**2. Chronic lymphocytic leukemia**

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the western world. The disease typically occurs in elderly patients and has a highly variable clinical progression. CLL is characterized by the clonal expansion and accumulation of mature CD19+, CD5+, and CD23+ B lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs [1]. CLL cells are phenotypically similar to antigen-experienced B cells and show gene expression profiles similar to memory B cells [2]. The cellular origin of CLL is still debated, but it is assumed that CLL cells originate either from unmutated mature CD5+ B cells or CD5+CD27+ post-germinal center B-cell subsets [3]. CLL cells recirculate between peripheral blood and secondary lymphoid organs, where they proliferate in distinct areas of tissue, termed "pseudofollicles," at a daily birth rate of approximately 1–2% of the entire clone size [4]. Survival of CLL cells strictly depends on a permissive microenvironment composed of cellular components such as monocyte-derived nurse-like cells, T cells, follicular dendritic cells, mesenchymal stromal cells, and endothelial cells. Such dynamic combination of components leads to the presence of molecules such as cytokines, chemokines, and angiogenic factors. Leukemic cells take advantage of these vital proteins by interacting with them via cell-surface receptors or cell adhesion molecules to further facilitate their proliferation and survival [5, 6]. CLL cells are also characterized by an often observed defect in apoptosis which allows peripheral blood B lymphocytes to survive [7].

Autoantigens and/or autonomous mechanisms activate the BCR and its signaling cascade in secondary lymphatic tissues, playing a central pathogenic role in CLL [8]. These events result in activation of multiple downstream regulators in B cells which ultimately mediate changes in cell proliferation, survival, and migration via both transcriptional modulation and phosphorylation. BCR signaling responses in CLL cells are heterogeneous, with effective activation of only a selected set of downstream responses [9]. Another key property of BCRs is that they exhibit somatic mutations in varying amounts; importantly, the degree of mutation has been found to inform the prognosis of disease [2, 10]. Furthermore, many cases of CLL (approximately one third) are characterized by a nearly indistinguishable subset of BCRs exhibiting shared antigens. This suggests a close link between these specific molecules and CLL pathogenesis.

**75**

progress of CLL.

**2.1 Incidence and risk**

younger than 55 years of age [16].

environmental risk for developing CLL [19].

**2.2 Symptoms and diagnosis**

of more than 5 × 109

*New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia*

CLL cells usually show constitutive phosphorylation of signaling proteins which promote their proliferation and survival, leading to pathological processes. Protein phosphorylation in lymphocytes is tightly associated with the regulation of a variety of protein activities, functional regulation, and cell signaling and may thus affect initiation and/or progression of the disease. As such, protein phosphorylation may be one of the most promising targets for the discovery of novel cancer-related protein markers and in turn their application in new approaches to molecular therapy. The constitutive activation of proteins by phosphorylation presents its potential for prognostic significance, as the identification of aberrant signal transduction in leukemic cells can become a potential target for novel agents. After BCR stimulation, CLL cells have shown a tendency toward impaired phosphorylation levels. Higher basal phosphorylation levels of PLCγ2 (pY759), p44/42 MAPK (pT202/Y204), p38 MAPK (pT180/Y182), NF-κB p65 (pS529), STAT5 (pY694), and STAT6 (pY641) were detected in CLL cells compared to normal B cells, predicting their impaired function [12]. As such, these markers may represent some of the novel protein targets involved in the development of efficient therapeutics. Cancer cells with constitutive STAT3 activation have been reported to have elevated levels of cell cycle regulation and antiapoptotic proteins, leading to apoptotic resistance. Constitutive serine phosphorylation of STAT1 and STAT3 has also been reported in CLL cells [13]. More recently, new phosphorylations on threonine (pThr314) and two serine residues (pSer254, pSer265) of CD23, which is overexpressed and abnormally regulated in CLL, were reported in B lymphocytes of B-CLL patients [14]. Regulation of these CD23, CLL-associated phosphorylation sites brings new insight to the involvement of this transmembrane protein marker in the onset and

CLL is the most common leukemia in western countries, with an estimated incidence of about 4.5 new cases per 100,000 individuals annually [1]. It is most frequent in white populations in the United States and the lowest in Eastern Asian populations [15]. Median age at diagnosis is usually 72 years, and more male than female patients (1.7:1) are affected. About 10% of CLL patients are reported to be

