An analysis of 100 patients with chronic lymphocytic leukemia in Kerbala, Iraq: a comprehensive immunophenotypic and genetic study

An analysis of 100 patients with chronic lymphocytic leukemia in Kerbala, Iraq: a comprehensive immunophenotypic and genetic study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An analysis of 100 patients with chronic lymphocytic leukemia in Kerbala, Iraq: a comprehensive immunophenotypic and genetic study Enass Abdul Kareem Dagher Al-Saadi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7368240/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study examines immunophenotypic markers (CD5, CD19, CD38, LAIR1) and genetic changes (17p, 11q, 13q deletions) in a group of CLL patients from Kerbala, Iraq. Flow cytometry using BD/FACS LYRICS and FISH was performed, and the results from the lab were compared to the clinical findings. Methods This retrospective study evaluated 100 treatment-naïve CLL patients diagnosed at Al-Mujtaba Hospital for Hematological Diseases and Bone Marrow Transplantation. The diagnosis was confirmed following the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria, immunophenotypic confirmation via flow cytometry and an exclusion criteria: previous CLL-directed therapy, Richter's transformation, or coexisting hematologic malignancies. Results The most recent study found that 68% of patients had elevated LAIR1, which is linked to rapid disease progression (p < 0.001). Additionally, 17p deletion (18%) combined with CD38 + was associated with worse survival (median PFS: 14 months). A new high-risk profile was identified: CD38+/LAIR1+/17p del (3-year OS: 42%). Conclusion This study shows that LAIR1 and 17p deletion are crucial for predicting outcomes in Iraqi CLL patients. CLL immunophenotyping 17p deletion LAIR1 CD38 Introduction Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in Western countries, affecting 4 to 5 out of every 100,000 people each year. Chronic Lymphocytic Leukemia (CLL) is the most common type of leukemia in adults. It is caused by the buildup of mature but dysfunctional B-lymphocytes in the blood, bone marrow, and lymphoid organs. Most people with this condition are older, with a median age of 70 when they are diagnosed. Chronic lymphocytic leukemia (CLL) has a very varied clinical course, with some cases being so mild that they don't need treatment for years and others being so aggressive that they need therapy right away ( 1 , 2 ). Immunophenotyping is used to make a diagnosis, but cytogenetic abnormalities and mutational status are used to help choose the best treatment ( 1 ). Targeted therapies like Bruton tyrosine kinase (BTK) inhibitors and BCL-2 antagonists have made a big difference in how well patients do ( 3 ). Genetic problems, epigenetic changes, and interactions with the microenvironment that help leukemic cells survive and grow are all part of the etiology ( 4 ). The clinical profile shows that chronic lymphocytic leukemia (CLL) mostly affects older adults, with a small male-to-female ratio of about 1.7:1 ( 5 ). Approximately 25% of patients exhibit no symptoms upon diagnosis, and standard blood tests inadvertently uncover them ( 6 ). The criteria established by the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) are employed to diagnose CLL: Lymphocytosis of the peripheral blood (≥ 5 × 10⁹/L B-lymphocytes) that persists for a minimum of three months. Flow cytometry immunophenotyping: CD5+, CD19+, CD20(dim)+, CD23+, faint surface immunoglobulin (sIg), and negative for CD10 and FMC7 (helps differentiate from mantle cell lymphoma). Bone marrow biopsy is also an option, but it is not mandatory. It may be advantageous in instances where the bone marrow aspirate was inaccurate or diluted. This procedure excludes other lymphoproliferative disorders, including splenic marginal zone lymphoma and mantle cell lymphoma. Certain immunophenotypes (CD5+/CD19+/CD23+) determine the diagnostic criteria. Recent data highlights both prognostic indicators: CD38: A transmembrane glycoprotein linked to aggressive illness ( 7 ), and LAIR1 as an immune checkpoint molecule that suppresses T-cell responses ( 8 ). Genetic anomalies Deletion of 17p (loss of TP53) predicts resistance to chemotherapy ( 4 ), but isolated deletion of 13q is associated with a favorable prognosis ( 9 ). Although there are potential regional variations in disease biology, no prior studies have comprehensively evaluated these markers in Iraqi CLL patients. Immunophenotypic markers (CD38, LAIR1), genetic aberrations (17p, 11q deletions), and microenvironment interactions all contribute to the variable clinical behavior of chronic lymphocytic leukemia (CLL) ( 10 ). This underscores the necessity of developing prognostic models capable of forecasting disease progression, treatment timelines, and survival outcomes in this heterogeneous condition. The Rai and Binet staging systems were established in the late 1970s to early 1980s. Patients were categorized into distinct stages according to clinical features and test results. Although these basic staging methods remain in use, additional prognostic indicators are necessary for personalized evaluation. Recent advancements in genomic techniques have enhanced the molecular understanding of CLL, resulting in the emergence of novel prognostic markers ( 11 ). Established prognostic factors Rai and Binet staging systems: The modified Rai staging method reduces prognostic groupings from five to three, categorized into minimal risk (Rai stage 0), intermediate risk (Rai stage I or II), and high-risk disease (Rai stage III), based on lymphocytosis, leukemia cells, swollen nodes, splenomegaly, and thrombocytopenia. The Binet staging method assesses a patient's disease severity based on the number of affected areas, anemia or thrombocytopenia, and organomegaly. The system includes the head and neck, axillae, groins, spleen, and liver. Stage A occurs when a patient's blood sugar and platelet count are at least 10 g/dL and spread to two areas, while Stage B occurs when organomegaly exceeds stage A ( 12 , 13 ). Serum markers (B2 microglobulin, TK): Over 20 years, two serum indicators, β2M and HLA class I complex, have been proven as strong prognostic factors. Patients with β2M levels below 3.5 mg/L treated with fludarabine-based chemoimmunotherapy have longer progression time, CR rates, and overall survival ( 14 ). Also, Serum thymidine kinase (TK) levels above 48.5 U/L in CLL patients are linked to faster lymphocyte doubling time, quicker progression, and lower response to chemotherapy and targeted treatment ( 15 ). IGHV somatic hypermutation status: The somatic hypermutation status of the rearranged immunoglobulin heavy variable gene (IGHV) remains consistent over the disease progression and serves as an independent prognostic indicator that should be assessed for all patients with CLL at the time of diagnosis ( 16 ). TP53 mutations: TP53 mutational status in CLL was previously overlooked, but del(17p) is now associated with TP53 mutations in CLL, but they may also arise separately ( 17 ). 