus un numr de 301 pacieni diagnosticai cu cancer colorectal
stadiul I-III, internai i operai cu viz radical n Clinica Chirurgie V a Spitalului Clinic
Municipal Cluj-Napoca n perioada ianuarie 1999-decembrie 2008.
Recidivele la distan s-au identificat prin examen local general, examinri
paraclinice (examinri de laborator, radiografie toracic, ecografie general, computer

tomografie, scintigrafie), iar recidivele locale s-au stabilit prin ecografie abdominal
i/sau endoscopie digestiv inferioar cu biopsie. S-a calculat perioada n luni de la
intervenia chirurgical pn la apariia recidivei loco-regionale sau la distan, aceasta
fiind considerat supravieuirea fr recidiv.

Rezultate
112 (37,2%) pacieni au dezvoltat recidive pe perioada de urmrire de 5 ani i
189 (62,8%) pacieni care nu au dezvoltat recidive.
Din cele 112 cazuri cu recidiv, la 43 (38,4%) cazuri s-au nregistrat recidive
locale i la 69 (61,6%) cazuri recidiv la distan.
La 159 de pacieni formaiunea tumoral a fost situat la nivelul colonului i sau nregistrat 46 de recidive din care 20 la distan. n 142 de cazuri (47,2%) tumora sa situat la nivelul rectului, din care au recidivat 66 cu 44 de cazuri la distan. Dintre
pacienii fr recidiv 76 (40,2%) au avut cancer rectal, iar 113 (59,8%) cancer
colonic.
Numrul de leucocite nregistrat la pacienii cu recidiv a fost nalt semnificativ
statistic mai mare (8600 (6800; 11000/mm3) vs. 7100 (6200; 8375)/mm3),
comparativ cu cel determinat la fr recidiv (p<0,001).
Raportul neutrofile/limfocite calculat la pacienii cu recidiv a fost nalt
semnificativ statistic mai mare dect la cei fr recidiv [3,4 (2,6; 5,9) vs. 2,6 (2,1; 3,4)]
(p<0,001).
Am determinat o diferen nalt semnificativ statistic ntre stadiile bolii n ceea
ce privete recidiva (p<0,001).
Din lotul studiat, 162 de pacieni au fost ncadrai n stadiul I i II (N0), dintre
acetia 33 au fcut recidive, din care 19 locale i 14 la distan. Din 139 de pacieni, 82
de pacieni au fost N1 din care au prezentat recidive 34 (41,4%) - 14 recidiv local i
20 la distan. Stadiul N2 au fost 57 de pacieni, 45 (57,7%) au prezentat recidiv local 10 i la distan 35 de cazuri (p<0,001).
Pentru pacienii aflai n stadiul III am calculat un LNR de 0,4 (0,21; 0,58) la cei
care au prezentat recidive i un LNR de 0,09 (0,07; 0,16) la cei fr recidive. Pacienii
care s-au situat ntr-un nivel mai mare al LNR au avut frecven mai mare a recidivelor
(p<0,001).
Rata recidivelor a fost nalt semnificativ statistic mai mare la pacienii cu scor
Klintrup mai mic (p<0,001). Rata recidivelor a fost nalt semnificativ statistic mai
mare la pacienii cu scor Petersen mai mare (p<0,001).
Invazia venoas a fost descris la 7 (3,7%) pacieni fr recidiv i la 67 (59,8%)
pacieni cu recidiv (p<0,001). Invazia perineural a fost descris la 2 (1,1%) pacieni
fr recidiv i la 30 (26,8%) pacieni cu recidiv (p<0,001).Pacienii care au prezentat
un scor de necroz mai mare au avut o rat mai mare a recidivelor (p<0,001). Pacienii
care au prezentat un scor al componentei mucinoase mai mare au avut o rat mai mare
a recidivelor (p<0,001). Pacienii care au prezentat un scor de desmoplazie mai mare
au avut o rat mai mic a recidivelor (p<0,001).
La analiza multivariata pacienii aflai n stadiul IIIC au avut probabilitatea mai
mare de a dezvolta recidive comparativ cu cei n stadiul I (HR - 9,75; IC 95% 1,2377,35; p=0,03). Pacienii cu scor Klintrup=<1 au avut probabilitatea mai mare de a
dezvolta recidive comparativ cu cei cu Kintrup >1 (HR - 0,10; IC 95% 0,04-0,25;
p<0,001). Pacienii cu scor Petersen >1 au avut probabilitatea mai mare de a dezvolta
recidive comparativ cu cei cu scor Petersen <1 (HR 1,92; IC 95% 1,17-3,61;
p=0,01).Pacienii cu scor de necroz 3 au avut probabilitatea mai mare de a dezvolta
recidive comparativ cu cei cu scor 0 (HR 2,84; IC 95% 1,31-6,16; p=0,008). Pacienii

cu scor desmoplazic 3 au avut probabilitatea mai mic de a dezvolta recidive dect cei
cu scor 1 (HR - 0,43; IC 95% 0,22-0,95; p=0,004).

Studiu 3. Analiza i evaluarea rolului prognostic al genelor MLH1,

MSH2, MSH6 i PMS2 n cancerul colorectal operat
Ipoteza de lucru
n literatura de specialitate exist un hiatus n ceea ce privete studiile care
cerceteaz diferenele fenotipice dintre cancerele colorectale instabile microsatelitar
sporadic i transmis genetic (motenit). Studiul propus are ca scop identificarea
diverselor caracteristici fenotipice i clinico-patologice cu rol prognostic n cancerele
colorectale instabile microsatelitar sporadice, comparativ cu cele motenite.

Material i metod
Au fost luai n studiu un numr de 103 pacieni diagnosticai cu cancer
colorectal stadiul I-III, internai i operai cu viz radical n Clinica Chirurgie V a
Spitalului Clinic Municipal Cluj-Napoca, n perioada mai 2005-decembrie 2008.
Studiul genetic s-a efectuat prin metode imunohistochimice pentru evidenierea
expresiei genelor MLH1, MLH2, MLH6, PMS2. Pentru probele care au prezentat lipsa
expresiei genice pentru MLH1, considernd c aceasta poate s apar att prin
hipermetilarea secvenei promoter a genei reparatoare MLH1 (sporadic), ct i prin
transmitere autosomal dominant, motenit, s-a efectuat o a doua examinare
constnd n testarea mutaiei genei BRAF V600E. La pacienii care asociaz lipsa
expresiei MLH1 cu prezena mutaiei V600E a BRAF se consider c mecanismul de
apariie a cancerului este prin hipermetilarea secvenei promoter a genei reparatoare
MLH1, deci sporadic.

