Annals of Clinical & Laboratory Science 37:34-38 (2007)
© 2007 Association of Clinical Scientists
Morphoproteomic Expression of H-ras (p21ras) Correlates with Serum Monoclonal Immunoglobulin Reduction in Multiple Myeloma Patients Following Pamidronate Treatment
Ping L. Zhang1,3,
Albert T. Quiery, Jr.2,
Thomas M. Blasick3 and
Robert E. Brown1
1 Division of Laboratory Medicine, 2 Department of Adult Hematology & Oncology, and 3 Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania
Address correspondence to: Ping L. Zhang, M.D., Ph.D., Division of Laboratory Medicine, Geisinger Medical Center, 100 North Academy Ave., Danville, PA 17822, USA; tel 570 271 6333; fax 570 271 6105; e-mail plzhang{at}geisinger.edu.
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Abstract
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Bisphosphonates have been used to treat lytic lesions of multiple myeloma because of their inhibitory effects on osteoclasts. However, their effects on myeloma cells, per se, are not known to be correlated with specific markers. The goal of this study was to assess molecular concomitants of myeloma that might serve as markers for predicting the pharmacologic impact of bisphosphonates on malignant plasma cells. We tested the correlation of serum monoclonal immunoglobulin (Ig) level (IgG and IgA classes) with therapies utilizing two aminobisphosphonates, pamidronate (Aredia) and/or zoledronate (Zometa), in 19 patients with multiple myeloma. Myeloma cells from bone marrow biopsies were immunohistochemically stained for H-ras (p21 ras), N-ras, and the 
subunit common to farnesyl and geranylgeranyl transferase (FT/GGT ). Elevated expression level of H-ras in myeloma cells, rather than N-ras or FT/GGT, was significantly associated with a decrease of serum monoclonal Ig level following pamidronate treatment. The data suggest that pamidronate may have a direct inhibitory effect on the proliferation of myeloma cells, thus causing reduction in serum monoclonal Ig level. H-ras expression in myeloma cells may prove to be valuable in predicting the therapeutic effects of pamidronate.
Keywords: multiple myeloma, bisphosphonates, pamidronate, zoledronate, H-ras, serum monoclonal Ig
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Introduction
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Bisphosphonates, analogues of farnesyl pyrophosphate, specifically inhibit farnesyl pyrophosphate synthase in the cholesterol biosynthesis pathway [1]. Inhibition of this enzyme by bisphosphonates leads to decreased production of isoprenoid intermediates like farnesyl that are needed for the prenylation of ras via farnesyl transferase (FT) [2]. Among the forms of ras proteins (H-ras, N-ras, K-ras), K-ras has been found to be resistant to FT inhibitors [3]. The basis for this resistance may be related to the high affinity of K-ras for FT and its capacity to be prenylated by the related enzyme geranylgeranyl transferase (GGT) in the presence of FT. Several bisphosphonates have been approved by the Food and Drug Administration (FDA) to treat malignant lytic lesions in bone [4,5].
Multiple myeloma is one of the most common hematologic neoplasms in the United States [6]. Multiple myeloma is a B-cell malignancy of plasma cells that generally produces a monoclonal immunoglobulin protein that serves as an index of the overall burden of myeloma cells. Because bisphosphonates inhibit osteoclastic activity and thus bone resorption, bisphosphonates have been used to treat myeloma patients with destructive bone lesions [5,7]. Currently, zoledronate (Zometa) is used more commonly than pamidronate (Aredia) for 3 reasons: First, zoledronate is more potent than pamidronate [5]. Second, zoledronate has proven effectiveness in patients with all types of bone lesions from osteolytic to osteoblastic [8]. Third, only 15 min is required for zoledronate infusion, while pamidronate infusion requires 2–4 hr [7].
