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Keywords:

  • chronic lymphocytic leukemia;
  • CD31;
  • CD38;
  • survival;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) is the ligand for CD38, a transmembrane glycoprotein that is expressed on the surface of leukemic cells in many patients with B-cell chronic lymphocytic leukemia (B-CLL). In a previous study, the authors showed that CD38 expression was correlated with a poor prognosis in patients with B-CLL. In the current study, blood samples from patients with B-CLL were examined to identify CD31 surface marker expression, and CD31 expression was correlated with several other known prognostic variables, including CD38.

METHODS

Using flow cytometry, peripheral blood samples from 120 patients with B-CLL were analyzed for CD31 and CD38 expression on CD19 positive leukemic B cells.

RESULTS

Thirteen of 120 patients (11%) had CD31 expression on < 20% of their B cells, and the remaining patients had various levels of CD31 expression. The median expression of CD31 was 76% of leukemic, CD19 positive cells. Levels of CD31 expression were not correlated with survival outcomes or with any of the known prognostic parameters when all patients were considered. Patients who had high CD38 expression (≥ 20%), as expected, had significantly shorter survival (P = 0.001) compared with patients who had low CD38 expression (< 20%). However, in patients with low CD38 expression, a subgroup with low CD31 expression (< 76%) had significantly longer survival compared with the survival for the entire group (P = 0.0001). Moreover, the survival pattern of patients with low CD38 expression and high CD31 expression was not significantly different from the survival pattern seen in patients with high CD38 expression.

CONCLUSIONS

CD31 expression further defined a subgroup of patients with B-CLL who had a different survival outcome. Defining the interaction between CD31 expression and CD38 expression in patients with CLL will require further exploration. Cancer 2003;97:1914–9. © 2003 American Cancer Society.

DOI 10.1002/cncr.11264

CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]) is a 130-kD member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, neutrophils, monocytes, and naïve B-lymphocytes.1–3 The extracellular portion is comprised of 574 amino acids and is organized into 6 Ig-like homology domains, each of which is encoded by a single exon.4–6 It has a short, single-pass transmembrane domain. The intracellular domain contains 118 amino acids encoded by 8 different exons that can be spliced alternatively, creating several isoforms. CD31 plays a key role in determining the number of adhesion-mediated biologic events. The expression of CD31 varies at different stages of B-cell maturation.1 Deaglio and colleagues identified CD31 as the ligand for CD38.7

Several reports have shown that human neoplastic cells are capable of expressing a receptor and its ligand.8–10 The clinical and biologic significance of this observation is not clear. However, a preferential survival advantage of the neoplastic cells has been suggested. An example of that observation is human myeloma cells that coexpress CD38 and its ligand, CD31.8 Similarly, it has been reported that B cells in patients with chronic lymphocytic leukemia (CLL) express CD40 and its ligand, CD154.9, 10 This coexpression serves as an autocrine stimulation loop, which prolongs B-cell survival and allows evasion of death by the CD95 (Fas) pathway.

Recent studies examined B cells in patients with CLL to determine expression levels of CD38 and the presence or absence of Ig gene mutation.11–16 Although the correlation between Ig gene mutation and CD38 expression is controversial, the correlation between CD38 expression and prognosis is not. Our group and others have shown that CD38 expression predicts poor outcome in patients with B-cell CLL (B-CLL).

In this study, we examined peripheral blood from patients with B-CLL for coexpression of CD38 and its ligand, CD31. We also correlated this expression with various clinical characteristics and outcome. CD31 expression further identified a subgroup of patients with CLL who had more aggressive disease among the patients with CD38 negative disease.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patient Population

One hundred twenty patients presented to The University of Texas M. D. Anderson Cancer Center (M. D. Anderson Cancer Center) were selected randomly. All samples were collected at the time of presentation to M. D. Anderson Cancer Center. Diagnosis was confirmed by studying peripheral blood samples and bone marrow samples using morphology, immunophenotyping, cytogenetics, and molecular studies (Ig heavy-chain and light-chain gene rearrangement and T-cell receptor rearrangement). Complete clinical data for patients were recorded from the time of presentation to M. D. Anderson Cancer Center. Informed consent was obtained from all patients, and samples were collected according to protocol approved by the M. D. Anderson Institutional Review Board.

Measurement of CD31 and CD38 Expression by Flow Cytometry

Cryopreserved peripheral blood samples obtained at time of diagnosis were used for flow cytometry analysis. Samples were prepared using a whole-blood lysing technique and a three-color staining method. Isotype-matched, negative control antibodies were used to separate positive cells from negative cells. Directly labeled monoclonal antibodies against the surface antigens CD19 peridinin chlorophyll protein, CD38 allophycocyanins, and CD31 phycoerythrins were used (Becton Dickinson Immunocytochemistry Systems, San Jose, CA). Immunophenotyping expression was measured with a FACScaliber device (Becton Dickinson). CD31 and CD38 expression levels in the CD19 positive, gated population, were expressed as the percent of positive cells. Fresh and frozen samples from each of 10 patients were analyzed to test the effect that freezing had on surface expression of CD19, CD31, and CD38. No significant changes in CD19, CD31, or CD38 expression levels were detected.

