Chronic Lymphocytic Leukemia and Immunoglobulin M

Introduction

Chronic lymphocytic leukemia (CLL) is a cancer of CD5+ B cells. This disorder is typified by the buildup of tiny, mature-looking neoplastic lymphocytes in tissues such as the bone marrow, blood, and secondary lymphoid tissues (1). The consequences of this buildup include enlargement of the spleen, lymphocytosis, lymphadenopathy, and leukemia cell infiltration of the marrow. The development of CLL is linked to genetic factors. It has been observed that CLL is the most prevalent adult leukemia in western countries. However, its incidence is low in people of Asian origin (1).

CLL is classified into two main subgroups that contrast in their clinical behavior. The basis of the distinction between the two subgroups is whether the diseased cells express transformed or untransformed immunoglobulin heavy-chain variable region gene (IGHV). This phenomenon reflects the phase of normal B cell differentiation from which they arise. CLL cells that produce an unchanged IGHV stem from a B cell that has not gone through segregation in germinal areas. In most cases, this group of patients displays a more aggressive form of the disease compared to patients with CLL cells that produce a transfigured IGHV. CLL cells with changed IGHV originate from B cell that secretes immunoglobulin that has experienced somatic hypermutation or immunoglobulin isotype switching such as that seen in healthy B cells in an immune reaction to an antigen. The somatic mutations observed in IGHV at the germinal sites is a normal part of affinity development of antibodies and is not morbid. Additionally, certain CLL cells resemble untransformed CLL cells but stem from B cells with constrained somatic mutations, for example, CLL with immunoglobulin heavy chains encoded by mutated IGHV3–21 genes. The cellular origin of CLL is illustrated in Figure 1.

The collection of immunoglobin molecules secreted by the CLL cells of all patients is significantly smaller than the range of immunoglobulin molecules a single person can produce. This observation points towards the prejudiced use of specific IGHV genes. The limited immunoglobulin selection in CLL is emphasized by the observation that approximately 1 in 75 CLL patients have diseased cells express identical immunoglobulin molecules. The narrow assortment of immunoglobins provides strong proof that CLL B cells are chosen according to the binding action of immunoglobulins expressed on their surfaces, which implies that B cell receptor (BCR) signaling affects CLL pathogenesis significantly. The sequence of BCR signaling is indicated in Figure 2.

Genetic experiments show that several genetic alterations are found in CLL, for example, single- nucleotide polymorphisms, chromosomal modifications and changes in noncoding RNA, for example, microRNA (miRNA). A few of these genetic modifications can be used to establish a prognosis and direct therapeutic approaches. Exchanges between CLL cells and their surroundings can stimulate the propagation of B cells and add to disease.

Purpose of the Study

The purpose of the study is to correlate CLL and IgM to understand the role of IgM in the manifestation and development of the disease.

Statement of the Research Problem

This study will determine the correlation between CLL and IgM. Estimates show that CLL accounts for approximately 19,000 of all newly detected cancers in the United States in 2016 (1). Men are two times more likely to develop the disease than women. Additionally, the risk of developing the disease increases with advancing age. Abnormalities in the modulation of B cell receptor (BCR) signaling is a common feature of CLL pathology (2). Therefore, investigations into the enzymes involved in the BCR pathway have paved way for significant therapeutic advances. Moreover, the stimulation of BCR in lymphoid organs is linked to the propagation and survival of CLL cells, which results in advancing disease.

CLL cells are reported to express IgD and IgM simultaneously. However, the levels expressed in these cells are lower than those reported in normal B cells. BCR signaling is triggered when surface immunoglobulins crosslink and phosphorylate the immunoreceptor tyrosine-based activation motifs present on CD79A and CD79B molecules. An operational BCR is needed for the subsistence of mature B cells and is upheld in most established B cell cancers such as CLL. It is presumed that the surface immunoglobulin of CLL B cells is occupied by autoantigens that cause constitutive BCR signaling. The value of this association is highlighted by the medical success of kinase inhibitors that impede BCR signaling. CLL is wide-ranging hence the consequence of BCR signaling varies from heightened B cell stimulation to B cell anergy. Augmented B cell stimulation is pronounced in U-CLL, whereas anergy prevails in most instances of CLL that produce mutated IGHV (M-CLL).

Consequently, the original cell that gives rise to CLL may determine the fate cell (activation as opposed to anergy) because cell types that give rise to CLL vary in their display of DNA methylation and responses to autoantigen. There is little information regarding BCR signaling that causes anergy, which is associated with various outcomes and disease progression in the affected individuals. Furthermore, the development of effective therapeutic agents for the management of CLL requires a detailed understanding of the role of each molecule that is expressed in the disease. The medical progression of the CLL is highly unpredictable, a phenomenon that has directed scientific investigations of the disease towards the identification of prognosis markers. CLL cells cannot grow in a culture in spite of their ability to build up in the blood. Consequently, there is a need to understand the downstream series of reactions involved in the development of CLL cells to direct the rational development of curative agents that control the pathways. Therefore, extensive research on the impact of IgM in the development, course, and prognosis of CLL patients is warranted. A thorough understanding of the role of these molecules in CLL would help to identify the inherent risk posed by the disease, hence facilitating the timely and effective treatment of the disorder.

Literature Review

CLL is characterized by the gradual buildup of cancerous monoclonal B lymphocytes in blood as well as primary and secondary lymphoid organs. Diseased cells look like developed B cells that express IgM and IgD on their exterior. CLL cells show varying extents of anergy, which is associated with a decreased expression of IgM as well as a signaling ability. Elevated BCR signaling following the activation of IgM and IgD is related to poor prognosis and advanced disease. Nonetheless, there is limited information regarding the functional disparities between these two types of CLL and their joint role in the disease.

