Xue et al., NPM1 mutation monitoring after allogeneic HSCT
Supplementary Information
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SUPPLEMENTARY FIGURE
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Supplementary Figure 1. ROC analysis for NPM1mut quantitative monitoring after allogeneic
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HSCT. ROC curve, showing sensitivity (Y-axis) and 1-specificity (X-axis) calculated taking into
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account the total 178 samples harvested in post-transplantation hematological remission from the 28
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AML patients included in our study. The reflection line (y=x) is the dashed black line.
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Accompanying sensitivity and specificity table is shown on the right. Based on this ROC analysis,
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the highest Youden index (indicated by the red arrow) was observed for values just above 10-6
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NPM1mut/ABL copies, which also represented the maximal sensitivity limit that we accepted for our
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results. Abbreviations: AUC, area under the curve; CI, confidence interval.
Xue et al., NPM1 mutation monitoring after allogeneic HSCT
Supplementary Information
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SUPPLEMENTARY METHODS
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Sample Collection and Processing
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Bone marrow aspirate samples were harvested in concomitance to routine diagnostic procedures
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and upon written informed consent approved by the San Raffaele Ethic Committee. As a center
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policy, bone marrow evaluations were performed starting from day +30 after allogeneic HSCT and
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carried out monthly in the first trimester, then once in a trimester for the first year, and yearly after
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that. Mononuclear cells were separated by Ficoll-PaqueTM PLUS (GE Healthcare) gradient
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centrifugation, and freshly processed for nucleic acid extraction. Genomic DNA was extracted
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using the Qiamp Blood Minikit (QIAGEN, Venlo, The Netherlands), checked for purity using a
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Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20°C
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for further analyses. For RNA extraction, samples were resuspended in Trizol® reagent (Invitrogen,
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Carlsbad, CA, USA) and either immediately processed or stored at -80°C for subsequent
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phenol/chloroform precipitation procedure according to the manufacturer’s recommendations,
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without further procedures for genomic DNA elimination. Total RNA was checked for quality using
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a Nanodrop spectrophotometer and stored at -80°C. When necessary 1 µg of total RNA was retro-
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transcribed into complementary DNA following a published protocol for reverse transcription mix
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(RT-Mix) preparation and cycling conditions1.
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NPM1mut screening and monitoring
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Screening for the presence of NPM1mut in bone marrow samples harvested at diagnosis was
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performed by conventional Sanger sequencing, using the primer and reaction conditions suggested
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by Dohner and collaborators2, and using genomic DNA as template.
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For the follow-up of NPM1mut-positive patients, transcript levels of the mutations of interest were
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monitored over time by TaqMan chemistry-based RT-qPCR assays, using bone marrow-derived
Xue et al., NPM1 mutation monitoring after allogeneic HSCT
Supplementary Information
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cDNA as template. For NPM1mutA-positive patients we employed the Ipsogen® NPM1 mutA
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MutaQuant Kit, following the manufacturer's indications for reaction mix preparation and cycling
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conditions, and using an Applied BioSystems 7500 thermocycler (Thermo Fisher Scientific,
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Waltham, MA, USA). For the longitudinal monitoring of the single patient carrying NPM1mut B, we
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followed published indications from Gorello and collaborators3. For both mutA and mutB
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monitoring, we employed 100 ng of RNA equivalent cDNA per well as template and ABL1 as
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reference gene: as cDNA quality control, a minimum of 10'000 copies of ABL were set as
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minimum limit for acceptance of results. Negative template controls were always included in plate
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design. Results were obtained upon interpolation of results from those obtained by standard curves
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obtained from serial dilutions of the relevant target plasmids into water: for NPM1mut standard
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curves, average values for slope and intercept were 3.47 and 40.15, respectively, whereas for ABL
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3.50 and 40.01, respectively. Each reaction was performed in duplicates. Results above 10-4 copies
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NPM1mut/ABL copy, reported by Gorello and others as the maximal reproducible sensitivity of
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these assays3, were considered positive without need of confirmation. In case of positive results in
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the range between the maximal reproducible sensitivity (10-4 NPM1mut copies/ABL copy) and the
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maximal sensitivity (10-6 NPM1mut copies /ABL copy), assays were repeated in triplicates for
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confirmation. Out of the 16 assays befalling in this range, repetition confirmed the initial results in
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11 (68%). Values below 10-6 NPM1mut copies /ABL copy were considered negative.
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Quantification of WT1 Transcript
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The quantitative assessment of WT1 transcript in bone marrow samples was performed using RT-
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qPCR through the WT1 ProfileQuant kit ELN (QIAGEN Sciences, Germantown, MD), based on
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TaqMan technology, developed on the bases of the assay from Van Dijk and collaborators4 and
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already validated in the context of a collaborative study led by a group of experts from the ELN
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consortium5. Also in this case, all experiments were performed in duplicate wells employing 100 ng
Xue et al., NPM1 mutation monitoring after allogeneic HSCT
Supplementary Information
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of RNA equivalent cDNA per well. Results were normalized with respect to the number of ABL
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transcripts and expressed as copies of WT1/104 copies of ABL. Based on previous work from
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Cilloni and collaborators5, a threshold of 250 copies of WT1/104 copies of ABL was used to define
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WT1 overexpression in bone marrow samples.
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Hematopoietic Chimerism analysis
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Host-specific hematopoietic chimerism was assessed using the AlleleSEQR® Chimerism Assay,
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(Celera Genomics, Alameda, CA, USA), according to the manufacturer recommendations, and
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using 50 ng genomic DNA as template for triplicate well experiments. Taking advantage of the
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ΔΔCt method, the relative amount of a host-specific marker was assessed by relating the Ct value
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derived from amplification of that marker to the Ct value of its respective endogenous control
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(RNaseP) both in pre-HSCT sample (calibrator ΔCtC) and in post-HSCT chimeric sample
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(unknown ΔCtU). In particular, based on our center experience and to published data6, we
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considered as threshold for relapse prediction a host-specific signal above 1%.
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Statistical analysis
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To select the best NPM1mut value to use as threshold for relapse prediction, a receiver operating
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characteristic (ROC) analysis was performed7, using the SPSS version 20 software (SPSS Inc./IBM,
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Armonk, NY, USA). Youden index was calculated as the highest sum of sensitivity plus specificity
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minus 1008.
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SUPPLEMENTARY REFERENCES
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Supplementary Information
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polymerase chain reaction of fusion gene transcripts for residual disease detection in
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leukemia - a Europe Against Cancer program. Leukemia 2003; 17(12): 2318-57.
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Dohner K, Schlenk RF, Habdank M, Scholl C, Rucker FG, Corbacioglu A et al. Mutant
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nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid
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leukemia and normal cytogenetics: interaction with other gene mutations. Blood 2005;
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Gorello P, Cazzaniga G, Alberti F, Dell'Oro MG, Gottardi E, Specchia G et al. Quantitative
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assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin
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Van Dijk JP, Knops GH, Van De Locht LT, Menke AL, Jansen JH, Mensink EJ et al.
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Cilloni D, Renneville A, Hermitte F, Hills RK, Daly S, Jovanovic JV et al. Real-time
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Metz CE. Basic principles of ROC analysis. Semin Nucl Med 1978; 8(4): 283-98.
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