review

J Korean Soc Coloproctol: Vol. 25, No. 2, 129-38, 2009 DOI: 10.3393/jksc.2009.25.2.129 REVIEW 대장암에서유전자불안정성의임상적의의 이강영 연세대학교의과대학외과학교실 Genomic Instability in Colorectal Cancer; from Bench to Bed Kang Young Lee, M.D. Departments of Surgery, Yonsei University College of Medicine, Seoul, Korea Colorectal cancer is a disease developed by the accumulation of genomic alteration. Two genomic instability pathways, chromosomal instability pathway and microsatellite instability pathway, are known as the main pathways of the development of colorectal cancer. These are almost always mutually exclusive and tumors developed through each pathways show distinct clinicopathologic features. For the reason, molecular markers which represent each genomic instability pathways have been a candidate for translational research to find out prognostic or predictive factors. Loss of heterozygosity and aneuploidy are the hallmark of chromosomal instability and regarded as poor prognostic markers, whereas tumors with high frequency of microsatellite instability show better prognosis than microsatellite stable tumor. As a predictive factor of response from chemotherapy, loss of heterozygosity seems to be associated with a survival benefit from 5-FU adjuvant therapy. MSI-H has been reported as a predictive factor for poor response to 5-FU adjuvant chemotherapy. However, these molecular markers are not accepted to use in the clinic yet, since some of this kind of studies reported contradictory results. Further study will be needed to make more concrete evidences for these markers and to identify new molecular markers for routine use in the clinic. Keywords: Colorectal cancer; Microsatellite instability; Chromosomal instability; Molecular marker 중심단어 : 대장암, 미소위성체불안정성, 염색체불안정성, 분자생물학적표지자 서 대장암은우리나라에서 2005년기준으로인구 10만명당 36.2명의새로운환자가발생하여발생빈도가가장빠르게증가하고있는암가운데하나이며발생빈도와암사망율모두 4번째로높은암이다. 1 대장암은대장의정상점막에서선종 (early adenoma) 이발생하고이형성증이동반된선종 (late adenoma) 에서선암 (adenocarcinoma) 으로진행하는단계적인형태학적변화의과정을거치며발생하는것으로알려져있다. 대장암의발생및진행과정에대한연구는단지대 Received : January 20, 2009 Accepted : April 10, 2009 Correspondence to : Kang Young Lee, M.D. Department of Surgery, Yonsei University College of Medicine, 134 Sinchon-dong, Seodaemun-gu, Seoul 120-752, Korea Tel : +82.2-2228-2123, Fax : +82.2-313-8289 E-mail : kylee117@yuhs.ac 론 The Korean Society of Coloproctology 장암의발생과정의이해뿐아니라대장암의예방, 치료표적및예후인자의발굴등대장암치료를위해서도중요하다. 지난수십년간의분자생물학연구결과의축적으로이와같은대장암의발생, 성장및전이의과정은다양한유전자변이의축적에의한것임이잘알려져있다. 2 지금까지알려진대표적인대장암발생기전은염색체불안정성 (chromosomal instability), 미소위성체불안정성 (microsatellite instability), CpG island 메틸화표현형 (CpG island methylator phenotype; CIMP) 등이알려져있다. 이처럼분자생물학적발암과정이서로다른암들은유전자변이의형태및임상병리학적인특성에도서로차이를보인다. 이러한이유로발암과정이서로다른암에서보이는분자생물학적인특성을기초로이를대장암의예후, 치료반응의예측등여러분야에서임상에적용하고자하는연구가계속되고있다. 이에본고찰에서는대장암의발암과정을살펴보고이의임상의적용을위한 129

