The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2012  |  Volume : 37  |  Issue : 2  |  Page : 102--110

Prognostic significance of CLLU1 expression in B-cell chronic lymphocytic leukemia


Nahla A. Nosair1, Salwa A. Essa1, Atef M. Taha2,  
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt

Correspondence Address:
Nahla A. Nosair
Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta
Egypt

Abstract

Background

B-cell chronic lymphocytic leukemia (B-CLL) results from the accumulation of slowly proliferating long-lived CD5-positive B cells with apoptotic defects. The clinical course of CLL is variable and several markers have been proposed to predict the outcome in CLL patients. CLL-upregulated gene 1 (CLLU1), a gene that is exclusively upregulated in CLL cells, was identified and mapped to chromosome 12q22.

Objective

The objective of this study was to evaluate the prognostic significance of CLLU1 expression in B-CLL patients and its relationship with other well-established prognostic markers.

Methodology

Forty newly diagnosed CLL patients were included in the study. The expression levels of CLLU1 were determined by quantitative real-time PCR, CD38 expression was determined by flow cytometry, and the presence of trisomy 12 was evaluated by the fluorescence in-situ hybridization technique. The relationship between CLLU1 expression levels and Binet’s clinical staging of the disease, CD38 expression, and trisomy 12 was evaluated. The response of CLL patients to therapy and progress of the disease were monitored in relation to CLLU1 expression levels.

Results

CLLU1 expression was found to be upregulated 0.4–1120.1-fold in B-CLL cells as compared with normal B cells. CLL patients were segregated into two groups on the basis of high and low CLLU1 expression levels. High expression of CLLU1 was significantly associated with higher absolute lymphocytic count and also higher serum β2 microglobulin and lactate dehydrogenase levels with no significant association detected between CLLU1 expression levels and age of the patients at diagnosis. Significant positive correlations could be detected between high CLLU1 expression levels and both the advanced clinical stages of the disease and the positive expression levels of CD38. High expression of CLLU1 occurred irrespective of trisomy 12 and was associated with failure to achieve complete remission and shorter overall survival in CLL patients.

Conclusion

The present study demonstrated that high CLLU1 expression in B-CLL cells is a significant predictor of disease progression and poor response to therapy. High expression levels of CLLU1 correlate significantly with the known prognostic markers of the disease, mainly the clinical staging and CD38 expression. CLLU1 expression levels are independent of trisomy 12.




How to cite this article:
Nosair NA, Essa SA, Taha AM. Prognostic significance of CLLU1 expression in B-cell chronic lymphocytic leukemia.Egypt J Haematol 2012;37:102-110


How to cite this URL:
Nosair NA, Essa SA, Taha AM. Prognostic significance of CLLU1 expression in B-cell chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2012 [cited 2019 Dec 9 ];37:102-110
Available from: http://www.ehj.eg.net/text.asp?2012/37/2/102/135063


Full Text

 Introduction



B-cell chronic lymphocytic leukemia (B-CLL) results from accumulation of small mature B lymphocytes, which have undergone monoclonal expansion, in blood, marrow, and lymphoid organs 1.

The clinical course of CLL is variable 2. For about half the patients the diagnosis of CLL will not affect morbidity or mortality, whereas for the rest of the patients CLL constitutes a malignant disease that substantially reduces the life expectancy 3.

Within the past decade, several markers have been proposed to predict the outcome of CLL patients 4. Although the old parameters such as clinical stage (Rai et al. 5 or Binet et al. 6] are unable to prospectively distinguish early-stage CLL that progresses rapidly to aggressive disease from disease destined to remain in an early stage for an extended time period, the new parameters have considerably improved disease risk classification 7.

The most well-investigated and commonly used biologic prognostic markers are the mutational status of the immunoglobulin heavy chain region (IgVH) 8, cytogenetic aberrations such as del(17)(p13), del(11)(q22) and trisomy 12 3,9, expression of intracellular 70 kDa &zgr;-associated protein (ZAP-70) 10–12, and expression of the transmembrane glycoprotein CD38 13–15.

