CD38 expression as a prognostic indicator in chronic lymphocytic ...

The Hematology Journal (2004) 5, 145–151 & 2004 The European Hematology Association All rights reserved 1466-4680/04 $25.00 www.nature.com/thj

CD38 expression as a prognostic indicator in chronic lymphocytic leukaemia Patrick D Thornton1, Cristina Fernandez1, Giada M Giustolisi1, Ricardo Morilla1, Shayne Atkinson1, Roger P A’Hern1, Estella Matutes1 and Daniel Catovsky*,1 1

Academic Department of Haematology and Cytogenetics, Institute of Cancer Research and Royal Marsden NHS Trust, London, UK

Staging systems and laboratory features help predict survival in chronic lymphocytic leukaemia but they do not distinguish patients who will progress from those whose disease will remain indolent. CD38 expression has emerged as an independent prognostic factor, yet there is debate as to what level of CD38 affects prognosis. We plotted the hazard ratios for the treatment-free interval (TFI) between the higher and lower groups defined by CD38 cut-offs from 0 to 100%. The maximum hazard ratio was achieved for a cut-off of 7%. We examined by triple colour analysis the values for CD38 in 289 untreated patients using both X30 and X7% as thresholds for prognosis. Using a X7% threshold (but not X30%), we showed a significant correlation with advanced stage and male sex. The interval from diagnosis to first therapy or TFI was longer (median 36 months) in patients with o7% CD38 positive cells than those with X 30% (8.7 months) or with intermediate values between 7 and 29% (Po0.00005). The o7% threshold also identified patients in stage A with a long TFI (P ¼ 0.0001). Multivariate analysis showed that CD38 has independent prognostic value for TFI in all patients. In 135 patients tested for deletions of p53, 13q14 and 11q23 and for trisomy 12, we showed a correlation between 13q14 deletion and o30%/o7% CD38 positive cells and a tendency for trisomy 12 to be associated with X30%/X7% CD38 positive cells. We conclude that 7% may be a more useful threshold for disease progression than higher values of CD38. The Hematology Journal (2004) 5, 145–151. doi:10.1038/sj.thj.6200360 Keywords:

CD38; CLL; prognosis

Introduction Chronic lymphocytic leukaemia (CLL) is the most commont leukaemia in the Western world.1 It is characterised by the uncontrolled proliferation of mature B-lymphocytes with a distinct morphology and immunological profile.2 CLL has a highly variable clinical outcome, with some affected individuals having a survival of months to a few years, while others have almost normal lifespan. According to the NCI-WG guidelines, patients with advanced stage require treatment but not necessarily patients with early-stage disease.3 The two most widely used staging systems by Rai and Binet have excellent prognostic value,4–6 and remain the strongest predictors of survival.7,8 However, neither system distinguishes early-stage patients who will progress from those whose disease will remain indolent and will not benefit from early treatment. The *Correspondence: Daniel Catovsky, Academic Department of Haematology and Cytogenetics, The Royal Marsden NHS Trust, 203 Fulham Road, London, UK; Tel.: þ 20 7808 2875/2880; Fax: þ 20 7351 6420; E-mail: [email protected] Received 16 May 2003; accepted 19 November 2003

lymphocyte doubling time and the French substages AI and AII have been shown to correlate with survival,9,10 but no single test can identify stage A CLL patients who will remain stable from the 20 to 30% who are likely to progress.11 Older age has been shown to confer a worse prognosis and females with CLL live longer than their male age-matched counterparts.12 Other proposed prognostic indicators are; high serum beta-2 microglobulin,13,14 atypical morphology and increased number of prolymphocytes,15,16 levels of soluble CD2317 serum thymidine kinase levels,18 bone marrow histology19,20 and cytogenetic abnormalities21,22 particularly when examined by fluorescence in situ hybridisation (FISH).23 Trisomy 12, 11q23 deletion, p53 deletion are associated with poor prognosis and 13q14 deletion confers good prognosis if it is the only abnormality.23 Recently, it has been suggested that the presence or absence of immunoglobulin (Ig VH) gene mutations could distinguish two groups of CLL. Those with an unmutated Ig VH were more likely to progress, require treatment and have a shorter survival than those with mutations.24 This was taken a step further by Dalme et al.25 who reported that individuals in whom

