Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma

Abstract

Background: Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells.

Design and methods: The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells.

Results: Cells with the phenotype of tumor plasma cells (CD38(++)CD19(-)CD45(-/+)CD56(-/+/++)) or memory B cells (CD38(-)/CD19(+)/CD27(+)) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified.

Conclusions: The increased expression of a specific "early" oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack "late" oncogene (K-RAS) mutations but express the "early" oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

Figure 1.

Blimp-1 and XBP-1 expression. Global RT-PCR products from plasma cells and memory B cells were analyzed by RT-PCR for expression of the transcription factors XBP-1 and Blimp-1. Results are shown for three myeloma patients and three healthy individuals, with 100 sorted cells per tube in each case. Lanes 1–3, oncogene-negative (healthy) memory B cells; lanes 4–6, oncogene-positive (myeloma) memory B cells; lanes 7–9, MM plasma cells; lanes 10–12, healthy plasma cells.

Figure 2.

Oncogene analyses of plasma cells and memory B cells: (A) from a CCND1-positive MM patient (MM-1) and (B) from a CCND1-positive MGUS patient (MGUS-2). Global RT-PCR products were screened for CCND1 expression using real-time PCR, with 100 sorted cells per tube in each case. CCND1-positive or negative real-time PCR products were run on a gel and bands corresponding to CCND1 were excised from the gel and sequenced. The oncogene/β-actin ratio determined by real-time PCR is shown below each positive case. P, positive control; N, negative control (H₂O); lanes 1 and 2, healthy memory B cells; lanes 3 and 4, oncogene-positive memory B cells; lane 5, RT control; lanes 6 and 7, myeloma plasma cells; lanes 8 and 9, healthy plasma cells.

Figure 3.

Identification of IGH-MMSET hybrid transcripts in memory B cells. (A) The FGFR3-positive MM patient (MM-3) was analyzed as described in Figure 2. (B) The CD38⁺⁺/CD19⁻/CD56⁺⁺ plasma cells were flow-sorted directly to PCR tubes in numbers of 50, 5 and 1 cell/PCR tube followed by nested RT-PCR for IGH-MMSET hybrid transcripts. (C) RT-PCR analysis of IGH-MMSET hybrid transcripts in sorted CD19⁺/CD38⁻/CD27⁺ memory B cells. Memory B cells were flow sorted in numbers of 500, 100 and 50 cells/PCR tube, with eight PCR tubes for each cell number followed by RT-PCR analysis of IGH-MMSET hybrid transcripts. N = negative control (H₂O).

Figure 4.

Screening memory B cells for K-RAS61 mutations. (A) Sensitivity of ACB-PCR when analyzing FACS-sorted plasma cells. FACS-sorting of 1, 2 or 10 MM plasma cells from a patient with a K-RAS61 mutation to PCR tubes containing 100 FACS-sorted CD19⁺ B cells from a healthy individual. The results are only shown for the CAA to CAC mutation, but similar results was obtained for the CAA to CTA mutation. In four out of five PCR tubes with a single FACS-sorted K-RAS61⁺ MM plasma cell among 100 healthy B cells, a K-RAS61⁺ MM plasma cell was detected. (B) From six patients with a K-RAS61 mutation, 100 CD19⁺/CD38⁻/CD27⁺ memory B cells were FACS sorted for each ACB-PCR analysis. For each of the MM patients the K-RAS61 mutation type is shown and the total number of CD19⁺/CD38⁻/CD27⁺ memory B cells available for analysis is given (n). P = positive control, 100 FACS-sorted plasma cells from a patient with a K-RAS61 mutation. N = negative control, 100 FACS-sorted CD19⁺ B cells from a healthy donor. WT = wild-type, an analysis for wild-type RAS was performed for each FACS-sorted memory B-cell population as a positive control.

Figure 5.

Simultaneous screening for CCND1 expression and K-RAS61 mutations. A cell lysate obtained from 100 FACS-sorted memory B cells from patient MM-13 was split into two aliquots, one for global RT-PCR followed by real-time PCR for CCND1 and one for ACB-PCR detection of K-RAS61 mutations. (A) In 5/96 PCR tubes CCND1⁺ memory B cells were identified, the measured CCND1 level is shown (n. 1–5). N. 6–10 show five representative negative CCND1 measurements. (B) Each memory B-cell lysate was analyzed for the KRAS61 CAA to CAC mutation present in the patients’ MM plasma cells. As a control for the presence of cDNA, each cell lysate was analyzed for the presence of wild-type (WT) RAS. Lanes 1–5, CCND1-positive memory B cells (see Online Supplementary Figure 2A). Lanes 6–10, CCND1-negative memory B cells. Lane 11, positive control, 100 FACS-sorted plasma cells from patient MM-13 with a K-RAS61 mutation. Lane 12, negative control, 100 FACS-sorted CD19⁺ B cells from a healthy donor. Lane 13, negative control (H₂O). M = molecular size marker. RAS mutations were absent from all memory B cells analyzed.