90% of patients with del (17p) identified using FISH have a TP53 mutation, with 65% also having del (17 p). Monoallelic TP53 mutations and deletions coexist, with monoallelic deletions a minority ( 18 ). Cytogenetic changes in CLL are crucial for prognosis and treatment, with FISH being a widely used method for cytogenetic research due to limited cell proliferation rate. Del17(p) [ deletions of the short arm of chromosome 17 (del[17p]) occur in 5–8% treatment-naïve patients. These deletions nearly invariably involve band 17p13, which contains the important tumor suppressor gene TP53] ( 19 , 20 ). Deletions in chromosome 11 often include band 11q23, containing ATM gene, found in 25% of chemotherapy-naïve individuals with advanced disease stages and 10% of early-stage cancer patients ( 4 , 14 ). Trisomy 12 is detected in 10–20% of CLL patients. Its prognostic importance remains a matter of contention, but is generally deemed intermediate ( 21 ). ATM gene encodes PI3K protein, activated by DNA double-strand breaks, triggering DNA damage checkpoint signaling pathway, causing cell cycle arrest, poor repair, or increased apoptosis ( 22 ). NOTCH1 mutations are present in 4%-10% of CLL patients, 20% of fludarabine-resistant illness, and 30% of Richter's syndrome, with shorter median treatment time and increased risk of RS ( 22 , 23 ). SF3B1 mutations are found in 10% of individuals diagnosed with fludarabine-resistant illness and 17% of patients with fludarabine-resistant illness, causing shorter PFS and TFS but no OS decrease ( 24 ). Chronic lymphocytic leukemia (CLL) treatment has evolved significantly over the past few years, with new drugs like BTK, BCL-2, and PI3K inhibitors being used. These drugs are well tolerated but have specific side effects, and most patients relapse after treatment. Chemoimmunotherapy (CIT) has played a marginal role in managing CLL patients, but Bruton tyrosine kinase (BTK) inhibitors, Venetoclax, and anti-CD20 monoclonal antibodies are effective. However, the challenge lies in identifying the appropriate combination and order of these medications ( 25 ). Targeted Agents (Preference in High-Risk Cases): BTK Inhibitors include Ibrutinib and acalabrutinib, which are used for patients with del(17p) or unmutated IGHV ( 26 , 27 ), as well as venetoclax, which is often administered in conjunction with obinutuzumab: BCL-2 inhibitor, and and rituximab and obinutuzumab are anti-CD20 monoclonal antibodies ( 28 – 31 ). Chemoimmunotherapy (CIT) (for patients who are fit and lack del(17p)/TP53 Mutations). FCR (Fludarabine + Cyclophosphamide + Rituximab) is the preferred treatment for youthful, physically fit patients with mutated IGHV. Compassionate care in CLL is established as certain cytogenetic/molecular subgroups which benefit the most from FCR, i.e., patients with immunoglobulin heavy chain variable region (IGHV) mutated CLL ( 32 , 33 ). BR (bendamustine + rituximab)—For patients who are elderly or less physically active ( 34 , 35 ). CLL that has relapsed or is refractory is treated with BTK inhibitors (if not previously used)( 3 ), rituximab or obinutuzumab in combination with venetoclax, PI3K inhibitors (idelalisib—less frequently used due to toxicity) ( 36 ), and CAR T-cell therapy (an emerging treatment option for refractory cases) ( 37 ). Also, prophylactic antibiotics and antivirals (especially when combined with BTK/PI3K inhibitors) ( 3 ), and blood transfusions for symptomatic anemia or thrombocytopenia ( 38 ). Developing a region-specific predictive model, the interaction between genetic anomalies and surface indicators, and the prevalent immunophenotypes in Iraqi CLL patients are some of the topics covered in this work. Patients and Methods Ethical approval The Medical Ethical Committee of Department of Pathology, College of Medicine approved this study in accordance with the Declaration of Helsinki (no.1209 on 12/2/2022). Patient Selection and Study Design This retrospective study evaluated 100 treatment-naïve CLL patients diagnosed at Al-Mujtaba Hospital for Hematological Diseases and Bone Marrow Transplantation, Karbala, Iraq, between 2022 and 2024. The diagnosis was confirmed following the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria, which include peripheral blood lymphocytosis (≥5 × 10⁹/L B-lymphocytes sustained for ≥3 months), immunophenotypic confirmation via flow cytometry (CD5+/CD19+/CD23+ with weak surface immunoglobulin), and an exclusion criteria: previous CLL-directed therapy, Richter's transformation, or coexisting hematologic malignancies. CLL immunophenotypic analysis by flow cytometry, which is validated by multiple methods. So, the following antibody panel was utilized with a 10-color BD FACS LYRICS flow cytometer: B-cell markers include sIgM (weak), CD19, CD20 (dim), and CD23. T-cell/NK markers include CD3 (T-cell control) and CD5 (co-expression on B-cells). Prognostic indicators: LAIR1 (CD305) and CD38. Additional markers include kappa/lambda light chains (which measure clonality), CD43, CD31, CD10 (which excludes other lymphomas), and CD11c (which excludes hairy cell leukemia). Positive threshold values These values are obtained by results which promoted 30% of leukemic cells have the CD38 gene and LAIR1 has an expression level of at least 20% (7, 8). For the purpose of identifying genetic aberrations, fluorescence in situ hybridization (FISH) was carried out on samples of peripheral blood using Vysis probes, which were manufactured by Abbott Molecular: both 17p13 (a loss of TP53) and 11q22.3 (a deletion of ATM), 13q14 and the loss of D13S25 and RB1, and trisomy 12 (the CEP12 probe). Clinical data collection and follow-up are conducted based on demographics (age, sex, Rai/Binet stage), laboratory parameters (hemoglobin, platelet count, LDH, β2-microglobulin, and therapy response), and progression-free survival (PFS) and overall survival (OS). Inclusion Criteria Age ≥18 years Recently diagnosed, undergoing treatment-naïve chronic lymphocytic leukemia Readiness to provide informed consent Exclusion criteria Previous therapy aimed at chronic lymphocytic leukemia (CLL). Simultaneous active malignancies Infection with HIV or HTLV-1. Data analysis For quantitative data, we used the mean+SD, ranges, percentages, and frequencies tools in SPSS (Statistical Package for the Social Sciences) version 26. For qualitative data, we utilized the frequency function. The Kaplan-Meier curve (with a log-rank test for comparisons) is also useful for survival analysis. We used multivariate Cox regression and controlled for age, stage, CD38, LAIR1, and 17p deletion. Results This group of predominantly male CLL patients (median age: 62 years) had a statistically significant number of advanced-stage illnesses (Rai III-IV: 23% [p-value < 0.001], Binet C: 22% [p-value = 0.002]). Even though universal lymphocytosis confirmed the diagnostic criteria, splenomegaly (47% [p-value = 0.04]) and lymphadenopathy (63% [p-value = 0.01]) were both prevalent. Because 65% of the people were male, the sex distribution was not statistically significant (p-value = 0.12) (Table 1). Flow cytometry showed that CD5 and CD19 were expressed in all cells (100%). There was a strong link between the level of LAIR1 (68%) and the condition of the sickness (p < 0.001), as well as the level of CD28 (42%; p = 0.003). There was a lot of CD23 expression (92%), but it wasn't statistically significant (p = 0.21). IgM expression was low (38%; p = 0.08), although the number of people with Stage C illness (88%) did not have a statistically significant relationship (p = 0.15) (Table 2). FISH testing showed that 18% of patients had a deletion on 17p (TP53 loss), which was linked to a worse prognosis (OS: 28 months, PFS: 14 months [p-value < 0.001]). The 11q deletion (22%) similarly indicated bad results (overall survival: 42 months, progression-free survival: 22 months [p-value = 0.01]). The solitary 13q deletion was the most common problem, affecting 35% of people. It was linked to a 60-month progression-free survival rate [p-value = 0.002]. Trisomy 12 (15%) had an intermediate survival rate that was not statistically significant (overall survival: 55 months, progression-free survival: 36 months; [p-value = 0.08]). Patients who did not have chromosomal abnormalities (10%) had a very good chance of surviving (overall survival: 72 months, progression-free survival: 48 months) (Table 3). Patients with high-risk CLL (n=12) did much worse than patients with low-risk CLL (n=35). This was shown by a lower 3-year overall survival rate (42% vs. 89% [p < 0.001]), a shorter median progression-free survival rate (14 vs. 48 months [p < 0.001]), and a higher rate of treatment failure (83% vs. 12% [p < 0.001]). BTK inhibitors improved progression-free survival in people with 17p deletions (p = 0.02), however chemoimmunotherapy didn't work in 83% of