Departments of 1 Hematology and Medical Oncology and 2 Pathology, Hospital Clínico Universitario, University of Valencia, Valencia 3 Hematology Section, Hospital Francesc de Borja, Gandia, Spain
Received 8 April 2002; revised 26 July 2002; accepted 9 September 2002
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Abstract |
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High serum levels of soluble intercellular adhesion molecule-1(s-ICAM-1/s-CD54) have been associated with adverse clinical features and poor outcome in chronic lymphocytic leukemia, Hodgkins disease and non-Hodgkins lymphoma, but their value in the different subtypes of non-Hodgkins lymphoma has not been well addressed.
Patients and methods:
Our aim was to study the serum levels of s-ICAM-1 in diffuse large B-cell lymphoma (DLBCL) and to correlate them with clinical characteristics and outcome. We analyzed the serum levels of s-ICAM-1 in a series of 55 patients with DLBCL diagnosed in a single institution. s-ICAM-1 levels were quantified by an immunoenzymatic assay. Median age was 62 years (range 2296); 29 (53%) were male. Twenty-eight (51%) presented with advanced clinical stage (III/IV), 32 (58%) had extranodal involvement, 28 (51%) had high serum lactate dehydrogenase (LDH) and 23 (43%) had high ß2-microglobulin levels. All patients received anthracycline-containing regimens. Correlation between clinical variables and s-ICAM-1 levels were tested with the MannWhitney U-test and survival was plotted by the KaplanMeier method, and curves compared with the log-rank test.
Results:
Serum levels of s-ICAM-1 were significantly increased in patients with DLBCL compared with normal controls (589 ± 487 versus 279 ± 65 ng/ml, respectively; P <0.001). Higher levels of s-ICAM-1 were present in patients with B symptoms, advanced stage and increased LDH and ß2-microglobulin. s-ICAM-1 levels also correlated with achievement of a complete response. Patients with s-ICAM-1 over 668 ng/ml had a shorter time to treatment failure (TTF) (3-year TTF, 59% versus 20%, respectively; P = 0.01) and overall survival (OS) (3-year OS, 58% versus 22%, respectively; P = 0.04) than the remainders. When only low and lowintermediate risk patients in the international prognostic index score were considered, those with s-ICAM-1 over 668 ng/ml also had worse TTF and OS.
Conclusions:
In DLBCL, s-ICAM-1 levels correlated with high tumor burden and lymphoma dissemination and may contribute to assessment of prognosis.
Key words: diffuse large B-cell lymphoma, prognosis, soluble intercellular adhesion molecule-1 (CD54)
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Introduction |
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There is some evidence that ICAM-1 is involved in neoplastic dissemination. It is expressed at high levels in solid tumors, such as hepatocellular carcinoma [5], melanoma [6] and bladder cancer [7], with this expression correlating with metastatic dissemination in these tumors. In B-cell lymphoproliferative disorders the tumoral expression of ICAM-1 is closely related to the degree of cell maturation [8, 9]. Thus, mantle-cell-derived lymphoproliferative diseases, such as chronic lymphocytic leukemia (CLL) or mantle-cell lymphoma, are often negative or weakly positive for ICAM-1, whereas ICAM-1 expression is more heterogeneous in germinal center cell lymphomas such as follicular or diffuse large-cell subtypes. It has been suggested that reduction of this molecule on the neoplastic lymphoid cells could impair the T-cell recognition with this contributing to neoplastic dissemination through a defective antitumor response [10]. In accord with this, we previously described a positive correlation between low expression of ICAM-1 and advanced stage, extranodal involvement, bone marrow infiltration, poor response to treatment and worse survival in patients with non-Hodgkins lymphoma, especially in the aggressive subgroup [11]. Recently, loss of ICAM-1 expression on lymphoma cells has been associated with decreased tumor-infiltrating T lymphocytes in diffuse large B-cell lymphoma (DLBCL) [12].
ICAM-1 can be shed from the cellular surface into serum, resulting in a soluble form (s-ICAM-1) which is also capable of recognizing its ligand LFA-1. Recently, several studies have pointed out the important prognostic value of serum levels of this molecule in Hodgkins disease [13], CLL [14] and non-Hodgkins lymphoma [15]. In the last setting, high ICAM-1 levels have been associated with adverse prognostic factors such as advanced stage, high ß2-microglobulin levels and poor response to treatment, specially in high-grade subgroups. However, most series contain several histological subtypes making the interpretation of results difficult.
The aim of the present study was to determine the serum levels of ICAM-1 at diagnosis in a series of patients consecutively diagnosed with DLBCL in our institution, and to correlate them with biological and clinical features, as well as response to treatment and outcome.
