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Keywords:

  • P2X7 receptor genotype;
  • B-cell chronic lymphocytic leukaemia;
  • Bcl-2 family expression;
  • in vitro drug resistance;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

Summary. A cohort of 121 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated for a single nucleotide polymorphism in the P2X7 receptor gene (1513 A[RIGHTWARDS ARROW]C), and the findings were correlated with clinical prognostic markers, in vitro sensitivity to fludarabine, expression of Bcl-2 family proteins and overall survival. The frequency of the polymorphism in B-CLL samples was not significantly different from that found in normal healthy controls (P = 0·27; Fisher's exact test). Furthermore, when the B-CLL patients were analysed according to P2X7 genotype (1513 A/A versus 1513 A/C), there was no significant difference in age at diagnosis, stage at diagnosis, lymphocyte doubling time, time to first treatment, progression-free survival and overall survival, and neither was there any evidence of bias in terms of VH gene mutational status, CD38 expression, in vitro sensitivity to fludarabine or expression of Bcl-2, Bax or Mcl-1 between the two groups. These results indicate that the 1513 A[RIGHTWARDS ARROW]C polymorphism of the P2X7 gene is unlikely to play a significant role in the pathogenesis or disease progression of B-CLL.

B-cell chronic lymphocytic leukaemia (B-CLL) is the most common leukaemia in the western world and is a quintessential example of a malignant condition that is caused by a failure of apoptotic cell death rather than a loss of proliferative control (Reed, 1998). It is typically characterized by the accumulation of CD5+ B lymphocytes in the blood, bone marrow, lymph nodes and spleen that are immunologically incompetent and are largely blocked in G0/G1 of the cell cycle (Foon et al, 1990). The clinical course of CLL is variable and remains difficult to predict. However, once disease progression is encountered and treatment is required, it is likely that drug resistance will ensue (Rozman & Montserrat, 1995).

Traditionally, B-CLL has been regarded as a neoplastic proliferation of naïve B cells of the mantle zone, but this view is now being revised in the light of data relating to somatic mutation of immunoglobulin variable heavy-chain (VH) genes. It is now well established that B-CLL is composed of two distinct subsets with either mutated or unmutated VH genes; cases with mutated VH genes having a more favourable prognosis and requiring less treatment than unmutated cases (Damle et al, 1999; Hamblin et al, 1999; Kröber et al, 2002; Oscier et al, 2002). In addition, there is growing evidence of a correlation between VH gene mutational status and CD38 expression in CLL, in which unmutated cases possess at least a subpopulation of CD38+ cells (Damle et al, 1999; Ghia et al, 2003).

The purinergic P2X7 receptor has recently become the focus of several studies in B-CLL cells, with one study emphasizing its ability to promote cellular growth (Adinolfi et al, 2002) and another study outlining the proapoptotic function of this receptor (Wiley et al, 2002). A single nucleotide polymorphism (1513 A[RIGHTWARDS ARROW]C) has been reported to be present in a proportion of cases of B-CLL resulting in a loss of function of the P2X7 receptor (Wiley et al, 2002). However, the incidence of this mutation and its relevance to the pathogenesis of B-CLL are still open to question. Wiley et al (2002) found that the frequency of the polymorphism was higher in B-CLL patients with indolent disease compared with normal individuals and suggested that the loss of function of this receptor might have an antiapoptotic effect, resulting in the accumulation of leukaemic clones. In contrast, the study by Thunberg et al (2002) found a significantly higher frequency of the polymorphism in normal controls compared with B-CLL patients with no bias in frequency of the 1513C allele between B-CLL patients with mutated or unmutated VH genes. Interestingly, overall survival was found to be significantly longer in B-CLL patients who were heterozygous for the A[RIGHTWARDS ARROW]C polymorphism compared with those whose genotype was homozygous for 1513A. In the present study, we analysed 121 B-CLL patients for P2X7 genotype and correlated these findings with a range of clinical parameters. As drug resistance is frequently encountered in those patients with progressive disease, we decided to investigate whether in vitro drug resistance to fludarabine correlated with P2X7 receptor genotype. In addition, as both in vitro and in vivo drug resistance has previously been associated with Bcl-2 family protein expression (Robertson et al, 1996; Pepper et al, 1997; Kitada et al, 1998), we quantified the ex vivo expression of Bcl-2, Bax and Mcl-1 and compared their expression patterns between the two subsets of our B-CLL patient group selected by genotype (1513 A/A versus 1513 A/C plus 1513 C/C).

