17p deletion , CD 80 and CD 86 expression in chronic lymphocytic leukemia

Limitation

The relatively small sample size in our study makes our significant results require further verification.

Conclusion:

CD80 plays a role in CLL pathogenesis and interfere with the immune system in CLL patients. Therefore, cases with enhanced CD80 expression require earlier therapeutic intervention and may indicate a worse prognosis, which can guide treatment strategies for this disease. The 17p deletion acts as a prognostic marker in CLL.

Recommendation:

Understanding the role of CD80 in CLL opens up potential avenues for therapeutic inter-ventions, diagnosis and prognosis. Further studies involving larger populations to validate these findings and if CD86 has role in CLL or not.

Acknowledgments

The authors thank the study participants.

Statement regarding data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Authors’ contributions:  K.M.S, N.I.O, N.H.A, A.S.A designed the study, conducted the medical evaluation, and collected samples. They also contributed to conceptualization, data curation, and formal analysis. K.M.S,  N.I.O, W.M.A, M.A.E, R.Y.A conducted investigation, developed methodology, mana-ged project administration, and conducted laboratory sample processing. K.M.S, N.I.O, N.H.A, A.S.A shared supervision respon-sibilities, validated the results, visualized data, and contributed to writing the original draft. N.I.O. and R.Y.A. performed statistical analyses. K.M.S, N.I.O, N.H.A, A.S.A  reviewed and edited the manuscript. All authors conceptualized, edited, and approved the final version of the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Introduction

Chronic lymphocytic leukemia (CLL) is defined by the clonal growth of mature B lymphocytes. The formation and progression of CLL are marked by the accumulation of these abnormal cells in the bone marrow, lymph nodes, and spleen ⁽¹⁾. The variable nature of CLL makes it challenging to predict survival outcomes due to its complex and varied clinical presentation ⁽²⁾

The Rai and Binet staging systems are commonly used to assess disease progression but are insufficient^(3,4). Chromosomal abnor-malities, such as 17p deletion, 11q deletion, trisomy 12, unmutated immuneglobulin heavy chain variable gene (IGHV), and ZAP-70 expression, have been studied as reliable indicators of poor prognosis. However, these tests can be expensive ^(5).

T cell immunological responses against tumors initiate with recognition by the T cell receptor (TCR) of tumor antigen-derived peptides in conjunction with MHC class I antigens. However, T cells require a second trans-membrane signal event to acquire effector cell roles. This second signal is provided by costimulatory molecules, such as CD80 (B7-1) and CD86 (B7-2), which are expressed on antigen-presenting cells (APCs) like dendritic cells and certain macrophages. These costimulatory molecules bind to their ligands on T cells, facilitating T cell activation ⁽⁶⁾.

The tumor suppressor gene TP53, located on 17p13, plays a crucial role in cell cycle arrest, DNA repair, genomic stability, and apoptosis. Although TP53 mutations are relatively rare in hematological malignancies, they are the most frequently mutated gene in human cancer ⁽⁷⁾.

 The TP53 gene encodes the p53 transcription factor protein, an essential regulator of the DNA damage response pathway⁽⁸⁾. Deletions of 17p13 are associated with poor treatment response and unfavorable outcomes in CLL, emphasizing the importance of detecting these changes for clinical management⁽⁹⁾. The presence of TP53 abnormalities in patients' lymphocytes suggests the need for immediate treatment initiation and may indicate potential resistance to chemotherapy ⁽¹⁰⁾.

We aimed to investigate the prognostic value of CD80 and CD86 expression and the significance of 17p deletion in CLL patients.

Patients and Methods

Patients

This prospective study was conducted at the Clinical Pathology and Oncology Departments of Minia University's Faculty of Medicine, Egypt, from September 2022 to September 2023. The study included 19 patients with newly diagnosed chronic lymphocytic leukemia (CLL) (Group Ia). The same patients were followed up on day 21 after 3 cycles of   induction therapy called: BTK inhibitor (acalaburtinib) (Group Ib) to assess treatment response.

Patients underwent thorough physical examination to detect lymphadenopathy, splenomegaly, or hepatomegaly and medical evaluations including laboratory tests, abdominal ultrasonography, and bone marrow aspiration. Clinical data were collected from hospital records and patient files. Blood samples were analyzed for complete blood count (CBC), immune-phenotyping, fluores-cence in situ hybridization (FISH) analysis and LDH.