Rezultate
Au rezultat astfel trei loturi de pacieni: un lot de 18 pacieni, reprezentnd cu
cancere instabile microsatelitar aprute sporadice, un al doilea lot de 16 pacieni cu
cancere microsatelitar instabile cu defecte genetice motenite, i un al treilea lot de 69
(67%) de pacieni cu cancere stabile microsatelitar (MSS).
Dintre pacienii din grupul MSI sporadic 2 (11.1%) au avut cancer rectal, iar
16 (88,9%) cancer colonic. Dintre pacienii din grupul MSI motenit 4 (25%) au avut
cancer rectal, iar 12 (75%) cancer colonic, iar n grupul MSS, 44 (63,7%) de pacieni au
avut cancer rectal i 25(36,3%) de pacieni cancer de colon.
Cea mai frecvent localizare n grupul MSI a fost la nivelul colonului drept, 9
(50%) cazuri si 5 (31,2%) cazuri in tumorile MSI mostenite.
Din cei 18 pacieni din grupul cu tumori MSI sporadice, 10 pacieni
reprezentnd 55,55% au fost stadializai n stadiul I i II, 7 pacieni (38,85%) n stadiul
IIIB, i un pacient n stadiul IIIC. n cazul tumorilor colonice MSI ereditare, 9 (56,2)
pacieni au fost clasificai n stadiile I i II, 7 pacieni (43,7%) au fost cuprini n stadiul
III. n grupul tumorilor MSS, 9 (13%)pacieni au fost n stadiul I, 31 de pacieni au fost
n stadiul II (45%) i respectiv 29 (42%) de pacieni n stadiul III.

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Am identificat diferene semnificative statistic n ceea ce privete aspectul

macroscopic vegetant al tumorii ntre grupul de pacieni cu MSI sporadic i grupul de
pacieni cu tumori stabile microsatelitar (p=0,05).
n cadrul pacienilor cu tumori MSI sporadice se observ o supravieuire la 5 ani
de 83,3% i o rat de recidiv de 5,6 %. La pacienii cu tumori colorectale MSI
transmise ereditar se evideniaz o rat de supravieuire la 5 ani de 56,2%, iar rata de
recidiv a fost de 37,5% (p=0,035 la testul Fisher). Pacienii din grupul cu tumori MSI
motenite au avut probabilitatea mai mare s prezinte recidive la 5 ani, comparativ cu
cei din grupul cu tumori MSI sporadice (6 (37,5%) vs. 1 (5,6%) (p=0,03).
n grupul pacienilor cu tumori colorectale stabile microsatelitar s-a obinut o
rat de supravieuire la 5 ani de 63,8% i o rat de recidiv de 40,6%.
Comparnd supravieuirile i recidivele ntre lotul cu tumori MSI sporadice i
lotul cu tumori MSS se obine o diferen semnificativ statistic n cazul recidivei
(p=0,004 la testul Fisher exact). Pacienii din grupul MSS au avut probabilitate mai
mare s prezinte recidive la 5 ani dect cei din grupul cu tumori MSI sporadice (28
(40,6)vs 1(5,6)) (p-0,01).
Analiznd n detaliu lotul de pacieni cu tumori colorectale MSI cu defecte
genetice transmise ereditar, observm c n 9 din 16 cazuri s-a pus n eviden gena
PMS2 singura ca fiind afectat. n aceste cazuri, supravieuirea la 5 ani a fost de 44,4%,
iar rata de recidiv fiind de 55,5%, cu 33,3% rata de recidiv la distan. n dou cazuri,
gena afectat PMS2 s-a nsoit i de defectul genei MLH1 n aceste situaii
supravieuirea a fost de 50% n condiiile n care atunci cnd este afectat doar gena
MLH1, supravieuirea este de 100%.

Studiu 4. Rolul analizei ganglionului santinel n cancerul

colorectal operat
Ipoteza de lucru
Pornind de la datele existente n literatur, ne-am propus s identificm i s
analizm ganglionul santinel la pacienii cu cancer colorectal operat cu viz curativ,
prin dou metode distincte, in vivo i ex vivo

Material i metod
Pentru realizarea obiectivelor propuse am efectuat un studiu prospectiv, pe un
numar de 22 de pacienti internati n Clinica Chirurgie V a Spitalului Clinic Municipal
Cluj-Napoca n perioada septembrie 2012-martie 2014. Studiul a inclus pacieni
diagnosticai cu cancer colorectal stadiul I-III, internai i operai cu viza radical.
Pentru identificarea ganglionului santinel am folosit dou tehnici diferite: metoda in
vivo i ex vivo, folosind colorant vital albastru de metil 1%.
Pentru pacienii n care ganglionii santinel i ceilali ganglioni au fost negativi,
s-a efectuat un studiu imunohistochimic pentru identificarea micrometastazelor.
Ne-am propus s studiem rata de identificare; acurateea; sensibilitatea; rata
rezultatelor fals negative; suprastadializarea, precum si s comparm cele dou
metode in vivo i ex vivo n identificarea ganglionului santinel la pacienii cu cancer
colorectal operat.

Rezultate
n urma aplicrii criteriilor de includere i excludere au rezultat un numr de 22
de pacieni care au intrat n studiu. La 11 pacieni am efectuat tehnica in vivo de
identificare a ganglionului santinel, iar la 11 pacieni tehnica ex vivo. Din punct de
vedere topografic, n 3 (13,6%) cazuri formaiunea tumoral s-a localizat la nivelul
cecului, n 3 (13,6%) cazuri la nivelul colonului ascendent, n 3 (13,6%) cazuri la
nivelul transversului, ntr-un caz la nivelul flexurii colice stngi, ntr-un caz la nivelul
descendentului, n 8 (36,6%) cazuri la nivelul sigmei i n 3 (13,6%) cazuri la nivelul
rectului.
Din cei 22 de pacieni, unul a fost stadializat T1, 5 n stadiul T2 i ceilali 16 n
stadiul T3. Cinci pacieni au fost ncadrai n stadiul I, conform stadializrii TNM, 6
pacieni n stadiul IIA, un pacient n stadiul IIIA, 9 pacieni stadializati IIIB i unul n
stadiul IIIC.
Identificarea ganglionilor santinel s-a realizat n 8 din 11 cazuri (72,7%) prin
tehnica in vivo i n 9 din 11 de cazuri (81,8%) prin tehnica ex vivo.
Nu s-au identificat diferene semnificative statistic ntre tehnica in vivo i cea ex
vivo n ceea ce privete rata de identificare, acurateea, sensibilitatea, rezultatele fals
negative i suprastadializarea. Pentru tehnica ex vivo, acurateea a fost de 88,8%,
sensibilitatea a fost de 83,3%, rezultatele fals negative au fost de 16,7%. Pentru tehnica
in vivo, acurateea a fost de 75%, sensibilitatea a fost de 75%, iar rezultatele fals
negative au fost de 25%.
n studiul nostru, la pacienii la care examinarea cu hematoxilin-eozin a
ganglionilor nu a identificat invazie ganglionar (N0), am efectuat o examinare
imunohistochimic pentru identificarea micrometastazelor. Atit prin tehnica in vivo,
cit si prin tehnica ex vivo, am identificat cite un caz cu micrometastaze la nivelul
ganglionilor santinel, a rezultat astfel fenomenul de suprastadializare ceea ce a
determinat creterea sensibilitii n ambele cazuri.
n timpul aplicrii tehnicii in vivo am identificat ntr-un caz un ganglion
santinel situat n afara ariei limfatice a segmentului colic respectiv i notat ca i drenaj
limfatic aberant.