Isolation of leukaemic lymphocytes.  Peripheral blood samples from 121 patients with B-CLL attending the clinic at Birmingham Heartlands Hospital (78 male and 43 female) were obtained with the patients' informed consent. A total of 39/121 (32%) had previously received a number of alternative therapeutic regimens, but none had been treated for at least 3 months before analysis. Patient selection was based purely on a definitive diagnosis of B-CLL and patient consent. Clinical staging was based on the Binet system (Binet et al, 1981). The median age at diagnosis was 68 years (range 36–92 years), and the median follow-up was 66 months (range 11–201 months). Peripheral blood was also obtained from 95 normal healthy individuals in order to determine the frequency of the 1513C polymorphism in individuals who did not have B-CLL. These samples were derived from antenatal clinics and from patients requiring surgery for non-diabetic cataracts (61 females and 34 males).

Genotyping of the P2X7 polymorphism.  DNA was extracted from 121 B-CLL patients and 95 anonymized age-matched healthy controls using a standard salting-out method (Miller et al, 1988). Polymerase chain reaction (PCR) of the P2X7 exon 13 was carried out as described previously (Gu et al, 2001), with modification of the cycling conditions (denaturing at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min). Subsequently, two 10 µl aliquots were taken from the PCR product. The first was digested using HhaI restriction enzyme (New England Biolabs, Hitchin, UK), as the 1513C allele introduces an additional restriction site (Thunberg et al, 2002). Digested products were run on an 8% polyacrylamide gel on a Hoeffer mighty Small II at 250 V for 1 h. The gel was stained with ethidium bromide and visualized using an ultraviolet transilluminator (Fig 1A). The second aliquot was digested using BseRI restriction enzyme (New England Biolabs) according to the manufacturer's instructions; the 1513C allele abolishes the restriction site and only the 1513A allele is cut. The digested products were run on a 2% agarose gel, stained with ethidium bromide and visualized (Fig 1B). The two restriction enzymes were used in parallel to provide complementary results as one cuts the 1513A allele while the other cuts the 1513C.

image

Figure 1. (A) Polyacrylamide gel (8%) of PCR products derived from amplification of exon 13 of the P2X7 gene after digestion using the HhaI restriction enzyme. Only those products that contain the 1513C allele are cut by this enzyme. (B) Agarose gel (2%) of PCR products from the same amplification reaction but digested using the BseRI restriction enzyme; this enzyme cuts those products that contain the 1513A allele. The contents of each lane are shown on the right.

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VH gene mutational analysis. The VH gene mutational status of 62/121 patients was analysed according to the method described previously (Stankovic et al, 2002). Samples from all 62 patients were assessed for VH gene mutational status as part of this study regardless of whether they had been analysed previously. The resulting PCR products were sequenced, and the sequences with a germline homology of 98% or higher were considered unmutated and those with a homology less than 98% mutated.

Cell culture conditions.  Freshly isolated peripheral blood lymphocytes (1 × 106/ml) were cultured in minimum essential medium (Invitrogen, Paisley, UK) supplemented with penicillin, streptomycin and 10% fetal calf serum (FCS). Cultures were incubated for 48 h at 37°C in a humidified 5% carbon dioxide atmosphere in the presence of fludarabine (1 × 10−7, 2·5 × 10−7, 5 × 10−7, 7·5 × 10−7, 1 × 10−6, 2·5 × 10−6 and 5 × 10−6 M). In addition, control cultures were carried out in which no drug was added to cultured B-CLL cells. Cells were subsequently harvested by centrifugation, and apoptosis was quantified by flow cytometry using the methods outlined below. All experiments were performed in duplicate.

Measurement of in vitro apoptosis.  In this study, changes in forward light scatter (FSC) and side light scatter (SSC) characteristics were used to quantify apoptotic and viable cell populations as described previously (Pepper et al, 1999). Typically, lymphocytes show a reduction in FSC (a function of cytoplasmic shrinkage) and an increase in SSC (due to increased granularity) when they undergo apoptosis (Ferlini et al, 1996). All LD50 values (the concentration of fludarabine required to kill 50% of the cells) were derived from dose–response curves. Duplicate samples were assessed using fluorescein isothiocyanate (FITC)-labelled annexin V (Dako, Ely, UK) to confirm the presence of apoptotic cells in the cell cultures and to validate the FSC/SSC quantification method (Vermes et al, 1995).