The etiology of CLL is still unknown. Genetics and environmental factors may play an important role. Over 25 gene polymorphisms have been identified as contributing to CLL from a familial standpoint. These include genes that play roles in apoptosis, B-cell biology, as well as regulation by microRNAs, all of which have been found to be involved in disease progression [17, 18]. As such, it is important to note that relative to the general population, a six- to ninefold greater risk of developing the disease exists in individuals who have or have had relatives with CLL. Consequent protein synthesis and the involvement of newly synthesized proteins in disease onset and progression are the focus of numerous current studies. Insecticide exposure and farming history have also been associated with a higher

According to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) 2008 guidelines [20], a CLL diagnosis is established by the presence

at least 3 months, co-expressing CD5-, CD19-, and CD23-positive and weakly expressing CD20- and CD79b-positive as well as surface immunoglobulins.

/L peripheral lymphocytes, which lasts for a duration of

*DOI: http://dx.doi.org/10.5772/intechopen.85449*

*New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia DOI: http://dx.doi.org/10.5772/intechopen.85449*

CLL cells usually show constitutive phosphorylation of signaling proteins which promote their proliferation and survival, leading to pathological processes. Protein phosphorylation in lymphocytes is tightly associated with the regulation of a variety of protein activities, functional regulation, and cell signaling and may thus affect initiation and/or progression of the disease. As such, protein phosphorylation may be one of the most promising targets for the discovery of novel cancer-related protein markers and in turn their application in new approaches to molecular therapy. The constitutive activation of proteins by phosphorylation presents its potential for prognostic significance, as the identification of aberrant signal transduction in leukemic cells can become a potential target for novel agents. After BCR stimulation, CLL cells have shown a tendency toward impaired phosphorylation levels. Higher basal phosphorylation levels of PLCγ2 (pY759), p44/42 MAPK (pT202/Y204), p38 MAPK (pT180/Y182), NF-κB p65 (pS529), STAT5 (pY694), and STAT6 (pY641) were detected in CLL cells compared to normal B cells, predicting their impaired function [12]. As such, these markers may represent some of the novel protein targets involved in the development of efficient therapeutics. Cancer cells with constitutive STAT3 activation have been reported to have elevated levels of cell cycle regulation and antiapoptotic proteins, leading to apoptotic resistance. Constitutive serine phosphorylation of STAT1 and STAT3 has also been reported in CLL cells [13]. More recently, new phosphorylations on threonine (pThr314) and two serine residues (pSer254, pSer265) of CD23, which is overexpressed and abnormally regulated in CLL, were reported in B lymphocytes of B-CLL patients [14]. Regulation of these CD23, CLL-associated phosphorylation sites brings new insight to the involvement of this transmembrane protein marker in the onset and progress of CLL.

#### **2.1 Incidence and risk**

*Advances in Hematologic Malignancies*

**2. Chronic lymphocytic leukemia**

peripheral blood B lymphocytes to survive [7].

specific molecules and CLL pathogenesis.

leukemia.

adults. CLL treatment tends to focus mainly on controlling the state of the disease and its associated symptoms, rather than on its definitive eradication. The specifics of treatment will largely depend on the patient's prognosis and the specific CLL subtype. Therefore, lifelong observation and follow-up are strongly recommended and supported for all the patients. The combination of chemotherapy and nonchemotherapeutic drugs has improved survival of CLL patients overall, leading to long-lasting remissions. The pathology of CLL is complex in that it is influenced by a number of genetic and molecular changes, the CLL microenvironment, as well as various signaling pathways, of which the B-cell receptor (BCR) signaling pathway is central to CLL activation. Signaling pathways that are identified as being affected in CLL patients can provide opportunities for the development of diseasespecific drugs to the extent that they may be applicable in future clinical testing and molecular treatments. In any type of cancer, molecular therapy which targets specific regulatory proteins or their disease-associated posttranslational modifications can make way for novel applications which provide even higher specificity and efficiency with regard to treatment. This approach certainly applies to any type of