Concluzii generale
Din cercetarea nostr se desprind urmtoarele concluzii generale:
Supravieuirea la 5 ani a fost superioar pentru pacienii cu cancer localizat la
nivelul colonului (71,9%), comparativ cu pacienii cu cancer localizat la nivelul
rectului (58,4%). Localizarea colonic a reprezentat un factor prognostic
pozitiv, semnificativ i independent;
n studiul nostru, componentele celulare care alctuiesc sistemul inflamator
general s-au asociat semnificativ statistic, la analiza univariat, cu
supravieuirea la 5 ani, demonstrnd un rol prognostic infaust;
Am demonstrat c scorul Glasgow modificat este un factor prognostic
independent, negativ pentru supravieuirea pacienilor cu cancer colorectal
operai cu viz curativ (p<0,001). Considerm c poate fi utilizat de rutin n

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identificarea pacienilor cu risc care ar putea beneficia de urmrire

postoperatorie mai frecvent i de tratament adjuvant;
Pacienii cu anemie au avut o probabilitate mai mare de deces la 5 ani i am
demonstrat c nu exist relaie de cauzatitate ntre transfuziile de snge i
supravieuire i c impactul negativ prognostic se datoreaz circumstanelor
care necesit transfuzii sanguine i nu transfuziilor n sine;
Am atras atenia asupra problemelor i limitrilor existente n cadrul clasificrii
TNM prin faptul c supravieuirea pacienilor cu cancer colorectal n stadiile IIB
i IIC este inferioar stadiului III A, explicnd necesitatea identificrii unor noi
factori prognostici, care individual sau mpreun cu clasificarea existent TNM
s identifice subgrupe de pacieni cu risc ce ar putea fi urmrii ndeaproape i
care ar beneficia de noi opiuni terapeutice;
LNR asigur o stratificare prognostic superioar comparativ cu numrul de
ganglioni pozitivi. Utilizarea LNR alturi de stadializarea TNM poate reprezenta
un factor prognostic independent la pacienii cu cancer colorectal n stadiul III;
Rezultatele studiului nostru arat c scorul Klintrup care contabilizeaz
rspunsul inflamator local, la nivelul marginii de invazie tumoral, este asociat
cu supravieuirea global, putnd fi folosit ca i factor prognostic independent;
Am demonstrat c rspunsul inflamator local i sistemic reprezint factori
predictivi importani la pacienii cu cancer colorectal operai cu viz radical.
Invazia venoas a reprezentat un factor prognostic infaust i independent n
determinismul supravieuirii la 5 ani n lotul studiat (p<0,001 i p=0,003);
Scorul componentei mucinoase folosit pentru prima dat de ctre noi n studiul
de fa, s-a asociat semnificativ statistic cu supravieuirea;
Scorul desmoplazic, folosit n cercetarea noastr, poate fi folosit ca factor
prognostic independent i pozitiv n supravieuirea la 5 ani a pacienilor cu
cancer colorectal operat cu intenie curativ;
La lotul studiat 37,2% din pacieni au dezvoltat recidive, 14,2% recidive locale;
Intervalul liber de boal median a fost de 20 de luni pentru recidivele locale si
de 24 de luni pentru metastazele la distan;
Recidivele au fost mai frecvente cnd localizarea formaiunii tumorale a fost la
nivelul rectului, iar dintre recidivele la nivelul rectului cele mai frecvente sunt
recidivele la distan,
Recidivele locale au fost mai numeroase dect metastazele la distan n stadiile
IIB i IIC;
Calcularea LNR ofer o mai bun predicie a dezvoltrii de recidive;
Am demonstrat rolul prognostic independent al scorului Klintrup n dezvoltarea
recidivelor. Scorul Petersen s-a dovedit a fi un factor independent att n
apariia recidivelor n general, ct i n apariia recidivelor locale n mod special;
Invazia venoas, scorul de necroz mare i scorul desmoplazic (folosit pentru
prima dat n aceast form n lucrarea de fa) s-au asociat cu dezvoltarea
recidivelor att la analiza univariat, ct i la analiza multivariat;
Tumorile colorectale instabile microsatelitar aprute prin mecanism sporadic,
apar mai frecvent la femei din mediul urban, se localizeaz la nivelul colonului

drept, nu se localizeaz la nivelul rectului inferior, iar simptomul dominant cel

mai frecvent sunt durerile abdominale. Tumorile MSI sporadice sunt mai
frecvent vegetante, bine difereniate i nu prezint invazie perineural;
Tumorile colorectale instabile microsatelitar aprute prin mecanism ereditar
(motenite) apar mai frecvent la brbai din mediul urban i sunt localizate mai
frecvent la nivelul colonului drept, dar n rare cazuri pot fi localizate i la nivelul
rectului inferior. La aceste tipuri de tumori valorile LNR-ului au fost mai mari,
iar dimensiunile tumorilor n axul lung al colonului sunt mai mari;
Pacienii din grupul cu tumori instabile microsatelitar sporadice au avut o
supravieuire la 5 ani superioar fa de pacienii cu tumori instabile
microsatelitar motenite (83,3% vs 56,2%), iar rata recidivei la 5 ani la aceti
pacieni a fost mai mic fa de cei cu tumori instabile microsatelitar motenite
(5,6% vs 37,5%);
Pacienii n stadiul III cu tumori MSI motenite au rspuns mai puin la
chimioterapie, comparativ cu pacienii cu tumori MSI sporadice;
n studiul nostru, n grupul cu tumori MSI motenite, cea mai frecvent mutaie
s-a produs la nivelul genei PMS2 dintre genele reglatoare ale sistemului MMR;
Tehnica identificrii ganglionului santinel este fezabil i reprezint o metod
simpl i ieftin cu o rat ridicat de identificare de 81,8% prin tehnica ex vivo
i 72,7% prin tehnica in vivo ;
Tehnicile in vivo i ex vivo n identificarea ganglionului santinel pot fi folosite
n egal msur cu rezultate comparabile;
Aplicnd tehnica in vivo pentru identificarea ganglionului santinel se pot
identifica situaii de drenaj limfatic aberant care determin lrgirea zonei
rezecate n scopul obinerii unei rezecii complete;
Utiliznd teste de imunohistochimie, pentru identificarea micrometastazelor,
am putut realiza o suprastadializare a pacienilor iniial stadializai N0, crescnd
astfel sensibilitatea tehnicilor de identificare a ganglionilor santinel.

