急性骨髓性白血病是一種具遺傳異質性的疾病 (heterogeneous disorder)，其基因異常會影響疾病的侵犯強度以及對治療的反應。而在急性骨髓性白血病，白血
病細胞常因染色體轉位或基因突變進而影響造血轉錄因子導致造血異常。白血病的產生有「兩擊模式」(two hit model )的假說：第一類突變，會激活造血相關的酪胺酸激酶，有利於白血病細胞增殖，例如費城染色體(BCR/ABL)，FMS 相關酪胺酸激酶3（FMS like tyrosine kinase 3, FLT3）活化突變；第二類突變，涉及染色體轉位或造血轉錄因子之突變，導致細胞的正常分化被阻斷，包含MLL 重組。其中比較有趣為MLL 重組，MLL 基因位於人類染色體11q23，其異常在急性白血病常見，MLL 基因與拍檔基因重組形成融合基因。目前已知有七十多種fusion transcripts被選殖出來，因重組未能而在常規染色體檢查時發現，且其預後差，因此用分子生物技術來檢測MLL 基因重組就非常重要。我們發展一個創新的檢測方法，可以使用有限的臨床檢體來檢測MLL 常見基因變異，或偵測到新的融合基因(novelfusion partners)，稱之為3’-RAFT (3’-Rapid Amplification of Fusion
Transcript)。另外我們利用螢光原位雜交法及反轉錄聚合酶連鎖反應等分子檢驗的方法，檢測染色體檢查正常的急性骨髓性白血病病人，因一些因素而未發現到融合基因及基因變異的情形，並將分子診斷結果將與臨床特徵、FAB 分類、染色體變化及治療結果等作關聯性分析，以評估MLL 基因重組所形成的各種融合基因之臨床意義和預後價值。未來希望可以更精確且靈敏地偵測MLL 基因重組及所產生的不同融合基因，其研究結果也有助於將來探究血癌的生物學，即MLL 基因重組的致癌機轉。

Acute myeloid leukemia (AML) is a highly heterogeneous disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation. In approximately 60% of cases, specific recurrent chromosomal
aberrations can be identified by modern cytogenetic techniques, and is an important indicator to classify patients into three prognostic categories: favorable, intermediate, and poor risk. Currently, favorable risk patients are usually treated with chemotherapy while poor risk patients receive allogeneic stem cell transplantation. However, the largest subgroup of AML patients (approximately 40%) has no identifiable cytogenetic abnormalities and is classified as intermediate risk. In this special subgroup of patients, a number of studies have demonstrated the relationship between different translocations involving the mixed lineage leukemia (MLL) gene and patient
prognosis. The heterogeneity of MLL-rearranged AML is reflected by the identification of more than 70 different fusion partners of this gene and the panel is continuously increasing. The aim of this study is to develop a sensitive molecular profiling test for relevant risk stratification that can help in the decision of treatment and/or follow-up strategy.

