대한혈액학회지 2003년 38권 4호 P

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1 대한혈액학회지 : 제 8 권제 호 년 원 저 ) 급성백혈병에서 Granulocyte Colony-Stimulating Factor (G-CSF) 수용체발현양상의임상적의의 고려대학교의과대학진단검사의학교실, 내과학교실, 단국대학교의과대학진단검사의학교실 윤수영 배숙영 서재홍 김병수 김영기 이갑노 임인수 Expression of Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) and Clinical Correlates in Acute Leukemia Soo-Young Yoon, Sook Young Bae, Jae Hong Seo, Byung Soo Kim, Young Kee Kim, Kap No Lee, and Insoo Rheem Departments of Laboratory Medicine, Internal Medicine, Korea University School of Medicine, Korea Department of Laboratory Medicine, Dankook University College of Medicine, Cheonan, Korea Background : Granulocyte colony-stimulating factor (G-CSF) is commonly used to reduce leukopenic period during treatment of malignancy including acute leukemia. Leukemic blasts expressing granulocyte colony-stimulating factor receptor (G-CSFR) were reported and also may proliferate in response to therapeutic administration of G-CSF. However, it is not clear whether G-CSFR expression on leukemic blasts is related to clinical outcome such as leukocyte recovery or leukemia relapse. Current study evaluated expression of G-CSFR in acute leukemia and correlated with hematologic and clinical parameters. Methods : Peripheral blood or bone marrow aspirate was evaluated from patients with acute myelogenous leukemia (AML) and with acute lymphoblastic leukemia (ALL), with acute undifferentiated leukemia (AUL), with acute biphenotypic leukemia (ABL), with acute mixed-lineage leukemia (AMLL). G-CSFR expression was analyzed using flow cytometry and was correlated with immunophenotype and response for chemotherapy. Results : More than % of blasts were positive for G-CSFR in % (/) of AML, % (/) of ALL, and all negative in ABL, AMLL, and AUL. Except that all monocytic lineage leukemias (M, M) and all three cases of ALL with CD expression were positive, no consistent correlation was observed among G-CSFR expression pattern, type of acute leukemia, response to induction therapy and relapse (P>.). Conclusion : Current study revealed G-CSFR was expressed on not only myelogenous leukemic cells but also lymphoid ones. Although our data suggest G-CSFR expression does not affect therapeutic outcome, it remains to be determined whether G-CSF therapy is safe in G-CSFR-positive acute leukemia. (Korean J Hematol ;8:~) Key Words : Granulocyte colony-stimulating factor (G-CSF) receptor, Acute leukemia, Flow cytometry 접수 : 년 월 일, 수정 : 년 월 일승인 : 년 월 일이논문은 학년도단국대학교대학연구비지원으로연구되었음. 책임저자 : 임인수, 단국대학교의과대학진단검사의학교실 Tel : )-8, Fax : )- insoo@dankook.ac.kr 서 Granulocyte colony-stimulating factor (G-CSF) 는급 론

