Early molecular response in chronic myeloid leukemia patients predicts future response status Sailaja Kagita, Sangeeta Jiwtani, Srihari Uppalapati, Vijay Gandhi Linga, Sadasivudu Gundeti & Raghunadharao Digumarti Tumor Biology Tumor Markers, Tumor Targeting and Translational Cancer Research ISSN 1010-4283 Tumor Biol. DOI 10.1007/s13277-013-1585-2
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Author's personal copy Tumor Biol. DOI 10.1007/s13277-013-1585-2
RESEARCH ARTICLE
Early molecular response in chronic myeloid leukemia patients predicts future response status Sailaja Kagita & Sangeeta Jiwtani & Srihari Uppalapati & Vijay Gandhi Linga & Sadasivudu Gundeti & Raghunadharao Digumarti
Received: 5 November 2013 / Accepted: 19 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Imatinib is the frontline therapy for chronic myeloid leukemia (CML) management. Most of the CML patients achieve major responses, but a proportion (nearly 25–35 %) of them develop drug resistance. Molecular monitoring using quantitative real-time PCR at regular intervals according to European LeukemiaNet (ELN) helps in the assessment of long-term outcomes in imatinib-treated CML patients. Eighty-four CML patient samples (42 at diagnosis and 42 at 3-month intervals from the same patients) were analyzed for Bcr-Abl transcript levels. Quantification results revealed that the patients with 10 % Bcr-Abl levels (P10 % Bcr-Abl levels were found to have 25.0 % of suboptimal response and 3.57 % of failure to imatinib at standard dose. Hence, the present study confirms that early molecular monitoring at 3 months after imatinib initiation helps in predicting the concurrent cytogenetic response and treatment optimization in CML patients. Keywords Chronic myeloid leukemia . Imatinib . Molecular response . Bcr-Abl . Real-time PCR
Introduction Imatinib (IM) is a tyrosine kinase inhibitor (TKI), an initial choice for treating Ph-positive chronic myeloid leukemia (CML) in India. Monitoring minimal residual disease by quantitative real-time PCR at regular intervals according to European LeukemiaNet (ELN) guidelines helps in the assessment of longS. Kagita : S. Jiwtani : S. Uppalapati : V. G. Linga : S. Gundeti : R. Digumarti (*) Nizams Institute of Medical Sciences, Punjagutta, Hyderabad 500082, Andhra Pradesh, India e-mail:
[email protected]
term outcomes in imatinib-treated CML patients [1–3]. Early reductions in Bcr-Abl can predict a subsequent cytogenetic response [4]. There are few reports from subcontinent looking at a molecular response using quantitative real-time PCR for patients on imatinib treatment. This is the first of its kind from subcontinent in which we tried to compare the molecular response with cytogenetic response. CML is the commonest adult leukemia in India with the median age of 38–40 years at diagnosis. The annual incidence ranges from 0.8 to 2.2 per 100,000 population for men and from 0.6 to 1.6 per 100,000 population for women and occurs at a decade earlier than in the Western world [5, 6]. The incidence in the West is 1 per 100,000 population [7]. The present study presents the results of a sustained monitoring of molecular response in CML patients on imatinib.
Materials and methods A total of 84 CML samples (42 newly diagnosed and 42 follow-up samples at 3 months from same patients) were included in the study. The study was approved by the institutional ethics committee, and an informed consent was obtained from every patient participating in the study. Twenty-six out of 42 were males and 16 were females. The median age at presentation was 33 years (range 9–63 years). All these patients were diagnosed in chronic phase, 38 were in early chronic (12 months after diagnosis). RNA extraction, cDNA synthesis, and real-time PCR RNA was extracted from 10 mL peripheral blood after the lysis of red blood cells using TRIzol method (Invitrogen, Karlsruhe, Germany). The concentration and purity of the RNA was measured in NanoDrop 1000 (Thermo Scientific). Total RNA (1 μg) was reversely transcribed into complementary DNA using high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA). Commercially available
