CXCL12 polymorphism and malignant cell ... - The FASEB Journal

Download PDF
1 downloads 0 Views 402KB Size Report
Comment
*CHRU de Tours, Laboratoires d'Hématologie et d'Immunologie et Service d'Hématologie et. Thérapie Cellulaire; †Université François Rabelais de Tours, ...
The FASEB Journal • FJ Express Full-Length Article

CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia Florence Dommange,* Guillaume Cartron,*,† Claire Espanel,* Nathalie Gallay,†,¶ Jorge Domenech,*,†,¶ Lotfi Benboubker,*,†,¶ Marc Ohresser,*,† Philippe Colombat,*,†,¶ Chistian Binet,*,†,¶ Herve Watier,*,† and Olivier Herault*,†,¶,1 for the GOELAMS Study Group *CHRU de Tours, Laboratoires d’He´matologie et d’Immunologie et Service d’He´matologie et The´rapie Cellulaire; †Universite´ Franc¸ois Rabelais de Tours, e´quipes EA-3853 et EA-3855; and ¶ Inserm, e´quipe ESPRI “Microenvironnement de l’he´matopoı¨e`se et cellules souches,” Tours, France Stromal cell-derived factor 1 (SDF-1), a chemokine abundantly produced by the bone marrow microenvironment, and its receptor CXCR4 have crucial roles in malignant cell trafficking. In acute myeloid leukemia (AML), blasts invade the bloodstream and may localize in extramedullar sites, with variations from one patient to another. We hypothesized that a polymorphism in the SDF-1 coding gene (CXCL12 G801A) could influence blast dissemination and tissue infiltration in AML. CXCL12 G801A polymorphism was determined in 86 adult patients and 100 healthy volunteers. The allelic status and CXCR4 expression on bone marrow blasts were analyzed in relation to peripheral blood blast (PBB) counts and frequency of extramedullar tumor sites. 801A carrier status (801G/A, 801A/A) was found to be associated with a higher PBB count compared with 801G/G homozygous patients (Pⴝ0.031) and higher frequency of extramedullar tumor sites (odds ratio 2.92, 95% confidence interval 1.18 –7.21, Pⴝ0.018). Moreover, the PBB count was correlated with CXCR4 expression (correlation coefficient 0.546, Pⴝ0.001) when considering 801A carriers. In conclusion, a polymorphism in the SDF-1 gene is shown for the first time to be associated with the clinical presentation of a malignant hematological disease and more generally with the risk of distant tissue infiltration by tumor cells.—Dommange, F., Cartron, G., Espanel, C., Gallay, N., Domenech, J., Benboubker, L., Ohresser, M., Colombat, P., Binet, C., Watier, H., Herault, O., for the GOELAMS Study Group. CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia. FASEB J. 20, E1296 –E1300 (2006)

ABSTRACT

Key Words: SDF-1 䡠 CXCR4 䡠 blast cells 䡠 microenvironment Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation within the bone marrow of myeloid progenitors arrested in their maturation process. In contrast to normal hematopoiesis, it is usually associated with egress of immature cells from the bone E1296

marrow into the circulation before anchoring in extramedullar locations. Peripheral blood blast (PBB) counts and the number of extramedullar tumor sites are extremely variable from one patient to another and depend in part on AML subtype. Chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are involved in the trafficking of malignant cells (1). SDF-1/CXCR4 signaling is active in many cancer cells, including those of solid tumors and hematological malignancies. CXCR4 and its ligand SDF-1 were recently shown to have an important role in breast, prostate, ovarian, and sympathetic nervous system cancer metastasis (2– 6), as well as in the in vitro migration assays of malignant cells from pancreatic cancers (7), non-Hodgkin B cell lymphomas (8), chronic lymphocytic leukemias, and acute leukemias (9 –12). SDF-1 is constitutively produced in the bone marrow by immature osteoblasts lining the endosteum region and by stromal and endothelial cells. SDF-1 is also produced by different hematopoietic cells as well as by AML blast cells, which express varying amounts of functionally active CXCR4 (10 –13). The mechanisms of blast dissemination are poorly understood and could partly mimic those involved in the egress of normal hematopoietic progenitor cells (HPCs) from bone marrow during mobilization in which SDF-1/CXCR4 axis has a pivotal role (14). The granulocyte colony-stimulating factor-induced mobilization process is associated with a decrease in medullary levels of SDF-1 and up-regulation of CXCR4 (15), and our group has reported an association between the mobilizing capacity of HPCs and a single nucleotide polymorphism (SNP) in CXCL12 (16), the SDF-1-encoding gene. This polymorphism is located at nucleotide position 801 (G to A transition, counting from the 1 Correspondence: Laboratoire d’He´matologie / Equipe Inserm ESPRI-EA3855, CHRU-Hoˆpital Bretonneau, 2 bd Tonnelle, Tours Cedex F-37044, France. E-mail: olivier.herault@ med.univ-tours.fr doi: 10.1096/fj.05-5667fje