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the western world. The disease typically occurs in elderly patients and has a highly variable clinical progression. CLL is characterized by the clonal expansion and accumulation of mature CD19+, CD5+, and CD23+ B lymphocytes in the peripheral blood, bone marrow, and secondary lymphoid organs [1]. CLL cells are phenotypically similar to antigen-experienced B cells and show gene expression profiles similar to memory B cells [2]. The cellular origin of CLL is still debated, but it is assumed that CLL cells originate either from unmutated mature CD5+ B cells or CD5+CD27+ post-germinal center B-cell subsets [3]. CLL cells recirculate between peripheral blood and secondary lymphoid organs, where they proliferate in distinct areas of tissue, termed "pseudofollicles," at a daily birth rate of approximately 1–2% of the entire clone size [4]. Survival of CLL cells strictly depends on a permissive microenvironment composed of cellular components such as monocyte-derived nurse-like cells, T cells, follicular dendritic cells, mesenchymal stromal cells, and endothelial cells. Such dynamic combination of components leads to the presence of molecules such as cytokines, chemokines, and angiogenic factors. Leukemic cells take advantage of these vital proteins by interacting with them via cell-surface receptors or cell adhesion molecules to further facilitate their proliferation and survival [5, 6]. CLL cells are also characterized by an often observed defect in apoptosis which allows

Autoantigens and/or autonomous mechanisms activate the BCR and its signaling cascade in secondary lymphatic tissues, playing a central pathogenic role in CLL [8]. These events result in activation of multiple downstream regulators in B cells which ultimately mediate changes in cell proliferation, survival, and migration via both transcriptional modulation and phosphorylation. BCR signaling responses in CLL cells are heterogeneous, with effective activation of only a selected set of downstream responses [9]. Another key property of BCRs is that they exhibit somatic mutations in varying amounts; importantly, the degree of mutation has been found to inform the prognosis of disease [2, 10]. Furthermore, many cases of CLL (approximately one third) are characterized by a nearly indistinguishable subset of BCRs exhibiting shared antigens. This suggests a close link between these

**74**

CLL is the most common leukemia in western countries, with an estimated incidence of about 4.5 new cases per 100,000 individuals annually [1]. It is most frequent in white populations in the United States and the lowest in Eastern Asian populations [15]. Median age at diagnosis is usually 72 years, and more male than female patients (1.7:1) are affected. About 10% of CLL patients are reported to be younger than 55 years of age [16].

The etiology of CLL is still unknown. Genetics and environmental factors may play an important role. Over 25 gene polymorphisms have been identified as contributing to CLL from a familial standpoint. These include genes that play roles in apoptosis, B-cell biology, as well as regulation by microRNAs, all of which have been found to be involved in disease progression [17, 18]. As such, it is important to note that relative to the general population, a six- to ninefold greater risk of developing the disease exists in individuals who have or have had relatives with CLL. Consequent protein synthesis and the involvement of newly synthesized proteins in disease onset and progression are the focus of numerous current studies. Insecticide exposure and farming history have also been associated with a higher environmental risk for developing CLL [19].

#### **2.2 Symptoms and diagnosis**

According to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) 2008 guidelines [20], a CLL diagnosis is established by the presence of more than 5 × 109 /L peripheral lymphocytes, which lasts for a duration of at least 3 months, co-expressing CD5-, CD19-, and CD23-positive and weakly expressing CD20- and CD79b-positive as well as surface immunoglobulins.

Immunophenotyping by flow cytometry is required to establish CLL diagnosis based on cell identity, clonality, and quantity [21].

Two clinical staging systems, the Rai et al. [22] and Binet et al. [23] systems, are used to group patients with CLL into risk groups with discrete clinical outcomes. These two staging systems are relatively simple and widely used, relying on a physical examination and standard laboratory tests. Notably, the clinical presentation of CLL at diagnosis is extremely variable. Approximately 60% of patients are asymptomatic, and it is possible to detect the presence of the disease via a routine blood cell count. Lymphadenopathy (80%) and splenomegaly (50%) may be observed. Hepatomegaly is less frequent. As the disease progresses, patients can have B symptoms (weight loss, fever, night sweats, weakness) and exhibit a higher risk of infections. Lymphocytosis is constantly present, but the absolute number of lymphocytes is extremely variable. Anemia and thrombocytopenia may be also observed in 15–30% of patients [22–24]. Monoclonal B lymphocytosis (MBL), which can be observed in 5% of patients who exhibit a regular blood count and no other characteristics of a lymphoproliferative disposition, is characterized by a monoclonal B lymphocyte number of less than 5 × 10<sup>9</sup> /L in circulating blood [25]. Advancement from MBL to CLL is seen in a frequency of 1–2% cases per year [26].

