BACKGROUND: Ph+ ALL is observed in about 20-30% 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%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. 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 and to detect early relapse. AIM: To investigate 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 AND PATIENTS: The 12.765 Digital array (Fluidigm) is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; 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. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. At the time of writing, we analyzed 22 Ph+ ALL samples (11 positive for the P190 BCR-ABL1 isoform and 11 for the P210 ) who were 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 µl volume of input cDNA was loaded and two panel for each dilution were used. Analysis parameter chosen for digital raw data processing were automated set threshold of 0.65 and target Ct range 20-40. 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, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.
A Lonetti , I Iacobucci , A Ferrari , C Papayannidis , S Paolini , D Cilloni , et al. (2010). DETECTION OF RARE COPIES OF BCR-ABL1 TRANSCRIPT IN PATIENTS WITH PHILADELPHIA POSITIVE (PH ) ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) WITH A HIGH SENSITIVE NANOFLUIDIC ARRAY.
DETECTION OF RARE COPIES OF BCR-ABL1 TRANSCRIPT IN PATIENTS WITH PHILADELPHIA POSITIVE (PH ) ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) WITH A HIGH SENSITIVE NANOFLUIDIC ARRAY
LONETTI, ANNALISA;IACOBUCCI, ILARIA;PAPAYANNIDIS, CRISTINA;PAOLINI, STEFANIA;MARTINELLI, GIOVANNI
2010
Abstract
BACKGROUND: Ph+ ALL is observed in about 20-30% 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%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. 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 and to detect early relapse. AIM: To investigate 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 AND PATIENTS: The 12.765 Digital array (Fluidigm) is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; 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. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. At the time of writing, we analyzed 22 Ph+ ALL samples (11 positive for the P190 BCR-ABL1 isoform and 11 for the P210 ) who were 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 µl volume of input cDNA was loaded and two panel for each dilution were used. Analysis parameter chosen for digital raw data processing were automated set threshold of 0.65 and target Ct range 20-40. 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, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