0892-6638/06/0020-1296 © FASEB

ATG start codon) in the 3⬘ untranslated region (3⬘UTR) of the SDF-1␤ transcript (GenBank accession number L36033). The ability of blasts to exit from the bone marrow microenvironment, circulate in the peripheral blood, and anchor in extramedullar locations might thus depend on the CXCL12 genotype. The purpose of our study was to determine whether CXCL12 G801A polymorphism is critical for the dissemination of malignant cells in de novo AML.

MATERIALS AND METHODS Cases Eighty-six consecutive adult Caucasian patients (37 female and 49 male) with newly diagnosed de novo AML and 100 healthy volunteers were included in this study after obtaining informed consent and approval by the ethics committee of the University Hospital of Tours. The French-AmericanBritish (FAB) cooperative group classification was applied to define the AML subtype and because of the heterogeneity, statistical analyses were performed grouping myeloid (FAB M0/M1/M2) and (myelo)monocytic subtypes (FAB M4/M5). Cytogenetic risk groups were classically defined as favorable, unfavorable and intermediate according to Grimwade et al. (17). Since all patients were treated in the same hospital, clinical data were homogeneous. Blast dissemination and tissue infiltration were evaluated by PBB count and the presence of at least one extramedullar tumor site (lymph nodes, liver, spleen, skin, gums, testicles, Waldeyer ring, vertebrae, and nervous system). CXCL12 genotype analysis Genomic DNA was extracted from marrow or blood samples, and CXCL12 G801A polymorphism was determined with a polymerase chain reaction-restriction fragment length polymorphism assay [according to the method described by Winkler et al. (18)]. The PCR primers were CAGTCAACCTGGGCAAAGCC(F) and AGCTTTGTGCCTGAGAGTCC(R). The PCR product was digested with the restriction endonuclease MspI (New England Biolabs, Beverly, MA), resulting in two fragments of 202 and 100 bp for the 801G allele and in one fragment of 302 bp for the 801A variant because of the elimination of the MspI restriction site. These fragments were identified by 6% PAGE (Fig. 1a). Flow cytometric analysis The expression of CXCR4 on the surface of bone marrow blasts was determined by flow cytometry (FACSCalibur, Becton-Dickinson, San Jose, CA) using anti CD45-APC (HI30), anti CXCR4-PE (12G5), and anti CD34-FITC (HPCA-2) monoclonal antibodies (mAbs) from BD Biosciences (San Jose, CA). The cells were incubated with saturating concentrations of these mAbs or isotypic controls for 30 min at 4°C and then washed twice with phosphate buffer. At least 5000 events were collected for each sample. CXCR4 expression was measured on leucoblast populations after setting a gate on the SSC/FL4 (CD45) scatter graph, according to Lacombe et al. (19) completed in certain cases with CD34 expression to optimize leucoblast gating (Fig. 1b). CXCR4 expression on gated events was evaluated by signal/noise (S/N) ratio of geometric mean fluorescence intensities (MFI) obtained with SDF1 GENE POLYMORPHISM AND LEUKEMIA DISSEMINATION

Figure 1. Representative experiment. a) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of CXCL12 G801A polymorphism in 3 different patients. PCR product was digested with the restriction endonuclease MspI. 801G allele resulted in 2 fragments of 202 and 100 pb and 801A variant in 1 fragment of 302bp. b) Flow cytometric analysis of CXCR4 expression on AML leucoblast cells. These results were obtained from a patient with newly diagnosed acute myelomonocytic leukemia (FAB-M4 AML). CXCR4 expression was measured on leucoblast populations after setting a gate on side scatter (SSC)/anti-CD45 APC scatter graph (gate R1), completed with CD34 expression to optimize leucoblast gating (gate R2). Intensity of CXCR4 expression is shown as signal/noise ratio defined as geometric mean fluorescence intensity (MFI) of CXCR4-expressing cells (signal) divided by MFI of cells stained with IgG1 isotypic control antibody (Ab) (noise). anti CXCR4 monoclonal antibody (mAb) and its isotypic control (G155–178, BD Biosciences), respectively. Statistical analysis The results were expressed as median (range), and number (percentage) of patients in 801A carrier (801A/A homozygous and 801A/G heterozygous patients or healthy volunteers) and 801G/G groups. Age, gender, diagnosis (FAB subtype), cytogenetics (risk group), bone marrow blast percentage, PPB count, extramedullar tumor sites, CXCR4 expression, and CXCL12 G801A polymorphism were considered as variables in univariate analysis performed using the Mann-Whitney U test, ␹2 contingency test, and Z test for non-zero correlation, as appropriate. To evaluate the influence of independent factors on the risk of extramedullar tumor site(s), multivariate analysis was performed using the multiple regression method, including the variables with P ⬍ 0.10 in the univariate tests. The level of significance was set at 0.05.