Small lymphocytic lymphoma (SLL), in which the same leukemic cell population is mostly restricted to the bone marrow and lymphoid tissues, is similarly managed but considered to be a single entity [27]. The transformation into Richter syndrome (most commonly diffuse large B-cell lymphoma) occurs in 5–10% of all CLL cases and usually has a very poor prognosis [16].

#### **2.3 Prognostic factors**

The most important prognostic factors aside from clinical Rai and Binet staging systems are serum markers including β2 microglobulin levels [28], thymidine kinase levels [29], soluble CD23 levels [30], cellular markers including CD38 [31] and ζ chain associated protein kinase 70 (ZAP70) [32], CD49d [33], chemokines CCL3 a CCL4, genetic parameters including the mutational status of IGHV genes [10], and cytogenetic aberrations [34]. Unfavorable prognostic factors also include the male gender, ≥65 years of age, poor performance status due to medical comorbidities, late-stage disease at diagnosis, an initial white blood cell count above 35 × 109 /L, lymphocyte doubling time of less than 6 months, and a diffuse histological pattern in bone marrow infiltration [35]. Elevated levels of beta-2 microglobulin, serum thymidine, and serum CD23 at diagnosis also result in a poor prognosis [36].

ZAP-70 is a cytoplasmic protein tyrosine kinase initially identified in T cells. ZAP-70 expression in CLL is associated with increased BCR signaling capacity and greater responsiveness to chemokines resulting in more pronounced CLL cell migration and activation. Patients with ZAP-70 expression in more than 20% of CLL cells have a relatively shorter median time from diagnosis to initial treatment [37], and ZAP-70 appears to be a risk factor that is closely linked to aggressive CLL [32]. CD38 is a transmembrane protein that supports B-cell interaction and differentiation through the binding of CD31 [38], a cell adhesion molecule expressed by cells of the CLL microenvironment. Patients with high CD38 expression experience faster progression and shorter life expectancy [31]. The expression of the surface molecule CD49d, the α4 subunit of the integrin heterodimer α4β1, promotes microenvironment-mediated proliferation of CLL leukemic cells and has been identified in a subgroup of patients characterized by

**77**

frequency [50].

**2.4 Therapy**

*New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia*

progression of disease and short survival [33]. Both CCL3 and CCL4 are members of a cluster of cytokines with function as chemoattractants for monocytes and lymphocytes. They promote the communication of survival and proliferation signals to malignant cells and are associated with worse clinical outcomes in CLL

Immunoglobulin heavy-chain variable region (IGHV) mutation status plays an important role in CLL prognosis. Based on the degree of somatic hypermutation IGHV segments, unmutated IGHV (98% or more sequence homology with the germline sequence) corresponds to CLL originating from B cells that have not undergone a somatic mutation. Such patients can be classified as "unmutated" (U-CLL). Mutated IGHV (less than 98% sequence homology) is referred to as "mutated" (M-CLL) cases [41]. The presence of unmutated IGHV predicts a more aggressive disease type and has traditionally been associated with significantly decreased survival rates compared with mutated IGHV, which is associated with slower disease progression and longer survival [10, 31]. The differences in clinical behavior between M-CLL and U-CLL are determined by differences in responsiveness to external signals (such as BCR responsiveness). U-CLL BCRs are polyreactive and mostly recognize autoantigens and other environmental antigens [42, 43]. In contrast, affinity-matured BCRs from M-CLL cases bind to a restricted set of more specific antigens that either occur infrequently or induce anergy. Consequently, the M-CLL clone remains stable overall or expands at a

More than 80% of patients with previously untreated CLL have cytogenetic abnormalities, most common of which is a deletion in chromosome del(13q) [del(13q14.1)] (55%), followed by del(11q) [del(11q22-23)] (10–25%), trisomy 12 (10–20%), and del(17p) [del(17p13)] (5–10%) [34, 46]. Recommended analyses include interphase cytogenetic analysis with FISH for the detection of the del(17p), which affects p53 expression. A positive outcome is often seen in individuals who have deletions in 13q. This is likely a result of two missing miRNAs typically found in 13q, miR-15-1, and miR-16-1, which exhibit strong activity in healthy B cells; miR-15-1 and miR-16-1 are thought to play a role in the downregulation of B-cell

