Despite of recent advances [1, 2], plasma cell myeloma (PCM) remains an incurable disease and new approaches that induce long-term tumor regression and improve disease outcome are needed. Immunotherapy that targets tumor-associated antigens (TAAs) has been shown to be an effective treatment approach in cancer therapy, particularly for diseases re-emerging after therapy, and a number of new agents have been developed. Brentuximab vedotin, a monoclonal antibody (mAb)-monomethyauristatin E conjugate that targets CD30, was recently approved by the Federal Drug Administration for use in patients with refractory Hodgkin lymphoma and anaplastic large-cell lymphoma . SGN-70, a humanized monoclonal antibody that specifically targets CD70, has been shown to exhibit potent anti-myeloma activity in vitro and significantly prolonged the survival of tumor-bearing mice in vivo [4, 5]. Humanized anti-CD70 antibody conjugated with the tubulin inhibitor auristatin induced complete regression of renal cell carcinoma in xenografted mouse model . CXCR4 (CD184), a chemokine receptor, and its ligand, stromal cell-derived factor-1 (SDF-1/CXCL12), serves as the key factor for stem cell and immune cell trafficking and plays a critical role in stem cell mobilization, human immunodeficiency virus infection, autoimmune diseases, cancer, and tissue regeneration . Interruption of the CXCR4-CXCL12 signaling can disrupt the adhesive tumor–stromal interaction that confers survival and drug-resistance signals, and therefore, makes the tumor cells accessible to conventional chemotherapy. CXCR4 antagonists are now in Phase I/II clinical studies for acute leukemia and chronic lymphocytic leukemia [8, 9]. AMD 3100, a specific small-molecule antagonist of CXCL12 binding to CXCR4, has been shown to be effective in inhibiting PCM cell survival and proliferation in vitro [10, 11]. Feng et al. found that myeloma stem cells expressed CD184, and obtained growth support from bone marrow microenvironment, at least partially via the CXCR4 signaling pathway. Kim et al.  found that AMD3100 targeting CD184 inhibited the survival and proliferation of myeloma cells. CD44 is a member of cell-surface proteoglycan family, and ligand binding through CD44 induces internalization and intracellular drug release . CD44v6 is found frequently expressed in multiple myeloma and associated with deletion of chromosome 13q. Bivatuzumab mertansine is a novel cytotoxic immunoconjugate specifically targeting the CD44 splice variant CD44v6 . Integrin-a4 (CD49d) is a major molecule serving as an anchor mediating physical interactions of PCM cells with the extracellular matrix (ECM), as well as cellular microenvironmental elements . Natalizumab, a monoclonal antibody against CD49d, is highly effective in multiple sclerosis , and has recently been shown to block tumor cell adhesion and chemosensitize PCM cells to bortezomib in vitro . Endoglin (CD105) expressed by endothelial cells, has been speculated to play a role in tumor-related angiogenesis and neovascularization . A phase 1 clinical trial with anti-CD105 (c-SN6j; also known as TRC105) is ongoing in patients with advanced or metastatic solid tumors .
In this study, we assessed 101 cases of PCM for expression of CD30, CD44, CD49d, CD70, CD105, and CD184 by multicolor flow cytometry immunophenotypic analysis. The goal of this study is to provide preclinical data that will be helpful for design of possible novel targeted therapies for patients with PCM.
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- MATERIALS AND METHODS
- LITERATURE CITED
In this study, we used multicolor flow cytometry immunophenotypic analysis to assess PCM cases for expression of some recently described biomarkers that have exhibited promise in targeted cancer therapy, including CD30, CD44, CD49d, CD70, CD105, and CD184. The advantage of flow cytometry is that the technology allows us to precisely assess surface marker expression in a large number of BM neoplastic plasma cells.
We identified uniform and bright CD49d expression in all cases of PCM analyzed, in keeping with results reported by Kraj et al. . This finding supports the concept that the interaction between CD49d and the BM microenvironment may be a requisite for proliferation of PCM cells., These results also support the idea that targeting CD49d, using agents such as Natalizumab, may inhibit PCM cell growth by disrupting the adhesion of PCM cells to non-cellular and cellular components of the microenvironment . We also show in this study that CD184, another molecule involved in cross-talk between PCM and BM stromal cells, is frequently expressed in PCM cases, in over 90% of cases. CD184 expression, however, is much more heterogeneous in a given case. The variation in CD184 expression did not correlate with disease status, but likely reflect individual case variability in the expressions of myeloma anchor proteins. Interestingly, we did observe a significantly higher CD184 expression in CD117+ PCM cases (see discussion below).
