Jeroen E. J. Guikema

CD27 is heterogeneously expressed in multiple myeloma: low CD27 expression in patients with high-risk disease

Summary. Expression of CD27 on malignant plasma cells (PC) (CD138+CD38++) was analysed in a cross‐sectional study of bone marrow (BM) samples from multiple myeloma (MM) patients (nu2003=u200328), monoclonal gammopathy of undetermined significance (MGUS) patients (nu2003=u20036) and BM PC from healthy donors (nu2003=u20034). MM PC expressed CD27 with a variable, lower intensity pattern compared with the consistent high expression in MGUS and healthy donors. MM patients in complete clinical remission displayed a higher percentage of CD27+ PC compared with patients at diagnosis, relapse or in partial remission. In MM, loss of CD27 correlated with loss of CD19 (R2u2003=u20030·4, Pu2003<u20030·0001). Human MM cell lines (nu2003=u20039) did not express CD27 whereas de novo plasma cell leukaemia (PCL) (nu2003=u20033) had a high expression. Re‐analysis of a cDNA microarray data set, generated from newly diagnosed MM patients (nu2003=u200374), demonstrated that the MM subgroup with the highest prevalence of poor prognostic factors had the lowest CD27 mRNA expression. Fluorescence‐activated cell sorting and allele‐specific oligonucleotide polymerase chain reaction showed that both CD27+ and CD27– PC subpopulations in MM can belong to the clonal disorder. In conclusion, CD27 is heterogeneously expressed on MM PC and loss of CD27 expression might have prognostic value in MM.

Multiple myeloma related cells in patients undergoing autologous peripheral blood stem cell transplantation

A high incidence of oligoclonal serum M‐components is observed in multiple myeloma (MM) patients treated with autologous stem cell transplantation (ASCT). To determine whether these M‐components are produced by myeloma clonally related cells or caused by an aberrant B‐cell regeneration we analysed by semi‐nested ASO‐RT‐PCR and DNA sequencing the immunoglobulin (Ig) variable genes (VH) obtained from bone marrow samples obtained before and after transplantation and peripheral blood stem cell (PBSC) samples from seven patients.

CD27‐triggering on primary plasma cell leukaemia cells has anti‐apoptotic effects involving mitogen activated protein kinases

Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was set out to assess the functional properties of CD27 expressed on PCL plasma cells by triggering with its ligand CD70. Using CD27‐expressing purified plasma cells from a PCL patient we demonstrated that CD27‐triggering modestly inhibited spontaneous and dexamethasone‐induced apoptosis. In vitro stimulation and Western blotting showed that activation of p38 and extracellular‐regulated kinase 1/2 (ERK1/2) mitogen‐activated protein kinases (MAPK) was associated with CD27‐mediated signal transduction. Specific inhibition of p38 and ERK1/2 MAPK abolished the anti‐apoptotic effects of CD27‐triggering. Interestingly, simultaneous inhibition of p38 and ERK1/2 strongly sensitized PCL cells for dexamethasone‐induced apoptosis. Finally, in dexamethasone‐treated PCL cells, CD27‐triggering was associated with persistent DNA‐binding activity of activator protein 1 (AP‐1) but not of nuclear factor‐κB. These findings suggest that, in primary PCL, specific anti‐apoptotic pathways exist that might provide novel therapeutic targets.

Myeloma clonotypic B cells are hampered in their ability to undergo B-cell differentiation in vitro

Summary.u2002 In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V(D)J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (nu2003=u200310) and the ability to differentiate in vitro was determined using the CD40‐culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotype‐specific allele‐specific oligonucleotide reverse transcription polymerase chain reaction (ASO‐RT‐PCR) after culturing with CD40L and interleukin 4 (IL‐4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B‐cell differentiation was studied by abrogating CD40 signalling accompanied by IL‐10 and IL‐2 stimulation, enhancing differentiation towards Ig‐secreting cells. The numbers of clonotypic B cells were determined by quantitative ASO‐PCR. An increase in cell number was observed upon CD40L and IL‐4 stimulation, whereas the relative number of clonotypic B cells was unaltered. In contrast, upon B‐cell differentiation the relative number of clonotypic B cells decreased. In conclusion, clonotypic B cells can be cultured and isolated in vitro using the CD40 system. Clonotypic B cells responded to CD40 triggering in a similar fashion as to non‐clonotypic normal B cells. However, the ability of clonotypic B cells to undergo in vitro activation and differentiation into Ig‐secreting cells is hampered.

Complex biallelic IGH rearrangements in IgM-expressing Z-138 cell line : Involvement of downstream immunoglobulin class switch recombination

Chromosomal translocations involving the immunoglobulin (Ig) receptor loci usually disrupt and silence these loci. On the basis of observations in follicular lymphoma (FL) with downstream Ig heavy chain (IGH) class switch recombination (CSR), we hypothesized that downstream CSR‐mediated chromosomal translocations would leave the V(D)J‐Cμ transcription unit intact, thereby still allowing IgM expression from the IGH allele involved in the translocation. To test this hypothesis, we analyzed biallelic IGH translocations in the IgM‐expressing cell line Z‐138 by interphase FISH, DNA fiber–FISH, long‐distance vectorette PCR, and DNA sequencing. One IGH allele was involved in a t(11;14), showing a break in the JH region that juxtaposed the Eμ enhancer and the 3′ Cα enhancers to the cyclin D1 gene. The other IGH allele contained a t(8;14) breakpoint involving the 3′ end of a Sγ region, whereas the reciprocal breakpoint at 8q24 was approximately 40 kb centromeric of MYC. Molecular analysis showed that this IGH allele harbored a normal V(D)J‐Cμ complex, which is responsible for IgM expression. These data show that chromosomal breakpoints such as the t(8;14) can occur in downstream IGH constant regions and do not necessarily interfere with Ig expression.

