The Korean Journal of Pathology 2010; 44: DOI: /KoreanJPathol 간전이를동반한대장암에서의 VEGF-A, VEGFR-1, VEGFR-2 발현양상과미세혈관밀도의비교 정은희 김영

The Korean Journal of Pathology 2010; 44: 571-80 DOI: 10.4132/KoreanJPathol.2010.44.6.571 간전이를동반한대장암에서의 VEGF-A, VEGFR-1, VEGFR-2 발현양상과미세혈관밀도의비교 정은희 김영 민병우 이경화김현수 1 이재혁 전남대학교의과대학병리학교실및 1 내과학교실소화기분과 접수 : 2010년 3월 23일게재승인 : 2010년 8월 13일 책임저자 : 이재혁우 519-809 전남화순군화순읍일심리 160 화순전남대학교병원병리과전화 : +82-61-379-7073 Fax: +82-61-379-7099 E-mail: jhlee@chonnam.ac.kr * 본논문은보건복지부암정복추진연구개발사업의지원에의해연구되었음 ( 과제번호 : 0720570). Differences in Expression of VEGF-A, VEGFR-1, VEGFR-2 and Microvessel Density in Colorectal Cancer with Liver Metastasis Eun Hui Jeong Young Kim Byeong Woo Min Kyung Hwa Lee Hyun Soo Kim 1 Jae Hyuk Lee Departments of Pathology and 1 Division of Gastroenterology, Department of Internal Medicine, Chonnam National University Medical School, Hwasun, Korea Background : Colorectal cancer (CRC) is one of the most common malignant neoplasms and is a leading cause of mortality worldwide. Metastasis to the liver is a frequent event in patients with CRC. An essential step in the metastatic cascade is angiogenesis. Methods : This study included 45 patients who underwent a partial colectomy with hepatic resection for CRC with hepatic metastases. Immunohistochemistry was performed using vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, VEGFR-2, and CD34 antibodies to examine the relationship between CRC with liver metastases and angiogenesis. Results : CRC showed significantly stronger expression of VEGF-A, VEGFR-1, and VEGFR-2 than liver metastases (p < 0.05). Microvessel density was also higher in CRC than in liver metastases (p < 0.05). Conclusions : Compared with previous studies, we found a higher expression of VEGF-A, VEGFR-1, VEGFR-2, and microvessel density in CRC than in liver metastases, which could be ascribed to a difference in vessel distribution and blood supply in each organ. Given its profuse blood supply and distinct cell populations, the liver might provide a rich milieu for tumor cell growth with less expression of angiogenesis-inducing agents. Key Words : Thymus gland; Neoplasms; Immunohistochemistry; World Health Organization; Histological classification 대장암은전세계적으로가장흔한암중하나이며, 현재우리나라에서도꾸준히증가추세에있다. 1 또한대장암은다른장기로전이를잘하는암으로알려져있는데, 그중간전이가흔하게동반된다. 실례로대장암진단당시약 25% 의환자에게서간전이가동반되어있다고알려져있고, 25% 이상의환자가진단후 2년이내에간전이가발생한다고보고되어있다. 2 또이런간전이는암관련사망의중요원인이되고있다. 3 암세포가원격전이를하려면암세포의유리, 국소침습, 이동, 신생혈관생성, 혈관침습, 혈관내상피세포로의유착, 그리고다른장기내에서의성장등여러단계를거쳐야하는데, 4 이중신생혈관생성은 2-3 mm 3 이상의암이성장을하기위해서는반드시필요한과정이다. 5 신생혈관은이미만들어져있는혈관에서부터새로운모세혈관이만들어지는것인데, 이과정은혈 관형성유도인자 (angiogenesis inducing agent) 의영향을받고, 이렇게만들어진혈관은암세포의성장, 암세포의혈관침습과전이등에도움을준다. 6 신생혈관생성에는많은혈관형성유도인자가관여를하는데그중에서도혈관내피세포성장인자 (vascular endothelial growth factor, VEGF) 가매우중요한역할을한다. 현재까지 VEGF 에는 5가지의아형 (VEGF-A, -B, -C, -D, and F) 이밝혀져있고, 각각의아형과반응하는수용체는 3가지 (VEGF receptor [VEGFR]-1, VEGFR-2, and VEGFR-3) 가존재한다고알려져있다. 7 VEGF-A는 VEGFR-1, -2를통해서신호전달체계를활성화시키는데, 이는혈관내피세포의증식, 이동, 생존을촉진시킨다. 8 또 VEGF-C와 D는 VEGFR-2, VEGFR-3와결합하여 571