Prejudiced usage of the IGHV in CLL highlights the value of antigen stimulation. Unmutated CLL (U-CLL) lean towards expressing autoreactive BCRs with a low affinity and multiple reactivities. Conversely, monoreactive BCRs with a high affinity are usually found in mutated CLL (M-CLL). Independent BCR signaling is a distinguishing trait in CLL. Studies involving transgenic murine CLL models have proposed that cell autonomy and outside diminished affinity for BCR associations play a role in the pathogenesis of CLL. This phenomenon has led to the development of disrupters of the BCR pathway, particularly the kinase enzymes, as CLL treatments. These agents have demonstrated immense clinical success, which is attributed to lymphocytosis brought about by the redeployment of diseased cells from their site of accumulation (lymphoid organs) into the periphery. This movement is founded on the opposition to relocation and preservation signals. As a result, chemokine-influenced migration and integrin stimulation of CLL cells were prevented by going for Syk and Btk. These findings indicate that blocking the interaction between CLL and protective signals found in their immediate microenvironment plays a role in the effectiveness of these treatment agents. The specific receptors that are involved in the movement and survival of CLLLs include chemokine receptors CXCR4, CXCR5, CCR7, CXCL12, and CCRL2 (2).

The expression of CXCR3 in CLL differs from one patient to another. Elevated levels of CXCR3 are correlated with a slow form of the disease. The reduced expression of CXCR4 and CXCR5 is common in high-risk instances following activation with arrested α-IgM antibodies. On the other hand, the effect of IgD stimulation on the expression prototype of CXCR4 and CXCR5 has not been assessed.

Estimations show that CLL comprised approximately 19,000 of all freshly diagnosed malignancies in the United States in 2016 (1). Men were more likely to develop the condition compared to women. Additionally, the likelihood of having CLL increased as one advanced in age. Various factors contribute to the predisposition to CLL, for example, genetics, environmental exposure, and immune status.

The influence of genetics on the development of CLL is indicated by the fact that a significant proportion of CLL patients (approximately 9%) have a close family relation with the disease. Additionally, having a first-degree relative with CLL increases one’s risk of developing the disease by 850% (1). The incidence of CLL is pronounced in monozygotic twins compared to dizygotic twins. Findings from genome-wide association studies indicate that single nucleotide polymorphisms in about 30 loci are linked to hereditary CLL, which shows that shared genetic variations play a part in the transmissible risk (3).

Distorted expression of genes that are found in or in the vicinity of CLL-linked SNPs can influence the development of the disease. For instance, SNPs in LEF1 could cause the increased expression of lymphoid enhancer-binding factor 1 that amplifies WNT signaling. It has been shown that there is an overexpression of LEF1 in CLL, which is associated with defiance of apoptosis. Furthermore, patients with CLL often have SNPs in the BCL2 gene. This gene codes for an anti-apoptotic protein that is produced in large quantities in the disease. On the other hand, SNPs that lower the expression of mir-15a and mir-16-1 are linked with hereditary CLL due to the impact of mir-15a and mir-16-1 in inhibiting the expression of the miRNAs BCL2 and ZAP70 (4). Low expression of these miRNAs leads to high expression of these genes, which code for proteins that augment the defiance to apoptotic signals or increase BCR signaling.

Making contact with Agent Orange is a known risk factor in the development of CLL. As a result, veterans with CLL who were exposed to the substance during their service in the military have been allowed by the US Department of Veterans Affairs to ask for benefits (5). Environmental exposure to insecticides and ionizing radiations are also thought to increase one’s risk of developing the disease. However, there is no evidence to show that dietary and lifestyle attributes play a role in CLL risk.

The development of CLL is associated with several genetic changes such as chromosomal modifications, transmutations, epigenetic alterations, as well as shifts in the expression of miRNAs. About 80% of CLL patients have one out of four widespread chromosomal changes such as obliterations in chromosome 13q, which manifest as del(13q), del(17p) or del(11q), and trisomy 12 (1). Del(13q) is prevalent in more than half of CLL patients and is associated with good prognosis. One crucial attribute that is found in the obliterated regions is the DLEU2-mir-15–16 cluster whose role is to control the production of proteins that hinder apoptosis or interfere with the advancement of the cell cycle. About 7% of CLL patients have del(17p), which interferes with the functions of the tumor suppressor gene (TP53). On the other hand, del(11q) is linked to changes in the ATM gene whose purpose is to produce proteins that fix DNA damage. These chromosomal changes lead to dire clinical upshots. About 16% of CLL patients may have trisomy 12, which has an intermediary prognosis.

CLL holds a high level of genetic variation. It has been shown that recurrent somatic mutations are prevalent in genes that function in DNA damage such as ATM and TP53, those involved in the processing of mRNA processing such as XPO1 and SF3B1, chromatin amendment such as CHD2, HIST1H1E, and ZMYM3, WNT and NOTCH1 signaling, and inflammatory pathways. Mutations in EGR2 or BRAF get in the way of B cell-associated signaling and transcription.

The practical role of a number of alleged driver mutations has been verified. For instance, stopping the genes involved in the WNT pathway in CLL cells has been shown to reduce the viability of the cells (6). POT1 serves in the safeguarding of telomeres. Changes in this gene have been shown to hinder the attachment of protective telomeric proteins to telomeric DNA, which caused chromosomal aberrances and anomalous telomeres. Additionally, changes in SF3B1 are connected with atypical RNA splicing and an impaired response to DNA damage. The identification of somatic mutations and their comparative incidences differs from patient to patient.

CLL is associated with changes in miRNA, particularly mir-15a and mir-16-1, which may be eliminated or downregulated in about 60% of patients. The two miRNAs work on BCL2 and MCL1, which code for the production of anti-apoptotic proteins belonging to the BCL-2 family. Low production or elimination of these miRNAs leads to increased production of the target genes. Specific miRNAs are also dysregulated or differentially in specific subgroups of CLL, leading to unique clinical characteristics. The elimination of these miRNAs or their reduced expression promotes the production of TCL1A, which encourages the advancement of CLL in transgenic mice when constitutively expressed in established B cells. In contrast, the heightened expression of mir-155 is connected to improved BCR signaling, B cell propagation, and formation of lymphomas.

