Frequency of TP53 Mutation in B-Cell Chronic Lymphocytic Leukaemia and its Association with Lymphocyte Doubling Time

By Syeda Mah Ali, Syed Muhammad Irfan, Naila Raza

Affiliations

1. Department of Haematology, Liaquat National Hospital, Karachi, Pakistan

doi: 10.29271/jcpsp.2023.11.1249

ABSTRACT
Objective: To determine the frequency of TP53 mutation at diagnosis of B-cell Chronic Lymphocytic Leukaemia (B-CLL) in Pakistani patients, and to investigate whether lymphocyte doubling time (LDT) of less than 1 year could be used as a surrogate marker for TP53 mutation.
Study Design: A cross-sectional descriptive study.
Place and Duration of the Study: Department of Haematology, Liaquat National Hospital, from January 2020 to December 2022.
Methodology: Patients diagnosed with B-CLL based on the criteria set by International Workshop on Chronic Lymphocytic Leukaemia were included in the study. Clinico-haematological parameters were recorded, and TP53 mutation analysis was performed by fluorescence in situ hybridisation. Patients were followed every 3-6 months after diagnosis with the recent complete blood count (CBC) reports to record CBC parameters and calculate LDT.
Results: A total of 128 B-CLL cases were evaluated, with a mean age of 62 years. Among these cases, 10 patients (7.8%) tested positive for TP53 mutation, while 118 patients (92.2%) tested negative. During the follow-up period, 26 patients were lost to follow-up, with only one patient from the TP53 positive group. In the TP53 positive group, 55.6% (n=5/9) patients had an LDT of less than 1 year, indicating aggressive disease compared to 30.1% (n=28/93) patients in the negative group (p <0.1).
Conclusion: TP53 mutations may be associated with shorter LDT, indicating aggressive disease. Further research is needed to fully comprehend the relationship between TP53 mutation and LDT in B-CLL.

Key Words: TP53 mutation, B-Cell chronic lymphocytic leukaemia (B-CLL), Lymphocyte doubling time (LDT).

INTRODUCTION

B-Cell Chronic Lymphocytic Leukaemia (B-CLL) is an indolent malignancy of mature lymphoid cells. In the Western countries, it is the most prevalent leukaemia in adults, accounting for 25-35% of all leukaemia cases.¹ However, in Asia, the prevalence of CLL is relatively low, comprising of only 10% of the cases observed in the Western countries.^2,3 In Pakistan, CLL is considered a rare haematological malignancy, with an overall prevalence of 0.9% and accounting for approximately 9.7% of leukaemia cases.^4,5 This disease primarily affects older individuals, with the incidence rate rising after the age of 60, and there is a slight male predominance.⁶

CLL  has  a  prolonged  clinical  course  that  spans  over  many years and displays considerable heterogeneity in clinical behaviour.

While some patients experience rapidly progressive disease and require chemo-immunotherapy upon diagnosis, others undergo years of observation without any treatment or change in the clinical status. This variable clinical course underscores the significance of prognostic and predictive markers in disease management. Clinical staging such as Rai and Binet's staging, and lymphocyte doubling time (LDT) are the most commonly used and cost-effective prognostic markers. However, additional serum markers, cytogenetics, and molecular markers have gained importance over time. According to the International CLL-IPI working group, TP53 mutation, serum Beta 2 Microglobulin >3.5 mg/L, and unmutated IGHV gene are independent prognostic factors that not only predict survival time but also provide insights into clinical behaviour, thus guiding treatment decisions.⁷

TP53  mutation  is  recognised  as  the  most  significant  prognostic marker in CLL, supported by numerous studies and prospective clinical trials.^8,9 These findings consistently demonstrated that patients with TP53 mutation are at a higher risk of developing rapidly progressive disease that is resistant to chemo-immunotherapy. As a result, these patients should be considered for targeted therapy when appropriate. The updated guidelines from the International Workshop on Chronic Lymphocytic Leukaemia (iwCLL) emphasised the importance of conducting TP53 mutation analysis prior to initiating treatment.¹⁰

In countries like Pakistan, performing molecular tests for every B-CLL patient undergoing treatment is a significant challenge due to the poor socioeconomic conditions. The objectives of this study were to assess the frequency of TP53 mutation at the time of B-CLL diagnosis in Pakistani patients and investigate its relationship with lymphocyte doubling time (LDT). The rationale was to determine whether an LDT of less than 1 year could serve as a surrogate marker for TP53 mutation.

