Diffuse Aggressive B-Cell Lymphomas

Histopathology

By definition, the malignant cells proliferate and infiltrate in a diffuse pattern. Usually there is complete effacement of the lymph node architecture, although occasionally there is partial involvement. In the lymph node, partial involvement can manifest as interfollicular or sinusoidal infiltration. Sclerosis can be seen in some cases and may even impart a pseudonodular pattern. Lack of true follicle formation can be confirmed with immunostaining showing lack of a follicular dendritic cell meshwork that is typical of follicular lymphomas. In extranodal sites, obliteration of the normal epithelial components is seen. In a mucosal site, such as the stomach, epithelial invasion by large cells with formation of lymphoepithelial lesions is sometimes seen. Although this is a feature of mucosa-associated lymphoid tissue (MALT)-type lymphomas, such LBCLs are currently considered and classified as extranodal DLBCL, NOS. In such cases an effort should be made to determine whether a conventional low-grade lymphoma (MALT lymphoma) is also present in the biopsy, indicating transformation rather than a de novo DLBCL, NOS.

DLBCL, NOS is cytologically heterogeneous, with morphologic variants recognized. Common to all is a nuclear chromatin pattern that is open and vesicular compared to the condensed chromatin pattern of small B-cell lymphomas. Nucleoli may be single or multiple and variably sized. Cells are usually large (compared to the nuclear size of a benign histiocyte) but occasionally can be intermediate in size (approximately the same size as a benign histiocyte). Three morphologic variants are recognized in the WHO classification, but it is not required to report them. They are the centroblastic, immunoblastic, and anaplastic variants ( Figs. 8.1 and 8.2 ). Centroblastic cells are characterized by round to oval large nuclei with vesicular chromatin and multiple (two to four) small nucleoli that are closely applied to the nuclear membrane. Cytoplasm is scant, but visible, particularly on Giemsa staining, in which it has a basophilic quality. In some cases, polylobated nuclei predominate as opposed to the typical round contours. The occurrence of polylobated nuclei typically imparts a smaller cell size and can make distinction from small B-cell lymphomas difficult. Multilobated cells are common in some extranodal lymphomas, such as primary lymphoma of bone. Immunoblasts are large cells with a single, prominent central nucleolus and more abundant basophilic cytoplasm. Plasmacytoid differentiation can be seen. Diagnosis of the immunoblastic variant is appropriate when more than 90% of cells are of this type. Some studies have suggested a worse outcome for patients with this variant. The anaplastic variant is composed of sheets of anaplastic, atypical cells with bizarre multilobated–multinucleated cells that may resemble Reed-Sternberg cells. This term reflects both cytologic features and pattern of growth. For example, anaplastic DLBCL, NOS may grow within lymph node sinuses, reminiscent of an epithelial tumor. Pleomorphic may be a better term than anaplastic, because the latter can be confused with anaplastic large-cell lymphoma of T-cell lineage.

Other unusual morphologic variants have been described, and it is useful to be aware of them primarily because they can initially be mistaken for other non-hematolymphoid tumors until immunophenotyping is performed. These variants include signet ring cell, microvillus, myxoid, and spindle cell variants ( Fig. 8.3 ).

Approximately 5% to 15% of patients with DLBCL, NOS have bone marrow involvement, and this is associated with an adverse outcome. The histology, however, is highly variable. The pattern can be paratrabecular, interstitial, or diffuse. The diffuse pattern is uncommon and is usually seen when the lymphomatous infiltrate is composed of large cells. The cytology is often discordant (approximately half of the cases), meaning the marrow infiltrate is composed of small lymphocytes or mixed small and large lymphocytes. Studies have shown that patients with concordant large cells in the bone marrow have a poor response to therapy and poor overall survival. This finding is independent of the International Prognostic Index (IPI) score. The presence of discordant marrow involvement at the time of staging (small B cells in the marrow) has been shown to be a predictor of inferior progression-free survival, but it is not independent of the clinical IPI variables. Discordant disease travels with older age, elevated serum lactate dehydrogenase (LDH), advanced stage, and increased number of extranodal sites.

