Association of serum protein electrophoresis with clinicopathological characteristics and its prognostic relevance in chronic lymphocytic leukaemia patients

Objective: To assess the association of serum protein electrophoresis abnormalities with clinicopathological characteristics, and its impact on overall survival in chronic lymphocytic leukaemia patients. Methods: The prospective study was conducted at Haematology and Immunology departments of the University of Health Sciences, Lahore, Pakistan, from 2019 to 2022, and comprised newly diagnosed chronic lymphocytic leukaemia patients. Lactate dehydrogenase and beta-2 microglobulin levels were measured by spectrophotometric principle, whereas serum protein electrophoresis was determined through commercially available capillary electrophoresis systems. Patients were followed up for 2 years post-diagnosis. Data was analysed using SPSS 21. Results: Of the 50 patients, 40(80%) were males and 10(20%) were females. The overall mean age was 60±11 years. Serum protein electrophoresis was available for 40(80%) patients, and, among them, 12(30%) patients had abnormal levels, while 29(72.5%) required treatment. Overall response rate was 25(86.2%), and median two-year overall survival was 16.5 months (95% confidence interval: 10-20 months). Abnormal serum protein electrophoresis was significantly associated with Binet stage C, lower mean haemoglobin levels and higher median levels of lactate dehydrogenase and beta-2 microglobulin (p<0.05)). Regarding overall survival, the survival curves of chronic lymphocytic leukaemia patients with normal and abnormal serum protein electrophoresis status differed significantly (p=0.04). Conclusion: Abnormal serum protein electrophoresis could be considered a surrogate marker for advanced chronic lymphocytic leukaemia disease.


Introduction
Chronic lymphocytic leukaemia (CLL) is characterised by clonal expansion of small relatively monomorphic B lymphocytes in peripheral blood, bone marrow and other secondary lymphoid organs. 1,2CLL represents a rare haematological malignancy in Asians (<5% of all leukaemia), whereas it is 10-fold more common in the Western hemisphere, accounting for >30% of all leukaemia cases. 3e clinical course of CLL patients is heterogeneous in nature.Some patients experience an aggressive disease progression, while others have an indolent course of disease.Clinico-haematological, biochemical and genetic features play a significant role in determining the clinical course and in predicting the treatment response and prognosis.Studies have shown that the standard Rai 4 and Binet 5 clinical staging systems are not very efficient in predicting prognosis of patients, especially in the early stage of the disease.Therefore, there is a need to identify more markers that may improve the existing staging systems for better prediction and risk stratification of CLL patients at diagnosis. 2,6 erous studies have reported that the variety of molecular abnormalities seen in CLL patients, which are ultimately responsible for the development and progression of this illness, reflect clinical heterogeneity. 7,8owever, for cytogenetic and molecular analysis of CLL, high degree of technical and medical expertise is required for each and every case, and the process is laborious and costly.To overcome these difficulties, many different biological laboratory markers such as beta-2 (β 2 ) microglobulin, serum soluble cluster of differentiation CD23, CD38 and zeta-chain-associated protein kinase (ZAP70), have been evaluated as prognostic markers in previous studies.Currently, these biomarkers are seldom used in the standard staging and follow-up of CLL patients. 9ong biological parameters, serum protein electrophoresis (SPE) has been studied and recommended for the initial staging of CLL patients.Different patterns on SPE can be detected that include either hypogammaglobulinaemia or hypergammaglobulinaemia, and immunoglobulin peaks, that are all considered abnormal SPE.Hypogammaglobulinaemia, the most prevalent among all abnormal SPE patterns, is assumed to be a predictor of infection and is correlated to tumour burden. 10Different prevalence rates of Hypogammaglobulinaemia, and monoclonal paraprotein have been reported. 11,12imilarly, the prognostic significance of abnormal SPE has been studied in CLL, but has shown contradictory findings regarding infection risk, treatment-free survival (TFS) and overall survival (OS). 10,13,14nce there is a lack of data regarding frequency of SPE abnormalities and its prognostic significance in local CLL patients, the current study was planned to fill the gap by assessing the association of SPE abnormalities with clinicopathological characteristics, and its impact on OS in CLL patients.

