Targeted Therapy in Blood cancers (I) 林建鴻 醫師 馬偕紀念醫院 血液暨腫瘤科 2009-10-28

「血液」的來源 & 分布 製造 分布 卵黃囊 : 胚胎 2 週後 肝臟﹝+ 脾臟﹞: 胚胎 6 週﹝10週﹞後 骨髓 : 胚胎 24 週後  成人 四肢腸骨造血 ( 20 歲以前 ) 分布 骨髓 循環系統 脾臟、肝臟

血球家譜 骨髓 髓幹細胞 幹細胞 T/NK T 細胞 淋巴 幹細胞 B 細胞 Plasma 細胞 RBC 紅血球 Neutrophil 中性球 Monocyte 單核球 Eosinophil 伊紅球 Basophil 嗜鹼球 Platelet 血小板 髓幹細胞 幹細胞 骨髓 T/NK T 細胞 細胞 NK 細胞 B 細胞 Plasma 細胞 淋巴 幹細胞 胸腺 淋巴結

血液組成 血液 佔體重 7% 細胞 45% volume 血漿 55% volume 3500cc /50Kg; 5000cc/ 70kg. 紅血球數 ~ 5,000K/ul (95%) 白血球數 ~ 5K/ul (0.1%) 血小板數 ~ 250K/ul (5%) 血漿 55% volume

WHO Classification of Myeloid Neoplasms in Adults - 2008 AML MDS MPN (Myeloproliferative Neoplasms) MPN MDS/ MPN MPN with Eosinophilia and Abnormalities of PDGFRA/B Abnormalities of FGFR1 Classic CML PV ET PMF Atypical CNL CEL (NOS)/HES SM MPN-U CMML JMML aCML MDS/MPD-U i.e. RARS-T Rearranged PDGFRA FGFR1 Tefferi, Vardiman Leukemia 2007; EPUB 9-2007

WHO Classification of Myeloid Neoplasms in Adults - 2008 AML MDS MPN (Myeloproliferative Neoplasms) Differentiation Impaired Normal Proliferation/survival Preserved Impaired Increased Tefferi, Vardiman Leukemia 2007; EPUB 9-2007

慢性骨髓性白血病 Chronic Myeloid Leukemia (CML)

Epidemiology of CML Incidence: Around 200 patients per year in Taiwan Median age range at presentation is 45-55 years CML accounts for 15%-20% of all adult leukemia Incidence increases with age Up to 30% of patients are aged >60 years Slightly higher incidence in males Male-to-female ratio—1.3:1 At presentation 50% diagnosed by routine laboratory tests 85% diagnosed during chronic phase 根據台灣癌症登記的統計, 每年新發生的CML病人大約為150人左右 出現臨床症狀之年齡中數為53歲，年齡範圍中數為45~55歲。 全球之慢性骨髓性白血病(CML)發生率在每10萬人中約有1~2個病例，佔所有成人白血病的15~20%。CML發生率會隨著年齡而上升，近30%的患者60歲以後才會出現臨床症狀，如此可能影響治療方式。兒童較少見CML，約10%的患者年齡小於20歲。 男性CML患者比女性稍多，男性與女性比例為1.3:1。 約50%的患者診斷時並無症狀，臨床上以常規實驗室血液檢測診斷CML。 85%的患者確定診斷時處於CML之慢性期。 Druker BJ. Cancer Cell. 2002;1:31-36; Greenlee RT et al. CA Cancer J Clin. 2001;51:15-36; Taiwan Cancer Registry Sawyers. N Engl J Med. 1999;340:1330; Faderl et al. Ann Intern Med. 1999;131:207. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Faderl S, Talpaz M, Estrov Z, Kantarjian HM. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med. 1999;131:207-219. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

Incidence and Mortality Associated With Leukemias (United States, 2008) 44,270 35,000 35,000 30,000 25,000 20,000 15,000 10,000 5000 Overall AML CLL CML ALL 30,000 25,000 21,710 20,000 15,110 15,000 13,290 10,000 8,820 可將最常見的白血病分為4大類：依照細胞族譜可分為—骨髓性或淋巴性；根據臨床病程之最終分化程度又可分為—急性或慢性。這4種白血病的診斷特性、病程與治療選擇都不同。 根據文獻記載，全球CML發病率在每10萬人中約1~2個病例，佔所有成人白血病的15~20%。 2008年美國44,270位白血症病例中，CML佔11% (n=4830)；慢性淋巴性白血病(CLL)佔34% (n=15,110)；急性骨髓性白血病(AML)佔30% (n=13,290)；急性淋巴性白血病(ALL)佔12% (n=5,430)；其他類型之白血病佔13% (n=5,610)。 5,430 4,830 4,390 5000 450 1,460 AML = acute myeloid leukemia; CLL = chronic lymphocytic leukemia; CML = chronic myelogenous leukemia; ALL = acute lymphoblastic leukemia. American Cancer Society. At: http://www.cancer.org/docroot/STT/stt_0.asp. Accessed Jan 2009. American Cancer Society. At: http://www.cancer.org/docroot/STT/st_0.asp. Accessed August 30, 2004. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Kantarjian HM, Giles FJ, O’Brien SM, Talpaz M. Clinical course and therapy of chronic myelogenous leukemia with interferon-alpha and chemotherapy. Hematol Oncol Clin North Am. 1998;12:31-80. American Cancer Society. At: http://www.cancer.org/docroot/STT/stt_0.asp. Accessed November 2004. Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics, 1997. CA Cancer J Clin. 1997;47:5-27.