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Patients and methods |
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Median age of the series was 62 years (range 2296). Twenty-nine patients (53%) were male. In all cases the diagnosis was based on histological criteria according to the REAL/WHO classification. The main initial characteristics of the patients are listed in Table 1. Advanced Ann Arbor stage was observed in 28 patients (51%) and extranodal involvement in 32 patients (58%), including bone marrow infiltration in five patients (9%). Nineteen cases had high or highintermediate risk according to the international prognostic index (IPI) [16]. The median follow-up of the alive patients was 40 months (range 14113).
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Determination of serum ICAM-1 levels
Serum samples from patients with DLBCL were obtained at diagnosis before treatment. Moreover, serum samples from 30 healthy donors were obtained in order to have a normal value. The soluble ICAM-1 (s-ICAM-1) levels in serum were determined by enzyme immunoassay kits purchased from R&D Systems (Abingdon, UK). This assay is based on the simultaneous reaction of s-ICAM-1 present in the sample or standard to two antibodies directed against different epitopes on the s-ICAM-1 molecule. The capture antibody is coated onto the walls of the microtiter wells and the other is conjugated to the enzyme horseradish peroxidase. s-ICAM-1 present in the serum forms a bridge between the two antibodies. After removal of unbound material by aspiration and washing, the amount of conjugate bound to the well is detected by reaction with a substrate (tetramethylbenzidine) specific for the enzyme, which yields a colored product proportional to the amount of conjugate, and thus s-ICAM-1 present in the serum. The colored product is quantified using a spectrophotometer set a 450 nm with a correction wavelength of 620 nm. By analyzing standards of known s-ICAM-1 concentration coincident with samples and plotting a curve of signal versus concentration, the concentration of unknown samples could be determined. The sensitivity of the assay was <0.35 ng/ml, the inter-assay coefficient of variation (CV) was <8%, and the intra-assay CV varied from 3.3% to 4.8%.
Statistical analysis
s-ICAM-1 levels are presented as mean ± standard deviation (SD). Comparison of s-ICAM-1 levels and clinical and biological variables were made by the MannWhitney U-test. Survival curves were built by the KaplanMeier method [19], and the curves compared with the long-rank test [20]. Univariate and multivariate analysis for response and survival were made by a logistic regression and the Cox regression model [21], respectively. A two-sided P value of 0.05 was considered to be statistically significant.
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Results |
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s-ICAM-1 and clinical features
The correlations between s-ICAM-1 levels and the main clinical and biological features are detailed in Table 2. s-ICAM-1 levels correlated with some factors depending on the patient: those with B symptoms had significantly higher levels of s-ICAM-1 than the others (P = 0.034). Those with non-ambulatory performance status also had higher levels of s-ICAM-1, although this difference did not reach statistical significance (P = 0.09). No correlation was observed between s-ICAM-1 levels and age.
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s-ICAM-1, response to treatment and outcome
In the univariate analysis, there was a significant negative correlation between the levels of s-ICAM-1 and achievement of a CR as either a continuous or dichotomized variable. Patients with low levels of s-ICAM-1 reached CR more frequently than those with normal s-ICAM-1 (CR rates 82% versus 42%, respectively; P = 0.023). When a logistic regression for response achieved was performed including IPI, ß2-microglobulin and s-ICAM-1 levels, only s-ICAM-1, as both a continuous or dichotomized variable, was predictive for response [relative risk (RR) 2.37, 95% confidence interval (CI) 1.035.48; P = 0.008].
Three-year time to treatment failure (TTF) and survival of the whole series were 50% and 49%, respectively. As both a continuous or dichotomized variable, s-ICAM-1 levels correlated with outcome. Patients with levels of s-ICAM-1 over 668 ng/ml (percentile 75) had a shorter TTF than the others (3-year TTF 59% versus 20%, respectively; P = 0.01) (Figure 1A). Moreover, patients with low s-ICAM-1 levels showed longer overall survival (OS) then the remainders (3-year OS, 58% versus 22%, respectively; P = 0.04) (Figure 1B). When only patients with low IPI scores were selected, patients with s-ICAM-1 levels over 668 ng/ml also had a shorter TTF (3-year TTF 67% versus 14%; P = 0.0002) (Figure 2A) and survival (3-year OS 68% versus 28%; P = 0.006) than the remainders (Figure 2B). In the univariate analysis, additional variables with prognostic value for survival were: advanced stage, LDH, ß2-microglobulin, performance status, extranodal involvement and the IPI. Differences in CR rate, 3-year TTF and 3-year OS based on each parameter of the IPI are detailed in Table 3. In the Cox regression analysis performed by pairs, s-ICAM-1 retained its prognostic significance apart from advanced stage, LDH, ß2-microgobulin and the IPI (low/lowintermediate versus high/highintermediate). When this test was performed taking into account IPI score (low/lowintermediate risk versus high/highintermediate), ß2-microglobulin and s-ICAM-1(<668 versus >668 ng/ml), only s-ICAM-1 retained its prognostic significance (RR 4.45, 95% CI 1.38114.28; P = 0.01).
