MOLECULAR DIAGNOSIS OF ACUTE PROMYELOCYTIC LEUKEMIA … · * 41 healthy donors, 10 CLL, 4 ALL, 5...
Transcript of MOLECULAR DIAGNOSIS OF ACUTE PROMYELOCYTIC LEUKEMIA … · * 41 healthy donors, 10 CLL, 4 ALL, 5...
MOLECULAR DIAGNOSIS OF ACUTE PROMYELOCYTIC LEUKEMIA IN 30 MINUTES BY SINGLE-STEP RETRO TRANSCRIPTION LOOP MEDIATED AMPLIFICATION REACTION (RT-LAMP).
Rigo F1, Minnucci G1, Amicarelli G1, Rizzo G1, Mesturini R1, Zanghì P2, Spinelli O2, Colotta F1, Rambaldi A2.
1DiaSorin SpA, Gerenzano (VA), Italy; 2Ospedali Riuniti di Bergamo, Bergamo, Italy
A rapid detection of PML-RARa translocation is crucial for effective management of patients affected by Acute Promyelocytic Leukemia (APL)[1]. This life-threatening disease, if diagnosed promptly, benefits from treatment with retinoids and arsenic trioxide that have revolutionized therapy over the last decade[2]. The molecular detection of the three PML-RARa transcripts (bcr1, bcr2, bcr3) is actually based on conventional RT-PCR, a long and laborious multi-step method often not suitable for clinical needs[3]. We have developed two ultra-rapid RT-LAMP (Retro Transcription Loop-mediated Isothermal Amplification) assays to overcome the limitations of traditional RT-PCR in order to obtain a diagnosis in real time within 30 minutes.
INTRODUCTION
References: [1] Diverio D, et al. Haematologica (1995) 80, 155-160; [2] de Thè H and Chen Z Nature Review (2010) 10, 775-783; [3] Van Dongen JJM et al. Leukemia (1999) 13, 1901-1928
Duplex RT-LAMP bcr2-GUSb
Reaction mix
TOTAL RNA EXTRACTION
METHODS
500ng 300ng
bcr2 5%
ONE ENZYME DNA polymerase with retro-transcription and strand-
displacement activities
FLUORESCENT DYES specific for the targets of interest (bcr1, 2, 3, GUSb),
monitorable in dedicated fluorescent channels
bcr1 bcr2 bcr3 Negative* TOT
bcr1 15 - - - 15
bcr2 - 2 - - 2
bcr3 - - 17 - 17
Negative * - - - 62 62
TOT 15 2 17 62 96
0
20
40
60
80
100
10 15 20 25
% Q
ue
nch
ing
minutes
Fluorescent channel specific for bcr1
undiluted
dilutedt 10-1
diluted 10-2
diluted 10-3
RESULTS
Negative cell line Replicates PML-RARa
Results TOM-1 18
Negative*
697 17 RS411 15 HL-60 115
KASUMI 17 K562 20 REH 7 MV4 26
TOT replicates 235
RT-LAMP is HIGHLY SENSITIVE
CLINICAL VALIDATION
The PML-RARa RT-LAMP assays were validated on RNA obtained from 34 clinical samples previously diagnosed at Ospedali Riuniti di Bergamo by using conventional RT-PCR (Biomed) [3].
RT-LAMP is HIGHLY SPECIFIC
The level of sensitivity was established on mutated RNA from positive patients diluted into negative cell line RNA (HL-60)
* 41 healthy donors, 10 CLL, 4 ALL, 5 AML, 1 PV, 1 CML.
RT-P
CR
RE
SU
LT
S
RT-LAMP RESULTS
The assays specificity was established on negative PML-RARa RNA extracted from 8 cell lines.
Level of sensitivity bcr1: 10-3
(confirmed also on RNA extracted from NB4 cell line diluted in HL-60)
100% specificity on 235 replicates
Level of sensitivity bcr3: 10-3 Level of sensitivity bcr2: 10-2
Real time amplification of different concentrations of the three transcripts bcr1 (A), bcr3 (B) and bcr2 (C). Interestingly it’s possible to observe, for all transcripts, a clear relationship between dose and amplification time.
A B C
* All negative results were validated through correct GUSb RNA amplification.
CONCLUSIONS FEATURES OF RT-LAMP VERSUS CONVENTIONAL RT-PCR
Two multiplex Fluorescent RT-LAMP assays have been developed to detect and distinguish the three fusions transcripts (bcr1, bcr2 and bcr3) of the PML-RARa translocation.
The PML-RARa RT-LAMP assays represent an ultra-rapid, specific, sensitive and cheap method for molecular confirmation of APL. The single-step format monitored in real-time simplifies the entire reaction set-up and ensure reliability. This 30 minutes reaction can significantly improve the outcome of APL patients allowing to start the therapy timely
MULTIPLE PRIMER SETS Different primer sets for the simultaneous detection
of the target sequences
Frequency
RT-LAMP REACTION consists in:
300ng
ONE STEP RT, amplification and signal detection in a single
homogeneous step
ISOTHERMAL Incubation at constant temperature (65°C)
ULTRA-RAPID detection of positive samples within 15’
OBJECTIVE RESULTS Clear discrimination of the specific transcript
REAL TIME Fluorescence signals monitored during the
reaction
VALIDATION of NEGATIVE RESULTS GUSb amplification within 30’
REACTION CONDITIONS
RT-LAMP RT-PCR (Biomed
protocol[3]) FEATURES IMPROVEMENT
ASSAY FORMAT MULTIPLEX SIMPLEX Simultaneous detection and discrimination of the fusion transcripts; easy assay set-up
CONTROL OF REACTION INTERNAL EXTERNAL Low risk of false negative results
STEPS 1 3 Low risk of contamination and errors; easy
assay set-up
TIME TO RESULTS 35 minutes 3 hours and 30 minutes Ultra rapidity
SENSITIVITY bcr1:10-3
bcr2:10-2
bcr3: 10-3
bcr1:10-3
bcr2:n.d.
bcr3: 10-2
Comparable (bcr1) and improved (bcr2 and bcr3) sensitivity suitable at the onset
SPECIFICITY 100% n.d No false positive nor false negative results
RESULTS INTERPRETATION OBJECTIVE SUBJECTIVE No need for specialized personnel
RT-LAMP significantly improves the diagnosis of APL in clinical laboratories
Triplex RT-LAMP bcr1-bcr3-GUSb
Reaction mix
Frequency
55% bcr1
40% bcr3
IC
100% agreement with conventional RT-PCR on 96 clinical samples
IC
INTERNAL CONTROL GUSb housekeeping mRNA:
• controls extraction procedure and quality of RNA • controls the presence of inhibitors • controls the correct reaction conditions