Cytogenetic Subclone Burden: A New Biomarker Predicting Chronic Lymphocytic Leukemia Patients Outcome

Background: Chronic lymphocytic leukemia is the most prevalent adult leukemia that occurs in older patients and presents a variable course of the disease. Risk stratification of CLL is a matter of continuous improvement. Thus, this study aimed to assess the impact of the quantification of 17p del and 11q del cytogenetic subclones on the outcome of patients with chronic lymphocytic leukemia. Patients and Methods: This is a prospective study that involved 100 subjects with CLL. For all included patients; assessment of the cytogenetic subclones burden for 17p del and 11q del using the FISH technique was carried out. Results: CLL patients with a high 17p del (>33%) cytogenetic subclone burden showed significantly shorter lymphocyte doubling time (LDT), time to first treatment (TTFT), and progression free survival (PFS) compared to those with a lower burden. On contrary 11q del subclone(>30%) burden had an insignificant impact on LDT, TTFT and PFS. Conclusion: Quantification of 17pdel burden (>vs.≤33%) could be used for refining risk stratification of CLL patients.


Introduction
Chronic lymphoproliferative disorders of B cells (BCLPD) are characterized by the accumulation of mature B lymphocytes in peripheral blood and bone marrow with progressive lymphadenopathy and splenomegaly.These disorders have been categorized according to their genetic changes, histopathological features and immunophenotype [1].The most prevalent type of leukemia in adults among B-CLPD is chronic lymphocytic leukemia (CLL), with an estimated incidence of 4 to 6 occurrences per 100,000 persons each year in western countries [2,3].CLL is diagnosed by the presence of more than 5,000 monoclonal B cells per microliter with specific immune phenotypes [4].Matutes Score was designed for CLL diagnosis [5].It depends on the expression pattern of CD5, CD23, FMC7, SmIg, and CD22.Most CLL cases had a score of more than 3, whereas non-CLL cases had RESEARCH ARTICLE Cytogenetic Subclone Burden: A New Biomarker Predicting Chronic Lymphocytic Leukemia Patients Outcome deletions of 11q or 17p, have poor prognosis [7][8][9][10] Many trials have been done to search for new biomarker for improvement risk stratification of CLL patients outcome [11][12][13].In this context, Recent reports indicated that the degree of positivity of cytogenetic findings by FISH could have different impact on CLL patients outcome [14].The purpose of this study was to assess the impact of quantification of 11qdel and 17pdel cytogenetic subclones burden on CLL patients outcome.

Patients
The present study is a prospective study conducted on 100 patients with CLL (61 men, 39 women); their mean age is 60.5 years, recruited from the inpatient and outpatient clinics of the Mansoura University Oncology Center for a two-year period from December 2019 to December 2021.This study was approved by the institutional review board of the Mansoura Faculty of Medicine and done according to declaration of Helsenki.All included patients gave their informed consent to participate in the current study.The sample size was calculated using an online sample size calculator (https://www.openepi.com/SampleSize/SSMean.htm) with anticipated prevalence of chronic

Samples
Bone marrow samples were collected at diagnosis by sterile procedure and distributed as follows.1.Two milliliters were added to dipotassium ethylene-diaminetetraacetate (EDTA) (1.2 mg of anhydrous salt per milliliter of blood) for performing a flow cytometric assay to detect antigens expression by lymphocytes 2. Two milliliters were delivered in heparinized tube for FISH (fluorescence in situ hybridization) studies.

Cytogenetic detection by FISH technique The FISH technique includes 4 steps
Culture, Hybridization.Microscopy and Interpretation.The interphase FISH technique was performed on heparinized samples after optimization of the protocol using commercially available probe from Cytocell UK as follows: Heparinized samples were inoculated in the culture medium and kept overnight incubation, then harvesting was done to obtain a clear pellet.Slides were prepared from harvested specimens and viewed under a phase contrast microscope.Then, the following fluorescent-labeled probes were added: LSI p53 (17p13), LSI ATM l(11q22.3), and LSI D13S319 (13q14.3)(all probes were from Cytocell, UK).The slides were added to the Lecia ThermoBrite instrument (Thermobrite Leica, Technoscient, Germany) to allow the denaturation of the sample and probe simultaneously, and then hybridization and posthybridizatin washes were applied.Finally, the slides were counterstained by 10µl of DAPI and examined with the aid of an Olympus BX 61 fluorescent microscope ( Optoscient company, Japan) that is fitted with filters suited for DAPI, rhodamine, and fluorescein.Using Cytovision Image Capture software and a monochrome digital camera, representative pictures were taken (Applied Imaging).

