Genetic Abnormalities and Patterns of Antigenic Expression in Multiple Myeloma

Myelomatous plasma cells show a high heterogeneity both in their immunophenotypic characteristics as well as in their cytogenetic features. Thus far, no extensive studies have been carried out to explore whether such antigenic diversity is associated with specific genetic characteristics. We have investigated the relationship between the immunophenotypic profile at plasma cell and both their DNA ploidy status (evaluated by flow cytometry) and specific genetic features (ascertained by fluorescence in situ hybridization) in a large series of 915 patients with newly diagnosed multiple myeloma. The non-hyperdiploid multiple myeloma group (n = 454, 52%) was associated with a significantly higher frequency of positivity for CD28 and CD20 as well as a higher incidence of CD56⁻ and CD117⁻ cases (P < 0.001). Remarkably, 13q deletion and immunoglobulin heavy chain (IGH) gene rearrangements, which were significantly more common in non-hyperdiploid multiple myeloma, showed a strong association with CD117⁻ cases. IGH translocation to 11q13 was associated with reactivity for CD20 (P < 0.001), down-regulation of CD56 (P < 0.001), and lack of expression of CD117 (P = 0.001). By contrast, IGH translocations to other chromosome partners were almost exclusively found among CD20⁻ and CD117⁻ cases (P < 0.001). These results suggest that genetic categories in multiple myeloma exhibit particular immunophenotypic profiles which in turn are strongly associated with the DNA ploidy status.

The immunophenotypic antigenic expression profile of myelomatous plasma cells is highly heterogeneous and clearly different from that of normal plasma cells (1–3). In some multiple myeloma patients, usually <30%, plasma cells retain the expression of progenitor cell– (CD117 or c-kit; refs. 4, 5) or myeloid- (CD33; ref. 3) associated markers as well as B-lineage–associated antigens [CD19 (6, 7) and CD20 (8, 9)] or CD45 marker (10, 11). CD28, a molecule involved in cell-cell interactions, is expressed in approximately a quarter of patients (12, 13), whereas CD56, an adhesion molecule that facilitates the anchorage between plasma cells and bone marrow stromal structures, is present in around two thirds of multiple myeloma patients (3, 14–16). This antigenic diversity may not only be associated with different clinical features but also, and probably more importantly, it may reflect variable genetic changes in plasma cells. The existence of an association between the immunophenotype of blast cells and their underlying genetic abnormalities has already been observed in other hematological malignancies with well-defined chromosomal rearrangements, such as PML/RARα, within the acute myeloid leukemias (17) and both BCR/ABL and TEL/AML1 in lymphoblastic leukemias (18, 19). Multiple myeloma presents a marked karyotypic complexity, which has delayed the identification of abnormalities involved in the pathogenesis of the disease. Although multiple myeloma shows certain heterogeneity in its genetic portrait, it seems apparent the immunoglobulin heavy chain (IGH) translocations are an initiating event in about half of cases, probably through deregulation of cyclin D (20). Another common genetic feature of multiple myeloma is the deletion of the RB gene present in between one third and half of the patients by interphase fluorescent in situ hybridization (FISH; refs. 21, 22). Both abnormalities are associated with non-hyperdiploid karyotypes (23, 24). Information about the potential relationship between genetic abnormalities and immunophenotypic markers is currently limited to the association found between t(11;14)(q13;q32) and CD20 expression (25). In fact, to the best of our knowledge, no systematic study has been conducted to analyze potential associations between specific genetic abnormalities, including 14q32 translocations, deletions of 13q [del(13q)], DNA hyperdiploidy and non-hyperdiploidy, and unique patterns of antigen expression in multiple myeloma patients. In the present study, conducted in a series of 915 patients prospectively analyzed at diagnosis, we have found that non-hyperdiploid cases more frequently display CD20 and CD28 expression together with a lower reactivity for CD56 and CD117. In addition, these two latter antigens were also down-regulated in patients with t(11;14) whereas expression of CD20 was higher. In turn, IGH translocations to other chromosomal partners were almost exclusively found among CD20⁻ and CD117⁻ cases. Finally, 13q was more frequently deleted among CD117 (c-kit)–negative cases.

