Neoplastic Hematopathology.docx

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Neoplastic Hematopatholo gy

PENYEBAB FUSI BCR-ABL PADA LEUKEMIA MYELOID KRONIS

TEKNIK PCR BCR-ABL

Leukemia granulositik kronis (LGK) adalah neoplasma mieloproliferatif yang ditandai oleh ekspansi sel sumsum tulang pluripoten yang tidak terkendali. Ciri khas dari penyakit ini adalah adanya translokasi resiprokal kromosom (9;22)(q34; q11.2), menghasilkan turunan kromosom 9q+ dan 22q- kecil, yang dikenal sebagai kromosom Philadelphia (Ph), menghasilkan gen fusi BCRABL dan produksi protein fusi BCR-ABL. BCR-ABL memiliki aktivitas tirosin kinase konstitutif dan cukup untuk menimbulkan penyakit.1,2 Dalam sebagian kecil kasus, (5-10%), kromosom Ph bersifat kriptogenetis, sering disebabkan oleh translokasi yang kompleks, dan diagnosis membutuhkan fluorescent in situ hybridization (FISH) untuk menunjukkan gen fusi BCR-ABL atau polymerase chain reaction (PCR) untuk menunjukkan transkrip mRNA BCR-ABL.Pada penegakan diagnosis LGK secara definitif diperlukan pemeriksaan baku emas berupa pemeriksaan sitogenetika untuk mendeteksi kromosom Philadelphia. Selain itu, juga dapat melalui pemeriksaan Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) untuk mendeteksi transkrip gen BCR-ABL terhadap sampel aspirasi sumsum tulang. Namun demikian, teknik pemeriksaan sitogenetika dan RT-PCR BCR-ABL bukanlah pemeriksaan yang mudah, sehingga hanya sedikit pusat laboratorium yang mampu melakukan pemeriksaan tersebut. Harga pemeriksaan tersebut pun masih relatif mahal dan belum ditanggung oleh program pemerintah, sehingga tidak semua pasien mampu melaksanakannya.1,2

ASPEK BIOMOLEKULAR PEMERIKSAAN BCR - ABL KUALITATIF

Pemeriksaan BCR - ABL mendeteksi ekspresi chimeric fusion protein BCR-ABL yang dapat digunakan untuk mengindentifikasi kromosom Philadelphia. Kromosom Philadelphia ditemukan pada lebih dari 90% pasien chronic myeloid leukemia (CML) dewasa, 15-30% orang dewasa dengan acute lymphoblastic leukemia (ALL) dan 2% acute myeloid leukemia (AML). Gen BCRABL merupakan fusi antara gen ABL (kromosom 9 wilayah q34) dengan gen BCR (breakpoint cluster region; kromosom 22 wilayah q11) pada kromosom Philadelphia yang terjadi akibat translokasi timbal balik antara kromosom 9 dan kromosom 22. Gen ini mengkode protein yang memiliki aktivitas tirosin kinase yang kuat dan aktif secara konsitutif (mengaktivasi sejumlah protein yang terlibat dalam regulasi siklus sel yang mempercepat pembelahan sel dan mempengaruhi DNA repair).4 Gen fusi BCR-ABL akan melakukan transkripsi mRNA yang mengkode suatu protein yang mempunyai aktivitas tyrosine kinase lebih kuat dibandingkan gen ABL yang normal. Bergantung kepada lokasi break point pada gen BCR dengan gen ABL, sampai saat ini telah ditemukan tiga lokasi break point pada gen BCR. Pertama, apabila break point terjadi pada M-BCR (major break point cluster region) maka lokasinya adalah intron ekson e13 dan ekson e14 dengan ekson a2 pada gen ABL (e13a2 dan e14a2) yang akan mengkode protein BCR-ABL p210. Kedua, break point yang terjadi pada minor break point cluster region lokasinya adalah intron ekson e1 yang bergabung dengan ekson a2 pada gen ABL (e1a) yang akan mengkode protein BCR-ABL p190. Ketiga, break point yang terjadi pada micro break point cluster region, maka lokasinya adalah intron ekson e19 dengan ekson a2 pada gen ABL (e19a2) yang akan mengkode protein BCR-ABL p230.1,2 Terjadi fusi gen BCR-ABL penting untuk patogenesis pada Chronic Myloid Leukemia (CML). Pemeriksaan ini mendeteksi fusi transkrip gen BCR-ABL (seperti major e14a2 (b3a2), e13a2 (b2a2), micro e1a2 dan minor e19a12 (c3a2)) pada pasien kromosom Philadelphia (t[(9:22)(q34;q11)]) positif leukemia. Translokasi ini terjadi pada ±95% pasien CML.