Immunophenotype

Immunophenotypically, the malignant B cells of DLBCL, NOS express the pan B-cell markers CD19, CD20, CD22, CD79a, and PAX5 in the vast majority of cases. Although lack of CD20 expression is rarely encountered in pretreatment cases, and CD20 may be lost in cases occurring following rituximab therapy. Beyond this DLBCL, NOS is an immunophenotypically heterogeneous disease. CD10 is expressed in 25% to 50% of cases, BCL6 is expressed 50% to 90% of cases, and MUM1 is expressed in 35% to 65% of cases. Surface immunoglobulin (Ig) is expressed in most cases, but in approximately 10% of cases it is undetectable by flow cytometry. Other variably expressed antigens include CD30 (10% to 40% of cases), CD5 (<10% of cases), and cyclin D1 (~2% of cases with weak and focal staining). Expression of Ki-67 is highly variable and can range from 30% to more than 95%.

Gene expression profiling (GEP) has shown that DLBCL, NOS can be classified into prognostically significant subtypes based on the cell of origin (COO)—namely, germinal center B-cell type (GCB) and activated B-cell (ABC) type—with the ABC-type DLBCL, NOS exhibiting a different biology and worse overall response to cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab (R-CHOP) therapy compared to the GCB type. For this reason the current WHO classification requires the identification of the COO subtype. Until recently GEP has required the use of fresh frozen tissue and has not been widely used in clinical laboratories. However, algorithms based on immunohistochemical (IHC) staining have been devised that can be routinely performed and can differentiate the COO subtypes of DLBCL, NOS with close concordance to the GEP classification scheme. The Hans algorithm uses the expression pattern of CD10, BCL6, and MUM1 and has approximately 80% to 86% concordance with the GEP classification. The Choi algorithm uses the expression pattern of CD10, BCL6, MUM1, GCET1, and FOXP1 and has approximately 93% concordance with the GEP classification ( Fig. 8.4 ). Another approach referred to as the tally algorithm uses a combination of IHC antibodies but without regard to order and shows the highest concordance with GEP. Germinal center markers (CD10 and GCET1) and activated B-cell markers (MUM1 and FOXP1) are scored (≥30% of cells), and cases are classified based on whether more germinal center or ABC markers are present. In case of a tie, LMO2 is used to determine subtype (≥30% + Cells = GCB; <30% + Cells = non-GCB).

MYC and BCL2 are two other proteins that are also variably expressed in DLBCL, NOS and approximately 25% to 30% of cases express both when using cut-offs of ≥40% and ≥50% for MYC and BCL2, respectively. These so-called dual expressers (also sometimes referred to as “double expressers”) are enriched in the ABC type and are associated with worse response to R-CHOP therapy, independent of the IPI, COO type, and underlying MYC and BCL2 gene rearrangements. Dual expression of MYC and BCL2 also defines a group of patients at high risk of central nervous system relapse. Importantly, dual expression of MYC and BCL2 is not a surrogate marker for dual rearrangement of the MYC and BCL2 genes (so-called double-hit lymphoma; see discussion below). Molecular genetic testing by fluorescence in situ hybridization (FISH) analysis is required to exclude underlying gene rearrangements of the MYC and BCL2 genes.

Molecular Genetics

Because DLBCLs are monoclonal B-cell neoplasms, Ig genes are usually clonally rearranged whereas T-cell receptor genes are typically in the germline configuration.

Our understanding of the molecular heterogeneity of DLBCL, NOS has been advanced by gene expression profiling. DLBCL, NOS can be segregated into at least three subgroups based on similarities to normal cell types: GCB (45% to 55%), ABC (35% to 45%), and the unclassifiable “Type 3” group (10%), which shows a probability of less than 90% based on gene expression of belonging to either GCB or ABC subtypes ( Fig. 8.5 ). The IHC classifiers use binary GCB versus ABC/non-GCB categories only, and thus the unclassifiable cases may be variably distributed within these two groups, making these algorithms less biologically meaningful. However, GEP has recently become possible in formalin-fixed paraffin-embedded (FFPE) tissue specimens, and assays, such as Lymph2Cx, have been developed to determine COO in DLBLC, NOS using FFPE with near equivalent concordance to the fresh frozen tissue–based assays. These FFPE-based assays are likely to find their way into routine clinical practice in the near future, particularly if the results of ongoing clinical trials with targeted drugs indicate that therapeutic decisions should be made based on COO. Mini-gene predictors, using two to six genes, have also been described that appear to be independent of the clinical IPI variables.

