REZUMATUL TEZEI DE DOCTORAT

Factori prognostici n cancerul colorectal operat

Doctorand Bogdan Vasile Micu

Conductor de doctorat Prof. dr. Aurel Andercou

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CUPRINS INTRODUCERE 15 STADIUL ACTUAL AL CUNOATERII 17 1. Factori prognostici n cancerul colorectal 19 1. 1. Factori clinici 21 1.1.1. Vrsta 21 1.1.2. Sexul 21 1.1.3. Simptomatologia 21 1.1.3.1. Durata simptomatologiei 22 1.1.4. Obstrucia i perforaia 22 1.1.5. Localizarea tumorii primare 22 1.1.6. Anemia i transfuziile sanguine perioperatorii 23 1. 2. Factori histopatologici 23 1.2.1. Stadiul tumoral 23 1.2.2. Factori aparinnd tumorii primare 25 1.2.2.1. Profunzimea invaziei tumorale 25 1.2.2.2. Dimensiunea tumorii primare 25 1.2.2.3. Aspectul macroscopic al tumorii primare 26 1.2.2.4. Aspectul histopatologic al tumorii primare 26 1.2.2.5. Marginile de rezecie - tumora rezidual 26 1.2.2.6. Configuraia marginilor de invazie tumoral 28 1.2.2.7. Budding-ul tumoral 28 1.2.2.8. Necroza tumoral 28 1.2.2.9. Gradul de difereniere al tumorii 28 1.2.3. Ganglionii limfatici regionali 29 1.2.3.1. Numrul nodulilor limfatici 29 1.2.3.2. Topografia nodulilor limfatici invadai 29 1.2.3.3. Limph node ratio (LNR) 29 1.2.3.4. Micrometastazele 30 1.2.3.5. Depozite tumorale izolate n nodulii limfatici 30 1.2.3.6. Invazia extracapsular a nodulilor limfatici 30 1.2.3.7. Ganglionul santinel 30 1.2.3.8. Adenopatia limfatic reactiv 31 1.2.4. Invazia vascular 31 1.2.4.1. Invazia venoas 31 1.2.4.1. Invazia venoas 31 1.2.5. Invazia perineural 31 1.2.6. Depozitele tumorale extramurale 32 1.2.7. Angiogeneza tumoral 32 1.2.8. Reacia imun peritumoral (rspunsul imun local al gazdei) 32 1. 3. Factori biologici 32 1.3.1. Markeri serici tumorali 32 1.3.1.1. Antigenul carcinoembrionar (ACE) 32 1.3.1.2. Antigenul carbohidrat 19-9 (CA 19-9) 32 1.3.1.3. Antigenul carbohidrat 242 (CA 242) 32

1.3.1.4. Antigenul carbohidrat 50 (CA50) 33 1.3.1.5. Antigenul carbohidrat 125 (CA 125) 33 1.3.2. Markeri biochimici tumorali 33 1.3.2.1. Timidilat sintetaza 33 1.3.3. Coninutul de ADN 33 1.4. Factori moleculari 33 1.4.1. Gene de supresie tumoral 33 1.4.1.1. Gena p53 33 1.4.1.2. Gena p27 33 1.4.1.3. 18qLOH (pierderea heterozigozitii) i Gena Deleiei n cancerul colorectal (DCC) 33 1.4.1.4. Instabilitatea microsatelitar 34 1.4.1.5. Hipermetilarea ADN-ului 34 1.4.2. Oncogenele 34 1.4.2.1. Ras 34 1.4.2.2. BRAF 34 2. Rspunsul inflamator al gazdei n cancerul colorectal 35 2. 1. Rspunsul inflamator sistemic 35 2. 2. Rspunsul inflamator local 36 3. Instabilitatea microsateliilor n cancerul colorectal 37 3. 1. Patogeneza apariiei instabilitii microsatelitare 37 3. 2. Metode de identificare a instabilitii microsatelitare 37 3. 3. Fenotipul cancerelor colorectale instabile microsatelitar 38 3. 4. Noi abordri n managementul cancerelor colorectale instabile microsatelitar 38 4. Ganglionul santinel n cancerul colorectal 41 4. 1. Argumente pentru identificarea ganglionului santinel n cancerul colorectal 41 4. 2. Tehnici de identificare a ganglionului santinel 42 4. 3 Examenul histopatologic 42 4. 4. Perspectivele identificrii ganglionului santinel n cancerul colorectal 42 CONTRIBUIA PERSONAL 45 1. Ipoteza de lucru/obiective 47 2. Metodologie general 47 3. Studiu 1. Evaluarea factorilor prognostici clinici i morfopatologici n cancerul colorectal operat

