Impact of Fluorescent In Situ Hybridization Aberrations and CLLU1 Expression on the Prognosis of Chronic Lymphocytic Leukemia: Presentation of 156 Patients from Turkey

Objective: This study evaluates the impact of CLLU1 expression and fluorescent in situ hybridization (FISH) analysis of a group of Turkish chronic lymphocytic leukemia (CLL) patients. Materials and Methods: A total of 156 CLL patients were analyzed by FISH method; 47 of them were also evaluated for CLLU1 expression. Results were correlated with clinical parameters. Results: FISH aberrations were found in 62% of patients. These aberrations were del13q14 (67%), trisomy 12 (27%), del11q22 (19%), del17p (8%), and 14q32 rearrangements (20%). Overall del11q22 and del17p were associated with the highest mortality rates, shortest overall survival (OS), and highest need for medication. Homozygous del13q14, 14q32 rearrangements, and higher CLLU1 expression correlated with shorter OS. Conclusion: Cytogenetics/FISH analysis is still indicated for routine evaluation of CLL. Special consideration is needed for the poor prognostic implications of del11q22, del17p, 14q32 rearrangements, and homozygous del13q14. The impact of CLLU1 expression is not yet clear and it requires more data before becoming routine in genetic testing in CLL patients.


Introduction
The clinical manifestation of chronic lymphocytic leukemia (CLL) is variable. While some patients are asymptomatic for years, others show a rapid progression of the disease [1]. Recent identifiers of high-risk patients include chromosomal abnormalities, immunoglobulin heavy chain variable gene, ZAP70, CD38, β2 microglobulin and lactate dehydrogenase (LDH), and CLL upregulated gene 1 (CLLU1) expression [2]. The chromosomal abnormality rate in CLL is 30%-50%; this rate reaches up to 70%-80% with the fluorescent in situ hybridization (FISH) method [3,4]. FISH results have shown that del13q14 is correlated with good prognosis whereas del11q22 and del17p indicate poor prognosis [5,6].
Unfortunately, CLL is genetically heterogeneous. Recently relevant new genomic abnormalities such as NOTCH1 and SF3B1 mutations as well as BIRC3 disruptions have been described [7,8], but none of these genetic markers are unique to CLL. CLLU1 is defined as the first gene specific to CLL. The high expression level of CLLU1 seems to be unique in CLL [9]. However, its relevance to prognosis is still unclear.
In this study, the distribution and prognostic impact of chromosomal aberrations via FISH as well as CLLU1 expression levels were analyzed in a group of North Anatolian CLL patients.

Patients
Interphase FISH analysis was applied to blood or bone marrow samples of 156 CLL patients. Of these, 47 were also evaluated for CLLU1 expression and compared with 35 healthy controls. Staging was done according to the modified Rai staging (MRS) system. The results of the β2 microglobulin, LDH, white blood cell (WBC) count, and absolute lymphocyte count were grouped as high or low risk (Table 1).
FISH analyses were conducted using an Olympus BX51 microscope equipped with a Progressive Scan Video Camera (Tokyo, Japan). Image analysis was carried out with CytoVision software (version 3.93; Applied Imaging, Grand Rapids, MI, USA). For each probe for optimization, a cut-off level was obtained by counting 300 cells. Results were considered clonal when the percentage of cells with any given chromosome abnormality exceeded the normal cut-off value.

CLLU1 Expression
For the analysis of CLLU1 expression, RNA was isolated (QIAGEN, Hilden, Germany); cDNA was synthesized using a cDNA Reverse Transcription Kit (Ipsogen, QIAGEN). CLLU1 expression was tested by real time-polymerase chain reaction (Rotor-Gene Q, QIAGEN) using primers/probes previously defined (Ipsogen, CLLU1 Profile Quant Kit). Analysis was performed using the comparative Ct method of relative quantification with β2 microglobulin as an endogenous control. The CLLU1 expression levels were measured as fold upregulation in relation to normal patients' cells and a cut-off value was defined to separate high from low expression levels [11].

Statistical Analysis
The chi-square test was applied to determine the relationship among clinical and laboratory parameters (LDH and β2 microglobulin, WBC, MRS, CLLU1 expression, and subsets of FISH abnormalities). Overall survival (OS) was tested by the Kaplan-Meier method. The survival curves were statistically compared using a log-rank test (p≤0.05).

Patient Population
Of 156 patients, 103 patients were male. Ages ranged from 36 to 90 years (median: 68 years). In total, 37 patients died during the study. The median OS time was 101±12 months.
The shortest survival was observed with del11q and del17p and trisomy 12; the longest survival was with del13q14 and in normal patients (p>0.05). The need for medication was significantly higher for del11q22 and del17p (p<0.05). Homozygous del13q14 showed twofold shorter OS (p>0.05) and was categorized in the high-risk group (p<0.05) ( Table 3). Positive 14q32 rearrangements showed a twofold increase in mortality and need for medication (p>0.05). They were categorized in the intermediate-to high-risk group (p<0.05).
FISH results were correlated with MRS. The 11q22 and 17p13 deletions had an advanced stage (p<0.05), as well as higher WBC and absolute lymphocyte counts (p<0.05). No difference was observed within groups with respect to β2 microglobulin and LDH and initiation of therapy (p>0.05) ( Table 1).

Results of CLLU1 Expression
CLLU1 expression represented a continuum ranging from 0.1 to 3900 and a median of 17.6-fold upregulation (Figure 1). In the group with high CLLU1 expression, survival time was twofold lower and the need for medication was twofold higher (p>0.05). High CLLU1 expression was associated with higher WBC count.  There was no correlation between CLLU1 expression and FISH anomalies, β2 microglobulin and LDH levels, or MRS (p>0.05).
Few studies refer to homozygote del13q14, and its contribution to prognosis is unclear. Some have reported that homozygote del13q14 is associated with an advanced stage [15,16], while Puiggros et al. [17] noted the opposite. In our study, homozygote del13q14 was correlated with advanced stage and shorter survival.
Previous studies reported that TP53, NOTCH, SF3B1, and BIRC3 mutations are accountable for poor prognosis [7,8]. The impact of CLLU1 expression as a new prognostic factor in CLL is unclear.
In the present report, high CLLU1 expression indicated shorter survival and higher need for treatment. Similar results were observed in the literature [11,18,19].
In our study, there was no correlation between CLLU1 expression and FISH aberrations. Some have reported that patients with del17p13 and del11q22 have significantly higher levels of CLLU1 [11,18]. Chen et al. [20] noted the opposite. Buhl et al. [21] reported no increase in the level of CLLU1 in patients with trisomy 12; Gonzalez et al. [19] noted the opposite. There was no correlation between trisomy 12 and CLLU1 expression in our study (Table 4).

Conclusion
A chromosomal evaluation is still needed for the genetic evaluation of CLL because it can identify unique translocations or aberrations in which breakpoints could lead to identification of new molecular markers. Application of a FISH panel including probes aiming to detect homozygous del13q14, del11q22, del17p, 14q32 rearrangements, and trisomy 12 should still be the routine. The impact of testing CLLU1 expression is not yet clear and there is a need for more relevant data.

Ethics
Ethics Committee Approval: This study had the permission of the Ondokuz Mayıs University Ethical Committee (approval number: 201/855).
Informed Consent: It was received.

Authorship Contributions
Surgical