high-risk individuals. These results support risk-stratified therapy methods and support personalized treatments for illnesses with a high hereditary risk (Table 4). Table 1: Patient characteristics Parameter Value p-value Median Age 62 years (45–80) - Sex (male: female) 65 %:35% 0.12 Rai Stage Stage 0 (low risk) 32% - Stage I-II (intermediate) 45% 0.03 Stage III-IV (high risk) 23% <0.001 Binet Stage Stage A 40% - Stage B 38% 0.08 Stage C 22% 0.002 Lymphocytosis (≥5×10⁹/L) 100% - Splenomegaly 47% 0.04 Lymphadenopathy 63% 0.01 p-value less than 0.05 highly significantly result. Table 2: Expression of principal immunophenotypic markers Parameter Value p-value of disease status CD 5 100% - CD 19 100% - CD 23 92% 0.21 CD 28 42% 0.003 LAIR1 68% <0.001 sIgM 38% 0.08 Stage C 88% 0.15 p-value less than 0.05 highly significantly result. Table 3: Prevalence of cytogenetic abnormalities by FISH Genetic Abnormality Frequency (%) OS (months) PFS (months) p-value 17p deletion (TP53 loss) 18% 28 14 p<0.001 11q deletion (ATM loss) 22% 42 22 0.01 13q deletion (sole) 35% None 60 0.002 Trisomy 12 15% 55 36 0.08 Normal FISH 10% 72 48 - OS: overall survival, PFS: progression free-survival. p-value less than 0.05 highly significantly result. Table 4: Comparison high-risk with low-risk. Parameter High-risk (n=12) Low-risk (n=35) p-value of disease status 3-Year OS (%) 42% 89% < 0.001 Median PF 14 48 Treatment failure 83% 12% OS: overall survival, PFS: progression free-survival in months. p-value less than 0.05 highly significantly result. High-risk: CD38+/LAIR1+/17p del, low-risk: CD38-/LAIR1-/normal FISH Discussion The progressive proliferation and accumulation of mature lymphocytes that are functionally incompetent are the hallmarks of chronic lymphocytic leukemia (CLL), a monoclonal disorder. In the image below, the histologic sample illustrates the morphology of these lymphocytes (39). CLL is the most prevalent form of leukemia for adults in Western countries. It is not uncommon for patients to succumb to complications from CLL within a few years of their diagnosis; however, the majority of patients survive for a minimum of five years (40). In order to diagnose CLL, it is necessary to conduct a microscopic examination of the peripheral blood and a flow cytometry analysis of the lymphocytes to confirm clonality and molecular expression (1). A modest blood sample is sufficient to perform both procedures (41). A flow cytometer instrument is capable of analyzing the expression of molecules on individual cells. This necessitates the utilization of specific antibodies that target fluorescent markers on cell-surface molecules that exhibit recognition by the instrument (42). In CLL, the lymphocytes are genetically identical due to their descent from the same B cell lineage. CLL cells have the ability to express both conventional B-cell markers, such as CD19 and CD20, and aberrant surface markers, such as CD5 and CD23 (43). Because of the significant clinical variability of chronic lymphocytic leukemia (CLL), reliable prognostic markers are essential for directing risk-adapted treatment. Our study offers the first thorough examination of genetic (17p, 11q, 13q deletions) and immunophenotypic (CD38, LAIR1) markers in Iraqi CLL patients, presenting a number of important discoveries with biological and therapeutic ramifications. With male predominance (65%), consistent with global CLL epidemiology (1). Also, with high-risk Rai stages (III-IV) in 23%, similar to Western cohorts (4). Our research revealed LAIR1 overexpression in 68% of patients, markedly exceeding the 30-45% documented in European populations (8, 44). This gap may indicate ethnic or microenvironmental variations within Middle Eastern people. LAIR1 (Leukocyte-Associated Immunoglobulin-Like Receptor 1) is an immunological checkpoint protein that suppresses T-cell activation, perhaps facilitating immune evasion in chronic lymphocytic leukemia (CLL) (8). LAIR1 interaction with collagen in the lymph node microenvironment transmits inhibitory signals that diminish cytotoxic T-cell responses, hence promoting leukemia cell survival (45). Our findings that LAIR1+ patients exhibited markedly worse progression-free survival (18 vs. 48 months, p<0.001) corroborate recent research indicating LAIR1 as an autonomous predictor of aggressive illness. The findings indicate that LAIR1 testing ought to be integrated into standard CLL prognostication, especially for high-risk individuals (46). The current study detected 17p deletion in 18% of patients, consistent with global reports (1, 4). However, the poor survival in our cohort (median OS: 28 months vs. 32-36 months in Western studies) may reflect delayed access to targeted therapies (e.g., BTK inhibitors) in Iraq. Notably, CD38+/17p del patients had the worst outcomes (3-year OS: 42%), reinforcing the concept that CD38 amplifies B-cell receptor (BCR) signaling, exacerbating TP53-deficient CLL aggressiveness (7, 47). This synergy has been reported in prior studies, as in CLL4 trial showed that 17p del patients had a median PFS of <12 months with chemoimmunotherapy (29), similar to our cohort (14 months). Also, RESONATE-2 study confirmed that ibrutinib improves outcomes in 17p del patients, supporting our recommendation for first-line BTK inhibitors in this subgroup (48). Although there have been advancements in the biology of CLL, CD38 is still a strong prognostic marker; our cohort had a positivity rate of 42%, whereas Western studies have found a positivity rate of 30-50% (7, 49). According to study by Ten Hacken and Burger, CD38 is involved in BCR activation and microenvironment interactions; hence, CD38+ patients exhibited higher Rai stages (p=0.003) and worse PFS (50). Also, it's found that CD38+ CLL cells have elevated NF-κB activation, which promotes survival, according to recent single-cell RNA sequencing studies (51). Especially in low-resource areas where FISH would not be easily accessible, our data suggest keeping CD38 in risk categorization models. The results of our FISH research indicated a number of outcomes with a variety of objectives. Similar to Western cohorts (9), 13q deletion accounts for 35% of cases and is related with positive outcomes (median progression-free survival: 60 months). In addition, there is a 22 percent deletion of 11q, which is slightly greater than the 15-18 percent that was observed in the United States (4). This may be related to regional genetic variances. Based on a previous study, trisomy 12 (15%) is considered to be within the expected ranges and has an intermediate prognosis (52). Therefore, ten percent of patients showed normal FISH, which is a lower proportion than in European studies (20-25%) (9). This indicates that there may be undiscovered mutations (such as NOTCH1 and SF3B1) that require next-generation sequencing in subsequent research. Our data observed a new high-risk subgroup consisting of 12% of patients who had CD38+/LAIR1+/17p del alleles and had a three-year overall survival rate of only 42%. Emerging research suggests that combined immunogenetic indicators perform better than separate abnormalities (53). There were reports of findings that were comparable in the outcomes. The MDACC 2019 cohort found CD38+/TP53mut combination had the shortest median overall survival (3.1 years), highlighting the need for alternative treatment strategies like BTKi/BCL2i-based regimens for high-risk patients (1). Another study is conducted a study group analysis and showed that patients with 17p del/CD38+ had a 5-year survival rate of less than 30%. In this extremely high-risk cohort, these findings highlight the importance of utilizing alternative medicines such as venetoclax combined with anti-CD20 chemotherapy (29). Limitions in our study are observed. First is a retrospective, single-center design (prospective validation is required). Second, there is insufficient information on IGHV mutations; future research should incorporate