51 3.1. Introducere 51 3.2. Ipoteza de lucru 51 3.3. Material i metod 52 3.4. Rezultate 52 3.5. Discuii 68 3.6. Concluzii 75 4. Studiu 2. Analiza factorilor prognostici ai recidivei n cancerul colorectal operat

79 4.1. Introducere 79 4.2. Ipoteza de lucru 79

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4.3. Material i metod 79 4.4. Rezultate 80 4.5. Discuii 95 4.6. Concluzii 99 5. Studiu 3. Analiza i evaluarea rolului prognostic al genelor MLH1, MSH2, MSH6 i PMS2 n cancerul colorectal operat

103 5.1. Introducere 103 5.2. Ipoteza de lucru 103 5.3 Material i metod 104 5.4 Rezultate 105 5.5 Discuii 115 5.6 Concluzii 118 6. Studiu 4. Rolul analizei ganglionului santinel n cancerul colorectal operat

121 6.1 Introducere 121 6.2 Ipoteza de lucru 121 6.3 Material i metod 122 6.4 Rezultate 125 6.5 Discuii 128 6.6 Concluzii 129 7. Concluzii generale 131 8. Originalitatea i contribuiile inovative ale tezei 135 REFERINE 137 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

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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 (11.000/mm3), numrul de limfocite (3000/mm3), numr de neutrofile (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 10-30%; 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 la0,61;

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 clinici i morfopatologici n cancerul colorectal operat 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

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Am determinat o diferen nalt semnificativ statistic n ceea ce privete supravieuirea pacienilor cu T>2 (93 (89,4%)), comparativ cu cei cu T=

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 s-au nregistrat 46 de recidive din care 20 la distan. n 142 de cazuri (47,2%) tumora s-a 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

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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.

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.

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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

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

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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.

ABSTRACT OF THE DOCTORAL THESIS

Prognostic factors in operated colorectal cancer

Doctoral candidate Bogdan Vasile Micu

Doctoral supervisor Prof. Dr. Aurel Andercou

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CONTENTS INTRODUCTION 15 CURRENT STATE OF KNOWLEDGE 17 1. Prognostic factors in colorectal cancer 19 1. 1. Clinical factors 21 1.1.1. Age 21 1.1.2. Sex 21 1.1.3. Symptomatology 21 1.1.3.1. Duration of symptomatology 22 1.1.4. Obstruction and perforation 22 1.1.5. Location of the primary tumor 22 1.1.6. Anemia and perioperative blood transfusion 23 1. 2. Histopathological factors 23 1.2.1. Tumor stage 23 1.2.2. Factors related to the primary tumor 25 1.2.2.1. Tumor invasion depth 25 1.2.2.2. Size of the primary tumor 25 1.2.2.3. Macroscopic appearance of the primary tumor 26 1.2.2.4. Histopathological appearance of the primary tumor 26 1.2.2.5. Resection margins residual tumor 26 1.2.2.6. Configuration of tumor invasion margins 28 1.2.2.7. Tumor budding 28 1.2.2.8. Tumor necrosis 28 1.2.2.9. Tumor differentiation degree 28 1.2.3. Regional lymph nodes 29 1.2.3.1. Number of lymph nodes 29 1.2.3.2. Topography of invaded lymph nodes 29 1.2.3.3. Lymph node ratio (LNR) 29 1.2.3.4. Micrometastases 30 1.2.3.5. Isolated tumor deposits in lymph nodes 30 1.2.3.6. Extracapsular lymph node invasion 30 1.2.3.7. Sentinel lymph node 30 1.2.3.8. Reactive lymphadenopathy 31 1.2.4. Vascular invasion 31 1.2.4.1. Venous invasion 31 1.2.5. Perineural invasion 31 1.2.6. Extramural tumor deposits 32 1.2.7. Tumor angiogenesis 32 1.2.8. Peritumoral immune reaction (local host immune response) 32 1. 3. Biological factors 32 1.3.1. Serum tumor markers 32 1.3.1.1. Carcinoembryonic antigen (CEA) 32 1.3.1.2. Carbohydrate antigen 19-9 (CA 19-9) 32 1.3.1.3. Carbohydrate antigen 242 (CA 242) 32 1.3.1.4. Carbohydrate antigen 50 (CA50) 33