CD38 as a prognostic marker in CLL PD Thornton et al

146

30% or greater of their CLL cells expressed CD38 by tricolour flow cytometry had unmutated Ig VH and this was associated with aggressive disease. In contrast, patients with mutated Ig VH had indolent disease and low levels of CD38 expression. Subsequent studies confirmed the prognostic value of CD38 expression, but not the correlation between Ig VH and CD38.26–28 In addition, it is not clear what level of CD38 expression should be regarded as positive and is more relevant to prognosis. While the original publication suggested a 30% threshold, others have used 20%29 and recently two other groups have suggested 7% as the best cut-off.30,31 Ghia et al.32 also suggest that the pattern of CD38 expression is relevant in predicting disease progression, while others have found changes in CD38 expression on sequential measurements.33 We have tested CD38 expression in samples from untreated patients using three-colour flow cytometry and assessed its prognostic value. By plotting the hazard ratios for CD38 expression from 0 to 100% against treatment-free intervals (TFI) we found that expression of greater than 7% was the most valuable cut-off for CD38 expression. We also examined correlations of chromosomal abnormalities by FISH with CD38 expression in some of these cases.

Materials and methods We studied 289 previously untreated patients with CLL, 209 were males and 80 females with an age range of 30– 84 years (median 62), diagnosed between January 1968 and March 2002. In all, 126 were stage A, of which 65 were progressive stage A, 109 stage B and 54 stage C. This included 207 patients entered into the CLL4 trial from participating UK centres and 82 patients attending for regular follow-up at the RMH. Patients entered into the CLL4 trial were tested at randomisation, whereas the others were tested at their out-patient review appointments. The median duration between diagnosis and testing was 9 months (0–386 months). TFI was estimated using date of diagnosis to either date of treatment or last follow-up for those who remained treatment free. Median follow-up for patients was 20 months (0–409 months). To assess the stability of CD38 expression, we repeated the CD38 expression on 17 of these patients. The diagnosis of CLL was confirmed by morphology assessment of peripheral blood lymphocytes and immunophenotype.34 Only fresh whole blood from the above patients was studied over a 2-year period (January 2000 to April 2002). Samples were analysed for surface expression of CD19/CD5/CD38 using the monoclonal antibodies labelled with the following flourochromes; anti-CD19 APC (Caltag, Burlingame, CA, USA) anti-CD5 FITC (Becton & Dickinson, San Jose, CA, USA) and anti-CD38 PE (Becton & Dickinson) as described previously.25 Controls included isotype-matched mouse antibody coupled to the corresponding flourochromes. Flow cytometric analysis was performed on a Becton & Dickinson FACScanner flow The Hematology Journal

cytometer. Measurements of forward and side scatter were used to gate on lymphocytes and exclude monocytes. The cell quest software system (Becton & Dickinson) was used to acquire and analyse the data. The CD38 expression was calculated by gating on CD5/ CD19 þ lymphocytes. FISH analysis was performed on 135 samples at the same time as the CD38 measurement. Mononuclear cells were obtained by centrifugation over a density gradient and fixed according to standard cytogenetic protocols. Commercially available probes for D13S25 (13q14), p53 and 12 Centromere were used to detect deletions of 13q14, p53 and trisomy 12 (Vysis, Richmond, UK). The YAC probe 755B11 (11q23) was kindly provided by Dr S Stilgenbauer (University of ULM, Germany) for interphase screening of 11q deletions. The 11q23 probe was amplified and purified further by Alu PCR following the method described by Lengauer et al.35 Dual colour FISH combining D13S25 (13q14) with a centromeric probe for chromosome 12 (CEP 12) and a p53 probe with a centromeric probe for chromosome 17 (CEP 17), to ensure that p53 deletions were not due to monosomy of chromosome 17, was performed for 16– 24 h at 371C. DNA from YAC755B11 (11q23) labelled with digoxigenin was combined with a differently labelled clone mapping to a chromosomal region rarely involved in CLL as an internal control. These were subsequently detected using flourochrome-conjugated antibodies. Signal numbers were enumerated in 200 nuclei for each flourochrome. Cells from 10 normal controls were used to establish cut-off levels for confident detection of abnormalities. Statistical analysis was carried out using the software SPSS (SPSS Inc, Chicago, USA). Pearson w2 analysis was applied to look for correlations with stage, age, sex, 11q23 deletions, trisomy 12, 13q14 (as a single abnormality) and p53 deletions on 135 patients. Multivariate analysis for stage, age gender and CD38 expression was performed using Cox’s proportional hazards model36 on all 289 patients. Kaplan–Meier curves were constructed for the TFIs and compared by log rank tests. Hazard ratios for low, intermediate and high levels of CD38 positivity were calculated using Cox’s proportional hazards model.36

