CLLU1 expression has prognostic value in chronic lymphocytic leukemia after first-line therapy in younger patients and in those with mutated IGHV genes

CLLU1, located at chromosome 12q22, encodes a transcript specific to chronic lymphocytic leukemia and has potential prognostic value. We assessed the value of CLLU1 expression in the LRF CLL4 randomized trial. Samples from 515 patients with chronic lymphocytic leukemia were collected immediately before the start of treatment. After RNA extraction and cDNA synthesis, CLLU1 expression was assessed by quantitative polymerase chain reaction. In total, 247 and 268 samples were identified as having low and high CLLU1 expression, respectively. The median follow-up was 88 months. High CLLU1 expression was significantly correlated with unmutated IGHV genes, ZAP-70 and CD38 positivity, and absence of 13q deletion (all r>0.2, P<0.0001). At 6 years, patients with high CLLU1 expression had significantly worse progression-free survival (9% versus 17%; P=0.03) and overall survival (42% versus 57%; P=0.0003) than patients with low CLLU1 expression. Among patients with mutated IGHV genes, overall survival at 6 years was 50% in those with high CLLU1 expression and 76% in those with low CLLU1 expression (P=0.005). However, CLLU1 expression was not an independent predictor of overall survival in a multivariate model including TP53 aberrations, beta-2 microglobulin level, age and IGHV mutation status. Nor did it predict response to treatment. CLLU1 expression analysis helps to refine the prognosis of patients with chronic lymphocytic leukemia who have mutated IGHV genes.

section_content: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable outcome depending on clinical and biological characteristics. Clinical staging is a relatively simple method of assessing the outcome of CLL patients based on clinical parameters. 1, 2 ] [14] [15] [16] [17] CLLU1 located at chromosome 12q22, encodes a novel transcript, which has been identified as a marker specific to CLL. 18 In particular, high CLLU1 expression (CLLU1-H) is significantly more frequent in unmutated IGHV and CD38 + CLL. 19, 20 CLLU1-H has been shown to be associated with shorter overall survival and shorter time to first treatment in patients with early stage CLL, 20 especially those younger than 70 years. 13 In addition, CLLU1 expression has been shown to be a specific and stable marker in CLL cells, 21 making it a promising marker not only for prognosis but also for minimal residual disease analysis. 22 However, the effect of CLLU1 expression on response to therapy is currently unknown and, to date, there are no data on the significance of CLLU1 expression with regards to progression-free survival and overall survival in patients receiving first-line therapy. We assessed the value of CLLU1 expression as a prognostic marker in the LRF CLL4 randomized controlled trial.
section_name: Introduction

section_name: Design and Methods

section_content: Pre-treatment blood samples were collected at the time of entry into the trial and were available for CLLU1 expression analysis from 515 of the 777 patients. The patients were previously untreated, 25% having Binet stage A-progressive disease, 45% stage B and 30% stage C. The male:female ratio was 3:1 and the median age was 65 years (range, 35-86 years). Patients were randomized to receive chlorambucil, fludarabine, or fludarabine with cyclophosphamide. The trial was approved by a multicenter research ethics committee and all patients gave informed consent.
section_name: Patients and samples

section_content: RNA was extracted using the RNeasy Mini Kit (Qiagen) after which Correspondence: david. gonzalez-de-castro@icr. ac. uk CLLU1, located at chromosome 12q22, encodes a transcript specific to chronic lymphocytic leukemia and has potential prognostic value. We assessed the value of CLLU1 expression in the LRF CLL4 randomized trial. Samples from 515 patients with chronic lymphocytic leukemia were collected immediately before the start of treatment. After RNA extraction and cDNA synthesis, CLLU1 expression was assessed by quantitative polymerase chain reaction. In total, 247 and 268 samples were identified as having low and high CLLU1 expression, respectively. The median follow-up was 88 months. High CLLU1 expression was significantly correlated with unmutated IGHV genes, ZAP-70 and CD38 positivity, and absence of 13q deletion (all r>0. 2, P<0. 0001). At 6 years, patients with high CLLU1 expression had significantly worse progression-free survival (9% versus 17%; P=0. 03) and overall survival (42% versus 57%; P=0. 0003) than patients with low CLLU1 expression. Among patients with mutated IGHV genes, overall survival at 6 years was 50% in those with high CLLU1 expression and 76% in those with low CLLU1 expression (P=0. 005). However, CLLU1 expression was not an independent predictor of overall survival in a multivariate model including TP53 aberrations, beta-2 microglobulin level, age and IGHV mutation status. Nor did it predict response to treatment. CLLU1 expression analysis helps to refine the prognosis of patients with chronic lymphocytic leukemia who have mutated IGHV genes. (controlled-trials. com identifier: ISRCTN58585610) cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (LifeTechnologies). CLLU1 mRNA expression levels were assessed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) using B2M as the endogenous control, as described previously. 20 In brief, CLLU1 and B2M were amplified in duplicate in a 7500 Fast Real-Time PCR stystem (LifeTechnologies) using 5 μL of cDNA in a total volume of 25 μL containing TaqMan Fast Universal PCR Master Mix (LifeTechnologies), and 200 μM of primers/probes. The ΔΔCt method was used, with RNA extracted from a normal B-cell pool as a calibrator, to calculate the relative expression of CLLU1. The assay was validated in a preliminary set of 15 samples from the original series 13 and a real-time relative quantification (RQ) of >40 was used to define cases with high CLLU1 expression, as in the original series.
section_name: RNA extraction and CLLU1 expression analysis