Patients. A total of 915 patients enrolled in the PETHEMA/GEM protocol (six alternating cycles of VBCMP/VBAD followed by autologous stem cell transplantation) were included in the study at diagnosis. The patient's characteristics at the time of diagnosis are shown in Table 1.

Immunophenotypic studies by flow cytometry. A total of 2 × 10⁶ freshly bone marrow cells were incubated with an appropriate panel of quadruple monoclonal antibody combinations: CD38/CD56/CD19/CD45, CD138/CD28/CD33/CD38, and CD20/CD117/CD138/CD38. The order of the monoclonal antibodies in these combinations correspond to the four fluorochromes used: FITC, phycoerythrin, pteridin chlorophyll protein-cyanin-5, and allophycocyanin. A negative control tube, only stained for CD38-pteridin chlorophyll protein-cyanin-5, was always included in the panel to establish a cutoff for positivity for FITC-, phycoerythrin, and allophycocyanin signals based on the level of the natural autofluorescence of plasma cells. Acquisition of information on 2 × 10⁴ stained cells corresponding to the whole bone marrow cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software program (BD Biosciences, San José, CA; Fig. 1A). If the percentage of bone marrow plasma cells was lower than 10% (286 cases, 31%), a highly sensitive second acquisition step, done through a “live-gate” drawn on a low/intermediate side scatter/CD38 strong-positive area, where plasma cells are located, was required (Fig. 1B). At least, 3 × 10³ plasma cells were recorded for analysis. Using the Paint-A-Gate-PRO software (BD Biosciences), plasma cells were specifically selected based on their high level of expression for CD38 and CD138 and their typical light scatter characteristics, forward side scatter and intermediate side scatter, after excluding other cellular components and debris (Fig. 1C and D). Evaluation of the antigenic profile of plasma cells was based on the following variables: (a) presence or absence of antigen expression, (b) pattern of antigen expression (homogeneous versus heterogeneous); (c) percentage of plasma cells with positive expression for each antigen; and (d) median amount of antigen expressed per cell within a given population of plasma cells as expressed by the median fluorescence intensity (arbitrary relative linear units scaled from 0 to 10⁴).

DNA ploidy measurements. In all 915 patients immunophenotypically analyzed, a double-staining procedure for nuclear DNA (with propidium iodide) and surface plasma cell antigens (with anti-CD38 and anti-CD138 monoclonal antibodies, provided both by Cytognos, SL, Salamanca, Spain) was used to specifically analyze the plasma cell DNA content—plasma cell DNA ploidy status (26). Measurements were done on a FACSCalibur flow cytometer (BD Biosciences) using the CellQUEST software after acquiring information on at least 2 × 10⁴ cells/sample. After excluding cell doublets in FL2-Area versus FL2-Width bivariate dot plot, plasma cells were clearly discriminated from normal residual bone marrow cells based on their CD38-CD138 strong-positive intensity using the Paint-A-Gate PRO program (BD Biosciences). For DNA ploidy analysis, DNA index was calculated as the ratio obtained between the modal channel of the G₀-G₁ of plasma cells and G₀-G₁ peak of the remaining normal residual cell populations present in the sample, using the ModFIT software program (Verity Software House, Topsham, ME), according to previously described criteria (27). DNA aneuploid cases were considered to be hypodiploid when the DNA index was ≤0.9, hyperdiploid when the DNA index ranged from 1.08 to 1.8, and tetraploid/near-tetraploid once the DNA index was >1.8. All remaining cases were being considered as DNA diploid.

