Expression of the Lung Resistance Protein Predicts Poor Outcome in Patients with Multiple Myeloma¹

Chemotherapy is the mainstay of treatment for MM,³ but resistance to anticancer drugs remains a major problem in the clinical management of MM patients. MDR is one important type of drug resistance that is clinically relevant in several solid tumors and leukemias (1). Different mechanisms can contribute to MDR, and some of them have already been studied in MM. MDR1/P-gp expression occurs with various frequencies in MM and is associated with clinical drug resistance to anthracyclines and Vinca alkaloids (1). Corresponding clinical trials to overcome this resistance by combining chemotherapy with resistance-modifiers have been performed (2, 3, 4). These studies suggested that, at least in some patients, modulation of P-gp did occur and was associated with improved outcome. However, the overall response rates were low and of short duration in these studies. MRP, another important factor involved in MDR, is also expressed in MM (5, 6), but its clinical relevance remains to be determined. Recent evidence suggests that alterations in apoptosis are involved in the drug resistance of MM (7, 8). Fas-mediated apoptosis may be important in MM patients (7), and MM patients with p53 deletions had shorter overall survival than those without such a deletion (8).

LRP is a newly described protein related to MDR. It was first detected in a non-P-gp multidrug-resistant lung cancer cell line (9). LRP is the human major vault protein (10). Vaults are complex ribonucleoprotein particles, which, in addition to the major vault protein, also contain several minor vault proteins and a small RNA. Vaults are located mainly in the cytoplasm but a small fraction is also associated with the nuclear membrane. They are believed to mediate intracellular and, in particular, nucleocytoplasmic transport (11). LRP expression of tumor cell lines is associated with resistance to doxorubicin, vincristine, carboplatin, cisplatin, and, of particular interest for MM, melphalan (12). LRP is physiologically overexpressed in colon tissue, lung tissue, renal proximal tubules, adrenal cortex, and macrophages; but its physiological function remains to be evaluated (13).

To determine whether LRP is of any predictive and/or prognostic significance in MM, we have studied LRP expression in plasma cells of MM patients and its association with both response to chemotherapy and survival of the patients.

Seventy-two previously untreated patients (47 females, 25 males) with MM were studied after having obtained informed consent according to institutional guidelines. Forty-two of these patients had been included in a previous study on the clinical significance of p53 deletions (8). The median age of the patients was 64 (range, 33–87) years. Sixty-one patients received induction chemotherapy. Melphalan-based regimens (melphalan/prednisone or VMCP) were administered in 53 patients. Four patients were treated with VAD, and four patients received VAD followed by high-dose melphalan. Nine patients were not treated with chemotherapy because of an early asymptomatic stage of the disease, and two patients died before chemotherapy. Fifty-eight patients were evaluable for response. Three patients were not evaluable for response because no follow-up data were available after the first two courses of chemotherapy. Response to induction chemotherapy was assessed according to standard criteria (14). For an objective response, a sustained decrease of the paraprotein to less than 50% of the pretreatment level, normalization of a preexisting hypercalcemia, and no increase in the number and/or size of lytic bone lesions were required.

Mononuclear cells were isolated from bone marrow aspirates by Ficoll-Hypaque (Sigma Inc., St. Louis, MO) gradient centrifugation. Cells were washed twice with PBS, treated with Carnoy’s fixative (methanol/glacial acetic acid, 3:1), and stored at −80°C.

Smears of cell lines and myeloma specimens were prepared, air-dried, and fixed in cold acetone (−20°C, 10 min). The myeloma specimens had been prefixed with Carnoy’s fixative, but this prefixation had no impact on the staining results. After two wash steps followed by a 20-min incubation with normal goat serum (Dako, Glostrup, Denmark; diluted 1:20), cells were incubated for 2 h with the monoclonal antibody LRP-56 (Alexis, Läufelfingen, Switzerland; dilution 1:25). Binding of the primary antibody was detected by the avidin-biotin-peroxidase method. Bound peroxidase was developed with 3-amino-9-ethylcarbazole (Sigma Inc., St. Louis, MO) and 0.1% H₂O₂ in acetate buffer (pH 5.2). The slides were counterstained with Mayer’s Hämalaun and mounted with Aquatex (Merck, Darmstadt, Germany). All of the washes were performed in PBS.