RESULTS Patient characteristics, CXCL12 genotypes, and the expression of CXCR4 on blast cells are presented in E1297

TABLE 1.

Patient characteristics and expression of CXCR4 on bone marrow blasts at diagnosis of de novo AML

FAB subtype

No. of patients

Age (yr)

M0 M1 M2 M3 M4 M5 M7 Total

3 15 23 9 22 13 1 86

41 关25–74兴 51 关18–74兴 48 关17–75兴 47 关22–75兴 51 关16–72兴 44 关22–78兴 37 47 关16–78兴

CXCL12 G801A polymorphism

A/A 0 3 2 0 1 0 0 6

A/G 0 6 6 4 9 6 0 31

G/G 3 6 15 5 12 7 1 49

Bone marrow blast %

Peripheral blood blast count (/␮l)

Patients with extramedullar tumor site(s)

90 关71–91兴 88 关38–98兴 54 关17–98兴 88 关72–98兴 75 关23–97兴 80 关35–97兴 87 79 关17–98兴

13.0 关6.1–15.0兴 4.3 关0.1–94.1兴 2.6 关0–33.0兴 5.5 关0.1–61.6兴 7.9 关0–99.3兴 15.3 关0.8–137.1兴 0.6 5.6 关0–137.1兴

1 (33%) 8 (55%) 6 (26%) 3 (33%) 6 (27%) 8 (62%) 0 32 (37%)

CXCR4 expression (S/N MFI)

1.7 关1.6–2.8兴 1.5 关1.0–16.0兴 2.1 关1.0–27.5兴 2.0 关1.0–3.0兴 2.9 关1.0–11.6兴 2.2 关1.0–37.4兴 1.5 2.2 关1.0–37.4兴

Data provided are median 关range兴 and number (%). FAB, French American British classification; S/N MFI, signal/noise ratio of geometric mean fluorescence intensities

Table 1 for each FAB subtype. The frequency of the 801A allele in our group of patients was the expected value for Caucasians (20) and was not different from those among healthy volunteers. Moreover the frequencies of this allele were similar in FAB groups (41 and 46% in M0-M1-M2 and M4-M5 groups, respectively). CXCR4 expression was not statistically different between FAB groups [1.7 (1–28) and 2.9 (1–37.4) in M0-M1-M2 and M4-M5 groups, respectively] and between CXCL12 genotypes [1.9 (1–28) and 2.5 (1.0 – 37.4) in 801A carriers and 801G/G patients, respectively]. The PBB count was not statistically influenced by FAB subtype [4.2 (0 –94.1) and 10.3 (0 –137.1) PBB/␮l in M0-M1-M2 and M4-M5 groups, respectively]. It was moderately correlated with the percentage of blasts in the bone marrow compartment (correlation coefficient 0.356, P⬍0.001), which was not different in 801A carriers and 801G/G patients [80 (23–98)% and 76 (17–98)%, respectively]. As presented in Fig. 2, the presence of the 801A allele was associated with an increased PBB count when comparing 801A carriers to 801G/G patients [10.4 (0.1–94.1) and 2.6 (0 –137.1) PBB/␮l, respectively, P⫽0.031]. Moreover, PBB count was correlated with CXCR4 expression on bone marrow blasts (Fig. 3) in 801A carriers (correlation coefficient 0.546, P⫽0.001), whereas such a correlation could not be evidenced in 801G/G patients (correlation coefficient 0.176). Patients presenting extramedullar tumor site(s) were characterized by a lower mean age [39 (18 –78) vs. 53 (16 –75) yr, P⫽0.005], a higher PBB count [10.9 (0.1– 137.1) vs. 2.5 (0 –99.3) PBB/␮l, P⫽0.002], and a higher percentage of 801A carriers (59.4 vs. 33.3%, P⫽0.018). CXCL12 801A carrier status was indeed highly associated with extramedullar locations, which were found in 51.4% of 801A carriers (66.7 and 48.4% of A/A and A/G patients, respectively) and in 26.5% of 801G/G patients (Fig. 2), with an odds ratio of 2.92 (95% confidence interval 1.18 –7.21). Interestingly, CXCR4 expression was not different in patients with and without extramedullar locations [S/N⫽2.2 (1–37.4) and 2.2 (1–11.6), respectively]. Age, PBB count, 801A carrier E1298