Epigenetic modifications that take place in the CLL epigenome include global hypomethylation alongside local hypermethylation, which has also been observed in other types of cancers. Increasing the heterogeneity of methylation is linked to enhanced genetic intricacy due to the attainment of subclonal transmutations, which connects genomic and methylomic progression in CLL. Methylation patterns lead to the classification of discrete clinical CLL subclasses. Methylation arrays are transmissible traits hence they can be used to identify the normal B cell that gave rise to the CLL. It has been noted that CLL cells of distinct patients arise from a range of B cell maturing states that are not confined to specific maturation conditions. However, U-CLL and M-CLL have characteristic methylation arrays that have some similarity to those of pre-germinal and post-germinal centers respectively. The assortment of possible origins of CLL cells underscores the genetic and phenotypic variations in this disorder. These findings also indicate that epigenetic programming based on transcription factors could play a vital role in the advancement of CLL.

Immune deficiency is a consequence of CLL and is attributed to the advancement of hypogammaglobulinemia resulting in an increased risk of infection. IL-10, an immunosuppressive factor obtained from T cells, is thought to play a role in this mechanism. CLL cells are hypothesized to produce IL-10 (1). Additionally, M-CLL is thought to produce larger quantities of IL-10 than U-CLL. Then again, the presence of malignant cells can also influence the expression of IL-10. Cancer cells are usually higher in patients with U-CLL compared to M-CLL, which may explain the immune deficiency observed in patients with M-CLL. Another plausible explanation for decreased immunity in CLL is that diseased cells produce large quantities of programmed cell death ligands (PD-L1 and PD-L2), which overwhelm the effector rejoinders T cells that produce programmed cell death protein 1 (PD-1) hence resulting in a compromised cellular immune function. The literature review shows that CLL is diverse and that different interactions lead to varying consequences in term of disease manifestation and prognosis. Therefore, there is a need to clarify the precise effect of IgM in CLL as a step towards a comprehensive understanding of the disease.

Hypotheses

The study was founded on two null hypotheses. The first hypothesis was that there is no relationship between IgM levels and CLL characteristics. The second hypothesis was that the relationship between IgM and CLL attributes did not affect the treatment of CLL.

Objectives of the Study

To achieve the overall purpose of the study, two main objectives were set. The first objective of the study was to correlate the production and characteristics of IgM and the incidence and prognosis of CLL. The second objective of the study was to determine the impact of IgM on the diagnosis, classification, and management of CLL.

Materials and Methods

The study employed quantitative research designs and built on background information using primary sources from reputable databases. The first step in the study was to identify the connection between CLL and IgM to determine the prognosis of patients with the disorder. The next approach was to review recent guidelines on the identification of risk and treatment of CLL. Therefore, the study delved deeper to understand the correlation between IgM and CLL and how this relationship affected the management of the disease. Three databases were used to search scholarly sources on the topic. They included PubMed, Google Scholar, and EbscoHost. The search based on predetermined keywords retrieved numerous articles, which were further reviewed based on their relevance to the objectives of the study.

Three articles were chosen, two focusing on the correlation between IgM and CLL and one about the diagnosis, risk classification, and treatment of CLL. The decision to use the three articles was guided by the exhaustiveness and reliability of the methods used to compile the data in the articles. Apart from the three primary sources that were used to determine the correlation of CLL and IgM with respect to risk stratification and management of CLL, other peer reviewed articles were used to support the findings of the study.

Different quantitative methods to determine the intricate biochemical relationships between IgM and CLL. For example, flow cytometry was used to determine the clonality of the cells. RT-PCR was used to distinguish between different types of CD79b. RT-PCR helps to amplify DNA and is used alongside DNA sequencing to study part of the genome. Such an approach was necessary given the influence of genetics on the development of CLL. Western blotting was used to characterize B-CLL cells, whereas two-dimensional electrophoresis was used to separate different protein components. Interphase fluorescence in situ hybridization (FISH) was used to identify chromosomal defects, whereas amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) and Sanger sequencing were employed in the identification of mutations (7).

The data derived from the studies were then analyzed using different statistical tools as required. For example, the expression levels of sIgM in different categories of CLL were conveyed as means ± the standard error of means (8). Comparisons between categorical variables were accomplished using the x² or the Fisher exact tests as appropriate. The Mann Whitney nonparametric test was used to make comparisons between continuous variables. The Kaplan-Meier method with the aid of log-rank statistics was used to conduct survival studies. All statistical analyses were conducted at α= 0.05 level of significance. The software used in the analyses included the Statistical Package for the Social Sciences (SPSS) and Graphpad Prism.

Results

Study 1: The Correlation Between IgM and CLL

Article 1

Nédellec, S., Renaudineau, Y., Bordron, A., Berthou, C., Porakishvili, N., Lydyard, P.M., Pers, J.O. and Youinou, P., 2005. B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients. The Journal of Immunology, 174 : 3749-3756.

Nédellec et al. (7) used surface IgM (sIgM) cross-linking to classify a group of 41 patients with CLL as non-responders (group I) or responders (group II). Responders were further grouped into induced proliferation (subgroup IIa with 7 patients) and apoptosis groups (subgroup IIb with 19 patients). It was noted that the cross-linking of sIgM on B-CLL cells resulted in no effect, proliferation, or apoptosis. Following the hypothesis that patients’ heterogeneity could elucidate the fate of B cell after sIgM cross-linking, the reactions to anti-µ were quantified in the subjects leading to the above classifications (group I and group II). Anti-µ did not influence apoptosis. Group II exhibited two conflicting changes: the proportion of AnV-binding cells reduced in subgroup IIa and increased in subgroup IIb. The decline in sIgM expression was more apparent in group I than the rest of the groups. The AnV data were corroborated by nuclear compression, DNA disintegration, and hypoploidy of the lymphoid cells in 15 patients selected arbitrarily.