130 Kang Young Lee: Genomic Instability in Colorectal Cancer; from Bench to Bed 연구결과들을고찰해보고자한다. 본론대장암의형성과정 (Colorectal tumorigenesis) 사람의정상체세포분열과정에서돌연변이의발생가능성은매우낮지만암세포에서는수천의유전자변이가발견되며이와같은유전자변이의축적에의하여암이발생하는것으로알려져있다. 암세포에서체세포돌연변이의빈도는암종에따라차이가있고대장암의경우 1.21/Mb의빈도로체세포돌연변이가관찰된다고한다. 3,4 대장암의발생은대장점막에서선종이발생하고이후선암이발생하는형태학적변화를거치며이는이와같은유전자변이의축적에의한다. 2 대장암의발생은염색체불안정성 (chromosomal instability), 미소위성체불안정성 (microsatellite instability) 에의한것으로알려져있고최근촉진유전자의과메틸화 (promoter hypermethylation) 를특징으로하는 CIMP도중요한부분으로알려져있다. 5 염색체불안정성 (chromosomal instability pathway) 산발성대장암의약 85% 는염색체불안정 (chromosomal instability) 으로인하여발생하는것으로알려져있다. 4 염색체불안정성에의한발암과정은유전자변이의축적으로 K- ras와같은종양유전자 (proto-oncogene) 의활성화와 APC (chromosome 5q), p53 (chromosome 17p), DCC, SMAD2, SMAD 4 (chromosome 18q) 등종양억제유전자 (tumor suppressor gene) 의불활성화에의하여정상점막에서선암으로진행되는과정이다. 2,6,7 이과정에의해발생한암은염색체의부분결실이나증폭으로홀배수체 (aneuploidy) 를보이며, 이형접합성소실 (loss of heterozygosity) 이흔히발견되고결과적으로유전자변이를유발한다. 8 이러한염색체이상은정상대장점막에서는발견되지않지만선종, 선암으로진행하며빈도가증가하고암의진행에따라더욱증가된다. 9 염색체불안정성이발생하는기전은아직명확히밝혀져있지는않지만다양한연구가진행되고있다. mitotic checkpoint protein (BUB1, BUB1b) 을 encoding하는유전자의변이와 centrosome-associataed serine thronine kinase (Aurora kinase A) 의과발현이염색체불안정성의기전이될수있다고보고된다. 7,10,11 또한 telomere dysfunctions, microtubule dynamic instability, kinetochore structure and function, chromosome condensation and sister chromatid cohesion 등이염색체불안정성의원인을설명하는기전으로제시되고있다. 12-14 이외에도 APC, TP53, FBXW7, CHFR 등이이과정에관여할가능성이제시되는등많은연구가진행되고있는분야이다. 15-18 염색체불안정성을확인하기위하여는미소위성체 (microsatellite) 를이용한 LOH검사 (Fig. 1), array based comparative genomic hybridization 등 19 다양한실험방법을쓸수있다. 연구방법의많은발전으로동시에많은검체의결과를얻을수있고정확도도증가되었지만아직검사방법의한계, 비용문제등의제한이있다. 예를들면 array CGH 방법은복제수의변화 (copy number variation) 를확인하는데매우예민한검사이지만다수의상호전좌 (reciprocal translocation) 가있는경우에는 allele copy number 또는 DNA 양에거의변화가없어서염색체불안정성이없는것으로보일수있다. 20 또한미소위성체 (microsatellite) 를이용한 LOH 진단의경우에도위음성, 또는 non-informative의경우에도검사결과에오류를가져올수있다. 종양의이질성과함께이러한연구방법의제한은연구결과를얻는데제한이되고있으나연구방법의발전과함께극복될수있으리라기대한다. 미소위성체불안정성 (microsatellite instability pathway) 미소위성체 (microsatellite) 는 1-6개정도의짧은염기서열이반복되는 DNA의일부를지칭하는말이다. 미소위성체불안정성 (microsatellite instability) 은이러한반복되는염기의삽입또는상실로인하여정상세포와비교하여암세포에서반복되는염기의길이변화가초래된상태를말한다. 21 DNA 복제과정에서미소위성체에서돌연변이의발생은여러가지요인에의하여결정되지만가장중요한것은 DNA 복제과정의이상을교정하는시스템의효율성에있으며염색체불일치복구유전자 (mismatch repair gene) 가정상적으로기능을하는경우미소위성체에서의돌연변이를 100-1,000배감소시킬수있다. 22 미소위성체불안전성은유전성비용종성대장암 (hereditary non-polyposis colorectal cancer) 의표지자로써발견되었으며이는특정유전자에만국한된현상이아니라종양세포의유전자에전체적으로발견되는이상이다. 23,24 유전성비용종성대장암은전체대장암가운데약 2-3% 의빈도로보고되며 hmlh1, hmsh2, hmsh6, hpms2, hpms1 등염색체불일치복구유전자의배선돌연변이 (germline mutation) 에의한염색체불일치복구유전자의기능상실로인하여미