Results The mean value for CD38 expression in all patients was 28.5% and for stage A patients was 26% (Figures 1 and 2). Hazard ratios for the effect of CD38 expression on TFI were used to determine the most valuable cut-off (Figure 3). We plotted the hazard ratio (95% confidence interval) for TFI between the higher and lower groups defined by differing cut-offs from 0 to 100% CD38 expression. (The greater the hazard ratio the better the discrimination between groups.) The maximum hazard ratio was achieved for a cut-off of 7%. These statistics were subsequently applied considering two thresholds for CD38 positivity: X30% and X7% CD38-positive cells.


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Correlation of CD38 expression with stage, gender, age The analysis of the correlation between stage and CD38 values showed that using a 30% threshold there was no statistically significant correlation (Table 1). On the other hand, using a 7% threshold there were significantly more stage B and C patients with X7% CD38positive cells. Furthermore, male sex was significantly associated with X7% but not X30% CD38 expression (Table 1). There was no significant correlation with age and CD38 expression.

Treatment-free interval Figure 1

Distribution of CD38 expression in all patients.

Figure 2

Distribution of CD38 expression in stage A patients.

All patients were untreated at time of testing and were analysed according to TFI from the time of diagnosis to the time of first treatment, as there have been few deaths so far. Of the 289 patients, 224 required treatment, 42% had a CD38 value of o7% and the rest X7% (Table 2). The median TFI for each group according to CD38 levels was as follows: 36 months for low expression (o7%); 3.5 months for intermediate expression (7–29%) and 8.7 months for high expression (X30%) (Table 2). These differences are statistically significant (Po0.00005). Separating patients into two groups: o7% and X7%, the median TFI for o7 was 36 months and for 47% was 6.2 months (Po0.00005) (Figure 4). When the analysis of TFI was done for values above and below 30% there was still a statistical difference, but with a greater P-value (P ¼ 0.0001) (Figure 5). Using Cox regression analysis it was apparent that there was an increasing risk for the need for treatment from 1 to 2.02 with increasing CD38 values. TFI analysis for stage A patients also showed that the median TFI was longer using the 7% threshold (Table 2 and Figure 6), this difference was statistically significant (P ¼ 0.0001) and confirmed by Cox regression analysis (Table 2). When the analysis of TFI was done for values above and below 30%, there was still a statistical difference, but with a higher P-value (P ¼ 0.0004) (Figure 7).

Multivariate analysis Multivariate analysis of age, stage gender and CD38 expression in the 289 patients showed that only stage and CD38 are independent prognostic variables for progression to treatment. (both Po0.0005). We compared our findings with those of two recent reports also using a 7% cut-off (Table 3). The inverse w2 method was used to combine significance levels37 across the two studies addressing TFI and the overall w2 values for the two cut-offs were then compared. The 7% cut-off was found to be a significantly better predictor of outcome (P ¼ 0.0005). Figure 3

The hazard ratio for a 7% cut-off was 2.00 (95% CI: 1.52– 2.62), that for a 30% cut-off was 1.70 (95% CI: 1.30–2.24), although the confidence intervals overlap this does indicate that the 7% cut-off is a better discriminant.

