Evaluation of prognostic variables in chronic lymphocytic leukemia and association with disease stage
- Authors:
- Published online on: March 13, 2021 https://doi.org/10.3892/mco.2021.2262
- Article Number: 100
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Copyright: © Attia et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as evidenced by the fact that a proportion of the patients have refractory disease and succumb within 2-3 years after the diagnosis, whereas others display an indolent disease course without need for treatment and may survive for decades (1). Due to this heterogeneity, CLL is considered suitable as a disease model for personalized medicine. In addition, malignant cells are easily obtainable, and the patient characteristics, including clinical parameters (e.g., age and comorbidities) and biological parameters (e.g., genomic aberrations and mutations), are diverse. A large number of cellular biomarkers have been found to be correlated with the prognosis of CLL patients. Certain genetic lesions, such as loss of function of the DNA-damage response by tumour protein P53 (TP53) gene (del17p), or ataxia-telangiectasia (ATM) gene (del11q), particularly with respect to DNA-damaging chemotherapy (e.g., fludarabine), have a highly negative prognostic impact (2). Furthermore, markers such as CD49d, CD38 and intracytoplasmic ZAP-70, which are not only correlated with prognosis, but also with genetic aberrations, are possibly involved in the mobilization and homing of CLL cells (3,4). CD49d is the α4 subunit of the integrin heterodimer α4β1. Its expression promotes microenvironment-mediated proliferation of CLL cells and identifies a subgroup of CLL patients who develop progressive disease (5,6). The levels of soluble serum markers, such as thymidine kinase, β2-microglobulin (B2M) and soluble CD23, are important predictors of prognostic relevance in patients with CLL (7,8). With the recent advent of new, more effective therapies, the known survival predictors, which require continuous research to properly stratify patients for treatment, are becoming obsolete (9). The aim of the present study was to evaluate the most commonly used genetic and serological prognostic markers known to have survival predictive value for identifying high-risk CLL patients with a higher tumor burden to ensure appropriate management, and to validate CD49d as a predictive prognosticator in a sample of Egyptian patients with CLL.
Patients and methods
Subjects
A total of 81 Egyptian patients with CLL were recruited from the National Cancer Institute and Kasr El-Aini Hospital between December 2017 and October 2019. A total of 75 healthy subjects who were age- and sex-matched to the studied patients were randomly recruited as the control group. All participants provided informed verbal consent before being included in the study, in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments involving human subjects. The study was approved by the Ethics Committee of the National Research Center (registration no. 17-147). The diagnosis of CLL was based on the World Health Organization classification of tumors of hematopoietic and lymphoid tissues (10). Full history taking and thorough clinical examination of the patients were performed to evaluate the presence of pallor, petechiae, jaundice, lymphadenopathy, splenomegaly or hepatomegaly. Treatment was initiated in 58% of patients with disease-related symptoms, advanced or progressive disease according to criteria described by recent guidelines (11). At least one of these criteria should be met: Evidence of progressive marrow failure as manifested by the development or worsening of anemia and/or thrombocytopenia; massive (≥6 cm below the left costal margin) or progressive or symptomatic splenomegaly; massive nodes (≥10 cm in longest diameter) or progressive or symptomatic lymphadenopathy; progressive lymphocytosis with an increase of ≥50% over a 2-month period, or lymphocyte doubling time <6 months; autoimmune complications, including anemia or thrombocytopenia, poorly responsive to corticosteroids; symptomatic or functional extranodal involvement; disease-related symptoms including unintentional weight loss ≥10% within the previous 6 months, significant fatigue, fever (≥38.0˚C) for ≥2 or weeks without evidence of infection, night sweats for ≥1 month without evidence of infection.
Patients were evaluated clinically according to the Binet and Rai staging systems (11) regardless of treatment type or disease duration.
Sample collection
Peripheral blood samples (6 ml) were collected in K2-EDTA, sodium heparin and plain blood collection tubes (Becton, Dickinson and Company), in order to perform diagnostic and prognostic evaluation of patients with CLL.
Flow cytometric analysis
Monoclonal antibodies for diagnostic and prognostic evaluation (all from Beckman Coulter Immunotech) included: CD19-PC7 (cat. no. IM3628U); CD5-FITC (cat. no. IM0468U); CD10-PC5.5 (cat. no. B16490); CD49d-FITC (cat. no. IM1404U); CD38-PE (cat. no. IM2371U) and ZAP-70-PE (cat. no. B57658). Monoclonal antibodies (all from Agilent Technologies, Inc.) included: CD20-FITC (cat. no. F0799); CD79b-PE (cat. no. R7272); anti-kappa-FITC (cat. no. F0434); and anti-lambda-PE light chains (cat. no. R0437).