Vol. 20

September 2006

status, and bone marrow blast percentages [87% (17–98) and 75% (23–98) in patients with and without tissue infiltration, respectively, P⫽0.090] were included in a multivariate analysis, and the variables found to be independently associated with risk of extramedullar tumor

Figure 2. Association between CXCL12 G801A polymorphism, peripheral blood blast count, and frequency of extramedullar tumor site(s). Each dot represents 1 patient. Horizontal bars represent median values of PBB counts: 10.4 PBB/␮l in 801A carriers and 2.6 PBB/␮l in 801G/G patients (P⫽0.031). Black dots represent patients presenting extramedullar tumor site(s), observed in 51.4% of 801A carriers and 26.5% of 801G/G patients (P⫽0.018).

The FASEB Journal

DOMMANGE ET AL.

Figure 3. Schematic diagram of critical role of CXCL12 G801A polymorphism for dissemination of marrow blast cells of adult patients suffering from de novo acute myeloid leukemia

site(s) were PBB count (P⫽0.012), age (P⫽0.029), and CXCL12 G801A polymorphism (P⫽0.042). These results were supported by an absence of difference between 801A carriers and 801G/G patients when considering age [44 (16 –72) and 51 (17–78) yr, respectively].

DISCUSSION The significance of SNPs in cancer is a recent finding. For example, SNPs are involved in the clinical presentation of malignant diseases, e.g., SNP of vascular endothelial growth factor (VEGF) in melanomas (21), and in the therapeutic response, e.g., SNP of FCGR3A in lymphomas (22). In this study, we report a genetic determinant associated with the risk of metastasis. Malignant cell migration, which is recognized as a critical step in metastasis, is a complex process mainly involving metalloproteases, adhesion molecules, and chemokines such as SDF-1. Recent reports indicate that the SDF-1/CXCR4 interaction may be important for the metastasis of solid tumors that express CXCR4 (2– 6). SDF-1 is the major chemokine released by the bone marrow microenvironment. It is encoded by the CXCL12 gene, and our study demonstrates that the CXCL12 801A allele is an independent risk factor for distant tissue infiltration by malignant cells in AML, concomitant with a higher circulating malignant cell count. These results are supported by our previous findings concerning mobilization of normal HPCs (16) and by the recent description of a higher frequency of splenomegaly and hepatomegaly in patients with chronic lymphocytic leukemia carrying this variant than in patients homozygous for the common 801G/G genotype (23). The functional significance of this polymorphism has not been characterized. Several polymorphisms have recently been found elsewhere in the CXCL12 gene region that are in linkage disequilibrium with CXCL12 G801A, and the authors suggested the presence of haplotypic variation in expression of SDF-1 transcripts (24). Nevertheless, this study was performed with Indonesian islanders and the results may be different in SDF1 GENE POLYMORPHISM AND LEUKEMIA DISSEMINATION

Caucasians. Direct evidence of an influence of CXCL12 G801A polymorphism on the production or transcript half-life of SDF-1 has not been obtained in vitro, because SDF-1 expression is limited to stromal cells and other tissues not easy to analyze. It was hypothesized in 1998 that this mutation could be associated with increased marrow stromal cell secretion of SDF-1 (18), without confirmation to date. On the other hand, it could be associated with lower secretion of SDF-1, an hypothesis supported by the lower SDF-1 level recently described in the plasma of normal homozygous 801A subjects (25) and in stromal layer supernatants of long-term marrow cultures established from 801A carriers patients suffering from lymphoid malignancies (unpublished observation from J. Domenech). This decreased production of SDF-1 might explain the increased capability of malignant cells to egress from the bone marrow microenvironment. In this context, the correlation observed between CXCR4 expression on bone marrow blasts and the PBB count observed in A carrier patients might result from weaker SDF-1-induced down-regulation of this receptor (26), which is not sufficiently effective to hold back the malignant cells in the marrow compartment. The lack of correlation between CXCR4 expression and PBB count in 801G/G patients might be explained by the existence of a critical intramedullary threshold of concentration of SDF-1 below which blasts leave the marrow, regardless of the influence of other factors involved in the migration process (adhesion molecules, etc.). Based on the hypothesis of the effect of CXCL12 polymorphism on the intramedullary production of SDF-1, 801A-carrier patients might have a concentration below this threshold and therefore present higher PPB count correlated with the expression of CXCR4. On the contrary, 801G/G patients might have a SDF-1 concentration above the threshold and the level of expression of CXCR4 might be a minor cofactor in the extramedullar dissemination of blasts, hence the lack of correlation. In conclusion, we report that CXCL12 G801A polymorphism is a genetic determinant involved in the clinical presentation of leukemia. This description of increased release of blasts from the bone marrow to the blood and higher frequency of distal dissemination in 801A carriers is the first report of an association between this polymorphism and the risk of tissue infiltration by malignant cells. It would be interesting to determine whether 801A carriers had reduced overall survival and a greater risk of recurrence of AML. Moreover, as the SDF-1/CXCR4 axis is involved in the migration process of various types of cancer cells, it could be hypothesized that the 801A variant constitutes a general risk factor for metastasis development and that assessment of CXCL12 G801A polymorphism should help in identifying patients at risk of metastasis. Further studies should clarify this question. We thank Danielle Truglio, Anne-Franc¸oise Pannier, Anne Mathieu, and Chloe´ Charroing for technical assistance. This E1299