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primers and TaqMan probe from Applied Biosystems (TaqMan Pre-Developed Assay Reagents for gene expression) were used for quantification of Bcr-Abl transcripts (b2a2 and b3a2) (Applied Biosystems, Foster City, CA). ABL gene as an endogenous control and a positive control K562 cell line cDNA were included in the reverse transcription (RT)-PCR. The reaction mixture of 20 μL contains the following: 0.25 μL of forward and reverse primers of each (250 nmol/L) and probe (150 nmol/L), 10.0 μL of expression assay mix, 7.0 μL nuclease-free water, and 2 μl of cDNA. The thermal cycling conditions were as follows: 2 min at 50 °C and 5 min at 95 °C, followed by 45 cycles of 15 s at 95 °C for 15 s and 60 °C for 1 min. All the samples were run in duplicate to minimize handling errors. Assays included negative controls in all stages of reactions to assess specificity and to rule out contamination. Relative quantification was performed by the ΔΔCT method, and the fold change of transcript levels was measured relative to the reference gene in terms of cycle threshold (CT) [8, 9]. Event-free survival (EFS) was measured from the start of treatment to the date of loss of complete hematologic response, loss of complete or major cytogenetic response, discontinuation of therapy for toxicity or lack of efficacy, progression to accelerated or blastic phases, or death at any time. Transformation-free survival (TFS) was measured from the start of therapy to the date of transformation, to accelerated or blastic phases while on therapy, or to the date of the last follow-up. Survival was measured from the time treatment was started to the date of death from any cause at any time or date of the last follow-up. Statistical analysis Correlation between the Bcr-Abl expression levels at a 3-month interval and different variables like cytogenetic response, Sokal score, EUTOS (European Treatment and Outcome Study) risk score, and clinical phase were evaluated using the Student’s t test and ANOVA test. Sokal score defines the risk of disease progression and survival time in newly diagnosed CML patients and the EUTOS score predicts complete cytogenetic response (CCyR) at 18 months in patients being treated with TKI therapy [10, 11]. Probabilities of EFS, TFS, and overall survival (OS) were calculated. Statistical analyses were performed using the GraphPad Prism software, version 6.0 (San Diego, CA).
Results Eighty-four CML samples (42 at diagnosis and 42 at 3-month intervals from the same patients) were analyzed for Bcr-Abl transcript levels using RT-PCR. Patient disease characteristics were presented in Table 1. Out of 42 cases, 90.47 % were in early chronic phase and 9.52 % in late chronic phase. All the patients were started on imatinib 400 mg. After 6 months of
Table 1 Baseline disease characteristics (n = 42)
Age (years) Hb (gm/dL) TLC (mm3) PC (lakh/mm3) PB blasts (%) BMA blasts (%) Eosinophils (%) Basophils (%) Spleen size (cm)
Median
Range
33 10.9 145,500 3.96 2 0 2.5 3 8
9–63 6.4–16 13,800–308,000 1.3–12.8 0–18 0–15 0–12 0–12 0–35
imatinib therapy, 80.95 % (34/42) of the patients had achieved a complete cytogenetic response and 16.66 % (7/42) partial cytogenetic response, and one patient had no response. With respect to Sokal score, 30.95 % of the patients had high risk, 40.47 % intermediate risk, and 28.57 % low risk. When EUTOS scores were considered, 26.19 % of patients had high risk and 73.80 % low risk. The 2-year EFS, TFS, and OS rates for the whole group were 61.90 %, 97.61 % and 97.61 %, respectively (Table 2). The Bcr-Abl quantification results were normalized to international scale (IS). Reductions in Bcr-Abl/Abl gene ratio are expressed as a percentage, and the IS standardized baseline was 100 %. A value of 0.1 % IS (equivalent to a 3-log reduction from the standardized baseline) is referred to as a major molecular response (MMR). A baseline value was Table 2 Clinical characteristics (n = 42) No. Cytogenetic response after 6 months of IM initiation CCyR 34 PCyR 7 LFU 1 Phase Early chronic 38 Late chronic 4 Sokal risk High 13 Intermediate 17 Low 12 EUTOS risk High 11 Low 31 2-year outcome EFS TFS OS
Percent
80.95 16.6 2.38 90.47 9.52 30.95 40.47 28.57 26.19 73.80 61.90 97.61 97.61
CCyR complete cytogenetic response, PCyR partial cytogenetic response, EFS event-free survival, TFS tumor-free survival, OS overall survival
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calculated for our laboratory using the median quantification of 30 pretreatment samples, and our standardized baseline was 115.4 %. MMR value from our baseline value was 0.115 (3-log reduction), and this value was adjusted to IS by dividing 0.1 by 0.1154 (0.866), used as a conversion factor [12]. Patients were grouped into two categories based on Bcr-Abl expression levels after 3 months of IM initiation: 33.33 % (14/42) of the patients exhibited 10 % expression (34.44±18.05). When the two groups of patients with 10 % Bcr-Abl levels were compared with cytogenetic response, 100 % of the patients in 10 % group had achieved CCyR. The probability of achieving a complete cytogenetic response at 6 months was significantly higher in patients with lower Bcr-Abl levels (