The association between trisomy 12 and prognosis is still not clear [47]. A deletion in 11q results in the ataxia telangiectasia mutated (ATM) gene, which has shown to be a predictor of poor clinical outcome [34]. Deletions of the short arm of chromosome 17 cause the loss of one tumor protein p53 (TP53) allele and are associated with inactivating mutations in the other allele in 80% of patients with CLL. This cytogenetic aberration is associated with the worst CLL prognosis. Patients have shown marked resistance against genotoxic chemotherapies which has forced clinicians to alter their first-line treatment [34, 48]. Further recurring gene alterations have been found in 5% of cases of CLL samples at time of diagnosis; via whole genome/exome sequencing, genes influencing NOTCH1 and myeloid differentiation primary response (MYD88) [49] have been identified alongside genes coding for splicing factor 3B subunit 1 (SF3B1) [50] and baculoviral IAP repeat containing 3 (BIRC3) [51]. Patients experiencing progressive/refractory CLL and Richter's syndrome were observed to exhibit these mutations in greater

CLL is an incurable disease with a highly heterogeneous clinical course. Previous studies have shown that early treatment with chemotherapeutic agents

lymphoma 2 (BCL2), which acts as an antiapoptotic molecule [34].

*DOI: http://dx.doi.org/10.5772/intechopen.85449*

[39, 40].

slower rate [44, 45].

progression of disease and short survival [33]. Both CCL3 and CCL4 are members of a cluster of cytokines with function as chemoattractants for monocytes and lymphocytes. They promote the communication of survival and proliferation signals to malignant cells and are associated with worse clinical outcomes in CLL [39, 40].

Immunoglobulin heavy-chain variable region (IGHV) mutation status plays an important role in CLL prognosis. Based on the degree of somatic hypermutation IGHV segments, unmutated IGHV (98% or more sequence homology with the germline sequence) corresponds to CLL originating from B cells that have not undergone a somatic mutation. Such patients can be classified as "unmutated" (U-CLL). Mutated IGHV (less than 98% sequence homology) is referred to as "mutated" (M-CLL) cases [41]. The presence of unmutated IGHV predicts a more aggressive disease type and has traditionally been associated with significantly decreased survival rates compared with mutated IGHV, which is associated with slower disease progression and longer survival [10, 31]. The differences in clinical behavior between M-CLL and U-CLL are determined by differences in responsiveness to external signals (such as BCR responsiveness). U-CLL BCRs are polyreactive and mostly recognize autoantigens and other environmental antigens [42, 43]. In contrast, affinity-matured BCRs from M-CLL cases bind to a restricted set of more specific antigens that either occur infrequently or induce anergy. Consequently, the M-CLL clone remains stable overall or expands at a slower rate [44, 45].

More than 80% of patients with previously untreated CLL have cytogenetic abnormalities, most common of which is a deletion in chromosome del(13q) [del(13q14.1)] (55%), followed by del(11q) [del(11q22-23)] (10–25%), trisomy 12 (10–20%), and del(17p) [del(17p13)] (5–10%) [34, 46]. Recommended analyses include interphase cytogenetic analysis with FISH for the detection of the del(17p), which affects p53 expression. A positive outcome is often seen in individuals who have deletions in 13q. This is likely a result of two missing miRNAs typically found in 13q, miR-15-1, and miR-16-1, which exhibit strong activity in healthy B cells; miR-15-1 and miR-16-1 are thought to play a role in the downregulation of B-cell lymphoma 2 (BCL2), which acts as an antiapoptotic molecule [34].

The association between trisomy 12 and prognosis is still not clear [47]. A deletion in 11q results in the ataxia telangiectasia mutated (ATM) gene, which has shown to be a predictor of poor clinical outcome [34]. Deletions of the short arm of chromosome 17 cause the loss of one tumor protein p53 (TP53) allele and are associated with inactivating mutations in the other allele in 80% of patients with CLL. This cytogenetic aberration is associated with the worst CLL prognosis. Patients have shown marked resistance against genotoxic chemotherapies which has forced clinicians to alter their first-line treatment [34, 48]. Further recurring gene alterations have been found in 5% of cases of CLL samples at time of diagnosis; via whole genome/exome sequencing, genes influencing NOTCH1 and myeloid differentiation primary response (MYD88) [49] have been identified alongside genes coding for splicing factor 3B subunit 1 (SF3B1) [50] and baculoviral IAP repeat containing 3 (BIRC3) [51]. Patients experiencing progressive/refractory CLL and Richter's syndrome were observed to exhibit these mutations in greater frequency [50].