study was supported by the “Comite´ d’Indre-et-Loire de la Ligue Nationale Contre le Cancer,” the Rotary Club of Blois (37), and the French “Les Sapins de l’Espoir contre le Cancer” and “CANCEN” associations.

15.

REFERENCES

16.

1. 2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Burger, J. A., and Kipps, T. J. (2006) CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 107, 1761–1767 Liang, Z., Yoon, Y., Votaw, J., Goodman, M. M., Williams, L., and Shim, H. (2005) Silencing of CXCR4 blocks breast cancer metastasis. Cancer Res. 65, 967–971 Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N., et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50 –56 Russell, H. V., Hicks, J., Okcu, M. F., and Nuchtern, J. G. (2004) CXCR4 expression in neuroblastoma primary tumors is associated with clinical presentation of bone and bone marrow metastases. J. Pediatr. Surg. 39, 1506 –1511 Scotton, C. J., Wilson, J. L., Scott, K., Stamp, G., Wilbanks, G. D., Fricker, S., Bridger, G., and Balkwill, F. R. (2002) Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res. 62, 5930 –5938 Taichman, R. S., Cooper, C., Keller, E. T., Pienta, K. J., Taichman, N. S., and McCauley, L. K. (2002) Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res. 62, 1832–1837 Koshiba, T., Hosotani, R., Miyamoto, Y., Ida, J., Tsuji, S., Nakajima, S., Kawaguchi, M., Kobayashi, H., Doi, R., Hori, T., et al. (2000) Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression. Clin. Cancer Res. 6, 3530 –3535 Arai, J., Yasukawa, M., Yakushijin, Y., Miyazaki, T., and Fujita, S. (2000) Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1. Eur. J. Haematol. 64, 323–332 Burger, J. A., Burger, M., and Kipps, T. J. (1999) Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 94, 3658 –3667 Mohle, R., Bautz, F., Rafii, S., Moore, M. A., Brugger, W., and Kanz, L. (1998) The chemokine receptor CXCR-4 is expressed on CD34⫹ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cellderived factor-1. Blood 91, 4523– 4530 Mohle, R., Schittenhelm, M., Failenschmid, C., Bautz, F., KratzAlbers, K., Serve, H., Brugger, W., and Kanz, L. (2000) Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia. Br. J. Haematol. 110, 563–572 Voermans, C., van Heese, W. P., de Jong, I., Gerritsen, W. R., and van Der Schoot, C. E. (2002) Migratory behavior of leukemic cells from acute myeloid leukemia patients. Leukemia 16, 650 – 657 Tavor, S., Petit, I., Porozov, S., Avigdor, A., Dar, A., Leider-Trejo, L., Shemtov, N., Deutsch, V., Naparstek, E., Nagler, A., and Lapidot, T. (2004) CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice. Cancer Res. 64, 2817–2824 Kucia, M., Reca, R., Miekus, K., Wanzeck, J., Wojakowski, W., Janowska-Wieczorek, A., Ratajczak, J., and Ratajczak, M. Z. (2005) Trafficking of normal stem cells and metastasis of cancer

