**4. Prognostic markers in DLBCL**

#### **4.1 Gene expression profile (GEP)**

Clinical and morphological differences in DLBCL suggest the biological significance of subtypes, which may help in adapting the therapy to be defined. The development of DNA microarray techniques provided the ability to identify gene expression of the tumor with a new molecular entity and a molecular predictor essential for survival. DNA microarray can analyze thousands of sites of previously identified genes. DLBCL has been identified by several tumor markers associated with unfavorable outcome after therapy and which can be linked to tumor cells and normal B lymphocytes. Phenotypic cells resemble normal B lymphocytes, but the clinical course is completely different [50–52].

Gene expression profile has shown that DLBCL can be divided into two molecular entities that differ in the gene expression profile. Hans et al. have identified two subgroups of DLBCL [34, 52]. One subgroup was called germinal center B-cell-like (GCB) and has a characteristic gene expression of normal GC B cells, in about 50% of cases, and the second group was called activated B-cell-like (ABC). Type-3 group is defined as nonclassified cases. According to the GEP classification, the observed prognosis distinguishes two major subtypes after the chemotherapy. Patients with GCB lymphoma have a more favorable outcome in 5-year survival than patients in the ABC group (68% vs. 24%) [52]. The distinction between GCB and ABC

#### *Normal and Malignant B-Cell*

subgroups and their effect on survival are independent of any clinical features of the disease, and these findings are further confirmed in other retrospective series with patients treated with chemotherapy [34, 42, 45–49].

In DLBCL, the ratio of *BCL2*-positive cases is highly variable, ranging from 24 to 80% among the study population in the previous studies that used immunohistochemistry (IHC); therefore, its prognostic relevance is controversial [16, 53–55].

Overexpression of the *BCL2* is associated with poor prognosis [55]. ABC-DLBCL subgroup is characterized by transcription expression of the *BCL2* gene [55], trisomy 3, and 18q21-q22 and loss of 6q21-q22 [56]. Activation of the antiapoptotic NF-ĸB signal pathway occurs at ABC but not at GCB DLBCL [56].

GCB DLBCL is characterized by frequent *REL* amplifications, *BCL2* translocations, and ongoing somatic hypermutation of the immunoglobulin genes [56].

Immunohistochemistry studies have a significant role, although complexity in the expression and performance of the interpretation of findings is very complex. They are used in the prognosis of DLBCL and in the therapeutic advantage of using rituximab [52].

DLBCL is a common type of aggressive non-Hodgkin's lymphoma with clear biological and clinical characteristics. Many studies have focused on the significance of internal tumor characteristics, such as the presence of proteins or the presence of gene expression and chromosomal translocation. Significant component of cell biology is inherited from non-transformed stem cells. Each of the B-cellular lymphoma inherits the traces of B-cell differentiation, although the extent to which this malignancy is maintained and the maintenance of molecular and physiological properties of normal B cells are not yet clear. Redistribution of immunoglobulin genes in DLBCL and other lymphomas carries mutations having somatic hypermutation characteristics, and the diversity of antibodies normally occurs only with the germinal center of the secondary lymphoid organ. This data suggest that DLBCL is generated from each of the germinal centers of the B cell or from the B cell at a later stage of differentiation [1, 25, 50, 52, 56].

#### *4.1.1 Germinal center B-cell-like (GCB)*

CB DLBCL appears to be generated from the normal germinal B cell, the translocation t (14, 18) involving *BCL2* [54], and the amplification of *c-REL* on the chromosome 2p was detected in 45% GCB DLBCL, whereas it was never detected at ABC subtype [56]. This finding suggests that different subgroups represent different diseases and the consequence is a different mechanism of malignant transformation and significantly different survival after chemotherapy. CD10 and *BCL-6* are considered as germinal center markers, while IRF4/MUM1 is expressed on plasma cells in atypical light zones of the germinal center. By morphological characteristics, the center is uniform form monomorphic. Expressions CD10, BCL-6, and MUM1 are used to classify GCB and ABC lymphoma subtype [1, 25, 46, 50, 56].

Many factors of B-cell differentiation in the germinal center affect the expression of transcription factors. The key B-cell transcription factor is PAX5 (PAX5 is a nuclear protein that has the ability to bind to wild-type PAX5 target loci) [53] and is required for regulating many B-cell-specific transcription factors and for B-cellular development from the B stage to mature B cells. The transcription factor PU.1 is an E26 transformation-specific family transcription factor that is required for the development of the immune system. PU.1 functions at both early and late stages of lymphoid and myeloid differentiation [57], and the IRF involved with Blim1/ PRDM1 was isolated from the family in further regulation of the transcriptional factor *BCL6*, which is essential for the formation and maintenance of the germinal center. Translocation t (3q27) is included in *BCL6* gene and is found in 20–40% diffuse lymphomas. IRF4/MUM1 expression is required for activation-induced cytidine deaminase (AID) expression, which is important for two significant functions

**85**

lymphomas [39].