論文審定書…………………………………………………………… i
誌謝…………………………………………………………………… ii
中文摘要………………………………………………………….….. iii
英文摘要………………………………………..……………………. iv
第 一 章 序論……………………………………………………… 1
1.1 急性骨髓性白病………………………………………… 1
1.1.1 急性骨髓性白血病的分類……………………………… 1
1.1.2 染色體分析的重要性…………………………………… 3
1.1.3 AML 中的基因轉位…………………………………….. 4
1.1.4 急性骨髓細胞白血病治療………………………………… 5
1.2 混合系白血病基因……………………………………… 7
1.2.1 轉錄調控因子、組織蛋白修飾及染色質重構………… 7
1.2.2 MLL 基因及其功能………………………………………… 9
1.2.3 MLL 融合基因(MLL fusion genes) …………………… 11
1.3 偵測MLL 融合基因的方法…………………………… 12
1.3.1 細胞遺傳技術(Cytogenetic technique) ………………… 13
1.3.2 螢光原位雜交法(FISH) ………………………………… 14
1.3.3 反轉錄聚合酶連鎖反(RT-PCR) ………………………… 15
1.4 染色體核型正常之急性骨髓性白血病………………… 16
1.4.1 染色體核型正常之急性骨髓性白血病發生率及預後…… 16
1.4.2 核仁磷酸蛋白、FMS 相關酪胺酸激酶3 基因突變……… 17
第 二 章 研究目標…………………………………………………… 18
第 三 章 實驗設計與材料方法.………………………………………19
3.1 實驗材料與實驗分組………………………………………19
3.2 染色體核型分析(Karyotyping) ……………………………19
3.3 螢光原位雜交法(FISH) …………………………………… 20
3.4 建構融合基因質體（Plasmid Constructions）………………21
3.5 建立Multiplex PCR 方法…………………………………… 26
3.6 MLL 融合基因的檢測方法………………………………… 28
3.7 BCR-ABL 融合基因的檢測方法……………………………29
3.8 AML-ETO 融合基因的檢測方法……………………………29
3.9 PML-RARα 融合基因的檢測方法………………………… 30
3.10 CEFβ-MYH11 融合基因inv(16)(p13;q22) …………………31
3.11 FMS 相關酪胺酸激酶3 (FLT3)基因突變的偵測……………31
3.12 核仁磷酸基因(NPM1) 基因突變的偵測…………………32
第 四 章 研究結果……………………………………………………33
4.1 以臨床案例為樣本，來瞭解成大醫學中心中，染色體檢查正常的AML 病人其基因變異的情形…………………………33
4.1.1 染色體核型分析方法分析……………………………………33
4.1.2 利用螢光原位雜交法FISH 偵測11q23 變異…………………33
4.1.3 建構融合基因質體（Plasmid Constructions）…………………34
4.1.4 MLL 融合基因檢測的結果……………………………………34
4.1.5 利用RT-PCR 的方法偵測病患各項的融合基因的結果………36
4.1.6 染色體正常的AML 細胞在核仁磷酸及FMS 相關酪胺酸激酶3 基因突變及預後…………………………………………37
4.2 發展一個創新的檢測方法- 3’-RAFT，可以使用有限的臨床
檢體來檢測MLL 常見基因變異，或偵測到新的融合基因
(novel fusion partners) …………………………………………38
4.2.1 利用MV4-11 細胞株來驗證3’-RAFT 方法……………………38
4.2.2 針對其它融合基因做RAFT 之後再做PCR 分析的結果……39
第 五 章 討論…………………………………………………………41
參考文獻……………………………………………………………… 45
圖……………………………………………………………………… 58
表……………………………………………………………………… 81
作者自述………………………………………………………………87

1. Shipley JL, Butera JN: Acute myelogenous leukemia. Exp Hematol 2009,
37(6):649-658.
2. Gregory TK, Wald D, Chen Y, Vermaat JM, Xiong Y, Tse W: Molecular
prognostic markers for adult acute myeloid leukemia with normal cytogenetics.
J Hematol Oncol 2009, 2:23.
3. Kelly LM, Gilliland DG: Genetics of myeloid leukemias. Annu Rev Genomics
Hum Genet 2002, 3:179-198.
4. 中華民國兒童癌症基金會: 兒童白血病衛教手冊.
5. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR,
Sultan C: Proposals for the classification of the acute leukaemias.
French-American-British (FAB) co-operative group. Br J Haematol 1976,
33(4):451-458.
6. Vardiman JW, Harris NL, Brunning RD: The World Health Organization (WHO)
classification of the myeloid neoplasms. Blood 2002, 100(7):2292-2302.
7. S.H. Swerdlow C, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele,J.,
Vardiman, J.W, : WHO Classification of Tumours of Haematopoietic
andLymphoid Tissues Fourth Edition, IARC, Lyon,. 2008.
8. McKenna RW: Multifaceted approach to the diagnosis and classification of
acute leukemias. Clin Chem 2000, 46(8 Pt 2):1252-1259.