572 정은희 김영 민병우외 3 인 신생림프관및혈관형성을유도한다. 9 그리고 VEGF는대장암에서높게나타나는데, 이중 VEGF-A는다른아형에비해특히더높게나타나며, 이렇게높은 VEGF-A는대장암의침윤성이나진행정도에큰영향을미친다. 9-11 본연구에서는간전이가없는대장암과간전이가있는대장암에서 VEGF-A, VEGFR-1 및 VEGFR-2의발현양상차이와그에따른미세혈관밀도의연관성을통해각단백들이대장암에서전이를예측할수있는표지자로서의기능을할수있는지그가능성에대해알아보고, 대장암의원발부위와간전이부위에서각단백들의발현양상차이와그에따른미세혈관밀도의차이및연관성, 임상병리학적변수사이의연관성을알아보고자하였다. vidin-horseradish peroxidase를각각 10분간작용시켰으며, horseradish peroxidase의발색은 diaminobenzidine을이용하였다. 발색반응이끝난다음헤마톡실린대조염색을시행하였는데, 염색의전과정에있어서부치온도는 45 로하였고음성대조군은일차항체대신항마우스 IgG 와항래빗 IgG 희석액을반응시킨슬라이드를동일한과정을거쳐실험에이용하였다. 13 VEGF- A, VEGFR-1, VEGFR-2는정상대조염색이되었던혈관내피세포에비해강하게발현된경우를강양성 (3+) 으로판단하였고, 대조염색과비슷하게발현된경우는중양성 (2+), 대조염색보다약하지만양성을확인할수있는정도로발현된경우는약양성 (1+), 음성인경우는 0으로판단하였다. 재료및방법 Table 1. Clinicopathological characteristics of selected patients 재료 Factors Control group Metastasis group n % n % 전남대학교병원과화순전남대학교병원에서 2002년 1월부터 2007년 12월까지대장암의간전이로간절제와부분적대장절제술을시행한 45예와간전이가없는 30예를대상으로임상적특징을조사하고병리학적소견을검토하였다. 또한 World Health Organization (WHO) 분류에근거하여조직학적분화도를나누고, 고분화와중등도분화는저등급으로, 저분화와미분화는고등급으로분류하였다. 종양병기와림프절병기는 American Joint Committee on Cancer의기준에따라분류하였다. 12 면역조직화학염색 10% 중성포르말린에고정시킨후제작한파라핀포매괴로부터 3 μm 두께의절편을만들고이를탈파라핀과함수과정을거친후 VEGF-A (A-20, Santa Cruz Biotech, Santa Cruz, CA, USA) 는펩신으로 10분간반응시킨후완충액으로세척하여조직항원을노출시켰고, VEGFR-1 (Y103, Abcam, Cambridge, UK), VEGFR-2 (KDR/EIC, Abcam), 그리고 CD34 (QBEND10, Dako, Carpinteria, CA, USA) 는가열처리항원유도 (antigen retrieval) 방법을사용하여조직항원이잘노출되도록하였다. 또 VEGFR-1, VEGFR-2는 EDTA buffer에서 125 로 15분간처리를하였고, CD34는 citrate buffer에서 125 로 15분간처리하였다. 그리고내재성 peroxidase를억제하기위해 3% 의 hydrogen peroxidase를반응시킨다음 VEGF-A, VEGFR-1, VEGFR- 2를각각 1:200으로희석하여일차항체로약 90분간작용을시켰고, CD34는 1:100으로희석하여일차항체로약 90분간작용을시킨후완충액으로세척하였다. 이때반응산물의검출을위해바이오틴이부착된항마우스 IgG와항래빗 IgG 및 strepta- Age (yr) 60 10 33.3 21 46.70 > 60 20 66.7 24 53.30 Sex Male 17 56.7 31 68.9 Female 13 43.3 14 31.1 Primary cancer site Ascending colon 4 13.3 6 13.3 Transverse colon 3 10.0 3 6.7 Descending colon 1 3.3 3 6.7 Sigmoid colon 4 13.3 12 26.7 Rectum 18 60.0 21 46.7 Gross type Fungating 2 6.7 8 17.8 Ulcerofungating 21 70.0 26 57.8 Ulcerative 3 10.0 8 17.8 Infiltrative 4 13.3 3 6.7 Differentiation WD 5 16.7 12 26.7 MD 20 66.7 26 57.8 PD-UD 5 16.7 7 15.5 Lymphovascular invasion Absent 24 80.0 29 64.4 Present 6 20.0 16 35.6 T stage T2 0 0.0 2 4.4 T3 28 93.3 20 44.4 T4 2 6.7 23 51.1 N stage N0 7 23.3 11 24.4 N1 13 43.3 13 28.9 N2 10 33.3 21 46.7 Liver mass type Solitary 0 0.0 24 53.3 Multiple 0 0.0 21 46.7 WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; UD, undifferentiated.