Group I patients did not show Ca²⁺ even though ionomycin assembled Ca²⁺ as normal. Conversely, subgroups IIa and IIb showed an upturn in intracellular Ca²⁺. Ag treatment triggered a persistent Ca²⁺ increase in group II. However, Ca²⁺ was activated fast but momentarily. The proportion of Ki-67-expressing B cells increased following activation by sIgM. Additionally, DNA flow outlines for subgroup IIa indicated that the cells were in the S phase of the cell cycle. This observation was linked to Ki-67 positivity at the end of 24 hours.

BCR signaling controlled the fate of B-CLL cells as demonstrated by the blocking of transduction factors. Obstruction of PI3K in subgroups IIa and IIb cells prohibited the initiation of Akt hence preventing anti-µ-influenced propagation of subgroup IIa cells. However, this blockade did not affect the antiapoptotic machinery of subgroups IIa and IIb cells. Inhibition studies revealed that the obstruction of PKC with bisindolylmaleimide stopped propagation and caused sIgM-prompted apoptosis in the cells of subgroup IIa. On the other hand, apoptosis was heightened in subgroup IIb cells, which showed that ERK was triggered in a PKC-dependent fashion and that PKC instinctively wields its antiapoptotic actions. Zap70 moved into LR following sIgM involvement showing that Zap70 played a part in BCR signal transduction.

Different patterns of reactions attributed to sIgM cross-linking were correlated with biological prognosis qualities and clinical stages of CLL. The expression of CD38 was linked to positive responses to sIgM cross-linking. Therefore, at least 30% of B cells in more subgroup IIb patients expressed CD38. The expression of TR-CD79b was lower in subgroups IIa and IIb as opposed to group I leukemia cells. The manifestation of Zap70 was confined to B cells from subgroup IIb subjects, which matched sequencing records from IgVH genes. The observed biological attributes matched the clinical stages of CLL. Group I comprised 11 and 4 patients from grade A and B respectively, whereas subgroup IIa had 2, 4, and 1 patients from grade A, B, and C in that order. On the other hand, subgroup IIb had 3, 10, and 6 grade A, B, and C patients correspondingly.

Article 2

D’Avola, A., Drennan, S., Tracy, I., Henderson, I., Chiecchio, L., Larrayoz, M., Rose-Zerilli, M., Strefford, J., Plass, C., Johnson, P.W. and Steele, A.J., 2016. Surface IgM expression and function associate with clinical behavior, genetic abnormalities and DNA methylation in CLL. Blood, 128 : 816-826.

D’Avola et al. (8) examined the relationship between IgM expression and clinical behavior, genetic aberrances, and DNA methylation in patients with CLL. The expression of sIgM was assessed in the form of the intracellular mobilization of calcium ions following stimulation. The quantities of sIgM levels and their signaling capability were examined in 270 CLL patients. These parameters varied across the subjects. There was a significant correlation between sIgM concentrations and signaling ability. sIgM levels exhibited minimal variation in U-CLL compared to M-CLL groups. However, the total concentrations were markedly higher in U-CLL than in M-CLL. Signaling ability also differed significantly between the two groups. The subclass of CLL using IGHV3-21showed elevated sIgM concentrations and signaling capacity compared to non-IGHV3-21 M-CLL notwithstanding the IGHV3-21 mutational status. IGHV3-21 is associated with a belligerent disease progression. M-CLL has many cases without detectable signaling capacities, which were related to very low expression, thereby showing a deeply anergic group that was absent in U-CLL. The quantities and signaling capacity of sIgD levels showed minimal variation and did not differ between the two subgroups of CLL. Additionally, the observed variations were not related to sIgM levels.

The levels of functional sIgM forecasted the advancement of CLL. High sIgM expression and sIgM signaling were linked to a rapid progression of the disease. A multivariate Cox regression corrected for sIgM concentrations, IGHV status, and sIgM signaling showed that sIgM levels and signaling forecasted the advancement of CLL in a manner that was unrelated to the IGHV status. A separate analysis of sIgM within U-CLL or M-CLL indicated that elevated levels were linked with a more destructive behavior. Elevated sIgM levels and signaling in M-CLL produced a progression that was as fast as the U-CLL with low sIgM levels and signaling. This intersection indicated that even though cell origin influenced tumor behavior significantly, other stimuli on BCR could affect clinical outcomes in a minority of cases. The analysis of sIgD did not reveal any significant differences in the survival of CLL cells with or low signaling levels.

sIgM levels and signaling were connected with low-risk genetic injuries, which were augmented in U-CLL than M-CLL. An analysis of FISH lesions indicated that that CLL subgroups with +12 or del(17p) defects expressed significantly higher levels of sIgM and signaling potential compared to those with del13q. This connection was apparent following separate analyses of U-CLL or M-CLL. NOTCH1_ΔCT was recognized in 13 patients (11 were U-CLL patients). Evaluation by ddPCR showed different allele frequencies for NOTCH1_ΔCT, which was associated with sIgM intensities and signaling capability. SF3B1 mutations were evident in 12 CLL (5 in U-CLL and 7 in M-CLL). There was no differential sIgM signaling in U-CLL, which was attributed to the presence of other genetic lesions. On the other hand, the samples with SF3B1 mutations in

M-CLL showed higher sIgM levels and signaling compared with those with single del13q or other mutations.

DNA methylation maturation status (MMS) demonstrated an inverse association with sIgM levels in M-CLL (8). The DNA MMS is a representation of the CLL epigenetic development and was markedly lower in U-CLL, which was in line with the findings of previous investigations. CLL with higher sIgM amounts had lower MMS than those with low sIgM signaling capacity. The analysis of U-CLL only did not show any significant correlations between sIgM levels and MMS. The M-CLL, on the other hand, showed an outstandingly high correlation between MMS and a decrease in sIgM levels or aptitude. Most of these M-CLL did not have any genetic lesions.