Clinical Presentation of Chronic Phase CML Asymptomatic in ~50% of cases Common symptoms Fatigue Weight loss/anorexia Abdominal fullness Common signs Palpable splenomegaly Common laboratory findings Abnormal differential Leukocytosis Thrombocytosis Anemia Basophilia 疲倦、體重減輕、腹脹與夜間盜汗等臨床症狀最常出現於CML的慢性期。50%的患者無臨床症狀。 50%以上的患者在身體檢查時可由觸診發現脾臟腫大。 一般實驗室檢查結果包括分化異常的白血球增多症、貧血、嗜鹼性球增多症與血小板增多症。 Faderl et al. Ann Intern Med. 1999;131:207. Goldman. Curr Opin Hematol. 1997;4:277. Faderl S, Talpaz M, Estrov Z, Kantarjian HM. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med. 1999;131:207-219. Goldman JM. Chronic myeloid leukemia. Curr Opin Hematol. 1997;4:277-285.

Biologic Properties of CML Proliferative disorder of hematopoietic stem cells Characterized by presence of a chromosomal abnormality (Ph chromosome) Arises from a single molecular abnormality, the Bcr-Abl tyrosine kinase Abnormality causes transformation of a hematopoietic progenitor into malignant clone 3 clinical stages Chronic phase Accelerated phase Blastic phase CML是一種造血先驅細胞增生的惡性疾病。此種疾病已經過廣泛研究，臨床分期與預後因子的定義非常清楚，並已有文獻報告記載多種療法之療效與安全性。 1961年，在CML患者的骨髓(BM)細胞中發現稱為費城(Ph)染色體之特殊染色體異常。Ph染色體是因為第9號與第22號染色體的長臂發生相互易位t (9;22)(q34;q11)，導致第22號染色體變短。 95%的CML患者都有Ph染色體，Ph染色體會使Bcr-Abl酪胺酸激酶異常持續活化而導致惡性轉化。因此Bcr-Abl激酶是研發新藥設計的主要目標。 CML按病程可以分為三期: 慢性期, 急性期, 急性轉化期 Ph = Philadelphia. Faderl et al. N Engl J Med. 1999;341:164. Sawyers. N Engl J Med. 1999;340:1330. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 1990;247:824-830. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 1999;341:164-172. Heisterkamp N, Jenster G, ten Hoeve J, Zovich D, Pattengale PK, Groffen J. Acute leukemia in bcr/abl transgenic mice. Nature. 1990;344:251-253. Nowell PC, Hungerford DA. Chromosome studies on normal and leukemic human leukocytes. J Nat Cancer Inst. 1960;25:85-109. Pasternak G, Hochhaus A, Schultheis B, Hehlmann R. Chronic myelogenous leukemia: molecular and cellular aspects. J Cancer Res Clin Oncol. 1998;124:643-660. Rowley JD. A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

Comparative Peripheral Blood Smear Normal Chronic Phase CML CML慢性期患者之典型Wright’s染色血液抹片可見骨髓細胞過多的特徵－主要是顆粒球增多，可發現包括芽球到所有成熟分化期的顆粒球。嗜鹼性球增多與輕微貧血也很常見。30%~50%的患者在診斷時血小板數目會增多。 BM可見骨髓系細胞明顯增生，有些患者會有纖維化現象。 Courtesy of John K. Choi, MD, PhD, University of Pennsylvania. Chronic leukaemia and myelodysplasia. In: Hoffbrand AV, Pettit JE, eds. Color Atlas of Clinical Hematology. 2nd ed. London, UK: Mosby-Wolfe; 1994:180-207. Cortes JE, Talpaz M, Kantarjian H. Chronic myelogenous leukemia: a review. Am J Med. 1996;100:555-570. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159.

Hematologic Parameters by Phase of CML Parameter Chronic Accelerated Blastic WBC count (/L) 20 × 109 — — Blasts (%) 1-15 15 30 Basophils (%)  20 — Platelets  or normal  or   Bone marrow Myeloid hyperplasia 確認CML與疾病分期之診斷檢查，對決定治療方法而言非常重要。 週邊血液的全血分類計數中，白血球(WBC)數目通常20×109/L，白血球數目隨著病情惡化還可能會增加。增加的白血球中，有許多是顆粒球，這些顆粒球成熟時期不一。 週邊血液芽球增加是CML進入加速期與急性轉化期的特徵，此時症狀與急性白血病類似。 雖然血小板的數目經常會在慢性期增加，但血小板減少症可能是末期疾病的跡象。 雖然淋巴球與單核球數目可能正常，但惡化的貧血與嗜鹼性球增多症是末期CML的特徵。 BM抽取液與切片檢體可見骨髓細胞增多，骨髓系細胞增生且多是未成熟的骨髓芽球；骨髓系與紅血球系細胞比例增加為10:1到30:1 (正常為2:1到5:1)。 嗜酸性球、嗜鹼性球與巨核球數目都會增加且發育不良。 細胞學檢查顯示：95%的患者都有Ph 染色體；但是病情惡化時可能會出現其他異常核型。可用高敏感之聚合酶鏈反應偵測bcr-abl RNA。 WBC = white blood cell. Faderl et al. Oncology (Huntingt). 1999;13:169. Sawyers. N Engl J Med. 1999;340:1330. Cortes JE, Talpaz M, Kantarjian H. Chronic myelogenous leukemia: a review. Am J Med. 1996;100:555-570. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