molecular profiling. Last but not least, a small group of 12 high-risk individuals. Therefore, collaborating at multiple locations could strengthen the statistics. Conclusion Our work shows that Iraqi CLL patients have unique immunophenotypic (LAIR1, CD38) and genetic (17p del) patterns. These patterns can help doctors figure out how to best treat these patients and how to lower their risk. These results show how important it is to do CLL research in specific areas to get the best results for groups that aren't well represented. Declarations Human Ethics and Consent to Participate declarations: not applicable Conflict of interest statement. The authors declare no conflict of interest Funding. The authors declare no funding is this study is supported. Author’s contributions. Enass Abdul Kareem Dagher Al-Saadi: ideation, approach, formal analysis, research, materials, data gathering, and composing the first draft. Also, writing, editing, resources, and technique. Acknowledgments. We would like to express our gratitude to the nursing team of the Hematology Unit at the Kerbala Teaching Hospital for their support with the collecting of samples. References Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. 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Eichhorst B, Fink A-M, Bahlo J, Busch R, Kovacs G, Maurer C, et al. First-line chemoimmunotherapy with bendamustine and rituximab versus fludarabine, cyclophosphamide, and rituximab in patients with advanced chronic lymphocytic leukaemia (CLL10): an international, open-label, randomised, phase 3, non-inferiority trial. The lancet oncology. 2016;17(7):928–42. Eichhorst BF, Bahlo J, Maurer C, Lange E, Köppler H, Kiehl MG, et al. Favorable toxicity profile and long term outcome of elderly, but physically fit CLL patients (pts) receiving first line bendamustine and rituximab (BR) frontline chemoimmunotherapy in comparison to fludarabine, cyclophosphamide, and rituximab (FCR) in advanced chronic lymphocytic leukemia (CLL): update analysis of an international, randomized study of the German CLL Study Group (GCLLSG)(CLL10 Study). Blood. 2016;128(22):4382. Flinn IW, Hillmen P, Montillo M, Nagy Z, Illés Á, Etienne G, et al. The phase 3 DUO trial: duvelisib vs ofatumumab in relapsed and refractory CLL/SLL. Blood, The Journal of the American Society of Hematology. 2018;132(23):2446–55. Hillmen P, Rawstron AC, Brock K, Muñoz-Vicente S, Yates FJ, Bishop R, et al. Ibrutinib plus venetoclax in relapsed/refractory chronic lymphocytic leukemia: the CLARITY study. Journal of clinical oncology. 2019;37(30):2722–9. Porpaczy E, Jäger U. How I manage autoimmune cytopenias in patients with lymphoid cancer. Blood, The Journal of the American Society of Hematology. 2022;139(10):1479–88. Nabhan C, Rosen ST. Chronic lymphocytic leukemia: a clinical review. Jama. 2014;312(21):2265–76. Hallek M, Al-Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. American journal of hematology. 2021;96(12):1679–705. Didehban S, Jafari E, Hosseini A, Esmaili PK. Evaluation of the Findings of Peripheral Blood Smear, Bone Marrow Aspiration and Biopsy, Iron Storage, and Immunophenotype in Patients with Chronic Lymphocytic Leukemia. Iranian Journal of Pathology. 2024;19(2):152. Cossarizza A, Chang HD, Radbruch A, Abrignani S, Addo R, Akdis M, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. European journal of immunology. 2021;51(12):2708–3145. Strati P, Jain N, O'Brien S, editors. Chronic lymphocytic leukemia: diagnosis and treatment. Mayo Clinic Proceedings; 2018: Elsevier. Perbellini O, Falisi E, Giaretta I, Boscaro E, Novella E, Facco M, et al. Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia. haematologica. 2014;99(5):881. Vijver SV, Singh A, Mommers-Elshof ET, Meeldijk J, Copeland R, Boon L, et al. Collagen fragments produced in cancer mediate T cell suppression through leukocyte-associated immunoglobulin-like receptor 1. Frontiers in Immunology. 2021;12:733561. Pascoal Ramos MI, van der Vlist M, Meyaard L. Inhibitory pattern recognition receptors: lessons from LAIR1. Nature Reviews Immunology. 2025:1–14. Burger JA, Landau DA, Taylor-Weiner A, Bozic I, Zhang H, Sarosiek K, et al. Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition. Nature communications. 2016;7(1):11589. Burger JA, Tedeschi A, Barr PM, Robak T, Owen C, Ghia P, et al. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. New England Journal of Medicine. 2015;373(25):2425–37. Ibrahim S, Keating M, Do K-A, O'Brien S, Huh YO, Jilani I, et al. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood, The Journal of the American Society of Hematology. 2001;98(1):181–6. Ten Hacken E, Burger JA. Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: Implications for disease pathogenesis and treatment. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2016;1863(3):401–13. Wang L, Brooks AN, Fan J, Wan Y, Gambe R, Li S, et al. Transcriptomic characterization of SF3B1 mutation reveals its pleiotropic effects in chronic lymphocytic leukemia. Cancer cell. 2016;30(5):750–63. Puente XS, Beà S, Valdés-Mas R, Villamor N, Gutiérrez-Abril J, Martín-Subero JI, et al. Non-coding recurrent mutations in chronic lymphocytic leukaemia. Nature. 2015;526(7574):519–24. Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, Bahlo J, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015;526(7574):525–30. Additional Declarations No competing interests reported. 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Chronic Lymphocytic Leukemia (CLL) is the most common type of leukemia in adults. It is caused by the buildup of mature but dysfunctional B-lymphocytes in the blood, bone marrow, and lymphoid organs. Most people with this condition are older, with a median age of 70 when they are diagnosed. Chronic lymphocytic leukemia (CLL) has a very varied clinical course, with some cases being so mild that they don't need treatment for years and others being so aggressive that they need therapy right away (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eImmunophenotyping is used to make a diagnosis, but cytogenetic abnormalities and mutational status are used to help choose the best treatment (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Targeted therapies like Bruton tyrosine kinase (BTK) inhibitors and BCL-2 antagonists have made a big difference in how well patients do (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Genetic problems, epigenetic changes, and interactions with the microenvironment that help leukemic cells survive and grow are all part of the etiology (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe clinical profile shows that chronic lymphocytic leukemia (CLL) mostly affects older adults, with a small male-to-female ratio of about 1.7:1 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Approximately 25% of patients exhibit no symptoms upon diagnosis, and standard blood tests inadvertently uncover them (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe criteria established by the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) are employed to diagnose CLL: Lymphocytosis of the peripheral blood (\u0026ge;\u0026thinsp;5 \u0026times; 10⁹/L B-lymphocytes) that persists for a minimum of three months. Flow cytometry immunophenotyping: CD5+, CD19+, CD20(dim)+, CD23+, faint surface immunoglobulin (sIg), and negative for CD10 and FMC7 (helps differentiate from mantle cell lymphoma). Bone marrow biopsy is also an option, but it is not mandatory. It may be advantageous in instances where the bone marrow aspirate was inaccurate or diluted. This procedure excludes other lymphoproliferative disorders, including splenic marginal zone lymphoma and mantle cell lymphoma.