이강영 : 대장암에서유전자불안정성의임상적의의 131 소위성체불안정성이발생하고이는상염색체우성 (autosomal dominant) 으로유전된다. 25,26 미소위성체불안정성은산발성대장암에서는약 10-15% 빈도로보고되며이는주로 hmlh1 유전자의후천적인촉진유전자의과메텔화 (acquired promoter hypermethylation) 에의한전사억제 (transcriptional silencing) 에의한것으로알려져있다. 27-29 MSI는전유전자에걸쳐여러부분에서동시에발생하지만특정유전자 (TGF-bRII, Bax, IGF II receptor, PTEN) 를암호화하는영역 (coding sequences) 에서발생하여이유전자의기능발현을억제하여암의진행에관여하기도한다. 30 미소위성체불안정성검사는암과정상조직에서얻어진 genomic DNA를중합효소연쇄반응 (polymerase chain reaction) 으로증폭한후전기영동 (electrophoresis) 을하여정상조직과암조직의미소위성체 (microsatellite) 길이를비교하여차이가있으면미소위성체불안정성으로판정한다 (Fig. 1). 검사를하는표지자의종류, 수는연구자에따라차이가있으나 Bethesda guideline에따르면 2개의 mononucleotide marker (BAT25, BAT26) 와 3개의 dinucleotide marker (D5S346, D2S123, D17S250) 을기준이되는표지자로제시하였고검사를시행한표지자가운데 40% 이상에서불안정성 (instability) 이있으면 MSI-H, 40% 미만인경우 MSI-L, 불안정성이없는경우 MSS로분류한다고하였다. 31 CIMP (CpG island methylator phenotype) 염색체불안정성, 미소위성체불안정성과함께 DNA 메틸화, 히스톤변형등유전자의후생학적변화 (epigenetic change) 가대장암의발암기전의하나로알려져있다. 5 정상조직과비교하여암조직에서는 DNA에전반적인저메틸화 (global hypomethylation) 와특정유전자와관련된부분에서과메텔화 (regional hypermethylation) 를보인다. 32 이가운데 DNA 과메틸화 (hypermethylation) 는 retinoblastoma 유전자촉진자 (promoter) 의과메틸화가보고된이래암생성과관련된많은유전자가과메틸화로인하여비활성화되어있음이증명되었다. 33 CpG island 는 DNA의 5 촉진자영역에존재하는 CpG 염기서열을다수포함하는 0.3-4 kb 길이의유전자를지칭하며약 50% 의유전자에서발견된다. 34 CpG islands는정상적으로메틸화가안된상태로존재하며메틸화가안된상태를유지하는기전은아직확실히알려져있지는않다. CpG islands의과메틸화는유전자의기능억제를유발하며 5 대장암에서 p16 (CDKN2A), p14/arf, hmlh1, MGMT 등많은유전자에서과메틸화와이로인한유전자의기능억제가확인되어있다. 35 이와같은 CpG islands의과메틸화의많은부분은노화과정에서나타나는현상으로정상대장조직에서도관찰되지만노화와관련된부분을제외하고나면대장암조직 A B Fig. 1. Microsatellite instability and loss of heterozygosity analysis with capillary electrophoresis. (A) microsatellite instability; change of allele length or aberrant peak compared to corresponding normal (B) loss of heterozygosity; complete loss of one allele or more than 50% loss of one allele compared to corresponding normal.

132 Kang Young Lee: Genomic Instability in Colorectal Cancer; from Bench to Bed 에서만과메틸화가관찰되는유전자군이있고 36 이들은임상병리학적으로뿐만아니라분자생물학적으로도독특한특성을대장암환자군을대표하게되며이를 CpG island methylator phenotype (CIMP) 양성으로분류한다. 37,38 유전자변이의임상적응용현재까지대장암의근치적수술후예후예측및치료방침의결정에가장널리쓰이고있는방법은 TNM 병기분류방법이다. 이는절제된암조직의조직병리검사에의하여암침윤깊이, 영역림프절전이, 원격전이여부등을종합하여병기를구분하는방법으로지금까지예후를비교할수있는가장좋은방법으로알려져있다. 하지만이방법에의해서도같은병기를가진환자에서서로상이한예후를보이는경우가있고치료반응은특히예측하기가어렵다. 