Mobilized peripheral blood (MPB) and bone marrow (BM) cells were collected from MM patients enrolled in high-dose chemotherapy programs, approved by IRB at HSR, after informed consent. Mononuclear cells were purified by density centrifugation using the lymphocyte separation medium Lymphoprep (Nycomed Pharma, Asker, Norway).
CD34+ cells were positively selected using the following immunomagnetic separation devices: mini-, and midi-MACS (CD34+ MultiSort Kit, Miltenyi Biotec, Bergisch Gladbach, Germany), cliniMACS (Miltenyi Biotec), ISOLEX300i (Baxter Healthcare, Irvine, CA, USA), according to the manufacturer's instructions. CD138+ cells were selected with mini-MACS device, using direct conjugated anti CD138 microbeads (Miltenyi Biotec). Immunoselected cells were analyzed by flow-cytometry, to determine the purity of selections.
Retroviral supernatant production
BML-1 Moloney Murine Leukemia Virus (MoMLV) based retrovirus, containing the ΔNGFR marker gene under the control of LTR promoter  was collected from producer cells, kindly provided by Roche Diagnostics GmbH. Producer cells were expanded in 200 μl/cm2 Iscove's modified Dulbecco's medium (IMDM, Bio Whittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (FCS, Euroclone, Wetherby, West Yorkshire, UK), 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine at 5 × 104 cells/cm2, at 37°C with 5% CO2. The medium was replaced after 48 hours, 72 hours and 84 hours reducing the volume to 66 μl/cm2 and the temperature to 33°C . Viral supernatant (SN) was collected with 12-hours serial collections, 0.45 filtered and -80°C frozen before use.
Culture, transduction and selection of transduced cells
CD34+ cells were cultured in X-Vivo 10 (Bio Whittaker) serum free medium containing the cytokines thrombopoietin (PeproTech, Rocky Hill, NJ, USA), FLT3-Iigand (PeproTech), stem cell factor (R&D Systems Minneapolis, MN, USA) at 50 ng/ml (thereafter designated complete medium) at 5–6 × 105cells/cm2 for 84 hours. Transduction of CD34+ cells were performed in complete medium, at the same cell concentration. Cells were prestimulated for 24 hours, and overnight transduced with the BML-1 retroviral vector on RetroNectin (TaKara Biomedicals, Kyoto, Japan)-coated non-tissue culture-treated plates, or T75 flasks, with 4:1 v/v SN/culture medium, supplemented with 5× concentrated cytokines. Fresh complete medium was replaced after virus withdrawal and cells were cultured for further 48 hours. At the end of the culture, cells were collected, washed, and incubated with anti-NGFR biotinylated antibody at 2.5 × 106 cell/ml for 30 minutes at 4°C and then with 10 μl streptavidin (SA)-conjugated microbeads (Miltenyi Biotec) for 15 minutes at 4°C. ΔNGFR+ cells were then isolated by mini-MACS immunoselection device. ΔNGFR positive and negative fractions were analyzed by flow-cytometry for CD34, CD45, CD138, and ΔNGFR antigens. Clonal transduction of CD138+ cells was performed in U-bottom 96-well plates with 4:1 SN/culture medium only in wells scored positive for 1 cell at light microscope. Prestimulation was performed with 40 μl of complete medium, and after 24 hours, 128 μl of virus, complemented with 32 μl of 5× cytokines, were directly added to each well. To avoid disturbance of the cells, transduction was stopped by substituting 150 μl of medium with fresh complete medium. Doubling of single cells was scored daily by microscopy and viability of cells using Trypan blue exclusion assessed at the end of transduction.
Immunofluorescence staining and flow cytometric analyses
Cells were stained with conjugated monoclonal antibodies (mAbs) in 100 μl PBS, 0,1% sodium-azide, 0,3% BSA (PBS FACS) at 4°C for 25 minutes, after the staining cells were washed and resuspended in PBS FACS. For biotinylated mAbs a secondary staining with streptavidin-PE/FITC was performed. The following mAbs were used: CD34-PE (Becton Dickinson, San Josè, California, USA), CD45-FITC/TC (Caltag, Burlingame, California, USA), CD138-FITC (Valter Occhiena, Torino, Italy), CD38-TC (Caltag), SA-FITC/PE/TC (Caltag), biotinylated-NGFR. Isotype-identical mAbs IgG1-FITC/PE/TC and biotinylated-IgG1 (Caltag) served as control. Samples were acquired with FacsScan device (Becton Dickinson). Data were analyzed using CellQuest software (Becton Dickinson).