The small cell lung cancer cell line SW1573 and its drug-resistant variant SW1573/2R120 (provided by Dr. R. J. Scheper, Free University Hospital, Amsterdam) were used as negative and positive controls for LRP expression, respectively. In addition, negative controls without the LRP-56 antibody were performed for each sample. In some cases, additional controls with an irrelevant isotype-specific antibody (IgG2b) were done. There was no difference in staining between the irrelevant isotype-specific antibody and the negative control without any primary antibody.

Staining of at least 200 plasma cells was evaluated independently by two investigators (M. F., G. P.) who had no previous knowledge of the clinical data of the patients. LRP expression was scored according to the staining intensity (no staining, −; low intensity, +; intermediate intensity, ++; or high intensity, +++) and to the percentage (0%, <10%, or ≥10%) of staining plasma cells.

FISH studies with a DNA probe specific for the p53 locus at 17p13 were performed to detect deletions of p53 as described previously (8).

Associations between clinical as well as laboratory parameters and LRP were assessed by either χ² test, Fisher‘s exact test, or Kruskal-Wallis test. Survival probabilities were calculated with the product limit method according to Kaplan-Meier (15). Overall survival time was defined as the period between the time of diagnosis and the time of death. Surviving patients were censored at the time of the last follow-up. Differences between survival curves were analyzed by means of the log-rank test.

LRP expression of previously untreated patients was immunocytochemically determined by means of the monoclonal antibody LRP-56. LRP staining was detected as characteristic granular cytoplasmic staining. LRP expression was scored according to both the percentage and the intensity of staining plasma cells (Table 1). According to the percentage of staining plasma cells, 8 (11%) samples had no LRP staining cells, 20 (28%) samples contained <10% staining cells and 44 (61%) showed ≥10% staining plasma cells. The percentage of staining plasma cells ranged from 0 to 90% (Fig. 1 B). The intensity of LRP staining was low, intermediate, and high in 11 (15%), 30 (42%), and 23 (32%) samples, respectively. In all of the samples with a high staining intensity, ≥10% staining plasma cells were observed. For additional analyses, samples were divided into positive (≥10% staining plasma cells) and negative (<10% staining plasma cells). Using this classification, we found that LRP was positive in 44 (61%) of 72 bone marrow samples at diagnosis.

Next, we addressed the question whether or not LRP expression correlated with clinical or laboratory parameters. The major clinical and laboratory findings of the patients are summarized in Table 2. There was no significant association between LRP expression and age, sex, type of the paraprotein, serum creatinine, stage, β₂-microglobulin, LDH, or CRP (Table 2). However, LRP expression was more frequently observed in patients with a p53 deletion detected by means of FISH than in those without a deletion of p53. Deletion of p53 was seen in 16 (22%) of 72 patients. LRP was positive in 88% of patients with a p53 deletion but in only 54% of patients without a p53 deletion (P = 0.01; Table 2).

Sixty-one patients received induction chemotherapy. The treatment protocols were equally distributed among LRP-negative and LRP-positive patients. Fifty-eight patients were evaluable for response to induction chemotherapy. The overall response rate of all of the evaluable patients was 67%. The response rate was 87% for patients without LRP expression but only 54% for patients with LRP expression (P = 0.01; Table 3). The response rate decreased with increasing percentages of LRP-positive plasma cells. The response rates were 87% for patients with <10% staining plasma cells, 62% for patients with 10–29% staining plasma cells, and 43% for patients with 30–100% staining plasma cells (Fig. 1,A). This difference was also statistically significant (P = 0.02). Responders to chemotherapy had significantly lower percentages of LRP-positive plasma cells than nonresponders (P = 0.0002; Fig. 1,B). With the exception of p53 deletions, there was no other standard prognostic variable sharing any correlation with response (Table 3).