The levels of sIgM could serve as an autonomous prognostic factor of time to progression requiring first treatment (TTFT) in CLL. The findings demonstrated that elevated sIgM levels were correlated with a fast TTFT, advanced stage, shorter time to lymphocyte doubling (LDT) and shorter overall survival (OS). TTFT was used as an indication of the possible function of sIgM as a clinical predictive stricture against recognized phenotypic, hereditary or methylation-risk classes. A univariate analysis amended for elevated sIgM levels and phenotypic pointers CD38 and ZAP-70 showed that raised sIgM levels were reliable prognostic strictures of short TTFT (8). On the other hand, adjusting for elevated sIgM quantities, U-IGHV status, and heritable factors showed that increased sIgM levels forecasted TTFT without the influence of genetics. Clinical associations with DNA methylation status were conducted against 3 distinct CLL clusters namely low-programmed CLL (LP-CLL), intermediate-programmed CLL (IP-CLL), and high-programmed CLLs (HP-CLL). All LP-CLL subjects belonged to U-CLL and were sIgM signalers while M-CLL consisted of IP-CLL or HP-CLL and had high or low sIgM signaling. It was also noted that high sIgM and IP-CLL standing envisaged the advancement of the disease.

Study 2: The Impact of IgM on the Diagnosis, Classification, and Management of CLL.

Article 3

Hallek, M., 2017. Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment. American Journal of Hematology, 92 :946-965.

Hallek (9) updated clinical guidelines on the identification, risk classification, and management of CLL. CLL is exemplified by the clonal propagation and buildup of developed, CD5-positive B cells in the bone marrow, blood, spleen, and lymph nodes. A recent report shows that the ability to produce clonal cells in CLL is developed in the hematopoietic stem cell (HSC) phase, which signifies that the main leukemogenic occurrence in CLL engages multipotent, self-renewing HSCs. Findings of the genomic environment of CLL in large groups indicate that the disorder may be initiated by the deficit or inclusion of sizeable chromosomal material (for example, deletion 11q, deletion 13q, and trisomy 12) followed by extra mutations that increase the aggressiveness of the disorder. Obliterations on the long arm of chromosome 13, particularly implicating band 13q14, which are denoted as del(13q14), correspond to the most common cytogenetic abnormality in CLL. This deletion is observed in approximately 55% of all CLL cases.

A single del(13q14) is exemplified by a nonthreatening course of the illness. It has been shown that two miRNAs (miR-15a and 16–1) are situated in the dangerous region of del(13q14). Erasures of the long limb of chromosome 11, which are referred to as del(11q) are noted in about 25% of CLL with advanced disease who have not yet undergone chemotherapy as well as a tenth of patients in the initial stages of the disease. Most of these obliterations usually involve band 11q23, which holds the gene ATM that codes for kinase ATM, an enzyme that corrects proximal DNA mutilation. Moreover, patients with del(11q) often display a large lymphadenopathy, quick advancement of the disease and a lower overall survival than patients without this deletion. However, a few of the poor predictive attributes of del(11q) can be circumvented using chemoimmunotherapy. About 10 to 20% of CLL patients show trisomy 12. Nonetheless, the genes that take part in the pathogenesis of CLL with trisomy 12 are not known.

On the other hand, omissions of the short limb of chromosome 17, which are known as del(17p), are present in approximately 5%-8% of CLL patients who have not undergone chemotherapy-naïve patients. These omissions must involve band 17p13 that holds TP53, a well-known tumor suppressor gene. Patients with del(17p) clone exhibit pronounced resistance against genotoxic chemotherapies, which cannot be circumvented by the inclusion of anti-CD20 antibodies in contemporary chemoimmunotherapy. Changes in TP53 are observed in 4% to 37% of patients with CLL. These changes are linked with a very poor prognosis in many studies. It has also been observed that most patients with verified del(17p) have transmutations in the rest of the TP53 allele. Consequently, mutations in TP53 are rare in instances where del(17p) is absent. However, these cases also have adverse effects on response to treatment and overall survival. TP53 mutations have been linked to increased genomic convolution in CLL, showing that a malfunctional DDR encourages a “mutator phenotype” in CLL.

The survival of CLL cells relies on an accommodating microenvironment that consists of cellular elements such as T cells, macrophages, or stromal follicular dendritic cells. These elements provide incentives for the detection of vital subsistence and pro-proliferative signaling pathways in altered cells. This setting supplies a number of essential proteins such as cytokines, chemokines, and angiogenic factors, which associate with leukemic cells through apposite adhesion molecules or surface receptors to promote the subsistence of CLL cells. Some of the novel treatment agents wield their pharmacological effects by altering the microenvironment of CLL cells. Therefore, the management of CLL is currently going through vital changes that began about a quarter a century ago.

The diagnosis of CLL is ascertained by tests such as blood counts, blood smears, as well as immunophenotyping of moving B lymphocytes. Moving lymphocytes identify a clonal B-cell population containing the CD5 antigen in addition to B-cell markers. The identification of CLL is done when there are 5000 or more B lymphocytes per milliliter of peripheral blood for at least 3 months. Flow cytometry is used to ascertain the clonality of the circulating B lymphocytes. Blood smears in CLL often show tiny, mature lymphocytes with a thin margin of cytoplasm and a compact nucleus without visible nucleoli and incompletely aggregated chromatin as indicated in Figure 3.

These cells may be seen together with larger or uncharacteristic cells, severed cells, or prolymphocytes that may form approximately 55% of the blood lymphocytes. The presence of more than 55% of prolymphocytes is an indication of prolymphocytic leukemia (B-cell PLL). Other distinguishing features in CLL include Gumprecht nuclear shadows, which are also referred to as smudge cells. These cells are usually found in the form of cell debris.

The staging of CLL uses two prognostic systems known as Rai and Binet staging systems (9). These staging systems are determined by conducting a physical exam and the findings of a blood count. The amended Rai staging system outlines low-risk CLL as patients displaying lymphocytosis with leukemia cells in the blood or marrow. The lymphoid cells should exceed 30%. This stage was originally known as Rai stage 0. Patients displaying lymphocytosis, engorged nodes, enlarged spleen or liver can be said to have an intermediate-risk disease, which was initially referred to as Rai stage I or II. High-risk disease occurs when patients have disease-related anemia marked by hemoglobin levels that are lower than 11 grams per deciliter (previously stage III). The incidence of thrombocytopenia, which is described by a platelet count of less than 100× 10⁹/L, was previously known as stage IV. A summary of the Rai staging system is indicated in Table 1.