위에서언급된두유전자불안정성 (genomic instability) 에의한대장암은한암종에서동시에두가지기전의특성을보이는경우는거의없으며서로다른임상병리학적인특징을갖는다 (Table 1). 39-42 이처럼종양발생기전이다른암종은생물학적인특성이달라서각각의과정에관여하는유전자들이나특징적인유전자변화를이용하여예후또는치료반응의예측을위한표지자로써사용하기위한연구들이진행되어왔다. 염색체불안정성 (chromosomal instability) 의임상적용 염색체불안정성 (Chromosomal instability) 에의한대장암은염색체의결실 (deletion), 삽입 (insertion), 또는재배열 (rearrangement) 등을특징으로하며이과정에서암억제유전자 (tumor suppressor gene) 의기능소실또는약화, 혹은암유전자 (oncogene) 의활성화 (amplification) 에의하여암 Table 1. Characteristics of CIN and MSI MSI-H MSS P value References Gender (M:F) 1:2.1 1:0.66 0.004 30 Proximal location 70-89% 18-37% <0.001 30, 31, 32 Lymph nodes 35.3% 57.5% <0.001 31 metastasis Poorly differentiated 26-43.8% 8-18% <0.001 30, 31, 32 Mucinous histology 31-67% 15-18% <0.05 30, 32 Crohn s like lymphoid 69.2% 44.4% <0.0007 32 reaction Absence of dirty 82.7% 23.4% <0.001 32 necrosis B-raf mutation 43.5% 5% 33 K-ras mutation 11.4% 34.7% 33 P53 mutation 12.3% 48.9% 33 발생에관여한다. 13 홀배수체 (anuploidy) 는염색체불안정성에의한대장암의특징중하나이며홀배수체 (anuploidy), 이형접합성소실 (loss of heterozygosity) 을동반한암은예후가불량하다. 43 대장암에서 18q 염색체의이형접합성소실 (loss of heterozygosity) 의빈도는 70% 까지보고되며이는대장암의발암과정에서뿐아니라예후인자로써도중요하다. 44 18q chromosome에는 DCC (18q21.3), SMAD2 (18q21.1), SMAD4 (18q21.1), Cables (18q11-12) 등의유전자가존재하며 45,46 이유전자들의발현결핍이있거나 18q LOH가있는대장암은예후가불량하다고보고된다 (Table 2). 47-52 또한 II기대장암환자에서 18q LOH가있는환자의예후가유의하게나쁨을보고하여 18q LOH가 II 기대장암환자에서수술후항암보조요법의적응이되는고위험환자를찾는지표가될수있다고하였다. 53 17p 염색체의이형접합성소실은대장암에서빈번히관찰되며 TP53 유전자변이는약 50% 의빈도로보고된다. 54 TP53 유전자는 17p13에위치하며세포주기 (cell cycle) 의조절, 세포자멸사 (apoptosis), 발암과정 (carcinogenesis) 등에관여한다. 55,56 예후인자로써 TP53 유전자변이는불량한예후를시사하며 52,57-59 이는다른예후인자들에독립적인예후인자로보고된다 (Table 3). 60 대장암에서이형접합성소실 (loss of heterozygosity) 은 5- FU 항암제에대한치료반응의예측인자로써가능성도제 Table 2. 18q LOH (DCC) as a prognostic factor in colorectal cancer Number of patients TNM stage Impact on prognosis Reference 42 IV (palliative) Poor prognosis Aschele et al. 38 170 II Poor prognosis Reymond et al. 39 132 II/III Poor prognosis Shibata et al. 40 460 II/III Poor prognosis Watanabe et al. 41 151 I-IV Shorter survival Ogunbiyi et al. 42 220 I-IV Shorter survival Diep et al. 43 145 II/III Shorter survival Jen et al. 44 Table 3. LOH of 17p (TP53) as a prognostic factor in colorectal cancer Number of patients TNM stage Impact on prognosis Reference 91 IV Poor prognosis Mollevi et al. 48 213 I-IV Poor prognosis Chang et al. 49 220 I-IV Shorter survival Diep et al. 43 73 II Shorter survival Choi et al. 50