The Ph Chromosome and the bcr-abl Gene: The t(9;22) Translocation Chromosome 9 q+ Chromosome 9 Philadelphia Chromosome (or 22q-) Chromosome 22 Ph染色體是由第9號與第22號染色體之長臂相互易位t (9;22)(q34;q11)而產生。 由染色體9q34區製造非接受器酪胺酸激酶的abl基因(Abelson老鼠白血病致癌基因原)轉移到染色體22q11區的bcr基因(斷裂點聚集區)，以產生異常之雜合bcr-abl基因。 bcr-abl基因會轉錄成雜合訊息RNA；此RNA的轉譯產物是異常融合的蛋白質酪胺酸激酶。 1960年，Ph染色體最初被認為是CML患者之骨髓細胞中變短的第22號染色體。這是有關人類癌症之專一性基因異常的第一篇報告。 95%的CML患者都有Ph染色體－所以Ph染色體是CML的特徵。 標準細胞學技術可在BM細胞之有絲分裂中期偵測到Ph染色體。 紅血球、顆粒球、單核球與巨核球等所有骨髓系細胞都有Ph染色體，有些淋巴系細胞也會有Ph染色體。這表示惡性CML轉化作用源自幹細胞。 老鼠如果接受感染帶有由Ph染色體反轉錄病毒之p210 Bcr-Abl激酶基因的BM細胞時，會產生惡性血液疾病，症狀類似CML慢性期的骨髓性增生。 bcr-abl bcr FUSION PROTEIN WITH CONSTITUTIVE TYROSINE KINASE ACTIVITY abl Melo. Blood. 1996;88:2375. Pasternak et al. J Cancer Res Clin Oncol. 1998;124:643. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 1990;247:824-830. Osarogiagbon UR, McGlave PB. Chronic myelogenous leukemia. Curr Opin Hematol. 1999;6:241-246. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 1999;341:164-172. Melo JV. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood. 1996;88:2375-2384. Pasternak G, Hochhaus A, Schultheis B, Hehlmann R. Chronic myelogenous leukemia: molecular and cellular aspects. J Cancer Res Clin Oncol. 1998;124:643-660. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

History The Philadelphia chromosome 1960 by Peter Nowell (University of Pennsylvania School of Medicine) and David Hungerford ( Fox Chase Cancer Center) 1973, Janet D. Rowley(University of Chicago) identified the mechanism by which the Philadelphia chromosome arises as a translocation.

* 2基因ABL與BCR重新接合成一新的融合基因BCR/ABL

Cytogenetics

FISH

Clinical Development of Imatinib Glivec的臨床研究update

Stage-Related Survival With SCT (1987-2001) 100 90 80 70 60 First chronic phase (n=1903) % surviving 50 40 Accelerated and second chronic phase (n=744) 30 20 同種異體幹細胞移植(SCT)是目前認為唯一可能治癒CML的療法；但是大多數患者並不適合作SCT。 適合作SCT的條件除了必須有HLA吻合的手足捐贈者外，還必須考慮捐贈者與患者年紀(一般而言<55歲)是否合適。最好能於診斷後1年內作移植手術。約15~20%的CML患者符合上述條件。 慢性期患者接受移植手術的5年存活率為54~70%；存活率隨著疾病期數的進展而降低。 CML慢性期的復發率為13~20%。 以捐贈骨髓登錄資料尋找SCT的無血緣捐贈者(MUD)可將適合SCT之CML患者增加到30%。分子檢查可比對HLA-A、HLA-B與DRB1。移植3年後的存活率是40~57%。但是患者之年齡、病期與移植時間點等因素對移植結果影響甚鉅。 可能會發生嚴重疾病(如GVHD與全身感染)。SCT手術引起的罹病率約為10%，但年齡和HLA吻合程度影響很大(如MUD移植之罹病率可能為25~50%)。 Blastic phase (n=159) 10 P=0.0001 0 1 2 3 4 5 Years after transplant National Marrow Donor Program overview slide presentation. At: http://www.marrow.org/NMDP/SLIDESET/sld031.htm. Accessed November 2004. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Goldman JM. Chronic myeloid leukemia. Curr Opin Hematol. 1997;4:277-285. Hansen JA, Gooley TA, Martin PJ, et al. Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med. 1998;338:962-968. Mughal TI, Goldman JM. Chronic myeloid leukaemia: a therapeutic challenge. Ann Oncol. 1995;6:637-644. National Marrow Donor Program overview slide presentation. Available at: http://www.marrow.org/NMDP/SLIDESET/sld031.htm. Accessed November 2004. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