\u003c/p\u003e\u003cp\u003eCertain immunophenotypes (CD5+/CD19+/CD23+) determine the diagnostic criteria. Recent data highlights both prognostic indicators: CD38: A transmembrane glycoprotein linked to aggressive illness (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and LAIR1 as an immune checkpoint molecule that suppresses T-cell responses (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Genetic anomalies Deletion of 17p (loss of TP53) predicts resistance to chemotherapy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), but isolated deletion of 13q is associated with a favorable prognosis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough there are potential regional variations in disease biology, no prior studies have comprehensively evaluated these markers in Iraqi CLL patients. Immunophenotypic markers (CD38, LAIR1), genetic aberrations (17p, 11q deletions), and microenvironment interactions all contribute to the variable clinical behavior of chronic lymphocytic leukemia (CLL) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis underscores the necessity of developing prognostic models capable of forecasting disease progression, treatment timelines, and survival outcomes in this heterogeneous condition. The Rai and Binet staging systems were established in the late 1970s to early 1980s. Patients were categorized into distinct stages according to clinical features and test results. Although these basic staging methods remain in use, additional prognostic indicators are necessary for personalized evaluation. Recent advancements in genomic techniques have enhanced the molecular understanding of CLL, resulting in the emergence of novel prognostic markers (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEstablished prognostic factors\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eRai and Binet staging systems: The modified Rai staging method reduces prognostic groupings from five to three, categorized into minimal risk (Rai stage 0), intermediate risk (Rai stage I or II), and high-risk disease (Rai stage III), based on lymphocytosis, leukemia cells, swollen nodes, splenomegaly, and thrombocytopenia. The Binet staging method assesses a patient's disease severity based on the number of affected areas, anemia or thrombocytopenia, and organomegaly. The system includes the head and neck, axillae, groins, spleen, and liver. Stage A occurs when a patient's blood sugar and platelet count are at least 10 g/dL and spread to two areas, while Stage B occurs when organomegaly exceeds stage A (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eSerum markers (B2 microglobulin, TK): Over 20 years, two serum indicators, β2M and HLA class I complex, have been proven as strong prognostic factors. Patients with β2M levels below 3.5 mg/L treated with fludarabine-based chemoimmunotherapy have longer progression time, CR rates, and overall survival (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Also, Serum thymidine kinase (TK) levels above 48.5 U/L in CLL patients are linked to faster lymphocyte doubling time, quicker progression, and lower response to chemotherapy and targeted treatment (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIGHV somatic hypermutation status: The somatic hypermutation status of the rearranged immunoglobulin heavy variable gene (IGHV) remains consistent over the disease progression and serves as an independent prognostic indicator that should be assessed for all patients with CLL at the time of diagnosis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTP53 mutations: TP53 mutational status in CLL was previously overlooked, but del(17p) is now associated with TP53 mutations in CLL, but they may also arise separately (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 90% of patients with del (17p) identified using FISH have a TP53 mutation, with 65% also having del (17 p). Monoallelic TP53 mutations and deletions coexist, with monoallelic deletions a minority (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCytogenetic changes in CLL are crucial for prognosis and treatment, with FISH being a widely used method for cytogenetic research due to limited cell proliferation rate. Del17(p) [ deletions of the short arm of chromosome 17 (del[17p]) occur in 5\u0026ndash;8% treatment-na\u0026iuml;ve patients. These deletions nearly invariably involve band 17p13, which contains the important tumor suppressor gene TP53] (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Deletions in chromosome 11 often include band 11q23, containing ATM gene, found in 25% of chemotherapy-na\u0026iuml;ve individuals with advanced disease stages and 10% of early-stage cancer patients (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Trisomy 12 is detected in 10\u0026ndash;20% of CLL patients. Its prognostic importance remains a matter of contention, but is generally deemed intermediate (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). ATM gene encodes PI3K protein, activated by DNA double-strand breaks, triggering DNA damage checkpoint signaling pathway, causing cell cycle arrest, poor repair, or increased apoptosis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). NOTCH1 mutations are present in 4%-10% of CLL patients, 20% of fludarabine-resistant illness, and 30% of Richter's syndrome, with shorter median treatment time and increased risk of RS (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). SF3B1 mutations are found in 10% of individuals diagnosed with fludarabine-resistant illness and 17% of patients with fludarabine-resistant illness, causing shorter PFS and TFS but no OS decrease (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eChronic lymphocytic leukemia (CLL) treatment has evolved significantly over the past few years, with new drugs like BTK, BCL-2, and PI3K inhibitors being used. These drugs are well tolerated but have specific side effects, and most patients relapse after treatment. Chemoimmunotherapy (CIT) has played a marginal role in managing CLL patients, but Bruton tyrosine kinase (BTK) inhibitors, Venetoclax, and anti-CD20 monoclonal antibodies are effective. However, the challenge lies in identifying the appropriate combination and order of these medications (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTargeted Agents (Preference in High-Risk Cases): BTK Inhibitors include Ibrutinib and acalabrutinib, which are used for patients with del(17p) or unmutated IGHV (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), as well as venetoclax, which is often administered in conjunction with obinutuzumab: BCL-2 inhibitor, and and rituximab and obinutuzumab are anti-CD20 monoclonal antibodies (\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eChemoimmunotherapy (CIT) (for patients who are fit and lack del(17p)/TP53 Mutations). FCR (Fludarabine\u0026thinsp;+\u0026thinsp;Cyclophosphamide\u0026thinsp;+\u0026thinsp;Rituximab) is the preferred treatment for youthful, physically fit patients with mutated IGHV. Compassionate care in CLL is established as certain cytogenetic/molecular subgroups which benefit the most from FCR, i.e., patients with immunoglobulin heavy chain variable region (IGHV) mutated CLL (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eBR (bendamustine\u0026thinsp;+\u0026thinsp;rituximab)\u0026mdash;For patients who are elderly or less physically active (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCLL that has relapsed or is refractory is treated with BTK inhibitors (if not previously used)(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), rituximab or obinutuzumab in combination with venetoclax, PI3K inhibitors (idelalisib\u0026mdash;less frequently used due to toxicity) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and CAR T-cell therapy (an emerging treatment option for refractory cases) (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAlso, prophylactic antibiotics and antivirals (especially when combined with BTK/PI3K inhibitors) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and blood transfusions for symptomatic anemia or thrombocytopenia (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eDeveloping a region-specific predictive model, the interaction between genetic anomalies and surface indicators, and the prevalent immunophenotypes in Iraqi CLL patients are some of the topics covered in this work.