E1300

Vol. 20

September 2006

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. Stem Cells 23, 879 – 894 Petit, I., Szyper-Kravitz, M., Nagler, A., Lahav, M., Peled, A., Habler, L., Ponomaryov, T., Taichman, R. S., Arenzana-Seisdedos, F., Fujii, N., et al. (2002) G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat. Immunol. 3, 687– 694 Benboubker, L., Watier, H., Carion, A., Georget, M. T., Desbois, I., Colombat, P., Bardos, P., Binet, C., and Domenech, J. (2001) Association between the SDF1–3⬘A allele and high levels of CD34(⫹) progenitor cells mobilized into peripheral blood in humans. Br. J. Haematol. 113, 247–250 Grimwade, D., Walker, H., Oliver, F., Wheatley, K., Harrison, C., Harrison, G., Rees, J., Hann, I., Stevens, R., Burnett, A., and Goldstone, A. (1998) The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 92, 2322–2333 Winkler, C., Modi, W., Smith, M. W., Nelson, G. W., Wu, X., Carrington, M., Dean, M., Honjo, T., Tashiro, K., Yabe, D. et al. (1998) Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. ALIVE Study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC). Science 279, 389 –393 Lacombe, F., Durrieu, F., Briais, A., Dumain, P., Belloc, F., Bascans, E., Reiffers, J., Boisseau, M. R., and Bernard, P. (1997) Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 11, 1878 –1886 Magierowska, M., Lepage, V., Boubnova, L., Carcassi, C., de Juan, D., Djoulah, S., El Chenawi, F., Grunnet, N., Halle, L., Ivanova, R., et al. (1998) Distribution of the CCR5 gene 32 base pair deletion and SDF1–3⬘A variant in healthy individuals from different populations. Immunogenetics 48, 417– 419 Howell, W. M., Bateman, A. C., Turner, S. J., Collins, A., and Theaker, J. M. (2002) Influence of vascular endothelial growth factor single nucleotide polymorphisms on tumour development in cutaneous malignant melanoma. Genes Immun. 3, 229 –232 Cartron, G., Dacheux, L., Salles, G., Solal-Celigny, P., Bardos, P., Colombat, P., and Watier, H. (2002) Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood 99, 754 –758 Poulain, S., Chevret, A. C., Stalnikiewicz L., Wierre, C., Simon, M., Pollet, J. P., Duthilleul, L., and Morel, P. (2003) Polymorphism in the SDF1 (stromal derived factor 1) gene is a new prognostic factor independent of Binet staging system in chronic lymphocytic leukemia (CLL). Blood 102, 669a Kimura, R., Nishioka, T., Soemantri, A., and Ishida, T. (2005) Allele-specific transcript quantification detects haplotypic variation in the levels of the SDF-1 transcripts. Hum. Mol. Genet. 14, 1579 –1585 Soriano, A., Martinez, C., Garcia, F., Plana, M., Palou, E., Lejeune, M., Arostegui, J. I., De Lazzari, E., Rodriguez, C., Barrasa, A., et al. (2002) Plasma stromal cell-derived factor (SDF)-1 levels, SDF1–3⬘A genotype, and expression of CXCR4 on T lymphocytes: their impact on resistance to human immunodeficiency virus type 1 infection and its progression. J. Infect. Dis. 186, 922–931 Amara, A., Gall, S. L., Schwartz, O., Salamero, J., Montes, M., Loetscher, P., Baggiolini, M., Virelizier, J. L., and ArenzanaSeisdedos, F. (1997) HIV coreceptor downregulation as antiviral principle: SDF-1alpha-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186, 139 –146

The FASEB Journal

Received for publication March 19, 2006. Accepted for publication April 17, 2006.

DOMMANGE ET AL.