2 대한혈액학회지 : 제 8 권제 호 년 성백혈병을포함한악성종양의치료에있어화학요법으로인한백혈구감소증을완화시키기위해흔히사용되어왔다. ) G-CSF는정상조혈세포에서조혈모세포의증식을유도할뿐만아니라, 아포프토시스 (apoptosis) 를억제하여세포의생존율을유지하고, 호중구로의분화를촉진한다. ) 백혈병세포에서도비슷한작용을보이는데, G-CSF 를투여했을때체내외실험모두에서아포프토시스가억제되고, ) DNA 합성이촉진되며, 백혈병세포의수가증가되고경우에따라서는백혈병세포의분화를유도하기도한다. ) 급성골수성백혈병의경우발현정도는다양하나대부분 G-CSF 수용체를발현하며,,) 드물게급성림프아구성백혈병세포역시 G-CSF 수용체를발현한다. ) 그러나이러한수용체발현이백혈병치료에보조적으로사용되는 G-CSF에의한백혈병세포의증식으로이어져재발의가능성을높이는지, 치료에대한반응이나특정백혈병종류와연관이있는지는아직잘알려져있지않다. 이에본연구에서는백혈병유형에따른 G-CSF 수용체발현양상을조사하고, 동시에시행한면역표현형과의연관및 G-CSF 수용체발현에따른유도요법과 G-CSF 치료에대한반응을비교함으로써이의임상적의의를평가하고자하였다. 대상및방법. 대상 명의급성골수성백혈병환자와 명의급성림프아구성백혈병환자, 명의급성미분화성백혈병 (acute undiffenrentiated leukemia, AUL), 명의급성혼합계열백혈병 (acute mixed-lineage leukemia, AMLL), 명의급성양성백혈병 (acute biphenotypic leukemia, ABL) 환자의총 명의진단시사용된말초혈액이나골수흡인을대상으로하였다 (Table ).. 백혈병진단및분류백혈병진단을위해말초혈액및골수흡인에서백혈병세포의백분율을구하였으며, myeloperoxidase (MPO), Sudan black B (SBB), periodic acid schiff (PAS), chloroacetate esterase (CAE), α-naphythyl butyrate esterase (ANBE), acid phosphatase (AP) 등의세포화학염색과 CD, CD, CD, CD, CD, CD, CD9, CD, CD, CD, CD, CD, CD, HLA-DR을이용한면역표지자검사를시행하였다. 급성골수성백혈병 Table. Characteristics of patients with acute leukemia B B precursor B precursor with myeloid marker M M M M M M basophilic unclassified Acute undifferentiated leukemia Acute biphenotypic leukemia Acute mixed-lineage leukemia N Sex (M/F) / / / / / / / / / / / / /9 / / / Age (range) ~ ~ ~, 9 ~9 ~9 ~ 9 ~9, Total / ~9 은 FAB 분류기준을적용하였고급성림프아구성백혈병은면역학적분류에따랐으며, 각군의특성은 Table 에정리하였다. 급성혼합계열백혈병과급성양성백혈병의분류는저자들이발표한논문에서제시한기준에따랐다. ). 유세포분석기를이용한면역표지자검사및 G-CSF 수용체분석 말초혈액이나골수흡인의단핵구 개를 phycoerythrin conjugate anti-g-csf receptor antibody (anti- CD, Serotec, UK) µl과 분간상온암소에서항온반응 (incubation) 후적혈구를용혈시키고 PBS로 회세척하였다. 유세포분석기는 FACScan (Becton Dickinson, USA) 을사용하였고 Lysis II program (Becton Dickinson, USA) 으로분석하였다. 전방산란 (forward scatter) 과측방산란 (side scatter) 의 dot plot에서아세포를 gate한후이중 % 와 % 이상이양성인경우를 G-CSF 수용체발현양성으로하여 (Fig. ), 치료에대한반응의차이및다른특정면역표현형과의연관성을분석하였다.. 화학치료에대한반응평가 추적이가능했던환자에서화학치료에대한관해율을비교하였으며재발여부를조사하였다. 관해후관해가유지