Fluorescence in situ hybridization analysis. Interphase-FISH studies were carried out only in those samples with a bone marrow plasma cell infiltration by flow cytometry above 10%. The presence of del(13q) was evaluated in 370 of the 915 patients with a specific probe for the 13q14 (LSI 13-RB1 probe, Vysis, Inc., Downers Grove, IL). To detect IGH rearrangements, a specific split-apart probe for the IGH region (14q32) was used in 326 cases: LSI IGH Dual Color, break apart rearrangement probe (Vysis). Patients with IGH translocations were further analyzed for the 11q13 (CCND1) partner by means of the LSI IGH/CCND1, dual color, dual fusion translocation probe (Vysis). The interphase-FISH procedure used has been previously described in detail (28). A total of 500 interphase nuclei were analyzed using the scoring criteria recommended by the manufacturer.

Statistical analyses. Statistical analyses were done using the SPSS statistical software (SPSS Inc., Chicago, IL). Descriptive data were shown as median and range values. The χ² and Fisher exact tests were used to assess the statistical significance of associations found between categorical variables. For continuous variables, a nonparametric test (Wilcoxon test for matched-paired variables) was employed. P values below 0.05 were considered to be associated with statistical significance.

A high heterogeneity in cell surface antigen expression is observed in myelomatous plasma cells.Table 2 summarizes the immunophenotypic results obtained in the whole series of 915 patients at diagnosis. As shown, myelomatous plasma cells completely lose (negative expression) the pan-B cell marker CD19 in the majority of multiple myeloma patients (93%), and only partially or completely retained in 4% and 3% of the cases, respectively. The CD20 marker was present in 17% of all multiple myeloma cases, most of them (13%) showing expression in all plasma cell, whereas in 4% of cases, only present in a subset of the myelomatous plasma cell. CD28 was detected in 38% of cases, with either a homogeneous (33% of cases) or heterogeneous (5%) pattern of expression. It should be noted that in normal plasma cell, CD28 is always negative or only weakly expressed in a small subset of plasma cells (<15%). As shown in Table 2, four different patterns of expression were observed for CD45: (a) negative (73% of cases), (b) homogeneous weak positive (10% cases), (c) homogeneous bright (9%), and (d) heterogeneous expression (8%); the two latter patterns are similar to CD45 expression in normal plasma cell, whereas the former two patterns are not observed in normal conditions. Concerning CD56, a high percentage of patients (70%) showed a homogeneous positive expression on all plasma cell, another 9% had lower and heterogeneous pattern of expression ranging from CD56-negative to CD56-positive plasma cells, whereas in the remaining 21% of the cases, reactivity for CD56 was completely absent. Other antigens, such as CD33 and CD117, were present in 20% and 36% of the multiple myeloma cases, respectively, with either a homogeneous (12% and 30%, respectively) or heterogeneous (8% and 6%, respectively) pattern of expression (Table 2). For further statistical analyses, those cases with antigenic patterns such as (+^w), (+), or (−/+) were grouped as “positive (+) multiple myeloma” for all antigens, except for CD45 and CD56 in which only those cases with (+, −/+) and (+^w;+), respectively, were grouped into positive (+) multiple myeloma.

DNA ploidy status by flow cytometry. DNA plasma cell contents were evaluable by flow cytometry in 869 of the 915 patients in which immunophenotypic studies were done (Table 3). In 370 patients (43%), myelomatous plasma cells showed an hyperdiploid DNA content (DNA index range, 1.08-1.79). Tetraploidy or near-tetraploidy, defined by a DNA index of 2 ± 0.3, was found in 18 cases (2%). Only 9 patients (1%) had a hypodiploid DNA cell content (DNA index range, 0.69-0.9) whereas 400 (46%) were diploid cases. The remaining 72 cases (8%) were considered biclonal cases in which two populations of myelomatous plasma cells with different DNA contents were observed: in 19 cases (26%), the two populations of plasma cells showed DNA hyperdiploidy, whereas in the remaining 53 cases a diploid plasma cell population coexisted with an aneuploid one. In these 53 latter cases, the aneuploid clone corresponded to a DNA tetraploid clone in 27 cases (38%), a DNA hyperdiploid population in 25 (35%), and a hypodiploid clone in one case (1%). For all further analyses, patients were grouped according to the DNA ploidy status into two categories: (a) “hyperdiploid multiple myeloma” (n = 414 cases, 48%) including the hyperdiploid, and those biclonal multiple myeloma showing one or two different DNA hyperdiploid plasma cell populations; (b) all other cases were considered as “non-hyperdiploid multiple myeloma” (n = 455, 52%; see Table 3).