The Binet staging system relies on the number of involved areas, which is indicated by the presence of distended lymph nodes whose diameters exceed 1 cm, enlarged organs, or the presence of anemia or thrombocytopenia. The body areas that are considered during the assessment include the head and neck, the Waldeyer ring, axillae, groins, palpable spleen, and a clinically enlarged liver. According to the Binet staging system, stage A CLL is highlighted by as hemoglobin levels equal to or greater than 10 g/dL and platelets equal to or greater than 100× 10⁹/L. On the other hand, stage B has similar conditions in addition to organomegaly that exceeds that of stage A with 3 or more regions of enlarged nodes or organs. Stage C has hemoglobin levels of less than 10 g/dL or a platelet count that is lower than 100× 10⁹/L. Details about the Binet staging system are shown in Table 2.

Different biological and heritable markers also have prognostic importance. For instance, obliterations of the short limb of chromosome 17 (del(17p)) as well as changes in the TP53 gene foretell resistance to available chemotherapies. Due to the current progress in CLL therapy, the first two clinical staging systems have become inadequate to differentiate three or more extrapolative subgroups. As a result, a more pertinent prognostic score has been formulated by an international conglomerate of study groups. This system is known as the CLL International Prognostic Index (CLL-IPI) and it uses inherited, biological, and medical variables to categorize CLL into very diverse risk groups. The CLL-IPI classes are indicated in Table 1.

The CLL-IPI provides a practical approach to treatment based on the symptoms manifested. Asymptomatic patients or those in the initial stages of the disease do not require any form of treatment. The decision to initiate treatment is influenced by the presence of active or symptomatic disease. It is recommended that patients in the early stages of the disease without any apparent symptoms should be observed without any treatment unless there is evidence of fast disease advancement. This decision is informed by the lack of evidence demonstrating the advantages of early therapeutic interventions.

Treatment can only be started when the patient presents with active disease.

Conditions that highlight active disease include gradual marrow failure as shown by anemia or thrombocytopenia, particularly in stages II and IV Rai or Binet stage C. Other symptoms include splenomegaly, symptomatic lymphadenopathy, gradual lymphocytosis, lymphocyte doubling time of fewer than 6 months, autoimmune anemia, unresponsive thrombocytopenia, disease-related indications such as unexplained weight loss over the last 6 months, fatigue, or fevers and chills devoid of other signs of infection for more than 30 days.

Monotherapy using alkylating agents has been used as the first front-line therapy for CLL. Chlorambucil has been used as the “gold standard” treatment for a long time. Its benefits include low levels of toxicity, cost-effectiveness, and expediency as an oral drug. However, its shortcomings include low to missing CR rate and certain side effects that arise with protracted use, for example, secondary acute leukemia, continued cytopenia, and myelodysplasia. These shortcomings have led to the development of combination treatments such as chlorambucil with anti-CD20 antibodies, which has demonstrated higher levels of efficiency. Consequently, the use of chlorambucil is restricted to cases involving weak, elderly patients, where treatment with a cheap cytostatic drug is the focus of therapy.

Three purine analogs used in CLL treatment include fludarabine, pentostatin, and cladribine. Monoclonal antibodies such as anti-CD20 antibodies (Rituximab, Ofatumumab, and Obinutuzumab), other monoclonal antibodies such as Alemtuzumab are also used (9). Agents directed at B-cell receptor signaling are also employed in treatment because B-cell receptor signaling is thought to play a crucial role in the survival of CLL cells. These agents include Idelalisib, Ibrutinib, and Acalabrutinib. BCL-2 inhibitors are also employed in treatment due to the involvement of proteins belonging to the B-cell lymphoma 2 family in the modulation of the apoptotic process. The main drug in this category is Venetoclax (ABT-199), which hampers the development of BCL-2-dependent tumors without affecting human platelets. One oral dose of the drug has been shown to cause tumor lysis within 24 hours in 3 patients with refractory CLL. Other chemotherapeutic agents include immunomodulatory drugs such as Lenalidomide, which has produced satisfactory outcomes as a maintenance treatment in patients with high risk CLL.

Notable progress in CLL treatment has been accomplished by the combined use of various treatment methods. Purine equivalents and alkylating agents have dissimilar modes of action and partly nonoverlapping toxicity outlines, which makes it logical to combine these two approaches to attain synergy. Chemoimmunotherapy is also used in the treatment of CLL. Several combination treatments have been tested, for example, combinations using rituximab, ofatumumab, obinutuzumab, alemtuzumab and other targeted agents (9).

Going by the numerous treatment choices in CLL, selecting an optimal therapeutic plan for patients can become a daunting task that requires experience, sound clinical judgment, and the correct use of diagnostic tools. Overall, a clinician should consider several parameters before initiating treatment. They include the clinical phase of the disorder, symptoms presented by the patient, the fitness and coexistent ailments of the patient, the heritable risk of leukemia, and the treatment state such as response or nonresponse to the previous treatment.

Discussion

The main purpose of this study was to determine the correlation between CLL and IgM and its impact on the prognosis and management of CLL. The findings reported in the previous section refute the null hypotheses that there was no relationship between IgM levels and CLL characteristics and that the relationship between IgM and CLL attributes did not affect the treatment of CLL. Nédellec et al. (7) sought to elucidate the role of sIgM in regulating unprompted apoptosis in B-CLL cells and relating differential reactions to anti-µ with recognized predictive features. The study sample was grouped into two main groups according to resistance or sensitivity. The extent of unprompted apoptosis was the same in both groups, which raised the question as to how the same type of interaction between a receptor and ligand result in two opposite effects (proliferation in one group and apoptosis in the other category).