Few patients have CLL with only prognostically favorable or unfavorable characteristics, whereas many have CLL with various combinations of favorable and unfavorable traits 16. Managing the treatment course for CLL patients cannot be planned without taking prognosis into account 7. Therefore, biologic prognostic markers have clinical value when they can lead to early identification of patients at high risk of developing progressive disease; the earlier the patients are identified the more effective the intervention 17.

CLL-upregulated gene 1 (CLLU1) was identified and was highly regulated in CLL cells without IgVH hypermutation 18. It was mapped to chromosome 12q22, within a cluster of genes that is active in germinal center B cells, suggesting that the region is accessible for transcription in B cells 19.

CLLU1 encodes six mRNAs with no sequence homology to any known gene, and most transcripts appear to be noncoding 18. Two transcripts, however, potentially encode a peptide with remarkable structural similarity to human interleukin-4 (IL-4) 20.

Appreciable levels of CLLU1 were detectable only in CLL cells and not in a panel of normal tissue extracts or in any other tested hematologic malignancy 18. This unique and restricted expression pattern suggests that CLLU1 is the first disease-specific gene identified in CLL 19.

The objective of this study was to evaluate the prognostic significance of CLLU1 expression in B-CLL patients and its relationship with other well-established prognostic markers.

 Patients and methods



This study was carried out (between February 2009 and December 2011) on 40 newly diagnosed CLL patients attending the Hematology Unit of the Internal Medicine Department of Tanta University Hospital. These included 22 men and 18 women whose ages ranged from 46 to 63 years with a male-to-female ratio of 1.2 : 1. They were treated with fludarabine and cyclophosphamide, then followed up for a period of 12–30 months to assess their clinical outcome.

Patients were diagnosed on the basis of the following:

Thorough history taking and clinical examination.Laboratory investigations including the following: Estimation of complete blood count using an Advia 60 cell counter (Bayer, Pennsylvania, USA) with examination of Leishman stained peripheral blood smears. Bone marrow aspiration and morphological examination with stress on bone marrow lymphocyte percentage. Immunophenotyping of whole peripheral blood using the FACS caliber flow cytometer [Becton Dickinson (BD), Ireland, UK]. The following panel of monoclonal antibodies (moAbs) was used: CD5, CD19, CD23, CD38, CD79b, FMC7, surface immunoglobulin (sIg), and κ and &lgr; light chains, labeled with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Liver and kidney function tests. Estimation of serum lactate dehydrogenase (LDH) and β2 microglobulin (β2M) levels.

CLL was distinguished from other chronic lymphoproliferative disorders according to the scoring system proposed by Moreau et al. 21.

Flow cytometric immunophenotyping

Flow cytometric immunophenotyping of lymphocytes was performed by the whole blood lysis method. A volume of 100 µl of fresh ethylenediaminetetraacetic acid peripheral blood samples was prepared for flow cytometry by adding 12 µl of the following antibodies or matched isotype controls: CD5 FITC/CD23 PE, CD19 PE/CD38 FITC, CD10 PE/FMC7 FITC, CD2 PE/CD79b FITC, sIg FITC, κ light chain FITC, and &lgr; light chain FITC. Then the tubes were incubated for 30 min at 4°C in the dark. The erythrocytes were lysed using 1× FACS lysis solution (BD). Cells were washed twice in PBS (pH 7.4) supplemented with 5% fetal calf serum and resuspended in PBS containing 2% formaldehyde and analyzed on a fluorescence-activated cell scanner (FACS caliber; BD). Acquisition and analysis were carried out using Cell Quest software (Sanjose, California, USA). A total of 10000 events were routinely acquired 22. The moAbs were supplied by Coulter Electronics (Hialeah, Florida, USA).

For analysis of CD38 expression, anti-CD38 moAb FITC was used, and CD38 expression level was assessed as a percentage of CD38-positive cells of the gated B cells (CD19+/CD5+). Patients were considered positive for CD38 when 20% or more leukemic cells (CD19+/CD5+) expressed it 23 [Figure 1].{Figure 1}

Quantitative reverse transcriptase-polymerase chain reaction

CLLU1 expression levels were quantified on the Gene Amp 5700 sequence detection system (Applied Biosystems, Foster City, California, USA) with the use of TaqMan one-step real-time PCR (RT-PCR) reagent (#4309169; Applied Biosystems).