Flow cytometric analysis of Bcl-2, Bax, Mcl-1 and CD38 protein expression.  Samples from 93/121 patients were analysed by triple immunofluorescence using CD5-FITC-, CD38-phycoerythrin (PE)- and CD19-PE cyanin 5 (Cy5)-conjugated antibodies and by dual immunofluorescent staining using combinations of Bcl-2, Bax and Mcl-1 antibodies in conjunction with CD19 (pan B-cell marker). Briefly, 1 × 106 cells were incubated with 10 µl of anti-CD19-PE Cy5-conjugated antibody or an isotype-matched negative control (Dako). The cells were then fixed using a commercially available kit (Dako) and resuspended in permeabilization solution together with titration-determined volumes of each antibody or isotype-matched negative control, i.e. Bcl-2-FITC (Dako), Bax and Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, a FITC-labelled secondary antibody was added to the Bax- and Mcl-1-labelled cells (Dako). The cells were then resuspended in 0·5 ml of 1% paraformaldehyde before flow cytometric analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). From each sample, at least 10 000 cells were analysed, and non-specific binding was excluded by gating using the isotype-negative control antibodies. Gating of the CD19+ (B cells) was performed in the analyses in order to quantify the apoptosis and specific protein expression in leukaemic B-lymphocyte subpopulations. The mean fluorescent intensity (MFI) was calculated for each individual protein using winmdi software (J. Trotter, Scripps Research Institute, La Jolla, CA, USA).

Statistical analysis.  Patients were grouped into two subsets depending on genotype (1513 A/A and 1513 A/C plus 1513 C/C), and statistical analysis was performed using parametric and non-parametric tools depending on whether the data obeyed a Gaussian distribution. CD38 expression, in vitro sensitivity to fludarabine and Bcl-2 family expression were analysed using the Student's t-test. Comparison of the allele frequency of the 1513 A[RIGHTWARDS ARROW]C polymorphism and the VH gene mutation status of the 1513 A/A and 1513 A/C plus 1513 C/C genotype subgroups was calculated by the Fisher's exact test. Stage at diagnosis and lymphocyte doubling time (greater or less than 12 months) was analysed by the chi-square test, and all other parameters were analysed using the Mann–Whitney U-test. Survival analysis was calculated according to the Kaplan–Meier method. All statistical analyses were performed using graphpad prism 3·0 software (Graphpad Software, USA).

Frequency of the 1513C polymorphism in B-CLL patients and healthy controls

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

In this study, 36 (29·8%) of the 121 B-CLL patients were found to be heterozygous for the 1513C allele, three (2·4%) were homozygous for the 1513C allele and 82 (67·8%) were homozygous for the 1513A genotype. The frequency of A[RIGHTWARDS ARROW]C polymorphism in this study bisected the two previously published studies of Wiley et al (2002) and Thunberg et al (2002). In contrast to the two previously published studies (Thunberg et al, 2002; Wiley et al, 2002), the frequency of the 1513C allele in 95 age-matched healthy controls [31 (33%) 1513 A/C and 5 (5%) 1513 C/C] was not significantly different from that found in the B-CLL patient group (P = 0·27; Fisher's exact test). The frequency data for the B-CLL patients and normal healthy control group are given in Table I. There was no sex bias in the frequency of the 1513C allele in either the B-CLL patient group or the normal healthy control group.

Table I.  Frequency of the P2X7 gene polymorphism (1513 A[RIGHTWARDS ARROW]C) in patients with B-CLL and healthy controls.
GenotypeB-CLL (n = 121)Controls (n = 95)
  • *

    P  = 0·27 calculated by Fisher's exact test.

1513 A/A83 (69%)59 (62%)
1513 A/C35 (29%)31 (33%)
1513 C/C3 (2%)5 (5%)
1513C allele frequency*  0·174  0·216

Clinical features and prognostic markers

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

The mean age at diagnosis was not significantly different between the homozygous (1513 A/A) genotype group and the heterozygous (1513 A/C) group (67·4 ± 10·6 years versus 64·1 years ± 10·8 years; P = 0·13). There was no significant difference between the stage at diagnosis, lymphocyte doubling time or the median follow-up time between the two groups (P = 0·85 and P = 0·47, chi-square test and P = 0·53, Student's t-test respectively). Furthermore, there was no significant difference between the time to first treatment, progression-free survival or overall survival (P = 0·93, 0·32 and 0·44 respectively; Mann–Whitney test). The data are summarized in Table II, and the overlaid Kaplan–Meier curves for overall survival of the heterozygous (1513 A/C) patient group and the homozygous (1513 A/A) patient group are shown in Fig 2.