Stability of CD38 expression To assess the stability of CD38 expression we repeated twice CD38 expression on 17 patients. we found that The Hematology Journal


148 Table 1 Correlation of CD 38 expression in 289 patients o30

X30%

P-value (w2)

o7%

X7%

P-value (w2)

Total

Stage A Stage B Stage C

87 (69%) 67 (61%) 35 (65%)

39 (31%) 42 (39%) 19 (35%)

0.47

72 (57%) 42 (38.5%) 25 (46%)

54 (43%) 67 (60.5%) 29 (54%)

0.02

126 (43%) 109 (38%) 54 (19%)

Male Female Age o60

135 (64.5%) 54 (67.5%) 89 (69%)

74 (35.5%) 26 (32.5%) 40 (31%)

0.64

93 (44%) 46 (57.5%) 68 (53%)

116 (56%) 34 (42.5%) 61 (47%)

0.05

209 (72%) 80 (28%) 129 (45%)

0.25

0.16

Table 2 TFI and CD38 expression Number of patients treated

Median TFI (months)

Hazard ratio

CD38 expression o7% 7–29% X30%

139 50 100

94 (68%) 42 (84%) 88 (88%)

36 3.5 8.7

1 1.95 2.02

1.35 1.50

2.82 2.74

o0.0005

Total

289

224

72 15 39

30 (42%) 9 (60%) 31 (79%)

1 2.188 2.649

1.032 1.593

4.636 4.405

¼ 0.001

126

70

Stage A patients only o7% 7–29% X30% Total

122 22 26

95% confidence interval for hazard ratio (Cox regression analysis)

P-value

Total number of patients

with 13q14 deletion as a single abnormality. In addition trisomy 12 was associated with CD38 expression of X7% and X30% approaching significant levels.

Discussion

Figure 4

TFI for all patients using a 7% threshold (Po0.00005).

there was little change in CD38 expression in 16/17 tested over a period from 0.5 to 12 months (median 4.6 months). The only observed change was in one patient whose CD38 expression changed from 17 to 60% and this was associated with disease progression.

Correlation with FISH analysis (Table 4) FISH study of 135 patients found no significant correlation with CD38 expression and deletions of p53 or deletions of 11q23. However, there was a correlation between o30% and o7% CD38 expression and cases The Hematology Journal

Recent studies have suggested that there may be two forms of CLL, one requiring treatment early with a short survival and the other with a more indolent disease course.24,25 Oscier et al.38 found an association between unmutated Ig VH genes and trisomy 12 and, between mutated Ig VH genes and 13q14 deletion. The same group confirmed that unmutated VH genes were associated with trisomy 12, atypical morphology, advanced stage and progressive disease.24 Simultaneously, Damle et al.25 demonstrated a negative prognostic impact of unmutated Ig VH genes in CLL and suggested a tight correlation between expression of CD38 in X30% of CLL cells and unmutated Ig VH. Although there is evidence that both mutational status of the Ig VH gene and CD38 expression are reliable prognostic factors in CLL, there is controversy as to whether the CD38 expression is a surrogate marker for mutational status. Subsequent studies have failed to confirm this association,27,28 while others have suggested that CD38 negativity may improve the adverse effect of an unmutated VH gene26 and a few others have shown that CD38 expression can occasionally be upregulated and may change with disease progression, whereas Ig VH gene mutations are irreversible.39 Nevertheless, high levels of CD38 expression have been shown to be


149 Table 3 Overview of publications using 7% cut-off P-values Krober (Survival)30 Wiestner (TFI)31 Current paper (TFI) Combined (TFI)37 Comparison (TFI)

o7 versus X7

o30 versus X30

0.02 0.001 0.0000007 0.00000003

0.13 0.003 0.0001 0.00001 0.0005

P-values for the three studies which have employed CD38 cut-offs of X7 and X30 are shown. Two studies examined TFI, these results have been combined to give single overall (two-sided) P-values, which were then compared. This indicated that the X7 cut-off was a highly significantly better discriminant.

Figure 5

TFI for all patients using a 30% threshold (P ¼ 0.0001).

Figure 6

TFI stage A patients using a 7% threshold (P ¼ 0.0001).

