The potential use of basigin (CD147) as a prognostic marker in B‐cell precursor acute lymphoblastic leukaemia (2010)

BSG (basigin), also known as CD147 or extracellular matrix metalloproteinase inducer (EMMPRIN), is a cell surface glycoprotein that belongs to the immunoglobulin (Ig) superfamily. Four mRNA transcripts of BSG exist. Protein expression for isoform 1 is restricted to the retina, whereas isoform 2 is expressed on all leucocytes, red blood cells, platelets and endothelial cells, and is highly expressed on cells with high metabolic activity such as lymphoblasts, and tumor cells. (Iacono et al, 2007) The main function of BSG is to induce production of matrix metalloproteins (MMPs), which can degrade extracellular matrix components, thereby promoting tumor cell invasiveness and metastasis. (Reimers et al, 2004). Several studies have suggested a role for BSG in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Gene expression profiling of paired diagnosis-relapse samples from BCP-ALL patients showed transcriptional upregulation of BSG at relapse, (Beesley et al, 2005) although this could not be confirmed in subsequent studies. (Bhojwani et al, 2006; de Haas et al, 2009) In addition, Beesley et al (2005) showed that increased BSG mRNA expression at diagnosis was associated with an increased risk of relapse in paediatric BCP-ALL patients. To investigate the potential use of BSG expression as a prognostic marker for paediatric BCP-ALL, we evaluated the expression of BSG in normal and malignant B-cell precursor cells in more detail. Furthermore, we hypothesized that BSG may be involved in the migration of ALL cells to extramedullary sites, such as the central nervous system (CNS), which occurs in 20–30% of ALL relapses. Gene expression profiling of 27 paired diagnosis-relapse bone marrow (BM) samples from paediatric BCP-ALL patients confirmed significantly increased BSG mRNA expression at relapse, although the fold change was only 1·3, compared to 2·0 reported in an earlier study of 11 patients. (Beesley et al, 2005) Real-time quantitative polymerase chain reaction (RQ-PCR) analysis specific for the four isoforms of BSG, performed in 15 paired diagnosis-relapse samples of BCP-ALL patients, showed that isoform 2 was predominantly expressed with a significant difference between diagnosis and relapse (Fig 1A), whereas expression of isoform 1, 3 and 4 was >100-fold lower and did not differ between diagnosis and relapse. BSG protein expression, analysed using six-color flow cytometry, was slightly but not statistically significantly increased at relapse (n = 10) compared to diagnosis (n = 41) samples (Fig 1B). This slight increase was mainly due to two patients who showed a remarkably high BSG protein expression at relapse. One of these patients was the only patient evaluated with an early relapse (during therapy) and the other patient was the only t(9;22) positive patient, suggesting that high BSG expression might be associated with poor risk ALL. In line with our data, Beesley et al (2008) recently reported a slight increase (not statistically significant) in BSG protein expression at relapse of BCP-ALL (n = 10) using Western Blot analysis. As BSG plays an important role in migration and metastasis, we hypothesized that high levels of BSG may lead to ‘escape’ of ALL cells to extramedullary sites, such as CNS or testis. Indeed, BSG protein expression was significantly higher on ALL cells derived from cerebrospinal fluid (CSF) compared to BM-derived ALL cells (Fig 1B), indicating that BSG may play a role in CNS localization. It remains to be evaluated whether high BSG expression predisposes to CNS localization or whether ALL cells upregulate BSG expression once located within the CNS. The prognostic value of BSG in BCP-ALL. (A) BSG isoform 2 mRNA expression was determined in 15 paired diagnosis (Dx)-relapse (R) BM samples of ALL patients using RQ-PCR. Data are expressed as −ΔCT, defined as CT(GUS) – CT(BSG). The mean fold change (2ΔΔCT) in mRNA expression between relapse and diagnosis was 1.3. (B) BSG protein expression was analysed in 41 primary Dx BM samples, 10 R BM samples, and 5 R CSF samples of ALL patients using six-color flow cytometry. For each patient, median BSG fluorescence intensity (MFI) of the malignant cell population is indicated in the figure. The mean fold change in protein expression was 1.2 between R BM and Dx BM, 1.9 between R BM and R CSF, and 2.3 between Dx BM and R CSF. Open symbols indicate relapse samples, filled symbols diagnosis samples. (C) BSG isoform 2 mRNA expression was determined in diagnostic samples of 19 BCP-ALL patients who remained in complete remission and in diagnostic samples of 18 patients who relapsed, as described in A. (D) BSG protein expression was determined in 33 BCP-ALL patients, classified as high risk (HR), medium risk (MR), or standard risk (SR), depending on prednisone response, presence of t (9;22) or t (4;11), and minimal residual disease levels at day 33 and 79. BSG protein expression was measured as described in B.