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ABSTRACT OF THE DOCTORAL THESIS

Prognostic factors in operated

colorectal cancer

Doctoral candidate Bogdan Vasile Micu

Doctoral supervisor Prof. Dr. Aurel Andercou

CONTENTS
INTRODUCTION
CURRENT STATE OF KNOWLEDGE

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1. Prognostic factors in colorectal cancer

1. 1. Clinical factors
1.1.1. Age
1.1.2. Sex
1.1.3. Symptomatology
1.1.3.1. Duration of symptomatology
1.1.4. Obstruction and perforation
1.1.5. Location of the primary tumor
1.1.6. Anemia and perioperative blood transfusion
1. 2. Histopathological factors
1.2.1. Tumor stage
1.2.2. Factors related to the primary tumor
1.2.2.1. Tumor invasion depth
1.2.2.2. Size of the primary tumor
1.2.2.3. Macroscopic appearance of the primary tumor
1.2.2.4. Histopathological appearance of the primary tumor
1.2.2.5. Resection margins residual tumor
1.2.2.6. Configuration of tumor invasion margins
1.2.2.7. Tumor budding
1.2.2.8. Tumor necrosis
1.2.2.9. Tumor differentiation degree
1.2.3. Regional lymph nodes
1.2.3.1. Number of lymph nodes
1.2.3.2. Topography of invaded lymph nodes
1.2.3.3. Lymph node ratio (LNR)
1.2.3.4. Micrometastases
1.2.3.5. Isolated tumor deposits in lymph nodes
1.2.3.6. Extracapsular lymph node invasion
1.2.3.7. Sentinel lymph node
1.2.3.8. Reactive lymphadenopathy
1.2.4. Vascular invasion
1.2.4.1. Venous invasion
1.2.5. Perineural invasion
1.2.6. Extramural tumor deposits
1.2.7. Tumor angiogenesis
1.2.8. Peritumoral immune reaction (local host immune response)
1. 3. Biological factors
1.3.1. Serum tumor markers
1.3.1.1. Carcinoembryonic antigen (CEA)
1.3.1.2. Carbohydrate antigen 19-9 (CA 19-9)
1.3.1.3. Carbohydrate antigen 242 (CA 242)
1.3.1.4. Carbohydrate antigen 50 (CA50)

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1.3.1.5. Carbohydrate antigen 125 (CA 125)

1.3.2. Biochemical tumor markers
1.3.2.1. Thymidylate synthase
1.3.3. DNA content
1.4. Molecular factors
1.4.1. Tumor suppression genes
1.4.1.1. p53 gene
1.4.1.2. p27 gene
1.4.1.3. 18qLOH (loss of heterozygozity) and the deleted in colorectal cancer
(DCC) gene
1.4.1.4. Microsatellite instability
1.4.1.5. DNA hypermethylation
1.4.2. Oncogenes
1.4.2.1. Ras
1.4.2.2. BRAF

2. Host inflammatory response in colorectal cancer

2. 1. Systemic inflammatory response
2. 2. Local inflammatory response

3. Microsatellite instability in colorectal cancer

3. 1. Pathogenesis of microsatellite instability
3. 2. Methods for the identification of microsatellite instability
3. 3. Phenotype of microsatellite unstable colorectal cancers
3. 4. New approaches in the management of microsatellite unstable colorectal cancers

4. Sentinel lymph node in colorectal cancer

4. 1. Arguments for the identification of the sentinel lymph node in colorectal cancer
4. 2. Techniques for the identification of the sentinel lymph node
4. 3. Histopathological examination
4. 4. Perspectives of the identification of the sentinel lymph node in colorectal cancer

PERSONAL CONTRIBUTION
1. Work hypothesis/objectives
2. General methodology
3. Study 1. Evaluation of clinical and pathomorphological factors in
operated colorectal cancer
3.1. Introduction
3.2. Work hypothesis
3.3. Material and method
3.4. Results
3.5. Discussions
3.6. Conclusions

4. Study 2. Analysis of the prognostic factors of recurrence in

operated colorectal cancer
4.1. Introduction
4.2. Work hypothesis
4.3. Material and method

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4.4. Results
4.5. Discussions
4.6. Conclusions

5. Study 3. Analysis and evaluation of the prognostic role of MLH1,

MSH2, MSH6 and PMS2 genes in operated colorectal cancer
5.1. Introduction
5.2. Work hypothesis
5.3. Material and method
5.4. Results
5.5. Discussions
5.6. Conclusions

6. Study 4. Role of the analysis of the sentinel lymph node in

operated colorectal cancer
6.1. Introduction
6.2. Work hypothesis
6.3. Material and method
6.4. Results
6.5. Discussions
6.6. Conclusions

7. General conclusions
8. Originality and innovative contributions of the thesis
REFERENCES

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Key words: colorectal cancer, prognostic factors, survival, recurrence, inflammatory

response, microsatellite instability, sentinel lymph node

Introduction
Colorectal cancer has become a major public health problem worldwide because
of its high prevalence, mortality and morbidity. In Romania, colorectal cancer ranks
third in terms of incidence and mortality from malignant tumors, while survival has
not significantly improved over the past 5 years.
Although therapeutic procedures are well established and are uniformly applied
to all patients, the results regarding survival and the incidence of local or distant
recurrences differ, most likely due to prognostic factors depending on the patient, as
well as on tumor characteristics.
After curative surgery, the tumor stage of patients with colorectal cancer can be
accurately established. Frequently, patients with early stages will subsequently
develop local and/or distant recurrences, while patients with initial advanced stages
will survive without recurrences, most probably due to the implication of factors that
play a role in the evolution of patients with radically operated colorectal cancer.
Approximately 15-20% of all colorectal cancers develop through an alternative
pathway of tumorigenesis, characterized by microsatellite instability (MSI). During
DNA replication, errors in microsatellite sequences may occur. The DNA MMR
(mismatch repair) system, which consists of genes that repair the mismatches of
nitrogenous bases, plays the role of repairing errors that occur during replication. A