*B Cell Lymphomagenesis*

*DOI: http://dx.doi.org/10.5772/intechopen.87241*

*4.1.2 Activated B-cell-like (ABC)*

tiation in the plasma cells [42].

*BCL6* and IRF4 / MUM1, they have co-expression [8, 59].

of the germinal center, namely, somatic hypermutation (SHM) and AID-mediated class switch recombination (CSR) [58]. They depend on hierarchical expression and the common regulation of transcription factors. For example, *BCL6* and IRF4/ MUM1 are expressed mutually exclusively in germinal center cells, but with DLBCL

The Epstein–Barr virus (EBV) is the second factor associated with disturbed GCB regulation. EBV is associated with numerous lymphoid neoplasms including Burkitt's lymphoma and classical Hodgkin's lymphoma and posttransplant lymphoproliferative disease. The transformation that leads through EBV is capable of major negative selection in the germinal center. EBV/LMP2A induces escape of the defective B cells of the germinal center of the apoptosis and does not only offer an intriguing model of lymphomagenesis but also confirms the significant role of exogenous factors such as viruses involved in forming the response of the germinal center [60]. EBV latent membrane protein 2A (LMP2A) is expressed on the membranes of B lymphocytes and blocks B-cell receptor (BCR) signaling in EBV-transformed B lymphocytes in vitro. In vivo studies indicate that LMP2A allows B-cell survival in the absence of normal BCR signals [60]. CD10 expression is associated with a more favorable prognosis, and MUM1 is normally found on plasma cells and in a small part of the atypical spot zone in the

germinal center and represents a marker for a non-germinal center [61].

properties of normal B cells that are activated over B-cell receptors.

Patients in this group have significantly better 5-year overall survival and survival without signs of disease activity after chemotherapy with combined drugs (68% for GCB vs. 24.7% for non-GCB with use of gene-expression profiling) [8, 52, 56].

ABC-DLBCL is generated from the postgerminal center of B cells during plasmacytic differentiation. ABC is characterized by frequent changes in chromosome arm 3p but not 3q [62]. ABC lymphoma has a subtype gene expression that has the

Trisomy 3 is a common chromosomal aberration and is found in 26% of respon-

ABC lymphomas can obtain genetic changes that interfere with Blimp-1, thereby blocking full differentiation into plasma cells. The block in differentiation represents an early step in the pathogenesis of ABC subtypes, but the nature of the cell precursor is unclear. These lymphomas contain high activation-induced cytidine deaminase (AID) values, and their IgH genes have severe mutations. However, most ABC lymphomas did not undergo a recombinant trigger, and they have IgM expression, unlike most of the normal B cells of the germinal center and other GCB

dents. The forkhead transcription factor (FOXP1) is the third most potentially monitored gene in trisomy 3. High FOXP1 expression can contribute to B-cell lymphomagenesis by promoting B-cell survival inhibiting plasma cell differentiation. The small FOXP1 isoform (FOXP1-iso) is predominantly expressed in ABC-DLBCL, and studying its oncogenic potential and transcriptional activity is directly compared to FOXP1-FL in DLBCL cell lines and primary human B cells [61–64]. ABC lymphomas have expression of the plasma cell genome, including transcriptional factor XBP1, which is the regulator of immunoglobulin secretion. XPB1 is a protein which in humans is encoded by the *XPB1* gene located on chromosome 22. The XBP1 protein is a transcription factor that regulates the expression of genes important of the immune system and in the cellular stress response. *C-MYC* is necessary and sufficient for activation of the IRE1/XBP1 pathway [65, 66]. The constituent part of the NF-κB activation causes ABC lymphoma with expressing the IRF4 transcription factor, and this may push them toward differen-

#### *B Cell Lymphomagenesis DOI: http://dx.doi.org/10.5772/intechopen.87241*

*Normal and Malignant B-Cell*

*4.1.1 Germinal center B-cell-like (GCB)*

subgroups and their effect on survival are independent of any clinical features of the disease, and these findings are further confirmed in other retrospective series

In DLBCL, the ratio of *BCL2*-positive cases is highly variable, ranging from 24 to 80% among the study population in the previous studies that used immunohistochemistry (IHC); therefore, its prognostic relevance is controversial [16, 53–55].

