Endothelial progenitor cells display clonal restriction in multiple myeloma

Twenty-three consecutive MM patients diagnosed by Southwest Oncology Group criteria [17] were studied according to protocols approved by the Institutional Review Board of State University of New York Downstate Medical Center, and informed consent was obtained in accordance with the Declaration of Helsinki. Clinical stage was determined according to Durie and Salmon [18]; in addition to stage, serum β₂-microglobulin and albumin and, when feasible, tumor cell karyotype (Wycoff Heights Medical Center, Brooklyn, NY) constituted clinical prognostic indicators [19] (Table 1). Fifteen healthy hospital staff served as controls.

Confluent EPCs were outgrown from peripheral blood mononuclear cells (PBMCs) and from BM for DNA extraction. EPCs outgrown from PBMCs were preferentially used; however, when DNA from EPCs derived from PBMCs was not sufficient for the outlined studies, DNA from EPCs grown from BM was used. The EPC DNA for each patient was obtained from only one source, i.e., either from peripheral blood or from BM aspirate, as indicated in Tables 1, 2, 3. To grow EPCs, heparinized peripheral blood (20 ml), or a single-cell suspension of bone marrow (10 ml) was separated by Ficoll-Hypaque (Sigma Chemicals, St. Louis, MO) density-gradient centrifugation within 6 hours of collection. The mononuclear cell layer was resuspended in EndoCult Basal Medium supplemented with 20% fetal bovine serum (StemCell Technologies, Vancouver, Canada) and antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin). Ficoll-separated mononuclear cells from peripheral blood or bone marrow (7 × 10⁶/well) were grown to confluence in laminin-coated, 6-well plates (Becton Dickinson Labware, Bedford, MA) as previously described [9, 20]. Under these culture conditions, EPC colonies are observed at 14 to 18 days, and confluence is reached 18 to 28 days after plating peripheral blood or bone marrow cells. To determine endothelial cell phenotype and purity of outgrown cells, EPCs were also grown on laminin-coated, 96-well plates (Becton Dickinson Labware) and subjected to two-color immunostaining with combinations of antibodies directed against endothelial cell markers, including vascular endothelial growth factor receptor-2 (kinase insert domain-containing receptor/fetal liver kinase-1 [KDR]), CD133, CD144 (vascular endothelial cadherin [VE-cadherin]), and von Willebrand factor (vWF) as we previously described [9]. Anti-CD38-PE (BD PharMingen, San Diego, CA) was employed to detect MM cells; TO-PRO-3 (Molecular Probes, Eugene, OR) was used to counterstain the nuclei of EPCs (Figure 1). Appropriate isotype-matched serum with each of the antibodies was used as negative control. Images of the stained cells were digitally recorded in the sequential mode on a confocal laser scanning microscope (Bio-Rad MRC 1024ES, Bio-Rad, Hercules, CA). Percent positive cells were quantitated in a minimum of 3 fields (original × 40) per well. In addition, viable EPCs, after detachment from laminin (Cell Dissociation Buffer, Invitrogen, Carlsbad, CA), were stained using direct immunofluorescence with indicated combinations of anti-vWF-FITC (Serotec, Raleigh, NC), anti-CD38-PE (BD PharMingen), anti-CD133-PE (Miltenyi Biotec, Auburn, CA), and anti-CD45-PECy5 (eBioscience, San Diego, CA), as well as isotype-specific control antibodies, and analyzed with a FACSort flow cytometer (BD Biosciences) as previously described [9]. To assure absence of contaminating plasma cells, bone marrow aspirates were also stained for the plasma cell marker CD38 using anti-CD38-PE and analyzed by flow cytometry (see Additional file 1).