Although pamidronate and zoledronate have become the standard of care for the treatment of lytic bone lesions associated with multiple myeloma, knowledge regarding direct inhibitory effects of the bisphosphonates on the malignant cells, per se, is limited to several case reports [9–11]. Therefore, it is possible that a reduction of serum total monoclonal immunoglobulin (Ig) level following pamidronate or zoledronate therapy may reflect the inhibitory effects of the drugs on the malignant myeloma cells. To date, it has not been established whether or not there is correlation between such effects of the bisphosphonates and the expression of presumed target markers such as the various types of ras and the farnesylation pathway.
In this investigation, we studied the serum total monoclonal Ig level in patients with myeloma as an index to evaluate the inhibitory effects of pamidronate and zoledronate. We used immunohistochemical staining of myeloma cells for H-ras, N-ras, and the -subunit common to farnesyl and geranylgeranyl transferase (FT/GGT) to assess their expression levels vis-à-vis the efficacy of pamidronate or zoledronate. We also investigated the impact of pamidronate and zoledronate therapy on serum total monoclonal Ig level in myeloma patients.
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Materials and Methods
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From April 1996 to June 2003, a total of 29 patients with multiple myeloma, seen at Geisinger Medical Center, had archival paraffin-embedded tissue blocks that were available for immunohistochemical studies. Nineteen of the myeloma patients were treated with pamidronate, administered iv at 90 mg per 2 hr infusion every 4 wk for up to 2 yr. Seven of the myeloma patients were shifted to zoledronate (Zometa) treatment when this drug became available. Zoledronate was administered iv at 4 mg per 15 min infusion every 4 wk for up to 2 yr. Serum monoclonal immunoglobulin IgG or IgA levels in the myeloma patients were obtained through the SunQuest laboratory information system of Geisinger Medical Center.
Tissue blocks of bone marrow biopsies from the patients with myeloma were collected in the Division of Laboratory Medicine. Primary antibodies against H-ras, N-ras, and the -subunit common to FT/GGT were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Blocks were routinely fixed in formalin and embedded in paraffin. For each block, a 5-µm section was dewaxed in xylene and rehydrated with graded concentrations of ethanol to water. Slides were stained immunohistochemically for the various markers using a DAKO autostainer (model E172566). The intensity of stained tumor cells was scored microscopically, ranging from 0 to 3+ (0, no staining; 1+, weak fine particular staining; 2+, moderate granular staining; 3+, strong and large granular staining).
The percent changes of serum monoclonal Ig level during 6 mo of therapy with pamidronate or zoledronate were compared in the 2 treatment groups. The correlations between the percent of decline of serum monoclonal Ig level and the expression score of H-ras, N-ras, and FT following pamidronate treatment were calculated using StatView software. A p value <0.05 was deemed statistically significant.
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Results
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Fig. 1
shows examples of 3+ staining of H-ras, N-ras, and FT/GGT in myeloma cells. Expression levels of N-ras, H-ras, and FT/GGT in the 29 myeloma cases are listed in Table 1. Approximately one-third of the cases had >1+ positivity for H-ras, whereas positivity >1+ for N-ras was seen in one-half of the cases and positivity >1+ for FT/GGT was seen in three-fourths of the cases.
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Table 1. Immunohistochemical expression scores of protein markers in myeloma cells from 29 patients with multiple myeloma.
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In the 19 patients with myeloma who received pamidronate, H-ras expression was significantly associated with reduction of serum monoclonal Ig level during pamidronate treatment from months 2 to 4 (Table 2). The association between reduction in serum monoclonal Ig level and H-ras expression was not significant at months 1, 5, or 6 during pamidronate treatment. There were no significant correlations between the expressions of N-ras or FT/GGT and changes of serum monoclonal Ig level during 6 months of pamidronate treatment.
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Table 2. Correlation (r value) between immunohistochemical expression of 3 protein markers and serum monoclonal immunoglobulin (Ig) changes during 6 months of pamidronate in 19 patients with multiple myeloma.