The Philadelphia chromosome or Philadelphia translocation (Ph) is a specific genetic abnormality in chromosome 22 of leukemia cancer cells (particularly chronic myeloid leukemia (CML) cells). This chromosome is defective and unusually short because of reciprocal translocation, t(9;22)(q34;q11), of genetic material between chromosome 9 and chromosome 22, and contains a fusion genecalled BCR-ABL1. This gene is the ABL1 gene of chromosome 9

juxtaposed onto the breakpoint cluster region BCR gene of chromosome 22, coding for a hybrid protein: a tyrosine kinase signalling protein that is "always on", causing the cell to divideuncontrollably by interrupting the stability of the genome and impairing various signaling pathways governing the cell cycle.[1] The presence of this translocation is a highly sensitive test for CML, since all cases of CML are positive for BCR-ABL1.[2] (Some cases are confounded by either a cryptic translocation that is invisible on G-banded chromosome preparations, or a variant translocation involving another chromosome or chromosomes as well as the long arm of chromosomes 9 and 22. Other similar but truly Ph-negative conditions are considered CML-like myeloproliferative neoplasms.[3]) However, the presence of the Philadelphia (Ph) chromosome is not sufficiently specific to diagnose CML, since it is also found in acute lymphoblastic leukemia[4] (aka ALL, 25–30% of adult cases and 2–10% of pediatric cases) and occasionally in acute myelogenous leukemia (AML) as well as mixedphenotype acute leukemia (MPAL).

. Manfaat Pemeriksaan BCR-ABL kualitatif adalah untuk menunjang diagnosis chronic myeloid leukemia (CML) atau leukemia lainnya seperti acute lymphoblastic leukemia (ALL) atau acute myeloid leukemia (AML) pada individu terduga CML atau leukemia lainnya. The chromosomal defect in the Philadelphia chromosome is a reciprocal translocation, in which parts of two chromosomes, 9 and 22, swap places. The result is that a fusion gene is created by juxtaposing the ABL1 gene on chromosome 9 (region q34) to a part of the BCR (breakpoint cluster region) gene on chromosome 22 (region q11). This is a reciprocal translocation, creating an elongated chromosome 9 (termed a derivative chromosome, or der 9), and a truncated chromosome 22 (the Philadelphia chromosome, 22q-).[5][6] In agreement with the International System for Human Cytogenetic Nomenclature (ISCN), this chromosomal translocation is designated as t(9;22)(q34;q11). The symbol ABL is derived from Abelson, the name of a leukemia virus which carries a similar protein. The symbol BCR is derived from breakpoint cluster region, a gene which encodes a protein that acts as a guanine nucleotide exchange factor for Rho GTPase proteins.[citation needed]

Translocation results in an oncogenic BCR-ABL gene fusion that can be found on the shorter derivative 22 chromosome. This gene encodes for a BCR-ABL fusion protein. Depending on the precise location of fusion, the molecular weight of this protein can range from 185 to 210 kDa. Consequently, the hybrid BCR-ABL fusion protein is referred to as p210 or p185. Three clinically important variants encoded by the fusion gene are the p190, p210, and p230 isoforms.[7] p190 is generally associated with B-cell acute lymphoblastic leukemia (ALL), while p210 is generally associated with chronic myeloid leukemia but can also be associated with ALL and AML.[8] p230 is usually associated with chronic myelogenous leukemia associated with neutrophilia and thrombocytosis (CML-N).[8] Additionally, the p190 isoform can also be expressed as a splice variant of p210.[9] The Abl gene expresses a membrane-associated protein, a tyrosine kinase, and the BCR-Abl transcript is also translated into a tyrosine kinase containing domains from both the BCR and ABL1 genes. The activity of tyrosine kinases is typically regulated in an auto-inhibitory fashion, but the BCR-Abl fusion gene codes for a protein that is "always on" or constitutively activated, leading to impaired DNA binding and unregulated cell division (i.e. cancer). This is due to the replacement of the myristoylated cap region, which when present induces a conformational change rendering the kinase domain inactive, with a truncated portion of the BCR protein.[10] Although the BCR region also expresses serine/threonine kinases, the tyrosine kinasefunction is very relevant for drug therapy. As