전이대장암에서혈관유도인자 573 광학현미경을통한미세혈관밀도측정 신생혈관밀도의측정은 Bosari 등 14 의방법에따라세정맥및모세혈관만을포함시켰는데, CD34에양성이면서인접한큰혈관과떨어져있고명백하게분리되어있는개개의내피세포또는내피세포의군집을하나의미세혈관으로간주하였다. 그리고 100배의광학현미경시야에서미세혈관형성이풍부한부위 10 곳을무작위로선택하여밀도를측정하였는데, 이때디지털카메라가부착된광학현미경을통해얻어진염색된표본슬라이드 Table 2. Comparison of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 expression in the control and metastasis groups Control group Metastasis group Total p-value VEGF 0 1 0 1 (1.3) 0.004* 1 8 3 11 (14.7) 2 7 9 16 (21.3) 3 14 33 47 (62.7) Total 30 (40.0) 45 (60.0) 75 (100) VEGFR-1 0 1 0 1 (1.3) 0.561 1 6 13 19 (25.3) 2 15 14 29 (38.7) 3 8 18 26 (34.7) Total 30 (40.0) 45 (60.0) 75 (100) VEGFR-2 0 2 0 2 (2.6) < 0.001* 1 28 22 50 (71.4) 2 0 13 13 (17.3) 3 0 10 10 (13.3) Total 30 (40.0) 45 (60.0) 75 (100) Values are presented as number (%). 의미세혈관단면적을화상분석소프트웨어 AnalySIS ver. 3.2 (Soft-Imaging Software GmbH, Munster, Germany) 를이용하여측정하였다. 종양내에존재하는미세혈관의밀도를 100배로관찰한뒤미세혈관의형성이가장풍부한부위를선택하여 200배를일정배율로 10필드이상을이동하면서측정하였고, 각증례당신생혈관의밀도는측정된값을전체면적으로나눈백분율 (%) 로나타내어평균값으로표시하였다. 13 통계학적분석 SPSS ver. 15.0 (SPSS Inc., Chicago, IL, USA) 을이용하여대조군과전이군의대장암에서 VEGF-A, VEGFR-1, VE GFR-2의발현정도를 linear-by-linear association test를통해비교하였고, 전이군내에서대장암과간전이에서의 VEGF-A, VEGFR-1, VEGFR-2의발현정도를 Wilcoxon signed ranks test를통하여비교하였다. 면역화학염색결과와각임상요소의연관성은 Pearson chisquare test 또는 Fisher s exact test를통해평가하였고, 각혈관형성인자들간의연관성은대장암에한해서 Kendall s tau-b test를통해평가하였다. 또한혈관형성인자들과미세혈관밀도와의관계는미세혈관밀도를평균값을기준으로두그룹으로구분한후 linear-by-linear association test를통해비교하였으며, 대장암과간전이에서미세혈관밀도는 paired t-test로비교하였고, 각임상요소와미세혈관밀도의연관성은 independent-samples t-test를이용하여분석하였는데, 이때 p값이 0.05 이하인경우를통계학적으 Table 3. Comparison of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 expression in colorectal cancer and liver metastases Liver 0 1 2 3 Total p-value Colon VEGF 0 0 0 0 0 0 (0.0) < 0.001* 1 0 1 1 1 3 (6.7) 2 0 5 1 3 9 (20.0) 3 2 15 9 7 33 (73.3) Total 2 (4.4) 21 (46.7) 11 (24.4) 11 (24.4) 45 (100.0) VEGFR-1 0 0 0 0 0 0 (0.0) < 0.001* 1 3 7 3 0 13 (28.9) 2 3 8 3 0 14 (31.1) 3 1 15 2 0 18 (40.0) Total 7 (15.6) 30 (66.7) 8 (17.8) 0 (0.0) 45 (100.0) VEGFR-2 0 0 0 0 0 0 (0.0) < 0.001* 1 8 13 1 0 22 (48.9) 2 4 8 1 0 13 (28.9) 3 2 8 0 0 10 (22.2) Total 14 (31.1) 29 (64.4) 2 (4.4) 0 (0.0) 45 (100.0) Values are presented as number (%).