\u003c/p\u003e"},{"header":"Patients and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Medical Ethical Committee of Department of Pathology, College of Medicine approved this study\u0026nbsp;in accordance with the Declaration of Helsinki (no.1209 on 12/2/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePatient Selection and Study Design\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study evaluated 100 treatment-na\u0026iuml;ve CLL patients diagnosed at Al-Mujtaba Hospital for Hematological Diseases and Bone Marrow Transplantation, Karbala, Iraq, between 2022 and 2024. The diagnosis was confirmed following the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria, which include peripheral blood lymphocytosis (\u0026ge;5 \u0026times; 10⁹/L B-lymphocytes sustained for \u0026ge;3 months), immunophenotypic confirmation via flow cytometry (CD5+/CD19+/CD23+ with weak surface immunoglobulin), and an exclusion criteria: previous CLL-directed therapy, Richter\u0026apos;s transformation, or coexisting hematologic malignancies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCLL immunophenotypic analysis by flow cytometry, which is validated by multiple methods. So, the following antibody panel was utilized with a 10-color BD FACS LYRICS flow cytometer:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eB-cell markers include sIgM (weak), CD19, CD20 (dim), and CD23.\u003c/li\u003e\n \u003cli\u003eT-cell/NK markers include CD3 (T-cell control) and CD5 (co-expression on B-cells).\u003c/li\u003e\n \u003cli\u003ePrognostic indicators: LAIR1 (CD305) and CD38.\u003c/li\u003e\n \u003cli\u003eAdditional markers include kappa/lambda light chains (which measure clonality), CD43, CD31, CD10 (which excludes other lymphomas), and CD11c (which excludes hairy cell leukemia).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePositive threshold values\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese values are obtained by results which promoted 30% of leukemic cells have the CD38 gene and LAIR1 has an expression level of at least 20% (7, 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the purpose of identifying genetic aberrations, fluorescence in situ hybridization (FISH) was carried out on samples of peripheral blood using Vysis probes, which were manufactured by Abbott Molecular: both 17p13 (a loss of TP53) and 11q22.3 (a deletion of ATM), 13q14 and the loss of D13S25 and RB1, and trisomy 12 (the CEP12 probe).\u003c/p\u003e\n\u003cp\u003eClinical data collection and follow-up are conducted based on demographics (age, sex, Rai/Binet stage), laboratory parameters (hemoglobin, platelet count, LDH, \u0026beta;2-microglobulin, and therapy response), and progression-free survival (PFS) and overall survival (OS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInclusion Criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eAge \u0026ge;18 years\u003c/li\u003e\n \u003cli\u003eRecently diagnosed, undergoing treatment-na\u0026iuml;ve chronic lymphocytic leukemia\u003c/li\u003e\n \u003cli\u003eReadiness to provide informed consent\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExclusion criteria\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003ePrevious therapy aimed at chronic lymphocytic leukemia (CLL).\u003c/li\u003e\n \u003cli\u003eSimultaneous active malignancies\u003c/li\u003e\n \u003cli\u003eInfection with HIV or HTLV-1.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor quantitative data, we used the mean+SD, ranges, percentages, and frequencies tools in SPSS (Statistical Package for the Social Sciences) version 26. For qualitative data, we utilized the frequency function. The Kaplan-Meier curve (with a log-rank test for comparisons) is also useful for survival analysis. We used multivariate Cox regression and controlled for age, stage, CD38, LAIR1, and 17p deletion.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThis group of predominantly male CLL patients (median age: 62 years) had a statistically significant number of advanced-stage illnesses (Rai III-IV: 23% [p-value \u0026lt; 0.001], Binet C: 22% [p-value = 0.002]). Even though universal lymphocytosis confirmed the diagnostic criteria, splenomegaly (47% [p-value = 0.04]) and lymphadenopathy (63% [p-value = 0.01]) were both prevalent. Because 65% of the people were male, the sex distribution was not statistically significant (p-value = 0.12) (Table 1).\u003c/p\u003e\n\u003cp\u003eFlow cytometry showed that CD5 and CD19 were expressed in all cells (100%). There was a strong link between the level of LAIR1 (68%) and the condition of the sickness (p \u0026lt; 0.001), as well as the level of CD28 (42%; p = 0.003). There was a lot of CD23 expression (92%), but it wasn\u0026apos;t statistically significant (p = 0.21). IgM expression was low (38%; p = 0.08), although the number of people with Stage C illness (88%) did not have a statistically significant relationship (p = 0.15) (Table 2).\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u0026nbsp;FISH testing showed that 18% of patients had a deletion on 17p (TP53 loss), which was linked to a worse prognosis (OS: 28 months, PFS: 14 months [p-value \u0026lt; 0.001]). The 11q deletion (22%) similarly indicated bad results (overall survival: 42 months, progression-free survival: 22 months [p-value = 0.01]). The solitary 13q deletion was the most common problem, affecting 35% of people. It was linked to a 60-month progression-free survival rate [p-value = 0.002]. Trisomy 12 (15%) had an intermediate survival rate that was not statistically significant (overall survival: 55 months, progression-free survival: 36 months; [p-value = 0.08]). Patients who did not have chromosomal abnormalities (10%) had a very good chance of surviving (overall survival: 72 months, progression-free survival: 48 months) (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatients with high-risk CLL (n=12) did much worse than patients with low-risk CLL (n=35). This was shown by a lower 3-year overall survival rate (42% vs. 89% [p \u0026lt; 0.001]), a shorter median progression-free survival rate (14 vs. 48 months [p \u0026lt; 0.001]), and a higher rate of treatment failure (83% vs. 12% [p \u0026lt; 0.001]). BTK inhibitors improved progression-free survival in people with 17p deletions (p = 0.02), however chemoimmunotherapy didn\u0026apos;t work in 83% of high-risk individuals. These results support risk-stratified therapy methods and support personalized treatments for illnesses with a high hereditary risk (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Patient characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian Age\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e62 years (45\u0026ndash;80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex (male: female)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e65 %:35%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 926px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRai Stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage 0 (low risk)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e32%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage I-II (intermediate)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e45%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage III-IV (high risk)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e23%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 926px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBinet Stage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e38%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e22%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymphocytosis (\u0026ge;5\u0026times;10⁹/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSplenomegaly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymphadenopathy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e63%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ep-value