-Patients with a hematological cancer diagnosis other than CLL were not included in our study.

Procedure for sampling

Under complete aseptic conditions, approximately six milliliters of venous blood were collected from each individual: Two milliliters of blood were evacuated in  tubes containing Ethylene Diamine Tetra acetic Acid (EDTA) for CBC and immunophenotyping, two milliliters evacuated in  tubes that had been heparinized for FISH analysis, and two milliliters in  a plain tube into which the blood was left to clot, centrifuged,  the expressed serum was utilized  for the purpose of determining Lactate dehydrogenase ( LDH).

In addition to bone marrow aspiration and examination for: Confirmation of diagnosis, assessment of bone marrow involvement, monitoring disease progression, detection of Richter's transformation and guiding treatment decisions.

Methodology:

Laboratory Procedure:

Complete blood count using an automated hematology analyzer (CELLTAC G, developed by Japan, NIHON KOHDEN CORPORATION). Differential leucocytic count analysis using peripheral blood smears stained with Leishman to identify lymphocyte percentage and morphology. Bone marrow aspiration and examination   using Klima-type marrow puncture needles and leishman-stained smear analysis.  Following the instructions provided by the manufacturer, LDH measurement using a commercially available kit utilizing clinical chemistry automation systems (SELECTRA PRO XL, ELITech group, Netherlands).

- Immunophenotyping of routine panel of chronic lymphocytic leukemia (Markers for diagnosis in CLL cases – CD19, CD5, CD79b and FMC7, CD23 and CD25 molecules) and CD80 and CD86 expressions were determined by Flowcytometer.

Expression of CD 80 and CD 86 staining steps:

For each sample, two tubes were used  , one was the test tube and the other tube was used for isotypic control then hundred  ul (100 ul) of blood samples were added in both tubes and  ten  ul of  phycoerythrin (PE) conjugated anti-CD80 and Fluorescein isothiocyanate (FITC) conjugated anti-CD86,  ten  μL of allophy-co-cianine (APC) Anti - CD5 monoclonal antibody, ten μL of anti CD19 PerCP-Cy5.5  conjugated antibody were added (only to the test tube). After that, we vortexed the tubes and let them sit in the dark at room temperature for 15 minutes. Then, we added 2 ml of lysing buffer solution to each tube, vortexed them again, and let them sit in the dark for 10 minutes. After that, the tubes were centrifuged at 1200 rpm for 5 minutes; after that, the supernatant was removed and discarded. Following that, each tube was washed with phosphate buffer saline (PBS). Tubes were prepared for data acquisition by flow cytometric analysis after cells were suspended in 300 ul of PBS. The kits were supplied by Kemet Medical. Readings were obtained using flow cytometry (BD-FACS Canto II Flow Cytometer, Becton, Dickinson and Company, USA) to evaluate CD80 and CD86 expression after lymphocyte population gating (CD5+/CD19+). BD FACS Diva software was used for data processing.

17p deletion by FISH method in CLL patients

Heparinized samples were subjected to the following: Mixed with Lymphocyte Sepa-ration Medium, a solution of Ficoll and sodium diatrizoate (Hypaque) with a density set at 1.077 g/ml. Its primary function was to separate mononuclear cells (MNCs) from blood samples. FISH was performed on MNCs that had been isolated. By directly labeling the probes with the fluorochrome dye, an epifluo-rescence fluorescence microscope could be used for visualization.

Adding 5 μl of 4′,6-diamidino-2-phenylindole (DAPI)/antifade solution [11] was used to counterstain the slide in that instance. Using a

Zeiss Axio Imager Z2 Fluorescence Micro-scope (model BX53).  Cameras were Dual camera ports Axiocam 506 color and Axiocam 506 mono and the microscope uses ISIS FISH imaging system (Meta Systems GmbH, Germany).  A total of 200 interphase nuclei were scored per probe per slide.

Ethical statement

The study was approved by the Minia University, Faculty of Medicine, Institutional Review Board (MUFMIRB, Approval number: 95:9/2021, Date of approval: 27 September 2021). Written informed consent was obtained from participants, and proce-dures followed the principles outlined in the Declaration of Helsinki.

Statistical analysis

The data was analyzed using IBM SPSS 26.0, a statistical tool developed by IBM and located in Armonk, New York, USA. Mean, standard deviation (SD), lowest, and maximum values for quantitative measurements were used to express the data. For non-parametric data, the two groups were compared using an indepen-dent sample t test. The Chi-square test or Fisher's exact test were employed for comparing categorical variables. P-values < 0.05 were considered significant.

