2.2. Cell of origin

The incidence of NHL is 19.5 per 100,000 men and women per year. Approximately 2.1% of men and women will be diagnosed with NHL at some point based on 2012–2014 SEER database [2] (SEER data). The incidence of DLBCL is approximately 7 cases per 100,000 persons per year. Males have a higher incidence with (M/F) incidence ratio of 1.5–1.6. Incidence is higher among whites than blacks or Asians and increases steeply with age. The overall incidence of DLBCL has declined 0.5% per year during 1992–2001 [2]. This decline was observed predominantly in men aged 25–54, on contrary in the elderly, the incidence of

DLBCL is a mature B cell neoplasm arising from germinal center and post germinal center B cells. Etiology of DLBCL is complex and multifactorial. DLBCL can arise de-novo or from transformation of indolent diseases like chronic lymphocytic lymphoma/small lymphocytic lymphoma (so-called Richter's transformation), follicular lymphoma, and marginal zone lymphoma. Those that originate from previous hematological malignancies are generally more aggressive and carry a poor prognosis. Chemical substances such as pesticides, herbicides, alkylating agents, ionizing radiation have been implicated as risk factors. An association has been found between inherited immune deficiency disorders such as ataxia-telangiectasia, Wiskott-Aldrich syndrome, common variable immunodeficiency, severe combined immunodeficiency, X-linked lymphoproliferative disorder [3]. Patients with acquired immunodeficiency states such as AIDS, organ or stem cell transplantation, autoimmune or rheumatologic

DLBCL is composed of large B cells with a diffuse growth pattern arising from mature B cells at different stages of differentiation. Normal B cell development takes place in the bone marrow and results in the transformation of a B-progenitor cell into mature B cell. Mature B cells have undergone immunoglobulin VDJ gene rearrangement and express a complete IgM antibody molecule on the cell surface [4]. After release from the bone marrow, antigen naïve mature B cells are exposed to antigen in the interfollicular area of the secondary lymphoid tissues. Majority then migrate into the germinal center. Mature antigen exposed B cells proliferate in the center of a primary follicle to form the germinal center. The centroblasts mature into centrocytes as they transition into light zone of the germinal center. In the germinal center, B cell undergoes class-switch recombination and somatic hypermutation. Centroblasts are thought to give rise to germinal center B cell (GCB) DLBCL. After transition through the germinal center, B cells can become memory cells or plasmablasts which undergo further development to become plasma cells. Plasmablasts are thought to give rise to activated B cell

DLBCL increased 1.4% per year during the same time-period.

diseases also have a higher incidence of DLBCL [4].

1.2. Etiology and risk factors

40 Hematology - Latest Research and Clinical Advances

2. Pathology and biology

2.1. Pathogenesis

like (ABC) DLBCL [4].

The cell of origin classification (COO) has been the most significant development in the understanding of DLBCL biology. The gold standard test to identify COO is Gene expression profiling (GEP). GEP divides DLBCL into germinal center B cell (GCB), activated B cell (ABC) or unclassifiable (Type 3) types. GCB DLBCL is derived from normal GC centroblasts. Various mechanisms have been elucidated that drives pathogenesis of these subclasses based on COO [5]. GCB like tumors contain the (14;18) translocation (IgH/BCL-2 fusion gene) typical of follicular lymphoma and amplifications of the locus on chromosome 2 which codes for the c-Rel oncogene. BCL2 is dysregulated by t (14;18), PTEN deletions and amplification of miR-17-92 leading to deregulation of PI3K/mTOR pathway which are activated further promoting cell survival, proliferation and growth. GCB is further characterized by gain or amplification of MDM2, a negative regulator of the tumor suppressor p53 as well as deletions of the known tumor suppressor genes TP73 and ING1 that lead to genomic stability. The GEP of ABC DLBCL suggests that it is derived from B cells that are in the process of differentiating into plasma cells. Most of the genes expressed by normal GCB cells are down regulated in ABC DLBCL. There is upregulation of many genes normally expressed by plasma cells including XBP-1, amplification of BCL2, CD79A/CD79B ITAM mutation/deletion leading to chronic active BCR signaling, CARD11/A20 mutation, MYD88 mutation, STAT3 activation which lead to NFkB activation, FOXP1 activation. ABC-like DLBCL is associated with loss of 6q21, trisomy 3, and gains of 3q and 18q21–22. This locus on 6q encodes suppressor gene PRDM1 (Blimp-1) which is a master regulator in the differentiation of mature B lymphocytes to plasma cells, loss of which may lead to inhibition of terminal differentiation. ABC DLBCL has high expression and constitutive activity of the nuclear factor kappa B (NF-ĸB) complex involved in the B cell receptor signaling pathway. This classification not only defined subgroups with distinct biology and pathogenesis but also identified groups of patients with different outcomes after treatment [4]. In a study done at British Columbia looking at outcomes in patients treated with R-CHOP, there were 56% patients with GCB subtype and 32% with ABC subtype. The ABC group experienced significantly inferior outcomes as compared to the GCB group [6]. A third type, type III DLBCL (Unclassifiable as per GEP profiling) is also identified in which tumor cells have gene expression profiles that do not resemble either germinal center or post germinal center B cells [4, 7].

