OBJECTIVE RESULTS Clear identification of the right transcript through automatic

elaboration of results; the instrument returns results in terms of positive negative or invalid:

SEMI-AUTOMATIC ULTRA RAPID DETECTION OF THE PML-RARa FUSION TRANSCRIPTS

BY RETRO-TRANSCRIPTION LOOP MEDIATED AMPLIFICATION (RT-LAMP) REACTION ON THE LIASON IAM INSTRUMENT Riccardo Mesturini 1, Giulia Minnucci 1, Giulia Amicarelli 1, Francesca Rigo 1, Giulia Rizzo1, Pamela Zanghì 2, Silvia Salmoiraghi 2, Orietta Spinelli 2,

Francesco Colotta 1, Alessandro Rambaldi 2

1Molecular Diagnostics, DiaSorin SpA, Gerenzano, 2USC Hematology, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

After the initial morphologic evaluation, the accurate and timely molecular identification of the Acute Promyelocitic Leukemia (APL) associated PML-RARa fusion gene is mandatory to start an appropriate treatment based on all-trans retinoic acid and to reduce the risk of potentially fatal hemorrhagic complications [1, 2]. To improve the molecular diagnosis of Acute Promyelocitic Leukemia, we developed a novel ultra rapid screening test, based on the Loop mediated isothermal AMPlification (LAMP), easy to be performed even in not specialized laboratories.

INTRODUCTION

METHODS

RESULTS

The system consists in two fluorescent multiplex assays, one specific for the most frequent transcripts (bcr1 and bcr3) and one for the more rare bcr2 starting from 500 and 300 ng of total RNA, respectively. To control the extraction procedure, RNA integrity, reaction functionality and absence of inhibitors, both the assays also detect the endogenous GUSb housekeeping RNA as internal control.

Amplification in Channel Results

500 nm Positive for bcr1 translocation

570 nm Positive for bcr3 translocation

530 nm Negative

No amplification Invalid run

Amplification in Channel Results

500 nm Positive for bcr2 translocation

530 nm Negative

No amplification Invalid run

Negative cell line Replicates PML-RARa

Results

TOM-1 18

Negative (GUSb RNA

amplification)

697 17

RS411 15

HL-60 114

KASUMI 17

K562 20

REH 7

MV4 26

TOT replicates 234

SENSITIVITY CLINICAL VALIDATION

bcr1 bcr2 bcr3 Negative* TOT

bcr1 15 - - - 15

bcr2 - 2 - - 2

bcr3 - - 17 - 17

Negative * - - - 62 62

TOT 15 2 17 62 96

CONCLUSIONS

The fluorescent PML-RARa RT-LAMP assays are highly specific, sensitive and rapid. The isothermal single-step format, monitorable in real-time, simplifies the entire reaction set-up and ensures reliability, also in not highly specialized laboratories. The ultra-rapid reaction can significantly reduce the time to diagnosis and thus the risk of hemorrhagic complications by allowing initiation of early treatment.

The PML-RARa RT-LAMP assays were validated on RNA obtained from 96 clinical samples previously diagnosed at Azienda Ospedaliera Papa Giovanni XXIII by using conventional RT-PCR (Biomed) [3].

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

SPECIFICITY 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-

PC

R R

ESU

LTS

RT-LAMP RESULTS

The assays specificity was established on negative PML-RARa RNA extracted from 8 cell lines.

100% agreement with conventional RT-PCR on 96 clinical samples

Level of sensitivity bcr1: 10-3

(confirmed also on RNA extracted

from NB4 cell line diluted in HL-60)

100% specificity (234 replicates, validated through IC)

Level of sensitivity bcr3: 10-3

Level of sensitivity bcr2: 10-2

Amplification of the three transcripts is monitorable in real time (panel A, B, C). For all transcripts, an inverse relationship between dose and amplification time is visible.

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Triplex assay: 500 nm channel

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dilutedt 10-1

diluted 10-2

diluted 10-3

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Triplex assay: 570 nm channel

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Duplex assay: 500 nm channel

undiluted

diluted 10-1

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Duplex bcr2-GUSb Triplex bcr1-bcr3-GUSb

Triplex bcr1-bcr3-

GUSb

Duplex bcr2-GUSb

FLUORESCENT DYES Specific for the targets of interest (bcr1, 2, 3, GUSb), monitorable in real-time

MULTIPLE PRIMER SETS Different primer sets for the simultaneous detection and distinction of the

three fusion transcripts

ONE STEP

RT, amplification and signal detection in a single homogeneous step

REAL TIME Fluorescence signal monitored during reaction by

dedicated channels

ULTRA-RAPID Detection of positive samples within 15’

VALIDATION OF NEGATIVE RESULTS GUSb amplification within 30’

1. PMLRARa RT-LAMP REACTION MIXES:

2. PATIENT’S RNA :

3. INCUBATION AT 65°C FOR 40 MINUTES ONTO THE LIAISON IAM

500 ng/reaction ( for the Triplex assay) 300 ng/reaction ( for the Duplex assay)