18

dysfunctional MMR system leads to the appearance of errors in microsatellites, a

phenomenon termed microsatellite instability, which is responsible for the
accumulation of somatic mutations in oncogenes or suppressor genes with an
important role in tumor initiation and progression. These tumors are classified as MSI
(microsatellite unstable), and those that do not show this phenomenon are considered
to be microsatellite stable (MSS).
The recurrence rate is over 30% in patients with colorectal cancer stage I and II
subjected to resection with a curative intent. An accurate staging would require
multiserial examinations with hematoxylin-eosin staining, immunohistochemistry
techniques and polymerase chain reaction. The concept of sentinel lymph node might
offer a solution, allowing to redefine staging and to identify a group of patients that
could benefit from adjuvant therapy. The procedures for the identification of the
sentinel lymph node are not standardized, the methods, materials, techniques and
selection of patients differing between institutions and surgeons.

Work hypothesis/objectives
Evaluation and analysis of prognostic factors involved in operated colorectal
cancer;
Identification and analysis of new prognostic factors in operated colorectal
cancer;
Prognostic value and role of local and general inflammatory response in
operated colorectal cancer;
Analysis and prognostic role of the lymph node ratio (LNR) in operated
colorectal cancer stage III;
Analysis and evaluation of phenotypic and prognostic differences between
microsatellite unstable colorectal tumors occurring through a sporadic or
hereditary mechanism and microsatellite stable colorectal tumors, in operated
colorectal cancer;
Identification of the sentinel lymph node in colorectal cancer using the in vivo
and the ex vivo method;
Evaluation and analysis of the prognostic role of the sentinel lymph node in
colorectal cancer surgery.

General methodology
To achieve the proposed objectives, we studied patients hospitalized and
operated in the Surgical Clinic of the Cluj-Napoca City Clinical Hospital, the 5th Surgical
Department of the Iuliu Haieganu University of Medicine and Pharmacy Cluj-Napoca,
and we collaborated with the Service of Pathological Anatomy of the Cluj-Napoca City
Clinical Hospital and the Santomar Laboratory of Pathological Anatomy Cluj-Napoca.
All patients included in the study signed an informed consent, and the study was
approved by the Ethics Commission of the Cluj-Napoca City Clinical Hospital. The
diagnosis of colorectal cancer was made preoperatively by clinical examination,
laboratory and paraclinical examinations (chest X-ray, abdominal ultrasound, lower
digestive endoscopy with biopsy). The exclusion criteria were as follows: patients with
distant metastases detected pre- or intraoperatively, synchronous primary tumors,
inflammatory intestinal disorders, patients with other histopathological types of
cancer than adenocarcinoma, patients operated in emergency who were administered
preoperative radiotherapy, patients deceased less than 30 days postoperatively, those
with incomplete data, and those who did not sign the informed consent.

The data were analyzed using the observation sheets, operative protocols and
anatomo-pathological reports. Thus, a data base including demographic data, clinicoanamnestic data, laboratory and paraclinical examinations, intraoperatively described
aspects, pathomorphological examination, number of examined lymph nodes, ratio
between the number of invaded lymph nodes and the number of examined lymph
nodes defined as the lymph node ratio (LNR) was created.
The following were included in the study: leukocyte count defined as three value
ranges (<8500/mm3; 8500-11000/mm3; >11000/mm3), lymphocyte count
(<1000/mm3; 1000-3000/mm3; >3000/mm3), neutrophil count (<7500/mm3;
>7500/mm3), ratio between neutrophils and lymphocytes, hemoglobin, hematocrit,
thrombocyte count (150000-370000/mm3), presence of anemia.
A new microscopic anatomo-pathological analysis was performed, with the
analysis of the tumor invasion margin and the revision of TNM staging according to the
latest edition, the 7th, of the AJCC Cancer Staging Manual, in force since 1 January
2010.
The Petersen index was calculated, which is constructed from the scores
assigned to four pathomorphological variables. The total score was calculated by the
cumulation of these scores, which resulted in a Petersen index ranging between 0 and
5, subdivided into low risk (0-1) and high risk (2-5).
Local inflammatory response at the tumor invasion margin was calculated using
the Klintrup criteria, by quantifying the local inflammatory infiltrate. The scores 0 and
1 were considered as reduced local inflammatory response, and the scores 3 and 4, as
increased local inflammatory response.
Tumor necrosis was also quantified, as follows: 0 for no necrosis; 1 for focal
necrosis, less than 10%; 2 for moderate necrosis, between 10-30%; 3 for extensive
necrosis, over 30%. The mucinous component was also assessed, so that 0 was
assigned to the absence of the mucinous component; 1 to a minimal mucinous
component, less than 10%; 2 to a moderate mucinous component, between 10-15%;
and 3 to an extensive mucinous component, over 50%. Desmoplasia score: 0 for no
desmoplasia, 1 for reduced or minimal desmoplasia, 2 for moderate desmoplasia, and
3 for marked/extensive desmoplasia. For stage III patients, the lymph node ratio (LNR)
was calculated by dividing the number of tumor-invaded lymph nodes to the total
number of removed lymph nodes. Based on this criterion, the patients were divided
into 5 groups, having the following value ranges: <0.10, 0.11-0.21, 0.22-0.36, 0.37-0.6,
and >0.61, respectively.
For a subgroup of patients, the preoperative systemic inflammatory response
was also calculated by using the modified Glasgow prognostic score (mGPS), calculated
depending on C-reactive protein and albumin values.
The patients were followed up during a 5-year period, at 3 and 6 months
postoperatively during the first year, then once a year.
Statistical analysis was performed using the SPSS software, version 20. Data
were considered as nominal, ordinal or quantitative. Quantitative data were tested for
the distribution normality with the Kolmogorov-Smirnov test. Quantitative variables
were described by median and 25th and 75th percentiles (non-normal distribution).
Nominal and ordinal variables were characterized by frequency and percentage.

20

Study 1. Evaluation of clinical and pathomorphological prognostic

factors in operated colorectal cancer
Work hypothesis
Starting from the available literature data, we aimed to evaluate and analyze the
prognostic factors involved in the survival of patients operated for colorectal cancer
with a curative intent. We also aimed to identify and analyze new prognostic factors
implicated in colorectal cancer.