Patients and Methods
The prospective study was conducted at the Haematology and Immunology departments of the University of Health Sciences (UHS), Lahore, Pakistan, from 2019 to 2022.The sample was raised using convenience sampling technique from among CLL patients presenting at the Institute of Nuclear Medicine & Oncology Lahore (INMOL) Cancer Hospital, Lahore, Pakistan.Those included were diagnosed CLL cases with at-least 2-year follow-up.The CLL diagnosis was done according to the International Workshop on Chronic Lymphocytic Leukaemia (IWCLL) criteria that is based on persistent lymphocytosis, typical lymphocyte morphology on peripheral blood smears and immunophenotyping results. 2 Patients with relapsed/refractory CLL, and acute or chronic lympho-proliferative disease were excluded, and so were those with a history of previous treatment.
The sample size was calculated as per the guidelines of the World Health Organisation (WHO) using the formula: In the formula, Z 1-α/2 was 95% confidence level 1.96, P was the anticipated frequency of abnormal SPE in CLL = 7% 15 , d was the margin of error = 7%, and n was the required sample size = 51.
After approval from the UHS ethics review committee, and informed consent from all the patients, baseline clinical and laboratory characteristics were retrieved from the patients' charts.Clinical stage of the patients was determined using the Binet staging system 5 .History and physical examination was conducted with help of clinical haematologist of INMOL Hospital, and data was noted on a predesigned proforma.Patients were followed up from the time of diagnosis to death or last follow-up, which was up to a maximum of 2 years, and was termed OS.Treatment indications and response assessments were done in line with IWCLL guidelines. 2 Peripheral blood samples were collected at baseline before the initiation of treatment.Of the 5ml venous blood sample collected by a trained phlebotomist using aseptic technique, 3ml was dispersed in ethylenediaminetetraacetic (EDTA) acid vacutainer for complete blood count (CBC) and peripheral smear examination, while 2ml was kept in plain vacutainers for serum separation.
Patients were divided into normal SPE group, and with abnormal SPE group with Hypogammaglobulinaemia, and hypergammaglobulinaemia patterns.Data was analysed using SPSS 21 and GraphPad Prism 8.0.Data was expressed as frequencies and percentages, or mean ± standard deviation, or median with interquartile range (IQR), as appropriate.Chi-square, Fisher's exact, student's t-test and Mann-Whitney U tests were used, as appropriate.Cox proportional hazard model was used to assess the association between 2-year OS and predictor variables.Survival curves were plotted using Kaplan-Meier estimates, and log-rank statistics were used to evaluate the differences between prognostic factors and 2-year OS.Two-sided p<0.05 was considered statistically significant.
All the identified significant risk variables in univariate analysis were subjected to multivariate analysis and none of them could retain the status as significant independent predictor of poor OS in CLL patients (Table 5).The survival curves of CLL patients with normal SPE differed significantly from CLL patients with abnormal SPE (p=0.04)(Figure 3).

