Semi-Automated Molecular Detection of BCR-ABL Major and Minor Transcripts by Isothermal RT-Loop Mediated Reaction on the Liaison IAM Instrument

Elena D’Agostini1, Giulia Minnucci, PhD1, Giulia Amicarelli, PhD1, Cinzia Pultrone1, Veronica Tettamanzi1, Chiara Boroni2, Orietta Spinelli2,Francesco Colotta, MD1, Alessandro Rambaldi, MD2

1DiaSorin SpA, Gerenzano (VA), Italy; 2USC HematologyOspedali Riuniti di Bergamo, Bergamo, Italy.

The molecular detection of the BCR-ABL fusion transcripts is necessary for the genetic confirmation of Chronic Myeloid Leukemia

(CML) diagnosis and for the risk classification of Acute Lymphoblastic Leukemia (ALL) [1,2]. The molecular identification for this

purpose is actually based on conventional RT-PCR [3]; the main limitations are long time-to-result and multi-step procedures that

often cause a slow-down of diagnostic laboratories routine activity. Here we present a novel molecular method, based on Loop

Mediated Amplification (LAMP) [4] reaction that, coupled with the Liaison IAM instrument (DiaSorin SpA), ensures semi-

automated rapid detection of BCR-ABL p190 and p210 fusion transcripts and of the endogenous GUSβ mRNA.

INTRODUCTIONINTRODUCTION RESULTSRESULTSRT-LAMP is HIGHLY SENSITIVE

RNA EXTRACTION500ng

METHODSMETHODS

BCR-ABL multiplex Fluorescent RT-LAMP assay has been developed to detect and distinguish the two fusion transcripts (p190 and

p210) of the BCR-ABL translocation.

BCRBCR--ABL RTABL RT--LAMPLAMP

The assay detects p210 and p190

dilutions down to 10-5 and 10-4,

respectively (Figure 1 A,B).

The level of sensitivity was

established on 500ng RNA extracted

from p210 and p190 positive cell

lines (K562 and TOM1 respectively)

serially diluted into negative cell line

RNA (from cell line HL60).

CConventional RT-PCR

(Biomed protocol [3])

A

Results by conventional RT-PCR

(Biomed) [3]

p190* p190* p210p210§§ NegativeNegative

^̂

TotTot

p190*p190* 30 - - 30

RNA EXTRACTION500ng

Fluorescence generation from amplification of

ISOTHERMAL

Incubation at constant temperature 65°C for

50 min50 min in Liaison IAM instrument (DiaSorin)

ONE HOMOGENEOUS STEP

Retrotranscription and amplification in a single

step using one single enzyme, a DNA pol with RT

and strand-displacement activities

MULTIPLE PRIMER SETS

3 primer sets for the simultaneous detection of

p190, p210 and GUSββββ (Internal Control)

RETROTRANSCRIPTION

PCR

Control (ABL)p190 p210

Liaison IAM instrument

CLINICAL VALIDATIONCLINICAL VALIDATIONRTRT--LAMP is HIGHLY SPECIFICLAMP is HIGHLY SPECIFIC

** (29 B-ALL, 1 CML)

§ (27 CML, 3 B-ALL)

^ 60 Healthy Donors

The assay specificity was established on negative BCR-ABL RNA

extracted from 7 cell lines (Table 1). All negative results have

been validated by amplification of the Internal Control (GUSβ).

Figure 1

1000ng

Triplex BCR-ABL

RT-LAMP

reaction mix

REAL TIME MONITORING

Interestingly a clear relationship

between target dose and

amplification time is observed (Fig 1

C,D)

D

(Biomed protocol )

B

The BCR-ABL RT-LAMP assay was validated on RNA obtained

from 120 clinical samples previously diagnosed at Ospedali

Riuniti di Bergamo by using conventional RT-PCR (Biomed) [3]

Re

sults b

y

RT

-LAM

P

p210p210§§ - 30 - 30

Negative^Negative^ - - 60 60

TotTot 30 30 60 120

50’

Fluorescence generation from amplification of

p190, p210 and Internal Control (GUSββββ) detected

in dedicated fluorescent channels:

BCR-ABL

RT-LAMP

RT-PCR

(Biomed protocol[3])

ASSAY FORMAT TRIPLEX SIMPLEX

TEMPERATURE ISOTHERMAL (65°C) PCR CYCLING

CONTROL OF REACTION INTERNAL (GUS β) EXTERNAL

STEPS 1 3

TIME TO RESULTS 50 min 3 h and 30 min

TARGETS RNA cDNA

SENSITIVITYp190: 10-4

p210: 10-5

p190: 10-3

p210: 10-4

SPECIFICITY 100% n.a.

RESULTS INTERPRETATIONOBJECTIVE

(automatic elaboration)SUBJECTIVE

Amplification in Channel Results

500 nm Positive p190

570 nm Positive p210

530 nm Negative

No amplification Invalid run

Automatic elaboration of

results: GEL SEPARATION

RESULTS

100% specificity on 275 replicates

CONCLUSIONSCONCLUSIONS

100% agreement with conventional RT-PCR on 120

clinical samples

REFERENCES:

[1] National Comprehensive Cancer Network 2010

[2] National Cancer Institute 2010

[3] Van Dongen JJM et al. Leukemia (1999) 13, 1901-1928

[4] Notomi T et al. NAR (2000) 15: 28 (12): E63

The triplex p190-p210-GUSβ RT-LAMP is a one-step

procedure for specific, highly sensitive and rapid molecular

detection of the BCR-ABL fusion transcripts.

The semi-automatic approach on the instrument Liaison

IAM allow to simplify the entire procedure, reducing the

contamination risks deriving from the conventional, multi

step RT-PCR, thus resulting in a significantly improvement of

the diagnostic lab routine.

From RNA to

interpreted result in

50 minutes

From RNA to result in From RNA to result in

3 hours and 30 minutes

Table 1 Table 2