Statistical Analyses

A Cox proportional hazards model was used to evaluate possible associations between overall survival and each risk factor in univariate and multivariate analyses. Kaplan–Meier survival curves and log-rank tests also were used to compare survival between groups. Survival was measured from the test date to the last follow-up date (censored) or to the date of death by any cause. The variables examined were age, gender, Rai and Binet stages, splenomegaly, hepatomegaly, hemoglobin level, serum β2 microglobulin level, white blood cell count, platelet count, lymphocyte count in the peripheral blood, lymphocyte percentage in bone marrow, cellularity of bone marrow, number of lymph node sites involved by disease, history of prior therapy, and survival. All data were collected from reviewing the patients' records and were entered into a data base. Spearman, Wilcoxon, and Kruskall–Wallis tests were used for testing correlations and for performing nonparametric hypotheses tests between subgroups.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

CD31 Expression Profiles of Patients with CLL

Patient characteristics are summarized in Table 1. Overall, our patient population was representative of the types of patients with B-CLL seen at our referral center. The ratio of men to woman was 1.5:1.0, and the median patient age was 63.2 years. Seventy-eight percent of patients has Rai and Binet Stage 0–II disease, and 64% of patients were untreated. CD31 expression varied between 0% and 100% (Fig. 1). Thirteen patients (11%) had CD31 expression in < 20% of their B cells. The median expression level was 76% of CD19 positive B-CLL cells (Fig. 1). Figure 2 shows no correlation in the pattern of CD31 expression and CD38 expression in patients with B-CLL.

Table 1. Clinical Characteristics of Patients with B-Cell Chronic Lymphocytic Leukemia
CharacteristicNo. of patients (%)
Age (yrs) 
 Mean63.2
 Range30–83
Gender 
 Male72 (60)
 Female48 (40)
White blood cells (× 103/μL) 
 Median62
 Range3–652
Lymphocytes (%) 
 Median86
 Range24–99
Platelets (× 103/μL) 
 Median152
 Range12–541
Hemoglobin (g/dL) 
 Median12.9
 Range4.0–16.8
β2 microglobulin (mg/dL) 
 Median3.0
 Range1.3–8.2
Bone marrow cellularity (%) 
 Median50
 Range20–95
Bone marrow lymphocytes (%) 
 Median79
 Range22–97
Rai and Binet stage 
 0–II94 (78)
 III–IV26 (22)
Lymph nodes involved 
 0–159 (49)
 2–3 or more61 (51)
No history of prior therapy 
 Hepatomegaly14 (12)
 Splenomegaly31 (26)
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Figure 1. Representative flow cytometry profiles of CD31 expression and CD38 expression in patients with B-cell chronic lymphocytic leukemia. (A) A profile that is strongly positive for CD31 and partially positive for CD38 expression. (B) A profile that is negative for CD31 expression and positive for CD38 expression. (C) A profile that is positive for CD31 expression and negative for CD38 expression. (D) A profile that is positive for CD31 expression and positive for CD38 expression. The antibodies used were labeled directly for CD19 peridinin chlorophyll protein (CD19 PerCP), CD31 phycoerythrins (CD31 PE), and CD38 allophycocyanins (CD38 APC).

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Figure 2. There was no correlation between percentages of CD31 positive and CD38 positive B cells in patients with B-cell chronic lymphocytic leukemia. R: correlation coefficient.

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Comparison of CD31 Expression with Other Prognostic Parameters

No correlation was found between the level of CD31 expression and Rai and Binet staging, splenomegaly, age, gender, white blood cell count, number of circulating lymphocytes, and level of bone marrow involvement. There was no correlation between CD31 expression and CD38 expression (Fig. 2).

Clinical Relevance of CD31 Expression

Using a Cox proportional hazards regression model with CD31 as a continuous variable, we found no significant correlation between CD31 expression and survival. Using cut-off points (median, upper, and lower quartiles) also showed no correlation with survival (Fig. 3A). In contrast to CD31, as expected, high CD38 expression (Fig. 3B) was correlated with poor survival outcomes. However, because CD31 expression represents a ligand for CD38, and because each molecule may influence the other's function, we divided patients into four groups according to their CD31 and CD38 expression levels as follows: low CD31/low CD38, low CD31/high CD38, high CD31/low CD38, and high CD31/high CD38. We used 20% as a cut-off point for CD38 expression and 76% (median) as a cut-off point for CD31 expression. Figure 4A,B shows that patients with the immunophenotype low CD31/low CD38 showed significantly better survival outcomes compared with other groups (P = 0.002). Unexpectedly, high CD31 expression/low CD38 expression identified a group of patients who had significantly shorter survival outcomes compared with patients who had low CD31 expression/low CD38 expression (P = 0.006). In fact, the survival pattern in patients with low CD38 expression/high CD31 expression was not significantly different from what was seen in patients with high CD38 expression (Fig. 4C). Similar results were obtained when we analyzed previously untreated patients only. Previously untreated patients with low CD38 expression/low CD31 expression had significantly longer survival compared with all other patients (P = 0.014) (Fig. 5A). There was no difference in survival among the previously untreated group between patients with low CD38 expression (< 20%) but with high CD31 expression (> 76%) and patients with high CD38 expression, irrespective of their CD31 expression levels (P = 0.66) (Fig. 5B).