the N-terminal Y177 and CC domains from BCR encode the constitutive activation of the ABL1 kinase, these regions are targeted in therapies to downregulate BCR-ABL kinase activity. Tyrosine kinase inhibitors specific to such domains as CC, Y177, and Rho (such as imatinib and sunitinib) are important drugs against a variety of cancers including CML, renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GISTs). The fused BCR-ABL protein interacts with the interleukin-3 receptor beta(c) subunit and is moderated by an activation loop within its SH1 domain, which is turned “on” when bound to ATP and triggers donwstream pathways. The ABL tyrosine kinase activity of BCR-Abl is elevated relative to wild-type ABL.[11] Since ABL activates a number of cell cycle-controlling proteins and enzymes, the result of the BCR-Abl fusion is to speed up cell division. Moreover, it inhibits DNA repair, causing genomic instability and potentially causing the feared blast crisis in CML. The BCR-ABL1 fusion gene and protein encoded by the Philadelphia chromosome affects multiple signaling pathways that directly affect apoptotic potential, cell division rates, and different stages of the cell cycle to achieve unchecked proliferation characteristic of CML and ALL.

JAK/STAT pathway[edit] Particularly vital to the survival and proliferation of myelogenous leukemia cells in the microenvironment of the bone marrow is cytokine and growth factor signaling. The JAK/STATpathway moderates many of these effectors by activating STATs, which are transcription factors with the ability to modulate cytokine receptors and growth factors. JAK2 phosphorylates the BCR-ABL fusion protein at Y177 and stabilizes the fusion protein, strengthening tumorigenic cell signaling. JAK2 mutations have been shown to be central to myeloproliferative neoplasms and JAK kinases play a central role in driving hematologic malignancies (JAK blood journal). ALL and CML therapies have targeted JAK2 as well as BCR-ABL using nilotinib and ruxolitinib within murine models to downregulate downstream cytokine signaling by silencing STAT3 and STAT5 transcription activation (appelmann et al). The interaction between JAK2 and BCR-ABL within these hematopoietic malignancies implies an important role of JAKSTAT-mediated cytokine signaling in promoting the growth of leukemic cells exhibiting the Ph chromosome and BCR-ABL tyrosine kinase activity. Though the centrality of the JAK2 pathway to direct proliferation in CML has been debated, its role as a downstream effector of the BCR-ABL tyrosine kinase has been maintained. Impacts on the cell cycle via JAK-STAT are largely peripheral, but by directly impacting the maintenance of the hematopoietic niche and its surrounding microenvironment, the BCR-ABL upregulation of JAK-STAT signaling plays an important role in maintaining leukemic cell growth and division.[12][13]

Ras/MAPK/ERK pathway[edit] The Ras/MAPK/ERK pathway relays signals to nuclear transcription factors and plays a role in governing cell cycle control and differentiation. In Ph chromosome-containing cells, the BCR-ABL tyrosine kinase activates the RAS/RAF/MEK/ERK pathway, which results in unregulated cell proliferation via gene transcription in the nucleus. The BCR-ABL tyrosine kinase activates Ras via phosphorylation of the GAB2 protein, which is dependent on BCR-located phosphorylation of Y177. Ras in particular is shown to be an important downstream target of BCR-ABL1 in CML, as Ras mutants in murine models disrupt the development of CML associated with the BCR-ABL1 gene (Effect of Ras inhibition in hematopoiesis and BCR/ABL leukemogenesis). The Ras/RAF/MEK/ERK pathway is also implicated in overexpression of osteopontin (OPN), which is important for maintenance of the hematopoietic stem cell niche, which indirectly influences unchecked proliferation characteristic of leukemic cells. BCR-ABL fusion cells also exhibit constitutively high levels of activated Ras bound to GTP, activating a Ras-dependent signaling pathway which has been shown to inhibit apoptosis downstream of BCR-ABL (Cortez et al). Interactions with the IL-3 receptor also induce the Ras/RAF/MEK/ERK pathway to phosphorylate transcription factors which play a role in driving the G1/S transition of the cell cycle.[14][15][16]