RNA was extracted from peripheral blood mononuclear cells using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions.

Amplification of cDNA1 sequence (which quantifies the level of splice variants 1 and 1a) was performed by the comparative method of relative quantification using β2M as an internal standard and a pool of purified normal B lymphocytes as the calibrator 24 [Figure 2].{Figure 2}

cDNA1 was recommended as a measure for the CLLU1 level because the cDNA1 quantitative real-time (QRT)-PCR reaction spans an exon–intron boundary and is thus not affected by contaminating DNA 18.

The primers and probe used for CLLU1 were as follows:

Forward primer, 5º-AGCTTGCAGATGGCAGATCA-3º

Reverse primer, 5º-CATAAAGGGCAGCGAAATGC-3º

6-carboxyfluorescein (FAM) probe, 5º-TATCTCCAGGCCTTTCATTGGGTCAGGT-3º

The primers and probe used for β2M were as follows:

Forward primer, 5º-TGACTTTGTCACAGCCCAAGATA-3º

Reverse primer, 5º-AATCCAAATGCGGCATCTTC-3º

FAM probe, 5º TGATGCTGCTTACATGTCTCGATCCCA-3º

The reactions were carried out under the following conditions: one cycle of 2 min at 95°C, 45 cycles of 30 s at 95°C, 1 min at 62°C, 1 min at 72°C, and finally, one cycle of 5 min at 72°C 18.

CLLU1 expression levels were measured as fold upregulation in relation to normal CD19-positive B cells from 10 healthy donors 19.

Detection of trisomy 12 by the fluorescence in-situ hybridization technique

Peripheral blood samples on preservative-free heparin were used for detection of trisomy 21 by the fluorescence in-situ hybridization (FISH) technique after 72 h of culturing with pokeweed stimulation. FISH analysis of interphase cells was carried out using the chromosome enumeration probe (CEP) 12 DNA probe (spectrum orange; Vysis, Illinois, USA) specific to the centromeric region of human chromosome 12 according to manufacturer’s instructions [Figure 3].{Figure 3}

FISH analysis was carried out by codenaturation in a Vysis Hybrit hybridization system (Illinois, USA), which allows denaturation of both the probe and the target DNA on slides at the same time (at 74°C), followed by overnight hybridization 37°C; thereafter, posthybridization washes were performed. DAPI counter stain was added and signals were detected using an epifluorescence Olympus BX60 microscope equipped with selective filters for fluorescein, rhodamine, and DAPI. The images were captured with an IAI camera and analyzed using the PC applied system analysis software (Vysis, Abbott Park, Illinois, USA). Trisomy 12 was diagnosed if more than 10% of interphase cells presented more than two signals of CEP 12. A total of 200 interphase cells were analyzed 25.

Statistical analysis

Data were analyzed using SPSS (version 16; SPSS Inc., Chicago, Illinois, USA). Quantitative data were expressed in the form of mean±SD; qualitative data were described in the form of number and percentage. Differences between groups were evaluated using Student’s t-test for quantitative data and the χ2-test for qualitative data. Pearson’s correlation was used to detect the relationship between CLLU1 expression and clinical staging, CD38 expression and trisomy 12. Survival analysis was carried out according to the Kaplan–Meier curve.

 Results



To evaluate the prognostic significance of CLLU1 in chronic lymphocytic leukemia, 40 newly diagnosed B-CLL patients were included in this study. Their ages ranged from 46 to 63 years (mean: 54.5±5.76 years); there were 22(55%) men and 18(45%) women with a male-to-female ratio of 1.2 : 1 [Table 1].{Table 1}

According to Binet’s clinical staging system 6, the proportions of patients at each stage at the time of diagnosis were as follows: 23 patients (57.5%) at stage A, 10 patients (25.0%) at stage B and seven patients (17.5%) at Binet stage C. The clinical and biological data of CLL patients at diagnosis are shown in [Table 1].