Historical Perspective on Therapeutic Options in CML Hydroxyurea / busulfan were early oral chemotherapeutics of choice for CML Allogeneic SCT remains a curative option for patients with CML Associated with mortality and GVHD IFN- therapy alone superior to busulfan Addition of ara-C to IFN- therapy improves hematologic and cytogenetic responses Hydroxyurea/busulfan是治療CML之早期口服化學治療用藥。雖然Busulfan副作用發生率比hydroxyurea高，但是仍用於同種異體SCT的準備療法。 同種異體SCT可治療CML，但是此療法的早期存活率比hydroxyurea與IFN-合併療法還低。此外，大約一半接受同種異體SCT的患者4年內會產生GVHD。 CML慢性期患者接受IFN-單一治療的OS比busulfan治療較佳，但與hydroxyurea治療差不多。在IFN-治療中加入ara-C可提高血液學與細胞學反應和OS。

Imatinib Mesylate: Background A selective tyrosine kinase inhibitor of KIT Bcr-Abl PDGFR-A/B First used in Ph+ CML C29H31N7O•CH4SO3 Molecular weight 589.7 試管與體內研究都顯示Imatinib mesylate (以前稱為STI571)。是可抑制特殊酪胺酸激酶活性的選擇性酪胺酸激酶抑制劑。 imatinib mesylate可有效抑制細胞內之Bcr-Abl酪胺酸激酶、穿膜酪胺酸激酶接受器(PDGFR)以及幹細胞生長因子的接受器KIT。 臨床研究已顯示Imatinib mesylate具有顯著的抗腫瘤活性，imatinib mesylate可用於治療 費城染色體(Ph+)陽性之CML慢性期成人新病例 IFN-治療失敗之Ph+ CML急性轉化期、加速期或慢性期 幹細胞移植(SCT)後復發的Ph+ CML小兒病例 對IFN-治療有抗藥性的Ph+ CML小兒病例 Class: Phenylaminopyrimidines PDGFR = platelet-derived growth factor receptor; Ph = Philadelphia chromosome. Druker et al. Nat Med. 1996;2:561. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med. 2002;346:645-652. O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2003;348:994-1004. Sawyers CL, Hochhaus A, Feldman E, et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood. 2002;99:3530-3539. Talpaz M, Silver RT, Druker BJ, et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood. 2002;99:1928-1937.

Normal Bcr-Abl Signaling Substrate The kinase domain activates a substrate protein, eg, PI3 kinase, by phosphorylation This activated substrate initiates a signaling cascade culminating in cell proliferation and survival Bcr-Abl Effector P P ADP P Bcr-Abl是調控不佳(disregualted)之酪胺酸激酶蛋白，可進行自體與受質之磷酸化作用。 具有開啟(活化)與關閉(自體抑制)兩種狀態之高度彈性結構。 狀態可透過分子間或由其他蛋白質調控。 腺核苷三磷酸(ATP)與Bcr-Abl之激酶作用區結合便啟動Bcr-Abl訊息，如此可讓結合受質會被磷酸化。磷酸根轉移之後，磷酸化的受質就可結合並活化下游作用分子。 ATP P P P SIGNALING ADP = adenosine diphosphate; ATP = adenosine triphosphate; P = phosphate. Savage and Antman. N Engl J Med. 2002;346:683 Scheijen and Griffin. Oncogene. 2002;21:3314. Hantschel O, Superti-Furga G. Regulation of the c-Abl and Bcr-Abl tyrosine kinases. Nat Rev Mol Cell Biol. 2004;5:33-44. Savage DG, Antman KH. Imatinib mesylate–a new oral targeted therapy. N Engl J Med. 2002;346:683-693. Scheijen B, Griffin JD. Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease. Oncogene. 2002;21:3314-3333.

Imatinib Mesylate: Mechanism of Action Imatinib mesylate occupies the ATP binding pocket of the Abl kinase domain This prevents substrate phosphorylation and signaling A lack of signaling inhibits proliferation and survival Bcr-Abl P ATP imatinib mesylate在分子層次會與Bcr-ABl之酪胺酸激酶區的特殊作用區結合。 imatinib mesylate是和ATP有類似結構的藥劑，可與Bcr-Abl結合。imatinib mesylate與Bcr-Abl的結合親和力比ATP和Bcr-Abl的ATP結合區更強。 Bcr-Abl的酪胺酸激酶活性取決於ATPase活性。 Bcr-Abl與imatinib mesylate結合之後會阻止Bcr-Abl和ATP的結合與水解，而影響Bcr-Abl的激酶活性。 阻斷Bcr-Abl下游需要磷酸化才能活化的受質。 然後阻斷Bcr-Abl所活化的下游訊息傳遞路徑。 Imatinib mesylate SIGNALING Savage and Antman. N Engl J Med. 2002;346:683. Savage DG, Antman KH. Imatinib mesylate–a new oral targeted therapy. N Engl J Med. 2002;346:683-693. Scheijen B, Griffin JD. Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease. Oncogene. 2002;21:3314-3333.