#### **2.4 Therapy**

CLL is an incurable disease with a highly heterogeneous clinical course. Previous studies have shown that early treatment with chemotherapeutic agents

*Advances in Hematologic Malignancies*

1–2% cases per year [26].

**2.3 Prognostic factors**

above 35 × 109

prognosis [36].

based on cell identity, clonality, and quantity [21].

CLL cases and usually has a very poor prognosis [16].

Immunophenotyping by flow cytometry is required to establish CLL diagnosis

Two clinical staging systems, the Rai et al. [22] and Binet et al. [23] systems, are used to group patients with CLL into risk groups with discrete clinical outcomes. These two staging systems are relatively simple and widely used, relying on a physical examination and standard laboratory tests. Notably, the clinical presentation of CLL at diagnosis is extremely variable. Approximately 60% of patients are asymptomatic, and it is possible to detect the presence of the disease via a routine blood cell count. Lymphadenopathy (80%) and splenomegaly (50%) may be observed. Hepatomegaly is less frequent. As the disease progresses, patients can have B symptoms (weight loss, fever, night sweats, weakness) and exhibit a higher risk of infections. Lymphocytosis is constantly present, but the absolute number of lymphocytes is extremely variable. Anemia and thrombocytopenia may be also observed in 15–30% of patients [22–24]. Monoclonal B lymphocytosis (MBL), which can be observed in 5% of patients who exhibit a regular blood count and no other characteristics of a lymphoproliferative disposition, is characterized by a monoclonal B lymphocyte number of less than 5 × 10<sup>9</sup>

in circulating blood [25]. Advancement from MBL to CLL is seen in a frequency of

Small lymphocytic lymphoma (SLL), in which the same leukemic cell population is mostly restricted to the bone marrow and lymphoid tissues, is similarly managed but considered to be a single entity [27]. The transformation into Richter syndrome (most commonly diffuse large B-cell lymphoma) occurs in 5–10% of all

The most important prognostic factors aside from clinical Rai and Binet staging systems are serum markers including β2 microglobulin levels [28], thymidine kinase levels [29], soluble CD23 levels [30], cellular markers including CD38 [31] and ζ chain associated protein kinase 70 (ZAP70) [32], CD49d [33], chemokines CCL3 a CCL4, genetic parameters including the mutational status of IGHV genes [10], and cytogenetic aberrations [34]. Unfavorable prognostic factors also include the male gender, ≥65 years of age, poor performance status due to medical comorbidities, late-stage disease at diagnosis, an initial white blood cell count

histological pattern in bone marrow infiltration [35]. Elevated levels of beta-2 microglobulin, serum thymidine, and serum CD23 at diagnosis also result in a poor

ZAP-70 is a cytoplasmic protein tyrosine kinase initially identified in T cells. ZAP-70 expression in CLL is associated with increased BCR signaling capacity and greater responsiveness to chemokines resulting in more pronounced CLL cell migration and activation. Patients with ZAP-70 expression in more than 20% of CLL cells have a relatively shorter median time from diagnosis to initial treatment [37], and ZAP-70 appears to be a risk factor that is closely linked to aggressive CLL [32]. CD38 is a transmembrane protein that supports B-cell interaction and differentiation through the binding of CD31 [38], a cell adhesion molecule expressed by cells of the CLL microenvironment. Patients with high CD38 expression experience faster progression and shorter life expectancy [31]. The expression of the surface molecule CD49d, the α4 subunit of the integrin heterodimer α4β1, promotes microenvironment-mediated proliferation of CLL leukemic cells and has been identified in a subgroup of patients characterized by

/L, lymphocyte doubling time of less than 6 months, and a diffuse

/L

**76**

was unable to demonstrate a benefit due to these therapeutic interventions in CLL patients [52]. The standard treatment for patients with early disease is a "watch-and-wait" strategy. Treatment should only be initiated in patients with progressive or symptomatic/active disease. In order to determine the best approach to treatment, crucial factors such as the stage of disease, physical status, and cytogenetic risk should be assessed on a per-patient basis [18]. Additionally, the "Go-Go," "Slow-Go," and "No-Go" comorbidity classifications present another important set of factors in determining the optimal avenue for treatment [53].