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Discussion |
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More recently, a large number of biological factors have also been described. A high proliferation rate evaluated by the expression of Ki-67 antigen on tumor cells identified a group of patients who had a very poor outcome [22]. Bcl-2 protein expression also impacts negatively on survival [23]. Among the soluble adhesion molecules, patients with high s-CD44 levels showed a significantly decreased overall and progression-free survival [24] compared with those with low levels. All these factors contribute to the assessment of prognosis in these patients, as they reflect biological aspects intrinsic to tumor biology, such as growth rate and sensitivity to chemotherapy, as well as other more complex aspects such as dissemination.
ICAM-1 is an adhesion molecule implicated in many physiological functions: it participates in the lymphocyte recirculation and homing and also play a role in the immune response as an adhesive and co-stimulator molecule. Low expression of ICAM-1 has been correlated with dissemination in low-grade lymphoproliferative disorders [25]. In our previous study, its absence or low positivity was associated with advanced stage, bone marrow infiltration and an adverse clinical course in diffuse large cell lymphoma [11]. A soluble form of this molecule has been identified and high levels of serum s-ICAM-1 correlated with adverse prognostic factors in non-Hodgkins lymphoma [2629], CLL [13] or Hodgkins disease [15]. In the last, s-CD54 was identified as an independent prognostic factor from other well-known factors in this entity. In high-grade non-Hodgkins lymphoma, Christiansen et al. [14] found a correlation between s-CD54 levels and disease activity as well as tumor burden and dissemination. Nevertheless, this group included patients of different histologies and phenotypes. In agreement with this, but limited to the DLBCL subtype, we report on a significant correlation between high levels of s-ICAM-1 and B symptoms, advanced stage, and increased levels of LDH and ß2-microglobulin. Also in agreement with Christiansen et al., we did not find any association with age or extranodal involvement.
In non-Hodgkins lymphoma, as a dichotomized variable, patients with increased levels of s-ICAM-1 had a shorter survival compared with those with low levels [14, 29], but these data have not been confirmed either as a continuous variable or in the multivariate analysis [14]. On the other hand, in primary extranodal lymphomas [25], high serum s-ICAM-1 associated significantly with shorter disease-free survival and OS, in both univariate and multivariate analysis. In our series, patients with levels of s-ICAM-1 higher than the 75th percentile had a worse prognosis in terms of TTF and survival. In DLBCL, serum ß2-microglobulin adds prognostic information to the IPI system [30], but it is not always determined in all institutions. In our series, in the multivariate analysis, s-ICAM-1 levels retained its prognostic significance apart from the IPI and ß2-microglobulin, although these data need to be confirmed in larger series due to the small number of patients. As has been suggested by other authors [31, 32], the performance status, the serum LDH value and extranodal involvement (two or more) are the main discriminating factors in terms of CRs and 3-year TTF and OS. Furthermore, s-ICAM-1 seemed to identify a group of patients with a worse outcome among those with low risk in the IPI system. All, these data suggest that this molecule adds prognostic significance, probably dependent on biological aspects of the disease.
The origin of s-ICAM-1 and its physiological role are not completely known. It is probably shed from the cell surface of activated endothelium and hematopoietic cells and retains its functional capability to recognize its ligand, LFA-1, present on the lymphocyte membrane. High levels of s-ICAM-1 may adhere to lymphocyte LFA-1, making neoplastic cells capable of avoiding the control of the immunological system. On the other hand, adhesion to bone marrow or lymph node extracellular matrix through ß1- and ß2-integrins impairs the apoptotic process, prolonging cell survival and contributing to chemoresistance in several lymphoid malignancies such as multiple myeloma [33] and CLL [34, 35]. This could also occur in diffuse large cell lymphoma, as it has been suggested by Fornarini et al. [36], although it has not been fully demonstrated.
In conclusion, in DLBCL, high serum levels of s-ICAM-1 correlate with disease activity, tumor burden and dissemination, low rate of response and adverse prognosis in terms of TTF and OS. Quantification of s-ICAM-1 levels may identify a subgroup of patients with worse prognosis in both the whole group and the low/lowintermediate risk group of the IPI.
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Acknowledgements |
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Footnotes |
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