History of Glivec Year Event 1992年 諾華藥廠製造出Gleevec® (註：美國稱為Gleevec，台灣稱為Glivec)。 1998年06月 Gleevec®使用於慢性骨髓性白血病的第一期臨床試驗開始進行。 2001年4月5日 Gleevec®使用於慢性骨髓性白血病的第一期臨床試驗結果於新英格蘭醫學雜誌(The New England Journal of Medicine)發表。 新英格蘭醫學雜誌也發表了一個案研究顯示Gleevec®在晚期之惡性胃腸道基質瘤(GISTs)病人可能有其效果存在。 2001年5月1日 諾華癌症研究治療領域處處長亞歷士‧邁特(Alex Matter)醫師因為其在Gleevec®研發中所佔的重要地位而獲得哈佛的Warren Aplert 基金會科學獎。 2001年5月10日 美國食品藥物管理局(U.S. Food and Drug Administration, FDA)在送請新藥申請不到三個月的時間，即核准Gleevec®使用於急性轉化期、加速期、干擾素-α治療失敗之慢性期的費城染色體陽性慢性骨髓性白血病病人。 2001年5月13日 在美國臨床癌症醫學會年會中發表的資料顯示Gleevec®使用於GIST的治療有效。 2001年10月3日 台灣行政院衛生署核准使用Glivec®治療急性轉化期、加速期、干擾素-α治療無效之慢性期的慢性骨髓性白血病病人。 2001年11月7日 歐盟核准Glivec®使用於急性轉化期、加速期、干擾素-α治療失敗之慢性期的費城染色體陽性慢性骨髓性白血病成年病人。

IRIS Study Design and Patient Status at 6 Years RANDOMI Z E 364 (66%) Imatinib n = 553 14 (3%) Crossover 359 (65%) IFN-a + Ara-C Discontinued study treatment 181 (33%) 13 (2%) 從2000年6月到2001年1月間共有1106位患者加入IRIS研究。這是最大型並且也是最快得到結果的第三期CML研究。 IRIS研究中，無療效、療效消失或對治療未耐受的患者可更換治療組別。 將研究數據做研究時間點分析之後，獨立數據監控委員會就會修改IRIS之起始研究步驟，讓採用IFN- + ara-C合併治療但在治療1年後(原來試驗步驟擬定之治療時間為2年)仍未達MCR之患者改採imatinib mesylate治療。 治療中之病情惡化定義為 WBC數目增加。 MCR或CHR消失。 加速期或急性轉化期。 死亡。 追蹤六年後, 原本的553位randomized到imatinib arm的受試者中, 仍有364位(66%)六年後還維持在imatinib arm; 而IFN-a + Ara-C的對照組則由原本的554位受試者只剩13位(2%)還維持在IFN-a + Ara-C arm n = 553 After patients discontinue study treatment, only survival and SCT information is collected Ara-C, cytarabine; SCT, stem cell transplantation. Hochhaus A et al. Blood. 2007;110:15a-16a. Abstract 25. ASH 2007 oral presentation.

Overall Survival (ITT Principle): Imatinib Arm 100 90 80 70 Estimated overall survival at 7 years is 86% (94% considering only CML-related deaths) 60 % Without Event 50 40 30 Survival: deaths associated with CML Imatinib 7年後的overall survival 約為86%, 如果將非CML相關的死亡排除, 其overall survival則可以提高為94%, 長期追蹤後imatinib的survival data仍然相當優異, 並沒有隨著時間的拉長而下降 20 Overall Survival 10 12 24 36 48 60 72 84 96 Months Since Randomization IRIS 7 year update 29

Molecular Response Rates Major molecular response (MMR) and the depth of molecular response increase over time 10 20 30 40 50 60 70 80 90 100 % of available samples 3 6 9 12 15 18 21 24 36 42 48 54 66 72 78 84 BCR-ABL% (International Scale) ≤0.1% (MMR) ≤0.01% IRIS七年的data追蹤病人MMR的狀況, 可以發現隨著時間的增加, 仍然持續有病人陸續達到MMR或是4個log reduction, 可以發現長期使用imatinib仍然是有臨床療效上改善的角色 Sample Analysis Timepoints (months) IRIS 7 year update

IRIS 7-Year Update: Conclusions Overall Survival 86%. Event Free Survival 81%; 7% progressed to AP/BC on imatinib 40% patients discontinued study imatinib CCR achieved by 456 of 553 (82%) of patients MMR rates and the depth of molecular responses in patients increase over time MMR obtained at any time point represents a “safe haven” for patients Both molecular and cytogenetic evaluations should be used to guide treatment decisions until CCyR is achieved, with molecular assessments measured thereafter No new safety issues observed Overall survival為86%. EFS為81%, 有7%progress到AP/BC 40%的病人停止”試驗用imatinib” 共有82%的病人達CCR 即使追蹤到第七年, 達到MMR的比例隨時間增加而增加 MMR可以作為safe heaven的指標 Molecular以及cytogenetic的evaluation皆應該作為治療時間測療效的工具 沒有發現新的safety issues 根據本項研究結果，FDA核准使用imatinib mesylate為治療CML慢性期的第一線療法。 31