Overexpression of the *BCL2* is associated with poor prognosis [55]. ABC-DLBCL

GCB DLBCL is characterized by frequent *REL* amplifications, *BCL2* translocations, and ongoing somatic hypermutation of the immunoglobulin genes [56]. Immunohistochemistry studies have a significant role, although complexity in the expression and performance of the interpretation of findings is very complex. They are used in the prognosis of DLBCL and in the therapeutic advantage of using rituximab [52]. DLBCL is a common type of aggressive non-Hodgkin's lymphoma with clear biological and clinical characteristics. Many studies have focused on the significance of internal tumor characteristics, such as the presence of proteins or the presence of gene expression and chromosomal translocation. Significant component of cell biology is inherited from non-transformed stem cells. Each of the B-cellular lymphoma inherits the traces of B-cell differentiation, although the extent to which this malignancy is maintained and the maintenance of molecular and physiological properties of normal B cells are not yet clear. Redistribution of immunoglobulin genes in DLBCL and other lymphomas carries mutations having somatic hypermutation characteristics, and the diversity of antibodies normally occurs only with the germinal center of the secondary lymphoid organ. This data suggest that DLBCL is generated from each of the germinal centers of the B cell or from the B cell at a later stage of differentiation [1, 25, 50, 52, 56].

subgroup is characterized by transcription expression of the *BCL2* gene [55], trisomy 3, and 18q21-q22 and loss of 6q21-q22 [56]. Activation of the antiapoptotic

CB DLBCL appears to be generated from the normal germinal B cell, the translocation t (14, 18) involving *BCL2* [54], and the amplification of *c-REL* on the chromosome 2p was detected in 45% GCB DLBCL, whereas it was never detected at ABC subtype [56]. This finding suggests that different subgroups represent different diseases and the consequence is a different mechanism of malignant transformation and significantly different survival after chemotherapy. CD10 and *BCL-6* are considered as germinal center markers, while IRF4/MUM1 is expressed on plasma cells in atypical light zones of the germinal center. By morphological characteristics, the center is uniform form monomorphic. Expressions CD10, BCL-6, and MUM1 are used to classify GCB and ABC lymphoma subtype [1, 25, 46, 50, 56]. Many factors of B-cell differentiation in the germinal center affect the expression of transcription factors. The key B-cell transcription factor is PAX5 (PAX5 is a nuclear protein that has the ability to bind to wild-type PAX5 target loci) [53] and is required for regulating many B-cell-specific transcription factors and for B-cellular development from the B stage to mature B cells. The transcription factor PU.1 is an E26 transformation-specific family transcription factor that is required for the development of the immune system. PU.1 functions at both early and late stages of lymphoid and myeloid differentiation [57], and the IRF involved with Blim1/ PRDM1 was isolated from the family in further regulation of the transcriptional factor *BCL6*, which is essential for the formation and maintenance of the germinal center. Translocation t (3q27) is included in *BCL6* gene and is found in 20–40% diffuse lymphomas. IRF4/MUM1 expression is required for activation-induced cytidine deaminase (AID) expression, which is important for two significant functions

NF-ĸB signal pathway occurs at ABC but not at GCB DLBCL [56].

with patients treated with chemotherapy [34, 42, 45–49].

**84**

of the germinal center, namely, somatic hypermutation (SHM) and AID-mediated class switch recombination (CSR) [58]. They depend on hierarchical expression and the common regulation of transcription factors. For example, *BCL6* and IRF4/ MUM1 are expressed mutually exclusively in germinal center cells, but with DLBCL *BCL6* and IRF4 / MUM1, they have co-expression [8, 59].

The Epstein–Barr virus (EBV) is the second factor associated with disturbed GCB regulation. EBV is associated with numerous lymphoid neoplasms including Burkitt's lymphoma and classical Hodgkin's lymphoma and posttransplant lymphoproliferative disease. The transformation that leads through EBV is capable of major negative selection in the germinal center. EBV/LMP2A induces escape of the defective B cells of the germinal center of the apoptosis and does not only offer an intriguing model of lymphomagenesis but also confirms the significant role of exogenous factors such as viruses involved in forming the response of the germinal center [60]. EBV latent membrane protein 2A (LMP2A) is expressed on the membranes of B lymphocytes and blocks B-cell receptor (BCR) signaling in EBV-transformed B lymphocytes in vitro. In vivo studies indicate that LMP2A allows B-cell survival in the absence of normal BCR signals [60].

CD10 expression is associated with a more favorable prognosis, and MUM1 is normally found on plasma cells and in a small part of the atypical spot zone in the germinal center and represents a marker for a non-germinal center [61].