Interpretation of results
The signal screening was evaluated in 200 cells.The cut-off point for positivity for single deletion was 10% for del(17p), del(11q).In a normal cell, this probe will appear as discrete red and green spots, one for each homologue (2G, 2R).In cells with a single deletion, one red signal with two green signals (2G, 1R) will appear.The percentage of positivity was evaluated in positive cases for subclone classification (Figure 1, 2).

Statistical analysis
The information was examined, coded, and tabulated with the help of IBM's Statistical Software for the Social Sciences (2017 release) (version 25.0 of IBM SPSS Statistics for Windows, IBM Corp, Armonk, New York).Each parameter's data was collected, and analysis was performed as needed.To check if the data were normally distributed, the Kolmogorov-Smirnov test was carried out.We utilised the mean and standard deviation for parametric data.The median and range for non-parametric numerical data were adopted.We relied on frequency and percentage to represent non-numerical parameters.The means of the 2 groups were compared using the Student T test.The Mann-Whitney U test was used to determine whether or not there was a significant difference in the means of 2 groups when testing a nonparametric variable (U test).The chi-square test was used to analyse the link among two qualitative variables.When the projected count was less than 5 in more than twenty percent of the cells, we used Fisher's exact test to look for a link among the two qualitative variables.To evaluate the sensitivity and specificity of quantitative diagnostic tests that

Results
The characteristics of CLL patients are shown in Table 1.Patients with CLL were stratified according to 17p del and 11q del into positive and negative ones.ROC curve was applied to identify the best cutoff that can sub-classify the positive ones into those with good or bad outcome (Figure 3).This analysis revealed that the best cut-off value of 17p del percent was (33%) ( sensitivity was 83.3%, specificity was 85.7%).On the other hand, the best cut-off value of 11q del percent was (30%) (sensitivity was 72.7%, specificity was 75%).In this context, all studied CLL cases were stratified according to 17p del into 3 subgroups cases with negative 17p del ( 33%).Likewise, according to 11q del detection levels, CLL cases was classified into 3 subcategories which includes negative 11q del ( 30%) (Table 2, Figure 3).
Patients with high positive 17p del (> 33%) had significantly shorter LDT, shorter TTFT and shorter PFS than those with low positive 17p del (≤33%) as well as those with wild type.While patients with wild 17p del did not differ significantly from those with low positive 17p del (≤33%).Furthermore, high smudge cells count were significantly associated with wild and low positive 17p del (≤33%).On the other hand, no significant association was found regarding positive CD38, and RAI stages with 17p del among CLL group (Table 3).
Patients with high positive 11q del (> 30%) had significantly shorter LDT, shorter TTFT and shorter PFS than those with wild type, but did not differ significantly from those with low positive 11q del (≤30%).Also, more smudge cells were significantly associated with wild and low positive 11q del(≤30%).While no significant association was found regarding CD38, and Rai stages with 11q del among CLL group (Table 4).

Discussion
In chronic lymphocytic leukemia, deletion of the short arm of chromosome 17 is associated with rapid disease progression, as well as a poor response to treatment [15][16][17].Previous studies stated that 17p del was positive in 5-10% at diagnosis [18,19].The present study revealed that 17p del was positive in 19%.The ROC curve of 17p del percent was performed to evaluate the validity of 17p del percent for the prediction of CLL patients outcome.High-accuracy AUC was found (AUC=0.923) at best cut-off value of 17pdel burden equal to ≥33% vs<33%.
We have found that patients with a lower percentage of cells with 17p del had significantly better outcome (better LDT, TTFT, PFS & OS) than those with a higher percentage of cells with 17p del, as well as those with wild type.Similarly, Strati et al. [20] showed that a higher rate complete remission (CR)/ non-complete remission(NCR) was associated with a "lower burden" of 17p del detected by FISH.Additionally, previous research showed that patients with 17p del as a low subclone burden had a survival advantage compared to patients with 17p del with a high burden [21,22].Yuan et al [14] demonstrated that the proportion of 17p del-positive cells and the size of 17p del subclones were significant indicators of prognosis in CLL.Furthermore, Li et al. [23] and Van Dyke et al. [24] found that CLL patients with a high proportion of cells