3 8 윤수영외 인 : 급성백혈성에서 G-CSF 수용체발현양상 A B C Fig.. G-CSF receptor (G-CSFR) analysis on leukemic blasts. A) Leukemic blast gating on dot plot of forward scatter and side scatter of bone marrow aspirate. Histogram for phycoerythrin conjugated G-CSFR shows either positive B) or negative C). White peak of leukemic blast signal was compared to red peak of isotypic control. Table. G-CSF receptor expression according to acute leukemic classification % % n Positive Positive B B precursor B precursor with myeloid marker M M M M M M basophilic unclassified 9 Acute undifferentiated leukemia Acute biphenotypic leukemia Acute mixed-lineage leukemia Total 된증례들과재발, 유도요법중사망, 관해실패한증례들의 G-CSF 수용체발현율을비교하였는데, 완전관해후관찰기간중재발이없는경우를반응이좋은 (good) 것으로하였고, 완전관해되지않았거나재발이있는경우를반응이나쁜 (poor) 것으로구분하여분석하였다.. 통계 SAS (SAS Institute Inc., Cary, NC, USA) 통계프로 그램을이용하여 Fisher's exact test 또는 Chi-square test 로검증하였으며유의수준은.로하였다. 결과전방산란과측방산란의 dot plot에서아세포를 gate한후이중 % 이상이양성인경우를 G-CSF 수용체발현양성으로하였을경우급성골수성백혈병에서 % (9/), 급성림프아구성백혈병에서 % (/) 이었으며, ABL

4 대한혈액학회지 : 제 8 권제 호 년 9 례와, AMLL 례, 급성미분화성백혈병 례는모두음성이었다. 이를 % 로낮추면급성골수성백혈병 %, 급성림프아구성백혈병 % 에서 G-CSF 수용체양성이었다 (Table ). 례의급성골수성백혈병 M, M에서모두 % 이상의 G-CSF 수용체양성을보였으며, 소아와성인에서의유의한차이는없었다 (Table, P>.). 급성림프아구성백혈병군에서 G-CSF 수용체양성인 례중 례에서골수성표지자 (myeloid marker) 인 CD이발현하여, CD을표현하는례에서 G-CSF 수용체양성율이높았으며 (P=.) 그외의특정면역표현형과는의의있는연관이없었다 (P>.). G-CSF 수용체양성이며 G-CSF를사용한 례의급성골수성백혈병중 례가완전관해되었고 례는부분관해를보였으며, G-CSF 수용체음성이며 G-CSF를사용한 례의급성골수성백혈병중 례는완전관해되었고 례는불변 (refractory) 을보였다. G-CSF 수용체양성이며 G-CSF를사용한급성림프아구성백혈병 례는완전관해되었으나후에재발되었으며, G-CSF 수용체음성이며 G-CSF를사용한급성림프아구성백혈병 례는완전관해되었다. 급성림프아구성백혈병과급성골수성백혈병에서 G-CSF 수용체양성여부에따른유도요법에대한반응이나예후의차이는보이지않았다 (Table, P>.). 고찰 G-CSF는골수에서호중구생산을촉진시키며성숙호중구의기능을조절한다. ) G-CSF 수용체는골수의정상골수성전구세포에분포하고있으며, 단구 (monocyte) 와 8) T-세포의일부에도 9) G-CSF 수용체가있어항염증작용이 Table. G-CSF receptor expression according to subtypes of acute leukemia % % n Positive Positive Adult Children Primary Relapsed Secondary 8 9 Table. G-CSF receptor expression and prognosis G-CSF Receptor N Response Good Poor BMT Complete remission Expire before induction chemotherapy Induction failure Relapse Transfer Positive Positive Acute undifferentiated leukemia Acute mixed-lineage leukemia Acute biphenotypic leukemia 9 Total 9

