Introduction. Ph+ ALL is observed in about 30% of adult ALL and is associated with a very poor prognosis and early relapse. Tyrosine kinase inhibitors have improved overall treatment results, with a rapid response and a complete remission (CR) rate ranging 90%. However most patients experienced hematological relapse in a short time. Molecular analysis based on quantitative assays (i.e. quantitative polymerase chain reaction, qPCR) provides detection of residual leukemic cells measuring BCR-ABL1 transcript level and becomes necessary in the monitoring of minimal residual disease to confirm molecular CR or to detect early relapse. We investigated the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies in Ph+ ALL patients who obtained molecular remission as assessed by conventional qPCR. Methods. The 12.765 Digital array (Fluidigm) is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Digital raw data are then processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per panel using the Poisson probabilistic distribution. We analyzed 22 Ph+ ALL samples expressing the P190 (11) or P210 (11) BCR-ABL1 isoform in complete (87%) or major (13%) molecular response (BCR-ABL1/ABL ratio ≤0.001 or <0.1, respectively) as assessed by conventional qPCR; RNA integrity was evaluated using the control gene ABL. Results. First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 P190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 mL volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates (P=0.0014, Paired t TEST analysis). We then analyzed duplicates of Ph+ ALL samples with a positive control for each chip: digital array resulted positive in 58% of complete molecular response samples with 5.5 as median number of copies detected (range 0.5-11). Conclusions. The Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript with greater accuracy than conventional qPCR, as demonstrated for samples in molecular remission, and could provide an accurate monitoring method for Ph+ ALL CR patients. Further studies to confirm these results are actually ongoing. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione, Università 2007-2009.

A HIGH SENSITIVE NANOFLUIDIC ARRAY IMPROVES THE DETECTION OF RARE COPIES OF BCR-ABL1 TRANSCRIPT IN PATIENTS WITH PHILADELPHIA POSITIVE (PH+) ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

LONETTI, ANNALISA;IACOBUCCI, ILARIA;Ferrari A;PAPAYANNIDIS, CRISTINA;PAOLINI, STEFANIA;ABBENANTE, MARIACHIARA;MARTINELLI, GIOVANNI
2010

Abstract

Introduction. Ph+ ALL is observed in about 30% of adult ALL and is associated with a very poor prognosis and early relapse. Tyrosine kinase inhibitors have improved overall treatment results, with a rapid response and a complete remission (CR) rate ranging 90%. However most patients experienced hematological relapse in a short time. Molecular analysis based on quantitative assays (i.e. quantitative polymerase chain reaction, qPCR) provides detection of residual leukemic cells measuring BCR-ABL1 transcript level and becomes necessary in the monitoring of minimal residual disease to confirm molecular CR or to detect early relapse. We investigated the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies in Ph+ ALL patients who obtained molecular remission as assessed by conventional qPCR. Methods. The 12.765 Digital array (Fluidigm) is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Digital raw data are then processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per panel using the Poisson probabilistic distribution. We analyzed 22 Ph+ ALL samples expressing the P190 (11) or P210 (11) BCR-ABL1 isoform in complete (87%) or major (13%) molecular response (BCR-ABL1/ABL ratio ≤0.001 or <0.1, respectively) as assessed by conventional qPCR; RNA integrity was evaluated using the control gene ABL. Results. First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 P190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 mL volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates (P=0.0014, Paired t TEST analysis). We then analyzed duplicates of Ph+ ALL samples with a positive control for each chip: digital array resulted positive in 58% of complete molecular response samples with 5.5 as median number of copies detected (range 0.5-11). Conclusions. The Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript with greater accuracy than conventional qPCR, as demonstrated for samples in molecular remission, and could provide an accurate monitoring method for Ph+ ALL CR patients. Further studies to confirm these results are actually ongoing. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione, Università 2007-2009.
2010
S3
S29
S29
Lonetti A; Iacobucci I; Ferrari A; Papayannidis C; Paolini S; Cilloni D; Abbenante M; Guadagnuolo V; Pane F; Baccarani M; Martinelli G
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/154763
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