*P < 0.05; **P < 0.01; ***P < 0.001; NS, not statistically significant. Mean expression levels are indicated with grey bars. To study the correlation between BSG expression and clinical outcome in more detail, we next analysed whether high BSG expression at diagnosis is associated with risk of relapse, as previously reported for patients treated according to the Australian national protocols. (Beesley et al, 2005) In a retrospective comparison of BSG mRNA expression levels between diagnostic samples from 18 BCP-ALL patients with a relapse versus 19 patients who remained in complete remission, no significant differences in BSG mRNA expression were found (Fig 1C). Second, in a prospective study, BSG protein expression was determined in 33 BCP-ALL patients. As an early measure of clinical outcome, patients were classified as high risk (HR), medium risk (MR), or standard risk (SR), depending on prednisone response, presence of t(9;22) or t(4;11), and minimal residual disease levels at day 33 and 79. (Conter et al, 2010) Although the risk of relapse differed considerably between these three risk groups (risk of relapse ∼80%, ∼30%, and <5% for HR, IR, and SR patients, respectively), no significant difference in BSG protein expression was observed (Fig 1D). These data indicate that BSG expression levels at diagnosis have no prognostic significance in paediatric BCP-ALL patients treated according to the Dutch national therapy protocols. Flowcytometric analysis of BSG protein expression during normal B cell differentiation showed that the expression of BSG protein gradually declined during B cell differentiation, with relatively high BSG expression in pro-B cells and relatively low BSG expression in mature B cells (Fig 2A). BSG expression levels were also high at the pre-B-II large stage, probably because of the proliferation of these cells, which has been suggested to be regulated by BSG. (Iacono et al, 2007) BSG protein expression of BCP-ALL cells was within the range observed for normal B-cell precursor cells, but no straightforward relation between BSG expression on ALL cells and the immunophenotypic (pro-, common-, pre-B-ALL) or immunogenotypic (based on the presence of Ig gene rearrangements) maturation status of the ALL cells could be demonstrated (Fig 2B). Relationship between BSG expression and maturation status of normal (A) and malignant (B) BCP cells. (A) BSG protein expression on different B-cell subsets in BM from six healthy donors was determined using six-color flow cytometry. Hierarchical order of immunoglobulin gene rearrangements during normal B cell differentiation is indicated in panel A. (van Zelm et al, 2005). (B) Based on immunoglobulin/T cell receptor gene rearrangement status, BCP-ALL patients were classified as early (IGHD@-IGHJ@ present, no IGK@ rearrangement), late (IGKV@-IGKJ@ or IGK@-IGKDEL rearrangement), or intermediate (all others), as indicated in panel B. ALL immunophenotypes (pro-, common- or pre-B-ALL) are indicated with separate symbols. Mean BSG protein expression is indicated with grey bars. In conclusion, although a slightly increased BSG mRNA expression was found in BM-derived BCP-ALL cells at relapse, we could not confirm the prognostic significance of BSG expression levels at diagnosis. However, high BSG expression in ALL cells derived from CSF indicate that BSG may be involved in extramedullary localization; this is currently under investigation. JFdV was responsible for the design of this study, performed experiments, analysed and interpreted the data, and wrote the manuscript. JGtM and HKW performed the experiments, analysed and interpreted the data and critically revised the manuscript. JJMvD and VHJvdV contributed to the conception and design of this study, interpretation of the data, and critical revision of the manuscript. All authors gave final approval of the version to be published. The authors declare that they have no potential conflicts of interest.