Heterogeneity in the Multiple Myeloma Tumor Clone

Multiple Myeloma (MM) is a plasma cell malignancy which is characterized by a very heterogeneous disease outcome. Heterogeneity in plasma cell characteristics, including morphology, maturation status, immunophenotype and genetic abnormalities partly account for the variable disease outcome. Although the plasma cell is the predominant cell type in MM, several studies have shown that less mature B cells, which are clonally related to the malignant plasma cells, are present in the bone marrow and peripheral blood of MM patients. The significance of these so-called myeloma clonotypic B cells in the disease process remains largely unknown. In this review the role of myeloma clonotypic B cells and myeloma tumor clone heterogeneity in relation to prognosis and clinical outcome are discussed.

MYC in diffuse large B-cell lymphoma: always the bad guy?

With immunochemotherapy, diffuse large B-cell lymphoma (DLBCL) can be cured in ∼ 60% of patients [1]. However, there is considerable variability in clinical course and prognosis of the disease. This clinical heterogeneity can increasingly be explained by underlying biological differences, such as the difference between germinal center type DLBCL (GCB-DLBCL) and non-GCB or activated B-cell type DLBCL (ABC-DLBCL). Within the generally more favorable GCB-type subgroup, rearrangements of MYC and BCL2 have been recognized as being associated with an unusually aggressive clinical course and therapy resistance [2]. In DLBCL, MYC aberrations are seen in ∼ 10% of all newly diagnosed cases, of which 20% are so-called “single-hit”, where only MYC is rearranged, and 60–80% are either double or triple hits, where MYC rearrangement occurs combined with BCL2 and/or BCL6 rearrangement. MYC translocations are found mostly in GCB-type DLBCL, either as a progression/transformation event in the case of preceding follicular lymphoma or as a de novo event [3]. MYC protein overexpression can, however, also be seen independent of gene alterations, suggesting that genes or pathways regulating MYC expression are affected. Combined overexpression of MYC and BCL2 protein (“double expressors”) also confers an unfavorable prognosis; this overexpression is not restricted to GCB-type DLBCL [4]. As recently addressed in a meta-analysis, several studies have looked at the effect of MYC rearrangement on the prognosis of DLBCL and, although most of these identified MYC as an unfavorable prognostic factor, some failed to show prognostic significance [5]. As yet, a robust prognostic analysis of the impact of single vs double or triple hit aberrations in DLBCL is lacking, mainly due to the fact that limited numbers of cases are present in each of the relevant subgroups. In this issue of Leukemia & Lymphoma the adverse prognostic significance of a single MYC aberration in DLBCL is challenged by Caponetti et al. [6]. In a retrospective analysis of 205 patients with DLBCL retrieved from the Nebraska Lymphoma Study Group Registry, the significance of MYC, BCL2 and BCL6 rearrangements and MYC copy number alterations was investigated with regard to overall and event-free survival. Caponetti et al. [6] report that event-free survival in patients with single MYC rearrangements was not inferior to that of the entire cohort of patients with DLBCL, whereas those with double or single hits did have a very poor outcome. Since the samples were collected over a long period of time, the question is how representative the outcome is for the overall population of DLBCL patients. In addition, only 22% of the patients in this study were treated with immunochemotherapy and the general long-term outcome of the patients was poor. Furthermore there also appears to be some imbalance between the groups, since a higher percentage of the single MYCue031 cases had received rituximab. The fact that some of the outcome analyses did not yield statistically significant results may also be related to the fact that the number of MYCue031/BCL2-/BCL6and double /triple hit cases was indeed relatively low. MYC is a central transcription factor that regulates 10–15% of all genes, including those involved in cell proliferation, differentiation and metabolism, but also those affecting apoptosis. MYC predominantly binds to active promoter regions of target genes and not to silent genes, thus activating or stimulating existing programs within a cell type [7]. Besides this “transcriptional amplifier” effect, MYC can also regulate more specific sets of genes. The latter may indeed explain the fact that it may have differential effects on cell proliferation across different lymphoma types associated with MYC translocation, such as Burkitt lymphoma and DLBCL. It has also been shown that overexpression of MYC induces apoptosis by triggering the BH3-only protein BIM, which is counteracted by BCL2 overexpression, thus explaining the poor outcome of concurrent MYC and BCL2 aberrations. Lymphoma cells may also circumvent the pro-apoptotic effects of MYC overexpression in several other ways, including MYC hotspot mutations that prevent BIM induction [8]. All the latter imply that focusing solely on gross genetic aberrations such as chromosomal translocations may in fact bias the definition of patient subgroups and their prognosis. A definitive answer to the question of whether single MYC aberrations are prognostically unfavorable should Leukemia & Lymphoma, November 2015; 56(11): 3003–3004