Adverse Events Management of Imatinib

Imatinib Mesylate: Nonhematologic Adverse Events Grade 3/4 Grade 1/2 imatinib mesylate治療患者並不常出現嚴重(3+級)非血液學症狀。 常見之的第1級或第2級副作用包括噁心、淺層水腫、肌肉痙攣、腹瀉、嘔吐、皮疹與疲倦。 所有病期之CML患者的副作用大致相同。 Nausea CP/AP/BP Superficial edema CP/AP/BP Muscle cramps CP/AP/BP Diarrhea CP/AP/BP Vomiting CP/AP/BP Rash CP/AP/BP Fatigue CP/AP/BP CP/AP/BP = chronic phase/accelerated phase/blastic phase. Kantarjian et al. N Engl J Med. 2002;346:645. Sawyers et al. Blood. 2002;99:3530. Talpaz et al. Blood. 2002;99:1928. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med. 2002;346:645-652. Sawyers CL, Hochhaus A, Feldman E, et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood. 2002;99:3530-3539. Talpaz M, Silver RT, Druker BJ, et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood. 2002;99:1928-1937.

Imatinib Mesylate: Grade 3/4 Hematologic Adverse Events 48 33 35 11 12 6 8 1 1 雖然imatinib mesylate治療末期患者較常出現嚴重(3+級)血液學副作用，但這些副作用可能是因為大部分處於細胞分裂狀態的細胞是Ph+，而由反應中潛藏之骨髓抑制作用所引起；或是由血小板減少症等與晚期疾病有關的自然病程潛在症狀所引起。 Neutropenia CP AP BP Thrombocytopenia CP AP BP Anemia CP AP BP Kantarjian et al. N Engl J Med. 2002;346:645. Sawyers et al. Blood. 2002;99:3530. Talpaz et al. Blood. 2002;99:1928. Deininger MW, O'Brien SG, Ford JM, Druker BJ. Practical management of patients with chronic myeloid leukemia receiving imatinib. J Clin Oncol. 2003;21:1637-1647. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med. 2002;346:645-652. Sawyers CL, Hochhaus A, Feldman E, et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood. 2002;99:3530-3539. Talpaz M, Silver RT, Druker BJ, et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood. 2002;99:1928-1937.

Myelosuppression in CML: Typically a Positive Therapeutic Outcome of Imatinib Mesylate Ph+ Ph– Imatinib mesylate In CML, Ph+ cells contribute to the majority of hematopoiesis Imatinib mesylate eliminates Ph+ cells; this therapeutic effect may result in myelosuppression CML患者的大部分的造血作用由Ph+細胞進行。 Imatinib mesylate治療可去除Ph+細胞，且有証據顯示imatinib mesylate不會抑制正常造血作用，所以推測骨髓抑制作用由imatinib mesylate治療所引起。 末期CML患者較常發生骨髓抑制作用。 支持性療法(如生長因子)、暫時調降劑量與/或中止治療是處理嚴重骨髓抑制作用的方式。 Myelosuppression is more common in advanced phases of CML Severe myelosuppression may be managed by supportive therapy, by temporary dose reduction, and/or treatment interruptions Ph = Philadelphia chromosome. Deininger et al. J Clin Oncol. 2003;21:1637. Deininger MW, O'Brien SG, Ford JM, Druker BJ. Practical management of patients with chronic myeloid leukemia receiving imatinib. J Clin Oncol. 2003;21:1637-1647.

Patient Management in CML: Summary Imatinib mesylate is generally well tolerated Nonhematologic toxicities are usually manageable Myelosuppression on imatinib mesylate may be managed with dose interruption Erythropoietin is effective in improving anemia in patients on imatinib mesylate therapy Imatinib mesylate通常耐受性良好。可以治療胃腸、肌肉骨骼毒性、皮膚皮疹與水腫等非血液學毒性。因為患者的造血作用主要來自Ph+細胞，所以早期出現暫時性骨髓抑制作用可能是少數必須停止治療的原因之一。最近已證實紅血球生成素可有效控制以imatinib mesylate治療的患者之貧血症狀。

Imatinib Resistance (1) Resistance (CML CP) Primary: Failure to meet “milestones” 3M CHR, 6M mCR, 12M MCR, 18M CCR Secondary: Progression after initial response Molecular, cytogenetic, hematological

Mechanisms of Resistance (2) Compliance 30% doses not taken during first year Plasma concentration -acidic glycoprotein, drug interactions, metabolic Intracellular concentration Influx - hOct-1 & OATP1A2 Efflux – ABCB1 (MDR-1) Increased expression BCR-ABL Duplication Ph, gene amplification, regulatory Abl KD mutations (~ 50%) CP 30% -> AP/BC 70%; Primary 30%, Secondary 60% BCR-ABL independence Lyn, Hck ? Autocrine/Paracrine ? Abrogation apoptosis mechanisms

Frequency of Mutations 1)Hochhaus A. et al. Leukemia 18:1321 (2004) 2) Soverini S. et al. GINEMA WP, Nilotinib Investigator Meeting Dublin, Oct 2008

Treatment Options for Imatinib Failure Dose escalation imatinib Switch to second generation TKI Allogeneic SCT Clinical trial