The findings of this study help to solve the inconsistency between reports regarding the responses of B cells from diverse B-CLL patients to anti-µ where it was alleged to extend their survival (10) or to fast-track their apoptosis. There is a likelihood that the group whose survival was prolonged corresponded to group IIa in this study, whereas those whose apoptosis was quickened matched group IIb. The functional divergence observed in group II patients also corresponded to the Ca²⁺ flux data given that the rejoinder was modest in proliferation and dynamic in apoptosis. The area of little variation was attributed to an extended release of intracellular Ca²⁺ as opposed to an ensuing Ca²⁺ inflow because the cells were grown in a Ca²⁺-free medium. Ca²⁺ mobilization outlines similar to those reported by Nédellec et al. (7) in B-CLL cells that demonstrated varying responses following anti-µ treatment have been reported in previous investigations. However, these studies did not link the observed behavior to proliferation or apoptosis.

Disparities in the relationships of stimulated kinases clarify the described heterogeneity. Nédellec et al. (7) limited the number of kinases, which led to the distinction of subgroups within group II of responding B-CLL. The study ascertained the constitutive activation of PI3K by showing that its inhibition permitted apoptosis. This kinase plays a vital role in the growth and development of B cells. For example, it promotes their survival and growth as demonstrated by wortmannin deterrence of their anti-µ-induced propagation. A number of molecules including Akt usually preclude the apoptosis of B cells. However, Akt is not phosphorylated in resting B cells. Nonetheless, the ligation of sIgM to the molecule leads to serine phosphorylation thereby controlling BCR-mediated propagation. ERK is also stimulated when bisindolylmaleimide is available and may play a role in forestalling apoptosis in B-CLL. The stimulation of ERK alongside the deactivation of p38 appear vital to the survival of B-CLL. Therefore, the proliferative rejoinder of B cells to the cross-linking of sIgM in subgroup IIa patients was probably because of the elevated activity of ERK as opposed to p38.

On the other hand, the involvement of sIgM was a precondition for the phosphorylation of PI3K in subgroup IIa. Therefore, a greater activity of p38 compared to ERK promoted the apoptotic reaction. Such disrupted modulation may arise from prolonged stimulation as shown by the sustenance of Ca²⁺ fluxes. The upshot of signal transduction via the BCR is influenced by its magnitude and extent. Zap70 can augment BCR’s ability to signal based on its connection with high BCR signal transduction in B-CLL. The findings of this study also show that Zap70 protein is useful in subgroup IIb cells because it goes through tyrosine phosphorylation and moves into the LR as a result of sIgM ligation. Curtailed signaling via the BCR is linked to reduced CD38 populations, transmutations in the IgVH genes, failed expression of Zap70, and favored utilization of TR-CD79b. B-CLL instances with undamaged BCR-signaling pathways can communicate effectively the signals required for propagation or programmed cell death. Subgroup IIb cells that undergo programmed cell death hold high densities of CD38, do not have mutations in IgVH genes, produce Zap70, and utilize FLCD79b preferentially (7).

Interestingly, subgroup IIa does not include patients with bad prognostic traits even though the signal transduction pathway is operational. These cells do not undergo apoptosis but instead propagate following ligation with sIgM. Therefore, the same ligand-receptor contact can stimulate contrasting upshots of B cell action on costimulatory signals and the differentiation phases of the cells. The cells from subgroup IIa patients that multiply were more segregated than those from the subgroup IIb that undergo apoptosis. This supposition is harmonious with the fact that most transformed B-CLL do not signal via sIgM even though their prognosis is better than that of unchanged B-CLL. Additionally, Zap70, which is linked to a poorer prognosis, augments sIgM signaling in B-CLL. These facts support the observation that the prognosis was better in the signal-resisting compared to the signal-sensitive group of patients. Future studies should look into the survival and response to treatment in the signal resisting and signal-sensitive patients.

The study conducted by D’Avola et al. (8) elucidates the critical role of anergy in CLL. The findings show that the extent of anergy working on sIgM (and not sIgD) has a strong relationship with slow disease advancement in CLL patients. The reduction of the expression of sIgM in tumor cells provides evidence regarding the dealings of CLL cells with assumed (super)antigen in vivo. This interaction can be reversed in vitro in the course of circulation after an engagement in different tissues. Healthy people have high levels of sIgM in B cells compared to those observed in CLL. Additionally, the levels of sIgM in memory B cells are higher than those seen in naïve B cells (8). However, these quantities are not reflected in the leukemic cells M-CLL and U-CLL. This observation is accounted for by the fact that the expression of sIgM in CLL is controlled by various events in vivo. The major disconcertion is anergy prompted by antigens, which is present in both subsets but is prevalent in M-CLL. Anergy is the probable cause of the differences in disease behavior between the two subsets.

Nonetheless, the extent of anergy varies between the two subsets and is broad in M-CLL. A further look at the variability in M-CLL shows that the level of sIgM plays an important role in disease progression. Genetic damage also influences the course of the disease. This study shows that sIgM levels or signaling vary across different genetic clusters in CLL, which insinuates that inherent tumor-related factors also contribute to the prognosis of the disease. This phenomenon is pronounced in U-CLL where genetic abrasions with poor prognosis such as +12, del(17p) and NOTCH1_ΔCT correlate with elevated levels of sIgM. CLL cells with mutations that lead to the loss of TP53 and NOTCH1_ΔCT have endurance and propagation advantages over those devoid of these lesions. The +12 translocation is also linked with increased quantities and signaling of integrins such as CD49d and CD38 (8). The findings of this study demonstrate a connection between sIgM and CD49d or CD38 levels in the U-CLL and M-CLL subtypes.

The global DNA methylation standing of CLL cells has a close connection with the origin of B cells. This phenomenon is preserved to a large extent post transformation in the course of disease progression. The extent of DNA methylation maturity in CLL, which can be determined by corresponding changes taking place in normal B-cell development, can expediently split CLL into 3 subgroups within U-CLL and M-CLL. These subgroups include LP-CLL, IP-CLL, and HP-CLL.

M-CLLs consisting of H-CLL and IP-CLL have the highest levels of DNA methylation variability. Evaluation of the corresponding sIgM levels shows a significant inverse association between the expression of sIgM and methylation maturation in M-CLL. Seemingly, cells obtained from more developed B cells within M-CLL are more predisposed to the initiation of anergy, which could be inherent to the normal equivalent of memory-like B cells. Then again, the initiation of anergy could be attributed to the nature of the autoantigens.

