Patients and samples
Bone marrow deoxyribonucleic acid (DNA) samples from 132 consecutive patients with newly diagnosed AML and MDS-EB during May 2014- April 2018 were used for performing TP53 mutation analysis, using MassArray® System (Agena Bioscience, CA, USA). All AML and MDS-EB patients recruited in this retrospective study had the routine study results of nucleophosmin 1 (NPM1), FMS like tyrosine kinase (FLT3) internal tandem duplication (ITD) and CCAAT/enhancer- binding protein alpha (CEBPA) analysis.
Chemotherapy Protocol
Younger AML patients (aged< 60 years) received induction chemotherapy with intravenous (i.v.) Ara-C 100 mg/m2/day for 7 consecutive days together with i.v. idarubicin 12 mg/m2/day for 3 consecutive days (7 + 3 regimen). Patient aged≥ 60 years or unfit patients were treated with 5 + 2 regimen; 5 days of i.v. Ara-C 100 mg/m2/day combined with 2 days of i.v. idarubicin 12 mg/m2/day. The bone marrow (BM) study was re-evaluated 28 days after induction therapy. Patients achieving CR received the first cycle of consolidation chemotherapy with the same regimen as induction therapy and then followed by 3 cycles of IDAC or HiDAC therapy for patients aged< 70 years.
AML patients aged≥ 70 years and MDS-EB patients were treated with azacitidine 100 mg/day subcutaneously for 7 consecutive days and repeated every 4 weeks. Disease response was re-evaluated after 4- 6 cycles of azacitidine. CR was defined as BM blasts< 5% with absolute neutrophil count≥ 1,000/μL, platelet count≥ 100,000/μL, absence of circulating blasts and absence of extramedullary disease.
Targeted Genomic Analyses
MassArray® System (Agena Bioscience, CA, USA) was utilized to perform targeted genomic confirmation in this study. Once the target sequence(s) were input, the software will automatically generate the set of three primers; forward, reverse & extension primer. Primers were designed to target hotspots mutation at codon 31, 126, 143, 175, 179, 194, 220, 238, 241, 248, 249, 255, 273, 280, 282, 290 and 310.
Multiplex PCR technique consisted of 3 steps;
- PCR amplification was done using polymerase chain reaction (PCR) primer mix (forward and reverse primers) at the concentration of 100 nM, MgCl2 solution, dNTP 500 μM, PCR buffer 1x concentration, PCR enzyme 0.2 unit/μL and 10-20 ng DNA. All to mix up to the total volume of 5μL. The thermal cycles were 2 minute-cycle of 95°C following 30 second-cycle of 95°C, 30 second-cycle of 56°C, then 1 minute-cycle of 72°C= 45 cycles and 5 minute-cycle of 72°C= 5 cycles.
- Eliminating excess dNTPs from the previous step by adding shrimp alkaline phosphatase (SAP). The protocol comprised 10X SAP buffer 0.17 μL, SAP Enzyme 0.3 μL, distilled water (HPLC grade) 1.53 μL, to incubate in the thermal cycles of 37°C= 40 minutes following by 85°C= 5 minutes.
- Adding a single nucleotide as the terminator bases (ddNTPs) at the 3’ position of the extend primers. By bringing the PCR products from former step, to add with extend primer mix 0.52-1.57 μM, IPLEX® buffer 0.222x, IPLEX® terminator mix 0.222x and IPLEX® enzyme 0.142 unit/μL, to make up to the total volume of 9 μL. The thermal cycles are; [95°C= 30 seconds + {95°C= 5 seconds+ 5x(52°C= 5 seconds + 80°C=5 seconds)}] = 40 cycles following 72°C= 3 minutes.
Since very small volume dispensed, either Automated Liquid Handler or Manual Dispensing was adopted by the Manufacturer’s certified technician.
- Data analysis report; to dispense the final analyze on the SpectroChip® and bring in to the MassArray® analyzer (Mass Spectrometry), the report generated via MassArray® Typer Software.
Outcome assessment
The objectives of this study were to evaluate the prevalence of TP53 mutation in AML and MDS-EB patients, and also overall survival (OS). OS was defined as the interval between the dates of diagnosis and death.
Statistical analysis
The factors which included age, white blood cell (WBC) count, cytogenetics, TP53 and other molecular data were compared between patients with and without TP53 mutation, using Chi-square. OS was calculated by the Kaplan–Meier method, difference between groups were calculated using the log-rank test for univariate analysis. Cox’s Regression model was used for multivariate survival analysis. All calculations were performed using the statistical package of social sciences software, SPSS statistics version 17 (Chicago: SPSS Inc; 2008).