이강영 : 대장암에서유전자불안정성의임상적의의 133 시되었다. UK AXIS trial에포함되었던 393명의 stage II, III 환자를대상으로한연구결과에서는 17p 또는 18q LOH 가 5-FU 치료에대하여좋은치료효과를예측할수있는예측인자로보고하였다. 61 미소위성체불안정성 (microsatellite instability) 의임상적용 미소위성체불안정성 (MSI-H) 을보이는종양은 MSI-L/ MSS 종양과비교하여독특한임상병리학적인특성을보이며 62 환자의예후도좋은것으로보고된다 (Table 4). 40,63-65 진단당시의병기분포, 국소림프절전이의빈도등대장암의진행정도를시사하는지표들을비교해보면 MSI-H 종양이 MSS 종양보다진행된암의빈도가낮으며, 예후인자로써 MSI-H는다른임상적인지표들에독립적인예후인자임을보인다. 40 MSI-H 종양의예후가좋은이유는아직확실하지는않지만면역학적인특성이이를설명하는기전이될수있다. MSI- H 종양을 H&E 염색을하여관찰할때특징중하나는 peritumoral lymphocyte infiltration (Crohn s like inflammation) 소견이다. 66-68 이러한염증반응이 MSI-H 종양의예후가좋은이유의하나로설명되며특히 granzyme B, perforin 등을발현하는 CD8-positive lymphocytes의세포독성이중요한역할을하는것으로생각된다. 67-69 염색체불일치복구유전자 (MMR) 는항암제의세포내작용에대하여도중요한역할을하는것으로알려져있다. Base analogue (6-TG), alkylating agent (MNNG), adduct-forming drug (cisplatin) 등과같은 DNA-damaging agent는염색체불일치복구유전자에결손이있는세포에서약물반응이저하되는것으로보고된다. 70-72 이러한항암제에의하여유발된 DNA의변화 (adduct formation, incorporation) 는그자체로세포사멸을유도하지는못하지만염색체불일치복구체계가이러한 DNA의변화를인식하여이를교정하고자한다. 하지만항암제에의한 DNA의변화는염색체불일치복구체계에의하여교정되어지지않기때문에결국세포에서는 cell cycle arrest가일어나고세포자멸사 (apoptosis) 가 Table 4. Microsatellite instability as a prognostic factor in colorectal cancer 유도되는것으로설명된다. 73 하지만염색체불일치복구유전자에변이가일어난세포에서는이러한과정이일어나지않기때문에항암제에저항성을가지는것으로이해된다. 대장암에서가장많이쓰여지는항암제인 5-FU의경우 5-FU 대사물이바로 DNA에결합하거나또는 dttp 생성억제에의한 dntp의불균형을초래하고이러한 DNA의이상이염색체불일치복구유전자에의하여확인되면 DNA 나선구조의손상, G2 arrest 등을유발하고궁극적으로세포사멸 (cell death) 을유발하는것으로생각된다 (Fig. 2). 염색체불일치복구기전에문제가있는경우이러한세포사멸로유도하는과정에장애가생겨 5-FU 항암제에저항성을갖게된다. 74 염색체불일치복구유전자와항암제내성과의연관성은 hmlh1 유전자가포함된정상 chromosome 3를삽입하거나, 5-Aza- 2 -deoxycytidine으로탈메틸화 (demethylation) 시켜서염색체불일치복구유전자의기능을복원함으로써항암제내성이없어지고효과를회복하는것으로도증명되어있다. 70,75 염색체복제유전자변이에의한항암제에대한내성은세포주실험에서뿐아니라대장암환자의검체를이용한실험에서도확인되고있다. Ribic 등은 5개의 randomized study 에서얻어진 570명의 stage II, III 환자를대상으로 MSI 상태가 5-FU 치료반응을예측할수있는지확인하였다. 76 이들의결과를보면 MSS/MSI-L 종양에서는 5-FU adjuvant therapy를한경우유의한생존율의증가를확인할수있었으나 MSI-H 종양에서는생존율의증가가없었고오히려 5- FU 치료를하지않은환자군의생존율이더좋았다. 이는 MSI-H 종양을가진환자의경우항암제치료에의한효과는없고항암제의부작용으로인하여예후가나쁠수있을것으로추정하였다. MSI-H 종양의 5-FU 치료에대한내성은다른연구들에의하여도세포주실험에서와같은결과가보고되었다. 50,77,78 하지만 5-FU 반응을예측하는인자로써 MSI- H에대한연구결과는보고에따라차이가있어 Kim 등은 NSABP에서진행되었던 4개의 randomized trial에서추출 Covalent complex with FdUMP 5-FU TS inhibition Accumulation of dump dutp incorporation MMR DNA Breaks Death Number of patients TNM stage Impact on prognosis Reference 607 I-IV Favorable Gryfe et al. 31 154 I-IV Favorable Samowitz et al. 54 255 III Favorable Wright et al. 55 dntp pool Imbalance by depletion of dttp Polymerase errors G2 arrest 216 I-IV Favorable Gafa et al. 56 Fig. 2. A model of the proposed role of MMR in 5-FU mediated cell death and cell cycle responses.

