1Hematology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt.
2Chemical Pathology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt.
3Department of Clinical Pathology, Medical Military Academy, Cairo, Egypt.
Therapeutic protocols used in adult acute lymphoblastic leukemia (ALL) are widely variable, and glucocorticoids (GCs) are essential components in ALL treatment. Therefore, this study aimed to evaluate the distribution of prominent glucocorticoid receptor (GR) gene polymorphic variants among adult ALL patients. We also investigated the association between GR messenger ribonucleic acid (mRNA) isoform expressions and the response to chemotherapy.
Fifty-two newly diagnosed Philadelphia-negative adult ALL patients and 30 healthy control subjects were enrolled in this study. Genotyping was carried out using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. GR mRNA isoform expressions were assayed by quantitative real-time PCR.
ALL patients in this study had a median age of 34 years (range, 18-75). GRα expression was associated with complete remission (
Our data suggest that the level of GR isoform expression may be useful in predicting GC response, achievement of complete remission, and better event-free survival in ALL patients. However, further evaluation with a larger cohort of patients is warranted.
Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell at one of several discrete stages of development. This disease causes leukemic cells to accumulate relentlessly because of their altered response to growth and death signals . While ALL is mostly curable in children, similar progress in the treatment of ALL in adults has lagged behind, and some patients suffer from refractory or recurrent diseases that cannot be cured with conventional chemotherapy .
Therapeutic protocols used in ALL are widely variable. However, glucocorticoids (GC) are essential components in ALL treatment. GCs exert their antileukemic effects through reduction of cell proliferation, promotion of G1 cell cycle arrest, and induction of apoptosis. To induce antileukemic effects, GCs have to bind their intracellular receptor, which belongs to the nuclear hormone receptor superfamily. The ligand-receptor complex then translocates to the nucleus and transactivates or transrepresses, or both, GC responsive genes. Therefore, in oncology, GC function is mainly based on the induction of cell death .
The glucocorticoid receptor (GR) gene consists of 9 exons and is located on chromosome 5. Alternative splicing of the primary GR transcript can result in GRα and GRβ, and alternative splicing of the large exon 9 generates mRNA coding for GRα and GRβ. GRα has been the primary focus of research because of its predominant expression, ligand-binding properties, and transcriptional activity. However, GRβ does not bind GCs and is transcriptionally inactive . Nonetheless, Oakley et al. demonstrated that GRβ, despite the fact that it is transcriptionally inactive, influences GC-mediated gene transcription . The authors showed that GRβ was capable of binding and inactivating GRα, thus inhibiting signal transduction. More recently, a third splice variant of the GR, GRγ, was identified. GRγ was produced as a result of alternative splicing and retains 3 base pairs (bp) from the intron separating exons 3 and 4. As a consequence, an additional amino acid (arginine) is located between the 2 zinc fingers of the DNA-binding domain .
The GR gene is also named nuclear receptor subfamily 3, group C, member 1 (
The aim of this study was to evaluate the distribution of the 3 most prominent GR gene polymorphic variants (
The present study examined 52 newly diagnosed Philadelphianegative adult ALL patients who presented to the hematology department at the Alexandria University Medical Research Institute and the Mostafa Kamel Military Hospital in Alexandria. Patients were excluded if they had a prior malignancy or comorbid organ function abnormality. Patients were diagnosed and classified according to the morphological and immunophenotypic characterization of blast cells in the bone marrow. Cytogenetic studies were performed for all patients. Informed consent was obtained from all patients. Thirty apparently healthy age- and sex-matched volunteers were included as controls.
All patients completed a detailed history and a thorough clinical examination. Chemotherapy was given according to the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL) 2003 protocol . Peripheral blood samples were collected from participants upon diagnosis and before starting therapeutic measures. A 10 mL venous blood sample was drawn into 2 ethylenediaminetetraacetic acidcontaining sterile vacutainers. Patients were followed up for a median of 24 months (range, 5 to 40 mo) and event-free survival was calculated.
Corticosteroid sensitivity was defined as a peripheral blood blast cell count <1,000/µL after the 7-day corticosteroid prephase. However, corticosteroid sensitivity could not be examined in 3 patients who had a pretreatment peripheral blood blast cell count <1,000/µL. Chemotherapy sensitivity was defined as a bone marrow blast cell percentage less than 5% after the first week of chemotherapy . Poor early responders were defined as patients with corticosteroid-resistant and/or chemotherapy-resistant ALL. Achievement of complete remission was evaluated according to the standard criteria .
Baseline high-risk factors included having a white blood cell (WBC) count ≥30×109/L for B-lineage ALL or >100×109/L for T-lineage ALL, an early T-cell phenotype, clinical and/or morphologic central nervous system (CNS) involvement, t(4;11) or t(1;19) translocation, hypodiploidy, -7 or +8 . All patients with at least 1 baseline high-risk factor were classified in the high-risk ALL subgroup. All other patients were in the standard-risk ALL subgroup.
