Disertasi

PROFIL IMUNOFENOTIP, MUTASI GEN FLT3-ITD, NPM1, DAN CEBPA PADA LEUKEMIA MIELOID AKUT, SERTA HUBUNGANNYA DENGAN LUARAN KLINIS. = THE PROFILE OF IMMUNOPHENOTYPE, FLT3-ITD, NPM1 AND CEBPA GENE MUTATION IN ACUTE MYELOID LEUKEMIA AND ITS RELANTIONSHIP WITH CLINICAL OUTCOME.

Latar Belakang: Leukemia mieloid akut (LMA) mempunyai karakteristik heterogenisitas derajat tinggi yang ditandai dengan kelainan kromosom/sitogenetik, mutasi gen, dan perubahan eskpresi gen. Mutasi gen telah diketahui mempunyai peran penting sebagai biomarker prognostik dan prediktif terkait dengan kesintasan, dan juga sebagai sasaran terapi. Mutasi gen yang terlibat cukup penting dalam prognosis pasien LMA adalah FLT3-ITD, NPM1, dan CEBPA. Tujuan: Untuk mengetahui frekuensi imunofenotip, mutasi gen FLT3-ITD, NPM1, dan CEBPA serta untuk mengetahui hubungannya dengan kesintasan tiga bulan pada pasien LMA. Metode Penelitian: Penelitian dilakukan dengan menggunakan desain longitudinal prospektif terhadap pasien LMA dewasa rawat jalan dan rawat inap RS Kanker Dharmais (RSKD) pada bulan Juli 2014 sampai Februari 2015. Sampel berupa 20 mL aspirat sumsum tulang atau darah perifer dengan antikoagulan EDTA. Penelitian dilakukan di Laboratorium Patologi Klinik dan Bagian Penelitian dan Pengembangan RSKD. Deteksi mutasi gen FLT3-ITD dilakukan dengan metode elektroforesis. Deteksi mutasi NPM1 dan CEBPA dilakukan dengan metode DHPLC yang kemudian dilanjutkan dengan pemeriksaan sekuens DNA untuk mengetahui jenis mutasi. Variabel bebas berupa usia, jenis kelamin, jumlah leukosit, ekspresi imunofenotip (CD34 dan aberrant), dan mutasi gen (FLT3-ITD, NPM1, dan CEBPA), sedangkan variabel tergantung adalah luaran klinis kesintasan dalam tiga bulan. Hasil Penelitian dan Pembahasan: Didapatkan total 66 subjek dengan rerata usia relatif lebih muda yaitu 42,37 + 15,75 dan sebanyak 83,3 % di bawah 60 tahun. Sebanyak 54 % subjek mempunyai ekspresi CD34, dan hanya 15,2 % mempunyai ekspresi aberrant. Terdapat 40 subjek yang menjalani kemoterapi induksi (60,5 %). Hanya 37 dari 66 subjek yang dilakukan pemeriksaan mutasi gen FLT3-ITD, NPM1, dan CEBPA. Frekuensi mutasi gen FLT3-ITD, NPM1 dan CEBPA berturut-turut 16,2 %, 40,5 %, dan 35,1 %. Pada keseluruhan subjek (n = 66) ditemukan bahwa FLT3 wild type dan CEBPA mutasi berhubungan bermakna terhadap kesintasan dalam tiga bulan yang lebih pendek dengan adjusted RR berturut-turut 2,21 (p 0,022; IK95 % 1,50 - 3,26) dan 2,08 (p 0,036; IK95% 1,06 - 4,07). Pada kelompok subjek yang mendapat kemoterapi (n = 40), hanya mutasi FLT3-ITD yang secara klinis mempunyai kecenderungan memengaruhi kesintasan tiga bulan dengan RR 1,73 (p 0,273; IK95 % 1,18 - 2,54). Sebagai hasil tambahan, ditemukan bahwa mutasi NPM1 berhubungan dengan ekspresi CD34 negatif (p 0,018) dan mutasi CEBPA berhubungan dengan jumlah leukosit lebih besar dari 50 x10 3 /μL (p 0,028). Kesimpulan: Usia LMA di Indonesia relatif lebih muda. Frekuensi mutasi FLT3-ITD relatif lebih rendah dan mutasi NPM1 dan CEBPA cenderung lebih tinggi. FLT3 wild type dan mutasi CEBPA berhubungan dengan kesintasan yang lebih pendek pada penelitian ini. Diperoleh data bahwa kejadian mutasi NPM1 behubungan dengan CD34 negatif dan mutasi CEBPA berhubungan dengan jumlah leukosit yang lebih tinggi.
Kata kunci: CEBPA, FLT3-ITD, kesintasan tiga bulan; Leukemia Mieloid Akut; NPM1.


Background: Acute Myeloid Leukemia (AML) has high degree of heterogeneity characteristic signed with chromosome/cytogenetic abnormality, gene mutation, and gene expression changes. The importance of gene mutation has been known as prognostic and predictive biomarker associated with survival and also as a targeted therapy. Gene mutation that is considered important in AML patient prognosis are FLT3-ITD, NPM1 and CEBPA. Purpose: To obtain frequency data of immunophenotype and gene mutation of FLT3-ITD, NPM1, and CEBPA, and its relationship with three-month overall survival. Method: This research was a prospective longitudinal study on adult AML patients who were admitted as outpatient and inpatient in Dharmais National Cancer Center (DNCH) on July 2014 until February 2015. Samples used were 20 mL of bone marrow aspirate or peripheral blood using EDTA anticoagulant. Research was done in Pathological Laboratory and Research and Development of DNCH. FLT3-ITD gene mutation was detected using electrophoresis and NPM1 and CEBPA mutation was detected using DHLPC method continued with DNA sequencing examination to know the type of mutation. The independent variables were age, sex, leukocyte, immunophenotype (CD34 and aberrant), gene mutations (FLT3-ITD, NPM2 and CEBPA). The dependent variable was three-months overall survival. Result and Discussion: a total of 66 subjects were collected with mean age relatively young, 39.78 years and 83.3 % of them were under 60 years. Fifty four percent subjects had positivity in CD34 expression and 15.2 % had aberrant expression. Forty subjects received induction chemotherapy (60.5 %). Only 37 of 66 subject were analyzed gene mutations. The frequency of FLT3, NPM1 and CEBPA mutation frequency was subsequently 16.2 %, 40.5 % and 35.1 %. From all subjects (n = 66), was found that wild type FLT3 and mutant CEBPA was statistically significant had twice mortality rate in three months than mutant FLT3-ITD (p 0,022; adjusted RR 2,21; IK95 % 1,50 - 3,26) and wild type CEBPA (p 0,036; adjusted RR 2,08; IK95 % 1,06 - 4,07). In treated groups (n = 40), there was no statistically significant association between independent variables with three-months survival. However, there was a clinically significant tendency that wild type FLT3 was consistently related with longer OS (RR 1.73, 95 % CI 1.18 - 2.54). As an additional result, it was found that the NPM1 mutation is associated with lack of CD34 expression (p 0.018) and CEBPA mutations were associated with leukocyte levels > 50 X103 / mL (p 0.028). Conclusion: The age of AML patients in Indonesia are relatively younger. Proportion of FLT3-ITD gene mutation is relatively lower, whereas proportion of NPM1 and CEBPA mutations tend to be higher. FLT3-ITD wild type and CEBPA mutation are related with shorter survival in this research. Incidence of NPM1 gene mutation is related to lack of CD34 and CEBPA mutation is related to higher leukocyte count.
Keyword: Acute Myeloid Leukemia; CEBPA, FLT3-ITD, NPM1,; three-months overall survival.