5 윤수영외 인 : 급성백혈성에서 G-CSF 수용체발현양상 나면역기능조절에도관여하는것으로보인다. 백혈병에서의 G-CSF 수용체는골수성백혈병및 B 세포계열의급성림프아구성백혈병에서발현되며, ) 난소암, 피부암등의고형암에서도 G-CSF 수용체가발견되어보고되었다. ) 골수이형성증후군에서는많은경우 G-CSF 수용체의발현에장애가있으며,,) congenital neutropenia, Shwachman-Diamond syndrome (SDS) 등의질환에서 G-CSF 수용체의유전학적변이에의해골수이형성증후군및골수성백혈병으로진행될수있다.,) G-CSF 수용체에는여러이형 (isomer) 이있는데이중 class Ⅳ 이형이급성골수성백혈병에서많이나타나고병인에관여하는것으로보인다.,) 본연구의급성골수성백혈병에서 G-CSF 수용체발현율은 % 의기준치 (cutoff value) 에의해판별하였을때 % 로유사한방법을사용한기존의다른연구 ) 의양성률과비슷하였다. 급성골수성백혈병의아형간의수용체발현양상은보고에따라다르기는하나 M, M에서다른아형에서보다비교적양성률이높은데,,,) 일반적으로 G-CSF 수용체의발현이과립성백혈구 (granular leukocytes) 에서많고성숙정도에비례하는것을감안하면당연한결과로추정된다.,,8) 한편본연구에서는특이하게 례의 M, M에서모두에서 G-CSF 수용체를표현하는아세포수가 % 이상으로 M, M에비해높은결과를보였는데다른연구결과와유의한차이인지는더많은대상의자료가필요할것으로보인다. G-CSF는 IL-나 GM-CSF (granulocyte-macrophage colony-stimulating factor) 와는달리 lineage-restricted CSF로여겨져왔고, 따라서골수성이아닌다른악성종양의경우에는화학요법후백혈구감소증을치료하는데효과적이고안전하다고생각되어왔다. 9,) 그러나대부분의급성림프아구성백혈병세포역시 G-CSF의수용체를표현하는것이알려져급성림프아구성백혈병에서의 G-CSF 사용역시급성골수성백혈병에서와마찬가지로주의하여관찰되어야한다는의견이제기되어왔다. 8,~) 례의급성림프아구성백혈병중 례가 G-CSF 수용체를표현했는데, B lineage 9례중 9례, T lineage 례중 례, T와 B 를모두표현하는 례중 례가수용체를표현하였고, 8) 례의 B 세포림프구성백혈병을대상으로한연구에서는 9% (/) 의양성률을보였다. ) 본연구의급성림프아구성백혈병은모두 B 세포계열이었으며 % (/) 에서 G-CSF 수용체양성을보여양성률이크게다르지않았다. 급성림프아구성백혈병세포가 G-CSF 수용체를발현할뿐아니라나아가 G-CSF에반응하여증식한다는증거도있는데, 급성림프아구성백혈병세포를대상으로한연구에서각각 례중 례, ) 례중 례에서 ) G-CSF에의해 colony 형성이증가하였다. 특정면역표현형이나유전자결함과는특별한상관이없는것으로보인다. ) 골수성백혈병세포와같이 9) 림프구성백혈병세포에서도 G-CSF 수용체발현정도와 G-CSF에의한세포증식이꼭일치하지않았고, 방법에따라수용체가검출되지않는경우에도 G-CSF에반응하여증식한경우가보고되었다. ) G-CSF는호중구감소증및골수이식시생착 (engraftment) 기간을단축하는목적으로흔히사용되며 G-CSF를다른화학요법에추가하였을때세포주기 (cell cycle) 에들어가는백혈병세포의부분을늘려화학요법의효과를증대시켜관해율을높이는목적으로도이용되고있다. 따라서 G-CSF 수용체발현여부는백혈병의치료에대한반응에도영향을줄것으로추정된다. 최근골수성백혈병환자를대상으로한연구에서 G-CSF 사용의적절성을판단하기위한 G-CSF 수용체분석은유용하지않다고보고하였다. ) 본연구결과에서도 G-CSF 수용체발현여부가치료에대한반응이나예후와는무관한것으로평가되었으나대상수가적어보다많은례에서추가적인연구가필요하다고생각된다. 또한본연구대상에서는실제로 G-CSF 치료를시행한경우는급성골수성백혈병에서 례, 급성림프아구성백혈병에서 례로건수가적어, 이에대한통계적으로유의한결과는도출하지못하였다. G-CSF 수용체에대해서는수용체의수및유무외에도환자상태에따른수용체수의변화, 수용체의여러가지이형및수용체의변이등의여러가지병인과관련될수있는요소가많아계속적인연구가필요하며급성림프아구성백혈병에서같은식으로작용할지등에대한연구도지속되어야한다. 국내에서의 G-CSF 수용체및 GM-CSF 수용체연구는대부분의골수성백혈병세포주와급성골수성백혈병환자에친화성이다른 GM-CSF 수용체가존재함을확인하였고, ) 정상인및감염등이있는환자를대상으로 G-CSF 수용체및 GM-CSF 수용체수및발현을연구하였는데호중구에서 G-CSF 수용체가가장많았고, 환자의상태에따라수용체수도변화될수있다고하였다. ) 백혈병을대상으로한 G-CSF 수용체에대한연구는본연구가국내에서는처음으로생각되며정상적인세포와는달리백혈병에서는 G-CSF 수용체의발현이골수계에국한되지않으며거의모든백혈병에서발현되고있음을알수있다. 본연구에서는대상환자수가충분하지못하고 G-CSF 수용체에관련되어추가로고려할요소들이적지않기때문에, 더많은예를대상으로 G-CSF수용체발현여부및병인과특정백혈병아형이나치료에대한반응, 예후등과의관련을확인하고 G-CSF사용의안전성여부에관한연구가더필요할것으로생각된다.