The FASEB Journal • FJ Express Summary

CXCL12 polymorphism and malignant cell dissemination/tissue infiltration in acute myeloid leukemia Florence Dommange,* Guillaume Cartron,*,† Claire Espanel,* Nathalie Gallay,†,¶ Jorge Domenech,*,†,¶ Lotfi Benboubker,*,†,¶ Marc Ohresser,*,† Philippe Colombat,*,†,¶ Chistian Binet,*,†,¶ Herve Watier,*,† and Olivier Herault*,†,¶,1 for the GOELAMS Study Group *CHRU de Tours, Laboratoires d’He´matologie et d’Immunologie et Service d’He´matologie et The´rapie Cellulaire; †Universite´ Franc¸ois Rabelais de Tours, e´quipes EA-3853 et EA-3855; and ¶ Inserm, e´quipe ESPRI “Microenvironnement de l’he´matopoı¨e`se et cellules souches,” Tours, France To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5667fje SPECIFIC AIMS Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation within the bone marrow of blast cells arrested in their maturation process. Peripheral blood blast (PBB) counts and the number of extramedullar tumor sites are extremely variable from one patient to another. The chemokine stromal-derived factor (SDF)-1 and its receptor CXCR4 regulate the trafficking of many cancer cells, including those of solid tumors and hematological malignancies. There is also growing evidence that SDF-1 has a pivotal role in the regulation of trafficking of normal hematopoietic progenitor cells (HPCs) and their homing/retention in bone marrow, and the mechanisms of blast dissemination could mimic those involved in the egress of normal HPCs from bone marrow during mobilization. The granulocyte colony-stimulating factor-induced mobilization process of HPCs is associated with a decrease in medullary levels of SDF-1 and up-regulation of CXCR4, and our group has reported an association between the mobilizing capacity of HPC and a single nucleotide polymorphism in CXCL12 (CXCL12 G801A), the SDF1-encoding gene. The ability of blasts to exit from the bone marrow microenvironment, circulate in the peripheral blood, and anchor in extramedullar locations might thus depend on the CXCL12 genotype. The purpose of our study was to determine whether CXCL12 G801A polymorphism is critical for the dissemination of malignant cells in de novo AML. PRINCIPAL FINDINGS 1. Frequency of the CXCL12 801A allele in de novo AML patients Eighty-six consecutive adult Caucasian patients with newly diagnosed de novo AML and 100 healthy volun0892-6638/06/0020-1913 © FASEB

teers were included in this study after obtaining informed consent and approval by the ethics committee of the University Hospital of Tours. The French-American-British (FAB) cooperative group classification was applied to define the AML subtype and because of the heterogeneity, statistical analyses were performed grouping myeloid (FAB M0/M1/M2) and (myelo)monocytic subtypes (FAB M4/M5). CXCL12 G801A polymorphism was determined with a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay (PCR-RFLP; Fig. 1a), and the frequency of the 801A allele in our group of patients (7% A/A and 36% A/G) was the expected value for Caucasians and was not different from those among healthy volunteers. Moreover the frequencies of this allele were similar in FAB groups (41% and 46% in M0-M1-M2 and M4-M5 groups, respectively). 2. Peripheral blood blast count and CXCL12 G801A polymorphism Blast dissemination was evaluated by PBB count. The PBB count was not statistically influenced by FAB subtype [4.2 (0 –94.1) and 10.3 (0 –137.1) PBB/␮l in M0-M1-M2 and M4-M5 groups, respectively]. It was correlated with the percentage of blasts in the bone marrow compartment (correlation coefficient 0.356, P⬍0.001), which was not different in 801A carriers (801A/A and 801A/G patients) and 801G/G patients [80 (23–98)% and 76 (17–98)%, respectively]. As presented in Fig. 2, the presence of the 801A allele was 1 Correspondence: Laboratoire d’He´matologie/Equipe Inserm ESPRI-EA3855, CHRU-Hoˆpital Bretonneau, 2 bd Tonnelle, Tours Cedex F-37044, France. E-mail: olivier.herault@ med.univ-tours.fr doi: 10.1096/fj.05-5667fje

1913

4. CXCR4 expression on marrow blast cells and leukemic dissemination in CXCL12 801A carriers The expression of CXCR4 on the surface of bone marrow blasts was determined by flow cytometry (Fig. 1b). CXCR4 expression was not statistically different between FAB groups [1.7 (1–28) and 2.9 (1–37.4) in M0-M1-M2 and M4-M5 groups, respectively] or between patients with and without extramedullar locations [S/N⫽2.2 (1–37.4) and 2.2 (1–11.6), respectively]. Considering CXCL12 G801A polymorphism, CXCR4 expression was correlated with PBB count in 801A carriers (correlation coefficient 0.546, P⫽0.001), whereas such a correlation could not be evidenced in 801G/G patients (correlation coefficient 0.176).

CONCLUSIONS AND SIGNIFICANCE Figure 1. Representative experiment. a) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of CXCL12 G801A polymorphism in 3 different patients. PCR product was digested with the restriction endonuclease MspI. 801G allele resulted in 2 fragments of 202 and 100 pb and 801A variant in 1 fragment of 302bp. b) Flow cytometric analysis of CXCR4 expression on AML leucoblast cells. These results were obtained from a patient with newly diagnosed acute myelomonocytic leukemia (FAB-M4 AML). CXCR4 expression was measured on leucoblast populations after setting a gate on side scatter (SSC)/anti-CD45 APC scatter graph (gate R1), completed with CD34 expression to optimize leucoblast gating (gate R2). Intensity of CXCR4 expression is shown as signal/noise ratio defined as geometric mean fluorescence intensity (MFI) of CXCR4-expressing cells (signal) divided by MFI of cells stained with IgG1 isotypic control antibody (Ab) (noise).