The range of CLLU1 expression in normal control B lymphocytes from the 10 healthy donors was 0.003–1.0-fold upregulated with the median value being 0.1-fold upregulated.

The studied CLL patients expressed CLLU1 with a wide range (from 0.4 to 1120.1-fold upregulation). Patients (5/40) with expression levels below the highest normal value (1.0) were included in the low-expression group of patients, whereas the median value of CLLU1 expression of the remaining patients (39.6-fold upregulation) was used as a cut-off value to categorize CLL patients into two groups of high and low CLLU1 expressions to facilitate the comparison of CLLU1 with other prognostic markers.

The group of high CLLU1 expression included 18 patients (45%) with expression levels at least 39.6 and a range of expression from 39.6 to 1120.1 (mean: 434.03±102.5-fold upregulation), whereas the low CLLU1 expression group included 22 patients (55%) with expression levels below 39.6 and a range of expression from 0.4 to 35.3 (mean: 14.11±2.71-fold upregulation). There was a high statistically significant difference (P=0.001) in CLLU1 expression levels between the two groups of patients [Table 2].{Table 2}

No significant difference (P=0.633) was noted between the high and low CLLU1 expression groups regarding the age of CLL patients at diagnosis [Table 3].{Table 3}

Comparing the two groups of CLLU1 expression as regards some known prognostic markers such as peripheral blood absolute lymphocytic count and serum levels of β2M and LDH, statistically significant differences were found between the two groups. There was a significant increase in absolute lymphocytic count (P=0.001) in the high CLLU1 expression group compared with the low expression one. Serum levels of both β2M and LDH were significantly higher (P=0.001) in the group of high CLLU1 expression compared with the group of low expression [Table 4].{Table 4}

Only four patients (17.39%) at Binet stage A expressed high CLLU1 levels, whereas the remaining 19 patients at stage A (82.61%) were in the low-expression group. Most of the stage B and C patients were in the high CLLU1 expression group (82.35%), and only three patients showing low CLLU1 expression were at stages B and C (17.65%). There was a statistically significant difference in CLLU1 expression levels of patients at different Binet stages, expression levels being higher at stages B and C when compared with stage A [Table 5]. A highly significant positive correlation (P=0.001) was found between CLLU1 expression levels and Binet clinical staging of CLL patients [Figure 4].{Table 5}{Figure 4}

We used the flow cytometric assay to measure the levels of CD38 expression in CLL patients. Sixteen patients (40%) were defined as CD38 positive and 24 (60%) as CD38 negative [Table 1]. Among the 16 patients with CD38-positive CLL, 11 (68.75%) expressed high levels of CLLU1, whereas only five patients (31.25%) expressed low CLLU1 levels. Within the group having CD38-negative CLL, 17 patients (70.83%) expressed low CLLU1 levels and only seven patients (29.17%) belonged to the high-expression group, with a statistically significant difference (P=0.013) between the two groups of CLLU1 expression regarding CD38 levels [Table 6]. There was a highly significant (P=0.001) positive linear correlation between the expression levels of both CD38 and CLLU1 in the studied CLL patients [Figure 5].{Table 6}{Figure 5}

The copy number of chromosome 12 was studied using the CEP 12 FISH probe, and seven patients with trisomy 12 (17.5%) were detected among the studied CLL patients. Three patients with trisomy 12 expressed high levels of CLLU1 (42.86%) and four patients (57.14%) expressed low levels of CLLU1 with no significant difference (P=0.900), or a significant correlation (P=0.328) could be detected between the two groups of CLLU1 expression as regards the presence or absence of trisomy 12 [Table 7] and [Figure 6].{Table 7}{Figure 6}

As regards the response to therapy and outcome of the studied CLL patients, 17 patients (42.5%) achieved complete remission (CR), whereas 23 patients (57.5%) did not. CR was achieved by only four patients (23.53%) of the high CLLU1 expression group and 13 patients (76.47%) of the low CLLU1 expression group. Although most of the patients in the high CLLU1 expression group (60.87%) failed to achieve CR in either a stable or progressive course of the disease, four patients (23.53%) were able to achieve CR [Table 8], with a statistically significant difference between the two groups of CLLU1 expression regarding the response to therapy and outcome in CLL patients (P=0.018).{Table 8}

The significant positive correlation between CLLU1 expression levels and failure to achieve CR confirmed the poor prognostic significance of CLLU1 expression in CLL.