Genomic DNA was extracted using a genomic DNA purification Kit (Fermentas, USA) according to the protocol supplied by the manufacturer. Polymerase chain reaction (PCR) amplification of DNA segments containing the polymorphic sites was carried out using primer sequences (Table 1) and the PCR reaction mixture described previously . The PCR protocol included denaturation at 95℃ for 5 minutes followed by 30 cycles of denaturation at 94℃ for 1 minute, annealing at 55℃ (except for N363S polymorphism at 51℃) for 1.5 minutes, extension at 72℃ for 1.5 minutes, and a final cycle at 72℃ for 5 minutes.
A restriction fragment length polymorphism analysis was performed to determine GR gene polymorphisms as described previously . To determine
To determine N363S polymorphism genotypes, 20 µL of relevant PCR product (248 bp) were digested with 1 unit of
The ER22/23EK polymorphism genotypes were determined by digesting 20 µL of relevant PCR product (482 bp) with 1.25 units of
Peripheral blood mononuclear cells from patients and controls were isolated using Ficoll gradient centrifugation (1.077 g/mL). Total RNA was extracted using the RNeasy RNA extraction Mini Kit (Qiagen, USA). The extracted RNA was reversely transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, USA) according to the manufacturer instructions. The GR α, β, γ primers and glyceraldehyde-3-phosphate dehydrogenase were designed, and the real-time PCR reactions were performed according to the Koga protocol . The threshold cycle (CT) was used to calculate the mRNA expression levels of the PCR targets. The 2-ΔΔCT method was used for relative quantification .
Data were fed to the computer using IBM SPSS software package version 20.0. Qualitative data were described using numbers and percents. Comparisons between different groups regarding categorical variables were tested using a chi-square test. When more than 20% of the cells had an expected count less than 5, a Monte Carlo correction was used. Parametric tests were applied for normally distributed data, while nonparametric tests were used for abnormally distributed data. Significance of the obtained results was judged at the 5% level. Kaplan-Meier survival estimates and log rank
Fifty-two newly diagnosed Philadelphia-negative adult ALL patients with a median age of 34 years (range, 18 to 75 yr) were enrolled in this study. Participants included 35 (67.3%) males and 17 (32.7%) females. Initial WBC counts ranged from 0.5-299×109/L (median, 33×109/L), platelet counts ranged from 5-457×109/L (median, 42×109/L), and hemoglobin levels ranged from 4-14 g/dL (median, 7.6 g/dL). Palpable lymphadenopathy was present in 36/52 patients (69%), 34 patients (65.3%) had splenomegaly, 26 patients (50%) had hepatomegaly, and 21 patients (40.3%) had mediastinal masses. CNS infiltration was found in 6 patients (11.5%). Thirty-eight cases had B-cell ALL while 14 had T-cell ALL. Thirty-four patients (65.6%) were classified as high-risk. Three patients (5.8%) had testicular disease at presentation. GR was encountered in 26/49 patients (53.1%) and 42 patients failed to achieve early response to treatment (80.8%). Thirty-four patients (65.6%) achieved complete remission. Laboratory and clinical findings in relation to the response to induction chemotherapy are shown in Table 2. The event-free survival was 48.1% during the study follow-up period (range, 5 to 40 mo, median, 24 mo).
Among ALL patients with the
Event-free survival was higher in patients with the wild type
No significant differences were found between ALL patients and normal patients regarding GRα and GRβ mRNA expression; however, GRγ mRNA expression was a bit higher in patients compared to controls (
Interactions among host, disease, and treatment factors determined treatment efficacy in the general ALL model . The effect of GR gene polymorphisms on the efficacy of treatment for childhood ALL has been considered in a few clinical studies but no studies, to the best of our knowledge, have reported results concerning adult ALL patients. The complete remission rate among the studied patients was 65.4%, which is lower than other reports utilizing the same protocol (GRAALL). This could be attributed to sample size issues, delayed diagnoses, or delayed referrals to a hematologist resulting in a stormier and less fruitful remission induction.
The incidence of
In the present work, the overall frequency of
As evidenced by work in transgenic mice with increased and decreased GR expression, the level of GR expression in apoptosis is important . Among the different isoforms of the GR, the α isoform was shown to be responsible for GC-mediated transcriptional activation, while the other isoforms were responsible for disrupting either the hormone binding domain (GRβ) or the DNA binding domain (GRγ) . In the current study, patients who achieved complete remission had higher pretreatment GRα and lower GRγ expression levels. Compared to GC-sensitive patients, the expression of GRγ mRNA was significantly higher in GC-resistant patients. In addition, α isoform expression in excess to β isoform expression was noted in patients with higher event-free survival. Similar results were found by Beger et al. and Haarman et al. . For example, Beger et al. found that GRγ expression was lower in cells from patients with a good response to GCs compared to patients with a poor response to GCs . Similarly, Gerdes et al. reported that the γ isoform was potentially associated with poor prednisone response in childhood ALL . These data were correlated with cell survival, demonstrating a more pronounced induction of apoptosis in cells from patients with a good response to GCs. This may be linked to the insertion of arginine within the DNA binding domain of the GR that may introduce conformational changes in the 2 zinc fingers thereby reducing DNA affinity .
In conclusion, the
Kaplan-Meier chart showing event-free survival for the