134 Kang Young Lee: Genomic Instability in Colorectal Cancer; from Bench to Bed 된 542명환자의검체를가지고동일한내용의연구를진행하였고이들의결과는 MSI 상태가 5-FU 반응을예측하지못한다고보고하였다. 79 지금까지미소위성체불안정성 (microsatellite instability) 에대한대부분의연구는 MSS/MSI-L 종양에대한 MSI-H 종양의특성에대한것이다. MSI-L 종양은 MSS와정의에서는다르지만 38 임상병리학적인특성에거의차이가없어서 MSS 종양과같은범주로여겨지는경우가많다. 하지만 MSI- L 종양에서 MSS에비하여 K-ras 유전자변이의빈도가높고 80,81 예후에서도차이가있다는보고가있다. 82,83 대장암에서 MSI-L의임상적의의에대하여는앞으로연구가필요한부분이다. CIMP (CpG island methylator phenotype) CIMP는 Toyota 등에의하여대장암에서기술된이후염색체불안정성, 미소위성체불안정성과함께대장암이발생하는한기전으로알려져있다. 36 또한 CIMP 기전에의한대장암은단지과메틸화 (hypermethylation) 의특징뿐아니라임상병리학적 ( 고령, 여성, 우측대장에호발 ), 분자생물학적 (MSI, K-ras, p53, B-raf 유전자변이와연관 ) 으로특징적인모양을보인다. 35,84,85 이와같은 CIMP 대장암의특성과염색체불안정성, 미소위성체불안정성과의관계등에기초하여 Jass는대장암의분자생물학적분류를제안하기도하였다. 86 분자생물학적인표지자로써 CIMP의역할에대한연구는최근에많은결과가보고되고있지만문제는 CIMP를진단하기위한표지자의표준화가안되어있다는것이다. CIMP는 MSI와같이유전자의여러부분을검사하여양성과음성을구분하기때문에사용된표지자의종류및수가다르게되면결과의차이를보일수있고결국이러한차이로인하여객관적인결과의해석이어렵게된다. 이와같은문제를해결하기위하여 CIMP 표지자의표준화를위한연구가계속되고있고 87-89 조만간표준화된 CIMP panel에대한동의가이루어질것으로기대된다. 이를바탕으로 CIMP에대한중재연구또한더욱활발해질것이다. 결론대장암에대한분자생물학적인이해가깊어지고실험기법이발전하면서대장암의발생및진행과정에대한분자생물학적이해를위한연구에서많은환자의검체들을가지고실제임상에적용하기위한표지자를찾고자하는중재연구가 활발히진행되고있다. 하지만실험실에서의세포주실험과는달리고형암의이질성, 많은임상변수등으로인하여연구결과들이서로상충되는경우가많다. 이러한이유로 American Society of Clinical Oncology (ASCO), European Group on Tumour Markers (EGTM) 에서분자생물학적표지자의임상활용가능성에대하여검토를하였으나아직임상에서사용을권고하기에는증거가부족한것으로결론지어졌다. 90,91 하지만이와같은문제점들은후속연구들에의하여극복될수있을것이며표준화된치료에근거한임상자료들과실험실연구성과의유기적인연결이대장암환자각각에게맞춤형치료를근간에실현시킬수있을것이다. REFERENCES 1. Ministry for Health, Welfare and Family Affairs. Annual Report of cancer incidence (2005) and survival (1993-2005) in Korea. Seoul: Ministry for Health, Welfare and Family Affairs; 2008. 2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67. 3. Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, et al. Patterns of somatic mutation in human cancer genomes. Nature 2007;446:153-8. 4. Wang TL, Rago C, Silliman N, Ptak J, Markowitz S, Willson JK, et al. Prevalence of somatic alterations in the colorectal cancer cell genome. Proc Natl Acad Sci 2002;99:3076-80. 5. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999; 21:163-7. 6. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-32. 7. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, et al. Mutations of mitotic checkpoint genes in human cancers. Nature 1998;392:300-3. 8. Grady WM. Genomic instability and colon cancer. Cancer Metastasis Rev 2004;23:11-27. 9. Tsafrir D, Bacolod M, Selvanayagam Z, Tsafrir I, Shia J, Zeng Z, et al. Relationship of gene expression and chromosomal abnormalities in colorectal cancer. Cancer Res 2006;66:2129-37. 10. Nishida N, Nagasaka T, Kashiwagi K, Boland CR, Goel A. High copy amplification of the Aurora-A gene is associated with chromosomal instability phenotype in human colorectal cancers. Cancer Biol Ther

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