Using Kaplan–Meier survival analysis it was determined that the overall survival was significantly shorter in high CLLU1 expression group of patients when compared with the low-expression group [Figure 7].{Figure 7}

 Discussion



CLL is a lymphoproliferative disorder caused by clonal expansion of B cells 26 and has an extremely variable clinical course 27. The disease runs an indolent course in some patients, and life expectancy is not shortened. In others, the disease is aggressive and progresses rapidly with short survival time after diagnosis 28.

The appearance of new therapies has shifted the therapeutic goal from control of leukocytosis to the achievement of molecular remission, especially in younger patients 29. Therefore, it is very important to identify factors that can predict a poor prognosis and the patients who will benefit from intensive therapy at an early stage of the disease.

CLLU1 at the 12q22 region was recently reported by Buhl et al. 18 to be a specific gene that is exclusively upregulated in CLL cells. CLLU1 is the first example of a CLL-specific gene with a highly restricted expression pattern. A low ‘background’ CLLU1 expression was found in B cells, cell lines and malignancies with a B-cell origin, but a lower or even undetectable expression in non-B cells was reported 18. In fact, the absence of expression of CLLU1 in all the tissues analyzed raised the possibility that it may not be required for normal human function and development 19.

The aim of this study was to evaluate the prognostic significance of CLLU1 expression in CLL patients and its relationship with other well-established prognostic markers.

In the present study, CLLU1 expression levels were measured using QRT-PCR and patients were categorized into two groups; the first of them consisted of 18 patients (45%) who expressed high levels of CLLU1 (high-expression group), whereas the second consisted of 22 patients (55%) who expressed lower levels of CLLU1 (low-expression group). There was a significant difference in the expression levels between the two groups. CLLU1 expression was found to be upregulated 0.4–1120.1-fold in B cells of CLL patients as compared with normal B cells.

In accordance with the results of the present study, Chen et al. 30 used semi-quantitative RT-PCR to detect the expression levels of CLLU1 in 50 Chinese CLL patients and found positive expression in 52% of the patients under study. In addition, Buhl et al. 19 measured the expression levels of CLLU1 in 59 CLL patients using QRT-PCR and found that CLLU1 levels ranged from 0.2 to 4257.4 with a median 27.27-fold above that of the normal B cells and that the high-expression group constituted 47.8% of the studied patients.

It was reported that, all CLL patients express CLLU1, some at very low levels, some at intermediate levels and some at several 1000-fold upregulated levels 19. The reason for the high expression of CLLU1 by CLL cells could be accidental activation of a promoter upstream of the CLLU1 gene 19. All the CLLU1 transcripts were clustered in a small region on chromosome 12q22. It is likely that all the differentially expressed mRNAs are derived from the same primary transcript, whereas alternative splicing and use of different polyadenylation sites then generated the different transcripts. This would explain the difference in expression pattern and the wide range of CLLU1 expression in CLL patients 18.

Many prognostic factors in CLL have been described and are associated with worse prognosis; these include the degree of absolute lymphocytosis and serum markers such as β2M and LDH 31. In the present study, a significant difference was detected between the two groups of high and low CLLU1 expressions with regard to these prognostic parameters. High expression of CLLU1 was significantly associated with higher absolute lymphocytic count and higher serum β2M and LDH levels.

No significant difference could be detected between both groups of CLLU1 expression and the age of CLL patients at diagnosis. This agreed with the findings of Buhl et al. 19, who did not find significant correlation between the patient’s age at diagnosis and CLLU1 gene expression levels. However, age-stratified analysis performed by Josefsson et al. 17 revealed that the prognostic significance of CLLU1 was particularly strong in patients younger than 70 years of age at diagnosis and absent in patients aged 70 years or older. The age of our studied patients ranged from 46 to 63 years and no one reached the age of 70 during the period of follow-up.