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Figure 3. (A) Kaplan–Meier survival curve showing no significant difference between patients with B-cell chronic lymphocytic leukemia (B-CLL) who had high CD31 expression levels (> 76%) and patients who had low CD31 expression levels (< 76%). The 76% expression level represents the median in the tested patients. (B) Kaplan–Meier survival curve comparing patients with B-CLL with CD38 positive samples (≥ 20%) and CD38 negative samples (< 20%). The difference was significant (P = 0.001).

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Figure 4. (A) Kaplan–Meier survival curve, based on variable CD31 and CD38 expression levels, comparing the length of survival among patients with B-cell chronic lymphocytic leukemia (B-CLL). Patients with low CD31 expression/low CD38 expression had significant differences in survival compared with other groups (P = 0.002). (B) Kaplan-Meier survival curve based on patients with low CD31 expression/low CD38 expression compared with all other groups combined. Patients with low CD31 expression/low CD38 expression had significantly longer survival compared with all other patients (P = 0.0001). (C) Patients with high CD31 expression/low CD38 expression showed no significant difference in survival compared with patients who were positive for CD38 expression, regardless of their CD31 expression status (P = 0.09).

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Figure 5. (A) Kaplan–Meier survival curve showing that previously untreated patients with low CD31 expression/low CD38 expression had a significantly longer survival compared with all other groups. (B) Previously untreated patients who had high CD31 expression levels but low CD38 expression levels had no significant difference in survival compared with patients who had CD38 positive chronic lymphocytic leukemia, regardless of their CD31 expression status.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The overall objective of this study was to examine the expression of CD31 and CD38 surface markers and to correlate the findings with clinical characteristics and survival outcomes in patients with B-CLL. We found that most patients with B-CLL had CD31 expression in most CLL cells. Overall, the median expression was 76% in CD19 positive B cells. Only 11% of patients had CD31 expression in < 20% of CD19 positive cells. CD31 expression did not appear to be an independent prognostic factor for survival outcomes. However, patients who had low CD38 expression/low CD31 expression had better outcomes compared with all other groups of patient with B-CLL. Patients with high expression levels of CD31 showed no significant difference in survival compared with patients who had high expression levels of CD38. This also was true when we analyzed only previously untreated patients.

CD31 was cloned and assigned to the Ig superfamily in 1990.3 It is expressed on endothelial cells, and its role as an adhesion molecule has been reported widely.17, 18 It also is expressed on a variety of hematopoietic cells, including neutrophils, monocytes, platelets, and lymphocytes.1, 2, 19, 20 However, 70% of naïve human B cells express CD31 at high levels.1 These B cells display a germline configuration of Ig variable-region genes.1 CD31 plays a significant role in hematopoietic cell adhesion and trafficking through endothelial cells, and it functions as a receptor for CD38.7 Analysis of the levels of expression of CD31 and CD38 may help in distinguishing patients with CLL who have naïve B cells with unmutated Ig from patients with mutated Ig; however, further study is needed.

We do not know why patients who have B-CLL with low expression levels of CD31 and CD38 are associated with a good prognosis. To our knowledge, only one abstract has been published comparing CD31 expression in normal peripheral blood B cells and B-CLL cells. In that abstract, a significant increase in CD31 expression was observed in neoplastic B cells compared with normal cells.21 Coexpression of both markers may allow a specific level of stimulation and may permit critical signal-transduction events that allow transendothelial trafficking. These events may provide a survival advantage to the malignant cells. High expression of CD31 on B-CLL cells, by itself, was not associated with a statistically significant difference in survival, whereas high CD38 expression did have such an association. However, low coexpression of both markers was associated with better survival outcomes. It is possible that low expression of both CD31 and CD38 is a better predictor of memory B cells and Ig gene mutation compared with low expression of CD38 alone. This remains to be investigated. At this point, our data suggest that both CD31 and CD38 should be tested in patients with B-CLL to separate those with low CD31 expression and CD38 expression levels, because those patients appear to have a fairly indolent course.

More recently, Jelinek and colleagues confirmed the prognostic value of CD38 surface-marker expression in patients with CLL; however, CD38 expression was independent of Ig gene mutation status.16 CD31 expression status adds complexity to the prognostic model, as determined by CD38 and Ig mutation, although it remains to be seen whether CD31 in combination with CD38 can replace the need for studying Ig mutation status, which is a complex and expensive procedure.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES