Elsevier

Leukemia Research

Volume 34, Issue 9, September 2010, Pages 1127-1131
Leukemia Research

Detection of circulating lymphoma cells in patients with non-Hodgkin lymphoma using MAGE-A3 gene expression in peripheral blood

https://doi.org/10.1016/j.leukres.2009.11.028Get rights and content

Abstract

Background/AIMS

Lymphoma-specific gene expression in peripheral blood reflects the presence of circulating lymphoma cells (CLCs). MAGE-A3 is widely expressed in solid tumors and is a potent candidate for immunotherapy. To determine whether MAGE-A3 expression would be a useful marker for CLCs in non-Hodgkin lymphoma (NHL), we assessed MAGE-A3 mRNA expression in the peripheral blood of NHL patients and controls.

Methods

We measured MAGE-A3 gene expression in ten lymphoma cell lines (Farage, RL, SU-DHL, Toledo, WSU-NHL, BJA-B, Daudi, Raji, Granta-519 and Jurkat) using nested RT-PCR, and determined detection sensitivity using mixtures of MAGE-A3-positive and -negative cells over a range of 1/106 to 106/106 cells. MAGE-A3 expression was determined in buffy coat samples of 40 controls and 95 NHL patients prior to treatment. Clinical characteristics (e.g., cell lineage) and international prognostic indices, including age, performance, LDH, stage and extra-nodal involvement, were evaluated and related to MAGE-A3 expression. Hazard ratios, reflecting risk for overall survival and progression-free survival, were also evaluated. Follow-up MAGE-A3 expression was evaluated at two time points: after 3–4 cycles of chemotherapy (80 patients) and after 6–8 cycles of chemotherapy (74 patients).

Results

MAGE-A3 mRNA was detected in four lymphoma cell lines – RL, Farage, Toledo and Raji – and was present in 45 of 95 (47.3%) patients with NHL, but in none of the 40 controls. The detection sensitivity was 1 in 1000 cells. MAGE-A3 expression prior to treatment was not associated with clinical features or patient survival. During follow-up, only six patients (7.5%) were positive for MAGE-A3 after 3–4 cycles of chemotherapy and three (4.1%) were positive after 6–8 cycles.

Conclusions

The results showed that MAGE-A3 gene expression was frequent in NHL patients and decreased after effective chemotherapy, suggesting that MAGE-A3 can be used as a tumor marker for CLCs in patients with NHL. However, MAGE-A3 expression showed no prognostic value in this group of patients.

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.

Section snippets

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).

References (21)

There are more references available in the full text version of this article.

Cited by (7)

View all citing articles on Scopus
1

These authors contributed equally to this study.

View full text