Evaluarea cazurilor de leucemie limfocitară cronică cu expresia proteinei izomorfe p53 utilizând testul ELISA

1. Introduction

Chronic lymphocytic leukemia (CLL) is characterized by variable clinical manifestations influenced by age, sex and genetic background. A key molecular determinant of disease progression is the dysfunction of the TP53 tumor suppressor gene, which regulates apoptosis and genomic stability⁽¹⁾. Mutations in TP53 gene result in structurally altered isoforms of the p53 protein. These variants not only impair DNA repair and cell cycle control, but also promote leukemic cell survival. In chronic lymphocytic leukemia, TP53 abnormalities are strongly associated with resistance to conventional chemotherapy and poorer survival outcomes⁽²⁾ (Figure 1).

P53 gene mutation is a common event in human neoplasia, and a single allele is responsible for hereditary cancer susceptibility syndrome (Li Fraumeni syndrome). This variant encodes distinct isoforms of the p53 protein, which may disrupt its transcriptional activity^(3-5).

Over the past decade, techniques such as immunohistochemistry (IHC) and ELISA have enabled the detection of mutant p53 isoforms in hematological malignancies. This study applied ELISA to measure mutant p53 isoforms in B-CLL patients, aiming to correlate the expression levels with clinical stage and treatment response.

2. Materials and method

Patient cohort. Blood samples were collected from 85 patients diagnosed with B-CLL at government hospitals. From this group, 20 patients were selected for detailed analysis based on poor response to standard chemotherapy and immunotherapy. All participants provided the informed consent.

The diagnosis of chronic lymphocytic leukemia along with clinical staging and assessment of treatment response were carried out according to the guidelines established by the International Workshop on CLL⁽⁶⁾. Patients were evaluated through a comprehensive physical examination to confirm B-cell chronic lymphocytic leukemia (B-CLL), presenting with symptoms that included persistent coughing, night sweats and retrosternal discomfort.

Diagnostic criteria. The diagnosis and staging followed the International Workshop on CLL (IWCLL) guidelines. Clinical evaluation included physical exa­mi­na­tion, imaging (CT/PET) and hematological analysis. Peripheral blood smears confirmed lymphocytosis (>5000 lymphocytes/µL) with <10% prolymphocytes. Flowcytometry immunophenotyping confirmed CLL with markers CD5+, CD19+, CD20+/− and CD23+ receptor^(7-10) (Figure 2).

Mutations in the TP53 gene, resulting in accumulation of dysfunctional p53 isoforms, are significant prognostic markers in chronic lymphocytic leukemia. ELISA detection of these isoforms provides a practical and reliable method for risk stratification^(11,12).

ELISA assay. Mutant p53 isoform levels were quantified using ELISA with PAb 240 monoclonal antibodies. The results were expressed in µg/dL, with a reference interval of 10-40 µg/dL.

Statistical analysis. Data were analyzed using descriptive statistics, Fisher’s exact test for categorical comparisons, and student’s t-test for continuous variables. The significance was set at p<0.05.

3. Results

Patient characteristics. Of the 20 patients studied, 14 were men (55-85 years old) and six were women (39-85 years old). All were undergoing cytostatic and/or immunotherapy regimens at the time of evaluation. More high pathological values, 50 µg/dL and 60 µg/dL in men, respectively 140 µg/dL in a female patient, from the study cohort, characterize clinical stages with unfavorable and unpredictable evolutions, in afflicted men and women, regardless of age or sex, in a ratio of approximately 2:1, as described in the specialized literature (Graphic 1).

Statistical interpretation. Female concentrations ranged from 10 to 140 µg/dL (mean = 39.6 µg/dL). Three patients (two men and one woman) exhibited pathological levels (>40 µg/dL). In this study, the mean concentration was determined to be 14.8 µg/dL, with a standard deviation (STDEV) of 6.46, a coefficient of variation (CV) of 0.4%, and a probability index (NORMDIST) of p=0.079. The frequency of protein isoforms within the study group was estimated at 17%. Transformation of CLL into diffuse large B-cell lymphoma occurred in 3.5% of the cases, consistent with findings reported in meta-analyses from the specialized literature. Comparisons between categorical and numerical variables were conducted using Fisher’s exact test. In the subset of 17 patients analyzed, the average concentration of p53 protein in CLL cases was found to be 16.76 µg/dL (Table 1).

The specificity of the method (SP) was calculated as the proportion of cases with normal p53 protein levels (range: 10-40 µg/dL) out of the total number of B-CLL patients (n=20).

This resulted in 17/20, or 85%, indicating good specificity within the expected range (70-90%; 95% CI). The positive predictive value (PPV) was determined as the number of true outliers for p53 (three cases with p53 variants) divided by the total number of abnormal cases above the cutoff, yielding 3/3, or 100% (95% CI).

For statistical testing, the t statistic was applied, resulting t=2.01. The corresponding critical values from the t distribution with 20 degrees of freedom, for a two-tailed test at a=0.05, were ±2.01. Thus, the observed result aligned with the threshold for statistical significance.

Correlation with disease stage:

• Elevated p53 isoform levels were strongly associated with advanced CLL (stages III/IV).
• Patients with pathological concentrations demonstrated significantly reduced treatment responsiveness (p=0.034).
• Transformation into diffuse large B-cell lymphoma (Richter’s syndrome) occurred in 3.5% of cases.

4. Discussion

This study confirms the clinical relevance of TP53 mutations in CLL. Elevated isoform expression was observed in approximately 15% of cases, consistent with international data. Patients with biallelic TP53 disruptions often exhibit resistance to therapy, highlighting the importance of early detection⁽¹³⁾.

ELISA proved sensitive for detecting p53 isoforms, complementing standard IHC-based methods. High concentrations of mutant isoforms were predictive of poor prognosis, aligning with the published literature linking TP53 abnormalities to chemotherapy resistance. Beyond prognosis, restoration of wild-type p53 function is emerging as a therapeutic strategy (Figure 3).

Research indicates that reactivating the functional form of the p53 protein may trigger the regression of some cancer cells without harming healthy cells.

Additionally, other studies have reported that eleva­ted levels of adenosine triphosphate (ATP) in malignant B lymphocytes in CLL can influence the P53 gene, promoting apoptosis in these cells⁽¹⁴⁾.

Antibodies specific for p53 and p53 for phosphorylated at three different sites in the field of activation were used in parallel analyses in investigations of CLL treatments and are in current clinical trials⁽¹⁵⁾. The role of autophagy in cancer, which could be changed by the p53 status, is expected to be developed in new anti-cancer therapies^(16,17).

5. Conclusions

Patients with elevated levels require closer monitoring and may benefit from alternative treatment approaches. Further research integrating molecular diagnostics with targeted therapies could substantially improve survival outcomes in this high-risk subgroup.   

Corresponding author: Ioan-Ovidiu Gheorghe E-mail: ovis45@yahoo.com

Conflict of interest: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY licence.