Detection of circulating lymphoma cells in patients with non-Hodgkin lymphoma using MAGE-A3 gene expression in peripheral blood
Introduction
Non-Hodgkin lymphoma (NHL) is a heterogeneous group of B- and T-cell tumors that exhibits a wide range of responses to treatment [1]. The introduction of combined rituximab plus CHOP therapy (cyclophosphamide/doxorubicin/vincristine/prednisone) has improved NHL treatment outcomes, but problems associated with relapse and refractory tumors remain [2], [3]. Thus, a biomarker capable of predicting subgroups with poor prognosis and detecting minimal residual disease after treatment would be an invaluable adjunct to chemotherapy.
Lymphoma-specific gene expression in peripheral blood reflects the presence of circulating lymphoma cells (CLCs). The ideal target gene for CLC detection in peripheral blood is one that is not expressed in normal lymphocytes. In this regard, MAGE-A3 has been highlighted as a potent target for immunotherapy because it appears to be restricted to tumors and is widely expressed in many types of tumors [4], [5], [6], [7]. In solid cancers, MAGE-A3 gene expression is detected not only in tumor tissue but also in circulating tumor cells, and has shown prognostic value [8], [9]. MAGE-A3 gene expression has been observed in certain hematologic malignancies, including leukemia and multiple myeloma [10], [11], but has not been evaluated in NHL. To determine the potential of MAGE-A3 expression as a marker for the presence CLCs in NHL, we assessed MAGE-A3 mRNA expression in the peripheral blood of controls and patients with NHL.
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MAGE-A3 expression in lymphoma cell lines
The U266 multiple myeloma cell line, which is known to express the MAGE-A3 gene, was used as positive control in reverse transcriptase-polymerase chain reaction (RT-PCR) assays [11]. Ten lymphoma cell lines were evaluated for MAGE-A3 mRNA expression, including the NHL cell lines, Farage, RL, SU-DHL, Toledo and WSU-NHL; the Burkitt lymphoma cell lines, BJA-B, Daudi and Raji; the mantle cell lymphoma cell line, Granta-519; and the T-cell leukemia cell line, Jurkat.
RNA preparation and cDNA synthesis
Total RNA was extracted from
MAGE-A3 expression in cell lines, and detection sensitivity of nested RT-PCR
MAGE-A3 mRNA expression was confirmed in the positive control cell line (U266) and in four of ten lymphoma cell lines: RL, Farage, Toledo and Raji (Fig. 1A). All cell lines positive for MAGE-A3 expression were of NHL origin, except the Raji line, which originated from a Burkitt lymphoma patient. The detection sensitivity, determined using positive (RL) and negative (Daudi) cell mixtures at ratios ranging from 1 cell/106 cells to 106 cells/106 cells, was 1 cell/1000 cells (Fig. 1B).
Using quantitative
Discussion
The present study showed that, because of its tumor-restricted expression pattern and demonstrated absence of expression in controls, MAGE-A3 mRNA in the peripheral blood of NHL patients could be used as a tumor marker for CLCs. Using a nested RT-PCR method, we demonstrated that the positivity rate of MAGE-A3 expression was as high as 47%. However, MAGE-A3 expression was not associated with clinical characteristics or prognosis, a result that differs from previous breast and lung cancer studies
Conflict of Interest
The authors have no conflict of interest.
Acknowledgments
This work was supported by grants from the National Cancer Center of Korea (0810440) and the Innovative Research Institute for Cell Therapy, Republic of Korea (A062260).
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These authors contributed equally to this study.