The significance of single nucleotide polymorphisms (SNPs) in cancer is a recent concept. For example, SNPs are involved in the clinical presentation of malig-

associated with an increased PBB count when comparing 801A carriers to 801G/G patients [10.4 (0.1–94.1) and 2.6 (0 –137.1) PBB/␮l, respectively, P⫽0.031]. 3. Extramedullar tumor sites and CXCL12 G801A polymorphism Tissue infiltration was evaluated by the presence of at least one extramedullar tumor site (lymph nodes, liver, spleen, skin, gums, testicles, Waldeyer ring, vertebrae, and nervous system). Since all patients were treated in the same hospital, clinical data were homogeneous. CXCL12 801A carrier status was highly associated with extramedullar locations, which were found in 51.4% of 801A carriers (66.7 and 48.4% of A/A and A/G patients, respectively) and in 26.5% of 801G/G patients (Fig. 2), with an odds ratio of 2.92 (95% confidence interval 1.18 –7.21). These results were reinforced by a multivariate analysis showing that the independent variables associated with risk of extramedullar tumor site(s) were PBB count (P⫽0.012), age (P⫽0.029), and CXCL12 G801A polymorphism (P⫽0.042). 1914

Vol. 20

September 2006

Figure 2. Association between CXCL12 G801A polymorphism, peripheral blood blast count, and frequency of extramedullar tumor site(s). Each dot represents 1 patient. Horizontal bars represent median values of PBB counts: 10.4 PBB/␮l in 801A carriers and 2.6 PBB/␮l in 801G/G patients (P⫽0.031). Black dots represent patients presenting extramedullar tumor site(s), observed in 51.4% of 801A carriers and 26.5% of 801G/G patients (P⫽0.018).

The FASEB Journal

DOMMANGE ET AL.

nant diseases, e.g., SNP of vascular endothelial growth factor (VEGF) in melanomas, and in the therapeutic response, e.g., SNP of FCGR3A in lymphomas. In this study, we report a genetic determinant associated with the risk of metastasis. Malignant cell migration, which is recognized as a critical step in metastasis, is a complex process mainly involving metalloproteases, adhesion molecules, and chemokines such as SDF-1. Recent reports indicate that the SDF-1/CXCR4 interaction may be important for the metastasis of solid tumors that express CXCR4. SDF-1 is the major chemokine released by the bone marrow microenvironment. It is encoded by the CXCL12 gene, and our study demonstrates that the CXCL12 801A allele is an independent risk factor for distant tissue infiltration by malignant cells in AML, concomitant with a higher circulating malignant cell count (Fig. 3). These results are supported by our previous findings concerning mobilization of normal HPC and by the recent description of a higher frequency of splenomegaly and hepatomegaly in patients with chronic lymphocytic leukemia carrying this variant than in patients homozygous for the common genotype. The functional significance of this polymorphism has not been characterized. It could be associated with lower secretion of SDF-1, a hypothesis supported by the description of lower SDF-1 level in the plasma of normal homozygous 801A subjects and in stromal layer supernatants of long-term marrow cultures established from 801A carriers patients suffering from lymphoid malignancies. This decreased production of SDF-1 might explain the increased capability of malignant cells to egress from the bone marrow microenvironment. In this context, the correlation observed between CXCR4 expression on bone marrow blasts and the PBB count observed in A carrier patients might result from weaker SDF-1-induced down-regulation of this receptor, which is not sufficiently effective to hold back the malignant cells in the marrow compartment. In conclusion, we report that CXCL12 G801A polymorphism is a genetic determinant involved in the clinical presentation of leukemia. This description of

SDF1 GENE POLYMORPHISM AND LEUKEMIA DISSEMINATION

Figure 3. Schematic diagram of critical role of CXCL12 G801A polymorphism for dissemination of marrow blast cells of adult patients suffering from de novo acute myeloid leukemia

increased release of blasts from the bone marrow to the blood and higher frequency of distal dissemination in 801A carriers is the first report of an association between this polymorphism and the risk of tissue infiltration by malignant cells. It would be interesting to determine whether 801A carriers had reduced overall survival and a greater risk of recurrence of AML. Moreover, as the SDF-1/CXCR4 axis is involved in the migration process of various types of cancer cells, it could be hypothesized that the 801A variant constitutes a general risk factor for metastasis development and that assessment of CXCL12 G801A polymorphism should help in identifying patients at risk of metastasis. Further studies should clarify this question.

1915