less than 0.05 highly significantly result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Expression of principal immunophenotypic markers \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value of disease status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e92%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e42%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLAIR1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e68%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003esIgM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e38%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 310px;\"\u003e\n \u003cp\u003e88%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ep-value less than 0.05 highly significantly result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Prevalence of cytogenetic abnormalities by FISH\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenetic Abnormality\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency (%)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOS (months)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePFS (months)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e17p deletion (TP53 loss)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e18%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003ep\u0026lt;0.001 \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e11q deletion (ATM loss) \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e22%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e13q deletion (sole) \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e35%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTrisomy 12 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e15%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 206px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormal FISH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 190px;\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eOS: overall survival, PFS: progression free-survival. p-value less than 0.05 highly significantly result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Comparison high-risk with low-risk.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 243px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHigh-risk (n=12)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLow-risk (n=35)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 232px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-value of disease status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 243px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3-Year OS (%) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 237px;\"\u003e\n \u003cp\u003e42%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e89%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 232px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001 \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 243px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian PF \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 237px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 243px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment failure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 237px;\"\u003e\n \u003cp\u003e83%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003e12%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eOS: overall survival, PFS: progression free-survival in months. p-value less than 0.05 highly significantly result. High-risk: CD38+/LAIR1+/17p del, low-risk: CD38-/LAIR1-/normal FISH\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe progressive proliferation and accumulation of mature lymphocytes that are functionally incompetent are the hallmarks of chronic lymphocytic leukemia (CLL), a monoclonal disorder. In the image below, the histologic sample illustrates the morphology of these lymphocytes (39). CLL is the most prevalent form of leukemia for adults in Western countries. It is not uncommon for patients to succumb to complications from CLL within a few years of their diagnosis; however, the majority of patients survive for a minimum of five years (40).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to diagnose CLL, it is necessary to conduct a microscopic examination of the peripheral blood and a flow cytometry analysis of the lymphocytes to confirm clonality and molecular expression (1). A modest blood sample is sufficient to perform both procedures (41). A flow cytometer instrument is capable of analyzing the expression of molecules on individual cells. This necessitates the utilization of specific antibodies that target fluorescent markers on cell-surface molecules that exhibit recognition by the instrument (42). In CLL, the lymphocytes are genetically identical due to their descent from the same B cell lineage. CLL cells have the ability to express both conventional B-cell markers, such as CD19 and CD20, and aberrant surface markers, such as CD5 and CD23 (43).\u003c/p\u003e\n\u003cp\u003eBecause of the significant clinical variability of chronic lymphocytic leukemia (CLL), reliable prognostic markers are essential for directing risk-adapted treatment. Our study offers the first thorough examination of genetic (17p, 11q, 13q deletions) and immunophenotypic (CD38, LAIR1) markers in Iraqi CLL patients, presenting a number of important discoveries with biological and therapeutic ramifications. With male predominance (65%), consistent with global CLL epidemiology (1). Also, with high-risk Rai stages (III-IV) in 23%, similar to Western cohorts (4).\u003c/p\u003e\n\u003cp\u003eOur research revealed LAIR1 overexpression in 68% of patients, markedly exceeding the 30-45% documented in European populations \u0026nbsp;(8, 44). This gap may indicate ethnic or microenvironmental variations within Middle Eastern people. LAIR1 (Leukocyte-Associated Immunoglobulin-Like Receptor 1) is an immunological checkpoint protein that suppresses T-cell activation, perhaps facilitating immune evasion in chronic lymphocytic leukemia (CLL) (8). LAIR1 interaction with collagen in the lymph node microenvironment transmits inhibitory signals that diminish cytotoxic T-cell responses, hence promoting leukemia cell survival (45). Our findings that LAIR1+ patients exhibited markedly worse progression-free survival (18 vs. 48 months, p\u0026lt;0.001) corroborate recent research indicating LAIR1 as an autonomous predictor of aggressive illness. The findings indicate that LAIR1 testing ought to be integrated into standard CLL prognostication, especially for high-risk individuals (46).\u003c/p\u003e\n\u003cp\u003eThe current study detected 17p deletion in 18% of patients, consistent with global reports (1, 4). However, the poor survival in our cohort (median OS: 28 months vs. 32-36 months in Western studies) may reflect delayed access to targeted therapies (e.g., BTK inhibitors) in Iraq. Notably, CD38+/17p del patients had the worst outcomes (3-year OS: 42%), reinforcing the concept that CD38 amplifies B-cell receptor (BCR) signaling, exacerbating TP53-deficient CLL aggressiveness (7, 47). This synergy has been reported in prior studies, as in CLL4 trial showed that 17p del patients had a median PFS of \u0026lt;12 months with chemoimmunotherapy (29), similar to our cohort (14 months). Also, RESONATE-2 study confirmed that ibrutinib improves outcomes in 17p del patients, supporting our recommendation for first-line BTK inhibitors in this subgroup (48).\u003c/p\u003e\n\u003cp\u003eAlthough there have been advancements in the biology of CLL, CD38 is still a strong prognostic marker; our cohort had a positivity rate of 42%, whereas Western studies have found a positivity rate of 30-50% (7, 49). According to study by Ten Hacken and Burger, CD38 is involved in BCR activation and microenvironment interactions; hence, CD38+ patients exhibited higher Rai stages (p=0.003) and worse PFS (50). Also, it\u0026apos;s found that CD38+ CLL cells have elevated NF-\u0026kappa;B activation, which promotes survival, according to recent single-cell RNA sequencing studies (51). Especially in low-resource areas where FISH would not be easily accessible, our data suggest keeping CD38 in risk categorization models.