Figure 1 (
Figure 1(a).Representative CLL FISH Image of Case with Negative 17p del Shows Two Red Dots Represent LSI p53(17p13) Probe and Two Green Dots Represent D17Z1(CEP17) Probe for Control, One for each Homologue.

Figures 1 ,
Figures 1, 2 displayed FISH photos of CLL cases with different grades of 17p del and 11q del. Figure 1(a): Represent CLL FISH image of a case with negative 17p del. Figure 1(b): Represent CLL FISH image of a case with 25% 17p del. Figure 1(c): Representative CLL FISH image of a case with 95% 17p del. Figure 2(a): Represent CLL FISH image of a case with 22% 11q del. Figure 2(b): Represent CLL FISH image of a case with 80% 11q del.

Figure 1 (
Figures 1, 2 displayed FISH photos of CLL cases with different grades of 17p del and 11q del. Figure 1(a): Represent CLL FISH image of a case with negative 17p del. Figure 1(b): Represent CLL FISH image of a case with 25% 17p del. Figure 1(c): Representative CLL FISH image of a case with 95% 17p del. Figure 2(a): Represent CLL FISH image of a case with 22% 11q del. Figure 2(b): Represent CLL FISH image of a case with 80% 11q del.
leukemia and level of absolute precision of 2% with alpha error of 5% and study power of 90%.All patient data including age, complete hemogram, and course of the disease were collected from the patient's medical record.The diagnosis was based on the morphological examination of peripheral blood and bone marrow smears 1Hematology Unit, Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt.2InternalMedicine Department, Mansoura Faculty of Medicine, Mansoura University, Mansoura, Egypt.*For Correspondence: salaharef@yahoo.comlymphocytic

Table 1 .
Patients Characteristics of CLL Patients at DiagnosisFigure 3. ROC Curve to Determine the Best Cut off Value that Discriminate the Cytogenetic Burden Better Classify CLL Patients into Good and Bad Outcomes Regarding 17pdel and 11q del., The high accuracy AUC was found (AUC=0923) at best cut of value of 17p del percent (=33%), sensitivity was 83.3%, while specificity 85.7%.Regarding 11q del moderate accuracy AUC was found (AUC=0.841) at best cut of value of 11p del percent (=30%), sensitivity was 72.7%, while specificity 75%.with 17p del had unfavorable results, whereas those with a low proportion (<20,<25% respectively) had good results similar to those without 17p del.Regarding 11q del, this is the first study to investigate the value of 11q del subclone identification.The present study showed that 11q del was positive in 15% of the cases.The ROC curve of 11q del percent was performed for the assessment of the validity of 11q del percent for

Table 2 .
Classification of Positive CLL Case Regarding 17pdel and 11qdel into High Positive Cases and Low Positive Cases according to the Finding Addressed by the ROC Curve.

Table 3 .
Comparison of LDT, TTFT, PFS, CD38, Smudge Cells and RAI Stages According to 17p del among CLL Group P1, comparison between wild, low positive, high positive 17p del; P2, comparison between wild, low positive 17p del; P3, comparison between wild, high positive 17p del; P4, comparison between low positive, high positive P1, comparison between wild, low positive, high positive 11q del; P2, comparison between wild, low positive 11q del; P3, comparison between wild, high positive 11q del; P4, comparison between low positive, high positive 11q del

Table 4 .
Comparison of LDT, TTFT, PFS, CD38, Smudge Cells and RAI Stages According to 11q del among CLL Group.theprediction of good patient outcome (CR, NCR) and bad patient outcome (SD and PD).A moderate accuracy AUC was found (AUC=0.841) at the best cut-off value of 11q del percent (30%).