#### 2.3. Double-hit lymphomas/double expressor

DLBCL with MYC gene rearrangement in addition to BCL2 and/or BCL6 gene rearrangements by Fluorescent in Situ Hybridization (FISH) or standard cytogenetics are known as double-hit lymphomas. MYC is rearranged in 5–15% of DLBCL and is frequently associated with BCL2 or to a lesser extent, BCL6 translocation. They are more aggressive and have a much poorer prognosis. Double-hit lymphomas are now recognized as a distinct entity in the 2016 WHO classification [8]. MYC and BCL2 are the key regulators of cellular proliferation and apoptosis, respectively. Dysregulation of MYC and BCL2 can cause chromosomal translocation or gene amplification but it may also occur by transcriptional upregulation downstream of NFkB pathway signaling [9]. This leads to a highly aggressive clinical behavior with very high Ki67 and overlapping pathologic features with Burkitt's lymphoma. These patients have an inferior prognosis compared to those with DLBCL without these mutations. FISH for MYC, BCL2 and BCL6 gene rearrangements should be tested for patients with expression of MYC and either BCL2 or BCL6 by IHC and a GCB like immunophenotype to identify these double- (or triplehit) lymphomas [10]. They have very poor outcomes with standard R-CHOP-based chemotherapy [11]. While the cases identified by FISH positive results are called double- (or triplehit) lymphomas, those patients with high expression of MYC and BCL2 protein by Immunohistochemistry (IHC) alone in the absence of gene rearrangements by FISH are labeled as double expressor and they seem to have an intermediate prognosis.

noncleaved cells, with round to oval nuclei with multiple nucleoli and thin rim of basophilic cytoplasm, the immunoblasts are larger cells with more prominent nucleoli and abundant cytoplasm. Other less common cytological variants include multilobated and anaplastic forms. However the clinical significance of less common forms is debatable. Due to inter- and intra-observer variability in the characterization of DLBCL based on the appearance of tumor cells, all the morphological subtypes are grouped as one category in the current WHO classification except for plasmablastic lymphoma. The plasmablastic lymphoma displays immunophenotype characteristics which allows its distinction from other morphological subtypes of DLBCL (Figures 1–3). Histopathological and immunohistochemistry slides of diffuse large B cell Lymphoma showing characteristics of DLBCL of germinal center origin (GC) in Figure 1 and activated B cell

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The revised WHO classification 2016 [8] classifies DLBCL NOS into GCB and ABC/non-GC subtypes based on cell of origin. The use of IHC algorithm is accepted [8]. We also now have a

Figure 2. Lymph node sections showing ABC type DLBCL (a) The H&E stained sections showing diffuse infiltration of large atypical lymphoid cells with irregular nuclear contours and vesicular chromatin (X40 magnification). On immunohistochemistry cells are (b) CD20 positive (X20 magnification) (c) MUM1 positive (X20 magnification) (d) CD 79A positive

origin (ABC) in Figure 2.

2.5. WHO classification

(X20 magnification).

In a study on 193 patients who were double expressor the 3 year OS rate was 46 vs. 75% and the 3 year PFS rate was 46 vs. 65% [11].