574 정은희 김영 민병우외 3 인 로의미있는것으로판정하였다. 결과임상병리학적특성 75예환자의연령분포는 29세에서 85세까지였으며, 평균연령은전이군이 60.7±10.9세대조군이 62.5±12.6세이며, 남녀성비는전이군이 2.21, 대조군이 1.30으로남성비율이더높았다. 또한대장암의위치를상행결장, 횡행결장, 하행결장, S자결장, 직장등으로구분하였을때대조군과전이군모두직장에서발생한예가각각 18예, 21예로가장많았다. 육안상표본의모양에따라대장암을구분하였을때궤양성융기형타입이전이군에서 57.8%, 대조군에서 70.0% 로가장많았고, 조직학적분화도를보았을때는양쪽모두중등도분화타입이가장많았다 (Table 1). 그리고대조군대장암의평균크기 는 4.93±2.0 cm, 전이군에서원발대장암의평균크기는 4.7± 1.9 cm, 간전이의평균크기는 3.1±2.6 cm이었으며, 간전이가있는 45예중 40예가동시성전이였고, 5예가이시성전이였다. VEGF-A, VEGFR-1, VEGFR-2의발현및미세혈관형성의비교 VEGF-A, VEGFR-1, VEGFR-2의발현은종양의침윤부위에서제일강한발현정도를보였고주변비종양성샘조직에서는발현이되지않거나미약하게발현되는양상을보였으며, 간세포의경우전이된종양보다강한발현정도를보였다. 종양의침윤부위를대상으로대조군과전이군의대장암에서 VEGF-A의발현정도를비교하였을때, 전이군에서강양성 (+3) 을보이는예의비율이 73.3% 로대조군보다높게나타났으며, VEGFR-2 역시전이군에서더높게나타나통계학적으로유의한차이가있었다 (p=0.004, p<0.001)(table 2). 전이군내에서원발대장암과간전이암에서 VEGF-A의발현정도를비교하였을때는원발대장암에서강양성 (+3) 을보 1+ A 2+ B 3+ C 1+ D 2+ E 3+ F Fig. 1. Expression of vascular endothelial growth factor (VEGF)-A in colorectal cancer and liver metastasis. VEGF-A is expressed in tumor epithelial cells. (A-C) VEGF-A expression in colorectal cancer along with increasing immunoreactivity intensity. (D-F) VEGF-A expression in liver metastases along with increasing immunoreactivity intensity.