Human androgen receptor gene assay (HUMARA) was used to examine XCI status of EPCs and non-endothelial cells to evaluate the clonal status of each population. Heterozygosity at the AR locus is a prerequisite for XCI evaluation by HUMARA. Androgen receptor locus was therefore examined in 18 female MM patients by polymerase chain reaction (PCR) assay in DNA extracted from PBMCs, as described previously [21]. Definitive polymorphism in the human AR locus was found in 11 of the 18 patients studied (Table 1, Cases 1–11). In the remaining 7 patients, the AR locus did not display obvious heterozygosity (data not shown) and therefore these patients were not candidates for evaluation of EPC clonality using HUMARA. Within a monoclonal cell population in a woman, all cells have the same combination of active and inactive X chromosomes. The maternally and paternally inherited X chromosomes can be distinguished by polymorphisms involving the number of tandemly repeated CAG repeats within the AR gene. Using AR-specific PCR primers that flank the CAG repeats, two PCR products of different sizes can be amplified from a heterozygous patient's genomic DNA. The primers that flank the AR CAG repeat sequence also span an HpaII restriction site (CCGG) that is methylated and thereby protected from digestion when present on an inactive X chromosome, but is unmethylated and thereby susceptible to digestion when present on an active X chromosome. Thus, when HpaII-treated DNA is subjected to PCR with AR-specific primers, copies of the inactive allele are amplified, whereas copies of the active allele are cleaved by HpaII and cannot yield a PCR product. As a result, a single product is obtained after amplification of DNA from a clonal cell population from a heterozygous female patient. DNA was extracted from confluent EPCs, intact hair roots, and PBMCs using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA). DNA samples were divided into two identical aliquots, one of which was incubated overnight at 37°C with the methylation-sensitive restriction enzyme HpaII for the digestion of unmethylated (or active) alleles. A second restriction enzyme, RsaI, which recognizes a four-base pair sequence not present in the amplified region of the AR locus, was also included in the reaction to facilitate the HpaII digestion process. Male DNA with cytogenetically verified 46XY karyotype was used as control for complete digestion.

Both digested and undigested DNA samples were amplified using AR-specific primers 5'-GTC CAA GAC CTA CCG AGG AG-3' and 5'-CCA GGA CCA GGT AGC CTG TG-3'. Amplicons were labeled by including a radioactive nucleotide (α-[³³P]-dCTP) (NEN, PerkinElmer Life Sciences, Boston, MA) in the PCR. PCR products were separated on 8% denaturing gels, followed by autoradiography. Densitometric analyses of the alleles, performed at least twice for each sample using MultiAnalyst version 1.1 software (Bio-Rad), included normalization of the ratios based upon the non-digested samples by dividing the allele ratio of the digested sample by the ratio of the non-digested sample from the same specimen.

One-step PCR as described by Deane and Norton [22] was used to determine V_H gene rearrangement within confluent EPCs and MM-cell-infiltrated BM aspirate samples. In each experiment, genomic DNA extracted from unseparated BM aspirate containing 10%-95% MM cells (Table 3) was used to identify the clonotypic IG V_H gene rearrangement within the neoplastic plasma cells. V_H genes were amplified from genomic DNA extracted from freshly obtained bone marrow samples and primary EPCs using a genomic DNA purification kit (Gentra Systems, Minneapolis, MN). A set of 7 V_H family-specific PCR primers was used to isolate the clonally expressed V_HD_HJ_H, as described previously [22]. The forward primers were from the first framework region of the most common V_H family members, and the single reverse J_H primer was common to the 3' end of all six J_H genes. PCR products were run on a 1.0% agarose gel, where the V_H gene rearrangement was identified as a single band of 350 bp. For cloning and sequencing, DNA was extracted from the resolved 350-bp bands using the QIAquick Gel Extraction Kit (QIAGEN). Dideoxy sequencing (GENEWIZ, North Brunswick, NJ) was performed using the same V_H primer that was added to the original PCR reaction. Nucleotide sequences of amplified DNA were identified by BLAST and compared by FASTA.