Table II.  Characteristics of B-CLL patients according to P2X7 genotype.
 All patients1513 A/A genotype1513 A/C genotype
  1. LDT, lymphocyte doubling time; TTFT, time to first treatment; PFS, progression-free survival.

No. of cases1218335
Mean age (years)666764
Sex M/F76/4252/3123/12
Median follow-up (months)666763
Binet stage A/B/C82/19/1756/15/1226/4/5
LDT (< 12/> 12 months)14/788/436/35
Mean TTFT (months)4443·844·3
Mean PFS (months)60·262·856·3
image

Figure 2. Comparison of the overall survival curves of individuals with B-CLL carrying the 1513 A/A (n = 82) or the 1513 A/C (n = 36) genotype.

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The VH gene mutational status of B-CLL patients is an important prognostic marker, with those patients with unmutated VH genes having significantly shorter survival times. In this study, we found no significant difference in VH gene mutational status between the two P2X7 genotypes; 47% of B-CLL patients with the 1513 A/A genotype and 44% with the 1513 A/C genotype had unmutated VH genes (P = 0·75; Fisher's exact test). Furthermore, it has been suggested that the presence of CD38 clones, regardless of the size of the CD38 population, is a poor prognostic marker in B-CLL (Ghia et al, 2003). We therefore analysed (51/121 patients) whether there was a different pattern of CD38 expression between the homozygous (1513 A/A) patient group (n = 28) and the heterozygous (1513 A/C) group (n = 23). Figure 3 shows the percentage of CD38+ clones in each of the P2X7 polymorphic patient groups; there was no significant difference between the two groups (P = 0·38).

image

Figure 3. Comparison of the percentage of CD38-expressing clones in B-CLL samples taken from patients with the 1513 A/A (n = 28) or the 1513 A/C (n = 23) genotype.

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In vitro drug sensitivity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

Eighty-five of the 121 B-CLL samples were assessed for in vitro drug sensitivity to fludarabine (61 homozygous for the 1513A allele and 24 heterozygous for the 1513C allele). The characteristic changes in the FSC and SSC resulting from cellular shrinkage described previously were used to quantify apoptosis (Ferlini et al, 1996). In addition, annexin V labelling was also performed in order to verify the light scatter data. Apoptosis was induced in all 85 patient samples after exposure to fludarabine with a mean LD50 value (± SD) of 1·3 (± 1·2) × 10−6 mol/l. Figure 4 shows that there was no significant difference in the LD50 values between the two patient groups defined by the presence or absence of the 1513C allele (P = 0·79; Student's t-test).

image

Figure 4. Comparison of the in vitro sensitivity to fludarabine in B-CLL samples taken from patients with the 1513 A/A (n = 61) or the 1513 A/C (n = 24) genotype. Cytotoxicity was compared using LD50 values (± SD) derived from in vitro cultures of B-CLL cells exposed to fludarabine (1 × 10−7−5 × 10−6 mol/l) for 48 h.

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Ex vivo Bcl-2 family expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

Previous studies have shown that high expression of Bcl-2 and Mcl-1 and high Bcl-2/Bax ratios are associated with drug resistance both in vitro and in vivo (Robertson et al, 1996; Pepper et al, 1997; Kitada et al, 1998). In this study, patient samples (93/121) were assessed for ex vivo expression of Bcl-2, Bax and Mcl-1 in order to determine whether any of these parameters were significantly different between homozygous (1513 A/A) B-CLL patients and heterozygous (1513 A/C) B-CLL patients. The expression profile of all these apoptosis-regulating proteins was not found to be significantly different between the two patient groups (Fig 5A–D).

image

Figure 5. Comparison of the ex vivo Bcl-2 family protein expression in B-CLL samples taken from patients with the 1513 A/A (n = 59) or the 1513 A/ C (n = 28) genotype. (A) Bcl-2 protein expression; (B) Bax protein expression; (C) Bcl-2/Bax ratios; and (D) Mcl-1 protein expression.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References