Monotherapy with alkylating agents (chlorambucil) and purine analogs (fludarabine, pentostatin, cladribine, bendamustine) has served as an initial, frontline therapy for CLL and was the therapeutic "gold standard" for several decades [52]. Compared to monotherapy, the combination of fludarabine with alkylating cyclophosphamide is more widely used, leading to an increased effect on malignant lymphocytes and greater remission inductions [54]. The onset of biological treatment using monoclonal antibodies has led to significant changes in the approach to treatment. As CD20 is expressed on most B-cell malignancies, the introduction of the anti-CD20 antibody rituximab improved the treatment of most CD20-positive non-Hodgkin lymphomas, including CLL. Rituximab is less active as a single agent; however, combinations of rituximab with chemotherapy have shown to be very efficacious therapies for CLL [55]. The combination of rituximab, fludarabine, and cyclophosphamide is considered to be the standard first-line therapy (FCR chemoimmunotherapy) [56]. Ofatumumab and obinutuzumab are another set of CD20 antibodies used for the treatment of patients with relapsed/refractory CLL [57, 58]. Alemtuzumab is a recombinant, fully humanized, monoclonal antibody against the CD52 antigen. Monotherapy with alemtuzumab is used in patients with advanced CLL or relapsed patients after second-line fludarabine therapy and with poor prognostic features [59]. Autologous stem-cell transplantation is not useful in CLL. Maintenance therapy in CLL patients with higher risk of relapse may have some benefit but is not generally recommended [18].

Lenalidomide is an immunomodulatory agent that induces only mild apoptosis of leukemic cells but also reduces CLL proliferation through a cereblon-/p21-dependent mechanism. Lenalidomide has pleiotropic effects on the CLL microenvironment: it increases CD4+ T-mediated antigen presentation, proliferation, and activity and enhances NK and CD4+ T-cell mediated antitumor immune responses [60]. It is active alone, in CLL relapsed/refractory patients, or as an initial treatment for elderly patients or in combination with rituximab [61].

The CXCR4/CXCL12 signaling axis represents another important therapeutic target in CLL. CXCR4 antagonists have been developed, including peptide CXCR4 antagonists (BKT140), small molecule CXCR4 antagonists (AMD3100, plerixafor), and antibodies to CXCR4 (MDX-1338) [62]. Plerixafor inhibits CXCL12-mediated signaling activation on CLL cells and is used in combination with rituximab in relapsed CLL patients [63].

Proteins in the Bcl-2 family are key regulators of the apoptotic process with proapoptotic and prosurvival activities. Venetoclax is a so-called BH3-mimetic drug designed to block the function of the Bcl-2 protein and inhibits the growth of BCL-2-dependent tumors in vivo. Monotherapy with this drug is active and well tolerated in patients with relapsed or refractory del(17p) CLL, providing a new therapeutic option for this very poor prognosis population [64].

B-cell receptor signaling seems to play an important role in the survival of CLL cells. Inhibitors targeting BCR-associated kinases have changed the landscape of

**79**

developed [78].

**3. Acute lymphocytic leukemia**

result in abnormal growth [80].

*New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia*

treatment for CLL patients, inducing durable remissions in relapsed/refractory patients, including those carrying unfavorable genetic alterations (e.g., del17p, del11q) [65]. Randomized trials comparing new drugs and/or their combinations with standard chemoimmunotherapy regimens are ongoing and will allow to better define optimal treatment strategies [66]. New light shed onto the mechanisms of BCR activation in CLL has enabled for the design and application of kinase inhibitors targeting BCR signaling kinases BTK, PI3K, and SYK. Bruton's tyrosine kinase, BTK, is a non-receptor tyrosine kinase that plays a central role in downstream activation of cell survival pathways such as NF-κB and MAP kinases via Src family kinases. Ibrutinib is the first human BTK inhibitor. The drug binds irreversibly to a cysteine residue (Cys-481) in the BTK kinase domain [67] and inhibits BTK phosphorylation and its enzymatic activity [68]. Ibrutinib inhibits CLL cell survival and proliferation, as well as leukemia cell migration toward the tissue homing chemokines [69]. Previous tests have shown that ibrutinib yielded durable remissions in CLL/SLL patients with relapsed, refractory, or high-risk disease and in previously untreated older patients [70]. Acalabrutinib, a potentially more selective, irreversible BTK inhibitor has been tested and is currently under early clinical development [71]. PI3Kδ is expressed by hematopoietic cells and plays a critical role in B-cell homeostasis and function. Idelalisib is a highly selective PI3Kδ inhibitor, which antagonizes CLL-survival signals coming from the microenvironment as well as BCR stimulation [72]. This drug inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells and also inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase activation [73]. Idelalisib has been tested as single agent or in combination strategies with clinical benefit in patients with relapsed/refractory CLL [74]. Additional PI3K inhibitors are currently under development, including duvelisib, a potent PI3K γδ inhibitor, which antagonizes BCR and microenvironment interactions in vitro [75]. Spleen tyrosine kinase (SYK), which belongs to the SYK/ZAP70 family of non-receptor kinases, has been implicated in tissue homing and retention of activated B cells due to its role as a downstream activator of BCR signaling (chemokine and integrin receptors) [76]. Up to this point, only limited responses have been seen in patients experiencing CLL relapse after introduction of fostamatinib disodium (FosD) to the treatment regimen [77]. FosD is currently the only available inhibitor of SYK on the market, with additional similar drugs being