D’Avola et al. (8) confirmed the connection between DNA methylation maturity and a good prognosis, which had been reported in previous studies (11). This observation was indicated by the marked increase of TTFT in HP-CLLs as opposed to IP-CLLs. It was evident that low expression of sIgM and elevated MMS in M-CLL resulted in a slow advancement of the disease. Therefore, this study points out the relevance of sIgM in the clinical progression of CLL, which informs the treatment options. In the study population, 55% of the patients needed chemoimmunotherapy. About 16% of the needy patients received inhibitors of the BCR pathway-related kinases, which increased their chances of survival. Consequently, the TTFT was used as a measure of disease advancement. An analysis of sIgM levels with recognized phenotypic, hereditary, or methylation extrapolative indicators of progression showed the autonomous role of sIgM levels. These three studies indicate that the production of IgM in CLL is correlated with the course of the disorder. Therefore, sIgM is a potential marker that can be used for the identification of CLL with belligerent behavior. However, there is a need for standardization and validation studies in various cohorts before sIgM levels can be used routinely as a prognostic marker in clinical scenarios.

Conclusion

The management of CLL is complicated by the lack of reliable markers to forecast the course of the disease in affected patients. The reviewed studies indicate that sIgM levels are elevated in specific groups of CLL patients and that this elevation is associated with aggressive outcomes. Therefore, sIgM is a valuable marker of tumor cell origin and behavior. It has been shown that sIgM levels and roles are reflections of the acute factors operating on CLL in vivo, for instance, genetic damage. IgM is a significant indicator of associations with antigen. Another valuable insight noted in the study is the impact of BCR cross-linking on CLL prognosis. Insensitivity facilitated by BCR cross-linking delineates patients with slow B-CLL, whereas the tendency to apoptose instead of propagating distinguishes patients with acute disease. Therefore, the reliability of the various signaling pathways can help in foretelling this upshot.

The current understanding of the mechanisms of CLL has provided headway in the treatment of the disease. New pharmacological agents have been developed, which have the capacity to boost the outcomes of CLL patients. It is expected that the simultaneous use of targeted and nonchemotherapeutic remedies will provide profound, molecular, and lifelong remissions. However, there is limited knowledge concerning the ideal use of these drugs in terms of the right combinations, succession, and length of treatment. Therefore, hematologists and oncologists should work together towards the longstanding management of CLL by treating affected patients in clinical trials. Future studies should also delve deeper into the BCR transduction pathways to guide the development of therapeutic agents that target BCR signaling.

Bibliography

Kipps, T.J., Stevenson, F.K., Wu, C.J., Croce, C.M., Packham, G., Wierda, W.G., O’Brien, S., Gribben, J. and Rai, K., 2017. Chronic lymphocytic leukaemia. Nature Reviews Disease Primers, 3 : 1-53.

Haerzschel, A., Catusse, J., Hutterer, E., Paunovic, M., Zirlik, K., Eibel, H., Krenn, P.W., Hartmann, T.N. and Burger, M., 2016. BCR and chemokine responses upon anti-IgM and anti-IgD stimulation in chronic lymphocytic leukaemia. Annals of Hematology, 95 : 1979-1988.

Berndt, S.I., Camp, N.J., Skibola, C.F., Vijai, J., Wang, Z., Gu, J., Nieters, A., Kelly, R.S., Smedby, K.E., Monnereau, A. and Cozen, W., 2016. Meta-analysis of genome-wide association studies discovers multiple loci for chronic lymphocytic leukemia. Nature Communications, 7 : 1-9.

Talukdar, V., Kar, A. and Majumder, P., 2016. A glimpse on chronic lymphocytic leukemia and its insights of molecular heterogeneity. IJAR, 2 : 643-652.

Mescher, C., Gilbertson, D., Randall, N.M., Tarchand, G., Tomaska, J., Baumann Kreuziger, L. and Morrison, V.A., 2018. The impact of Agent Orange exposure on prognosis and management in patients with chronic lymphocytic leukemia: a National Veteran Affairs Tumor Registry Study. Leukemia & Lymphoma, 59 : 1348-1355.

Laco, F., Low, J.L., Seow, J., Woo, T.L., Zhong, Q., Seayad, J., Liu, Z., Wei, H., Reuveny, S., Elliott, D.A. and Chai, C.L., 2015. Cardiomyocyte differentiation of pluripotent stem cells with SB203580 analogues correlates with Wnt pathway CK1 inhibition independent of p38 MAPK signaling. Journal of Molecular and Cellular Cardiology, 80 : 56-70.

Nédellec, S., Renaudineau, Y., Bordron, A., Berthou, C., Porakishvili, N., Lydyard, P.M., Pers, J.O. and Youinou, P., 2005. B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients. The Journal of Immunology, 174 : 3749-3756.

D’Avola, A., Drennan, S., Tracy, I., Henderson, I., Chiecchio, L., Larrayoz, M., Rose-Zerilli, M., Strefford, J., Plass, C., Johnson, P.W. and Steele, A.J., 2016. Surface IgM expression and function associate with clinical behavior, genetic abnormalities and DNA methylation in CLL. Blood, 128 : 816-826.

Hallek, M., 2017. Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment. American Journal of Hematology, 92 : 946-965.

Alhakeem, S.S., Sindhava, V.J., McKenna, M.K., Gachuki, B.W., Byrd, J.C., Muthusamy, N. and Bondada, S., 2015. Role of B cell receptor signaling in IL‐10 production by normal and malignant B‐1 cells. Annals of the New York Academy of Sciences, 1362 : 239-249.

Oakes, C.C., Seifert, M., Assenov, Y., Gu, L., Przekopowitz, M., Ruppert, A.S., Wang, Q., Imbusch, C.D., Serva, A., Koser, S.D. and Brocks, D., 2016. DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia. Nature Genetics, 48 : 253-264.