DNA binding and apoptosis[edit] The c-Abl gene in wild-type cells is implicated in DNA binding, which affects such processes as DNA transcription, repair, apoptosis, and other processes underlying the cell cycle. While the nature of this interaction has been debated, evidence exists to suggest that c-Abl phosphorylates HIPK2, a serine/threonine kinase, in response to DNA damage and promotes apoptosis in normal cells. The BCR-ABL fusion, in contrast, has been shown to inhibit apoptosis, but its effect on DNA binding in particular is unclear.[17] In apoptotic inhibition, BCR-ABL cells have been shown to be resistant to drug-induced apoptosis but also have a proapoptotic expression profile by increased expression levels of p53, p21, and Bax. The function of these pro-apoptotic proteins, however, is impaired, and apoptosis is not carried out in these cells. BCR-ABL has also been implicated in preventing caspase 9 and caspase 3 processing, which adds to the inhibitory effect.[18][19][20] Another factor preventing cell cycle progression and apoptosis is the deletion of the IKAROS gene, which presents in >80% of Ph chromosome positive ALL cases. The IKAROS gene is critical to Pre-B cell receptor-mediated cell cycle arrest in ALL cells positive for Ph, which when impaired provides a mechanism for unchecked cell cycle progression and proliferation of defective cells as encouraged by BCR-ABL tyrosine kinase signaling

Nomenclature[edit] The Philadelphia chromosome is designated Ph (or Ph') chromosome and designates the shortened chromosome 22 which encodes the BCR-ABL fusion gene/protein kinase. It arises from the translocation, which is termed t(9;22)(q34.1;q11.2), between chromosome 9 and chromosome 22, with breaks happening in region (3), band (4), sub-band (1) of the long arm (q) of chromosome 9 and region (1), band (1), sub-band (2) of the long arm (q) of chromosome 22. Hence the chromosome breakpoints are written as (9q34.1) and (22q11.2), respectively, using ISCN standards.

Therapy Tyrosine kinase inhibitors in the late 1990s, STI-571 (imatinib, Gleevec/Glivec) was identified by the pharmaceutical company Novartis (then known as Ciba Geigy) in high-throughput screens for tyrosine kinase inhibitors. Subsequent clinical trials led by Dr. Brian J. Druker at Oregon Health & Science University in collaboration with Dr. Charles Sawyers and Dr. Moshe Talpaz demonstrated that STI571 inhibits proliferation of BCR-ABL-expressing hematopoietic cells. Although it did not eradicate CML cells, it did greatly limit the growth of the tumor clone and decreased the risk of the feared "blast crisis".[citation needed] In 2000 Dr. John Kuriyan determined the mechanism by which STI-571 inhibits the Abl kinase domain.[22] It was marketed in 2001 by Novartis as imatinib mesylate(Gleevec in the US, Glivec in Europe). Other pharmacological inhibitors are being developed, which are more potent and/or are active against the emerging Gleevec/Glivec resistant BCR-abl clones in treated patients. The majority of these resistant clones are point-mutations in the kinase of BCR-abl. New inhibitors include dasatinib and nilotinib, which are significantly more potent than imatinib and may overcome resistance. Combination therapies with nilotinib and ruxolitnib have also shown success in suppressing resistance by targeting the JAK-STAT and BCR-ABL stages simultaneously. Small molecule inhibitors, like arsenic trioxide and geldanamycinanalogues, have also been identified in downregulating BCR-ABL kinase translation and promoting its degradation by protease.[23][24] Axitinib, a drug used to treat renal cell carcinoma, has been shown to be effective at inhibiting the Abl kinase activity in patients with BCR-ABL1(T315I).[25] The T315I mutation in the fusion gene confers resistance to other tyrosine kinase inhibitors such as imatinib, however axitinib has been

successfully been used to treat a patient with ALL carrying this mutation, as well as CML cells in culture. Treatment of pediatric Ph+ ALL with a combination of standard chemotherapy and RTK inhibitors may result in remission,[citation needed] but the curative potential is unknown.