The cell-of-origin distinctions also have underpinnings in biology, with molecular genetics and next-generation sequencing having been helpful in furthering our understanding of the molecular alterations underlying these disease subtypes. GCB-type DLBCL harbors rearrangements of BCL2 exclusively, usually in the form of a t(14;18)(q32q21) ( IGH-BCL2 ), which is seen in approximately 35% to 45% of cases. MYC rearrangements are also largely restricted to the GCB-type occurring in approximately 10% of all DLBCL, NOS cases. Co-occurrence of BCL2 and MYC rearrangements is seen in approximately 5% of all DLBCL cases, called double-hit lymphoma, and is associated with aggressive behavior. DLBCL cases that harbor a double-hit abnormality are re-classified as high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements (discussed later); thus laboratories must have a strategy to find these cases. Some have elected to preform FISH testing on all cases of diffuse aggressive B-cell lymphomas, whereas others have elected screening strategies that involve MYC IHC. GCB-type DLBCL is also enriched with recurrent mutations or deletions in genes associated with epigenetic regulation ( EZH2 , CREBBP , EP300 , MLL2 , MLL3 , MEF2B , and TET2 ). Recurrent mutations in genes associated with B-cell receptor, Toll-like receptor, and NF-κB signaling pathways ( CARD11, CD79A , CD79B , MYD88, TNFPAI3 , and NFKBIE ) are also seen in a minority of cases. In addition, recurrent mutations in DNA repair/genomic integrity pathways are also identified ( TP53 ) in 10% to 20% of cases. The EZH2 mutation is exclusive to GCB-type DLBCL, NOS and is found in approximately 20% of cases. It is a gain-of-function mutation that leads to increased expression of the EZH2 protein, which is responsible for methylation of histone H3 at lysine 27 leading to transcriptional repression and gene silencing. ABC-type DLBCL is enriched with 3q27/ BCL6 gene rearrangements as well as gains/amplifications of BCL2 and MYC ; double-hit lymphoma is rare in this type of DLBCL. ABC-type DLBCL, NOS is enriched with recurrent mutations or deletions in genes associated with Toll-like receptor signaling, NF-κB signaling, and B-cell receptor signaling pathways ( MYD88 , TNFPAI3 , CD79A , CD79B , CARD11, and NFKBIE ), although recurrent mutations and deletions in genes related to epigenetic modification ( CREBBP , TET2 , EP300 , MLL2 , and MLL3 ) are occasionally observed. Recurrent mutations in DNA repair/genomic integrity pathways are also identified ( TP53 ) in 10% to 25% of cases. PRDM1/BLIMP1 mutations are seen exclusively in ABC-type DLBCL, NOS and are found in approximately 25% of cases. It is a loss-of-function mutation that prevents terminal differentiation of the malignant B cells.

In aggregate, the survival data and molecular genetic findings underscore the rationale behind distinguishing DLBCL, NOS cases based on cell of origin. Constitutive alterations in specific pathways in these subtypes, such as epigenetic regulation and NF-κB signaling in GCB and ABC-type lymphomas, respectively, support the potential effects of specific biological therapies for patients with GCB versus ABC-type DLBCLs.

Histopathology

Lymph nodes involved by TC/HRBCL show architectural effacement by a diffuse or vaguely nodular cellular infiltrate consisting predominantly (>90%) of small T cells and nonepithelioid histiocytes. Malignant cells are scattered singly among the cellular background, although in some cases they may cluster. They are large, highly atypical cells that are often multilobated with variably prominent nucleoli ( Fig. 8.6 ). In some cases the cells can resemble the lymphocyte-predominant (LP) cells characteristic of nodular lymphocyte predominance Hodgkin lymphoma (NLPHL) or Hodgkin and Reed-Sternberg cells characteristic of classic Hodgkin lymphoma (HL). Some cases may have cells that have more conventional immunoblastic or centroblastic features.