METHODOLOGY

This was a cross-sectional, descriptive study, conducted at Liaquat National Hospital, Karachi. A total of 128 B-CLL patients who attended the haematology outpatient department (OPD) between January 2020 and December 2022 were enrolled and monitored. Inclusion criteria encompassed Pakistani patients aged between 30 to 90 years who were recently diagnosed with B-CLL, according to iwCLL criteria, which relied on immuno-phenotyping results.¹⁰ Patients who had previously been diagnosed with other forms of cancer, whether haematological or non-haematological, and already had a confirmed TP53 gene mutation were not included in the study. Additionally, patients who had undergone chemotherapy or radiotherapy as part of their treatment for any type of cancer were also excluded from the study.

Written informed consent was obtained from each patient for their participation in the study, including data collection and laboratory investigations. A detailed medical history, physical examination, ECOG performance status, and Binet's staging were documented for each patient. Diagnostic and prognostic investigations included complete blood count (CBC), peripheral blood smear, immunophenotyping via flowcytometry on peripheral blood, abdominal ultrasound/computed tomography (CT) scan of the whole body, serum creatinine, serum lactate dehydrogenase levels, serum uric acid, serum alanine transaminase levels, serum calcium and direct antiglobulin test. Additionally, TP53 mutation analysis was performed using the Fluorescence in situ Hybridisation (FISH) technique right after diagnosis.

Lymphocyte doubling time (LDT) refers to the duration required for lymphocytes to double in number from the count observed at diagnosis. An LDT of 12 months or less indicated rapidly progressive disease with a poor prognosis, while an LDT of more than 12 months suggested slowly progressive disease and a favourable prognosis.^10,11 Patients were followed up every 3-6 months after diagnosis, with recent CBC reports. Absolute Lymphocyte Count (ALC) was recorded at the initial visit (baseline) and during each subsequent follow-up visit to calculate the LDT and evaluate disease progression. The median time from diagnosis to the follow-up visits was approximately 10 months (range: 7-13 months), which was used to calculate the LDT.

The sample size (n=82) for the study was determined using the open epi sample size calculator, considering a 95% confidence level. The prevalence of the outcome variable was derived from a previous study. Non-probability consecutive sampling was employed as the sampling technique.

Data compilation and analysis were performed using IBM SPSS version 22. Qualitative variables were presented in frequencies and percentages, while quantitative variables were expressed as mean ± SD. The relationship between categorical variables was assessed using the Pearson Chi-square (χ2) test or Fisher's exact test. A p-value of less than 0.05 was considered statistically significant.

RESULTS

A total of 128 patients who had recently been diagnosed with B-CLL participated in the study. Among these patients, 89 (69.5%) were males, and 39 (30.5%) were females. The mean age of the patients was 62 years, ranging from 35 to 88 years. TP53 mutation analysis was conducted on all patients using the FISH technique with peripheral blood samples. The results showed that 10 patients (7.8%) tested positive for the mutation, while 118 patients (92.2%) tested negative.

Table I presents the clinical and laboratory parameters of patients diagnosed with B-CLL, with and without TP53 mutation. A comparison between these groups revealed significant associations between TP53 mutation and age over 62 years (p=0.03), advanced clinical stage (p=0.03), baseline ALC (p=0.007), and follow-up ALC between 12-18 months after diagnosis (p=0.001). However, TP53 mutation did not show a significant association with gender (p=1.0), LDT (p=0.1), LDH (p=0.7), direct antiglobulin test (p=1.0), serum creatinine level (p=0.3), serum ALT levels (p=0.7), and serum calcium (p=0.3).