BCR/ABL Inhibitors Imatinib, Nilotinib, Dasatinib, Bosutinib (SK-606)

Comparison of Kinase Inhibitors Imatinib (STI571) Gleevec* Nilotinib (AMN 107) Tasigna* Dasatinib (BMS354825) Sprycel* Bosutinib (SK606) Route Oral Potency (Phos) 1 ~25 ~300 ~ 200 t½ (hours) 20 15 5½ 24 Dose 400 mg QD 400 mg BID 100 mg QD 500 mg QD Metabolism CYP4A4 CYP2D6 CYP3A4 CYP2C8 CYP2C9 FMO3 Abl KD Inactive Inactive & Active

Weisberg et al Nat Rev Cancer 7:345 (2007) TKIs in the ATP Pocket Weisberg et al Nat Rev Cancer 7:345 (2007)

Selectivity of Tyrosine Kinase Inhibitors Imatinib (Phos. IC50) PDGFR 72 nM > Kit 99 nM BcrAbl 221 nM Src >1000 nM Nilotinib 20 nM 75 nM 209 nM Dasatinib 0.1 nM 1.8 nM 2.9 nM 18 nM Bosutinib 3 nM 85 nM >3000 >10000 nM 1. Manley PW, et al. Proc Am Assoc Cancer Res 2007;48:772. 2. Weisberg E, et al. Cancer Cell 2005;7:1129. 3. Remsing Rix LL, et al. Leukemia 2009;23:477.

Considerations in Long-Term Therapeutic Management of CML: Summary Resistance to imatinib mesylate may be multifactorial Bcr-Abl mutations or clonal evolution Dose escalation to 800 mg/d recaptures responses in patients who relapsed on 400 mg/d Combination therapy with imatinib mesylate or other clinical studies Escalation of imatinib should be the primary therapeutic consideration for patients with CML-CP who do not initially achieve the expected response to standard therapy 許多因素會引起imatinib mesylate抗性。Bcr-Abl基因突變與單株細胞演化都是可能引起抗性的因素。對每天接受400 mg的imatinib mesylate治療產生抗性之患者，其Bcr-Abl激酶仍有活性，所以Bcr-Abl可成為有效的治療標的。將每天劑量提高到800 mg可使接受400 mg治療但疾病復發的患者達到反應。臨床研究可考慮使用包括imatinib mesylate之合併治療的創新療法。 對於imatinib resistance 或是只達suboptimal response的病人, imatinib 加劑量可使病人獲得療效上的明顯改善

Acute Myeloid Leukemia (AML)

AML: definition / concept Clonal expansion of myeloid blasts in blood marrow (BM), peripheral blood (PB) or other tissue Minimum threshold of blast cells for defining AML (BM): >30% blasts (FAB classification) >20% blasts (OMS, 2001) Proliferation + differentiation block / maturation arrest

AML: epidemiology Incidence: 2 - 3 pts /100.000 inhab - year Overall: 1.2% (US) Lineal increase with age: <35: < 1 /100.000 inhab - year >65: >10 /100.000 inhab - year Median age: 60 - 70 year-old

AML: clinical symptoms Bone marrow failure Anemia Neutropenia Thrombocytopenia Extramedullary involvement (skin, gums, CNS, other) Proliferative symptoms Coagulopathy Leukostasis Metabolic disorders (lysis tumor syndrome)

Normal Bone Marrow: Cell Heterogeneity

BM in AML: Monomorphous Cell Appearance

AML: gums (gingival) infiltration

AML: skin infiltration (granulocytic sarcoma)

AML: prognosis in younger population 38 + 4% After a median follow-up of seventeen months the overall survival of whole series (siries) is 38% at 3 years

AML: outcome according to cytogenetics (MRC) Grimwade et al, 1998

AML: main cytogenetic abnormalities Favorable: t(15;17), t(8;21), inv(16) Intermediate risk: normal karyotype Unfavorable: abn 5 (del/-5), abn 7 (del/-7), inv(3q)/t(3;3), complex karyotype (≥5 abn), abn 11q, t(6;9), del(17p)

Figure 1 Clinical and molecular characteristics of APL Chromosomal translocation t(15;17)(q22;q21) (C) with the resultant fusion transcripts between PML and RAR (D). Wang, Z.-Y. et al. Blood 2008;111:2505-2515 Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

Figure 2 ATRA in treating APL Wang, Z.-Y. et al. Blood 2008;111:2505-2515 Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

Survival of APL Chemotherapy: ATRA+CT regimens: median duration of remission ranged: 11-25 months Only 35% to 45% of the patients could be cured by CT alone (by 5-year DFS). ATRA+CT regimens: CR rate up to 90% to 95% 6-year DFS up to 86% (± 10%) in low-risk patients in a report

Classic Myeloproliferative Neoplasms (MPN)

Blood . 2008;112:2190-98

Natural History of MPDs Overt PMF Post ET/PV MF PV ET Early PMF Progressive constitutional symptoms Progressive cytopenias Progressive organomegaly/EMH Short term: Vascular events Leukemic transformation Premature death Lead time: Typically years (>10) to  Time: Variable 3-5 years common CP1266735-1