Acute lymphocytic leukemia (ALL), also known as acute lymphoblastic leukemia or acute lymphoid leukemia, is the most common malignancy in children and the least common type of leukemia in adults. It is an acute type of cancer invading blood and spreading throughout the body to other organs, such as the liver, spleen, lymph nodes, and central nervous system. Without treatment, it can be fatal within a few months. ALL is characterized by a malignant transformation and proliferation of lymphoid progenitor cells in the bone marrow, blood, and extramedullary sites, which replace normal blood cells [79]. The exact causes of ALL remain largely unknown, but it is thought to result from genetic alterations such as structural chromosome rearrangements, aneuploidy, and mutations in genes that encode for transcription factors regulating lymphoid development, tumor suppressors, proteins that regulate cell cycle progression, and epigenetic modifiers. Such defects

*DOI: http://dx.doi.org/10.5772/intechopen.85449*

*New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia DOI: http://dx.doi.org/10.5772/intechopen.85449*

treatment for CLL patients, inducing durable remissions in relapsed/refractory patients, including those carrying unfavorable genetic alterations (e.g., del17p, del11q) [65]. Randomized trials comparing new drugs and/or their combinations with standard chemoimmunotherapy regimens are ongoing and will allow to better define optimal treatment strategies [66]. New light shed onto the mechanisms of BCR activation in CLL has enabled for the design and application of kinase inhibitors targeting BCR signaling kinases BTK, PI3K, and SYK. Bruton's tyrosine kinase, BTK, is a non-receptor tyrosine kinase that plays a central role in downstream activation of cell survival pathways such as NF-κB and MAP kinases via Src family kinases. Ibrutinib is the first human BTK inhibitor. The drug binds irreversibly to a cysteine residue (Cys-481) in the BTK kinase domain [67] and inhibits BTK phosphorylation and its enzymatic activity [68]. Ibrutinib inhibits CLL cell survival and proliferation, as well as leukemia cell migration toward the tissue homing chemokines [69]. Previous tests have shown that ibrutinib yielded durable remissions in CLL/SLL patients with relapsed, refractory, or high-risk disease and in previously untreated older patients [70]. Acalabrutinib, a potentially more selective, irreversible BTK inhibitor has been tested and is currently under early clinical development [71]. PI3Kδ is expressed by hematopoietic cells and plays a critical role in B-cell homeostasis and function. Idelalisib is a highly selective PI3Kδ inhibitor, which antagonizes CLL-survival signals coming from the microenvironment as well as BCR stimulation [72]. This drug inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells and also inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase activation [73]. Idelalisib has been tested as single agent or in combination strategies with clinical benefit in patients with relapsed/refractory CLL [74]. Additional PI3K inhibitors are currently under development, including duvelisib, a potent PI3K γδ inhibitor, which antagonizes BCR and microenvironment interactions in vitro [75]. Spleen tyrosine kinase (SYK), which belongs to the SYK/ZAP70 family of non-receptor kinases, has been implicated in tissue homing and retention of activated B cells due to its role as a downstream activator of BCR signaling (chemokine and integrin receptors) [76]. Up to this point, only limited responses have been seen in patients experiencing CLL relapse after introduction of fostamatinib disodium (FosD) to the treatment regimen [77]. FosD is currently the only available inhibitor of SYK on the market, with additional similar drugs being developed [78].