To rule out false-positive results due to contaminating plasma cells, the sensitivity of the PCR system to detect IGH rearrangement in EPCs was evaluated by quantitating the minimum contamination with tumor DNA required for IGH gene rearrangement. Thus, DNA samples from human umbilical vein endothelial cells (HUVECs), which do not have IGH rearrangement, were spiked with decreasing percentages of DNA obtained from the MM cell line U266, which has a clonotypic IGH rearrangement. In these experimental conditions, 2% or more of contaminating plasma cells were necessary to give positive results for IGH gene rearrangement (data not shown). As flow cytometry showed positivity of 0.1 ± 0.01% (n = 3) in EPCs for the myeloma cell antigen CD38, IGH rearrangement observed in these cultures was concluded to be a tumor-specific feature of EPCs. In addition to DNA extracted from HUVECs, DNA from EPCs outgrown from a healthy control were studied for V_H rearrangement as negative controls for these experiments.

To further rule out that plasma cell contamination in EPCs could be responsible for IGH rearrangement found in primary cultures, quantitation of EPCs containing the IGH rearrangement was performed using SYBR Green I real-time PCR. In B cell malignancies, quantitative real-time PCR assays have been established that use tumor-specific IGH rearrangements as quantitative markers of tumor cells [23]. The amount of fluorescence produced by this real-time PCR reaction is proportional to the starting DNA target number during the early phases of amplification. DNA from primary EPCs from Case 15, which contained the clonotypic rearrangement found in BM cells, was PCR-amplified using V_H4A forward and J_H reverse primers [22], and cloned using a TA Cloning Kit (Invitrogen, Carlsbad, CA) following recommended procedures. Briefly, about 5 ng of fresh PCR product was ligated to 50 ng of pCR2.1 at equal molar concentrations overnight at 16°C. Competent DH5α Escherichia coli were transformed with 20% of the ligation reaction and plated on LB agar with 100 μg/mL of ampicillin. Recombinant plasmid containing the ligated PCR-amplified IGH gene product was recovered with a QIAGEN Miniprep DNA purification system (QIAGEN) and digested with EcoRI to confirm presence of insert. Sequencing of cloned plasmid DNA using the M13 reverse primer (GENEWIZ) also confirmed the inserted patient-specific IGH rearrangement (data not shown).

Plasmid DNA containing human apolipoprotein B100 (ApoB) [24] was obtained from Kezhi Dai for use as an internal standard to normalize genomic DNA levels of IGH rearrangement in EPCs and BM cells from the same patient, as described for detection of minimal residual disease in acute lymphoblastic leukemia [25, 26]. Quantitative PCR was performed using the qPCR Core Kit for SYBR Green I (Eurogenetec, San Diego, CA). Reactions performed in duplicate for IGH quantitation contained 10 ng of either EPC or BM cell genomic DNA from Case 15, and 2 × qPCR master mix containing 8 μM of either IGH primers (V_H4 forward 5'-TCGGAGACCCTGTCCCTCACCTGCA-3', patient allele-specific reverse 5'-CCAGAGATCGAAGAACCAGTCTTC-3') or ApoB primers (ApoB forward 5'-GCAAG CAGAA GCCAG AAGTG A-3', ApoB reverse 5'-CCATT TGGAG AAGCAGTTTG G -3'). Amplification conditions, using an ABI Prism 7000 sequence detector equipped with a 96-well thermal cycler (Applied Biosystems, Foster City, CA), were 10 minutes at 95°C (to activate the enzyme) and 40 cyclesof denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. Data were collected and analyzed with ABI Prism SDS software v1.0 (Applied Biosystems, Foster City, CA). To obtain a standard curve for quantifying patient BM and EPC DNA, IGH and ApoB plasmids were diluted in 4-fold increments and subjected to quantitative PCR in duplicate as previously described [25]. Standard curves plotting cycle threshold (C_T) versus log number of copies were produced with correlation coefficients greater than or equal to 0.97. Copy numbers of ApoB and rearranged IGH loci in EPCs and BM were obtained from these standard curves. Percent of cells bearing the IGH rearrangement was calculated as 100% × (log copy numbers of IGH/log copy numbers of ApoB).

Data are presented as means ± standard deviations. Student's t test, two-tailed, was used to compare indicated groups. Associations between variables were evaluated by Pearson's r. Significance was set at p ≤ .05.