section_name: Statistical methods

section_content: CLLU1-H or CLLU1-L, respectively) and other relevant clinical and biological variables were assessed using the Pearson's productmoment correlation coefficient and multiple regression analysis. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Overall survival was calculated from randomization to death while progression-free survival was calculated from randomization to relapse or death, censored at the date of latest follow-up for patients remaining event-free. The follow-up was to 31 st October 2010, with the median follow-up of survivors being 88 months (range, 48 -141 months). Six years was the time-point chosen for comparing survival between groups, because follow-up data were available for all survivors up to this point, except for two patients lost to follow-up at 48 and 50 months. Cox regression was used for analysis of variables associated with survival. P values ≤0. 05 were considered statistically significant. All analyses were performed using the Statistica software package (StatSoft, Tulsa, OK, USA).
section_name: Correlations between CLLU1 expression values (high or low;

section_name: Results

section_content: The demographic, clinical and biological characteristics of the 515 trial patients with CLLU1 mRNA expression data were not significantly different from those of the 262 patients without available data (Online Supplementary Table S1 ), demonstrating that the series of 515 cases was representative of the whole trial population. The RQ values of CLLU1 expression ranged from 0. 02 to 46,757 (median 47). Various cut-offs to differentiate between high and low expression were investigated. However, no important differences were seen (data not shown) and the value used by Josefsson et al. 13 (RQ=40) was, therefore, selected for this analysis. A total of 247 and 268 samples were identified as CLLU1-H (RQ>40) and CLLU1-L (RQ<40), respectively.
section_name: CLLU1 expression in patients in the LRF CLL4 trial

section_content: The variables most closely correlated with high CLLU1 mRNA expression were unmutated IGHV genes, ZAP-70 positivity (defined as >10% of cells 8 ), CD38 positivity (defined as ≥7% of cells 8 ), and absence of 13q deletion (all r>0. 2, P<0. 0001, Table 1 ). Higher CLLU1 expression was also moderately correlated with serum b 2 microglobulin ≥4 mg/L and trisomy of chromosome 12, the location of CLLU1 (both r>0. 1, P<0. 05, Table 1
section_name: Correlations between CLLU1 expression, biological and clinical variables

section_content: 4] Patients with CLLU1-H had significantly shorter overall survival from randomization than patients with CLLU1-L. The 6-year overall survival rate was 42% [95% confidence interval (95% CI) 36-48%] in patients with CLLU1-H versus 57% (95% CI: 51-63%) in patients with CLLU1-L [hazard ratio (HR) 1. 48 (95% CI: 1. 20-1. 84); P=0. 0003; Figure 1A ]. CLLU1 mRNA expression was also significantly associated with progression-free survival. At 6 years the progression-free survival rate was 9% (95% CI: 6-12%) for patients with CLLU1-H versus 17% (95% CI: 13-22%) for patients with CLLU1-L [HR 1. 23 (95% CI: 1. 03-1. 47); P=0. 03; Figure 1B ]. However, CLLU1 expression did not predict response to treatment. The overall response rate was 79% in patients with CLLU1-H and 78% in those with CLLU1-L. The rate of complete responses was 16% in both groups. Moreover, there were no differences in response rates between patients with CLLU1-H and CLLU1-L expression within any of the treatment groups (Table 2 ). 

CLLU1 mRNA expression was significantly associated with overall survival in patients with mutated IGHV genes. Within this group, the overall survival rate at 6 years was 50% (95% CI: 36-62%) for patients with CLLU1-H versus 76% (95% CI: 67-83%) for patients with CLLU1-L [HR 1. 93 (95% CI: 1. 24-3. 00); P=0. 005; Figure 2A ]. On the other hand, in patients with unmutated IGHV genes there was no difference. In this group the overall survival rate at 6 years was 40% (95% CI: 33-47%) for patients with CLLU1-H versus 35% (95% CI: 26-45%) for those with CLLU1-L [HR 0. 91 (95% CI: 0. 69-1. 20); P=0. 5]. Altogether, for patients with unmutated IGHV genes, the overall survival rate at 6 years was significantly worse than for patients with mutated IGHV who had CLLU1-H [39% versus 50%, HR 1. 52 (95% CI: 1. 06-2. 18); P=0. 02]. 