PV -Hypercellular -Panmyelosis with normal maturation pattern -Megakaryocytes of variable sizes, lacking significant cytologic dysplasia, often loosely clustered -Reticulin varies, usually 0-1 WHO Revised 2008: Minor Criteria “Hypercellularity for Age Trilineage Growth (panmyelosis) Prominent erythroid, granulocytic, and megakaryocytic proliferation” PV Courtesy of James Vardiman, MD

JAK2V617F James et. al., Nature 2005;434:1143 Levine et. al., Cancer Cell 2005;7:387 Kralovics et. al., NEJM 2005;352:1779-90 Baxter et. al., Lancet 2005;365:1054 Goldman JM, NEJM 2005;352:1744

Blood . 2008;112:2190-98

Blood . 2008;112:2190-98

PV ET PMF ? OTHER MUTATIONS 95-97% JAK2V617F 50-60% JAK2 EXON 12 1-3% 30% 40% 95-97% JAK2V617F 50-60% MPL MUTATIONS 5-10% 1% PV ET PMF

Normal C Cytokines EPO/TPO/ G-CSF STAT PI3K MAPK JAK2 Inhibition MPL W515L W515K S204P IVS 11/12; 10/11 JAK2V617F (Exon 14) Or JAK2 Exon 12 Mutants Cytokines EPO/TPO/ G-CSF Normal Membrane Membrane JAK2 JAK2 P JAK2 P P Stat Stat Stat STAT PI3K MAPK JAK2 Inhibition Nucleus Activation of genes of proliferation and survival Adapted from Levine et. al. Nature Cancer Reviews 2007;7:673

Prevent vascular event Myeloproliferative Treatment Goals ?JAK2 inhibitors? Cure Increase survival Delay disease progression Alleviate symptoms Prevent vascular event CP1266735-2

Treatment Algorithm in PV Risk Phlebotomy Cytoreduction Aspirin Low Yes No Yes High Yes Yes Yes Indeterminate Yes ? Depends* Hct <45% *may be used if acquired VWD is ruled out Pearson et al. Lancet 2:1219, 1978 Thomas et al. Lancet 2:161, 1977 Low-risk: no history of thrombosis and age < 60 years and Plt. ct. < 1 million and no CV risk factors High-risk: history of thrombosis or age  60 years Indeterminate-risk: Neither low- nor high-risk CP1044663-59

Targeting Myelofibrosis AML Therapies MDS Therapies TKI in MF Imatinib: Blood 2004 Dasatinib: Did not start INCB018424 (Ongoing) XL019 (Closed to toxicity) TG101348 (Ongoing) LBH589 Myelofibrosis Marrow Environment JAK2 Inhibition 74

Tyrosine Kinase Inhibitors Aurora Kinase Inhibitors JAK2 Inhibitors Tyrosine Kinase Inhibitors Aurora Kinase Inhibitors JAK2 JAK3 JAK1 TYK2 Involved with T cell function Innate Immunity 75

JAK2 Crystal Structure w/TG101348 TG101348 co-crystallized with JAK2 JH1 domain produced in SF9 insect cells Bound in ATP pocket Key interactions confirmed Pardanani et. al. ASH 2008; a97

INCB18424 19 322 4.5 2.7 IC50 (nM) Tyk2 JAK3 JAK2 JAK1 INCB18424 Eligibility: Advanced PMF or post-PV/ET MF patients Platelets >100 x 109/L ANC >1 x 109/L Primary objectives: safety and determination of MTD Secondary objectives: PK, PD, safety of extended dosing, and preliminary efficacy Administration: oral, twice daily, continuously Verstovsek et. al. Blood 2007;110(11):a558 77

INCB18424 Results in Rapid and Profound Reduction in Spleen Size NOTE: Data is censored after a dose change or dose interruption Verstovsek et. al ASH 2008 a1762 78

Improvement in Constitutional Symptoms 25 mg BID 10 mg BID Includes only patients with assessment for all time points Verstovsek et. al ASH 2008 a1762 79

Improvement in Body Weight } Weight Gain Loss of ascites and/or organomegaly } NOTE: Data is censored after a dose change or dose interruption Mesa et. al ASH 2008 a1760 80

JAK2 Inhibitor Efficacy Anemia Spleen Const Sx Pruritus JAK2 Burden Myelo- proliferation INCB018424 X 10% XL019 CEP701 TG101348 ITF2357 8-12% PEG Inf 2a Up to 100% Hydroxyurea 21-45% 81

Targeted Inhibitors of JAK2 In Vitro Murine Phase I Phase II Phase III 82

MPD Therapy Timeline CML ET PV PMF 2008 JAK2 Inhibitor 1960 Ph Chrom 2001 Imatinib 2005 JAK2V617F CML ET PV PMF ?

Conclusions Improved pathogenetic understanding has made a major impact in treatment of CML, HES, MPN associated mutations have led to explosive investigations into these disorders Multiple MPN associated mutations appear to signal through the JAK-STAT pathway JAK2 inhibitors undergoing testing with promising (but early) results Role in JAK2 in thrombosis, disease progression uncertain

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