1.3.1.5. Carbohydrate antigen 125 (CA 125) 33 1.3.2. Biochemical tumor markers 33 1.3.2.1. Thymidylate synthase 33 1.3.3. DNA content 33 1.4. Molecular factors 33 1.4.1. Tumor suppression genes 33 1.4.1.1. p53 gene 33 1.4.1.2. p27 gene 33 1.4.1.3. 18qLOH (loss of heterozygozity) and the deleted in colorectal cancer (DCC) gene 33 1.4.1.4. Microsatellite instability 34 1.4.1.5. DNA hypermethylation 34 1.4.2. Oncogenes 34 1.4.2.1. Ras 34 1.4.2.2. BRAF 34 2. Host inflammatory response in colorectal cancer 35 2. 1. Systemic inflammatory response 35 2. 2. Local inflammatory response 36 3. Microsatellite instability in colorectal cancer 37 3. 1. Pathogenesis of microsatellite instability 37 3. 2. Methods for the identification of microsatellite instability 37 3. 3. Phenotype of microsatellite unstable colorectal cancers 38 3. 4. New approaches in the management of microsatellite unstable colorectal cancers 38 4. Sentinel lymph node in colorectal cancer 41 4. 1. Arguments for the identification of the sentinel lymph node in colorectal cancer 41 4. 2. Techniques for the identification of the sentinel lymph node 42 4. 3. Histopathological examination 42 4. 4. Perspectives of the identification of the sentinel lymph node in colorectal cancer 42 PERSONAL CONTRIBUTION 45 1. Work hypothesis/objectives 47 2. General methodology 47 3. Study 1. Evaluation of clinical and pathomorphological factors in operated colorectal cancer

51 3.1. Introduction 51 3.2. Work hypothesis 51 3.3. Material and method 52 3.4. Results 52 3.5. Discussions 68 3.6. Conclusions 75 4. Study 2. Analysis of the prognostic factors of recurrence in operated colorectal cancer

79 4.1. Introduction 79 4.2. Work hypothesis 79 4.3. Material and method 79

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4.4. Results 80 4.5. Discussions 95 4.6. Conclusions 99 5. Study 3. Analysis and evaluation of the prognostic role of MLH1, MSH2, MSH6 and PMS2 genes in operated colorectal cancer

103 5.1. Introduction 103 5.2. Work hypothesis 103 5.3. Material and method 104 5.4. Results 105 5.5. Discussions 115 5.6. Conclusions 118 6. Study 4. Role of the analysis of the sentinel lymph node in operated colorectal cancer

121 6.1. Introduction 121 6.2. Work hypothesis 121 6.3. Material and method 122 6.4. Results 125 6.5. Discussions 128 6.6. Conclusions 129 7. General conclusions 131 8. Originality and innovative contributions of the thesis 135 REFERENCES 137 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

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.

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The data were analyzed using the observation sheets, operative protocols and anatomo-pathological reports. Thus, a data base including demographic data, clinico-anamnestic 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 (11000/mm3), lymphocyte count (3000/mm3), neutrophil count (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: 0.61, 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.

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

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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=1 had a better prognosis compared to those with a Klintrup score =

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

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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 5-year 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

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;

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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

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.