6 대한혈액학회지 : 제 8 권제 호 년 요 배경 : Granulocyte colony-stimulating factor (G-CSF) 는급성백혈병을포함한악성종양의치료에있어화학요법으로인한백혈구감소증을완화시키기위해흔히사용되어왔다. 일부백혈병에서아세포가 G-CSF 수용체를발현하고, G-CSF 투여시이에반응하여아세포가증식된예가보고되었으나이의임상적의의에관해서는아직확실한결론이없다. 이에본연구에서는백혈병유형에따른 G-CSF 수용체발현양상을조사하고이의임상적의의를분석하고자하였다. 방법 : 명의급성골수성백혈병환자와 명의급성림프아구성백혈병환자, 명의급성미분화성백혈병, 명의급성혼합계열백혈병환자, 명의환자의말초혈액이나골수흡인을 G-CSF 수용체항체로염색한후유세포분석기로검사하였다. G-CSF 수용체양성과다른면역표현형과의연관성과 G-CSF 수용체발현에따른치료에대한반응의차이를비교하였다. 결과 : 전방산란과측방산란의 dot plot에서아세포를 gate한후이중 % 이상이양성인경우를 G-CSF 수용체의발현양성으로하였을경우급성골수성백혈병에서 % (9/), 급성림프아구성백혈병에서 % (/) 이었으며, 급성양성백혈병 례와, 급성혼합계열백혈병 례, 급성미분화성백혈병 례는모두음성이었다. 이를 % 로낮추면급성골수성백혈병 %, 급성림프아구성백혈병 % 에서 G-CSF 수용체양성이었다. 례의급성골수성백혈병 M, M에서모두 % 이상의 G-CSF 수용체양성을보였다. CD을표현하는급성림프아구성백혈병군에서 G-CSF 수용체양성률이높으며 (P=.), 그외의특정면역표현형과는의의있는연관이없었다. G-CSF 수용체양성인군과음성인군에서유도요법에대한반응이나예후의차이는없었다 (P>.). 결론 : 정상세포와는달리백혈병에서는 G-CSF 수용체의발현이골수계에국한되지않음을알수있었으며특정유형과의연관은찾을수없었다. G-CSF수용체발현여부가치료에대한반응이나예후와는무관한것으로보이나, G-CSF 수용체양성인예에서 G-CSF 사용의안전성여부에관한연구가더필요할것으로생각된다. 약 참고문헌 ) Demetri GD, Griffin JD : Granulocyte colony-stimulating factor and its receptor. Blood 8:9-88, 99 ) Bradbury D, Zhu YM, Russell N:Regulation of Bcl- expression and apoptosis in acute myeloblastic leukaemia cells by granulocyte-macrophage colony-stimulating factor. Leukemia 8:8-9, 99 ) Baer MR, Bernstein SH, Brunetto VL, Heinonen K, Mrozek K, Swann VL, Minderman H, Block AW, Pixley LA, Christiansen NP, Fay JW, Barcos M, Rustum Y, Herzig GP, Bloomfield CD : Biological effects of recombinant human granulocyte colonystimulating factor in patients with untreated acute myeloid leukemia. Blood 8:8-9, 99 ) Kondo S, Okamura S, Asano Y, Harada M, Niho Y: Human granulocyte colony-stimulating factor receptors in acute myelogenous leukemia. Eur J Haematol :-, 99 ) Kawada H, Sasao T, Yonekura S, Hotta T:Clinical significance of granulocyte colony-stimulating factor (G-CSF) receptor expression in acute myeloid leukemia. Leuk Res :-, 998 ) Handa A, Kashimura T, Takeuchi S, Yamamoto A, Murohashi I, Bessho M, Hirashima K:Expression of functional granulocyte colony-stimulating factor receptors on human B-lymphocytic leukemia cells. Ann Hematol 9:-, ) Yoon SY, Kwon JA, Lee CK, Cho YJ, Kim YG, Lee KN : A Case of Biphenotypic Acute Leukemia. Korean J Hematol :-9, 99 8) Boneberg EM, Hartung T:Molecular aspects of anti-inflammatory action of G-CSF. Inflamm Res :9-8, 9) Franzke A, Piao W, Lauber J, Gatzlaff P, Konecke C, Hansen W, Schmitt-Thomsen A, Hertenstein B, Buer J, Ganser A:G-CSF as immune regulator in T cells expressing the G-CSF receptor : Implications for transplantation and autoimmune diseases. Blood :-9, ) Ninci EB, Brandstetter T, Meinhold-Heerlein I, Bettendorf H, Sellin D, Bauknecht T:G-CSF receptor expression in ovarian cancer. Int J Gynecol Cancer :9-, ) Kudo K, Nagai H, Numata S, Ichihara M, Kinoshita T, Horibe K, Kato K, Matsuyama T, Kodera Y, Kojima S:Absence of mutations in the granulocyte colony-stimulating factor (G-CSF) receptor gene in patients with myelodysplastic syndrome/acute myeloblastic leukaemia occurring after treatment of aplastic anaemia with G-CSF. Br J Haematol :-8, ) Awaya N, Uchida H, Miyakawa Y, Kinjo K, Matsushita H, Nakajima H, Ikeda Y, Kizaki M: Novel variant isoform of G-CSF receptor involved in induction of proliferation of FDCP- cells : Relevance to the pathogenesis of myelodysplastic syndrome. J Cell Physiol 9:-, ) Hunter MG, Avalos BR : Deletion of a critical internalization domain in the G-CSFR in acute