Splenic involvement occurs in a multifocal and micronodular pattern with expansion of the white pulp by an infiltrate of cells similar in composition to that described in the lymph node. The red pulp is not usually involved. Within the liver the lymphomatous infiltrate is confined principally to the portal tracts. Bone marrow involvement in TC/HRBCL occurs at a significantly higher rate than de novo DLBCL, NOS (60%). Involvement is often paratrabecular with a polymorphous infiltrate of small T cells, histiocytes, and scattered large atypical CD20+ B cells. Small B cells are typically scarce.

Immunophenotype

As noted, most cells in the infiltrate are reactive CD3+ T cells or CD68+ histiocytes. Only occasional CD20+ large B cells should be seen, and small CD20+ B cells should be absent or only rarely seen ( Fig. 8.7 ). The immunophenotype of the malignant cells is somewhat heterogeneous. The malignant cells are positive for CD45 and the pan B-cell markers CD20, CD79a, and PAX5; however, expression of CD10, BCL6, BCL2, and EMA is variable. Some have suggested, based on BCL6 expression, that a germinal center origin can be entertained, but others could not replicate these findings. It is likely that TC/HRBCL, as currently described, is heterogeneous, in part because of a lack of precise diagnostic criteria. Many believe that a subset of cases within the category of TC/HRBCL might define a specific entity, closely related to NLPHL. Evidence in support of this theory includes the occurrence of the two lymphomas in the same patient and an identical immunophenotype of large B cells in both (CD20+, CD79a+, BCL6+, EMA+, and CD75+). EBV is absent, as are follicular dendritic cell meshworks when using stains for CD21, CD23, or CD35. Background T cells are typically CD8+ and express cytotoxic markers, including TIA1, granzyme B, and perforin. The distinguishing features between TC/HRBCL and NLPHL rely primarily on the surrounding non-neoplastic microenvironment.

Molecular Genetics

Immunoglobulin genes are clonally rearranged and somatically hypermutated in the majority of cases suggestive of a germinal center B-cell stage of differentiation. Few studies have investigated the molecular genetics landscape of TC/HRBCL, but there appears to be an average of approximately five genetic abnormalities per tumor, with reported abnormalities including gains of 2p11, 4q13, 18q21, Xq, and Xp21 and losses of 9p11 and 17p.

Histopathology

The tumor resembles other DLBCLs cytologically. In a series of 62 PCNSL cases in immunocompetent patients, 87% were centroblastic and 13% were immunoblastic. The pattern of involvement is diffuse with a propensity for perivascular involvement ( Fig. 8.8 ). A diffuse pattern is more readily seen when a large mass is biopsied; perivascular involvement may be the only pattern seen in smaller stereotactic biopsy specimens. Necrosis and surrounding gliosis are commonly associated features.

Immunophenotype

The lymphoma cells express pan B-cell markers CD19, CD20, CD79a, and PAX5. CD10 is expressed in less than 20% of cases, BCL6 in 60% to 80% of cases, and MUM1 in more than 90% of cases. The reported expression of BCL2 is variable but is seen in at least 50% of cases, with dual expression of MYC and BCL2 identified in upward of 80% of cases in some series. Using the Hans algorithm, approximately 80% to 95% of cases are classified as ABC/non-GCB type; this is due to the frequent expression of MUM1. Because many cases co-express BCL6, it has been suggested that this tumor is derived from a B cell in the late germinal center to early post-germinal center stage of development. EBV is usually absent.

Molecular Genetics

Monoclonal immunoglobulin gene rearrangement is present. There are no specific diagnostic molecular genetic abnormalities identified in PCNSL, but abnormalities of 1q21, 6q, 7q, and 14q are reported. Comparative genomic hybridization studies have shown that gains outnumber losses, with chromosome 12q showing the most frequent gains (63%). The most commonly deleted locus is located at chromosome 6q (47%), with 6q21-22 being the commonly deleted region. Rearrangement of the BCL6 gene is also seen in a significant minority of cases (23% to 37%). Gene expression profiling shows that PCNSL is a tumor of late germinal center B cells, and its transcriptional signature closely resembles that of systemic DLBCL, NOS. Using an integrative analysis combining GEP and high-resolution array comparative genomic hybridization, it has been shown that 65% of PCNSLs harbor deletions of 9p21.3 involving CDKN2A as well as a number of previously unrecognized deletions associated with diminished expression of the candidate genes. Several recent studies have identified highly recurrent somatic mutations affecting the NF-κB pathway in the majority of PCNSL, including mutations in MYD88 , TBL1XR1 , and CD79B . These mutations occur with greater frequency than is seen in DLBCL, NOS, likely reflecting the paucity of antigen stimuli from the surrounding immune-privileged environment and serving as a genetic hallmark for this disease.