Del(13q) and 14q32 translocations by interphase fluorescence in situ hybridization: correlation with DNA ploidy. Del(13q) was detected by interphase FISH in 120 of the 370 cases analyzed (32%). Translocations involving the IGH gene were observed in 104 of 326 cases (32%), 48 of these cases (15%) with IGH/CCND1 gene rearrangements. A significant association between the presence of IGH translocations and 13q⁻ was found: 53% of patients with IGH gene rearrangements had del(13q) versus 23% of patients with normal IGH (P < 0.001). Once the cases with IGH translocations were grouped according to the presence or not of t(11;14), del(13q) was significantly more common among cases with IGH translocations different from t(11;14) than in the t(11;14) group (60% versus 40%, P < 0.0001). On correlating these chromosomal changes detected by FISH with the plasma cell DNA cell contents evaluated by flow cytometry, it was observed that multiple myeloma patients with DNA hyperdiploidy were associated with a significantly lower incidence of both del(13q) and IGH gene rearrangements, including both the t(11;14) and the no t(11;14) cases, as compared with non-hyperdiploid multiple myeloma [19% versus 45% (P < 0.001) and 10% versus 49% (P < 0.0001), respectively; Table 4].

Associations between the plasma cell DNA ploidy status and their patterns of antigenic expression. On correlating the DNA ploidy status with the immunophenotypic characteristics of myelomatous plasma cell, we observed that the non-hyperdiploid multiple myeloma group was associated with a significantly (P = 0.001) higher frequency of positivity for CD28 (61% versus 39%) and CD20 (72% versus 28%) as well as a higher incidence of CD56⁻ (65% versus 35%) and CD117⁻ (64% versus 36%), as compared with hyperdiploid multiple myeloma (Table 5). In contrast, reactivity for the CD33 antigen was more frequently observed in cases with DNA hyperdiploidy (63% versus 37%, P = 0.001). No statistically significant associations were observed for CD19 or CD45 antigens.

Associations between chromosomal abnormalities and patterns of antigenic expression on plasma cells. Interphase-FISH analysis also showed interesting correlations between specific chromosomal abnormalities and the pattern of antigen expression on plasma cells. Accordingly, del(13q) and IGH gene rearrangements were significantly more common in CD117-negative cases than in positive ones [37% versus 26% (P = 0.02) and 38% versus 19% (P = 0.0001), respectively]. The t(11;14) was associated with CD20 expression (P < 0.001), low reactivity for CD56 (P < 0.001), and lack of CD117 (P = 0.001; Table 6). In fact, t(11;14) was observed four times more frequently among the CD20 positive cases as compared with the negative ones (32% versus 8%, P < 0.001). The IGH translocations other than t(11,14) were almost exclusively associated with absence of expression of both CD20 (of 58 cases with other IGH translocations, 49 were CD20 negative) and CD117 (46 of 59 cases). For the remaining surface antigens analyzed, no association with recurrent chromosomal abnormalities studied was observed.

Our study represents the most well-documented observation in myeloma of a relationship between antigenic characteristics of plasma cell, DNA ploidy and genetic abnormalities. Many groups, including our own, have reported on the antigenic characteristics of myelomatous plasma cells (1, 6, 9, 13, 29–32). However, some of these studies were relatively old and based only on single or double antigen stainings without appropriate identification of the plasma cell clone, which is critical in neoplasias where the tumor cell infiltration is frequently low. The present report is the largest series of multiple myeloma patients in which the immunophenotypical characteristics of plasma cells analyzed by multiparametric flow cytometry are described. Our results show that the immunophenotypic profile of myelomatous plasma cells is variable not only between different patients but also within each patient. These findings may either suggest that the neoplastic clone is able to undergo a certain degree of differentiation (10, 33) or that intrinsic clonal evolution frequently occurs in multiple myeloma (34). Thus, only a minority of multiple myeloma patients retain B-lineage associated antigens, one third expresses the CD28 costimulatory receptor, and intriguingly, a similar proportion of patients express the stem cell factor receptor (c-kit antigen, CD117). CD56, a molecule involved in anchorage of myelomatous plasma cells to bone marrow stroma, is absent in approximately one third of the cases. The possibility that this antigenic heterogeneity could result from the occurrence of different genetic portraits sounds attractive and will be discussed later on.