During the follow-up period, 26 patients were lost to follow-up, with one patient from the TP53 positive group and 25 patients from the negative group. In the TP53 positive group, 55.6% (n=5/9) of patients had an LDT of less than 1 year, indicating aggressive disease, compared to 30.1% (n=28/93) of patients in the negative group (55.6% vs. 30.1%; p <0.1). Similarly, in the TP53 mutation with B-CLL group, 44.4% (n=4/9) of patients had an LDT of more than 1 year, while 69.9% (n=65/93) of patients in the TP53 negative group had an LDT of more than 1 year (44.4% vs. 69.9%; p <0.1). Figure 1 shows the study variable (TP53 mutation status) and their associations with LDT.

Figure 1: Association of TP53 mutation status with lymphocyte doubling time.

Table I: Characteristics of patients according to TP53 mutation status.

Patients’ characteristics	All n (%)	TP53 mutation positive n (%)	TP53 mutation negative n (%)	p-value
Patients	128	10(7.8)	118(92.2)
Age (mean)	62.37	69	61.8	0.03
Gender    Male    Female	89(69.5) 39(30.5)	7(70) 3(30)	82(69.5) 36(30.5)	1.0
Binet’s stage    A    B    C	81(63.3) 18(14.1) 29(22.7)	3(30) 3(30) 4(40)	78(66.1) 15(12.7) 25(21.2)	0.03
ALC at presentation (mean)	70.4x109/L	110 x109/L	67.3 x109/L	0.002
ALC (b/w 12-18 months)	65.5 x109/L	130.5 x109/L	59.5 x109/L	0.005
LDT    ≤12 months    >12 months	33(32) 69(67.6)	5(55.6) 4(44.4)	28(30.1) 65(69.9)	0.1
Direct antiglobulin test Positive Negative	4(3.4) 115(96)	0(0) 9(100)	4(3.6) 106(96.4)	1.0
LDH (Mean)	298U/L	279 U/L	300 U/L	0.7
Uric acid (mean)	5.8mg/dl	6.2mg/dl	5.4mg/dl	0.1
Serum creatinine (mean)	0.9 mg/dl	0.9 mg/dl	0.9 mg/dl	0.6
Serum calcium (mean)	9.98 mg/dl	16.67 mg/dl	9.42 mg/dl	0.3
ALT (mean)	25.26 U/L	21.56 U/L	25.57 U/L	0.7

DISCUSSION

The present study aimed to investigate the frequency of TP53 mutation in patients with B-CLL at the time of diagnosis and assess its association with LDT. In this study, 10 patients (7.8%) tested positive for TP53 mutation. This frequency aligned with the western studies reporting a range of 3.7 to 8.5% of patients detected positive for TP53 in CLL.^12,13 These findings contrast with a local study showing a relatively high number of TP53 -positive patients, i.e. 13.7%.¹⁴ Another local study also demonstrated similar findings, with 18.5% of B-CLL patients having 17p deletion.¹⁵ Similarly from the neighbouring country, India, Kadam Amare et al. reported 22% frequency of TP53 in CLL.¹⁶

The relatively low frequency observed in this study may be attributed to the fact that mutation status was examined in newly diagnosed patients regardless of their clinical stage or treatment indication. These findings support a previously published Swedish study where Zainuddin et al. reported a 3.7% frequency (n=10/268) of CLL patients detected positive for TP53 mutation, concluding that TP53 mutation is rare at disease onset.¹³ These results endorsed the notion that TP53 mutation develops during disease progression. However, in resource-limited countries, the use of TP53 mutation analysis is limited due to its high cost which has made its utility debatable.

