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Dr. Marco Ladetto - Improved IgH-based MRD detection by using droplet digital PCR: a comparison with real time quantitative PCR in MCL and MM

Marco Ladetto, MD
Division of Hematology
Department of Molecular Biotechnologies and Health Sciences
University of Torino
Torino, Italy


Program: Oral and Poster Abstracts
Session: 622. Non-Hodgkin Lymphoma: Biology, excluding Therapy: Poster III

Monday, December 9, 2013, 6:00 PM-8:00 PM, Hall G (Ernest N. Morial Convention Center)

Daniela Drandi1*, Lenka Kubiczkovà2*, Nadia Dani3*, Simone Ferrero1*, Luigia Monitillo4*, Barbara Mantoan4*, Elisa Genuardi1*, Daniela Barbero4*, Manuela Gambella4*, Davide Barberio3*, Paola Ghione4*, Guglielmo Guarona1*, Elona Saraci4*, Paola Omedè5*, Roberto Passera6*, Roman Hajek2,7, Sergio Cortelazzo, MD8*, Antonio Palumbo9, Mario Boccadoro, MD1, Giorgio Inghirami10* and Marco Ladetto4

1Division of Hematology, Department of Molecular Biotechnologies and Health Sciences, University of Torino, Torino, Italy
2Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
3Bioclarma, Torino, Italy
4University of Torino, Department of Molecular Biotechnology and Health Sciences, Hematology division at A.O.U. San Giovanni Battista, Torino, Italy
5Division of Hematology, A.O.U. S.Giovanni Battista, Torino, Italy
6Division of Nuclear Medicine, A.O.U. San Giovanni Battista, Statistical Consultant, University of Torino, Torino, Italy
7Dept. of Hematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic
8Hematology department, S. Maurizio Regional Hospital, Bolzano, Bozen, Italy
9Myeloma Unit, Division of Hematology, University of Torino, Torino, Italy
10Department of Pathology and Center for Experimental Research and Medical Studies (CeRMS), University of Turin, Turin, Italy, Turin, Italy


In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM).  RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above.


Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log.


Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed  major qualitative discordance.  28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR.


Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment.

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