Figure 7 TFI for stage A patients using a 30% threshold (P ¼ 0.0004).

associated with shorter progression-free survival following treatment40 and treatment resistance.41 The original report by Dalme et al chose a cut-off of 430% CD38-positive cells based on observations from a scatter plot of 47 analysed patients. In the present study of 289 patients, we observed that there are a significant percentage of cases whose CD38 expression is clustered around 30% and therefore we find it difficult to segregate those patients into those with a positive or negative CD38 expression. Our experience is similar to that of Ghia et al.32 who described three different groups of CD38 expression; entirely negative, associated with mutated Ig VH genes and good prognosis, entirely positive and an intermediate group with bimodal expression ranging from 8 to 76% both associated with unmutated Ig VH genes and poor prognosis. Recently, the MD Anderson group found significantly shorter survival times using 20% CD38 expression as threshold29 and Kro¨ber et al.30 could not demonstrate a survival advantage in CD38 negative patients using a 30% cut-off yet demonstrated a prognostic influence using a 7% cut-off value, which is in keeping with another recent publication by Weistner et al.31 To address the problem of which value to use, we plotted the hazard ratio for the TFI between the higher and lower groups defined by differing cut-offs from 0 to 100% CD38 expression. The maximum hazard ratio is achieved for a cut-off of X7%, so we believe this value as a better discriminator for prognosis in CLL. Our results show a significant correlation with advanced stage, and a shorter TFI using a 7% CD38 threshold rather than 30% in a large cohort of untreated patients. There is a significantly longer TFI in patients with o7% CD38-positive cells than both those with 7–29% and X30%. In fact, the median TFI in patients with 7–29% expression is close to that of those with X30% expression. We also assessed the value of CD38 as a prognostic marker in stage A in order to discriminate those who needed treatment early from those who would remain stable. We showed a statistically significant finding similar to that of the whole group with median TFI for o7% expression longer than those with values of 7– 29% and with X30% expression. Therefore, we suggest that the X7% threshold may be best to determine which stage A CLL patients may progress. Whether CD38 will be an independent prognostic value in patients with The Hematology Journal


150 Table 4 Correlation of CD38 expression with FISH in 135 patients o30 11 q23 deletion Trisomy 12 13q14 (sole deletion) P53 deletion

10/79 3/79 46/79 6/79

(13%) (4%) (58%) (8%)

X30%

P-value (w2)

o7%

X7%

P-value (w2)

Total

12/56 (21%) 7/56 (12.5%) 19/56 (34%) 2/56 (4%)

0.17 0.06 0.005 0.33

6/51 (12%) 1/51 (2%) 30/51 (59%) 4/51 (8%)

16/84 (19%) 9/84 (11%) 35/84 (42%) 4/84 (5%)

0.34 0.06 0.05 0.46

22/135 10/135 65/135 8/135

stage A progressive, B and C is currently being tested in the CLL4 trial. Although not all patients in this study tested at diagnosis, and others have suggested that CD38 expression may change with disease evolution or treatment,33 we observed that CD38 expression remains stable all but one out of 17 patients. Recently, others have found a correlation between CD38 expression and other established prognostic factors in CLL such as stage, morphology, LDH, B2 microglobulin, pattern of marrow infiltration and chromosomal aberrations such as deletion of 11q23.30,42 It is well established that 11q23, p53 and trisomy 12 are associated with worse disease in CLL whereas 13q14, if the single abnormality, with good prognosis.23 We observed a significant correlation between 13q14 deletions and o30%/o7% values of CD38 expression. Deletion of 13q14 is the commonest deletion seen in CLL and is also associated with Ig VH mutational status.38 It is likely that the mechanisms responsible to confer good prognosis following deletion of 13q14 may involve VH mutations and low CD38

expression. In addition, we found a significant correlation with male gender and X7% CD38 expression a trend for trisomy 12 and higher levels (X7/X30%) of CD38 expression. These findings are similar to previously reported associations seen with unmutated Ig VH and may be explained by the concordance of CD38 expression and Ig VH in around 2/3 of cases.31,39 In conclusion, we have found that in patients whose CD38 expression is X7%, there is a significant correlation with advanced stage, male sex and a shorter TFI, and that it is an independent prognostic variable for treatment requirement in previously untreated patients. We suggest that the 7% threshold should be examined further as a prognostic indicator for in CLL. Acknowledgements PT was supported by a Clinical Fellowship from the Leukaemia Research Fund. We are grateful to Dr S Stilgenbauer University of ULM, Germany for the gift of the YAC 755b11 and the participants of the LRF CLL4 trial who provided samples for this study.

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