CD44, another adhesion molecule, was found expressed in approximately 75% of PCM cases and significantly higher in patients with persistent/recurrent disease. Similarly, Kraj et al.  reported a heterogeneous CD44 expression in PCM in contrast with a higher and more uniform expression of CD44 in plasma cell leukemia. CD44 serves as a cell surface receptor for extracellular matrix components including hyaluronan, and mediates interactions of PCM cells and BM stroma. Ohwada et al. showed that CD44 engagement by an anti-CD44 mAb led to phosphorylation and degradation of IkappaB-alpha, and contributed to myeloma cell resistance to Dexamethasone . Our findings suggest that CD44 expression might be upregulated via therapy or as a manifestation of disease progression or, cases with a higher CD44 expression might be more resistant to dexamethasone treatment. Similar to CD184, we observed a significantly higher expression of CD44 in CD117+ PCM cases. A recent study showed that CD117 expression in PCM might act as an additional anchor molecule, resulting in alterations of both BM myeloid and lymphoid maturation, and contributing to the greater maintenance of the homeostatic role of residual normal plasma cells and a more limited spread of (mono)clonal plasma cells . However, based on current understanding, we cannot explain the underlying biological connection between the higher expressions of CD44 and CD184 and CD117 expression in myeloma cells. We did not observe an association of CD44 and CD184 with CD28 and CD56; nor with conventional cytogenetics or FISH results in PCM. Liebisch et al. observed that CD44v6 expression in myeloma cells correlated with del(13)q cytogenetic abnormalities. In this study, we did not find any correlation with the expression of standard form of CD44 (P = 0.495).
In this study, CD30, CD70, and CD105 were expressed infrequently in PCM cells. In physiologic states, CD30 expression is restricted in subpopulations of activated B- and T-cells . By immunohistochemistry (IHC), CD30 immunostaining is often observed in a variable number of normal plasma cells at a low level; however, CD30 was found much less often expressed in neoplastic plasma cells by IHC . Our results using flow cytometry are in accord with IHC results and suggest that neoplastic plasma cells might lose their capability to express CD30 as one of the normal functions. CD105 (endoglin), is predominantly expressed in angiogenic endothelial cells and is upregulated by hypoxia. Recently, we found that CD105 was highly expressed in myeloblasts in certain subtypes of acute myeloid leukemia (unpublished data); and in normal bone marrow cells, CD105 is expressed in immature erythroid precursors. Our study showed that CD105 expression was nearly absent in PCM cells. Interestingly, Tsirakis et al. reported increased soluble (serum) CD105 (sCD105) in PCM patients and the levels were correlated positively with advanced stage of disease of PCM . Our findings suggest that increased sCD105 in advanced stage of PCM likely reflects an increased tumor angiogenesis, but not directly related to the number of neoplastic plasma cells. CD70 is a member of the Tumor necrosis factor family that is aberrantly expressed by a number of hematological malignancies and some carcinomas. McEarchern et al. performed IHC on PCM tissue microarray slides and reported a variable CD70 expression in 13 of 31 (42%) cases of PCM . They described that the staining pattern for CD70 was both membranous and cytoplasmic. In our study, the median CD70 expression level was 0.2% (range, 0–50.7%) and only 3 of 101 (3%) PCM cases had 20% or more cells that were CD70 positive. It is noteworthy that we used flow cytometry method that allowed us to measure the surface CD70 expression only; and to specifically assess its expression on neoplastic plasma cells. Additionally, we obtained a large number of tumor cells (median, 3400 PCM cells) for assessment. Our results indicate that CD70 may not be a suitable marker to target in majority of the myeloma patients. Other biomarkers currently under investigation for potential target therapy include CD56, CD138, CD200, and CS-1 [27, 28]. Elotuzumab, an anti-CS1 monoclonal antibody, has recently achieved clinically meaningful responses when combined with lenalidomide or bortezomib in patients with relapsed and relapsed/refractory PCM .
In summary, we showed that CD49d, CD44, and CD184 are highly expressed in plasma cell myeloma. All three of these antigens are involved in interactions between PCM and the BM microenvironment. CD44 expression is significantly higher in persistent/relapsed PCM than untreated patients. Both CD44 and CD184 are expressed at significantly higher levels in CD117+ PCM cases. In contract, CD30, CD70, and CD105 are very infrequently expressed on PCM. These data provide useful information for the future design of potential novel targeted therapies.