Assessment of CD49d, CD38 and cytoplasmic ZAP-70 (cytoZAP-70) markers was performed using Beckman Coulter Navios Flow Cytometer, as described by Chen et al (12). Labeled Mo Ab (20 µl) were dispensed into appropriately labeled tubes, followed by the addition of 100 µl sample and incubation in the dark for 20 min. Three and half milliliter Lysing reagent (3.5 ml) was added to each tube, inverted once and kept for 5 min. Centrifugation was performed at 300 x g for 3 min at room temperature, 3.5 ml PBS was added, and centrifugation was repeated at 300 x g for 3 min. CD49d and CD38 were tested in two different tubes and were gated on CD19+ B cells. IntraPrep Permeabilization Reagent (cat. no. A07803; Beckman Coulter Immunotech) was used for cytoZAP-70 detection. Specific isotypic controls for FITC and PE-conjugated monoclonal antibodies were used. The expression results were reported as the percentage of CD49d+/CD38+/cytoZAP-70+ cells over the corresponding isotypic control within the gated CD19+ B-cell population for each patient and control subject.
Fluorescence in situ hybridization (FISH)
FISH was performed for all fresh heparinized peripheral blood samples obtained from the patients according to the manufacturer's instructions using the Vysis CLL FISH Probe Kit (cat. no. 04N02-021; Abbott Molecular) (13). The SpectrumGreen-labeled (LSI) ATM probe specific for del11q22.3 contains the ATM gene, the SpectrumOrange-labeled LSI-D13S319 probe is located at 13q14.3 and the SpectrumOrange-labeled LSI TP53 probe is located at 17p13.1. In addition, the SpectrumGreen-labeled CEP 12 probe is located at the centromere of chromosome 12. A total of 200 interphase cells were examined for each probe. The number of observed orange and green signal patterns from each of two readers were added together to generate the count for 200 nuclei for each specimen. Normal/abnormal determination was made for each specimen by comparing the number of nuclei with observed abnormal patterns per 200 scoreable nuclei to the normal determined cut-off value. Cut-off points were determined by the main laboratory of Kasr El-Aini Hospital from cytogenetically normal controls among patients' relatives.
Serology
Serum samples from all recruited subjects were quantitatively analyzed for sCD23 and B2M levels using the human sCD23 ELISA kit (Invitrogen; Thermo Fisher Scientific, Inc.; Bender MedSystems GmbH; cat no. BMS 227-2) and the B2M ELISA kit, ORGENTEC Diagnostika GmbH; cat. no. ORG 5BM), according to the manufacturer's protocols.
Statistical analysis
Data were coded and entered into the SPSS analysis program (version 25; IBM Corp.). Data are presented as mean with standard deviation, minimum and maximum for quantitative data and as frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were performed using the non-parametric Kruskal-Wallis and Mann-Whitney U tests (14). Categorical data were compared using the χ2 test. Fisher's exact test was used instead when the expected frequency was <5(15). Correlations between quantitative variables were assessed using Spearman's correlation coefficient. P<0.05 was considered to indicate statistically significant differences.
Results
Patient characteristics
The clinical data of patients with CLL are summarized in Table I. A total of 32 patients (39.51%) had an indolent disease course during follow-up, whereas 39 patients (48.14%) had progressive disease with clinical symptoms. A total of 10 patients (12.35%) were newly diagnosed at the time of recruitment. According to the Binet staging system, 50.6% of the patients were stage C, 35.8% were stage B and 13.6% were stage A at diagnosis. In the present study, patients with CLL had clonal neoplastic cells with the morphological characteristics of small mature B lymphocytes in the peripheral blood. The hematological and serological findings of patients with CLL and the age- and sex-matched controls are listed in Table II. The wide range in these parameters in the CLL group was expected due to the presence of different categories of patients as regards disease stage and therapeutic interventions. i) On flow cytometry, the patient samples displayed 100% positive expression of CD19, 25 and 20, as well as clonal restriction (λ-restricted, 37.04% and κ-restricted, 62.96% of the samples). ii) Prognostic workup: The expression levels (%) of prognostic markers by flow cytometry are demonstrated in Table III and Fig. 1. Representative flow cytometric dot plots of different prognostic variables on CD19+ B-CLL cases are shown in Fig. 2A-F. A total of 54 patients with CLL (66.6%) exhibited genetic aberrations using the FISH technique (Table IV and Fig. 3A-D).