9. Xiao Z, Greaves MF, Buffler P, Smith MT, Segal MR, Dicks BM, Wiencke JK,
Wiemels JL: Molecular characterization of genomic AML1-ETO fusions in
childhood leukemia. Leukemia 2001, 15(12):1906-1913.
10. Kogan SC, Bishop JM: Acute promyelocytic leukemia: from treatment to
genetics and back. Oncogene 1999, 18(38):5261-5267.
11. Rudkin CT, Hungerford DA, Nowell PC: DNA Contents of Chromosome Ph1
and Chromosome 21 in Human Chronic Granulocytic Leukemia. Science 1964,
144(3623):1229-1231.
12. Rowley JD: Letter: A new consistent chromosomal abnormality in chronic
myelogenous leukaemia identified by quinacrine fluorescence and Giemsa
staining. Nature 1973, 243(5405):290-293.
13. Shtivelman E, Lifshitz B, Gale RP, Canaani E: Fused transcript of abl and bcr
genes in chronic myelogenous leukaemia. Nature 1985, 315(6020):550-554.
14. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G,
Gottardi E, Rambaldi A, Dotti G, Griesinger F et al: Standardized RT-PCR
analysis of fusion gene transcripts from chromosome aberrations in acute
leukemia for detection of minimal residual disease. Report of the BIOMED-1
Concerted Action: investigation of minimal residual disease in acute leukemia.
Leukemia 1999, 13(12):1901-1928.
15. Marschalek R: Mixed lineage leukemia: roles in human malignancies and
potential therapy. FEBS J 2010, 277(8):1822-1831.
16. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY: Use
of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood
1988, 72(2):567-572.
17. Berman E, Heller G, Santorsa J, McKenzie S, Gee T, Kempin S, Gulati S,
Andreeff M, Kolitz J, Gabrilove J et al: Results of a randomized trial comparing
idarubicin and cytosine arabinoside with daunorubicin and cytosine arabinoside
in adult patients with newly diagnosed acute myelogenous leukemia. Blood 1991,
77(8):1666-1674.
18. Weick JK, Kopecky KJ, Appelbaum FR, Head DR, Kingsbury LL, Balcerzak SP,
Bickers JN, Hynes HE, Welborn JL, Simon SR et al: A randomized
investigation of high-dose versus standard-dose cytosine arabinoside with
daunorubicin in patients with previously untreated acute myeloid leukemia: a
Southwest Oncology Group study. Blood 1996, 88(8):2841-2851.
19. Li YM, Broome JD: Arsenic targets tubulins to induce apoptosis in myeloid
leukemia cells. Cancer Res 1999, 59(4):776-780.
20. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville
R, Talpaz M: Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in
the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia
with the Philadelphia chromosome. N Engl J Med 2001, 344(14):1038-1042.
21. Rabbitts TH: Chromosomal translocations in human cancer. Nature 1994,
372(6502):143-149.
22. Lemon B, Tjian R: Orchestrated response: a symphony of transcription factors
for gene control. Genes Dev 2000, 14(20):2551-2569.
23. Goll MG, Bestor TH: Histone modification and replacement in chromatin
activation. Genes Dev 2002, 16(14):1739-1742.
24. Berger SL: Histone modifications in transcriptional regulation. Curr Opin Genet
Dev 2002, 12(2):142-148.
25. Klose RJ, Bird AP: Genomic DNA methylation: the mark and its mediators.
Trends Biochem Sci 2006, 31(2):89-97.
26. Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, Fuks F, Lo
Coco F, Kouzarides T, Nervi C et al: Methyltransferase recruitment and DNA
hypermethylation of target promoters by an oncogenic transcription factor.
Science 2002, 295(5557):1079-1082.
27. Sharma RP: Schizophrenia, epigenetics and ligand-activated nuclear receptors: a
framework for chromatin therapeutics. Schizophr Res 2005, 72(2-3):79-90.
28. Yeates TO: Structures of SET domain proteins: protein lysine
methyltransferases make their mark. Cell 2002, 111(1):5-7.