전이대장암에서혈관유도인자 575 이는예의비율이 73.3% 로, 간전이암에서의 24.4% 에비해훨씬높게나타났으며이는통계학적으로도유의한차이가있었다 (p<0.001)(fig. 1). VEGFR-1의경우에도중양성과강양성을보이는예의비율이원발대장암에서각각 31.1% 와 40.0% 로간전이에서의 17.8% 와 0.0% 에비하여유의하게높았으며 (p< 0.001), VEGFR-2 역시비슷하게통계적으로유의한차이를보였다 (p<0.001)(table 3, Figs. 2, 3). 면역조직화학염색상 CD34에염색되는미세혈관은 200배시야에서쉽게확인되었으며대부분풍부한미세혈관들을관찰할수있었다 (Fig. 4). 대조군과전이군의대장암에서광학현미경과화상분석장치를이용하여측정한미세혈관밀도에있어서대조군의미세혈관밀도는 2.21±1.27%, 전이군의미세혈관밀도는 4.45±2.28% 로전이군의미세혈관밀도가통계학적으로유의하게높았다 (p=0.001). 또한전이군의원발부위와간전이에서측정한미세혈관밀도는각각 4.45±2.28%, 2.24±0.84% 로원발대장암에서의미세혈관밀도가통계학적으로유의하게높았다 (p=0.001)(fig. 5). 혈관형성인자들간의연관성및혈관형성인자와미세혈관밀도와의연관성 대조군과전이군의대장암총 75예를대상으로발현된혈관형성인자들간의연관성을비교해보았을때 VEGF-A의발현증가는 VEGFR-1, VEGFR-2 발현증가와유의한관계를보였으며 (p=0.013, p=0.023), VEGFR-1과 VEGFR-2의발현역시통계학적으로유의한것으로나타났다 (p=0.016)(table 4). 또한혈관형성인자들의발현이증가할수록미세혈관밀도도증가하는경향을보였다 (Table 5). 혈관형성인자들과임상병리학적변수간의연관성 임상병리학적변수에따른혈관형성인자들의연관성을확인하기위한비교에서통계학적편의를위하여 VEGF-A, VEGFR- 1, VEGFR-2의발현정도를두그룹, 즉약양성이하 (0-1+) 를저발현 (low expression) 그룹, 중양성이상 (2+-3+) 을고발 1+ A 2+ B 3+ C 1+ D 2+ E Fig. 2. Expression of vascular endothelial growth factor receptor (VEGFR)-1 in colorectal cancer and liver metastases. (A-C) VEGFR-1 expression in colorectal cancer along with increasing immunoreactivity intensity. (D, E) VEGFR-1 expression in liver metastases along with increasing immunoreactivity intensity.

576 정은희 김영 민병우외 3 인 1+ A 2+ B 3+ C 1+ D 2+ E Fig. 3. Expression of vascular endothelial growth factor receptor (VEGFR)-2 in colorectal cancer and liver metastases. (A-C) VEGFR-2 expression in colorectal cancer along with increasing immunoreactivity intensity. (D, E) VEGFR-2 expression in liver metastases along with increasing immunoreactivity intensity. A B A B Fig. 4. Microvessel density in colorectal cancer and liver metastases. Microvessel distribution is manifested by CD34 immunohistochemical staining in colorectal cancer (A) and liver metastases (B). This photomicrograph reveals a higher number of microvessels in colorectal cancer than in liver metastases. Fig. 5. Measure of microvessel density by the AnalySIS 3.2 program. (A) Vascular endothelial cells are CD34 positive. (B) Microvessels are highlighted in green fluorescent color and are calculated as a percentage of microvessel density per unit area.