The patients were categorized according to the Binet staging system, and a significant increase in the expression of sCD23, CD49d and ZAP-70 was detected in group C (P=0.008, 0.034 and 0.017, respectively) when compared to both groups A and B, as shown in Table V and Figs. 4 and 5. Elevated B2M (>3.5 µg/ml) was more frequently detected in group B and C patients (91.7 and 97.1%, respectively; P=0.004). In addition, patients under treatment with advanced disease and aggressive course exhibited higher B2M levels compared with those with an indolent course (P=0.025). The B2M levels were significantly elevated in group C patients when compared with group A patients (P=0.034).
 | Table VSignificant prognostic variables in CLL patient groups according to the Binet staging system. |
Higher frequency of CD49d positivity was found in patients with positive CD38 expression (P=0.027). CD49d was considered positive when its level was >30% according to Bulian et al (3). Higher incidence of hepatomegaly and splenomegaly was detected among CD49d+ patients (57.1 and 85.7% with P=0.049 and 0.016 respectively). Furthermore, CD49d+ patients exhibited significantly higher CD38 levels (P=0.002; Table VI and Fig. 6). Additionally, CD49+ patients exhibited higher expression of trisomy 12 (P=0.015) and lower expression of del13q14 (P=0.001), as shown in Table VI and Fig. 7.
| Table VISignificant prognostic variables in patients with CLL according to CD49d (CD49d+, n=21; CD49d-, n=60) and trisomy 12 (trisomy 12+, n=113; trisomy 12-, n=68) expression. |
Variations in the levels of CD38 and ZAP-70 in relation to the different categories of sCD23 and CD49d are shown in Fig. 8. Higher CD38 expression was observed in patients with levels of sCD23 >95 U/ml and CD49d positivity >30% (P=0.012).
In trisomy 12+ patients (n=13; 16% of patients with CLL), CD49d positivity was more frequently observed (75%; P=0.015), as was a higher mean level of CD49d (P=0.004) and lower mean expression of del13q (P=0.000; Table VI and Fig. 9).
Patients positive for CD38 had a higher total leukocyte count (TLC), absolute lymphocyte count (ALC) and sCD23 levels (P=0.038, 0.02 and 0.000, respectively).
Patients with both CD49d+ and B2M>3.5 µg/ml exhibited higher TLC (P=0.048), higher ALC (P=0.036), higher expression of CD38 (P=0.002) and trisomy 12 (P=0.034), and lower expression of del13q14 (P=0.002; Fig. 10).
In correlation studies, s-CD23 and CD49d were positively correlated with CD38 (r=0.267 and 0.371; P=0.037 and 0.002, respectively); sCD23 was positively correlated with B2M (r=0.344, P=0.003); and CD38 was positively correlated with the presence of del11q (r=0.314, P=0.036).
Discussion
The present study investigated different clinical and biological prognosticators in patients with CLL. Patients on novel therapeutic agents were included in the study.
Patients were categorized according to the Binet clinical staging system. sCD23, CD49d and ZAP-70 exhibited significantly higher expression in group C when compared with groups A and B. Therefore, the expression of these prognostic markers in the early stages and throughout the disease management may help identify progression. Popova et al (16) reported that flow cytometric markers were negative in the early stages of CLL and underwent changes in 10-25% of cases with disease progression. Previously, Saka et al (17) found that sCD23 determination at diagnosis and during the course of the disease may help identify patients who rapidly progress to more advanced stages.
Detection of CD49d by flow cytometry was considered superior to CD38 and ZAP-70 in predicting overall survival (18). In the present study, the frequency of positive CD49d and CD38 cases was 25.9 and 28.4% of the total number of cases, whereas that of ZAP-70 was 8.6%. Due to the lower frequency of positive ZAP-70 expression among CLL patients, the interpretation of the results was easier for CD49d and CD38.
Patients with CLL were stratified according to their CD49d levels to identify variations in different prognosticators. In CD49d+ patients, higher incidence of hepatomegaly and splenomegaly was observed, whereas higher expression of CD38, sCD23 and trisomy 12 and significantly lower expression of del13q14 were detected by FISH. These findings were in agreement with those of previous investigators who found that positive CD49d expression >30% was associated with advanced clinical stage and aggressive CLL course (3,18-20). Recently, Tissino et al identified a bimodal expression of CD49d with distinct clinicobiological characteristics in 20% of CLL cases. They recommended that the pattern of CD49d expression should also be considered to improve its prognostic impact in CLL (21).