Overall survival was estimated according to Kaplan-Meier in 72 patients. Twenty-seven patients died (7 LRP-negative patients, 20 LRP-positive patients). Overall survival was shorter in patients with LRP expression (Fig. 2). At a median follow-up of 29 months, median overall survival was 33 months for LRP-positive patients and was not reached for LRP-negative patients (P = 0.04). This difference in survival was also observed in the group of patients treated with chemotherapy (n = 61). In this group of treated patients, median overall survival was 33 months for LRP-positive patients and was not reached for LRP-negative patients (Fig. 3). Similar results were observed when patients treated with high-dose melphalan (n = 4) were excluded from the analysis (data not shown).

In our study, we have demonstrated the clinical significance of LRP in MM on an unselected patient population. LRP positivity—defined as ≥10% staining plasma cells—was observed in 61% of MM patients at diagnosis. LRP expression was associated with poor response to induction chemotherapy and shorter overall survival of MM patients. Similar results have recently been reported by Raaijmakers et al. (16), who, using the same cutoff level as we did, found LRP expression as a predictive and prognostic factor with regard to chemotherapy response, event-free survival, and overall survival in patients treated with conventional-dose melphalan (n = 38) but not in patients receiving high-dose melphalan (n = 32). In that study (16), the independent prognostic significance was shown in multivariate analyses that included age, plasma cell labeling index, β₂-microglobulin, and LDH. A similar predictive and prognostic value of LRP expression has previously been reported for acute myeloid leukemia (17, 18, 19), acute lymphoblastic leukemia (20), and advanced ovarian cancer (21).

A high plasma cell labeling index and an elevated lactate dehydrogenase level have been described as predictors of poor response to chemotherapy and shorter survival of the patients (22). Other prognostic factors in MM include β₂-microglobulin and soluble interleukin-6 receptor (22). P-gp is expressed at very low levels and has no predictive and prognostic value in untreated MM (23). However, P-gp is highly expressed in MM refractory to VAD treatment (23).

LRP expression correlated with deletion of p53 (Table 2). This deletion was associated with poor response to chemotherapy (Table 3) and shorter survival (data not shown), which is consistent with a previous report (8). The correlation between LRP and p53 deletion raises the possibility that p53 may be involved in the regulation of LRP expression. A potential impact of p53 on the expression of drug resistance factors has previously been shown for MDR1 and MRP (24, 25). In the case of MDR1, mutant p53 has been shown to specifically stimulate the MDR1 promoter in vitro, whereas wild-type p53 represses its activity (24). In the case of MRP, wild-type p53 acts as a negative regulator of MRP transcription, and a loss of wild-type p53 function contributes to an up-regulation of the MRP gene (25). Thus, additional studies on the possible involvement of p53 in the regulation of LRP gene expression are warranted. Other potential regulatory factors for LRP expression include tumor necrosis factor-α (26) and estradiol (27). When colon carcinoma cell lines were transfected with a vector containing tumor necrosis factor-α cDNA, reduced levels of LRP RNA or protein and increased MRP RNA or protein were observed (26). Previously, tumor necrosis factor-α treatment was shown to suppress P-gp expression and to sensitize these cells to anticancer drugs (28, 29). In the breast cancer cell line MCF-7, estradiol treatment increased the amount of LRP present in the nuclear extract and LRP coimmunoprecipitated with the estrogen receptor (27). These results suggest that the interaction between vaults and estrogen receptor is probably related to the intracellular transport of the receptor molecule.

It has been speculated that vaults mediate the bidirectional transport of molecules between cytoplasm and nucleus (30). Vaults may also be involved in the transport of anticancer drugs out of the cell or into cytoplasmatic vesicles (10). The involvement of vaults in the transport of anticancer drugs is supported by the recent findings that vault synthesis is directly linked to drug resistance of cells (31). Nevertheless, it remains to be clarified, e.g., in antisense experiments that specifically block LRP expression, whether LRP or vaults are directly involved in drug resistance.

In conclusion, LRP expression is a predictive factor for poor response to induction chemotherapy in MM and a prognostic factor with regard to survival of the patients. From a clinical point of view, high-dose melphalan, which was shown to be superior to conventional-dose chemotherapy in MM (32, 33), might, thus, be particularly beneficial for patients with LRP-expressing myeloma cells.