The most commonly used prognostic factor in patients with B-CLL is the clinical staging 32. In the present study, a significant positive correlation was found between CLLU1 expression levels and Binet stages, with the highest levels in advanced clinical stages (B and C). This was consistent with the findings of many other studies 17,19,30.

CD38 has been reported as an independent predictor of prognosis for CLL patients 33. Its high expression correlates with a shorter median survival and necessity for early treatment 34.

In this study, CD38 was expressed in 40% of the studied patients and its expression levels were significantly higher in the group of high CLLU1 expression when compared with the low CLLU1 expression group. There was a highly significant positive correlation between the expression levels of both CD38 and CLLU1. This positive correlation was also reported by Chen et al. 30, who found positive CD38 expression in nearly half of the studied CLL patients and a higher CLLU1-positive expression in CD38-positive patients than in CD38-negative patients. Buhl et al. 19 reported that CLLU1 was significantly upregulated in the CD38-positive CLL group.

It was shown that the interaction of CD38 with its ligand CD31 on nurse-like cells results in the transmission of survival and antiapoptotic signals to the CLL cells, which may explain why high CD38 levels are associated with poor outcome 35.

Trisomy 12 represents the third most frequent chromosomal aberration in CLL and often occurs as the sole cytogenetic lesion 3. Trisomy 12 as a single aberration confers an intermediate prognostic risk 36. As CLLU1 is mapped to chromosome 12, one interesting question is whether high expression of CLLU1 is associated with amplification of chromosome 12.

In the present study, trisomy 12 was detected by FISH analysis in seven CLL patients (17.5%), four of whom (57.14%) expressed low CLLU1 levels with no significant relation between CLLU1 expression levels and the presence or absence of trisomy 12. Another study found that high expression of CLLU1 in CLL samples was independent of amplification of chromosome 12 30.

High expression of CLLU1 gene is restricted to B-CLL. It was also proven that CLLU1 expression level is an intrinsic and constant parameter of the CLL clone 37.

To study the prognostic role of CLLU1 in B-CLL patients in the present work, the response of CLL patients to therapy and the progress of the disease were monitored in relation to the levels of CLLU1 expression. CR rates were significantly higher in patients with low CLLU1 expression levels when compared with those in the high-expression group. The higher the expression of CLLU1, the more likely the failure to achieve CR with more progressive course of the disease. Survival analysis was carried out according to the Kaplan–Meier method and showed a highly significant shorter overall survival associated with higher CLLU1 expression levels when compared with lower expression levels.

Previous studies indicated that patients with high CLLU1 expression have shorter time intervals from diagnosis to initiation of therapy and decreased overall survival times 17,19,30.

CLLU1 may play a role in the pathogenesis of CLL 19. The possibility that CLLU1 encodes an IL-4-like polypeptide, mainly expressed in CLL patients with poor prognosis, is in line with current CLL disease models, which suggest that CLL cells are stimulated by autocrine factors 26. It is currently believed that CLL cells receive stimuli in their local environment, which makes them less susceptible to apoptosis and prolongs their life span 18. Survival signals may be mediated by soluble factors such as IL-4 38, or they may be transmitted by cell–cell contact in the microenvironment 39.

Activation of the IL-4-signaling pathway can increase CLL cell survival and decrease cell sensitivity to apoptotic stimuli 40. Thus, if CLLU1 can act as an IL-4 agonist, it could be involved in the molecular pathogenesis of CLL and its response to therapy 18.

The unique and exclusive expression pattern of CLLU1 in B-CLL cells makes this gene a target for CLL-specific therapy.

 Conclusion



The present study demonstrated that high CLLU1 expression by B-CLL cells is a significant predictor of disease progression and poor response to therapy. High expression levels of CLLU1 correlate significantly with the known prognostic markers of the disease, mainly the clinical staging and CD38 expression. CLLU1 expression levels are independent of trisomy 12. Further studies to evaluate the possible role of CLLU1 in the pathogenesis of CLL and the possibility of its use as a target for CLL-specific therapy are required.[40]

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