In the subgroup of CD38+ patients, higher tumor burden with higher TLC, ALC and increased sCD23 levels were detected. These associations were in agreement with previous findings (22). Serological markers have been considered as predictors of tumor burden in addition to disease progression. In the present study, when patients were stratified according to sCD23 expression levels, high levels were associated with increased expression of CD38 and CD49d, indicating its role in disease progression. In patients with high levels of sCD23, higher CD38 expression was observed in the presence of positive CD49d expression compared with patients negative for CD49d expression. Patients with B2M levels >3.5 µg/ml and positive CD49d expression exhibited increased tumor burden, higher expression of CD38 and trisomy 12. These findings highlighted that integrating both serological markers and CD49d may add to the prognostic value of each marker alone. A number of researchers have investigated the integration of prognostic markers into a combined risk score due to discrepancies found in the same patient, with some prognostic factors suggesting shorter time to first treatment and overall survival compared with others (23-25).
Trisomy12+ CLL patients represent a distinctive subset of patients with CLL who exhibit higher expression of CD49d and lower expression of del13q14. The association between trisomy 12 and CD49d expression was previously explained (21). Trisomy 12+ CLL is characterized by high mutation frequency of the NOTCH1 gene, and an association between higher CD49d expression and the presence of NOTCH1 mutations through nuclear factor-κB pathway involvement has also been reported (26,27). In the future, the assessment of TP53 aberrations, in the form of both gene mutations and del17p, may become mandatory, as patients carrying such gene disruptions are defined as high-risk. In addition, the analysis of TP53 mutations can identify 30-40% of CLL patients who only carry the gene mutations and may be missed by FISH. Cohen et al proposed that TP53 aberrations, unmutated IGHV, mutated NOTCH1 and CD49d expression are powerful prognosticators and have predictive significance in CLL before treatment initiation to guide therapeutic decisions (28). IGHV mutational status is a stable CLL prognostic marker and can be measured at any time throughout the disease course. It may also help with the selection of novel targeted therapies in unmutated cases. Further studies are recommended on a larger scale to assess and integrate the IGHV mutational status and TP53 mutations to optimize patient stratification.
Expression of the sCD23, CD49d and ZAP-70 prognostic markers in early-stage CLL and throughout the disease management may help identify any progression to a more advanced stage. CD49d is considered superior as a prognostic marker and CLL patients positive for CD49d expression exhibited higher incidence of hepatomegaly and splenomegaly, higher expression of CD38, sCD23 and trisomy 12, and significantly lower expression of del13q14. CD38 expression was associated with higher tumor burden, higher TLC and ALC, and increased sCD23 levels. CLL patients with high levels of sCD23 exhibited higher CD38 expression in the presence of CD49d-positive expression compared with CD49d-negative patients. Patients with high B2M levels (>3.5 µg/ml) and CD49d-positive expression exhibited increased tumor burden, higher expression of CD38 and trisomy 12. Therefore, integrating serological markers and CD49d expression by flow cytometry may add to the prognostic value of each marker alone and help identify high-risk patients with a higher tumor burden.
Acknowledgements
Not applicable.
Funding
The present study was supported by the Science, Technology and Innovation Funding Authority (STDF), Egypt, through Targeted Health call grant no. 22918.
Authors' contributions
HRMA and AHA contributed to the project preparation, study design and submission for funding. HRMA, MHI and SHAEA were responsible for the management of purchasing tasks and schedules. MHI coordinated specimen collection and transport and implemented a quality policy throughout the laboratory analysis workflow. MHI and SHAEA contributed to serological analysis and tabulation of final result. Patient selection and data collection were performed by NMH, MES, RA, RR and OYAED. FISH was performed by MEY and HAAH. Flow cytometric analysis was performed by RAO. NE contributed to appropriate selection of CD markers and supervision of flow cytometric procedures. Analysis of data and the first draft of the manuscript were prepared by HRMA. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
All participants provided verbal informed consent prior to being included in the present study in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments on humans and the study was approved by ethical committee of National Research Center, Egypt, (registration no. 17-147).
Patient consent for publication
Not applicable.
Availability of data and materials
The datasets generated and/or analyzed in the present study are available from the corresponding author upon reasonable request.
Competing interests
The authors declare that they have no competing interests.
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