Several recent studies have suggested that the presence of a single nucleotide polymorphism in the P2X7 gene in B-CLL may have prognostic significance (Thunberg et al, 2002; Wiley et al, 2002). However, the data presented in these papers is somewhat contradictory. Wiley et al (2002) suggested that a loss of function polymorphic mutation in the P2X7 gene might result in failed apoptotic signalling through this receptor and contribute to the pathogenesis of B-CLL. In this regard, they demonstrated a threefold greater frequency of the 1513C allele in their study of Australian B-CLL patients compared with healthy individuals. In contrast, in a much larger Swedish study, Thunberg et al (2002) found a significantly higher frequency of the 1513C allele in normal healthy controls compared with B-CLL patients. Overall, survival was significantly longer in the B-CLL patients with the 1513 A/C genotype compared with the 1513 A/A genotype. Furthermore, although the frequency of the 1513C allele was not significantly different between cases of B-CLL with mutated and unmutated VH genes, patients with mutated VH genes who also possessed the 1513C allele were shown to have a longer median survival than those with the 1513 A/A genotype. In the present study, we found no significant difference in the frequency of the 1513C allele in B-CLL patients and normal healthy controls (P = 0·27), and the 1513C allele had no prognostic impact, even in patients with mutated VH genes. Interestingly, the frequency of the 1513C allele in the B-CLL patient group in our study was not significantly different from that found in the Australian study (P = 0·23; Fisher's exact test). Furthermore, the frequency of the 1513C allele in the normal healthy control group was not significantly different from that found in the Swedish study (P = 0·14; Fisher's exact test). One explanation for the difference between our study and the Australian study in terms of the discrepancy in frequency of the 1513C allele in the control groups may be attributable to natural variation of the polymorphism within the gene pool from different geographical locations and the relatively small sample size of the Australian study. Furthermore, the difference in frequency of the 1513C allele between the B-CLL patients in the Swedish study and our study may be caused partly by a referral centre effect; most of the Swedish study patients presented with progressive disease with a significant numerical bias in the patient group with unmutated VH genes. Indeed, despite a shorter median follow-up compared with the present study (56 months versus 66 months), the Swedish study showed a 50% survival rate of 72 months for the 1513 A/A genotype and 104 months for the 1513 A/C genotype, whereas in our study, the 50% survival rate has not yet been reached for either genotypic group.

It has been suggested that the P2X7 receptor may have a function in growth promotion, and loss of function would correlate with a more indolent course of disease and longer survival. Alternatively, the P2X7 receptor may be required for efficient removal of lymphocytes from the circulation, and the poor function of the P2X7 receptor in those individuals who inherit the 1513C allele would favour lymphoaccumulation and lead to earlier diagnosis (Di Virgilio & Wiley, 2002). If the first hypothesis were true, then we would expect to see a bias in frequency of the 1513C allele in patients with mutated VH genes and a significantly increased overall survival in all patients who possessed the 1513C allele. This was not found to be the case in the study of Thunberg et al (2002) or in the present study. If the second hypothesis were true, then there would be a trend towards a younger age at diagnosis in those patients carrying the 1513C allele; again, this was not borne out in the present study.

Adinolfi et al (2002) suggested that increased expression and function of the P2X7 receptor might correlate with ‘evolutive’ disease. Presumably, this would require ‘evolutive’ patients to possess the 1513 A/A genotype because the presence of the 1513C allele would confer at least a partial loss of function to the receptor. However, in the present study, we found no evidence of a bias in ‘evolutive’ disease in those patients with the 1513 A/A genotype compared with those patients with the 1513 A/C genotype as measured by lymphocyte doubling time, time to first treatment or progression-free survival. In addition, the poor prognostic markers, as defined by unmutated VH genes and CD38 expression, were not significantly different in B-CLL patients with the 1513 A/A genotype and B-CLL patients with the 1513 A/C genotype. Furthermore, there was no significant difference between these two genotypic subpopulations in terms of in vitro sensitivity to fludarabine and no evidence of altered expression of the apoptosis-controlling Bcl-2 family proteins.

In conclusion, we have found no evidence that the P2X7 gene polymorphism 1513 A[RIGHTWARDS ARROW]C plays a significant role in the pathogenesis, disease progression or in vitro responsiveness to fludarabine in this cohort of 121 UK B-CLL patients.

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  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Frequency of the 1513C polymorphism in B-CLL patients and healthy controls
  6. Clinical features and prognostic markers
  7. In vitro drug sensitivity
  8. Ex vivo Bcl-2 family expression
  9. Discussion
  10. References
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