Material and method

To achieve the proposed objectives, we performed a retrospective observational
study including 301 patients diagnosed with colorectal cancer stage I-III, hospitalized
and radically operated in the 5th Surgical Clinic of the Cluj-Napoca City Clinical
Hospital, in the period January 1999 - December 2008.

Results
The study included 197 patients (66.4%) who survived for a 5-year follow-up
period, and 104 patients (34.6%) who deceased.
The leukocyte count recorded in the deceased patients was highly statistically
significantly higher compared to the values measured in survivors, 8700 (6925;
11000)/mm3 vs. 7050 (6100; 8275)/mm3 (p<0.001).
The neutrophil/lymphocyte ratio calculated in deceased patients was highly
statistically significantly higher than in survivors [3.3 (1.5; 6) vs. 2.7 (2.1; 3.4)]
(p<0.001).
Of the survivors, 83 (42.1%) had rectal cancer and 114 (57.9%) had colon
cancer. Of the deceased patients, 59 (56.7%) had rectal cancer and 45 (43.3%) had
colon cancer. Of patients with colonic neoplasms, 54 (34%) had right side location. In
these patients, there were 21 deaths (46.7%), a statistically significantly higher
frequency compared to other locations (p=0.03).
Survival by stages was 87.8% for stage I, 81.5% for stage IIA, 63.33% for stage
IIB, 44.4% for stage IIC. For stage III, the following results were obtained: 80% for
stage IIIA, 51.38% for stage IIIB, and 34.88% for stage IIIC.
A highly statistically significant difference was found regarding the survival of
patients with T>2 [93 (89.4%)], compared to those with T=<2 [11 (10.6%)] (p<0.001).
A highly statistically significant difference was evidenced regarding the survival
of patients with N>0 [67 (64.4%)], compared to those with N=0 [37 (35.6%)]
(p<0.001). The survival rate was statistically significantly higher for group N1
compared to group N2 (p<0.001).
We determined a HR of 12.3 (95% CI 4.9-31) for patients with a LNR >0.61
compared to those with a LNR <0.1 (p<0.001).
A high Klintrup score was found in 9 patients (8.7%) of the deceased group,
while 136 survivors (69%) had a high Klintrup score (p<0.001). A high risk of the
Petersen score was determined in 52 patients of the deceased group (50%) and in 11
surviving patients (5.6%) (p<0.001).
Venous invasion was described in 14 surviving patients (7.1%) and in 60
deceased patients (57.7%) (p<0.001). Perineural invasion was reported in 32 patients,
of which 8 (4.1%) were surviving patients and 24 (23.1%) were deceased patients
(p<0.001).
Patients with a higher necrosis score had a higher death rate (p<0.001).
Patients with a higher score of the mucinous component had a higher death rate
(p<0.001). Patients with a lower desmoplasia score had a higher death rate (p<0.001).

In multivariate analysis, colon cancer location was associated with a better

survival than rectal location (HR 0.57; 95% CI 0.35-0.94; p=0.02). Patients with T>2
had an infaust prognosis compared to those with T=<2 (HR 2.23; 95% CI 1.11-4.47;
p=0.02). Patients with a Klintrup score >1 had a better prognosis compared to those
with a Klintrup score = <1 (HR 0.20; 95% CI 0.09-0.44; p<0.001). Patients with
venous invasion had a worse prognosis compared to those without venous invasion
(HR 2.26; 95% CI 1.31-3.91; p=0.003). Patients with a desmoplasia score of 3 had a
lower death rate than those with a score of 1 (HR 0.42; 95% CI 0.22-0.85; p=0.01).

Study 2. Analysis of the prognostic factors of recurrence

in operated colorectal cancer
Work hypothesis
Starting from the literature data, we aimed to evaluate and analyze the
prognostic factors involved in local and distant recurrences in patients with curative
surgery for colorectal cancer. We also aimed to identify and analyze new prognostic
factors involved in the recurrence of colorectal cancer operated with a curative intent.

Material and method

To achieve the proposed objectives, we performed a retrospective observational
study that included 301 patients diagnosed with colorectal cancer stage I-III,
hospitalized and radically operated in the 5th Surgical Clinic of the Cluj-Napoca City
Clinical Hospital in the period January 1999 - December 2008.
Distant recurrences were identified by general local examination, paraclinical
examinations (laboratory examinations, chest X-ray, general ultrasound, computed
tomography, scintigraphy), and local recurrences were determined by abdominal
ultrasound and/or lower digestive endoscopy with biopsy. The period from surgery to
the development of locoregional or distant recurrences was calculated in months and
was considered recurrence-free survival.

Results
112 patients (37.2%) developed recurrences over the 5-year follow-up period,
and 189 patients (62.8%) did not develop recurrences.
Of the 112 recurrence cases, 43 (38.4%) were local recurrences and 69 (61.6%)
were distant recurrences.
In 159 patients, the tumor mass was located in the colon, and there were 46
recurrences, of which 20 distant recurrences. In 142 cases (47.2%), the tumor was in
the rectum, and 66 of the cases had recurrences, of which 44 distant recurrences. Of
the patients without recurrences, 76 (40.2%) had rectal cancer and 113 (59.8%) had
colon cancer.
The leukocyte count recorded in patients with recurrences was highly
statistically significantly higher compared to that found in patients without
recurrences, 8600 (6800; 11000)/mm3 vs. 7100 (6200; 8375)/mm3 (p<0.001).
The neutrophil/lymphocyte ratio calculated in patients with recurrences was
highly statistically significantly higher than in patients without recurrences [3.4 (2.6;
5.9) vs. 2.6 (2.1; 3.4)] (p<0.001).
We determined a highly statistically significant difference between the stages of
the disease regarding recurrence (p<0.001).