29. Zegerman P, Canas B, Pappin D, Kouzarides T: Histone H3 lysine 4 methylation
disrupts binding of nucleosome remodeling and deacetylase (NuRD) repressor
complex. J Biol Chem 2002, 277(14):11621-11624.
30. Stass S, Mirro J, Melvin S, Pui CH, Murphy SB, Williams D: Lineage switch in
acute leukemia. Blood 1984, 64(3):701-706.
31. Mirro J, Zipf TF, Pui CH, Kitchingman G, Williams D, Melvin S, Murphy SB,
Stass S: Acute mixed lineage leukemia: clinicopathologic correlations and
prognostic significance. Blood 1985, 66(5):1115-1123.
32. Mirro J, Kitchingman GR, Williams DL, Murphy SB, Zipf TF, Stass SA: Mixed
lineage leukemia: the implications for hematopoietic differentiation. Blood 1986,
68(2):597-599.
33. Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R, 3rd, Patel Y, Harden
A, Rubinelli P, Smith SD, LeBeau MM, Rowley JD et al: Identification of a
gene, MLL, that spans the breakpoint in 11q23 translocations associated with
human leukemias. Proc Natl Acad Sci U S A 1991, 88(23):10735-10739.
34. Huret JL, Dessen P, Bernheim A: An atlas of chromosomes in hematological
malignancies. Example: 11q23 and MLL partners. Leukemia 2001,
15(6):987-989.
35. Nilson I, Lochner K, Siegler G, Greil J, Beck JD, Fey GH, Marschalek R:
Exon/intron structure of the human ALL-1 (MLL) gene involved in
translocations to chromosomal region 11q23 and acute leukaemias. Br J
Haematol 1996, 93(4):966-972.
36. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G: Genome
regulation by polycomb and trithorax proteins. Cell 2007, 128(4):735-745.
37. Hsieh JJ, Cheng EH, Korsmeyer SJ: Taspase1: a threonine aspartase required for
cleavage of MLL and proper HOX gene expression. Cell 2003, 115(3):293-303.
38. Zeleznik-Le NJ, Harden AM, Rowley JD: 11q23 translocations split the
"AT-hook" cruciform DNA-binding region and the transcriptional repression
domain from the activation domain of the mixed-lineage leukemia (MLL) gene.
Proc Natl Acad Sci U S A 1994, 91(22):10610-10614.
39. Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O, Meyerson M,
Cleary ML: The menin tumor suppressor protein is an essential oncogenic
cofactor for MLL-associated leukemogenesis. Cell 2005, 123(2):207-218.
40. Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD, Levine SS, Lee JC,
Hayes DN, Shanmugam KS, Bhattacharjee A, Biondi CA et al: Menin
associates with a trithorax family histone methyltransferase complex and with
the hoxc8 locus. Mol Cell 2004, 13(4):587-597.
41. Jin S, Zhao H, Yi Y, Nakata Y, Kalota A, Gewirtz AM: c-Myb binds MLL
through menin in human leukemia cells and is an important driver of
MLL-associated leukemogenesis. J Clin Invest 2010, 120(2):593-606.
42. Thiel AT, Blessington P, Zou T, Feather D, Wu X, Yan J, Zhang H, Liu Z, Ernst
P, Koretzky GA et al: MLL-AF9-induced leukemogenesis requires coexpression
of the wild-type Mll allele. Cancer Cell 2010, 17(2):148-159.
43. Yang Y, Gurung B, Wu T, Wang H, Stoffers DA, Hua X: Reversal of
preexisting hyperglycemia in diabetic mice by acute deletion of the Men1 gene.
Proc Natl Acad Sci U S A 2010, 107(47):20358-20363.
44. Karnik SK, Chen H, McLean GW, Heit JJ, Gu X, Zhang AY, Fontaine M, Yen
MH, Kim SK: Menin controls growth of pancreatic beta-cells in pregnant mice
and promotes gestational diabetes mellitus. Science 2007, 318(5851):806-809.