전이대장암에서혈관유도인자 577 Table 4. Association between vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 VEGF 0 1 2 3 VEGFR-1 0.013* 0 0 1 0 0 1 (1.3) 1 0 5 5 9 19 (25.3) 2 1 3 7 18 29 (38.7) 3 0 2 4 20 26 (34.7) Total 1 (1.3) 11 (14.7) 16 (21.3) 47 (62.7) 75 (100) VEGFR-2 0.023* 0 0 2 0 0 2 (2.6) 1 1 9 10 30 50 (71.4) 2 0 0 3 10 13 (17.3) 3 0 0 3 7 10 (13.3) Total 1 (1.3) 11 (14.7) 16 (21.3) 47 (62.7) 75 (100) VEGFR-1 0 1 2 3 VEGFR-2 0.016* 0 0 0 1 1 2 (2.6) 1 1 16 20 13 50 (71.4) 2 0 3 5 5 13 (17.3) 3 0 0 3 7 10 (13.3) Total 1 (1.3) 11 (14.7) 16 (21.3) 47 (62.7) 75 (100) Values are presented as number (%). Total p-value 현 (high expression) 그룹으로묶었으며, 임상병리학적변수들도두그룹으로단순화하여분석하였다. Table 5. Association between microvessel density and the expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 Microvessel density 3.5 > 3.5 VEGF 0.028* 0 1 0 1 (1.3) 1 10 1 11 (14.7) 2 10 6 16 (21.3) 3 26 21 47 (62.7) Total 47 (62.7) 28 (37.3) 75 (100) VEGFR-1 0.017* 0 1 0 1 (1.3) 1 15 4 19 (25.3) 2 19 10 29 (38.7) 3 12 14 26 (34.7) Total 47 (62.7) 28 (37.3) 75 (100) VEGFR-2 0.019* 0 2 0 2 (2.6) 1 36 14 50 (71.4) 2 4 9 13 (17.3) 3 5 5 10 (13.3) Total 47 (62.7) 28 (37.3) 75 (100) Values are presented as number (%). Total p-value Table 6. Association between clinicopathological variables and the expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 in colorectal cancer No. of cases C-VEGF C-VEGFR-1 C-VEGFR-2 Low High p - Low High p - Low High p - 12 63 value 20 55 value 52 23 value Age (yr) 60 31 4 27 0.75 6 25 0.23 19 12 0.21 > 60 44 8 36 14 30 33 11 Sex Male 48 6 42 0.27 15 33 0.23 31 17 0.23 Female 27 6 21 5 22 21 6 Colon mass size (cm) 4.0 39 4 35 0.21 9 30 0.46 24 15 0.13 > 4.1 36 8 28 11 25 28 8 Primary cancer site Proximal colon 16 1 15 0.42 6 10 0.27 13 3 0.36 Distal colon 59 11 48 14 45 39 20 Gross type Fungating 57 10 47 0.72 12 45 0.05 38 19 0.56 Infiltrative 18 2 16 8 10 14 4 Differentiation LG 63 11 52 0.68 16 47 0.72 43 20 0.75 HG 12 1 11 4 8 9 3 Lymphovascular invasion Absent 53 10 43 0.49 14 39 0.94 39 14 0.22 Present 22 2 20 6 16 13 9 T stage T2 + T3 50 8 42 1.00 11 39 0.20 36 14 0.60 T4 25 4 21 9 16 16 9 N stage N0 18 2 16 0.72 2 16 0.13 9 9 0.04* N1 + N2 57 10 47 18 39 43 14 C-VEGF, colorectal cancer VEGF; LG, low grade; HG, high grade.