7 윤수영외 인 : 급성백혈성에서 G-CSF 수용체발현양상 myelogenous leukemia preceded by severe congenital neutropenia. Blood 9:-, 999 ) Freedman MH, Alter BP : Risk of myelodysplastic syndrome and acute myeloid leukemia in congenital neutropenias. Semin Hematol 9:8-, ) White SM, Ball ED, Ehmann WC, Rao AS, Tweardy DJ : Increased expression of the differentiation-defective granulocyte colony-stimulating factor receptor mrna isoform in acute myelogenous leukemia. Leukemia :899-9, 998 ) Kutlay S, Beksac M, Dalva K, Ilhan O, Koc H, Akan H, Ozcan M:The detection of flow cytometric G-CSF receptor expression and it's effect on therapy in acute myeloid leukemia. Leuk Lymphoma :9-9, ) Motoji T, Watanabe M, Uzumaki H, Kusaka M, Fukamachi H, Shimosaka A, Oshimi K, Mizoguchi H:Granulocyte colony-stimulating factor (G-CSF) receptors on acute myeloblastic leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay. Br J Haematol :- 9, 99 8) Shinjo K, Takeshita A, Ohnishi K, Ohno R: Granulocyte colony-stimulating factor receptor at various differentiation stages of normal and leukemic hematopoietic cells. Leuk Lymphoma :-, 99 9) Budel LM, Touw IP, Delwel R, Lowenberg B: Granulocyte colony-stimulating factor receptors in human acute myelocytic leukemia. Blood :8-, 989 ) Piao YF, Okabe T:Receptor binding of human granulocyte colony-stimulating factor to the blast cells of myeloid leukemia. Cancer Res :-, 99 ) Ohyashiki JH, Ohyashiki K, Miyauchi J, Fujieda H, Tauchi T, Saito M, Nakazawa S, Kimura N, Clark SC, Toyama K:Immunogenotypes and clonal culture analysis in B-precursor acute lymphoblastic leukemia. Leukemia :-, 99 ) Drach D, Estrov Z, Zhao S, Drach J, Cork A, Collins D, Kantarjian H, Andreeff M:Granulocytecolony stimulating factor, granulocyte-macrophage colony stimulating factor, PIXY-, stem cell factor, interleukin-, and interleukin- : Receptor binding and effects on clonogenic proliferation in acute lymphoblastic leukemia. Leuk Lymphoma : 9-88, 99 ) Cha KE, Yoon SY, Lee KN : Effect of granulocyte colony-stimulating factor on chemotherapeutic activity of cytosine arabinoside in acute leukemic cell lines. Int J Hematol :99-, ) Kim ES, Seol JG, Lee YY, Kim BK, Lee HH : Identification of Granulocyte-Macrophage Colony Stimulating Factor receptor assay on Leukemic cells. Korean J BRM :9-, 99 ) Lee KY, Suh BG, Kim JW, Lee W, Kim SY, Kim YY, Lee J, Lim J, Kim M, Kang CS, Han K: Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes. Exp Mol Med :-,

Jkbcs016(92-97).hwp

Jkbcs016(92-97).hwp Expression of bcl-2 and Apoptosis and Its Relationship to Clinicopathological Prognostic Factors in Breast Cancer - A Study with Long Term Follow-up correlated with the survival rate.(journal of Korean