Results

This study included 19 patients with chronic lymphocytic leukemia (CLL), consisting of 15 males and 4 females with ages ranging from 58 to 70 years. The patients were evaluated before (Group Ia) and after induction therapy (Group Ib). All patients presented with lymph-adenopathy and splenomegaly. According to Rai staging, 9 cases (47.36%) were classified as stage II, and 10 cases (52.63%) were classified as stage III (Table 1).

Discussion

Chronic lymphocytic leukemia (CLL) patients exhibit diverse clinical outcomes, ranging from aggressive to indolent ⁽¹²⁾. Various antigen-presenting cells, including dendritic cells and macrophages, express CD80 and CD86 proteins, which are crucial for costimulation and leukemia treatment ⁽¹³⁾.  The presence of 17p deletion in CLL is associated with poor response to chemotherapy and unfavorable outcomes ⁽⁹⁾.

Our research aimed to investigate the expression of CD80 and CD86 cell surface markers and 17p deletion in CLL patients. We found a positive correlation between CD80% and LDH level, consistent with André et al.,⁽¹⁴⁾. This correlation may be attributed to CD80/CD86 signaling regulating LDH activity and lactate production in cancer cells.

We also observed a statistically significant positive correlation between CD86% and total leukocyte count (TLC), consistent with Arruga et al.,⁽¹⁵⁾. This may be explained by increased CD86% expression on antigen-presenting cells stimulating stronger T-cell activation against CLL cells.

Our findings contrandicted those of Takács et al.,⁽¹⁶⁾, who found that patients with high CD86 expression in their samples had a lower lymphocyte count at diagnosis. Additionally, compared to patients with lower CD86-expressing CLL cells, these patients required therapeutic intervention sooner and had higher serum LDH levels.

A highly statistically significant positive correlation was found between CD80% and absolute lymphocyte count, consistent with the data and explanation provided by Esensten et al.,^[17], who explained that CD80 expression is facilitated by TCR engagement and is dependent on the expression of CD80 on APCs, which increases with an increase in absolute lymphocyte count.

The co-expression of CD80% and CD86% may contribute to tumor progression, also, our results aligned with Dakappagari et al.,⁽¹⁸⁾, who linked high CD80% and CD86% expression to poor prognosis.

A potential explanation for the downregulation could be that immunoglobulin synthesis is suppressed due to the release of soluble CD80 (sCD80) after B-cell lysis. As demonstrated by Garcia et al.,⁽¹⁹⁾, this kind of system would be able to anticipate consequences in individuals, especially those with active or advanced stages of the disease. It is still unclear if CD80% expression mediates leukemic cell clearance or is only a marker of lenalidomide's undeter-mined biological effects on CLL cell biology. To that end, it's worth noting that Aue et al. ⁽²⁰⁾ demonstrated that CD80, a signaling protein, can send signals to B-cell lymphoma cells that promote cell death and restrict proliferation.

Patients with 17p deletion are challenging to treat due to poor response to standard treat-ment and unfavorable outcomes. Early diagn-osis of 17p deletion can enhance survival rates by identifying high-risk individuals who may benefit from alternative therapies ⁽²¹⁾.

This study compared CLL patients with 17p deletion to those without and found that patients with 17p deletion had significantly higher total leukocyte counts (TLC). This finding is consistent with Mahmood et al.,⁽²²⁾, who reported elevated total lymphocytic count and absolute lymphocytic count in patients with 17p deletion -positive CLL. Additionally, Yokusa et al.,⁽²³⁾ found a significant positive correlation between absolute lymphocyte count and Rai stages in 17p deletion patients, which aligns with our study's findings.

Our results showed that raised LDH levels at diagnosis were more in CLL patients with 17p deletion than negative FISH patients. This was in consistent with Autore et al.,⁽²⁴⁾ who found elevation in LDH level in 17p deletion patients compared to negative FISH patients and this suggests also the prognostic value of LDH in CLL.

When looking at CD86 and CD80 expression in patients with 17p deletion, those with positive 17p deletion showed a greater expression than those with negative 17p deletion. Prior work by Herman et al.,⁽²⁵⁾  provided an explanation for this phenomenon by demonstrating elevated signaling pathway activity as predicted by the expression of representative target genes and supported by flow cytometry for active signaling molecules CD86% and CD80%, respectively .