578 정은희 김영 민병우외 3 인 Table 7. Association between clinicopathological variables and the expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor (VEGFR)-1, and VEGFR-2 in liver metastases No. of cases L-VEGF L-VEGFR-1 L-VEGFR-2 Low High p - (n = 23) (n = 22) value Low High p - (n = 37) (n = 8) value Low High p - (n = 43) (n = 2) value Age (yr) 60 21 12 9 0.45 16 5 0.32 21 0 0.49 > 60 24 11 13 21 3 22 2 Sex Male 31 18 13 0.17 26 5 0.69 30 1 0.53 Female 14 5 9 11 3 13 1 Colon mass size (cm) 4.0 26 16 10 0.10 21 5 1.00 26 0 0.17 > 4.1 19 7 12 16 3 17 2 Liver mass size (cm) 2.5 23 15 8 0.053 20 3 0.46 22 1 1.00 > 2.6 22 8 14 17 5 21 1 Primary cancer site Proximal colon 9 5 4 1.00 9 0 0.18 8 1 0.36 Distal colon 36 18 18 28 8 35 1 Gross type Fungating 34 18 16 0.67 28 6 1.00 33 1 0.43 Infiltrative 11 5 6 9 2 10 1 Differentiation LG 38 18 20 0.41 30 8 0.32 36 2 1.00 HG 7 5 2 7 0 7 0 Lymphovascular invasion Absent 29 13 16 0.26 22 7 0.23 28 1 1.00 Present 16 10 6 15 1 15 1 T stage T2 + T3 22 16 6 0.005* 20 2 0.24 22 0 0.49 T4 23 7 16 17 6 21 2 N stage N0 11 8 3 0.17 9 2 1.00 11 0 1.00 N1 + N2 34 15 19 28 6 32 2 Liver mass type Solitary 24 13 11 0.66 22 2 0.12 22 2 0.49 Multiple 21 10 11 15 6 21 0 L-VEGF, liver metastases VEGF; LG, low grade; HG, high grade. Table 8. Comparison of microvessel density in colorectal cancer and liver metastasis Colon Liver LG, low grade; HG, high grade. No. of cases Mean p-value No. of cases Mean p-value Total cases 75 45 Age (yr) 60 31 3.49 ± 2.38 0.32 21 2.27 ± 0.91 0.78 > 60 44 3.59 ± 2.12 24 2.20 ± 0.80 Sex Male 48 3.89 ± 2.38 0.033* 31 2.26 ± 0.85 0.81 Female 27 2.96 ± 1.78 14 2.19 ± 0.87 Colon mass size (cm) 4.0 39 3.60 ± 2.06 0.96 26 2.18 ± 0.85 0.61 > 4.1 36 3.50 ± 2.40 19 2.31 ± 0.85 Primary cancer site Proximal colon 16 2.94 ± 2.08 0.82 9 2.00 ± 0.53 0.35 Distal colon 59 3.72 ± 2.24 36 2.30 ± 0.90 Gross type Fungating 57 3.53 ± 2.28 0.90 34 2.22 ± 0.86 0.83 Infiltrative 18 3.64 ± 2.07 11 2.29 ± 0.82 Differentiation LG 63 3.63 ± 2.30 0.48 38 2.29 ± 0.88 0.34 HG 12 3.14 ± 1.78 7 1.95 ± 0.58 Lymphovascular invasion Absent 53 3.48 ± 2.08 0.28 29 2.37 ± 0.79 0.16 Present 22 3.73 ± 2.58 16 2.99 ± 0.90 T stage T2 + T3 50 2.79 ± 1.54 0.001* 22 1.83 ± 0.48 0.001* T4 25 5.08 ± 2.60 23 2.63 ± 0.94 N stage N0 18 3.01 ± 1.80 0.23 11 1.98 ± 0.60 0.25 N1 + N2 57 3.73 ± 2.32 34 2.32 ± 0.90 Liver mass type Solitary 24 2.08 ± 0.88 0.18 Multiple 21 2.42 ± 0.79