Analysis of the plasma cell DNA contents by flow cytometry allows an easy discrimination between hyperdiploid and non-hyperdiploid multiple myeloma (27, 35, 36). As far as hypodiploid cases are concerned, their frequency by flow cytometry is usually underestimated because the DNA content of the largest chromosomes represents <4% of the total DNA and, therefore, single or balanced chromosomal losses usually go undetected by flow cytometry. Therefore, some of our DNA diploid cases may actually correspond to hypodiploid multiple myeloma by conventional cytogenetics; for this reason, both groups of patients were analyzed together as non-hyperdiploid multiple myeloma. Conventional cytogenetic studies have shown that non-hyperdiploid multiple myeloma are associated with a higher frequency of IGH translocations and del(13q) (23, 24). Here, using flow cytometry, we confirm and extend the observations about the occurrence of associations between ploidy and specific chromosomal changes. Accordingly, whereas del(13q) occurred in 60% of non-hyperdiploid cases, it was observed in only 27% of patients with a hyperdiploid plasma cell DNA content. Similarly, 14q32 translocations, including both the t(11;14) and no t(11;14), were detected in half of non-hyperdiploid patients but only in a minority (10%) of the hyperdiploid multiple myeloma. Our results also show an association between 13q⁻ and IGH gene rearrangements, confirming previous reports which suggested that there was a correlation between del(13q) and 14q32 translocations not involving 11q13 (37).

The potential correlation between genetic lesions and the antigenic profile of tumor cells is an area of increasing interest. In this large series of 915 untreated multiple myeloma patients, we have shown for the first time that the non-hyperdiploid multiple myeloma display unique phenotypic profiles. Thus, the association found between non-hyperdiploid multiple myeloma and an increased expression of both CD20 and CD28 in the absence of reactivity for CD56⁻ and CD117⁻ contributes to the identification of a distinct subgroup of multiple myeloma patients. As far as specific genetic/phenotypic associations are concerned, our group and others have shown that blast cells from adult precursor B-ALL carrying BCR/ABL rearrangements (18), as well as children with TEL/AML1⁺ precursor B-ALL and PML/RARα⁺ AML (17) patients, display unique aberrant phenotypes. In multiple myeloma, Robillard et al. (25) have reported on the existence of a strong association between t(11;14) and CD20 expression: 10 of 12 patients (83%) with CD20 expression had t(11;14). Here, in a much larger series of patients we have confirmed such an association although the proportion of positive cases was not as high. In addition, our data show that t(11;14) is also associated with down-regulation of CD56 and lack of reactivity for the CD117 antigen, which altogether delineate a unique antigenic profile for this genetic group of multiple myeloma patients. Of note, the lack of c-kit (CD117) was not only associated with t(11;14) and non-hyperdiploid DNA content but also with del(13q) and IGH translocations other than t(11;14). Further studies on the functional role of c-kit antigen in multiple myeloma could be of interest. Regarding the prognostic implications of these markers, it should be noted that t(11;14), which is a favorable genetic marker (37), was associated with two antigens, CD56 and CD117, also related to prolonged survival.⁹

Further studies on the functional role of c-kit antigen in multiple myeloma, as well as the prognostic significance of these finding, would be of interest. Overall, the present results indicate that the aforementioned relationship between flow cytometry immunophenotyping, DNA ploidy, and genetic profiles offers a new insight into the biological characterization of the different multiple myeloma subtypes.

We thank Mark Anderson for his help with the English language, and Gloria Ercilla, Amador Crego, and Ma. Angeles Hernández for excellent technical assistance in flow cytometry and FISH processing.