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In the 7 myeloma patients who received sequential treatments with pamidronate followed by zolendronate, the average age was 70 ± 3 yr. Their hematologic indices included hematocrit, 30.4 ± 0.9 %; hemoglobin, 10 ± 0.3 g/dl; white blood cells, 5,200 ± 500/µl; mean corpuscular volume; 91.3 ± 2.8 µm3; and platelets, 166,200 ± 22,500/µl. Table 3 shows that the 7 patients had greater reduction of serum monoclonal Ig level during pamidronate therapy, compared to that during zoledronate therapy, after 3–6 month treatment periods with the respective drugs. In these patients, there was 40% reduction of mean serum monoclonal Ig level after 6 months of pamidronate treatment, whereas after zoledronate treatmentfor6months, the mean serum monoclonal Ig level increased 54%, rather than diminished.
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Table 3. Percent change in serum monoclonal immunoglobulin (Ig) level in response to pamidronate (P) versus zolendronate (Z) during 6 months of tandem therapy in 7 patients with multiple myeloma.
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Discussion
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Pamidronate is an inhibitor of farnesyl diphosphate synthase, which can suppress ras farnesylation related to G-protein coupling and bone resorption. This inhibitor has been used to treat lytic lesions resulting from metastatic breast cancer and multiple myeloma [12,13]. In a previous study, we found that the in vitro effects of pamidronate did not correlate with protein expression of the farnesylation pathway markers, FT/GGT, in breast cancer cells [14]. Among the forms of ras proteins (H-ras, N-ras, K-ras4A, and K-ras4B), K-ras has been found to be resistant to inhibitors of farnesyl diphosphate synthase [3]. Our previous data on breast carcinoma cell lines also showed that antitumor effects of pamidronate appear to be associated with relative expression levels of H-ras and N-ras [14]. In the current study, we found that intensity of H-ras (p21ras) expression was correlated with reduction in the mean serum monoclonal Ig level following pamidronate therapy in 19 patients with myeloma. The data suggest that pamidronate blocked an H-ras-related growth pathway that resulted in inhibition of myeloma cell production of serum monoclonal Ig. N-ras and FT expression did not show any correlation with the monoclonal Ig level. This finding raises the question whether H-ras expression might be used to predict the treatment effects of pamidronate.
Pamidronate and zoledronate have both been approved to treat osteolytic lesion in multiple myeloma patients [4,5]. They may also exert direct anti-tumor activity in patients with multiple myeloma [9–11]. Our data showed a significant reduction of serum monoclonal Ig level during pamidronate treatment, supporting the direct inhibition of pamidronate on myeloma cells. However, when patients were shifted to zoledronate treatment after initial treatment with pamidronate, the serum monoclonal Ig changes following zoledronate treatment were significantly less than following pamidronate treatment. The most likely explanation is that the initial use of pamidronate led to the development of drug resistance (possible via clonal evolution or induction of its target enzyme) to zoledronate in patients with multiple myeloma. However the mechanism for developing the drug resistance is unclear. A second possibility is that pamidronate may have a greater antitumor effect on malignant plasma cells compared to that of zoledronate. This possibility seems less likely, since zoledronate is the bisphosphonate reported to be more potent [5]. Since our study involved a limited number of patients, additional studies are needed to compare the changes in serum monoclonal Ig level following pamidronate and zoledronate treatments in patients with multiple myeloma.
In summary, our study showed expression of H-ras, N-ras, and FT/GGT in myeloma cells using an immunohistochemical method. High level of H-ras expression was associated with reduction in serum monoclonal Ig level, implying a role of H-ras expression in predicting the effects of bisphosphonate treatment. Our data suggest that pamidronate may have a direct inhibitory effect on the proliferation of myeloma cells, thereby causing reduction of the serum monoclonal Ig level.
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Acknowledgments
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The study was supported by an intramural grant of Geisinger Health System. The authors thank Ms. Sharon Stroh for excellent secretarial support.
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Abstract
Introduction