22

Of the studied group, 162 patients were classified as stage I and II (N0); of these,
33 had recurrences, 19 local and 14 distant recurrences. Of 139 patients, 82 were N1, of
which 34 (41.4%) had recurrences - 14 local recurrences and 20 distant recurrences.
Stage N2 was assigned to 57 patients, of which 45 (57.7%) had recurrences - local
recurrences 10 and distant recurrences 35 cases (p<0.001).
For patients with stage III, we calculated a LNR of 0.4 (0.21; 0.58) in those with
recurrences, and a LNR of 0.09 (0.07; 0.16) in those without recurrences. Patients with
a higher LNR had a higher frequency of recurrences (p<0.001).
The recurrence rate was highly statistically significantly higher in patients with
a lower Klintrup score (p<0.001). The recurrence rate was highly statistically
significantly higher in patients with a higher Petersen score (p<0.001).
Venous invasion was described in 7 patients without recurrences (3.7%) and in
67 patients with recurrences (59.8%) (p<0.001). Perineural invasion was reported in 2
patients without recurrences (1.1%) and in 30 patients with recurrences (26.8%)
(p<0.001). Patients with a higher necrosis score had a higher recurrence rate
(p<0.001). Patients with a higher score of the mucinous component had a higher
recurrence rate (p<0.001). Patients with a higher desmoplasia score had a lower
recurrence rate (p<0.001).
In multivariate analysis, patients with stage IIIC had a higher probability to
develop recurrences compared to patients with stage I (HR 9.75; 95% CI 1.23-77.35;
p=0.03). Patients with a Klintrup score =<1 had a higher probability to develop
recurrences compared to those with a Klintrup score >1 (HR 0.10; 95% CI 0.04-0.25;
p<0.001). Patients with a Petersen score >1 had a higher probability to develop
recurrences compared to those with a Petersen score <1 (HR 1.92; 95% CI 1.17-3.61;
p=0.01). Patients with a necrosis score of 3 had a higher probability to develop
recurrences compared to those with a score of 0 (HR 2.84; 95% CI 1.31-6.16;
p=0.008). Patients with a desmoplasia score of 3 had a lower probability to develop
recurrences than those with a score of 1 (HR 0.43; 95% CI 0.22-0.95; p=0.004).

Study 3. Analysis and evaluation of the prognostic role of MLH1,

MSH2, MSH6 and PMS2 genes in operated colorectal cancer
Work hypothesis
There is a gap in the literature regarding studies on the phenotypic differences
between sporadic and genetic (inherited) microsatellite unstable colorectal cancers.
The proposed study aims to identify the various phenotypic and clinico-pathological
characteristics with a prognostic role in sporadic compared to inherited microsatellite
unstable colorectal cancers.

Material and method

The study included 103 patients diagnosed with colorectal cancer stage I-III,
hospitalized and radically operated in the 5th Surgical Clinic of the Cluj-Napoca City
Clinical Hospital, in the period January 1999 December 2008.
The genetic study was performed by immunohistochemical methods in order to
evidence MLH1, MLH2, MLH6, PMS2 gene expression. For the samples showing a lack
of MLH1 gene expression, considering that this might occur both through the
hypermethylation of the MLH1 repair gene promoter sequence (sporadic) and through
autosomal dominant inheritance, a second examination consisting of the testing of the
V600E mutation in the BRAF gene was performed. In patients with a lack of MLH1
expression associated with the presence of the V600E mutation in BRAF, the

mechanism of cancer is considered to be through the hypermethylation of the MLH1

repair gene promoter sequence, consequently sporadic.

Results
Thus, three groups of patients resulted: a group of 18 patients, representing
sporadic microsatellite unstable cancers, a second group of 16 patients, with
microsatellite unstable cancers with inherited genetic defects, and a third group of 69
patients (67%) with microsatellite stable (MSS) cancers.
Of the patients in the sporadic MSI group, 2 (11.1%) had rectal cancer, and 16
(88.9%) had colon cancer. Of the patients in the inherited MSI group, 4 (25%) had
rectal cancer and 12 (75%) had colon cancer, while in the MSS group, 44 patients
(63.7%) had rectal cancer and 25 patients (36.3%) had colon cancer.
The most frequent location in the MSI group was in the right colon, 9 cases
(50%) for sporadic MSI tumors and 5 cases (31.2%) for inherited MSI tumors.
Of the 18 patients in the group with sporadic MSI tumors, 10 patients
representing 55.55% were graded stage I and II, 7 patients (38.85%) stage IIIB, and
one patient stage IIIC. In the case of inherited MSI colon tumors, 9 patients (56.2) were
classified as stage I and II, and 7 patients (43.7%) were classified as stage III. In the
group of MSS tumors, 9 patients (13%) had stage I, 31 patients (45%) had stage II, and
29 patients (42%) had stage III.
Statistically significant differences were found regarding the macroscopic
vegetative appearance of the tumor between the sporadic MSI cancer group and the
group of patients with microsatellite stable tumors (p=0.05).
Among patients with sporadic MSI tumors, there was a 5-year survival of 83.3%
and a recurrence rate of 5.6%. In patients with inherited MSI colorectal tumors, the 5year survival rate was 56.2% and the recurrence rate was 37.5% (p=0.035 on Fishers
exact test). Patients in the group with inherited MSI tumors had a higher probability to
develop recurrences at 5 years compared to those in the group with sporadic MSI
tumors, 6 (37.5%) vs. 1 (5.6%) (p=0.03).
In the group of patients with microsatellite stable colorectal tumors, a 5-year
survival rate of 63.9% and a recurrence rate of 40.6% were obtained.
By comparing the survival and recurrence rates between the group with
sporadic MSI tumors and the group with MSS tumors, a statistically significant
difference was obtained in the case of recurrences (p=0.004 on Fishers exact test).
Patients in the MSS group had a higher probability to develop recurrences at 5 years
compared to those in the group with sporadic MSI tumors (28 (40.6) vs. 1(5.6))
(p=0.01).
A detailed analysis of the group of patients with MSI colorectal tumors with
inherited genetic defects showed that in 9 of 16 cases, the PMS2 gene was the only one
affected. In these cases, the 5-year survival rate was 44.4%, and the recurrence rate
was 55.5%, with a 33.3% distant recurrence rate. In two cases, the affected PMS2 gene
was accompanied by an MLH1 gene defect, resulting in a 50% survival rate, under the
conditions in which survival is 100% when the MLH1 gene alone is affected.

Study 4. Role of the analysis of the sentinel lymph node in operated

colorectal cancer
Work hypothesis
24

Starting from the available literature data, we aimed to identify and analyze the
sentinel lymph node in patients with colorectal cancer operated with a curative intent,
using two distinct in vivo and ex vivo methods.

Material and method

To achieve the proposed objectives, we performed a retrospective study on 22
patients admitted to the 5th Surgical Clinic of the Cluj-Napoca City Clinical Hospital in
the period September 2012 March 2014. The study included patients diagnosed with
colorectal cancer stage I-III, hospitalized and operated with a radical intent. To identify
the sentinel lymph node, two different techniques were used: the in vivo and the ex vivo
method, using a vital stain methylene blue 1%.
For patients with negative sentinel and other lymph nodes, an
immunohistochemical study was performed for the identification of micrometastases.
We aimed to study the identification rate, accuracy, sensitivity, the rate of false
negative results, overstaging, as well as to compare the two in vivo and ex vivo methods
for the identification of the sentinel lymph node in patients with operated colorectal
cancer.