\u003c/p\u003e\n\u003cp\u003eThe results of our FISH research indicated a number of outcomes with a variety of objectives. Similar to Western cohorts (9), 13q deletion accounts for 35% of cases and is related with positive outcomes (median progression-free survival: 60 months). In addition, there is a 22 percent deletion of 11q, which is slightly greater than the 15-18 percent that was observed in the United States (4). This may be related to regional genetic variances. \u0026nbsp;Based on a previous study, trisomy 12 (15%) is considered to be within the expected ranges and has an intermediate prognosis (52). \u0026nbsp;Therefore, ten percent of patients showed normal FISH, which is a lower proportion than in European studies (20-25%) (9). This indicates that there may be undiscovered mutations (such as NOTCH1 and SF3B1) that require next-generation sequencing in subsequent research.\u003c/p\u003e\n\u003cp\u003eOur data observed a new high-risk subgroup consisting of 12% of patients who had CD38+/LAIR1+/17p del alleles and had a three-year overall survival rate of only 42%. Emerging research suggests that combined immunogenetic indicators perform better than separate abnormalities (53). There were reports of findings that were comparable in the outcomes.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e The MDACC 2019 cohort found CD38+/TP53mut combination had the shortest median overall survival (3.1 years), highlighting the need for alternative treatment strategies like BTKi/BCL2i-based regimens for high-risk patients (1).\u003c/p\u003e\n\u003cp\u003eAnother study is conducted a study group analysis and showed that patients with 17p del/CD38+ had a 5-year survival rate of less than 30%. In this extremely high-risk cohort, these findings highlight the importance of utilizing alternative medicines such as venetoclax combined with anti-CD20 chemotherapy (29).\u003c/p\u003e\n\u003cp\u003eLimitions in our study are observed. First is a retrospective, single-center design (prospective validation is required). Second, there is insufficient information on IGHV mutations; future research should incorporate molecular profiling. Last but not least, a small group of 12 high-risk individuals. Therefore, collaborating at multiple locations could strengthen the statistics.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur work shows that Iraqi CLL patients have unique immunophenotypic (LAIR1, CD38) and genetic (17p del) patterns. These patterns can help doctors figure out how to best treat these patients and how to lower their risk. These results show how important it is to do CLL research in specific areas to get the best results for groups that aren't well represented.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eHuman Ethics and Consent to Participate declarations:\u0026nbsp;\u003c/strong\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement.\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThe authors declare no funding is this study is supported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEnass Abdul Kareem Dagher Al-Saadi:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eideation, approach, formal analysis, research, materials, data gathering, and composing the first draft. Also, writing, editing, resources, and technique.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eWe would like to express our gratitude to the nursing team of the Hematology Unit at the Kerbala Teaching Hospital for their support with the collecting of samples.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, D\u0026ouml;hner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood, The Journal of the American Society of Hematology. 2018;131(25):2745\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKikushige Y. Pathogenesis of chronic lymphocytic leukemia and the development of novel therapeutic strategies. Journal of clinical and experimental hematopathology. 2020;60(4):146\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eByrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. New England Journal of Medicine. 2013;369(1):32\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026ouml;hner H, Stilgenbauer S, Benner A, Leupolt E, Kr\u0026ouml;ber A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. New England Journal of Medicine. 2000;343(26):1910\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA: a cancer journal for clinicians. 2023;73(1).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEichhorst B, Ghia P. 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The clinical significance of NOTCH1 and SF3B1 mutations in the UK LRF CLL4 trial. Blood, The Journal of the American Society of Hematology. 2013;121(3):468\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Kzayer LaFY, Saeed RM, Ghali HH, Tanaka M, Al-Jadiry MF, Faraj SA, et al. Comprehensive genetic analyses of childhood acute leukemia in Iraq using next-generation sequencing. Translational Pediatrics. 2023;12(5):827.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBraish J, Cerchione C, Ferrajoli A. An overview of prognostic markers in patients with CLL. Frontiers in Oncology. 2024;14:1371057.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBinet J, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. 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Nature. 2015;526(7574):525\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CLL, immunophenotyping, 17p deletion, LAIR1, CD38","lastPublishedDoi":"10.21203/rs.3.rs-7368240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7368240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThis study examines immunophenotypic markers (CD5, CD19, CD38, LAIR1) and genetic changes (17p, 11q, 13q deletions) in a group of CLL patients from Kerbala, Iraq. Flow cytometry using BD/FACS LYRICS and FISH was performed, and the results from the lab were compared to the clinical findings.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis retrospective study evaluated 100 treatment-na\u0026iuml;ve CLL patients diagnosed at Al-Mujtaba Hospital for Hematological Diseases and Bone Marrow Transplantation. The diagnosis was confirmed following the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria, immunophenotypic confirmation via flow cytometry and an exclusion criteria: previous CLL-directed therapy, Richter's transformation, or coexisting hematologic malignancies.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe most recent study found that 68% of patients had elevated LAIR1, which is linked to rapid disease progression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, 17p deletion (18%) combined with CD38\u0026thinsp;+\u0026thinsp;was associated with worse survival (median PFS: 14 months). A new high-risk profile was identified: CD38+/LAIR1+/17p del (3-year OS: 42%).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study shows that LAIR1 and 17p deletion are crucial for predicting outcomes in Iraqi CLL patients.\u003c/p\u003e","manuscriptTitle":"An analysis of 100 patients with chronic lymphocytic leukemia in Kerbala, Iraq: a comprehensive immunophenotypic and genetic study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 14:51:18","doi":"10.21203/rs.3.rs-7368240/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9256f09f-e73a-4143-919f-2b66e1c6923f","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-09T00:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 14:51:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7368240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7368240","identity":"rs-7368240","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}