45. Gao SB, Hua X, Jin GH: Menin regulates endocrine diseases by controlling
histone modification and gene transcription. Ann Endocrinol (Paris) 2008,
69(5):426-432.
46. Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I, Herr
W, Cleary ML: Leukemia proto-oncoprotein MLL forms a SET1-like histone
methyltransferase complex with menin to regulate Hox gene expression. Mol
Cell Biol 2004, 24(13):5639-5649.
47. Hess JL, Yu BD, Li B, Hanson R, Korsmeyer SJ: Defects in yolk sac
hematopoiesis in Mll-null embryos. Blood 1997, 90(5):1799-1806.
48. Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J, Ben Abdelali R,
Macintyre E, De Braekeleer E, De Braekeleer M et al: New insights to the MLL
recombinome of acute leukemias. Leukemia 2009, 23(8):1490-1499.
49. Felix CA, Hosler MR, Slater DJ, Parker RI, Masterson M, Whitlock JA,
Rebbeck TR, Nowell PC, Lange BJ: MLL genomic breakpoint distribution
within the breakpoint cluster region in de novo leukemia in children. J Pediatr
Hematol Oncol 1998, 20(4):299-308.
50. Ono R, Nosaka T, Hayashi Y: Roles of a trithorax group gene, MLL, in
hematopoiesis. Int J Hematol 2005, 81(4):288-293.
51. La P, Desmond A, Hou Z, Silva AC, Schnepp RW, Hua X: Tumor suppressor
menin: the essential role of nuclear localization signal domains in coordinating
gene expression. Oncogene 2006, 25(25):3537-3546.
52. Krivtsov AV, Armstrong SA: MLL translocations, histone modifications and
leukaemia stem-cell development. Nat Rev Cancer 2007, 7(11):823-833.
53. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden
MD, Sallan SE, Lander ES, Golub TR, Korsmeyer SJ: MLL translocations
specify a distinct gene expression profile that distinguishes a unique leukemia.
Nat Genet 2002, 30(1):41-47.
54. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R, Behm FG,
Raimondi SC, Relling MV, Patel A et al: Classification, subtype discovery, and
prediction of outcome in pediatric acute lymphoblastic leukemia by gene
expression profiling. Cancer Cell 2002, 1(2):133-143.
55. Dimartino JF, Cleary ML: Mll rearrangements in haematological malignancies:
lessons from clinical and biological studies. Br J Haematol 1999,
106(3):614-626.
56. Dreyling MH, Schrader K, Fonatsch C, Schlegelberger B, Haase D, Schoch C,
Ludwig W, Loffler H, Buchner T, Wormann B et al: MLL and CALM are fused
to AF10 in morphologically distinct subsets of acute leukemia with translocation
t(10;11): both rearrangements are associated with a poor prognosis. Blood 1998,
91(12):4662-4667.
57. Rubnitz JE, Raimondi SC, Tong X, Srivastava DK, Razzouk BI, Shurtleff SA,
Downing JR, Pui CH, Ribeiro RC, Behm FG: Favorable impact of the t(9;11) in
childhood acute myeloid leukemia. J Clin Oncol 2002, 20(9):2302-2309.
58. Tamai H, Inokuchi K: 11q23/MLL acute leukemia : update of clinical aspects. J
Clin Exp Hematop 2010, 50(2):91-98.
59. Mitelman F, Johansson B, Mertens F: The impact of translocations and gene
fusions on cancer causation. Nat Rev Cancer 2007, 7(4):233-245.
60. Gray JW, Collins C: Genome changes and gene expression in human solid
tumors. Carcinogenesis 2000, 21(3):443-452.
61. Frohman MA, Dush MK, Martin GR: Rapid production of full-length cDNAs
from rare transcripts: amplification using a single gene-specific oligonucleotide
primer. Proc Natl Acad Sci U S A 1988, 85(23):8998-9002.