Patients in this group have significantly better 5-year overall survival and survival without signs of disease activity after chemotherapy with combined drugs (68% for GCB vs. 24.7% for non-GCB with use of gene-expression profiling) [8, 52, 56].

#### *4.1.2 Activated B-cell-like (ABC)*

ABC-DLBCL is generated from the postgerminal center of B cells during plasmacytic differentiation. ABC is characterized by frequent changes in chromosome arm 3p but not 3q [62]. ABC lymphoma has a subtype gene expression that has the properties of normal B cells that are activated over B-cell receptors.

Trisomy 3 is a common chromosomal aberration and is found in 26% of respondents. The forkhead transcription factor (FOXP1) is the third most potentially monitored gene in trisomy 3. High FOXP1 expression can contribute to B-cell lymphomagenesis by promoting B-cell survival inhibiting plasma cell differentiation. The small FOXP1 isoform (FOXP1-iso) is predominantly expressed in ABC-DLBCL, and studying its oncogenic potential and transcriptional activity is directly compared to FOXP1-FL in DLBCL cell lines and primary human B cells [61–64].

ABC lymphomas have expression of the plasma cell genome, including transcriptional factor XBP1, which is the regulator of immunoglobulin secretion. XPB1 is a protein which in humans is encoded by the *XPB1* gene located on chromosome 22. The XBP1 protein is a transcription factor that regulates the expression of genes important of the immune system and in the cellular stress response. *C-MYC* is necessary and sufficient for activation of the IRE1/XBP1 pathway [65, 66].

The constituent part of the NF-κB activation causes ABC lymphoma with expressing the IRF4 transcription factor, and this may push them toward differentiation in the plasma cells [42].

ABC lymphomas can obtain genetic changes that interfere with Blimp-1, thereby blocking full differentiation into plasma cells. The block in differentiation represents an early step in the pathogenesis of ABC subtypes, but the nature of the cell precursor is unclear. These lymphomas contain high activation-induced cytidine deaminase (AID) values, and their IgH genes have severe mutations. However, most ABC lymphomas did not undergo a recombinant trigger, and they have IgM expression, unlike most of the normal B cells of the germinal center and other GCB lymphomas [39].

#### *Normal and Malignant B-Cell*

NF-ĸB is present as a latent, inactive, IĸB-bound complex in the cytoplasm. When a cell accepts any of a large number of extracellular signals, NF-ĸB quickly enters the nucleus and activates gene expression. Key step for controlling NF-ĸB activity is the regulation of the I kappa B kinase (IKK) [42, 55, 62].

Various genetic abnormalities activate CARD11 in ABC subtype lymphoma. In normal B cells, antigen-receptor-induced NF-kB activation requires CARD11, a cytoplasmic scaffolding protein. To determine whether CARD11 contributes to tumorigenesis, we sequenced the CARD11 gene in human DLBCL tumors [67]. In about 10% of patients, CARD11 is an oncogene with acquired mutations that activate NF-κB and can prolong cell survival. Mutant CARD11 generates large protein aggregates in the cytoplasm, which represent possible NF-κB activation sites. The survival of lymphoma ABC with the wild-type CARD11 depends on B-cell receptors and kinases [67].

ABC lymphomas have expression of the plasma cell genome, including transcriptional factor XBP1, which is the main regulator of immunoglobulin secretion. ABC lymphoma has presented expression of genes characteristic of mitogen-activated B cells in the blood and is associated with a worse prognosis [66]. ABC-DLBCL is characterized by gain of 3q, 18q, and 19q and loss of 6q and 9p21, and GCB DLBCL is characterized by gain of 1q, 2p, 7q, and 12q; the genomic imbalances characteristic of the CD5(+) and CD5(−)CD10(+) groups were similar to those of the ABC and GCB types [68].

Lymphoma ABC does not have one expression of the germinal center gene, but has gene expression that is normally present in B cells after B-cell receptor stimulation. MUM1 is found on plasma cells at later stage B-cellular differentiation and is associated with the gene profile of activated B-cellular DLBCL. The presence of *BCL2* and Cyklin D2 is adverse prognostic parameters. Polymorphic centroblasts and immunoblasts are more commonly present in activated B-cell DLBCL. This group is characterized by a group of genes that are characteristic of plasma cells, in part, added to the endoplasmic reticulum and Golgi's proteins involved in secretion. There is a significantly higher percentage of patients in this group [69].

#### **5. Conclusion**

The contribution of GC selection to phenomena such as immunodominance and original antigenic guilt has also not been established and is likely to be of considerable interest. Answering these questions will be crucial to our quest to produce more broad and new medications and will undoubtedly bring novel insights into the general functioning of GC, as well as of the immune system in general.