Results
Following the application of inclusion and exclusion criteria, 22 patients were
enrolled in the study. In 11 patients, the in vivo technique for the identification of the
sentinel lymph node was used, and in 11 patients, the ex vivo technique was applied.
From a topographic point of view, the tumor was located in 3 cases (13.6%) in the
cecum, in 3 cases (13.6%) in the ascending colon, in 3 cases (13.6%) in the transverse
colon, in one case in the left colic flexure, in one case in the descending colon, in 8 cases
(36.6%) in the sigmoid colon, and in 3 cases (13.6%) in the rectum.
Of the 22 patients, one was staged T1, 5 T2, and the other 16 T3. Five patients
were classified as stage I, according to TNM staging, 6 patients as stage IIA, one patient
as stage IIIA, 9 patients as stage IIIB, and one patient as stage IIIC.
Sentinel lymph nodes were identified in 8 of 11 cases (72.7%) using the in vivo
technique, and in 9 of 11 cases (81.8%) using the ex vivo technique.
There were no statistically significant differences between the in vivo and the ex
vivo technique regarding the identification rate, accuracy, sensitivity, false negative
results and overstaging. For the ex vivo technique, accuracy was 88.8%, sensitivity was
83.3%, the rate of false negative results was 16.7%. For the in vivo technique, accuracy
was 75%, sensitivity was 75%, and the rate of false negative results was 25%.
In our study, patients in whom the hematoxylin-eosin examination of lymph
nodes detected no lymph node invasion (N0) were subjected to immunohistochemical
examination for the identification of micrometastases. The in vivo and the ex vivo
technique identified one case each with micrometastases in the sentinel lymph nodes,
which resulted in overstaging, determining an increase of sensitivity in both cases.
During the application of the in vivo technique, we identified in one case a
sentinel lymph node situated outside the lymphatic area of the colic segment
concerned, which was noted as aberrant lymphatic drainage.

General conclusions
Survival at 5 years was better for patients with cancer located in the colon
(71.9%) compared to patients with cancer located in the rectum (58.4%). Colon
location was a significant and independent positive prognostic factor;

 In our study, the cellular components of the general inflammatory system were
statistically significantly associated, in univariate analysis, with 5-year survival,
demonstrating an infaust prognostic role;
We demonstrated that the modified Glasgow score was an independent negative
prognostic factor for the survival of patients with colorectal cancer operated
with a curative intent (p<0.001). We consider that it can be used as routine for
the identification of patients at risk who might benefit from more frequent
postoperative follow-up and adjuvant treatment;
Patients with anemia had a higher probability of death at 5 years and we
demonstrated that there was no causal relationship between blood transfusion
and survival and that the negative prognostic impact was due to the
circumstances requiring blood transfusion, not to transfusion itself;
We drew attention to the problems and limitations of the TNM classification
through the fact that the survival of patients with colorectal cancer stage IIB and
IIC was lower compared to stage IIIA, and we explained the need to find new
prognostic factors that, individually or in association with the existing TNM
classification, might identify subgroups of patients at risk, who could be
followed up closely and could benefit from new therapeutic options;
LNR ensures a better prognostic stratification compared to the number of
positive lymph nodes. The use of LNR along with TNM staging may be an
independent prognostic factor in patients with colorectal cancer stage III;
The results of our study show that the Klintrup score which assesses local
inflammatory response at the tumor invasion margin is associated with overall
survival and can be used as an independent prognostic factor;
We demonstrated that local and systemic inflammatory response was an
important predictive factor in patients with colorectal cancer operated with a
radical intent;
Venous invasion was an infaust and independent prognostic factor for 5-year
survival in the studied group (p<0.001 and p=0.003);
The score of the mucinous component, used for the first time by us in this study,
was statistically significantly associated with survival;
The desmoplasia score, used in our research, can be used as an independent
positive prognostic factor for 5-year survival in patients with colorectal cancer
operated with a curative intent;
In the studied group, 37.2% of the patients developed recurrences, 14.2% being
local recurrences;
The median disease-free survival was 20 months for local recurrences and 24
months for distant metastases;
Recurrences were more frequent when the tumor was located in the rectum,
and among recurrences in the rectum, distant recurrences were the most
frequent;
Local recurrences were more numerous than distant metastases in stages IIB
and IIC;
The calculation of LNR allows for a better prediction of the development of
recurrences;
We demonstrated the independent prognostic role of the Klintrup score in the
development of recurrences. The Petersen score proved to be an independent
factor both for the development of recurrences in general and local recurrences
in particular;

26

 Venous invasion, a high necrosis score and desmoplasia score (used for the first
time in this form in this study) were associated with the development of
recurrences both in univariate analysis and multivariate analysis;
Microsatellite unstable colorectal tumors occurring by sporadic mechanism are
more frequent in women from urban environment, they are located in the right
colon, they are not located in the lower rectum, and the most frequent dominant
symptom is abdominal pain. Sporadic MSI tumors are more frequently
vegetative, well differentiated and have no perineural invasion;
Microsatellite unstable colorectal tumors by inherited mechanism are more
frequent in men from urban environment and are more frequently located in the
right colon, but in rare cases, they can also be located in the lower rectum. In
these types of tumors, LNR values were higher, and tumor dimensions along the
long axis of the colon were larger;
Patients in the group with sporadic microsatellite unstable tumors had a better
5-year survival than patients with inherited microsatellite unstable tumors
(83.3% vs. 56.2%), and the 5-year recurrence rate in these patients was lower
compared to patients with inherited microsatellite unstable tumors (5.6% vs.
37.5%);
Patients with inherited MSI tumors stage III had a poorer response to
chemotherapy compared to patients with sporadic MSI tumors;
In our study, in the group of inherited MSI tumors, the most frequent mutation
occurred in the PMS2 gene among the regulatory genes of the MMR system;
The technique for the identification of the sentinel lymph node is feasible and
represents an easy and inexpensive method with a high identification rate of
81.8% using the ex vivo technique and 72.7% using the in vivo technique;
The in vivo and ex vivo techniques for the identification of sentinel lymph nodes
can be equally used with comparable results;
The in vivo technique for the identification of the sentinel lymph node allows
the detection of aberrant lymphatic drainage situations, which leads to an
extension of the resected area in order to obtain complete resection;
By using immunohistochemistry tests for the identification of micrometastases,
we were able to perform an overstaging of patients initially staged N0,
increasing in this way the sensitivity of the techniques for the identification of
sentinel lymph nodes.