Similarly, the difference in overall survival according to CLLU1 expression appeared more marked in younger than in older patients. In patients aged <70 years, the 6year survival rate was 51% (95% CI: 43-58) in those with CLLU1-H versus 68% (95% CI: 60-74%) in those with CLLU1-L [HR 1. 62 (95% CI: 1. 23 CLLU1 mRNA expression was not an independent predictor of overall survival in a multivariate model including the variables previously shown to be independent predictors of overall survival in this trial: TP53 aberrations (deletion and/or mutation), b 2 -microglobulin level, age and IGHV mutational status. 8, 24 Treatment group did not predict survival but was also included in the model (Table 3 ).
section_name: Association between CLLU1 expression and clinical outcomes

section_content: The prognostic value of CLLU1 mRNA expression has previously been investigated in four retrospective studies, 13, 15, 16, 20 including between 59 and 252 patients each and at a median follow-up from diagnosis of between 43 and 102 months. The end-points in all four studies were time to first treatment and overall survival from diagnosis. Our study, reported here, is the first investigation of the prognostic value of CLLU1 mRNA expression in a controlled randomized trial. In this trial CLLU1 expression was analyzed in samples collected at baseline from 515 previously untreated patients and the median follow-up from randomization was 88 months. We analyzed the effect of CLLU1 expression across three different therapies, including an alkylating agent (chlorambucil) and a purine analog (fludarabine) given alone or in combination with cyclophosphamide. In addition to overall survival, we included response to treatment and progression-free survival as end-points. Time to first treatment was not an end-point as all patients required treatment from the outset of the study. The cut-off used to distinguish between low and high CLLU1 expression remained the same as in the study by Josefsson et al., 13 making it possible to com- pare our results directly with theirs and to validate their findings prospectively. We were able to confirm that patients who had high CLLU1 mRNA expression had significantly shorter overall survival than those with low expression. They also had significantly shorter progression-free survival. The prognostic effect of CLLU1 expression on overall survival has been previously suggested to be greatest in patients under the age of 70 years and in IGHV mutated cases 13. We confirmed these findings in this randomized trial with respect to both younger patients and the IGHV mutated group. It was previously shown that younger age and mutated IGHV genes were indicators of good prognosis in the LRF CLL4 trial. 8 As suggested by Josefsson et al., a possible explanation for the greater adverse effect of high CLLU1 expression in these groups with good prognosis, compared with its effect in the corresponding groups with poorer prognosis, 13, 15 may be that high CLLU1 expression exerts its clinical effect in the longer term and does not, therefore, affect patients who have shorter survival because of older age or unmutated IGHV genes. This could also explain why CLLU1 expression did not predict overall survival in the study by Stamatopoulos et al. in 170 patients with a median follow-up of only 64 months from diagnosis. 15 In this context, it is interesting that in our trial CLLU1 expression did not affect response to treatment, in any treatment group. Unlike overall and progression-free survival, the rates of these shorter-term end-points (overall response rate and complete response rate) were no different between patients with high or low CLLU1 expression. 

9] [20] We were able to confirm these findings, particularly with regards to unmutated IGHV status, ZAP-70 and CD38 positivity, absence of 13q deletion and raised levels of b 2 -microglobulin. 

Our findings suggest that CLLU1 mRNA expression may be useful as a prognostic indicator in patients requiring treatment, specifically within the groups with longer life expectancy defined by younger age or mutated IGHV genes. Within these groups CLLU1 expression was able to identify patients with particularly good prognosis. However, we cannot recommend CLLU1 expression as a prognostic test in all patients. b 2 -microglobulin level, which can be measured by a simple laboratory test, retained independent significance in a multivariate analysis of factors predicting overall survival, while CLLU1 expression did not. b 2 -microglobulin level distinguished between subgroups with relatively good and poor prognosis not only in patients with mutated IGHV genes but also in those whose IGHV genes were unmutated. It was perhaps to be expected that CLLU1 expression would not be an independent predictor of overall survival in a multivariate model which included IGHV mutation status, because of the correlation between these two variables. However, Kaderi et al. 16 also found that CLLU1 expression did not independently predict overall survival, in 252 newly diagnosed patients (median follow-up of 102 months), in a multivariate model including other RNA-based markers. They found that LPL expression was the most significant of the markers included and, like CLLU1 expression, it was particularly able to discriminate between subgroups within the groups with good prognosis, such as those with mutated IGHV genes. b 2 -microglobulin level was not used as a comparator in their analysis. 

We have confirmed that the level of CLLU1 mRNA expression provided prognostic information in patients with CLL in the LRF CLL4 trial. We recommend the inclusion of CLLU1 expression in the risk stratification of patients with CLL, in particular in those defined by other markers as having a good prognosis.
section_name: Discussion

section_content: Funding This work was supported by the Leukaemia and Lymphoma Research ( LRF CLL4 trial core grant), the National Institute of Health Biomedical Research Centre at the Royal Marsden Hospital and The Institute of Cancer Research. The Clinical Trial Service Unit received research support from the Medical Research Council and Cancer Research UK. ME was supported by the Arbib Foundation.

section_name: Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.

section_content: The CLLU1 gene: prognostic impact haematologica | 2013; 98 (2)
section_name: Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.