The results of this study showed that patients with TP53 mutation had a higher proportion of patients (55.6%) with LDT less than 1 year compared to those without TP53 mutation (30.1%). Conversely, patients without TP53 mutations had a higher proportion (69.9%) of patients with LDT more than 1 year compared to 44.4% of patients with TP53 mutation. Although these results suggested that TP53 mutation may be associated with a shorter LDT in CLL but statistical significance was not observed. Therefore, LDT may complement TP53 aberrations in CLL prognostification but it cannot replace them.

Furthermore, TP53 positive patients were older and had higher baseline ALC. Additionally, significant association between positive TP53 mutation and the doubling of ALC count between 12 to 18 months indicated a rapidly progressive nature of the disease in mutation-positive patients. These findings emphasised the crucial role of early determination of mutation status, particularly in patients with a high baseline ALC count, age over 60 years, and high-risk disease (such as Binet Stage C). Patients who test positive for TP53 mutation should be closely monitored under strict surveillance. These findings were consistent with some of the previously reported studies.^17,18 This study contributed to the growing body of literature supporting the association between TP53 mutations and advanced-stage disease, which may have implications for the clinical management and treatment decisions for patients with CLL.^19,20

However, it is important to acknowledge the limitations of this study, including the small sample size and the loss of 26 patients to follow-up. The exclusion of these patients may have potentially impacted the result. Moreover, the impact of TP53 mutation on LDT may be influenced by other factors, such as age, gender, and stage of the disease. Further studies with larger sample size and longer follow-up periods are needed to confirm findings and explore the potential mechanism underlying the association between TP53 mutation and LDT in CLL. The clinical implications of this finding warrant additional investigation, as it could potentially impact treatment decision-making and patient prognosis. 

CONCLUSION

There was a relatively low frequency of TP53 mutation at the time of B-CLL diagnosis, specifically at 7.8%. Patients with a positive TP53 mutation tended to experience an earlier increase in lymphocyte count, with lymphocyte count doubling between 12 to 18 months, compared to patients without the TP53 mutation. TP53 mutations may be linked to a shorter LDT in patients with CLL. 

ETHICAL APPROVAL:
An approval was obtained from the Ethical Review Committee (ERC) of Liaquat National Hospital and Medical College with approval number 0581-2020 LNH – ERC. Data confidentiality was guaranteed that under no circumstances, patient’s name, identity and any other associated information will be disclosed to any other person.

PATIENTS’ CONSENT:
All participants provided written informed consents prior to participation.

COMPETING INTEREST:
The authors declared no competing interest.

AUTHORS' CONTRIBUTION:
SMA: Conception of the work, design, data collection, interpretation, and analysis, drafting of the initial version.
SMI: Conception, data analysis, review, and editing.
NR: Review and editing.
All authors approved the final version of the manuscript to be published.
 

REFERENCES

1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics 2021. Ca Cancer J Clin 2021; 71(1):7-33. doi:10.3322/caac. 21654.
2. Yang S, Varghese AM, Sood N, Chiattone C, Akinola NO, Huang X, et al. Ethnic and geographic diversity of chronic lymphocytic leukaemia. Leukemia 2021; 35(2):433-9. doi: 10.1038/s41375-020-01057-5.
3. Miranda-Filho A, Piñeros M, Ferlay J, Soerjomataram I, Monnereau A, Bray F. Epidemiological patterns of leukaemia in 184 countries: A population-based study. Lancet Haematol 2018; 5(1):e14-e24. doi: 10.1016/ S2352-3026(17)30232-6.
4. Dodhy M, Zafar H, Aslam W. Chronic lymphocytic leukaemia: An experience of a decade at a tertiary care hospital. Ann Pak Inst Med Sci 2011; 7(1):196-9.
5. Zeeshan R, Irfan SM, Sultan S, Bhimani S. ZAP-70 protein expression in B-cell Chronic Lymphoid Leukaemia: A single center experience from Pakistan. Asian Pac J Cancer Prev 2015; 16(4):1587-90. doi: 10.7314/apjcp.2015.16.4.1587.
6. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics 2022. Cancer J Clin 2022; 72(1):7-33. doi: 10.3322/caac. 21708.
    