Histopathology

The tumor infiltrates the dermis and hypodermis and is composed of diffuse sheets of medium to large centroblasts and immunoblasts without admixed small centrocytes ( Fig. 8.9 ). The cells have a characteristic round nuclear shape. There is little stromal reaction, and few infiltrating, small non-neoplastic lymphocytes are present. Mitoses are easily seen. The epidermis is uninvolved in most cases and separated from the tumor by a Grenz zone.

Immunophenotype

The tumor is positive for the pan B-cell markers CD20, CD79a, and PAX5. Most cases (>85%) express BCL2, MUM1, and FOXP1 and lack expression of CD10. BCL6 expression is seen in a variable number of cases. p63 is also variably expressed but seen in most cases. Cytoplasmic IgM with or without coexpression of IgD is reported in virtually all cases. EBV is negative. Follicular dendritic cell meshworks are absent.

Molecular Genetics

Monoclonal Ig rearrangement is present. The tumor lacks t(14;18); however, translocations involving BCL6 , MYC, and IGH are common. Recurring genetic abnormalities include amplification of chromosome 18q21, where the BCL2 and MALT1 genes are located; deletion of chromosome 9p21.3, where the CDKN2A gene is located; deletion of 6q, where the BLIMP1 gene is located; and recurrent somatic mutation of MYD88 , which is seen in approximately 60% to 70% of cases. Amplification of BCL2 is believed to be the mechanism responsible for the frequent expression of BCL2 protein seen in this tumor. Gene expression profiling shows that the tumor is similar to activated B-cell–like DLBCL, NOS.

Histopathology

Epstein-Barr–positive DLBCL, NOS is morphologically heterogeneous. It can be divided into polymorphous and large-cell lymphoma subtypes; however, these are of no clinical significance, and usually both morphologies can be seen in the same specimen when examined thoroughly. In all cases, the tumor consists of a polymorphic or monomorphic infiltrate of immunoblast-like transformed cells and Hodgkin and Reed-Sternberg (HRS)-like cells that efface normal architecture ( Fig. 8.10 ). Inflammatory cells, including small reactive lymphocytes, plasma cells, histiocytes, and epithelioid cells, are admixed in polymorphic areas. Foci of geographic necrosis are also common and reminiscent of posttransplant lymphoproliferative disorder. In younger patients, cases often resemble T-cell/histiocyte-rich large B-cell lymphoma.

Immunophenotype

The malignant cells in EBV+ DLBCL, NOS express the pan B-cell antigens CD20, CD79a, and PAX5 and exhibit a nongerminal center immunophenotype, being negative for CD10 and BCL6 and positive for MUM1. Approximately three fourths of cases express CD30, and this is more commonly seen in Southeast Asian and Latin cases. CD15 is universally absent. PD-L1 is frequently expressed. EBV is present in all cases, typically seen in more than 50% of malignant cells, and best detected by in situ hybridization for EBV-encoded RNA (EBER).

Molecular Genetics

Epstein-Barr virus–positive (EBV+) DLBCL, NOS is a monoclonal disorder, and a clonal rearrangement of the IGH gene is usually detectable. A clonal EBV genome is also usually identified. Rearrangements of BCL2 , BCL6 , and MYC are absent. GEP studies have identified enhanced activity of the NF-κB and JAK/STAT pathways within this disease; however, mutations affecting these pathways ( CD79B , CARD11 , MYD88 ) are only rarely detected, which is suggestive of EBV-mediated activation of these pathways. In addition, gains of 9p24 ( PD-L2 ) have been identified, which promote immune evasion.