In vitro clonogenic cell assays
Long-term marrow cultures (LTMC) were performed according to a described procedure . Briefly, 70000 CD34+/ΔNGFR+ cells were seeded on top of MS5 murine stromal cells in Myelocult medium (StemCell Technologies, Vancouver, BC, CA), added with 10 ng/ml IL6 (Pepro Tech) and half of the medium was weekly replaced. After three weeks of culture, cells were resuspended in Methocult GF medium (StemCell Technologies) and plated in duplicate in a (Colony Forming Units-Cells) CFU-C assay. Colonies were scored two weeks later, individually picked and DNA was extracted for PCR analysis. DNA extraction was performed adding, for each colony, 25 μl of KCl lysis buffer and protease K at 50 μg/ml. Colonies were incubated overnight at 37°C and heat inactivated for 15' at 94°C. KCl lysis buffer consists of 1:1 v/v mix of solution A (100 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2) and solution B (10 mM Tris pH 8.3, 2.5 mM MgCl2, 1% Tween-20, 1% NP-40). DNA was then analyzed for the presence of ΔNGFR transgene, and patient-specific myeloma markers.
To determine the presence of ΔNGFR transgene, DNA was amplified with primers 5'-LΔ1: GGCCGTTGGATTACACGGTC and 3'-MAGO: CCTACAGGTGGGGTCTTTCA. PCR reactions were carried out in a final volume of 25 μl, with 1 μM primers, 2.5 mM MgCl2, 0.25 mM dNTPs and 1.25 U Taq Gold (Perkin Elmer, Wellesley, MA, USA), at following conditions: 7 minutes at 94°C, 50 seconds at 94°C, 50 seconds at 60°C and 50 seconds at 72°C for 40 cycles, 5 minutes at 72°C. As a control for the presence of DNA, HLA genes were amplified (primer 5':GTGCTGCAGGTGTAAACTTGTACCAG and primer 3':CACGGATCCGGTAGCAGCGGTAGAGTTG). PCR conditions were the same as above, except for annealing temperature that was 56°C.
To detect tumor cells, the clonal variable region (VDJ) rearrangements of the IgH genes of each patient were amplified using 5' consensus primers derived from the conserved sequences of the variable region and 3' consensus primer derived from the joining region as previously described . PCR products were sequenced and sequences from CDR2 and CDR3 regions were used to design patient-specific tumor primers. Oligonucleotides were subsequently tested for specificity using polyclonal DNA from normal individuals as negative controls. For each patient, CD34+ cells before and after culture, and after ΔNGFR immunoselection were amplified to detect residual myeloma cells. Cells were directly resuspended at 1 × 105/50 μl in KCl lysis solution for genomic DNA extraction (as described above) and PCR amplifications were performed using 5, or 10 μl of DNA. A first round PCR was performed using patient specific VH family primers and a second round was performed amplifying 1 μl of the first PCR product with patient-specific primers. The sensitivity was 10-4-10-5 .
Limiting dilution assays were performed serially diluting DNA with water, in 0.5 log increments and each dilution was subsequently amplified with patient-specific primers. At least five PCR reactions were performed for each dilution level from the level in which reactions were positive up to the level in which all reactions were negative for clonal markers. Polyclonal DNA was always included as negative control. This technique has been previously validated by diluting a known number of tumor cells in normal marrow or peripheral blood cells. An indirect quantification of the tumor load in the positive samples of the dilution was performed according to the statistical methods of likelihood maximization and χ2 minimization (MC), derived from the single-hit Poisson model, as described by Taswell . Combined frequency estimates were obtained by analyzing with MC estimator the pooled frequency determinations. Comparison of frequencies between groups was performed through evaluation of confidence interval (CI) and probability value, and CI for the ratio of 2 frequencies. All calculations were performed using MATLAB (version 5, The Math Works Inc.).