전이대장암에서혈관유도인자 579 비교결과총 75예의대장암에서림프절전이만이 VEGFR- 2의고발현과연관이있었으며 (p=0.041), 다른임상병리학적변수와혈관형성인자들간의유의성은보이지않았다 (Table 6). 또 45예의간전이에서는종양병기 (T stage) 만이 VEGF-A의고발현과유의한상관관계를보였으며 (p=0.005)(table 7), 전이군내원발대장암의경우간전이의크기가작은그룹일수록 VEGFR-1과 VEGFR-2의고발현비율이높게나타났다 (p= 0.003, p=0.002). 또한종양병기 3인 (T3, n=48) 예에서대조군과전이군의혈관형성인자발현은 VEGF-A, VEGFR-2가전이군에서고발현을보였고 (p=0.01, p=0.00), 림프절병기 2인 (N2, n=31) 예에서는 VEGF-A가전이군에서고발현을보였는데 (p=0.046), VEGFR-2의경우통계적유의성은보이지않았으나 (p=0.058) 전이군에서더높은발현을보이는경향성이있는걸로나타났다. 한편임상병리학적변수에따른대장암미세혈관밀도의차이를보았을때남성이더높은미세혈관밀도를보였으며 (p= 0.03), 종양병기가높은그룹에서미세혈관밀도가더높았다 (p=0.001). 간전이에서미세혈관밀도의경우도종양병기가높은그룹에서유의하게높게나타났다 (p=0.001)(table 8). 그리고종양병기가 3인예 (T3) 에서전이군의미세혈관밀도가더높았으며 (p=0.016), 림프절병기가 2인예 (N2) 에서도전이군의미세혈관밀도가높았다 (p=0.004). 고찰대장암은현재우리나라에서증가추세에있는암중하나로다른장기로전이를잘하는암이다. 이대장암은주로 60 내지 70대에호발하며, 50세이전에대장암이발생하는경우는 20% 미만으로알려져있다. 본연구에서도대조군과전이군의평균연령이각각 62.5세와 60.7세로노년층에서발생한예가다수포함되어있었고, 알려진바와같이직장과 S자결장에서발생한예가주로많았다. 대장암은주변장기로직접침윤할뿐아니라림프관과혈관을따라간, 폐, 뼈등으로흔하게원격전이를한다. 간은두가지이유에서원격전이가잘되는데그이유는첫째, 간으로하부식도, 위, 비장, 소장등복부장기들의정맥혈류가유입되어여과되는데, 간이차지하는심장박출량은 30% 로폐다음으로여과되는혈류량이많다는것이다. 둘째, 생리학적으로암세포성장에충분한환경을제공할수있는간세포, 혈관내피세포, 쿠퍼 (Kupffer) 세포, 섬유모세포, 혈관주위세포, 염증세포등이다양하게구성되어있다는것이다. 15 혈류에서파괴되지않은암세포는간에도달해성장을하고, 이전이된암세포주변의동모양혈관내피세포의모세혈관화가일어나는과정을거쳐전이된세포가성장하게된다. 16,17 이런일련의과정에영향을미치는인자들에대한다양한연구가이루어 지고있고본연구의경우혈관형성인자가대장암이간으로전이할때의연관성및예후를예측할수있는표지자로서의가능성을확인하고, 대장암과간전이에서혈관형성인자와의연관성뿐만아니라미세혈관밀도와의관계도알아보고자하였다. Cascinu 등 18 의연구에의하면 T2 대장직장암에서림프절전이가없는군보다림프절전이가있는군에서 VEGF 증가를보여혈관형성인자가림프절전이와관련이있다고하였다. 일부연구에서는 VEGF-A가직장, 결장암의발생에의미있는영향을주지만, 림프절전이나원격전이에는영향이적을것이라고보고하고있다. 그러나본연구에서는간전이의여부와 VEGF-A, VEGFR-2의고발현이연관이있었으며, 미세혈관밀도역시간전이와관련이있는것으로나타났다. 19 또한 Duff 등 20 의연구에따르면원발대장암과림프절전이사이에 VEGF-A, VEGFR-2의발현은연관성이없고간전이에서종양의침윤부위보다 VEGFR-2의발현이감소됨을보고하고있는데, 본연구에서도전이군의원발대장암과간전이간혈관형성인자와의발현차이는간전이가아닌원발대장암에서더높게나타났고, 미세혈관밀도역시원발대장암에서더높게나타났다. Cascinu 등 18 의연구에서도다른복부의전이보다간전이의경우 VEGF의발현이낮게나타나는것을보여주고있으며, Dirix 등 21 은대장암의전이양상에따라 VEGF의혈장농도를측정하여전이장소간에혈관형성인자발현의이형성이있음을확인하였다. 이런이형성은전이장소에따른혈관분포와혈류량, 산소공급등미세환경상태에따라차이가있기때문으로생각된다. 20 특히저산소상태의경우 VEGF의발현이높아지는데, 앞서언급했듯간은폐다음으로여과되는혈류량이많은장기이고, 암세포가성장할수있는산소와영양분을정맥과동맥에서동시에공급을받을수있으며또원래구성되어있는동모양혈관조직의변형이궁극적으로는일반적인신생혈관등을생성하여산소와영양분을공급할수있는다양한기전을가진독특한혈관구조를가지고있다. 따라서저산소상태라는자극의부족으로 VEGF의합성이억제되어간전이의성장에혈관형성인자가끼치는영향이복막이나림프절등의다른전이장소에비해감소되는것으로생각된다. 18,22-24 또한임상병리학적인자와혈관형성인자와의발현연관성을보았을때, 종양병기가높을수록 VEGF의발현이높고미세혈관밀도가더높게나타나혈관형성인자가대장암의성장및침윤과연관이있음을알수있다. 본연구결과, 간전이가동반된대장암의경우혈관형성인자나미세혈관밀도가간전이의진행정도를확인할수있게하는연관성이없어간전이후예후를가늠할수있는표지자로서의가능성이없는것으로생각된다. 그러나간전이의여부에따라혈관형성인자나미세혈관밀도가큰차이를보임으로써낮은병기의대장암에서높은혈관형성인자의발현이나미세혈관밀도가전이를예측하거나나쁜예후를보여주는표지자로서의사용이가능하다고판단된다.

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