Blood or marrow transplants[edit] A potentially curative, but risky, option for pediatric Ph+ ALL or Ph+ CML is bone marrow transplant or cord blood transplant, but chemotherapy is favored by some for achieving first remission (CR1). For some, bone marrow transplant from a matched sibling donor or a matched, unrelated donor may be favored when remission is obtained. Cord blood transplant is favored by some when a 10/10 bone marrow match is not available, and cord blood transplant may have some advantages, including a reduced incidence of graft-vs-host disease (GVHD), which is a common and significant complication of transplant. However, transplant with cord blood sometimes requires longer periods of time for engraftment, which may increase the potential for complications due to infection. Regardless of the type of transplant, transplant-related mortality and relapse are possible, and the rates may change as treatment protocols improve. For second remission (CR2), if achieved, both chemotherapy and transplant options are possible, and many physicians prefer transplant.[citation needed]

History[edit] The Philadelphia chromosome was first discovered and described in 1959 by David Hungerford from Fox Chase Cancer Center (then the Institute for Cancer Research) and Peter Nowell from the University of Pennsylvania School of Medicine, and was named after the city in which both facilities are located.[1][26][27] Hungerford was writing his doctoral thesis on chromosomes in a genetics lab at Fox Chase Cancer Center, and detected a tiny flaw in chromosomes from the blood cells of patients with a type of leukemia. It was the first genetic defect linked with a specific human cancer. Nowell was a pathologist at the University of Pennsylvania, studying leukemia cells under the microscope when he noticed cells in the act of dividing. To his surprise, their chromosomes—usually an indistinct tangle— were visible as separate structures. Nowell searched for an expert on chromosomes in the area to work with and found Hungerford. While conducting his microscopic studies, Hungerford made the observation that certain leukemia cells had an abnormally short chromosome 22. The mutation became known as the Philadelphia chromosome. In 1973, Janet Rowley at the University of Chicago identified the mechanism by which the Philadelphia chromosome arises as a translocation.[1][28][29]

PROSEDUR ASPIRASI SUMSUM TULANG Aspirasi sumsum tulang adalah prosedur untuk mendapatkan sampel dari tulang besar manusia. Sumsum tulang terdiri dari stem sel yaitu sel-sel primitif yang belum berdiferensiasi dan dilindungi oleh stroma. Stem sel ini terdiri dari dua jenis sel yaitu sel yang akan berdiferensiasi menjadi sel darah dan sel yang akan berdiferensiasi menjadi jaringan ikat. Sampel yang didapatkan pada aspirasi sumsum tulang terdiri dari komponen sel dan fragmen jaringan ataupun gabungan dari keduanya (Gambar ). Sampel ini biasanya digunakan untuk kepentingan pemeriksaan sitologi untuk menentukan morfologi dan hitung jenis sel. Lebih jauh lagi, sampel ini digunakan untuk pemeriksaan sitogenetika, biologi molekular, mikrobiologi, imunohistokimia dan sitometri. Interpretasi akhir dari aspirasi sumsum tulang ini akan berguna untuk kepentingan klinis dan penegakan diagnosis beberapa kelainan pada sel darah ataupun sumsum tulang. Metode dalam persiapan, proses dan pelaporan hasil aspirasi sumsum tulang ini sangat bervariasi yang pada akhirnya akan mempengaruhi hasil diagnosis dan tata laksana.

Gambar . Pengambilan sampel aspirasi sumsum tulang.

2. 3. Philip A. Thompson. Diagnosis and Treatment of Chronic Myeloid Leukemia (CML) in 2015. Mayo Clin Proc. 2015 October; 90(10): 1440–1454. 4. Prodia. 2019. BCR-ABL Kualitatif. http://m.prodia.co.id/id/produklayanan/pemeriksaanlaboratoriumdetails/bcr-abl-kualitatif. Diakses: 19 Maret 2019. 5. Tri Agusti Sholikah. 2017. Fusion gene bcr-abl : from etiopathogenesis to the management of Chronic Myeloid Leukemia. Jurnal Kedokteran dan Kesehatan Indonesia;Volume 8 (1). Hal: 29-36. 6. Wikipedia. 2019. Philadelphia Chromosome. https://en.wikipedia.org/wiki/Philadelphia_chromosome. Diakses: 19 Maret 2019. 7. Elias J. Jabbour. 2014. CME Information: Chronic myeloid leukemia: 2014 update on diagnosis, monitoring, and management. American Journal of Hematology, Vol. 89, No. 5, May 2014. Hal: 547-554

8. Junita. 2017. Aspirasi Sumsum Tulang. https://www.alomedika.com/tindakanmedis/hematologi-dan-onkologi/aspirasi-sumsum-tulang. Diakses: 19 Maret 2019. 9.