7. Group IC-IW. An international prognostic index for patients with chronic lymphocytic leukaemia (CLL-IPI): A meta-analysis of individual patient data. Lancet Oncol 2016; 17(6):779-90. doi: 10.1016/S1470-2045(16)30029-8.
8. Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE, et al. Multivariate analysis of prognostic factors in CLL: Clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 2002; 100(4):117784. doi:10. 1182/blood.v100.4.1177.h81602001177_1177_1184.
9. Quijano S, López A, Rasillo A, Sayagués JM, Barrena S, Sánchez ML, et al. Impact of trisomy 12, del (13q), del (17p), and del (11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B‐cells in chronic lymphocytic leukaemia. J Intern Society Analytical Cytol 2008; 74(3):139-49. doi: 10.1002/cyto.b.20390.
10. 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. Blood 2018; 131(25): 2745-60. doi: 10.1182/blood-2017-09-806398.
11. Montserrat E, Sanchez-Bisono J, Viñolas N, Rozman C. Lymphocyte doubling time in chronic lymphocytic leukaemia: analysis of its prognostic significance. British J Haematol 1986; 62(3):567-75. doi: 10.1111/j.1365-2141. 1986.tb02969.x.
12. Zenz T, Eichhorst B, Busch R, Denzel T, Häbe S, Winkler D, et al. TP53 mutation and survival in chronic lymphocytic leukaemia. J Clin Oncol 2010; 28(29):4473-9. doi: 10.1200/ JCO.2009.27.8762.
13. Zainuddin N, Murray F, Kanduri M, Gunnarsson R, Smedby KE, Enblad G, et al. TP53 Mutations are infrequent in newly diagnosed chronic lymphocytic leukaemia. Leukaemia Research 2011; 35(2):272-4. doi: 10.1016/j.leukres.2010. 08.023.
14. Qadir H, Nasir N, Qadir N, Adil SN, Tanzeem H, Qadir A. Frequency of TP53 gene mutation in patients with chronic lymphocytic leukaemia. J Ayub Med Coll Abbottabad 2020; 32(4):523-6.
15. Mahmood R, Khan SA, Altaf C, Malik HS, Khadim MT. Clinicohaematological parameters and outcomes in a cohort of chronic lymphocytic leukaemia patients with Deletion 17p from Pakistan. Blood Res 2018; 53(4):276-80. doi: 10.5045/br.2018.53.4.276
16. Kadam Amare PS, Gadage V, Jain H, Nikalje S, Manju S, Mittal N, et al. Clinico-pathological impact of cytogenetic subgroups in B-cell chronic lymphocytic leukaemia: Experience from India. Indian J Cancer 2013; 50(3):261-7. doi: 10.4103/0019-509X.118730.
17. Dicker F, Herholz H, Schnittger S, Nakao A, Patten N, Wu L, et al. The detection of TP53 mutations in chronic lymphocytic leukaemia independently predicts rapid disease progression and is highly correlated with a complex aberrant karyotype. Leukaemia 2009; 23(1):117-24. doi: 10.4103/ 0019-509X.118730.
18. Rossi D, Khiabanian H, Spina V, Ciardullo C, Bruscaggin A, Famà R, et al. Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukaemia. Blood 2014; 123(14):2139-47. doi: 10.1182/blood-2013-11-539726.
19. Stilgenbauer S, Schnaiter A, Paschka P, Zenz T, Rossi M, Döhner K, et al. Gene mutations and treatment outcome in chronic lymphocytic leukaemia: Results from the CLL8 trial. Blood 2014; 123(21):3247-54. doi: 10.1182/blood- 2014-01-546150.
20. Baliakas P, Jeromin S, Iskas M, Puiggros A, Plevova K, Nguyen-Khac F, et al. Cytogenetic complexity in chronic lymphocytic leukaemia: definitions, associations, and clinical impact. Blood 2019; 133(11):1205-16. doi: 10.1182/ blood-2018-09-873083.