62. von Bergh AR, Wijers PM, Groot AJ, van Zelderen-Bhola S, Falkenburg JH,
Kluin PM, Schuuring E: Identification of a novel RAS GTPase-activating
protein (RASGAP) gene at 9q34 as an MLL fusion partner in a patient with de
novo acute myeloid leukemia. Genes Chromosomes Cancer 2004,
39(4):324-334.
63. Osaka M, Rowley JD, Zeleznik-Le NJ: MSF (MLL septin-like fusion), a fusion
partner gene of MLL, in a therapy-related acute myeloid leukemia with a
t(11;17)(q23;q25). Proc Natl Acad Sci U S A 1999, 96(11):6428-6433.
64. Frohman MA: On beyond classic RACE (rapid amplification of cDNA ends).
PCR Methods Appl 1994, 4(1):S40-58.
65. Bertling WM, Beier F, Reichenberger E: Determination of 5' ends of specific
mRNAs by DNA ligase-dependent amplification. PCR Methods Appl 1993,
3(2):95-99.
66. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J,
Hann I, Stevens R, Burnett A et al: The importance of diagnostic cytogenetics
on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10
trial. The Medical Research Council Adult and Children's Leukaemia Working
Parties. Blood 1998, 92(7):2322-2333.
67. Bienz M, Ludwig M, Leibundgut EO, Mueller BU, Ratschiller D, Solenthaler M,
Fey MF, Pabst T: Risk assessment in patients with acute myeloid leukemia and a
normal karyotype. Clin Cancer Res 2005, 11(4):1416-1424.
68. Akagi T, Ogawa S, Dugas M, Kawamata N, Yamamoto G, Nannya Y, Sanada M,
Miller CW, Yung A, Schnittger S et al: Frequent genomic abnormalities in acute
myeloid leukemia/myelodysplastic syndrome with normal karyotype.
Haematologica 2009, 94(2):213-223.
69. de Oliveira FM, Tone LG, Simoes BP, Falcao RP, Brassesco MS,
Sakamoto-Hojo ET, dos Santos GA, Marinato AF, Jacomo RH, Rego EM:
Acute myeloid leukemia (AML-M2) with t(5;11)(q35;q13) and normal
expression of cyclin D1. Cancer Genet Cytogenet 2007, 172(2):154-157.
70. Gilliland DG, Griffin JD: The roles of FLT3 in hematopoiesis and leukemia.
Blood 2002, 100(5):1532-1542.
71. Sheikhha MH, Awan A, Tobal K, Liu Yin JA: Prognostic significance of FLT3
ITD and D835 mutations in AML patients. Hematol J 2003, 4(1):41-46.
72. Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF,
Haferlach T, Hiddemann W, Falini B: Nucleophosmin gene mutations are
predictors of favorable prognosis in acute myelogenous leukemia with a normal
karyotype. Blood 2005, 106(12):3733-3739.
73. John P. Greer MMW: Wintrobe’s clinical hematology” 12th edition
74. Andersson A, Hoglund M, Johansson B, Lassen C, Billstrom R, Garwicz S,
Nilsson PG, Mitelman F, Fioretos T: Paired multiplex reverse-transcriptase
polymerase chain reaction (PMRT-PCR) analysis as a rapid and accurate
diagnostic tool for the detection of MLL fusion genes in hematologic
malignancies. Leukemia 2001, 15(8):1293-1300.
75. Cuthbert G, Thompson K, Breese G, McCullough S, Bown N: Sensitivity of
FISH in detection of MLL translocations. Genes Chromosomes Cancer 2000,
29(2):180-185.
76. Mardis ER: Next-generation DNA sequencing methods. Annu Rev Genomics
Hum Genet 2008, 9:387-402.
77. Sanger F, Coulson AR: A rapid method for determining sequences in DNA by
primed synthesis with DNA polymerase. J Mol Biol 1975, 94(3):441-448.
78. von Bubnoff A: Next-generation sequencing: the race is on. Cell 2008,
132(5):721-723.
79. Kahvejian A, Quackenbush J, Thompson JF: What would you do if you could
sequence everything? Nat Biotechnol 2008, 26(10):1125-1133.