Discussion
CLL is a heterogeneous disease characterised by a variable clinical course.Some individuals present with aggressive condition that needs treatment right away, while others have a more stable, non-progressive disease that may not require treatment for years.Over the past 20 years, a number of prognostic markers have been discovered and evaluated to identify individuals with a poor prognosis in order to optimize the clinical care of CLL patients with closer follow-up periods 16 .The present study evaluated the prognostic impact of abnormal SPE in a geographical novel sample set of Pakistani CLL patients.The study confirmed the previously reported significant impact of abnormal SPE as it was found associated with poor prognostic markers, such as advanced Binet stage, high LDH and high β-2microglobulin levels.Moreover, in the present CLL sample set, a significant trend of association of abnormal SPE with survival outcomes was also noted.
The present study found that frequency of abnormal SPE was 30% in the CLL cohort, out of which 10(25%) cases were with hypogammaglobulinaemia and 2(5%) had hypogammaglobulinaemia, while no patient was seen having monoclonal band.This was in agreement with a previous study, 11 but differed from other European and Egyptian.studies. 9,12,15,16Overall, 56% cases in a study 9 had abnormal SPE, while in another study, the corresponding value was 28%. 15So wide variation in overall frequency of abnormal SPE has been reported in the literature ranging from as low as 7-13% to as high as 30-80%. 15e mean age at diagnosis in the current CLL cohort was 60 years, with 40% of patients presenting at a younger (≤55 years) age.This age distribution in CLL was consistent with other Pakistani and Indian studies. 17,180][21] This may reflect referral bias, but may also be suggestive of a genetic predisposition for Indo-Pak CLL patients to present at a younger age, especially considering higher rates of consanguinity in this ethnic group, and can be of significance as this earlier-age CLL may represent a severe disease with worse prognosis and inferior survival outcomes. 22[19][20][21][22][23][24] CLL has a highly variable natural course with OS ranging from a few years to several decades.The Binet and Rai staging systems still provide a simplified way based on a few simple clinical and laboratory variables, especially in resource-limited healthcare systems, to predict CLL disease progression at diagnosis despite being almost half-a-century old. 4,5On the contrary, cytogenetic and complex molecular testing has been recommended by the IWCLL guidelines for CLL prognostication. 2 Among all genetic biomarkers, only immunoglobulin heavy variable (IGHV) gene mutation status and deletion 17p (Del 17p), which is a form of tumour protein 53 (TP53) genetic aberration, are recommended at present to predict CLL progression and chemo-resistance. 23 the current study, 72% of the CLL patients presented with advanced Binet stages B and C that seems to be a hallmark of the disease in under-developed regions of the world. 17,18,24The representation of advanced Binet stages among CLL patients decreases sharply for Chinese (43.9%) and European (18.8%) patients. 9,19 the present study, CLL cases with abnormal SPE were significantly associated with Binet stage C which was in line with previous studies. 9,16,25While considering other laboratory parameters, statistically significant lower mean Hb levels and low platelet count were found in present CLL sample with abnormal SPE, which was contrary to a study. 9Moreover, high β2-microglobulin and higher LDH levels were present in the current CLL patients with abnormal SPE, as suggested by previous reports 16,25 .
In the current study, higher β2-microglobulin levels appeared to be a prognostic clinicopathological marker of poor 2-year OS, which has already been reported. 26,27he relative percentage of CLL patients with elevated β2microglobulin was 10.7% in Europeans, 36% in a local study 28 and 70-75.4% in Indians. 9,18 the present study, while considering the impact of abnormal SPE on 2-year OS, significant association was observed (p=0.04).This was in contrast to earlier findings showing no significant impact on OS in CLL patients with any kind of immunoglobulin (Ig) abnormality on SPE 15 .Rather patients with abnormal SPE/Ig aberrations had significantly reduced TFS, which was not measured in the current study in which only first-line treatment outcomes were measured.
Moreover, multivariate analysis of the risk variables, including elevated β2-microglobulin, anaemia, thrombocytopenia and abnormal SPE showed that no factor retained the status of being a significant independent predictor of poor OS in the current CLL patients.The reason for this might be the small sample size with shorter follow-up.
Among genetic features, only Del 17p data was available for less than half of the current patients, and only 2(11.8%) harboured it, which is concordant with detection rates of 11-18.5% in other studies 18,28 , while European and Chinese studies displayed lower frequencies of 5-7.8%. 19,20me of these studies 19,20,28 also suggested significant prognostic relevance of Del 17p positivity, which the current study did not determine because of limited Del 17p testing facilities.
A vast majority (64.9-80%) of Indo-Pak CLL patients needed treatment at the time of diagnosis as reflected in the present and other such studies from the region. 17,18  the contrary, only about half or a quarter of CLL cases necessitated therapeutic interventions among Chinese and Western cohorts. 9,19These observations in Indo-Pak CLL patients were consistent with published literature 17,18,28 showing higher proportions of younger patients in the region with aggressive disease behaviour and shorter time to first treatment.As suggested earlier, another reason for this could be delay in making proper diagnosis and referring patients to appropriate cancer care centres. 22e current study has limitations as it had a small sample size, comprised single-centre data and lacked molecular analysis.Despite the limitations, however, the current study, to the best of our knowledge, is the first to report the prognostic significance of SPE and its associations with clinicopathological parameters in Pakistani CLL patients.

Conclusion
Abnormal SPE could be used as surrogate biomarker of advanced disease as CLL cases with abnormal SPE presented with enrichment of poor prognostic markers, such as Binet stage C, lower mean Hb levels, higher median LDH and β2-microglobulin levels.Abnormal SPE could be a significant predictor of poor OS in CLL patients.

Figure- 1 :
Figure-1: Patient characteristics stratified by serum protein electrophoresis (SPE) status in chronic lymphocytic leukaemia (CLL) patients.These include age (a), haemoglobin (Hb) (b), platelet count (c), absolute lymphocyte count (ALC) (d), beta-2 (β2) microglobulin (e) and lactate dehydrogenase (LDH) levels (f).The X-axis displays the SPE status as normal or abnormal, while the Y-axis represents different variables in question.The number of samples analysed in both groups is shown below the SPE status.In a-c, the columns represent mean values with standard deviation (SD) bars, while for d-f, each group tested is represented as a box and whisker plot covering the interquartile range (IQR), median values as horizontal lines within the box, and error bars representing minimum and maximum values.

Table - 1
: Demographic and clinical characteristics of the patients (n=50).

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Abnormal SPE was not significantly associated with gender, presence of B symptoms, lymphadenopathy, organomegaly and bone marrow (BM) infiltration (p>0.05).A significant association was observed for Binet stage C compared to patients with normal SPE (Table4).